The Interactive Fly

Zygotically transcribed genes

Cytoskeleton

  • What is cytoskeleton?

    The Actin-Based Cytoskeleton
  • Molecular requirements for actin-based lamella formation in Drosophila S2 cells
  • GMF promotes leading-edge dynamics and collective cell migration in vivo
  • The core and conserved role of MAL is homeostatic regulation of actin levels
  • WHAMY is a novel actin polymerase promoting myoblast fusion, macrophage cell motility and sensory organ development
  • New insights into the regulatory function of CYFIP1 in the context of WAVE- and FMRP-containing complexes
  • The chromokinesin Klp3a and microtubules facilitate acentric chromosome segregation
  • Absence of the Drosophila jump muscle actin Act79B is compensated by up-regulation of Act88F
  • Nuclear F-actin and myosins drive relocalization of heterochromatic breaks
  • Myosin1D is an evolutionarily conserved regulator of animal left-right asymmetry
  • SETD3 protein is the actin-specific histidine N-methyltransferase
  • Differential lateral and basal tension drive folding of Drosophila wing discs through two distinct mechanisms

    The Microtubule-Based Cytoskeleton
  • Decoding cilia function: defining specialized genes required for compartmentalized cilia biogenesis
  • Microtubule-induced nuclear envelope fluctuations control chromatin dynamics in Drosophila embryos
  • The roles of microtubule-based motor proteins in mitosis: comprehensive RNAi analysis in the Drosophila S2 cell line
  • Misato controls mitotic microtubule generation by stabilizing the tubulin chaperone protein-1 complex
  • A PAR-1-dependent orientation gradient of dynamic microtubules directs posterior cargo transport in the Drosophila oocyte
  • Shot and Patronin polarise microtubules to direct membrane traffic and biogenesis of microvilli in epithelia
  • Digitor/dASCIZ has multiple roles in Drosophila development
  • Scaling of cytoskeletal organization with cell size in Drosophila
  • The Drosophila orthologue of the INT6 onco-protein regulates mitotic microtubule growth and kinetochore structure
  • Tau and spectraplakins promote synapse formation and maintenance through Jun kinase and neuronal trafficking
  • Polarized microtubule dynamics directs cell mechanics and coordinates forces during epithelial morphogenesis
    Genes and proteins of the cytoskeleton and cell motility

    What is cytoskeleton?

    The skeleton of the cell can be thought of as a maze of tubes and ropes. The tubes are composed of the protein tubulin, the basis of the microtubular cytoskeleton, and the ropes are actin, a component of muscle that forms the actin based cytoskeleton. If the tubulin and actin "ropes" are like the rigging of a ship, then the centrioles are analogous to the mast of the ship, providing a central organizing element for the microtubular filaments. Additional elements of the cytoskeleton are present immediately under the cell membrane, and serve a supportive function, like the ribs of a ship, to stretch the analogy.

    Cytoskeleton is an important aspect of cell motility, assuring that a motile cell has a front and back as it moves along a substratum. Dynamic changes in cytoskeleton, in this case the actin based cytoskeleton, take place during differentiation processes. Dorsal closure is one example of a developmental process involving cell motility and the actin based cytoskeleton. Hemipterous is involved in a signaling process that affects cell motility in dorsal closure. The process of dorsal closure is described at the Hemipterous site.

    The region immediately beneath the cell membrane is known as the cortex. Here a completely different set of cytoskeletal elements establish and maintain cell polarity, help to maintain cell shape, and serve to anchor proteins embedded in the cell surface. Thus the cortex has a major role in cell-cell communication mediated by cell surface proteins. The cortex serves as an anchor for Oskar, a key protein in establishing oocyte polarity, and also anchors Prospero and Numb, proteins important in neural cell polarity.

    Microtubules (MTs), built of tubulin, are the highways on which dynein and kinesin motors travel. Microtubules are hollow, cylindrical polymers of alpha and beta tubulin heterodimers. During polymerization, the dimers assemble head-to-tail into typically 13 protofilaments arranged parallel to the long axis of the MT. The asymmetry of the individual dimers imparts an intrinsic polarity to the MT, which is displayed as kinetic differences between the two ends. The plus end, originally defined as the end of the axon's microtubule distal to the cell body, elongates 2-3 times faster than the minus end, or the end proximal to the cell body. Dependent on cell type, MTs may be arrayed in a variety of configurations, and since motors are unidirectional, the arrangements of MTs dictates the effective direction of motor movement. Kinesins are plus end directed motors, while dyneins are minus end directed motors. In mitosis, the minus end of microtubules are associated with centrosomes. Gamma Tubulin is involved in the nucleation of mitotic microtubules. Two antiparallel, overlapping MT arrays are generated with the plus ends of each interdigitating in the overlap zone (or in some cases interacting with chromosomal kinetochores) (Walker, 1993).

    One of the major functions of cytoskeleton carried out by microtubules during mitosis is the equitable distribution of chromosomes to the two poles of the cell. The cytoskeleton is also responsible for structuring the cytoplasm of the cell so that proteins and nucleic acids can be carried from one site to another. The polar distribution of Bicoid mRNA and Oskar mRNA in the oocyte is accomplished through employment of the oocyte microtubular cytoskeleton.

    Cytoplasmic streaming, another function of the microtubular based cytoskeleton, takes place in egg chambers during stage 12 and stage 13. cappuccino and spire are required to repress this microtubule-based ooplasmic streaming in the oocyte and to ensure the proper partitioning of molecular determinants within the oocyte. In mutants, the bundling of the microtubules at the cortex of the oocyte and the streaming of the oocyte cytoplasm occurs prematurely, by stage 8 in oogenesis. It is thought that this movement within the oocyte is necessary to mix the oocyte cytoplasm with the cytoplasm being rapidly added from the nurse cells, by an actin based cytoskeleton mechanism (Theurkauf, 1994). It is likely that subcortical nurse-cell microfilaments play a role in cytoplasmic flow into the oocyte. A nonmuscle myosin is found associated with subcortical actin but not with cytoplasmic networks. These subcortical actin filaments are very sensitve to cytochalasin treatment. Contraction of the subcortical actin could play a role in the bulk movement of nurse cells into the oocyte (Cooley, 1992 and references).

    Prior to late vitellogenesis, characterized by the bulk flow of cytoplasm into the oocyte, during previtellogenesis, a different type of intercellular transport occurs. During this phase, treatment with colchicine, which inhibits microtubular transport mechanisms, does not change transport processes through the ring canals. However, when the microfilament inhibitor cytochalasin B is applied, the transfer of particles through the ring canals is completely inhibited. When an inhibitor of myosin-driven motility is applied to follicles, all movements within the cytoplasm stop. It is therefore thought that cytoplasmic myosin and the actin based microfilament network play a decisive role in particle movements during previtellogenesis (Bohrmann, 1994).

    The actin based microfilament cytoskeleton plays an additional role in a process known as cytoplasmic dumping. At stage 11, the nurse cells dump their contents into the oocyte through cytoplasmic bridges termed ring canals. Microfilament bundles form in the nurse cells during this process and are apparently required to hold the nurse cell nuclei in place so that they do not obstruct the ring canals and allow rapid flow of nurse cell cytoplasm into the oocyte. Mutants in chickadee, quail and singed affect actin bundle formation. Profilin, encoded by chickadee, is presumably required for the polymerization of the actin filaments that compose the bundles (Cooley, 1992), while a villin-related protein encoded by quail and a fascin-related protein encoded by singed are thought to be required to cross-link the actin filaments to form the bundles. Two components of the actin-lined ring canals have also been identified - an adducin-like protein encoded by hu-li tai shao and a protein containing scruin repeats encoded by kelch (Manseau, 1996 and references).

    It appears that there is an interaction between the actin and tubulin based components of cytoskeleton. Profilin, encoded by chickadee, a component of the actin based cytoskeleton, physically interacts with Cappuccino, involved in the microtubule based cytoskeleton. Mutants in chickadee resemble cappuccino in that they fail to localize Staufen protein and Oskar mRNA in the posterior pole of the developing oocyte. A strong allele of cappuccino has multinucleate nurse cells, similar to those described for chickadee (Manseau, 1996).

    Molecular requirements for actin-based lamella formation in Drosophila S2 cells

    Cell migration occurs through the protrusion of the actin-enriched lamella. The effects of RNAi depletion of approximately 90 proteins implicated in actin function on lamella formation have been investigated in Drosophila S2 cells. Similar to in vitro reconstitution studies of actin-based Listeria movement, it has been found that lamellae formation requires a relatively small set of proteins that participate in actin nucleation (Arp2/3 and SCAR), barbed end capping (capping protein), filament depolymerization (cofilin and Aip1), and actin monomer binding (profilin and cyclase-associated protein). Lamellae are initiated by parallel and partially redundant signaling pathways involving Rac GTPases and the adaptor protein Nck, which stimulate SCAR, an Arp2/3 activator. RNAi of three proteins (kette, Abi, and Sra-1) known to copurify with and inhibit SCAR in vitro leads to SCAR degradation, revealing a novel function of this protein complex in SCAR stability. These results have identified an essential set of proteins involved in actin dynamics during lamella formation in Drosophila S2 cells (Rogers, 2003).

    Under routine culture conditions, S2 cells display a roughly spherical morphology with a diameter of ~10 µm. These cells are not motile and exhibit no obvious morphological polarity, but time-lapse microscopy of cells expressing GFP-actin reveals that their surfaces are dynamic and continuously extend and absorb membrane ruffles. S2 cells may be induced to undergo a dramatic change in their morphology when plated on glass coverslips coated with the lectin concanavalin A (con A). Within 20 to 30 min after plating on this substrate, these cells avidly attach, flatten, and spread to adopt a discoid morphology of approximately double their normal diameter (20 µm). Spread cells resemble a 'fried egg' with a domed central region containing the nuclei and majority of organelles surrounded by a thin, organelle-free zone (Rogers, 2003).

    To better understand the organization of actin in S2 cells, con A-adhered S2 cells expressing GFP-actin were fixed and stained with Texas red X-phalloidin, a probe that selectively binds to filamentous actin. When examined by fluorescence microscopy, most S2 cells (90%) exhibited a highly developed, radially symmetrical actin cytoskeleton that could be divided into three zones: a dense peripheral network at the extreme periphery of the cells (~1 µm wide), a second central zone (4-6 µm wide) of lower actin density composed of filaments, and a third circular bundle of filaments that surrounded the nucleus. Arp3, cofilin, and capping protein were enriched in this first actin-dense zone at the leading edge, especially at membrane ruffles. Enabled/VASP was further restricted to the extreme edge of the periphery (<1 µm). In contrast, immunolocalization of profilin/chickadee revealed puncta that were distributed throughout the cell and particularly abundant in the inner nuclear and organelle-rich domain. These puncta were not associated with adhesion structures; immunofluorescent staining against phosphotyrosine failed to stain the ventral surface of the cells. The distributions of these well-characterized actin-binding proteins are generally similar to those described in other cell types that form actin-rich lamellae (Rogers, 2003).

    A small proportion (<10%) of cells did not exhibit such well-spread lamella but rather possessed numerous and dynamic filopodia evenly spaced around their circumference. These short (1-2 µm) projections exhibited cycles of elongation and retraction. Interconversion of the two cell morphologies has not been observed. RNAi studies were restricted to the predominant population of cells that spread and form lamella on the con A-coated surfaces (Rogers, 2003).

    Also, actin dynamics were directly visualized in the lamellae of living S2 cells expressing GFP-actin after plating on con A. Membrane ruffles formed at the cell periphery, folded back toward the cell center, and ultimately fused with the dorsal surface of the cell. Such ruffling activity was more or less symmetrically distributed around the cell, and polarized morphologies or cell movement was rarely observed. At sufficiently low levels of protein induction, a speckled pattern of GFP-actin was observed, and time-lapse imaging revealed a centripetal flow of actin from the periphery toward the center of the cell at a rate of ~4.0 ± 0.44 µm/min, which is somewhat faster than described in other systems, such as migrating fibroblasts or neuronal growth cones. In summary, imaging of actin and actin-binding proteins indicates that con A-induced spreading of S2 cells constitutes an attractive model system for understanding the molecular basis of lamella formation (Rogers, 2003).

    To dissect the molecular basis of lamella formation, the susceptibility of S2 cells to RNAi was exploited to identify proteins involved in this process. A candidate list of ~90 proteins implicated in aspects of actin function or in cell motility during neuronal development and dorsal closure during Drosophila embryogenesis was compiled (Rogers, 2003).

    DNA microarray analysis demonstrated that only five genes in this list are not expressed above background levels in S2 cells. Since very low expressing genes nevertheless may be important for cell function, these genes were still subjected to RNAi analysis. A 7-day RNAi treatment was used to deplete proteins before assaying the cells for lamella formation on con A-treated coverslips. Filamentous actin was visualized with rhodamine-phalloidin, and DNA was stained with DAPI to screen for multiple nuclei reflecting cytokinesis defects. For every treatment, at least 500 cells were examined. The efficacy of the RNAi treatments was verified by immunoblotting extracts from dsRNA-treated cells using a panel of antibodies to 13 proteins. Immunoblotting for those tested revealed that RNAi reduced protein expression by at least 90% of endogenous levels and in many cases was not detectable. This immunoblot analysis included five proteins for which RNAi did not elicit an obvious phenotype. Greater than 90% reduction in the levels of 10 motor proteins subjected to RNAi was achieved and no case was encountered where RNAi had failed to reduce protein levels. It is, therefore, speculated that dsRNAs against proteins that could not be quantitated most likely produced a similar degree of inhibition (Rogers, 2003).

    Of the ~90 genes tested, RNAi produced obvious aberrant morphologies in 19 cases. The observed defects can be categorized into seven phenotypic classes that will be described below (Rogers, 2003).

  • Class 1: p20 subunit of Arp2/3, SCAR, kette, Abi, and Sra-1

    The Arp2/3 complex was inactivated by targeting its crucial p20 subunit (Arc-p20 ), which mediates protein-protein interactions within the Arp 2/3 complex and, therefore, is essential for stability and actin-nucleating activity. After p20 RNAi, >90% of S2 cells exhibited a striking morphological defect when plated on con A. Instead of the circular, symmetrical shape usually induced on this substrate, p20-depleted cells adopted a stellate, radially asymmetrical cell morphology. Phalloidin staining revealed that these cells rarely formed lamellae; instead filamentous actin was enriched in the distal tips of a variable number of tapered projections. The presence of actin filaments could be due to residual Arp2/3 or to alternative actin-nucleating activities. In addition, actin filaments were sometimes observed to run radially from the center of the cell body along the lengths of these projections. These processes were also enriched in microtubules that often extended to their distal regions. The frequency of multinucleate cells was approximately the same as control cells, indicating that inhibition of Arp2/3 does not affect cytokinesis (Rogers, 2003).

    Cells contain actin nucleation-promoting factors that activate the Arp2/3 complex. Genetic analysis in Drosophila has shown that one of these factors, SCAR, is essential for numerous actin-based processes during development, while WASP, another activator, mediates a subset of Arp2/3 functions in neuronal cell fate determination. WASP RNAi did not alter cell morphology or actin organization in S2 cells. In contrast, RNAi against SCAR exactly duplicated the morphological defects observed with RNAi of the p20 subunit of Arp2/3 in >80% of the cells. Interestingly, RNAi for three proteins (Kette, Sra-1, and Abi) that were recently identified to copurify with SCAR produced a phenotype indistinguishable from SCAR or p20. Thus, it is concluded that lamella formation in S2 cells is a SCAR-Arp2/3-dependent process (Rogers, 2003).

  • Class 2: profilin and cyclase-associated protein

    The second category of RNAi-induced morphological defect was typified by inhibition of profilin (Chickadee), an actin monomer-binding protein. After this treatment, >85% of cells failed to spread on con A and instead retained their spherical shape. Phalloidin staining was diffuse throughout these cells, however, individual filaments could not be resolved. These cells also were defective in cytokinesis, as revealed by the high incidence of multiple nuclei (39%). A similar morphology also was generated by RNAi against cyclase-associated protein (CAP/Act up), another monomeric actin-binding protein that plays an important role in actin organization in Drosophila. When bound to monomeric actin, profilin acts to restrict actin incorporation to the barbed-end of actin filaments and mediates exchange of ADP for ATP. It is speculated that the accumulation of f-actin in profilin and CAP RNAi cells, along with the failure to form lamellae, may reflect nonproductive polymerization of actin filaments from both the barbed and pointed ends (Rogers, 2003).

  • Class 3: cofilin and Aip1

    The actin-binding protein cofilin/Twinstar is essential for actin-based functions in many cell types, and in vitro and in vivo studies indicate a role for cofilin in actin filament severing and turnover. Inhibition of cofilin by RNAi prevented S2 cell spreading on con A in >95% of treated cells. These cells retained their spherical morphology, and phalloidin staining revealed a dramatic cortical accumulation of filamentous actin as well as a wrinkled "raisin-like" texture to the surface of the cell. The abnormal accumulation of filamentous actin within the cells suggests that actin turnover is inhibited in S2 cells depleted of either of these two proteins. Cofilin-inhibited S2 cells exhibited a high incidence of multinucleate cells, implicating a role in cytokinesis. This morphology and actin distribution was mimicked by RNAi inhibition of Aip1, a protein that acts cooperatively with cofilin in disassembling actin in Xenopus and budding yeast. Aip1 also produced a cytokinesis defect. These results indicate that both cofilin and Aip1 are essential for actin remodeling during lamella formation and that, despite the similarities in cell morphology produced by RNAi against either of them, these two proteins have distinct roles in actin regulation (Rogers, 2003).

  • Class 4: slingshot

    Slingshot is a protein phosphatase that activates the actin-severing activity of cofilin; loss-of-function experiments in Drosophila have demonstrated that tissues mutant for slingshot exhibit abnormal accumulations of f-actin. S2 cells treated with dsRNA to inhibit slingshot are able to attach and spread efficiently on con A. However, the lamellae in >50% of these cells exhibited structural abnormalities as compared with controls. The distribution of f-actin was uniformly dense from the cell periphery to the center of the cell and did not show the typical distal enrichment commonly observed in spread S2 cells. Cells exhibiting this morphology typically had prominent radial bundles of actin that spanned the entire width of the lamellae. It is speculated that this cellular morphology is produced by a partial loss of cofilin activity, leading to inefficient disassembly of the dendritic array of actin filaments at the rear of the lamellae and thus producing a lamellipod that is radially wider than normal. Cytokinesis defects were not observed in these cells (Rogers, 2003).

  • Class 5: capping protein

    Capping protein is an important regulatory factor that binds to the barbed ends of actin filaments to prevent actin monomer addition. Recent studies have suggested that a functional antagonism between capping protein and enabled/VASP regulates the length and polymerization rate of actin filaments in the lamella. This balance controls the rate of lamella protrusion in motile cells. S2 cells treated with dsRNA against capping protein adher and spread normally, but ~80% had lamellae exhibiting a hyper-ruffled shape. Lamellae in S2 cells lacking capping protein also exhibited an accumulation of filamentous actin at the periphery that extended 2-3 µm inwards from the cell perimeter, as compared with ~1 µm in untreated cells. An explanation for the abnormal lamella morphology has been suggested. In the absence of capping protein, enabled/VASP-mediated actin filament elongation favors the formation of abnormally long filaments at the cell margin. These filaments push against the membrane, fueling protrusion, until compressive forces exceed the flexural rigidity of long filaments, causing them to buckle and the membrane to retract. This hypothesis explains the hyper-ruffled phenotype as well as the accumulation of f-actin at the margin of the cell. No accumulation of multinucleated cells was observed, suggesting that capping protein is dispensable for cytokinesis (Rogers, 2003).

  • Class 6: Cdc42

    A sixth category of morphological defect was produced by depletion of Cdc42 by RNAi. Cdc42, a member of the Rho family of small G proteins, regulates actin organization and is generally thought to mediate the formation of filopodia during cellular migration. Inhibition of Cdc42 prevented formation of a normal lamella in ~50% of the cells. Instead, actin was organized into long, thin processes that projected from the entire periphery of the cell. These processes did not resemble the filopodia that spontaneously form on some S2 cells or that form in response to overexpression of constitutively active Cdc42V12, because they were typically >10 µm in length and possessed a uniform diameter. This morphology is difficult to reconcile with what is known about Cdc42 functions, although a cellular null phenotype for Cdc42 in metazoan cells has not been reported (Rogers, 2003).

  • Class 7: myosin II, Rho1, AcGAP, diaphanous, citron kinase, anillin, scraps, and Rho1

    A seventh category was failure of cytokinesis without inhibition of cell spreading on con A-coated surfaces. Cells in this category (>95%) possessed multiple nuclei and were much larger in diameter than control cells. Phalloidin staining revealed that, despite their larger size, cells were able to form lamellae with normal architecture. Inhibition of Rho1 and its downstream effectors citron kinase, diaphanous, AcGAP, and myosin II typified this defect. Many of these molecules were recently identified in a similar S2-based RNAi screen for genes specifically involved in cytokinesis, but Aip1, CAP, citron kinase, and diaphanous were not tested in this study (Rogers, 2003).

    In addition to producing cytokinesis defects, however, cells depleted of cytoplasmic myosin II sometimes failed to form normal lamellae, in addition to producing cytokinesis defects. These cells contained abundant filamentous actin, as judged by phalloidin staining, but the actin cytoskeleton displayed an overall lack of organization with filaments criss-crossing the width of the cell in an apparently random manner. These results reveal a role for myosin II in the organization of actin in the lamellae (Rogers, 2003).

    The SCAR-associated proteins kette, Sra-1, and Abi prevent degradation of SCAR: Native SCAR exists in a trans-inhibited state in a complex with the Kette, Sra-1, and Abi proteins. Given the demonstrated role of these proteins in suppressing SCAR activity in vitro, it was surprising that RNAi-mediated depletion of Sra-1, Abi, or Kette resulted in a SCAR-like phenotype rather than in excessive actin polymerization. One hypothesis to account for these observations was that SCAR is either not localized at the membrane or degraded in the absence of members of the kette-Sra-1-Abi complex. To test these ideas, Kette, Abi, or Sra-1 RNAi-treated cells were stained with anti-SCAR antibodies and the overall staining intensities were observed to be reduced or eliminated. Quantitative immunoblotting was performed and it was found that Kette, Sra-1, and Abi RNAi treatments caused a considerable reduction of SCAR levels in S2 cells. Depletion of Abi, Kette, and Sra-1 reduced SCAR protein levels to 34.3 ± 18, 17.3 ± 9.5, and 9.6 ± 2.6%, respectively. In contrast, cells treated with dsRNA versus Diaphanous did not show reduced SCAR levels. From these observations, it is concluded that the kette-Sra-1-Abi complex is required for SCAR stability (Rogers, 2003).

    The small G proteins Rac1/2 and Mtl and the adaptor protein Nck mediate cell spreading and lamella formation via two independent pathways: Activation of SCAR proteins is generally thought to be mediated by Rac GTPases. However, RNAi of Drosophila Rac 1, Rac 2, and the Rac-like protein Mtl did not prevent cell spreading or lamella formation. Genetic evidence has demonstrated that these small G proteins are functionally redundant in many tissues in the fly. Furthermore, in vitro experiments show that the inhibitory SCAR complex can be activated either by Rac1 or the SH2-SH3 adaptor protein Nck. To test whether this is the case in S2 cells, cells were treated with dsRNA designed to simultaneously inhibit Rac1 and Rac2 (Rac1/2) and Mtl for 7 d. Unexpectedly, phalloidin staining revealed that these dsRNA-treated cells spread and formed a normal lamella when plated on con A (Rogers, 2003).

    Next, the in vitro finding that either Rac or Nck is able to activate SCAR was tested by simultaneously inhibiting various combinations of Rac1/2, Mtl, and the Drosophila orthologue of Nck (Dreadlocks). This treatment produced three different cell morphologies: cells with normal lamellae, cells that spread but exhibited an abnormal serrated edge, and cells exhibiting the stellate morphology observed after RNAi of Arp2/3 and SCAR. The serrated cell shape likely represents an intermediate morphology caused by incomplete inhibition of the signaling pathway. In control RNAi-treated cells, >95% of the cells formed normal lamellae with <5% of the cells exhibiting a serrated cell margin. Stellate cells were never observed in control cultures. Inhibition of Nck alone by RNAi caused a reduction in the number of S2 cells with normal lamellae to ~65% and an increase in serrate cells to ~30% and stellate cells to 5%. Double RNAi treatments to inhibit Nck and Rac1/2 or Nck and Mtl produced moderate increases in the number of serrate cells compared with Nck alone. However, simultaneous application of dsRNAs against Nck, Rac1/2, and Mtl induced a dramatic increase in serrate and stellate cells to ~30% and ~20%, respectively. These observations suggest that the Rac-like proteins and Nck are partially redundant for lamella formation in S2 cells (Rogers, 2003).

  • GMF promotes leading-edge dynamics and collective cell migration in vivo

    Lamellipodia are dynamic actin-rich cellular extensions that drive advancement of the leading edge during cell migration. Lamellipodia undergo periodic extension and retraction cycles, but the molecular mechanisms underlying these dynamics and their role in cell migration have remained obscure. This study shows that glia-maturation factor (GMF), which is an Arp2/3 (see Arpc1) complex inhibitor and actin filament debranching factor, regulates lamellipodial protrusion dynamics in living cells. In cultured S2R+ cells, GMF silencing resulted in an increase in the width of lamellipodial actin filament arrays. Importantly, live-cell imaging of mutant Drosophila egg chambers revealed that the dynamics of actin-rich protrusions in migrating border cells is diminished in the absence of GMF. Consequently, velocity of border cell clusters undergoing guided migration was reduced in GMF mutant flies. Furthermore, genetic studies demonstrated that GMF cooperates with the Drosophila homolog of Aip1 (flare) in promoting disassembly of Arp2/3-nucleated actin filament networks and driving border cell migration. These data suggest that GMF functions in vivo to promote the disassembly of Arp2/3-nucleated actin filament arrays, making an important contribution to cell migration within a 3D tissue environment (Poukkula, 2014).

    The core and conserved role of MAL is homeostatic regulation of actin levels

    The transcription cofactor MAL (Myocardin-related transcription factor or Mrtf) is regulated by free actin levels and thus by actin dynamics. MAL, together with its DNA-binding partner, SRF, is required for invasive cell migration and in experimental metastasis. Although MAL/SRF has many targets, this study provides genetic evidence in both Drosophila and human cellular models that actin is the key target that must be regulated by MAL/SRF for invasive cell migration. By regulating MAL/SRF activity, actin protein feeds back on production of actin mRNA to ensure sufficient supply of actin. This constitutes a dedicated homeostatic feedback system that provides a foundation for cellular actin dynamics (Salvany, 2014).

    The transcription cofactor MAL is regulated by cellular actin dynamics and confers this regulation on the activity of its DNA-binding partner, SRF. Free G-actin directly binds to MAL via RPEL motifs at the N terminus of MAL and negatively regulates its activity. This regulation is conserved from mammals to insects (Somogyi 2004). In the physiological context of the animal, the function of MAL and related proteins (MRTF-A and MRTF-B in mammals and mal-d/mrtf in Drosophila) appears conserved as well, related to active changes in the cytoskeleton. For example, initiation of invasive cell migration is essentially abolished in the absence of MAL or SRF in border cell migration in the Drosophila ovary (Somogyi 2004) or in mouse bipolar neurons exiting the subventricular zone of the brain and for cancer cells in three-dimensional (3D) invasion assays and experimental metastasis (Medjkane, 2009; Salvany, 2014).

    Actin is a very abundant and exquisitely conserved protein in eukaryotic cells. Cycling of actin between G-actin and F-actin pools is controlled by a vast array of regulators, which have been the focus of considerable attention. Actin protein synthesis is also a regulated process. Actin mRNA localization and localized protein synthesis are important for cell migration and axonal growth and guidance. This study presents evidence that regulation of actin gene transcription is itself a key regulatory step in the control of invasive cell migration. Using a genome-wide approach, Actin5C was identified as a major target of the MAL/SRF transcription factor complex. Loss or reduction of MAL activity impairs invasive migration in Drosophila and human cancer cell models. It was found that restoring actin expression can be sufficient to replace the requirement for MAL to support invasive migration in these models. Thus, actin and MAL form a conserved homeostatic feedback system to ensure that actin levels are appropriate to support the actin dynamics required for complex cell behavior (Salvany, 2014).

    To understand why MAL is essential for invasive migration and whether the apparent similarity of its role in different organisms reflects a conserved molecular mechanism, attempts were made to identify Drosophila MAL target genes at the genome level. A combination was used of chromatin immunoprecipitation (ChIP) and gene expression analysis and focus was placed on MAL, as SRF has functions independent of MAL/mrtf in mammals and flies. To perform analysis in the relevant tissue context, a mutant in the single Drosophila mal-d gene was used that abolishes expression in the ovary (mal-dΔ7) (Somogyi, 2004). This specifically blocked invasive migration by border cells and caused overall ovary growth defects. Ubiquitous expression of a GFP-tagged version of Mal-d completely rescued the mal-d mutant phenotypes, showing that the fusion protein provides normal Mal-d function. The MAL-GFP transgene also allowed efficient identification of MAL-GFP-bound regions in the Drosophila genome by immunoprecipitation with GFP. Key MAL-GFP-bound regions were confirmed in independent samples, and their recovery was dependent on the presence of the transgene. In parallel, genome-wide expression analysis of wild-type versus mal-d mutant ovaries identified genes whose expression was dependent on MAL. These two complementary data sets allowed genome-wide identification of MAL target genes (Salvany, 2014).

    There were two key findings from this genome-wide analysis. First, only a small number of genes qualified as direct MAL targets, with MAL-GFP bound to the regulatory region and a significant decrease of mRNA expression in the mutant: the cytoplasmic Actin5C gene and five other genes, most encoding heat-shock proteins. A few additional genes encoding cytoskeletal proteins or regulators were identified as potential targets in the MAL-GFP-bound set. Second, three of the four most enriched MAL-bound regions in the whole genome were associated with Actin5C. The MAL-bound regions were conserved in other species, suggesting functional importance, and in each case, these sites bracketed gene-free upstream regions of ~10 kb. The latter is noteworthy because the Drosophila genome is dense, with most 'housekeeping genes' closely spaced, and large regulatory regions generally confined to developmental regulators. These findings focused the attention of this study on Actin5C (Salvany, 2014).

    The gene expression arrays indicated a modest decrease of Actin5C mRNA levels in the mal-d mutant. Quantitative RT–PCR of carefully matched ovary samples showed a twofold decrease of mature Actin5C mRNA and a threefold to fourfold decrease of primary transcript in mal-d mutants, with no change in the closely related, but less highly expressed, Actin42A gene. In FACS-sorted migratory cells, including border cells, Actin5C levels were fourfold reduced. Analysis of the Actin5C promoter and upstream region in luciferase reporter assays showed robust promoter activity and 200-fold to 600-fold up-regulation by coexpression of SRF and activated MAL (mal-d ΔN -). Conversely, knockdown of MAL or SRF by RNAi gave 50-fold to 100-fold reduction in basal Actin5C expression. The Actin5C regulatory region also conferred responsiveness to drugs affecting actin dynamics, specifically induction by Cytochalasin D and inhibition by Latrunculin B, as observed for mammalian MAL/SRF-regulated genes. This type of reporter assay generally reveals regulatory potential at the transcriptional level. The large magnitude of regulation of the Actin5C promoter/enhancer region by MAL/SRF is consistent with the abundant binding of MAL to this region. In vivo, compensatory mechanisms may contribute to sustaining Actin5C mRNA levels upon loss of MAL activity. Thus, MAL and actin dynamics have the potential to regulate Actin5C transcription over a large dynamic range (Salvany, 2014).

    The genomic data raised the possibility that Actin5C might not be just one of many cytoskeletal target genes for MAL regulation but the key target gene. If a transcription factor has one key target gene in vivo, re-expression of this gene should replace the need for the transcription factor. In genetic terms, expression of the target gene should rescue the phenotype of complete loss of function for the transcription factor in specific cells (Salvany, 2014).

    To investigate this hypothesis functionally, the severe defect in invasive migration observed in mal-d mutant border cells was examined. Actin5C is the major cytoplasmic actin gene, and, as expected, mutating it perturbs border cell migration. To determine whether Actin5C was the sole required target gene of MAL, whether re-expression of Actin5C in cells that appear to be null for mal-d (mal-d S2) (Somogyi, 2004) could restore invasive migration was tested. Fly strains were used in which the Actin5C gene has a Gal4-responsive transposon, an 'EP element,' in the promoter region. Surprisingly, migration was indeed restored to normal when Actin5C was activated by Gal4 in mal-d mutant cells. Thus, as long as the Actin5C gene is induced at an adequate level, border cells do not need MAL to invade and migrate (Salvany, 2014).

    In fully mutant ovaries (mal-d&Delta:7 homozygous females), restricted expression of Actin5C in terminally differentiated outer border cells using slbo-Gal4 provided significant but less efficient rescue of migration despite normal expression levels. This suggested that MAL also acts in other cells, consistent with the general oogenesis phenotype. The ORF of Actin5C in a Gal4-reponsive transgene (UAS-Actin-ORF) also provided some rescue of migration, whereas a construct with stop codons present did not. This confirmed that actin protein expression was responsible for the activity of the Actin5C locus in border cells (Salvany, 2014).

    To test whether this finding extended to other tissues, another mal-d mutant phenotype, bent bristles, was examined. Bristles are 'hairs' organized by actin-rich structures and are characteristically defective in mal-d mutants (Somogyi, 2004). This effect of MAL deficiency was also restored to normal by ectopic expression of the Actin5C, accomplished by placing Actin5C cDNA under the control of a heat-shock promoter and rearing at 29°C. Thus, the requirement for MAL in these contexts reflects a specific need for MAL-driven induction of Actin5C expression. Regulation of other targets, direct or indirect, is not required. These findings indicate that the primary role of Drosophila MAL is to regulate actin levels in response to free actin fluctuations, a homeostatic feedback regulation (Salvany, 2014).

    It was next asked whether this role of MAL as regulator of actin homeostasis is conserved in mammalian cells. The cytoplasmic β-actin and γ-actin genes are regulated by MAL/SRF in mammalian cells. As for Drosophila Actin5C, β-actin is essential for embryonic development and proper cell migration, whereas the related γ-actin can be compensated for. However, many other genes are more dramatically regulated by SRF and mrtfs, and some of these are important for cell migration and related functions. It is therefore assumed that MAL and SRF exert their function by regulating a battery of cytoskeletal genes. For SRF, the number of direct target genes is estimated at 200-300. For MAL and MRTFs, this is less clear, as systematic ChIP analysis is missing. Based on the findings in the fly model, the hypothesis was tested that a single target gene (β-actin) could be the key effector of mammalian MAL and that its expression could replace the need for MAL-driven gene regulation in an assay of invasive cell migration (Salvany, 2014).

    The requirement for MAL is most pronounced in cases of active cell shape change and cytoskeletal challenge, such as tissue or matrix invasion (Somogyi, 2004; Medjkane, 2009; Pinheiro, 2011). Therefore, a simple cellular assay was sought that could test the ability of cells to invade into a confined environment. Migration under agarose provides mechanical resistance to movement and has been used to study migration of eukaryotic cells in a constrained space. The assay can easily be adapted to human tumor cells such as MDA-MB-231 breast cancer cells. MAL activity in these cells is provided by mrtf-a and mrtf-b, and reducing their expression severely reduces invasion in an organotypic assay and in experimental metastasis in mice (Medjkane, 2009). Simultaneous knockdown of mrtf-a and mrtf-b in MDA-MB-231 cells by siRNA reduced β-actin transcript levels. It also produced severe attenuation of migration under agarose, confirming that this assay interrogates MAL-dependent cell movement (Salvany, 2014).

    To assess the importance of cytoplasmic actin downstream from MAL, stable cell lines were derived from MDA-MB-231 that allowed the ORF of human β-actin, or an N-terminally Flag-tagged version of β-actin, to be induced from the strong CMV promoter by the TET-ON system. These cell lines showed some baseline expression of the transgenes and six-fold to 10-fold induction upon Tet addition. Under agarose, migration was similar to the parental cell line and not changed by Tet addition. Knockdown of mrtf-a and mrtf-b largely attenuated migration under agarose. Remarkably, ectopic induction of β-actin or Flag-β-actin in the mrtf-a/b-depleted cells rescued migration to control levels. This indicates that human cancer cells require MAL activity to perform invasive migration for the exact same reason that Drosophila border cells do: to regulate cytoplasmic actin gene expression. Regulation of any other potential MAL target genes may not be required for this cell behavior. These experiments rely on siRNA-mediated reduction of mrtf activity; thus, it remains possible that other target genes are important but require only low levels of mrtf activity (Salvany, 2014).

    Elegant experiments have shown how activity of SRF and its transcriptional cofactor, MAL, is regulated by the level of free actin and thereby by the dynamics of the actin cytoskeleton. This study has shown that the key role of MAL is to regulate the expression level of cytoplasmic actin. It is suggested that this simple feedback system allows cells to produce sufficient free actin to meet the needs of their changing cellular cytoskeletal dynamics. The extensive MAL-decorated regulatory region displayed by Actin5C may serve to tune actin gene expression sensitively and accurately. This study provides evidence that cytoplasmic actin is the sole critical target gene for invasive migration in Drosophila and possibly also in human cells. It is therefore proposed that this regulation is the ancestral function of the MAL/SRF complex in animal cells. Additional target genes for MAL have been acquired in different species and are likely to contribute to cell fitness. Examples include other cytoskeletal proteins (Medjkane, 2009) as well as genes encoding heat-shock proteins, some of which may interact with actin. While some of these genes appear more dramatically regulated by MAL when considering relative mRNA levels, actin may be the 'most regulated' gene if considering the number of transcripts induced. In any case, identification of actin as the core, essential target of MAL reveals the core of this transcription regulatory 'network' to be a simple and logical feedback system (Salvany, 2014).

    Why is the MAL-driven regulation of actin particularly critical for invasively migrating cells? The stimuli inducing a robust F-actin-based cytoskeleton when initiating migration into constrained space is likely to convert free G-actin into F-actin-rich structures. Maintaining the appropriate free actin pool for further cytoskeletal buildup or for other cellular functions then requires new production of actin. Other dramatic shape changes, such as cells rounding up in the stratified epidermal layer of the skin, may induce similar sudden actin pool depletion and therefore require MAL and SRF. It has been suggested that MAL forms part of a mechanical feedback system for invasive cells (Somogyi, 2004) whereby mechanical tension induces MAL activity in order to make 'robust' cells. Related concepts of tension-driven responsiveness have recently been indicated for mammalian MAL/SRF function as well (Connelly, 2010; McGee, 2011). The mechanical feedback logic is fully compatible with the simple molecular feedback system presented here. It can be regarded as an alternate point of perturbation impinging on actin homeostasis; namely, stretching or stressing cells to provoke a biomechanical cytoskeletal response. Robust feedback systems such as this one driven by MAL are likely to be well conserved through animal evolution, when the target is a crucial one. The ability of cells to occasionally migrate and invade is a characteristic of animal systems, and the dynamic actin cytoskeleton is central to this behavior (Salvany, 2014).

    WHAMY is a novel actin polymerase promoting myoblast fusion, macrophage cell motility and sensory organ development

    Wiskott-Aldrich syndrome proteins (WASP) are nucleation promoting factors (NPF) that differentially control the Arp2/3 complex. In Drosophila, three different family members, SCAR/WAVE, WASP and WASH, have been analyzed so far. This study characterizes WHAMY, the fourth Drosophila WASP family member. whamy originated from a wasp gene duplication and underwent a sub-neofunctionalization. Unlike WASP, WHAMY specifically interacts with activated Rac1 through its two CRIB domains that are sufficient for targeting WHAMY to lamellipodial and filopodial tips. Biochemical analyses showed that WHAMY promotes exceptionally fast actin filament elongation, while it does not activate the Arp2/3 complex. Loss- and gain-of function studies revealed an important function of WHAMY in membrane protrusions and cell migration in macrophages. Genetic data further imply synergistic functions between WHAMY and WASP during morphogenesis. Double mutants are late-embryonic lethal and show severe defects in myoblast fusion. Trans-heterozygous mutant animals show strongly increased defects in sensory cell fate specification. Thus, WHAMY is a novel actin polymerase with an initial partitioning of ancestral WASP functions in development and subsequent acquisition of a new function in cell motility during evolution (Brinkmann, 2015).

    The actin cytoskeleton plays a central role in a number of different cellular functions, such as cell shape changes, cell motility and membrane trafficking. Members of the Wiskott–Aldrich syndrome protein (WASP) family are conserved nucleation-promoting factors (NPF) that activate the Arp2/3 complex, a major actin nucleator in eukaryotic cells. In mammals, the WASP protein family consists of eight different members: the two Wiskott-Aldrich syndrome proteins WASP and N-WASP (also known as WAS and WASL, respectively), the related WASP family Verprolin homologous proteins WAVE1–WAVE3 (also known as SCAR1–SCAR3 and WASF1–WASF3, the Wiskott–Aldrich syndrome protein and SCAR homolog WASH (also known as WASH1), and the WHAMM and JMY proteins. WASP proteins share a conserved C-terminal Arp2/3-complex-activating WCA module. This module consists of either one or multiple actin-monomer-binding WH2 (W) domains, a central domain (C) and an acidic (A) domain, which mediate Arp2/3 binding. Apart from the catalytic WCA module, WASP proteins often share a proline-rich region and a basic region, which bind SH3-domain containing proteins and acidic phosphoinositides, respectively. WASP proteins are regulated by similar molecular principles. Under resting conditions NPFs are primarily inactive and become activated upon binding of the Rho GTPases Cdc42 and Rac1. Additionally, a variety of factors further modulate proper activation and recruitment of WASP proteins (Brinkmann, 2015).

    In Drosophila, only three WASP subfamily members have been described, namely WAVE, WASP and WASH (also known as CG13176). Insects like Drosophila have subsequently lost a WHAMM/JMY gene, although the common ancestor first arose in invertebrates. Genetic studies indicate that WAVE and WASP are the central activators of the Arp2/3 complex, differentially regulating most aspects of Arp2/3 function in Drosophila. These studies highlight distinct, but also overlapping cellular requirements of WAVE and WASP during development. WAVE function is in particular essential for cell shape and morphogenetic cell movements during development. By contrast, WASP function is needed for cell fate specification of sensory organ precursors (SOPs) and spermatid Both, WASP and WAVE are required for myoblast fusion (Brinkmann, 2015).

    Loss of maternal and zygotic WASP results in late-embryonic lethality due to strong defects in cell fate decisions of neuronal cell lineages and myoblast fusion defects. Remarkably, animals lacking zygotic WASP function survive until early adulthood. Thus, maternally provided WASP protein is sufficient for proper embryonic and larval development. Mutant wasp flies show no strong morphological defects except a partial loss of sensory bristles. Loss of zygotic Arp2/3 function results in a similar, albeit stronger, neurogenic phenotype suggesting an involvement of additional factors in Arp2/3-dependent SOP development (Rajan et al., 2009). The loss of sensory bristles in wasp and arp2/3 mutants phenocopies Notch loss-of-function and is caused by a pIIa-to-pIIb cell fate transformation. This results in an excess of neurons at the expense of bristle sheath, shaft and socket cells. Recent work further suggests that the WASP–Arp2/3 pathway rather plays an important role in the trafficking of Delta-positive vesicles from the basal area to the apical cortex of the signal-sending pIIb cell (Brinkmann, 2015).

    Remarkably, rescue experiments have implied that established activators of WASP, such as Cdc42 or phosphatidylinositol 4,5-bisphosphate (PIP2), are not required for WASP function, neither for the myoblast fusion process nor for SOP development. The identity of an independent activator that might act cooperatively to control Arp2/3 function in these contexts is unknown. This study presents a functional analysis of WHAMY, a new WASP-like protein that regulates cell motility of Drosophila blood cells but also synergizes with WASP during embryonic muscle formation and cell fate specification of adult SOPs (Brinkmann, 2015).

    The identification of all WASP family homologs in all sequenced organisms allows a detailed phylogenetic analysis of the origin of diverse subfamilies evolving differential cellular functions. WASP proteins are multi-domain proteins. They share functions that are encoded by similar domains at the C-termini, whereas different N-terminal domains mainly define their diverse cellular processes. Gene duplication and domain shuffling are two important mechanisms driving novel and increasingly complex developmental programs during evolution. It is thought that this boost in domain shuffling is responsible for the apparent disconnection between greatly increased phenotypic complexity and a relatively small difference in gene number between humans and Drosophila (Brinkmann, 2015).

    The whamy gene is an excellent example for how gene duplication and subsequent domain shuffling can create new gene functions after initial gene duplication. It arose through a duplication of wasp at the base of the genus Drosophila. Although the encoded protein has evolved a new function in cell motility, it also functions synergistically with WASP in muscle formation and sensory organ development. In the latter, WHAMY can even partially substitute for WASP, indicating that it has kept functionality following the duplication. This duality is reflected in the sequence of the WHAMY CRIB domains. As there is an overlap in function with WASP, selective pressure has been reduced since the duplication, leading to the observed increase of evolutionary rate. Following the duplication of the CRIB domains within WHAMY, a similar trend can be found. Whereas one domain has kept the function of binding to Cdc42-GTP, the other has lost the ability to interact. This is reflected in domain-specific conserved substitutions. The duplication of the wasp gene and subsequent subneofunctionalization of whamy might have occurred at the same time as the loss of a true WHAMM/JMY ancestor during insect evolution (Veltman, 2010). Like Drosophila WHAMY, the common ancestor of WHAMM/JMY proteins in invertebrates also lacks the characteristic C-terminal tryptophan residue in their VCA domains that is crucial for Arp2/3 binding and activation (Veltman, 2010). This further implies a primary Arp2/3-independent function of the common ancestor of invertebrate WHAMM/JMY proteins (Brinkmann, 2015).

    WHAMY shows no Arp2/3-activating nucleation promoting factor (NPF) activity in vitro. However, different from WASP, WHAMY itself is able to promote fast elongation of linear actin filaments from actin-rich clusters. With respect to its activity, WHAMY resembles the WH2-domain containing Ena/VASP polymerases that actively drive processive actin-filament elongation and promote assembly of both lamellipodial and filopodia actin networks. Notably, Ena/VASP proteins are tetramers, and their oligomerization is mandatory to allow for polymerase activity in experiments in solution, as used in this study. Since fast filament elongation was exclusively observed from WHAMY clusters in total internal reflection fluorescence (TIRF) experiments, and consistent with the size exclusion chromatography experiments, it is proposed that WHAMY requires oligomerization to acquire actin polymerase activity. Concerning previously analyzed proteins of the WASP family, the filament elongation activity of WHAMY is therefore rather unique, and when compared to other fast actin polymerases, only the Drosophila formin Diaphanous achieves comparable high elongation activity in vitro. As evidenced from the pyrene data, the activity of WHAMY can further be increased by Rac1 (Brinkmann, 2015).

    Rac1 seems to act on both the activity and the localization of WHAMY at lamellipodial tips. Both of the two CRIB domains of WHAMY bind equally to activated Rac1, and only loss of both CRIB domains abolishes Rac1 binding and the localization to the leading edge. Therefore, it currently remains unclear why WHAMY contains two CRIB domains and whether they differentially mediate distinct cellular functions. They might contribute to a local clustering of WHAMY and Rac1 at the leading edge. The most prominent Rac1 effector represents the WAVE regulatory complex (WRC) that drives Arp2/3-mediated branched actin nucleation. Rac1 directly binds and activates the WRC by allosterically releasing the bound Arp2/3-activating WCA domain of WAVE. Overexpression of WHAMY leads to a strong induction of filopodia, presumably due to the filament elongation activity of WHAMY. Additionally, competition between WHAMY and the WRC for Rac1 could disturb the balance between nucleation and elongation activity, and therefore might contribute to the observed overexpression phenotype. Different from WHAMY, WRC function is essential for lamellipodia formation and cell migration in most eukaryotic cells. By contrast, loss of WHAMY function does not impair lamellipodia formation but rather regulates cell spreading and contributes to cell motility (Brinkmann, 2015).

    WHAMY does not compete but rather functions together with WASP in Drosophila morphogenesis. Previous studies have revealed that the major established activators of WASP, such as Cdc42 and PIP2, are not required for the function of WASP in sensory organ development or myoblast fusion. This observation already suggests that additional components, such as WHAMY, might act together with WASP in sensory organ development and myoblast fusion. Consistent with this, further reduction of whamy function in wasp mutants was found to phenocopy loss of arp2/3 function, resulting in an excess of neurons and a near absence of bristle sheath, shaft and socket cells. Rescue data further indicate that WHAMY can partially substitute for WASP function. Thus, WHAMY cooperates with WASP rather than acting redundantly in sensory organ development. Based on TIRF microscopy data, it is suggested that WHAMY might potentially generate mother filaments in close vicinity of Arp2/3 complex facilitating Arp2/3-mediated actin assembly (Brinkmann, 2015).

    How might WHAMY and WASP act on actin dynamics during sensory organ development? Recent work suggests that the WASP–Arp2/3 pathway is not involved in Notch receptor endocytosis or its processing in the signal-receiving cell (pIIa) but rather plays an important role in the trafficking of Delta-positive vesicles from the basal area to the apical cortex of the signal-sending pIIb cell. This model also implies that recycled Notch ligands such as Delta and Serrate are active at apical junctions with actin-rich structures induced by WASP and the Arp2/3 complex, which in turn activate apical Notch receptor in pIIa. In vivo, WHAMY localizes at dynamic vesicles during sensory organ precursor formation and, together with WASP, becomes strongly enriched at apical junctions shortly after SOP division. Thus, a scenario is proposed in which WASP and WHAMY might act either on the assembly of actin-rich structures or directly promote apical trafficking of Delta through Rab11-recycling endosomes (Brinkmann, 2015).

    A dynamic reorganization of the actin cytoskeleton into distinct cellular structures is also necessary to ensure successful myogenesis. Filopodial protrusions are crucial for the attachment of FCMs to the founder cell and growing myotube, and for the initiation of the fusion process. The recognition and adhesion of myoblasts depends on members of the immunoglobulin superfamily (IgSF) that are expressed specifically in myoblasts in a ring-like structure. The interaction of these proteins leads to the formation of a cell communication structure, which has been termed fusion-restricted myogenic adhesive structure (FuRMAS) or podosome-like structure. The cytodomains of the IgSFs trigger the activation of WAVE in founder cells, and of WAVE and WASP in FCMs. In FCMs, WAVE- and WASP-mediated Arp2/3 activation results in the formation of a dense F-actin focus that accumulates at the interface of adhering myoblasts. Electron microscopy studies have revealed that WASP is required for the formation of fusion pores at apposing myoblasts during embryonic and indirect flight muscle development. These fusion pores expand until full cytoplasmic continuity is achieved, and WASP has implicated to be required for fusion pore expansion. It has been discussed that WASP is required for the removal of membrane residuals during membrane vesiculation. WHAMY might contribute to this process, but the detailed mechanistic contribution of WHAMY in fusion pore formation needs to be addressed in future studies by ultrastructural analyses (Brinkmann, 2015).

    New insights into the regulatory function of CYFIP1 in the context of WAVE- and FMRP-containing complexes

    CYtoplasmic FMRP Interacting Protein 1 (CYFIP1) is a candidate gene for intellectual disability (ID), autism, schizophrenia and epilepsy. It is a member of a family of proteins that is very conserved during evolution, sharing high homology with dCYFIP, its Drosophila homolog. CYFIP1 interacts with the Fragile X Mental Retardation Protein (FMRP), whose absence causes the Fragile X Syndrome, and with the translation initiation factor eIF4E. It is a member of the WAVE Regulatory Complex (WRC), thus representing a link between translational regulation and actin cytoskeleton. Data is presented showing a correlation between mRNA levels of CYFIP1 and other members of the WRC. This suggests a tight regulation of the levels of the WRC members not only by post-translational mechanisms, as previously hypothesized. Moreover, the impact of loss of function of both CYFIP1 and FMRP on neuronal growth and differentiation in was studied in two animal models, fly and mouse. These two proteins antagonize each other's function not only during neuromuscular junction growth in the fly but also during new neuronal differentiation in the olfactory bulb of adult mice. Mechanistically, FMRP and CYFIP1 modulate mTor signaling in an antagonistic manner, likely via independent pathways, supporting the results obtained in mouse as well as in fly at the morphological level. Collectively, these results illustrate a new model to explain the cellular roles of FMRP and CYFIP1 and the molecular significance of their interaction (Abekhoukh, 2017).

    The chromokinesin Klp3a and microtubules facilitate acentric chromosome segregation

    Although poleward segregation of acentric chromosomes is well documented, the underlying mechanisms remain poorly understood. This study demonstrates that microtubules play a key role in poleward movement of acentric chromosome fragments generated in Drosophila melanogaster neuroblasts. Acentrics segregate with either telomeres leading or lagging in equal frequency and are preferentially associated with peripheral bundled microtubules. In addition, laser ablation studies demonstrate that segregating acentrics are mechanically associated with microtubules. Finally, this study shows that successful acentric segregation requires the chromokinesin Klp3a. Reduced Klp3a function results in disorganized interpolar microtubules and shortened spindles. Normally, acentric poleward segregation occurs at the periphery of the spindle in association with interpolar microtubules. In klp3a mutants, acentrics fail to localize and segregate along the peripheral interpolar microtubules and are abnormally positioned in the spindle interior. These studies demonstrate an unsuspected role for interpolar microtubules in driving acentric segregation (Karg, 2017).

    Absence of the Drosophila jump muscle actin Act79B is compensated by up-regulation of Act88F

    Actins are structural components of the cytoskeleton and muscle, and numerous actin isoforms are found in most organisms. However, many actin isoforms are expressed in distinct patterns allowing each actin to have a specialized function. Numerous studies have demonstrated that actin isoforms both can and cannot compensate for each other under specific circumstances. This allows for an ambiguity of whether isoforms are functionally distinct. This study analyzed mutants of Drosophila Act79B, the predominant actin expressed in the adult jump muscle. Functional and structural analysis of the Act79B mutants found the flies to have normal jumping ability and sarcomere structure. Analysis of actin gene expression determined that expression of Act88F, an actin gene normally expressed in the flight muscles, was significantly up-regulated in the jump muscles of mutants. This indicated that loss of Act79B caused expansion of Act88F expression. When double mutants were created of Act79B and Act88F, this abolished the jump ability of the flies and resulted in severe defects in myofibril formation. These results indicate that Act88F can functionally substitute for Act79B in the jump muscle, and that the functional compensation in actin expression in the jump muscles only occurs through Act88F (Dohn, 2018).

    Nuclear F-actin and myosins drive relocalization of heterochromatic breaks

    Heterochromatin mainly comprises repeated DNA sequences that are prone to ectopic recombination. In Drosophila cells, 'safe' repair of heterochromatic double-strand breaks by homologous recombination relies on the relocalization of repair sites to the nuclear periphery before strand invasion. The mechanisms responsible for this movement were unknown. This study shows that relocalization occurs by directed motion along nuclear actin filaments assembled at repair sites by the Arp2/3 complex. Relocalization requires nuclear myosins associated with the heterochromatin repair complex Smc5/6 and the myosin activator Unc45, which is recruited to repair sites by Smc5/6. ARP2/3, actin nucleation and myosins also relocalize heterochromatic double-strand breaks in mouse cells. Defects in this pathway result in impaired heterochromatin repair and chromosome rearrangements. These findings identify de novo nuclear actin filaments and myosins as effectors of chromatin dynamics for heterochromatin repair and stability in multicellular eukaryotes (Caridi, 2018).

    Myosin1D is an evolutionarily conserved regulator of animal left-right asymmetry

    The establishment of left-right (LR) asymmetry is fundamental to animal development, but the identification of a unifying mechanism establishing laterality across different phyla has remained elusive. A cilia-driven, directional fluid flow is important for symmetry breaking in numerous vertebrates, including zebrafish. Alternatively, LR asymmetry can be established independently of cilia, notably through the intrinsic chirality of the acto-myosin cytoskeleton. This study shows that Myosin1D (Myo1D), a previously identified regulator of Drosophila LR asymmetry, is essential for the formation and function of the zebrafish LR organizer (LRO), Kupffer's vesicle (KV). Myo1D controls the orientation of LRO cilia and interacts functionally with the planar cell polarity (PCP) pathway component VanGogh-like2 (Vangl2; see Drosophila Van Gogh), to shape a productive LRO flow. These findings identify Myo1D as an evolutionarily conserved regulator of animal LR asymmetry, and show that functional interactions between Myo1D and PCP are central to the establishment of animal LR asymmetry (Juan, 2018).

    SETD3 protein is the actin-specific histidine N-methyltransferase

    Protein histidine methylation is a rare post-translational modification of unknown biochemical importance. In vertebrates, only a few methylhistidine-containing proteins have been reported, including beta-actin as an essential example. The evolutionary conserved methylation of beta-actin H73 is catalyzed by an as yet unknown histidine N-methyltransferase. This study reports that the protein SETD3 is the actin-specific histidine N-methyltransferase. In vitro, recombinant rat and human SETD3 methylated beta-actin at H73. Knocking-out SETD3 in both human HAP1 cells and in Drosophila melanogaster resulted in the absence of methylation at beta-actin H73 in vivo, whereas beta-actin from wildtype cells or flies was > 90% methylated. As a consequence, it was shown that Setd3-deficient HAP1 cells have less cellular F-actin and an increased glycolytic phenotype. In conclusion, by identifying SETD3 as the actin-specific histidine N-methyltransferase, this work pioneers new research into the possible role of this modification in health and disease and questions the substrate specificity of SET-domain-containing enzymes (Kwiatkowski. 2018).

    Differential lateral and basal tension drive folding of Drosophila wing discs through two distinct mechanisms

    Epithelial folding transforms simple sheets of cells into complex three-dimensional tissues and organs during animal development. Epithelial folding has mainly been attributed to mechanical forces generated by an apically localized actomyosin network, however, contributions of forces generated at basal and lateral cell surfaces remain largely unknown. This study shows that a local decrease of basal tension and an increased lateral tension, but not apical constriction, drive the formation of two neighboring folds in developing Drosophila wing imaginal discs. Spatially defined reduction of extracellular matrix density results in local decrease of basal tension in the first fold; fluctuations in F-actin lead to increased lateral tension in the second fold. Simulations using a 3D vertex model show that the two distinct mechanisms can drive epithelial folding. This combination of lateral and basal tension measurements with a mechanical tissue model reveals how simple modulations of surface and edge tension drive complex three-dimensional morphological changes (Sui, 2018).

    This work has uncovered two new mechanisms of epithelial fold formation. First, a locally defined basal decrease of surface and edge tension, associated with local reduction of ECM density, leads to basal cell expansion and folding. Second, a lateral increase of surface tension at the future fold location, associated with F-actin flows and pulsatile contractions, leads to a local reduction of tissue height and fold formation. It is conceivable that both mechanisms may also operate in combination during epithelial folding (Sui, 2018).

    A simplified picture resulting from mechanical analysis of how basal tension reduction can induce fold formation is as follows. Higher basal tension in the cells outside the fold compared to cells inside the fold stretches the basal surface areas of fold cells. Consequently, fold cells widen basally and reduce cell height to maintain cell volume. The new force balance state is characterized by apical indentation and wedge-shaped, shortened cells. How is ECM depletion linked to a decrease in basal cell edge and surface tension? In one scenario, following ECM depletion, the actomyosin network lacks stabilization via binding to integrins, reducing the active tension it can generate with myosin molecular motors. Alternatively, the ECM and cortical actomyosin network, linked together via integrins and other molecules, can be seen as a single composite material under tension. Elastic straining of the ECM, e.g. during tissue growth, could give rise to a passive mechanical tension within the ECM. As the ECM is depleted, the composite material is reorganized and passive ECM stress due to ECM straining could be lost, also contributing to the overall decrease in basal tension in the fold (Sui, 2018).

    Lateral tension increase can also induce fold formation. This can be outlined in a simplified picture (see Differential lateral and basal tension drive folding of Drosophila wing discs through two distinct mechanisms). Increased lateral tension leads to a reduction in cell height. Since basal tension is high, the shortened cells deform the apical surface inwards, while the basal surface resists deformation. As the cells resist volume changes, they widen. Conceivably, increased apical tension in the fold cells favors further basal expansion of the fold cells (Sui, 2018).

    Folding requires the transition of cells from a columnar to a wedge-shape where the apical surface is smaller than the basal surface. Previous work has stressed the role of mechanical stresses generated by apical actomyosin networks driving apical constriction during folding. This work shows that for the epithelial folds, in the case of the wing, apical constriction is not important. Instead, they rely either on the basal widening of cells due to the decrease of basal tension or alternatively on increased lateral tension. Interestingly, two fundamentally different mechanisms generate similar morphologies of neighboring folds. This implies that the mechanical processes shaping a tissue cannot be deduced from the tissue morphology alone. Cell shortening and an active role for the ECM is also required for the folding of the zebrafish embryonic brain. Basal decrease of tension and lateral increase of tension may therefore represent two important mechanisms driving the folding of epithelia in different organisms (Sui, 2018).

    Decoding cilia function: defining specialized genes required for compartmentalized cilia biogenesis

    The evolution of the ancestral eukaryotic flagellum is an example of a cellular organelle that became dispensable in some modern eukaryotes while remaining an essential motile and sensory apparatus in others. To help define the repertoire of specialized proteins needed for the formation and function of cilia (see Kimball's Cilia and Flagella site or UTMB Cell Biology Topics), comparative genomics was used to analyze the genomes of organisms with prototypical cilia, modified cilia, or no cilia and 200 genes were identified that are absent in the genomes of nonciliated eukaryotes but are conserved in ciliated organisms. Importantly, over 80% of the known ancestral proteins involved in cilia function are included in this small collection. Using Drosophila as a model system, a novel family of proteins (OSEGs: outer segment) was then characterized that is essential for ciliogenesis. Osegs encode components of a specialized transport pathway unique to the cilia compartment and are related to prototypical intracellular transport proteins (Avidor-Reiss, 2004).

    Cilia are microtubule-rich, hair-like cellular extensions that perform essential motile and sensory functions. In sperm and in unicellular eukaryotes, a motile form of cilia called flagellum propels cells to their destination, while in epithelial cells, multiple motile cilia beat synchronously to stir extracellular fluid. In vertebrate photoreceptor cells and invertebrate mechano- and chemoreceptor neurons, the entire sensory transduction machinery is housed in a specialized cellular compartment derived from the cilium. This domain, known as the outer segment, is a hallmark of these sensory neurons and an outstanding example of subcellular compartmentalization as a strategy to optimize function. The ancestral nature of the eukaryotic cilia is evident by its presence in organisms from both lineages: D. melanogaster (Dm), H. sapiens (Hs), T. brucei (Tb), and C. reinhardtii (Cr). In three independent events (indicated by red stars), cilia were lost in lineages leading to A. thaliana (At), D. discoideum (Dd), and S. cerevisiae (Sc). Similarly, compartmentalized cilia were lost in P. falciparum (Pf), while motile cilia were lost in C. elegans (Ce) (Avidor-Reiss, 2004).

    Given the wide range of cells and tissues that contain cilia, and the extraordinary diversity of roles performed by cilia, a basic question in cell biology is how ciliogenesis is orchestrated and to what extent common mechanisms underlie this process. Cilia formation begins when the basal body, a centriole-related structure, serves as a template for the assembly of the axoneme. This process can proceed through two different mechanisms. In most motile and sensory cilia, the basal body docks to the plasma membrane, and a bud-like structure containing the axoneme and the ciliary membrane projects out from the cell body; since the ciliary membrane and the axoneme are assembled concurrently as a compartment separated from the cell body, this process is referred to as compartmentalized ciliogenesis. In a few cases, however, such as in the sperm cells of Drosophila and the flagella of the parasite Plasmodium, the entire axoneme is first assembled inside the cytosol and only later is either extruded or matures into a flagellum (i.e., cytosolic biogenesis) (Avidor-Reiss, 2004).

    Unlike cytosolic biogenesis, the process of compartmentalized ciliogenesis requires that cilia, flagella, and outer segments transport their building blocks -- proteins and metabolites -- from the cell soma. Genetic and biochemical studies in the biflagellated green alga Chlamydomonas have singled out kinesin II, dynein 1b, and 17 additional proteins named intraflagellar transport (IFT) particle proteins as candidate proteins involved in flagella biogenesis. IFT particle proteins are proposed to function as macromolecular rafts traveling up and down the flagellum, via kinesin and dynein, transporting axonemal precursor proteins to their growing tips. Consistent with this postulate, mutations in the Chlamydomonas IFT particle proteins IFT88 and IFT52 produce very short flagella. Similar results are seen in C. elegans mutants defective in the IFT orthologs OSM-5 and OSM-6 (Avidor-Reiss, 2004 and references therein).

    This study reports the development of a novel bioinformatics approach to identify genes involved in ciliogenesis. The strategy is based on the hypothesis that the ancestral eukaryote was a ciliated unicellular organism, and that cilia and flagella were independently lost throughout evolution from several eukaryotic groups. By comparing the genomes of ciliated and nonciliated organisms, a collection of candidate genes important for cilia formation and function was identified. In addition, by phylogenetically examining orthologs in organisms with 'compartmentalized' versus 'cytosolic' axonemes, a large subgroup selectively expressed in Drosophila sensory outer segments, but not in sperm, a novel family of proteins (OSEGs: outer segment) essential for compartmentalized ciliogenesis was isolated and characterized. Together, these studies establish a compelling bioinformatics strategy to help decode gene function and lay the foundation for a comprehensive dissection of eukaryotic ciliogenesis and outer segment development (Avidor-Reiss, 2004).

    In order to identify specialized genes essential for cilia biogenesis and function, a phylogenetic screen was undertaken that identified genes conserved in the genomes of ciliated organisms but absent in nonciliated eukaryotes. It was reasoned that gene loss can be used as a powerful tactic to map gene function, particularly if the biological process in question (e.g., cilia biogenesis in this case) is conserved in distantly related species and if it was lost more than once during evolution. Eight species were chosen representing the two major lineages of eukaryotic evolution, and which included nodes where cilia were lost or modified during the evolution of eukaryotes (Avidor-Reiss, 2004).

    Because Drosophila contains experimentally tractable motile and sensory cilia and has an extensively annotated genome, it was selected as the anchor for these studies. BLAST searches were performed against the proteome of five ciliated (H. sapiens [Hs], C. elegans [Ce], P. falciparum [Pf], C. reinhardtii [Cr], and T. brucei [Tb]) and three nonciliated organisms (A. thaliana [At], S. cerevisiae [Sc], D. discoideum [Dd]) and orthologs were sought of the 14,000 Drosophila genes in each of these species using a 'reciprocal best hit' algorithm. Because the T. brucei and C. reinhardtii genomes are incomplete, a ciliary protein was considered as conserved in Bikonts (ancestorally biciliate eukaryotes) if it was present in either of those two species (Cr/Tb). Similarly, a partial draft of the Dictyostelium discoideum (Dd) proteome is now available; this organism displays exquisite motility, yet it lacks ciliated structures, thus providing a robust bioinformatics counterscreen (Avidor-Reiss, 2004).

    Because all ciliated organisms have an axoneme but may differ in their mode of ciliogenesis, or whether they have motile or nonmotile cilia, it is suspected that distinct sets of proteins might be required during biogenesis of the various forms of cilia. Therefore, the screening strategy was applied to four different search routines: (1) to identify genes involved in processes common to all cilia, like axoneme formation, all ciliated versus all nonciliated eukaryotes were compared (i.e., genes conserved in Hs, Dm, Pf, Cr/Tb, and Ce but not in At, Sc, or Dd); (2) to identify genes involved in cilia motility (either of compartmentalized or cytosolic origin), organisms with motile cilia versus those with nonmotile or no-cilia were compared (i.e., genes conserved in Hs, Dm, Pf, and Cr/Tb but not in Ce, At, Sc, or Dd); (3) to identify genes involved in cilia compartmentalization, organisms with compartmentalized cilia biogenesis versus cytosolic biogenesis were compared (e.g., genes conserved in Hs, Dm, Ce, and Cr/Tb but not in Pf, At, Sc, or Dd) and (4) to identify genes that may be unique to organisms that have both motile and compartmentalized cilia, genes were sought that are shared between Drosophila and organisms with prototypical cilia (i.e., Hs and Cr/Tb but not in Ce, Pf, At, Sc, or Dd) (Avidor-Reiss, 2004).

    From a total of 121,243 predicted transcriptional units and 141,000 ESTs (ESTs were used in Chlamydomonas due to the lack of an assembled partial proteome), a total of 187 ancestral genes were identified: (1) 16 conserved in all ciliated organisms, but absent in nonciliated (all-cilia subset); (2) 18 present only in organisms with motile cilia (motility subset); (3) 103 common only to organisms with compartmentalized cilia biogenesis (compartment subset), and (4) 50 shared only between organisms with prototypical cilia (both motile and compartmentalized; prototypical-cilia subset) (Avidor-Reiss, 2004).

    To evaluate the performance of the screen, it was asked whether known genes implicated in ciliogenesis are indeed enriched in this collection. A search of the literature revealed that there are 36 genes that have been implicated in ciliogenesis in either flies or in other organisms and were part of the likely ancestral repertoire of genes in the primitive eukaryotic cell (e.g., conserved in organisms from both ancestral eukaryotic lineages). This set includes specialized genes whose primary role is in cilia biogenesis and function (e.g., dynein arms, IFTs), as well as genes that may also participate in other cellular processes (e.g., dynein light chains: see Dynein and intracellular transport). Remarkably, 30 out of the 36 known genes (>80%) are included in the 187 ancestral gene collection obtained in the bioinformatics screen; of the remaining six, five also function outside the cilia and were filtered out because they are present in nonciliated organisms (four dynein subunits and myosin VIIA), and one (left/right-dynein) was eliminated because it did not have an ortholog in Tb or Cr (Avidor-Reiss, 2004).

    The selectivity of the screen is also illustrated by examining the genes in the motility subset: all six known ciliary genes recovered in this collection, in fact, encode proteins involved in motility (four axonemal dynein subunits, a radial spoke protein, and Mbo2, a protein important for flagella waveform). In addition, of the remaining 12 candidate motility genes in this subgroup, five are specifically expressed in testis, a tissue highly enriched in motile cells. Taken together, these results substantiate the logic of the approach and the search criteria that were used (Avidor-Reiss, 2004).

    Of particular interest was the formation of sensory outer segments, therefore focus was placed on the genes in the cilia-compartment subset both as a platform for gene discovery and for dissecting mechanisms of outer segment biogenesis. Curation of the 103 candidates in this group suggested that several may not have a direct role in ciliogenesis, yet they cosegregated with the selection criteria. These included ion channels, signal-transduction components, transcription factors, and metabolic enzymes. In order to extract 'ciliary' genes from this subset, it was demanded that candidates meet two additional search criteria. (1) Many genes involved in sensory cilia formation share an upstream regulatory sequence known as the X box, often at 150 to 50 nucleotides upstream from the translation start site. A general search of the D. melanogaster and C. elegans genomes for the presence of the 14 nucleotides consensus X box motif has demonstrated that this sequence is much too abundant to be used as a primary screen (for instance, 2449 of Dm and 1897 of Ce genes contain such a motif); however, as a secondary screen, it selected 41 candidates from the cilia-compartment subset. Notably, over 90% of the known ciliary genes in the compartment subset (14/15) are included in these 41 genes. (2) Compartmentalized cilia in Drosophila are found only in chemo- and mechano-sensory neurons. Because these neurons are scattered all over the fly body and comprise a minute fraction of the fly cells, available EST databases contain none, or very few, representatives ESTs. Based on this premise, the compartment subset was searched for genes that contained 0-4 ESTs and 48 candidates were identified. Importantly, these 48 candidates contain nearly all of the known ciliary genes in the original collection (13/15). Together, these two secondary screens identified a total of 30 genes that overlapped both the X box and EST filters: these were chosen for biological validation (Avidor-Reiss, 2004).

    Genes involved in compartmentalized ciliogenesis should satisfy two important requirements: (1) the genes should be expressed in ciliated sensory cells; (2) the proteins must be essential for outer segment formation or function. The genes selected in the compartment subset encode members of several protein groups, including IFT proteins, Bardet-Biedl syndrome (BBS)-related polypeptides, C2 domain-containing proteins, small G proteins, a group of 'coil-coil' proteins, and a family of six WD-domain proteins (OSEGs: outer segment). Below a short summary of these families is provided (Avidor-Reiss, 2004).

    BBS is a heterogeneous genetic disorder that is characterized by retinal dystrophy, renal malformation, learning disabilities, and obesity. Six BBS genes have been cloned, and several were recently implicated in ciliogenesis. Drosophila has three BBS orthologs, and all three were selected in this screen (BBS1 and BBS8 as part of the compartment subset, and BBS4 as part of the prototypical-cilia subset). Interestingly, this collection also includes two additional proteins (CG5142 and CG4525) sharing a similar domain organization; it is suggested these proteins encode new BBS members (Avidor-Reiss, 2004).

    The C2 domain is a 120 amino acid sequence that functions as a Ca2+-dependent membrane-targeting module in proteins involved in signal transduction (e.g., protein kinase C, cytosolic phospholipase A2) or transport processes (e.g., synaptotagmin I, rabphilin). The analysis identified three novel C2 domain-containing proteins (CG18631, CG9227, and CG14870). Given the central role of calcium in regulating cilia function, as well as processes as diverse as membrane fusion, protein transport, and protein breakdown, these are worthy candidates for sensors of the calcium signals. Small G proteins are known to function as universal molecular switches in a wide range of intracellular processes. Recently, Leishmania ARL3 (LdARL-3A) was implicated in flagellum biogenesis. Notably, the screen identified ARL3 and ARL6, two Arf-like proteins, as components of the compartment group. The cilia-compartment subset also contains orthologs of all seven known IFT particle proteins. In addition, this group also contains two novel WD domain-containing proteins (OSEGs) and three novel coiled-coiled candidate IFT members. OSEGs are a family of six related polypeptides sharing the same predicted topology and signature sequences: an N terminus with seven tandem WD repeats (300 residues), a β sheet rich interdomain (300 residues), and multiple TPR-like repeats (tetratricopeptide repeats; 300 residues). WD repeats are 44-60 residue sequence motifs that fold as parts of two adjacent blades of a typically seven blade propeller structure. TPR-like repeats comprise a TPR-related sequence motif that folds into two antiparallel α helices; these in turn assemble into large right-handed helices. WD- and TPR-like-repeats are often found in large macromolecular assemblies and are thought to function as structural platforms for reversible protein-protein interactions (Avidor-Reiss, 2004).

    To identify the cells that express the candidate ciliary compartment genes, 15 genes representing the various gene families were selected, plus a control each from the all-cilia (Tctex2) and prototypical-cilia subsets (BBS4), and transgenic flies were generated expressing Gal4 promoter fusions. Individual lines were crossed to flies containing UAS reporters and examined for GFP expression in larvae and adult animals (Avidor-Reiss, 2004).

    In Drosophila, there are three types of ciliated cells: sperm, mechanosensory, and chemosensory neurons. Mechanosensory and chemosensory cilia are assembled through compartmentalized ciliogenesis, while the sperm tail is assembled via cytoplasmic ciliogenesis. Refined specificity is demonstrated in the anatomical sites of expression of all 17 genes: each transgene is restricted to ciliated cells, with BBS4 and the 15 candidate compartment genes expressed exclusively in neurons of mechanosensory and chemosensory organs. The remaining one, Tctex2/LC2 (a dynein light chain subunit from dynein arms and cytosolic dyneins), was also expressed in sperm cells. No other sites of expression were observed for any of the transgenes. Taken together, these results strongly authenticate the bioinformatics strategy, provide a new perspective into the evolution of cilia, and set the foundation for a comprehensive use of this approach in other biological processes (Avidor-Reiss, 2004).

    To gain insights into the biology of outer segment biogenesis, mutants defective in candidate cilia-compartment genes were sought. Drosophila mutants with outer segment defects are expected to be mechanosensory defective. Mutagenized F3 lines were screened for the presence of mechanoinsensitive flies and mechanoreceptor currents (MRC) and transepithelial potentials (TEP) were recorded from candidate lines. Mutations that affect the cilia are predicted to show defective MRC. In contrast, mutations that affect the function or development of the support and accessory cells should abolish both the MRC and the TEP. MRCs and TEPs were recorded from multiple bristles in various uncoordinated lines and complementation groups were selected with normal TEP but defective MRC and they were tested in chemo-sensory and sperm motility assays. Two complementation groups with abnormal mechano- and chemosensory responses but normal sperm motility mapped near the location of oseg1 and oseg2, respectively. It is expected that mutant alleles would carry missense or nonsense mutations, and that introduction of the wild-type gene into mutant animals should rescue their behavioral and physiological phenotype. Indeed, oseg1179 and oseg110 alleles had stop codons in oseg1, and the oseg2 allele contained a nonconservative substitution in the oseg2 gene. More importantly, introduction of the wild-type oseg1 and oseg2 genes by germline transformation rescued the uncoordinated and MRC defects of oseg1 and oseg2 mutants (Avidor-Reiss, 2004).

    To analyze the phenotype of oseg1 and oseg2 mutants in detail, the ultra-structure of the sensory cilium was examined by EM serial section analyses. Wild-type mechano- and chemosensory dendrites contain a striated rootlet, two basal bodies, a connecting cilium, and the outer segment. oseg1 and oseg2 mutants have normal inner segments and an intact rootlet, basal bodies, and connecting cilium. However, both mutants display dramatic defects in outer segment morphology: In mechanoreceptor neurons, oseg1 has a striking reduction of the distal-most end of the outer segment (the dendritic tip and tubular body), while oseg2 has a total loss of the tubular body. In chemoreceptors, oseg1 and oseg2 both show severely shortened outer segments. Together, these results firmly implicate oseg genes in ciliogenesis, and outer segment formation (Avidor-Reiss, 2004).

    HMM analyses and secondary structure predictions indicate that OSEGs are related to α- and β'-coatomer, two proteins involved in intracellular trafficking. Significantly, clathrin heavy chain (Chc) also displays prominent domain similarity to OSEG family members. Because outer segments (and cilia) are separated from the rest of the cell by a connecting cilium, they need to import their proteins from the cell soma and therefore might be expected to require specialized machinery to assemble a functional compartment (Avidor-Reiss, 2004).

    If the OSEG proteins were essential for the transport of selective macromolecules into ciliary compartments, they would be expected to meet several criteria. (1) In contrast to structural or signal transduction components of the outer segment, OSEGs should travel in and out of the outer segment, while concentrating primarily at the base of the cilia. This region of the cell is considered the cilium's 'hub', a strategic place between the cell soma and the outer segment, and is hypothesized to function as the site where molecules targeted to the cilium are loaded and transported via the microtubule-based motors. (2) Ciliary cargo should be transported normally from the cell soma to the cilia base of oseg mutants, but it should be unable to enter the cilia and therefore may accumulate near the cilia base (Avidor-Reiss, 2004).

    To examine the subcellular localization of OSEG proteins, translational fusions between all six OSEG family members and GFP were engineered. Each fusion protein was then targeted to ciliated sensory cells using a pan-neuronal promoter. In order to mark the position of the cilium, the cells were co-labeled with mab21A6, a monoclonal antibody that labels the base of the cilium at the inner/outer segment boundary. As predicted, all GFP-tagged OSEG proteins localize primarily at the base of the cilium and can be found inside the sensory cilia (Avidor-Reiss, 2004).

    To examine transport into outer segments, it was necessary to identify a candidate cargo protein, ideally one that requires either of the available mutants (oseg1 or oseg2). Mechanosensory outer segments contain at their distal-most end a unique microtubule-rich structure known as the tubular body; this is the proposed site of channel anchoring and force generation in mechanosensory bristles. The α-tubulin isoform in the tubular body is encoded by the α1tub84B gene in Drosophila. Therefore, it was hypothesized that α1tub84B would be an ideal OSEG cargo. Overexpression of a plain GFP reporter, or even a membrane tagged-GFP, does not label the outer segment of ciliated neurons. However, if GFP is linked to α1tub84B (i.e., a GFP-α1tub84B fusion protein), it is now co-targeted with tubulin and functions as a robust reporter of α1tub84B transport into the outer segment (Avidor-Reiss, 2004).

    Next, the GFP-α1tubulin 84B reporter was introduced into oseg mutant backgrounds and its localization was examined. The GFP-α1tub84B cargo completely fails to enter the outer segment of oseg2 mutants, but is efficiently transported to the outer segments of controls and oseg1 mutants. Furthermore, EM examination of oseg2 mutant cells revealed a dramatic accumulation of microtubules at the base of the cilium. These results prove that oseg2, but not oseg1, is essential for tubulin transport into the cilium and illustrate an important aspect of OSEGs function: OSEGs may play distinct roles, and different cargo are likely to be matched to specific OSEG members. Notably, the N-terminal WD domains of α-coatomers and clathrin have been implicated in cargo recognition and sorting. The identification of six OSEG members with distinct N-terminal WD domains may provide the structural basis for selective cargo recognition within this family (Avidor-Reiss, 2004).

    The bioinformatics approach also identified two kinesin II subunits as cilia-compartment genes. <Kinesin II has been shown to be required for cilia assembly in a variety of organisms and was proposed to function as the anterograde motor carrying cargo from the base of the cilia to its distal tip. If OSEGs mediate the kinesin-based intraciliary transport, and if this transport were specifically required for outer segment formation, it was reasoned that mutations in Kinesin-like protein at 64D (klp64D), the central component of Drosophila kinesin II, should generate in vivo phenotypes that resemble oseg defects. Thus, flies were generated defective in klp64D function and mechano- and chemo-sensory physiology and the transport and accumulation of α1tub84B into sensory cilia were examined. klp64D mutant animals share all of the hallmarks of oseg2 mutants: (1) severe chemoinsensitivity, (2) a total loss of mechanoreceptor currents, (3) GFP-α1tub84B completely failing to enter the outer segments, and (4) microtubules dramatically accumulating at the base of the cilia. Furthermore, klp64D animals, just like oseg2 mutants, have an almost complete loss of the tubular body, but have normal basal bodies and connecting cilia; thus, kinesin II is also not essential for the assembly of the proximal ciliary structures, including axoneme components. Together, these results substantiate kinesin II as a critical player in OSEG function and validate the fundamental importance of intraciliary transport in outer segment (compartmentalized cilia) biogenesis (Avidor-Reiss, 2004).

    In this study, a novel bioinformatics screen was used, relying on evolutionary gene conservation and gene loss as a paradigm to discover loci selectively involved in cilia formation and function. This strategy efficiently identified a wide spectrum of known ciliary proteins and dramatically enriched the repertoire of candidate ciliary genes. Because of the focus on identifying ciliary genes of the ancestral eukaryotic cell (e.g., by selecting ciliary genes found in both Bikonts and Unikonts lineages), the recovery of genes unique to specific lineages was not expected. However, by using selective combinations of genomes in the search algorithm, it was possible to define and distinguish between genes involved in cilia motility versus cilia compartmentalization: as additional genomes are completed, it should be possible to target new categories (Avidor-Reiss, 2004).

    Approximately 200 genes were selected in the four searches described in this paper. The cilia-compartment subset was analyzed in detail and 27 genes were identified as strong ciliary compartment candidates. Fifteen were selected for detailed in vivo expression studies and it was demonstrated that all were specifically expressed in compartmentalized cilia. Many of the genes in the motility and prototypical-cilia subsets were also examined using a spectrum of curation strategies. This analysis identified an additional collection of novel candidate ciliary genes. It will be of great interest to determine whether mutations in the human orthologs of these genes underlie cilia-based sensory, developmental, or reproductive disorders (Avidor-Reiss, 2004).

    Ciliary genes that serve multiple cellular functions were not selected in this screen, mainly because they are still present in organisms that have lost ciliated structures. For example, dyneins are critical components of the ciliary motility apparatus, yet many were filtered out in these screens because they are also involved in intracellular transport in nonciliated organisms. Indeed, it is suggested that the reason so few candidate genes were recovered in the 'all ciliated organisms' subgroup is because proteins common to all cilia, like those involved in axoneme assembly, are also required in basic cellular processes and therefore conserved in nonciliated organisms (e.g., α-tubulin, β-tubulin, γ-tubulin, centrin, pericentrin, etc.) (Avidor-Reiss, 2004).

    What do cilia-compartment genes do? At a basic level, these genes should encode components of the intraciliary transport system and the cilia pore, a supramolecular structure that forms the gate into the cilia (see Scholey Intraflagellar transport motors in Caenorhabditis elegans neurons). Indeed, the current screen identified all of the known IFT homologs found in Drosophila, including novel OSEG members. By extension, it is suggested that the compartment group also contains the molecular components of the cilia pore complex (Avidor-Reiss, 2004).

    This study shows that oseg1 and oseg2 have distinct roles in ciliogenesis, but neither oseg1 nor oseg2, or even kinesin II, are required for formation of the connecting cilium. These results demonstrate that the assembly of outer segment is orchestrated independently of the connecting cilia (and its axoneme). It will be of great interest to determine which cilia-compartment genes have a role in the biogenesis of this structure (Avidor-Reiss, 2004).

    OSEGs are characterized by the presence of two major protein-protein interaction domains, WD and TPR repeats, implicated in the assembly of multiprotein complexes. Significantly, the most closely related proteins outside of the family are α- and β'-coatomer, two cargo-carrying proteins intimately involved in intracellular trafficking. Furthermore, clathrin heavy chains display striking domain similarity to the OSEG family: an N terminus, consisting of 7 WD repeats and a C terminus consisting of 35 TPR-like repeats known as CHCR motifs. Interestingly, coatomers and clathrin-mediated transport systems use small G proteins of the Arf subfamilies as regulators of the transport process. Notably, the screen also identified ARL3 and ARL6, two Arf-like proteins, as components of the ciliary compartment group, with ARL6 expression restricted to mechano- and chemo-sensory neurons (Avidor-Reiss, 2004 and references therein).

    What do OSEGS do? The Drosophila oseg2 gene shares significant similarity with a 20 amino acid tryptic peptide from Chlamydomonas IFT172. IFTs were originally identified as a group of proteins enriched in the flagella of Chlamydomonas dynein-1b mutants and absent in the flagella of kinesin II mutants. Because anterograde transport is blocked in kinesin mutants, and retrograde transport is abolished in dynein mutants, IFT particle proteins were proposed to function as molecular rafts transporting cargo up and down the axoneme. Multiple lines of evidence strongly support the proposal that OSEGs function as ciliary transport proteins: (1) OSEGs are specifically expressed in ciliated cells, and the proteins are selectively localized to the cilia and cilia base; (2) OSEGs share structural similarity to prototypical intracellular transport proteins (e.g., clathrin, COP1); (3) oseg2 mutants have specific defects in intraciliary transport; (4) Drosophila OSEGS are required for compartmentalized ciliogenesis (sensory cilia) but not for cytosolic ciliogenesis (sperm tail), and (5) flies defective in oseg2 have nearly the same phenotype as mutants defective in klp64D, the ciliary motor. While there is very limited available data on oseg orthologs in Chlamydomonas, several of the oseg orthologs in C. elegans genes map at, or near, the location of worm mutations leading to sensory cilia defects and implicated in cilia formation and maintenance. For example, OSEG2 and OSEG5 are orthologs of OSM-1 and CHE-2, and OSEG1 and OSEG3 are probably orthologs of DAF-10 and CHE-3, respectively. Surprisingly, the integration of these proteins into a group of genes related to the main families of intracellular transport proteins had escaped notice. These results illustrate a common foundation in the organization of intracellular transport systems, whether mediating internalization of surface proteins, transferring cargo between organelles, or delivering components from the cell body to distal ciliary compartments (Avidor-Reiss, 2004 and references therein).

    Microtubule-induced nuclear envelope fluctuations control chromatin dynamics in Drosophila embryos

    Nuclear shape is different in stem cells and differentiated cells and reflects important changes in the mechanics of the nuclear envelope (NE). The current framework emphasizes the key role of the nuclear lamina in nuclear mechanics and its alterations in disease. Whether active stress controls nuclear deformations and how this stress interplays with properties of the NE to control NE dynamics is unclear. This study addresses this problem in the early Drosophila embryo, in which profound changes in NE shape parallel the transcriptional activation of the zygotic genome. Microtubule (MT) polymerization events are shown to produce the elementary forces necessary for NE dynamics. Moreover, large-scale NE deformations associated with groove formation require concentration of MT polymerization in bundles organized by Dynein. However, MT bundles cannot produce grooves when the farnesylated inner nuclear membrane protein Kugelkern (Kuk) is absent. Although it increases stiffness of the NE, Kuk also stabilizes NE deformations emerging from the collective effect of MT polymerization forces concentrated in bundles. Finally, it is reported that MT-induced NE deformations control the dynamics of chromatin and its organization at steady state. Thus, the NE is a dynamic organelle, fluctuations of which increase chromatin dynamics. It is proposed that such mechanical regulation of chromatin dynamics by MTs might be important for gene regulation (Hampoelz, 2011).

    In summary, these results indicate that nuclear shape in Drosophila embryos is not simply determined by nuclear factors that control deformability but instead requires the interplay between active stresses exerted by polymerization of MTs organized in bundles and properties of the NE. Surprisingly, MTs do not shape the NE like a static scaffold that constrains inherent dynamics of the NE. Rather, it was found that MT dynamics is essential. Polymerization of MTs produces small high-frequency fluctuations of the NE but is not capable of large-scale deformations into grooves. Groove formation requires MT polymerization within bundles, a property which depends on Dynein. It is thus proposed that pushing forces emanating from MT polymerization events are the fundamental active process underlying nuclear deformations. However, their organization in bundles is essential for lobulation. Bundling of growing MTs along a stationary core probably increases their ability to produce force. In vitro experiments and simulations showed that MTs in a bundle reach pushing forces much higher than the stall force of individual MTs. Moreover, relaxation of grooves might be facilitated within bundles, as pushing of bundles towards obstacles can facilitate collective catastrophe. Bundle integrity is ensured by cytoplasmic Dynein. Although no enrichment of Dynein was observed at the NE, Dynein could be localized at the NE, where it would allow bundle cohesion and attachment. In Drosophila photoreceptors and other systems, Dynein is recruited to the NE by the LINC (linker of nucleoskeleton and cytoskeleton) complex. The LINC complex consists of a KASH domain protein and a SUN domain protein, and spans the NE. In contrast to Dynein inhibition, reduced activity of the Drosophila LINC complex component CG3287/Klaroid did not affect nuclear morphology. Instead, this enhanced the MT-induced fluctuations of the NE, indicating that MT polymerization forces are less efficiently buffered. By bridging the NE, LINC proteins are believed to dampen cytoskeletal forces at the lamina or heterochromatin. The observations made in this study are consistent with this interpretation (Hampoelz, 2011).

    The LINC complex also transmits cytoskeletal forces across the NE to direct chromosome movement during meiosis. In fission yeast, LINC proteins mediate MT- and Dynein-dependant oscillating movements of whole nuclei. This results in nucleoplasmic agitation, which promotes pairing of homologous regions. By analogy, MT-induced fluctuations of the NE could serve as a means to generally enhance chromatin mobility at the onset of zygotic transcription. This would increase the probability of interactions with other loci or the NE and could tune cis-regulatory interactions. The recently established concept of transcription factories where active loci are pulled into pre-assembled sites of mRNA production demands a mobile chromatin. Interestingly, transcription is affected in kuk mutants, including up- and downregulation of predominantly early zygotic genes. Although a specific role in directly regulating these target genes cannot be excluded, a more likely scenario is a global contribution of Kuk to transcription at this stage owing to its effects on NE mechanics and dynamics as well as chromatin mobility. It will be important to study this quantitatively through direct visualization of dynamics at specific loci and transcription with fluorescence in situ hybridization (Hampoelz, 2011).

    Although necessary, MT polymerization forces are not sufficient to produce grooves in the NE. These deformations require specific material or structural properties of the NE. This work sheds new light on this process. Comparison of human embryonic stem cells and differentiated cells indicates that deformability is usually increased when stiffness is reduced (and vice versa), for instance owing to absence or knockdown of A-type lamins. A Drosophila A-type lamin is not expressed during cellularization, and nuclear deformability is instead controlled by the farnesylated, INM-protein Kuk. Kuk increases the stiffness of the NE, and it is required for large deformations probably because stiffness is required for the pre-stressed NE to buckle. However, Kuk is likely to control other properties of the NE as its depletion cannot be rescued by elevated levels of the B-type lamin Dm0, which also increase NE stiffness. Likewise, overexpressed Dm0 does not enhance lobulation. Kuk could stabilize transient and small deformations imposed by MTs. Stabilization of NE curvature would work as a ratchet and allows the temporal integration of small polymerization forces contributed by individual MTs in bundles. The relative amounts of MT polymerization forces and NE stiffness would define the threshold above which buckling is possible (Hampoelz, 2011).

    The roles of microtubule-based motor proteins in mitosis: comprehensive RNAi analysis in the Drosophila S2 cell line

    Kinesins and dyneins play important roles during cell division. Using RNA interference (RNAi) to deplete individual (or combinations of) motors followed by immunofluorescence and time-lapse microscopy, the mitotic functions were examined of cytoplasmic dynein and all 25 kinesins in Drosophila S2 cells. Four kinesins are involved in bipolar spindle assembly, four kinesins are involved in metaphase chromosome alignment, Dynein plays a role in the metaphase-to-anaphase transition, and one kinesin is needed for cytokinesis. Functional redundancy and alternative pathways for completing mitosis were observed for many single RNAi knockdowns, and failure to complete mitosis was observed for only three kinesins. As an example, inhibition of two microtubule-depolymerizing kinesins initially produced monopolar spindles with abnormally long microtubules, but cells eventually formed bipolar spindles by an acentrosomal pole-focusing mechanism. From this phenotypic data, a model is constructed for the distinct roles of molecular motors during mitosis in a single metazoan cell type (Goshima, 2003).

    Before beginning functional analysis, the sequences of all Drosophila kinesin superfamily proteins were identified and analyzed. A BLAST search was performed on the fly database using the conserved motor domain of fly conventional kinesin (1-340 aa). 25 genes emerged as exhibiting significant (E-value < 1e-15) sequence homology. Sequence alignments of the motor and non-motor domains with kinesins from other organisms were used to assign the Drosophila kinesins to different subfamilies. This analysis identified clear subfamilies and mammalian homologues for 21 of the 25 genes. The remaining four are divergent kinesins that have no homology in their tail domains to kinesins in other organisms. Five kinesins may not be present or are expressed at very low levels in S2 cells. Nevertheless, RNAi was performed for all 25 kinesins so as not to miss a potential mitotic involvement of a low copy number kinesin (Goshima, 2003).

    RNAi of eight kinesins and cytoplasmic DHC caused a variety of mitotic defects, including monopolar spindle formation, chromosome misalignment, anaphase delay, and cytokinesis failure. The involvement of other motors in mitosis cannot be ruled out due to several experimental caveats. (1) Due to unavailability of antibodies, the reduction of 16 motors by RNAi could not be confirmed. However, for the 10 motors that were examined, drastic (>90%) protein reductions by RNAi were confirmed. Moreover, similar reductions of 24 other cytoskeleton-related proteins was confirmed by immunoblotting and/or immunostaining after dsRNA treatment, regardless of the appearance of phenotypes, no case has been encountered where protein levels were not dramatically reduced. Based on such results, it is highly likely that all motors were reduced to very low levels by RNAi. (2) Even the small amount of residual protein that remains after RNAi treatment may be sufficient for cellular function. (3) Functional redundancy may obscure a mitotic phenotype for some motors. This could be potentially examined by exhaustive double or triple RNAi for many combinations of motor proteins (Goshima, 2003).

    The results provide several general insights into the roles of molecular motors in mitosis. First, cells appear to have redundant or alternate mechanisms for completing mitosis, despite the absence of a kinesin motor. These experiments, for example, uncovered a novel rescue mechanism for converting monopolar to bipolar spindles by an acentrosomal pole-focusing mechanism. By performing multiple RNAi, it could also be show that several kinesin act in a partially redundant manner to ensure congression of chromosomes to the metaphase plate. Only three kinesins appear to be absolutely essential for completing mitosis; Klp61F [BimC/Eg5] and Klp67A [Kip3] RNAi cells could not proceed into anaphase, and Pavarotti [MKLP1]-depleted cells could not execute cytokinesis. The phenotypes from comprehensive RNAi analysis enable a model to be derived for the roles of microtubule-based motors in the sequential steps of mitosis in S2 cells (Goshima, 2003).

    The establishment of spindle bipolarity, the first step of mitosis, requires Klp61F [BimC/Eg5], a bipolar homotetrameric kinesin. Time-lapse imaging results are consistent with proposals that Klp61F [BimC/Eg5] cross-links and slides apart antiparallel spindle microtubules (Goshima, 2003).

    Ncd [Kin C] depletion causes defects in spindle pole integrity. MTOC fusion is inhibited, and multiple poles, frequently acentrosomal, are produced. Time-lapse observation of GFP-tubulin in Ncd [Kin C] RNAi cells suggests that microtubules are frequently released from the existing pole in prometaphase, and these released microtubules can build additional acentrosomal spindles. Ncd [Kin C] may move toward the microtubule minus ends and physically tether the microtubules to the poles. The S2 Ncd [Kin C] RNAi phenotype is quite different from the Drosophila Ncd [Kin C] null mutant, in which meiotic spindle defects are observed, but spindle formation defects in mitotic cells are either not severe or not observed. Variations in phenotype after inhibition of Kin C kinesins are also evident in comparing results from different experimental systems. For example, antibody inhibition of XCTK2 induced accumulation of asters and half spindle in Xenopus egg extract. It is speculated that Ncd [Kin C] may serve as a primary motor for spindle pole coalescence in S2 cells, whereas in most of the fly tissues, Ncd [Kin C]'s function may be secondary and/or compensated by cytoplasmic dynein (Goshima, 2003).

    The regulation of microtubule dynamics by motor proteins is essential for efficient spindle assembly. RNAi of Klp10A [Kin I], a member of the microtubule-destabilizing Kin I kinesins, resulted in excessively long microtubules emanating radially from the MTOC, suggesting that Klp10A [Kin I] destabilizes most, if not all, spindle microtubules. Monopolar spindles were frequently formed, as found in fixed images after protein inhibition of the vertebrate homologue KCM1/MCAK. Live-cell imaging clearly shows that separated MTOCs fuse together after NEB, despite the presence of Klp61F [BimC/Eg5]. Longer than normal microtubules may be physically difficult for Klp61F [BimC/Eg5] to cross-link into antiparallel bundles. Alternatively, pushing forces against the cell cortex exerted by long astral microtubules might overcome the forces generated by Klp61F [BimC/Eg5]. However, Klp10A [Kin I] RNAi cells eventually succeed in forming monastral bipolar spindles, which segregate sister chromatids in anaphase, strongly suggesting that stable kinetochore-spindle interaction can be achieved in the absence of Klp10A [Kin I]. A mitotic function was not detected for the two other Kin I motor proteins (Klp59C and Klp59D), although another analysis involving antibody microinjection found anaphase A defects for Klp59C in fly embryos (Goshima, 2003).

    Klp67A [Kip3] was originally localized at mitochondria. However, a mitochondria transport defect after Klp67A [Kip3] RNAi was not detected in S2 cells Instead, RNAi of Klp67A [Kip3] produces long mitotic microtubules, indicating that it destabilizes microtubules. This result is consistent with sequence analysis showing that Klp67A [Kip3] is related to S. cerevisiae Kip3 and Schizosaccharomyces pombe Klp5/Klp6, null mutants of which have longer than normal spindle microtubules. Excessively long microtubules may give rise to monopolar spindle formation in Klp67A [Kip3] RNAi cells by the mechanism described above for Klp10A [Kin I]. However, unlike Klp10A [Kin I], which destabilizes all spindle microtubules, Klp67A [Kip3] appears to act selectively on microtubules between the poles and chromosomes; astral microtubules have normal length in Klp67A [Kip3] RNAi cells. Thus, Klp67A [Kip3] and Klp10A [Kin I] have distinct roles as microtubule-destabilizing proteins in the spindle (Goshima, 2003).

    Three and possibly four chromosomal kinesins are important for chromosome movement. Previous reports have shown that the kinetochore-localized CENP-meta [CENP-E] is essential for chromosome congression at early developmental stages. The genetic phenotypes of the two chromosome-associated kinesins, No distributive disjunction [Nod] and Klp3A [chromokinesin], for mitotic chromosome alignment are less evident. Nod [Kid] is important for chromosome positioning in meiosis, but is nonessential for fly viability. Klp3A [chromokinesin] is also nonessential for fly viability, and null mutation causes cytokinesis failure in meiosis. However, the data suggest that these two chromokinesins act redundantly in mitotic cells for prometaphase chromosome movement; double RNAi of Klp3A [chromokinesin] and Nod [Kid] causes more severe chromosome misalignment phenotypes than single RNAi treatments. The survival of adult fly without either Nod [Kid] or Klp3A [chromokinesin] might be due to this redundancy. However, phenotypic analyses show that the actions of these two chromokinesins are distinct. The chromosome arms were abnormally extended along the direction of spindle axis during prometaphase and metaphase after Nod [Kid] RNAi, suggesting that Nod [Kid] functions to transport chromosomal arms away from the pole. In contrast, Klp3A [chromokinesin] may be needed for both kinetochore and arm-directed chromosome motility. The redundant function of chromatin- and kinetochore-localized kinesins for chromosome congression may be a general feature of eukaryotes (Goshima, 2003).

    The Klp67A [Kip3] RNAi phenotype also suggests an additional role for this motor in proper chromosome-spindle interaction, most probably at the kinetochore. After depletion of Klp67A [Kip3], chromosomes are scattered in the spindle (in contrast to Klp10A [Kin I] RNAi), possibly reflecting unbalanced spindle tension on chromosomes. Additionally, very few cells enter anaphase, presumably because of activation of the spindle checkpoint that monitors spindle-kinetochore interaction. In S. pombe, two Klp67A [Kip3]-like proteins, Klp5 and Klp6, are localized at mitotic kinetochores, and have been proposed to generate stable kinetochore-spindle interactions and tension at kinetochores. Because RNAi phenotypes of Klp67A [Kip3] are similar in many aspects to Klp5/Klp6 mutants, Klp67A [Kip3] also might act at kinetochores (Goshima, 2003).

    In addition to Klp67A [Kip3], cytoplasmic dynein plays a role in the metaphase-to-anaphase transition. Dhc64C (cytoplasmic dynein) may control the timing of anaphase onset, possibly by transporting Rod or other checkpoint proteins away from kinetochores as proposed for the fly embryo. A similar checkpoint inactivation model was proposed for mammalian dynein/dynactin based on inhibition analyses (Goshima, 2003).

    The roles of motors in anaphase A (chromosome to pole motion) and anaphase B (spindle elongation) are less clear from this paper, since no defects were detected by fixed cell images after RNAi. This may be due to the fact that RNAi depletion of a single motor protein may cause kinetic defects in anaphase, which may be difficult to detect in fixed population images. For example, recent live-cell imaging of anaphase in living Drosophila embryos shows that antibodies against Kin I motors slow down rather than completely block anaphase. It is also possible that Klp61F [BimC/Eg5] or Klp67A [Kip3] play roles in anaphase; however, RNAi of these motors arrests cells earlier in mitosis (Goshima, 2003).

    Finally, central spindle formation and cytokinesis in S2 cells require Pavarotti [MKLP1]. However, it has not been clear whether Pav is needed for the formation or maintenance of the central spindle. Live-cell observations of Pav [MKLP1] RNAi cells shows that some cells formed central spindles, but could not maintain them, whereas others showed a complete failure in formation (Goshima, 2003).

    An interesting phenomenon is the conversion of a monopolar spindle to a monastral bipolar spindle, in which one of the poles lacks a centrosome. Anastral bipolar spindles, in which both poles lack centrosomes, are commonly observed in higher plant mitosis and animal female meiosis, but the formation and relevance of acentrosomal poles in animal mitotic cells have been less clear. It has been found that bipolar spindles containing one centrosomal and one acentrosomal pole can be formed if one centrosome is destroyed by laser ablation. In addition, there has been a previous report of asymmetrical bipolar spindles in nonmeiotic cells) (Goshima, 2003).

    The live-cell analysis provides a mechanism for monastral bipolar spindle formation. Initially, microtubules emerge from the chromosomal region in the monopolar spindle. Similar observations of chromosome-directed microtubule formation have been made for acentrosomal spindle formation in meiosis. Simultaneous RNAi experiments show that Klp61F [BimC/Eg5], but not cytoplasmic dynein, is required for monastral bipolar spindle formation. Bipolar kinesins may be necessary for bundling the chromosome-generated microtubules. The role of Ncd [Kin C] during monopolar to bipolar conversion is ambiguous by double the RNAi method, since Ncd [Kin C] depletion dominantly inhibits monopolar spindle formation. However, considering Ncd [Kin C]'s role in MTOC fusion in early prometaphase and pole maintenance, it also may be needed for acentrosomal pole-focusing in the final step of the conversion. Klp67A [Kip3] may also play a minor role in this process because monopolar spindles generated after Klp67A [Kip3] RNAi do not always form an acentrosomal pole, perhaps due to problems in chromosome-microtubule interaction as discussed above (Goshima, 2003).

    Monopolar to monastral bipolar spindle conversion also appears to occur in untreated cells as well, because both monopolar (<5% of mitotic cells) and monastral bipolar spindles (10% of bipolar spindles) were observed in untreated S2. Moreover, it has been reported that 0.9% of mitotic spindles of the wild-type Drosophila larval neuroblasts display monastral bipolar spindles. In the monastral bipolar spindle of Klp10A [Kin I] RNAi and untreated cells, anaphase chromosome movement can occur. Thus, the mitotic checkpoint is activated during the monopolar spindle phase, but is properly down-regulated once metaphase is achieved in monastral bipolar spindle. Thus, in the presence of a functional spindle checkpoint system, monastral bipolar spindle formation enables completion of mitosis in rare cases when the spindle gets trapped in a monopolar state after nuclear envelope breakdown. Although originally believed to be a property of plant and meiotic animal cell, it is proposed that acentrosomal pole formation constitutes a general backup mechanism for mitosis in somatic animal cells (Goshima, 2003).

    A PAR-1-dependent orientation gradient of dynamic microtubules directs posterior cargo transport in the Drosophila oocyte

    Cytoskeletal organization is central to establishing cell polarity in various cellular contexts, including during messenger ribonucleic acid sorting in Drosophila melanogaster oocytes by microtubule (MT)-dependent molecular motors. However, MT organization and dynamics remain controversial in the oocyte. This study used rapid multichannel live-cell imaging with novel image analysis, tracking, and visualization tools to characterize MT polarity and dynamics while imaging posterior cargo transport. All MTs in the oocyte were highly dynamic and were organized with a biased random polarity that increased toward the posterior. This organization originated through MT nucleation at the oocyte nucleus and cortex, except at the posterior end of the oocyte, where PAR-1 suppressed nucleation. These findings explain the biased random posterior cargo movements in the oocyte that establish the germline and posterior (Parton, 2011).

    Despite the importance of MTs in the oocyte, how they are organized and to what extent they are dynamic have remained highly controversial. Moreover, the prevailing models for MT organization have mostly relied on a static view of MT distribution and on indirect measures of polarity, such as the steady-state distribution of motor fusions and cargoes. By using live-cell imaging and developing novel image analysis and global visualization tools, the dynamics and polarity of MTs were characterized directly in living oocytes. MTs were found to form a dynamic cortical network extending into the posterior with a bias in net orientation that increases toward the posterior. It was established that posterior-directed cargo is actively transported on these dynamic MTs, with no evidence for preferential transport by a subpopulation of more stable, posttranslationally modified MTs. Significantly, the magnitude and distribution of the observed bias in cargo movements parallels closely the polarity of the MT network. These findings explain the previously reported subtle biased random transport of posterior cargoes and lead to the proposal of the following model for posterior cargo localization: posterior cargo is transported on the entire dynamic MT network and the overall net bias in MT orientation directs the net movement of cargo to the posterior cap, where it becomes anchored (Parton, 2011).

    The results reveal that the establishment of the biased MT network is dependent on a specific distribution of MT nucleation sites around the oocyte cortex, with a critical, PAR-1-dependent exclusion of MT nucleation from the posterior cortex. This extends upon previous observations that PAR-1 affects MT organization, leading to an increased density of MTs at the posterior. By using highly sensitive imaging techniques in live oocytes, it was demonstrated that, in contrast to previous work, in stage 9 oocytes, MT nucleation is also distributed along the anterior and lateral cortexes. Initiation of MTs is predominantly from the anterior of the oocyte with a sharp decrease in nucleation along the posterior two thirds of the oocyte cortex. Those MTs nucleating along the anterior are constrained to grow in a more posterior orientation, whereas nucleation along the lateral cortex is more random in orientation. The combination of these two contributions to the network of MTs present in the oocyte results in a slight excess of plus ends extending in a posterior direction, which increases in magnitude closer to the posterior. Despite the fact that, at the extreme posterior, there are no MT nucleation sites, it was found that, even at the extreme posterior, a percentage of MTs appear to orient toward the anterior. This is caused by MTs bending around as they extend into the posterior. Importantly, this detailed analysis of cargo movements reveals a bias in cargo movement directionality at the posterior that matches precisely the bias in MT orientation (Parton, 2011).

    It is interesting to consider how the PAR-1 kinase might prevent the nucleation of MTs at the posterior cortex. The PAR genes are conserved polarity determinants with common functions in a variety of organisms. PAR-1 is known to function in association with other PAR proteins, so it is possible that the other PAR proteins also function together with PAR-1 to inhibit MT nucleation in the oocyte. However, several other factors may also be involved. PAR-1 could affect MTs through its association with Tau, which has been shown in mammalian cells and proposed in the Drosophila oocyte, but this remains contentious, as the presence of Tau is not absolutely required for PAR-1 function. Another possibility is that PAR-1 could act through the components of the γ-TuRC complex or some other MT nucleation components, rather than through a direct affect on MTs. Whatever the molecular mechanism of PAR-1 inhibition of MT nucleation, it is most likely to involve the phosphorylation of a downstream target of the PAR-1 kinase (Parton, 2011).

    The live-imaging results highlight the role of a dynamic MT network in establishing cell polarity in the oocyte, in which no stable, posttranslationally modified MTs were detected. This raises the question of why this should be the case when, in some other cells, subsets of either stably bundled or completely stable, posttranslationally modified MTs have been observed and proposed to have functional roles in directing cell polarity. Moreover, in many polarized cell types, including the blastoderm embryo and secretory columnar epithelial cells of egg chambers, MTs are organized with a very strong apical-basal polarity and include stable MTs. This makes functional sense in both cases, as cargoes have to be transported very rapidly either apically or basally. In contrast, in the oocyte, MTs perform three key functions that are not necessarily all compatible with having a very strict apical-basal polarity. First, they provide a means of randomly distributing generic components, such as mitochondria and lipid droplets, throughout the cytoplasm. Second, they provide a network to gather cargoes and redistribute them to distinct intracellular destinations, initiating and maintaining cell polarity. Third, they provide a scaffold that maintains structural integrity. It is proposed that the dynamic, subtly biased network of MTs in the oocyte provides an efficient compromise for dealing with these multiple conflicting biological requirements. During mid-oogenesis, the oocyte undergoes a huge expansion in size, when many cellular components are transported from the nurse cells or secreted from the overlying follicle cells. Although generic cellular components, such as Golgi, mitochondria, and lipid droplets, must be kept distributed throughout the ooplasm, the nucleus and specific mRNAs and proteins must be transported to different poles to establish the embryonic axes. A biased random network of MTs enables the mixing of generic components by continuous transport using molecular motors with opposing polarities, while, at the same time, allowing specific components to be transported by motors with single polarities to the anterior or posterior poles for anchoring. Furthermore, a network of highly dynamic MTs would allow efficient capturing of cargo by the motors throughout the entire ooplasm in a rapidly growing and developing oocyte. The fact that the MT network is highly dynamic also makes considerable functional sense for such a rapidly developing system. The MT cytoskeleton is reorganized extensively during Drosophila oogenesis but most dramatically during stage 7. This fits well with observations in other cell types showing that MTs are highly dynamic in nature and are often reorganized to direct cellular polarization (Parton, 2011).

    MTs certainly play critical roles in driving cell polarization and extension in many kinds of eukaryotic cells, for example, during guidance of extending neuronal growth cones, in migratory cells, in dorsal closure, and in fields of bristles with planar polarity in fly wings. In all these cases, the polarity and dynamics of MTs have tended to be studied quite crudely because of an inability to follow the subtleties of global MT polarity and dynamics. Therefore, it is highly likely that MTs in such cells are more complex and subtle than previously thought. Interestingly, at least in Xenopus laevis oocytes in which hook decoration and EM were used as the previous gold standard for determining the polarity of individual MTs, a network of MTs is nucleated at the cortex, leading to a bias of polarity rather than an absolute polarity. The methods used in this study are significantly easier to apply technically than hook decoration methods and are, therefore, more generally applicable to study the orientation and dynamics of MTs in most cells. For example, it has been possible to apply these analysis tools to examine subtleties of MT organization in migratory border cells (Parton, 2011).

    It is proposed that during cellular reorganization and repolarization, as in the oocyte, the establishment of a dynamic, subtly biased MT network is a widespread phenomenon and provides a general mechanism by which strong cell polarity can be initiated and maintained while efficiently handling the transport requirements of cargoes distributed throughout the cytoplasm. The tools developed in this study to quantitate global or local bias in a complex field of MTs can now be applied widely to other oocytes and other cell types to test the generality of the proposed biased random model for MT organization (Parton, 2011).

    Shot and Patronin polarise microtubules to direct membrane traffic and biogenesis of microvilli in epithelia

    In epithelial tissues, polarisation of microtubules and actin microvilli occurs along the apical-basal axis of each cell, yet how these cytoskeletal polarisation events are coordinated remains unclear. This study examines the hierarchy of events during cytoskeletal polarisation in Drosophila and human epithelia. Core apical-basal polarity determinants polarise the Spectrin cytoskeleton to recruit the microtubule-binding proteins Patronin (CAMSAP1/2/3 in humans) and Shortstop (Shot; MACF1/BPAG1 in humans) to the apical membrane domain. Patronin and Shot then act to polarise microtubules along the apical-basal axis to enable apical transport of Rab11 endosomes by the Nuf-Dynein microtubule motor complex. Finally, Rab11 endosomes are transferred to the MyoV actin motor to deliver the key microvillar determinant Cadherin99C to the apical membrane to organise the biogenesis of actin microvilli (Khanal, 2016).

    These results reveal a mechanism linking determinants of cell polarity with stepwise polarisation of the spectrin cytoskeleton, microtubule cytoskeleton and biogenesis of actin microvilli through apical trafficking of Cad99C. The results suggest that polarisation of the apical spectrin βH-Spectrin is dependent on polarity determinants, likely through interactions with the FERM domain proteins and the apical polarity determinant Crb. The spectraplakin Shot is highly similar to βH-Spectrin, and is able to bind to and colocalise with it at the apical domain of epithelial cells, suggesting that the two proteins might have a similar function. βH-Spectrin is linked to microtubules through Patronin, whereas Shot can directly bind microtubules. Consequently, redundancy is anticipated between βH-Spectrin and Shot, or between Patronin and Shot. Accordingly, this study found that mutation of βH-spectrin only had a mild phenotype, whereas mutation of α-spectrin simultaneously disrupted both pairs of proteins in parallel and caused a drastic phenotype, completely disrupting the apical trafficking of Cad99C and microvillar biogenesis. More importantly, double mutants for shot and βH-spectrin had a more severe effect on microtubule and Cad99C localisation than either alone, therefore demonstrating that the two proteins act in a redundant fashion (Khanal, 2016).

    Downstream of the spectrin cytoskeleton, Patronin and Shot are required in parallel to drive apical-basal polarisation of microtubules, which is then responsible for orienting the apical transport of Cad99C, within Rab11 endosomes, by the Dynein motor protein. Eliminating microtubules from cells by overexpressing Katanin60 results in loss of Nuf-Dynein-based apical Rab11 endosome transport and failure to efficiently deliver Cad99C to the apical membrane. The effect on Cad99C polarisation is not an indirect effect of loss of polarity due to impaired Rab11 and Dynein function in localising the apical polarity determinant Crumbs to the apical membrane because, firstly, polarity is maintained in cells expressing Rab11 or Dynein RNAi, as indicated by the normal localisation of aPKC and, secondly, loss of Crb does not strongly affect cell polarity in the follicle cell epithelium owing to redundancy with Bazooka. The results indicate that even under conditions with severe depletion of microtubules, the overall shape of the follicle cell epithelium is relatively normal, indicating that polarised microtubules are required to influence formation of apical microvilli, rather than for other functions of the actin cytoskeleton in epithelial cells. Similarly, no strong effects are seen on cell shape upon loss of either Patronin or Shot (or both), raising questions over the claimed requirement for Patronin homologs and microtubules in formation or maintenance of adherens junctions epithelial cells in culture (Khanal, 2016).

    The final step in delivery of Cad99C to the apical membrane also requires actin-based transport through the action of Rip11-MyoV complex. Compromising normal MyoV function in Drosophila follicle cells by expressing a dominant-negative version of the protein, results in loss of Rab11 polarisation from the apical membrane and its abnormal accumulation in the sub-apical region. This phenotype in Drosophila shows similarities with the human microvillus inclusion disease, where mutations in the Myo5b gene also cause loss of Rab11 endosomes from the apical membrane (Khanal, 2016).

    In summary, these results reveal how the spectrin cytoskeleton acts to polarise microtubules in epithelial cells, and how polarised microtubules then direct trafficking of Rab11 endosomes carrying Cad99C to the apical membrane. This process relies on a hierarchy of events, and disruption at any stage can lead to failure in delivering Cad99C to the apical membrane, resulting in defective biogenesis of microvilli. These findings are directly relevant to human diseases such as Usher's Syndrome Type 1 and microvillus inclusion disease, helping to outline the molecular and cellular basis for these conditions (Khanal, 2016).

    Misato controls mitotic microtubule generation by stabilizing the tubulin chaperone protein-1 complex

    Mitotic spindles are primarily composed of microtubules (MTs), generated by polymerization of α- and β-Tubulin hetero-dimers. Defects in Tubulin polymerization dramatically affect spindle formation and disrupt chromosome segregation. Recently studies have described a role for the product of the conserved misato (mst) gene in regulating mitotic MT generation in flies, but the molecular function of Mst remains unknown. This study used affinity purification mass spectrometry (AP-MS) to identify interacting partners of Mst in the Drosophila embryo. Mst was shown to associate stoichiometrically with the hetero-octameric Tubulin Chaperone Protein-1 (TCP-1) complex, with the hetero-hexameric Tubulin Prefoldin complex, and with proteins having conserved roles in generating MT-competent Tubulin. RNAi-mediated in vivo depletion of any TCP-1 subunit phenocopies the effects of mutations in mst or the Prefoldin-encoding gene merry-go-round (mgr), leading to monopolar and disorganized mitotic spindles containing few MTs. Crucially, it was demonstrated that Mst, but not Mgr, is required for TCP-1 complex stability and that both the efficiency of Tubulin polymerization and Tubulin stability are drastically compromised in mst mutants. Moreover, structural bioinformatic analyses indicate that Mst resembles the three-dimensional structure of Tubulin monomers and might therefore occupy the TCP-1 complex central cavity. Collectively, these results suggest that Mst acts as a co-factor of the TCP-1 complex, playing an essential role in the Tubulin-folding processes required for proper assembly of spindle MTs (Palumbo, 2015).

    Digitor/dASCIZ has multiple roles in Drosophila development
    This study provides evidence that the spindle matrix protein Skeletor in Drosophila interacts with the human ASCIZ (also known as ATMIN and ZNF822) ortholog, Digitor. This interaction was first detected in a yeast two-hybrid screen and subsequently confirmed by pull-down assays. The study also confirms a previously documented function of Digitor as a regulator of Dynein light chain/Cut up expression. Digitor was shown to be a nuclear protein that localizes to interband and developmental puff chromosomal regions during interphase but redistributes to the spindle region during mitosis. Its mitotic localization and physical interaction with Skeletor suggests the possibility that Digitor plays a direct role in mitotic progression as a member of the spindle matrix complex. Furthermore, a true null Digitor allele results in complete pupal lethality when homozygous, indicating that Digitor is an essential gene. Digitor plays critical roles in regulation of metamorphosis and organogenesis as well as in the DNA damage response. In the Digitor null mutant larvae there are greatly elevated levels of γH2Av, indicating accumulation of DNA double-strand breaks. Furthermore, reduced levels of Digitor decrease the resistance to paraquat-induced oxidative stress resulting in increased mortality in a stress test paradigm. It was shown that an early developmental consequence of the absence of Digitor is reduced third instar larval brain size although overall larval development appears otherwise normal at this stage. Altogether the data shows that Digitor is a nuclear protein that performs multiple roles in Drosophila larval and pupal development (Sengupta, 2016).

    Scaling of cytoskeletal organization with cell size in Drosophila

    Spatially organized macromolecular complexes are essential for cell and tissue function, but the mechanisms that organize micron-scale structures within cells are not well understood. Microtubule-based structures such as mitotic spindles scale with cell size, but less is known about the scaling of actin structures within cells. Actin-rich denticle precursors cover the ventral surface of the Drosophila embryo and larva and provide templates for cuticular structures involved in larval locomotion. Using quantitative imaging and statistical modeling, denticle number and spacing were demonstrated to scale with cell size over a wide range of cell lengths in embryos and larvae. Denticle number and spacing are reduced under space-limited conditions, and both features robustly scale over a ten-fold increase in cell length during larval growth. The relationship between cell length and denticle spacing can be recapitulated by specific mathematical equations in embryos and larvae, and accurate denticle spacing requires an intact microtubule network and the microtubule minus-end-binding protein, Patronin. These results identify a novel mechanism of microtubule-dependent actin scaling that maintains precise patterns of actin organization during tissue growth (Spencer, 2017).

    The Drosophila orthologue of the INT6 onco-protein regulates mitotic microtubule growth and kinetochore structure

    INT6/eIF3e is a highly conserved component of the translation initiation complex that interacts with both the 26S proteasome and the COP9 signalosome, two complexes implicated in ubiquitin-mediated protein degradation. The INT6 gene was originally identified as the insertion site of the mouse mammary tumor virus (MMTV), and later shown to be involved in human tumorigenesis. Here we show that depletion of the Drosophila orthologue of INT6 (Int6) results in short mitotic spindles and deformed centromeres and kinetochores with low intra-kinetochore distance. Poleward flux of microtubule subunits during metaphase is reduced, although fluorescence recovery after photobleaching (FRAP) demonstrates that microtubules remain dynamic both near the kinetochores and at spindle poles. Mitotic progression is delayed during metaphase due to the activity of the spindle assembly checkpoint (SAC). Interestingly, a deubiquitinated form of the kinesin Klp67A (a putative orthologue of human Kif18A) accumulates near the kinetochores in Int6-depleted cells. Consistent with this finding, Klp67A overexpression mimics the Int6 RNAi phenotype. Furthermore, simultaneous depletion of Int6 and Klp67A results in a phenotype identical to RNAi of just Klp67A, which indicates that Klp67A deficiency is epistatic over Int6 deficiency. It is proposed that Int6-mediated ubiquitination is required to control the activity of Klp67A. In the absence of this control, excess of Klp67A at the kinetochore suppresses microtubule plus-end polymerization, which in turn results in reduced microtubule flux, spindle shortening, and centromere/kinetochore deformation (Renda, 2017).

    Tau and spectraplakins promote synapse formation and maintenance through Jun kinase and neuronal trafficking

    The mechanisms regulating synapse numbers during development and aging are essential for normal brain function and closely linked to brain disorders including dementias. Using Drosophila, this study demonstrates roles of the microtubule-associated protein Tau in regulating synapse numbers, thus unravelling an important cellular requirement of normal Tau. In this context, it was found that Tau displays a strong functional overlap with microtubule-binding spectraplakins, establishing new links between two different neurodegenerative factors. Tau and the spectraplakin Short Stop act upstream of a three-step regulatory cascade ensuring adequate delivery of synaptic proteins. This cascade involves microtubule stability as the initial trigger, JNK signalling as the central mediator, and kinesin-3 (see Drosophila Unc-103) mediated axonal transport as the key effector. This cascade acts during development (synapse formation) and aging (synapse maintenance) alike. Therefore, these findings suggest novel explanations for intellectual disability in Tau deficient individuals, as well as early synapse loss in dementias including Alzheimer's disease (Voelzmann, 2016).

    The correct formation and subsequent maintenance of synapses is a key prerequisite for brain development, function and longevity. Precocious loss of synapses is observed in late onset neurodegenerative diseases including Alzheimer's disease (AD) and Frontotemporal Dementia (FTD), likely contributing to the cognitive decline and neuronal decay observed in patients. Therefore, the characterisation of mechanisms maintaining synapses during ageing would have major implications for understanding of dementias (Voelzmann, 2016).

    The development of synapses and their maintenance during ageing is dependent on sustained transport of synaptic proteins from the distant soma, driven by motor proteins which trail along the bundles of microtubules in axons and dendrites. Microtubules are regulated by microtubule binding proteins which are therefore in a key position to regulate synapse formation and maintenance (Voelzmann, 2016).

    Tau is a microtubule associated protein (MAP) discovered in the mid-seventies. Reduction in Tau levels has been linked to intellectual disability and a class of brain disorders termed 'dementias which lack distinctive histopathology' (DLDH). Tau detachment from MTs is linked to prominent neurodegenerative diseases such as Alzheimer's disease, Frontotemporal Dementia and some forms of Parkinson's disease. In vitro, Tau has the ability to regulate microtubule properties including stability, cross-linkage and polymerisation. Through such functions, Tau would be expected to regulate multiple aspects of neuronal cell biology, but its physiological roles are still not understood and highly debated. This might partly be due to experimental challenges posed by functional redundancy, where other MAPs are proposed to mask physiological roles of Tau (Voelzmann, 2016).

    A good model in which to deal with functional redundancy is the fruit fly Drosophila melanogaster. As is ideal for studies of Tau, Drosophila neurons provide access to powerful genetics, they are readily established for research on the neuronal cytoskeleton, on neuronal transport and on synapses. Importantly, concepts and mechanisms gained from work in flies are often well conserved in higher organisms (Voelzmann, 2016).

    Work in Drosophila suggested that the spectraplakin Short Stop (Shot), a large actin-MT linker molecules and potent regulators of microtubules, could display potential functional overlap with Tau during microtubule stabilisation. This hypothesis is attractive because the well-conserved mammalian spectraplakin Dystonin is already linked to a neurodegenerative disease (type VI hereditary sensory autonomic neuropathy; OMIM #614653), and its paralogue ACF7/MACF1 plays important roles during brain development). Since ACF7 continues to be expressed in the brain, it is tempting to speculate that it might be required for neuronal maintenance (Voelzmann, 2016).

    This study used Drosophila neurons, in culture and in vivo alike, to demonstrate novel roles of Tau in regulating the formation and maintenance of synapses during ageing, by coordinating the intracellular trafficking of synaptic proteins. Thus, this study shows that the role of Tau in synapse regulation occurs in functional overlap with Shot. The robust shot-tau double-mutant phenotypes enabled study of the mechanistic cascade composed of three steps: microtubule stability as the trigger, the JNK signalling pathway as the mediator and kinesin-3 mediated axonal transport of synaptic proteins as the key effector. It is propose that a new mechanism based on the loss of Tau function which could explain intellectual disability in MAPT (the human tau gene) mutant individuals and precocious synapse loss in tau-related neurodegeneration (Voelzmann, 2016).

    The aim of these studies was to understand the role of endogenous Tau in neurons with particular attention to synapses. This effort was essentially aided by the finding that Tau and Shot are functionally redundant, and the subsequent incorporation of Shot into these studies. The robust phenotypes of shot-tau double-mutant neurons enabled this study to demonstrate roles of Shot-Tau during the formation and maintenance of pre-synaptic sites in axons, and unravel the underlying mechanistic cascade which involves three major steps. Firstly, the absence of Shot-Tau causes microtubule destabilisation. Secondly, this cytoskeletal stress causes aberrant JNK activity patterns with upregulation in somata and downregulation at axon tips. Thirdly, aberrant JNK activation leads to a somatic roadblock for kinesin-3 mediated transport, thus inhibiting the delivery of synaptic proteins and eventually causing synapse loss. Depending on whether the functions of Tau and/or Shot are removed during development or ageing, either the formation or the maintenance of synapses are affected, respectively (Voelzmann, 2016).

    The model explaining the function of Tau and Shot in synapse establishment and maintenance by regulating intracellular transport, is supported by loss- and gain-of-function experiments, genetic interactions and cross-rescue experiments. The initial finding that shot-tau mutant neurons had reduced branch numbers, could have suggested that defects on synapse numbers is indirect. However, experiments with double knock-down in culture and in the adult brain clearly showed strong synapse reduction whilst maintaining normal branch patterns, and Unc-104 rescued synapse reduction in shot-tau mutant neurons without major increases of the branch pattern in these neurons. These results clearly demonstrate that changes in neuronal morphology are not the cause of changes in synapse number (Voelzmann, 2016).

    Notably, the synaptic function of Tau described in this study for Drosophila might be conserved in higher animals or humans, since also aged Tau knock-out mice develop a reduction of synaptic proteins from the hippocampus (Voelzmann, 2016 and references therein).

    These findings provide potential new mechanistic explanations for various tau related brain disorders. For example, microdeletions in the region of MAPT (the human tau gene) cause intellectual disability, and Tau's synapse-promoting roles may well contribute to this pathology. Furthermore, various tauopathies are characterised by precocious pathological loss of synapses. The currnet data suggest that loss of tau could lead to defective synapse maintenance and eventually synapse loss. For example, a prominent group of dementias which lacks distinctive histopathology (DLDH) are characterised by the loss of Tau. Further tauopathies including Alzheimer disease, typically involve hyper-phosphorylation and aggregate formation of Tau. In this scenario, there are two parallel, non-exclusive modalities through which Tau can cause pathology. Firstly, detached hyper-phosphorylated tau attains gain-of-function roles in the cytoplasm damaging neurons through a number of mechanisms. Secondly, hyper-phosphorylation of tau causes a loss-of-function condition by depleting Tau from microtubules. However, since Tau knock-out mouse models mostly failed to show significant phenotypes and the neuronal functions of endogenous tau remain little understood, the pathological importance of Tau loss from microtubules has been marginalised. The current results now re-emphasise the notion that loss of Tau from microtubules could contribute to neurodegenerative pathology and deliver mechanistic explanations (Voelzmann, 2016).

    To unravel pathomechanisms caused by the loss of Tau, a combined depletion of Shot and Tau gave strong phenotypes, ideal for short-term experimental approaches. However, similar, yet milder phenotypes were found if only Tau was depleted, suggesting that the mechanisms described in this study could well contribute to slow disease progression in tauopathies. The discovery that spectraplakins are MAPs which functionally overlap with Tau, opens up new experimental avenues for Tau studies. So far, spectraplakins have been linked to the degeneration of sensory and autonomous neurons, and it remains to be elucidated whether they may have similar roles also in the brain. These results clearly hint at this possibility (Voelzmann, 2016).

    The loss of Tau and/or Shot inhibits kinesin-3 mediated transport leading to accumulation of synaptic proteins in the soma of neurons. A road-block mechanism is proposed suppressing the initiation of axonal transport in somata of Shot-Tau depleted neurons, which is caused indirectly through microtubule stress and mediated by JNK (Voelzmann, 2016).

    The involvement of microtubules in causing a transport block is supported by experiments using microtubule stabilising and de-stabilising drugs which rescued or mimicked the shot-tau mutant phenotypes, respectively. Similarly, axonal transport defects and cognitive deficits of PS19Tg mice (expressing the P301S mutant form of human tau) and various other mouse and fly tauopathy models were shown to be rescued by microtubule-stabilising drugs, suggesting that the mechanisms described may be conserved and relevant to disease (Voelzmann, 2016).

    The somatic road-block is a novel mechanism through which the loss of Tau can interfere with the transport of synaptic proteins and provides potential explanations also for somatic accumulations of postsynaptic proteins such as PSD-95, AMPA and NMDA receptors observed in mouse tauopathy models. A likely mechanism causing a roadblock in intracellular transport could be the direct inactivation of Unc-104 or its associated adaptor proteins, for example through JNK or other kinases within its pathway. This mode of regulation has a clear precedent in kinesin-1 and its adaptor Jip which are directly phosphorylated by JNK leading to transport inhibition. Unfortunately, extensive attempts to co-immunoprecipitate JNK and Kinesin-3 were unsuccessful, leaving open for now the exact molecular mechanism (Voelzmann, 2016).

    It is proposed that aberrant JNK activation downstream of microtubule destabilisation or stress is the ultimate cause for the defective delivery of synaptic proteins in Tau and/or Shot loss of function. Also in mouse, microtubule stress leads to somatic activation of the JNK pathway, suggesting this mechanism is likely to be conserved with vertebrates (Voelzmann, 2016).

    The JNK pathway is emerging as a central player in neurodegenerative diseases. Its activation is prompted by various neurodegeneration risk factors including oxidative stress, inflammation, and ageing. Furthermore, JNK is activated in AD patients and in several AD models where it triggers progression of the pathology. The new link between Tau/spectraplakins, JNK and synapses proposed in this study, is therefore likely to provide mechanistic explanations for synaptic pathology observed in AD and other tauopathies (Voelzmann, 2016).

    This study has delivered an important conceptual advance by revealing a new mechanistic cascade which can explain synaptic decay as the consequence of Tau loss from microtubules. Furthermore, a previously unknown functional redundancy with spectraplakins was identified as a promising new avenue for research on Tau. These findings emphasize that Tau detachment from microtubules can be an important aspect contributing to the pathology of tauopathies in parallel to roles of hyper-phosphorylated Tau in the cytoplasm. Synaptic decay, axonal transport and alterations in the JNK pathway are emerging as central players in a wider range of adult-onset neurodegenerative diseases, and here this study has aligned these factors into a concrete mechanistic cascade (Voelzmann, 2016).

    Polarized microtubule dynamics directs cell mechanics and coordinates forces during epithelial morphogenesis

    Coordinated rearrangements of cytoskeletal structures are the principal source of forces that govern cell and tissue morphogenesis. However, unlike for actin-based mechanical forces, knowledge about the contribution of forces originating from other cytoskeletal components remains scarce. This study has establish microtubules as central components of cell mechanics during tissue morphogenesis. Individual cells were found to be mechanically autonomous during early Drosophila wing epithelium development. Each cell contains a polarized apical non-centrosomal microtubule cytoskeleton that bears compressive forces, whereby acute elimination of microtubule-based forces leads to cell shortening. It was further established that the Fat planar cell polarity (Ft-PCP) signalling pathway couples microtubules at adherens junctions (AJs) and patterns microtubule-based forces across a tissue via polarized transcellular stability, thus revealing a molecular mechanism bridging single cell and tissue mechanics. Together, these results provide a physical basis to explain how global patterning of microtubules controls cell mechanics to coordinate collective cell behaviour during tissue remodelling. These results also offer alternative paradigms towards the interplay of contractile and protrusive cytoskeletal forces at the single cell and tissue levels (Singh, 2018).

    During development individual cells assemble into complex tissues and organs with specialized forms and functions. Tissue morphogenesis is driven by mechanical forces that are generated by the cytoskeleton within cells and transmitted in a coordinated manner through adhesion molecules across neighbouring cells. The best-studied cytoskeletal component is actin, which, together with other proteins, forms protrusive and contractile arrays, a fundamental constituent of rearrangements on the single cell and tissue levels. Recent work has suggested that microtubules, similar to actin, can also generate forces in cells. However, understanding of the contribution of microtubules to cell mechanics, cell shape changes and force coordination during morphogenesis remains poor. This is mainly due to the fact that many current models describing the mechanical state of tissues during shape changes focus on actomyosin dynamics and/or rely on continuum mechanics. These studies, which are based on coarse-grain observations of cell movements or cell shape changes, reveal only part of the physical mechanisms that drive morphogenesis and do not directly investigate the physicomechanical context of tissue remodelling. To understand the relationship between cell mechanics, force patterning and molecular structure, this study investigated the mechanical properties of microtubules at high spatiotemporal resolution using wing development in Drosophila melanogaster as a paradigm (Singh, 2018).

    During pupal wing development, non-centrosomal microtubules form an apical array of parallel microtubule bundles that are globally aligned along the proximal-distal (P-D) axis. Patterning of the microtubule cytoskeleton depends on the Ft-PCP signalling pathway and occurs during the early phase of wing reshaping (that is, between 14 and 18 h after puparium formation, or APF). This patterning is associated with extensive changes in cell shape, cell divisions and cell-cell rearrangements. In the Drosophila wing, the Ft-PCP pathway further orients cell elongation and cell divisions along the P-D axis to induce wing tissue elongation. Intriguingly, rescue of the Hippo pathway in Ft-PCP mutant animals, in which microtubule alignment is impaired, aberrant development results in perturbed cell elongation and an abnormal rounded wing shape, suggesting that there is an interdependence between these events. Therefore this study explored the possibility that microtubule-based cell mechanics control cell and tissue shape during early wing development between 16 and 18 h APF (Singh, 2018).

    Tissue remodelling is driven by intrinsic and extrinsic mechanisms, and it has previously been shown that extrinsic mechanical forces act during the late phase of wing reshaping (starting 18 h APF). These forces are generated by hinge contraction of the wing that is attached to the cuticle on the distal side. This study evaluated the mechanical autonomy of individual cells before hinge contraction at an earlier developmental stage (that is, between 16 and 18 h APF). This was done by isolating a single cell (or a small patch of cells) using a single-pulse multipoint procedure to cut AJs, thus mechanically uncoupling individual cells from their surrounding. Strikingly, the shape of individual isolated cells did not change significantly upon laser ablation at 17-18 h APF, when cells in the wing are already elongated. The same result was obtained when patches of cells were isolated. Additional analyses of the Feret's diameter before and after ablation showed a small isotropic decrease in cell size, providing evidence that at this early stage, individual cells are not influenced by the neighbouring cells or by tissue-scale forces in a polarized fashion. Consistently, analysis of animals expressing a mutant form of dumpy protein, an extracellular matrix protein associated with tissue remodelling at later developmental stages, showed no substantial differences in wing shape compared to wild-type wings at 18 h APF. Together, these experiments argue that unlike later stages, cell autonomous forces are the major drivers of initial cell shape changes between 16 and 18 h APF (Singh, 2018).

    To identify the molecular mechanism underlying cell autonomous shape formation, the distribution and dynamics of two cytoskeletal force-generators were investigated: microtubules and non-muscle myosin II (MyoII) as a component of the actomyosin cytoskeleton. MyoII was detected at the apical cell cortex at the level of AJs. A subsequent analysis of the signal distribution within single cells revealed a planar polarized distribution of MyoII along the P-D axis, which correlated with increased tension along the same junctions. As MyoII provides contractile forces, this should result in P-D junctional shortening upon laser ablation. However, this is inconsistent with the current ablation experiments, suggesting that there is an opposing force present. Interestingly, staining of microtubules showed planar polarized apical microtubules along the P-D axis at the level of AJs. Microtubules are the stiffest cytoskeletal filaments, with a persistence length on the order of millimetres. Microtubules are therefore well suited to balance the tension generated by actomyosin contraction. Consistently, the distribution of microtubules and MyoII in mechanically isolated cells remain polarized. In addition, microtubule and MyoII polarity was preserved in dumpy mutant wings at 18 h APF, indicating that they are polarized in a cell autonomous fashion. The possible role of the atypical myosin Dachs, a downstream component of the Fat signalling pathway, was also analyzed. Dachs mutant wings showed no change in cell elongation or microtubule polarity, which is consistent with recent work reporting that recombinant Dachs does not have ATPase activity and can therefore not function as a molecular motor. Together, these observations argue that planar polarized microtubules may balance actomyosin tensional forces that pull on P-D junctions and stabilize cell shape (Singh, 2018).

    To validate this hypothesis, and to elucidate the dynamic and functional role of microtubules in cell mechanics, their properties were investigated during wing development. Live cell imaging of EOS-α-tubulin (EOS-Tub) showed that microtubules were not static but engaged in complex and dynamic rearrangements. An analysis of microtubule straightness showed that in wing cells, virtually all microtubules along the P-D axis were bent, consistently undergoing short wavelength buckling (~3 μm) near the cell cortex. It was further observed that growing microtubules remain straight and only start to buckle after they reach the cell cortex, exhibiting local short wavelength buckling near these sites. This result indicates that microtubule polymerization can generate considerable compressive forces to induce microtubule buckling (Singh, 2018).

    Next, whether buckling of microtubules in Drosophila wing epithelium is indeed a result of forces acting on microtubules was also investigated, as suggested by the current experiments and in vitro studies, or whether the cellular environment yields more flexible microtubules. This is important, as buckling of flexible microtubules would rule out a role in balancing actomyosin contractility. To probe the forces of single microtubule filaments in vivo, individual microtubules were cut by laser ablation and the subsequent relaxation was monitored using live imaging. Previously curved microtubules rapidly straighten out, thus verifying that microtubules are indeed loaded with compressive forces. Finally, it was also observed that local ablation of microtubules triggers a rapid translocation of the adjacent junction. This finding supports the idea that non-centrosomal microtubules continuously generate pushing forces via polymerization that may then be stored as compressive forces in a polarized fashion to balance contractile forces generated by junctional actomyosin (Singh, 2018).

    How are microtubules polarized along the P-D axis? While the molecular mechanism has remained elusive, previous work has established that the Ft-PCP signalling pathway aligns the apical microtubule network along the P-D axis by regulating association sites of microtubules with AJs. Considering the observed stability of aligned microtubules, whether directional differences in microtubule dynamics could serve as a mechanism for the planar polarization of microtubules was tested. Monitoring of EB1 tagged with green fluorescent protein (EB1-GFP) revealed two populations of microtubule-plus ends: fast growing microtubules with a growth velocity of 24.43 ± 0.43 7mi;m min-1 (mean ± s.e.m.), and slow growing microtubules with a velocity of 17.06 ± 0.26 7mi;m min-1. A further analysis showed that the microtubule growth rates depended on relative localization within cells as well as the growth angle relative to the P-D axis. Microtubule growth rates in the cell interior were higher compared to the cell cortex. Similarly, microtubules along the P-D axis grew faster than microtubules growing perpendicular to the P-D axis along the A-P axis, establishing a spatial gradient in microtubule growth velocity. The lower growth rate along the A-P axis close to the cell periphery suggests that there is more frequent pausing and switching between polymerization and depolymerization of microtubules, thus indicating a decreased stability of A-P oriented microtubules (Singh, 2018).

    It was reasoned that over time, such differences in dynamics and stability may result in predominantly P-D aligned microtubules. To test this hypothesis, the cortical residence time was analyzed of microtubules as a function of their angle with respect to the P-D axis. Intriguingly, it found that microtubules that interact with the P-D cell cortex have a longer lifetime than microtubules interacting with the A-P cortex. Upon closer inspection, dynamic cycles of short-lived interactions of microtubules with A-P junctions were noted followed by depolymerization. Importantly, A-P oriented microtubules do not show buckling behaviour, which is in contrast to P-D oriented microtubules, but rather undergo catastrophe soon after interaction with A-P oriented cell junctions. This result suggests that microtubule-plus ends are less stable at these sites and thus cannot sustain long-lasting interactions with the cell cortex, which are required to generate compressive forces. Building on these observations, in silico probing was performed to see whether the angular difference in lifetime may indeed be sufficient for microtubule polarization. Assuming a random orientation for de novo formed microtubules, the lifetime of each microtubule was defined as a function of the angle with a maximal lifetime along the P-D axis. Upon expiration, individual microtubules were re-introduced into the system at random angles, therefore keeping the total number of microtubules constant. Consistent with the in vivo observations, the simulation reached a steady-state at which a constant fraction of microtubules polarized along the P-D axis. Taken together, these observations point to a mechanism whereby microtubule stability regulates the planar alignment of the microtubule cytoskeleton along the P-D axis, which in turn directs cell mechanics along this axis. These data place directional microtubule stability upstream of proposed mechanisms of how cell shape influences microtubule alignment. Furthermore, these results are consistent with previous findings that microtubule association with P-D oriented AJs during the initial stage of wing development depends on Ft-PCP signalling (Singh, 2018).

    Having established that planar polarized microtubule-based forces shape single cells, their mechanical coupling and integration into tissue-level mechanics were investigated. In a first round of experiments, transcellular coupling of microtubules were investigated on the ultrastructural level using transmission electron microscopy (TEM). In agreement with previous work, AJs were juxtaposed in neighbouring cells associated with microtubule filaments that span across cells in wild-type wings, forming supracellular cables analogous to myosin cables. Notably, no such association was observed in ftl(2)fdd1 / ftl(2)fd dGC13 (ft d) and ftl2 fd/ftGRV;ActP-Gal4/UAS-FtΔECDΔN-1 (N1) mutant wings, in which microtubules are randomly oriented in wing cells, therefore providing structural support for the Ft-PCP-dependent stabilization of microtubule-based forces at P-D oriented AJs. Consistently, ft mutant clones showed a fragmented microtubule cytoskeleton, arguing that there is Ft-PCP-dependent stabilization of microtubules via coupling at AJs (Singh, 2018).

    To further validate the role of polarized transcellular microtubule stability in tissue mechanics and organization, tissue shape changes were observed upon acute perturbation of microtubule-based forces. To control microtubule dynamics in a precise spatial and temporal manner, he recently developed photostatin (PST1)35, a photo-switchable analogue of combretastatin A-4 (CA4)36 was used. The drug was applied to directly test the requirement of microtubules for cell shape maintenance. Notably, it was found that the exposed wing area contracted along the P-D axis upon microtubule inhibition. Quantitative cell shape analysis showed a small but significant reduction in the elongation index (EI) in selective regions where the drug was activated, arguing that polarized tissue stabilization is via microtubule-based forces. Finally, overexpression of the microtubule-severing protein Spastin increased cell shape heterogeneity. These results are consistent with the hypothesis that an intact polarized microtubule cytoskeleton is not only required for the maintenance of anisotropic cell shape but also critically involved in shaping the whole tissue during morphogenesis via polarized transcellular force stability (Singh, 2018).

    Understanding the role of microtubules during animal development has so far been limited, especially because of a shortage of methods suitable to demonstrate causality in vivo. Taking advantage of complementary genetic, chemical, numerical and microscopy approaches, these experiments unveil polarized microtubule-based compressive forces as an alternative principle for stabilizing and maintaining cell and tissue shape during morphogenesis. Alignment of microtubules along the P-D axis was found to be based on increased longevity and polymerization of microtubules interacting with P-D oriented AJs compared to non-polarized microtubules. The result of this microtubule patterning along the P-D axis is an asymmetric distribution of protruding forces, which are stored in a polarized fashion via compressive loads on microtubules. Considering that actomyosin and microtubules are both planar polarized, it is plausible to envision that the observed compressive load on microtubules plays an active role in balancing actin-based contractile forces, resulting in the cell mechanical autonomy observed in the laser ablation experiments. Intriguingly, it was recently shown that acetylation of microtubules increases their mechanical resistance and that microtubules undergo self-repair upon damage. These important features support the role of the microtubule cytoskeleton as a site of long-term compressive force storage. Finally, evidence is provided that planar polarized microtubules are coupled at AJs across individual cells, bridging forces on the tissue level via polarized transcellular stability. Although the molecular identity remains elusive, the data suggests an involvement of AJ-associated proteins organized by the Ft-PCP pathway in this process (Singh, 2018).

    Collectively, this work provides evidence that PCP-based planar patterning of the microtubule cytoskeleton not only results in polarized cell-autonomous forces but also coordinates global force patterning during tissue morphogenesis. The proposed mechanism establishes the Ft-PCP pathway at the onset of cell and wing elongation, before shape changes, due to extrinsic mechanical forces. Consistently, in a Ft-PCP mutant, in which initial elongation fails, consecutive remodelling by extrinsic tensile forces cannot rescue these length defects, therefore leading to shorter and rounder adult wings. Considering that the Ft-PCP signalling pathway controls a variety of dynamic cell population in vertebrates, the microtubule-based mechanism described in this study is likely to be physiologically relevant beyond wing development (Singh, 2018).

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    list of genes involved in cytoskeleton


    Zygotically transcribed genes

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