The Interactive Fly

Zygotically transcribed genes

Cytoskeleton

What is cytoskeleton?

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

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

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

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


Genes and proteins of the cytoskeleton and cell motility

Genes affecting the cytoskeleton

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).

  • 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).

    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 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).

    References

    Avidor-Reiss, T., Maer, A. M., Koundakjian, E., Polyanovsky, A., Keil, T., Subramaniam, S. and Zuker, C. S. (2004). Decoding cilia function: defining specialized genes required for compartmentalized cilia biogenesis. Cell 117(4): 527-39. 15137945

    Bohrmann, J. and Biber, K. (1994). Cytoskeleton-dependent transport of cytoplasmic particles in previtellogenic to mid-vitellogenic ovarian follicles of Drosophila. time-lapse analysis using video-enhanced contrast microscopy. J. Cell Sci. 107: 849-858. 8056841

    Cooley, L., Verheyen, E. and Ayers, K. (1992). chickadee encodes a profilin required for intercellular cytoplasm transport during Drosophila oogenesis. Cell 69: 173-84. 1339308

    Goshima, G. and Vale, R. D. (2003). The roles of microtubule-based motor proteins in mitosis : comprehensive RNAi analysis in the Drosophila S2 cell line. J. Cell Bio. 162: 1003-1016. 12975346

    Hampoelz, B., et al. (2011). Microtubule-induced nuclear envelope fluctuations control chromatin dynamics in Drosophila embryos. Development 138(16): 3377-86. PubMed Citation: 21752932

    Manseau, L., Calley, J. and Phan, H. (1996). Profilin is required for posterior patterning of the Drosophila oocyte. Development 122: 2109-16. 8681792

    Rogers, S. L., Wiedemann, U., Stuurman, N. and Vale, R. D. (2003). Molecular requirements for actin-based lamella formation in Drosophila S2 cells. J. Cell Biol. 162: 1079-1088. 12975351

    Theurkauf, W. E. (1994). Premature microtubule-dependent cytoplasmic streaming in cappuccino and spire mutant oocytes. Science 265: 2093-96. 8091233

    Walker, R. A. and Sheetz, M. P. (1993). Cytoplasmic microtubule-associated motors. Annu. Rev. Biochem. 62: 429-451

    list of genes involved in cytoskeleton


    Zygotically transcribed genes

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