Drosophila RhoGEF2 associates with microtubule plus ends in an EB1-dependent manner

Members of the Rho/Rac/Cdc42 superfamily of GTPases and their upstream activators, guanine nucleotide exchange factors (GEFs), have emerged as key regulators of actin and microtubule dynamics. In their GTP bound form, these proteins interact with downstream effector molecules that alter actin and microtubule behavior. During Drosophila embryogenesis, a Gα subunit (Concertina) and a Rho-type guanine nucleotide exchange factor (DRhoGEF2) have been implicated in the dramatic epithelial-cell shape changes that occur during gastrulation. Using Drosophila S2 cells as a model system, this study shows that DRhoGEF2 induces contractile cell shape changes by stimulating myosin II via the Rho1 pathway. Unexpectedly, it was found that DRhoGEF2 travels to the cell cortex on the tips of growing microtubules by interaction with the microtubule plus-end tracking protein EB1. The upstream activator Concertina, in its GTP but not GDP bound form, dissociates DRhoGEF2 from microtubule tips and also causes cellular contraction. It is proposed that DRhoGEF2 uses microtubule dynamics to search for cortical subdomains of receptor-mediated Gα activation, which in turn causes localized actomyosin contraction associated with morphogenetic movements during development (Rogers, 2004).

The cellular functions of the microtubule plus-end binding protein EB1 has been characterized in Drosophila S2 cells; this protein plays an important role in regulating microtubule dynamics and in the assembly and dynamics of the mitotic spindle (Elliott, 2005). In order to learn more about EB1's functions, attempts were made to identify EB1 binding partners with affinity purification. Recombinant Drosophila GST-EB1 bound to glutathione Sepharose beads was used as an affinity chromatography matrix to bind interacting partners from S2 cell extracts. Bound proteins were eluted from the beads and separated by SDS-PAGE, and excised bands were subjected to tryptic digestion and mass spectrometry fingerprinting (Rogers, 2004).

Twenty 'EB1-specific' proteins were identified over the course of five independent pull-down experiments. However, of these, only six candidates were identified in all five trials: CLIP190, the Drosophila ortholog of vertebrate CLIP-170, which localizes to the plus ends of microtubules, Orbit/MAST, a microtubule plus-end-associated protein that interacts with CLIP-170, nonmuscle myosin II heavy chain, the minus-end-directed kinesin, Ncd, Shortstop, a member of the spectraplakin family of actin/microtubule cross-linking proteins, and DRhoGEF2. This paper focuses on DRhoGEF2 for further study (Rogers, 2004).

The association of DRhoGEF2 with EB1 in vitro raised the possibility that this protein may localize to the tips of microtubules. To test this idea, polyclonal antibodies were generated against the C-terminal 720 amino acid residues of DRhoGEF2. These antibodies recognized a ~280 kDa polypeptide on immunoblots of S2 cell extracts; this polypeptide was eliminated after DRhoGEF2 RNAi treatment, indicating that the antibodies were reacting with the correct polypeptide (Rogers, 2004).

By immunofluorescence, anti-DRhoGEF2 antibodies recognized punctate structures distributed throughout the cell. Superimposed upon this punctate pattern, however, were short (~1 μm) linear tracks that colocalized with the tips of microtubules. Moreover, immunofluorescent staining of DRhoGEF2 in S2 cells expressing low amounts of EB1-EGFP indicated that these two proteins colocalize exactly at microtubule tips. In the perinuclear region of many cells, DRhoGEF2 antibodies also stained larger spots that costained with γ-tubulin, a centrosome marker. Depletion of DRhoGEF2 with RNAi eliminated antibody staining of all of these structures in S2 cells. Thus, these immunofluorescence experiments reveal that DRhoGEF2 exists in three pools within S2 cells: punctate throughout the cell, at microtubule tips, and on centrosomes (Rogers, 2004).

DRhoGEF2 tagged with green fluorescent protein (GFP) was tested to examine its dynamic behavior through time-lapse imaging with a spinning-disk microscope. As predicted from immunofluorescence data, 'comet-like' structures of DRhoGEF2-GFP moved from the cell center toward the periphery in a manner that was very similar to that observed for EB1-GFP. In many cells, an intense spot of DRhoGEF2-GFP was observed near the perinuclear region. This spot likely corresponded to the centrosome staining because the tips of nucleated microtubules emanated from this point in a radial pattern. Microtubule dynamics are essential for this movement because it could be eliminated with either 10 μM colchicine or 10 μM taxol. Thus, it is concluded that DRhoGEF2 associates with the tips of growing microtubules and exhibits plus-end tracking that is qualitatively similar to that described for EB1 (Rogers, 2004).

Because DRhoGEF2 was isolated based upon its association, direct or indirect, with EB1, EB1 was deleted from cells with RNAi and whether the association of DRhoGEF2 with microtubule tips was examined. In cultures treated with control dsRNA, scoring of fixed cells stained for DRhoGEF2 and microtubules revealed that 94% of the cells (n = 300) had DRhoGEF2 associated with the microtubule tip. In contrast, in S2 cells treated for 7 days with EB1 dsRNA, only 5% of the cells retained DRhoGEF2 at the plus ends. These results demonstrate that targeting of DRhoGEF2 to growing microtubule plus ends is an EB1-dependent process (Rogers, 2004).

To further understand DRhoGEF2 functions, how overexpression and depletion of the protein affects the morphology of S2 cells was examined. When S2 cells are plated on concanavalin A, they adopt a 'fried-egg' appearance with a dome-like central domain defined by the nucleus and perinuclear organelle-rich region and an extended, symmetrical lamella. In contrast, overexpressing DRhoGEF2 caused many cells to adopt a smaller, contracted footprint on the substrate and to become significantly taller than control cells. These overexpressing cells formed a skirt of abnormally large membrane ruffles that tapered to the base of a raised, organelle-rich compartment, and the overall morphology resembled a 'bonnet' shape. This result suggests that DRhoGEF2 can induce contractility, in agreement with the genetic phenotype of DRhoGEF2 mutations (Rogers, 2004).

Several genetic studies implicate DRhoGEF2 as a positive regulator of Rho1. To test whether Rho activation is involved in generating the unusual phenotype associated with DRhoGEF2 overexpression, cells were transfected with constitutively active Rho1V14 and transfected cells were identified with an antibody raised against Drosophila Rho1. As predicted, most of the Rho1V14-expressing cells duplicated the morphology produced by DRhoGEF2 overexpression. In order to next test if inhibition of Rho1 prevented DRhoGEF2-induced shape change, DRhoGEF2-EGFP was transfected into cells that had been treated with Rho RNAi. Depletion of Rho1 by RNAi produced large multinucleate cells that did not contract in response to DRhoGEF2 overexpression. In contrast, RNAi inhibition of the other six Rho family members did not block DRhoGEF2-induced contraction (Rogers, 2004).

Active Rho is known to stimulate nonmuscle myosin II, and a genetic interaction has been demonsrated between DRhoGEF2 and myosin II during Drosophila morphogenesis. One well-characterized mechanism by which Rho1 activates myosin II is Rho kinase (DROK in Drosophila) stimulation, which activates the motor by phosphorylating the myosin light chain and by inactivating myosin light chain phosphatase. In order to determine if DROK is indeed downstream of DRhoGEF2, DROK was depleted with RNAi or kinase activity was inhibited with Y-27632, a pharmacological inhibitor, and then cell morphology was examined after DRhoGEF2 overexpression. Both treatments significantly reduced the numbers of cells exhibiting the contracted morphology. From these data, it is concluded that DRhoGEF2 changes S2 cell morphology through Rho1 and its downstream effector, DROK (Rogers, 2004).

To confirm that myosin II is a downstream effector in the DRhoGEF2 pathway in this system, the behavior of GFP-tagged myosin II in control S2 cells on concanavalin A was compared with that of S2 cells overexpressing DRhoGEF2. To perform this analysis, a stable cell line was generated expressing the myosin II regulatory light chain (RLC), known by Drosophila nomenclature as Spaghetti squash, under the control of the gene's endogenous promoter. Ectopic expression of RLC-GFP did not produce observable defects in actin organization or behavior; its distribution exactly coincided with the myosin II distribution determined by immunofluorescence staining of the same cells. RLC-GFP typically incorporated into punctae in the cell periphery and into higher-order structures in the central region of the cells. Time-lapse spinning-disk confocal microscopy revealed that punctae of RLC-GFP formed in the distal cell periphery and then translocated centripetally at a constant rate of 4.0 ± 0.3 μm/min toward the cell center. Such behavior of RLC-GFP is qualitatively very similar to the behavior of fluorescently labeled myosin II in cultured mammalian cells (Rogers, 2004).

Upon overexpression of DRhoGEF2, punctae of RLC-GFP were rarely observed. Instead, the majority of RLC-GFP signal was present in circular 'purse string' structures surrounding the organelle-dense region at the center of the cell. Time-lapse observation revealed that peripheral formation of RLC-GFP punctae and retrograde flow were infrequent in DRhoGEF2-overexpressing cells and that these RLC-GFP-containing purse strings were stable over a span of hours. The location and concentration of the myosin II suggests that actomyosin contraction is responsible for producing the bonnet-shaped appearance of these cells. From these observations, it is propose that DRhoGEF2 regulates myosin II dynamics and contractility in S2 cells (Rogers, 2004).

Genetic analyses of epithelial-sheet invagination in the early Drosophila embryo suggest that DRhoGEF2 may act downstream of the heterotrimeric Gα protein Concertina (Cta). To examine directly whether Concertina can activate DRhoGEF2, cells were transfected either with Myc-tagged wild-type Cta or Myc-tagged Cta bearing a constitutively activating point mutation (R277H) that inactivates GTPase activity, and the morphology of the transfected cells was examined. Cells expressing Myc-Cta were morphologically indistinguishable from untransfected cells, and only 3% of cells displayed a mildly contracted phenotype. In contrast, the majority of cells expressing Myc-CtaR277H exhibited the contracted morphology and myosin II purse string reminiscent of DRhoGEF2 overexpression. Similar results were obtained with three other constitutively activated Concertina constructs. However, the shape change was prevented in 88% of these cells (if they were pretreated for 7 days with dsRNA so that DRhoGEF2 was depleted. These results suggest that Concertina can act upstream of DRhoGEF2 to regulate S2 cell morphology (Rogers, 2004).

Next it was determined whether activation of DRhoGEF2 through Concertina affected its association with the microtubule cytoskeleton. Cells expressing Myc-Cta or Myc-CtaR277H were fixed and double stained for the Myc epitope tag and for DRhoGEF2. Overexpression of wild-type Concertina did not affect DRhoGEF2 association with microtubule plus ends or with the centrosome. However, constitutively activated Concertina resulted in DRhoGEF2 dissociation from microtubule tips; only 10% of the cells showed any colocalization of DRhoGEF2 with microtubule plus ends. Instead, DRhoGEF2 exhibited a diffuse staining pattern throughout the cell; this pattern likely represents association with the plasma membrane. Targeting of EB1 to the plus ends was not perturbed by CtaR277H, suggesting that Concertina signaling regulates the interactions between DRhoGEF2 and factors at microtubule tips (Rogers, 2004).

In an attempt to identify novel cellular factors that interact with EB1, this study unexpectedly discovered that DRhoGEF2, a key regulator of morphogenesis in Drosophila, associates with the tips of growing microtubules. This interesting type of intracellular motility required EB1 in a manner analogous to the EB1-dependent microtubule plus-end tracking of the vertebrate adenomatous polyposis coli (APC) tumor suppressor protein. This finding represents the first example of a regulator of the actin cytoskeleton that tracks along microtubule plus ends. Moreover, the dissociation of DRhoGEF2 from microtubule tips upon activation of Concertina also represents the first example of a regulated association of a protein with the microtubule plus end (Rogers, 2004).

The dissection of the DRhoGEF2 pathway at a cellular level is also consistent with genetic studies of Drosophila morphogenesis. These studies implicate Concertina in myosin II contractility through the Rho/Rho kinase pathway. The Rho1/Rho kinase/myosin II system is a widely employed module for bundling and contraction of actin filaments; it is involved in the formation of adhesion structures and stress fibers, retraction of the trailing edge in migrating cells, muscular contraction, morphogenetic cell shape changes, and construction of the cleavage furrow at the end of mitosis. Context- and location-specific activation of the Rho1/Rho kinase/myosin II module is likely to reside in the activation of specific RhoGEFs, over 20 of which reside within the Drosophila genome. This hypothesis is consistent with observations that inhibition of Rho1 or its downstream effectors causes a dramatic cytokinesis failure in S2 cells and embryos, but inhibition of DRhoGEF2 does not. Instead, DRhoGEF2 has been implicated in morphogenetic cell shape changes only in epithelial cells. Thus, it is believed that the signaling pathway that was engineered in S2 cells recapitulates events involved in the cellular shape changes preceding gastrulation in Drosophila blastula epithelia cells (Rogers, 2004).

However, in Drosophila development, this signaling pathway must be activated in a polarized manner by an unidentified receptor and its ligand so that myosin contraction occurs locally at the apical surface. In such a setting of asymmetric signaling, it is proposed that the intracellular transport of DRhoGEF2 on microtubule plus ends may play an important role in localized activation of the pathway. It is speculated that inactive DRhoGEF2 interacts with the tips of microtubules, whereupon these growing microtubules deliver 'packets' of DRhoGEF2 in the vicinity of the actin cortex. If DRhoGEF2 does not receive an activating input, it diffuses back into the cytoplasm to begin the transport cycle anew. However, if DRhoGEF2 is delivered to a subcortical region containing a high concentration of receptor-activated Concertina, DRhoGEF2 can locally activate the Rho1/Rho kinase/myosin II module. Moreover, because DRhoGEF2 possesses potential lipid (pleckstrin homology) and protein-protein (PDZ, RGS, and DH [Dbl homology]) interaction domains, microtubule-delivered DRhoGEF2 may be retained at the cortex if activated by Concertina. Although a microtubule-assisted activation of the Rho pathway during cellular shape changes during morphogenesis (such as in epithelial cells) is proposed, similar models that account for small GTPase activation during cellular motility have been suggested as well (Rogers, 2004).

In principle, interactions between DRhoGEF2 and its cortical activators could occur through diffusion within the cytoplasm. The evolution of this elaborate microtubule polymerization-based transport mechanism undoubtedly reflects some important property of the signaling pathway that is not yet understood. Perhaps the amount of DRhoGEF2 carried on the tip of a microtubule represents some quanta -- a critical concentration of the protein required either to respond to upstream inputs or to locally activate Rho1 in a cortical subdomain. This idea is supported by the observation that, at very low expression levels and without Concertina signaling, DRhoGEF2-GFP efficiently tracks microtubule ends without activating cellular contraction. Alternatively, it is possible that interaction with EB1 or some other protein at the microtubule plus end primes DRhoGEF2 for activation at the cortex. A third possibility is that microtubule dynamic instability is not uniform within a polarized cell but is locally modulated in order to deliver DRhoGEF2 to the cortex in a nonrandom manner. Testing between these hypotheses will require identification of the signaling components (i.e., the ligand-receptor pair) that act upstream of Concertina, reconstitution of the complete pathway in S2 cells, and the selective disruption of the association of DRhoGEF2 with microtubule tips in Drosophila embryos (Rogers, 2004).

Reconstitution of dynamic microtubules with Drosophila XMAP215, EB1, and Sentin

Dynamic microtubules (MTs) are essential for various intracellular events, such as mitosis. In Drosophila melanogaster S2 cells, three MT tip-localizing proteins, Msps/XMAP215, EB1, and Sentin (an EB1 cargo protein), have been identified as being critical for accelerating MT growth and promoting catastrophe events, thus resulting in the formation of dynamic MTs. However, the molecular activity of each protein and the basis of the modulation of MT dynamics by these three factors are unknown. This paper shows in vitro that XMAP215msps has a potent growth-promoting activity at a wide range of tubulin concentrations, whereas Sentin, when recruited by EB1 to the growing MT tip, accelerates growth and also increases catastrophe frequency. When all three factors are combined, the growth rate is synergistically enhanced, and rescue events are observed most frequently, but frequent catastrophes restrain the lengthening of the MTs. It is propose that MT dynamics are promoted by the independent as well as the cooperative action of XMAP215msps polymerase and the EB1-Sentin duo (Li, 2012).

This study reveals the in vitro growth-accelerating and catastrophe-promoting activities of Drosophila EB1 and Sentin toward growing MTs, in the presence or absence of XMAP215msps, which has been shown to be a potent MT growth promoter. The combination of these two machineries, which likely interact with each other at the MT plus ends, produced MTs with the greatest dynamicity; this observation is consistent with the fact that either of the two machineries is critical for the formation of dynamic MTs in S2 cells. Furthermore, the localization data may reinforce the proposal that Sentin, for which no homologous genes have been identified thus far outside the insect species, might be the functional homologue of the mammalian Slain2 protein that was recently shown to be important for MT growth in cells and was observed to interact with XMAP215 as well as EB1 (van der Vaart, 2011). Thus, this study takes a step forward in understanding the molecular mechanism underlying the regulation of MT dynamics by multiple nontubulin proteins and, moreover, suggests that the mechanism may be widely conserved in animal cells (Li, 2012).

The growth-promoting activity of XMAP215msps is generally consistent with that of the best-characterized Xenopus orthologue. The approximately twofold increase in MT growth rate detected in this study might be an underestimation, given that pig brain tubulins were used for Drosophila proteins. However, together with the appearance of severe RNAi phenotypes, it is highly probable that XMAP215msps is the major MT polymerase of Drosophila (Li, 2012).

EB1 and Sentin had two activities, one that overlaps with the activity of kinesin-13 (catastrophe promotion) and the other with that of XMAP215 (growth acceleration). It is believed that these activities would be associated with their in vivo functions because the RNAi of EB1 or Sentin in the S2 cells decreased both growth speed and catastrophe frequency. However, the data do not go against the view that XMAP215 and kinesin-13 are the primary growth- and catastrophe-promoting factors, respectively. In fact, it is proposed that the EB1-Sentin machinery would act as a supporter of the core XMAP215-kinesin-13 machinery by accelerating MT growth as well as MT aging for catastrophe and that S2 cells do need both mechanisms to make MTs fully dynamic. This hierarchical relationship might be conserved in vertebrates because MT phenotypes observed after EB1 depletion are rescued by the overexpression of XMAP215 in Xenopus egg extracts (Li, 2012).

The growth acceleration on addition of both EB1 and Sentin was comparable with that observed after addition of XMAP215msps. However, the EB1-Sentin complex is not considered to be a processive polymerase like XMAP215 for a few reasons. First, growth-promoting activity is not seen at a low tubulin concentration in the presence of EB1 and Sentin. Second, unlike the case of XMAP215, data could not be obtained to indicate that Sentin stably binds to soluble tubulins. Third, the known residence time of XMAP215 and EB1 on the MT ends is very different. The average end residence time of a single XMAP215 molecule is 3.8 s, which is long enough to processively add 25 tubulin dimers to the end of each MT protofilament. In contrast, the residence time of yeast and mammalian EB1 family proteins is <0.4 s, which is too short to processively add multiple tubulins. A recent structural study showed that EB1 recognizes a structural feature on the MT surface near the plus end and catalyses the transition of tubulin from the recognized state at growing ends to the state tubulin adopts in the lattice distant from the ends, which is assumed to be the basis of the EB1 activity. Sentin bound to EB1 might modulate the plus-end structure for favorable tubulin incorporation, rather than acting as a polymerase. An interesting future experiment would be to determine the structural basis of EB1-recruiting Sentin in this context (Li, 2012).

When XMAP215msps, EB1, and Sentin were all combined, the most dynamic MTs thus far were observed; growth rate and catastrophe frequency were maximized, and rescue events were occasionally observed. Interestingly, the dramatic increase in the growth rate and rescue frequency might be because of the synergistic effects of EB1-Sentin and XMAP215msps. On the basis of the experiments using the truncated Sentin fragments, it is tempting to speculate that the physical interaction of Sentin with XMAP215msps facilitates localization of both proteins at the MT ends and modulates their activities, thereby contributing to these synergistic effects. However, other models, including a model in which the XMAP215msps and EB1-Sentin machineries work independently, cannot yet be excluded at this stage (Li, 2012).

The key features of MT dynamic instability (growth, shrinkage, catastrophe, and rescue) were obtained by in vitro combination of three plus-end factors. The reconstitution, however, still failed to precisely reproduce what happens in vivo. For example, pausing MTs are frequently observed in cells, particularly in interphase (-40%), but this behavior was rarely seen in any conditions in the in vitro assay. In S2 cells, at least two other factors, kinesin-13 depolymerase and the CLASP rescue factor, are known to significantly affect interphase MT dynamics probably in an EB1-independent manner. Although CLASP is mostly concentrated at the kinetochore during mitosis and perhaps does not significantly affect the astral MT dynamics, kinesin-13 acts on every MT in every stage of the cell cycle. A next step toward understanding the mechanism controlling MT plus-end dynamics would be to include these two factors in the assay and determine whether they can better reproduce the cellular MT behavior that has, for example, frequent pause events (Li, 2012).

γ-Tubulin ring complexes and EB1 play antagonistic roles in microtubule dynamics and spindle positioning

γ-Tubulin is critical for microtubule (MT) assembly and organization. In metazoa, this protein acts in multiprotein complexes called γ-Tubulin Ring Complexes (γ-TuRCs). While the subunits that constitute γ-Tubulin Small Complexes (γ-TuSCs), the core of the MT nucleation machinery, are essential, mutation of γ-TuRC-specific proteins in Drosophila causes sterility and morphological abnormalities via hitherto unidentified mechanisms. This study demonstrates a role of γ-TuRCs in controlling spindle orientation independent of MT nucleation activity, both in cultured cells and in vivo and examines a potential function for γ-TuRCs on astral MTs. γ-TuRCs locate along the length of astral MTs, and depletion of γ-TuRC-specific proteins increases MT dynamics and causes the plus-end tracking protein EB1 to redistribute along MTs. Moreover, suppression of MT dynamics through drug treatment or EB1 down-regulation rescues spindle orientation defects induced by γ-TuRC depletion. Therefore, a role is preposed for γ-TuRCs in regulating spindle positioning by controlling the stability of astral MTs (Bouissou, 2014).

Using cultured cells and Drosophila neuroblasts, this study has demonstrated a novel role of γ-TuRCs in spindle positioning. It is proposed that spindle positioning is controlled by a balance created by antagonistic factors, exerting stabilizing and destabilizing effects on astral MTs. γ-TuRCs and EB1 were characterized as representative examples for these two types of factors. Moreover, EB1 redistribution was shown to be is concomitant with an increase of GTP-tubulin islands along MTs. This suggests that γ-TuRC-dependent changes of MT dynamics involve switches of tubulin conformation that could affect EB1 localization (Bouissou, 2014).

γ-TuRCs were shown to stabilize astral MTs. These results are consistent with previous studies on γ-TuRCs associated to the lattice of interphase MTs that regulate dynamics by preventing MT depolymerization beyond the sites of γ-TuRC attachment. In comparison to interphase, the relative increase of MT dynamics upon γ-TuRC depletion is weaker during mitosis. Cell cycle differences, such as the composition of the cortical area or the balance of MT-associated proteins, may affect MT behaviour. Consistent with a role of γ-TuRCs in regulating astral MT dynamics, regular spindle orientation can be restored in the absence of γ-TuRCs at least partially, when cells are simultaneously treated with drugs that reduce MT dynamics, or when the level of plus-end-binding proteins that promote MT dynamics is lowered (Bouissou, 2014).

To investigate the mechanisms by which γ-TuRCs regulate the stability of astral MTs, the localization of γ-TuRCs was studied in mitotic cells. Astral localization of γ-TuRCs was observed, evidenced by different immunofluorescence procedures and confirmed by live-imaging. Such a localization pattern has not been described before, likely for three reasons. First, this localization may have been obscured by a large background of cytoplasmic γ-TuRCs. Second, only a small fraction of γ-tubulin is localized on MTs, and the faint, punctuate staining may have been overlooked previously. Third, astral MTs mostly appear as individual MTs, not organized in bundles like kinetochore fibers, and consequently associated proteins appear less concentrated. This study also demonstrates that the recruitment of γ-TuRCs to astral MTs in mammalian cells is, at least partially, dependent on the augmin complexes, whereas augmin proteins, required for centrosome-independent microtubule generation within the spindle, are still present along MTs after γ-TuRC disassembly. These data suggest a recruitment of the augmin complexes on astral MTs prior to γ-tubulin complexes, or a pre-requisite of large complexes (including augmin and γ-TuRCs) forming in the cytoplasm before γ-TuRC recruitment. In addition to augmins, the protein Cdk5Rap2 may also be involved in binding γ-tubulin complexes to the MTs, since Cdk5Rap2 is known to interact with γ-TuRCs and concentrates partially at MT plus-ends, where it contributes to the regulation of MT dynamics (Bouissou, 2014).

So far, the main function that has been attributed to γ-TuRCs attached to the MT surface via augmin complexes is the nucleation of secondary MTs, to increase the density of kinetochore fibers. However, this study as well as previously published data on plant cells argue against a sole function of MT-bound γ-TuRCs in secondary nucleation and suggest that these complexes have additional functions. First of all, measurements of a homogeneous tubulin immunofluorescence intensity along astral MTs indicate that no additional MTs have been nucleated at these specific sites. Moreover in plant cells, the majority of γ-TuRC foci associated to interphase MTs are not nucleating any other MTs, and the subset of γ-tubulin complexes active in generating new MTs appear enriched in the homolog of Mozart1. Finally, a recently published study in Arabidopsis shows that the augmin subunit 8 binds to the MT plus-ends, regulates the dynamics of MT plus-ends and by this way controls MT reorientation in hypocotyls (Bouissou, 2014).

All these data lead to a proposal of certain heterogeneity or plasticity in γ-TuRCs associated to the MT surface. In addition to their nucleation activity, some γ-TuRCs, by attaching to MTs, could exert a stabilizing effect on individual MTs, independent of their dormant potential to nucleate secondary MTs. γ-TuRCs along the MT surface may function in an analogous manner as a Microtubule Associated Protein (MAP), or if localized to the plus-end, increase stability similar to a cap. It is suggested that the spindle orientation defects that were observe upon γ-TuRC-disassembly are not due to altered mechanisms of MT nuleation. First, it was shown that the activity of nucleation at the poles is comparable in control and γ-TuRC-deficient cells and mitotic MTs in cells lacking the full γ-TuRC proteins possess a regular structure, with 13 protofilaments per diameter. Moreover, suppression of MT dynamics is sufficient to restore spindle orientation. Besides, data in yeast suggest that mutations in individual components of the γ-tubulin complexes influence MT dynamics in a post-nucleation manner. In addition, depletion of Dgp71WD or of an augmin subunit does not modify the quantity and the elongation of de novo nucleated MTs associated with acentriolar centers but does affect their stability. This is consistent with previous data showing that in Drosophila cells the soluble pool of α/β tubulin is not significantly changed after depletion of individual γ-TuRC grip-motif proteins. Even if the soluble pool were slightly increased, the effects on MT dynamics would probably be minimal, since MT dynamics in cells, in contrast to the situation described in vitro, appear much more sensitive to the regulation by MT-associated proteins than to the concentration of free α/β tubulin (Bouissou, 2014).

Consistent with the hypothesis on altered MT dynamics, a change was observed in EB1 localization following depletion of γ-TuRC-specific subunits. EB1 is no longer concentrated at the MT plus-ends but rather distributed along MT side walls. Similar EB1 redistribution has been reported in other experimental setups affecting MT dynamics, for example following depletion of EB1 interactors, such as the XMAP215/Dis protein family or the p150Glued subunit of dynactin, or immunodepletion of γ-tubulin from frog egg extracts. However, a mechanistic explanation is lacking so far. One possibility would be that reduced affinity of EB1 to MT tips increases the pool of EB1 that might now bind to lower affinity sites on the MT lattice. Alternatively, and not mutually exclusive, changes in the size and intensity of EB1 staining after γ-TuRC depletion may result from alterations in GTP caps or GTP-remnants on MTs. This possibility was explored using the antibody hMB11, known to recognize a conformational state of tubulin, acquired upon binding of GTP. EB1 and hMB11 antibodies have higher affinity for GTP-MTs compared to GDP-MTs in vitro and both can colocalize as patches on the lattice of axonal MTs when EB1 is overexpressed. So, the extended MT surface that was stained with both EB1 and hMB11 after γ-TuRC depletion may be due to altered tubulin conformation or a modified guanosine nucleotide status. This interpretation makes sense with a recent study that proposes that the effects between EB1 and the MT-associated protein XMAP215 on MT dynamics don't rely on any direct interaction but rather on allosteric interaction through MT ends. Abnormal loading of EB1 to the MTs after γ-TuRC disassembly may have itself a feedback on the balanced loading of +TIP complexes and also on the MT binding to the cell cortex. Therefore it may enhance any primary effects following the loss of γ-TuRCs, especially on spindle orientation. In any of the above discussed scenarios, γ-TuRCs may not interact directly with EB1. This is based on the observations (1) that EB1 is generally not enriched at sites of γ-TuRC localization, (2) that a decrease of γ-TuRC along MTs leads to an increase of the length of EB1 staining, and (3) that EB1 appears absent from sucrose gradient fractions corresponding to γ-TuRCs (Bouissou, 2014).

Oriented cell divisions appear critical for proper development and maintenance of tissue homeostasis. Moreover, emerging evidence reveals a link between spindle mis-orientation and a number of developmental diseases as well as tumorigenesis. The study of spindle positioning is therefore fundamental to both developmental biology and human pathologies. This study provide new mechanistic information for understanding this key event. These results invoke γ-TuRCs as novel players in spindle orientation by indirectly controlling localization of EB1, a key protein involved in the physical interaction between mitotic spindle and cell cortex (Bouissou, 2014).

Protein Interactions

Shortstop recruits EB1/APC1 and promotes microtubule assembly at the muscle-tendon junction

Shortstop (Shot) is a Drosophila Plakin family member containing both Actin binding and microtubule binding domains. In Drosophila, it is required for a wide range of processes, including axon extension, dendrite formation, axonal terminal arborization at the neuromuscular junction, tendon cell development, and adhesion of wing epithelium. To address how Shot exerts its activity at the molecular level, the molecular interactions of Shot with candidate proteins was investigated in mature larval tendon cells. Shot colocalizes with the complex between EB1 and APC1 and with a compact microtubule array extending between the muscle-tendon junction and the cuticle. It is suggested that EB1 and APC1 become associated with the muscle-tendon basal hemiadherens junction in postmitotic tendon cells following their association with Shot. Shot forms a protein complex with EB1 via its C-terminal EF-hands and GAS2-containing domains. In tendon cells with reduced Shot activity, EB1/APC1 dissociate from the muscle-tendon junction, and the microtubule array elongates. The resulting tendon cell, although associated with the muscle and the cuticle ends, loses its stress resistance and elongates. These results suggest that Shot mediates tendon stress resistance by the organization of a compact microtubule network at the muscle-tendon junction. This is achieved by Shot association with the cytoplasmic faces of the basal hemiadherens junction and with the EB1/APC1 complex (Subramanian, 2003).

Tendon cells undergo maturation during larval stages. In third instar larvae, different tendon cells acquire distinct shapes according to their orientation and the type of muscles to which they are connected. Initially, the localization of Shot was characterized relative to MT and F-actin organization in mature tendon cells of flat, opened third instar larvae. Shot and Tubulin staining overlapped within the entire cell. A unique domain at the focal plane of the muscle-tendon junction exhibits a compact MT array, which overlapped Shot staining. This domain was not detected when the optical section was taken 0.5 microm more internal to the junction focal plane. An optical cross-section perpendicular to the muscle-tendon junction site shows that the MT-Shot array extends from the muscle-tendon junction to the cuticle. Moreover, the MT array is oriented in the same direction as the microfilaments of the muscle cells, as shown by EM analysis. Thus, Shot and MTs are colocalized within a unique subcellular domain in the tendon cell that connects the muscle-tendon junction and the cuticle (Subramanian, 2003).

These immunofluorescent localization studies suggest a distinct abundance of MTs and MFs at both sides of the muscle-tendon junction. While MFs are highly enriched at the muscle side, MTs are detected mainly at the tendon side. To address whether this organization reflects differences in the distribution of additional junction-associated proteins, the larval flat preps were stained for PSß-integrin, Paxillin, and P-tyrosine, and their relative distribution at the focal plane of the muscle-tendon junction was analyzed. In all preparations, Shot marks the outlines of the tendon cell. About half of the PSß-integrin staining overlaps Shot staining, whereas the other half is located at the muscle membrane. Paxillin is more abundant on the muscle side. Interestingly, staining for P-tyrosine, which marks the extent of tyrosine-phosphorylated proteins (including Paxillin, Src, and others) is restricted to the tendon side of the junction and is tightly associated with the plasma membrane. Thus, although PSß-integrin distribution appears to be equal at both sides of the muscle-tendon hemiadherens junction, the molecular composition on both sides of these junctions appears to be distinct, as demonstrated for EB1, APC1, Paxillin, and the extent of P-tyrosine reactivity. It is tempting to speculate that these unequal protein distributions might relate to the enrichment of MTs and Shot on the tendon cell side (Subramanian, 2003).

EB1 is an evolutionarily conserved protein that binds the plus ends of growing MTs. It was first identified as a binding partner for the adenomatous polyposis coli tumor suppressor, APC. The EB1/APC complex is involved in regulation of MT polymerization and MT association with distinct subcellular domains. For example, in yeast, the EB1 homolog (BIM1) has been shown to modulate MT dynamics and link MTs to the cortex. Drosophila EB1 (Elliott, 2005) is important for the proper assembly, dynamics, and positioning of the mitotic spindle. Its association with APC2 in apical cell-cell adherens junctions is suggested to be essential for parallel spindle orientation (Lu, 2002) and for neuroblast asymmetric cell division (Subramanian, 2003).

Biochemical data suggest that the association of the C-terminal EF-GAS2 domain with EB1 is MT independent. A direct physical interaction between the C-terminal EF-GAS2 domain and alpha-tubulin had been suggested by a yeast two-hybrid screen and by the ability to precipitate purified Tubulin by the GAR and GSR domains (both included within the C terminus of the mammalian ACF7). These domains lack the EF-hands motif. EB1 is detected in association with the entire Shot C-terminal domain containing EF-hands. At this stage, no additional information is available regarding the site responsible for EB1 association (Subramanian, 2003).

The data suggest that Shot association with the basal muscle-tendon junction is EB1 independent (since it is detected even in the absence of EB1/APC1); hence, it is suggested that EB1 and APC1 become associated with the muscle-tendon basal hemiadherens junction in postmitotic tendon cells following their association with Shot. The assembly of the MT-rich domain may be induced by either their direct association with Shot or with EB1/APC1, or with both. Interestingly, EB1, APC1, and Shot are not observed at the cell-cell adherens junctions formed between the tendon cell and its neighboring ectodermal cells (Subramanian, 2003).

There is no direct evidence for the polarity of MTs within the compact Shot/MT-rich domain, since no differential EB1 localization was detected in this domain. Other studies suggest that MTs in the entire epidermis are arranged at a polar orientation in which their plus ends face the basal pole and their minus ends face the cuticle. Similarly, in the pupal wing, MTs have been shown to be arranged with their plus ends facing the basal hemiadherens junctions. Thus, it is likely that in the tendon cells (which are part of the epidermal layer), MTs are similarly arranged, i.e., with their plus ends facing the basal hemiadherens junction (Subramanian, 2003).

The experiments show that reduced Shot activity leads to a significant tendon cell elongation, occurring presumably following muscle contractions. What is the mechanism allowing the MTs to elongate in the mutant tendon cell? An interesting possibility is that the MTs are connected to the muscle-tendon junction through their plus ends via their association with EB1 and Shot, and that this arrangement arrests further MT polymerization and maintains the MTs in a polarized arrangement. Following dissociation of the Shot/EB1/APC1 complex and the reduction of Shot activity, MTs undergo further polymerization and extension, leading to the significant elongation of the tendon cell. The newly formed MTs are not well connected to the cell cortex, thus leading to cell breakdown upon further muscle contraction. Support for the involvement of Shot in mediating MT-polarized organization emerges from recent analysis of Shot function in mushroom body neurons of the Drosophila adult brain. Distinct Nod-ßgal reactivity suggests that MT polarity within the axons is distinct from that of dendrites in wild-type mushroom body neurons. In neurons mutant for shot, the polarity of MTs in the axons is reversed and resembles that of dendrites (Subramanian, 2003).

Shot may perform a similar function in the organization of a compact and polarized array of MTs in the adult wing epithelium, as well as within the ligament cells of embryonic sensory chordotonal organs (Subramanian, 2003).

Studies with the different Shot domains show that the Actin binding domain, but not the Plakin domain, is capable of driving specific localization of both domains to the F-actin layer at the muscle-tendon junction. The thin Actin layer at the muscle-tendon junction may therefore be essential for the recruitment of Shot into the cytoplasmic faces of the hemiadherens domains. The C terminus containing the EF-hands and GAS2 domains is also capable of localizing at the muscle-tendon junction domain. This localization may be attributed to its association with endogenous EB1, as well as with MTs that are already arranged in the larval tendon cells, or with endogenous Shot. The Plakin domain on its own did not show specific subcellular localization, suggesting that it does not bind to proteins that are highly localized in the tendon cell. Alternatively, proteins that may form a complex with this domain may be engaged in existing protein complexes and therefore are not accessible to the exogenous Plakin protein. None of the Shot structural domains show a dominant-negative effect when overexpressed in tendon cells or in wing imaginal discs; this finding suggests that the proteins to which they bind are not present in limited amounts (Subramanian, 2003).

Interestingly, in larvae tendon cells, both the Actin-Plakin domain of Shot and the Shot C-terminal EF-GAS2 domain exhibit similar distribution. When transfected into Schneider cells, each domain shows a distinct subcellular localization. The Actin-Plakin-GFP was detected at the leading edge and in most cases did not overlap EB1, while the EF-GAS2 domain overlapped EB1 and decorated MTs. Similar studies with Drosophila Shot and ACF-7, the mammalian Shot ortholog, show that the Actin binding domain and the EF-GAS2 domain are associated with Actin MFs and with MTs, respectively, in transfected cells. However, the M1 domain in ACF-7 (similar to the Plakin domain) has been shown to be associated with MTs in the transfected cells, while the Shot-Plakin domain does not exhibit significant association with MTs. These differences may reflect differential distribution of yet uncharacterized ACF-7 binding proteins within the mammalian cells (Subramanian, 2003).

What could be the connection between Shot activity and reduced F-actin content? Recent studies suggest that MT disassembly activates Rho by the release of GEFs that are specifically associated with and inhibited by MTs. In tendon cells, no unique association was detected of GEF (Pebble) or Rho with the MT-rich domain. Therefore, the relevance of these factors is not clear. Recently, it was shown that in Drosophila embryonic tracheal cells, activated RhoA mimicks the Shot loss-of-function phenotype; this finding suggests a similar inverse correlation between Actin polymerization by RhoA and the loss of shot function. Thus, the activity of Shot in organizing MTs to special subcellular sites via its association with EB1/APC1, and the inhibition of F-actin in these sites, may be relevant to other tissues in which Shot plays an essential role (Subramanian, 2003 and references therein).

The MT network is essential for a wide array of cellular functions. Shot, a multidomain Plakin family member, is essential for arranging a compact network of MTs in tendon cells. This is achieved by the association of Shot with the cytoplasmic faces of the muscle-tendon junction and presumably by the subsequent recruitment of the EB1/APC1 complex to these sites. In tendon cells, this unique MT organization is essential to resist muscle contraction (Subramanian, 2003).

Distinct mechanisms govern the localisation of Drosophila CLIP-190 to unattached kinetochores and microtubule plus-ends

CLIP-170 was the first microtubule plus-end-tracking protein to be described, and is implicated in the regulation of microtubule plus-ends and their interaction with other cellular structures. The cell-cycle-dependent mechanisms which localise the sole Drosophila melanogaster homologue CLIP-190 have been studied. During mitosis, CLIP-190 localises to unattached kinetochores independently of spindle-checkpoint activation. This localisation depends on the dynein-dynactin complex and Lis1 which also localise to unattached kinetochores. Further analysis revealed a hierarchical dependency between the proteins with respect to their kinetochore localisation. An inhibitor study also suggested that the motor activity of dynein is required for the removal of CLIP-190 from attached kinetochores. In addition, CLIP-190 association to microtubule plus-ends is regulated during the cell cycle. Microtubule plus-end association is strong in interphase and greatly attenuated during mitosis. Another microtubule plus-end tracking protein, EB1, directly interacts with the CAP-Gly domain of CLIP-190 and is required to localise CLIP-190 at microtubule plus-ends. These results indicate distinct molecular requirements for CLIP-190 localisation to unattached kinetochores in mitosis and microtubule ends in interphase (Dzhindzhev, 2005; full text of article).

The CLIP family of proteins is implicated in regulating microtubule dynamics and linking microtubule plus-ends with other cellular structures. To understand these functions, it is crucial to elucidate where and how these proteins localise within cells. This study investigated the molecular mechanisms of CLIP-190 localisation using RNAi in Drosophila cells, rather than using the expression of dominant proteins, to gain a clearer and more comprehensive view. The study revealed that distinct, cell-cycle-dependent mechanisms localise CLIP-190 to microtubule plus-ends and unattached kinetochores (Dzhindzhev, 2005).

CLIP and EB1 proteins are two major families of microtubule plus-end-tracking proteins. This study is the first demonstration of both a physical interaction and localisation-dependency between CLIP and EB1 proteins in higher eukaryotes. CLIP-190 requires EB1 to localise to microtubule plus-ends, and CLIP-190 directly interacts with EB1 through its CAP-Gly domain, which also binds to microtubules. Considering that this has previously been reported for fission yeast homologues, these findings demonstrate that this interaction and dependency are conserved among eukaryotes. The most obvious interpretation would be that EB1 simply bridges microtubule plus-ends and CLIP-190. However, this is thought not be the case. First, CLIP-170 has been shown to bind directly to microtubule plus-ends in vitro. Secondly, localisation studies show incomplete overlapping of the two proteins on microtubule plus-ends. Also, other EB1 interacting proteins, such as RhoGEF2 and the spectraplakin Short stop, which both require EB1 for their localisation to microtubule plus-ends (Rogers, 2004; Slep, 2005), show distinct localisation from that of EB1. Therefore, it is more likely that EB1 acts as a loading factor for these proteins, rather than a simple bridge. In addition, it seems that multiple microtubule plus-end-binding proteins, such as CLIPs, CLASPs, p150Glued, EB1, Lis1, Dynein, Short stop, APC and RhoGEF2, directly interact with each other and with microtubules. It is an exciting challenge to understand the regulatory network acting on microtubule plus-ends (Dzhindzhev, 2005).

In addition, it was found that the association of CLIP-190 to microtubule plus-ends is greatly reduced during mitosis. This is in contrast to EB1, which is associated with plus-ends throughout the cell cycle. This cell-cycle-regulation has not been described in other systems, possibly because of a lack of co-examination with EB1, and leads to the conclusion that it is not the consequence of a change in microtubule dynamics. It might be possible that CLIP-190 is modified during the cell cycle. Since EB1 is essential for CLIP-190 localisation to microtubule ends, EB1 activity or interaction between EB1 and CLIP-190 might also be regulated. Alternatively, other inhibitory proteins might be activated during mitosis to attenuate CLIP-190 association with microtubule ends. Phosphorylation of the EB1 homologue mal3p and its inhibitory effects on the interaction with the CLIP-190 homologue tip1p have been shown in fission yeast (Busch, 2004). It remains to be examined whether this phosphorylation is cell-cycle-regulated. Cell-cycle regulation might be important for releasing CLIP-190 for kinetochore function or preventing the plus-end-binding activity from interfering with CLIP-190 function at kinetochores. This report is the first to describe the cell-cycle regulation of the plus-end-binding of CLIP proteins. Elucidation of the precise mechanism and significance of this regulation may lead to further understanding of the temporal and spatial regulation of microtubules in cells (Dzhindzhev, 2005).

CLIP-190 localises to unattached kinetochores in mitosis. The localisation of CLIP proteins to kinetochores has been shown in mammalian, Drosophila and budding-yeast cells. Studies of CLIP-170 localisation to kinetochores in mammalian cells suggest an intricate physical and functional relationship with the dynein-dynactin complex. CLIP-170 binds directly to and requires Lis1 for kinetochore localisation. In turn, Lis1 interacts with multiple subunits of dynein-dynactin and is displaced from kinetochores when the motor complex is disrupted. Most of these studies relied on the overexpression of dominant-negative proteins (Dzhindzhev, 2005).

A previously unreported dependency was found, namely the requirement of Lis1 for dynein localisation to kinetochores. In mammalian cells, dynein is required for Lis1 localisation, whereas the overexpression of full-length or truncated Lis1 does not prevent dynein localisation to kinetochores. These results lead to the idea that Lis1 might be an auxiliary protein that bridges the dynein complex to cargo proteins. The RNAi results clearly indicate that Lis1 is required for dynein localisation to kinetochores. Combined with previous results, Lis1 and dynein seem to depend on each other for their localisation. This is the first report of such dependency in any eukaryote, and it gives the Lis1 protein a more integral part in dynein function (Dzhindzhev, 2005).

The results also suggest that microtubule attachment directly removes CLIP-190 from kinetochores rather than through spindle-checkpoint signalling. Dynein seems to be responsible for the removal of CLIP-190 from kinetochores in addition to its role in localising CLIP-190 to kinetochores. It has been shown that dynein removes several kinetochore proteins along microtubules upon the attachment of microtubules. This study provides the first evidence that a member of the CLIP family also utilises dynein-motor-activity to leave attached kinetochores. Interestingly, it was found that unlike in interphase, EB1 is not required for the mitotic localisation of CLIP-190 to unattached kinetochores. This is intriguing in the light of recent evidence (Tirnauer, 2002b) that EB1 associates with attached kinetochores when the kinetochore microtubules are polymerizing (Dzhindzhev, 2005).

In conclusion, these results indicate that CLIP-190 localisation is regulated during the cell cycle and requires distinct mechanisms in mitosis and interphase. Spatial and temporal regulation of CLIP-190 localisation probably play crucial roles in the regulation of microtubule dynamics and their interaction with other cellular structures (Dzhindzhev, 2005).

Functionally distinct kinesin-13 family members cooperate to regulate microtubule dynamics during interphase: KLP10A is deposited on microtubules by the plus-end tracking protein EB1

Regulation of microtubule polymerization and depolymerization is required for proper cell development. Two proteins of the Drosophila melanogaster kinesin-13 family, KLP10A and KLP59C, cooperate to drive microtubule depolymerization in interphase cells. Analyses of microtubule dynamics in S2 cells depleted of these proteins indicate that both proteins stimulate depolymerization, but alter distinct parameters of dynamic instability; KLP10A stimulates catastrophe (a switch from growth to shrinkage) whereas KLP59C suppresses rescue (a switch from shrinkage to growth). Moreover, immunofluorescence and live analyses of cells expressing tagged kinesins reveal that KLP10A and KLP59C target to polymerizing and depolymerizing microtubule plus ends, respectively. The data also suggest that KLP10A is deposited on microtubules by the plus-end tracking protein, EB1. These findings support a model in which these two members of the kinesin-13 family divide the labour of microtubule depolymerization (Mennella, 2005).

Structural determinants for EB1-mediated recruitment of APC and spectraplakins to the microtubule plus-end

EB1 is a member of a conserved protein family that localizes to growing microtubule plus ends. EB1 proteins also recruit cell polarity and signaling molecules to microtubule tips. However, the mechanism by which EB1 recognizes cargo is unknown. This study defined a repeat sequence in adenomatous polyposis coli (APC) that binds to EB1's COOH-terminal domain and identified a similar sequence in members of the microtubule actin cross-linking factor (MACF) family of spectraplakins. MACFs directly bind EB1 and exhibit EB1-dependent plus end tracking in vivo. To understand how EB1 recognizes APC and MACFs, the crystal structure of the EB1 COOH-terminal domain was solved. The structure reveals a novel homodimeric fold comprised of a coiled coil and four-helix bundle motif. Mutational analysis reveals that the cargo binding site for MACFs maps to a cluster of conserved residues at the junction between the coiled coil and four-helix bundle. These results provide a structural understanding of how EB1 binds two regulators of microtubule-based cell polarity (Slep, 2005; full text of article).

This study has elucidated a motif shared by mammalian APC and several mammalian spectraplakin MACFs. Although these two families bear no apparent sequence homology outside the identified EB1 binding site, they do share a similar composite domain architecture and cellular functions. Both proteins are extremely large (1,000-5,000 amino acids) due to repeated domains in their central regions (ß-catenin and axin binding sites in APC and dystrophin-like spectrin repeats in MACFs. Both proteins also are characterized by microtubule binding motifs followed immediately after by the COOH-terminal EB1 recognition motif. In recent studies, both APC and the spectraplakins ACF7 and Shot have been shown to play roles in axon growth, cell polarity, and migration. In these studies, overexpression of the EB1 COOH-terminal domain resulted in a dominant-negative disruption of polarity signaling, indicative that an interaction with full-length EB1 is required for proper signal transduction/cytoskeletal stabilization by mammalian APC and spectraplakins. GSK-3ß, a kinase implicated in cell polarity signal transduction pathways, also has been implicated in the regulation of APC and MACFs. It is possible that this kinase might, at least in part, control polarity by regulating EB1 interaction because in vitro studies have shown that phosphorylation of APC abrogates binding to EB1 (Slep, 2005).

Interestingly, the two APC genes in Drosophila have the ß-catenin/axin binding domains, but lack the COOH-terminal region in mammalian APC that contains the EB1 binding motif. To date, Drosophila APCs have not been observed tracking along microtubule tips, although DmAPC2 has been found to tether mitotic spindles to cortical actin. Furthermore, the spectraplakin interaction with EB1 and microtubule tips appears to be widely conserved in all metazoans. Previous work has implicated the MACF Shot as an EB1-associated factor based on colocalization and coimmunoprecipitation, but a direct interaction has not been shown. This study definitively demonstrates that the in vivo association of Shot with the tips of microtubules is mediated by EB1. Thus, it is speculated that the spectraplakins may have represented the original metazoan invention of a microtubule tracking, cell polarity factor and that vertebrates subsequently incorporated this feature into APC by duplication and gene integration. Consistent with this idea, the EB1 binding motifs are more similar between vertebrate MACF and APC than they are between MACFs from vertebrates and invertebrates (Slep, 2005).

In addition to the EB1 interaction, localization studies suggest other features about the mechanism by which spectraplakins and APC interact with microtubules. Shot colocalizes only with the third of EB1 furthest from the microtubule plus end. This pattern, which is not observed for other proteins such as RhoGEFs, suggests that Shot does not coassemble with EB1 on the growing end of the microtubule but recognizes EB1 after a temporal delay, perhaps also suggesting some additional mechanism of regulation (Slep, 2005).

X-ray crystallographic studies show that the conserved EB1 COOH-terminal domain constitutes a unique coiled coil-four-helix bundle motif that confers both dimerization and cargo binding. Although EB1 and Bim1p were suggested to have a coiled coil domain based on sequence analysis, this is the first physical evidence that EB1 proteins are indeed dimers. Dimerization appears to rely not only on the coiled coil but also on the four-helix bundle motif, which provides nearly half of the buried surface area formed in dimerization. Consistent with this idea, destabilization of the four-helix bundle with the I224A mutation abolishes dimerization (Slep, 2005).

Alanine scanning mutagenesis, based on the crystal structure, was used to uncover a bivalent cargo binding site in the EB1 COOH-terminal domain. Both subunits of the homodimer appear to contribute residues to the cargo binding site, which is also supported by the finding that abrogating dimerization prevents cargo binding. Although the EB1 COOH-terminal domain is highly negatively charged, many of the key determinants in MACF2 binding are hydrophobic and form a discrete exposed hydrophobic pocket in the center of the domain. In comparison to the high conservation of EB1's COOH-terminal domain (from yeast to man), the repeat binding motifs found in APC and MACF2 are less well conserved, especially if one compares Drosophila to human. However, general features are the presence of several hydrophobic residues (mainly prolines) and a net positive charge. It is speculated that these prolines and other hydrophobics dock into EB1's hydrophobic pocket, whereas the charged residues may augment the binding interaction by providing a peripheral gasket (Slep, 2005).

The conservation of solvent-exposed residues in EB1's COOH-terminal domain extends from vertebrates down to the yeast homologues Bim1 and Mal3. Kar9 is functionally homologous to APC and Shot in its general role of microtubule search and capture; however, Kar9 does not have a repeat motif architecture similar to APC/spectraplakins. A weak segment of homology with hydrophobic character between regions of Kar9 and the EB1 binding motif has been reported, but this site has not been experimentally confirmed. It will be interesting to compare and contrast the molecular basis for the EB1-APC interaction and the Bim1-Kar9 interaction to determine if a similar or unique set of determinants govern these binding interactions (Slep, 2005).

The results also help to explain previous mutagenesis results of EB1 that were performed before the availability of a crystal structure. Wen (2004) mutated conserved hydrophilic residues within aa 211-229 in EB1 and found that the mutations E211A/E213A, E211A/D215A, K220A/R222A, and E211A/E213A/K220A/R222A inhibited EB1-APC interaction in vitro and yielded supporting phenotypic behavior in vivo in an mDia-mediated cell polarity pathway. The current results predict the strongest effect for constructs containing the K220A mutation and milder effects for the one with the E213A mutation, which agrees with the observations by Wen (2004). This correlation between EB1 mutants binding to MACF21595-1637 and APC also substantiates the likelihood of a similar binding architecture between these two classes of proteins and the EB1 COOH-terminal domain (Slep, 2005).

Several EB1 family members exist in metazoan genomes (four in Drosophila and three in humans. Although the crystal structures reported in this paper describe a homodimeric structure, the possibility that EB1 heterodimers exist and are used for diverse cargo recognition cannot be ruled out. The length of the coiled coil domains and the residues in the COOH-terminal domain that constitute the hydrophobic core and mediate dimer contacts are highly conserved across family members, especially within species. Because each bivalent cargo binding site is formed from residues contributed by each of the EB1 molecules, heterodimers could confer complex, pair-wise cargo recognition motifs. Thus, the possibility of whether or not heterodimers of EB1 can form in vitro and within cells merits investigation (Slep, 2005).

The EB1 COOH-terminal domain also may contain cargo binding sites other than the one that defined in this study for APC and MACF at the coiled coil-four-helix bundle junction. Several proteins, including the dynein-dynactin complex, Ncd, the microtubule-destabilizing kinesin Klp10A, and RhoGEF2, have been reported to track along microtubule tips and associate with EB1. However, only in the cases of the p150Glued dynactin subunit and Klp10A has a direct interaction with EB1 been shown. In the case of p150Glued, deletion studies have suggested that p150Glued and APC use overlapping yet unique binding sites on EB1's COOH-terminal domain. The p150Glued binding site maps to EB1's coiled-coil-four-helix bundle but also requires EB1's COOH terminus (13 residues not included in the crystallization construct). Thus, it is possible that EB1 contains multiple cargo binding sites, some of which might be independent and others which might be mutually exclusive. The crystal structure reported in this study should provide a valuable tool for probing these future questions concerning EB1-cargo interactions (Slep, 2005).

Structural basis of microtubule plus end tracking by XMAP215, CLIP-170, and EB1

Microtubule plus end binding proteins (+TIPS) localize to the dynamic plus ends of microtubules where they stimulate microtubule growth and recruit signaling molecules. Three main +TIP classes have been identified (XMAP215, EB1 and CLIP-170), but whether they act upon microtubule plus ends through a similar mechanism has not been resolved. This study reports crystal structures of the tubulin binding domains of XMAP215 (yeast Stu2p and Drosophila Msps), EB1 (yeast Bim1p and human EB1), and CLIP-170 (human), which reveal diverse tubulin binding interfaces. Functional studies, however, reveal a common property that native or artificial dimerization of tubulin binding domains (including chemically-induced heterodimers of EB1 and CLIP-170) induces tubulin nucleation/assembly in vitro and, in most cases, plus end tracking in living cells. It is proposed that +TIPs, although diverse in structure, share a common property of multimerizing tubulin, thus acting as polymerization chaperones that aid in subunit addition to the microtubule plus end (Slep, 2007).

This work analyzes the biochemical mechanisms of three plus end tracking protein families (EB1, CLIP-170 and XMAP215) using x-ray crystallography, in vitro tubulin binding/assembly assays, and in vivo plus end tracking assays. Five crystal structures are presented of +TIP tubulin binding domains across multiple species and families illustrating structural conservation within families and functional convergence across families. In vitro and in vivo analysis reveal an underlying general theme to these unrelated domains. While tubulin binding can be conferred by a single +TIP domain, the promotion of microtubule nucleation and polymerization in vitro and plus end tracking in vivo require multiple domains acting in concert. In the case of EB1 and CLIP-170 members, two domains are required for robust plus end localization and these two structurally unrelated domains can even cooperate if artificially joined together to achieve this activity. XMAP215 members are more complicated, perhaps due to the relative weaker association between TOG domains and tubulin in higher eukaryotic members. However, multimerization of TOG domains clearly facilitates tubulin nucleation in vitro and overall microtubule association in living cells. From these data, a model is presented in which unrelated tubulin binding domains have convergently evolved a similar mechanism for enhancing tubulin assembly on growing microtubule ends by acting as multivalent tubulin polymerization chaperones (Slep, 2007).

The tubulin binding domains of these three +TIP proteins are quite diverse in their architecture and show no evidence of a common evolutionary origin. However, the structural conservation within a +TIP class is very high, as evidenced by the near superimposition of yeast Bim1p with human EB1 and yeast Stu2p with Drosophila Mini spindles. The different architectures also suggest varying interactions modes with tubulin and potential synergy between the +TIPs. XMAP215 has a very flat binding interface created by a series of rigid, highly conserved loops on one face of the TOG domain; the length of the TOG domain is fairly closely matched to a tubulin monomer (α or β) and likely makes extensive contacts through a combination of hydrophobic and electrostatic interactions. In contrast to the elongated TOG domain, the CH domain of EB1 is spherical and approximately half the size. Mutagenesis suggests that several residues around one hemisphere contribute to tubulin binding. This data is consistent with recent work suggesting that EB1 might nestle in the groove between tubulin protofilaments. CLIP-170 also has a globular structure and the data suggests that it might bind tubulin’s disordered C-terminal tail as part of its binding interface (Slep, 2007).

Comparison of the +TIPS and protein engineering studies suggest a considerable structural variation in how +TIP proteins can be interconnected to achieve their activities in plus end tracking and promoting tubulin assembly. In some cases, tubulin binding domains are arrayed in tandem along a polypeptide chain with presumably unstructured linkers in between (e.g., Mini spindles, Ch-TOG, CLIP-170). The cis linking domains within a polypeptide appears to be critical, since it is shown that TOG domains added in trans do not reconstitute function. In other cases, single α/β tubulin binding domains are found in a polypeptide and multivalent tubulin binding is achieved by polypeptide dimerization [e.g., EB1 family and Bik1 (the CLIP-170 homologue from yeast)]. Hybrid strategies are also employed (e.g. CLIP-170, which has tandem Cap-Gly domains and is dimerized via a coiled coil and Stu2p which has tandem TOG domains, a dimerization domain and an additional C-terminal microtubule binding domain). It was also found that native dimerization sequences are not essential, as a variety of artificial dimerization strategies (e.g. GCN4 leucine zippers, gluthione S-transferase, and FKBP-rapamycin-FRB) are capable of reconstituting +TIP protein function (Slep, 2007).

Collectively, these studies reveal structural variation in how multiple +TIP tubulin binding domains can be combined to promote tubulin oligomerization for microtubule assembly. However, cis versus trans arrangements of +TIP domains may produce certain unique outcomes. For example, the linear arrays of TOG domains in the extended XMAP215 structure may generate a pseudo protofilament-like arrangement that is particularly effective for microtubule nucleation. Indeed, XMAP215 has a potent microtubule nucleation ability compared with CLIP-170, potentially related to an in vivo nucleation activity given XMAP215’s TACC-dependent localization to the centrosome. Analogous mechanisms exist for the actin cytoskeleton where the Spire protein utilizes several arrayed actin binding domains to template the nucleation of an actin filament (Slep, 2007).

The data show that single +TIP tubulin binding domains do not promote microtubule nucleation or growth in vitro (and in some cases are inhibitory), nor do they localize to microtubule plus ends in vivo. In contrast, multimerized +TIP tubulin binding domains are potent microtubule nucleator in vitro, promote microtubule growth in vitro and are requisite in vivo for microtubule plus end localization. For EB1 and CLIP-170, it was dynamically shown that plus end tracking requires an ability to bind more than one tubulin subunit. These results suggest a model in which +TIPs bind multiple tubulin dimers in solution and then deliver these larger tubulin oligomers to the ends of microtubules. This general idea was first introduced two decades ago by (Gard, 1987) to explain the high rates of tubulin assembly induced by XMAP215. Multimerization overcomes the inherent polymerization barrier tubulin heterodimers face due to single longitudinal and lateral tubulin:tubulin affinities estimated to be on the order of mM and M respectively. +TIP-induced multimerization of tubulin would increase the effective affinity for the microtubule lattice through cooperative binding, thereby decreasing the critical concentration for polymerization. By stabilizing tubulin-tubulin interactions prior to their full incorporation into a mature, cylindrical lattice, the +TIPs would act as polymerization chaperones. Physiologically, such chaperones would become particularly important for enhancing the growth of microtubules when free tubulin dimers are below the critical concentration of microtubule assembly or when microtubule destabilizing proteins are active (e.g in mitosis). This mechanism also enhances the spatial and temporal regulation of microtubule assembly in the cell, in part by regulating the localization and/or activity of +TIPs without modifying tubulin, the basic unit of polymerization (Slep, 2007).

Support for this general model comes from a number of other laboratories. Tandem Cap-Gly domains in CLIP-170 have been shown to plus end track whereas single Cap-Gly domains of the homolog CLIP-115 show greatly reduced microtubule association. Optical trapping studies analyzing microtubule growth showed step-wise growth that increased in size in the presence of full length XMAP215, a result they interpreted as XMAP215-facilitated incorporation of tubulin oligomers onto the microtubule end (Slep, 2007).

An apparent exception to the multiple tubulin binding rule for +TIPs is the CLASP family, which contains only one TOG domain at its N-terminus. However, informed by TOG structures and secondary structure predictions several TOG-like (TOGL) domains can be identified in the CLASP family. Two additional dodeca-helical domains are predicted with alternating loops that exhibit high homology to TOG domain intra-HEAT loops. If folded into a HEAT-like structure, the conservation profile of alternating loops would be localized to one face of the domain, suggesting that TOGL domains may bind tubulin by a mechanism similar to what is described in this study for the TOG domain. Thus, although poorly conserved at the primary structure level, XMAP215 and CLASP appear to be ancient +TIP relatives that may employ a similar general strategy for plus end tracking. Support for CLASP’s possible polymerization chaperone role comes from studies that observed CLASP-dependent microtubule subunit incorporation into fluxing kinetochore fibres (Slep, 2007).

The model for +TIPs as multimeric tubulin chaperones undoubtedly oversimplifies the complexity of interactions that are occurring at the microtubule plus end in vivo. +TIP families show divergent effects on microtubule dynamics, some promoting growth while others act as anti-pause, destabilization, rescue and perhaps even nucleation factors. Some of these differences in activity may reside in variations in the affinity constants of +TIP domains with tubulin monomers, oligomers and microtubules. As an example, non-EB1 family microtubule-binding CH domain proteins, including CLAMP and HEC1 appear to have higher affinity for the microtubule lattice than EB1 and localize along the entire length of the microtubule rather than just at plus ends. The spatial arrangement of +TIP domains, arrayed versus dimerized, may lead to distinct effects on microtubule dynamics by preferentially stabilizing lateral versus longitudinal tubulin-tubulin interactions. Finally, the intricate web of +TIP-+TIP interactions may generate unique outcomes on tubulin assembly. Multi-component in vitro assays, high resolution EM analysis of microtubules, and x-ray structures of +TIP-tubulin complexes will be required to further understand how +TIPs regulate the microtubule lattice (Slep, 2007).

The spectraplakin Short stop is an actin-microtubule cross-linker that contributes to organization of the microtubule network.

The dynamics of actin and microtubules are coordinated in a variety of cellular and morphogenetic processes; however, little is known about the molecules mediating this cytoskeletal cross-talk. Short stop (Shot), the sole Drosophila spectraplakin, os a model actin-microtubule cross-linking protein. Spectraplakins are an ancient family of giant cytoskeletal proteins that are essential for a diverse set of cellular functions; yet, little is known about the dynamics of spectraplakins and how they bridge actin filaments and microtubules. This study describes the intracellular dynamics of Shot and a structure-function analysis of its role as a cytoskeletal cross-linker. Shot was found to interact with microtubules using two different mechanisms. In the cell interior, Shot binds growing plus ends through an interaction with EB1. In the cell periphery, Shot associates with the microtubule lattice via its GAS2 domain, and this pool of Shot is actively engaged as a cross-linker via its NH(2)-terminal actin-binding calponin homology domains. This cross-linking maintains microtubule organization by resisting forces that produce lateral microtubule movements in the cytoplasm. The results provide the first description of the dynamics of these important proteins and provide key insight about how they function during cytoskeletal cross-talk (Applewhite, 2010).

This analysis revealed that Shot is able to interact with microtubules via two distinct mechanisms, association with the microtubule lattice mediated by the GAS2 domain of Shot, and an interaction with growing plus ends that is mediated by EB1, through multiple EB1-interacting motifs at the COOH terminus of Shot. The interaction of Shot with the microtubule lattice, but not with the plus end, requires an intact network of F-actin and a functional actin-binding domain at its NH2 terminus. The data also demonstrate that the actin- and microtubule-binding domains cooperate to regulate microtubule dynamics by cross-linking the two cytoskeletal networks. Shot cross-linking couples microtubules to actin in order to resist motor-mediated lateral sliding movements. The data suggest that Shot can exist in at least two different states. In the first state, observed after actin depolymerization or with the C isoform that exhibits low affinity for actin, Shot preferentially interacts with microtubule plus ends. In the second state, observed with the A, B, or Shot-ΔRod isoforms in the actin-rich region of the peripheral lamella, Shot is bound to the microtubule lattice via the GAS2 domain. The results suggest that the pool of Shot that is actively serving as a cross-linker is the lattice-bound population. It is speculated that the interconversion between these two states represents a regulated conformational change, so that the timing and placement of Shot-mediated cross-linking is precisely controlled within the cell. Understanding how Shot is regulated will be an important next step, but several potential mechanisms immediately present themselves. First, it is interesting to note that the Shot-GAS2-CTD construct decorated the microtubule lattice despite the presence of the EB1-interaction motif. This result suggests that GAS2 exerts a dominant effect when present in the same molecule and further suggests that it must be actively repressed when Shot is at microtubule plus ends. The hypothesis is favored that Shot is regulated by an intramolecular interaction that simultaneously represses the GAS2 and actin-binding CH domains during tip tracking. On activation, the molecule opens and is able to bind the lattice and actin. Second, the EF-hand motifs are closely positioned to the GAS2 domain, suggesting that calcium may participate in regulation of microtubule lattice binding. Third, there is evidence that Shot may act downstream of the small GTPase Rho; perhaps activation of this pathway leads to activation of the cross-linking activity. Fourth, binding of the CH domains to actin may itself be sufficient to trigger the transition from tip-tracker to lattice-binder. Understanding when and where Shot is activated to cross-link will be key to understanding its cellular functions (Applewhite, 2010).

Why does Shot possess these dual mechanisms (tip vs. lattice) for microtubule interaction? Microtubule dynamic instability is used as a mechanism to search intracellular space for docking and stabilization during kinetochore microtubule capture and polarization of migrating cells. Perhaps association with the microtubule plus end allows spectraplakins to search for activating inputs at the cell cortex or in actin-rich subregions of the cell. Different modes of microtubule association may also allow spectraplakins to regulate microtubule dynamics in different ways. A common theme emerging from studies of other +TIPs is that many of them function is as regulators of microtubule dynamic instability. Because the majority of subunit addition and loss occurs at the plus end, +TIPs are perfectly positioned to influence the conformation of tubulin at this site. This study presents evidence that Shot also can influence microtubule dynamics through a different mechanism, by cross-linking microtubules to the actin network to prevent lateral microtubule displacement by motor proteins (Applewhite, 2010).

Previous studies of the role of mouse ACF7 have clearly demonstrated a role for this protein as a 'guide' to direct microtubule growth along actin stress fibers to assist them in targeting to focal adhesions (Wu, 2008). The current data show that, in S2 cells, Shot-mediated cross-linking performs an additional function by cross-linking microtubules to actin in order to resist whip-like lateral displacements by motor proteins. Many Drosophila cell types exist without a dominant interphase microtubule organizing center, instead organizing acentriolar networks to organize their cytoplasm. It is speculated that, in the absence of a structural anchor at the minus end, acentriolar cells rely upon actin-microtubule cross-linking to maintain their organization and to resist forces produced by motor proteins. These forces may be generated during organelle transport, especially if the motors associated with the surface of one organelle engage multiple microtubules. Shot is, therefore, likely to play an important role in maintaining microtubule organization for the efficient motor-mediated delivery of organelles and other cargo to specific destinations within the cell. Furthermore, this activity is consistent with the observation that the microtubule network in mushroom body neurons in Shot mutants exhibits gross disorganization and an overall loss of microtubule polarity. Based on the current data a speculative model has been developed in which 'inactive' Shot associates with a growing microtubule plus end by its interaction with EB1. Dynamic instability allows microtubules to probe the cytoplasm for an activating signal in a 'search-and-capture' mechanism. On receiving the activating stimulus, Shot changes its conformation, releases from EB1, and engages actin with is dual CH domains and the microtubule lattice with its GAS2 domain. In this conformation, it is able to cross-link microtubules to actin to influence their growth trajectory or to stabilize the network against forces produced by motors. Testing these ideas will require careful observation of Shot dynamics in the developing embryo where cells are responding to extracellular cues, as well as genetic analyses to determine how the EB1-Shot interaction contributes to development (Applewhite, 2010).

EB1 promotes microtubule dynamics by recruiting Sentin in Drosophila cells

Highly conserved EB1 family proteins bind to the growing ends of microtubules, recruit multiple cargo proteins, and are critical for making dynamic microtubules in vivo. However, it is unclear how these master regulators of microtubule plus ends promote microtubule dynamics. This paper identifies a novel EB1 cargo protein, Sentin (FlyBase designation: Short spindle 2). Sentin depletion in Drosophila S2 cells, similar to EB1 depletion, resulted in an increase in microtubule pausing and led to the formation of shorter spindles, without displacing EB1 from growing microtubules. Sentin's association with EB1 is critical for its plus end localization and function. Furthermore, the EB1 phenotype os rescued by expressing an EBN-Sentin fusion protein in which the C-terminal cargo-binding region of EB1 is replaced with Sentin. Knockdown of Sentin attenuates plus end accumulation of Msps (Mini spindles), the orthologue of XMAP215 microtubule polymerase. These results indicate that EB1 promotes dynamic microtubule behavior by recruiting the cargo protein Sentin and possibly also a microtubule polymerase to the microtubule tip (Li, 2011).

In a genome-wide RNAi screen using S2 cells, several genes were identified whose knockdown leads to the shortening of the metaphase spindle, which was similar to EB1 or XMAP215Msps RNAi. Among them, one uncharacterized gene, CG9028/ssp2, was named 'Sentin' from the Japanese word Sentan (tip) in the present study. After RNAi treatment, Sentin levels were reduced by 90%, and the metaphase spindle length was 72% of the control. Centrosome/centriole-free spindles constructed using Sak (Plk4) RNAi were also shorter in the absence of Sentin than in the control cells. Thus, the formation of shorter spindles is likely to be caused by the presence of shorter microtubules in the Sentin-depleted spindles (Li, 2011).

This study has identified Sentin as an EB1 cargo protein. Furthermore, a series of truncation and gene fusion experiments raised an intriguing possibility that Sentin is the dominant cargo of EB1 for microtubule dynamics promotion in S2 cells. This idea is consistent with the fact that no other known cargo proteins phenocopy EB1 as closely as Sentin in S2 cells and that EB1 protein alone does not show growth-promoting activity in vitro in several studies. However, it is not ruled out that the cytoplasmic pool of other EB1 cargo proteins is also required to suppress microtubule pausing (Li, 2011).

How might Sentin promote plus end dynamics? The first possible model is that the EB1-Sentin complex alters the structure of the plus end of microtubules, e.g., closing the sheet or promoting a specific lattice configuration, so that tubulin dimers are favorably added to or removed from the end. However, the known structural changes of the plus end can be made by the EB1 protein alone. In the second model, Sentin catalyses the supply and removal of tubulin dimers at the microtubule plus ends, which is similar to the case of XMAP215 polymerase. These two activities may be responsible for the antipause activity of the EB1-Sentin complex observed in vivo. In the third model, EB1-Sentin makes microtubules dynamic by further recruiting XMAP215Msps polymerase to the tip. This model is consistent with the phenotypic similarity among EB1, Sentin, and XMAP215Msps and also the observation in Xenopus laevis egg extracts in which the EB1 depletion phenotype is rescued by overexpression of XMAP215 (Kronja, 2009). The latter two models, which are not mutually exclusive, might be experimentally tested through in vitro reconstitution of microtubule polymerization dynamics with purified EB1, Sentin, and XMAP215Msps (Li, 2011).

Kebab: kinetochore and EB1 associated basic protein that dynamically changes its localisation during Drosophila mitosis

Microtubule plus ends are dynamic ends that interact with other cellular structures. Microtubule plus end tracking proteins are considered to play important roles in the regulation of microtubule plus ends. Recent studies revealed that EB1 is the central regulator for microtubule plus end tracking proteins by recruiting them to microtubule plus ends through direct interaction. This study reports the identification of a novel Drosophila protein, which was called Kebab (kinetochore and EB1 associated basic protein; CG31672), through in vitro expression screening for EB1-interacting proteins. Kebab fused to GFP shows a novel pattern of dynamic localisation in mitosis. It localises to kinetochores weakly in metaphase and accumulates progressively during anaphase. In telophase, it associates with microtubules in central-spindle and centrosomal regions. The localisation to kinetochores depends on microtubules. The protein has a domain most similar to the atypical CH domain of Ndc80, and a coiled-coil domain. The interaction with EB1 is mediated by two SxIP motifs but is not required for the localisation. Depletion of Kebab in cultured cells by RNA interference did not show obvious defects in mitotic progression or microtubule organisation. Generation of mutants lacking the kebab gene indicated that Kebab is dispensable for viability and fertility (Meireles, 2011).

EB1 regulates microtubule plus ends through interaction with multiple proteins. This study identified a novel EB1-interating protein, Kebab, which shows a dynamic localisation to kinetochores and microtubules in mitosis. Previously several studies have successfully identified EB1-interacting proteins by mass-spectrometry after pull down using bacterially produced EB1 protein. One drawback of this approach is that the chance of a protein being identified depends on its level in a particular tissue or cell line. Kebab was identified by in vitro expression cloning. In vitro expression cloning was originally developed, using random cDNAs from Xenopus eggs. It was later adapted for use with a collection of annotated unique Drosophila cDNAs. This study looked for substrates of a kinase by examining the shift in gel mobility after kinase reaction. This method was further adapted for a pull down assay to identify interacting proteins. The advantage of this approach over a mass-spectrometry based one would be that low abundance proteins or those expressed in specific cell types have equal chance of being identified, as long as cDNA has been isolated from some tissues (Meireles, 2011).

Kebab localises to kinetochores during mitosis. The relative position of Kebab to the Drosophila CenpA orthologue Cid, suggests it localises to outer kinetochores. The most notable feature of Kebab is that it progressively accumulates on kinetochores during anaphase. Although this behaviour is unusual among kinetochore proteins, a group of centromere proteins, including CenpA, CenpC and Mis18, have been reported to progressively accumulate on centromeres during anaphase. These proteins interact with each other to define the centromere identity through loading of CenpA, a Histone 3 variant, on DNA. It is still not understood why this occurs during anaphase. Interestingly, Kebab is not colocalised with CenpA, rather is localised outside of CenpA. Although all kinetochore proteins require CenpA for their localisation, generally they do not increase in intensity during anaphase. It is possible that Kebab recruitment to kinetochores may be somehow linked to CenpA loading, or the dose of CenpA on centromeres, rather than other kinetochore proteins. Alternatively, Kebab may be regulated by a previously unknown mechanism (Meireles, 2011).

The second interesting property of Kebab is that microtubules are required for its kinetochore localisation. This property is unusual but not unique among kinetochore proteins. EB1 localisation to kinetochores in PtK1 cells was previously reported to depend on microtubules. It was found to localise to only one of the sister kinetochores moving away from poles, which is coupled to microtubule polymerisation. In contrast, Kebab localisation appears to be symmetrical in metaphase and become more prominent when microtubules are depolymerising in anaphase. Other kinetochore proteins such as Ska1 and Ajuba are also reported to depend on microtubules for their localisation. The mechanism and significance of this microtubule dependency are still under speculation. Kebab may recognise kinetochore-associated microtubule plus ends regardless of the polymerisation state. Alternatively, Kebab is transported to kinetochores along microtubules. In either case, it is a very interesting and unusual property of a kinetochore protein (Meireles, 2011).

The localisation of Kebab dynamically changes during mitotic progression. In late anaphase, Kebab starts localising to spindle microtubules or centrosomal regions. The association with microtubules becomes more prominent in telophase. This changing pattern of localisation is unique among previously reported kinetochore proteins or microtubule-associated proteins. Nevertheless, other types of proteins are known to change localisation during mitotic progression. For example, the chromosomal passenger complex localises to centromeres/kinetochores until metaphase and relocates to microtubules in the spindle midzone at the onset of anaphase. This change in localisation is considered to be crucial for the change in kinetochore behaviour at the onset of anaphase, and stabilisation of the spindle midzone in telophase. Dynamic localisation of Kebab may subtly influence a change in behaviour of kinetochore or spindle microtubules (Meireles, 2011).

Kebab can directly bind to EB1 in vitro. Interaction with EB1 is mediated by two SxIP motifs located near the CH domain. The SxIP motif is a linear motif found in many EB1 binding proteins. Mutations in both SxIP did not abolish the localisation of Kebab, suggesting interaction with EB1 is not essential for localisation. Consistently it was found that EB1 depletion did not disrupt the localisation of Kebab. It is possible that EB1-independent localisation masks the EB1 dependent localisation in a specific location. Alternatively EB1 interaction may be important for Kebab function rather than the localisation. Further studies are needed to clarify the significance of EB1 interaction (Meireles, 2011).

Kebab contains a domain which is most similar to the atypical CH domain of another kinetochore protein, Ndc80. Ndc80 is one of the critical proteins which connect kinetochores to microtubules. This domain of Ndc80 is considered to be the microtubule binding domain. The CH domain of Ndc80 is quite distinct from typical CH domains found in other proteins, and was only recognised after the crystal structure was determined. The discovery of the second member of this atypical CH domain group may shed light on how the essential kinetochore protein Ndc80 interacts with microtubules (Meireles, 2011).

No obvious functions have been revealed by RNAi or generation of null mutants. Although there are no obvious paralogues in the Drosophila genome, there may be other proteins that function redundantly with Kebab. Hundreds of proteins in each cell type can bind to microtubules, and collectively determine their behaviour. These consist of a diverse array of proteins, with only a small minority containing known microtubule binding motifs. It is likely that many structurally distinct proteins can function redundantly to regulate microtubules. For example, microtubule bundling can be achieved by many proteins or protein complexes which contain multiple microtubule binding sites. It is a challenge in biology to understand a system that involves many redundancies, such as microtubule regulation. Future identification of proteins that have overlapping function with Kebab will shed light on the function and regulation of Kebab protein in mitosis (Meireles, 2011).

The microtubule lattice and plus-end association of Drosophila mini spindles is spatially regulated to fine-tune microtubule dynamics

Individual microtubules (MTs) exhibit dynamic instability, a behavior in which they cycle between phases of growth and shrinkage while the total amount of MT polymer remains constant. Dynamic instability is promoted by the conserved XMAP215/Dis1 family of microtubule-associated proteins (MAPs). In this study, an in vivo structure-function analysis was conducted of the Drosophila homologue, Mini spindles (Msps). Msps exhibits EB1-dependent and spatially regulated MT localization, targeting to microtubule plus ends in the cell interior and decorating the lattice of growing and shrinking microtubules in the cell periphery. RNAi rescue experiments revealed that Msps' NH(2)-terminal four TOG domains function as paired units and are sufficient to promote microtubule dynamics and EB1 comet formation. TOG5 and novel inter-TOG linker motifs were identifed that are required for targeting Msps to the microtubule lattice. These novel microtubule contact sites are necessary for the interplay between the conserved TOG domains and inter-TOG MT-binding that underlies the ability of Msps to promote MT dynamic instability (Currie, 2011).

Growing evidence from several model systems indicates that members of the XMAP215/Dis1 protein family enhance microtubule dynamic instability and mitotic spindle assembly. These multi-domain proteins are highly conserved across taxa and there is particular interest in the human homologue, ch-TOG, as its overexpression has been documented in several cancer cell types. Despite the importance of these proteins, relatively little is known about their dynamics in living cells or about how their domain structure relates to their function in vivo. In this study, the Drosophila XMAP215 homologue, Mini spindles (Msps), was used as a model to address these questions in living S2 cells. It was found that Msps exhibits a complex and dynamic behavior in vivo. It localizes both to growing and shrinking microtubule plus ends. In the actin-rich cell periphery, Msps-GFP punctae exhibit a discontinuous and dynamic association with the microtubule lattice. Consistent with roles for both Msps and EB1 as key regulators of dynamic instability, it was observed that depletion of either of these proteins lead to alteration of the other' dynamics at microtubule plus ends (Currie, 2011).

Remarkably, microtubule dynamics and EB1 velocity could be partially restored by replacing endogenous Msps with the NH2-terminal four TOG domains TOG1-4. Structure/function analysis also revealed the presence of novel microtubule lattice-binding domains, which include sequences in two of the inter-TOG linker domains. These linkers contain novel motifs responsible for this interaction and are important for the normal dynamics of Msps as mutation of these motifs causes a loss of Msps from the lattice of peripheral microtubules and an alteration to the morphology and dynamics of the microtubule cytoskeleton. Thus, the data demonstrate that Msps is able to interact with microtubules through at least two mechanisms and that this bimodal interaction is required for normal dynamic instability (Currie, 2011).

This study, the first detailed examination of a Msps/Dis1 family member in a living animal cell, revealed that Msps exhibits a complex pattern of dynamics. Throughout the cell, Msps localizes to the plus ends of growing microtubules, consistent with other published descriptions of Msps behavior in early Drosophila embryos) and in vivo dynamics of homologues Stu2. Observations in S2 cells are in line with family members acting as microtubule polymerases that promote the incorporation of free tubulin at the plus end. Msps also exhibits dynamics similar to the in vitro behavior of XMAP215 as both proteins remain associated with microtubules during phases of growth and shrinkage. At present, the biological significance of Msps association with shrinking microtubules is not understood, however, Stu2 and XMAP215 do destabilize microtubules under specific in vitro experimental conditions. Persistent interaction with shrinking microtubules may reflect a physiologically important role for Msps during depolymerization in vivo. A model based on this interaction with both growing and shrinking microtubules is elaborated, in which Msps acts, not only as a polymerase, but as an anti-pause factor that is capable of 'catalyzing' the transition to either polymerization or depolymerization. Although it has primarily been shown to enhance the growth of microtubules, Msps is also necessary for their transition to disassembly in vitro and in vivo. These two seemingly opposing roles are mediated by 1) the TOG domains and plus end localization which enhance microtubule growth; and 2) microtubule lattice binding sites within linker2-TOG3 and linker4- TOG5 which enhance the transition between dynamic and non-dynamic states and also influence microtubule disassembly rates. Regulation and balance of these two domains maintain normal microtubule dynamics. In the future, it will be interesting to learn if these in vivo dynamics also apply to XMAP215 or human chTOG, which remain largely uncharacterized in cells (Currie, 2011).

One of the central observations from this work was that the NH2-terminal TOG domains of Msps are the key determinants of the protein's activity in vivo. Several previous studies have indicated a role for TOG domains as primary binding sites for tubulin in XMAP215/Dis1 proteins. Biochemical analysis of the TOG domains from Msps revealed that TOG1-2 was the minimal construct that was able to bind tubulin. Moreover, a construct consisting of a tandem array of TOG1212 functioned as a potent microtubule nucleator in vitro, suggesting that multiple pairs of TOG domains act in concert to promote polymerization. A fragment of Msps containing TOG1-4 was sufficient to rescue many aspects of microtubule dynamic instability in living S2 cells depleted of endogenous Msps, both in terms of interior EB1-GFP growth velocities and persistence as well as the frequencies of catastrophe and rescue. Consistent with the in vitro data, constructs embodying TOG1-2 imparted only a slight rescue of microtubule growth in S2 cells, while TOG3-4 did not. These data imply that individual pairs of TOG domains may contribute unequally to the activity of the protein, but do function cooperatively to promote persistent microtubule growth. This is supported by recent work that found that TOG1 and 2, specfically, play a key role in XMAP215‘s polymerase activity in vitro (Widlund, 2011). The lack of rescue by TOG1212 also indicates that each pair of TOG domains is uniquely suited to fulfill the cooperative function of tubulin addition. Although TOG1-4 exerted a strong effect on tubulin incorporation and loss from the plus end as measured by GFP-Tubulin and EB1-GFP growth rates, the TOG1-4 protein exhibited a predominantly cytoplasmic localization and did not accumulate at microtubule tips. These data suggest a mechanism in which TOG1-4 is able to associate with one or more tubulin heterodimers to transiently promote their addition or loss from the plus end without interacting in a processive manner or associating with the microtubule lattice (Currie, 2011).

Another key observation is the identification of novel microtubule interaction sites spanning the TOG3 and TOG5 and their preceding inter-TOG linkers. These sites are necessary for association with the microtubule lattice and for the lattice-associated diffusive movements exhibited by full-length Msps. This analyses identified the presence of conserved motifs in the inter-TOG regions that were enriched in basic amino acid residues, which are predicted form an electrostatic interaction with the negative COOH-terminus of tubulin. The linkers between Msps' TOG domains are predicted to be disordered stretches without secondary structure that were found to cooperate with the highly structured TOG3 or TOG5 to mediate microtubule binding (Currie, 2011).

The cooperation between ordered and disordered domains to mediate microtubule binding has become a common trend as more of the structural determinants of MAP-microtubule association have recently been elucidated. These disordered regions seem to allow a diffusion-capable attachment to microtubules in addition to the ability to 'titer' the interaction strength by charged residue addition or modification (i.e., phosphorylation) (Currie, 2011).

Beyond the structural basis for microtubule lattice-association, it was observed that Msps employs this mechanism in a spatially restricted manner in the actin-rich lamella of S2 cells. In other cell types, microtubules that enter this subcellular compartment exhibit decreased catastrophe rates, due to the small GTPase Rac1 regulating MAPs that influence microtubule dynamics. In this regard, the spatial transition from plus end binding to MAP lattice association has also been observed in mammalian epithelial cells for the +TIP CLASP. The Drosophila CLASP homolog, Orbit/MAST, when tagged with GFP displayed plus end dynamics similar to that of EB1 and did not differentially localize in the periphery of the cell. One possibility is that Msps functions in an orthologous manner to mammalian CLASP, which could be due to their shared structure as TOG domain-containing proteins. In any case, it is speculated that this transition from tip-tracking to lattice-binding reflects a regulated change in the conformation of Msps. Given the observation that TOG1-5 decorates the microtubule lattice constitutively, it is also hypothesized that the COOH-terminus of the protein is involved in this regulation and acts to 'gate' the microtubule binding activity of linker4-TOG5 in the full length protein. Future studies will address the basis of this regulation (Currie, 2011).

Ablating these microtubule lattice association sites in the full-length molecule had a dramatic effect on the dynamics and morphology of the microtubule cytoskeleton. Although lattice association is commonly a stabilizing property of most MAPs, it was surprising to find that without its normal lattice association, Msps Double Mutant produced very stable microtubules that exhibited fewer transitions and continuous growth after microtubules encountered the cell cortex. In contrast, although Msps dsRNA also produces very stable microtubules, these microtubules are seldom able to persistently grow against the actin retrograde flow in the peripheral lamella. These data, in conjunction with the increased shrinkage rates observed when cells expressed TOGs1-4 or TOG1-5, lead to a proposal that these lattice association sites may influence the catalysis between dynamic states and the shrinkage rates of peripheral microtubules. Microtubules in peripheral regions of S2 cells exhibit increased dynamic instability as compared to the interior of the cell and the data suggest a mechanism through which Msps may regulate these peripheral behaviors through its lattice association (Currie, 2011).

It is hypothesized that this influence on dynamics could be caused by three non-mutually exclusive mechanisms. Firstly, Msps lattice association could act to 'strip' Msps from the plus end, potentially taking heterodimers with it and lowering the concentration of this polymerase from the microtubule tip. Secondly, Msps- lattice binding domains could act to slightly perturb lateral interactions between heterodimers along the decorated protofilament. This cascade of 22 small perturbations could act to prime the microtubule lattice for depolymerization several microns distal to the plus end. Finally, lattice-bound Msps could be acting in concert with other +TIPs, such as kinesin-13 depolymerases, to influence catastrophe and shrinkage (Currie, 2011).

Msps dependence on EB1 for plus end localization in interphase has recently become more clear based on two recent studies identifying novel EB1 adaptor proteins, SLAIN/Sentin. In mammalian cells, SLAIN binds both chTOG and EB1 and is required for ch-TOG plus end tracking (van der Vaart, 2011). In Drosophila, a similar protein Sentin has also been described (Currie, 2011).

Although it has not yet been shown to bind Msps, RNAi depletion of Sentin alters the plus end localization of Msps. Depletion of EB1 removed Msps from both the plus end and lattice of microtubules. Although the mechanism for how Msps is lost from both plus end and lattice is not known, it suggests that the ability of full-length Msps to recognize the microtubule lattice is in some way tied to its plus end association. One possibility could be a whole-molecule conformational change that occurs when Msps interacts with EB1 at the growing end, which might be required to license Msps for association along the microtubule lattice. This most likely is concurrent with some other spatially regulated control that gives Msps a bimodal function between the cell interior and the periphery. EB1's plus end localization does not require Msps, as a population of EB1 remains on microtubule tips following Msps depletion, however, its dynamics are drastically altered. Rescue of EB1 velocities using the TOG domain region of Msps suggests that EB1 relies on the polymerase activity of Msps for its normal dynamics. This also seems true since Msps fragments that rescue EB1-GFP comet formation do not seem to localize to the microtubule plus end, although the possibility that there may be transient pools of TOG domain-fragments at the plus end cannot be ruled out. Instead, a model is favored where these TOG domain fragments act en masse to chaperone tubulin onto the plus end, subtly influencing the on and off rates of heterodimer addition. This creates a plus end structure with sufficient binding sites to support more normal EB1 comets (Currie, 2011).

Based on the data, Msps exhibits at least two modes of interaction with microtubules, both of which are essential to promote normal parameters of dynamic instability. A cycle of interactions is envisioned that begins upon association between soluble Msps and one or more tubulin heterodimers. The Msps-tubulin complex then recognizes the microtubule plus end and associates transiently. The molecular basis of this recognition is unknown, but may reflect a conformation of tubulin at the plus end (e.g. a growing sheet), a chemical signature (e.g., a cap of GTP tubulin), the presence of another +TIP such as EB1, or some combination of these. Upon binding, Msps delivers its tubulin 'cargo' to promote polymerization. In the cell interior, Msps then dissociates, thus behaving as a typical +TIP. In the cell periphery, however, following tubulin delivery, Msps receives a signal that causes it to engage its lattice-binding activity and to diffuse along the microtubule surface. Msps also associates with microtubule plus ends upon transition to catastrophe, perhaps working cooperatively with destabilizing factors such as kinesin-13 proteins and stathmin/OP18 (Currie, 2011).

Spectraplakins promote microtubule-mediated axonal growth by functioning as structural microtubule-associated proteins and EB1-dependent +TIPs (tip interacting proteins)

The correct outgrowth of axons is essential for the development and regeneration of nervous systems. Axon growth is primarily driven by microtubules. Key regulators of microtubules in this context are the spectraplakins, a family of evolutionarily conserved actin-microtubule linkers. Loss of function of the mouse spectraplakin ACF7 or of its close Drosophila homolog Short stop/Shot similarly cause severe axon shortening and microtubule disorganization. How spectraplakins perform these functions is not known. This study shows that axonal growth-promoting roles of Shot require interaction with EB1 (End binding protein) at polymerizing plus ends of microtubules. Binding of Shot to EB1 requires SxIP motifs in Shot's C-terminal tail (Ctail), mutations of these motifs abolish Shot functions in axonal growth, loss of EB1 function phenocopies Shot loss, and genetic interaction studies reveal strong functional links between Shot and EB1 in axonal growth and microtubule organization. In addition, it is reported that Shot localizes along microtubule shafts and stabilizes them against pharmacologically induced depolymerization. This function is EB1-independent but requires net positive charges within Ctail which essentially contribute to the microtubule shaft association of Shot. Therefore, spectraplakins are true members of two important classes of neuronal microtubule regulating proteins: +TIPs (tip interacting proteins; plus end regulators) and structural MAPs (microtubule-associated proteins). From these data a model is deduced that relates the different features of the spectraplakin C terminus to the two functions of Shot during axonal growth (Alves-Silva, 2012).

The regulation of MT networks is essential for many neuronal functions and processes, ranging from axonal growth to neurodegeneration. However, understanding how MTs are regulated in neurons remains a major challenge. Many MT-binding proteins have been identified and their potential roles in regulating the key processes of MT stabilization, MT polymerization, MT-based transport and MT-actin cross talk have been highlighted. But how these different functions merge into coordinated MT network regulation is poorly understood (Alves-Silva, 2012).

This work indicates spectraplakins as key integrators of different MT regulatory processes (see Model of Shot function in neuronal MT network organization). First, Shot stabilizes MTs, a role generally assigned to structural MAPs, such as Tau, MAP2 or MAP1b. Second, Shot regulates MT polymerization dynamics and guides them in the direction of axonal growth. In this function, Shot interacts with EB1, firmly establishing Shot-EB1 interaction as an important determinant of microtubule guidance as a crucial mechanism underpinning axon growth. In addition, Shot acts as an actin-MT linker during axonal growth, adding a third crucial MT regulatory role to the list. Therefore, spectraplakins work at the cross-roads of structural MAPs, +TIPs and actin-binding proteins, strongly suggesting that work on spectraplakins will provide exciting new opportunities to unravel the complexity of MT regulation in neurons (Alves-Silva, 2012).

At the molecular level, this work reveals the importance of the Ctail for Spectraplakin function. While GRD had previously been established as the domain of Shot which has the widest functional requirements, the current work suggested Ctail to be similarly important, as deduced from its crucial roles both in MT stabilization and guidance and its requirements in both neurons and tendon cells (Alves-Silva, 2012).

Shot localization along the shafts of axonal MTs is important for MT-stabilization, suggesting that spectraplakins might functionally overlap with structural MAPs. This finding might have important implications. For example, loss of structural MAP functions has relatively mild phenotypes even in double knock-out mice, and this could be caused by functional compensation through spectraplakins. It remains to be seen whether such potential functional overlap is general or context-specific. For example, different MAPs and spectraplakins might display individual traits, such as dependence on different MT modifications or selective antagonism to different destabilizing factors (Alves-Silva, 2012).

In support of these findings with Shot, MT-stabilizing roles of spectraplakins appear conserved in mammals. Thus, MT-stabilizing roles in neurons have similarly been reported for the Shot homolog BPAG1. Furthermore, axon shortening caused by knock-down of ACF7 in primary cortical neurons or N2A cells was rescued by taxol application. In agreement with these findings, also the domain requirements underlying MT stabilization are conserved. Thus, studies in fibroblasts have shown that related C termini of the mammalian spectraplakin ACF7/MACF1 and of human Gas2-like1/hGAR22 and Gas2-like2/hGAR17 all display MT-stabilizing properties that are dependent on their GRDs, and in each case the Ctails enhance their MT association. Therefore, this mechanistic principle appears conserved across a range of homologous proteins, and this work has provided first experimental proof that it is functionally relevant in vivo, such as in growing axons and tendon cells (Alves-Silva, 2012).

Despite their obvious functional conservation, Ctails are not conserved at the protein sequence level but display other commonalities instead. They are all unlikely to form an ordered secondary or tertiary structure, they all display a high content of arginines, serines and glycines, and most contain MtLS motifs. This study has identified a role for the arginines of Ctail and proposes that this positive net charge attracts Ctail to negatively charged MTs. Such a mechanisms is consistent with other models for MT association and can explain why Ctails are not conserved at the amino acid sequence level. In support of this model, the C terminus of mouse ACF7 has recently been demonstrated to detach from MTs, when negative surface charges of MTs were enzymatically removed (Alves-Silva, 2012).

The second subcellular role of Shot in MT guidance establishes EB1 and the EB1-Shot complex as important determinants of axonal growth. In the absence of Shot-EB1 complex function, MTs are disorganized and axons extend shorter. Similar phenotypes of curled, criss-crossed MTs correlating with axon shortening have been described in mammalian neurons lacking ACF7 function but also defective for Dynein/Lis1, GSK3 (as a regulator of APC downstream of Wnt3a/Dvl1 and of CLASP), or Spinophilin/Doublecortin (see Drosophila Spinophilin). The favored explanation for why MT disorganization attenuates axon growth is that MTs are less efficient in pushing along the axonal axis in the direction of axon growth. Notably, it has been shown previously that roles of Shot in MT guidance and axon growth also essentially require its linkage to actin. It is therefore proposed that Shot performs its MT guiding roles by linking MT plus ends to actin structures, such as the actin cortex in the axons or actin networks or bundles in growth cones. Such a function of spectraplakins is likely to coexist with parallel mechanisms of actin-MT linkage. For example, the F-actin-binding factor drebrin was shown to interact with EB3, and this interaction is likewise believed to be required to steer MT polymerization events in elongating axons (Alves-Silva, 2012).

Current models propose that +TIPs compete among each other for interaction with EB1 at MT plus ends. Other +TIPs, such as CLASP, APC, Dynein/Lis and CLIPs, are present in neurons and contribute to axonal growth regulation. Therefore, the +TIP functions of spectraplakins identified in this study can now be analyzed in the context of other +TIPs, providing new opportunities to gain an understanding of regulatory +TIP networks in the context of axonal growth (Alves-Silva, 2012).

Shot works at the cross-roads of different mechanisms of MT regulation, and this study has unraveled the underpinning mechanisms using a genetically and experimentally amenable and functionally well conserved Drosophila model of axon growth. These findings do not only advance the principal understanding of cytoskeletal regulation during axonal growth, but can be extrapolated to other functions of spectraplakins. Thus, spectraplakins play roles in clinically relevant cellular processes including neurodegeneration, skin blistering and cell migration in wound healing and brain development. The two modes of Shot function proposed in this study may very well be applicable. For example, the model for MT guidance displays interesting commonalities with models for ACF7 function in migrating keratinocytes during wound healing, where ACF7 is suggested to guide MTs along actin stress fibers to focal adhesions. Furthermore, MT-stabilizing roles are likely to explain Shot function in Drosophila tendon cells. Just like MT-stabilizing roles of Shot in axons and fibroblasts, GRD and Ctail are essential in tendon cells, where they enhance each others localization, whereas MtLS motifs and actin-binding calponin-homology domains of Shot are dispensable. Notably, tendon cells have been proposed as a cellular model for support cells of the vertebrate inner ear that are known to express the Shot homolog BPAG1. Therefore, the subcellular mechanisms of spectraplakins described in this study do not only have implications for the understanding of axonal growth but also for their other functions in neurons and non-neuronal cells (Alves-Silva, 2012).

Nesprin provides elastic properties to muscle nuclei by cooperating with spectraplakin and EB1

Muscle nuclei are exposed to variable cytoplasmic strain produced by muscle contraction and relaxation, but their morphology remains stable. Still, the mechanism responsible for maintaining myonuclear architecture, and its importance, is currently elusive. This study uncovered a unique myonuclear scaffold in Drosophila melanogaster larval muscles, exhibiting both elastic features contributed by the stretching capacity of MSP300 (nesprin) and rigidity provided by a perinuclear network of microtubules stabilized by Shot (spectraplakin) and EB1. Together, they form a flexible perinuclear shield that protects myonuclei from intrinsic or extrinsic forces. The loss of this scaffold resulted in significantly aberrant nuclear morphology and subsequently reduced levels of essential nuclear factors such as lamin A/C, lamin B, and HP1. Overall, a novel mechanism is proposed for maintaining myonuclear morphology, and its critical link to correct levels of nuclear factors in differentiated muscle fibers is revealed. These findings may shed light on the underlying mechanism of various muscular dystrophies (Wang, 2015).

Eb1: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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