TIP150 interacts with and targets MCAK at the microtubule plus ends

The microtubule (MT) cytoskeleton orchestrates the cellular plasticity and dynamics that underlie morphogenesis and cell division. Growing MT plus ends have emerged as dynamic regulatory machineries in which specialized proteins—called plus-end tracking proteins (+TIPs)—bind to and control the plus-end dynamics that are essential for cell division and migration. However, the molecular mechanisms underlying the plus-end regulation by +TIPs at spindle and astral MTs have remained elusive. Here, we show that TIP150 is a new +TIP that binds to end-binding protein 1 (EB1) in vitro and co-localizes with EB1 at the MT plus ends in vivo. Suppression of EB1 eliminates the plus-end localization of TIP150. Interestingly, TIP150 also binds to mitotic centromere-associated kinesin (MCAK), an MT depolymerase that localizes to the plus end of MTs. Suppression of TIP150 diminishes the plus-end localization of MCAK. Importantly, aurora B-mediated phosphorylation disrupts the TIP150–MCAK association in vitro. We reason that TIP150 facilitates the EB1-dependent loading of MCAK onto MT plus ends and orchestrates the dynamics at the plus end of MTs.     

Microtubule-binding sites of the CH domain of EB1 and its autoinhibition revealed by NMR

End-binding protein 1 (EB1) is one of the best studied plus-end tracking proteins. It is known that EB1 specifically binds the plus ends of microtubules (MTs) and promotes MT growth. EB1 activity is thought to be autoinhibited by an intramolecular interaction. Recent cryo-EM analyses showed that the CH domain of Mal3p (Schizosaccharomyces pombe EB1 homolog) binds to GMPCPP-MT (Sandblad, L. Cell 127 (2006) 1415-24), and strongly binds GTPγS-MT which is proposed to mimic MT plus ends better than GMPCPP-MT (Maurer S.P. et al. Cell 149 (2012) 371–82). Here, we report on the MT binding sites of the CH domain of EB1 as revealed by NMR using the transferred cross-saturation method. In this study, we used GMPCPP-MT and found that the MT binding sites are very similar to the binding site for GTPγS-MT as suggested by cryo-EM (Maurer S.P. et al. Cell 149 (2012) 371–82). Notably, the N-terminal tip of helix α6 of the CH domain did not make contact with GMPCPP-MT, in contrast to the cryo-EM study which showed that it is closely located to a putative switch region of β-tubulin in GTPγS-MT (Maurer S.P. et al. Cell 149 (2012) 371-82). Further, we found that the intramolecular interaction site of EB1 overlaps the MT binding sites, indicating that the MT binding sites are masked by interaction with the C-terminal domain. We propose a structural view of autoinhibition and its release mechanism through competition binding with binding partners such as adenomatous polyposis coli protein.     

CLASP1 and CLASP2 bind to EB1 and regulate microtubule plus-end dynamics at the cell cortex

CLIP-associating protein (CLASP) 1 and CLASP2 are mammalian microtubule (MT) plus-end binding proteins, which associate with CLIP-170 and CLIP-115. Using RNA interference in HeLa cells, we show that the two CLASPs play redundant roles in regulating the density, length distribution and stability of interphase MTs. In HeLa cells, both CLASPs concentrate on the distal MT ends in a narrow region at the cell margin. CLASPs stabilize MTs by promoting pauses and restricting MT growth and shortening episodes to this peripheral cell region. We demonstrate that the middle part of CLASPs binds directly to EB1 and to MTs. Furthermore, we show that the association of CLASP2 with the cell cortex is MT independent and relies on its COOH-terminal domain. Both EB1- and cortex-binding domains of CLASP are required to promote MT stability. We propose that CLASPs can mediate interactions between MT plus ends and the cell cortex and act as local rescue factors, possibly through forming a complex with EB1 at MT tips.     

Interaction between EB1 and p150glued is required for anaphase astral microtubule elongation and stimulation of cytokinesis

In animal cells, microtubules (MTs) of the mitotic apparatus (MA) communicate with the cell cortex to stimulate cytokinesis; however, the molecular nature of this stimulus remains elusive . A signal for cytokinesis likely involves the MT plus end binding family of proteins, which includes EB1, p150glued, APC, LIS1, and CLIP-170. These proteins modulate MT dynamics and facilitate interactions between growing MTs and their intracellular targets, including kinetochores, organelles, and the cell cortex . The dynein-dynactin complex mediates many of these microtubule capture events . We report that EB1 and p150glued interactions are required for stimulation of cytokinesis in dividing sea urchin eggs. Injected antibodies against EB1 or p150glued suppressed furrow ingression but did not prevent elongation of anaphase astral MTs toward the cortex, suggesting that EB1 and dynactin are both required for communication between the MA and the cortex. Targeted disruption of the interaction between EB1 and p150glued suppressed anaphase astral MT elongation and resulted in a delay of cytokinesis that could not be overcome by manipulation of the asters toward the cortex. We conclude that EB1 and dynactin participate in stimulation of the cleavage furrow, and their interaction promotes elongation of astral MTs at anaphase onset.     

EB1 is required for primary cilia assembly in fibroblasts

EB1 is a small microtubule (MT)-binding protein that associates preferentially with MT plus ends and plays a role in regulating MT dynamics. EB1 also targets other MT-associated proteins to the plus end and thereby regulates interactions of MTs with the cell cortex, mitotic kinetochores, and different cellular organelles [1, 2]. EB1 also localizes to centrosomes and is required for centrosomal MT anchoring and organization of the MT network [3, 4]. We previously showed that EB1 localizes to the flagellar tip and proximal region of the basal body in Chlamydomonas[5], but the function of EB1 in the cilium/flagellum is unknown. We depleted EB1 from NIH3T3 fibroblasts by using siRNA and found that EB1 depletion causes a approximately 50% reduction in the efficiency of primary cilia assembly in serum-starved cells. Expression of dominant-negative EB1 also inhibited cilia formation, and expression of mutant dominant-negative EB1 constructs suggested that binding of EB1 to p150(Glued) is important for cilia assembly. Finally, expression of a C-terminal fragment of the centrosomal protein CAP350, which removes EB1 from the centrosome but not MT plus ends [6], also inhibited ciliogenesis. We conclude that localization of EB1 at the centriole/basal body is required for primary cilia assembly in fibroblasts.     

Adenomatous polyposis coli on microtubule plus ends in cell extensions can promote microtubule net growth with or without EB1

In interphase cells, the adenomatous polyposis coli (APC) protein accumulates on a small subset of microtubules (MTs) in cell protrusions, suggesting that APC may regulate the dynamics of these MTs. We comicroinjected a nonperturbing fluorescently labeled monoclonal antibody and labeled tubulin to simultaneously visualize dynamics of endogenous APC and MTs in living cells. MTs decorated with APC spent more time growing and had a decreased catastrophe frequency compared with non-APC-decorated MTs. Endogenous APC associated briefly with shortening MTs. To determine the relationship between APC and its binding partner EB1, we monitored EB1-green fluorescent protein and endogenous APC concomitantly in living cells. Only a small fraction of EB1 colocalized with APC at any one time. APC-deficient cells and EB1 small interfering RNA showed that EB1 and APC localized at MT ends independently. Depletion of EB1 did not change the growth-stabilizing effects of APC on MT plus ends. In addition, APC remained bound to MTs stabilized with low nocodazole, whereas EB1 did not. Thus, we demonstrate that the association of endogenous APC with MT ends correlates directly with their increased growth stability, that this can occur independently of its association with EB1, and that APC and EB1 can associate with MT plus ends by distinct mechanisms.     

Microtubule plus end: a hub of cellular activities

Microtubules (MTs) are highly dynamic polymers, which control many aspects of cellular architecture. Growing MT plus ends accumulate a specific set of evolutionary conserved factors, the so-called MT plus-end-tracking proteins (+TIPs). +TIPs regulate MT dynamics and the reciprocal interactions of MTs with the cell cortex, mitotic kinetochores or different cellular organelles. Most +TIPs can directly bind to MTs, but the molecular mechanisms of their specific targeting to the growing plus ends remain poorly understood. Recent studies suggest that the members of one particular +TIP family, EB1 and its homologues, are present in all eucaryotic kingdoms, interact directly with the majority of other known plus-end-associated proteins and may be responsible for their specific accumulation at the MT tips.     

Cytoplasmic linker proteins promote microtubule rescue in vivo

The role of plus end-tracking proteins in regulating microtubule (MT) dynamics was investigated by expressing a dominant negative mutant that removed endogenous cytoplasmic linker proteins (CLIPs) from MT plus ends. In control CHO cells, MTs exhibited asymmetric behavior: MTs persistently grew toward the plasma membrane and displayed frequent fluctuations of length near the cell periphery. In the absence of CLIPs, the microtubule rescue frequency was reduced by sevenfold. MT behavior became symmetrical, consisting of persistent growth and persistent shortening. Removal of CLIPs also caused loss of p150Glued but not CLIP-associating protein (CLASP2) or EB1. This result raised the possibility that the change in dynamics was a result of the loss of either CLIPs or p150Glued. To distinguish between these possibilities, we performed rescue experiments. Normal MT dynamics were restored by expression of the CLIP-170 head domain, but p150Glued was not recruited back to MT plus ends. Expression of p150Glued head domain only partially restored MT dynamics. We conclude that the CLIP head domain is sufficient to alter MT dynamics either by itself serving as a rescue factor or indirectly by recruiting a rescue factor. By promoting a high rescue frequency, CLIPs provide a mechanism by which MT plus ends may be concentrated near the cell margin.     

Interactions between EB1 and Microtubules: DRAMATIC EFFECT OF AFFINITY TAGS AND EVIDENCE FOR COOPERATIVE BEHAVIOR*

Plus end tracking proteins (+TIPs) are a unique group of microtubule binding proteins that dynamically track microtubule (MT) plus ends. EB1 is a highly conserved +TIP with a fundamental role in MT dynamics, but it remains poorly understood in part because reported EB1 activities have differed considerably. One reason for this inconsistency could be the variable presence of affinity tags used for EB1 purification. To address this question and establish the activity of native EB1, we have measured the MT binding and tubulin polymerization activities of untagged EB1 and EB1 fragments and compared them with those of His-tagged EB1 proteins. We found that N-terminal His tags directly influence the interaction between EB1 and MTs, significantly increasing both affinity and activity, and that small amounts of His-tagged proteins act synergistically with larger amounts of untagged proteins. Moreover, the binding ratio between EB1 and tubulin can exceed 1:1, and EB1-MT binding curves do not fit simple binding models. These observations demonstrate that EB1 binding is not limited to the MT seam, and they suggest that EB1 binds cooperatively to MTs. Finally, we found that removal of tubulin C-terminal tails significantly reduces EB1 binding, indicating that EB1-tubulin interactions are mediated in part by the same tubulin acidic tails utilized by other MAPs. These binding relationships are important for helping to elucidate the complex of proteins at the MT tip.     

Microtubule dynamics and signal transduction at the immunological synapse: new partners and new connections

EMBO J (2012) 31 21, 4140–4152 doi:10.1038/emboj.2012.242; published online August242012Antigen recognition induces T cells to polarize towards antigen presenting cells (APC) generating an organized cell interface named the immunological synapse. T-cell microtubules (MTs) reorient the MT-organizing centre (MTOC) to the immunological synapse central region, while MT irradiate towards the synapse periphery. Martín-Cófreces et al (2012) describe in this issue that the MT plus-end-binding protein 1 (EB1) interacts with TCR cytosolic regions and mediate the organization of an immunological synapse fully functional to transduce activation signals.The pioneer work of Kupfer and Singer (1989) established that T-cell MTs rearrange in response to specific TCR engagement by APCs, resulting in MTOC orientation to the APC contact site in helper and cytotoxic T cells. MTOC reorientation was shown to be the result of a MT polymerization dynamic process involving MT posttranslational modifications (Kuhn and Poenie, 2002; Serrador et al, 2004). MT reorganization during T-cell antigen recognition is functionally linked to T-cell effector functions, like the polarized secretion of helper cytokines to B cells (Kupfer et al, 1991; Huse et al, 2006), or cytotoxic granules to target cells (Stinchcombe et al, 2006). MTs also transport TCR-carrying endosomes during synapse formation (Das et al, 2004) and TCR signalling complexes at the immunological synapse (Lasserre et al, 2010; Hashimoto-Tane et al, 2011). Altogether, these findings show that the dynamic reorganization of MTs and its related molecular transport are critical for the organization and function of the immunological synapse.Martín-Cófreces et al (2012) present here interesting new insights, unveiling a link between EB1 and the TCR complex. EB1 is one of a series of MT plus-end-associated proteins critical for MT polymerization dynamics (Slep, 2010). The first important finding initially issued from a two-hybrid screening was that EB1 could directly interact with TCR complex cytosolic regions. By GST pull-down and co-immunoprecipitation experiments, the authors narrowed down this interaction to two of the TCR complex subunits, ζ and ɛ, in their ITAM (immuno-receptor tyrosine-based activation motif)-containing regions, and within the C-terminal 82 amino-acid region on EB1. In T cells, EB1–TCR interaction could occur without TCR stimulation, suggesting that EB1 plays a role in TCR dynamics previous to TCR engagement. The authors then investigated EB1 localization and its involvement in synapse organization and function. Live cell imaging showed intense EB1 movement in the synapse area, with MTs growing from the MTOC to the synapse periphery, leading to an apparent concentration of EB1 at the T cell–APC interface. To analyse the relationship between MT dynamics and intracellular transport, the authors followed EB1–GFP and TCRζ–Cherry by total internal reflection fluorescence (TIRF) microscopy in synapses formed on anti-CD3-coated cover slips. They observed transient coincident spots between EB1 and TCRζ+ vesicles, suggesting that growing MTs transport TCRζ-carrying vesicles towards the immunological synapse. Consistently, EB1-silenced cells displayed altered TCRζ vesicle dynamics and TCRζ clustering at the synapse. Likewise, vesicle transport to the synapse of the signalling scaffold molecule LAT and its clustering at the synapse were altered. Finally, they observed transient encounters between TCRζ- and LAT-carrying vesicles inhibited by EB1 silencing. These observations point out to a crucial role of EB1 and MT dynamics in the organization of the immunological synapse.Immunological synapse organization has been related with its capacity to regulate TCR signal transduction. Therefore, Martín-Cófreces et al (2012) investigated how EB1 silencing impacted TCR signalling. EB1-silenced cells were indeed impaired in key TCR signalling events, like LAT tyrosine phosphorylation, which allows LAT interaction with activation effectors, like the phospholipase C (PLC)γ, promoting TCR signal propagation. Consistently, PLCγ activation was impaired in EB1-silenced cells. However, upstream activation events, like tyrosine phosphorylation of TCRζ and of its associated protein tyrosine kinase ZAP70, were not altered. This suggests that MT-dependent LAT vesicle traffic is key for LAT phosphorylation and the generation of TCR signalling complexes.Altogether, Martín-Cófreces'' findings reinforce the idea that polarized vesicle transport via organized MT networks is key to set up the immunological synapse as a signal transduction platform. EB1 interaction with two TCR subunits may link the TCR complex with MTs dynamics. It remains unanswered, however, whether EB1 also interacts with LAT, facilitating the merging at the synapse of distinct TCRζ- and LAT-carrying vesicles.Vesicle traffic on MTs generally occurs via molecular motors from the dynein and kinesin families. The former are associated with minus end-oriented transport, whereas the later mostly ensures plus-end-associated transport. The immunological synapse may use both types of transport. Thus, cytotoxic granule delivery to the synapse may mainly involve dynein-mediated vesicle traffic, since the MTOC translocates very close to the immunological synapse (Stinchcombe et al, 2006). Likewise, centripetal movements of signalling microclusters at the synapse involve dynein (Hashimoto-Tane et al, 2011). Martín-Cófreces et al (2012) show that TCRζ- and LAT-carrying vesicles are transported towards MT plus ends in an EB1-dependent manner. It remains uncertain whether EB1 could play a direct transport role at the immunological synapse, helping the attachment of TCRζ vesicles to growing MT plus ends. Alternatively, EB1 could mediate MT interactions with TCR complexes present at the plasma membrane. Initial TCR clustering at the synapse would help capturing EB1-positive MT plus ends, orienting MTs and MT-mediated traffic of TCRζ- and LAT-carrying vesicles to the synapse by a kinesin-based transport (Figure 1), and promoting TCRζ and LAT encountering and clustering at the synapse. EB1 silencing would perturb MT–plasma membrane interactions impairing this MT orientation and transport loop. MT polymerization kinetic studies on immunological synapses formed by EB1-silenced versus control T cells may help to clarify this mechanism. Although further studies will be necessary to elucidate the detailed mechanism, the work by Martín-Cófreces et al (2012) already highlights the importance of MT dynamics and vesicle traffic in the formation of a functional immunological synapse, raising novel and interesting questions on how the MT network helps to set up complex signal transduction machineries.Open in a separate windowFigure 1Model of the role of EB1 in MT dynamics and TCR signal transduction at the immunological synapse. (A) Initial T cell–APC contact. TCR initial clustering would favour the capture of EB1-containing MT plus ends at the T cell–APC contact. (B) Immune synapse formation. The increase capture of MTs plus ends by TCR clusters would promote the arrival of TCRζ- and LAT-carrying vesicles leading to increase TCR and LAT clustering and encountering at the synapse. Alternatively, EB1 interaction with TCR could also be directly involved in TCRζ vesicle transport to the synapse. In turn, increase TCR clustering would promote additional MT and capture, building an amplification loop for MT dynamics and vesicle transport. (C) Established immunological synapse. A structured MT network would facilitate the continuous arrival of TCRζ- and LAT-carrying vesicles through the MT plus ends at the immunological synapse periphery. Then the centripetal movement of TCR signalling complexes towards the MT minus end at the MTOC close to the synapse centre would bring signalling complexes to signal extinction sites (i.e., endosomes). The right panel in C represents a xy section of the immunological synapse, as it is observed on stimulatory cover slips.     

Dynamic behavior of GFP-CLIP-170 reveals fast protein turnover on microtubule plus ends

Microtubule (MT) plus end-tracking proteins (+TIPs) specifically recognize the ends of growing MTs. +TIPs are involved in diverse cellular processes such as cell division, cell migration, and cell polarity. Although +TIP tracking is important for these processes, the mechanisms underlying plus end specificity of mammalian +TIPs are not completely understood. Cytoplasmic linker protein 170 (CLIP-170), the prototype +TIP, was proposed to bind to MT ends with high affinity, possibly by copolymerization with tubulin, and to dissociate seconds later. However, using fluorescence-based approaches, we show that two +TIPs, CLIP-170 and end-binding protein 3 (EB3), turn over rapidly on MT ends. Diffusion of CLIP-170 and EB3 appears to be rate limiting for their binding to MT plus ends. We also report that the ends of growing MTs contain a surplus of sites to which CLIP-170 binds with relatively low affinity. We propose that the observed loss of fluorescent +TIPs at plus ends does not reflect the behavior of single molecules but is a result of overall structural changes of the MT end.     

Microtubule length and dynamics: Boundary effect and properties of extended radial array

In interphase cells, microtubules (MT) form an extended radial array. The length of individual MTs in living cells exhibits substantial stochastic fluctuations, while the average length distribution in a cell remains nearly constant. We present a quantitative model that describes the relation of the MT length and dynamics in the steady state in the cell using the minimal set of parameters (cell radius, tubulin concentration, critical concentration for plus-end elongation and the number of nucleation sites). The MT array is approximated as a radial system, where minus-ends of MTs are associated with nucleation sites on the centrosome, while plus ends grow and shorten. Dynamic instability of MT plus ends is approximated as a random walk process with boundary conditions; the behavior of an MT array is quantified using diffusion and drift coefficients (Vorobjev et al., 1997; Vorobjev et al., 1999). We show that the establishment of the extended steady-state array could be accomplished solely by the limitation of MT growth by the cell margin. For the cell radius, tubulin concentration, critical concentration for plus-end elongation, and the number of nucleation sites we determined the reference point in the parameter space where plus ends of individual MTs, on average, neither elongate nor shorten. In this case, the average MT length is equal to the half of the cell radius. When any parameter is shifted from its reference value, MTs become longer or shorter and, consequently, acquire a positive or negative drift of their plus ends. In the vicinity of the reference point, a change in any parameter has a major effect on the MT length and a rather small effect on the drift. When the average MT length is close to the cell radius, the drift of free plus ends becomes substantial, resulting in processive growth of individual MTs in the internal cytoplasm, accompanied by the apparent stabilization of plus ends at the cell margin. Under these conditions small changes in parameters have a significant impact on the magnitude of the drift. Experimental analysis of MT plus-end dynamics in different cultured cells shows that, in most cases, plus ends display positive drift, which, in the framework of the presented model, is in agreement with the simultaneous presence of long MTs.     

TTBK2 with EB1/3 regulates microtubule dynamics in migrating cells through KIF2A phosphorylation

Microtubules (MTs) play critical roles in various cellular events, including cell migration. End-binding proteins (EBs) accumulate at the ends of growing MTs and regulate MT end dynamics by recruiting other plus end–tracking proteins (+TIPs). However, how EBs contribute to MT dynamics through +TIPs remains elusive. We focused on tau-tubulin kinase 2 (TTBK2) as an EB1/3-binding kinase and confirmed that TTBK2 acted as a +TIP. We identified MT-depolymerizing kinesin KIF2A as a novel substrate of TTBK2. TTBK2 phosphorylated KIF2A at S135 in intact cells in an EB1/3-dependent fashion and inactivated its MT-depolymerizing activity in vitro. TTBK2 depletion reduced MT lifetime (facilitated shrinkage and suppressed rescue) and impaired HeLa cell migration, and these phenotypes were partially restored by KIF2A co-depletion. Expression of nonphosphorylatable KIF2A, but not wild-type KIF2A, reduced MT lifetime and slowed down the cell migration. These findings indicate that TTBK2 with EB1/3 phosphorylates KIF2A and antagonizes KIF2A-induced depolymerization at MT plus ends for cell migration.     

EBs clip CLIPs to growing microtubule ends

Proteins that track growing microtubule (MT) ends are important for many aspects of intracellular MT function, but the mechanism by which these +TIPs accumulate at MT ends has been the subject of a long-standing controversy. In this issue, Bieling et al. (Bieling, P., S. Kandels-Lewis, I.A. Telley, J. van Dijk, C. Janke, and T. Surrey. 2008. J. Cell Biol. 183:1223–1233) reconstitute plus end tracking of EB1 and CLIP-170 in vitro, which demonstrates that CLIP-170 plus end tracking is EB1-dependent and that both +TIPs rapidly exchange between a soluble and a plus end–associated pool. This strongly supports the hypothesis that plus end tracking depends on a biochemical property of growing MT ends, and that the characteristic +TIP comets result from the generation of new +TIP binding sites through MT polymerization in combination with the exponential decay of these binding sites.     

Microtubule Dynamic Instability Controls Podosome Patterning in Osteoclasts through EB1, Cortactin,and Src

In osteoclasts (OCs) podosomes are organized in a belt, a feature critical for bone resorption. Although microtubules (MTs) promote the formation and stability of the belt, the MT and/or podosome molecules that mediate the interaction of the two systems are not identified. Because the growing “plus” ends of MTs point toward the podosome belt, plus-end tracking proteins (+TIPs) might regulate podosome patterning. Among the +TIPs, EB1 increased as OCs matured and was enriched in the podosome belt, and EB1-positive MTs targeted podosomes. Suppression of MT dynamic instability, displacement of EB1 from MT ends, or EB1 depletion resulted in the loss of the podosome belt. We identified cortactin as an Src-dependent interacting partner of EB1. Cortactin-deficient OCs presented a defective MT targeting to, and patterning of, podosomes and reduced bone resorption. Suppression of MT dynamic instability or EB1 depletion increased cortactin phosphorylation, decreasing its acetylation and affecting its interaction with EB1. Thus, dynamic MTs and podosomes interact to control bone resorption.     

MAP1B regulates microtubule dynamics by sequestering EB1/3 in the cytosol of developing neuronal cells

MAP1B, a structural microtubule (MT)‐associated protein highly expressed in developing neurons, plays a key role in neurite and axon extension. However, not all molecular mechanisms by which MAP1B controls MT dynamics during these processes have been revealed. Here, we show that MAP1B interacts directly with EB1 and EB3 (EBs), two core ‘microtubule plus‐end tracking proteins’ (+TIPs), and sequesters them in the cytosol of developing neuronal cells. MAP1B overexpression reduces EBs binding to plus‐ends, whereas MAP1B downregulation increases binding of EBs to MTs. These alterations in EBs behaviour lead to changes in MT dynamics, in particular overstabilization and looping, in growth cones of MAP1B‐deficient neurons. This contributes to growth cone remodelling and a delay in axon outgrowth. Together, our findings define a new and crucial role of MAP1B as a direct regulator of EBs function and MT dynamics during neurite and axon extension. Our data provide a new layer of MT regulation: a classical MAP, which binds to the MT lattice and not to the end, controls effective concentration of core +TIPs thereby regulating MTs at their plus‐ends.     

Kif18B interacts with EB1 and controls astral microtubule length during mitosis

Regulation of microtubule (MT) dynamics is essential for proper spindle assembly and organization. Kinesin-8 family members are plus-end-directed motors that modulate plus-end MT dynamics by acting as MT depolymerases or as MT plus-end capping proteins. In this paper, we show that the human kinesin-8 Kif18B functions during mitosis to control astral MT organization. Kif18B is a MT plus-tip-tracking protein that localizes to the nucleus in interphase and is enriched at astral MT plus ends during early mitosis. Knockdown of Kif18B caused spindle defects, resulting in an increased number and length of MTs. A yeast two-hybrid screen identified an interaction of the C-terminal domain of Kif18B with the plus-end MT-binding protein EB1. EB1 knockdown disrupted Kif18B targeting to MT plus ends, indicating that EB1/Kif18B interaction is physiologically important. This interaction is direct, as the far C-terminal end of Kif18B is sufficient for binding to EB1 in vitro. Overexpression of this domain is sufficient for plus-end MT targeting in cells; however, targeting is enhanced by the motor domain, which cooperates with the tail to achieve proper Kif18B localization at MT plus ends. Our results suggest that Kif18B is a new MT dynamics regulatory protein that interacts with EB1 to control astral MT length.     

Stabilization of overlapping microtubules by fission yeast CLASP

Many microtubule (MT) structures contain dynamic MTs that are bundled and stabilized in overlapping arrays. CLASPs are conserved MT-binding proteins implicated in the regulation of MT plus ends. Here, we show that the Schizosaccharomyces pombe CLASP, cls1p/peg1p, mediates the stabilization of overlapping MTs within the mitotic spindle and interphase bundles. cls1p localizes to these regions but not to interphase MT plus ends. Inactivation of cls1p leads to the rapid depolymerization of spindle midzone MTs. cls1p also stabilizes a subset of MTs within interphase bundles. cls1p prevents disassembly of the entire microtubule, while still allowing for plus-end growth. It has no measurable effects on MT nucleation, polymerization, catastrophe, or bundling. A direct interaction with ase1p (PRC1/MAP65) targets cls1p to regions of antiparallel MT overlap. These findings show how a MT-stabilizing factor attached to specific sites on MTs can help to generate MT structures that have both dynamic and stable components.     

On the relation of the microtubule length and dynamics: boundary effect and properties of an extended radial array

In interphase cells, microtubules (MT) are long and form extended radial array. The length of individual MTs in living cells exhibits substantial stochastic fluctuations while the average length distribution in a cell remains nearly constant. We present a quantitative model that describes relation of the MT length and dynamics in the steady state in the cell using the minimal set of parameters (cell radius, tubulin concentration, critical concentration for plus end elongation, and the number of nucleation sites). The MT array is approximated as a radial system, where MT minus ends are associated with the nucleation sites on the centrosome, while plus ends grow and shorten. Dynamic instability of MT plus ends is approximated as a random walk process with boundary conditions and the behavior of MT array is quantified using diffusion and drift coefficients (Vorobjev et al., 1997, 1999). We show that establishment of the extended steady-state array could be accomplished solely by the limitation of the MT growth by the cell margin. We determined for the cell radius, tubulin concentration, critical concentration for plus end elongation, and number of nucleation sites the reference point in the parameter space where plus ends of individual MT on average neither elongate nor shorten. In this case average length of MT is equal to the half of cell radius. When any parameter is shifted from its reference value MTs become longer or shorter and consequently acquire positive or negative drift of their ends. In the vicinity of reference point, change in any parameter has major effect on the MT length and rather small effect on the drift. When mean length of the MTs is close to the cell radius the drift of the free plus ends becomes substantial, resulting in processive growth of individual MTs in the internal cytoplasm accompanied by apparent stabilization of the plus ends at the cell margin. Under these conditions small changes in parameters have significant impact on the magnitude of drift. Experimental analysis of the MT plus ends dynamics in different cultured cells shows that in most cases plus ends display positive drift, which, in the framework of the presented model, is in agreement with the simultaneous presence of long MTs.     

Phosphoregulation of the budding yeast EB1 homologue Bim1p by Aurora/Ipl1p

EB1 (end binding 1) proteins have emerged as central regulators of microtubule (MT) plus ends in all eukaryotes, but molecular mechanisms controlling the activity of these proteins are poorly understood. In this study, we show that the budding yeast EB1 protein Bim1p is regulated by Aurora B/Ipl1p-mediated multisite phosphorylation. Bim1p forms a stable complex with Ipl1p and is phosphorylated on a cluster of six Ser residues in the flexible linker connecting the calponin homology (CH) and EB1 domains. Using reconstitution of plus end tracking in vitro and total internal reflection fluorescence microscopy, we show that dimerization of Bim1p and the presence of the linker domain are both required for efficient tip tracking and that linker phosphorylation removes Bim1p from static and dynamic MTs. Bim1 phosphorylation occurs during anaphase in vivo, and it is required for normal spindle elongation kinetics and an efficient disassembly of the spindle midzone. Our results define a mechanism for the use and regulation of CH domains in an EB1 protein.