A plus-end raft to control microtubule dynamics and function

Cells require a properly oriented and organised microtubule array to transmit positional information. Recent data have revealed a heterogeneous population of microtubule-binding proteins that accumulates mainly at distal ends of polymerising microtubules. Two mechanisms may account for this concentration: transient immobilisation, which involves association of proteins with growing ends, followed by release more proximally; and deposition at ends via a molecular motor. As with lipid rafts, protein concentration at distal ends may allow a cascade of interactions in the restricted area of a microtubule plus end. This may, in turn, control the dynamic behaviour of this cytoskeletal network and its anchoring to other structures.     

Drosophila katanin is a microtubule depolymerase that regulates cortical-microtubule plus-end interactions and cell migration

Regulation of microtubule dynamics at the cell cortex is important for cell motility, morphogenesis and division. Here we show that the Drosophila katanin Dm-Kat60 functions to generate a dynamic cortical-microtubule interface in interphase cells. Dm-Kat60 concentrates at the cell cortex of S2 Drosophila cells during interphase, where it suppresses the polymerization of microtubule plus-ends, thereby preventing the formation of aberrantly dense cortical arrays. Dm-Kat60 also localizes at the leading edge of migratory D17 Drosophila cells and negatively regulates multiple parameters of their motility. Finally, in vitro, Dm-Kat60 severs and depolymerizes microtubules from their ends. On the basis of these data, we propose that Dm-Kat60 removes tubulin from microtubule lattice or microtubule ends that contact specific cortical sites to prevent stable and/or lateral attachments. The asymmetric distribution of such an activity could help generate regional variations in microtubule behaviours involved in cell migration.     

The microtubule plus-end tracking protein Bik1 is required for chromosome congression

During mitosis, sister chromatids congress on both sides of the spindle equator to facilitate the correct partitioning of the genomic material. Chromosome congression requires a finely tuned control of microtubule dynamics by the kinesin motor proteins. In Saccharomyces cerevisiae, the kinesin proteins Cin8, Kip1, and Kip3 have a pivotal role in chromosome congression. It has been hypothesized that additional proteins that modulate microtubule dynamics are involved. Here, we show that the microtubule plus-end tracking protein Bik1—the budding yeast ortholog of CLIP-170—is essential for chromosome congression. We find that nuclear Bik1 localizes to the kinetochores in a cell cycle–dependent manner. Disrupting the nuclear pool of Bik1 with a nuclear export signal (Bik1-NES) leads to slower cell-cycle progression characterized by a delayed metaphase–anaphase transition. Bik1-NES cells have mispositioned kinetochores along the spindle in metaphase. Furthermore, using proximity-dependent methods, we identify Cin8 as an interaction partner of Bik1. Deleting CIN8 reduces the amount of Bik1 at the spindle. In contrast, Cin8 retains its typical bilobed distribution in the Bik1-NES mutant and does not localize to the unclustered kinetochores. We propose that Bik1 functions with Cin8 to regulate kinetochore–microtubule dynamics for correct kinetochore positioning and chromosome congression.     

The regulation of microtubule dynamics in Saccharomyces cerevisiae by three interacting plus-end tracking proteins

Microtubule plus-end tracking proteins (+TIPs) are a diverse group of molecules that regulate microtubule dynamics and interactions of microtubules with other cellular structures. Many +TIPs have affinity for each other but the functional significance of these associations is unclear. Here we investigate the physical and functional interactions among three +TIPs in S. cerevisiae, Stu2, Bik1, and Bim1. Two-hybrid, coimmunoprecipitation, and in vitro binding assays demonstrate that they associate in all pairwise combinations, although the interaction between Stu2 and Bim1 may be indirect. Three-hybrid assays indicate that these proteins compete for binding to each other. Thus, Stu2, Bik1, and Bim1 interact physically but do not appear to be arranged in a single unique complex. We examined the functional interactions among pairs of proteins by comparing cytoplasmic and spindle microtubule dynamics in cells lacking either one or both proteins. On cytoplasmic microtubules, Stu2 and Bim1 act cooperatively to regulate dynamics in G1 but not in preanaphase, whereas Bik1 acts independently from Stu2 and Bim1. On kinetochore microtubules, Bik1 and Bim1 are redundant for regulating dynamics, whereas Stu2 acts independently from Bik1 and Bim1. These results indicate that interactions among +TIPS can play important roles in the regulation of microtubule dynamics.     

Induction of cytokinesis is independent of precisely regulated microtubule dynamics

Astral microtubules (MTs) emanating from the mitotic apparatus (MA) during anaphase are required for stimulation of cytokinesis in eggs. We have used green fluorescent protein-labeled EB1 to observe MT dynamics during mitosis and cytokinesis in normal sea urchin eggs. Analysis of astral MT growth rates during anaphase shows that MTs contact the polar cortex earlier than the equatorial cortex after anaphase onset but that a normal cleavage furrow is not induced until contact with MTs has been achieved throughout the cortex. To assess the role of MT dynamics in initiation of cytokinesis, we used a collection of small molecule drugs to affect dynamics. Hexylene glycol resulted in rapid astral elongation due to decreased MT catastrophe and precocious furrowing. Taxol suppressed MT dynamics but did not inhibit furrow induction when the MA was manipulated toward the cortex. Urethane resulted in short, highly dynamic astral MTs with increased catastrophe that also stimulated furrowing upon being brought into proximity to the cortex. Our findings indicate that astral MT contact with the cortex is necessary for furrow initiation but that the dynamic state of astral MTs does not affect their competency to stimulate furrowing.     

MCAK-independent functions of ch-Tog/XMAP215 in microtubule plus-end dynamics

The formation of a functional bipolar mitotic spindle is essential for genetic integrity. In human cells, the microtubule polymerase XMAP215/ch-Tog ensures spindle bipolarity by counteracting the activity of the microtubule-depolymerizing kinesin XKCM1/MCAK. Their antagonistic effects on microtubule polymerization confer dynamic instability on microtubules assembled in cell-free systems. It is, however, unclear if a similar interplay governs microtubule behavior in mammalian cells in vivo. Using real-time analysis of spindle assembly, we found that ch-Tog is required to produce or maintain long centrosomal microtubules after nuclear-envelope breakdown. In the absence of ch-Tog, microtubule assembly at centrosomes was impaired and microtubules were nondynamic. Interkinetochore distances and the lengths of kinetochore fibers were also reduced in these cells. Codepleting MCAK with ch-Tog improved kinetochore fiber length and interkinetochore separation but, surprisingly, did not rescue centrosomal microtubule assembly and microtubule dynamics. Our data therefore suggest that ch-Tog has at least two distinct roles in spindle formation. First, it protects kinetochore microtubules from depolymerization by MCAK. Second, ch-Tog plays an essential role in centrosomal microtubule assembly, a function independent of MCAK activity. Thus, the notion that the antagonistic activities of MCAK and ch-Tog determine overall microtubule stability is too simplistic to apply to human cells.     

Competence to develop sensory organs is temporally and spatially regulated in Drosophila epidermal primordia.

The Drosophila adult cuticle displays a stereotyped pattern of sensory organs (SOs). Its deployment requires the expression of the achaete (ac) and scute (sc) genes. Their products confer to cells of epidermal primordia (imaginal discs and histoblasts) the ability to become SO precursors (SOPs). In imaginal discs, ac and sc expression is spatially restricted to cell clusters within which one or a few cells become SOP(s). With the help of ubiquitous sc expression provided at different developmental times by a heat shock-sc (HSSC) chimeric gene, we have analyzed the response of epidermal primordia to the proneural action of the sc product, and have tested whether the patterned distribution of ac/sc products is necessary to position SOs correctly within the epidermis. Each primordium responds to HSSC expression by developing SOs only during a characteristic developmental period. In the absence of the endogenous ac and sc genes, most SOs induced by HSSC are of the correct type and are located in wild type positions. These results indicate that the capacity of primordia to respond to sc is temporally and spatially regulated, that specification of the type of SO does not depend on ac/sc, and that SO positioning utilizes topological information independent of the spatially restricted distribution of ac/sc products.     

Alternative splicing of the Drosophila Dscam pre-mRNA is both temporally and spatially regulated

The Drosophila melanogaster Down syndrome cell adhesion molecule (Dscam) gene encodes an axon guidance receptor that can express 38,016 different mRNAs by virtue of alternative splicing. The Dscam gene contains 95 alternative exons that are organized into four clusters of 12, 48, 33, and 2 exons each. Although numerous Dscam mRNA isoforms can be synthesized, it remains to be determined whether different Dscam isoforms are synthesized at different times in development or in different tissues. We have investigated the alternative splicing of the Dscam exon 4 cluster, which contains 12 mutually exclusive alternative exons, and found that Dscam exon 4 alternative splicing is developmentally regulated. The most highly regulated exon, 4.2, is infrequently used in early embryos but is the predominant exon 4 variant used in adults. Moreover, the developmental regulation of exon 4.2 alternative splicing is conserved in D. yakuba. In addition, different adult tissues express distinct collections of Dscam mRNA isoforms. Given the role of Dscam in neural development, these results suggest that the regulation of alternative splicing plays an important role in determining the specificity of neuronal wiring. In addition, this work provides a framework to determine the mechanisms by which complex alternative splicing events are regulated.     

Evolutionary dynamics of a spatially structured host-parasite association: Drosophila innubila and male-killing Wolbachia

The mode and tempo of host-parasite evolution depend on population structure and history and the strength of selection that the species exert on each other. Here we genetically and epidemiologically characterize populations of the mycophagous fly Drosophila innubila and its male-killing Wolbachia endosymbiont, with the aim of integrating the local through global nature of this association. Drosophila innubila inhabit the forested "sky island" regions of the of the southwestern United States and northern Mexico, where its distribution is highly fragmented. We examine geographically isolated sky island populations of D. innubila, surveying the frequency and expression of Wolbachia infection as well as the distribution of genetic variation within and among populations of the host and parasite. In all populations, Wolbachia infection is associated with virtually complete male-killing, thus providing no evidence for the evolution of population-specific interaction phenotypes or local resistance. Although Wolbachia infection occurs in each of the main populations, there is variation among populations in the prevalence of infection and the resulting population-level sex ratio of D. innubila. Among these populations, the nuclear genes of D. innubila show moderate, though significant, differentiation. In contrast, the host mitochondrial DNA (mtDNA), which shares transmission with Wolbachia, exhibits substantially greater geographic differentiation, even after accounting for differences in transmission between nuclear and mitochondrial genes. We suggest that this pattern is caused by local Wolbachia--but not D. innubila--fluctuations in prevalence that increase the severity of drift experienced only by the mtDNA. Overall, our data suggest that the association between D. innubila and male-killing Wolbachia is ecologically dynamic within local populations, but evolutionarily coherent across the species as a whole.     

The disappearance of cyclin B at the end of mitosis is regulated spatially in Drosophila cells

We have followed the behaviour of a cyclin B-green fluorescent protein (GFP) fusion protein in living Drosophila embryos in order to study how the localization and destruction of cyclin B is regulated in space and time. We show that the fusion protein accumulates at centrosomes in interphase, in the nucleus in prophase, on the mitotic spindle in prometaphase and on the microtubules that overlap in the middle of the spindle in metaphase. In cellularized embryos, toward the end of metaphase, the spindle-associated cyclin B-GFP disappears from the spindle in a wave that starts at the spindle poles and spreads to the spindle equator; when the cyclin B-GFP on the spindle is almost undetectable, the chromosomes enter anaphase, and any remaining cytoplasmic cyclin B-GFP then disappears over the next few minutes. The endogenous cyclin B protein appears to behave in a similar manner. These findings suggest that the inactivation of cyclin B is regulated spatially in Drosophila cells. We show that the anaphase-promoting complex/cyclosome (APC/C) specifically interacts with microtubules in embryo extracts, but it is not confined to the spindle in mitosis, suggesting that the spatially regulated disappearance of cyclin B may reflect the spatially regulated activation of the APC/C.     

The microtubule plus-end proteins EB1 and dynactin have differential effects on microtubule polymerization

Several microtubule-binding proteins including EB1, dynactin, APC, and CLIP-170 localize to the plus-ends of growing microtubules. Although these proteins can bind to microtubules independently, evidence for interactions among them has led to the hypothesis of a plus-end complex. Here we clarify the interaction between EB1 and dynactin and show that EB1 binds directly to the N-terminus of the p150(Glued) subunit. One function of a plus-end complex may be to regulate microtubule dynamics. Overexpression of either EB1 or p150(Glued) in cultured cells bundles microtubules, suggesting that each may enhance microtubule stability. The morphology of these bundles, however, differs dramatically, indicating that EB1 and dynactin may act in different ways. Disruption of the dynactin complex augments the bundling effect of EB1, suggesting that dynactin may regulate the effect of EB1 on microtubules. In vitro assays were performed to elucidate the effects of EB1 and p150(Glued) on microtubule polymerization, and they show that p150(Glued) has a potent microtubule nucleation effect, whereas EB1 has a potent elongation effect. Overall microtubule dynamics may result from a balance between the individual effects of plus-end proteins. Differences in the expression and regulation of plus-end proteins in different cell types may underlie previously noted differences in microtubule dynamics.     

Modulated microtubule dynamics enable Hklp2/Kif15 to assemble bipolar spindles

Activity of the sliding motor Eg5 and coordinated microtubule dynamics are both essential for mitotic spindle pole separation. It is still a matter of controversy if changes in microtubule dynamics can compensate inhibition of Eg5 activity and re-enable bipolarization. Using a consistent live cell-imaging approach, we show that perturbation of microtubule dynamics can compensate inhibition of Eg5 through a spindle formation process reminiscent of meiosis: In Eg5-inhibited mammalian somatic cells, alteration of microtubule dynamics through depletion of TOGp or low doses of nocodazole induces the formation of multiple acentrosomal spindle poles which pass through an intermediate multipolar state followed by bipolarization. Pole separation depends on Hklp2/Kif15, an otherwise dispensable plus end-directed spindle motor and results in spindles with two centrosomal poles. Once bipolar, spindles do not rely on altered microtubule dynamics to maintain their bipolarity anymore and are functional in chromosome segregation. We conclude that altered microtubule dynamics enable Hklp2/Kif15 to replace Eg5 in pole separation through a mechanism involving the formation of acentrosomal poles. Our observations suggest that combination chemotherapy regimens involving microtubule-targeting drugs and Eg5 inhibitors might be less effective than expected.     

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.     

Structure and regulation of the microtubule plus-end tracking protein Kar9

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Mini spindles, the XMAP215 homologue, suppresses pausing of interphase microtubules in Drosophila

Drosophila Mini spindles (Msps) protein belongs to a conserved family of microtubule-associated proteins (MAPs). Intriguingly, this family of MAPs, including Xenopus XMAP215, was reported to have both microtubule stabilising and destabilising activities. While they are shown to regulate various aspects of microtubules, the role in regulating interphase microtubules in animal cells has yet to be established. Here, we show that the depletion or mutation of Msps prevents interphase microtubules from extending to the cell periphery and leads to the formation of stable microtubule bundles. The effect is independent of known Msps regulator or effector proteins, kinesin-13/KinI homologues or D-TACC. Real-time analysis revealed that the depletion of Msps results in a dramatic increase of microtubule pausing with little or no growth. Our study provides the first direct evidence to support a hypothesis that this family of MAPs acts as an antipausing factor to exhibit both microtubule stabilising and destabilising activities.     

Kinetochore microtubule dynamics and attachment stability are regulated by Hec1

Mitotic cells face the challenging tasks of linking kinetochores to growing and shortening microtubules and actively regulating these dynamic attachments to produce accurate chromosome segregation. We report here that Ndc80/Hec1 functions in regulating kinetochore microtubule plus-end dynamics and attachment stability. Microinjection of an antibody to the N terminus of Hec1 suppresses both microtubule detachment and microtubule plus-end polymerization and depolymerization at kinetochores of PtK1 cells. Centromeres become hyperstretched, kinetochore fibers shorten from spindle poles, kinetochore microtubule attachment errors increase, and chromosomes severely mis-segregate. The N terminus of Hec1 is phosphorylated by Aurora B kinase in vitro, and cells expressing N-terminal nonphosphorylatable mutants of Hec1 exhibit an increase in merotelic attachments, hyperstretching of centromeres, and errors in chromosome segregation. These findings reveal a key role for the Hec1 N terminus in controlling dynamic behavior of kinetochore microtubules.