The microtubule plus end-tracking proteins mal3p and tip1p cooperate for cell-end targeting of interphase microtubules

BACKGROUND: CLIP-170 and EB1 protein family members localize to growing microtubule tips and link spatial information with the control of microtubule dynamics. It is unknown whether these proteins operate independently or whether their actions are coordinated. In fission yeast the CLIP-170 homolog tip1p is required for targeting of microtubules to cell ends, whereas the role of the EB1 homolog mal3p in microtubule organization has not been investigated. RESULTS: We show that mal3p promotes the initiation of microtubule growth and inhibits catastrophes. Premature catastrophes occur randomly throughout the cell in the absence of mal3p. mal3p decorates the entire microtubule lattice and localizes to particles along the microtubules and at their growing tips. Particles move in two directions, outbound toward the cell ends or inbound toward the cell center. At cell ends, the microtubule tip-associated mal3p particles disappear followed by a catastrophe. mal3p localizes normally in tip1-deleted cells and disappears from microtubule tips preceding the premature catastrophes. In contrast, tip1p requires mal3p to localize at microtubule tips. mal3p and tip1p directly interact in vitro. CONCLUSIONS: mal3p and tip1p form a system allowing microtubules to target cell ends. We propose that mal3p stimulates growth initiation and maintains growth by suppressing catastrophes. At cell ends, mal3p disappears from microtubule tips followed by a catastrophe. mal3p is involved in recruiting tip1p to microtubule tips. This becomes important when microtubules contact the cell cortex outside the cell ends because mal3p dissociates prematurely without tip1p, which is followed by a premature catastrophe.     

The minus end-directed motor Kar3 is required for coupling dynamic microtubule plus ends to the cortical shmoo tip in budding yeast

The budding yeast shmoo tip is a model system for analyzing mechanisms coupling force production to microtubule plus-end polymerization/depolymerization. Dynamic plus ends of astral microtubules interact with the shmoo tip in mating yeast cells, positioning nuclei for karyogamy. We have used live-cell imaging of GFP fusions to identify proteins that couple dynamic microtubule plus ends to the shmoo tip. We find that Kar3p, a minus end-directed kinesin motor protein, is required, whereas the other cytoplasmic motors, dynein and the kinesins Kip2p and Kip3p, are not. In the absence of Kar3p, attached microtubule plus ends released from the shmoo tip when they switched to depolymerization. Furthermore, microtubules in cells expressing kar3-1, a mutant that results in rigor binding to microtubules [2], were stabilized specifically at shmoo tips. Imaging of Kar3p-GFP during mating revealed that fluorescence at the shmoo tip increased during periods of microtubule depolymerization. These data are the first to localize the activity of a minus end-directed kinesin at the plus ends of microtubules. We propose a model in which Kar3p couples depolymerizing microtubule plus ends to the cell cortex and the Bim1p-Kar9p protein complex maintains attachment during microtubule polymerization. In support of this model, analysis of Bim1p-GFP at the shmoo tip results in a localization pattern complementary to that of Kar3p-GFP.     

Lamin A/C speckles mediate spatial organization of splicing factor compartments and RNA polymerase II transcription

The cylindrical shape of the fission yeast cell is generated by linear polarized growth from its cell ends. Using immunofluorescence and live imaging microscopy, we have investigated the roles of the cell end marker tea1p in generating linear polarized growth. We found that tea1p is primarily transported on plus ends of microtubules from the vicinity of the nucleus to the cell ends, and that its movement near the nucleus is independent of the kinesin tea2p. Deletion analysis identified a coiled-coil domain in tea1p essential for its retention at cell ends, and demonstrated that tea1p exerts different functions dependent on its location. On the tips of microtubules, tea1p prevents the curling of microtubules around the cell ends, whereas it is required for maintaining linear cell growth and for retention of polarity factors such as the Dyrk kinase pom1p, the CLIP170-like tip1p, and tea2p at the cell ends. We propose that tea1p has roles in organizing the microtubule cytoskeleton on the tips of microtubules, and in the retention of factors at the cell ends necessary for the cell to grow in a straight line.     

Tea2p kinesin is involved in spatial microtubule organization by transporting tip1p on microtubules

The positioning of growth sites in fission yeast cells is mediated by spatially controlled microtubule dynamics brought about by tip1p, a CLIP-170-like protein, which is localized at the microtubule tips and guides them to the cell ends. The kinesin tea2p is also located at microtubule tips and affects microtubule dynamics. Here we show that tea2p interacts with tip1p and that the two proteins move with high velocity along the microtubules toward their growing tips. There, tea2p and tip1p accumulate in larger particles. Particle formation requires the EB1 homolog, mal3p. Our results suggest a model in which kinesins regulate microtubule growth by transporting regulatory factors such as tip1p to the growing microtubule tips.     

Regulation of a formin complex by the microtubule plus end protein tea1p

The plus ends of microtubules have been speculated to regulate the actin cytoskeleton for the proper positioning of sites of cell polarization and cytokinesis. In the fission yeast Schizosaccharomyces pombe, interphase microtubules and the kelch repeat protein tea1p regulate polarized cell growth. Here, we show that tea1p is directly deposited at cell tips by microtubule plus ends. Tea1p associates in large "polarisome" complexes with bud6p and for3p, a formin that assembles actin cables. Tea1p also interacts in a separate complex with the CLIP-170 protein tip1p, a microtubule plus end-binding protein that anchors tea1p to the microtubule plus end. Localization experiments suggest that tea1p and bud6p regulate formin distribution and actin cable assembly. Although single mutants still polarize, for3Deltabud6Deltatea1Delta triple-mutant cells lack polarity, indicating that these proteins contribute overlapping functions in cell polarization. Thus, these experiments begin to elucidate how microtubules contribute to the proper spatial regulation of actin assembly and polarized cell growth.     

CLIP-170 family members: a motor-driven ride to microtubule plus ends

CLIP-170 family proteins regulate microtubule plus end dynamics. Two reports published in this issue of Developmental Cell show that Bik1 and tip1p, the CLIP-170-like proteins of budding and fission yeast, are carried to microtubule plus ends by kinesin motor proteins. These findings indicate a complex interplay between microtubule-associated proteins and suggest a novel mechanism by which kinesin proteins stabilize microtubules.     

Asymmetric microtubule pushing forces in nuclear centering

Dynamic properties of microtubules contribute to the establishment of spatial order within cells. In the fission yeast Schizosaccharomyces pombe, interphase cytoplasmic microtubules are organized into antiparallel bundles that attach to the nuclear envelope and are needed to position the nucleus at the geometric center of the cell. Here, we show that after the nucleus is displaced by cell centrifugation, these microtubule bundles efficiently push the nucleus back to the center. Asymmetry in microtubule number, length, and dynamics contributes to the generation of force responsible for this unidirectional movement. Notably, microtubules facing the distal cell tip are destabilized when the microtubules in the same bundle are pushing from the proximal cell tip. The CLIP-170-like protein tip1p and the microtubule-bundling protein ase1p are required for this asymmetric regulation of microtubule dynamics, indicating contributions of factors both at microtubule plus ends and within the microtubule bundle. Mutants in these factors are defective in nuclear movement. Thus, cells possess an efficient microtubule-based engine that produces and senses forces for centering the nucleus. These studies may provide insights into mechanisms of asymmetric microtubule behaviors and force sensing in other processes such as chromosome segregation and cell polarization.     

Multistep and multimode cortical anchoring of tea1p at cell tips in fission yeast

The fission yeast cell-polarity regulator tea1p is targeted to cell tips by association with growing microtubule ends. Tea1p is subsequently anchored at the cell cortex at cell tips via an unknown mechanism that requires both the tea1p carboxy-terminus and the membrane protein mod5p. Here, we show that a tea1p-related protein, tea3p, binds independently to both mod5p and tea1p, and that tea1p and mod5p can also interact directly, independent of tea3p. Despite their related structures, different regions of tea1p and tea3p are required for their respective interactions with an essential central region of mod5p. We demonstrate that tea3p is required for proper cortical localization of tea1p, specifically at nongrowing cell tips, and that tea1p and mod5p are independently required for tea3p localization. Further, we find that tea3p fused to GFP or mCherry is cotransported with tea1p by microtubules to cell tips, but this occurs only in the absence of mod5p. These results suggest that independent protein-protein interactions among tea1p, tea3p and mod5p collectively contribute to tea1p anchoring at cell tips via a multistep and multimode mechanism.     

Mcp1p tracks microtubule plus ends to destabilize microtubules at cell tips

Microtubule plus ends are dynamically regulated by a wide variety of proteins for performing diverse cellular functions. Here, we show that the fission yeast Schizosaccharomyces pombe uncharacterized protein mcp1p is a microtubule plus-end tracking protein which depends on the kinesin-8 klp6p for transporting along microtubules towards microtubule plus ends. In the absence of mcp1p, microtubule catastrophe and rescue frequencies decrease, leading to an increased dwell time of microtubule plus ends at cell tips. Thus, these findings suggest that mcp1p may synergize with klp6p at microtubule plus-ends to destabilize microtubules.     

Microtubule interactions with the cell cortex causing nuclear movements in Saccharomyces cerevisiae

During mitosis in budding yeast the nucleus first moves to the mother-bud neck and then into the neck. Both movements depend on interactions of cytoplasmic microtubules with the cortex. We investigated the mechanism of these movements in living cells using video analysis of GFP-labeled microtubules in wild-type cells and in EB1 and Arp1 mutants, which are defective in the first and second steps, respectively. We found that nuclear movement to the neck is largely mediated by the capture of microtubule ends at one cortical region at the incipient bud site or bud tip, followed by microtubule depolymerization. Efficient microtubule interactions with the capture site require that microtubules be sufficiently long and dynamic to probe the cortex. In contrast, spindle movement into the neck is mediated by microtubule sliding along the bud cortex, which requires dynein and dynactin. Free microtubules can also slide along the cortex of both bud and mother. Capture/shrinkage of microtubule ends also contributes to nuclear movement into the neck and can serve as a backup mechanism to move the nucleus into the neck when microtubule sliding is impaired. Conversely, microtubule sliding can move the nucleus into the neck even when capture/shrinkage is impaired.     

Determinants of S. cerevisiae dynein localization and activation: implications for the mechanism of spindle positioning

BACKGROUND: During anaphase in budding yeast, dynein inserts the mitotic spindle across the neck between mother and daughter cells. The mechanism of dynein-dependent spindle positioning is thought to involve recruitment of dynein to the cell cortex followed by capture of astral microtubules (aMTs). RESULTS: We report the native-level localization of the dynein heavy chain and characterize the effects of mutations in dynein regulators on its intracellular distribution. Budding yeast dynein displays discontinuous localization along aMTs, with enrichment at the spindle pole body and aMT plus ends. Loss of Bik1p (CLIP-170), the cargo binding domain of Bik1p, or Pac1p (LIS1) resulted in diminished targeting of dynein to aMTs. By contrast, loss of dynactin or a mutation in the second P loop domain of dynein resulted in an accumulation of dynein on the plus ends of aMTs. Unexpectedly, loss of Num1p, a proposed dynein cortical anchor, also resulted in selective accumulation of dynein on the plus ends of anaphase aMTs. CONCLUSIONS: We propose that, rather than first being recruited to the cell cortex, dynein is delivered to the cortex on the plus ends of polymerizing aMTs. Dynein may then undergo Num1p-dependent activation and transfer to the region of cortical contact. Based on the similar effects of loss of Num1p and loss of dynactin on dynein localization, we suggest that Num1p might also enhance dynein motor activity or processivity, perhaps by clustering dynein motors.     

Polarity determinants Tea1p, Tea4p, and Pom1p inhibit division-septum assembly at cell ends in fission yeast

Correct positioning of the cell-division plane is crucial for cell function in all organisms. The fission yeast Schizosaccharomyces pombe divides by utilizing an actomyosin-based contractile ring and is an attractive model for the study of cytokinesis. The metazoan anillin-related protein Mid1p stimulates medial assembly of the division septum by recruiting actomyosin-ring components to the medial cortex. Here, we describe an inhibitory mechanism, involving the cell-end-localized polarity determinants Tea1p, Tea4p/Wsh3p, and Pom1p (tip complex), which prevents division-septum assembly at the cell ends. While Mid1p and the tip complex are dispensable for cell viability, their simultaneous loss leads to lethality. The FER/CIP homology protein Cdc15p, which organizes the actomyosin ring and cell membranes during cytokinesis, is a candidate for regulation by the tip complex. Since dual regulation of division-site placement is also seen in nematodes, such regulation might be a general feature of eukaryotic cytokinesis.     

An InCytes from MBC Selection: The Mating-specific Gα Interacts with a Kinesin-14 and Regulates Pheromone-induced Nuclear Migration in Budding Yeast

As a budding yeast cell elongates toward its mating partner, cytoplasmic microtubules connect the nucleus to the cell cortex at the growth tip. The Kar3 kinesin-like motor protein is then thought to stimulate plus-end depolymerization of these microtubules, thus drawing the nucleus closer to the site where cell fusion and karyogamy will occur. Here, we show that pheromone stimulates a microtubule-independent interaction between Kar3 and the mating-specific Gα protein Gpa1 and that Gpa1 affects both microtubule orientation and cortical contact. The membrane localization of Gpa1 was found to polarize early in the mating response, at about the same time that the microtubules begin to attach to the incipient growth site. In the absence of Gpa1, microtubules lose contact with the cortex upon shrinking and Kar3 is improperly localized, suggesting that Gpa1 is a cortical anchor for Kar3. We infer that Gpa1 serves as a positional determinant for Kar3-bound microtubule plus ends during mating.     

Microtubule tips redirect actin assembly

In fission yeast, a protein complex that rides on the plus end of growing microtubules regulates establishment of new sites of actin cable assembly, necessary for cell growth, from one to both ends of the rod-shaped cells. Martin et al. describe a direct molecular link between the microtubule tip complex and the formin for3p, the nucleator for assembly of actin cables, which is necessary for this regulatory switch.     

Establishment of a cellular axis in fission yeast

Recent studies in fission yeast Schizosaccharomyces pombe reveal how cells establish a cellular axis that specifies domains as the functional 'ends' and 'middle' of the cell. During interphase, dynamic microtubules position the nucleus at the middle of the cell and orientate microtubule 'plus' ends towards the ends of the cell. At the cell ends, the microtubule plus ends might establish a zone of polarized cell growth and actin assembly by depositing factors such as Tea1p. At the cell middle, the nucleus might specify the position of the actin contractile ring and the future cell division site by positioning cytokinesis factors such as Mid1p.     

Shaping Fission Yeast with Microtubules

For cell morphogenesis, the cell must establish distinct spatial domains at specified locations at the cell surface. Here, we review the molecular mechanisms of cell polarity in the fission yeast Schizosaccharomyces pombe. These are simple rod-shaped cells that form cortical domains at cell tips for cell growth and at the cell middle for cytokinesis. In both cases, microtubule-based systems help to shape the cell by breaking symmetry, providing endogenous spatial cues to position these sites. The plus ends of dynamic microtubules deliver polarity factors to the cell tips, leading to local activation of the GTPase cdc42p and the actin assembly machinery. Microtubule bundles contribute to positioning the division plane through the nucleus and the cytokinesis factor mid1p. Recent advances illustrate how the spatial and temporal regulation of cell polarization integrates many elements, including historical landmarks, positive and negative controls, and competition between pathways.One of the ultimate goals in cell biology is to understand how cells are assembled. As in the development of multicellular organisms, single cells need to form distinct spatial domains with specific form, structure, and functions. How do cells organize themselves in space to form a specific shape and size?The fission yeast Schizosaccharomyces pombe is an attractive, simple unicellular model organism for studying cell morphogenesis. These are nonmotile cells with highly invariant shape 8–14 µm long and 3 µm in diameter. The relative simplicity of the cells and the powers of genetic approaches and live cell imaging facilitate rigorous and quantitative studies.Here, we review the current understanding of spatial regulation in fission yeast. The cell defines distinct cortical domains at each of the cell tips, along the sides of cells, and at the cell division plane. Each cortical domain is characterized by different sets of molecules, which impart distinct functions. In particular, as it proceeds through its cell cycle, the cell delineates distinct actin-rich cortical regions at cell tips for polarized cell growth and at the middle for cell division. In both cases, a self-organizing network of microtubules directly or indirectly contributes to the proper localization of these markers. In cell polarity, microtubule ends transport polarity factors to the plasma membrane, where they function to recruit protein complexes involved in actin assembly. In cytokinesis, a medial cortical site is marked by an interacting system of microtubules, the nucleus, and cell tip factors, and functions to organize actin filaments into a cytokinetic ring. This reliance on microtubules contrasts with polarity mechanisms in budding yeast in which spatial cues are dependent on septins and actin, but not microtubules. As many of these processes involve conserved proteins, this work in fission yeast contributes toward understanding the more complex microtubule-based regulation of cell migration, cytokinesis, and cell shape regulation in animal cells. This work in fission yeast thus provides a paradigm for how a self-organizing system can shape a cell.     

Stu2p,the budding yeast member of the conserved Dis1/XMAP215 family of microtubule-associated proteins is a plus end-binding microtubule destabilizer

The Dis1/XMAP215 family of microtubule-associated proteins conserved from yeast to mammals is essential for cell division. XMAP215, the Xenopus member of this family, has been shown to stabilize microtubules in vitro, but other members of this family have not been biochemically characterized. Here we investigate the properties of the Saccharomyces cerevisiae homologue Stu2p in vitro. Surprisingly, Stu2p is a microtubule destabilizer that binds preferentially to microtubule plus ends. Quantitative analysis of microtubule dynamics suggests that Stu2p induces microtubule catastrophes by sterically interfering with tubulin addition to microtubule ends. These results reveal both a new biochemical activity for a Dis1/XMAP215 family member and a novel mechanism for microtubule destabilization.     

Ase1p organizes antiparallel microtubule arrays during interphase and mitosis in fission yeast

Proper microtubule organization is essential for cellular processes such as organelle positioning during interphase and spindle formation during mitosis. The fission yeast Schizosaccharomyces pombe presents a good model for understanding microtubule organization. We identify fission yeast ase1p, a member of the conserved ASE1/PRC1/MAP65 family of microtubule bundling proteins, which functions in organizing the spindle midzone during mitosis. Using fluorescence live cell imaging, we show that ase1p localizes to sites of microtubule overlaps associated with microtubule organizing centers at both interphase and mitosis. ase1Delta mutants fail to form overlapping antiparallel microtubule bundles, leading to interphase nuclear positioning defects, and premature mitotic spindle collapse. FRAP analysis revealed that interphase ase1p at overlapping microtubule minus ends is highly dynamic. In contrast, mitotic ase1p at microtubule plus ends at the spindle midzone is more stable. We propose that ase1p functions to organize microtubules into overlapping antiparallel bundles both in interphase and mitosis and that ase1p may be differentially regulated through the cell cycle.     

The dynamic behavior of the APC-binding protein EB1 on the distal ends of microtubules

Adenomatous polyposis coli protein (APC) is a well-characterized tumor suppressor protein [1] [2] [3]. We previously showed that APC tagged with green fluorescent protein (GFP) in Xenopus A6 epithelial cells moves along a subset of microtubules and accumulates at their growing plus ends in cell extensions [4]. EB1, which was identified as an APC-binding protein by yeast two-hybrid analysis [5], was also reported to be associated with microtubules [6] [7] [8]. To examine the interaction between APC and EB1 within cells, we compared the dynamic behavior of EB1-GFP with that of APC-GFP in A6 transfectants. Time-lapse microscopy of live cells at interphase revealed that EB1-GFP was concentrated at all of the growing microtubule ends throughout the cytoplasm and abruptly disappeared from the ends when microtubules began to shorten. Therefore, EB1 appeared to be co-localized and interact with APC on the growing ends of a subset of microtubules. When APC-GFP was overexpressed, endogenous EB1 was recruited to APC-GFP, which accumulated in large amounts on microtubules. On the other hand, when microtubules were disassembled by nocodazole, EB1 was not co-localized with APC-GFP, which was concentrated along the basal plasma membrane. During mitosis, APC appeared to be dissociated from microtubules, whereas EB1-GFP continued to concentrate at microtubule growing ends. These findings showed that the APC-EB1 interaction is regulated within cells and is allowed near the ends of microtubules only under restricted conditions.     

Architecture of the Dam1 kinetochore ring complex and implications for microtubule-driven assembly and force-coupling mechanisms

The Dam1 kinetochore complex is essential for chromosome segregation in budding yeast. This ten-protein complex self-assembles around microtubules, forming ring-like structures that move with depolymerizing microtubule ends, a mechanism with implications for cellular function. Here we used EM-based single-particle and helical analyses to define the architecture of the Dam1 complex at 30-A resolution and the self-assembly mechanism. Ring oligomerization seems to be facilitated by a conformational change upon binding to microtubules, suggesting that the Dam1 ring is not preformed, but self-assembles around kinetochore microtubules. The C terminus of the Dam1p protein, where most of the Aurora kinase Ipl1 phosphorylation sites reside, is in a strategic location to affect oligomerization and interactions with the microtubule. One of Ipl1's roles might be to fine-tune the coupling of the microtubule interaction with the conformational change required for oligomerization, with phosphorylation resulting in ring breakdown.