Real-Time Imaging of Single γTuRC-Mediated Microtubule Nucleation Events In Vitro by TIRF Microscopy.

The γ-tubulin ring complex (γTuRC) is the major microtubule nucleator in cells. How γTuRC nucleates microtubules, and how nucleation is regulated is not understood. To gain an understanding of γTuRC activity and regulation at the molecular level, it is important to measure quantitatively how γTuRC interacts with tubulin and potential regulators in space and time. Here, we describe a total internal reflection fluorescence microscopy-based assay on chemically functionalized glass slides for the in vitro study of surface immobilized purified γTuRC. The assay allows to measure microtubule nucleation by γTuRC in real time and at a single molecule level over a wide variety of assay conditions, in the absence and presence of potential regulators. This setup provides a previously unavailable opportunity for quantitative studies of the kinetics of microtubule nucleation by γTuRC.

Structural transitions in the GTP cap visualized by cryo-electron microscopy of catalytically inactive microtubules.

Microtubules (MTs) are polymers of αß-tubulin heterodimers that stochastically switch between growth and shrinkage phases. This dynamic instability is critically important for MT function. It is believed that GTP hydrolysis within the MT lattice is accompanied by destabilizing conformational changes and that MT stability depends on a transiently existing GTP cap at the growing MT end. Here, we use cryo-electron microscopy and total internal reflection fluorescence microscopy of GTP hydrolysis-deficient MTs assembled from mutant recombinant human tubulin to investigate the structure of a GTP-bound MT lattice. We find that the GTP-MT lattice of two mutants in which the catalytically active glutamate in α-tubulin was substituted by inactive amino acids (E254A and E254N) is remarkably plastic. Undecorated E254A and E254N MTs with 13 protofilaments both have an expanded lattice but display opposite protofilament twists, making these lattices distinct from the compacted lattice of wild-type GDP-MTs. End-binding proteins of the EB family have the ability to compact both mutant GTP lattices and to stabilize a negative twist, suggesting that they promote this transition also in the GTP cap of wild-type MTs, thereby contributing to the maturation of the MT structure. We also find that the MT seam appears to be stabilized in mutant GTP-MTs and destabilized in GDP-MTs, supporting the proposal that the seam plays an important role in MT stability. Together, these structures of catalytically inactive MTs add mechanistic insight into the GTP state of MTs, the stability of the GTP- and GDP-bound lattice, and our overall understanding of MT dynamic instability.

Microtubule Nucleation Properties of Single Human γTuRCs Explained by Their Cryo-EM Structure.

The γ-tubulin ring complex (γTuRC) is the major microtubule nucleator in cells. The mechanism of its regulation is not understood. We purified human γTuRC and measured its nucleation properties in a total internal reflection fluorescence (TIRF) microscopy-based real-time nucleation assay. We find that γTuRC stably caps the minus ends of microtubules that it nucleates stochastically. Nucleation is inefficient compared with microtubule elongation. The 4 Å resolution cryoelectron microscopy (cryo-EM) structure of γTuRC, combined with crosslinking mass spectrometry analysis, reveals an asymmetric conformation with only part of the complex in a "closed" conformation matching the microtubule geometry. Actin in the core of the complex, and MZT2 at the outer perimeter of the closed part of γTuRC appear to stabilize the closed conformation. The opposite side of γTuRC is in an "open," nucleation-incompetent conformation, leading to a structural asymmetry explaining the low nucleation efficiency of purified human γTuRC. Our data suggest possible regulatory mechanisms for microtubule nucleation by γTuRC closure.

The speed of GTP hydrolysis determines GTP cap size and controls microtubule stability.

Microtubules are cytoskeletal polymers whose function depends on their property to switch between states of growth and shrinkage. Growing microtubules are thought to be stabilized by a GTP cap at their ends. The nature of this cap, however, is still poorly understood. End Binding proteins (EBs) recruit a diverse range of regulators of microtubule function to growing microtubule ends. Whether the EB binding region is identical to the GTP cap is unclear. Using mutated human tubulin with blocked GTP hydrolysis, we demonstrate that EBs bind with high affinity to the GTP conformation of microtubules. Slowing-down GTP hydrolysis leads to extended GTP caps. We find that cap length determines microtubule stability and that the microtubule conformation changes gradually in the cap as GTP is hydrolyzed. These results demonstrate the critical importance of the kinetics of GTP hydrolysis for microtubule stability and establish that the GTP cap coincides with the EB-binding region.

Determinants of Polar versus Nematic Organization in Networks of Dynamic Microtubules and Mitotic Motors.

During cell division, mitotic motors organize microtubules in the bipolar spindle into either polar arrays at the spindle poles or a "nematic" network of aligned microtubules at the spindle center. The reasons for the distinct self-organizing capacities of dynamic microtubules and different motors are not understood. Using in vitro reconstitution experiments and computer simulations, we show that the human mitotic motors kinesin-5 KIF11 and kinesin-14 HSET, despite opposite directionalities, can both organize dynamic microtubules into either polar or nematic networks. We show that in addition to the motor properties the natural asymmetry between microtubule plus- and minus-end growth critically contributes to the organizational potential of the motors. We identify two control parameters that capture system composition and kinetic properties and predict the outcome of microtubule network organization. These results elucidate a fundamental design principle of spindle bipolarity and establish general rules for active filament network organization.

Ensembles of Bidirectional Kinesin Cin8 Produce Additive Forces in Both Directions of Movement.

Most kinesin motors move in only one direction along microtubules. Members of the kinesin-5 subfamily were initially described as unidirectional plus-end-directed motors and shown to produce piconewton forces. However, some fungal kinesin-5 motors are bidirectional. The force production of a bidirectional kinesin-5 has not yet been measured. Therefore, it remains unknown whether the mechanism of the unconventional minus-end-directed motility differs fundamentally from that of plus-end-directed stepping. Using force spectroscopy, we have measured here the forces that ensembles of purified budding yeast kinesin-5 Cin8 produce in microtubule gliding assays in both plus- and minus-end direction. Correlation analysis of pause forces demonstrated that individual Cin8 molecules produce additive forces in both directions of movement. In ensembles, Cin8 motors were able to produce single-motor forces up to a magnitude of ∼1.5 pN. Hence, these properties appear to be conserved within the kinesin-5 subfamily. Force production was largely independent of the directionality of movement, indicating similarities between the motility mechanisms for both directions. These results provide constraints for the development of models for the bidirectional motility mechanism of fission yeast kinesin-5 and provide insight into the function of this mitotic motor.

Structural insight into TPX2-stimulated microtubule assembly.

During mitosis and meiosis, microtubule (MT) assembly is locally upregulated by the chromatin-dependent Ran-GTP pathway. One of its key targets is the MT-associated spindle assembly factor TPX2. The molecular mechanism of how TPX2 stimulates MT assembly remains unknown because structural information about the interaction of TPX2 with MTs is lacking. Here, we determine the cryo-electron microscopy structure of a central region of TPX2 bound to the MT surface. TPX2 uses two flexibly linked elements ('ridge' and 'wedge') in a novel interaction mode to simultaneously bind across longitudinal and lateral tubulin interfaces. These MT-interacting elements overlap with the binding site of importins on TPX2. Fluorescence microscopy-based in vitro reconstitution assays reveal that this interaction mode is critical for MT binding and facilitates MT nucleation. Together, our results suggest a molecular mechanism of how the Ran-GTP gradient can regulate TPX2-dependent MT formation.

Combinatorial regulation of the balance between dynein microtubule end accumulation and initiation of directed motility.

Cytoplasmic dynein is involved in a multitude of essential cellular functions. Dynein's activity is controlled by the combinatorial action of several regulatory proteins. The molecular mechanism of this regulation is still poorly understood. Using purified proteins, we reconstitute the regulation of the human dynein complex by three prominent regulators on dynamic microtubules in the presence of end binding proteins (EBs). We find that dynein can be in biochemically and functionally distinct pools: either tracking dynamic microtubule plus-ends in an EB-dependent manner or moving processively towards minus ends in an adaptor protein-dependent manner. Whereas both dynein pools share the dynactin complex, they have opposite preferences for binding other regulators, either the adaptor protein Bicaudal-D2 (BicD2) or the multifunctional regulator Lissencephaly-1 (Lis1). BicD2 and Lis1 together control the overall efficiency of motility initiation. Remarkably, dynactin can bias motility initiation locally from microtubule plus ends by autonomous plus-end recognition. This bias is further enhanced by EBs and Lis1. Our study provides insight into the mechanism of dynein regulation by dissecting the distinct functional contributions of the individual members of a dynein regulatory network.

Microtubule nucleation: beyond the template.

Microtubules are cytoskeletal filaments central to a wide range of essential cellular functions in eukaryotic cells. Consequently, cells need to exert tight control over when, where and how many microtubules are being made. Whereas the regulation of microtubule dynamics is well studied, the molecular mechanisms of microtubule nucleation are still poorly understood. Next to the established master template of nucleation, the γ-tubulin ring complex, other microtubule-associated proteins that affect microtubule dynamic properties have recently been found to contribute to nucleation. It has begun to emerge that the nucleation efficiency is controlled not only by template activity but also by, either additionally or alternatively, the stabilization of the nascent microtubule 'nucleus'. This suggests a simple conceptual framework for the mechanisms regulating microtubule nucleation in cells.

Complementary activities of TPX2 and chTOG constitute an efficient importin-regulated microtubule nucleation module.

Spindle assembly and function require precise control of microtubule nucleation and dynamics. The chromatin-driven spindle assembly pathway exerts such control locally in the vicinity of chromosomes. One of the key targets of this pathway is TPX2. The molecular mechanism of how TPX2 stimulates microtubule nucleation is not understood. Using microscopy-based dynamic in vitro reconstitution assays with purified proteins, we find that human TPX2 directly stabilizes growing microtubule ends and stimulates microtubule nucleation by stabilizing early microtubule nucleation intermediates. Human microtubule polymerase chTOG (XMAP215/Msps/Stu2p/Dis1/Alp14 homologue) only weakly promotes nucleation, but acts synergistically with TPX2. Hence, a combination of distinct and complementary activities is sufficient for efficient microtubule formation in vitro. Importins control the efficiency of the microtubule nucleation by selectively blocking the interaction of TPX2 with microtubule nucleation intermediates. This in vitro reconstitution reveals the molecular mechanism of regulated microtubule formation by a minimal nucleation module essential for chromatin-dependent microtubule nucleation in cells.

The multiple talents of kinesin-8.

Multiple activities cooperate to determine the architecture of the mitotic spindle. Kip3 is a kinesin-8 motor protein in budding yeast that acts as a microtubule depolymerase. Now Kip3 is shown to also crosslink and slide antiparallel microtubules, providing additional insights into how kinesin-8 motors control spindle integrity.

End-binding proteins and Ase1/PRC1 define local functionality of structurally distinct parts of the microtubule cytoskeleton.

The microtubule cytoskeleton is crucial for the intracellular organization of eukaryotic cells. It is a dynamic scaffold that has to perform a variety of very different functions. This multitasking is achieved through the activity of numerous microtubule-associated proteins. Two prominent classes of proteins are central to the selective recognition of distinct transiently existing structural features of the microtubule cytoskeleton. They define local functionality through tightly regulated protein recruitment. Here we summarize the recent developments in elucidating the molecular mechanism underlying the action of microtubule end-binding proteins (EBs) and antiparallel microtubule crosslinkers of the Ase1/PRC1 family that represent the core of these two recruitment modules. Despite their fundamentally different activities, these conserved families share several common features.

Directional switching of the kinesin Cin8 through motor coupling.

Kinesin motor proteins are thought to move exclusively in either one or the other direction along microtubules. Proteins of the kinesin-5 family are tetrameric microtubule cross-linking motors important for cell division and differentiation in various organisms. Kinesin-5 motors are considered to be plus-end-directed. However, here we found that purified kinesin-5 Cin8 from budding yeast could behave as a bidirectional kinesin. On individual microtubules, single Cin8 motors were minus-end-directed motors, whereas they switched to plus-end-directed motility when working in a team of motors sliding antiparallel microtubules apart. This kinesin can thus change directionality of movement depending on whether it acts alone or in an ensemble.

Cell cycle control of spindle elongation.

Different organisms employ a variety of strategies to segregate their chromosomes during mitosis. Despite these differences, however, the basic regulatory principles that govern this intricate process are evolutionarily conserved. Above all, rapid dephosphorylation of mitotic phosphoproteins upon the metaphase-to-anaphase transition has proven to be essential for proper function of the mitotic spindle and accurate chromosome segregation in all eukaryotes. Recently, a central midzone component, the microtubule crosslinker Ase1/PRC1 (anaphase spindle elongation 1/protein regulating cytokinesis 1), was uncovered as a universal target of such control mechanism. Depending on its phosphorylation status, Ase1 either restrains spindle elongation in metaphase or promotes it after anaphase onset via recruitment of kinesin motor proteins to the midzone. Here we discuss the potential role of Ase1/PRC1 as a central regulatory platform that interconnects distinct functions of the midzone such as spindle stability, spindle elongation and cytokinesis. Additionally, we provide a comparative overview of the chromosome segregation strategies used by the main model organisms.

Phosphorylation-dependent protein interactions at the spindle midzone mediate cell cycle regulation of spindle elongation.

The metaphase-to-anaphase transition is one of the most dramatic and highly regulated steps in cell division. At anaphase onset the protease separase dissolves sister chromatid cohesion. Simultaneously, the mitotic spindle elongates as interpolar microtubules (iMTs) slide apart at the spindle midzone, ensuring chromosome segregation. However, it remains unclear how spindle elongation is coordinated with cell cycle progression. Here we demonstrate that phosphorylation of the midzone organizer Ase1 controls localization and function of Cin8, a kinesin-5 that slides iMTs relative to each other. Phosphorylation of Ase1 by Cdk1 (cyclin-dependent kinase) inhibits Cin8 binding to iMTs, preventing bending and collapse of the metaphase spindle. In anaphase Ase1 dephosphorylation by the separase-activated phosphatase Cdc14 is necessary and sufficient for Cin8 recruitment to the midzone, where it drives spindle elongation. Our results reveal that sliding forces at the midzone are activated by separase and explain how spindle elongation is triggered with anaphase entry.

Cell division in Escherichia coli cultures monitored at single cell resolution.

BACKGROUND: A fundamental characteristic of cells is the ability to divide. To date, most parameters of bacterial cultures, including cell division, have been measured as cell population averages, assuming that all bacteria divide at a uniform rate. RESULTS: We monitored the division of individual cells in Escherichia coli cultures during different growth phases. Our experiments are based on the dilution of green fluorescent protein (GFP) upon cell division, monitored by flow cytometry. The results show that the vast majority of E. coli cells in exponentially growing cultures divided uniformly. In cultures that had been in stationary phase up to four days, no cell division was observed. However, upon dilution of stationary phase culture into fresh medium, two subpopulations of cells emerged: one that started dividing and another that did not. These populations were detectable by GFP dilution and displayed different side scatter parameters in flow cytometry. Further analysis showed that bacteria in the non-growing subpopulation were not dead, neither was the difference in growth capacity reducible to differences in stationary phase-specific gene expression since we observed uniform expression of several stress-related promoters. The presence of non-growing persisters, temporarily dormant bacteria that are tolerant to antibiotics, has previously been described within growing bacterial populations. Using the GFP dilution method combined with cell sorting, we showed that ampicillin lyses growing bacteria while non-growing bacteria retain viability and that some of them restart growth after the ampicillin is removed. Thus, our method enables persisters to be monitored even in liquid cultures of wild type strains in which persister formation has low frequency. CONCLUSION: In principle, the approaches developed here could be used to detect differences in cell division in response to different environmental conditions and in cultures of unicellular organisms other than E. coli.

Cdc14-regulated midzone assembly controls anaphase B.

Spindle elongation in anaphase of mitosis is a cell cycle-regulated process that requires coordination between polymerization, cross-linking, and sliding of microtubules (MTs). Proteins that assemble at the spindle midzone may be important for this process. In this study, we show that Ase1 and the separase-Slk19 complex drive midzone assembly in yeast. Whereas the conserved MT-bundling protein Ase1 establishes a midzone, separase-Slk19 is required to focus and center midzone components. An important step leading to spindle midzone assembly is the dephosphorylation of Ase1 by the protein phosphatase Cdc14 at the beginning of anaphase. Failure to dephosphorylate Ase1 delocalizes midzone proteins and delays the second, slower phase of anaphase B. In contrast, in cells expressing nonphosphorylated Ase1, anaphase spindle extension is faster, and spindles frequently break. Cdc14 also controls the separase-Slk19 complex indirectly via the Aurora B kinase. Thus, Cdc14 regulates spindle midzone assembly and function directly through Ase1 and indirectly via the separase-Slk19 complex.