Spatial and Temporal Scaling of Microtubules and Mitotic Spindles

During cell division, the mitotic spindle, a macromolecular structure primarily comprised of microtubules, drives chromosome alignment and partitioning between daughter cells. Mitotic spindles can sense cellular dimensions in order to adapt their length and mass to cell size. This scaling capacity is particularly remarkable during early embryo cleavage when cells divide rapidly in the absence of cell growth, thus leading to a reduction of cell volume at each division. Although mitotic spindle size scaling can occur over an order of magnitude in early embryos, in many species the duration of mitosis is relatively short, constant throughout early development and independent of cell size. Therefore, a key challenge for cells during embryo cleavage is not only to assemble a spindle of proper size, but also to do it in an appropriate time window which is compatible with embryo development. How spatial and temporal scaling of the mitotic spindle is achieved and coordinated with the duration of mitosis remains elusive. In this review, we will focus on the mechanisms that support mitotic spindle spatial and temporal scaling over a wide range of cell sizes and cellular contexts. We will present current models and propose alternative mechanisms allowing cells to spatially and temporally coordinate microtubule and mitotic spindle assembly.

Factor quinolinone inhibitors alter cell morphology and motility by destabilizing interphase microtubules

Factor quinolinone inhibitors are promising anti-cancer compounds, initially characterized as specific inhibitors of the oncogenic transcription factor LSF (TFCP2). These compounds exert anti-proliferative activity at least in part by disrupting mitotic spindles. Herein, we report additional interphase consequences of the initial lead compound, FQI1, in two telomerase immortalized cell lines. Within minutes of FQI1 addition, the microtubule network is disrupted, resulting in a substantial, although not complete, depletion of microtubules as evidenced both by microtubule sedimentation assays and microscopy. Surprisingly, this microtubule breakdown is quickly followed by an increase in tubulin acetylation in the remaining microtubules. The sudden breakdown and partial depolymerization of the microtubule network precedes FQI1-induced morphological changes. These involve rapid reduction of cell spreading of interphase fetal hepatocytes and increase in circularity of retinal pigment epithelial cells. Microtubule depolymerization gives rise to FH-B cell compaction, as pretreatment with taxol prevents this morphological change. Finally, FQI1 decreases the rate and range of locomotion of interphase cells, supporting an impact of FQI1-induced microtubule breakdown on cell motility. Taken together, our results show that FQI1 interferes with microtubule-associated functions in interphase, specifically cell morphology and motility.

Heat shock factor 1 suppression induces spindle abnormalities and sensitizes cells to antimitotic drugs

Background Heat shock factor 1 (HSF1) is the master regulator of the heat shock response and supports malignant cell transformation. Recent work has shown that HSF1 can access the promoters of heat shock proteins (HSPs) and allow HSP expression during mitosis. It also acts as a mitotic regulator, controlling chromosome segregation. In this study, we investigated whether the transactivation activity of HSF1 is required for the assembly of mitotic spindles. Results Our results showed that phosphorylation of HSF1 at serine 326 (S326) and its transactivation activity were increased during mitosis. Inhibition of the transactivation activity of HSF1 by KRIBB11 or CCT251263 during mitosis significantly increased the proportion of mitotic cells with abnormal spindles. It also hampered the reassembly of spindle microtubules after nocodazole treatment and washout by impeding the formation of chromosomal microtubule asters. Depletion of HSF1 led to defects in mitotic spindle assembly, subsequently attenuating cell proliferation and anchorage-independent cell growth (AIG). These HSF1 depletion-induced effects could be rescued by ectopically expressing wild-type HSF1 or a constitutively active mutant (∆202-316, caHSF1) but not the S326A or dominant negative (∆361-529, dnHSF1) mutants. In addition, overexpression of HSP70 partially reduced HSF1 depletion-induced spindle abnormalities. These results indicate that HSF1 may support cell proliferation and AIG by maintaining spindle integrity through its transactivation activity. Furthermore, inhibition of HSF1 transactivation activity by KRIBB11 or CCT251236 can enhance diverse anti-mitosis drug-induced spindle defects and cell death. Conclusions The increased transactivation activity of HSF1 during mitosis appears to be required for accurate assembly of mitotic spindles, thereby supporting cell viability and probably AIG. In addition, inhibition of the transactivation activity of HSF1 may enhance the mitotic errors and cell death induced by anti-mitosis drugs.

Mitotic Outcomes in Fibrous Environments

During mitosis, cells round up and generate outward forces to create space and orient the mitotic spindles. Here, using suspended ECM-mimicking nanofiber networks, we recapitulate in vivo adhesion organization and confinement to interrogate mitotic outcomes for various interphase cell shapes. Elongated cells attached to single fibers through two focal adhesion clusters (FACs) at their extremities result in perfect spherical mitotic cell bodies that undergo large 3D displacement while being held by retraction fibers. Increasing the number of parallel fibers increases cellular extremity FACs and retraction fiber-driven stability, leading to reduced 3D cell-body movement, metaphase plate rotations, and significantly faster division times. Interestingly, interphase kite shapes on a crosshatch pattern of four fibers undergo mitosis resembling single-fiber outcomes due to rounded bodies being primarily held in position by retraction fibers from two perpendicular suspended fibers. We develop a cortex-astral microtubule analytical friction and force model to capture retraction-fiber-driven stability of the metaphase plate rotations. We report that reduced orientational stability results in increased monopolar mitotic defects. In the case of cells attached to two parallel fibers, rounded mitotic cells can get confined between the suspended fibers, allowing estimation of the mitotic forces through measurement of the outward deflection of the fibers. Interestingly, confinement causes rotated mitotic spindles similar to those reported in dense tissues. Overall, we establish dynamics of mitosis in fibrous environments governed by fiber arrangement and architecture-driven differences in interphase cell shapes, adhesion geometries, and varying levels of mechanical confinement.

Distinctive Kinesin-14 Motors Associate with Midzone Microtubules to Construct Mitotic Spindles with Two Convergent Poles in Arabidopsis

Microtubule (MT) motors in the Kinesin-14 subfamily proliferated in photosynthetic organisms and they often incorporated sequences bearing novel structural features. To gain insights into the functions of diversified Kinesin-14 motors from an evolutionary perspective, we performed phylogenetic analyses across different eukaryotic kingdoms. Compared to fungi that have a single class of Kinesin-14, the early divergent protist Giardia possesses two classes and the motile green alga Chlamydomonas produces four classes (Kinesin-14A to Kinesin-14D). The fifth class Kinesin-14E first appeared among immotile green algae and the sixth Kinesin-14F emerged in mosses, concomitantly with the display of 3D growth. The conservation of Kinesin-14D from green algae prompted us to investigate its function in Arabidopsis in which three such motors functioned in cell cycle-dependent manners. They localized on selective spindle MTs and/or sometimes kinetochore-like structures, and later all became conspicuous on MT bundles in the spindle midzone following sister chromatid segregation. Genetic dissection of Kinesin-14D1 showed that its loss led to hypersensitivity to low doses of the MT-depolymerizing herbicide oryzalin. Kinesin-14D1 association with the midzone MTs in both prophase and mitotic spindles. The oryzalin treatment left behind discrete kinetochore fibers attached to randomly positioned chromosomes in the mitotic kinesin-14d1 cells but prevented the pole convergence of bipolar mitotic spindles. This function of Kinesin-14D1 in the spindle midzone is likely dependent on an MT-binding domain at the C-terminus to the catalytic motor domain. Therefore, our results revealed a novel Kinesin-14D-dependent mechanism that regulates the formation of bipolar spindle apparatus with converged acentrosomal poles.

Three-dimensional structure of the kinetochore-fibers in human mitotic spindles

During cell division, kinetochore microtubules (KMTs) provide a physical linkage between the spindle poles and the chromosomes. KMTs in mammalian cells are organized into bundles, so-called kinetochore-fibers (k-fibers), but the ultrastructure of these fibers is currently not well characterized. Here we show by large-scale electron tomography that each k-fiber in HeLa cells in metaphase is composed of approximately nine KMTs, only half of which reach the spindle pole. Our comprehensive reconstructions allowed us to analyze the three-dimensional (3D) morphology of k-fibers in detail, and we find that they exhibit remarkable variation. K-fibers display differences in circumference and KMT density along their length, with the pole-facing side showing a splayed-out appearance. We further observed that the association of KMTs with non-KMTs predominantly occurs in the spindle pole regions. Our 3D reconstructions have implications for models of KMT behavior and k-fiber self-organization as covered in a parallel publication applying complementary live-cell imaging in combination with biophysical modeling (Conway et al., 2021). The presented data will also serve as a resource for further studies on mitosis in human cells.

Self-organization of kinetochore-fibers in human mitotic spindles

During eukaryotic cell division, chromosomes are linked to microtubules (MTs) in the spindle by a macromolecular complex called the kinetochore. The bound kinetochore microtubules (KMTs) are crucial to ensuring accurate chromosome segregation. Recent electron tomography reconstructions (Kiewisz et al. 2021) captured the positions and configurations of every MT in human mitotic spindles, revealing that many KMTs in these spindles do not reach the pole. Here, we investigate the processes that give rise to this distribution of KMTs using a combination of analysis of the electron tomography reconstructions, photoconversion experiments, quantitative polarized light microscopy, and biophysical modeling. Our results indicate that in metaphase, KMTs grow away from the kinetochores along well-defined trajectories, continually decreasing in speed as they approach the poles. The locations of KMT minus ends, and the turnover and movements of tubulin in KMTs, are consistent with models in which KMTs predominately nucleate de novo at kinetochores and are inconsistent with substantial numbers of non-KMTs being recruited to the kinetochore in metaphase. Taken together, this work leads to a mathematical model of the self-organization of kinetochore-fibers in human mitotic spindles.

Expansion Microscopy Reveals Plasmodium falciparum Blood-Stage Parasites Undergo Anaphase with A Chromatin Bridge in the Absence of Mini-Chromosome Maintenance Complex Binding Protein

The malaria parasite Plasmodium falciparum undergoes closed mitosis, which occurs within an intact nuclear envelope, and differs significantly from its human host. Mitosis is underpinned by the dynamics of microtubules and the nuclear envelope. To date, our ability to study P. falciparum mitosis by microscopy has been hindered by the small size of the P. falciparum nuclei. Ultrastructure expansion microscopy (U-ExM) has recently been developed for P. falciparum, allowing the visualization of mitosis at the individual nucleus level. Using U-ExM, three intranuclear microtubule structures are observed: hemispindles, mitotic spindles, and interpolar spindles. A previous study demonstrated that the mini-chromosome maintenance complex binding-protein (MCMBP) depletion caused abnormal nuclear morphology and microtubule defects. To investigate the role of microtubules following MCMBP depletion and study the nuclear envelope in these parasites, we developed the first nuclear stain enabled by U-ExM in P. falciparum. MCMBP-deficient parasites show aberrant hemispindles and mitotic spindles. Moreover, anaphase chromatin bridges and individual nuclei containing multiple microtubule structures were observed following MCMBP knockdown. Collectively, this study refines our understanding of MCMBP-deficient parasites and highlights the utility of U-ExM coupled with a nuclear envelope stain for studying mitosis in P. falciparum.

Contribution of cytoplasm viscoelastic properties to mitotic spindle positioning

Cells are filled with macromolecules and polymer networks that set scale-dependent viscous and elastic properties to the cytoplasm. Although the role of these parameters in molecular diffusion, reaction kinetics and cellular biochemistry is being increasingly recognized, their contributions to the motion and positioning of larger organelles, such as mitotic spindles for cell division remain unknown. Here, using magnetic tweezers to displace and rotate mitotic spindles in living embryos, we uncovered that the cytoplasm matrix can impart viscoelastic reactive forces that move spindles, or passive objects with similar size, back to their original position. These forces are independent of cytoskeletal force generators, yet reach hundreds of piconewtons and scale with cytoplasm crowding. Spindle motion causes the cytoplasm to shear and rearrange, dissipating elastic energy and limiting spindle recoils with functional implications for asymmetric and oriented divisions. These findings suggest that bulk cytoplasm material properties may constitute important control elements for the regulation of division positioning and cellular organization.

Precise control of microtubule disassembly in living cells

Microtubules (MTs) are components of the evolutionarily conserved cytoskeleton, which tightly regulates various cellular activities. Our understanding of MTs is largely based on MT-targeting agents, which, however, are insufficient to dissect the dynamic mechanisms of specific MT populations due to their slow effects on the entire pool of MTs in cells. To address this limitation, we have used chemogenetics and optogenetics to disassemble specific MT subtypes by rapid recruitment of engineered MT-cleaving enzymes. Acute MT disassembly swiftly halted vesicular trafficking and lysosome dynamics. We also used this approach to disassemble MTs specifically modified by tyrosination and several MT-based structures including primary cilia, mitotic spindles, and intercellular bridges. These effects were rapidly reversed by inhibiting the activity or MT association of the cleaving enzymes. The disassembly of targeted MTs with spatial and temporal accuracy enables to uncover new insights of how MTs precisely regulate cellular architectures and functions.