Cytokinesis and the Spindle Midzone

The mitotic spindle positions the cytokinesis furrow. The cytokinesis furrow then forms and ingresses at the site of the mitotic spindle, between the spindle poles. Two populations of spindle microtubules are implicated in cytokinesis furrow positioning: radial microtubule arrays called asters and bundled non-kinetochore microtubules called the spindle midzone. Here I will discuss our recent results that provided examples of how aster-positioned and midzone-positioned cytokinesis can be mechanically and genetically separated. These experiments illustrate how asters and midzone contribute to cytokinesis. ASS (asymmetric spindle severing) is a mechanical way to spatially separate the aster and midzone signals. In Caenorhabditis elegans embryos, asters and midzone provide two consecutive signals that position the cytokinesis furrow. The first signal is positioned midway between the microtubule asters; the second signal is positioned over the spindle midzone. Aster and midzone contribution can also be genetically separated. Mutants in spd-1 have no detectable midzone and are defective in midzone-positioned but not aster-positioned cytokinesis. Disruption of the function of LET-99 and the heterotrimeric G-proteins GOA-1/GPA-16 and their regulator GPR-1/2 causes defects in aster-positioned cytokinesis but not in midzone-positioned cytokinesis. In order to understand aster-positioned cytokinesis we have to understand how microtubule asters spatially control the activity of LET-99, GPR-1/2 and GOA-1/GPA-16 and how the activity of these G-protein pathway components control the assembly of a contractile ring.

v-Src causes delocalization of Mklp1, Aurora B, and INCENP from the spindle midzone during cytokinesis failure

During each cell cycle, the mitotic spindle is efficiently assembled to achieve chromosome segregation and then rapidly disassembled as cells enter cytokinesis. Although much has been learned about assembly, how spindles disassemble at the end of mitosis remains unclear. Here we demonstrate that nucleocytoplasmic transport at the membrane domain surrounding the mitotic spindle midzone, here named the midzone membrane domain (MMD), is essential for spindle disassembly in Schizosaccharomyces pombe cells. We show that, during anaphase B, Imp1-mediated transport of the AAA-ATPase Cdc48 protein at the MMD allows this disassembly factor to localize at the spindle midzone, thereby promoting spindle midzone dissolution. Our findings illustrate how a separate membrane compartment supports spindle disassembly in the closed mitosis of fission yeast.

Prior to the cytokinesis, the cell-matrix interactions should be disrupted, and the mitotic cells round up. Prerequisite of mitosis, the centrosomes duplicate, spindle fibers are generated and move away from each other to opposite sides of the cells marking the cell poles. Later, an invagination in the plasma membrane is formed a few minutes after anaphase. This furrow ingression is driven by a contractile actomyosin ring, whose assembly is regulated by RhoA GTPase. At the completion of cytokinesis, the two daughter cells are still connected by a thin intercellular bridge, which is subjected to abscission, as the terminal step of cytokinesis. Here, it is overviewed, how syndecan-4, a transmembrane, heparan sulfate proteoglycan, can contribute to these processes in a phosphorylation-dependent manner.

Segregation of chromosomes during cell division requires correct formation of mitotic spindles. Here, we show that a scaffold attachment factor A (SAF-A), also known as heterogeneous nuclear ribonucleoprotein-U, contributes to the attachment of spindle microtubules (MTs) to kinetochores and spindle organization. During mitosis, SAF-A was localized at the spindles, spindle midzone and cytoplasmic bridge. Depletion of SAF-A by RNA interference induced mitotic delay and defects in chromosome alignment and spindle assembly. We found that SAF-A specifically co-immunoprecipitated with the chromosome peripheral protein nucleolin and the spindle regulators Aurora-A and TPX2, indicating that SAF-A is associated with nucleolin and the Aurora-A-TPX2 complex. SAF-A was colocalized with TPX2 and Aurora-A in spindle poles and MTs. Elimination of TPX2 or Aurora-A from cells abolished the association of SAF-A with the mitotic spindle. Interestingly, SAF-A can bind to MTs and contributes to the targeting of Aurora-A to mitotic spindle MTs. Our finding indicates that SAF-A is a novel spindle regulator that plays an essential role in kinetochore-MT attachment and mitotic spindle organization.

Cytokinesis occurs by the ingression of an actomyosin ring that separates the cell into two daughter cells. The mitotic spindle, comprising astral and central spindle microtubules, couples contractile ring ingression with DNA segregation. Cues from the central spindle activate RhoA, the upstream regulator of the contractile ring. However, additional cues from the astral microtubules also reinforce the localization of active RhoA. Using human cells, we show that astral and central spindle microtubules independently control the localization of contractile proteins during cytokinesis. Astral microtubules restrict the accumulation and localization of contractile proteins during mitosis, whereas the central spindle forms a discrete ring by directing RhoA activation in the equatorial plane. Anillin stabilizes the contractile ring during cytokinesis. We show that human anillin interacts with astral microtubules and that this interaction is competed by the cortical recruitment of anillin by active RhoA. Anillin restricts the localization of myosin to the equatorial cortex and that of NuMA (part of the microtubule-tethering complex that regulates spindle position) to the polar cortex. The sequestration of anillin by astral microtubules might alter the organization of cortical proteins to polarize cells for cytokinesis.

Kinetochore Orientation during Meiosis Is Controlled by Aurora B and the Monopolin Complex

Accurate chromosome segregation during cell division relies on coordinated actions of microtubule (MT)-based motor proteins in the mitotic spindle. Kinesin-14 motors play vital roles in spindle assembly and maintenance by crosslinking antiparallel MTs at the spindle midzone and anchoring spindle MTs’ minus ends at the poles. In this study, we investigate the force generation and motility of the Kinesin-14 motors HSET and KlpA. Our findings reveal that both motors are non-processive, producing single load-dependent power strokes per MT encounter, with estimated load-free power strokes of ~30 and ~35 nm, respectively. Each homodimeric motor generates forces of ~0.5 pN, but when assembled in teams, they cooperate to generate forces of 1 pN or more. Notably, the cooperative activity among multiple motors leads to increased MT-sliding velocities. These results quantitatively elucidate the structure-function relationship of Kinesin-14 motors and underscore the significance of cooperative behavior in their cellular functions.

Recent work has underscored the importance of membrane trafficking events during cytokinesis. For example, targeted membrane secretion occurs at the cleavage furrow in animal cells, and proteins that regulate endocytosis also influence the process of cytokinesis. Nonetheless, the prevailing dogma is that endosomal membrane trafficking ceases during mitosis and resumes after cell division is complete. In this study, we have characterized endocytic membrane trafficking events that occur during mammalian cell cytokinesis. We have found that, although endocytosis ceases during the early stages of mitosis, it resumes during late mitosis in a temporally and spatially regulated pattern as cells progress from anaphase to cytokinesis. Using fixed and live cell imaging, we have found that, during cleavage furrow ingression, vesicles are internalized from the polar region and subsequently trafficked to the midbody area during later stages of cytokinesis. In addition, we have demonstrated that cytokinesis is inhibited when clathrin-mediated endocytosis is blocked using a series of dominant negative mutants. In contrast to previous thought, we conclude that endocytosis resumes during the later stages of mitosis, before cytokinesis is completed. Furthermore, based on our findings, we propose that the proper regulation of endosomal membrane traffic is necessary for the successful completion of cytokinesis.

Cells with blocked microtubule polymerization are delayed in mitosis, but eventually manage to proliferate despite substantial chromosome missegregation. While several studies have analyzed the first cell division after microtubule depolymerization, we have asked how cells cope long-term with microtubule impairment. We allowed 24 clonal populations of yeast cells with beta-tubulin mutations preventing proper microtubule polymerization, to evolve for ˜150 generations. At the end of the laboratory evolution experiment, cells had regained the ability to form microtubules and were less sensitive to microtubule-depolymerizing drugs. Whole-genome sequencing identified recurrently mutated genes, in particular for tubulins and kinesins, as well as pervasive duplication of chromosome VIII. Recreating these mutations and chromosome VIII disomy prior to evolution confirmed that they allow cells to compensate for the original mutation in beta-tubulin. Most of the identified mutations did not abolish function, but rather restored microtubule functionality. Analysis of the temporal order of resistance development in independent populations repeatedly revealed the same series of events: disomy of chromosome VIII followed by a single additional adaptive mutation in either tubulins or kinesins. Since tubulins are highly conserved among eukaryotes, our results have implications for understanding resistance to microtubule-targeting drugs widely used in cancer therapy.

Mitotic spindles are mechanical structures that play a critical role in cell division by generating forces to separate chromosomes. They are ordered assemblages of proteins that make up microtubules (MT) and microtubule connectors whose mechanical properties are responsible for their structural integrity under mitotic forces. We use a continuum mechanics approach to study the stability of equilibrium of a mitotic spindle as a whole. We create and apply a finite element (FE) parameterised model of interpolar MTs, astral MTs and MT connectors varying the number of MT filaments and the arrangement of their interconnections. The model is based on the experimental data on Fission Yeast spindles in late anaphase B and mitotic HeLa cells [1]–[3]. We account for the complex interactions between interpolar MTs, astral MTs, connectors and centrosomes through mechanical coupling. Comparing the results with experiments and Molecular dynamics-based simulations [1], we demonstrate the great potential of Structural mechanics methods to address the stability of spindles. Here we report how buckling states of the spindle get localised towards either of centrosomes due to the irregular placement of microtubules and irregularities in MT coupling. In certain cases, such behaviour may result in nuclear misplacement, asymmetric division or other abnormalities. [1] J. J. Ward, H. Roque, C. Antony, and F. Nedelec, “Mechanical design principles of a mitotic spindle,” eLife, vol. 3, p. e03398, 2014. [2] F. Pampaloni, G. Lattanzi, A. Jonas, T. Surrey, E. Frey, and E.-L. Florin, “Thermal fluctuations of grafted microtubules provide evidence of a length-dependent persistence length,” Proc. Natl. Acad. Sci., vol. 103, no. 27, pp. 10248–10253, 2006. [3] F. M. Nixon, C. Gutierrez-Caballero, F. E. Hood, D. G. Booth, I. A. Prior, and S. J. Royle, “The mesh is a network of microtubule connectors that stabilizes individual kinetochore fibers of the mitotic spindle,” eLife, vol. 4, no. JUNE2015, pp. 1–21, 2015.

ABSTRACTDuring closed mitosis inS. pombe, the nuclear envelope and mitotic spindle must work together mechanically and biochemically to ensure successful nuclear division. Previous work has demonstrated that mechanical force from the nuclear envelope, transmitted through spindle pole bodies, can re-shape the spindle. However, it remains unclear how force might alter spindle biochemistry. Here, we investigate how force reprograms the spindle with two approaches: chronically increasing nuclear envelope tension via the lipid synthesis inhibitor cerulenin, and acutely applying force through an optical trap. Both perturbations slow elongation dynamics and reduce microtubule number. Despite this reduction, key spindle proteins Ase1 and Klp5 increase their density at the spindle midzone, indicating inward force from the nuclear envelope can alter spindle biochemistry. We find that while motor proteins Klp5 and Klp6 only minimally affect the spindle’s response to increased nuclear envelope force, the combination of removing Ase1 and increasing nuclear envelope force together rescue spindle stability. Together, our findings reveal that nuclear force on the spindle does not merely alter its shape, but is key in regulating its biochemistry to maintain force balance.

Successful reactivation in vitro of anaphase B has recently been achieved with mitotic spindles isolated from the diatom Stephanopyxis turris. When a population of isolated spindles was studied indirectly by using immunofluorescence, nearly all of them were found to have elongated; however, when studied directly by using video microscopy, only a small proportion of spindles elongated. We report here conditions that allow nearly all of the spindles to elongate when observed directly with video microscopy. These direct observations validate previous ones made using indirect immunofluorescence. In addition, we find that the isolated spindles elongate with a linear rate, that the elongation is unchanged after the chromatin surrounding the spindles is digested with DNase I, and that during elongation a phase-dense matrix may accumulate in the spindle midzone.

Through mutational analysis in Drosopjila we have identified the gene multiple asters (mast), which encodes a new 165 kDa protein. mast mutant neuroblasts are highly polyploid and show severe mitotic abnormalities including the formation of mono- and multi-polar spindles organized by an irregular number of microtubule-organizing centres of abnormal size and shape. The mast gene product is evolutionarily conserved since homologues were identified from yeast to man, revealing a novel protein family. Antibodies against Mast and analysis of tissue culture cells expressing an enhanced green fluorescent protein-Mast fusion protein show that during mitosis, this protein localizes to centrosomes, the mitotic spindle, centromeres and spindle midzone. Microtubule-binding assays indicate that Mast is a microtubule-associated protein displaying strong affinity for polymerized microtubules. The defects observed in the mutant alleles and the intracellular localization of the protein suggest that Mast plays an essential role in centrosome separation and organization of the bipolar mitotic spindle.

Previous genetic and biochemical studies have led to the hypothesis that the essential mitotic bipolar kinesin, KLP61F, cross-links and slides microtubules (MTs) during spindle assembly and function. Here, we have tested this hypothesis by immunofluorescence and immunoelectron microscopy (immunoEM). We show that Drosophila embryonic spindles at metaphase and anaphase contain abundant bundles of MTs running between the spindle poles. These interpolar MT bundles are parallel near the poles and antiparallel in the midzone. We have observed that KLP61F motors, phosphorylated at a cdk1/cyclin B consensus domain within the BimC box (BCB), localize along the length of these interpolar MT bundles, being concentrated in the midzone region. Nonphosphorylated KLP61F motors, in contrast, are excluded from the spindle and display a cytoplasmic localization. Immunoelectron microscopy further suggested that phospho-KLP61F motors form cross-links between MTs within interpolar MT bundles. These bipolar KLP61F MT-MT cross-links should be capable of organizing parallel MTs into bundles within half spindles and sliding antiparallel MTs apart in the spindle midzone. Thus we propose that bipolar kinesin motors and MTs interact by a “sliding filament mechanism” during the formation and function of the mitotic spindle.