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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2006.12.030 Byline: Marcel E. Janson (1), Rose Loughlin (2), Isabelle LoA[macron]odice (1), Chuanhai Fu (1), Damian Brunner (2), Francois J. Nedelec (2), Phong T. Tran (1) Abstract: Microtubule (MT) nucleation not only occurs from centrosomes, but also in large part from dispersed nucleation sites. The subsequent sorting of short MTs into networks like the mitotic spindle requires molecular motors that laterally slide overlapping MTs and bundling proteins that statically connect MTs. How bundling proteins interfere with MT sliding is unclear. In bipolar MT bundles in fission yeast, we found that the bundler ase1p localized all along the length of antiparallel MTs, whereas the motor klp2p (kinesin-14) accumulated only at MT plus ends. Consequently, sliding forces could only overcome resistant bundling forces for short, newly nucleated MTs, which were transported to their correct position within bundles. Ase1p thus regulated sliding forces based on polarity and overlap length, and computer simulations showed these mechanisms to be sufficient to generate stable bipolar bundles. By combining motor and bundling proteins, cells can thus dynamically organize stable regions of overlap between cytoskeletal filaments. Author Affiliation: (1) Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA 19104, USA (2) Cell Biology and Biophysics Program, European Molecular Biology Laboratory, 69117 Heidelberg, Germany Article History: Received 29 June 2006; Revised 4 October 2006; Accepted 26 December 2006 Article Note: (miscellaneous) Published: January 25, 2007

Ingestion or injection of gossypol suppresses sperm production and sperm motility in vivo (reviewed in ref. 1). We report here an in vitro assay for observing gossypol action on individual [...]

Microtubules exhibit dynamic instability in vivo and in vitro (with purified tubulin), stochastically switching between phases of persistent growth and shortening (reviewed in ref. 1). The growth phase is governed [...]

The polarized light microscope has traditionally been an important tool for studying fine structure in living cells (1, 2). However, conventional design allows for retardance imaging only in a narrow [...]

Cellular morphogenesis relies partly on cell polarization by the cytoskeleton. In the fission yeast Schizosaccharomyces pombe, it is well established that microtubules (MTs) deliver the spatial cue Teal, a kelch repeat protein, to the tip regions to direct the growth machinery at the cell tips driving the linear extension of the rod-shaped organism to maintain a straight long axis. Here, we report the characterization of Knk1 (kink), a previously unidentified member of the superfamily of ATPases associated with various cellular activities (AAA+), whose deletion causes a unique morphological defect characterized by the formation of kinks close to cell tips. Through genetic analysis, we place Knk1 into a novel pathway controlling cell shape independently of MTs and Teal. Knk1 localizes at cell tips. Its localization is mediated by the Knk1 N terminus and is enhanced upon ATP binding to the C-terminal ATPase domain. Furthermore, Knk1 tip recruitment is regulated by SRC-like adaptor 2 (Sla2) and cell division cycle 42 (Cdc42) independently of Sla2's role in endocytosis. Finally, we discovered that Knk1 shows an anticorrelated oscillatory behavior between the two cell tips at a periodicity that is different from the reported oscillatory Cdc42 dynamics. [ABSTRACT FROM AUTHOR]

The fission yeast Schizosaccharomyces pombe serves as a good genetic model organism for the molecular dissection of the microtubule (MT) cytoskeleton. However, analysis of the number and distribution of individual MTs throughout the cell cycle, particularly during mitosis, in living cells is still lacking, making quantitative modelling imprecise. We use quantitative fluorescent imaging and analysis to measure the changes in tubulin concentration and MT number and distribution throughout the cell cycle at a single MT resolution in living cells. In the wild-type cell, both mother and daughter spindle pole body (SPB) nucleate a maximum of 23 ± 6 MTs at the onset of mitosis, which decreases to a minimum of 4 ± 1 MTs at spindle break down. Interphase MT bundles, astral MT bundles, and the post anaphase array (PAA) microtubules are composed primarily of 1 ± 1 individual MT along their lengths. We measure the cellular concentration of αβ-tubulin subunits to be ~5 µM throughout the cell cycle, of which one-third is in polymer form during interphase and one-quarter is in polymer form during mitosis. This analysis provides a definitive characterization of αβ-tubulin concentration and MT number and distribution in fission yeast and establishes a foundation for future quantitative comparison of mutants defective in MTs. [ABSTRACT FROM AUTHOR]

Cellular processes rely on proteins with temperature-dependent stability and activity. While thermosensitivity in biological networks is well-explored, the effect of temperature on complex mechanochemical assemblies, like the spindle, is rarely studied. We examined fission yeast spindle dynamics and chromosome segregation from 15⁰C to 40⁰C. Our findings reveal that these parameters follow U-shaped temperature-dependent curves but reach their minima at different temperatures. Specifically, spindle dynamics peak around 35⁰C, whereas chromosome segregation defects are minimized at 25⁰C. This suggests a scenario in which mitotic errors are tolerated to expedite rapid cell cycle progression., Competing Interests: The authors declare that there are no conflicts of interest present., (Copyright: © 2024 by the authors.)

Mitosis is usually shorter than other phases of the cell cycle and maintains a consistent duration despite variations in cell size and spindle size. This suggests the existence of a compensatory mechanism that ensures a short duration, possibly as a protective measure against irreversible damage, such as DNA damage. To explore the link between prolonged mitosis and DNA damage, we develop a microscopy-based assay utilizing Rad52-GFP as a marker for mitotic DNA damage. Through this assay, we provide evidence that mutants with prolonged mitosis exhibit increased Rad52 puncta, indicating an elevation in endogenous DNA damage., Competing Interests: The authors declare that there are no conflicts of interest present., (Copyright: © 2023 by the authors.)

We previously showed that the silkworm holocentric spindles are square-shaped, compared to the canonical oval shape of human monocentric spindles (Vanpoperinghe et al. 2021). Further, while kinesin-5 depletion resulted in monopolar spindles in both cells, kinesin-14 depletion affected only the silkworm cells, resulting in mal-shaped spindles (Vanpoperinghe et al. 2021). We now extend our study to quantify the effect of kinesin-5 and kinesin-14 on spindle assembly dynamics and chromosome segregation in holocentric silkworm BmN4 cells. We find that mal-shaped spindle and prolonged mitosis duration are highly correlated with chromosome segregation error, leading to aneuploidy and cell death in BmN4 cells. Further, double RNAi-mediated depletion of kinesin-5 and kinesin-14 partially rescue the monopolar spindle and mal-shaped spindle phenotypes in kinesin-5 and kinesin 14-depleted cells, respectively., (Copyright: © 2022 by the authors.)

Human retinal pigment epithelium RPE-1 cells are immortalized diploid wild-type cells. RPE-1 is increasingly used for studies of spindle assembly dynamics and chromosome segregation. Here, we imaged living RPE-1 cells using the spinning disk confocal microscope and report their complete spindle assembly dynamic parameters. Live-cell experiments enabled ascribing precise timing of function of the kinesin-5 Eg5 and kinesin-14 HSET throughout different phases of mitosis. Eg5 functions at prophase and metaphase, to assemble and maintain spindle bipolarity, respectively. Eg5 inhibition results in spindle collapse during prophase and metaphase, resulting in monoastral/monopolar spindles. HSET functions throughout mitosis to maintain spindle length. HSET degradation results in shorter spindles through all phases of mitosis. Double-inhibition of Eg5 and HSET produces only monoastral/monopolar spindles, indicating that Eg5 and HSET may not be antagonistic in wild-type RPE-1 cells, contrary to previous studies using cancer cells. In the context of spindle assembly, our results highlight potential important differences between RPE-1 and other cancer-derived cell lines., (Copyright: © 2022 by the authors.)

Proper chromosome segregation during mitosis requires both the assembly of a microtubule (MT)-based spindle and the assembly of DNA-centromere-based kinetochore structure. Kinetochore-to-MT attachment enables chromosome separation. Monocentric cells, such as found in human, have one unique kinetochore per chromosome. Holocentric cells, such as found in the silkworm, in contrast, have multiple kinetochore structures per chromosome. Interestingly, some human cancer chromosomes contain more than one kinetochore, a condition called di- and tricentric. Thus, comparing how wild-type mono- and holocentric cells perform mitosis may provide novel insights into cancer di- and tricentric cell mitosis. We present here live-cell imaging of human RPE1 and silkworm BmN4 cells, revealing striking differences in spindle architecture and dynamics, and highlighting differential kinesin function between mono- and holocentric cells., (Copyright: © 2021 by the authors.)

To segregate the chromosomes faithfully during cell division, cells assemble a spindle that captures the kinetochores and pulls them towards opposite poles. Proper spindle function requires correct interplay between microtubule motors and non-motor proteins. Defects in spindle assembly or changes in spindle dynamics are associated with diseases, such as cancer or developmental disorders. Here, we compared mitotic and meiotic spindles in fission yeast. We show that, even though mitotic and meiotic spindles underwent the typical three phases of spindle elongation, they have distinct features. We found that the relative concentration of the kinesin-14 family protein Pkl1 is decreased in meiosis I compared to mitosis, while the concentration of the kinesin-5 family protein Cut7 remains constant. We identified the second kinesin-14 family protein Klp2 and microtubule dynamics as factors necessary for proper meiotic spindle assembly. This work defines the differences between mitotic and meiotic spindles in fission yeast Schizosaccharomyces pombe , and provides prospect for future comparative studies.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)