A nuclear-derived proteinaceous matrix embeds the microtubule spindle apparatus during mitosis

Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011 Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461 Instituto de Biologia Molecular e Celular, Universidade do Porto, 4150-180 Porto, Portugal.
Molecular biology of the cell (Impact Factor: 4.47). 08/2012; 23(18):3532-41. DOI: 10.1091/mbc.E12-06-0429
Source: PubMed


The concept of a spindle matrix has long been proposed. Whether such a structure exists, however, and what its molecular and structural composition are have remained controversial. In this study, using a live-imaging approach in Drosophila syncytial embryos, we demonstrate that nuclear proteins reorganize during mitosis to form a highly dynamic, viscous spindle matrix that embeds the microtubule spindle apparatus, stretching from pole to pole. We show that this "internal" matrix is a distinct structure from the microtubule spindle and from a lamin B-containing spindle envelope. By injection of 2000-kDa dextran, we show that the disassembling nuclear envelope does not present a diffusion barrier. Furthermore, when microtubules are depolymerized with colchicine just before metaphase the spindle matrix contracts and coalesces around the chromosomes, suggesting that microtubules act as "struts" stretching the spindle matrix. In addition, we demonstrate that the spindle matrix protein Megator requires its coiled-coil amino-terminal domain for spindle matrix localization, suggesting that specific interactions between spindle matrix molecules are necessary for them to form a complex confined to the spindle region. The demonstration of an embedding spindle matrix lays the groundwork for a more complete understanding of microtubule dynamics and of the viscoelastic properties of the spindle during cell division.

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Article: A nuclear-derived proteinaceous matrix embeds the microtubule spindle apparatus during mitosis

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    • "Based on this criterion, several spindle matrix proteins have been identified and studied in the context of spindle assembly and chromosome segregation. For example, among the Drosophilia spindle matrix proteins (Fabian et al., 2007; Johansen et al., 2011; Qi et al., 2004, 2005; Rath et al., 2004; Walker et al., 2000; Yao et al., 2012, 2014), Megator regulates spindle assembly checkpoints (SAC) (Lince-Faria et al., 2009). A conserved protein, BuGZ, which was identified as part of the lamin-B (LB) spindle matrix in Xenopus (Tsai et al., 2006; Ma et al., 2009), has recently been shown to facilitate chromosome alignment by controlling both stability and kinetochore loading of the SAC component Bub3 (Jiang et al., 2014; Toledo et al., 2014). "
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    ABSTRACT: Spindle assembly required during mitosis depends on microtubule polymerization. We demonstrate that the evolutionarily conserved low-complexity protein, BuGZ, undergoes phase transition or coacervation to promote assembly of both spindles and their associated components. BuGZ forms temperature-dependent liquid droplets alone or on microtubules in physiological buffers. Coacervation in vitro or in spindle and spindle matrix depends on hydrophobic residues in BuGZ. BuGZ coacervation and its binding to microtubules and tubulin are required to promote assembly of spindle and spindle matrix in Xenopus egg extract and in mammalian cells. Since several previously identified spindle-associated components also contain low-complexity regions, we propose that coacervating proteins may be a hallmark of proteins that comprise a spindle matrix that functions to promote assembly of spindles by concentrating its building blocks.
    Cell 09/2015; 163:1-15. DOI:10.1016/j.cell.2015.08.010 · 32.24 Impact Factor
    • "When proteins like Megator, soluble tubulin, or Mad2, which are restricted to the nucleus or the cytoplasm during interphase, diffusively equilibrate in all available spaces within the cell after NEB, this consequently leads to an enrichment of these proteins in the less crowded spindle region (Fig. 5 D). While other roles cannot be excluded , the spindle envelope might act as a selective barrier to impede the invasion of the spindle region by large organelles, while retaining Megator complexes (Fig. 5 E) that might show gel-like properties (Yao et al., 2012). Importantly, cyclin B/ Cdk1 and many other mitotic regulators, such as Polo/Plk1, Mad2, and Fizzy/Cdc20, as well as soluble tubulin and chromosomes , accumulate in the spindle region during mitosis (Moutinho-Santos et al., 1999; Raff et al., 2002; Lince-Faria et al., 2009; Hayashi et al., 2012). "
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    ABSTRACT: The mitotic spindle is a microtubular assembly required for chromosome segregation during mitosis. Additionally, a spindle matrix has long been proposed to assist this process, but its nature has remained elusive. By combining live-cell imaging with laser microsurgery, fluorescence recovery after photobleaching, and fluorescence correlation spectroscopy in Drosophila melanogaster S2 cells, we uncovered a microtubule-independent mechanism that underlies the accumulation of molecules in the spindle region. This mechanism relies on a membranous system surrounding the mitotic spindle that defines an organelle-exclusion zone that is conserved in human cells. Supported by mathematical modeling, we demonstrate that organelle exclusion by a membrane system causes spatio-temporal differences in molecular crowding states that are sufficient to drive accumulation of mitotic regulators, such as Mad2 and Megator/Tpr, as well as soluble tubulin, in the spindle region. This membranous "spindle envelope" confined spindle assembly, and its mechanical disruption compromised faithful chromosome segregation. Thus, cytoplasmic compartmentalization persists during early mitosis to promote spindle assembly and function. © 2015 Schweizer et al.
    The Journal of Cell Biology 08/2015; 210(5). DOI:10.1083/jcb.201506107 · 9.83 Impact Factor
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    • "We also studied the relationship between the spindle microtubules and the spindle matrix (Yao et al., 2012; Zheng, 2010) in lamin-A, LAP2α and BAF1 triple-knockdown cells. We observed that the spindle matrix was not only small and morphologically abnormal, but also that the spindle was obviously swollen and separated from the lamin-B spindle matrix in the knockdown cells due to the spindle matrix shrinkage (Fig. 5A,B). "
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    ABSTRACT: Some nuclear proteins crucial in interphase relocate to other places during the G2-M phase transition to take their mitotic functions. However, how they perform these functions and the underlying mechanisms remain largely unknown. Here we report that a fraction of the nuclear periphery proteins lamin-A/C, LAP2α and BAF1 relocate to the spindle and the cell cortex in mitosis. RNAi knockdown of these proteins induces short and fluffy spindle formation and disconnection of the spindle with the cell cortex. Disrupting the microtubule assembly leads to accumulation of these proteins in the cell cortex, whereas depolymerizing the actin microfilaments results in the short spindle formation. We further demonstrate that these proteins are part of a stable complex that links the mitotic spindle to the cell cortex and the spindle matrix by binding with the spindle-associated dynein and the actin filaments in the cell cortex and the spindle matrix. Together, our findings unveil a unique mechanism that the nuclear periphery proteins lamin-A/C, LAP2α and BAF1 are assembled into a protein complex in mitosis to regulate the mitotic spindle assembly and positioning. © 2015. Published by The Company of Biologists Ltd.
    Journal of Cell Science 06/2015; 128(15). DOI:10.1242/jcs.164566 · 5.43 Impact Factor
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