Filling a gap in open mitosis

Mitotic entry in higher eukaryotes is always accompanied by fenestration of the nuclear envelope, allowing mitotic regulators to passively diffuse from the cytoplasm into the "nuclear" space and vice versa. Curiously, several of these proteins have been shown to accumulate in the "nuclear" region although a stationary binding substrate could not be identified. Our recent findings shed light on the underlying mechanism and reveal its importance for accurate cell division.

HFSP Young Investigator Grant holders Iain Cheeseman, Helder Maiato and Matthias Weiss and colleagues
authored on Tue, 13 October 2015

Subcellular compartmentalization into organelles is the defining feature of eukaryotic cells. Among these different compartments, the nucleus is the most prominent structure as it confines the genome by its double-membrane, known as the nuclear envelope (NE). In higher eukaryotes the NE disassembles at least partially at the onset of mitosis. This not only permits centrosome-nucleated microtubules to enter the “nuclear” space, but also allows for the passive influx of mitotic regulators that are required for proper formation of the mitotic spindle, a microtubular assembly that acts as the key mediator of chromosome segregation. Interestingly, several mitotic regulators have been shown to accumulate in the spindle region in a microtubule-independent manner (forming a “spindle matrix”) in different systems, despite the absence of an apparent diffusion barrier or stationary binding substrate. These include soluble α-β-tubulin dimers, the basic building blocks of microtubules, or Mad2 and the conserved nucleoporin Tpr, which are implicated in the spindle assembly checkpoint (SAC), a signalling pathway that prevents premature chromosome segregation.

Figure: Drosophila S2 cell expressing a membrane tagged GFP-CD8 (white) together with mRFP-a-tubulin (cyan) and histone H2B-GFP (magenta). The spindle envelope surrounding the spindle region (magenta) is highlighted. Mitochondria outside the spindle region are also depicted (blue).

In our studies we set out to investigate how proteins are targeted to the spindle region in a microtubule-independent manner in systems where the NE does not remain intact during mitosis and, furthermore, we analyzed whether spatial confinement is coupled to mitotic fidelity. To this end, we combined several experimental approaches, such as live-cell imaging, laser microsurgery, Fluorescence Recovery after Photobleaching or Fluorescence Correlation Spectroscopy, in Drosophila and human culture cells. We found that accumulation of proteins in the spindle region is caused by a membrane system surrounding the spindle that acts as a molecular sieve: while proteins such as Mad2 or soluble tubulin are small enough to freely pass through the holes, membranous organelles and likely other large assemblies are retained in the cytoplasmic compartment. An “excluded volume effect” explains the apparent accumulation of e.g., soluble tubulin and Mad2 in the spindle region: upon NE fenestration small molecules diffuse in all available spaces within the cell and these are, due to the presence of membranous organelles, rarer in the cytoplasmic compartment. Thus, persistent cytoplasmic compartmentalization during mitosis underlies the microtubule-independent accumulation of mitotic regulators in the spindle region [1].

To investigate if confinement of proteins to the spindle region is required for proper chromosome segregation, we used laser microsurgery to disrupt the “spindle envelope” in Drosophila culture cells. We observed that spatial confinement of soluble tubulin and Megator (Drosophila Tpr orthologue) significantly decreased upon this procedure and, moreover, that spindle assembly and chromosome segregation were severely impaired. We thus not only identified a mechanism that underlies the microtubule-independent accumulation of proteins in the spindle region, but also demonstrated that spatial confinement is required for proper cell division [1]. Notably, if accumulation of molecules in the spindle region is merely caused by persistent cytoplasmic compartmentalization, it implies that certain molecules that embed the mitotic spindle might not necessarily be involved in mitotic processes or their confinement might be irrelevant during this stage of the cell cycle. For instance, although Megator is highly concentrated in the spindle region during mitosis in Drosophila cells, we have shown previously that human Tpr promotes the stability of SAC proteins during interphase at the NE and this is required to mount and sustain a robust checkpoint response during mitosis [2]. Together, our findings demonstrate that the spindle region is equipped with unique biochemical properties even in systems undergoing open forms of mitosis and that this is a basic requirement for accurate cell division.

References

[1] An organelle-exclusion envelope assists mitosis and underlies distinct molecular crowding in the spindle region. Schweizer N, Pawar N, Weiss M and Maiato H. J Cell Biol. (2015) 210(5):695-704.

Pubmed link [1]

[2] Spindle assembly checkpoint robustness requires Tpr-mediated regulation of Mad1/Mad2 proteostasis. Schweizer N, Ferrás C, Kern DM, Logarinho E, Cheeseman IM and Maiato H. J Cell Biol. (2013) 203(6):883-93

Pubmed link [2]

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