Membrane-based mechanisms of mitotic spindle assembly

Abstract

Chromosome segregation during mitosis is mediated by the mitotic spindle. Formation of this microtubular structure relies on distinct processes such as microtubule nucleation and growth and the consequent focusing of these filaments into spindle poles. Here, we discuss our recent finding that a size-exclusion spindle envelope promotes mitotic fidelity in Drosophila cells in light of distinct spindle assembly mechanisms.

During mitosis a replicated set of chromosomes needs to be accurately segregated to opposite sides of the cell. This process is mediated by the mitotic spindle, a structure composed of microtubules and a variety of associated proteins. Microtubule polymerization requires specific microtubule nucleators which regulate microtubule assembly from the so-called microtubule organizing centers (MTOCs).¹ The major microtubule nucleator is the γ-tubulin ring complex (γTuRC), a multi-protein complex assembled from γ-tubulin and gamma complex proteins (GCPs) 2-6 which have been proposed to laterally associate and coordinate a helical arrangement of γ-tubulin molecules that acts as a template for the addition of α/β-tubulin heterodimers, the basic building blocks of microtubules.² The major MTOC in animal somatic cells is the centrosome, an organelle that replicates during interphase to organize into 2 opposite spindle poles during mitosis. However, microtubule nucleation can additionally be initiated at acentrosomal sites to which γTuRC is recruited.³ For instance, microtubules can be nucleated along pre-existing (spindle) microtubules, a process that depends on the augmin complex.⁴ More recently, membranous organelles or structures such as the Golgi or endosomes, which have a well-established role as an MTOC during interphase or in membrane trafficking, respectively, have been implicated in spindle formation by acting as mitotic MTOCs.^5-7 Importantly, our recent findings in Drosophila and human culture cells revealed that membranous organelles are excluded from the spindle region by a membrane system that surrounds the mitotic spindle.⁸ This elastic “spindle envelope” that derives from the endoplasmic reticulum/nuclear envelope (NE) might additionally help focusing microtubules into spindle poles.^9,10 Furthermore, mathematical modeling predicts that spatial confinement of microtubules and molecular motors, possibly by mitotic membranes, is required for proper spindle formation.^10,11 Importantly, we have demonstrated that the “spindle envelope” in Drosophila cells neither provides a diffusion barrier for nuclear-derived mitotic regulators, such as the spindle assembly checkpoint (SAC) protein Mad2, nor for soluble tubulin dimers that passively enter the “nuclear” space at the prophase-prometaphase transition.⁸ Interestingly, these proteins accumulate in the spindle region in a microtubule-independent manner, without binding to a stationary substrate. Although the “spindle envelope” is irregularly fenestrated and open at spindle poles allowing the passive influx/efflux of e.g. soluble tubulin or Mad2, large assemblies such as membranous organelles (or their fragments) are retained in the cytoplasm. Upon NE fenestration at the onset of mitosis, 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, the mitotic cytoplasm cannot be regarded as a homogenous visco-elastic fluid as generally assumed, but the spindle region provides a distinct biochemical milieu. We further demonstrated that the spindle region in Drosophila cells is equipped with certain properties that favor spindle assembly.⁸ Severe disruption of the spindle envelope during early mitosis by laser microsurgery not only led to a significant decrease in spatial confinement of e.g., soluble tubulin, but also triggered acentrosomal microtubule nucleation in the proximity of the cut. These microtubules grew into the “nuclear” region and into the cytoplasm which seriously affected spindle assembly and consequently chromosome segregation. Thus, a size-exclusion spindle envelope might be as important for spindle assembly in Drosophila cells as a RanGTP gradient is required for proper spindle formation in other systems, where high levels of RanGTP stimulate microtubule nucleation/growth in the vicinity of chromatin.¹² In the future it will be important to elucidate how exactly persistent cytoplasmic compartmentalization underlies the spatial confinement of spindle assembly and, moreover, whether additional mitotic processes rely on the presence of a size-exclusion spindle envelope.

Figure 1.

Model of how membranous structures contribute to mitotic spindle assembly. (A) A fenestrated, membranous spindle envelope (red) excludes large membranous structures (green) from the spindle region (bright yellow) where the chromosomes (white) are located. This drives accumulation of small molecules (e.g. soluble tubulin) in the spindle region and equips this area with unique biochemical properties that favor the nucleation/growth of microtubules (black). The spindle envelope might also help focusing microtubules into 2 spindle poles. Membranous structures which are located in the cytoplasmic compartment (light yellow) might also contribute to spindle formation directly. (B) Drosophila S2 cell expressing mRFP-α-tubulin (red), histone H2B-GFP (green) and GFP-CD8 (green), marking the mitotic spindle, chromosomes and the spindle envelope, respectively.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed

Funding

Work in the H. Maiato laboratory is funded by the seventh framework program grant PRECISE from the European Research Council, the FLAD Life Science 2020 and the Louis-Jeantet Young Investigator Career Award.