Separating centrosomes interact in the absence of associated chromosomes during mitosis in cultured vertebrate cells

Abstract

We detail here how "free" centrosomes, lacking associated chromosomes, behave during mitosis in PtK(2) homokaryons stably expressing GFP-alpha-tubulin. As free centrosomes separate during prometaphase, their associated astral microtubules (Mts) interact to form a spindle-shaped array that is enriched for cytoplasmic dynein and Eg5. Over the next 30 min, these arrays become progressively depleted of Mts until the two centrosomes are linked by a single bundle, containing 10-20 Mts, that persists for > 60 min. The overlapping astral Mts within this bundle are loosely organized, and their plus ends terminate near its midzone, which is enriched for an ill-defined matrix material. At this time, the distance between the centrosomes is not defined by external forces because these organelles remain stationary when the bundle connecting them is severed by laser microsurgery. However, since the centrosomes move towards one another in response to monastrol treatment, the kinesin-like motor protein Eg5 is involved. From these results, we conclude that separating asters interact during prometaphase of mitosis to form a spindle-shaped Mt array, but that in the absence of chromosomes this array is unstable. An analysis of the existing data suggests that the stabilization of spindle Mts during mitosis in vertebrates does not involve the chromatin (i.e., the RCC1/RanGTP pathway), but instead some other chromosomal component, e.g., kinetochores.

Kinesin 6 family member Subito participates in mitotic spindle assembly and interacts with mitotic regulators

“…A defect in the centrosome-independent spindle assembly pathway may go unnoticed because of the ability of the centrosomes to form bipolar spindles, even in the absence of chromosomes (e.g. Brunet et al, 1998;Bucciarelli et al, 2003;Faruki et al, 2002;Walczak et al, 1998).…”

HR22C16: A Potent Small‐Molecule Probe for the Dynamics of Cell Division

“…However, it has been reported that antimitotic agents, including the Eg5-inhibitor monastrol, take 10-15 minutes to achieve inhibitory concentrations inside cells. [10] Thus, greater temporal control in perturbing this process is critical. Light can be controlled with outstanding temporal and spatial precision to localize photochemistry noninvasively in a biological context.…”

Regulated Assembly of the Mitotic Spindle: A Perspective from Two Ends

“…In C. elegans embyros, Ran is required for spindle assembly, but RCC1 is not (Askjaer et al, 2002). It is likely that centrosomes dominate spindle assembly when they are present and can override any defects in a Ran-dependent stabilization pathway (e.g., Faruki et al, 2002).…”

“…While sufficient to initiate the association between two half spindles, polar microtubule cross-linkages may not be sufficient to maintain a spindle structure. For instance, separated asters devoid of chromosomes initially form a Regulated Assembly of the Mitotic Spindle 105 bipolar spindle-like array, but this structure decays over time (Faruki et al, 2002). Thus, chromosomes remain an important contender in both spindle assembly and maintenance.…”