The Spatial Arrangement of Chromosomes during Prometaphase Facilitates Spindle Assembly

Abstract

Error-free chromosome segregation requires stable attachment of sister kinetochores to the opposite spindle poles (amphitelic attachment). Exactly how amphitelic attachments are achieved during spindle assembly remains elusive. We employed photoactivatable GFP and high-resolution live-cell confocal microscopy to visualize for the first time complete 3-D movements of individual kinetochores throughout mitosis in non-transformed human cells. Combined with electron microscopy, molecular perturbations, and immunofluorescence analyses, this approach reveals unexpected new details of chromosome behavior. Our data demonstrate that unstable lateral interactions between kinetochores and microtubules dominate during early prometaphase. These transient interactions lead to the reproducible arrangement of chromosomes in an equatorial ring on the surface of the nascent spindle. A computational model predicts that this toroidal distribution of chromosomes exposes kinetochores to a high-density of microtubules which facilitates subsequent formation of amphitelic attachments. Thus, spindle formation involves a previously overlooked stage of chromosome prepositioning which promotes formation of amphitelic attachments.

Contributions of Microtubule Dynamic Instability and Rotational Diffusion to Kinetochore Capture

“…The original search-and-capture model supposed that KC attachments to MTs are end-on, consistent with the results of Kalinina et al (24), while others observed primarily lateral attachments (14,19,20,23,25). Therefore, we studied how the probability of lateral or end-on attachment varied with MT length and the presence or absence of rotational diffusion in the model (Fig.…”

“…The original picture of search and capture posited that MTs undergo dynamic instability nucleated from centrosomes until they form end-on attachments with KCs. Additional effects such as diffusion of KCs (12,15), a bias in MT growth toward chromosomes (16)(17)(18), MT nucleation from KCs (15,21,22), spatial distribution and rotation of chromosomes (15,19,20), and KC size decreases after capture (20) can make search and capture more rapid. MT rotational diffusion and lateral capture were found to be important mechanisms in fission yeast (24).…”

“…However, the simplest search-and-capture mechanism does not appear rapid enough to capture multiple chromosomes quickly enough to match measured time in mitosis. Extensions to the search-and-capture mechanism that can make KC capture more rapid in large cells or cell extracts include KC diffusion (12,15), MT growth that is spatially biased toward chromosomes (16)(17)(18), chromosome spatial arrangements and rotation (15,19,20), and KC-initiated MTs that can interact with searching MTs (15,21,22). KCs in human cells change size and shape during mitosis, which can both speed up capture and minimize errors (20).…”

Prime movers: the mechanochemistry of mitotic kinesins

“…This force is thought to be mediated by the polymerization of microtubules against the chromosome and by chromosome-bound motors known as chromokinesins [G] (for review see 132 ). Of these, KIF22 (also known as NOD or KID), a member of the kinesin-10 family, is the most likely mediator of the polar ejection force 133 , although the extent to which KIF22 contributes to chromosome congression is unclear 112,[133][134][135][136][137][138] . Optical trapping microscopy shows KIF22 to be a non-processive motor that steps towards the microtubule plus-end, consistent with a role in mediating the ejection force 139 .…”

Pre-anaphase chromosome oscillations are regulated by the antagonistic activities of Cdk1 and PP1 on Kif18A

“…Upon nuclear envelope breakdown (NEBD), chromosomes attach to the forming spindle and move towards the spindle equator 1 . This motion termed congression 2 results in the alignment of chromosomes at the metaphase plate midway between the two spindle poles.…”