Format

Send to

Choose Destination
Methods Cell Biol. 2018;145:173-190. doi: 10.1016/bs.mcb.2018.03.022. Epub 2018 May 1.

Analyzing the micromechanics of the cell division apparatus.

Author information

1
Center for Frontier Research, National Institute of Genetics, Mishima, Shizuoka, Japan. Electronic address: abdesai@ucsd.edu.
2
Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY, United States. Electronic address: kapoor@mail.rockefeller.edu.

Abstract

Cell division involves mechanical processes, such as chromosome transport and centrosome separation. Quantitative micromanipulation-based approaches have been central to dissecting the forces driving these processes. We highlight two biophysical assays that can be employed for such analyses. First, an in vitro "mini-spindle" assay is described that can be used to examine the collective mechanics of mitotic motor proteins cross-linking two microtubules. In the spindle, motor proteins (e.g., kinesin-5, kinesin-14, and dynein) can localize to overlapping microtubules that slide relative to each other, work as an ensemble, and equilibrate between cytoplasm and the microtubules. The "mini-spindle" assay can recapitulate these features and allows measurements of forces generated between adjacent microtubules and their dependence on filament orientation, sliding speed, overlap length, and motor protein density. Second, we describe a force-calibrated microneedle-based "whole-spindle" micromechanics assay. Microneedle-based micromanipulation can be a useful technique to examine cellular scale mechanics, but its use has been restricted by the difficulty in getting probes to penetrate the plasma membrane without disrupting cell physiology. As detailed here, the use of cell-free extracts prepared from metaphase-arrested Xenopus eggs can address this limitation. These micromanipulation studies also benefit from the use of frozen stocks of Xenopus egg extract. Together, these approaches can be used to decipher how micromechanics and biochemical activities ensure successful cell division.

KEYWORDS:

Force measurement; Force-calibrated microneedles; Frozen extract; In vitro reconstitution; Kinesin-5; Microtubules; Optical tweezers; Spindle assembly; Xenopus egg extract

PMID:
29957203
PMCID:
PMC6029715
[Available on 2019-05-01]
DOI:
10.1016/bs.mcb.2018.03.022
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center