Abstract/Details

Mechanics of mitotic spindle poles and polymerization dynamics of microtubules


2011 2011

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Abstract (summary)

During cell division, chromosomes must faithfully segregate to maintain genome integrity, and this dynamic mechanical process is driven by the macromolecular machinery of the mitotic spindle. However, little is known about spindle mechanics. For example, spindle microtubules are organized by numerous cross-linking proteins yet the mechanical properties of those cross-links remain unexplored. To examine the mechanical properties of microtubule cross-links we applied optical trapping to mitotic asters that form in mammalian mitotic extracts. These asters are foci of microtubules, motors, and microtubule-associated proteins that reflect many of the functional properties of spindle poles and represent centrosome-independent spindle-pole analogs. We observed bidirectional motor-driven microtubule movements, showing that microtubule linkages within asters are remarkably compliant (mean stiffness 0.025 pN/nm) and mediated by only a handful of cross-links. Depleting the motor Eg5 reduced this stiffness, indicating that Eg5 contributes to the mechanical properties of microtubule asters in a manner consistent with its localization to spindle poles in cells. We propose that compliant linkages among microtubules provide a mechanical architecture capable of accommodating microtubule movements and distributing force among microtubules without loss of pole integrity—a mechanical paradigm that may be important throughout the spindle.

Microtubule assembly and disassembly are vital for many fundamental cellular processes. Our current understanding of microtubule assembly kinetics is based on a one-dimensional assembly model, which assumes identical energetics for subunits exchanging at the tip. In this model, the subunit disassociation rate from a microtubule tip is independent of free subunit concentration. Using total-internal-reflection fluorescence (TIRF) microscopy and an optical tweezers assay to measure in vitro microtubule assembly with nanometer resolution, we find that the subunit dissociation rate from a microtubule tip increases at higher free subunit concentrations. This is because, as predicted by Hill, there is a shift in microtubule tip structure from relatively blunt at low free subunit concentrations to relatively tapered at high concentrations, which we confirmed experimentally by TIRF microscopy. Because both the association and the dissociation rates increase with free subunit concentrations, we find that the kinetics of microtubule assembly are an order of magnitude faster than currently estimated in the literature.

Indexing (details)


Subject
Cellular biology;
Biomedical engineering;
Biophysics
Classification
0379: Cellular biology
0541: Biomedical engineering
0786: Biophysics
Identifier / keyword
Applied sciences; Biological sciences; Kinesin; Microtubules; Mitotic spindles; Optical tweezers
Title
Mechanics of mitotic spindle poles and polymerization dynamics of microtubules
Author
Charlebois, Blake D.
Number of pages
176
Publication year
2011
Degree date
2011
School code
0127
Source
DAI-B 73/04, Dissertation Abstracts International
Place of publication
Ann Arbor
Country of publication
United States
ISBN
9781267133700
Advisor
Hunt, Alan J.
University/institution
University of Michigan
University location
United States -- Michigan
Degree
Ph.D.
Source type
Dissertations & Theses
Language
English
Document type
Dissertation/Thesis
Dissertation/thesis number
3492754
ProQuest document ID
918226818
Copyright
Database copyright ProQuest LLC; ProQuest does not claim copyright in the individual underlying works.
Document URL
http://search.proquest.com/docview/918226818
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