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The molecular function of Ase1p: evidence for a MAP-dependent midzone-specific spindle matrix. Microtubule-associated
Scott C Schuyler1, Jenny Y Liu, David Pellman
1Department of Pediatric Oncology, The Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA.
Abstract:
The midzone is the domain of the mitotic spindle that maintains spindle bipolarity during anaphase and generates forces required for spindle elongation (anaphase B). Although there is a clear role for microtubule (MT) motor proteins at the spindle midzone, less is known about how microtubule-associated proteins (MAPs) contribute to midzone organization and function. Here, we report that budding yeast Ase1p is a member of a conserved family of midzone-specific MAPs. By size exclusion chromatography and velocity sedimentation, both Ase1p in extracts and purified Ase1p behaved as a homodimer. Ase1p bound and bundled MTs in vitro. By live cell microscopy, loss of Ase1p resulted in a specific defect: premature spindle disassembly in mid-anaphase. Furthermore, when overexpressed, Ase1p was sufficient to trigger spindle elongation in S phase-arrested cells. FRAP revealed that Ase1p has both a very slow rate of turnover within the midzone and limited lateral diffusion along spindle MTs. We propose that Ase1p functions as an MT cross-bridge that imparts matrix-like characteristics to the midzone. MT-dependent networks of spindle midzone MAPs may be one molecular basis for the postulated spindle matrix.
Insights
Budding yeast Ase1p, a microtubule-associated protein (MAP), functions as a cross-bridge in the spindle midzone. Loss of Ase1p causes premature spindle disassembly, while its overexpression promotes spindle elongation.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- The mitotic spindle midzone is crucial for maintaining spindle structure and function during cell division.
- Microtubule (MT) motor proteins are known contributors to midzone function, but the roles of microtubule-associated proteins (MAPs) are less understood.
Purpose of the Study:
- To investigate the role of Ase1p, a conserved midzone-specific MAP, in budding yeast spindle organization and function.
- To elucidate the molecular mechanisms by which Ase1p contributes to spindle midzone integrity and dynamics.
Main Methods:
- Biochemical assays including size exclusion chromatography and velocity sedimentation to characterize Ase1p.
- In vitro microtubule binding and bundling assays.
- Live cell microscopy and Fluorescence Recovery After Photobleaching (FRAP) to analyze Ase1p dynamics in vivo.
Main Results:
- Ase1p exists as a homodimer and binds/bundles microtubules in vitro.
- Loss of Ase1p leads to premature spindle disassembly during mid-anaphase.
- Overexpression of Ase1p induces spindle elongation even in S phase-arrested cells.
- Ase1p exhibits slow turnover and limited lateral diffusion within the spindle midzone.
Conclusions:
- Ase1p acts as a microtubule cross-bridge, conferring matrix-like properties to the spindle midzone.
- Networks of midzone MAPs, including Ase1p, may form the basis of a postulated spindle matrix, essential for spindle stability and elongation.