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Antibodies to the kinesin motor domain and CENP-E inhibit microtubule depolymerization-dependent motion of
V A Lombillo1, C Nislow, T J Yen
1Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder 80309.
Abstract:
Chromosomes can move with the ends of depolymerizing microtubules (MTs) in vitro, even in the absence of nucleotide triphosphates (Coue, M., V. A. Lombillo, and J. R. McIntosh. 1991. J. Cell Biol. 112:1165-1175.) Here, we describe an immunological investigation of the proteins important for this form of motility. Affinity-purified polyclonal antibodies to kinesin exert a severe inhibitory effect on depolymerization-dependent chromosome motion. These antibodies predominantly recognize a polypeptide of M(r) approximately 250 kD on immunoblots of CHO chromosomes and stain kinetochores as well as some vesicles that are in the chromosome preparation. Antibodies to CENP-E, a kinetochore-associated kinesin-like protein, also recognize a 250-kD electrophoretic component, but they stain only the kinetochroe region of isolated chromosomes. Polyclonal antibodies that recognize specific domains of the CENP-E polypeptide affect MT disassembly-dependent chromosome motion in different ways; antibodies to the head or tail portions slow motility threefold, while those raised against the neck region stop motion completely. Analogous antibodies that block conventional, ATP-dependent motility of cytoplasmic dynein (Vaisberg, G., M. P. Koonce, and J. R. McIntosh. 1993. J. Cell Biol. 123:849-858) have no effect on disassembly-dependent chromosome motion, even though they bind to kinetochores. These observations suggest that CENP-E helps couple chromosomes to depolymerizing MTs. A similar coupling activity may allow spindle MTs to remain kinetochore-bound while their lengths change during both prometaphase and anaphase A.
Insights
Kinesin-like protein CENP-E is crucial for chromosome movement during cell division by coupling chromosomes to depolymerizing microtubules. This interaction is essential for chromosome alignment and segregation in mitosis.
Area of Science:
- Cell Biology
- Molecular Biology
- Biophysics
Background:
- Chromosomes move along depolymerizing microtubules (MTs) in vitro without nucleotide triphosphates.
- Understanding the proteins involved in this motility is key to cell division mechanisms.
Purpose of the Study:
- To investigate the proteins responsible for depolymerization-dependent chromosome motility.
- To elucidate the role of kinesin and CENP-E in chromosome-microtubule interactions.
Main Methods:
- Immunological investigation using affinity-purified polyclonal antibodies.
- Immunoblotting to identify protein targets on CHO chromosomes.
- Functional assays to assess the effect of antibodies on chromosome motion.
Main Results:
- Antibodies to kinesin significantly inhibited depolymerization-dependent chromosome motion.
- CENP-E, a kinetochore-associated kinesin-like protein, was identified as a 250-kD component on chromosomes.
- Domain-specific antibodies against CENP-E differentially affected chromosome motility, with neck region antibodies halting motion.
- Antibodies against cytoplasmic dynein did not affect this specific motility.
Conclusions:
- CENP-E plays a critical role in coupling chromosomes to depolymerizing microtubules.
- This coupling mechanism is essential for chromosome dynamics during prometaphase and anaphase A.
- The findings suggest CENP-E is vital for proper chromosome segregation.