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Mitotic HeLa cells contain a CENP-E-associated minus end-directed microtubule motor
D A Thrower1, M A Jordan, B T Schaar
1Department of Biological Sciences, University of California, Santa Barbara 93106.
Abstract:
A minus end-directed microtubule motor activity from extracts of HeLa cells blocked at prometaphase/metaphase of mitosis with vinblastine has been partially purified and characterized. The motor activity was eliminated by immunodepletion of Centromere binding protein E (CENP-E). The CENP-E-associated motor activity, which was not detectable in interphase cells, moved microtubules at mean rates of 0.46 micron/s at 37 degrees C and 0.24 micron/s at 25 degrees C. The motor activity co-purified with CENP-E through several purification procedures. Motor activity was clearly not due to dynein or to kinesin. The microtubule gliding rates of the CENP-E-associated motor were different from those of dynein and kinesin. In addition, the pattern of nucleotide substrate utilization by the CENP-E-associated motor and the sensitivity to inhibitors were different from those of dynein and kinesin. The CENP-E-associated motor had an apparent native molecular weight of 874,000 Da and estimated dimensions of 2 nm x 80 nm. This is the first demonstration of motor activity associated with CENP-E, strongly supporting the hypothesis that CENP-E may act as a minus end-directed microtubule motor during mitosis.
Insights
Researchers identified a novel minus end-directed microtubule motor activity linked to Centromere binding protein E (CENP-E) in mitotic HeLa cells. This CENP-E motor function, distinct from dynein and kinesin, plays a role in mitosis.
Area of Science:
- Cell Biology
- Molecular Motors
- Mitosis
Background:
- Microtubule motor proteins are crucial for cellular processes, including chromosome segregation during mitosis.
- Existing motors like dynein and kinesin have distinct roles and characteristics.
- The specific motor proteins involved in precise mitotic movements, particularly those associated with kinetochores, are not fully elucidated.
Purpose of the Study:
- To identify and characterize novel microtubule motor activities present in mitotic cell extracts.
- To determine the molecular identity of a minus end-directed motor activity observed during mitosis.
- To investigate the potential role of Centromere binding protein E (CENP-E) as a microtubule motor.
Main Methods:
- Partial purification and characterization of microtubule motor activity from vinblastine-arrested HeLa cell extracts.
- Immunodepletion using antibodies against Centromere binding protein E (CENP-E) to assess its involvement.
- Microtubule gliding assays to measure motor velocity, nucleotide substrate utilization, and inhibitor sensitivity.
- Determination of the molecular weight and dimensions of the purified motor complex.
Main Results:
- A minus end-directed microtubule motor activity was isolated from mitotic HeLa cell extracts.
- This motor activity was specifically eliminated upon immunodepletion of CENP-E, indicating its association.
- The CENP-E-associated motor exhibited distinct kinetic properties (velocity, substrate use, inhibitor sensitivity) compared to dynein and kinesin, and was not detected in interphase cells.
- The motor complex had a high molecular weight (874,000 Da) and specific dimensions (2 nm x 80 nm).
Conclusions:
- This study provides the first evidence of motor activity directly associated with CENP-E.
- The findings strongly support the hypothesis that CENP-E functions as a minus end-directed microtubule motor during mitosis.
- CENP-E's motor function likely contributes to the precise movements of chromosomes and other structures during cell division.