Removal of the projections from cytoplasmic microtubules in vitro by digestion with trypsin
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Published on: March 16, 2014
Abstract:
Trypsin was found to selectively destroy the high molecular weight (HMW) proteins associated with microtubules. Both major groups of high molecular weight bands observed by sodium dodecylsulfate-polyacrylamide gel electrophoresis were destroyed at the same rate (t 1/2 = 1 min), while tubulin was little affected under the conditions employed. Destruction of the high molecular weight bands was correlated with the disappearance of the lateral projections observed on the surface of microtubules. The trypsin-treated protein was capable of reassembly to form microtubules and showed characteristic ring-shaped structures at low temperature. Analysis of fragments formed from the high molecular weight proteins indicated the initial appearance of a fragment of M = 255,000 which failed to bind to microtubules. These results suggest that a small fragment of the high molecular weight protein molecule may remain bound to the microtubule after removal of the lateral projection, and that this fragment may retain the ability to promote microtubule assembly.
Insights
Trypsin digestion selectively removed high molecular weight (HMW) proteins from microtubules, which are crucial for their structure. Despite HMW protein removal, the remaining tubulin could still reassemble, suggesting a key role for HMW proteins in microtubule assembly.
Area of Science:
- Cell Biology
- Structural Biology
- Biochemistry
Background:
- Microtubules are essential cytoskeletal components involved in various cellular processes.
- High molecular weight (HMW) proteins are associated with microtubules, but their specific roles are not fully understood.
- Understanding the function of HMW proteins is key to comprehending microtubule dynamics and assembly.
Purpose of the Study:
- To investigate the role of high molecular weight (HMW) proteins in microtubule structure and assembly.
- To determine the effect of selective enzymatic degradation of HMW proteins on microtubule integrity.
- To analyze the reassembly capacity of microtubules after HMW protein removal.
Main Methods:
- Selective enzymatic digestion of microtubules using trypsin.
- Analysis of protein components using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
- Observation of microtubule structure and reassembly using electron microscopy and low-temperature assays.
Main Results:
- Trypsin selectively and rapidly degraded HMW proteins associated with microtubules (t 1/2 = 1 min).
- Degradation of HMW proteins correlated with the loss of lateral projections on microtubule surfaces.
- Tubulin, the main component of microtubules, was largely unaffected by the trypsin treatment.
- Trypsin-treated microtubules retained the ability to reassemble into characteristic ring structures.
- A 255,000 M.W. fragment of HMW proteins was identified, which did not bind to microtubules.
Conclusions:
- HMW proteins, particularly their lateral projections, are crucial for microtubule structural integrity.
- A smaller fragment of HMW proteins may remain bound to tubulin and play a role in promoting microtubule assembly.
- These findings shed light on the molecular mechanisms governing microtubule assembly and dynamics.
