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MCAK associates with EB1
T Lee1, K J Langford, J M Askham
1CRUK Clinical Centre at Leeds, Division of Cancer Medicine Research, St James's University Hospital, Leeds, UK.
Abstract:
The microtubule (MT)-associated protein EB1 localizes to and promotes growth at MT plus ends. The MT depolymerizing kinesin MCAK has also been reported to track growing MT plus ends. Here, we confirm that human MCAK colocalizes with EB1 at growing MT ends when expressed as a GFP fusion protein in transfected cells. We show that MCAK associates with the C-terminus of EB1 and EB3 but much less efficiently with RP1. EB1 associates with the N-terminal localization and regulatory domain in MCAK but not with the motor domain of the protein. The interaction is competitive with the binding of other EB1 ligands and does not require MTs. Knockdown of EB1 expression using siRNA impaired the ability of GFP-MCAK to localize to MT tips in transfected cells. We propose that MCAK is targeted to growing MT ends by EB1, that MCAK is held in an inactive conformation when associated with EB1 and that this could provide the basis for a mechanism that facilitates rapid switching between phases of MT growth and depolymerization.
Insights
Microtubule-associated protein EB1 targets the kinesin MCAK to growing microtubule (MT) plus ends. This interaction may keep MCAK inactive, facilitating rapid switching between MT growth and depolymerization.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Microtubules (MTs) are dynamic polymers crucial for cell division and intracellular transport.
- EB1 is a key plus-end tracking protein that promotes MT growth.
- MCAK is a kinesin that depolymerizes MTs and has been observed at growing MT plus ends.
Purpose of the Study:
- To investigate the interaction between EB1 and MCAK at MT plus ends.
- To elucidate the mechanism by which MCAK is targeted to MT plus ends.
- To understand how this interaction influences MT dynamics.
Main Methods:
- Expression of GFP-tagged MCAK in transfected cells.
- Co-immunoprecipitation assays to study protein interactions.
- RNA interference (siRNA) to knockdown EB1 expression.
- Confocal microscopy to visualize protein localization at MT plus ends.
Main Results:
- Human MCAK colocalizes with EB1 at growing MT plus ends.
- MCAK binds to the C-terminus of EB1 and EB3, and EB1 binds to the N-terminal domain of MCAK.
- The EB1-MCAK interaction is competitive with other EB1 ligands and does not require MTs.
- EB1 knockdown impairs MCAK localization to MT tips.
Conclusions:
- EB1 targets MCAK to growing MT plus ends.
- MCAK associates with EB1 in an inactive conformation.
- This interaction provides a mechanism for rapid switching between MT growth and depolymerization phases.
