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Cytoplasmic linker proteins promote microtubule rescue in vivo
Yulia A Komarova1, Anna S Akhmanova, Shin-Ichiro Kojima
1Department of Cell and Molecular Biology, N University Medical School, Chicago, IL 60611, USA. y-komarova@northwestern.edu
Abstract:
The role of plus end-tracking proteins in regulating microtubule (MT) dynamics was investigated by expressing a dominant negative mutant that removed endogenous cytoplasmic linker proteins (CLIPs) from MT plus ends. In control CHO cells, MTs exhibited asymmetric behavior: MTs persistently grew toward the plasma membrane and displayed frequent fluctuations of length near the cell periphery. In the absence of CLIPs, the microtubule rescue frequency was reduced by sevenfold. MT behavior became symmetrical, consisting of persistent growth and persistent shortening. Removal of CLIPs also caused loss of p150Glued but not CLIP-associating protein (CLASP2) or EB1. This result raised the possibility that the change in dynamics was a result of the loss of either CLIPs or p150Glued. To distinguish between these possibilities, we performed rescue experiments. Normal MT dynamics were restored by expression of the CLIP-170 head domain, but p150Glued was not recruited back to MT plus ends. Expression of p150Glued head domain only partially restored MT dynamics. We conclude that the CLIP head domain is sufficient to alter MT dynamics either by itself serving as a rescue factor or indirectly by recruiting a rescue factor. By promoting a high rescue frequency, CLIPs provide a mechanism by which MT plus ends may be concentrated near the cell margin.
Insights
Cytoplasmic linker proteins (CLIPs) regulate microtubule dynamics. Removing CLIPs reduces microtubule rescue frequency, leading to symmetrical growth and shortening, with the CLIP head domain sufficient to restore normal dynamics.
Area of Science:
- Cell Biology
- Cytoskeletal Dynamics
Background:
- Microtubule (MT) dynamics are crucial for cellular processes.
- Plus end-tracking proteins, like cytoplasmic linker proteins (CLIPs), are key regulators of MT dynamics.
Purpose of the Study:
- To investigate the role of CLIPs in regulating MT dynamics at the plus end.
- To determine if CLIPs or associated proteins like p150Glued are responsible for observed MT dynamic changes.
Main Methods:
- Expression of a dominant-negative mutant to remove endogenous CLIPs from MT plus ends in CHO cells.
- Analysis of MT behavior, including growth, shortening, and rescue frequency.
- Rescue experiments using head domains of CLIP-170 and p150Glued.
Main Results:
- Absence of CLIPs reduced MT rescue frequency sevenfold, leading to symmetrical MT growth and shortening.
- CLIP removal caused loss of p150Glued, but not CLASP2 or EB1.
- Restoration of MT dynamics was achieved by expressing the CLIP-170 head domain alone, indicating CLIPs' sufficiency.
Conclusions:
- The CLIP head domain is sufficient to alter MT dynamics, acting as a rescue factor or recruiting one.
- CLIPs promote high MT rescue frequency, concentrating MT plus ends near the cell margin.