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Subunit interactions within the Chlamydomonas flagellar spokehead.

Takahiro Kohno¹, Ken-ichi Wakabayashi, Dennis R Diener

¹Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo, Japan.

Cytoskeleton (Hoboken, N.J.)

|March 11, 2011

Summary

Researchers investigated protein interactions within the radial spoke (RS)/central pair (CP) system of cilia and flagella. They identified specific interactions and a protein complex, proposing a model for spokehead assembly and RS-CP control of motor protein function.

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Area of Science:

Cell Biology
Biochemistry
Structural Biology

Background:

Cilia and flagella movement is driven by dynein motor proteins.
The radial spoke (RS)/central pair (CP) system regulates dynein force generation.
The precise interactions within the RS-CP system, particularly the spokehead, remain largely unknown.

Purpose of the Study:

To investigate protein-protein interactions between RS spokehead and spoke stalk proteins.
To identify functional interactions and characterize the assembly of spokehead components.
To propose a model for RS-CP interactions controlling ciliary/flagellar motility.

Main Methods:

Recombinant protein expression and purification.
Electroporation-mediated protein delivery into mutant cells.
In vitro glutathione S-transferase pulldown assays.
In situ chemical crosslinking of axonemes.
Sucrose density gradient centrifugation.

Main Results:

Three recombinant proteins demonstrated physiological activity in vivo.
Ten protein-protein interactions were detected in vitro between spokehead and stalk proteins.
Seven interactions between RS subunits were identified in situ.
A 7-10S complex formed by RSP1, RSP4, RSP6, and RSP9 suggests partial spokehead assembly.

Conclusions:

The study provides initial insights into the protein interactions governing the RS/CP system.
A model for spokehead subunit interactions and their role in regulating ciliary/flagellar mechanics is proposed.
These findings contribute to understanding the structural basis of ciliary/flagellar motility.