Total Internal Reflection Fluorescence (TIRF) Microscopy of Chlamydomonas Flagella

Overview

Journal Methods Cell Biol

Specialty Cell Biology

Date 2010 Apr 23

PMID 20409817

Citations 24

Authors

Benjamin D Engel

Karl-Ferdinand Lechtreck

Tsuyoshi Sakai

Mitsuo Ikebe

George B Witman

Wallace F Marshall

Affiliations

Soon will be listed here.

Abstract

The eukaryotic flagellum is host to a variety of dynamic behaviors, including flagellar beating, the motility of glycoproteins in the flagellar membrane, and intraflagellar transport (IFT), the bidirectional traffic of protein particles between the flagellar base and tip. IFT is of particular interest, as it plays integral roles in flagellar length control, cell signaling, development, and human disease. However, our ability to understand dynamic flagellar processes such as IFT is limited in large part by the fidelity with which we can image these behaviors in living cells. This chapter introduces the application of total internal reflection fluorescence (TIRF) microscopy to visualize the flagella of Chlamydomonas reinhardtii. The advantages and challenges of TIRF are discussed in comparison to confocal and differential interference contrast techniques. This chapter also reviews current IFT insights gleaned from TIRF microscopy of Chlamydomonas and provides an outlook on the future of the technique, with particular emphasis on combining TIRF with other emerging imaging technologies.

Citing Articles

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Distribution and bulk flow analyses of the intraflagellar transport (IFT) motor kinesin-2 support an "on-demand" model for Chlamydomonas ciliary length control.

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Conversion of anterograde into retrograde trains is an intrinsic property of intraflagellar transport.

Nievergelt A, Zykov I, Diener D, Chhatre A, Buchholz T, Delling M Curr Biol. 2022; 32(18):4071-4078.e4.

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In vivo imaging shows continued association of several IFT-A, IFT-B and dynein complexes while IFT trains U-turn at the tip.

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