Studying plus-end tracking at single molecule resolution using TIRF microscopy

Methods in Cell Biology
Ram Dixit, Jennifer L Ross

Abstract

The highly dynamic microtubule plus-ends are key sites of regulation that impact the organization and function of the microtubule cytoskeleton. Much of this regulation is performed by the microtubule plus-end tracking (+TIP) family of proteins. +TIPs are a structurally diverse group of proteins that bind to and track with growing microtubule plus-ends in cells. +TIPs regulate microtubule dynamics as well as mediate interactions between microtubule tips and other cellular structures. Most +TIPs can directly bind to microtubules in vitro; however, the mechanisms for their plus-end specificity are not fully understood. Cellular studies of +TIP activity are complicated by the fact that members of the +TIP family of proteins interact with each other to form higher-order protein assemblies. Development of an in vitro system, using minimal components, to study +TIP activity is therefore critical to unequivocally understand the behavior of individual +TIP proteins. Coupled with single molecule imaging, this system provides a powerful tool to study the molecular properties that are important for +TIP function. In this chapter, we describe a detailed protocol for in vitro reconstitution of +TIP activity at single molecule resolution usin...Continue Reading

Citations

Oct 5, 2012·The Journal of Biological Chemistry·Xiwei WangXuebiao Yao
Dec 15, 2015·Scientific Reports·J BergmanM Vershinin
Apr 20, 2016·Nature Communications·Noelia Blas-RusFrancisco Sánchez-Madrid
Nov 4, 2015·The Journal of Biological Chemistry·Han LiuXiaoyang Wu
Sep 26, 2013·Integrative Biology : Quantitative Biosciences From Nano to Macro·Megan E BaileyJennifer L Ross
Apr 14, 2019·Cytoskeleton·Liudmila BelonogovJennifer L Ross
Aug 16, 2013·Journal of Physics. Condensed Matter : an Institute of Physics Journal·Joshua PringleJennifer L Ross
Jun 23, 2020·The Journal of Cell Biology·Pablo MartinezCarolyn G Rasmussen

Related Concepts

Fluorochromes
Immunofluorescence Microscopy
Microtubules
Staining and Labeling
Imaging, Three-Dimensional, Computer Assisted
Protein Multimerization
Limit of Detection
Microscopy
Microscopy, Fluorescence
Microtubules

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