The use of fluorescence redistribution after photobleaching for analysis of cellular microtubule dynamics.
Microtubules (MTs) are highly dynamic polymers that serve as tracks for vesicular movement during interphase and as structural components of the mitotic spindle, which is used to segregate the genetic material. MT dynamics are highly regulated wherein MTs turnover differentially between interphase and mitosis. Within the mitotic spindle, there are distinct classes of MTs with different dynamic properties. To understand how cellular proteins regulate the dynamics of MTs, it is necessary to have methods to assess their turnover properties. In this chapter we present approaches to assess MT dynamics in cultured mammalian cells using fluorescence redistribution after photobleaching. We include a discussion of cell culture and imaging conditions that maintain cell viability. We also provide an extensive discussion of both data collection and analysis that are utilized to estimate the turnover dynamics of MTs.
Walczak
Claire E
CE
Medical Sciences, Indiana University, Bloomington, Indiana 47405, USA.
Rizk
Rania S
RS
Shaw
Sidney L
SL
D000818:Animals; D002470:Cell Survival / Q000502:physiology; D002477:Cells / Q000378:metabolism*; D002478:Cells, Cultured; D003627:Data Interpretation, Statistical; D005453:Fluorescence; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods*; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods; D008870:Microtubules / Q000378:metabolism*; D038761:Photobleaching