Analysis of dynamic instability of steady-state microtubules in vitro by video-enhanced differential interference contrast microscopy with an appendix by Emin Oroudjev.
Microtubules are major constituents of the cytoskeleton which display dynamic properties. They exhibit dynamic instability which is defined as the stochastic switching between growing and shortening at microtubule ends. Dynamic instability plays an important role in diverse cellular functions including cell migration and mitosis. Many successful antimitotic drugs and microtubule-associated proteins (MAPs) are known to modulate microtubule dynamics, and it is important to analyze the in vitro dynamic instability of microtubules to study the mechanism of action of microtubule-targeted therapeutics and MAPs. In this chapter, we describe a method to analyze the in vitro dynamic instability of microtubules at steady state using video-enhanced differential contrast (VE-DIC) microscopy in detail. In this method, microtubules are assembled to steady state at 30 degrees C with MAP-free tubulin in a slide chamber in the presence of GTP, using sea urchin axonemes as nucleating seeds. Images of microtubules are enhanced and recorded in real time by a video camera and an image processor connected to a DIC microscope which is maintained at 30 degrees C. We use two software programs to track and analyze the growing and shortening of plus or minus ends of microtubules in the real-time images recorded using VE-DIC. In this chapter, we describe the instructions to use the tracking software Real Time Measurement II (RTM II) program. The instructions to use the analysis software Microtubule Life History Analysis Procedures (MT-LHAP) in Igor Pro software have been described in detail in an appendix (Oroudjev, 2010) following this chapter.
Yenjerla
Mythili
M
Department of Molecular, Cellular, and Developmental Biology, The Neuroscience Research Institute, University of California, Santa Barbara, California 93106, USA.
Lopus
Manu
M
Wilson
Leslie
L
D000818:Animals; D006801:Humans; D008857:Microscopy, Interference / Q000295:instrumentation* / Q000379:methods*; D008870:Microtubules / Q000737:chemistry* / Q000378:metabolism*; D008954:Models, Biological; D055503:Protein Multimerization; D055550:Protein Stability; D012984:Software; D014404:Tubulin / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D014741:Video Recording / Q000379:methods
D014404:Tubulin