Biology Reference
In-Depth Information
CHAPTER
4
Detection and Quantification
of Microtubule Detachment
from Centrosomes and
Spindle Poles
Anutosh Ganguly
*
, Hailing Yang
{
and Fernando Cabral
{
*
Department of Physiology and Pharmacology, Snyder Institute for Chronic Disease,
University of Calgary, Calgary, Alberta, Canada
{
MD Anderson Cancer Center, Houston, Texas, USA
{
Department of Integrative Biology and Pharmacology, University of Texas Medical School,
Houston, Texas, USA
CHAPTER OUTLINE
Introduction .............................................................................................................. 50
4.1 Choice of Cell Line............................................................................................. 51
4.2 Microtubule Detachment in Spindles ................................................................... 52
4.3 Labeling Microtubules........................................................................................ 53
4.3.1 Rhodamine Tubulin Microinjection..................................................... 53
4.3.2 GFP-tubulin Expression..................................................................... 54
4.3.3 GFP-MAP4 Expression ..................................................................... 54
4.4 Measuring Detachment ....................................................................................... 54
4.4.1 Equipment....................................................................................... 54
4.4.2 Cell Preparation ............................................................................... 55
4.4.3 Data Collection................................................................................. 55
4.5 Microtubule Fragments as a Surrogate for Detachment ......................................... 57
4.6 Mechanism of Detachment.................................................................................. 59
Summary .................................................................................................................. 60
Acknowledgments ..................................................................................................... 60
References ............................................................................................................... 60
Abstract
Microtubule detachment from microtubule organizing centers is an important cellu-
lar process required for normal cell proliferation. When cells enter mitosis, micro-
tubule turnover
increases along with a concurrent
increase in microtubule
