Biology Reference
In-Depth Information
CHAPTER
10
Imaging GTP-Bound
Tubulin: From Cellular
to In Vitro Assembled
Microtubules
H ´ l ` ne de Forges
*
,{,1
, Antoine Pilon
{,},1
, Christian Po ¨ s
{,},2
and Franck Perez
*
,{,2
*
Institut Curie, Paris Cedex 05, France
{
CNRS UMR144, Paris Cedex 05, France
{
EA4530, Dynamique des microtubules en physiopathologie, Faculte´ de Pharmacie,
Universite´ Paris-Sud, Chaˆtenay-Malabry, France
}
Unite´ d'Hormonologie et Immunoanalyse, Poˆle de Biologie Me´dicale et Pathologie,
Hoˆpitaux Universitaires Est Parisien, APHP, Paris, France
}
Laboratoire de Biochimie-Hormonologie, Hoˆpitaux Universitaires Paris-Sud,
APHP, Clamart, France
CHAPTER OUTLINE
Introduction ............................................................................................................ 141
10.1 Imaging GTP Islands in Permeabilized Cells .....................................................142
10.1.1 Permeabilization ........................................................................ 143
10.1.2 MB11 Staining........................................................................... 144
10.1.3 Cell Fixation and Colabeling ........................................................ 144
10.2 Imaging GTP Caps and GTP Islands Using Centrosome-Based Microtubule
Assembly or Endogenous Microtubule Elongation in Permeabilized Cells ...........145
10.2.1 Cell Plating, Culture, and Treatment ............................................ 145
10.2.1.1 Prior to Assembly of Centrosome-Nucleated Microtubules .. 145
10.2.1.2 Prior to Extracellular Microtubule Elongation ....................... 145
10.2.2 Microtubule Growth .................................................................... 145
10.3 Imaging GTP Islands in Microtubules Assembled In Vitro...................................147
10.3.1 Microtubules Assembled in Suspension with GTP or GMPCPP........ 147
10.3.1.1 Separate Preparation of GMPCPP and GTP Microtubules ... 147
10.3.1.2 Mixed GMPCPP and GTP Microtubules and Staining
with MB11 Antibody......................................................................... 147
1,2
Equal contributions
