Live cell-imaging techniques for analyses of microtubules in Dictyostelium.
Dictyostelium amoebae provide a popular model system for analyses of cell and cytoskeletal dynamics. Yet, the sensitivity of Dictyostelium cells to phototoxic effects, their rapid cell movement, and the extraordinary motility of their microtubule system are specific challenges for live cell imaging. The protocols outlined in this chapter are optimized to minimize these challenges, using Dictyostelium cells expressing green fluorescent tubulin or microtubule plus-end markers such as TACC. We describe suitable specimen preparations, treatments with microtubule-depolymerizing drugs, and applicable settings on wide-field and confocal microscopy systems for four-dimensional time-lapse and fluorescence recovery after photobleaching analyses of microtubule dynamics.
Samereier
Matthias
M
Department of Cell Biology, Institut for Biochemistry and Biology, University of Potsdam, Potsdam-Golm 27708, Germany D-14476.
Meyer
Irene
I
Koonce
Michael P
MP
Gräf
Ralph
R
D002468:Cell Physiological Phenomena; D004023:Dictyostelium / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure*; D005453:Fluorescence; D007700:Kinetics; D008853:Microscopy / Q000379:methods*; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D008954:Models, Biological; D055503:Protein Multimerization / Q000502:physiology