Microinjection as a tool to explore small GTPase function.
Microinjection overcomes the plasma membrane barrier to the introduction of charged or large nonlipid soluble molecules into cells by the direct insertion of a hollow capillary micropipette into the cell. With the application of pressure, aqueous solution is then directly transferred into either the cytosol or the nucleus. I give specific examples of the application of this approach to the functional study of small GTPases of the Sar1, ARF, and rab family in membrane trafficking between the Golgi apparatus and endoplasmic reticulum (ER). The principles illustrated by these examples should be generally applicable to other small GTPases. Detailed protocols for capillary microinjection using semiautomated equipment are given.
Storrie
Brian
B
Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, USA.
D000818:Animals; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum / Q000502:physiology; D020558:GTP Phosphohydrolases / Q000502:physiology*; D006056:Golgi Apparatus / Q000502:physiology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D008845:Microinjections / Q000295:instrumentation / Q000379:methods*; D008858:Microscopy, Phase-Contrast; D020559:Monomeric GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000502:physiology; D010641:Phenotype; D014709:Vero Cells; D020691:rab GTP-Binding Proteins / Q000502:physiology
C094135:Rab6 protein; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D020558:GTP Phosphohydrolases; D020559:Monomeric GTP-Binding Proteins; D020691:rab GTP-Binding Proteins