The spatial and temporal dynamics of pleckstrin homology domain binding at the plasma membrane measured by imaging single molecules in live mouse myoblasts.
Pleckstrin homology (PH) domains act to target proteins to the plasma membrane and intracellular vesicles by binding to specific phosphoinositol phospholipids. We have investigated the binding kinetics of PH domains found in the tail region of the molecular motor, myosin X. Using total internal reflection fluorescence microscopy, we observed binding and release of individual PH domains fused to green fluorescent protein at the plasma membrane of living cells. Individual spots of light corresponding to single fluorescently tagged molecules were imaged onto a sensitive camera system, and digital image processing was then used to identify each fluorophore and store its trajectory in time and space. The PH domains bound with an apparent on-rate of 0.03 microm(-1) microm(-2) s(-1) and a detachment rate constant of 0.05 s(-1). The average residency time of the domains at the plasma membrane was about 20s. We found very limited movement of the membrane-bound PH domains in the mouse myoblast cells that we studied. This implies that the PH domains must either be attached to the cytoskeleton or corralled in a lipid compartment. Localization of the PH domains together with their rapid detachment rate is probably important in controlling the response of myosin X to signaling events and in regulating its cellular function.
Mashanov
Gregory I
GI
Medical Research Council National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom.
Tacon
Daryl
D
Peckham
Michelle
M
Molloy
Justin E
JE
D000818:Animals; D001798:Blood Proteins / Q000737:chemistry*; D019556:COS Cells; D002138:Calibration; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D049452:Green Fluorescent Proteins; D007091:Image Processing, Computer-Assisted; D007700:Kinetics; D008027:Light; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D032342:Muscle Cells / Q000378:metabolism*; D009218:Myosins / Q000737:chemistry*; D010743:Phospholipids / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection
D001798:Blood Proteins; D018076:DNA, Complementary; D008164:Luminescent Proteins; C409064:Myo10 protein, mouse; D010743:Phospholipids; D010750:Phosphoproteins; C039077:platelet protein P47; D049452:Green Fluorescent Proteins; D009218:Myosins