The Spatial and Temporal Dynamics of Pleckstrin Homology Domain Binding at the Plasma Membrane Measured by Imaging Single Molecules in Live Mouse Myoblasts

Gregory I Mashanov,Daryl Tacon,Michelle Peckham,Justin E Molloy

doi:10.1074/jbc.m312140200

Gregory I Mashanov, Daryl Tacon + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m312140200

Copy DOI

Abstract

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.

Highlights

Pleckstrin homology (PH) domains are found in over 250 proteins in the human genome (1) and are known to bind specific phosphoinositol phospholipids (PtdIns).[1]
We are interested in the behavior of myosin X, which is a dimeric molecular motor consisting of a highly conserved motor domain that interacts with actin to produce force and movement and a modular tail region that is thought to bind to membranes via its PH domains
The spatial intensity distribution produced by individual fluorophores should correspond to an Airy disc pattern with a characteristic point spread function determined by the imaging system

Summary

EXPERIMENTAL PROCEDURES

Cloning—The full-length myosin X cDNA was amplified from mouse myoblasts by long reverse transcription-PCR using primers based on the mouse sequence (GenBankTM accession number AJ249706) (14). Cell Culture, Transfection, and Expression Levels—Mouse myoblasts isolated from the “immortal mouse” (H2kb-tsA58) were cultured as described previously (15). They were transfected with the eGFP constructs using FuGENE 6 (Roche Diagnostics) or Genejuice (Novagen) following the recommended protocol. The final concentration of eGFP fusion protein in cells used in the TIRF experiments was in the nanomolar range (i.e. ϳ1 molecule/␮m3; 5000 molecules/cell). This was confirmed by independent measurements using confocal microscopy where fluorescence of a range of concentrations of eGFP was compared with that of live transfected cells. Under equivalent conditions in neutrophils, the concentration of target lipid, PtdIns(3,4,5)P3, in the plasma membrane was ϳ50 nM (local concentration of 5 ␮M) (16)

Objective

RESULTS

DISCUSSION