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

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 M ؊1 m ؊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.PH domains are found in over 250 proteins in the human genome (1) and are known to bind specific phosphoinositol phospholipids (PtdIns). 1 The binding of PH domains to their target phospholipids is controlled by phosphorylation of the inositol moiety of the lipids. Because there are five available phosphorylation positions, this could give rise to 32 different PtdIns forms (for example, a 5-bit binary encoding). Although binding affinities of different PH domains have been measured in vitro and their localization has been observed in vivo, little is known regarding the membrane binding dynamics of these domains. Recently, several laboratories have developed optical methods to make wide field observations of individual fluorophores in aqueous solution and these techniques enable the dynamic behavior (e.g. kinetics of membrane binding and release, diffusion times, and trafficking) of proteins, ligands, and viruses (2-7) to be measured simply by making steady-state observations. However, one of the main problems in imaging single fluorophores within a living cell is the high autofluorescence that overlaps the emission spectrum of the enhanced (S65T mutant) green fluorescent protein (eGFP) used in many such studies (8). To overcome this problem, we exploited total internal reflection fluorescence microscopy (TIRFM) to observe single fluorophores in living cells. TIRFM is a wide-field method (e.g. illuminates the entire cross-section of a specimen) that relies on near-field excitation (by the evanescent wave); thus, only a thin layer of the cell that lies close to the glasswater interface is illuminated (9). This technique enables the activity of individual molecules to be observed directly allowing membrane binding dynamics...

PtdIns(3,4,5)P3 is a regulator of myosin-X localization and filopodia formation

“…Although the Myo10 head, neck and coiled-coil domains are sufficient for filopodial tip localization, the full-length Myo10 constructs possessed additional domains that obviously contribute additional localization-regulating properties. Indeed, a construct corresponding to the three PH domains of Myo10 has also been indicated to localize at the plasma membrane (Mashanov et al, 2004).We therefore conclude that the C-terminal tail of Myo10 also contributes to Myo10 localization by binding to the membrane lipid PtdIns(3,4,5)P 3 .…”

Myosin-X: a MyTH-FERM myosin at the tips of filopodia

“…Although the PH2 domain of Myo10 preferentially binds to PtdIns(3,4,5)P 3 in lipid-binding assays, the isolated PH1 or PH2 domains show little interaction with other phospholipids (Park et al, 2008;Plantard et al, 2010). Importantly, a construct consisting of all three PH domains is sufficient for targeting to the plasma membrane, where it has a mean residence time of ~20 seconds (Mashanov et al, 2004). Experiments with macrophages show that endogenous Myo10 is recruited to the phagocytic cup downstream of PI3K and that Myo10 is required for Fc-mediated phagocytosis (Cox et al, 2002).…”

Differential trafficking of Kif5c on tyrosinated and detyrosinated microtubules in live cells

“…Only sequences captured at this faster frame rate and at this higher spatial resolution were used in the tracking analysis to calculate GFP-Kif5c velocities. Particle tracking analyses were conducted on original datasets using Kinetic Imaging Motion Analysis software, or using GMview (kindly provided by Gregory Mashanov, NIMR, London, UK; (Mashanov et al, 2004). GFP-Kif5c puncta were tracked from frame to frame.…”