No 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...

The LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1) scavenger receptor regulates vascular responses to oxidized-low-density-lipoprotein particles implicated in atherosclerotic plaque formation. LOX-1 is closely related to C-type lectins, but the mechanism of ligand recognition is not known. Here we show that human LOX-1 recognizes a key cellular phospholipid, PS (phosphatidylserine), in a Ca2+-dependent manner, both in vitro and in cultured cells. A recombinant, folded and glycosylated LOX-1 molecule binds PS, but not other phospholipids. LOX-1 recognition of PS was maximal in the presence of millimolar Ca2+ levels. Mg2+ was unable to substitute for Ca2+ in LOX-1 binding to PS, indicating a Ca2+-specific requirement for bivalent cations. LOX-1-mediated recognition of PS-containing apoptotic bodies was dependent on Ca2+ and was decreased to background levels by bivalent-cation chelation, LOX-1-blocking antibodies or PS-containing liposomes. The LOX-1 membrane protein is thus a Ca2+-dependent phospholipid receptor, revealing novel recognition of phospholipids by mammalian lectins.

Cellular motors (kinesin, dynein and myosin) are ubiquitous. A major task in cell biology is to determine how they function in cells. Here we focus on myosin 10, an intrafilopodial motor, and show how imaging green fluorescent protein fused to myosin 10 or its tail domains can help us understand the function of this myosin.