Kinetics of Myosin-I-Membrane Detachment Under Load

Abstract

Myo1c is a single-headed motor that links cell membranes to the underlying actin cytoskeleton. Actin binding occurs via the motor domain, while the tail domain interacts with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) through a putative pleckstrin homology (PH) domain. In this study, we measured the strength and lifetime of the myo1c attachment to spherical supported lipid bilayers (SSL) composed of 1 μm diameter silica or polystyrene beads coated with various mole fractions of PI(4,5)P2, dioleoyl-phosphatidylcholine (DOPC), and dioleoyl-phosphatidylserine (DOPS). The SSLs trapped by a laser beam were brought into contact with immobilized spherical (2 μm) silica pedestals sparsely labeled with the myo1c tail domain. Upon repeated contact and retraction cycles, binding events and subsequent rupture forces were measured. The most probable rupture forces (< 10 pN) from membranes containing 2% PI(4,5)P2 were found to be largely independent of the loading rate (pN/s). Nevertheless, the frequency of single myo1c molecules interacting with membranes containing 2% PI(4,5)P2 - 98% DOPC was considerably higher than with membranes that contained 20 - 80% DOPS. Lifetime measurements of myo1c attachment to 2% PI(4,5)P2 - 98% DOPC membranes under constant pulling forces (1 - 3 pN) were fitted to Bell's equation and the extrapolated duration of the bond at zero force was found to be ∼ 25 ms. Although phosphoinositide binding is crucial for the proper cellular targeting of myo1c, our results suggest that it is unlikely that this connection is a suitable anchor for force generation.