Visualizing Collective Dynamics of Nonprocessive Motors in Membrane Tube Formation

Abstract

The emergent collective behavior of motor proteins plays an important role in intracellular transport. For example, processive motors (kinesins) work in concert to extract membrane tubes from membrane compartments. In this case, it has been shown that motor proteins form dynamic clusters that can collectively generate enough force to extract membrane tubes (Koster et al, PNAS 2003, Leduc et al, PNAS 2004). Recent in vitro experiments have shown that nonprocessive motors (ncds) can also extract membrane tubes: here, tubes show distinct phases of persistent growth, retraction, and an intermediate regime characterized by dynamic switching between the two (Shaklee et al, PNAS 2008). The physical mechanism by which nonprocessive motors collectively mediate membrane tube formation has, however, not yet been experimentally investigated.We use a minimal in vitro model system where motors are specifically attached to a fluorescently labeled lipid on Giant Unilamellar Vesicles (GUVs) to examine motor behavior during membrane tube formation. Motors collectively extract membrane tubes from the GUV as they walk on underlying microtubules. FCS and FRAP experiments reveal a directed flow as processive motors walk at typical speeds (<500nm/s) along the underlying microtubule and accumulate at the tip of the growing membrane tube. However, fluorescence correlations in time show that nonprocessive motors exhibit purely diffusive behavior, decorating the entire length of the microtubule lattice with diffusion constants at least 10 times smaller than that of a lipid-motor complex freely diffusing in a lipid bilayer (1um^2/s); FRAP experiments confirm this longer timescale for exchange of motors in the tube. These results suggest that membrane-bound motor proteins interacting with a microtubule are restricted in their diffusive motion, potentially due to fast local binding/unbinding to the microtubule lattice. This restriction likely promotes dynamic motor accumulation needed for membrane tube regulation.