Transport efficiency of membrane-anchored kinesin-1 motors depends on motor density and diffusivity

Abstract

In eukaryotic cells, membranous vesicles and organelles are transported by ensembles of motor proteins. These motors, such as kinesin-1, have been well characterized in vitro as single molecules or as ensembles rigidly attached to nonbiological substrates. However, the collective transport by membrane-anchored motors, that is, motors attached to a fluid lipid bilayer, is poorly understood. Here, we investigate the influence of motors' anchorage to a lipid bilayer on the collective transport characteristics. We reconstituted "membrane-anchored" gliding motility assays using truncated kinesin-1 motors with a streptavidin-binding peptide tag that can attach to streptavidin-loaded, supported lipid bilayers. We found that the diffusing kinesin-1 motors propelled the microtubules in the presence of ATP. Notably, we found the gliding velocity of the microtubules to be strongly dependent on the number of motors and their diffusivity in the lipid bilayer. The microtubule gliding velocity increased with increasing motor density and membrane viscosity, reaching up to the stepping velocity of single motors. This finding is in contrast to conventional gliding motility assays where the density of surfaceimmobilized kinesin-1 motors does not influence the microtubule velocity over a wide range. We reason that the transport efficiency of membrane-anchored motors is reduced because of their slippage in the lipid bilayer, an effect that we directly observed using singlemolecule fluorescence microscopy. Our results illustrate the importance of motor-cargo coupling, which potentially provides cells with an additional means of regulating the efficiency of cargo transport. molecular motors | lipid bilayers | transport efficiency | motor-cargo coupling | streptavidin-binding peptide I ntracellular transport of membrane-bound vesicles and organelles is a process fundamental to many cellular functions including morphogenesis, signaling, and growth (1-4). Active cargo transport inside eukaryotic cells is mediated by ensembles of motor proteins, such as kinesins and dynein, walking on microtubule tracks (5), and myosins walking on actin filaments (6). Gaining mechanistic insight into the functioning of these motors inside the complex environment of cells is challenging. Several studies have thus used in vitro approaches to investigate transport mediated by groups of same or different motors attached to cargos such as silica beads (7), quantum dots (8), glass coverslips (9, 10), or DNA scaffolds (11,12). Although these approaches provide us with knowledge about the collective dynamics of multimotor transport, a key anomaly in these in vitro systems is the use of rather nonphysiological rigid cargo. Vesicular cargo transport by molecular motors requires their attachment to a fluid lipid bilayer either directly or via different adaptor molecules. The anchoring of motors in a diffusive lipid environment induces loose intermotor coupling along with the motors diffusing within the lipid bilayer, thereby increasing the flexibility of the system....

A new actin depolymerase: a Myosin 1 motor

“…3C, Table S1). This is in line with the results by Grover et al 16 showing a decreased gliding velocity of membrane-anchored kinesins due to their slippage in the lipid bilayer.…”

High-Resolution Imaging of a Single Gliding Protofilament of Tubulins by HS-AFM

“…This difference in velocity is in agreement to a previous report where a difference in velocity between MTs and protofilament bundles (PFBs) was observed under fluorescence microscope 19 . Such difference in velocity of MTs and PFs might be related to the fluctuation of kinesins on the surface, and consequent difference in the extent of force production by the kinesins at MTs or PFs 25 .

Figure 1HS-AFM observation of gliding MTs. ( a ) Schematic illustration of in vitro gliding assay of MTs on kinesins fixed to a mica supported lipid bilayer through streptavidin/biotin interaction.

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Geometry of antiparallel microtubule bundles regulates relative sliding and stalling by PRC1 and Kif4A

“…These proteins include 5 molecular motors that utilize the energy from ATP hydrolysis to mediate the transport of 6 one microtubule over another (referred to as 'relative sliding') [3][4][5]. Motor proteins 7 frequently act in conjunction with non-motor microtubule crosslinking proteins that 8 oppose relative sliding and regulate both the stability and the size of the arrays [1, 2, 6]. 9 The activities of motor and non-motor proteins are in turn modulated by the microtubule 10 cytoskeleton.…”