The motor proteins kinesin and dynein transport organelles, mRNA, proteins, and signaling molecules along the microtubule cytoskeleton. In addition to serving as tracks for transport, the microtubule cytoskeleton directs intracellular trafficking by regulating the activity of motor proteins through the organization of the filament network, microtubule-associated proteins, and tubulin posttranslational modifications. However, it is not well understood how these factors influence motor motility, and in vitro assays and live cell observations often produce disparate results. To systematically examine the factors that contribute to cytoskeleton-based regulation of motor protein motility, we extracted intact microtubule networks from cells and tracked the motility of single fluorescently labeled motor proteins on these cytoskeletons. We find that tubulin acetylation alone does not directly affect kinesin-1 motility. However, acetylated microtubules are predominantly bundled, and bundling enhances kinesin run lengths and provides a greater number of available kinesin binding sites. The neuronal MAP tau is also not sensitive to tubulin acetylation, but enriches preferentially on highly curved regions of microtubules where it strongly inhibits kinesin motility. Taken together, these results suggest that the organization of the microtubule network is a key contributor to the regulation of motor-based transport.

The cell relies on an intricate system of molecular highways and motors to transport proteins, organelles and other vesicular cargoes to their proper locations. Microtubules, long filaments that form a network throughout the cell, act as highways. The motor proteins kinesin and dynein associate with cargoes and transport them along microtubules. Rather than simply acting as passive tracks, microtubules contain signals that regulate kinesin and dynein to target cargoes to specific locations in the cell. These signals include the organization of the microtubule network, chemical modifications that alter the microtubule surface properties and mechanics, and microtubuleassociated proteins that modulate the motility of motor proteins and microtubule polymerization.

Microtubule-associated proteins (MAPs) modulate the motility of kinesin and dynein along microtubules to control the transport of vesicles and organelles. The neuronal MAP tau inhibits kinesin-dependent transport. Phosphorylation of tau at tyrosine 18 by fyn kinase results in weakened inhibition of kinesin-1. We examined the motility of early endosomes and lysosomes in cells expressing wild-type (WT) tau and phosphomimetic Y18E tau. We quantified the effects on motility as a function of the tau expression level. Lysosome motility is strongly inhibited by tau. Y18E tau preferentially inhibits lysosomes in the cell periphery, while centrally located lysosomes are less affected. Early endosomes are more sensitive to tau than lysosomes, and are inhibited by both WT and Y18E tau. Our results show that different cargoes have disparate responses to tau, likely governed by the types of kinesin motors driving their transport. In support of this model, kinesin-1 and -3 are strongly inhibited by tau while kinesin-2 and dynein are less affected. In contrast to kinesin-1, we find that kinesin-3 is strongly inhibited by phosphorylated tau. [Media: see text] [Media: see text] [Media: see text] [Media: see text]

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Endogenous electric fields are present in many biological processes including at the edge of healing wounds and can direct cell migration. Here we measure the effect of DC electric fields on actin polymerization dynamics. We find that actin waves are guided by DC electric fields for multiple cell types. Giant Dictyostelium discoideum cells (produced via electrofusion) allow us to measure electric field response of actin waves located far from the leading or trailing edge of a cell, and thus decouple cell polarity and shape from actin wave responses. We also use nanoridges, fabricated substrate nanotopographies, which alter the actin waves from the broad, two-dimensional waves seen on flat substrates to one-dimensional waves traveling along the nanoridges. Our data reveal new insights into the mechanism behind cell migration via DC electric fields with actin waves playing a key role in cell response and control.