Opposite-polarity motors activate one another to trigger cargo transport in live cells

Mechanical interactions between any two opposite-polarity motors are necessary and sufficient for bidirectional organelle transport in live cells

Regulation of Kinesin-1 Motor Activity by Ensconsin, a Microtubule-Binding Protein

The kinesin-1 motor drives the transport of numerous cellular cargoes toward the plus ends of microtubules. Kinesin-1 can inhibit its own activity via a direct interaction between its C-terminal tail and enzymatic motor domains. In the cell, kinesin-1 can be further regulated by interactions with cargoes as well as with the microtubule track. We investigated the effect of a microtubule-associated protein, ensconsin, on kinesin-1 activity. RNAi-mediated depletion and live cell imaging studies in Drosophila S2 cells revealed that ensconsin is required for the primary function of kinesin-1, organelle transport, as well as microtubule-microtubule sliding, a newly described function of the motor. We found that ensconsin is required for organelle transport in Drosophila neurons, and Drosophila homozygous for ensconsin gene deletion are unable to survive to adulthood, highlighting the importance of this MAP in the animal's development. We performed rescue experiments using truncated ensconsin mutants, and determined that a C-terminal region of ensconsin, while unable to bind microtubules, is required for kinesin-1-dependent organelle transport. This finding suggests that microtubule binding, via a separate, dedicated domain, may act as a mechanism to localize ensconsin's motor-activating domain near the microtubule surface. Through analysis of kinesin-1 mutants, we demonstrated that a "hingeless" mutant of kinesin-1, which mimics the active conformation of the motor, does not require ensconsin for cargo transport in vivo. This suggests that ensconsin plays a role in relieving autoinhibition of kinesin-1. Together, this work leads to a model in which ensconsin provides regulatory control over kinesin-1 in vivo near the microtubule surface, and adds to a growing body of evidence demonstrating that microtubules act not only as passive tracks for transport, but may also directly influence motor proteins and cargo delivery

Statistics of Active Transport in Xenopus Melanophores Cells

The transport of cell cargo, such as organelles and protein complexes in the cytoplasm, is determined by cooperative action of molecular motors stepping along polar cytoskeletal elements. Analysis of transport of individual organelles generated useful information about the properties of the motor proteins and underlying cytoskeletal elements. In this work, for the first time (to our knowledge), we study collective movement of multiple organelles using Xenopus melanophores, pigment cells that translocate several thousand of pigment granules (melanosomes), spherical organelles of a diameter of ∼1 μm. These cells disperse melanosomes in the cytoplasm in response to high cytoplasmic cAMP, while at low cAMP melanosomes cluster at the cell center. Obtained results suggest spatial and temporal organization, characterized by strong correlations between movement of neighboring organelles, with correlation length of ∼4 μm and pair lifetime ∼5 s. Furthermore, velocity statistics revealed strongly non-Gaussian velocity distribution with high velocity tails demonstrating exponential behavior suggestive of strong velocity correlations. Depolymerization of vimentin intermediate filaments using a dominant-negative vimentin mutant or actin with cytochalasin B reduced correlation of behavior of individual particles. Based on our analysis, we concluded that steric repulsion is dominant, but both intermediate filaments and actin microfilaments are involved in dynamic cross-linking organelles in the cytoplasm

The dynamic properties of intermediate filaments during organelle transport

Intermediate filament (IF) dynamics during organelle transport and their role in organelle movement were studied using Xenopus laevis melanophores. In these cells, pigment granules (melanosomes) move along microtubules and microfilaments, toward and away from the cell periphery in response to α-melanocyte stimulating hormone (α-MSH) and melatonin, respectively. In this study we show that melanophores possess a complex network of vimentin IFs which interact with melanosomes. IFs form an intricate, honeycomb-like network that form cages surrounding individual and small clusters of melanosomes, both when they are aggregated and dispersed. Purified melanosome preparations contain a substantial amount of vimentin, suggesting that melanosomes bind to IFs. Analyses of individual melanosome movements in cells with disrupted IF networks show increased movement of granules in both anterograde and retrograde directions, further supporting the notion of a melanosome-IF interaction. Live imaging reveals that IFs, in turn, become highly flexible as melanosomes disperse in response to α-MSH. During the height of dispersion there is a marked increase in the rate of fluorescence recovery after photobleaching of GFP-vimentin IFs and an increase in vimentin solubility. These results reveal a dynamic interaction between membrane bound pigment granules and IFs and suggest a role for IFs as modulators of granule movement