Molecular and Cellular Approaches Toward Understanding Dynein-Driven Motility

Active transport is integral to organelle localization and their distribution within the cell. Kinesins, myosins and dynein are the molecular motors that drive this long range transport on the actin and microtubule cytoskeleton. Although several families of kinesins and myosins have evolved, there is only one form of cytoplasmic dynein driving active retrograde transport in cells. While dynactin is an essential co-factor for most cellular functions of dynein, the mechanistic basis for this evolutionarily well conserved interaction remains unclear. Here, I use single molecule approaches with purified dynein to reconstitute processes in vitro, and implement an optogenetic tool in neurons to further dissect regulatory mechanisms of dynein-driven transport in cells. I demonstrate for the first time, at the single molecule level, that dynactin functions as a tether to enhance the initial recruitment of dynein onto microtubules but also acts as a brake to slow the motor. I then extend this work in neurons to understand regulation of the dynein motor at the cellular level. Neurons are particulary dependent on long-range transport as organelles and macromolecules must be efficiently moved over the extended length of the axon and further, have mechanisms in place for the compartment-specific regulation of trafficking in axons and dendrites. I use a light-inducible dimerization tool to recruit motor proteins or motor adaptors to organelles in real time to examine downstream effects of organelle motility and compartment-specific regulation of motors. I find that while dynein works efficiently in both axons and dendrites, kinesins are differentially regulated in a compartment-specific manner. I further demonstrate that dynein-driven motility in neurons is largely governed by microtubule orientation and requires microtubule dynamics for efficient navigation in axons and dendrites. Together, this work sheds light on the molecular and cellular mechanisms of dynein function both in vitro and in vivo using a combination of approaches. My findings converge to a model wherein dynactin enhances the recruitment of dynein onto microtubule plus ends, leading to efficient minus-end directed motility of dynein. This becomes especially critical in neuronal growth cones and dendrites owing to the large number of highly dynamic microtubules in these compartments

Development and Cell Biology of the Blood-Brain Barrier

The vertebrate vasculature displays high organotypic specialization, with the structure and function of blood vessels catering to the specific needs of each tissue. A unique feature of the central nervous system (CNS) vasculature is the blood-brain barrier (BBB). The BBB regulates substance influx and efflux to maintain a homeostatic environment for proper brain function. Here, we review the development and cell biology of the BBB, focusing on the cellular and molecular regulation of barrier formation and the maintenance of the BBB through adulthood. We summarize unique features of CNS endothelial cells and highlight recent progress in and general principles of barrier regulation. Finally, we illustrate why a mechanistic understanding of the development and maintenance of the BBB could provide novel therapeutic opportunities for CNS drug delivery. </jats:p

Dynein efficiently navigates the dendritic cytoskeleton to drive the retrograde trafficking of BDNF/TrkB signaling endosomes

The efficient transport of cargoes within axons and dendrites is critical for neuronal function. Although we have a basic understanding of axonal transport, much less is known about transport in dendrites. We used an optogenetic approach to recruit motor proteins to cargo in real time within axons or dendrites in hippocampal neurons. Kinesin-1, a robust axonal motor, moves cargo less efficiently in dendrites. In contrast, cytoplasmic dynein efficiently navigates both axons and dendrites; in both compartments, dynamic microtubule plus ends enhance dynein-dependent transport. To test the predictions of the optogenetic assay, we examined the contribution of dynein to the motility of an endogenous dendritic cargo and found that dynein inhibition eliminates the retrograde bias of BDNF/TrkB trafficking. However, inhibition of microtubule dynamics has no effect on BDNF/TrkB motility, suggesting that dendritic kinesin motors may cooperate with dynein to drive the transport of signaling endosomes into the soma. Collectively our data highlight compartment-specific differences in kinesin activity that likely reflect specialized tuning for localized cytoskeletal determinants, whereas dynein activity is less compartment specific but is more responsive to changes in microtubule dynamics. </jats:p

Pericyte-to-endothelial cell signaling via vitronectin-integrin regulates blood-CNS barrier

SummaryEndothelial cells of blood vessels of the central nervous system (CNS) constitute blood-CNS barriers. Barrier properties are not intrinsic to these cells; rather they are induced and maintained by CNS microenvironment. Notably, the abluminal surface of CNS capillaries are ensheathed by pericytes and astrocytes. However, extrinsic factors from these perivascular cells that regulate barrier integrity are largely unknown. Here, we establish vitronectin, an extracellular-matrix protein secreted by CNS pericytes, as a regulator of blood-CNS barrier function via interactions with its integrin receptor, α5 in endothelial cells. Genetic ablation of vitronectin or mutating vitronectin to prevent integrin binding as well as endothelial-specific deletion of integrin α5 causes barrier leakage. Furthermore, vitronectin-integrin α5 signaling maintains barrier integrity by actively inhibiting transcytosis in endothelial cells. These results demonstrate that signaling from perivascular cells to endothelial cells via ligand-receptor interactions is a key mechanism to regulate barrier permeability.</jats:p

Optogenetic control of organelle transport using a photocaged chemical inducer of dimerization

SummaryCell polarity, growth and signaling require organelle transport by cytoskeletal motor proteins that are precisely regulated in time and space. Probing these complex, dynamic processes requires experimental techniques with comparable temporal and spatial precision. Inducible dimerization offers the ability to recruit motor proteins to organelles in living cells. Approaches include rapamycin-induced dimerization of motors and cargo-bound binding partners [1] or the recent application of the TULIP light-inducible dimerization system [2,3]. In the latter system, motor recruitment is activated by blue light, and relaxes to an OFF state in the dark within seconds. While rapid relaxation is desirable for some applications, many experiments require sustained motor recruitment. Here, we use a photocaged chemical dimerizer to achieve sustained, spatially-defined motor recruitment to individual organelles with a single pulse of light. We demonstrate the general applicability of the system by recruiting microtubule plus end-directed kinesin-1 and minus end-directed dynein motors to peroxisomes and mitochondria in HeLa cells and primary neurons, leading to alterations in organelle transport on timescales from <10 seconds to >10 minutes after photoactivation