The Dynamic Localization of Cytoplasmic Dynein in Neurons Is Driven by Kinesin-1 (original) (raw)
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Cell Motility and the Cytoskeleton, 2006
We examined the respective roles of dynein and kinesin in axonal transport of neurofilaments (NFs). Differentiated NB2a/d1 cells were transfected with green fluorescent protein-NF-M (GFP-M) and dynein function was inhibited by co-transfection with a construct expressing myc-tagged dynamitin, or by intracellular delivery of purified dynamitin and two antibodies against dynein's cargo domain. Monitoring of the bulk distribution of GFP signal within axonal neurites, recovery of GFP signal within photobleached regions, and real-time monitoring of individual NFs/punctate structures each revealed that pertubation of dynein function inhibited retrograde transport and accelerated anterograde, confirming that dynein mediated retrograde axonal transport, while intracellular delivery of two anti-kinesin antibodies selectively inhibited NF anterograde transport. In addition, dynamitin overexpression inhibited the initial translocation of newly-expressed NFs out of perikarya and into neurites, indicating that dynein participated in the initial anterograde delivery of NFs into neurites. Delivery of NFs to the axon hillock inner plasma membrane surface, and their subsequent translocation into neurites, was also prevented by vinblastine-mediated inhibition of microtubule assembly. These data collectively suggest that some NFs enter axons as cargo of microtubues that are themselves undergoing transport into axons via dynein-mediated interactions with the actin cortex and/or larger microtubules. C-terminal NF phosphorylation regulates motor association, since anti-dynein selectively coprecipitated extensively phosphorylated NFs, while anti-kinesin selectively coprecipitated less phosphorylated NFs. In addition, however, the MAP kinase inhibitor PD98059 also inhibited transport of a constitutivelyphosphorylated NF construct, indicating that one or more additional, non-NF phosphorylation events also regulated NF association with dynein or kinesin. Cell Motil.
Differential roles of kinesin and dynein in translocation of neurofilaments into axonal neurites
Journal of Cell Science, 2011
Neurofilament (NF) subunits translocate within axons as short NFs, non-filamentous punctate structures (‘puncta’) and diffuse material that might comprise individual subunits and/or oligomers. Transport of NFs into and along axons is mediated by the microtubule (MT) motor proteins kinesin and dynein. Despite being characterized as a retrograde motor, dynein nevertheless participates in anterograde NF transport through associating with long MTs or the actin cortex through its cargo domain; relatively shorter MTs associated with the motor domain are then propelled in an anterograde direction, along with any linked NFs. Here, we show that inhibition of dynein function, through dynamitin overexpression or intracellular delivery of anti-dynein antibody, selectively reduced delivery of GFP-tagged short NFs into the axonal hillock, with a corresponding increase in the delivery of puncta, suggesting that dynein selectively delivered short NFs into axonal neurites. Nocodazole-mediated deplet...
Molecular Biology of the Cell, 2009
We have tested the hypothesis that kinesin-1A (formerly KIF5A) is an anterograde motor for axonal neurofilaments. In cultured sympathetic neurons from kinesin-1A knockout mice, we observed a 75% reduction in the frequency of both anterograde and retrograde neurofilament movement. This transport defect could be rescued by kinesin-1A, and with successively decreasing efficacy by kinesin-1B and kinesin-1C. In wild-type neurons, headless mutants of kinesin-1A and kinesin-1C inhibited both anterograde and retrograde movement in a dominant-negative manner. Because dynein is thought to be the retrograde motor for axonal neurofilaments, we investigated the effect of dynein inhibition on anterograde and retrograde neurofilament transport. Disruption of dynein function by using RNA interference, dominantnegative approaches, or a function-blocking antibody also inhibited both anterograde and retrograde neurofilament movement. These data suggest that kinesin-1A is the principal but not exclusive anterograde motor for neurofilaments in these neurons, that there may be some functional redundancy among the kinesin-1 isoforms with respect to neurofilament transport, and that the activities of the anterograde and retrograde neurofilament motors are tightly coordinated.
Dynein Regulator NDEL1 Controls Polarized Cargo Transport at the Axon Initial Segment
Neuron, 2016
The development and homeostasis of neurons relies heavily on the selective targeting of vesicles into axon and dendrites. Microtubule-based motor proteins play an important role in polarized transport; however, the sorting mechanism to exclude dendritic cargo from the axon is unclear. We show that the dynein regulator NDEL1 controls somatodendritic cargo transport at the axon initial segment (AIS). NDEL1 localizes to the AIS via an interaction with the scaffold protein Ankyrin-G. Depletion of NDEL1 or its binding partner LIS1 results in both cell-wide and local defects, including the non-polarized trafficking of dendritic cargo through the AIS. We propose a model in which LIS1 is a critical mediator of local NDEL1-based dynein activation at the AIS. By localizing to the AIS, NDEL1 facilitates the reversal of somatodendritic cargos in the proximal axon.
Dynein is required for polarized dendritic transport and uniform microtubule orientation in axons
Nature Cell Biology, 2008
Axons and dendrites differ in both microtubule (MT) organization and in the organelles and proteins they contain. Here we show that the MT motor dynein plays a critical role in polarized transport and in controlling the orientation of axonal MTs in fly dendritic arborisation (da) neurons. Changes in organelle distribution within the dendritic arbors of dynein mutant neurons correlate with a proximal shift in dendritic branch position. Dynein is also necessary for the dendrite-specific localization of Golgi outposts and the ion channel Pickpocket. Axonal MTs are normally oriented uniformly plus end-distal, but without dynein axons contain both plus and minus end-distal MTs. These data suggest that dynein is required for the distinguishing properties of the axon and dendrites: without dynein, dendritic organelles and proteins enter the axon and the axonal MTs are no longer uniform in polarity.
F1000 - Post-publication peer review of the biomedical literature, 2008
Axons and dendrites differ in both microtubule (MT) organization and in the organelles and proteins they contain. Here we show that the MT motor dynein plays a critical role in polarized transport and in controlling the orientation of axonal MTs in fly dendritic arborisation (da) neurons. Changes in organelle distribution within the dendritic arbors of dynein mutant neurons correlate with a proximal shift in dendritic branch position. Dynein is also necessary for the dendrite-specific localization of Golgi outposts and the ion channel Pickpocket. Axonal MTs are normally oriented uniformly plus end-distal, but without dynein axons contain both plus and minus end-distal MTs. These data suggest that dynein is required for the distinguishing properties of the axon and dendrites: without dynein, dendritic organelles and proteins enter the axon and the axonal MTs are no longer uniform in polarity. The differential distribution of organelles and proteins to distinct compartments within cells is critical to their specialized functions. Proteins and organelles are transported to different subcellular compartments by the MT motors dynein and kinesin. The multi-subunit dynein complex travels towards MT minus ends whereas the majority of kinesins travel towards MT plus ends. Cargo localization depends on motor activity and MT organization 1. In neurons, the signal-sending axons contain MTs that are oriented uniformly plus end-distal, whereas the signal-receiving dendrites have MTs whose orientation is mixed 2. How might this difference in MT orientation be created? Dynein and kinesin not only move along MTs, but can also transport MTs 3. Without the kinesin CHO1/MKLP the orientation of dendritic MTs are uniformly plus end-distal, rather than mixed, raising the possibility that MT motors may regulate MT polarity 4, 5. Whether dynein contributes to MT orientation in neurons remains an outstanding question. Similar to most mammalian neurons, the fly dendritic arborisation (da) neurons have distinct axonal and dendritic compartments 6-9 , and their MT organization resembles that in typical mammalian neurons 6,7. In a genetic screen we uncovered mutations in components of the dynein complex, dynein light intermediate chain 2 (dlic2) and dynein intermediate chain (dic, also called short wing), that cause a proximal shift in both organelle distribution and branch position within mutant dendritic arbours. These dynein mutations cause dendritic cargo to be mislocalized to axons and result in mixed orientation of axonal MTs. Our results provide Correspondence should be addressed to Yuh Nung Jan
Dynactin Is Required for Coordinated Bidirectional Motility, but Not for Dynein Membrane Attachment
Molecular Biology of The Cell, 2007
To test directly and rigorously the hypothesis that dynactin is required to attach dynein to membranes, we used both a Drosophila mutant and RNA interference to generate organisms and cells lacking the critical dynactin subunit, actin-related protein 1. Contrary to expectation, we found that apparently normal amounts of dynein associate with membrane compartments in the absence of a fully assembled dynactin complex. In addition, anterograde and retrograde organelle movement in dynactin deficient axons was completely disrupted, resulting in substantial changes in vesicle kinematic properties. Although effects on retrograde transport are predicted by the proposed function of dynactin as a regulator of dynein processivity, the additional effects we observed on anterograde transport also suggest potential roles for dynactin in mediating kinesin-driven transport and in coordinating the activity of opposing motors (King and Schroer, 2000).
mNUDC is required for plus-end-directed transport of cytoplasmic dynein and dynactins by kinesin-1
The EMBO Journal, 2009
Lissencephaly is a devastating neurological disorder caused by defective neuronal migration. The LIS1 (or PAFAH1B1) gene was identified as the gene mutated in lissencephaly patients, and was found to regulate cytoplasmic dynein function and localization. In particular, LIS1 is essential for anterograde transport of cytoplasmic dynein as a part of the cytoplasmic dynein-LIS1-microtubule complex in a kinesin-1-dependent manner. However, the underlying mechanism by which a cytoplasmic dynein-LIS1-microtubule complex binds kinesin-1 is unknown. Here, we report that mNUDC (mammalian NUDC) interacts with kinesin-1 and is required for the anterograde transport of a cytoplasmic dynein complex by kinesin-1. mNUDC is also required for anterograde transport of a dynactin-containing complex. Inhibition of mNUDC severely suppressed anterograde transport of distinct cytoplasmic dynein and dynactin complexes, whereas motility of kinesin-1 remained intact. Reconstruction experiments clearly demonstrated that mNUDC mediates the interaction of the dynein or dynactin complex with kinesin-1 and supports their transport by kinesin-1. Our findings have uncovered an essential role of mNUDC for anterograde transport of dynein and dynactin by kinesin-1.
Journal of Biological Chemistry, 2010
A single amino acid change, F580Y (Legs at odd angles, Loa, Dync1h1 Loa ), in the highly conserved and overlapping homodimerisation, intermediate chain, and light intermediate chain binding domain of the cytoplasmic dynein heavy chain can cause severe motor and sensory neuron loss in mice. The mechanism by which the Loa mutation impairs the neuron-specific functions of dynein is not understood. To elucidate the underlying molecular mechanisms of neurodegeneration arising from this mutation, we applied a cohort of biochemical methods combined with in vivo assays to systemically study the effects of the mutation on the assembly of dynein and its interaction with dynactin.