Dynein and kinesin motor proteins transport cellular cargos toward opposite ends of microtubule tracks. In neurons, microtubules are abundantly decorated with microtubule-associated proteins (MAPs) such as tau. Motor proteins thus encounter MAPs frequently along their path. To determine the effects of tau on dynein and kinesin motility, we conducted single molecule studies of motor proteins moving along tau-decorated microtubules. Dynein tended to reverse direction whereas kinesin tended to detach at patches of bound tau. Kinesin was inhibited at ~ 10-fold lower tau concentration than dynein and the microtubule-binding domain of tau was sufficient to inhibit motor activity. The differential modulation of dynein and kinesin motility suggests that MAPs can spatially regulate the balance of microtubule-dependent axonal transport.Active transport of cytoplasmic material along microtubules is critical for cell organization and function, and defects in this process are associated with dysfunction and disease (1). Much of the active transport in cells depends on the molecular motor proteins cytoplasmic dynein and kinesin-1, which transport cargo toward the minus-end (toward the cell center) and plusend of microtubules (toward the cell periphery), respectively. Dynein and kinesin have very different structures and translocation mechanisms (2). Kinesin has a compact motor domain and walks unidirectionally along single protofilaments with 8-nm steps (2). In contrast, dynein has a larger, more complex motor domain and is capable of variable step sizes, lateral steps across the microtubule surface, and processive runs toward both the minus-and plus-end of the microtubule (3-5). Cytoplasmic dynein function in vivo also requires an accessory complex, dynactin. This large, multiprotein complex is thought to facilitate dynein processivity (6) and may also regulate dynein activity (5). Within the cell, the balance between oppositely directed transport determines the steady-state distribution of organelles and biomolecules.In the crowded cell environment, dynein and kinesin compete with non-motile microtubuleassociated proteins (MAPs) for binding to the microtubule surface. MAPs bound to microtubules might also block the path of motor proteins. Thus, MAPs can provide spatiotemporal regulation of motor proteins in vivo. Tau, a neuronal MAP, inhibits kinesin activity in vivo and in vitro (7-10); however, its effect on dynein activity is not well understood. Our aim was to directly observe individual encounters between single dynein or kinesin motors and tau on microtubule tracks to determine how structurally distinct motors respond to obstacles in their path. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptTau is expressed in neurons as multiple splice forms in a developmentally regulated manner (11). These isoforms differ in the number of microtubule binding repeats and the length of the projection domain (Fig. 1A). Here we focused on the shortest and longest tau isoforms, tau23 and tau40, to comp...

Autophagosome biogenesis and maturation in primary neurons is a constitutive process that is spatially and temporally regulated along the axon.

Mitophagy is a cellular quality control pathway in which the E3 ubiquitin ligase parkin targets damaged mitochondria for degradation by autophagosomes. We examined the role of optineurin in mitophagy, as mutations in optineurin are causative for amyotrophic lateral sclerosis (ALS) and glaucoma, diseases in which mitochondrial dysfunction has been implicated. Using live cell imaging, we demonstrate the parkin-dependent recruitment of optineurin to mitochondria damaged by depolarization or reactive oxygen species. Parkin's E3 ubiquitin ligase activity is required to ubiquitinate outer mitochondrial membrane proteins, allowing optineurin to stably associate with ubiquitinated mitochondria via its ubiquitin binding domain; in the absence of parkin, optineurin transiently localizes to damaged mitochondrial tips. Following optineurin recruitment, the omegasome protein double FYVE-containing protein 1 (DFCP1) transiently localizes to damaged mitochondria to initialize autophagosome formation and the recruitment of microtubule-associated protein light chain 3 (LC3). Optineurin then induces autophagosome formation around damaged mitochondria via its LC3 interaction region (LIR) domain. Depletion of endogenous optineurin inhibits LC3 recruitment to mitochondria and inhibits mitochondrial degradation. These defects are rescued by expression of siRNA-resistant wild-type optineurin, but not by an ALS-associated mutant in the ubiquitin binding domain (E478G), or by optineurin with a mutation in the LIR domain. Optineurin and p62/SQSTM1 are independently recruited to separate domains on damaged mitochondria, and p62 is not required for the recruitment of either optineurin or LC3 to damaged mitochondria. Thus, our study establishes an important role for optineurin as an autophagy receptor in parkin-mediated mitophagy and demonstrates that defects in a single pathway can lead to neurodegenerative diseases with distinct pathologies.amaged mitochondria are selectively turned over in eukaryotic cells via mitophagy, a process in which double-membraned autophagosomes sequester and ultimately degrade mitochondria via lysosomal fusion (1, 2). This process is regulated by phosphatase and tensin homolog-induced putative kinase protein 1 (PINK1) and parkin, two proteins linked to hereditary forms of Parkinson's disease (3, 4). PINK1 is stabilized on the outer membrane of damaged mitochondria and recruits the E3 ubiquitin ligase parkin, which ubiquitinates proteins on the outer mitochondrial membrane (OMM) (5-13). Parkin-mediated ubiquitination of damaged mitochondria is followed by autophagosome formation and engulfment of mitochondria (1, 2). However, the proteins involved in dynamically recruiting autophagic machinery to ubiquitinated damaged mitochondria still remain elusive.Optineurin is an autophagy receptor, characterized by its ability to bind ubiquitin via its ubiquitin binding in ABIN (A20 binding and inhibitor of NF-κB) and NEMO (NF-κB essential modulator) (UBAN) domain (14) and the autophagosome-associated protein LC3 (microtub...

Axonal transport is essential for neuronal function, and many neurodevelopmental and neurodegenerative diseases result from mutations in the axonal transport machinery. Anterograde transport supplies distal axons with newly synthesized proteins and lipids, including synaptic components required to maintain presynaptic activity. Retrograde transport is required to maintain homeostasis by removing aging proteins and organelles from the distal axon for degradation and recycling of components. Retrograde axonal transport also plays a major role in neurotrophic and injury response signaling. This review provides an overview of the axonal transport pathway and discusses its role in neuronal function.

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Summary The microtubule motors kinesin and dynein function collectively to drive vesicular transport. High resolution tracking of vesicle motility in the cell indicates that transport is often bidirectional, characterized by frequent directional changes. However, the mechanisms coordinating the collective activities of oppositely-oriented motors bound to the same cargo are not well understood. To examine motor coordination, we purified neuronal transport vesicles and analyzed their motility using automated particle tracking with nanometer resolution. The motility of purified vesicles reconstituted in vitro closely models the movement of Lysotracker-positive vesicles in primary neurons, where processive bidirectional motility is interrupted with frequent directional switches, diffusional movement and pauses. Quantitative analysis indicates that vesicles co-purify with a low number of stably-bound motors: 1–5 dynein and 1–4 kinesin motors. These observations compare well to predictions from a stochastic tug-of-war model, where transport is driven by the force-dependent kinetics of teams of opposing motors in the absence of external regulation. Together, these observations indicate that vesicles move robustly with a small complement of tightly-bound motors, and suggest an efficient regulatory scheme for bidirectional motility where small changes in the number of engaged motors manifest in large changes in the motility of cargo.

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Summary Autophagy is an essential degradative pathway in neurons, yetlittle is known about the mechanisms driving this process in highly polarized cells. Here, we use dual-color live-cell imaging to investigate the neuron-specific mechanisms of constitutiveautophagosome biogenesis in primary DRG and hippocampal cultures. Under basal conditions autophagosomes are continuously generated in the axon tip. There is an ordered assembly of proteins recruited with stereotypical kinetics onto the developing autophagosome. Plasma- or mitochondrial-derived membraneswere not incorporated into nascent autophagosomes in the distal axon. Rather, autophagosomes are generated at DFCP1-positive subdomains of the endoplasmic reticulum, distinct from ER exit sites. Biogenesis events arehighly enriched distally; autophagosomes form infrequently in dendrites, the cell soma or mid-axon, consistent with a highly compartmentalized pathway for constitutive autophagy in primary neurons. This distal biogenesis may facilitate the degradation of damaged mitochondria and long-lived cytoplasmic proteins that reach the axon tip via slow axonal transport.