Kinesin Regulation in the Proximal Axon is Essential for Dendrite-selective Transport.

Neurons are polarized and typically extend multiple dendrites and one axon. To maintain polarity, vesicles carrying dendritic proteins are arrested upon entering the axon. To determine whether kinesin regulation is required for terminating anterograde axonal transport, we overexpressed the dendrite-selective kinesin KIF13A. This caused mistargeting of dendrite-selective vesicles to the axon and a loss of dendritic polarity. Polarity was not disrupted if the kinase MARK2/Par1b was coexpressed. MARK2/Par1b is concentrated in the proximal axon, where it maintains dendritic polarity-likely by phosphorylating S1371 of KIF13A, which lies in a canonical 14-3-3 binding motif. We probed for interactions of KIF13A with 14-3-3 isoforms and found that 14-3-3ß and 14-3-3ζ bound KIF13A. Disruption of MARK2 or 14-3-3 activity by small molecule inhibitors caused a loss of dendritic polarity. These data show that kinesin regulation is integral for dendrite-selective transport. We propose a new model in which KIF13A that moves dendrite-selective vesicles in the proximal axon is phosphorylated by MARK2. Phosphorylated KIF13A is then recognized by 14-3-3, which causes dissociation of KIF13A from the vesicle and termination of transport. These findings define a new paradigm for the regulation of vesicle transport by localized kinesin tail phosphorylation, to restrict dendrite-selective vesicles from entering the axon.

Polarized transport requires AP-1-mediated recruitment of KIF13A and KIF13B at the trans-Golgi.

Neurons are polarized cells that require accurate membrane trafficking to maintain distinct protein complements at dendritic and axonal membranes. The Kinesin-3 family members KIF13A and KIF13B are thought to mediate dendrite-selective transport, but the mechanism by which they are recruited to polarized vesicles and the differences in the specific trafficking role of each KIF13 have not been defined. We performed live-cell imaging in cultured hippocampal neurons and found that KIF13A is a dedicated dendrite-selective kinesin. KIF13B confers two different transport modes, dendrite- and axon-selective transport. Both KIF13s are maintained at the trans-Golgi network by interactions with the heterotetrameric adaptor protein complex AP-1. Interference with KIF13 binding to AP-1 resulted in disruptions to both dendrite- and axon-selective trafficking. We propose that AP-1 is the molecular link between the sorting of polarized cargoes into vesicles and the recruitment of kinesins that confer polarized transport.

Poly(pro-curcumin) Materials Exhibit Dual Release Rates and Prolonged Antioxidant Activity as Thin Films and Self-Assembled Particles.

Curcumin is a natural polyphenol that exhibits remarkable antioxidant and anti-inflammatory activities; however, its clinical application is limited in part by its physiological instability. Here, we report the synthesis of curcumin-derived polyesters that release curcumin upon hydrolytic degradation to improve curcumin stability and solubility in physiological conditions. Curcumin was incorporated in the polymer backbone by a one-pot condensation polymerization in the presence of sebacoyl chloride and polyethylene glycol (PEG, Mn = 1 kDa). The thermal and mechanical properties, surface wettability, self-assembly behavior, and drug-release kinetics all depend sensitively on the mole percentage of curcumin incorporated in these statistical copolymers. Curcumin release was triggered by the hydrolysis of phenolic esters on the polymer backbone, which was confirmed using a PEGylated curcumin model compound, which represented a putative repeating unit within the polymer. The release rate of curcumin was controlled by the hydrophilicity of the polymers. Burst release (2 days) and extended release (>8 weeks) can be achieved from the same polymer depending on curcumin content in the copolymer. The materials can quench free radicals for at least 8 weeks and protect primary neurons from oxidative stress in vitro. Further, these copolymer materials could be processed into both thin films and self-assembled particles, depending on the solvent-based casting conditions. Finally, we envision that these materials may have potential for neural tissue engineering application, where antioxidant release can mitigate oxidative stress and the inflammatory response following neural injury.

KAP is the neuronal organelle adaptor for Kinesin-2 KIF3AB and KIF3AC.

Kinesin-driven organelle transport is crucial for neuron development and maintenance, yet the mechanisms by which kinesins specifically bind their organelle cargoes remain undefined. In contrast to other transport kinesins, the neuronal function and specific organelle adaptors of heterodimeric Kinesin-2 family members KIF3AB and KIF3AC remain unknown. We developed a novel microscopy-based assay to define protein-protein interactions in intact neurons. The experiments found that both KIF3AB and KIF3AC bind kinesin-associated protein (KAP). These interactions are mediated by the distal C-terminal tail regions and not the coiled-coil domain. We used live-cell imaging in cultured hippocampal neurons to define the localization and trafficking parameters of KIF3AB and KIF3AC organelle populations. We discovered that KIF3AB/KAP and KIF3AC/KAP bind the same organelle populations and defined their transport parameters in axons and dendrites. The results also show that ∼12% of KIF3 organelles contain the RNA-binding protein adenomatous polyposis coli. These data point toward a model in which KIF3AB and KIF3AC use KAP as their neuronal organelle adaptor and that these kinesins mediate transport of a range of organelles.

Propofol attenuates kinesin-mediated axonal vesicle transport and fusion.

Propofol is a widely used general anesthetic, yet the understanding of its cellular effects is fragmentary. General anesthetics are not as innocuous as once believed and have a wide range of molecular targets that include kinesin motors. Propofol, ketamine, and etomidate reduce the distances that Kinesin-1 KIF5 and Kinesin-2 KIF3 travel along microtubules in vitro. These transport kinesins are highly expressed in the CNS, and their dysfunction leads to a range of human pathologies including neurodevelopmental and neurodegenerative diseases. While in vitro data suggest that general anesthetics may disrupt kinesin transport in neurons, this hypothesis remains untested. Here we find that propofol treatment of hippocampal neurons decreased vesicle transport mediated by Kinesin-1 KIF5 and Kinesin-3 KIF1A ∼25-60%. Propofol treatment delayed delivery of the KIF5 cargo NgCAM to the distal axon. Because KIF1A participates in axonal transport of presynaptic vesicles, we tested whether prolonged propofol treatment affects synaptic vesicle fusion mediated by VAMP2. The data show that propofol-induced transport delay causes a significant decrease in vesicle fusion in distal axons. These results are the first to link a propofol-induced delay in neuronal trafficking to a decrease in axonal vesicle fusion, which may alter physiological function during and after anesthesia.

Visualizing Vesicle-Bound Kinesins in Cultured Hippocampal Neurons.

Eukaryotic cells use microtubule-based vesicle transport to exchange molecules between compartments. Kinesin family members mediate all microtubule plus end-directed vesicle transport. Of the 45 kinesins expressed in humans, some 20 mediate microtubule plus-end directed vesicle transport. Here we describe a technique to visualize vesicle-bound kinesins in cultured hippocampal neurons. The method involves the expression of the vesicle-binding tail domain while minimizing the cytoplasmic pool. Using this approach drastically improves vesicle labeling compared to full-length kinesins. This tool is useful for systematically comparing the localization of different kinesins in the same cell type and for identifying cargo proteins that reside in vesicles moved by a specific kinesin family member. While we describe the assay in cultured hippocampal neurons, we expect it to be easily transferable to other eukaryotic cell types.

NgCAM and VAMP2 reveal that direct delivery and dendritic degradation maintain axonal polarity.

Neurons are polarized cells of extreme scale and compartmentalization. To fulfill their role in electrochemical signaling, axons must maintain a specific complement of membrane proteins. Despite being the subject of considerable attention, the trafficking pathway of axonal membrane proteins is not well understood. Two pathways, direct delivery and transcytosis, have been proposed. Previous studies reached contradictory conclusions about which of these mediates delivery of axonal membrane proteins to their destination, in part because they evaluated long-term distribution changes and not vesicle transport. We developed a novel strategy to selectively label vesicles in different trafficking pathways and determined the trafficking of two canonical axonal membrane proteins, neuron-glia cell adhesion molecule and vesicle-associated membrane protein-2. Results from detailed quantitative analyses of transporting vesicles differed substantially from previous studies and found that axonal membrane proteins overwhelmingly undergo direct delivery. Transcytosis plays only a minor role in axonal delivery of these proteins. In addition, we identified a novel pathway by which wayward axonal proteins that reach the dendritic plasma membrane are targeted to lysosomes. These results redefine how axonal proteins achieve their polarized distribution, a crucial requirement for elucidating the underlying molecular mechanisms.

A novel labeling strategy reveals that myosin Va and myosin Vb bind the same dendritically polarized vesicle population.

Neurons are specialized cells with a polarized geometry and several distinct subdomains that require specific complements of proteins. Delivery of transmembrane proteins requires vesicle transport, which is mediated by molecular motor proteins. The myosin V family of motor proteins mediates transport to the barbed end of actin filaments, and little is known about the vesicles bound by myosin V in neurons. We developed a novel strategy to visualize myosin V-labeled vesicles in cultured hippocampal neurons and systematically characterized the vesicle populations labeled by myosin Va and Vb. We find that both myosins bind vesicles that are polarized to the somatodendritic domain where they undergo bidirectional long-range transport. A series of two-color imaging experiments showed that myosin V specifically colocalized with two different vesicle populations: vesicles labeled with the transferrin receptor and vesicles labeled by low-density lipoprotein receptor. Finally, coexpression with Kinesin-3 family members found that myosin V binds vesicles concurrently with KIF13A or KIF13B, supporting the hypothesis that coregulation of kinesins and myosin V on vesicles is likely to play an important role in neuronal vesicle transport. We anticipate that this new assay will be applicable in a broad range of cell types to determine the function of myosin V motor proteins.

Smart motors and cargo steering drive kinesin-mediated selective transport.

Neurons are polarized cells, with dendrites and axons that require different complements of membrane proteins to fulfill their specialized functions. Membrane proteins are synthesized in the somatodendritic domain and delivered to their target membranes via long-range vesicle transport. Most anterograde vesicle transport is mediated by kinesin motors, but it is unclear how kinesins are targeted to axons or dendrites. Two main models have been proposed to explain kinesin selectivity. In the smart motor model, kinesin selectivity is conferred by a preference of the kinesin motor domain for specific subsets of microtubules. In the cargo steering model, kinesin selectivity is modulated by the vesicular cargo to which the motor is bound. We evaluate the evidence for both models and conclude that while the smart motor model may explain axonal selectivity, cargo steering is required for dendritic selectivity. Future work will determine the relative contributions of these models to polarized transport in living neurons.

A novel strategy to visualize vesicle-bound kinesins reveals the diversity of kinesin-mediated transport.

In mammals, 15 to 20 kinesins are thought to mediate vesicle transport. Little is known about the identity of vesicles moved by each kinesin or the functional significance of such diversity. To characterize the transport mediated by different kinesins, we developed a novel strategy to visualize vesicle-bound kinesins in living cells. We applied this method to cultured neurons and systematically determined the localization and transport parameters of vesicles labeled by different members of the Kinesin-1, -2, and -3 families. We observed vesicle labeling with nearly all kinesins. Only six kinesins bound vesicles that undergo long-range transport in neurons. Of these, three had an axonal bias (KIF5B, KIF5C and KIF13B), two were unbiased (KIF1A and KIF1Bß), and one transported only in dendrites (KIF13A). Overall, the trafficking of vesicle-bound kinesins to axons or dendrites did not correspond to their motor domain preference, suggesting that on-vesicle regulation is crucial for kinesin targeting. Surprisingly, several kinesins were associated with populations of somatodendritic vesicles that underwent little long-range transport. This assay should be broadly applicable for investigating kinesin function in many cell types.

The cellular mechanisms that maintain neuronal polarity.

As polarized cells, neurons maintain different sets of resident plasma membrane proteins in their axons and dendrites, which is consistent with the different roles that these neurites have in electrochemical signalling. Axonal and dendritic proteins are synthesized together within the somatodendritic domain; this raises a fundamental question: what is the nature of the intracellular trafficking machinery that ensures that these proteins reach the correct domain? Recent studies have advanced our understanding of the processes underlying the selective sorting and selective transport of axonal and dendritic proteins and have created potential avenues for future progress.

Analyzing kinesin motor domain translocation in cultured hippocampal neurons.

Neuronal microtubules are subject to extensive posttranslational modifications and are bound by MAPs, tip-binding proteins, and other accessory proteins. All of these features, which are difficult to replicate in vitro, are likely to influence the translocation of kinesin motors. Here we describe assays for evaluating the translocation of a population of fluorescently labeled kinesin motor domains, based on their accumulation in regions of the cell enriched in microtubule plus ends. Neurons lend themselves to these experiments because of their microtubule organization. In axons, microtubules are oriented with their plus ends out; dendrites contain a mixed population of microtubules, but those near the tips are also plus end out. The assays involve the expression of constitutively active kinesins that can walk processively, but that lack the autoinhibitory domain in the tail that normally prevents their binding to microtubules until they attach to vesicles. The degree to which such motor domains accumulate at neurite tips serves as a measure of the efficiency of their translocation. Although these assays cannot provide the kind of quantitative kinetic information obtained from in vitro assays, they offer a simple way to examine kinesin translocation in living neurons. They can be used to compare the translocation efficiency of different kinesin motors and to evaluate how mutations or posttranslational modifications within the motor domain influence kinesin translocation. Changes to motor domain accumulation in these assays can also serve as readout for changes in the microtubule cytoskeleton that affect kinesin translocation.

A Novel Assay to Identify the Trafficking Proteins that Bind to Specific Vesicle Populations.

Here we describe a method capable of identifying interactions between candidate trafficking proteins and a defined vesicle population in intact cells. The assay involves the expression of an FKBP12-rapamycin binding domain (FRB)-tagged candidate vesicle-binding protein that can be inducibly linked to an FKBP-tagged molecular motor. If the FRB-tagged candidate protein binds the labeled vesicles, then linking the FRB and FKBP domains recruits motors to the vesicles and causes a predictable, highly distinctive change in vesicle trafficking. We describe two versions of the assay: a general protocol for use in cells with a typical microtubule-organizing center and a specialized protocol designed to detect protein-vesicle interactions in cultured neurons. We have successfully used this assay to identify kinesins and Rabs that bind to a variety of different vesicle populations. In principle, this assay could be used to investigate interactions between any category of vesicle trafficking proteins and any vesicle population that can be specifically labeled.

A novel assay reveals preferential binding between Rabs, kinesins, and specific endosomal subpopulations.

Identifying the proteins that regulate vesicle trafficking is a fundamental problem in cell biology. In this paper, we introduce a new assay that involves the expression of an FKBP12-rapamycin-binding domain-tagged candidate vesicle-binding protein, which can be inducibly linked to dynein or kinesin. Vesicles can be labeled by any convenient method. If the candidate protein binds the labeled vesicles, addition of the linker drug results in a predictable, highly distinctive change in vesicle localization. This assay generates robust and easily interpretable results that provide direct experimental evidence of binding between a candidate protein and the vesicle population of interest. We used this approach to compare the binding of Kinesin-3 family members with different endosomal populations. We found that KIF13A and KIF13B bind preferentially to early endosomes and that KIF1A and KIF1Bß bind preferentially to late endosomes and lysosomes. This assay may have broad utility for identifying the trafficking proteins that bind to different vesicle populations.

Apoptosis-linked gene-2 (ALG-2)/Sec31 interactions regulate endoplasmic reticulum (ER)-to-Golgi transport: a potential effector pathway for luminal calcium.

Luminal calcium released from secretory organelles has been suggested to play a regulatory role in vesicle transport at several steps in the secretory pathway; however, its functional roles and effector pathways have not been elucidated. Here we demonstrate for the first time that specific luminal calcium depletion leads to a significant decrease in endoplasmic reticulum (ER)-to-Golgi transport rates in intact cells. Ultrastructural analysis revealed that luminal calcium depletion is accompanied by increased accumulation of intermediate compartment proteins in COPII buds and clusters of unfused COPII vesicles at ER exit sites. Furthermore, we present several lines of evidence suggesting that luminal calcium affected transport at least in part through calcium-dependent interactions between apoptosis-linked gene-2 (ALG-2) and the Sec31A proline-rich region: 1) targeted disruption of ALG-2/Sec31A interactions caused severe defects in ER-to-Golgi transport in intact cells; 2) effects of luminal calcium and ALG-2/Sec31A interactions on transport mutually required each other; and 3) Sec31A function in transport required luminal calcium. Morphological phenotypes of disrupted ALG-2/Sec31A interactions were characterized. We found that ALG-2/Sec31A interactions were not required for the localization of Sec31A to ER exit sites per se but appeared to acutely regulate the stability and trafficking of the cargo receptor p24 and the distribution of the vesicle tether protein p115. These results represent the first outline of a mechanism that connects luminal calcium to specific protein interactions regulating vesicle trafficking machinery.

The clathrin adaptor complex responsible for somatodendritic protein sorting.

Neuronal proteins contain "address labels" that govern their localization. In this issue of Neuron, Farías et al. (2012) identify the machinery that recognizes one class of dendritic localization signals and establish its role in the polarization of dendritic proteins, including several postsynaptic receptors.

A novel split kinesin assay identifies motor proteins that interact with distinct vesicle populations.

Identifying the kinesin motors that interact with different vesicle populations is a longstanding and challenging problem with implications for many aspects of cell biology. Here we introduce a new live-cell assay to assess kinesin-vesicle interactions and use it to identify kinesins that bind to vesicles undergoing dendrite-selective transport in cultured hippocampal neurons. We prepared a library of "split kinesins," comprising an axon-selective kinesin motor domain and a series of kinesin tail domains that can attach to their native vesicles; when the split kinesins were assembled by chemical dimerization, bound vesicles were misdirected into the axon. This method provided highly specific results, showing that three Kinesin-3 family members-KIF1A, KIF13A, and KIF13B-interacted with dendritic vesicle populations. This experimental paradigm allows a systematic approach to evaluate motor-vesicle interactions in living cells.

Alpha-synuclein delays endoplasmic reticulum (ER)-to-Golgi transport in mammalian cells by antagonizing ER/Golgi SNAREs.

Toxicity of human alpha-synuclein when expressed in simple organisms can be suppressed by overexpression of endoplasmic reticulum (ER)-to-Golgi transport machinery, suggesting that inhibition of constitutive secretion represents a fundamental cause of the toxicity. Whether similar inhibition in mammals represents a cause of familial Parkinson's disease has not been established. We tested elements of this hypothesis by expressing human alpha-synuclein in mammalian kidney and neuroendocrine cells and assessing ER-to-Golgi transport. Overexpression of wild type or the familial disease-associated A53T mutant alpha-synuclein delayed transport by up to 50%; however, A53T inhibited more potently. The secretory delay occurred at low expression levels and was not accompanied by insoluble alpha-synuclein aggregates or mistargeting of transport machinery, suggesting a direct action of soluble alpha-synuclein on trafficking proteins. Co-overexpression of ER/Golgi arginine soluble N-ethylmaleimide-sensitive factor attachment protein receptors (R-SNAREs) specifically rescued transport, indicating that alpha-synuclein antagonizes SNARE function. Ykt6 reversed alpha-synuclein inhibition much more effectively than sec22b, suggesting a possible neuroprotective role for the enigmatic high expression of ykt6 in neurons. In in vitro reconstitutions, purified alpha-synuclein A53T protein specifically inhibited COPII vesicle docking and fusion at a pre-Golgi step. Finally, soluble alpha-synuclein A53T directly bound ER/Golgi SNAREs and inhibited SNARE complex assembly, providing a potential mechanism for toxic effects in the early secretory pathway.

Vesicular calcium regulates coat retention, fusogenicity, and size of pre-Golgi intermediates.

The significance and extent of Ca(2+) regulation of the biosynthetic secretory pathway have been difficult to establish, and our knowledge of regulatory relationships integrating Ca(2+) with vesicle coats and function is rudimentary. Here, we investigated potential roles and mechanisms of luminal Ca(2+) in the early secretory pathway. Specific depletion of luminal Ca(2+) in living normal rat kidney cells using cyclopiazonic acid (CPA) resulted in the extreme expansion of vesicular tubular cluster (VTC) elements. Consistent with this, a suppressive role for vesicle-associated Ca(2+) in COPII vesicle homotypic fusion was demonstrated in vitro using Ca(2+) chelators. The EF-hand-containing protein apoptosis-linked gene 2 (ALG-2), previously implicated in the stabilization of sec31 at endoplasmic reticulum exit sites, inhibited COPII vesicle fusion in a Ca(2+)-requiring manner, suggesting that ALG-2 may be a sensor for the effects of vesicular Ca(2+) on homotypic fusion. Immunoisolation established that Ca(2+) chelation inhibits and ALG-2 specifically favors residual retention of the COPII outer shell protein sec31 on pre-Golgi fusion intermediates. We conclude that vesicle-associated Ca(2+), acting through ALG-2, favors the retention of residual coat molecules that seem to suppress membrane fusion. We propose that in cells, these Ca(2+)-dependent mechanisms temporally regulate COPII vesicle interactions, VTC biogenesis, cargo sorting, and VTC maturation.

SNARE status regulates tether recruitment and function in homotypic COPII vesicle fusion.

In mammals, coat complex II (COPII)-coated transport vesicles deliver secretory cargo to vesicular tubular clusters (VTCs) that facilitate cargo sorting and transport to the Golgi. We documented in vitro tethering and SNARE-dependent homotypic fusion of endoplasmic reticulum-derived COPII transport vesicles to form larger cargo containers characteristic of VTCs ( Xu, D., and Hay, J. C. (2004) J. Cell Biol. 167, 997-1003). COPII vesicles thus appear to contain all necessary components for homotypic tethering and fusion, providing a pathway for de novo VTC biogenesis. Here we demonstrate that antibodies against the endoplasmic reticulum/Golgi SNARE Syntaxin 5 inhibit COPII vesicle homotypic tethering as well as fusion, implying an unanticipated role for SNAREs upstream of fusion. Inhibition of SNARE complex access and/or disassembly with dominant-negative alpha-soluble NSF attachment protein (SNAP) also inhibited tethering, implicating SNARE status as a critical determinant in COPII vesicle tethering. The tethering-defective vesicles generated in the presence of dominant-negative alpha-SNAP specifically lacked the Rab1 effectors p115 and GM130 but not other peripheral membrane proteins. Furthermore, Rab effectors, including p115, were shown to be required for homotypic COPII vesicle tethering. Thus, our results demonstrate a requirement for SNARE-dependent tether recruitment and function in COPII vesicle fusion. We anticipate that recruitment of tether molecules by an upstream SNARE signal ensures that tethering events are initiated only at focal sites containing appropriately poised fusion machinery.