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.

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 Development of Neuronal Polarity: A Retrospective View.

In 1988, Carlos Dotti, Chris Sullivan, and I published a paper on the establishment of polarity by hippocampal neurons in culture, which continues to be frequently cited 30 years later (Dotti et al., 1988). By following individual neurons from the time of plating until they had formed well developed axonal and dendritic arbors, we identified the five stages of development that lead to the mature expression of neuronal polarity. We were surprised to find that, before axon formation, the cells pass through a multipolar phase, in which several, apparently identical short neurites undergo periods of extension and retraction. Then one of these neurites begins a period of prolonged growth, becoming the definitive axon; the remaining neurites subsequently become dendrites. This observation suggested that any of the initial neurites were capable of becoming axons, a hypothesis confirmed by later work. In this Progressions article, I will try to recall the circumstances that led to this work, recapture some of the challenges we faced in conducting these experiments, and consider why some of today's neuroscientists still find this paper relevant.

Anterograde Transport of Rab4-Associated Vesicles Regulates Synapse Organization in Drosophila.

Local endosomal recycling at synapses is essential to maintain neurotransmission. Rab4GTPase, found on sorting endosomes, is proposed to balance the flow of vesicles among endocytic, recycling, and degradative pathways in the presynaptic compartment. Here, we report that Rab4-associated vesicles move bidirectionally in Drosophila axons but with an anterograde bias, resulting in their moderate enrichment at the synaptic region of the larval ventral ganglion. Results from FK506 binding protein (FKBP) and FKBP-Rapamycin binding domain (FRB) conjugation assays in rat embryonic fibroblasts together with genetic analyses in Drosophila indicate that an association with Kinesin-2 (mediated by the tail domain of Kinesin-2α/KIF3A/KLP64D subunit) moves Rab4-associated vesicles toward the synapse. Reduction in the anterograde traffic of Rab4 causes an expansion of the volume of the synapse-bearing region in the ventral ganglion and increases the motility of Drosophila larvae. These results suggest that Rab4-dependent vesicular traffic toward the synapse plays a vital role in maintaining synaptic balance in this neuronal network.

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.

Axonal transport plays a crucial role in mediating the axon-protective effects of NmNAT.

Deficits in axonal transport are thought to contribute to the pathology of many neurodegenerative diseases. Expressing the slow Wallerian degeneration protein (Wld(S)) or related nicotinamide mononucleotide adenyltransferases (NmNATs) protects axons against damage from a broad range of insults, but the ability of these proteins to protect against inhibition of axonal transport has received little attention. We set out to determine whether these proteins can protect the axons of cultured hippocampal neurons from damage due to hydrogen peroxide or oxygen-glucose deprivation (OGD) and, in particular, whether they can reduce the damage that these agents cause to the axonal transport machinery. Exposure to these insults inhibited the axonal transport of both mitochondria and of the vesicles that carry axonal membrane proteins; this inhibition occurred hours before the first signs of axonal degeneration. Expressing a cytoplasmically targeted version of NmNAT1 (cytNmNAT1) protected the axons against both insults. It also reduced the inhibition of transport when cells were exposed to hydrogen peroxide and enhanced the recovery of transport following both insults. The protective effects of cytNmNAT1 depend on mitochondrial transport. When mitochondrial transport was inhibited, cytNmNAT1 was unable to protect axons against either insult. The protective effects of mitochondrially targeted NmNAT also were blocked by inhibiting mitochondrial transport. These results establish that NmNAT robustly protects the axonal transport system following exposure to OGD and reactive oxygen species and may offer similar protection in other disease models. Understanding how NmNAT protects the axonal transport system may lead to new strategies for neuroprotection in neurodegenerative diseases.

Selective microtubule-based transport of dendritic membrane proteins arises in concert with axon specification.

The polarized distribution of membrane proteins to axonal or somatodendritic neuronal compartments is fundamental to nearly every aspect of neuronal function. The polarity of dendritic proteins depends on selective microtubule-based transport; the vesicles that carry these proteins are transported into dendrites but do not enter the axon. We used live-cell imaging of fluorescently tagged dendritic and axonal proteins combined with immunostaining for initial segment and cytoskeletal markers to evaluate different models of dendrite-selective transport in cultured rat hippocampal neurons. In mature neurons, dendritic vesicles that entered the base of the axon stopped at the proximal edge of the axon initial segment, defined by immunostaining for ankyrinG, rather than moving into the initial segment itself. In contrast, axonal vesicles passed through the initial segment without impediment. During development, dendrite-selective transport was detected shortly after axons formed, several days before initial segment assembly, before the appearance of a dense actin meshwork in the initial segment, and before dendrites acquire microtubules of mixed polarity orientation. Indeed, some elements of selective transport were detected even before axon specification. These findings are inconsistent with models for selective transport that depend on the presence of an F-actin-based cytoplasmic filter in the initial segment or that posit that transport into dendrites is mediated by dyneins translocating along minus-end out microtubules. Instead our results suggest that selective transport involves the coordinated regulation of the different motor proteins that mediate dendritic vesicle transport and that the selectivity of motor-microtubule interactions is one facet of this process.

Inhibition of fast axonal transport by pathogenic SOD1 involves activation of p38 MAP kinase.

Dying-back degeneration of motor neuron axons represents an established feature of familial amyotrophic lateral sclerosis (FALS) associated with superoxide dismutase 1 (SOD1) mutations, but axon-autonomous effects of pathogenic SOD1 remained undefined. Characteristics of motor neurons affected in FALS include abnormal kinase activation, aberrant neurofilament phosphorylation, and fast axonal transport (FAT) deficits, but functional relationships among these pathogenic events were unclear. Experiments in isolated squid axoplasm reveal that FALS-related SOD1 mutant polypeptides inhibit FAT through a mechanism involving a p38 mitogen activated protein kinase pathway. Mutant SOD1 activated neuronal p38 in mouse spinal cord, neuroblastoma cells and squid axoplasm. Active p38 MAP kinase phosphorylated kinesin-1, and this phosphorylation event inhibited kinesin-1. Finally, vesicle motility assays revealed previously unrecognized, isoform-specific effects of p38 on FAT. Axon-autonomous activation of the p38 pathway represents a novel gain of toxic function for FALS-linked SOD1 proteins consistent with the dying-back pattern of neurodegeneration characteristic of ALS.

Culture of primary rat hippocampal neurons: design, analysis, and optimization of a microfluidic device for cell seeding, coherent growth, and solute delivery.

We present the design, analysis, construction, and culture results of a microfluidic device for the segregation and chemical stimulation of primary rat hippocampal neurons. Our device is designed to achieve spatio-temporal solute delivery to discrete sections of neurons with mitigated mechanical stress. We implement a geometric guidance technique to direct axonal processes of the neurons into specific areas of the device to achieve solute segregation along routed cells. Using physicochemical modeling, we predict flows, concentration profiles, and mechanical stresses within pertiment sections of the device. We demonstrate cell viability and growth within the closed device over a period of 11 days. Additionally, our modeling methodology may be generalized and applied to other device geometries.

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.

Differential neurite outgrowth is required for axon specification by cultured hippocampal neurons.

Formation of an axon is the first morphological evidence of neuronal polarization, visible as a profound outgrowth of the axon compared with sibling neurites. One unsolved question on the mechanism of axon formation is the role of axon outgrowth in axon specification. This question was difficult to assess, because neurons freely extend their neurites in a conventional culture. Here, we leveraged surface nano/micro-modification techniques to fabricate a template substrate for constraining neurite lengths of cultured neurons. Using the template, we asked (i) Do neurons polarize even if all neurites cannot grow sufficiently long? (ii) Would the neurite be fated to become an axon if only one was allowed to grow long? A pattern with symmetrical short paths (20 µm) was used to address the former question, and an asymmetrical pattern with one path extended to 100 µm for the latter. Axon formation was evaluated by tau-1/MAP2 immunostaining and live-cell imaging of constitutively-active kinesin-1. We found that (1) neurons cannot polarize when extension of all neurites is restricted and that (2) when only a single neurite is permitted to grow long, neurons polarize and the longest neurite becomes the axon. These results provide clear evidence that axon outgrowth is required for its specification.

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.

Oxidative stress inhibits axonal transport: implications for neurodegenerative diseases.

BACKGROUND: Reactive oxygen species (ROS) released by microglia and other inflammatory cells can cause axonal degeneration. A reduction in axonal transport has also been implicated as a cause of axonal dystrophies and neurodegeneration, but there is a paucity of experimental data concerning the effects of ROS on axonal transport. We used live cell imaging to examine the effects of hydrogen peroxide on the axonal transport of mitochondria and Golgi-derived vesicles in cultured rat hippocampal neurons. RESULTS: Hydrogen peroxide rapidly inhibited axonal transport, hours before any detectable changes in mitochondrial morphology or signs of axonal degeneration. Mitochondrial transport was affected earlier and was more severely inhibited than the transport of Golgi-derived vesicles. Anterograde vesicle transport was more susceptible to peroxide inhibition than retrograde transport. Axonal transport partially recovered following removal of hydrogen peroxide and local application of hydrogen peroxide inhibited transport, suggesting that the effects were not simply a result of nerve cell death. Sodium azide, an ATP synthesis blocker, had similar effects on axonal transport, suggesting that ATP depletion may contribute to the transport inhibition due to hydrogen peroxide. CONCLUSIONS: These results indicate that inhibition of axonal transport is an early consequence of exposure to ROS and may contribute to subsequent axonal degeneration.

Axonal transport analysis using Multitemporal Association Tracking.

Multitemporal Association Tracking (MAT) is a new graph-based method for multitarget tracking in biological applications that reduces the error rate and implementation complexity compared to approaches based on bipartite matching. The data association problem is solved over a window of future detection data using a graph-based cost function that approximates the Bayesian a posteriori association probability. MAT has been applied to hundreds of image sequences, tracking organelle and vesicles to quantify the deficiencies in axonal transport that can accompany neurodegenerative disorders such as Huntington's Disease and Multiple Sclerosis and to quantify changes in transport in response to therapeutic interventions.

General considerations for live imaging of developing hippocampal neurons in culture.

Dissociated cell cultures of the rodent hippocampus have become a standard model for studying many facets of neural development, including the development of polarity, axonal and dendritic growth, and synapse formation. The cultures are quite homogeneous--∼90% of the cells are pyramidal neurons--and it is relatively easy to express green fluorescent protein (GFP)-tagged proteins by transfection. This article describes the cultures and the key features of the system used to image them. It also includes suggestions on labeling cells with GFP-tagged proteins. It concludes with a discussion of the advantages and disadvantages of this culture system.

Long-term time-lapse imaging of developing hippocampal neurons in culture.

Dissociated cell cultures of the rodent hippocampus have become a standard model for studying many facets of neural development. The cultures are quite homogeneous and it is relatively easy to express green fluorescent protein (GFP)-tagged proteins by transfection. Studying developmental processes that occur over many hours or days--for example, dendritic branching--involves capturing images of a cell at regular intervals without compromising cell survival. This approach is also useful for studying events of short duration that occur asynchronously across the cell population. For such studies, it is highly desirable to use a computer-controlled microscope with an automated stage, to follow multiple cells at different locations in the culture, moving sequentially from one to the next and capturing an image at each location. A method to correct for focal drift is also required. For long-term imaging, we culture neurons in a medium without phenol red, which is thought to give rise to toxic substances following exposure to light. To label cells with GFP-tagged proteins for long-term imaging, we usually use nucleofection (rather than lipid-mediated transfection); this gives a high transfection efficiency, which makes it easier to find the right cell for imaging. Our protocol for long-term imaging is given here, along with appropriate methods to express GFP-tagged proteins. Examples illustrate how the protocol can be used to image cytoskeletal dynamics during axon specification and to study kinesin motor dynamics in stage 2 neurons (when minor neurites extend).

Short-term high-resolution imaging of developing hippocampal neurons in culture.

Dissociated cell cultures of the rodent hippocampus have become a standard model for studying many facets of neural development. The cultures are quite homogeneous and it is relatively easy to express green fluorescent protein (GFP)-tagged proteins by transfection. Because the cultures are essentially two dimensional, there is no need to acquire images at multiple focal planes. For capturing rapid subcellular events at high resolution, as described here, one must maximize weak signals and reduce background fluorescence. Thus, these methods differ in several respects from those used for time-lapse imaging. Lipofectamine-mediated transfection yields a higher level of expression than does transfection with a nucleofection device. Images are usually collected with a spinning-disk confocal microscope, which improves the signal-to-noise ratio. In addition, we use an imaging medium designed to minimize background fluorescence rather than to enhance long-term cell survival. It is also important to select cultures at an appropriate stage of development. In our hands, lipofectamine-based transfection works best on cells between 3 and 10 d after plating. GFP-based fluorescence can be observed as early as 4 h after adding the DNA/lipid complexes to the cells, but expression usually increases over the next ∼12 h and remains steady for days. The ratio of DNA to lipid is critical; to lower expression levels of the tagged construct, we use a combination of expression vector and empty plasmid, keeping the DNA amount constant. An example is included to illustrate the imaging of the microtubule-based vesicular transport of membrane proteins.