Don B. Arnold

Myosin-dependent targeting of transmembrane proteins to neuronal dendrites.

The distinct electrical properties of axonal and dendritic membranes are largely a result of specific transport of vesicle-bound membrane proteins to each compartment. How this specificity arises is unclear because kinesin motors that transport vesicles cannot autonomously distinguish dendritically projecting microtubules from those projecting axonally. We hypothesized that interaction with a second motor might enable vesicles containing dendritic proteins to preferentially associate with dendritically projecting microtubules and avoid those that project to the axon. Here we show that in rat cortical neurons, localization of several distinct transmembrane proteins to dendrites is dependent on specific myosin motors and an intact actin network. Moreover, fusion with a myosin-binding domain from Melanophilin targeted Channelrhodopsin-2 specifically to the somatodendritic compartment of neurons in mice in vivo. Together, our results suggest that dendritic transmembrane proteins direct the vesicles in which they are transported to avoid the axonal compartment through interaction with myosin motors.

An evolutionarily conserved dileucine motif in Shal K + channels mediates dendritic targeting

The molecular mechanisms underlying polarized sorting of proteins in neurons are poorly understood. Here we report the identification of a 16 amino-acid, dileucine-containing motif that mediates dendritic targeting in a variety of neuronal cell types in slices of rat brain. This motif is present in the carboxy (C) termini of Shal-family K+ channels and is highly conserved from C. elegans to humans. It is necessary for dendritic targeting of potassium channel Kv4.2 and is sufficient to target the axonally localized channels Kv1.3 and Kv1.4 to the dendrites. It can also mediate dendritic targeting of a non-channel protein, CD8.

MOLECULAR DETERMINANTS FOR SUBCELLULAR LOCALIZATION OF PSD-95 WITH AN INTERACTING K+ CHANNEL

Ion channels and PSD-95 are colocalized in specific neuronal subcellular locations by an unknown mechanism. To investigate mechanisms of localization, we used biolistic techniques to express GFP-tagged PSD-95 (PSD-95:GFP) and the K(+)-selective channel Kv1.4 in slices of rat cortex. In pyramidal cells, PSD-95:GFP required a single PDZ domain and a region including the SH3 domain for localization to postsynaptic sites. When transfected alone, PSD-95:GFP was present in dendrites but absent from axons. When cotransfected with Kv1.4, PSD-95:GFP appeared in both axons and dendrites, while Kv1.4 was restricted to axons. When domains that mediate the interaction of Kv1.4 and PSD-95 were disrupted, Kv1.4 localized nonspecifically. Our results provide evidence that Kv1.4 itself may determine its subcellular location, while an associated MAGUK protein is a necessary but not sufficient cofactor.

A Role for Kif17 in Transport of Kv4.2

Although kinesins are known to transport neuronal proteins, it is not known what role they play in the targeting of their cargos to specific subcellular compartments in neurons. Here we present evidence that the K+ channel Kv4.2, which is a major regulator of dendritic excitability, is transported to dendrites by the kinesin isoform Kif17. We show that a dominant negative construct against Kif17 dramatically inhibits localization to dendrites of both introduced and endogenous Kv4.2, but those against other kinesins found in dendrites do not. Kv4.2 colocalizes with Kif17 but not with other kinesin isoforms in dendrites of cortical neurons. Native Kv4.2 and Kif17 coimmunoprecipitate from brain lysate, and introduced, tagged versions of the two proteins coimmunoprecipitate from COS cell lysate, indicating that the two proteins interact, either directly or indirectly. The interaction between Kif17 and Kv4.2 appears to occur through the extreme C terminus of Kv4.2 and not through the dileucine motif. Thus, the dileucine motif does not determine the localization of Kv4.2 by causing the channel to interact with a specific motor protein. In support of this conclusion, we found that the dileucine motif mediates dendritic targeting of CD8 independent of Kif17. Together our data show that Kif17 is probably the motor that transports Kv4.2 to dendrites but suggest that this motor does not, by itself, specify dendritic localization of the channel.

Networks of Polarized Actin Filaments in the Axon Initial Segment Provide a Mechanism for Sorting Axonal and Dendritic Proteins

Trafficking of proteins specifically to the axonal or somatodendritic membrane allows neurons to establish and maintain polarized compartments with distinct morphology and function. Diverse evidence suggests that an actin-dependent vesicle filter within the axon initial segment (AIS) plays a critical role in polarized trafficking; however, no distinctive actin-based structures capable of comprising such a filter have been found within the AIS. Here, using correlative light and scanning electron microscopy, we visualized networks of actin filaments several microns wide within the AIS of cortical neurons in culture. Individual filaments within these patches are predominantly oriented with their plus ends facing toward the cell body, consistent with models of filter selectivity. Vesicles carrying dendritic proteins are much more likely to stop in regions occupied by the actin patches than in other regions, indicating that the patches likely prevent movement of dendritic proteins to the axon and thereby act as a vesicle filter.

Differential Trafficking of Transport Vesicles Contributes to the Localization of Dendritic Proteins

In neurons, transmembrane proteins are targeted to dendrites in vesicles that traffic solely within the somatodendritic compartment. How these vesicles are retained within the somatodendritic domain is unknown. Here, we use a novel pulse-chase system, which allows synchronous release of exogenous transmembrane proteins from the endoplasmic reticulum to follow movements of post-Golgi transport vesicles. Surprisingly, we found that post-Golgi vesicles carrying dendritic proteins were equally likely to enter axons and dendrites. However, once such vesicles entered the axon, they very rarely moved beyond the axon initial segment but instead either halted or reversed direction in an actin and Myosin Va-dependent manner. In contrast, vesicles carrying either an axonal or a nonspecifically localized protein only rarely halted or reversed and instead generally proceeded to the distal axon. Thus, our results are consistent with the axon initial segment behaving as a vesicle filter that mediates the differential trafficking of transport vesicles.

A Role for Myosin VI in the Localization of Axonal Proteins

In neurons polarized trafficking of vesicle-bound membrane proteins gives rise to the distinct molecular composition and functional properties of axons and dendrites. Despite their central role in shaping neuronal form and function, surprisingly little is known about the molecular processes that mediate polarized targeting of neuronal proteins. Recently, the plus-end-directed motor Myosin Va was shown to play a critical role in targeting of transmembrane proteins to dendrites; however, the role of myosin motors in axonal targeting is unknown. Here we show that Myosin VI, a minus-end-directed motor, plays a vital role in the enrichment of proteins on the surface of axons. Engineering non-neuronal proteins to interact with Myosin VI causes them to become highly concentrated at the axonal surface in dissociated rat cortical neurons. Furthermore, disruption of either Myosin VI function or expression leads to aberrant dendritic localization of axonal proteins. Myosin VI mediates the enrichment of proteins on the axonal surface at least in part by stimulating dendrite-specific endocytosis, a mechanism that has been shown to underlie the localization of many axonal proteins. In addition, a version of Channelrhodopsin 2 that was engineered to bind to Myosin VI is concentrated at the surface of the axon of cortical neurons in mice in vivo, suggesting that it could be a useful tool for probing circuit structure and function. Together, our results indicate that myosins help shape the polarized distributions of both axonal and dendritic proteins.

The role of Kif5B in axonal localization of Kv1 K+ channels

Here we present evidence that the kinesin, Kif5B, is involved in the transportation and axonal targeting of Kv1 channels. We show that a dominant negative variant of Kif5B specifically blocks localization to the axon of expressed, tagged versions of Kv1.3 in cultured cortical slices. In addition, the dominant negative variant of Kif5B blocks axonal localization of endogenous Kv1.1, Kv1.2, and Kv1.4 in cortical neurons in dissociated cultures. We also found evidence that Kif5B interacts with Kv1 channels. Endogenous Kv1.2 colocalized with Kif5B in cortical neurons and coimmunoprecipitated with Kif5B from brain lysate. The T1 domain of Shaker K+ channels has been shown to play a critical role in targeting the channel to the axon. We have three pieces of evidence to suggest that the T1 domain also mediates interaction between Kv1 channels and Kif5B: Addition of the T1 domain to a heterologous protein, TfR, is sufficient to cause the resulting fusion protein, TfRT1, to colocalize with Kif5B. Also, the T1 domain is necessary for interaction of Kv1.3 with Kif5B in a coimmunoprecipitation assay. Finally, dominant negative variants of Kif5B block axonal targeting of TfRT1, but have no effect on dendritic localization of TfR. Together these data suggest a model where Kif5B interacts with Kv1 channels either directly or indirectly via the T1 domain, causing the channels to be transported to axons.

Actin and Microtubule-Based Cytoskeletal Cues Direct Polarized Targeting of Proteins in Neurons

Delivery of proteins to axons or dendrites depends on interactions between molecular motors and the cytoskeleton. Neuronal proteins are transported to either the axon or dendrites through the action of kinesin motors; however, understanding of how cytoskeletal elements steer these cargo-motor complexes to one compartment or the other has remained elusive. Three recent developments—the discovery of an actin-based filter within the axon initial segment, the identification of the pivotal role played by myosin motors in dendritic targeting, and the determination of the properties of a kinesin motor that cause it to prefer axonal to dendritic microtubules—have now provided a structural framework for understanding polarized targeting in neurons.

The T1 domain of Kv1.3 mediates intracellular targeting to axons.

Shaker K+ channels play an important role in modulating electrical excitability of axons. Recent work has demonstrated that the T1 tetramerization domain of Kv1.2 is both necessary and sufficient for targeting of the channel to the axonal surface [ Gu, C., Jan, Y.N. & Jan, L.Y. (2003)Science,301, 646–649]. Here we use a related channel, Kv1.3, as a model to investigate cellular mechanisms that mediate axonal targeting. We show that the T1 domain of Kv1.3 is necessary and sufficient to mediate targeting of the channel to the axonal surface in pyramidal neurons in slices of cortex from neonatal rat. The T1 domain is also sufficient to cause preferential axonal localization of intracellular protein, which indicates that the domain probably does not work through compartment‐specific endocytosis or compartment‐specific vesicle docking. To determine whether the T1 domain mediates axonal trafficking of transport vesicles, we compared the trafficking of vesicles containing green fluorescent protein‐labelled transferrin receptor with those containing the same protein fused with the T1 domain in living cortical neurons. Vesicles containing the wild‐type transferrin receptor did not traffic to the axon, in accord with previously published results; however, those containing the transferrin receptor fused to T1 did traffic to the axon. These results are consistent with the T1 domain of Kv1.3 mediating axonal targeting by causing transport vesicles to traffic to axons and they represent the first evidence that such a mechanism might underlie axonal targeting.