Myosin-dependent targeting of transmembrane proteins to neuronal dendrites

Abstract

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.The differential trafficking of proteins synthesized in the secretory pathway allows the establishment and maintenance of membrane domains with distinct functional and morphological properties at opposite ends of polarized cells 1 . A first step in this process has been well defined in epithelial cells and involves the sorting of proteins at the trans-Golgi membrane into vesicles bound for either the apical or basolateral domain through interaction with clathrin adaptor proteins 2 . In neurons proteins bound for either the dendritic or axonal domains are also loaded into specific vesicles 3 . Moreover, this sorting step is likely to proceed by a similar mechanism in the two cell types, as AP4β, a constituent of a clathrin adaptor complex, is necessary for dendritic targeting of AMPA receptors 4 .Subsequent steps in polarized targeting of neuronal proteins are much less well defined. However, their properties can be inferred from the observations that vesicles containing dendritic proteins traffic specifically to dendrites, avoiding axonal processes 3 , and that kinesin motors, which interact with microtubules, are responsible for movement of vesicles within dendrites 5 . Taken together, these results suggest that after sorting at the Golgi membrane, vesicles carrying dendritic proteins preferentially interact with microtubules that project to NIH Public Access RESULTS Myosin Va is necessary for dendritic targetingTo investigate possible roles of myosin in polarized targeting, we examined the contributions of Myosins Va and Vb to dendritic targeting of the AMPA receptor GluR1, to which both myosins bind 11,15 . We used dissociated cultures of cortical neurons taken from embryonic day 18 rat embryos and transfected between 12 and 18 d in vitro with plasmid constructs encoding tagged transmembrane proteins. When transfected into dissociated rat cortical neurons, GluR1 tagged with green fluorescent protein (GFP) (Glu...

Specific Sorting and Post-Golgi Trafficking of Dendritic Potassium Channels in Living Neurons

“…By combining this approach with a number of quantitative analyses, we provided evidence that two dendritic ion channels, Kv2.1 and Kv4.2, are precisely sorted into distinct pools of transport vesicles that are targeted to specific dendritic subcompartments by different molecular mechanisms. Previous work has demonstrated that membrane proteins targeted to either axonal or somatodendritic compart- ments, such as the transferrin receptor and neuroglia cell adhesion molecule (NgCAM) are sorted in distinct populations of transport vesicles (1), a result that has also been supported by recent studies (37,38). Our study further extends this compartment-specific trafficking model to ion channels that are localized to specific subcompartments within the dendrites.…”

Delineating cooperative responses of processive motors in living cells

“…This expectation is consistent with results from live-cell motility assays showing that the addition of myosinVb motors to cargos can arrest kinesin-2-dependent vesicle motions (31). Such responses are also consistent with observations that myosinV motors play key roles in neuronal trafficking processes while interacting with kinesins (32). Fully confirming such behavior will require further investigations and assays using additional cell types.…”

Theoretical principles underlying optical stimulation of a channelrhodopsin-2 positive pyramidal neuron

“…For example, ChETA is a ChR2 genetic variant that has faster opening and closing rates than ChR2, with a reduced photocurrent amplitude (Gunaydin et al 2010), whereas ChEF demonstrates slower temporal kinetics, but increased light sensitivity (Lin et al 2009). ChR2 is also being targeted to specific regions of the neuron, such as the dendrites (Lewis et al 2009) and the axon (Lewis et al 2011). When ChR2 was limited to the axon initial segment (Grubb and Burrone 2010), action potentials could not be generated, presumably due to the small number of channels available for recruitment.…”

“…Our model predicts that redistributing ChR2 to the apical dendrites for apical directed stimulation may decrease irradiance levels by up to threefold. Recent advances in the development of not only neuron-specific, but also neuron section-specific, promoters for ChR2 (Lewis et al 2009) suggest that such channel distributions are possible. This concept could be especially advantageous for large volume optical stimulation with LEDs that rest on the cortical surface, while still targeting selective activation of layer V pyramidal neurons.…”