Characterizing how discrete functional domains in neurons are created by investigating how proteins are trafficked; combining in vitro approaches with in vivo live-cell imaging in the developing fruit fly
Neurons are functionally and structurally polarized cells, with distinct cellular projections that are specialized to receive and send signals (dendrites and axons, respectively). Axons and dendrites are essential for transmitting signals within a neuronal circuit, yet the molecular mechanisms that create these distinct structures have remained elusive. Our lab is addressing how neuronal polarity is created and maintained by focusing on the microtubule cytoskeleton, which has a dual function within cells: microtubules provide morphological structure and also serve as the major “highway” for the transport of proteins and organelles that are integral to neuronal function. We are combining genetic, molecular, live-cell imaging and biochemical approaches to delineate the microtubule-based mechanisms that create a polarized neuron, using the developing fruit fly as a model. Reflecting the importance of the microtubule cytoskeleton in neuronal development, multiple human neurodevelopmental disorders, including classic lissencephaly, are linked to microtubule defects. One of our central goals is to identify the molecular and cellular etiology of human disorders arising from microtubule defects, and determine how changes in the microtubule cytoskeleton impact neuronal structure and function.
The impact of microtubule post-translational modifications on neuronal polarization
The neuronal microtubule cytoskeleton is particularly complex, and differences in microtubule polarity, microtubule associated proteins (MAPs) and post-translational modifications (PTMs) are key features differentiating axons and dendrites. Of these features, PTMs have been little studied, and many critical questions, including what functional role microtubule PTMs have in specifying axon versus dendrite, remain unanswered. To address these questions, we are utilizing new genomic engineering techniques to create a novel system to manipulate tubulin and the microtubule cytoskeleton in vivo and test how changes in microtubule PTMs affect neuronal polarization. Our approach integrates genomic engineering with live-cell imaging to visualize how changes in the microtubule cytoskeleton affect neuronal morphology and microtubule dynamics within an intact, developing animal.
Effects of microtubule post-translational modifications on motor behavior
Within neurons, many different proteins and organelles are localized specifically to either dendrites or axons through the activity of the microtubule-based motors dynein and kinesin. Yet it remains unclear how motor proteins recognize where they are within cells and how they transport cargo to specific cellular destinations. The interaction between motors and the microtubule cytoskeleton can be regulated in part by microtubule-associated proteins (MAPs) and, as proposed more recently, microtubule post-translational modifications (PTMs). As MAPs and PTMs are differentially enriched in axons and dendrites, they are proposed to function as molecular “signposts” to guide motors to deliver their cargo to particular destinations within cells. We are addressing how microtubule PTMs affect the behavior of molecular motors, focusing on dynein, and are employing a combination of genetics and single molecule analysis. A significant advantage of using Drosophila is that we can easily move between probing the biophysical properties of the motor in single molecule assays in vitro to characterizing how it functions in neurons in vivo using genetics and live-cell imaging.
The functional architecture of sensory neuron dendrites
We rely on our senses to collect a rich array of information about our surroundings. Sensory neurons play an essential role in our lives, yet surprisingly little is known about the functional organization of sensory neuron dendrites, particularly about the trafficking of the receptors that mediate responses to environmental stimuli. Although receptor protein localization plays a significant role in sensory neuron function, the mechanisms regulating receptor localization and membrane insertion within a sensory dendritic arbor have not been investigated. Microtubule stability and microtubule PTMs have been proposed to correlate with receptor localization, but this model has not yet been tested. By integrating genomic engineering and protein labeling approaches, we plan to determine which features of the microtubule cytoskeleton (e.g. PTMs, stability) direct receptor localization and/or membrane insertion within sensory neuron dendrites.