Neurons communicate with each other via junctions known as synapses. Several components, such as synaptic vesicles, mitochondria, and active zone proteins, are necessary for information transfer to occur at synapses. Because most protein synthesis and cargo biogenesis takes place in the neuronal cell body, these components have to be transported to the synapse, which can be far from the cell body. The axon connecting the cell body and the synapse provides a highway for such long-distance transport. Axonal transport occurs in two directions: anterograde, from the cell body to the synapse, and retrograde, from the synapse to the cell body. Transport in both directions is important for neuronal function, and defects in transport contribute to several neurodegenerative diseases such as amyotrophic lateral sclerosis and Charcot-Marie-Tooth2A.
My laboratory seeks to understand the molecular mechanisms of long-distance axonal transport in vivo using C. elegans as a model. The axon contains microtubule tracks on which molecular motors walk, ferrying their cargo between the cell body and the synapse in both directions. Such transport is a complex multistep process. For example, in the anterograde direction, the motor has to recognize and bind its cargo, enter the axon, and start walking on the microtubules. Subsequently, cargo has to be directed to the correct destination, it has to be released from the motor upon reaching its destination, and then free motors have to be regulated. To understand this process, my laboratory is investigating which regulatory molecules are involved in each step and how they act together. This knowledge could contribute to possible disease intervention strategies.
My laboratory has focused on anterograde transport of two major cargoes: synaptic vesicles and mitochondria. To study how their transport is regulated, we have taken a primarily genetic approach using genetic screens, which provide an excellent means to identify molecules involved in regulating biological processes in vivo. We use the well-developed genetic model system C. elegans for our studies. In addition, the transparency of C. elegans allows live imaging of biological processes in intact living animals. This approach is particularly valuable for studying axonal transport because it is a long-distance dynamic process that occurs over minutes. For example, transport defects in mutants are often easy to detect and to describe by studying movies of cargo motion.
Synaptic Vesicle Transport
Synaptic vesicles are neurotransmitter-filled packages manufactured in the neuronal cell body. Because release of neurotransmitters is required for communication between neurons, synaptic vesicle transport is vital for synaptic function. The anterograde motor kinesin-3 carries synaptic vesicles to the synapse. To identify other molecules that regulate synaptic vesicle transport, my laboratory carried out genetic screens looking for second-site mutations in kinesin-3/unc-104 mutant backgrounds. Among the resulting complementation groups, we identified an allele of jip-3/unc-16, a gene encoding a JNK-interacting protein. JIP3 was previously known to play a role in transport as an adapter for another motor, kinesin-1. Our work points to a novel motor-independent role for JIP3/UNC-16 in trafficking of synaptic vesicle proteins.
In another study, variable cargo-binding ability of a series of kinesin-3/unc-104 alleles led us to directly examine the fate of this kinesin-3 motor at synapses. We found that kinesin-3 motors are degraded at synapses through ubiquitination (Figure 1), likely after they drop off their synaptic vesicle cargo. Our findings suggest that motor degradation could be a mechanism that ensures correct directionality of axonal transport. For example, without degradation, a motor may inappropriately attach to retrograde cargo and impede the cargo's intended passage to the cell body.
Transport of Mitochondria
My laboratory has begun to investigate the axonal transport of mitochondria. Mitochondria have a multiplicity of roles, a major one in this context being to provide energy for neurotransmitter release at the synapse. We observed that the nature of motion and the distribution of mitochondria in the axon differ greatly from those of synaptic vesicles. Mitochondria show much more frequent local reversal of motion compared with synaptic vesicles. Furthermore, the latter are largely localized to synapses, whereas the former are distributed throughout the axon. We think it likely that different regulatory mechanisms underlie the observed differences between the distribution of the two types of cargo. By looking at mutants showing altered mitochondrial distributions and by using quantitative analysis of observed motion, we are attempting to understand how motors and motor regulators drive the observed mitochondrial distributions.
Tools and Interdisciplinary Approaches
My laboratory studies axonal transport using concepts and tools from other disciplines as well. First, the ability to make high-quality movies allows the generation of a large amount of quantitative data measuring parameters such as cargo flux, velocity, and pause frequency, among others. In collaboration with a physicist, we are building mathematical models of cargo motion incorporating some of these parameters. Such a model has the potential to complement our genetic approach, for example, by generating testable predictions that we can experimentally verify.
Second, in collaboration with engineers, we have developed two methods to expand the scope of imaging studies. We built microfluidic devices to immobilize C. elegans noninvasively, permitting precise live imaging of subcellular processes (Figure 2). We are continuing to develop other microfluidic platforms that allow us, for instance, to track individual organisms throughout their development. We have also developed a laser-axotomy-based assay to track bulk flow of cargo in the axon over longer time scales.
Figure 2: Imaging synaptic vesicle transport in a microfluidic device. An adult C. elegans trapped and visualized in bright field (16fps, scale bar 200um). Synaptic vesicle transport transport imaged at 5fps, scale bar 5µm. Koushika lab members Sudip Mondal, Bikash Choudhary, and Jyoti Dubey
Third, although retrograde axonal transport is also an important process, it is harder to study than anterograde transport. We have addressed part of the difficulty by developing an assay to specifically tag and follow endogenous retrograde cargo in living C. elegans. We hope to expand this method and use it to study regulation in the retrograde pathway.
Grants from the government of India through the Department of Science and Technology, Department of Biotechnology, and Council of Scientific and Industrial Research provided partial support for these projects.
As of January 17, 2012