Regulation of retrograde cargo transport in axons

The formation and maintenance of neural circuits relies on the active transport of proteins and organelles throughout developing and mature neurons. This is a particularly important and challenging process in neurons that have long axonal projections extending great distances from the cell body. The primary driver of organelle and protein transport throughout axonal processes are molecular motors of the Kinesin and Dynein families. These motors use microtubules as tracks and are guided by microtubule polarity. The superfamily of Kinesin motor proteins (~45 in humans) is responsible for anterograde axonal transport towards microtubule plus ends oriented towards axon terminals. Conversely, a single molecular motor, cytoplasmic dynein, is the primary motor proteins complex responsible for microtubule minus end (cell body) directed transport of cargos. How unique cargos attach to the single retrograde motor for transport to new locations is largely unknown but likely relies on adaptor proteins that link them conditionally to the motor for transport. Our lab uses genetics, live imaging of cargo transport, and biochemistry in zebrafish embryos and larvae to identify novel regulators of dynein-mediated cargo transport in axons. 

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In Vivo analysis of Axonal traIn Vivo analysis of Axonal transport. (Top) Schematic of the in vivo transport setup in the zebrafish posterior lateral line (pLL). Neuromasts (NM; blue) are innervated by the pLL nerve (green) which extends from the pLL ganglion (pLLg) located just behind the ear. For transport analyses, zygotes are injected with DNA at the 1 cell stage resulting in the mosaic cellular inheritance of the expression construct. Larvae expressing tagged cargo (red) are then imaged. Right inset shows innervation of a NM by 2 axons, 1 expressing the tagged cargo. (Bottom left) Still of mitochondrial transport in the zebrafish pLL. Arrows point to the mitochondria that moved in the anterograde (pink) and retrograde (yellow) directions during imaging.

Mitochondria-dynein interaction during retrograde transport

Mitochondria are essential organelles in axons, necessary for the localized production of ATP as well as serving an important role in calcium buffering. Consequently, the proper positioning of mitochondria in axons is crucial for the formation and maintenance of active neural circuits. While elegant work has begun to  elucidate how anterograde mitochondrial transport is regulated by Kinesin motors, how retrograde mitochondrial transport is modulated is largely unknown. In a forward genetic screen we identified a zebrafish mutant with abnormal mitochondrial localization in axons due to disrupted retrograde movement of this organelle. A primary goal for our lab is to define the mechanism of mitochondria-dynein interaction for regulated transport of this organelle in axons. Additionally, we use functional studies to address how disrupted mitochondrial localization and transport impacts both mitochondrial potential, mitochondrial calcium buffering capacity, and neural circuit activity. 

Image of mitochondria-dynein interaction during retrograde transport; wildtype vs mutant; color and black and white images shown.
Cargo accumulation in axon terminals: a sign of disrupted cargo transport?

Cargo-specific retrograde axonal transport and neural circuit function

In a forward genetic screen we identified additional mutant lines with phenotypes indicative of retrograde transport disruptions. While the genetic lesions have been identified in these lines, the specific cellular defects caused by these point mutations is still unknown. Using these lines and additional strains found with further forward and reverse genetic screening, we will work to define the mechanisms of cargo-specific retrograde transport in axons. In addition, we will use these lines to then define the impact of retrograde transport disruptions on sensory and motor neural circuit function in vivo.

Comparison of mutant lines during retrograde transport disruptions.


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