Neurons are the fundamental unit of the nervous system, their function being to receive and propagate electrochemical signals. Due to this role, neurons can be exceptionally large cells with specialized subdomains known as dendrites and axons. To perform their signal reception and propagation functions, dendrites and axons contain unique complements of proteins which must be transported and delivered to their required destination. These proteins are packaged into membrane-bound organelles which molecular motors, such as kinesins, bind and transport. Kinesin-driven organelle transport is crucial for neuron development and maintenance, yet the mechanisms by which kinesins recognize and bind their specific organelle cargoes remain poorly defined. The neuronal function and specific organelle adaptors of heterodimeric Kinesin-2 family members KIF3AB and KIF3AC remain unknown. I developed a novel microscopy-based assay to define protein–protein interactions in intact neurons. The experiments revealed that KIF3AB and KIF3AC both bind kinesin-associated protein (KAP) and that these interactions are mediated by the distal C-terminal tail regions and not the coiled-coil domain. I used live-cell imaging in cultured hippocampal neurons to define the localization and trafficking parameters of KIF3AB and KIF3AC organelle populations. KIF3AB/KAP and KIF3AC/KAP bind the same organelle populations, and I defined their transport parameters in axons and dendrites. The results also show that ~12% of KIF3 organelles contain the RNA binding protein, adenomatous polyposis coli. These data point towards a model in which KIF3AB and KIF3AC use KAP as their neuronal organelle adaptor and that these kinesins mediate transport of a range of organelles.
In a separate project, I described a novel strategy to allow for consistent visualization of kinesin-bound organelles in live mammalian neurons. Previous attempts to label kinesin-bound organelles in live cells utilized expression of fluorophore-fused full-length kinesins. This strategy results in a diffuse, cytosolic expression pattern which obscures labeled organelles. This large fraction of cytosolic expression is hypothesized to be due to unbound autoinhibited motor. Therefore, reduction of this cytosolic, unbound kinesin pool is crucial for visualization of organelle-bound kinesins.
I describe two strategies that improve visualization of vesicle-bound kinesins. The first is a truncation strategy where only the organelle-binding tail domain of kinesins are expressed. Truncated kinesins only expressing organelle-binding tail domains are unable to form an autoinhibited conformation, increasing the amount of exogenously expressed protein available to bind to organelles. The second is a transcriptional control technique where constructs are designed with a nuclear localization signal and a zinc finger domain that acts as a plasmid-specific transcription repressor. Upon translation, any unbound kinesin tail is targeted to the nucleus where it represses its own transcription. Using these strategies drastically improves the imaging conditions for organelle-bound kinesins in live hippocampal neurons.;
December2022; School of Science
Biochemistry and Biophysics Program;
Rensselaer Polytechnic Institute, Troy, NY
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