Polarized transport requires ap-1-mediated recruitment of kif13a and kif13b at the trans-golgi

Authors
Montgomery, Andrew, Carver
ORCID
https://orcid.org/0009-0008-1989-4848
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Other Contributors
Gilbert, Susan, P
Forth, Scott
Gilbert, Ryan
Bentley, Marvin
Issue Date
2023-08
Keywords
Biology
Degree
PhD
Terms of Use
This electronic version is a licensed copy owned by Rensselaer Polytechnic Institute (RPI), Troy, NY. Copyright of original work retained by author.
Full Citation
Abstract
The nervous system is comprised of neurons, which send and receive electrochemicalsignals. To facilitate this function, they possess an exotic morphology containing complementary extensions: the signal-sending axon and signal-receiving dendrites. Unique complements of membrane proteins facilitate these functions, making neurons polarized. Membrane trafficking maintains this polarity but is poorly understood. Dendritically polarized membrane trafficking requires the sorting of dendritic proteins into vesicles at the Golgi, which must recruit the correct molecular motors to confer transport to the dendrites. The related kinesin-3 molecular motors, KIF13A and KIF13B, transport dendrite-selective vesicles. Whether there are differences between these motors and their binding and transport of dendrite-selective vesicles is unknown. I used quantitative fluorescence imaging to determine that KIF13s differ in their colocalization and cotransport with dendrite-selective transferrin receptor (TfR) vesicles: KIF13A is specialized for dendrite-selective transport, with KIF13B assisting but additionally transporting axon-selective vesicles containing neuron-glia cell adhesion molecule (NgCAM). I found that both motors are recruited to Golgi-derived vesicles at the transGolgi network (TGN) by binding the heterotetrameric clathrin adaptor protein (AP) complex-1. Critically, disrupting this interaction reduces dendrite- and axon-selective transport of TfR and NgCAM. However, AP-1 does not serve as the long-term kinesin adaptor for either vesicle population. In this model, KIF13s mediate polarized transport of distinct and overlapping vesicle populations, with AP-1 performing initial recruitment of KIF13s to these vesicles at the TGN. This project was only possible because of the development of a novel method to visualize organelle-bound kinesins. Before this method, researchers usually expressed full-length kinesins fused to a fluorophore that provided poor and inconsistent organelle labeling. Often there would be a soluble population of kinesin that likely concealed kinesin-bound organelles. The current model argues that this soluble pool consists of inhibited kinesins whose motor domains are bound to their cargo-binding tails. To overcome this problem, we expressed only the cargo-binding tails of the kinesins implicated in neuronal vesicle transport. For the smaller Kinesin-1s, we controlled transcription by incorporating a nuclear localization signal and zinc finger domain in the tail constructs. Kinesin-1 motors not bound to organelles would enter the nucleus where they would bind the plasmid and halt transcription. This strategy, combined with short expression times, gave a prodigious improvement in vesicle labeling for many transport kinesins. We now possess a powerful method to directly observe the organelles that kinesins bind.
Description
August2023
School of Science
Department
Dept. of Biological Sciences
Publisher
Rensselaer Polytechnic Institute, Troy, NY
Relationships
Rensselaer Theses and Dissertations Online Collection
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Users may download and share copies with attribution in accordance with a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 license. No commercial use or derivatives are permitted without the explicit approval of the author.
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