Polarized transport requires ap-1-mediated recruitment of kif13a and kif13b at the trans-golgi
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Authors
Montgomery, Andrew, Carver
Issue Date
2023-08
Type
Electronic thesis
Thesis
Thesis
Language
en_US
Keywords
Biology
Alternative Title
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
School of Science
Full Citation
Publisher
Rensselaer Polytechnic Institute, Troy, NY