# Organization of Microtubule Polarity During Neuronal Axon Development in vivo

> **NIH NIH F32** · UNIVERSITY OF WISCONSIN-MADISON · 2020 · $20,820

## Abstract

PROJECT SUMMARY
Proper organization of microtubules (MTs) is critical for development and function of neural circuits. MTs form
the tracks on which the molecular motors kinesin and dynein transport cargo to specific cell locations. The
inherent plus end/minus end polarity of MTs directs motor transport. In axons MTs are arranged with their plus
ends distal to the cell body. MT polarity must be established during initial axon formation, and also during axon
branching events. The mechanisms regulating MT polarity in axons and branches are poorly understood, but a
body of evidence suggests that molecular motors are crucial to MT polarity. Kinesin and dynein contribute to
MT polarity and are hypothesized to act by transport of MTs. However, visualizing MT polymer transport is
challenging and has only been done in vitro or cultured neurons.
Our lab showed previously that Clstn1, a kinesin-1 adaptor, regulates axon branching in sensory neurons. Our
preliminary data show that Clstn1 is also required for MT polarity in axons. How a kinesin adaptor, known
primarily to mediate cargo transport, also regulates MT polarity is unknown. My goal is to reveal the underlying
mechanisms. Clstn1 is known to bind and activate KLC, which activates KHC and kinesin motor activity, and
inhibits KHC tail binding to MTs. I hypothesize that Clstn1 activation of KLC prevents excessive MT
crosslinking by KHC tail-MT binding. In the absence of Clstn1, this crosslinking may oppose dynein’s ability to
remove misoriented MTs from the axon, leading to an increase in mispolarized MTs. I will use high-speed in
vivo 4D imaging approaches to test these hypotheses.
In Aim 1, I will first test if activation of KLC using a Clstn1 W-acidic domain peptide is sufficient to rescue MT
polarity in Clstn1-/- mutants. Second, I will use two approaches to disrupt the binding of the KHC tail to MTs to
reduce KHC crosslinking to MTs, and test for rescue of MT polarity Clstn1-/- mutants. Finally, I will test the roles
of KLC phosphorylation and proteolytic cleavage of Clstn1 in MT polarity.
In Aim 2 I propose to use advanced imaging technologies in collaboration with the Laboratory of Optical and
Computational Instrumentation to image MT dynamics in vivo. I will label MT polymers and plus/minus ends
with multiple fluorophores, and use fluorescence recovery after photobleaching to determine whether MT
transport contributes to polarity in vivo. I will test whether Clstn1 loss affects MT transport, and thereby
influences polarity. I will also test the hypothesis that Clstn1 functions to organize MT polarity during axon
branching by imaging MT dynamics at branch points. These experiments will enhance our understanding of the
cellular and molecular mechanisms that establish axon MT polarity.

## Key facts

- **NIH application ID:** 10267658
- **Project number:** 5F32NS098689-04
- **Recipient organization:** UNIVERSITY OF WISCONSIN-MADISON
- **Principal Investigator:** Elizabeth Haynes
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $20,820
- **Award type:** 5
- **Project period:** 2017-06-01 → 2020-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10267658

## Citation

> US National Institutes of Health, RePORTER application 10267658, Organization of Microtubule Polarity During Neuronal Axon Development in vivo (5F32NS098689-04). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10267658. Licensed CC0.

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