# Molecular Mechanisms of Motility Deduced from in Vitro Reconstituted Microtubule- and Actin-Based Motor Complexes

> **NIH NIH R35** · UNIVERSITY OF VERMONT & ST AGRIC COLLEGE · 2021 · $390,000

## Abstract

Our overall approach is to focus on multi-component in vitro reconstitutions that will provide insight into complex
biological processes such as cargo transport and cytokinesis. Expressed proteins used in the reconstitutions will
be biochemically characterized, and single-molecule and biochemical/biophysical techniques will assess motor
function. Cytoplasmic dynein-1 and kinesins drive long-distance motion on microtubules, which is required for
cell polarity and function. Dynein moves to the minus-end of the polar MT and drives retrograde transport, while
kinesins of class 1, 2 and 3 power motion to the opposite plus-end and drive anterograde transport. The biological
cargoes of these motors include membrane-bound vesicles, organelles and mRNA. Defects in trafficking
contribute to developmental and neurodegenerative diseases (e.g. Huntington’s and amyotrophic lateral
sclerosis). Bidirectional motion of cellular cargoes as well as purified organelles are driven by motors of opposite
directionality in many organisms and cell types. Dynein requires both dynactin and an activating adaptor for full
motor activity, and these adaptors are emerging as scaffolds for coupling both dynein and kinesin motors. A
major goal is to build on our in vitro reconstituted complex containing dynein-dynactin, the adaptor protein
Bicaudal D, the mRNA-binding protein Egalitarian, and mRNA cargo by the addition of kinesin-1. Preliminary
data show that this complex recapitulates the bidirectional motion seen in the cell. We will use biophysical and
single molecule techniques (TIRF and iSCAT microscopy) to determine the stepping patterns and force
dependence of these complexes to understand how the motors co-ordinate and/or compete to achieve this
motion. We will determine if coupling dynein with different classes of transporting kinesins (kinesin-1, kinesin-2,
or kinesin-3) affects the outcome, and how microtubule-associated proteins (MAPs) regulate these transport
complexes. To generalize findings, we will reconstitute a dynein-kinesin-1 complex based on the scaffolding
protein huntingtin, because it plays a causative role in Huntington’s disease. A second goal is to further our
biochemical/biophysical characterization of fission yeast myosins involved in cytokinesis. A major driving force
for cytokinesis is the interaction between myosin and actin that powers constriction of the contractile ring. The
complexity of this process in animal cells has led to the use of fission yeast as a favored model system. To
propose a more detailed molecular mechanism for cytokinesis in fission yeast it is essential to have an in depth
characterization of the principal contractile components. Here we will use biochemical/biophysical techniques to
characterize the two class II myosins involved in cytokinesis (Myo2 and Myp2), and determine how light chain
phosphorylation regulates their speed and force output. Lastly, we will pursue via collaboration how track
geometry influences transport ...

## Key facts

- **NIH application ID:** 10133095
- **Project number:** 5R35GM136288-02
- **Recipient organization:** UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
- **Principal Investigator:** KATHLEEN M TRYBUS
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $390,000
- **Award type:** 5
- **Project period:** 2020-04-01 → 2025-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10133095

## Citation

> US National Institutes of Health, RePORTER application 10133095, Molecular Mechanisms of Motility Deduced from in Vitro Reconstituted Microtubule- and Actin-Based Motor Complexes (5R35GM136288-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10133095. Licensed CC0.

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