# The Mechanism and Regulation of Cytoplasmic and Ciliary Dyneins

> **NIH NIH R35** · UNIVERSITY OF CALIFORNIA BERKELEY · 2021 · $617,252

## Abstract

Project Summary
Dyneins are AAA+ motors responsible for minus-end-directed motility along microtubules (MTs) and play
fundamental roles in cargo transport, mitosis, and ciliary beating. Dynein is currently the focus of the motor field
as the mechanism of its movement is not well understood in comparison to plus-end-directed kinesins. Despite
rapid transport of dynein-driven cargos in cells, previous in vitro studies identified mammalian dynein as a weak
motor, exhibiting slow motility and producing lower forces than kinesin. Recently, in vitro reconstitution of the
dynein-dynactin machinery revealed that mammalian dynein is autoinhibited when not transporting cargo, and
motility is activated when dynein forms a 2.5 MDa ternary complex with its cofactor dynactin and a cargo binding
adaptor. Therefore, all of the previous in vitro work on mammalian dynein used inactive motor and their
conclusions do not reflect how active dynein-dynactin machinery transports cargos in cells.
Our future goals are to dissect the mechanism of active cytoplasmic dynein complexes and determine how
dynein activation and motility are regulated across multiple scales using single molecule imaging, optical
trapping, MD simulations, and cryoEM. Specifically, we will determine how Lis1 plays a role in the activation of
and regulation of dynein motility. We will also study dynein motility in physiologically relevant conditions and ask
whether MT-associated proteins, MAP7 and Tau, inhibit dynein motility by sterically blocking its tubulin binding
site or by excluding its MT binding via liquid-liquid demixing. We will also characterize the motility of dynein and
dynactin disease mutants to reveal the molecular mechanism of neuropathies associated with these mutations.
Finally, we will reconstitute the entire MT transport machinery using cargo adaptors identified by in vivo studies
of mitochondria, autophagosomes, and vesicle transport, but not yet characterized in vitro. Using this approach,
we will dissect how cargo adaptors regulate motors to control the bidirectional transport of these cargos.
We will also study ciliary dyneins that slide parallel array of axonemal MTs to power ciliary beating. Several
models have been proposed to explain how the sliding activity of dyneins is self-regulated to orchestrate ciliary
oscillations. Predictions that these models make about the mechanism of ciliary dyneins have not been directly
tested. Recently, a recombinant expression system was developed for Tetrahymena outer-arm dynein (OAD),
enabling us to perform in-depth structural and biophysical studies of ciliary dyneins. Unlike cytoplasmic dynein,
OAD forms a heterodimer and is not processive. Using this system, we will characterize the mechanism of OAD
motility and force generation. We will then directly test the predictions of each model by constructing in vitro
geometries that mimic dynein/MT interactions in a beating cilium. Finally, we will identify structural components
that give ...

## Key facts

- **NIH application ID:** 10133096
- **Project number:** 5R35GM136414-02
- **Recipient organization:** UNIVERSITY OF CALIFORNIA BERKELEY
- **Principal Investigator:** Ahmet Yildiz
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $617,252
- **Award type:** 5
- **Project period:** 2020-04-01 → 2025-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10133096

## Citation

> US National Institutes of Health, RePORTER application 10133096, The Mechanism and Regulation of Cytoplasmic and Ciliary Dyneins (5R35GM136414-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10133096. Licensed CC0.

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