# Isolation, Characterization and Reconstruction of Vertebrate Striated Muscle Myosin Filaments

> **NIH NIH R21** · FLORIDA STATE UNIVERSITY · 2021 · $192,059

## Abstract

Project Summary
The long term goal of this research project is to understand the molecular mechanism of force production
through 3-D visualization of myosin molecular motors in their natural environment. This research project
focuses on extending methods used and results obtained in structural studies of muscle filaments isolated from
the large waterbug Lethocerus sp. to vertebrate striated muscle, specifically skeletal muscles obtained from
rabbits, Oryctolagus cuniculus. We have obtained a near atomic resolution 3-D image of thick filaments from
Lethocerus flight muscle, which have a helical structure with 4-fold rotational symmetry. No coiled-coil
protein of the size of myosin had been imaged previously at the resolution we have achieved (4.2Å) in the
backbone of the myosin filament. There is now more known about the 3-D structure of Lethocerus thick
filaments than those from any other animal. Recent advancements in detector technology, robotic electron
microscopes and high throughput data collection, have made this possible. We now propose to extend these
methods to the much more difficult vertebrate skeletal muscle thick filament, which is not a helical assembly
and has only 3-fold rotational symmetry. The high-resolution structure of Lethocerus thick filaments suggests
that studies of invertebrate thick filaments can inform familial muscle diseases caused by myosin rod
mutations. About 40% of disease-causing myosin mutations occur in the myosin coiled-coil domain. However,
how well invertebrate thick filaments can inform human disease depends on how similar their filament
backbones are structured like those of vertebrates. In the current funding period, we have obtained
unprecedented resolution and detail of the relaxed state of thick filaments from Lethocerus flight muscle. This
advance provides opportunity to investigate the mechanism whereby myosin rod mutations can affect muscle
function. The head folding of myosin II produces a head conformation called the interacting heads motif that
sequesters the myosin heads from interaction with the thin filament. In filaments of smooth and non-muscle
myosin, the head folding leads to filament instability and formation of a soluble conformation, called 10S,
incapable of polymerizing. This phenomenon has been hypothesized to be due to changes in the rod structure
brought on by the head folding. Put simply, the structure of the myosin rod and the myosin heads are coupled
in some way. Recent muscle research has pointed to the possibility that tension applied either internally by
myosin heads or externally by a stretch, can affect the structure of the thick filament. Thus, the thick filament
may function as a tension transducer, but the molecular mechanism by which this occurs is unknown. We
hypothesize that tension applied to the thick filament affects the structure of the myosin heads and vice versa,
that the myosin heads affect the structure of the myosin tails. This hypothesis can be tested using ...

## Key facts

- **NIH application ID:** 10268975
- **Project number:** 5R21AR077802-02
- **Recipient organization:** FLORIDA STATE UNIVERSITY
- **Principal Investigator:** Jose Renato Pinto
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $192,059
- **Award type:** 5
- **Project period:** 2020-09-24 → 2023-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10268975

## Citation

> US National Institutes of Health, RePORTER application 10268975, Isolation, Characterization and Reconstruction of Vertebrate Striated Muscle Myosin Filaments (5R21AR077802-02). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10268975. Licensed CC0.

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