Project Summary
The long term goal of this research project has been and continues to be an understanding of the molecular
mechanism of muscle function. The existing project, funded continuously since 1983, has evolved in parallel
with the capabilities of both microscopes and methods for 3-D image reconstruction from electron
microscopes. Originally focused on understanding myosin-actin interactions in situ in muscle using
chemically fixed, plastic embedded and sectioned muscle, it now proposes to take advantage of the resolution
revolution in cryoEM to study the major structural elements of the muscle at the highest resolution possible
using isolated components, followed by a return to imaging actin-myosin interactions in situ in frozen live
muscle cells. Striated muscles have four major components: actin-containing thin filaments, myosin-
containing thick filaments, a Z-disk to crosslink antiparallel thin filaments and a connecting filament to link
the thick filaments to the Z-disk. The least understood of these four elements are the thick filament, the Z-disk
and the connecting filament, whose interactions with the thick filament and Z-disk are its least understood
elements. Thus, our study of Z-disk and thick filament can make major contributions to an understanding of
all three elements. Following a major breakthrough of ours that showed that coiled-coil tail domain of myosin
can be imaged at subnanometer resolution, even near atomic resolution, the project concentrates initially on
subnanometer resolution imaging of thick filaments from several species, to examine the generality and
structural conservation of the “curved molecular crystalline layers” across species and muscle types. The
project will utilize the fruit fly, Drosophila melanogaster, to investigate how genetic removal of certain
component proteins affects how the myosin tails interact with non-myosin proteins to affect thick filament
properties. We will utilize mutations in the myosin tail of Drosophila that correspond to established disease
causing mutations in human striated muscle. The Z-disk will be studied using methodology developed in our
lab to isolate Z-disks from invertebrates applied to determination of the Drosophila melanogaster Z-disk. The
experimental system will facilitate decoration of the Z-disks with various signaling proteins. Ultimately, the
utility of these studies on components needs development within the myofibril. We will investigate by
cryoelectron tomography frozen-hydrated myofibrils of Lethocerus and Drosophila thinned using FIB/SEM in
states produced using various nucleotides to see how myosin heads interact with the thin filament in situ.
Ultimately, we will apply what we have learned methodologically from these myofibril studies to studies of
live cultured smooth and cardiac muscle cells fast frozen, thinned via FIB/SEM to visualize active interactions
between thick and thin filaments. This work will open to future structural investigation all the structures
present in a muscle cell within their natural context.