Regulation of the dynamics at actin's ends is of central importance in the assembly of thin filaments in the striated muscle sarcomeres, as well as in smooth muscle and non-muscle cells. An actin filament has two distinct ends: a fast-growing barbed end and a slower growing pointed end. Tropomodulin (Tmod) is a tropomyosin (TM) binding protein that binds to and regulates the dynamics of the pointed end. This proposal focuses on defining structure-function relationships in Tmod, and proteins that bind to and regulate its function. Of the many possible mechanisms that might influence dynamics at the actin pointed end, three that appear to be the most likely will be explored: isoform dependence in Tmod/TM interaction (Aim 1), identification of still unknown Tmod binding partners that change Tmod functional abilities (Aim 2) and Tmod phosphorylation (Aim 3). Actin capping is tight in in vitro experiments;yet, in living myocytes capping is transient. There must be a mechanism to regulate Tmod, either a protein that regulates by binding Tmod, or covalent modification.
In Aim 1, model peptides of the N-terminus of TM that bind to Tmod, and of the two defined TM binding sites on Tmod will be used to learn the structural basis of the specificity of binding of different TMs to different Tmods. Binding will be assayed using native gel electrophoresis, cross-linking, and circular dichroism spectroscopy. The specificities will then be evaluated in pointed end elongation assays and in rescue experiments using Tmod1 null embryonic stem cells.
Aim 2 tests the hypothesis: The Leucine-Rich- Repeat (LRR) domain in Tmod binds factors that regulate its function. Impure actin preparations from skeletal muscle contain a factor that negatively regulates Tmod capping. The activity depends on the presence of the LRR domain, a motif typically involved in protein-protein interactions.
The aim will be to purify, identify and characterize the regulatory factor.
In Aim 3 phosphorylation of Tmod by TRPM7 kinase will be studied. The kinase phosphorylates Tmod1 in multiple sites, Ser2, Thr54 and Ser163. Antibodies against phosphorylated sites will be used to identify modified Tmod in cells. Residues in the sites will be mutated to Ala and Asp or Glu to create an unphosphorylatable and a phosphomimetic proteins, correspondingly. The influence of these mutations on Tmod1 function will be assayed in TM-binding, nebulin-binding and actin-capping experiments. The effects of Tmod modifications on myofibrillogenesis will be checked in myocytes. Factors regulating Tmod capping activity are of great importance in proper myofibril formation. Determining the regulatory factors, whether they are a known or unknown entity, will provide a "missing link" in our understanding of control of the pointed end, and its upstream regulation.
Tropomodulin is a tropomyosin-binding protein which caps the slow-growing (pointed) end of the actin filament and therefore regulates its dynamics. Its overexpression in the heart of mice leads to myofibril degeneration and dilated cardiomyopathy. In a tropomodulin-null mouse, heart defects lead to embryonic lethality. Factors regulating Tmod function are of great importance in proper myofibril formation. The information obtained in this study will help us to understand the formation of the capping complex at the pointed end and its function in regulating dynamics of the actin filament in cells and tissues.
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