Our long term goal is to understand how contractile machinery of muscle cells assembles during development, how it dissembles during remodeling of muscle tissues, how external force and extracellular matrices affect gene expression and muscle structure and function, how the force is transmitted via the Z bands of the sarcomere, and how muscles malfunction in nemaline myopathy and other muscle diseases. The cytoplasm of striated muscle cells contains, besides actin and myosin filaments, contains at least two interconnected lattices. An intermediate filament lattice envelops and links all sarcomeres to the membrane skeleton, mitochondria, nuclei, and sarcoplasmic reticulum. Inside the sarcomere, a cytoskeletal matrix consisted of a set of elastic titin filaments and a set of inextensible nebulin filaments provides structural continuity. Both lattices generate restoring force. Active force and elastic force are transmitted through specialized anchor structures of the sarcomere. 1: Structure and function of the Z bands: One important stress-bearing structure is the Z line, a dense and narrow structure that anchors and organizes four major filaments: actin, titin, nebulin and desmin filaments. The Z lines play important roles in the structural organization of sarcomere, the transmission of mechanical forces as well as the stress signaling pathways. We are addressing the following questions: 1. what are the roles of titin, nebulin (skeletal muscles), nebulette (a nebulin-like protein in the heart) in the assembly and integrity of the Z line in vertebrate muscle? 2. What are the composition and structure of the unusually broad Z line of sonic muscle of Midshipman fish? 3. What are the roles of protein kinases, nonmuscle myosin and other signaling proteins in the function of the Z lines? 4. What is its relationship to the anomalous nemaline rod Z bodies found in aging heart muscle, in diseased skeletal muscle known as nemaline myopathy? (2) The roles of Xin family of proteins: In our search for binding partners of nebulin by yeast two-hybrid, we discovered that nebulin-SH3 binds to a large proline-rich region of human and mouse Xin protein, an adherens junction protein required for cardiac morphogenesis and looping (Wang, et al, 1999, Sinn, et al, 2002). Further characterization of this binding partner indicated that it was a heretofore unknown member of the Xin family that consists of two genes: aXin (first described by Jim Lin at Iowa) and bXin. Both aXin and bXin proteins have similar domain organization with an N-terminal repeat domain and a proline-rich domain. We are investigating the function of bXin in human and mouse. The human bXin (Chr. 2, 371 kb, 15 kb of 15 exons) and mouse bXin (Chr. 2, 330 kb, 12 kb of 13 exons) are expressed as complex, alternatively-spliced transcripts (7 for human bXin;5 for mouse bXin). One major product of the bXin gene is the bXin protein consisting of 3000 residues, with 94% being encoded by one long exon. Other alternatively-spliced variants exclude this exon and incorporate a LIM domain. Exon 7 and 13 of human bXin gene and exon 5 and 11 of mouse bXin gene are muscle-specific. The expression of bXin proteins is high in skeletal muscle and heart and low in lung and testis. bXin protein expression increases after differentiation in C2C12 myoblasts and after birth in mouse skeletal and heart muscle. In cultured mouse embryonic cardiomyocytes, bXin is localized at the stress fibers connecting myofibrils to focal adhesion and non-striated region along myofibrils. bXin appears to be important in the myosin isoform switching from nonmuscle myosin to sarcomeric myosin during myofibrillogenesis. Loss of bXin transcript and protein, by siRNA knock down technique, resulted in aberrant formation of myofibrils and caused the formation of extensive membrane protrusions. (3): Muscle assembly and disassemmbly in muscle cells subjected to external force: Since muscle cells both generate force internally and respond to force externally, we are investigating the cellular responses of muscle cells mechanical stress and how they alter their gene expression and programs of assembly and disassembly. The distribution of titin, nebulin, nebulette, Xin proteins and nonmuscle myosin IIA and IIB and into the myofibrils and the Z lines are being studied with siRNA knock down techniques and fluorescence localization techniques in live cells in culture. The muscle cells are subjected to cyclic strains on elastic substrate of defined stiffness. (4): The role of nebulin in nemaline myopathy animal model: We are collaborating with Dr. Ju Chen at UCSD to characterize the muscle on the further characterization of striated muscles in the nebulin knock out mice that they generated. We are determining determined the thin filament length distribution in skeletal muscles by electron and immunoelectron microscopy to assess the distribution of actin/tropomyosin/troponin, capping proteins such as tropomodulin, myopallidin and CapZ along the thin filaments as well as the glycogen granule proteins. We also plan to study the calcium activation of contraction of these muscles subjected to cyclic mechanical activity in vivo and as isolated muscle fibers to evaluate their extent of mechanical damage and their force pCa and force ATPase curves. Additionally, the rate of formation of nemaline rods will be monitored to assess the effect of external force on the nemaline rod formation.
We aim to understand the molecular basis of the formation of nemaline rods and whether the formation of inclusion bodies in muscle diseases such as nemaline myopathy is a coping mechanism to deal with weakened sarcomeres or force transmission.
| Wang, Paul C; Blumenthal, Robert P; Zhao, Yuliang et al. (2009) Building scientific progress without borders: nanobiology and nanomedicine in China and the U.S. Cancer Res 69:5294-5 |
| Yadavalli, Vamsi K; Forbes, Jeffrey G; Wang, Kuan (2009) Nanomechanics of full-length nebulin: an elastic strain gauge in the skeletal muscle sarcomere. Langmuir 25:7496-505 |
