RESEARCH INTERESTS
Deciphering the signals used for both macromolecular assembly and tissue morphogenesis are important biological problems, and embryonic muscle is an excellent system for studying both problems - the former in culture and the latter in vivo. For example, since myofibrils develop discrete long-axis and parallel alignment, sarcomeric spacing, and transverse sarcomeric registration, both qualitative and quantitative assessments are easily achieved.
Currently, we are examining the role of intra- and
intercellular calcium signals in the differentiation of
Xenopus myocytes
both in culture and in vivo
(J.
Cell Biol. 141: 1349-1356;
Dev. Biol. 178(2):484-497). We are examining the calcium-dependent
downstream mechanisms responsible for proper sarcomere assembly using real-time
approaches (the image shown here is a Xenopus myocyte expressing and
incorporating GFP-actin into I bands). We utilize immunochemical, biochemical,
molecular, and digital fluorescence imaging approaches to determine how calcium
signals influence muscle differentiation both in culture and in vivo.
In the future, we plan to utilize both electrocytes and muscle to understand myofibrillogenesis. Electrocytes are muscle-derived cells found in strongly and weakly electric fish. Depending on species, electrocytes show a range of myofibrillar organization, from a complete absence of contractile proteins to several organized myofibrils. This phylogenetic diversity forms the basis of a comparative approach using electrocytes that is likely to reveal key structural and signaling components necessary for myofibrillar organization.
Myofibrils are not only inherently interesting as the end effector of the muscle cell, but are also the ultimate site of functional degeneration in myopathies. Calcium signals necessary for myofibrillogenesis arise from ryanodine receptor (RyR) stores, and in swine and some human families a mutation in the RyR has been identified and associated with central core disease and susceptibility to malignant hyperthermia. This indicates that defective calcium regulation in mature muscle results in both metabolic and cytoskeletal disruptions. Furthermore, acute quadriplegic myopathy may involve the depletion of myosin via altered calcium homeostasis leading to calpain-mediated proteolysis. Our work indicates that calcium elevations are crucial signals in developing skeletal muscle, since blocking normal calcium transients leads to disrupted myofibrillogenesis. Part of this disruption occurs in thick filaments because calcium is required to activate myosin light chain kinase (MLCK), the enzyme which phosphorylates regulatory light chains (RLCs). In humans, mutations in either the essential or regulatory light chains of myosin have been associated with a rare myopathy in heart and skeletal muscle, and this pathology might be understood in terms of the central role played by the RLCs in thick filament assembly.
In
the intact myotome, production of calcium transients is correlated with
development along the A-P axis; transients are highest in the unsegmented
mesoderm and region of somite segmentation (shown in image) but absent in mature
somites (Dev.
Biol. 213(2):269-282.). This activity shows complex spatiotemporal patterns
and clustering that corresponds to future somitic furrows. Blocking the
production of these transients disrupts somite development. Since both the Wnt
and Notch-Delta intercellular signaling pathways are involved in muscle
development and somite segmentation; it is possible some of this signaling is
achieved via this calcium transient activity.
At least a dozen calcium-dependent proteins are involved in myofibrillogenesis, thus providing a reasonable framework for calcium-mediated sarcomere assembly. Remaining questions include: What are the AM/FM signaling parameters (in terms of amplitude, duration, frequency, and/or total calcium load) required for normal myocyte development in culture and in vivo? Will the disrupted myofibril phenotype seen when endogenous transients are blocked be rescued by reimposition of artificial transients? Do transients control gene expression as well as directly modulate protein interactions? Are morphogenetic movements and differentiation of myocytes in vivo directed by transients?