High Resolution Live Cell Fluorescence Imaging of Dysferlin

Tomas Kirchhausen, PhD

Harvard Medical School (Boston, MA)

Dr. Kirchhausen is a Professor in the Department of Cell Biology at Harvard Medical School and Senior Investigator of the Immune Disease Institute (Boston, MA)

Past Projects

Objective: 
The main objective of our studies is to help develop an understanding of how dysferlin participates in membrane repair, using advanced live cell imaging tools.

Project description

Skeletal muscle is amongst tissues subject to a high level of mechanical stress. Dysferlin is highly expressed in skeletal muscle, where it is important for maintenance of the plasma membrane integrity. Despite numerous efforts, the mechanism(s) responsible to resolve the acute membrane damage generated in muscle fibers is still unknown. 
Membrane repair is a favorable system for analysis, because events at the cell surface are especially accessible to advanced live-cell imaging methods that make it possible to observe at defined locations individual molecular events directly monitored by observing the recruitment dynamics of fluorescently labeled proteins like dysferlin. 
With support from the Foundation, we successfully used gene editing to generate, for the first time, muscle cells expressing physiological amounts of fluorescently tagged dysferlin. With this unique cellular reagent in hand, we will use our unique advanced live-cell imaging methods with high temporal and spatial resolution to study the molecular mechanisms underlying dysferlinopathy, particularly in the context of the intracellular traffic of dysferlin and its role(s) for membrane repair.
Complementary to this goal, we will use our imaging methods to adress the general question of whether the muscles in zebrafish embryos can be considered a model system for muscle membrane repair mediated by dysferlin.

Final Project Summary: 

Skeletal muscle is amongst tissues subject to a high level of mechanical stress. Dysferlin is highly expressed in skeletal muscle, where it is important for maintenance of the plasma membrane integrity. Despite numerous efforts, the mechanism(s) responsible to resolve the acute membrane damage generated in muscle fibers is still unknown. 
Membrane repair is a favorable system for analysis, because events at the cell surface are especially accessible to advanced live-cell imaging methods that make it possible to observe at defined locations individual molecular events directly monitored by observing the recruitment dynamics of fluorescently labeled proteins like dysferlin. 
With support from the Foundation, we successfully used CRISPR/CAS9 gene editing methods to generate, for the first time, muscle cells expressing physiological amounts of fluorescently tagged dysferlin. With this unique cellular reagent in hand, we combined laser ablation with live-cell imaging methods with high temporal and spatial resolution to study how dysferlin participates in plasma membrane repair.  These studies uncovered the existence of two complementary and mutually exclusive repair process, one based on dysferlin and specific for muscle cells and a second independent of dysferlin and constitutive for all cells including muscle.