of the Max - MDC

Neuromuscular and
Cardiovascular Cell Biology
Michael Gotthardt
Our long-term goal is to establish how mechanical input is translated into molecular signals. We focus on titin,
the largest protein in the human body and the multifunctional coxsackie-adenovirus receptor (CAR).
To lay the groundwork for the in vivo analysis of titin’s multiple signaling, elastic, and adaptor domains, we have
generated various titin deficient mice (knock-in and conditional knockout animals) and established a tissue culture
system to study titin’s muscle and non-muscle functions. We utilize a combination of cell-biological, biochemical,
and genetic tools to establish titin as a stretch sensor converting mechanical into biochemical signals.
Using a comparable loss of function approach we have created a conditional knockout of the coxsackie-adenovirus
receptor. With these mice, we have demonstrated that CAR is crucial for embryonic development and determines the
electrical properties of the heart.
Titin based mechanostransduction
Agnieszka Pietas, Michael Radke, Katy Raddatz,
Thirupugal Govindarajan
Titin is a unique molecule that contains elastic spring elements
and a kinase domain, as well as multiple phosphorylation
sites. Therefore, it has been frequently speculated
that titin and invertebrate giant titin-like molecules could
act as a stretch sensor in muscle. More recently, this concept
has been supported by studies on human dilative cardiomyopathies
which suggest an impaired interaction of
titin with its regulatory ligands Tcap/telethonin and MLP
protein. However, so far it has remained unknown how the
stretch signal is processed, i.e. how the mechanical stimulus
stretch is converted into a biochemical signal.
To investigate the stretch signaling pathway, we apply
mechanical strain in vivo (plaster cast for skeletal muscle;
aortic banding for the heart) and in tissue culture (cultivation
of primary cells on elastic membranes). The resulting
changes in protein expression and localization in our titin
kinase and spring element deficient animals are used to
map the mechanotransduction pathway.
Sarcomere assembly
Agnieszka Pietas, Thirupugal Govindarajan, Stefanie
Weinert*
Overlapping titin molecules form a continuous filament
along the muscle fiber. Together with the multiple binding
sites for sarcomeric proteins, this makes titin a suitable
blueprint for sarcomere assembly. The use of transgenic
techniques does not only allow us to address the function of
titin’s individual domains in sarcomere assembly, but also
to follow sarcomere assembly and disassembly using fluorescently
tagged proteins. Understanding the structural and
biomechanical functions of titin will help elucidate the
pathomechanisms of various cardiovascular diseases and
ultimately aid the development of suitable therapeutic
strategies.
Smooth muscle and non-muscle titins
Agnieszka Pietas, Nora Bergmann
Only recently, the muscle protein titin has been proposed to
perform non-muscle functions following its localization to
various cell compartments such as the chromosomes of
drosophila neuroblasts and the brush border of intestinal
epithelial cells. Titin has been implicated in cytokinesis
through localization to stress fibers/cleavage furrows and in
chromosome condensation through localization to mitotic
chromosomes. Drosophila melanogaster deficient in the titin
homologue D-titin show chromosome undercondensation,
premature sister chromatid separation, and aneuploidity.
Our preliminary data indicate that titin is present in virtually
every cell-type tested. Nevertheless, our knockout of
titin’s M-band exon 1 and 2 does not show an obvious nonmuscle
phenotype, such as a defect in implantation or in
cell-migration. Accordingly, we have extended the analysis
of our titin knockout animals to actin-filament dependent
functions (assembly of the brush border) and generated
additional titin deficient animals to establish the role of
titin in non-muscle cells.
46 Cardiovascular and Metabolic Disease Research