Molecular Genetics of Telethonin/ T-cap

Ruilin Zhang, Xiaolei Xu

Cell Biology and Genetics
Matriculated in 2003, zhang.ruilin@mayo.edu, Thesis Advisor – Xiaolei Xu

T-cap has been previously proposed as a component of the Z-discs stretch sensing complex which also includes titin and MLP. Our finding raised an interesting hypothesis that stretch may be able to regulate the expression of the sensor of its own, forming some kind of feed back loop

Telethonin/ T-cap is a small sarcomeric protein resided in the peripheral of Z-discs. First identified as a structure protein that interacts with N-terminal of the giant protein Titin and is important for the integrity of Z-discs, T-cap may also play a role in many signaling processes such as stretch sensing, mechano-electrical coupling in cardiomyocytes, and skeletal muscle growth. Mutations in T-cap have been found to be responsible for autosomal recessive limb-girdle muscular dystrophy 2G (LGMD2G) and cardiomyopathies patients. Despite its strong clinical relevance, there are no functional studies of T-cap in a genetic tractable animal model. We have cloned two zebrafish T-cap homologues and have taken the following four approaches to investigate functions of T-cap in this vertebrate model. 1) By morpholino knock down experiments, we found that Z-discs fail to align and become uncoupled with the T-tubule system. This discovery led us to further investigate the functions of T-caps in Z-disc/T-tubule system interaction, which may uncover novel mechanisms of mechano-electrical coupling system. 2) By whole mount in situ hybridization, we observed that the transcriptional level of T-cap1 but not T-cap2 can be induced by stretch. This discovery raised an exciting hypothesis that T-cap1 is involved in a feedback loop during the stretch sensing pathway. 3) To confirm the feedback loop hypothesis, we have dissected T-cap1 enhancer sequences and identified a 6kb enhancer that is sufficient to respond to the stretch signal. Using the Ventricular Myosin Heavy Chain (VMHC) enhancer as a paradigm, we will show that it is feasible to quickly identify minimal cis-acting elements by transient GFP assay and to confirm the finding by establishing stable transgenic fish lines. Similar technologies can be applied to further dissect T-cap1 enhancer to identify transcriptional factors that regulate the T-cap1 induction in response to stretch. 4) Taking advantage of a forward mutagenesis screen that is undergoing in the lab, we plan to identify mutants that affect genes that regulate the induction of T-cap1 in response to stretch.