Cardiac Calsequestrin Phosphorylation And Trafficking In The Mammalian Cardiomyocyte

CARDIAC CALSEQUESTRIN PHOSPHORYLATION AND TRAFFICKING IN THEMAMMALIAN CARDIOMYOCYTEbyTIMOTHY P. MCFARLANDMay 2011Advisor: Dr. Steven E. CalaMajor: PhysiologyDegree: Doctor of Philosophy Cardiac CSQ (CSQ2) is a multifaceted protein, capable of binding significantquantities of Ca and altering ryanodine receptor activity at the junctional sarcoplasmicreticulum (SR). Little is known about the trafficking of CSQ2 from its unknown site ofbiosynthesis, which appears to be of importance as its structure changes in a trafficking-dependent manner in various types of heart failure. Through the use of multipleantibodies specific to classic rough ER markers, and with the creation of CSQ-DsRedtetramer fusion protein, we were able to establish a juxtanuclear localization of roughER in cardiomyocytes. Using fluorescence confocal microscopy, the transloconcomplex proteins TRAP-α and TRAM, along with the ribosomal protein S6, were allvisualized and found to encapsulate both myonuclei. Additionally, time course studiesof CSQ-DsRed, in conjunction with anti-DsRed immunostaining, highlighted aperinuclear rough ER site of biosynthesis for CSQ2. The fusion protein exhibited atetramerization-dependent trafficking predicted to be very similar to CSQ2polymerization-dependent trafficking with high and low secretory compartment Caconcentrations leading to polymerization and monomerization, respectively.