John Jeffers
Pronouns: he/him
Research Mentor(s): Sivakumar Jeyarajan
Research Mentor School/College/Department: Pharmacology / Medicine
Program: UROPF
Session: Session 3 (11:00am – 11:50am)
Authors: John Jeffers, Sivakumar Jeyarajan, Les Satin
Abstract
The concentration and movement of calcium ions (Ca2+) within insulin-secreting pancreatic beta cells have recently been under investigation to understand its contribution to cellular communication and dysfunction in diabetic cells. To study the movement and relative concentrations of Ca2+ in real time, we utilized INS-1 832/13 pancreatic beta cells and simultaneously transfected three vectors containing genetically encoded intensitometric proteins that fluoresce when complexed with Ca2+. Cells that expressed all three of the fluorescent proteins, namely G-CAMP-er (for measuring endoplasmic reticulum Ca2+), R-CEPIA-mt (for measuring mitochondrial Ca2+), and B-GECO (for measuring cytosolic Ca2+), were subsequently imaged in a confocal microscope to track the levels of Ca2+ in real time via relative fluorescence. The transfected cells were imaged in a standard 11mM glucose imaging solution as a control, and to test the movement of Ca2+, we added cyclopiazonic acid (CPA), a known inhibitor of the SERCA (Smooth Endoplasmic Reticulum Ca2+-ATPase) to lower ER Ca2+ and raise cytosolic and mitochondrial Ca2+. Our results demonstrated consistency with previously discovered cellular responses for the transportation of Ca2+ within pancreatic beta cells. The aim of our lab is to successfully produce a single vector containing all three of these proteins that would allow for fixed stoichiometric expression of the probes in the cell that gives a more reliable real-time image of Ca2+ movement and reduce the inefficiency of subsequent transfections.
