20.109:Module 2

Module 2

Instructors: Alan Jasanoff and Natalie Kuldell

TA: Yoon Sung Nam

Once proteins are synthesized, their localization and conformation are critical to their function, but without “X-ray vision” to peer into a cell, these can be hard to detect. Fluorescence is one of the best tools in our toolkit for measuring proteins in living cells, allowing cellular functions to be probed unobtrusively. In this experimental module we will measure calcium fluctuations in living cells by tagging a calcium sensing protein with a fluorescent one. In the presence of calcium, the fluorescent protein will fold and the cells should appear green. We can transfect the genetically encoded calcium sensor into cells of interest, in this case mouse embryonic stem cells. Changes in fluorescence will allow us to quantitatively measure how chemical and physical perturbations affect calcium signaling in these cells.

Calmodulin structure visualized with Cn3D for PDB file 1DMO
Mouse embryonic stem cells expressing genetically-encoded Ca2+ sensor
images from N. Kuldell

Module 2 Day 1: Start-up signal measurement
Module 2 Day 2: Measuring calcium in vitro
Module 2 Day 3: Lipofection
Module 2 Day 4: Measuring calcium in vivo
Module 2 Day 5: Calcium signaling in vivo
Module 2 Day 6: Student presentations

TA notes, mod 2