IMAGING CALCINEURIN DYNAMICS IN YEAST: DEVELOPMENT AND APPLICATION OF A NOVEL PROBE OF CALCINEURIN ACTIVITY
Aronin, Tovah Yaffa Honor
MetadataShow full item record
Calcineurin (Cn) is a highly conserved calcium-responsive phosphatase that is crucial for many cellular pathways, especially those that control the response to outside stimuli. Misregulation of Cn has been associated with pathological conditions including cardiac hypertrophy, Down Syndrome, and Alzheimer’s Disease. Previous studies of Cn and its downstream effectors have largely relied on assays that lack single-cell or temporal resolution. Herein, I describe my efforts to develop a fluorescent probe based on a truncated version of the Cn-responsive transcription factor Crz1 and use it to investigate Cn-Crz1 activation dynamics in single cells in real time. Crz1 activates its own transcription as well as transcription of Cn regulators, thus participating in multiple feedback loops. The truncated version of Crz1 used for the probe does not bind DNA and is therefore inert with regards to feedback. Using this probe, I reveal a new phenomenon of Cn activity in the absence of stimulation named “flickering.” Flickering is a low level of Cn activation that stimulates brief probe translocation in steady-state conditions. I also use the probe to investigate feedback loops involved in Cn regulation. The vacuolar calcium transporters Pmc1 and Vcx1 are both necessary to maintain cytosolic calcium concentration and prevent hyperactivation of Cn in both stimulating and non-stimulating conditions. The putative Cn chaperone Rcn1p is needed for normal Cn activity both before and after signaling. My studies present a new way to measure Cn activation which can be applied to clinical and basic study of this crucial signaling factor.