IMAGING CALCINEURIN DYNAMICS IN YEAST: DEVELOPMENT AND APPLICATION OF A NOVEL PROBE OF CALCINEURIN ACTIVITY
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Date
2016-02-04
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Johns Hopkins University
Abstract
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.
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Keywords
yeast, calcium, calcineurin, signaling, NFAT, Crz1, imaging