Multidisciplinary studies of Ca2+-mediated cellular processes
Subtitle: sequence to structure to cellular dynamics.
Published: 2004
Language: eng
Number of pages: 259
Description: Through the highly regulated events of intracellular Ca2+ homeostasis, often seen as a Ca2+, wave or spike, intracellular Ca2+ acts as a second messenger that regulates numerous physiological cellular phenomena including fertilization, development and apoptosis. To study these Ca2+-mediated cellular processes, multidisciplinary approaches were employed combining information ranging from sequence to structure to cellular dynamics. First, the impressive growth of sequence databases inspired a bioinformatics approach to studying the function of large Ca2+-mediated protein superfamilies. To improve function prediction, a bioinformatics algorithm was developed for classifying subfamilies of large protein superfamilies and was demonstrated on the EF-hand superfamily. Using this algorithm combined with an understanding of the domain architecture of subfamilies, a classification tool and web-accessible database was created for the cadherin superfamily of Ca2+-dependent adhesion molecules (http://calcium.uhnres.utoronto.ca/cadherin ). Though rapid, these sequence-based approaches can only vaguely predict Ca2+-mediated functions, while a higher resolution understanding would require spatio-temporal imaging of Ca2+, signals in living cells. Thus, the second part of this thesis is focused on imaging Ca2+-related signaling events using FP (fluorescent protein)-based ratiometric FRET (fluorescence resonance energy transfer) probes (hereafter, FP-based probes). Using the atomic structure of the CaM (calmodulin) in complex with CKKp (CaM kinase kinase peptide), a new FP-based probe was modeled and created with improved characteristics for imaging of Ca 2+ signaling. In in vivo imaging, this new design resulted in a two-fold enhancement in the FRET efficiency, thereby expanding the ability of Ca2+ FP-based probe to visualize Ca2+  mobilization in living cells where a higher sensitivity is required for detection. Similarly, probes were created for detecting caspase proteolytic cleavage and using a co-culture of cells expressing different FP-based probes, simultaneous Ca2+ signaling and caspase cleavage events were imaged in cell death induced by an oxidative burst modeled by a bolus addition of [H2O2]f = 10 mM. When COS-7 cells were induced by an oxidative burst, caspase activation was observed within 2 minutes. This is faster than previously thought and could not been observed using standard in vitro caspase cleavage kits.
Notes: Adviser:  Mitsuhiko Ikura.

Thesis (Ph.D.)--University of Toronto, 2004.

Electronic version licensed for access by U. of T. users.

Source: Dissertation Abstracts International, Volume: 65-05, Section: B, page: 2192.