Intracellular Calmodulin Availability Accessed with Two-Photon Cross-Correlation

The availability and interactions of signaling proteins are tightly regulated in time and space to produce specific and localized effects. For calmodulin (CaM), a key transducer of intracellular Ca2+signaling, binding to its variety of targets initiates signaling cascades and regulates its subcellul...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2004-01, Vol.101 (1), p.105-110
Main Authors: Kim, Sally A., Heinze, Katrin G., Waxham, M. Neal, Schwille, Petra
Format: Article
Language:English
Subjects:
Animals
Atoms & subatomic particles
Autocorrelation
Biological Sciences
Biophysical Phenomena
Biophysics
Calcium
Calcium Signaling
Calcium-Calmodulin-Dependent Protein Kinase Type 2
Calcium-Calmodulin-Dependent Protein Kinases - chemistry
Calcium-Calmodulin-Dependent Protein Kinases - genetics
Calcium-Calmodulin-Dependent Protein Kinases - metabolism
Calmodulin - chemistry
Calmodulin - genetics
Calmodulin - metabolism
Cell Line
Cell lines
Cells
Enzymes
Fluorescence
HEK293 cells
Humans
Intracellular Fluid - metabolism
Molecular interactions
Molecular probes
Molecular spectroscopy
Molecules
Mutagenesis, Site-Directed
Neurons
Phosphorylation
Photons
Protein Binding
Proteins
Rats
Recombinant Fusion Proteins - chemistry
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - metabolism
Signal Transduction
Spectrometry, Fluorescence
Spectrum analysis
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Summary:The availability and interactions of signaling proteins are tightly regulated in time and space to produce specific and localized effects. For calmodulin (CaM), a key transducer of intracellular Ca2+signaling, binding to its variety of targets initiates signaling cascades and regulates its subcellular localization, thereby making it unavailable for subsequent binding interactions. Among CaM's numerous targets, Ca2+/CaM-dependent protein kinase II is one of the most striking due to its unique ability to increase its affinity for CaM by autophosphorylation and to translocate when bound to Ca2+/CaM. Two-photon fluorescence correlation spectroscopy and cross-correlation spectroscopy were utilized to compare mobility and molecular interactions between CaM and Ca2+/CaM-dependent protein kinase II in solution and in living cells. These techniques revealed that CaM availability in cells could be altered by a change in intracellular conditions. Two-photon fluorescence cross-correlation spectroscopy exemplifies a generally applicable approach for studying protein-protein interactions in living cells that allows access to the behavior of signaling molecules within their native environment to probe for heterogeneities in signaling pathways in different cellular compartments.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.2436461100