Spatial diffusivity and availability of intracellular calmodulin.
Calmodulin (CaM) is the major pathway that transduces intracellular Ca2+ increases to the activation of a wide variety of downstream signaling enzymes. CaM and its target proteins form an integrated signaling network believed to be tuned spatially and temporally to control CaM's ability to appropriately pass signaling events downstream. Here, we report the spatial diffusivity and availability of CaM labeled with enhanced green fluorescent protein (eGFP)-CaM, at basal and elevated Ca2+,quantified by the novel fluorescent techniques of raster image scanning spectroscopy and number and brightness analysis. Our results show that in basal Ca2+ conditions cytoplasmic eGFP-CaM diffuses at a rate of 10 microm(2)/s, twofold slower than the noninteracting tracer, eGFP, indicating that a significant fraction of CaM is diffusing bound to other partners. The diffusion rate of eGFP-CaM is reduced to 7 microm(2)/s when a large (646 kDa) target protein Ca2+/CaM-dependent protein kinase II is coexpressed in the cells. In addition, the presence of Ca2+/calmodulin-dependent protein kinase II, which can bind up to 12 CaM molecules per holoenzyme, increases the stoichiometry of binding to an average of 3 CaMs per diffusive molecule. Elevating intracellular Ca2+ did not have a major impact on the diffusion of CaM complexes. These results present us with a model whereby CaM is spatially modulated by target proteins and support the hypothesis that CaM availability is a limiting factor in the network of CaM-signaling enzymes.
Sanabria
Hugo
H
Department of Neurobiology and Anatomy, University of Texas Health Science Center at Houston, Texas 77030, USA.
Digman
Michelle A
MA
Gratton
Enrico
E
Waxham
M Neal
MN
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D004058:Diffusion; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D042541:Intracellular Space / Q000378:metabolism*; D012146:Rest; D013997:Time Factors; D014162:Transfection; D014783:Viscosity
D002147:Calmodulin; D049452:Green Fluorescent Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium