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Spatial diffusivity and availability of intracellular calmodulin.

Sanabria H, Digman MA, Gratton E, Waxham MN - Biophys. J. (2008)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology and Anatomy, University of Texas Health Science Center at Houston, Texas 77030, USA.

ABSTRACT
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.

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N&B analysis of eGFP-CaM coexpressed with nonlabeled CaMKII. Transfected HEK293 cells in basal conditions row I and with a media exchange for high Ca2+ (II). (A) A pseudocolor intensity profile of the whole cell. (B) The ROI from which RICS and N&B were determined. (C) The spatial map of the molecular brightness of 100 frames after subtraction of the slowly moving component (see Materials and Methods). (D) B-histogram where two Gaussian distributions are shown (green and blue lines). The total number of pixels (y axis) is plotted against B (x axis) scaled from 0.8 to 2. Note the long and asymmetric profile of the second (blue) component, which indicates a family of diffusing molecules with molecular brightness 3–12 times that of eGFP.
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fig6: N&B analysis of eGFP-CaM coexpressed with nonlabeled CaMKII. Transfected HEK293 cells in basal conditions row I and with a media exchange for high Ca2+ (II). (A) A pseudocolor intensity profile of the whole cell. (B) The ROI from which RICS and N&B were determined. (C) The spatial map of the molecular brightness of 100 frames after subtraction of the slowly moving component (see Materials and Methods). (D) B-histogram where two Gaussian distributions are shown (green and blue lines). The total number of pixels (y axis) is plotted against B (x axis) scaled from 0.8 to 2. Note the long and asymmetric profile of the second (blue) component, which indicates a family of diffusing molecules with molecular brightness 3–12 times that of eGFP.

Mentions: To examine the influence of CaM-binding targets on the brightness and mobility of eGFP-CaM, similar experiments to those just described were done but now coexpressing eGFP-CaM and nonlabeled αCaMKII in HEK293 cells. An example of single-cell analysis is presented in Fig. 6 where rows I and II represent the analysis of the same cell under basal conditions and at elevated Ca2+, respectively. Similar images from a second cell are presented in Fig. S3 (Data S1), but in that case we changed from basal to lower Ca2+ using ionomycin in the presence of the Ca2+-chelator EGTA. Column A represents the selected cell, and column B is the ROI from which the N&B analysis was done. The B-map and its histogram are shown in columns C and D, respectively. The brightness map (column C) shows that in a substantial fraction of pixels, eGFP-CaM exists as a monomer, particularly in the nuclear region, consistent with the data described above (Fig. 5).


Spatial diffusivity and availability of intracellular calmodulin.

Sanabria H, Digman MA, Gratton E, Waxham MN - Biophys. J. (2008)

N&B analysis of eGFP-CaM coexpressed with nonlabeled CaMKII. Transfected HEK293 cells in basal conditions row I and with a media exchange for high Ca2+ (II). (A) A pseudocolor intensity profile of the whole cell. (B) The ROI from which RICS and N&B were determined. (C) The spatial map of the molecular brightness of 100 frames after subtraction of the slowly moving component (see Materials and Methods). (D) B-histogram where two Gaussian distributions are shown (green and blue lines). The total number of pixels (y axis) is plotted against B (x axis) scaled from 0.8 to 2. Note the long and asymmetric profile of the second (blue) component, which indicates a family of diffusing molecules with molecular brightness 3–12 times that of eGFP.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2599858&req=5

fig6: N&B analysis of eGFP-CaM coexpressed with nonlabeled CaMKII. Transfected HEK293 cells in basal conditions row I and with a media exchange for high Ca2+ (II). (A) A pseudocolor intensity profile of the whole cell. (B) The ROI from which RICS and N&B were determined. (C) The spatial map of the molecular brightness of 100 frames after subtraction of the slowly moving component (see Materials and Methods). (D) B-histogram where two Gaussian distributions are shown (green and blue lines). The total number of pixels (y axis) is plotted against B (x axis) scaled from 0.8 to 2. Note the long and asymmetric profile of the second (blue) component, which indicates a family of diffusing molecules with molecular brightness 3–12 times that of eGFP.
Mentions: To examine the influence of CaM-binding targets on the brightness and mobility of eGFP-CaM, similar experiments to those just described were done but now coexpressing eGFP-CaM and nonlabeled αCaMKII in HEK293 cells. An example of single-cell analysis is presented in Fig. 6 where rows I and II represent the analysis of the same cell under basal conditions and at elevated Ca2+, respectively. Similar images from a second cell are presented in Fig. S3 (Data S1), but in that case we changed from basal to lower Ca2+ using ionomycin in the presence of the Ca2+-chelator EGTA. Column A represents the selected cell, and column B is the ROI from which the N&B analysis was done. The B-map and its histogram are shown in columns C and D, respectively. The brightness map (column C) shows that in a substantial fraction of pixels, eGFP-CaM exists as a monomer, particularly in the nuclear region, consistent with the data described above (Fig. 5).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology and Anatomy, University of Texas Health Science Center at Houston, Texas 77030, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus