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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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RICS analysis of eGFP-CaM in HEK cells. (A) A confocal slice through a HEK293 cell expressing eGFP-CaM. (B) The average intensity of 100 frames of the ROI identified in A is shown with the pseudocolor scale from 0 to 255. (C) The spatial autocorrelation of the intensity data from the red box shown in B. The profile of the correlation function is indicative of multiple diffusive components. (D) The fit of the autocorrelation with a two-component model for this cytoplasmic region of the cell gives values of 13.2 μm2/s and 0.036 μm2/s. (E) and (F) The spatial map of the two components scaled to be centered on the diffusion of each component (D1 and D2) in μm2/s. A mask of some of the intracellular boundaries is overlaid as topographical maps and a guide where diffusion takes place. Diffusion maps were obtained by scanning a 32 × 32 pixel sequentially over the whole data shown on B with a step of 16 pixels. The fitted values were smoothed for visualization.
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fig4: RICS analysis of eGFP-CaM in HEK cells. (A) A confocal slice through a HEK293 cell expressing eGFP-CaM. (B) The average intensity of 100 frames of the ROI identified in A is shown with the pseudocolor scale from 0 to 255. (C) The spatial autocorrelation of the intensity data from the red box shown in B. The profile of the correlation function is indicative of multiple diffusive components. (D) The fit of the autocorrelation with a two-component model for this cytoplasmic region of the cell gives values of 13.2 μm2/s and 0.036 μm2/s. (E) and (F) The spatial map of the two components scaled to be centered on the diffusion of each component (D1 and D2) in μm2/s. A mask of some of the intracellular boundaries is overlaid as topographical maps and a guide where diffusion takes place. Diffusion maps were obtained by scanning a 32 × 32 pixel sequentially over the whole data shown on B with a step of 16 pixels. The fitted values were smoothed for visualization.

Mentions: Our primary objective in this study was to establish the mobility and availability of CaM inside cells. Because of the numerous binding partners for CaM inside cells, it was expected that the mobility and distribution of eGFP-CaM would be complex. RICS analysis of eGFP-CaM expressed in HEK293 cells in basal conditions of Ca2+ is shown in Fig. 4. A confocal slice is shown of the selected cell in Fig. 4 A; note that the fluorescence is relatively uniform in the cytoplasm with the nucleus exhibiting less fluorescence. Then a smaller ROI was selected with the zoom tool (red box in Fig. 4 A) and 100 frames were collected (the average intensity is shown in Fig. 4 B, which includes an area of the nucleus and cytoplasm). Then RICS was applied to the ROI shown in Fig. 4 B, and after removing the immobile fraction we produced the spatial autocorrelation shown in Fig. 2 C. A two-component diffusion model was required to properly fit the data (Fig. 2 D). For this particular cell, the two components of diffusion of eGFP-CaM in the cytoplasm were 13.2 μm2/s and 0.036 μm2/s.


Spatial diffusivity and availability of intracellular calmodulin.

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

RICS analysis of eGFP-CaM in HEK cells. (A) A confocal slice through a HEK293 cell expressing eGFP-CaM. (B) The average intensity of 100 frames of the ROI identified in A is shown with the pseudocolor scale from 0 to 255. (C) The spatial autocorrelation of the intensity data from the red box shown in B. The profile of the correlation function is indicative of multiple diffusive components. (D) The fit of the autocorrelation with a two-component model for this cytoplasmic region of the cell gives values of 13.2 μm2/s and 0.036 μm2/s. (E) and (F) The spatial map of the two components scaled to be centered on the diffusion of each component (D1 and D2) in μm2/s. A mask of some of the intracellular boundaries is overlaid as topographical maps and a guide where diffusion takes place. Diffusion maps were obtained by scanning a 32 × 32 pixel sequentially over the whole data shown on B with a step of 16 pixels. The fitted values were smoothed for visualization.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: RICS analysis of eGFP-CaM in HEK cells. (A) A confocal slice through a HEK293 cell expressing eGFP-CaM. (B) The average intensity of 100 frames of the ROI identified in A is shown with the pseudocolor scale from 0 to 255. (C) The spatial autocorrelation of the intensity data from the red box shown in B. The profile of the correlation function is indicative of multiple diffusive components. (D) The fit of the autocorrelation with a two-component model for this cytoplasmic region of the cell gives values of 13.2 μm2/s and 0.036 μm2/s. (E) and (F) The spatial map of the two components scaled to be centered on the diffusion of each component (D1 and D2) in μm2/s. A mask of some of the intracellular boundaries is overlaid as topographical maps and a guide where diffusion takes place. Diffusion maps were obtained by scanning a 32 × 32 pixel sequentially over the whole data shown on B with a step of 16 pixels. The fitted values were smoothed for visualization.
Mentions: Our primary objective in this study was to establish the mobility and availability of CaM inside cells. Because of the numerous binding partners for CaM inside cells, it was expected that the mobility and distribution of eGFP-CaM would be complex. RICS analysis of eGFP-CaM expressed in HEK293 cells in basal conditions of Ca2+ is shown in Fig. 4. A confocal slice is shown of the selected cell in Fig. 4 A; note that the fluorescence is relatively uniform in the cytoplasm with the nucleus exhibiting less fluorescence. Then a smaller ROI was selected with the zoom tool (red box in Fig. 4 A) and 100 frames were collected (the average intensity is shown in Fig. 4 B, which includes an area of the nucleus and cytoplasm). Then RICS was applied to the ROI shown in Fig. 4 B, and after removing the immobile fraction we produced the spatial autocorrelation shown in Fig. 2 C. A two-component diffusion model was required to properly fit the data (Fig. 2 D). For this particular cell, the two components of diffusion of eGFP-CaM in the cytoplasm were 13.2 μm2/s and 0.036 μm2/s.

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