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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 transfected HEK293 cell. (A) The concentration map of eGFP-CaM of the same cell shown in Fig. 4. (B) The concentration histogram as the total number of pixels (y axis) plotted against concentration in μM (x axis). (C) The brightness map as the variance/intensity is scaled over the range from 0.8 to 2.0. (D) Brightness histograms of the data shown in (C); the total number of pixels (y axis) are plotted against B (x axis). On top of the B-histogram, the green Gaussian represents the contribution of a single eGFP, and the blue Gaussian represents the pixels representing two eGFP-CaMs per complex. These pixels are shown on red as a binary map on E and F, respectively.
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fig5: N&B analysis of eGFP-CaM transfected HEK293 cell. (A) The concentration map of eGFP-CaM of the same cell shown in Fig. 4. (B) The concentration histogram as the total number of pixels (y axis) plotted against concentration in μM (x axis). (C) The brightness map as the variance/intensity is scaled over the range from 0.8 to 2.0. (D) Brightness histograms of the data shown in (C); the total number of pixels (y axis) are plotted against B (x axis). On top of the B-histogram, the green Gaussian represents the contribution of a single eGFP, and the blue Gaussian represents the pixels representing two eGFP-CaMs per complex. These pixels are shown on red as a binary map on E and F, respectively.

Mentions: The N&B analysis of the cell presented in Fig. 4 is shown in Fig. 5. In Fig. 5 A the concentration distribution of eGFP-CaM is shown below a mask constructed from Fig. 4 B. The concentration histogram of this image is presented in Fig. 5 B. The B-map (brightness map) and its histogram are shown in Fig. 5, C and D, respectively. From the B-map we can see that in the nucleus and in large regions of the cytosol eGFP-CaM has the brightness of a single eGFP. This is evident in Fig. 5 E, where a binary map of the pixels showing the brightness of the monomeric eGFP is shown in red. This threshold value is selected by overlaying a Gaussian profile (green Gaussian) on top of the B-histogram (Fig. 5 D) and setting the threshold for the image to the peak of the profile. The blue Gaussian corresponds to a threshold value that would represent the “dimeric” state of eGFP-CaM, likely representing molecules with two bound CaM molecules; the map of such a selection is shown in Fig. 5 F as a binary map. From here, we see that almost no such complexes exist in the nuclear region, but in the cytoplasm we find that complexes are found near the perinuclear region and close to borders of other membranes. Even complexes with higher brightness are found (the additional pixels in Fig. 5 D at 1.4 variance/intensity and beyond), but they do not contribute significantly to the overall concentration of eGFP-CaM.


Spatial diffusivity and availability of intracellular calmodulin.

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

N&B analysis of eGFP-CaM transfected HEK293 cell. (A) The concentration map of eGFP-CaM of the same cell shown in Fig. 4. (B) The concentration histogram as the total number of pixels (y axis) plotted against concentration in μM (x axis). (C) The brightness map as the variance/intensity is scaled over the range from 0.8 to 2.0. (D) Brightness histograms of the data shown in (C); the total number of pixels (y axis) are plotted against B (x axis). On top of the B-histogram, the green Gaussian represents the contribution of a single eGFP, and the blue Gaussian represents the pixels representing two eGFP-CaMs per complex. These pixels are shown on red as a binary map on E and F, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: N&B analysis of eGFP-CaM transfected HEK293 cell. (A) The concentration map of eGFP-CaM of the same cell shown in Fig. 4. (B) The concentration histogram as the total number of pixels (y axis) plotted against concentration in μM (x axis). (C) The brightness map as the variance/intensity is scaled over the range from 0.8 to 2.0. (D) Brightness histograms of the data shown in (C); the total number of pixels (y axis) are plotted against B (x axis). On top of the B-histogram, the green Gaussian represents the contribution of a single eGFP, and the blue Gaussian represents the pixels representing two eGFP-CaMs per complex. These pixels are shown on red as a binary map on E and F, respectively.
Mentions: The N&B analysis of the cell presented in Fig. 4 is shown in Fig. 5. In Fig. 5 A the concentration distribution of eGFP-CaM is shown below a mask constructed from Fig. 4 B. The concentration histogram of this image is presented in Fig. 5 B. The B-map (brightness map) and its histogram are shown in Fig. 5, C and D, respectively. From the B-map we can see that in the nucleus and in large regions of the cytosol eGFP-CaM has the brightness of a single eGFP. This is evident in Fig. 5 E, where a binary map of the pixels showing the brightness of the monomeric eGFP is shown in red. This threshold value is selected by overlaying a Gaussian profile (green Gaussian) on top of the B-histogram (Fig. 5 D) and setting the threshold for the image to the peak of the profile. The blue Gaussian corresponds to a threshold value that would represent the “dimeric” state of eGFP-CaM, likely representing molecules with two bound CaM molecules; the map of such a selection is shown in Fig. 5 F as a binary map. From here, we see that almost no such complexes exist in the nuclear region, but in the cytoplasm we find that complexes are found near the perinuclear region and close to borders of other membranes. Even complexes with higher brightness are found (the additional pixels in Fig. 5 D at 1.4 variance/intensity and beyond), but they do not contribute significantly to the overall concentration of eGFP-CaM.

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