Limits...
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.

Show MeSH

Related in: MedlinePlus

RICS and N&B analysis of eGFP-CaMKII in HEK cells. (A) A confocal slice through HEK293 cells expressing eGFP-CaMKII. (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 concentration map obtained from the N&B analysis. (D) The spatial map of the brightness “B”. (E) Brightness histograms of the data shown in D; the total number of pixels (y axis) are plotted against B (xaxis). The green Gaussian represents the contribution with the molecular brightness of eGFP, and the blue Gaussian represents the contribution of molecular complexes with an average of ∼3 eGFP-CaMKII subunits presumably assembled into the holoenzyme complex. (F) The RICS analysis of the ROI shown in B. (G) The fit of the autocorrelation with a two-component model for this cytoplasmic region of the cell gives values of 2.6 μm2/s and 0.14 μm2/s.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2599858&req=5

fig3: RICS and N&B analysis of eGFP-CaMKII in HEK cells. (A) A confocal slice through HEK293 cells expressing eGFP-CaMKII. (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 concentration map obtained from the N&B analysis. (D) The spatial map of the brightness “B”. (E) Brightness histograms of the data shown in D; the total number of pixels (y axis) are plotted against B (xaxis). The green Gaussian represents the contribution with the molecular brightness of eGFP, and the blue Gaussian represents the contribution of molecular complexes with an average of ∼3 eGFP-CaMKII subunits presumably assembled into the holoenzyme complex. (F) The RICS analysis of the ROI shown in B. (G) The fit of the autocorrelation with a two-component model for this cytoplasmic region of the cell gives values of 2.6 μm2/s and 0.14 μm2/s.

Mentions: We present a typical example of data analyzed from an eGFP-CaMKII expressing HEK293 cell using the same layout as in Fig. 2. Fig. 3 A represents a confocal scan of the selected cell and Fig. 3 B represents the average intensity of the scanned region. Fig. 3 C shows the concentration map that clearly shows that eGFP-CaMKII is not expressed in the nuclear region. The concentration reported in Fig. 3 C is that of eGFP as determined for Fig. 2. The B-map (Fig. 3 D) shows that some values are ≤1, which correspond to the background counts and are areas where eGFP-CaMKII is not present, such as inside the nucleus. In the cytoplasm there is a large distribution of brightness ranging from one eGFP to complexes that contain five to eight copies of eGFP. This is expected because CaMKII is a multisubunit enzyme. This is clearly evident by the broadening in the histogram (Fig. 3 E), where complexes are responsible for the significant second Gaussian component (blue profile). This oligomeric complex of CaMKII expressed in the HEK293 cells has on average at least three fluorescent eGFP-CaMKII subunits per molecule. The heterogeneity in the second component presumably reflects the stochastic assembly of eGFP-CaMKII subunits with the endogenous unlabeled subunits in the HEK293 cells and/or incomplete maturation to produce the fluorescent state of the eGFP molecule. Since αCaMKII was not observed in untransfected HEK293 cells (see Fig. 1), eGFP-CaMKII subunits most likely are associated with one, or a combination, of the other three mammalian isoforms of CaMKII (β, γ, or δ) (35).


Spatial diffusivity and availability of intracellular calmodulin.

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

RICS and N&B analysis of eGFP-CaMKII in HEK cells. (A) A confocal slice through HEK293 cells expressing eGFP-CaMKII. (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 concentration map obtained from the N&B analysis. (D) The spatial map of the brightness “B”. (E) Brightness histograms of the data shown in D; the total number of pixels (y axis) are plotted against B (xaxis). The green Gaussian represents the contribution with the molecular brightness of eGFP, and the blue Gaussian represents the contribution of molecular complexes with an average of ∼3 eGFP-CaMKII subunits presumably assembled into the holoenzyme complex. (F) The RICS analysis of the ROI shown in B. (G) The fit of the autocorrelation with a two-component model for this cytoplasmic region of the cell gives values of 2.6 μm2/s and 0.14 μm2/s.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: RICS and N&B analysis of eGFP-CaMKII in HEK cells. (A) A confocal slice through HEK293 cells expressing eGFP-CaMKII. (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 concentration map obtained from the N&B analysis. (D) The spatial map of the brightness “B”. (E) Brightness histograms of the data shown in D; the total number of pixels (y axis) are plotted against B (xaxis). The green Gaussian represents the contribution with the molecular brightness of eGFP, and the blue Gaussian represents the contribution of molecular complexes with an average of ∼3 eGFP-CaMKII subunits presumably assembled into the holoenzyme complex. (F) The RICS analysis of the ROI shown in B. (G) The fit of the autocorrelation with a two-component model for this cytoplasmic region of the cell gives values of 2.6 μm2/s and 0.14 μm2/s.
Mentions: We present a typical example of data analyzed from an eGFP-CaMKII expressing HEK293 cell using the same layout as in Fig. 2. Fig. 3 A represents a confocal scan of the selected cell and Fig. 3 B represents the average intensity of the scanned region. Fig. 3 C shows the concentration map that clearly shows that eGFP-CaMKII is not expressed in the nuclear region. The concentration reported in Fig. 3 C is that of eGFP as determined for Fig. 2. The B-map (Fig. 3 D) shows that some values are ≤1, which correspond to the background counts and are areas where eGFP-CaMKII is not present, such as inside the nucleus. In the cytoplasm there is a large distribution of brightness ranging from one eGFP to complexes that contain five to eight copies of eGFP. This is expected because CaMKII is a multisubunit enzyme. This is clearly evident by the broadening in the histogram (Fig. 3 E), where complexes are responsible for the significant second Gaussian component (blue profile). This oligomeric complex of CaMKII expressed in the HEK293 cells has on average at least three fluorescent eGFP-CaMKII subunits per molecule. The heterogeneity in the second component presumably reflects the stochastic assembly of eGFP-CaMKII subunits with the endogenous unlabeled subunits in the HEK293 cells and/or incomplete maturation to produce the fluorescent state of the eGFP molecule. Since αCaMKII was not observed in untransfected HEK293 cells (see Fig. 1), eGFP-CaMKII subunits most likely are associated with one, or a combination, of the other three mammalian isoforms of CaMKII (β, γ, or δ) (35).

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