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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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Related in: MedlinePlus

Immunolabeling of transfected HEK293 cells. Red channel in most cases corresponds to the signal coming from Alexa-568 labeled goat anti-mouse secondary following labeling with a monoclonal antibody to αCaMKII. The green channel is eGFP or the eGFP construct signal. The overlay of the two channels is shown in the third column. (A) HEK293 cells expressing GFP; we see no evidence of endogenous αCaMKII, evidenced by the lack of signal in the red channel. (B) eGFP-CaM expressed on HEK293 cells when no antibody treatment was performed, showing little cross talk signal from the green channel into the red channel. eGFP-CaM is distributed homogeneously within the cells with slightly lower expression in the nucleus. (C) Coexpression of eGFP-CaM and the nonlabeled form of αCaMKII; we see similar results to those in B, plus we see signal from the red channel, indicating expression of αCaMKII in each cell that is also expressing eGFP-CaM. CaMKII is largely excluded from the nucleus, whereas eGFP and eGFP-CaM are not. The appearance of very bright spots possibly reflects recycling vesicles due to overexpression of eGFP-CaM. (D) Expression of eGFP-CaMKII, with the primary monoclonal antibody specific for αCaMKII was omitted from the staining protocol. Note the very weak signal in the red channel, which represents background fluorescence and/or some modest bleed-through of the green channel similar to row B. (E) eGFP-CaMKII expressed in HEK293 cells stained with monoclonal antibody specific to αCaMKII. Note that both signals overlay almost perfectly, as expected.
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fig1: Immunolabeling of transfected HEK293 cells. Red channel in most cases corresponds to the signal coming from Alexa-568 labeled goat anti-mouse secondary following labeling with a monoclonal antibody to αCaMKII. The green channel is eGFP or the eGFP construct signal. The overlay of the two channels is shown in the third column. (A) HEK293 cells expressing GFP; we see no evidence of endogenous αCaMKII, evidenced by the lack of signal in the red channel. (B) eGFP-CaM expressed on HEK293 cells when no antibody treatment was performed, showing little cross talk signal from the green channel into the red channel. eGFP-CaM is distributed homogeneously within the cells with slightly lower expression in the nucleus. (C) Coexpression of eGFP-CaM and the nonlabeled form of αCaMKII; we see similar results to those in B, plus we see signal from the red channel, indicating expression of αCaMKII in each cell that is also expressing eGFP-CaM. CaMKII is largely excluded from the nucleus, whereas eGFP and eGFP-CaM are not. The appearance of very bright spots possibly reflects recycling vesicles due to overexpression of eGFP-CaM. (D) Expression of eGFP-CaMKII, with the primary monoclonal antibody specific for αCaMKII was omitted from the staining protocol. Note the very weak signal in the red channel, which represents background fluorescence and/or some modest bleed-through of the green channel similar to row B. (E) eGFP-CaMKII expressed in HEK293 cells stained with monoclonal antibody specific to αCaMKII. Note that both signals overlay almost perfectly, as expected.

Mentions: To assess the distribution of each of the eGFP constructs used in this study, HEK293 cells were transfected with plasmids expressing eGFP, eGFP-CaM, eGFP-CaMKII, or the nonlabeled form of αCaMKII and were fixed, immunolabeled, and imaged on the LSM 510 (see Materials and Methods). Fig. 1 shows antibody labeling of αCaMKII in the red channel, and the green channel shows the expression of the various eGFP constructs. The final column shows image overlays of the two channels. It is clear from row A that there is no endogenous αCaMKII in HEK293 cells (absence of signal in the red channel). Note that eGFP is expressed throughout both the nucleus and cytoplasm of the cell (green channel) with some apparent enrichment in the nucleus. The bleed-through between channels is observed on row B, where no antibody treatment was added to HEK cells expressing eGFP-CaM. eGFP-CaM is distributed homogeneously within the cells with slightly lower expression in the nucleus. Strong immunostaining is evident when nonlabeled αCaMKII is expressed in the cells (row C) that disappear when the primary antibody is omitted (row D). αCaMKII is uniformly distributed in the cytoplasm but is largely excluded from the nucleus whether expressed without (row C) or with (row E) the eGFP tag. In cells expressing both eGFP-CaM and nonlabeled CaMKII (row C), GFP-CaM can be detected throughout the cytoplasm and the nucleus.


Spatial diffusivity and availability of intracellular calmodulin.

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

Immunolabeling of transfected HEK293 cells. Red channel in most cases corresponds to the signal coming from Alexa-568 labeled goat anti-mouse secondary following labeling with a monoclonal antibody to αCaMKII. The green channel is eGFP or the eGFP construct signal. The overlay of the two channels is shown in the third column. (A) HEK293 cells expressing GFP; we see no evidence of endogenous αCaMKII, evidenced by the lack of signal in the red channel. (B) eGFP-CaM expressed on HEK293 cells when no antibody treatment was performed, showing little cross talk signal from the green channel into the red channel. eGFP-CaM is distributed homogeneously within the cells with slightly lower expression in the nucleus. (C) Coexpression of eGFP-CaM and the nonlabeled form of αCaMKII; we see similar results to those in B, plus we see signal from the red channel, indicating expression of αCaMKII in each cell that is also expressing eGFP-CaM. CaMKII is largely excluded from the nucleus, whereas eGFP and eGFP-CaM are not. The appearance of very bright spots possibly reflects recycling vesicles due to overexpression of eGFP-CaM. (D) Expression of eGFP-CaMKII, with the primary monoclonal antibody specific for αCaMKII was omitted from the staining protocol. Note the very weak signal in the red channel, which represents background fluorescence and/or some modest bleed-through of the green channel similar to row B. (E) eGFP-CaMKII expressed in HEK293 cells stained with monoclonal antibody specific to αCaMKII. Note that both signals overlay almost perfectly, as expected.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Immunolabeling of transfected HEK293 cells. Red channel in most cases corresponds to the signal coming from Alexa-568 labeled goat anti-mouse secondary following labeling with a monoclonal antibody to αCaMKII. The green channel is eGFP or the eGFP construct signal. The overlay of the two channels is shown in the third column. (A) HEK293 cells expressing GFP; we see no evidence of endogenous αCaMKII, evidenced by the lack of signal in the red channel. (B) eGFP-CaM expressed on HEK293 cells when no antibody treatment was performed, showing little cross talk signal from the green channel into the red channel. eGFP-CaM is distributed homogeneously within the cells with slightly lower expression in the nucleus. (C) Coexpression of eGFP-CaM and the nonlabeled form of αCaMKII; we see similar results to those in B, plus we see signal from the red channel, indicating expression of αCaMKII in each cell that is also expressing eGFP-CaM. CaMKII is largely excluded from the nucleus, whereas eGFP and eGFP-CaM are not. The appearance of very bright spots possibly reflects recycling vesicles due to overexpression of eGFP-CaM. (D) Expression of eGFP-CaMKII, with the primary monoclonal antibody specific for αCaMKII was omitted from the staining protocol. Note the very weak signal in the red channel, which represents background fluorescence and/or some modest bleed-through of the green channel similar to row B. (E) eGFP-CaMKII expressed in HEK293 cells stained with monoclonal antibody specific to αCaMKII. Note that both signals overlay almost perfectly, as expected.
Mentions: To assess the distribution of each of the eGFP constructs used in this study, HEK293 cells were transfected with plasmids expressing eGFP, eGFP-CaM, eGFP-CaMKII, or the nonlabeled form of αCaMKII and were fixed, immunolabeled, and imaged on the LSM 510 (see Materials and Methods). Fig. 1 shows antibody labeling of αCaMKII in the red channel, and the green channel shows the expression of the various eGFP constructs. The final column shows image overlays of the two channels. It is clear from row A that there is no endogenous αCaMKII in HEK293 cells (absence of signal in the red channel). Note that eGFP is expressed throughout both the nucleus and cytoplasm of the cell (green channel) with some apparent enrichment in the nucleus. The bleed-through between channels is observed on row B, where no antibody treatment was added to HEK cells expressing eGFP-CaM. eGFP-CaM is distributed homogeneously within the cells with slightly lower expression in the nucleus. Strong immunostaining is evident when nonlabeled αCaMKII is expressed in the cells (row C) that disappear when the primary antibody is omitted (row D). αCaMKII is uniformly distributed in the cytoplasm but is largely excluded from the nucleus whether expressed without (row C) or with (row E) the eGFP tag. In cells expressing both eGFP-CaM and nonlabeled CaMKII (row C), GFP-CaM can be detected throughout the cytoplasm and the nucleus.

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