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Radial localization of inositol 1,4,5-trisphosphate-sensitive Ca2+ release sites in Xenopus oocytes resolved by axial confocal linescan imaging.

Callamaras N, Parker I - J. Gen. Physiol. (1999)

Bottom Line: Most puffs, however, exhibited a greater radial spread (3.25 micrometer), likely involving recruitment of radially neighboring release sites.The radial organization of puff sites a few micrometers inward from the plasma membrane may have important consequences for activation of calcium-dependent ion channels and "capacitative" calcium influx.However, on the macroscopic (hundreds of micrometers) scale of global calcium waves, release can be considered to occur primarily within a thin, essentially two-dimensional subplasmalemmal shell.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular and Molecular Neurobiology, Department of Psychobiology, University of California Irvine, Irvine, California 92697-4550, USA.

ABSTRACT
The radial localization and properties of elementary calcium release events ("puffs") were studied in Xenopus oocytes using a confocal microscope equipped with a piezoelectric focussing unit to allow rapid (>100 Hz) imaging of calcium signals along a radial line into the cell with a spatial resolution of <0.7 micrometer. Weak photorelease of caged inositol 1,4,5-trisphosphate (InsP3) evoked puffs arising predominantly within a 6-micrometer thick band located within a few micrometers of the cell surface. Approximately 25% of puffs had a restricted radial spread, consistent with calcium release from a single site. Most puffs, however, exhibited a greater radial spread (3.25 micrometer), likely involving recruitment of radially neighboring release sites. Calcium waves evoked by just suprathreshold stimuli exhibited radial calcium distributions consistent with inward diffusion of calcium liberated at puff sites, whereas stronger flashes evoked strong, short-latency signals at depths inward from puff sites, indicating deep InsP3-sensitive stores activated at higher concentrations of InsP3. Immunolocalization of InsP3 receptors showed punctate staining throughout a region corresponding to the localization of puffs and subplasmalemmal endoplasmic reticulum. The radial organization of puff sites a few micrometers inward from the plasma membrane may have important consequences for activation of calcium-dependent ion channels and "capacitative" calcium influx. However, on the macroscopic (hundreds of micrometers) scale of global calcium waves, release can be considered to occur primarily within a thin, essentially two-dimensional subplasmalemmal shell.

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Morphology of the  oocyte and correspondence of  puff sites with localization of the  ER and of InsP3 receptors. (A–C)  Confocal sections into the vegetal hemisphere of oocytes, with  the vertical axis representing  depth into the cell (z axis) and  the horizontal axis lateral (y) position. Images were obtained by  rapidly scanning in the x axis using a galvanometer mirror while  slowly advancing the microscope  focus using a synchronous motor. The aspect ratio provided  square pixels. (A) Resting fluorescence of an oocyte loaded  with Oregon green-1. (B) Distribution of ER stained with the lipophyllic dye fast DiI. (C) Distribution of InsP3 receptors visualized using an anti–InsP3 receptor  antibody and an FITC-conjugated secondary antibody. (D–F)  Images derived from those in  A–C, by enhancement to partially  compensate for the fall off in fluorescence at increasing depths as  a result of light scattering and absorption. Fluorescence in all images was scaled by a correction factor increasing linearly from 1 at the level of the pigment granules (about  one third the way down each image) to 3 at the bottom of the images. (G) Profile of fluorescence (arbitrary scale) as a function of depth,  averaged across a 15-μm section in the y axis of A. (H) Histogram shows frequency of puff occurrence as a function of depth into the cell,  replotted from Fig. 4 A, after scaling to match the dimensions of the images. (I) Distribution of InsP3 receptors in the animal hemisphere  of a physically sectioned oocyte. The image shows a lateral (x–y) confocal scan focused a few micrometers below the cut surface of the cell.  Receptors were visualized using an anti–InsP3 receptor antibody and Cy3-conjugated secondary antibody. The staining in this image is  more dense and less obviously punctate than that in C, probably because the animal hemisphere shows a higher density of InsP3 receptors  and more closely packed functional release sites than does the vegetal hemisphere (Callamaras et al., 1998b). Diffuse fluorescence at the  lower left arose primarily through incomplete blocking of autofluorescence by the barrier filter. Scale bar applies to all images in the figure.
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Figure 7: Morphology of the oocyte and correspondence of puff sites with localization of the ER and of InsP3 receptors. (A–C) Confocal sections into the vegetal hemisphere of oocytes, with the vertical axis representing depth into the cell (z axis) and the horizontal axis lateral (y) position. Images were obtained by rapidly scanning in the x axis using a galvanometer mirror while slowly advancing the microscope focus using a synchronous motor. The aspect ratio provided square pixels. (A) Resting fluorescence of an oocyte loaded with Oregon green-1. (B) Distribution of ER stained with the lipophyllic dye fast DiI. (C) Distribution of InsP3 receptors visualized using an anti–InsP3 receptor antibody and an FITC-conjugated secondary antibody. (D–F) Images derived from those in A–C, by enhancement to partially compensate for the fall off in fluorescence at increasing depths as a result of light scattering and absorption. Fluorescence in all images was scaled by a correction factor increasing linearly from 1 at the level of the pigment granules (about one third the way down each image) to 3 at the bottom of the images. (G) Profile of fluorescence (arbitrary scale) as a function of depth, averaged across a 15-μm section in the y axis of A. (H) Histogram shows frequency of puff occurrence as a function of depth into the cell, replotted from Fig. 4 A, after scaling to match the dimensions of the images. (I) Distribution of InsP3 receptors in the animal hemisphere of a physically sectioned oocyte. The image shows a lateral (x–y) confocal scan focused a few micrometers below the cut surface of the cell. Receptors were visualized using an anti–InsP3 receptor antibody and Cy3-conjugated secondary antibody. The staining in this image is more dense and less obviously punctate than that in C, probably because the animal hemisphere shows a higher density of InsP3 receptors and more closely packed functional release sites than does the vegetal hemisphere (Callamaras et al., 1998b). Diffuse fluorescence at the lower left arose primarily through incomplete blocking of autofluorescence by the barrier filter. Scale bar applies to all images in the figure.

Mentions: The endoplasmic reticulum (ER) was visualized by confocal scanning of live oocytes injected ∼12 h previously with a bolus of 20 nl of vegetable oil saturated with fast DiI. Diffusion of the lipophyllic fluorescent dye within continuous membrane structures provides specific labeling of the ER (Terasaki and Jaffe, 1993), and images were obtained in the vegetal hemisphere ∼200 μm distant from sites of injections made on the equator (see Fig. 7, B and E).


Radial localization of inositol 1,4,5-trisphosphate-sensitive Ca2+ release sites in Xenopus oocytes resolved by axial confocal linescan imaging.

Callamaras N, Parker I - J. Gen. Physiol. (1999)

Morphology of the  oocyte and correspondence of  puff sites with localization of the  ER and of InsP3 receptors. (A–C)  Confocal sections into the vegetal hemisphere of oocytes, with  the vertical axis representing  depth into the cell (z axis) and  the horizontal axis lateral (y) position. Images were obtained by  rapidly scanning in the x axis using a galvanometer mirror while  slowly advancing the microscope  focus using a synchronous motor. The aspect ratio provided  square pixels. (A) Resting fluorescence of an oocyte loaded  with Oregon green-1. (B) Distribution of ER stained with the lipophyllic dye fast DiI. (C) Distribution of InsP3 receptors visualized using an anti–InsP3 receptor  antibody and an FITC-conjugated secondary antibody. (D–F)  Images derived from those in  A–C, by enhancement to partially  compensate for the fall off in fluorescence at increasing depths as  a result of light scattering and absorption. Fluorescence in all images was scaled by a correction factor increasing linearly from 1 at the level of the pigment granules (about  one third the way down each image) to 3 at the bottom of the images. (G) Profile of fluorescence (arbitrary scale) as a function of depth,  averaged across a 15-μm section in the y axis of A. (H) Histogram shows frequency of puff occurrence as a function of depth into the cell,  replotted from Fig. 4 A, after scaling to match the dimensions of the images. (I) Distribution of InsP3 receptors in the animal hemisphere  of a physically sectioned oocyte. The image shows a lateral (x–y) confocal scan focused a few micrometers below the cut surface of the cell.  Receptors were visualized using an anti–InsP3 receptor antibody and Cy3-conjugated secondary antibody. The staining in this image is  more dense and less obviously punctate than that in C, probably because the animal hemisphere shows a higher density of InsP3 receptors  and more closely packed functional release sites than does the vegetal hemisphere (Callamaras et al., 1998b). Diffuse fluorescence at the  lower left arose primarily through incomplete blocking of autofluorescence by the barrier filter. Scale bar applies to all images in the figure.
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Related In: Results  -  Collection

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Figure 7: Morphology of the oocyte and correspondence of puff sites with localization of the ER and of InsP3 receptors. (A–C) Confocal sections into the vegetal hemisphere of oocytes, with the vertical axis representing depth into the cell (z axis) and the horizontal axis lateral (y) position. Images were obtained by rapidly scanning in the x axis using a galvanometer mirror while slowly advancing the microscope focus using a synchronous motor. The aspect ratio provided square pixels. (A) Resting fluorescence of an oocyte loaded with Oregon green-1. (B) Distribution of ER stained with the lipophyllic dye fast DiI. (C) Distribution of InsP3 receptors visualized using an anti–InsP3 receptor antibody and an FITC-conjugated secondary antibody. (D–F) Images derived from those in A–C, by enhancement to partially compensate for the fall off in fluorescence at increasing depths as a result of light scattering and absorption. Fluorescence in all images was scaled by a correction factor increasing linearly from 1 at the level of the pigment granules (about one third the way down each image) to 3 at the bottom of the images. (G) Profile of fluorescence (arbitrary scale) as a function of depth, averaged across a 15-μm section in the y axis of A. (H) Histogram shows frequency of puff occurrence as a function of depth into the cell, replotted from Fig. 4 A, after scaling to match the dimensions of the images. (I) Distribution of InsP3 receptors in the animal hemisphere of a physically sectioned oocyte. The image shows a lateral (x–y) confocal scan focused a few micrometers below the cut surface of the cell. Receptors were visualized using an anti–InsP3 receptor antibody and Cy3-conjugated secondary antibody. The staining in this image is more dense and less obviously punctate than that in C, probably because the animal hemisphere shows a higher density of InsP3 receptors and more closely packed functional release sites than does the vegetal hemisphere (Callamaras et al., 1998b). Diffuse fluorescence at the lower left arose primarily through incomplete blocking of autofluorescence by the barrier filter. Scale bar applies to all images in the figure.
Mentions: The endoplasmic reticulum (ER) was visualized by confocal scanning of live oocytes injected ∼12 h previously with a bolus of 20 nl of vegetable oil saturated with fast DiI. Diffusion of the lipophyllic fluorescent dye within continuous membrane structures provides specific labeling of the ER (Terasaki and Jaffe, 1993), and images were obtained in the vegetal hemisphere ∼200 μm distant from sites of injections made on the equator (see Fig. 7, B and E).

Bottom Line: Most puffs, however, exhibited a greater radial spread (3.25 micrometer), likely involving recruitment of radially neighboring release sites.The radial organization of puff sites a few micrometers inward from the plasma membrane may have important consequences for activation of calcium-dependent ion channels and "capacitative" calcium influx.However, on the macroscopic (hundreds of micrometers) scale of global calcium waves, release can be considered to occur primarily within a thin, essentially two-dimensional subplasmalemmal shell.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular and Molecular Neurobiology, Department of Psychobiology, University of California Irvine, Irvine, California 92697-4550, USA.

ABSTRACT
The radial localization and properties of elementary calcium release events ("puffs") were studied in Xenopus oocytes using a confocal microscope equipped with a piezoelectric focussing unit to allow rapid (>100 Hz) imaging of calcium signals along a radial line into the cell with a spatial resolution of <0.7 micrometer. Weak photorelease of caged inositol 1,4,5-trisphosphate (InsP3) evoked puffs arising predominantly within a 6-micrometer thick band located within a few micrometers of the cell surface. Approximately 25% of puffs had a restricted radial spread, consistent with calcium release from a single site. Most puffs, however, exhibited a greater radial spread (3.25 micrometer), likely involving recruitment of radially neighboring release sites. Calcium waves evoked by just suprathreshold stimuli exhibited radial calcium distributions consistent with inward diffusion of calcium liberated at puff sites, whereas stronger flashes evoked strong, short-latency signals at depths inward from puff sites, indicating deep InsP3-sensitive stores activated at higher concentrations of InsP3. Immunolocalization of InsP3 receptors showed punctate staining throughout a region corresponding to the localization of puffs and subplasmalemmal endoplasmic reticulum. The radial organization of puff sites a few micrometers inward from the plasma membrane may have important consequences for activation of calcium-dependent ion channels and "capacitative" calcium influx. However, on the macroscopic (hundreds of micrometers) scale of global calcium waves, release can be considered to occur primarily within a thin, essentially two-dimensional subplasmalemmal shell.

Show MeSH
Related in: MedlinePlus