Limits...
Connexin composition in apposed gap junction hemiplaques revealed by matched double-replica freeze-fracture replica immunogold labeling.

Rash JE, Kamasawa N, Davidson KG, Yasumura T, Pereda AE, Nagy JI - J. Membr. Biol. (2012)

Bottom Line: Currently, only serial thin-section transmission electron microscopy and freeze-fracture replica immunogold labeling have sufficient resolution to assign connexin proteins to either or both sides of gap junction plaques.However, freeze-fracture replica immunogold labeling has been limited because conventional freeze fracturing allows retrieval of only one of the two membrane fracture faces within a gap junction, making it difficult to identify connexin coupling partners in hemiplaques removed by fracturing.We now summarize progress in ascertaining the connexin composition of two coupled hemiplaques using matched double-replicas that are labeled simultaneously for multiple connexins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA. john.rash@colostate.edu

ABSTRACT
Despite the combination of light-microscopic immunocytochemistry, histochemical mRNA detection techniques and protein reporter systems, progress in identifying the protein composition of neuronal versus glial gap junctions, determination of the differential localization of their constituent connexin proteins in two apposing membranes and understanding human neurological diseases caused by connexin mutations has been problematic due to ambiguities introduced in the cellular and subcellular assignment of connexins. Misassignments occurred primarily because membranes and their constituent proteins are below the limit of resolution of light microscopic imaging techniques. Currently, only serial thin-section transmission electron microscopy and freeze-fracture replica immunogold labeling have sufficient resolution to assign connexin proteins to either or both sides of gap junction plaques. However, freeze-fracture replica immunogold labeling has been limited because conventional freeze fracturing allows retrieval of only one of the two membrane fracture faces within a gap junction, making it difficult to identify connexin coupling partners in hemiplaques removed by fracturing. We now summarize progress in ascertaining the connexin composition of two coupled hemiplaques using matched double-replicas that are labeled simultaneously for multiple connexins. This approach allows unambiguous identification of connexins and determination of the membrane "sidedness" and the identities of connexin coupling partners in homotypic and heterotypic gap junctions of vertebrate neurons.

Show MeSH

Related in: MedlinePlus

Comparison of limits of resolution of light microscopy (a) with ultrastructural resolution (b, c). a Neurons double-stained for Cx43 (green fluorescence) and the neuronal marker MAP-2 (red fluorescence) but without visualization of intervening glial cells. Without companion bright-field or differential interference optics to reveal other cell types, it is not possible to assign Cx43 unambiguously to the visualized cells, regardless of apparent close proximity. This deficiency is implicit in representative thin-section TEM images. b Modified from Peters et al. (1991). The limits of resolution in the blue and red wavelengths are indicated by superimposed red and blue discs, each of which overlaps cell margins of all three cell types, as well as multiple cytoplasmic membranes. c Two neuronal dendritic processes (red overlays), with a gap junction linking two thin intervening astrocyte processes (blue overlays). The astrocyte gap junction (shown at higher magnification in the inset) is double-labeled for Cx26 (12 nm gold) and Cx30 (20 nm gold). The limits of resolution in the x, y and z axes are indicated by the inscribed three-dimensional box, which corresponds to a single voxel (volume pixel) at the limit of resolution of confocal LM. If this region had been visualized by LM, with neurons stained red, astrocytes and oligodendrocytes not stained and connexins visualized using green fluorescence (as in a), Cx26 and Cx30 would have appeared to be localized to the decussating, small-diameter neuronal processes. Crossing red arrows indicate the limit of LM resolution in the red wavelength, suggesting that these two neuronal processes would have been in direct contact, with no room for intervening astrocyte processes. Barred Circle = gold bead on top of replica, as "noise" (Rash and Yasumura 1999). d, e “Serial sections in which gold–silver labeling for Cx32 (straight open arrows) was identified on the cytoplasmic surface of a peroxidase-labeled TH dendrite and in an apposed glial process (asterisks) that separates two TH-positive dendrites from one another” (Alvarez-Maubecin et al. 2000). However, we note that Cx32 is an oligodendrocyte connexin and is not found in astrocytes, nor has it been detected in ultrastructurally defined neuronal gap junctions, so we consider these images to represent background “noise” on two nonserial sections, each showing an astrocyte process between two different sets of TH neurons. Calibration bars 0.2 μm. f Comparison FRIL image of two neuronal gap junctions (red overlays) in adult rat retina that were immunogold-labeled for Cx36 (13- and three 20-nm gold beads). Unlabeled glutamate receptor postsynaptic density (yellow overlay). Modified from Rash et al. (2001). Calibration bars 0.1 μm, unless otherwise indicated
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3401501&req=5

Fig2: Comparison of limits of resolution of light microscopy (a) with ultrastructural resolution (b, c). a Neurons double-stained for Cx43 (green fluorescence) and the neuronal marker MAP-2 (red fluorescence) but without visualization of intervening glial cells. Without companion bright-field or differential interference optics to reveal other cell types, it is not possible to assign Cx43 unambiguously to the visualized cells, regardless of apparent close proximity. This deficiency is implicit in representative thin-section TEM images. b Modified from Peters et al. (1991). The limits of resolution in the blue and red wavelengths are indicated by superimposed red and blue discs, each of which overlaps cell margins of all three cell types, as well as multiple cytoplasmic membranes. c Two neuronal dendritic processes (red overlays), with a gap junction linking two thin intervening astrocyte processes (blue overlays). The astrocyte gap junction (shown at higher magnification in the inset) is double-labeled for Cx26 (12 nm gold) and Cx30 (20 nm gold). The limits of resolution in the x, y and z axes are indicated by the inscribed three-dimensional box, which corresponds to a single voxel (volume pixel) at the limit of resolution of confocal LM. If this region had been visualized by LM, with neurons stained red, astrocytes and oligodendrocytes not stained and connexins visualized using green fluorescence (as in a), Cx26 and Cx30 would have appeared to be localized to the decussating, small-diameter neuronal processes. Crossing red arrows indicate the limit of LM resolution in the red wavelength, suggesting that these two neuronal processes would have been in direct contact, with no room for intervening astrocyte processes. Barred Circle = gold bead on top of replica, as "noise" (Rash and Yasumura 1999). d, e “Serial sections in which gold–silver labeling for Cx32 (straight open arrows) was identified on the cytoplasmic surface of a peroxidase-labeled TH dendrite and in an apposed glial process (asterisks) that separates two TH-positive dendrites from one another” (Alvarez-Maubecin et al. 2000). However, we note that Cx32 is an oligodendrocyte connexin and is not found in astrocytes, nor has it been detected in ultrastructurally defined neuronal gap junctions, so we consider these images to represent background “noise” on two nonserial sections, each showing an astrocyte process between two different sets of TH neurons. Calibration bars 0.2 μm. f Comparison FRIL image of two neuronal gap junctions (red overlays) in adult rat retina that were immunogold-labeled for Cx36 (13- and three 20-nm gold beads). Unlabeled glutamate receptor postsynaptic density (yellow overlay). Modified from Rash et al. (2001). Calibration bars 0.1 μm, unless otherwise indicated

Mentions: The third source of connexin misassignment arises from the limited resolution of light microscopy (LM). Because of inherent limits of LM resolution, current immunocytochemical methods applied to complex CNS tissues are unable to discern whether specific connexins, reportedly identified either by diffuse cytoplasmic staining (Colwell 2000) or by the presence of both punctate immunolabeling for connexins and widespread cell-surface immunofluorescence (Nadarajah et al. 1996, 1997), link either neurons or glia or both. When only a single cell type (neuron) is visualized by immunofluorescence in CNS tissue, without companion bright-field or differential interference optics to reveal glial cells (Fig. 2a), it is not possible to assign connexins unambiguously to the visualized neuron, regardless of apparent close proximity of connexin labels. This failure to account for CNS tissue complexity is implicit in representative thin-section transmission electron microscopic (TEM) images (Fig. 2b), wherein all spaces between neurons are seen to be completely filled with the two primary types of macroglial cells (astrocytes and oligodendrocytes) found throughout the neuropil and by their even more pervasive thin processes that are also below the limit of LM resolution. For further clarification, the limits of resolution in the blue and red wavelengths are superimposed on the TEM image (Fig. 2b, blue and red discs), revealing that a single pixel at the limit of LM resolution in those wavelengths would overlap multiple plasma membranes of multiple cell types, with the blue dot overlapping with a neuronal plasma membrane, two astrocyte fingers and an oligodendrocyte soma and nucleus. This image suggests that, in the absence of companion ultrastructural examination, complex interdigitations of neuronal and glial processes preclude or make questionable the LM assignment of specific connexins to specific cell types in convoluted CNS tissue. Of course, this problem of assigning proteins to specific cell margins applies equally well to subcellular localization of all other membrane proteins.Fig. 2


Connexin composition in apposed gap junction hemiplaques revealed by matched double-replica freeze-fracture replica immunogold labeling.

Rash JE, Kamasawa N, Davidson KG, Yasumura T, Pereda AE, Nagy JI - J. Membr. Biol. (2012)

Comparison of limits of resolution of light microscopy (a) with ultrastructural resolution (b, c). a Neurons double-stained for Cx43 (green fluorescence) and the neuronal marker MAP-2 (red fluorescence) but without visualization of intervening glial cells. Without companion bright-field or differential interference optics to reveal other cell types, it is not possible to assign Cx43 unambiguously to the visualized cells, regardless of apparent close proximity. This deficiency is implicit in representative thin-section TEM images. b Modified from Peters et al. (1991). The limits of resolution in the blue and red wavelengths are indicated by superimposed red and blue discs, each of which overlaps cell margins of all three cell types, as well as multiple cytoplasmic membranes. c Two neuronal dendritic processes (red overlays), with a gap junction linking two thin intervening astrocyte processes (blue overlays). The astrocyte gap junction (shown at higher magnification in the inset) is double-labeled for Cx26 (12 nm gold) and Cx30 (20 nm gold). The limits of resolution in the x, y and z axes are indicated by the inscribed three-dimensional box, which corresponds to a single voxel (volume pixel) at the limit of resolution of confocal LM. If this region had been visualized by LM, with neurons stained red, astrocytes and oligodendrocytes not stained and connexins visualized using green fluorescence (as in a), Cx26 and Cx30 would have appeared to be localized to the decussating, small-diameter neuronal processes. Crossing red arrows indicate the limit of LM resolution in the red wavelength, suggesting that these two neuronal processes would have been in direct contact, with no room for intervening astrocyte processes. Barred Circle = gold bead on top of replica, as "noise" (Rash and Yasumura 1999). d, e “Serial sections in which gold–silver labeling for Cx32 (straight open arrows) was identified on the cytoplasmic surface of a peroxidase-labeled TH dendrite and in an apposed glial process (asterisks) that separates two TH-positive dendrites from one another” (Alvarez-Maubecin et al. 2000). However, we note that Cx32 is an oligodendrocyte connexin and is not found in astrocytes, nor has it been detected in ultrastructurally defined neuronal gap junctions, so we consider these images to represent background “noise” on two nonserial sections, each showing an astrocyte process between two different sets of TH neurons. Calibration bars 0.2 μm. f Comparison FRIL image of two neuronal gap junctions (red overlays) in adult rat retina that were immunogold-labeled for Cx36 (13- and three 20-nm gold beads). Unlabeled glutamate receptor postsynaptic density (yellow overlay). Modified from Rash et al. (2001). Calibration bars 0.1 μm, unless otherwise indicated
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3401501&req=5

Fig2: Comparison of limits of resolution of light microscopy (a) with ultrastructural resolution (b, c). a Neurons double-stained for Cx43 (green fluorescence) and the neuronal marker MAP-2 (red fluorescence) but without visualization of intervening glial cells. Without companion bright-field or differential interference optics to reveal other cell types, it is not possible to assign Cx43 unambiguously to the visualized cells, regardless of apparent close proximity. This deficiency is implicit in representative thin-section TEM images. b Modified from Peters et al. (1991). The limits of resolution in the blue and red wavelengths are indicated by superimposed red and blue discs, each of which overlaps cell margins of all three cell types, as well as multiple cytoplasmic membranes. c Two neuronal dendritic processes (red overlays), with a gap junction linking two thin intervening astrocyte processes (blue overlays). The astrocyte gap junction (shown at higher magnification in the inset) is double-labeled for Cx26 (12 nm gold) and Cx30 (20 nm gold). The limits of resolution in the x, y and z axes are indicated by the inscribed three-dimensional box, which corresponds to a single voxel (volume pixel) at the limit of resolution of confocal LM. If this region had been visualized by LM, with neurons stained red, astrocytes and oligodendrocytes not stained and connexins visualized using green fluorescence (as in a), Cx26 and Cx30 would have appeared to be localized to the decussating, small-diameter neuronal processes. Crossing red arrows indicate the limit of LM resolution in the red wavelength, suggesting that these two neuronal processes would have been in direct contact, with no room for intervening astrocyte processes. Barred Circle = gold bead on top of replica, as "noise" (Rash and Yasumura 1999). d, e “Serial sections in which gold–silver labeling for Cx32 (straight open arrows) was identified on the cytoplasmic surface of a peroxidase-labeled TH dendrite and in an apposed glial process (asterisks) that separates two TH-positive dendrites from one another” (Alvarez-Maubecin et al. 2000). However, we note that Cx32 is an oligodendrocyte connexin and is not found in astrocytes, nor has it been detected in ultrastructurally defined neuronal gap junctions, so we consider these images to represent background “noise” on two nonserial sections, each showing an astrocyte process between two different sets of TH neurons. Calibration bars 0.2 μm. f Comparison FRIL image of two neuronal gap junctions (red overlays) in adult rat retina that were immunogold-labeled for Cx36 (13- and three 20-nm gold beads). Unlabeled glutamate receptor postsynaptic density (yellow overlay). Modified from Rash et al. (2001). Calibration bars 0.1 μm, unless otherwise indicated
Mentions: The third source of connexin misassignment arises from the limited resolution of light microscopy (LM). Because of inherent limits of LM resolution, current immunocytochemical methods applied to complex CNS tissues are unable to discern whether specific connexins, reportedly identified either by diffuse cytoplasmic staining (Colwell 2000) or by the presence of both punctate immunolabeling for connexins and widespread cell-surface immunofluorescence (Nadarajah et al. 1996, 1997), link either neurons or glia or both. When only a single cell type (neuron) is visualized by immunofluorescence in CNS tissue, without companion bright-field or differential interference optics to reveal glial cells (Fig. 2a), it is not possible to assign connexins unambiguously to the visualized neuron, regardless of apparent close proximity of connexin labels. This failure to account for CNS tissue complexity is implicit in representative thin-section transmission electron microscopic (TEM) images (Fig. 2b), wherein all spaces between neurons are seen to be completely filled with the two primary types of macroglial cells (astrocytes and oligodendrocytes) found throughout the neuropil and by their even more pervasive thin processes that are also below the limit of LM resolution. For further clarification, the limits of resolution in the blue and red wavelengths are superimposed on the TEM image (Fig. 2b, blue and red discs), revealing that a single pixel at the limit of LM resolution in those wavelengths would overlap multiple plasma membranes of multiple cell types, with the blue dot overlapping with a neuronal plasma membrane, two astrocyte fingers and an oligodendrocyte soma and nucleus. This image suggests that, in the absence of companion ultrastructural examination, complex interdigitations of neuronal and glial processes preclude or make questionable the LM assignment of specific connexins to specific cell types in convoluted CNS tissue. Of course, this problem of assigning proteins to specific cell margins applies equally well to subcellular localization of all other membrane proteins.Fig. 2

Bottom Line: Currently, only serial thin-section transmission electron microscopy and freeze-fracture replica immunogold labeling have sufficient resolution to assign connexin proteins to either or both sides of gap junction plaques.However, freeze-fracture replica immunogold labeling has been limited because conventional freeze fracturing allows retrieval of only one of the two membrane fracture faces within a gap junction, making it difficult to identify connexin coupling partners in hemiplaques removed by fracturing.We now summarize progress in ascertaining the connexin composition of two coupled hemiplaques using matched double-replicas that are labeled simultaneously for multiple connexins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA. john.rash@colostate.edu

ABSTRACT
Despite the combination of light-microscopic immunocytochemistry, histochemical mRNA detection techniques and protein reporter systems, progress in identifying the protein composition of neuronal versus glial gap junctions, determination of the differential localization of their constituent connexin proteins in two apposing membranes and understanding human neurological diseases caused by connexin mutations has been problematic due to ambiguities introduced in the cellular and subcellular assignment of connexins. Misassignments occurred primarily because membranes and their constituent proteins are below the limit of resolution of light microscopic imaging techniques. Currently, only serial thin-section transmission electron microscopy and freeze-fracture replica immunogold labeling have sufficient resolution to assign connexin proteins to either or both sides of gap junction plaques. However, freeze-fracture replica immunogold labeling has been limited because conventional freeze fracturing allows retrieval of only one of the two membrane fracture faces within a gap junction, making it difficult to identify connexin coupling partners in hemiplaques removed by fracturing. We now summarize progress in ascertaining the connexin composition of two coupled hemiplaques using matched double-replicas that are labeled simultaneously for multiple connexins. This approach allows unambiguous identification of connexins and determination of the membrane "sidedness" and the identities of connexin coupling partners in homotypic and heterotypic gap junctions of vertebrate neurons.

Show MeSH
Related in: MedlinePlus