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. 1998 Sep 29;95(20):11981-6.
doi: 10.1073/pnas.95.20.11981.

Direct immunogold labeling of aquaporin-4 in square arrays of astrocyte and ependymocyte plasma membranes in rat brain and spinal cord

J E Rash  1 , T Yasumura, C S Hudson, P Agre, S Nielsen
Affiliations

Direct immunogold labeling of aquaporin-4 in square arrays of astrocyte and ependymocyte plasma membranes in rat brain and spinal cord

J E Rash et al. Proc Natl Acad Sci U S A. .

Abstract

Aquaporin (AQP) water channels are abundant in the brain and spinal cord, where AQP1 and AQP4 are believed to play major roles in water metabolism and osmoregulation. Immunocytochemical analysis of the brain recently revealed that AQP4 has a highly polarized distribution, with marked expression in astrocyte end-feet that surround capillaries and form the glia limitans; however, the structural organization of AQP4 has remained unknown. In freeze-fracture replicas, astrocyte end-feet contain abundant square arrays of intramembrane particles that parallel the distribution of AQP4. To determine whether astrocyte and ependymocyte square arrays contain AQP4, we employed immunogold labeling of SDS-washed freeze-fracture replicas and stereoscopic confirmation of tissue binding. Antibodies to AQP4 directly labeled approximately 33% of square arrays in astrocyte and ependymocyte plasma membranes in rat brain and spinal cord. Overall, 84% of labels were present beneath square arrays; 11% were beneath particle clusters that resembled square arrays that had been altered during fixation or cleaving; and 5% were beneath the much larger areas of glial plasma membrane that were devoid of square arrays. Based on this evidence that AQP4 is concentrated in glial square arrays, freeze-fracture methods may now provide biophysical insights regarding neuropathological states in which abnormal fluid shifts are accompanied by alterations in the aggregation state or the molecular architecture of square arrays.

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Figures

Figure 1
Figure 1
(A) Immunoblot of membrane fractions from spinal cord, cerebellum, and cerebral cortex (plus hippocampus). The blot was probed with affinity-purified anti-AQP4 and shows 29- and 31-kDa bands (splice variants) and a higher molecular mass band (undissociated-AQP4 oligomers). (B) Thin cryosection (0.9 μm) of spinal cord. Extensive AQP4-immunolabeling is observed on glial processes adjacent to capillaries (arrows) and adjacent to neurons (arrowhead). (×670.) (C) Ultrathin Lowicryl section of cerebellum. AQP4-labeling is associated with the glial process (G) on the side directly facing the capillary endothelium (E). (×72,000.)
Figure 2
Figure 2
Replica of ependymal-cell plasma membrane in section of suprachiasmatic nucleus after double-labeling with anti-Cx43/20-nm gold beads and anti-AQP4/10 nm-gold beads. (A) The single gap junction seen in this image has 22 large gold beads beneath or within 20 nm of P face IMPs and E face pits. No other 20-nm gold beads are present. Calibration bars in all freeze-fracture images represent 0.1 μm. (×85,000.) (B) Inscribed area from Fig. 2A that contains four anti-AQP4/10-nm gold particles beneath five square arrays (black arrows). Three additional square arrays are unlabeled (white arrows). The high angle of shadowing in this area resulted in reduced specimen contrast. (×130,000.) (C) AQP4/10-nm gold-labeled square array adjacent to Cx43/20-nm gold-labeled gap junction between ependymal cells in spinal cord. The white arrow points to AQP4/10-nm gold-labeled square array (shown at higher magnification in the inset image). Black arrows point to cryptic labeling of an unreplicated portion of the gap junction (see text). (×70,000; Inset ×135,000.)
Figure 3
Figure 3
High-specificity anti-AQP4/10-nm gold labeling of square arrays in astrocyte end-feet. (A) Gold labels are confined almost exclusively to the astrocyte end-feet. No gold beads are seen on other membranes or attached to the replicated extracellular matrix (on the left side of the image). (×70,000.) (B) Stereoscopic image of the area inscribed in A reveals 17 gold particles directly beneath square arrays. (×135,000.) (C–F) High-magnification stereoscopic images of labeled AQP4 square arrays (C and D printed with white shadows; E and F printed with black shadows). In images printed with black shadows, 2-nm crossbridges (indicated by an arrow in E) are resolved linking the subunit IMPs (see text and ref. 21). (×450,000.)
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