Ultrastructure of the Squid Axon Membrane as Revealed by Freeze-Fracture Electron Microscopy

The structure of the axolemma of the squid giant axon was studied by freeze-fracture electron microscopy. Three types of preparations were examined: intact axons, axons with their Schwann cell sheaths stripped off prior to freezing, and axons with their Schwann cell sheaths chemically detached but not mechanically removed. Because of a problem of cross-fracturing, the first two types of preparations revealed very few membrane faces of the axolemma. This cross-fracturing problem, however, was eliminated when we used a complementary replication method to fracture the third type of preparation. We found that the E-face of the axon membrane was smooth relative to the P-face, which showed many prominent intramembrane particles (IMP). The diameters of the typical IMP range from 6 to 15 nm. The P-face of the adjacent Schwann cells also showed many large IMP. The sizes and heights of the Schwann-cell IMP, however, appear to be more homogeneous than the P-face axolemma.     

Specificity of membrane specializations in mechanoreceptors of birds—A freeze-etching study

The detailed knowledge of the molecular process of mechanotransduction is still an unsolved question. The investigation of the intramembranous structure of the cutaneous mechanoreceptors may play an important role in elucidating this problem. In this relation, Herbst sensory corpuscles in ducks were studied for the first time using the freeze-etching and thin sectioning techniques. Herbst corpuscles have the basic structural components valid for most of the encapsulated mechanoreceptors in mammals: a capsule made of perineural cells, a lamellar complex of modified Schwann cells, surrounding the non-myelinated part of the receptor nerve fiber and its ending. Freeze-etching replicas reveal that the plasmalemmae of the capsule cells, modified Schwann cells and axolemmae of parts of the nerve fiber differ in both density and pattern of distribution of intramembranous particles (IMPs) as well as IMP size. On all the plasmalemmae the IMP density is higher on the P-face (2000–3300?µm^?2) than the respective E-face (800–1500?µm^?2). The axolemma of the ending of the receptor nerve fiber expresses higher density of IMPs than its shaft. The mean IMP size for all the plasmalemmae varies between 5.5 and 7.5?nm. Many tight junctions occur between the capsule cells. These results indicate that the non-myelinated axolemma as well as the plasmalemmae of other components of Herbst corpuscles are specialized in terms of content and distribution of IMPs. The IMPs may represent various kinds of mechanosensitive channel proteins or related membrane-bound proteins participating in the process of mechanotransduction.     

Gliogenesis and myelination in the optic nerve of trisomy 19 mice. A quantitative electron-microscopic study

Trisomy 19 (ts19) of the mouse permits detailed studies on the influence of an extra autosome upon the postnatal development of the central nervous system. To examine gliogenesis and myelinogenesis, the optic nerves of 19 ts19 pugs aged 1-15 days have been examined by light and electron microscopy and compared to those of litter-mate controls. Differentiation of astrocytes and oligodendrocytes, myelinogenesis as well as the opening of the eyes are each delayed by about 2 days. Myelin sheaths are normally structured in ts19. There is a decrease in the percentage of myelinated fibres. The cross-sectional area of the ts19 optic nerve is reduced. The fibre density, which decreases with age both in ts19 and control mice, is higher in ts19 mice. Both with ts19 and control animals, the distribution of fibre diameters of myelinated axons overlaps with that of promyelinated and unmyelinated fibres, but myelinated axons cannot be observed below a diameter of 0.3 micron, and unmyelinated axons are always smaller than 1 micron. The mean diameter of promyelinated axons is identical in ts19 and control animals. Myelination is therefore not severely disturbed in the ts19 optic nerve. As retinal differentiation in ts19 is delayed by 2 days as well, reports on an asynchronous development of neurons and myelin sheaths cannot be confirmed for the visual system.     

A freeze-fracture study of perinatal changes of intramembranous particles in microvilli of absorptive cells in mouse small intestine

Summary Perinatal changes in the appearance of intramembranous particles (IMPs) of microvilli of enterocytes were analyzed quantitatively. In both the jejunum and the ileum, the IMP density on the P-face showed no significant changes from day 17 of gestation to day 5 of postnatal life. It increased between day 5 and day 12, reached a maximum at day 21, and thereafter decreased slightly. The IMP density on the E-face remained almost constant during the perinatal period in both intestinal parts. Measurements of particle diameters proved that neither the P-face nor the E-face membrane showed significant differences in either mean value or size distribution among different age groups.This study has revealed that the perinatal change in the IMP density on the P-face of microvilli correlates well with changes in the activity of certain enzymes found in the membranes of microvilli, e.g. disaccharidase and aminopeptidase.This study was supported by a Grant-in-Aid for Scientific Research to T. Yamamoto from the Ministry of Education, Science and Culture of Japan     

Components of the plasma membrane of growing axons. I. Size and distribution of intramembrane particles

The plasmalemma of mature and growing olfactory axons of the bullfrog has been studied by freeze-fracture. Intramembrane particles (IMPs) of mature olfactory axons are found to be uniformly distributed along the shaft. However, during growth, a decreasing gradient of IMP density is evident along the somatofugal axis. The size histograms of axolemmal IMPs from different segments of growing nerve reveal regional differences in the particle composition. The distribution of each individual size class of particles along the growing nerve forms a decreasing gradient in the somatofugal direction; the slope of these gradients varies directly with particle diameter. These size-dependent density gradients are consistent with a process of lateral diffusion of membrane components that are inserted proximally into the plasma membrane. The membrane composition of the growth cone, however, appears to be independent of these diffusion gradients; it displays a mosaic pattern of discrete domains of high and low particle densities. The relative IMP profiles of these growth cone regions are similar to one another but contain higher densities of large IMPs than the neighboring axonal shaft. The shifting distributions of intramembrane particles that characterize the sprouting neuron give new insights into cellular processes that may underlie the establishment of the functional polarity of the neuron and into the dynamics of axolemmal maturation.     

Components of the plasma membrane of growing axons. III. Saxitoxin binding to sodium channels

The density of sodium channels was measured in growing and mature axons of the olfactory nerve of the bullfrog, using as a probe the drug saxitoxin (STX). The toxin binds to control nerves from adult animals in a saturable manner with a dissociation constant of approximately 23 nM at 4 degrees C and a capacity of 72 fmol/mg wet weight, equivalent to about five sites per square micrometer of axolemma. In growing nerves, obtained from adult frogs 4-5 wk following removal of the original nerve, the STX-binding capacity per wet weight of tissue is markedly reduced, to approximately 25% of control values, and appears to decrease in the proximodistal direction. STX-binding data, expressed as STX/mg wet weight, was converted to STX/micron 2 of axolemma using stereologically derived values of membrane area per milligram wet weight of nerve. The axolemmal content (area/mg wet weight) of all regions of growing nerve is substantially decreased compared to controls, but increases in the proximodistal direction by 60%. These changes in axolemmal area result in calculated STX receptor densities (per unit axolemmal area) which, in distal regions, are approximately at the level of the mature nerve and, in proximal regions, are actually increased above controls by 50 to 70%. Upon comparing the axolemmal density of intramembrane particles, reported in the companion paper, with the calculated density of STX receptors in both mature and growing nerves, we find a correlation between STX receptors and intramembrane particles with diameters of 11.5-14.0 nm. The growing axon's gradient of sodium channels and the shift from this gradient to a uniform distribution in the mature axon suggest (a) that sodium channels are inserted into the perikaryal plasmalemma and diffuse from there into the growing axolemma, and (b) that the axolemma undergoes functional maturation during growth.     

Freeze-fracture study of plasma membranes in wild type and daunorubicin-resistant Ehrlich ascites tumor and P388 leukemia cells

The plasma membrane is considered to play a major role in the development and maintenance of the multidrug resistance (MDR) phenotype, a role which may in part be mediated by an inducible 170 kD transmembrane protein (P-170). The present freeze-fracture study of plasma membranes of daunorubicin-resistant Ehrlich ascites and P388 leukemia cells demonstrated a significant increase in the density of intramembrane particles (IMP) in the P-face, but not the E-face, of resistant sublines compared with wild type cells. Furthermore, a three-dimensional histogram plot of the diameters of P-face IMPs in Ehrlich ascites tumor cells showed the emergence of a subpopulation of 9 X 11 nm IMPs not found in wild type cells. The size of these IMPs would be consistent with a MW of approximately 340 kD, thus indicating that P-170, shown to be present in both resistant cell lines by Western blot analysis and immunohistochemical staining, exists as a dimer in the plasma membrane. Incubation with the calcium channel blocker verapamil, in concentrations known to inhibit daunorubicin efflux in resistant cells, showed evidence of membrane disturbance in the form of IMP clustering in both wild type and resistant Ehrlich ascites tumor cells. However, incubation with daunorubicin itself did not alter the freeze-fracture morphology of the plasma membranes.     

Freeze-fracture study of plasma membranes in wild type and daunorubicin-resistant Ehrlich ascites tumor and P388 leukemia cells

The plasma membrane is considered to play a major role in the development and maintenance of the multidrug resistance (MDR) phenotype, a role which may in part be mediated by an inducible 170 kD transmembrane protein (P-170). The present freeze-fracture study of plasma membranes of daunorubicin-resistant Ehrlich ascites and P388 leukemia cells demonstrated a significant increase in the density of intramembrane particles (IMP) in the P-face, but not the E-face, of resistant sublines compared with wild type cells. Furthermore, a three-dimensional histogram plot of the diameters of P-face IMPs in Ehrlich ascites tumor cells showed the emergence of a subpopulation of 9 × 11 nm IMPs not found in wild type cells. The size of these IMPs would be consistent with a MW of approximately 340 kD, thus indicating that P-170, shown to be present in both resistant cell lines by Western blot analysis and immunohistochemical staining, exists as a dimer in the plasma membrane. Incubation with the calcium channel blocker verapamil, in concentrations known to inhibit daunorubicin efflux in resistant cells, showed evidence of membrane disturbance in the form of IMP clustering in both wild type and resistant Ehrlich ascites tumor cells. However, incubation with daunorubicin itself did not alter the freeze-fracture morphology of the plasma membranes.     

The plasma-membrance IMP pattern as related to animal/vegetal polarity in the amphibian egg

Freeze-fracture electron microscopy of the plasma membrane of the fertilized, uncleaved Xenopus egg shows that intramembranous particles (IMPs) range in size from ca. 50 to 200 Å and that more IMPs are attached to the E-face than to the P-face. The overall IMP densities of the animal and the vegetal hemisphere do not differ significantly. IMP-free regions (?, ca. 0.1 μm) on the tips of surface protrusions were irregularly distributed in the animal and the vegetal half (E-face) occupying ca. 8.5 and 2%, respectively of the free area. The relative densities for 16 different IMP sizes have been compared, on the basis of seven animal and seven vegetal halves, counting (E-faces only) ca. 10,000 IMPs in each hemisphere. For IMP sizes of ≤81 Å, a significant difference (P < 0.0005) was found, more small IMPs being present in the animal half. Some evidence for IMP-associated thin elements was found. These findings are discussed in relation to plasma membrane anisotropy and the morphogenetic role of the egg cortex.     

Specificity of membrane specializations in mechanoreceptors of birds--A freeze-etching study

The detailed knowledge of the molecular process of mechanotransduction is still an unsolved question. The investigation of the intramembranous structure of the cutaneous mechanoreceptors may play an important role in elucidating this problem. In this relation, Herbst sensory corpuscles in ducks were studied for the first time using the freeze-etching and thin sectioning techniques. Herbst corpuscles have the basic structural components valid for most of the encapsulated mechanoreceptors in mammals: a capsule made of perineural cells, a lamellar complex of modified Schwann cells, surrounding the non-myelinated part of the receptor nerve fiber and its ending. Freeze-etching replicas reveal that the plasmalemmae of the capsule cells, modified Schwann cells and axolemmae of parts of the nerve fiber differ in both density and pattern of distribution of intramembranous particles (IMPs) as well as IMP size. On all the plasmalemmae the IMP density is higher on the P-face (2000-3300 microm(-2)) than the respective E-face (800-1500 microm(-2)). The axolemma of the ending of the receptor nerve fiber expresses higher density of IMPs than its shaft. The mean IMP size for all the plasmalemmae varies between 5.5 and 7.5 nm. Many tight junctions occur between the capsule cells. These results indicate that the non-myelinated axolemma as well as the plasmalemmae of other components of Herbst corpuscles are specialized in terms of content and distribution of IMPs. The IMPs may represent various kinds of mechanosensitive channel proteins or related membrane-bound proteins participating in the process of mechanotransduction.     

Membrane structure of vesiculotubular complexes in developing axons in rat optic nerve: freeze-fracture evidence for sequential membrane assembly

Intra-axonal vesiculotubular complexes, located within developing axons in the optic nerve of eight-day-old rats, were examined by freeze-fracture electron microscopy. The clusters usually fill most of the cross section of the axon and extend for approximately 1 micron along the fibre axis. As seen in freeze-fracture, the E- and P-faces of the membranes comprising these clusters exhibit a paucity of intramembranous particles (i.m.ps). This i.m.p.-poor membrane structure is different from that of the axolemma per se, which contains i.m.p. densities of ca. 120 micron-2 on the E-face and ca. 400 micron-2 on the P-face. Since earlier studies indicate that the vesiculotubular complexes fuse with the axon membrane so as to contribute to membrane growth, it is suggested that axonal differentiation involves a sequential mode of membrane development, in which an initial growth of a relatively undifferentiated membrane bilayer is followed by in situ insertion of specialized proteins within specific membrane domains.     

Immunocytochemical Demonstration of Na+,K+-ATPase in Internodal Axolemma of Myelinated Fibers of Rat Sciatic and Optic Nerves

We used postembedding electron microscopic immunocytochemistry with colloidal gold to determine the ultrastructural distribution of Na+,K(+)-ATPase in the sciatic and optic nerves of the rat. Using a polyclonal antiserum raised against the denatured catalytic subunit of brain Na+,K(+)-ATPase, we found immunoreactivity along the internodal axolemma of myelinated fibers in both nerves. This antiserum did not produce labeling of nodal axolemma. These results suggest that an important site of energy-dependent sodium-potassium exchange is along the internodal axolemma of myelinated fibers in the mammalian CNS and PNS and that there may be differences between the internodal and nodal forms of the enzyme.     

Initiation of sodium channel clustering at the node of Ranvier in the mouse optic nerve

Ion fluxes in mammalian myelinated axons are restricted to the nodes of Ranvier, where, in particular, voltage-gated Na⁺ channels are clustered at a high density. The node of Ranvier is separated from the internode by two distinct domains of the axolemma, the paranode and the juxtaparanode. Each axonal domain is characterized by the presence of a specific protein complex. Although oligodendrocytes and/or myelin membranes are believed to play some instructive roles in the organization of axonal domains, the mechanisms leading to their localized distribution are not well understood. In this paper we focused on the involvement of myelin sheaths in this domain organization and examined the distribution of axonal components in the optic nerves of wild type, hypomyelinating jimpy mice and demyelinating PLP transgenic mice. The results showed that the clustering of Na⁺ channels does not require junction-like structures to be formed between the glial processes and axons, but requires mature oligodendrocytes to be present in close vicinity.     

An isoform of ankyrin is localized at nodes of Ranvier in myelinated axons of central and peripheral nerves

Two variants of ankyrin have been distinguished in rat brain tissue using antibodies: a broadly distributed isoform (ankyrinB) that represents the major form of ankyrin in brain and another isoform with a restricted distribution (ankyrinR) that shares epitopes with erythrocyte ankyrin. The ankyrinR isoform was localized by immunofluorescence in cryosections of rat spinal cord gray matter and myelinated central and peripheral nerves to: (a) perikarya and initial axonal segments of neuron cells, (b) nodes of Ranvier of myelinated nerve with no detectable labeling in other areas of the myelinated axons, and (c) the axolemma of unmyelinated axons. Immunogold EM on ultrathin cryosections of myelinated nerve showed that ankyrinR was localized on the cytoplasmic face of the axolemma and was restricted to the nodal and, in some cases, paranodal area. The major isoform of ankyrin in brain (ankyrinB) displayed a broad distribution on glial and neuronal cells of the gray matter and a mainly glial distribution in central myelinated axons with no significant labeling on the axolemma. These results show that (a) ankyrin isoforms display a differential distribution on glial and neuronal cells of the nervous tissue; (b) an isoform of ankyrin codistributes with the voltage-dependent sodium channel in both myelinated and unmyelinated nerve fibers. Ankyrin interacts in vitro with the voltage-dependent sodium channel (Srinivasan, Y., L. Elmer, J. Davis, V. Bennett, and K. Angelides. 1988. Nature (Lond.). 333:177-180). A specific interaction of an isoform of ankyrin with the sodium channel thus may play an important role in the morphogenesis and/or maintenance of the node of Ranvier.     

Rows of dimeric-particles within the axolemma and juxtaposed particles within glia, incorporated into a new model for the paranodal glial- axonal junction at the node of Ranvier

Using freeze-fracture techniques, we have analyzed the glial-axonal junction (GAJ) between Schwann cells and axons in the peripheral nervous system, and between oligodendrocytes and axons in the central nervous system of the rat. We have identified a new set of dimeric- particles arranged in circumferential rows within the protoplasmic fracture faces (P-faces) of the paranodal axolemma in the region of glial-axonal juxtaposition. These particles, 260 A in length, composed of two 115-A subunits, are observed in both aldehyde-fixed and nonfixed preparations. The rows of dimeric-particles within the axonal P-face are associated with complementary rows of pits within the external fracture face (E-face) of the paranodal axolemma. These axonal particles are positioned between rows of 160-A particles that occur in both fracture faces of the glial loops in the same region. We observed, in addition to these previously described 160-A particles, a new set of 75-A glial particles within the glial P-faces of the GAJ. These 75-A particles form rows that are centered between the rows of 160-A particles and are therefore superimposed over the rows of dimeric- particles within the paranodal axolemma. Our new findings are interpreted with respect to methods of specimen preparation as well as to a potential role for the paranodal organ in saltatory conduction. We conclude that this particle-rich junction between axon and glia could potentially provide an intricate mechanism for ion exchange between these two cell types.     

Plasma Membrane Domains and the Site of Sperm Entrance in Discoglossus pictus (Anura) Eggs

Freeze-fracture quantitative analysis reveals three different plasma membrane (PM) domains in the unfertilized egg of the anuran Discoglossus pictus . One of these is specific to the sperm entrance site (D1). where the plasma membrane shows a larger number of intramembranous particles (IMP) than the rest of the egg surface. Such an increment is due to a markedly higher number of the IMPs anchored to the P-face. The two other domains (D2 and D3) are characterized by a lower IMP density at the P-face with respect to D1. The IMP density decreased within 10 min after fertilization by about 33% in all domains observed, probably due to the insertion of new membrane through exocytosis. The possibility that the IMPs located in D1 may represent putative plasma membrane proteins playing a role in sperm-egg interaction and/or in egg activation is discussed.     

Dolphin peripheral visual pathway in chronic unilateral ocular atrophy: Complete decussation apparent

Components of the peripheral visual pathway were examined in two bottlenose dolphins, Tursiops truncatus, each with unilateral ocular degeneration and scarring of 3 or more years' duration. In both animals, the optic nerve associated with the blind eye right eye in Tg419 and left eye in Tt038 had a translucent, gel-like appearance upon gross examination. This translucency was also evident in the optic tract contralateral to the affected eye. In Tg419, myelinated axons of varying diameters were apparent in the left optic nerve, whereas the right optic nerve, serving the blind eye, appeared to be devoid of axons. In Tt038, myelinated axons were associated with the right optic nerve (serving the functional eye) and left optic tract but were essentially absent in the left optic nerve and right optic tract. Examined by light microscopy in serial horizontal sections, the optic chiasm of Tt038 was arranged along its central plane in segregated, alternating pathways for the decussation of right and left optic nerve fibers. Ventral to this plane, the chiasm was comprised of fibers from the left optic nerve, whereas dorsal to the central plane, fibers derived from the right optic nerve. Because of this architectural arrangement, the right and left optic nerves grossly appeared to overlap as they crossed the optic chiasm with the right optic nerve coursing dorsally to the left optic nerve. At the light and electron microscopic levels, the optic nerves and tracts lacking axons were well vascularized and dominated by glial cell bodies and glial processes, an expression of the marked glial scarring associated with postinjury axonal degeneration. The apparent absence of axons in one of the optic tract pairs (right in Tt038 and left in Tg419) supports the concept of complete decussation of right and left optic nerve fibers at the optic chiasm in the bottlenose dolphin. © 1994 Wiley-Liss, Inc.     

Myelinating glia of earthworm giant axons: thermally induced intramembranous changes

The median and lateral giant axons in the ventral nerve cord of the earthworm Lumbricus terrestris are ensheathed by extensive spiral glial cell wrappings which resemble vertebrate myelin. The other, smaller, axons are encompassed by attenuated glial processes, as is typical of invertebrates. The fine structural details of the glial cells have been studied in thin sections and in replicas produced by freeze-fracturing where the intramembranous particle (IMP) populations within the lipid bilayer are visible. These consist of both low-profile IMPs as well as prominent ones 6-8 nm in diameter, scattered at random over the lipid interface in the myelinating glia. The larger IMPs on both P and E faces number about 80/mum2 at 16 degrees C in contrast to the IMP density of 400/mum2 in the other glial membranes. After acclimation to 5, 16 and 26 degrees C, the loose myelin glial membranes show variations in the density of their larger IMP population; in animals acclimated over 3 or more weeks to 5 degrees C, the number of these IMPs is significantly (P less than 0.001) less per unit area than in animals acclimated to 16 or 26 degrees C. The size of the particles at 5 degrees C is significantly (P less than 0.001) smaller than those at 16 or 26 degrees C. When animals are subjected to a sudden differential in ambient temperature, from 26 or 16 to 5 degrees C, or from 5 to 26 degrees C, and their giant axons with encompassing glia are fixed and frozen 30 min after this temperature change, the IMP population of the glial membranes remaining does not appear to alter. The differences in the IMP population of the myelinating glial membranes at different temperatures may reflect the extent to which they insulate and/or influence the velocity of impulse propagation.     

Intramembrane particles and the organization of lymphocyte membrane proteins

An experimental system was developed in which the majority of all lymphocyte cell-surface proteins, regardless of antigenic specificity, could be cross-linked and redistributed in the membrane to determine whether this would induce a corresponding redistribution of intramembrane particles (IMP). Mouse spleen cells were treated with P-diazoniumphenyl- β-D-lactoside (lac) to modify all exposed cell-surface proteins. Extensive azo- coupling was achieved without significantly reducing cell viability or compromising cellular function in mitogen- or antigen-stimulated cultures. When the lac-modified cell- surface proteins were capped with a sandwich of rabbit antilactoside antibody and fluorescein-goat anti-rabbit Ig, freeze-fracture preparations obtained from these cells revealed no obvious redistribution of IMP on the majority of fracture faces. However, detailed analysis showed a statistically significant 35 percent decrease (P less than 0.01) in average IMP density in the E face of the lac-capped spleen cells compared with control cells, whereas a few E-face micrographs showed intense IMP aggregation. In contrast, there was no significant alteration of P-face IMP densities or distribution. Apparently, the majority of E-face IMP and virtually all P-face IMP densities or distribution. Apparently, the majority of E-face IMP and virtually all P-face IMP do not present accessible antigenic sites on the lymphocyte surface and do not associate in a stable manner with surface protein antigens. This finding suggests that IMP, as observed in freeze-fracture analysis, may not comprise a representative reflection of lymphocyte transmembrane protein molecules and complexes because other evidence establishes: (a) that at least some common lymphocyte surface antigens are indeed exposed portions of transmembrane proteins and (b) that the aggregation of molecules of any surface antigen results in altered organization of contractile proteins at the cytoplasmic face of the membrane.     

Tetrameric assembly of CHIP28 water channels in liposomes and cell membranes: a freeze-fracture study

Channel forming integral protein of 28 kD (CHIP28) functions as a water channel in erythrocytes, kidney proximal tubule and thin descending limb of Henle. CHIP28 morphology was examined by freeze-fracture EM in proteoliposomes reconstituted with purified CHIP28, CHO cells stably transfected with CHIP28k cDNA, and rat kidney tubules. Liposomes reconstituted with HPLC-purified CHIP28 from human erythrocytes had a high osmotic water permeability (Pf0.04 cm/s) that was inhibited by HgCl2. Freeze-fracture replicas showed a fairly uniform set of intramembrane particles (IMPs); no IMPs were observed in liposomes without incorporated protein. By rotary shadowing, the IMPs had a diameter of 8.5 +/- 1.3 nm (mean +/- SD); many IMPs consisted of a distinct arrangement of four smaller subunits surrounding a central depression. IMPs of similar size and appearance were seen on the P-face of plasma membranes from CHIP28k-transfected (but not mock-transfected) CHO cells, rat thin descending limb (TDL) of Henle, and S3 segment of proximal straight tubules. A distinctive network of complementary IMP imprints was observed on the E-face of CHIP28-containing plasma membranes. The densities of IMPs in the size range of CHIP28 IMPs, determined by non-linear regression, were (in IMPs/microns 2): 2,494 in CHO cells, 5,785 in TDL, and 1,928 in proximal straight tubules; predicted Pf, based on the CHIP28 single channel water permeability of 3.6 x 10(-14) cm3/S (10 degrees C), was in good agreement with measured Pf of 0.027 cm/S, 0.075 cm/S, and 0.031 cm/S, respectively, in these cell types. Assuming that each CHIP28 monomer is a right cylindrical pore of length 5 nm and density 1.3 g/cm3, the monomer diameter would be 3.2 nm; a symmetrical arrangement of four cylinders would have a greatest diameter of 7.2 nm, which after correction for the thickness of platinum deposit, is similar to the measured IMP diameter of approximately 8.5 nm. These results provide a morphological signature for CHIP28 water channels and evidence for a tetrameric assembly of CHIP28 monomers in reconstituted proteoliposomes and cell membranes.