The structure of the cytoplasm of lens fibers as determined by conical tomography.

Studies using conventional electron microscopy describe the cytoplasm of lens fiber cells as having essentially an amorphous structure. We hypothesized that significant structural detail might have been lost as a result of projecting the entire thickness of the section (50-100 nm) onto a single plane (the "projection artifact"). To test this hypothesis, we studied the 3D-structure of rat lens cortical fibers before and after extracting the "soluble" crystallins with low ionic strength buffers to make "ghosts." Tomographic series in conical geometry were collected at 55 degrees tilts and by 5 degrees rotations until completing a 360 degrees turn by low dose methods. They were aligned using fiduciary points, reconstructed with the weighted back projection algorithm and refined by projection matching. Analysis of the 3D-maps included semiautomatic density segmentation using a computer program based on the watershed algorithm. We found that the cytoplasm of cortical fibers, though appearing amorphous in regions of the highest density, was in fact comprised of an ordered structure resembling a "clustered matrix." The matrix was comprised of thin ( approximately 6 nm diameter) filaments bent sharply at 110-120 degrees angles and studded with cube-shaped particles (the "beaded" filaments). In cortical fibers, the particles measured a=14+/-2, b=13+/-2 and c=10+/-2.4 nm (n=30, mean+/-SD) and were spaced at distances measuring 27.5+/-2.4 nm apart (n=8, mean+/-SD), center-to-center. The matrix was formed as "beaded" filaments, bound to clusters of "soluble" proteins, crossed each other at nearly perpendicular angles. The matrix also made contact with the plasma membrane at a large number of distinct regions. We thus concluded that the cytoplasm of cortical lens fibers is comprised of a cytoskeletal matrix of "beaded" filaments that organize the "soluble" crystallins in separate regions. The association of this matrix with the plasma membrane allows the lens to maintain its structural integrity, while its association with crystallins yields its long-term transparency. Loss of either function likely would play a significant role in cataract formation.

The structural organization and protein composition of lens fiber junctions.

The structural organization and protein composition of lens fiber junctions isolated from adult bovine and calf lenses were studied using combined electron microscopy, immunolocalization with monoclonal and polyclonal anti-MIP and anti-MP70 (two putative gap junction-forming proteins), and freeze-fracture and label-fracture methods. The major intrinsic protein of lens plasma membranes (MIP) was localized in single membranes and in an extensive network of junctions having flat and undulating surface topologies. In wavy junctions, polyclonal and monoclonal anti-MIPs labeled only the cytoplasmic surface of the convex membrane of the junction. Label-fracture experiments demonstrated that the convex membrane contained MIP arranged in tetragonal arrays 6-7 nm in unit cell dimension. The apposing concave membrane of the junction displayed fracture faces without intramembrane particles or pits. Therefore, wavy junctions are asymmetric structures composed of MIP crystals abutted against particle-free membranes. In thin junctions, anti-MIP labeled the cytoplasmic surfaces of both apposing membranes with varying degrees of asymmetry. In thin junctions, MIP was found organized in both small clusters and single membranes. These small clusters also abut against particle-free apposing membranes, probably in a staggered or checkerboard pattern. Thus, the structure of thin and wavy junctions differed only in the extent of crystallization of MIP, a property that can explain why this protein can produce two different antibody-labeling patterns. A conclusion of this study is that wavy and thin junctions do not contain coaxially aligned channels, and, in these junctions, MIP is unlikely to form gap junction-like channels. We suggest MIP may behave as an intercellular adhesion protein which can also act as a volume-regulating channel to collapse the lens extracellular space. Junctions constructed of MP70 have a wider overall thickness (18-20 nm) and are abundant in the cortical regions of the lens. A monoclonal antibody raised against this protein labeled these thicker junctions on the cytoplasmic surfaces of both apposing membranes. Thick junctions also contained isolated clusters of MIP inside the plaques of MP70. The role of thick junctions in lens physiology remains to be determined.

Cysteine string proteins: a potential link between synaptic vesicles and presynaptic Ca2+ channels.

Presynaptic calcium channels are key regulators of neurotransmitter release. Oocyte expression studies suggest that cysteine string proteins are essential subunits or modulators of these channels. Subcellular fractionation revealed that cysteine string proteins copurify with synaptic vesicles. An average vesicle had eight protein monomers with both the amino and carboxyl termini detected on the cytoplasmic face. Thus, docked synaptic vesicles may regulate presynaptic calcium channels and neurotransmitter release.

Functional and morphological correlates of connexin50 expressed in Xenopus laevis oocytes.

Electrophysiological and morphological methods were used to study connexin50 (Cx50) expressed in Xenopus laevis oocytes. Oocytes expressing Cx50 exhibited a new population of intramembrane particles (9.0 nm in diameter) in the plasma membrane. The particles represented hemichannels (connexin hexamers) because (a) their cross-sectional area could accommodate 24 +/- 3 helices, (b) when their density reached 300-400/microm2, they formed complete channels (dodecamers) in single oocytes, and assembled into plaques, and (c) their appearance in the plasma membrane was associated with a whole-cell current, which was activated at low external Ca2+ concentration ([Ca2+]o), and was blocked by octanol and by intracellular acidification. The Cx50 hemichannel density was directly proportional to the magnitude of the Cx50 Ca2+-sensitive current. Measurements of hemichannel density and the Ca2+-sensitive current in the same oocytes suggested that at physiological [Ca2+]o (1-2 mM), hemichannels rarely open. In the cytoplasm, hemichannels were present in approximately 0.1-microm diameter "coated" and in larger 0.2-0.5-microm diameter vesicles. The smaller coated vesicles contained endogenous plasma membrane proteins of the oocyte intermingled with 5-40 Cx50 hemichannels, and were observed to fuse with the plasma membrane. The larger vesicles, which contained Cx50 hemichannels, gap junction channels, and endogenous membrane proteins, originated from invaginations of the plasma membrane, as their lumen was labeled with the extracellular marker peroxidase. The insertion rate of hemichannels into the plasma membrane (80, 000/s), suggested that an average of 4,000 small coated vesicles were inserted every second. However, insertion of hemichannels occurred at a constant plasma membrane area, indicating that insertion by vesicle exocytosis (60-500 microm2 membranes/s) was balanced by plasma membrane endocytosis. These exocytotic and endocytotic rates suggest that the entire plasma membrane of the oocyte is replaced in approximately 24 h.

Characterisation of the major intrinsic protein (MIP) from bovine lens fibre membranes by electron microscopy and hydrodynamics.

The major intrinsic protein (MIP) from bovine lens fibre membranes has been purified from unstripped membranes using a single ion-exchange chromatography step (MonoS) in the non-ionic detergent octyl-beta-D-glucopyranoside (OG). SDS-PAGE has confirmed the purity of the preparation and thin-layer chromatographic analysis has shown that the protein is virtually lipid-free. To establish a stable and monodisperse protein sample, we exchanged OG with decyl-beta-D-maltopyranoside (DeM), another non-ionic detergent, by gel-filtration column chromatography. We conclude that the resulting protein/detergent complex is composed of four copies of MIP (a tetramer) and a detergent micelle. This conclusion is based on: (1) measurement of the weight-average molecular mass (Mw,app) of the protein moiety in the protein/detergent complex by sedimentation equilibrium; (2) measurement of the apparent molecular mass of the complexes formed by MIP in OG, in DeM, in dodecyl-beta-D-maltopyranoside (DoM) and in sodium dodecylsulphate (SDS) by gel filtration; (3) measurement of the apparent molecular mass of pure detergent micelles; (4) measurement of the predicted change in the molecular mass of the MIP/DeM complex after partial enzymatic proteolysis; and (5) measurement of the size and shape of the MIP/detergent complex by electron microscopy and single-particle analysis. Therefore, the tetragonal arrangement of MIP observed in both plasma membranes and junctional membranes in lens fibre cells is maintained in solution with non-ionic detergents.

Morphology of fungiform papillae in canine lingual epithelium: location of intercellular junctions in the epithelium.

The localization and structure of intercellular junctions and barriers in the extragemmal epithelium of canine fungiform papillae were determined by using both morphological and electrophysiological methods. Gap junctions were located in all epithelial strata with the exception of the stratum corneum, suggesting that the epithelium functions as a syncytium. The extracellular space of the stratum corneum was composed of a discontinuous, three-dimensional network of tight junctions, modified desmosomes, and lamellar bodies. A zonula occludens, which stops the penetration of lanthanum, is present in the uppermost layer of the stratum granulosum. In freeze-fracture replicas, tight junctions appear as extended networks of ridges of variable thickness on the PF fracture face and complementary grooves on the EF fracture face. The relatively high resistance pathway resulting from the layers of corneocytes and networks of tight junctions and lamellar bodies in the stratum corneum is bypassed by the low-resistance pathway provided by the taste pore.

Transcellular and paracellular pathways in lingual epithelia and their influence in taste transduction.

The lingual epithelium is innervated by special sensory (taste) and general sensory (trigeminal) nerves that transmit information about chemical stimuli introduced into the mouth to the higher brain centers. Understanding the cellular mechanisms involved in eliciting responses from these nerves requires a detailed understanding of the contributions of both the paracellular and transcellular pathways. In this paper we focus on the contribution of these 2 pathways to the responses of salts containing sodium and various organic anions in the presence and absence of amiloride. Electrophysiological recordings from trigeminal nerves, chorda tympani nerves, and isolated lingual epithelia were combined with morphological studies investigating the location (and permeability) of tight junctions, the localization of amiloride-inhibitable channels, and Na-K-ATPase in taste and epithelial cells. Based on these measurements, we conclude that diffusion across tight junctions can modulate chorda tympani and trigeminal responses to sodium-containing salts and rationalize the enhancement of taste responses to saccharides by NaCl.

How many channels should there be in a gap junction?

The junctional resistance of the gap junctions between the lateral giant axons of the crayfish has been measured in coupled and uncoupled conditions and the number of gap junction particles has been counted under the same conditions. From these two sets of data it can be concluded that the plaque-forming particles observed in freeze fracture replicas of the synaptic area are all gap junctional particles and that their channels are open. Based on this and published results from other isolated pairs of coupled cells, an inverse relationship has been found between the input resistance of the cells and the calculated number of open gap junctional channels between them. The relationship applies to both excitable and nonexcitable cells.

Conical electron tomography of a chemical synapse: vesicles docked to the active zone are hemi-fused.

We have used thin sectioning and conical electron tomography to determine the three-dimensional structure of synaptic vesicles that were associated (docked) at release sites of the presynaptic membrane, called active-zones. Vesicles docked at the active zone occupied a strategic location: they formed regions of contact with the plasma membrane on one side and with that of one or more vesicles located deeper within the presynaptic terminal on the other side. The region of contact with the active zone measured approximately 15 nm in diameter ( approximately 2% of the vesicle's surface) and contained a smaller approximately 6 nm region where the proximal leaflets merged (hemi-fused). Hemi-fusion was only observed on the side of vesicles in contact with the active zone; at the side of contact between neighboring vesicles, the membranes were not hemi-fused. Approximately three-fourths of the docked vesicles contained hemi-fused regions. Vesicles fully fused to the active zone (exhibiting pores that appeared as interruptions of a single membrane) were less frequently observed ( approximately 1 of 10 hemi-fused vesicles). In conclusion, our observations in cortical synapses strengthen the hypothesis that hemi-fusion is a stable intermediary that precedes full fusion and release.

Conical tomography II: A method for the study of cellular organelles in thin sections.

We have used conical electron tomography in order to reconstruct neuronal organelles in thin sections of plastic embedded rat somato-sensory cortical tissue. The conical tilt series were collected at a 55 degrees tilt and at 5 degrees rotations, aligned using gold particles as fiduciary markers, and reconstructed using the weighted back projection algorithm. After a refinement process based on projection matching, the 3D maps showed the "unit membrane pattern" along the entire reconstructed volume. This pattern is indicative of the bilayer arrangement of phospholipids in biological membranes. Based on Fourier correlation methods as well as the visualization of the "unit membrane" pattern, we estimated resolutions of approximately 4 nm. To illustrate the prospective advantages of conical tomography, we segmented "coated" vesicles in the reconstructed volumes. These vesicles were comprised of a central core enclosing a small lumen, and a protein "coating" extending into the cytoplasm. The "coated" vesicle was attached to the plasma membrane through a complex structure shaped as an arch where the ends are attached to the membrane and the crook is connected to the vesicle. We concluded that conical electron tomography of thin-sectioned specimens provides a powerful experimental approach for studying thin-sectioned neuronal organelles at resolution levels of approximately 4 nm.

Conical tomography of freeze-fracture replicas: a method for the study of integral membrane proteins inserted in phospholipid bilayers.

We have used conical tomography to study the structure of integral proteins in their phospholipid bilayer environments. Complete conical series were collected from replicas of the water channel aquaporin-0 (AQP0), a 6.6 nm side tetramer with a molecular weight of approximately 120 kDa that was purified and reconstituted in liposomes. The replicas were tilted at 38 degrees , 50 degrees or 55 degrees and rotated by 2.5 degrees , 4 degrees , or 5 degrees increments until completing 360 degrees turns. The elliptical paths of between 6 and 12 freeze-fracture particles aligned the images to a common coordinate system. Using the weighted back projection algorithm, small volumes of the replicas were independently reconstructed to reconstitute the field. Using the Fourier Shell Correlation computed from reconstructions of even and odd projections of the series, we estimated a resolution of 2-3 nm, a value that was close to the thickness of the replica (approximately 1.5 nm). The 3D reconstructions exhibited isotropic resolution along the x-y plane, which simplified the analysis of particles oriented randomly in the membrane plane. In contrast to reconstructions from single particles imaged using random conical tilt [J. Mol. Biol. 325 (2003) 210], the reconstructions using conical tomography allowed the size and shape of individual particles representing the AQP0 channel to be identified without averaging or imposing symmetry. In conclusion, the reconstruction of freeze-fracture replicas with electron tomography has provided a novel experimental approach for the study of integral proteins inserted in phospholipid bilayers.

Purification of bovine lens cell-to-cell channels composed of connexin44 and connexin50.

Cell-to-cell channels composed of connexin44 and connexin50 were purified from plasma membranes of calf and fetal bovine lenses. The channels were treated with the nonionic detergents octyl-beta-D-glucopyranoside and decyl-beta-D-maltopyranoside, and the channel/detergent complexes purified by ion and gel filtration column chromatography. In negative staining, the channels appeared as annuli 11 +/- 0.6 nm (s.d., n = 105) in diameter and as 16 +/- 0.8 nm (s.d., n = 96) long particles which corresponded to top and side views of 'complete' cell-to-cell channels. The purified cell-to-cell channels were composed principally of a protein, called MP70, that appeared as a diffuse 55-75 kDa band in SDS-PAGE. Dephosphorylation with alkaline phosphatase transformed the diffuse 55-75 kDa band into two distinct bands of almost equal intensity. Immunoblotting showed the bands to be connexin44 and connexin50, respectively. The antibodies also recognized weaker bands composed of the unphosphorylated form of both connexins. The connexins appear to be processed independently 'in vivo'. The unphosphorylated form of connexin50 was present in channels and membranes from fetal, calf and adult bovine lenses, while unphosphorylated connexin44 only in channels purified from fetal lenses. Therefore, lens cell-to-cell channels are composed principally of equal amounts of phosphorylated connexins 44 and 50 that appear to be assembled in the same channel ('hybrid').

Polyhedral protein cages encase synaptic vesicles and participate in their attachment to the active zone.

In an effort to elucidate the interactions between synaptic vesicles and the membrane of the active zone, we have investigated the structure of interneuronal asymmetric synapses in the neocortex of adult rats using thin-sectioning, freeze-fracture, and negative staining electron microscopy. We identified three subtypes of spherical synaptic vesicles. Type I were agranular vesicles of 47.5 +/- 3.8 nm (mean SD, n = 24) in diameter usually seen aggregated in clusters in the presynaptic bouton. Type II synaptic vesicles were composed of a approximately 45-nm-diameter lipid bilayer sphere encased in a cage 77 +/- 4.6 nm (mean SD, n = 42) in diameter. The cage was composed of open-faced pentamers 20-22 nm/side arranged as a regular polyhedron. Type II caged vesicles were found in clusters at the boutons, adhered to the active zone, and were also present in axons. Type III synaptic vesicles appeared as electron-dense spheres 60-75 nm in diameter abutted to the membrane of the active zone. Clathrin-coated vesicles and pits of 116.6 +/- 9 nm (mean SD, n = 14) in diameter were also present in both the pre- and postsynaptic sides. Freeze-fracture showed that some intrinsic membrane proteins in the active zone were arranged as pentamers exhibiting the same dimension of those forming cages (approximately 22 nm/side). From these data, we concluded that: (a) the presynaptic bouton contains a heterogeneous population of "caged" and "plain" synaptic vesicles and (b) type II synaptic vesicles bind to receptors in the active zone. Therefore, current models of transmitter release should take into account the substantial heterogeneity of the vesicle population and the binding of vesicular cages to the membrane of the active zone.

Epithelial organization of the mammalian lens.

To understand the structural organization responsible for lens function, we have studied the three-dimensional arrangement of cells in the lens, and the location and molecular composition of specialized junctions controlling the paracellular and transcellular pathways. The lens is formed by a single layer of polarized cells that elongate along their apical-basal axis from the anterior to the posterior pole to form the cortex, and fold inward at the posterior pole to form the nucleus. The basal surfaces of all cells of the cortex (approximately two thirds of all lens cells) are bathed by the aqueous and vitreous humors. Therefore, their metabolism is not limited by diffusion of nutrients into the avascular lens. The apical surfaces of all cortical fibers are directed toward the interior of the lens, where they form two distinct structures here referred to as the 'apical interface' and the 'modiolus'. The apical interface is located at a point close to the anterior pole, and is formed by the association of the apical surface of anterior cortical cells and the apical surface of cortical fibers extending from the posterior pole. The modiolus is located close to the equator at the lateral edge of the apical interface, and is formed by the tapered apical ends of equatorial cortical fibers. The plasma membrane of cortical cells at the anterior pole are connected through 'leaky' tight junctions and small gap junctions. Extensive gap junction plaques composed of connexin43 connect equatorial fibers at the modiolus and posterior cortical fibers at the apical interface. Single cell-to-cell channels composed of connexin46 and connexin50 connect the lateral surfaces of equatorial and posterior cortical fibers. The lateral surfaces of these fibers also contain extensive junctions composed of aquaporin-0. The nucleus is connected to the humors through the paracellular pathway represented by the anterior (apical) and posterior (basal) suture lines. Therefore, the metabolic needs of nuclear fibers cannot be fulfilled by simple diffusion and requires the cell-to-cell pathway formed by specialized junctions. The lateral surfaces of nuclear fibers contain extensive wavy junctions composed of aquaporin-0, probably for the control of the permeability of the paracellular pathway. We propose a simple epithelium model for the lens in which nutrients move into the nucleus through the paracellular pathway represented principally by the suture lines, and the transcellular pathway represented by an extensive network of gap junction plaques composed of connexin43 at the apical surface, and single or small plaques of cell-to-cell channels composed of connexin46 and connexin50 in the lateral surfaces.

Single-membrane and cell-to-cell permeability properties of dissociated embryonic chick lens cells.

Ion channels are believed to play an important role in the maintenance of lens transparency. In order to ascribe junctional and nonjunctional permeability properties to specific lens cell types, embryonic chick lenses were enzymatically dissociated into cell clusters, cell pairs and single cells, and both cell-to-cell and single-membrane permeability properties were characterized with the patch-clamp technique. Double patch-clamp experiments and single patch-clamp experiments with Lucifer yellow in the pipette demonstrated that the cells in the dissociated preparation were well coupled, the average conductance between pairs being 42 +/- 27 nS. Double patch-clamp experiments also revealed single cell-to-cell channel events with a predominant unitary conductance of 286 +/- 38 pS. Whole-cell measurements of surface membrane conductance indicate heterogeneity within the population of dissociated embryonic chick lens cells: 63% of the cells have a voltage-independent leak current, 14% of the cells have a potassium-selective inward-rectifier current, and 23% of the cells have a current which turns off with positive voltage on a time scale on the order of seconds. The time constant for this turnoff is voltage dependent.

The Kohonen self-organizing map: a tool for the clustering and alignment of single particles imaged using random conical tilt.

An important step in determining the three-dimensional structure of single macromolecules is to bring common features in the images into register through alignment and classification. Here, we took advantage of the striking computational properties of the Kohonen self-organizing map (SOM) to align and classify images of channels obtained by random conical geometry into more homogeneous subsets. First, we used simulations with artificially created images to deduce simple geometrical rules governing the mapping of bounded (differing in size and shape) and unbounded (differing in in-plane orientation) variations in the output plane. Second, we measured the effect of noise on the accuracy of the algorithm to separate homogeneous subsets. Finally, we applied the rules ascertained in the previous steps to separate freeze-fracture images of the cytoplasmic and external domains of the small (approximately 118 kDa) aquaporin-0 water channel. Comparison with the results obtained from a similar input set using alignment-through-classification showed that both methods converged to stable classes exhibiting the same overall shapes (tetragonal and octagonal) for the cytoplasmic and external views of the channel. Processing with the SOM, however, was simplified by the utilization of the geometric rules governing the mapping of bounded and unbounded variations as well as the lack of subjectivity in selecting the reference images during alignment.

Purified lens junctional protein forms channels in planar lipid films.

Junctions isolated from bovine lenses were solubilized with the detergent octyl glucoside, and their protein(s) was reconstituted in unilamellar vesicles. The protein(s) appears as annular-shaped intramembrane particles approximately equal to 10 nm in diameter on the vesicles' fracture faces. The addition of the vesicle-containing junctional protein(s) to both sides of preformed lipid films induced voltage-dependent channels. The channels have a conductance of 200 pS in 0.1 M salt solutions and are thus large enough to account for the electrical coupling observed between intact lens fibers; they turn off when the magnitude of the voltage is increased and in the presence of octanol. Although the identity of the reconstituted channels as the communicating pathway between lens fibers remains to be proven, it is most likely that the reconstituted channels are formed by MIP-26, the major protein component of the isolated lens junctions.

Pentameric assembly of a neuronal glutamate transporter.

Freeze-fracture electron microscopy was used to study the structure of a human neuronal glutamate transporter (EAAT3). EAAT3 was expressed in Xenopus laevis oocytes, and its function was correlated with the total number of transporters in the plasma membrane of the same cells. Function was assayed as the maximum charge moved in response to a series of transmembrane voltage pulses. The number of transporters in the plasma membrane was determined from the density of a distinct 10-nm freeze-fracture particle, which appeared in the protoplasmic face only after EAAT3 expression. The linear correlation between EAAT3 maximum carrier-mediated charge and the total number of the 10-nm particles suggested that this particle represented functional EAAT3 in the plasma membrane. The cross-sectional area of EAAT3 in the plasma membrane (48 +/- 5 nm(2)) predicted 35 +/- 3 transmembrane alpha-helices in the transporter complex. This information along with secondary structure models (6-10 transmembrane alpha-helices) suggested an oligomeric state for EAAT3. EAAT3 particles were pentagonal in shape in which five domains could be identified. They exhibited fivefold symmetry because they appeared as equilateral pentagons and the angle at the vertices was 110 degrees. Each domain appeared to contribute to an extracellular mass that projects approximately 3 nm into the extracellular space. Projections from all five domains taper toward an axis passing through the center of the pentagon, giving the transporter complex the appearance of a penton-based pyramid. The pentameric structure of EAAT3 offers new insights into its function as both a glutamate transporter and a glutamate-gated chloride channel.

Calmodulin acts as an intermediary for the effects of calcium on gap junctions from crayfish lateral axons.

Lateral axons from the abdominal nerve cord of crayfish were internally perfused with the calcium receptor calmodulin (CaM) in solutions with low (pCa greater than 7.0) or high (pCa 5.5) calcium concentrations and studied electrophysiologically and morphologically. Results from these experiments show that when the internal solution contains calcium-activated calmodulin (Ca2+-CaM) the junctional resistance between the axons increases from control values of about 60 to 500-600 k omega in 60 min. In contrast, axons perfused with calmodulin in low calcium solutions maintain their junctional resistance at control levels during the 60-min perfusion. Similar results are obtained when only one or both coupled axons are perfused. The morphological study shows that in the perfused axons the axoplasmic organelles are replaced or grossly perturbed by the perfusion solution up to the region of the synapses. Additionally, in axons perfused with Ca2+-CaM there are regions where the synaptic gap between the membranes decreases from a control 4-6 to 2-3 nm. Both electrophysiological and morphological results can be interpreted as indicating that calcium-activated calmodulin acts directly on the junctional channels to induce their closure.