Properties of Membranes from the Halophyte Suaeda maritima: I. CYTOCHEMICAL STAINING OF MEMBRANES IN RELATION TO THE VALIDITY OF MEMBRANE MARKERS

The fine structure and certain cytochemical properties of theshoots from the halophyte Suaeda maritima have been examined.No major fine structural differences were observed between plantsgrown in the presence or absence of salt. Considerable evidenceof vesiculation was observed, particularly in cells of the stelartissue from the stems. The specificity of the phosphotungsticacid-chromic acid stain for plasma membranes was examined andsome doubt raised as to its widespread applicability as a plasmamembrane marker in plant cells. The suitability of other membranemarkers such as ruthenium red and ATPase activity were investigated.     

HELA CELL PLASMA MEMBRANES : I. 5'-Nucleotidase and Ouabain-Sensitive ATPase as Markers for Plasma Membranes

A method for the preparation of HeLa cell plasma membrane ghosts is described. The purity of the plasma membrane fraction was examined by phase contrast and electron microscopy, by chemical analysis, and by assay of marker enzymes. Data on the composition of the plasma membrane fraction are given. It was observed that the distribution pattern of 5'-nucleotidase activity among the subcellular fractions differed from that of ouabain-sensitive ATPase. In addition, the specific activity of 5'-nucleotidase did not follow the distribution of the membrane ghosts. Thus, this enzyme would seem unsuitable as a plasma membrane marker. A complete balance sheet for marker enzyme activities during the fractionation is necessary for the calculation of increase in specific activity because the activities of both 5'-nucleotidase and ouabain-sensitive ATPase might change during the fractionation procedures.     

Transcytosis of HIV-1 through Vaginal Epithelial Cells Is Dependent on Trafficking to the Endocytic Recycling Pathway

Background

While it is accepted that viruses can enter epithelial cells by endocytosis, the lack of an established biological mechanism for the trafficking of infectious virions through vaginal epithelial cells and their release from the plasma membrane has contributed to ongoing controversy about whether endocytosis is a mere artifact of some cell culture systems and whether squamous vaginal epithelial cells are even relevant as it pertains to HIV-1 transmission.

Methodology/Principal Findings

In this study, we investigated the intracellular trafficking pathway that HIV-1 exploits to transcytose vaginal epithelial cells. The reduction of endosome tubulation by recycling endosome inhibitors blocked transcytosis of HIV-1 in a cell culture and transwell system. In addition, we demonstrate that although heat-inactivated virus was endocytosed as efficiently as native virus, heat-inactivated virus was trafficked exclusively to the lysosomal pathway for degradation following endocytosis. Lysosomal protease-specific inhibitors blocked the degradation of inactivated virions. Immunofluorescence analysis not only demonstrated that HIV-1 was inside the cells but the different colocalization pattern of native vs. heat inactivated virus with transferrin provided conclusive evidence that HIV-1 uses the recycling pathway to get across vaginal epithelial cells.

Conclusions/Significance

Altogether, our findings demonstrate the precise intracellular trafficking pathway utilized by HIV-1 in epithelial cells, confirms that HIV-1 transcytosis through vaginal epithelial cells is a biological phenomenon and brings to light the differential intracellular trafficking of native vs heat-inactivated HIV-1 which with further exploration could prove to provide valuable insights that could be used in the prevention of transcytosis/transmission of HIV-1 across the mucosal epithelia.     

GROWTH AND DIFFERENTIATION OF CYTOPLASMIC MEMBRANES IN THE COURSE OF LIPOPROTEIN GRANULE SYNTHESIS IN THE HEPATIC CELL : I. Elaboration of Elements of the Golgi Complex

The synthesis of "very low" density lipoprotein in liver cells is characterized by the fact that the synthesized products, mostly triglycerides, are processed in the form of discrete, size-limited granules or globules, about 400 A in diameter. The present investigation has been made possible in part by the use of a fixative (OsO₄ in bidistilled H₂O at pH 6.0, in the absence of electrolytes) particularly effective in preserving cytoplasmic membranes and lipids, and giving them high stainability and differential contrast. Under these technical conditions, the lipoprotein granules retain their morphology and high density to electrons practically unaltered, and may serve as tracers in determining their route of transport from the sites of synthesis, starting at the rough-smooth ER junctions, to the lumen of Golgi concentrating vesicles. From the observations, it may be deduced that, along with lipoprotein granule synthesis and transport, there are also production and transfer of new membranes in the form of tubular extensions of smooth ER network which, by progressive fusion and coalescence, participate in the elaboration of fenestrated plates and solid Golgi sacs. In contradistinction to the entire process of liver lipoprotein granule synthesis, transport, and segregation, as reported in the present paper, appears to constitute a developmental sequence which includes the following communicating compartments, in consecutive order: cisternae of rough ER where proteins and possibly phospholipids are synthesized, smooth ER network where triglycerides are synthesized and transported in the form of dense granules, fusion of smooth ER tubular extensions into Golgi fenestrated plates, and further coalescence into solid Golgi sacs, ending in the segregation of the granules in appended concentrating vesicles, or detached "secretory vesicles." It seems that it is this progressive evolution in growth and configuration of membranes which is reflected in the so called polarity, from forming to mature faces, of the Golgi apparatus.     

Preparative Scale Isolation of Basal-Lateral Plasma Membranes from Rat Intestinal Epithelial Cells

A simple, efficient procedure is described for the preparative scale isolation of basal-lateral membranes from the rat intestinal epithelium. The intestinal mucosa was mildly homogenized and soluble protein and RNA were separated from the homogenate by differential centrifugation. The basal-lateral membranes were then separated from nuclei, mitochondria, and brush border membranes by differential centrifugation in a medium close to the equilibrium density of the basal-lateral membranes. Final purification of the basal-lateral membranes was achieved on a linear density gradient in a high-capacity zonal rotor. The final product (usually at least 40 mg protein) represented a 34% yield of basal-lateral membranes purified 18-fold with respect to protein, 26-fold with respect to brush border membranes, and 53-fold with respect to mitochondria.     

The Fine Structure of the Epithelial Cells of the Mouse Prostate : I. Coagulating Gland Epithelium

The fine structure of the epithelial cells of the anterior lobe, or coagulating gland, of the mouse prostate has been investigated by electron microscopy. This organ is composed of small tubules, lined by tall, simple cuboidal epithelium surrounded by connective tissue and smooth muscle. The epithelial cells are limited by a distinct plasma membrane, which covers minute projections of the cytoplasm into the lumen. The cell membranes of adjacent cells are separated by a narrow layer of structureless material of low density. The cavities of the endoplasmic reticulum are greatly dilated, and the cytoplasmic matrix is reduced to narrow strands, in which the various organelles are visible. The content of the cavities of the endoplasmic reticulum appears as structureless material of lesser density than the cytoplasmic matrix. Material which may be interpreted as secretion products can be seen in the lumina of the tubules. The possible nature of the material inside the cisternal spaces and the secretory mechanisms in these cells is discussed.     

Heterogeneity of Volume-sensitive Chloride Channels in Basolateral Membranes of A6 Epithelial Cells in Culture

A new technique allowing single-channel patch-clamp recordings from basolateral membranes of A6 renal epithelial cells in culture was developed. Using this technique we studied the chloride channels activated in these basolateral membranes during hypo-osmotic stress. Four different types of channel were identified and classified according to their current/voltage (I/V) relationships as observed in the on-cell configuration of the patch-clamp technique. Three of these channels had linear I/V relationships with unitary conductances of 12, 30 and 42 pS. The fourth type had an outwardly rectifying I/V curve with inward and outward conductances of 16 and 57 pS respectively. The kinetic properties of each class of channel were studied and kinetic models developed for two of them: the 42 pS channel and the outward rectifier. These models permitted the study of the evolution of the kinetic parameters during hypo-osmotic shock and revealed two different kinetic schemes of channel activation. The results of experiments made on the basolateral membranes were also compared with those of a set of analogous patch-clamp experiments carried out on isolated A6 cells. In these latter, the frequency of successful observations of active channels in a patch was 13%, whereas it was 31% for basolateral membranes. Also, of the four types of channel observed in basolateral membranes, two were never found in isolated cells, only the 12 pS channel and the outward rectifier were present in these isolated cells. Received: 17 April 1996/Revised: 26 June 1996     

HDAC6 Regulates Epidermal Growth Factor Receptor (EGFR) Endocytic Trafficking and Degradation in Renal Epithelial Cells

We present for the first time that histone deacetylase 6 (HDAC6) regulates EGFR degradation and trafficking along microtubules in Pkd1 mutant renal epithelial cells. HDAC6, the microtubule-associated α-tubulin deacetylase, demonstrates increased expression and activity in Pkd1 mutant mouse embryonic kidney cells. Targeting HDAC6 with a general HDAC inhibitor, trichostatin (TSA), or a specific HDAC6 inhibitor, tubacin, increased the acetylation of α-tubulin and downregulated the expression of EGFR in Pkd1 mutant renal epithelial cells. HDAC6 was co-localized with EGF induced endocytic EGFR and endosomes, respectively. Inhibition of the activity of HDAC6 accelerated the trafficking of EGFR from early endosomes to late endosomes along the microtubules. Without EGF stimulation EGFR was randomly distributed while after stimulation with EGF for 30 min, EGFR was accumulated around α-tubulin labeled microtubule bundles. These data suggested that the Pkd1 mutation induced upregulation of HDAC6 might act to slow the trafficking of EGFR from early endosomes to late endosomes along the microtubules for degradation through deacetylating α-tubulin. In addition, inhibition of HDAC activity decreased the phosphorylation of ERK1/2, the downstream target of EGFR axis, and normalized EGFR localization from apical to basolateral in Pkd1 knockout mouse kidneys. Thus, targeting HDAC6 to downregulate EGFR activity may provide a potential therapeutic approach to treat polycystic kidney disease.     

LOW RESISTANCE JUNCTIONS IN CRAYFISH : I. Two Arrays of Globules in Junctional Membranes

The ultrastructure of low resistance junctions between segments of lateral giant fibers in crayfish is studied in sections from specimens fixed either by conventional methods or by glutaraldehyde-H₂O₂ or by glutaraldehyde-lanthanum. Cross sections through junctions fixed by conventional glutaraldehyde display the usual trilaminar profile of two parallel membranes separated by a narrow gap. Most of the junctional regions appear covered by 500–800 Å vesicles which lie on both sides of the junction in rows adjacent to the membranes. Gross sections through junctions fixed by glutaraldehyde-H₂O₂ display, in regions containing vesicles, membranes with a beaded profile. The beads correspond to globules ~125 Å in width and ~170 Å in height arranged in a hexagonal pattern with a unit cell of ~200 Å. The globules of one membrane match precisely with those of the adjacent membrane, and opposite globules seem to come in contact with each other at the center of the junction. The membrane of the vesicles also contains globules. Occasionally the globules of the vesicles seem to join with those of the junctional membranes, apparently forming intracellular junctions. Injunctions negatively stained by lanthanum the globules are seen organized into two arrangements. Areas containing globules in a hexagonal array with a unit cell of ~200 Å (swollen pattern) are seen adjacent to areas in which the globules are more closely and disorderly packed (close packing), the minimum center-to-center distance between adjacent globules being ~125 Å. At higher magnification each globule appears composed of six subunits arranged in a circle around a central region occupied by lanthanum (possibly a pit).     

Acute and Subacute Effects of Thiamine Deficiency On the Morphometry and Cell Kinetics of Jejunal and Ileal Epithelial Cells

Abstract. The effects of acute and subacute thiamine deficiency on jejunal and ileal epithelial cells were studied in rats, using crypt and villus cell population, crypt cell production per crypt (CCPC), crypt growth fraction (I_p) and crypt cell cycle time (T_c) as parameters. In acute thiamine deficiency there was marked jejunal hypoplasia of the crypt and villus, but in the ileum there was hypoplasia only of the crypt. the jejunal epithelium of the subacute thiamine deficiency (STD) group showed no morphometric changes. In contrast, in the ileal epithelium of STD rats there was decreased crypt depth and villus cell population. Thiamine deficiency had no significant effect on CCPC, I_p and T_c.     

OPTICAL DIFFRACTION STUDIES OF CRYSTALLINE STRUCTURES IN ELECTRON MICROGRAPHS : I. Theoretical Considerations

Determination of the unit cell of crystalline particles by optical diffraction analysis of electron micrographs may establish the identity and help in approximating the molecular weight of the substances contained in the crystal. This technique may be particularly helpful when isolation and purification of the crystalline material cannot be accomplished.     

ASSEMBLY OF LIPIDS INTO MEMBRANES IN ACANTHAMOEBA PALESTINENSIS : II. The Origin and Fate of Glycerol3H-Labeled Phospholipids of Cellular Membranes

The membranes of Acanthamoeba palestinensis were studied by examination in fixed cells, and then by following the movements of glycerol-³H-labeled phospholipids by cell fractionation. Two previously undescribed structures were observed: collapsed cytoplasmic vesicles of cup shape, and plaques in food vacuole and plasma membrane similar in size to the collapsed vesicles. It appeared that the plaques formed by insertion of collapsed vesicles into membranes and/or that collapsed vesicles formed by pinching off of plaques. Fractions were isolated, enriched with nuclei, rough endoplasmic reticulum (RER), plasma membrane, Golgi-like membranes, and collapsed vesicles. The changes in specific activity of glycerol-³H-labeled phospholipids in these membranes during incorporation, turnover, and after pulse-labeling indicated an ordered sequence of appearances of newly synthesized phospholipids, first in nuclei and RER, then successively in Golgi membranes, collapsed vesicles, and finally, plasma membrane. In previous work we had found no large nonmembranous phospholipid pool in A. palestinensis. These observations are consistent with the hypothesis that membrane phospholipids are synthesized, perhaps as integral parts of membranes, in RER and nuclei. Subsequently, some of the newly synthesized phospholipids are transported to the Golgi complex to become integrated into the membranes of collapsed vesicles, which are precursors of the plasma membrane. Collapsed vesicles from the plasma membrane by inserting into it as plaques. When portions of the plasmalemma from food vacuoles, collapsed vesicles pinch off from their membranes and are recycled back to the cell surface.     

Complex N-Glycans Influence the Spatial Arrangement of Voltage Gated Potassium Channels in Membranes of Neuronal-Derived Cells

The intrinsic electrical properties of a neuron depend on expression of voltage gated potassium (Kv) channel isoforms, as well as their distribution and density in the plasma membrane. Recently, we showed that N-glycosylation site occupancy of Kv3.1b modulated its placement in the cell body and neurites of a neuronal-derived cell line, B35 neuroblastoma cells. To extrapolate this mechanism to other N-glycosylated Kv channels, we evaluated the impact of N-glycosylation occupancy of Kv3.1a and Kv1.1 channels. Western blots revealed that wild type Kv3.1a and Kv1.1 α-subunits had complex and oligomannose N-glycans, respectively, and that abolishment of the N-glycosylation site(s) generated Kv proteins without N-glycans. Total internal reflection fluorescence microscopy images revealed that N-glycans of Kv3.1a contributed to its placement in the cell membrane while N-glycans had no effect on the distribution of Kv1.1. Based on particle analysis of EGFP-Kv proteins in the adhered membrane, glycosylated forms of Kv3.1a, Kv1.1, and Kv3.1b had differences in the number, size or density of Kv protein clusters in the cell membrane of neurites and cell body of B35 cells. Differences were also observed between the unglycosylated forms of the Kv proteins. Cell dissociation assays revealed that cell-cell adhesion was increased by the presence of complex N-glycans of Kv3.1a, like Kv3.1b, whereas cell adhesion was similar in the oligomannose and unglycosylated Kv1.1 subunit containing B35 cells. Our findings provide direct evidence that N-glycans of Kv3.1 splice variants contribute to the placement of these glycoproteins in the plasma membrane of neuronal-derived cells while those of Kv1.1 were absent. Further when the cell membrane distribution of the Kv channel was modified by N-glycans then the cell-cell adhesion properties were altered. Our study demonstrates that N-glycosylation of Kv3.1a, like Kv3.1b, provides a mechanism for the distribution of these proteins to the cell body and outgrowths and thereby can generate different voltage-dependent conductances in these membranes.     

Centrioles in the cell cycle. I. Epithelial cells

A study was made of the structure of the centrosome in the cell cycle in a nonsynchronous culture of pig kidney embryo (PE) cells. In the spindle pole of the metaphase cell there are two mutually perpendicular centrioles (mother and daughter) which differ in their ultrastructure. An electron-dense halo, which surrounds only the mother centriole and is the site where spindle microtubules converge, disappears at the end of telophase. In metaphase and anaphase, the mother centriole is situated perpendicular to the spindle axis. At the beginning of the G1 period, pericentriolar satellites are formed on the mother centriole with microtubules attached to them; the two centrioles diverge. The structures of the two centrioles differ throughout interphase; the mother centriole has appendages, the daughter does not. Replication of the centrioles occurs approximately in the middle of the S period. The structure of the procentrioles differs sharply from that of the mature centriole. Elongation of procentrioles is completed in prometaphase, and their structure undergoes a number of successive changes. In the G2 period, pericentriolar satellites disappear and some time later a fibrillar halo is formed on both mother centrioles, i.e., spindle poles begin to form. In the cells that have left the mitotic cycle (G0 period), replication of centrioles does not take place; in many cells, a cilium is formed on the mother centriole. In a small number of cells a cilium is formed in the S and G2 periods, but unlike the cilium in the G0 period it does not reach the surface of the cell. In all cases, it locates on the centriole with appendages. At the beginning of the G1 period, during the G2 period, and in nonciliated cells in the G0 period, one of the centrioles is situated perpendicular to the substrate. On the whole, it takes a mature centriole a cycle and a half to form in PE cells.     

COMPOSITION OF CELLULAR MEMBRANES IN THE PANCREAS OF THE GUINEA PIG : I. Isolation of Membrane Fractions

The subcellular components involved in the synthesis, transport, and discharge of secretory proteins in the guinea pig pancreatic exocrine cell have been isolated from gland homogenates by differential and gradient centrifugation. They include rough and smooth microsomes derived respectively from the rough endoplasmic reticulum and Golgi periphery, a zymogen granule fraction consisting mainly of mature zymogen granules and a smaller population of condensing vacuoles, and a plasmalemmal fraction. Membrane subfractions were obtained from the particulate components by treatment with mild (pH 7.8) alkaline buffers which extract the majority (>95%) of the content of secretory proteins, allowing the membranes to be recovered from the extracting fluid by centrifugation. The purity of the fractions was assessed by electron microscopy and by assaying marker enzymes for cross-contaminants. The rough and smooth microsomes were essentially free of mitochondrial contamination; the smooth microsomes contained <15% rough contaminants. The zymogen granule fraction and its derived membranes were free of rough microsomes and contained <3% contaminant mitochondria. The plasmalemmal fraction was heterogeneous as to origin (deriving from basal, lateral, and apical poles of the cell) and contained varying amounts of adherent fibrillar material arising from the basement membrane and terminal web. The lipid and enzymatic composition of the membrane fractions are described in the following reports.