Alignment of cholesterol in the membrane bilayer

Freeze-fracture replicas of filipin-treated samples of guinea pig colon mucosa reveal areas in the membrane of the goblet cell granules labeled by filipin-cholesterol complexes (FCC) intermingled with regions patterned by "lines." The FCC and "lines" are arranged in an approximately rhombic pattern. Other membranes of the same cell or of other cells display either FCC only, aligned and occasionally ordered in "rhombs," "lines" only, with a similar pattern, or randomly distributed FCC. Optical diffraction was used to analyze and compare replicas of membranes with ordered FCC and "lines", as well as randomly distributed FCC. The results demonstrate that all these structures are reciprocally related through a common distribution pattern in the membrane. This observation supports the assumption that cholesterol has a preferential ordered distribution within the membrane bilayer.     

Reorganization of membrane cholesterol during membrane fusion in myogenesis in vitro: a study using the filipin-cholesterol complex

Using filipin and freeze-fracture electron microscopy, we examined the distribution of membrane cholesterol during the fusion of myoblasts in vitro. The early stages of fusion were characterized by the depletion of cholesterol from the membrane apposition sites, at which the plasma membranes of two adjacent cells were in close contact. At first, filipin-cholesterol complexes were absent from the plasma membrane of one cell only and were distributed homogeneously on the membrane of the other cell. Eventually, both of the closely apposed membranes became almost completely free the filipin-cholesterol complexes. Membrane fusion took place at several points within the filipin-cholesterol complex-free areas. In later stages, the cytoplasms of the fusing cells became confluent by fenestration of the plasma membranes formed with the filipin-cholesterol complex-free regions. Our observations suggest that membrane cholesterol is reorganized at these fusion sites and that fusion initiated by the juxtaposition of the cholesterol-free areas of each plasma membrane of the adjacent cells.     

The tight junction as a barrier to cholesterol in canine epithelial cells

Filipin has been used to test several models of continuity or flow of lipid components through the tight junction. Cultured canine kidney cells (MDCK) were fixed and incubated in the presence of filipin. Freeze-fracture replicas were analyzed and densities of filipin-cholesterol complexes measured. Fractures of membranes linked with tight junctions were compared statistically to determine whether filipin-cholesterol complexes (protrusions and pits, independently) were randomly distributed between the two membranes of cells separated by the tight junction. The results indicate that filipin-cholesterol complexes are not randomly distributed across the tight junction. If the density of filipin-cholesterol complexes is an accurate indication of membrane cholesterol concentration, then there is a difference in the cholesterol concentration between leaflets of membranes joined by tight junctions and models of the tight junction which suggest leaflet continuity across the junction are in error.     

Heterogeneous distribution of filipin-sterol complexes in nuclear membranes

Filipin, a sterol-specific polyene antibiotic, has been shown by electron microscopy to form complexes in membranes of mouse urinary bladder cells. Following instillation of a glutaraldehyde-filipin-dimethylsulfoxide solution into the bladder lumen, filipin-cholesterol complexes appear as membrane corrugations in thin sections and as 20–25 nm protuberances and depressions on PF and EF faces in freeze-fracture replicas. The complexes are observed in plasmalemma, Golgi membrane, rough endoplasmic reticulum and nuclear membrane of five different cell types (urothelial, endothelial, mesothelial, smooth muscle and fibroblasts). In the present report, we direct particular attention to the localization of numerous filipin-cholesterol complexes present in the nuclear envelopes of these cells. Our results suggest that enrichment of cell membranes with cholesterol occurs at an earlier stage in the flow-differentiation process than previously suspected. In addition, the unequal distribution of complexes in favor of the outer nuclear membrane suggests that it has a higher cholesterol content than the inner membrane.     

Membrane-associated proteins affect the formation of filipin-cholesterol complexes in viral membranes

The presence and distribution of cholesterol in biological membranes can be visualized by complex formation with the polyene antibiotic filipin after or during fixation with glutaraldehyde. In the envelopes of budding and immature retroviruses no filipin-cholesterol complexes are formed, but in the plasma membrane of host cells and in the envelopes of mature viruses filipin-cholesterol complexes are easily detected. However, after treatment of glutaraldehyde-fixed cells with pepsin, the presence of cholesterol in the envelopes of budding and immature viral particles could also be demonstrated. This indicates that in these structures the reaction of cholesterol with filipin is inhibited by proteins associated with the cholesterol-containing membrane. Treatment of fixed cells with trypsin, and of unfixed cells with cytochalasin B (CB) had no effect on detectability of cholesterol in these structures. On no occasion were cholesterol-filipin complexes formed in coated pits. The present findings call for caution when interpretating data on absence of filipincholesterol complexes in those membrane domains that are characterized by the presence of closely associated proteins.     

Heterogeneous Distribution of Membrane Cholesterol at the Attachment Site of Cryptosporidium muris to Host Cells

ABSTRACT. Distribution of membrane cholesterol at the attachment site of Cryptosporidium muris was investigated by freeze-fracture cytochemistry using a polyene antibiotic filipin. Since the host plasma membrane enveloped C. muris , the inner and outer membranes were continuous with the parasite plasma membrane at the annular ring and with host membrane at the dense band, respectively. Although many filipin-cholesterol complexes were observed on the plasma membrane of host cells and parasites, a line showing no complexes was evident at the above two membrane junctures. These observations indicate that parasitic infection of C. muris altered the organization of membrane cholesterol.     

Variability of the topography of low-density lipoprotein (LDL) receptors in the plasma membrane of cultured human skin fibroblasts as revealed by gold-LDL conjugates in conjunction with the surface replication technique

In this investigation the membrane-perturbing effect of filipin, a polyene antibiotic which reacts specifically with cholesterol or cholesterol-like compounds in cell membranes, has been exploited to study the distribution of coated pits in cultured human skin fibroblasts. The coated pits, showing no filipin-cholesterol complexes, occurred singly or in clusters without apparent localization of either type to a particular region of the fibroblast membrane. Colloidal gold, conjugated to low-density lipoprotein, has proven to be an excellent marker, allowing the localization of low-density lipoprotein receptors on the surface of cultured cells. A pattern similar to that for the coated pits in the plasma membrane fracture faces was observed in the distribution of gold-low-density lipoprotein conjugates in surface replicas, indicating that the low-density lipoprotein receptors are associated with these coated pits. It was shown that there is an apparent heterogeneity in the distribution of low-density lipoprotein receptors, from cell to cell and even among different areas of the same cell membrane. The binding capacity for gold-low-density lipoprotein complexes, as represented by the extent of surface labeling, was directly related to the cell variety within the culture or to the particular experimental procedure. The observation of differences in the distribution of gold-low-density lipoprotein conjugates, even among adjacent coated pits, provides evidence for various stages of activity of the low-density lipoprotein receptors corresponding to incorporation, mobility, and internalization.     

Topology of morphologically detectable protein and cholesterol in membranes of polypeptide-secreting cells

The freeze-fracture morphology of intracellular and plasma membranes in endocrine and exocrine polypeptide-secreting cells has been studied to detect changes while these membranes interact during secretion. A qualitative and quantitative evaluation of intramembrane particles and filipin binding as indicators of protein and cholesterol content of the membranes, respectively, reveals the following changes. From the forming of the maturing pole of the Golgi complex, membranes lose morphologically detectable protein and gain morphologically detectable cholesterol. The protein-poor, cholesterol-rich secretory granule membrane then interacts with a richly particulate plasma membrane in endocrine cells and with a moderately particulate luminal membrane in exocrine cells. The site of interaction between secretory granule and plasma membrane is characterized by a local clearing of intramembrane particles; by contrast, filipin-binding sites revealing cholesterol are present in this area. In exocrine cells, the fused secretory granule, which is initially rich in filipin-cholesterol complexes and poor in particles, appears to lose progressively its filipin labelling to resemble the poorly labelled luminal membrane. These findings, although they cannot be interpreted definitely at present, clearly show impressive changes of membrane structure along the secretory pathway and suggest that a corresponding degree of functional specialization is needed for proper interaction to occur.     

Compactin (ML-236B) reduces the content of filipin-cholesterol complexes in the plasma membrane of chronic lymphocytic leukemia cells

The polyene antibiotic filipin was used to visualize the presence and distribution of cholesterol in the plasma membrane of glutaraldehyde-fixed human chronic lymphocytic leukemia (CLL) cells. Both compactin (ML-236B), a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, and 25-hydroxycholesterol reduced the content of filipin-cholesterol complexes in the plasma membrane of CLL cells grown in media supplemented with either 15% delipidized horse serum or 15% normal (whole) horse serum. The reduction due to compactin was reversed by the concomitant addition of mevalonolactone. The ability of compactin to reduce the relative cholesterol content (as judged by filipin labeling) in CLL cells grown in lipoprotein-containing (normal) serum suggest that either CLL cells are different from other cells in that they predominantly utilize endogenously synthesized cholesterol for incorporation into the plasma membrane, or that a separate pool of endogenously synthesized cholesterol provides cholesterol for the plasma membrane.     

Electron microscopy of the tracheal ciliated mucosa in rat

Summary The structure of the tracheal epithelial cells from rat has been studied by electron microscopy on approximately 200 Å thick sections with a resolution of better than 30 Å.The epithelium is found to be of a simple columnar type composed of ciliated cells, mucus producing (goblet) cells, basal cells and a fourth kind of cell, here called brush cell. A great number of non-ciliated cells has also been encountered. It has been proved that these represent goblet cells in different stages of intracellular synthesis of mucous granules. The ciliated cells have approximately 8–9 cilia per square micron and there are about 270 cilia on each cell, the calculated surface area being 33 square microns. They are covered by a 70 Å thick membrane. The ciliary filaments are arranged in a pattern of 2 separate ones in the center and a ring of 9 peripheral ones, each divided into 2 subfilaments by a wall with same thickness as the filamentous wall itself, this being 60 Å. The peripheral filaments are continuous with the basal corpuscles. The structure of the corpuscles as compared with earlier findings is discussed. A number of 0.05 micron thick and 1 micron long filiform projections emerge from the cell surface. No cuticle is present.The cell membrane facing adjacent cells is 90 Å and separated from their cell membrane by a 105 Å wide space, this space, being expanded towards a level corresponding to the proximal parts of the cell. A structure that represents terminal bar has been encountered. The cytoplasm is loose and composed of 160 Å thick granules. Spaces enclosed by 50 Å thick membranes with attached 160 Å thick granules (-cytomembranes) are rare. The Golgi zone is analyzed and its regular composition of -cytomembranes, granules and vacuoles is confirmed. The mitochondria with a mean width of 0.23 micron differ to their inner structure from the common type in that the triple layered membranes are highly interconnected. Large opaque granules are encountered in the cytoplasm. Ring-shaped, 850 Å wide, structures are present in the nuclear membrane. The goblet cells are not as abundant as the ciliated cells, the ratio being 14. Small filiform projections covered by a 95 Å thick membrane protrude from the cell surface. This membrane is continuous with the cell membrane, the latter with the same dimensions as in the ciliated cells. Terminal bars are present. The cytoplasm is very opaque due to a dense packing of the 165 Å opaque granules, many in clusters of 4–6. The -cytomembranes have the same dimensions as mentioned above for those present in the ciliated cells. The Golgi zone is of regular composition. There is a suggestion that the Golgi vacuoles and the -cytomembranes are involved in the formation of mucus. In the stage of cellular activity with but few mucous granules, there is a great number of large opaque granules, the size varying from 0.4–1 micron. The mitochondria with a mean width of 0.23 micron have an outer triple layered membrane with a total thickness of 180 Å. The central less opaque layer is 70 Å and the opaque layer on either side is 55 Å. The inner membranes are arranged parallel to each other and have a triple layered composition where the central less opaque layer is 65 Å and the opaque layers each 60 Å. The brush cells belong to the non-ciliated cells. They are encountered singly, surrounded by goblet cells. The surface structures are shaped like brushes or clumsy protrusions which emerge from the distal end of the cell, and are covered by a 95 Å thick membrane. There have been no suggestions of the brushes being cilia in a stage of growth, nor is it probable that they represent stereocilia. They most nearly resemble the intestinal brush border extensions and thus might serve as a resorbing structure.The cytoplasm of the brush cells appears of medium opacity between the ciliated cells and goblet cells and is composed of 155 Å opaque granules. The -cytomembranes are very rare. The Golgi zone is diminutive though of regular composition. The mitochondria are abundant and small with a mean width of 0.14 micron. The outer and inner membranes are triple layered with approximately the same dimensions as reported for the mitochondria of the ciliated and goblet cells. The inner membranes are very few, often only one or two are present. Some of the large opaque granules have inside a very regular arrangement of small 60 Å thick opaque granules arranged in a crystallinic pattern. In the cytoplasm 0.5–1 micron long bundles of 30–40 Å wide fibrils are encountered. The nucleolus shows a characteristic structure of concentrically arranged thin membranes. The basal cells are believed to represent lymphocytes or white blood cells. They sometimes rest on the basement membrane, sometimes are encountered in the distal part of the intercellular spaces. They are bordered by a 110 Å thick cell membrane and have a rather opaque cytoplasm characterized by 160 Å thick opaque granules. A very small Golgi zone is present. The mitochondria, the mean width being 0.14 micron, have triple layered outer and inner membranes, where the less opaque central layer is 65–70 Å and the opaque layers 45–50 Å each. The basement membrane has a thickness of 600 Å. No inner structure has been resolved. The basement membrane is separated from adjacent parts of the ciliated, goblet, brush, and basal cells by a 250 Å wide less opaque space. Below the basement membrane is the lamina propria of the trachea, which is composed of collagen and elastin fibers together with fibroblasts, white blood cells and lymphocytes. The relationship between different types of tracheal epithelial cells in rat has been analyzed. There has been found no indication of a transformation of any type of cells observed into a different type of cell. The development of basal cells via supporting cells or intermediate cells to goblet cells or ciliated cells has not been noticed. On the contrary, all cells that in light microscopy could have been considered to be supporting or intermediate cells, we have been able to recognize as brush cells or as goblet cells to a varying degree filled with mucous granules. If the cells did not seem to reach the cell surface it has been found to be due to a diagonal direction of the sectioning. In this connection it should be emphasized that this relationship is valid only in rat where it is known that the epithelium is of a simple columnar type as distinct from the conditions in man, that epithelium being of a pseudostratified columnar type.This paper is based on a report given at the meeting of Deutsche Gesellschaft für Elektronenmikroskopie in Münster, March 28–31, 1955 and at the Scandinavian Electron Microscope Society Meeting in Stockholm, May 13, 1955.     

Effect of polyene antibiotics on the lectin-induced agglutination of transformed and untransformed cell lines.

Treatment of transformed Py3T3, SV101-3T3, and L1210 cells, as well as mitotic and Pronase-treated untransformed 3T3 cells, with the polyene antibiotics filipin, nystatin, and amphotericin B inhibited agglutination by wheat germ agglutinin. The effect of polyene antibiotic treatment was lectin and cell specific. Concanavalin A induced agglutination was not inhibited, wheat germ agglutination induced agglutination of untransformed 3T3 interphase cells was not influenced, and other aggregation phenomena, including those of erythrocytes with blood group specific antibodies or divalent cations, were unaffected by polyene treatments. This suggests that the formation of polyene-cholesterol complexes in transformed and erythrocyte cell membranes may specifically affect wheat germ agglutinin receptors and/or secondary events necessary for wheat germ agglutinin induced agglutination. Fluorescence studies of membrane filipin-cholesterol complexes showed that pretreating the cells with wheat germ agglutinin, but not concanavalin A, perturbed the fluorescence properties of filipin. Electron spin resonance studies with spin-labeled fatty acids revealed at best only a slight decrease in fatty acyl chain flexibility following filipin treatment. These studies indicate that there are not only quantitative differences between the agglutinability of transformed and untransformed cells with wheat germ agglutinin but that qualitative differences exist as well.     

Lectin histochemistry of 1,2-dimethylhydrazine-induced rat colon neoplasia

Lectins linked to fluorescein were used as carbohydrate probes to examine the goblet cell mucin and epithelial cell surface glycoconjugate alterations in an experimental rodent model of colonic neoplasia induced with parenteral 1,2-dimethylhydrazine dihydrochloride. Lectins derived from Triticum vulgare (WGA), Ricinus communis (RCA1), and Limulus polyphemus (LPA) showed reduced labeling of goblet cell mucin in these tumors, while binding with peanut lectin from Arachis hypogaea (PNA), a lectin ordinarily failing to bind to mucin in normal colon, was positive. In addition, RCA1 and LPA showed increased cell surface labeling of neoplastic epithelial cells. Finally, alterations were observed in lectin binding to "transitional" colonic mucosa adjacent to colonic tumors from carcinogen-treated rats. These findings indicate that significant alterations in both membrane and mucin glycoconjugates occur in colonic tumors and mucosa adjacent to tumors in a chemically induced experimental animal model of human colon cancer.     

Structural and biochemical differentiation of the guinea-pig colon during foetal development

Summary We have studied some aspects of the morphological and biochemical differentiation of the foetal guinea-pig colonic epithelium. At day 40 the epithelium was organised in ridges and appeared pseudo-stratified. Folding of the epithelium, followed by villus formation, occurred between days 45 and 55, and by day 50 mucus-secreting goblet cells appeared at the bases of the colonic villi. By day 55 most epithelial cells, including goblet cells, possessed numerous microvilli which, by day 65, had become organised into well developed brush-borders. Between day 55 and term (day 65–68) mucosal depth increased markedly and the colon attained its final glandular morphology.Biochemical studies showed the specific activities of the microvillar hydrolases to be much lower in the washed colon than in either foetal meconium or small intestine at all times during development. Furthermore, a membrane fraction highly enriched in microvillus hydrolase activities was prepared from foetal colonic meconium using techniques originally devised to isolate the foetal small intestinal microvillus membrane. This meconial subfraction was almost identical in polypeptide composition to the highly-purified foetal small intestinal microvillus membrane. Identification of the colonic microvillus membrane was hampered by the absence of reliable membrane markers. Nevertheless, a fraction 14-fold enriched in aminopeptidase activity was prepared from day 40 foetal colon and its polypeptide composition compared by SDS-PAGE to that of the small intestinal microvillus membrane at the same age.     

Expression and localization of Muc4/sialomucin complex (SMC) in the adult and developing rat intestine: implications for Muc4/SMC function

Muc4/sialomucin complex (SMC) is a high molecular mass heterodimeric membrane mucin, encoded by a single gene, and originally discovered in a highly metastatic ascites rat mammary adenocarcinoma. Subsequent studies have shown that it is a prominent component of many accessible and vulnerable epithelia, including the gastrointestinal tract. Immunoblot and immunofluorescence analyses demonstrated that Muc4/SMC expression in the rat small intestine increases from proximal to distal regions and is located predominantly in cells at the base of the crypts. These cells were postulated to be Paneth cells, based on their location, morphology, and secretory granule content. Immunohistochemistry indicated the presence of Muc4/SMC in these granules. Muc4/SMC expression was higher in the rat colon than small intestine and was abundantly present in colonic goblet cells, but not in goblet cells in the small intestine. Immunohistochemistry also suggested the presence of MUC4 in human colonic goblet cells. Biochemical analyses indicated that rat colonic Muc4/SMC is primarily the soluble form of the membrane mucin. Analyses of Muc4/SMC during development of the rat gastrointestinal tract showed its appearance at embryonic day 14 of the esophagus and at day 15 at the surface of the undifferentiated stratified epithelium at the gastroduodenal junction, then later at cell surfaces in the more distal regions of the differentiated epithelium of the small intestine, culminating in expression as an intracellular form in the crypts of the small intestine at about day 21. Limited expression in the colon was observed during development before birth at cell surfaces, with expression as an intracellular form in the goblet cells arising during the second week after birth. These results suggest that membrane mucin Muc4/SMC serves different functions during development of the intestine in the rat, but is primarily a secreted product in the adult animal.     

The histochemistry of glycoconjugates in the colonic epithelium of the chicken

Summary In the colonic epithelium of the chicken, glycoconjugates have been studied by means of selected histochemical methods of light and electron microscopy. According to the results obtained, most of the colonic goblet cells contained acidic and neutral glycoconjugates with sulphate and vicinal diol groupings, -D-mannose and -D-glucose residues and sialic acid-galactose dimers. These goblet cells were found to undergo changes in histochemical reactivity during upward migration along the crypts; -D-mannose and -D-glucose residues and terminal sialic acidgalactose dimers increased in amount. The striated border of the colonic columnar cells has, likewise, been found to contain such glycoconjugates as were similar in reactivity to those of the goblet cells. The histophysiological significances of glycoconjugates involved in the chicken colonic epithelium have been discussed with special reference to the functional activities of the carbohydrates.     

Blue native/SDS-PAGE analysis reveals reduced expression of the mClCA3 protein in cystic fibrosis knock-out mice

Cystic fibrosis (CF) is a frequent autosomal recessive disorder caused by mutation of a gene encoding a multifunctional transmembrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), located in the apical membrane of epithelial cells lining exocrine glands. In an attempt to get a more complete picture of the pleiotropic effects of the CFTR defect on epithelial cells and particularly on the membrane compartment, a bidimensional blue native (BN)/SDS-PAGE-based proteomic approach was used on colonic crypt samples from control and CFTR knock-out mice (cftr-/-). This approach overcomes the difficulties of membrane protein analysis by conventional two-dimensional PAGE and is able to resolve multiprotein complexes. Used here for the first time on crude membrane proteins that were extracted from murine colonic crypts, BN/SDS-PAGE allows effective separation of protein species and complexes of various origins, including mitochondria, plasma membrane, and intracellular compartments. The major statistically significant difference in protein maps obtained with samples from control and cftr-/- mice was unambiguously identified as mClCA3, a member of a family of calcium-activated chloride channels considered to be key molecules in mucus secretion by goblet cells. On the basis of this finding, we evaluated the overall expression and localization of mClCA3 in the colonic epithelium and in the lung of mice by immunoblot analysis and immunohistochemistry. We found that mClCA3 expression was significantly decreased in the colon and lung of the cftr-/- mice. In an ex vivo assay, we found that the Ca2+-dependent (carbachol-stimulated) glycoprotein secretion strongly inhibited by the calcium-activated chloride channel blocker niflumic acid (100 microm) was impaired in the distal colon of cftr-/- mice. These results support the conclusion that a ClCA-related function in the CF colon depends on CFTR expression and may be correlated with the impaired expression of mClCA3.     

Filipin-cholesterol complexes in plasma membranes and cell junctions of Tenebrio molitor epidermis

The polyene antibiotic filipin combines with cholesterol in membranes to form complexes that are readily identifiable in the electron microscope. The distribution of filipin-cholesterol (FC) complexes is most easily studied by freeze-fracture. Larval epidermis of Tenebrio molitor (Insecta, Coleoptera) was maintained in vitro for 48 hr, since the electrophysiological properties of the cells are best characterized under these conditions. The cells were fixed in buffered 3.0% glutaraldehyde at RT for 15 min, transferred to fresh fixative containing 1% DMSO and filipin (final concentration; 0.5 mg/ml) for 3 hr RT. Control cells were treated in fixative containing 1% DMSO only. In freeze fracture replicas, FC complexes appear on the plasma membrane as large circular protrusions measuring 26.5 +/- 6.8 nm (x +/- s.d.) n = 50, in diameter and 17.1 +/- 2.8 nm, n = 50, in height and 11.7 +/- 2.6 nm, n = 25, in depth. Protrusions are about two times more frequent on the E face while pits are several times more frequent on the P face. FC complexes are most abundant (greater than 50/mu m2) on the basal membrane surface of the cells but are excluded from regions of hemidesmosomal plaques that anchor the cells to the basal lamina. FC complexes are also abundant on the apical surfaces of the cells where cuticle secretion occurs. In the lateral regions below the junctional belt, FC complexes are less numerous but often appear to increase in frequency in a graded fashion away from the junctional region. The septate junctions are relatively free of FC complexes except in regions where they open to form islands. These islands often contain gap junctions but the FC complexes rarely invade the particle domains of the gap junctions. Single FC complexes were seen in three out of a total of 97 gap junctions. Exposure of the epidermis to 20-hydroxyecdysone for 24 hr in vitro did not induce the appearance of FC complexes within the cell junctions.     

Type C Niemann-Pick disease. Lysosomal accumulation and defective intracellular mobilization of low density lipoprotein cholesterol

The intracellular accumulation of unesterified cholesterol was examined during 24 h of low density lipoprotein (LDL) uptake in normal and Niemann-Pick C fibroblasts by fluorescence microscopy with filipin staining and immunocytochemistry. Perinuclear fluorescence derived from filipin-sterol complexes was observed in both normal and mutant cells by 2 h. This perinuclear cholesterol staining reached its peak in normal cells at 6 h. Subsequent development of fluorescence during the remaining 18 h of LDL incubation was primarily limited to the plasma membrane region of normal cells. In contrast, mutant cells developed a much more intense perinuclear fluorescence throughout the entire 24 h of LDL uptake with little enhancement of cholesterol fluorescence staining in the plasma membranes. Direct mass measurements confirmed that internalized LDL cholesterol more readily replenishes the plasma membrane cholesterol of normal than of mutant fibroblasts. Perinuclear filipin-cholesterol fluorescence of both normal and mutant cells was colocalized with lysosomes by indirect immunocytochemical staining of lysosomal membrane protein. Abnormal sequestration of LDL cholesterol in mutant cells within a metabolically latent pool is supported by the finding that in vitro esterification of cellular cholesterol could be stimulated in mutant but not in normal cell homogenates by extensive disruption of the intracellular membranous structures of cells previously cultured with LDL. Deficient translocation of exogenously derived cholesterol from lysosomes to other intracellular membrane sites may be responsible for the delayed homeostatic responses associated with LDL uptake by mutant Niemann-Pick Type C fibroblasts.     

Loss of SNAP29 Impairs Endocytic Recycling and Cell Motility

Intracellular membrane trafficking depends on the ordered formation and consumption of transport intermediates and requires that membranes fuse with each other in a tightly regulated and highly specific manner. Membrane anchored SNAREs assemble into SNARE complexes that bring membranes together to promote fusion. SNAP29 is a ubiquitous synaptosomal-associated SNARE protein. It interacts with several syntaxins and with the EH domain containing protein EHD1. Loss of functional SNAP29 results in CEDNIK syndrome (Cerebral Dysgenesis, Neuropathy, Ichthyosis and Keratoderma). Using fibroblast cell lines derived from CEDNIK patients, we show that SNAP29 mediates endocytic recycling of transferrin and β1-integrin. Impaired β1-integrin recycling affected cell motility, as reflected by changes in cell spreading and wound healing. No major changes were detected in exocytosis of VSVG protein from the Golgi apparatus, although the Golgi system acquired a dispersed morphology in SNAP29 deficient cells. Our results emphasize the importance of SNAP29 mediated membrane fusion in endocytic recycling and consequently, in cell motility.     

Detection of plasma membrane cholesterol by filipin during microvillogenesis and ciliogenesis in quail oviduct

Using filipin as a probe for the presence of membrane cholesterol, the evolution of cholesterol distribution in the apical plasma membrane was studied during estrogen-induced ciliogenesis in quail oviduct and compared with the distribution of intramembrane particles (IMPs). Ciliary growth is preceded by the first step of microvillus differentiation. Microvilli emerge in membrane domains rich in IMPs and devoid of filipin-cholesterol (f-c) complexes. However growing microvillus membrane shows f-c complexes. During ciliary growth, microvilli lengthen from 0.5 to 2 microns, indicating that the microvillar membrane is not a membrane reservoir for ciliogenesis. During ciliary growth, the characteristic ciliary necklace IMP rows appear progressively at the base of cilia. The first IMP row is organized in a membrane circlet lacking of f-c complexes, whereas the new shaft membrane in the middle of the circlet exhibits numerous complexes. These two different domains of the cilia keep their specificity during ciliary growth. Only the ciliary tip shows fewer complexes than the shaft membrane. The apical membrane of differentiated ciliated cells is thus composed of various domains, the ciliary shaft full of f-c complexes and poor in IMPs, the ciliary necklace is devoid of f-c complexes and rich in IMPs, the microvilli membrane is rich in both IMPs and f-c complexes, and the interciliary membrane is poor in both f-c complexes and IMPs, whereas the undifferentiated cells exhibit an apical membrane in which f-c complexes and IMPs are distributed homogeneously.