Reconstitution of ciliary membranes containing tubulin

Membranes from the gill cilia of the mollusc Aequipecten irradians may be solubilized readily with Nonidet P-40. When the detergent is removed from the solution by adsorption to polystyrene beads, the proteins of the extract remain soluble. However, when the solution is frozen and thawed, nearly all of the proteins reassociate to form membrane vesicles, recruiting lipids from the medium. The membranes equilibrate as a narrow band (d = 1.167 g/cm3) upon sucrose density gradient centrifugation. The lipid composition of reconstituted membranes (1:2 cholesterol:phospholipids) closely resembles that of the original extract, as does the protein content (45%). Ciliary calmodulin is the major extract protein that does not associate with the reconstituted membrane, even in the presence of 1 mM calcium ions, suggesting that it is a soluble matrix component. The major protein of reconstituted vesicles is membrane tubulin, shown previously to differ hydrophobically from axonemal tubulin. The tubulin is tightly associated with the membrane since extraction with 1 mM iodide or thiocyanate leaves a vesicle fraction whose protein composition and bouyant density are unchanged. Subjecting the detergent-free membrane extract to a freeze-thaw cycle in the presence of elasmobranch brain tubulin or forming membranes by warming the extract in the presence of polymerization-competent tubulin yields a membrane fraction with little incorporated brain tubulin. This suggests that ciliary membrane tubulin specifically associates with lipids, whereas brain tubulin preferentially forms microtubules.     

Evidence for tubulin-binding sites on cellular membranes: plasma membranes, mitochondrial membranes, and secretory granule membranes

We describe the interaction of pure brain tubulin with purified membranes specialized in different cell functions, i.e., plasma membranes and mitochondrial membranes from liver and secretory granule membranes from adrenal medulla. We studied the tubulin-binding activity of cellular membranes using a radiolabeled ligand-receptor assay and an antibody retention assay. The tubulin-membrane interaction was time- and temperature-dependent, reversible, specific, and saturable. The binding of tubulin to membranes appears to be specific since acidic proteins such as serum albumin or actin did not interfere in the binding process. The apparent overall affinity constant of the tubulin- membrane interaction ranged between 1.5 and 3.0 X 10(7) M-1; similar values were obtained for the three types of membranes. Tubulin bound to membranes was not entrapped into vesicles since it reacted quantitatively with antitubulin antibodies. At saturation of the tubulin-binding sites, the amount of reversibly bound tubulin represents 5-10% by weight of membrane protein (0.4-0.9 nmol tubulin/mg membrane protein). The high tubulin-binding capacity of membranes seems to be inconsistent with a 1:1 stoichiometry between tubulin and a membrane component but could be relevant to a kind of tubulin assembly. Indeed, tubulin-membrane interaction had some properties in common with microtubule formation: (a) the association of tubulin to membranes increased with the temperature, whereas the dissociation of tubulin- membrane complexes increased by decreasing temperature; (b) the binding of tubulin to membranes was prevented by phosphate buffer. However, the tubulin-membrane interaction differed from tubulin polymerization in several aspects: (a) it occurred at concentrations far below the critical concentration for polymerization; (b) it was not inhibited at low ionic strength and (c) it was colchicine-insensitive. Plasma membranes, mitochondrial membranes, and secretory granule membranes contained tubulin as an integral component. This was demonstrated on intact membrane and on Nonidet P-40 solubilized membrane protein using antitubulin antibodies in antibody retention and radioimmune assays. Membrane tubulin content varied from 2.2 to 4.4 micrograms/mg protein. The involvement of membrane tubulin in tubulin-membrane interactions remains questionable since erythrocyte membranes devoid of membrane tubulin exhibited a low (one-tenth of that of rat liver plasma membranes) but significant tubulin-binding activity. These results show that membranes specialized in different cell functions possess high- affinity, large-capacity tubulin-binding sites...     

Fusion between disk membranes and plasma membrane of bovine photoreceptor cells is calcium dependent.

Disk membranes and plasma membrane vesicles were prepared from bovine retinal rod outer segments (ROS). The plasma membrane vesicles were labeled with the fluorescent probe octadecylrhodamine B chloride (R18) to a level at which the R18 fluorescence was self-quenched. At pH 7.4 and 37 degrees C and in the presence of micromolar calcium, an increase in R18 fluorescence with time was observed when R18-labeled plasma membrane vesicles were introduced to a suspension of disks. This result was interpreted as fusion between the disk membranes and the plasma membranes, the fluorescence dequenching resulting from dilution of the R18 into the unlabeled membranes as a result of lipid mixing during membrane fusion. While the disk membranes exposed exclusively their cytoplasmic surface, plasma membrane vesicles were found with both possible orientations. These vesicles were fractionated into subpopulations with homogeneous orientation. Plasma membrane vesicles that were oriented with the cytoplasmic surface exposed were able to fuse with the disk membranes in a Ca(2+)-dependent manner. Fusion was not detected between disk membranes and plasma membrane vesicles oriented such that the cytoplasmic surface was on the interior of the vesicles. ROS plasma membrane-disk membrane fusion was stimulated by calcium, inhibited by EGTA, and unaffected by magnesium. Rod photoreceptor cells of vertebrate retinas undergo diurnal shedding of disk membranes containing the photopigment rhodopsin. Membrane fusion is required for the shedding process.     

Electron microscope demonstration of tubulin in cilia and basal bodies of rat tracheal epithelium by the use of an antitubulin antibody

It has been previously demonstrated that both cytoplasmic microtubules and the microtubules of cilia, flagella, and sperm tail contain tubulin. Although the morphology of cytoplasmic microtubules and that of axonemes differs in cells from which they have been isolated, the tubulin of the two structures shares physical and chemical properties. In some mammalian tissues, such as tracheal epithelium, cilia and basal bodies are difficult to isolate and characterize. The use of an enzyme- labeled immunoglobulin probe would facilitate identification and in situ localization of such proteins. Tubulin prepared from porcine brain by ion-exchange chromatography and from rat brain by the method of cyclic polymerization and depolymerization with subsequent disk gel electrophoresis with SDS were injected intravenously into rabbits. The animals were intermittently bled and the antisera extracted. The specificity of the antisera was proved by indirect immunofluorescence staining of the mitotic spindle, specific blocking of spindle staining by purified tubulin and not by other proteins, staining of 3T3 cytoplasmic microtubules, single line on immunoelectrophoresis, failure of control antisera to show any of these, and precipitation of antibody with all tubulin preparations and not with actin. We have shown by electron microscopy of ciliated cells of the tracheal epithelium stained with antitubulin by the indirect enzyme-labeled antibody method that the basal bodies, outer doublets, and central pair of the cilia contain tubulin. This indicates that tubulin in microtubules of cilia and basal bodies of rat tracheal epithelium is antigenically similar to tubulin extracted from cytoplasmic neurotubules of brains from the same species and from a different mammalian species. No other axonemal structures stained with the antitubulin. Three different preparations of tubulin from pigs and rats were used to immunize rabbits. All elicited similar antisera which gave identical staining patterns. The specificity of the staining was demonstrated by the absence of staining with immune serum absorbed with purified tubulin, the absence of staining with preimmune serum, and the absence of staining if any of the reagents were omitted during the staining reaction.     

Membrane events involved in myoblast fusion

Myoblast fusion has been studied in cultures of chick embryonic muscle utilizing ultrastructural techniques. The multinucleated muscle cells (myotubes) are generated by the fusion of two plasma membranes from adjacent cells, apparently by forming a single bilayer that is particle-free in freeze-fracture replicas. This single bilayer subsequently collapses, and cytoplasmic continuity is established between the cells. The fusion between the two plasma membranes appears to take place primarily within particle-free domains (probably phospholipid enriched), and cytoplasmic unilamellar, particle-free vesicles are occasionally associated with these regions. These vesicles structurally resemble phospholipid vesicles (liposomes). They are present in normal myoblasts, but they are absent in certain fusion-arrested myoblast popluations, such as those treated with either 5-bromo-deoxyuridine (BUdR), cycloheximide (CHX), or pospholipase C (PLC). The unilamellar, particle-free vesicles are present in close proximity to the plasma membranes, and physical contact is observed frequently between the vesicle membrane and the plasma membrane. The regions of vesicle membrane-plasma membrane interaction are characteristically free of intramembrane particles. A model for myoblast fusion is presented that is based onan interpretation of these observations. This model suggests that the cytoplasmic vesicles initiate the generation of particle-depleted membrane domains, both being essential components in the fusion process.     

Isolation of plasma membrane fractions from green wheat leaves by free-flow electrophoresis or two-phase partition alone or in combination

Plasma membrane vesicles were isolated from shoots of light-grown wheat seedlings by preparative free-flow electrophoresis, aqueous polymer two-phase partition or both. Plasma membrane vesicles were identified from staining of thin sections prepared for electron microscopy with phosphotungstic acid at low pH. The orientation of the plasma membrane vesicles was determined from latency and trypsin sensitivity of K⁺ Mg²⁺ATPase and of glucan synthase II, and concanavalin A-peroxidase binding and membrane asymmetry visualized by electron microscopy. The K⁺Mg²⁺ATPase and of glucan synthase II activities of plasma membrane fractions isolated by two-phase partition were latent and trypsin resistant. The vesicles bound concanavalin A-peroxidase strongly and exhibited a cytoplasmic side-in morphology. These fractions of cytoplasmic side-in vesicles were less than 10% contaminated by cytoplasmic side-out vesicles. By free-flow electrophoresis, two populations of vesicles which stained with phosphotungstic acid at low pH, designated D and E, were obtained. The vesicle population with the lower electrophoretic mobility, fraction E, contained plasma membrane vesicles with properties similar to those of the plasma membrane vesicles obtained after two-phase partition. The phosphotungstic-reactive vesicles with greater electrophoretic mobility, fraction D, were concanavalin A unreactive with the cytoplasmic membrane leaflet outwards. Less than 50% of the K⁺Mg²⁺-ATPase activity of this fraction was latent and trypsin sensitive. The vesicles of fraction D appeared to be preferentially cytoplasmic side-out. The electrophoretic mobilities of cytoplasmic side-out (non-latent glucan synthase II activity) and cytoplasmic side-in (latent glncan synthase II activity) plasma membrane vesicles isolated from a frozen and thawed wheat plasma membrane fraction, corresponded with the mobilities of fraction D and E, respectively, again showing that the plasma membrane vesicles with the lesser electrophoretic mobility were cytoplasmic side-in. The cytoplasmic side-in and cytoplasmic side-out vesicles therefore showed opposite eletrophoretic mobilities compared with a previous free-flow electrophoretic separation of soybean plasma membranes. The majorities of the plasma membrane vesicles of both fractions D and E entered the upper phase upon two-phase partition with the phase composition used for purification of wheat plasma membranes. Thus, neither electrophoretic mobility nor phase partitioning characteristics can be used as the only criteria for assignment of vesicle orientation.     

Synaptic Plasma Membrane Tubulin May Be an Integral Constituent

Abstract: Mild detergent extraction of chick brain synaptic plasma membranes followed by gel electrophoresis suggests that synaptic plasma membrane tubulin is an integral component. Although some of the synaptic plasma membrane tubulin might be aggregates, that possibility is not supported by the observation that tubulin aggregates that are added to synaptosomes before synaptic subfractionation do not partition with synaptic plasma membranes during membrane isolation.     

Cytoplasmic carboxypeptidase 5 regulates tubulin glutamylation and zebrafish cilia formation and function

Glutamylation is a functionally important tubulin posttranslational modification enriched on stable microtubules of neuronal axons, mitotic spindles, centrioles, and cilia. In vertebrates, balanced activities of tubulin glutamyl ligase and cytoplasmic carboxypeptidase deglutamylase enzymes maintain organelle- and cell type–specific tubulin glutamylation patterns. Tubulin glutamylation in cilia is regulated via restricted subcellular localization or expression of tubulin glutamyl ligases (ttlls) and nonenzymatic proteins, including the zebrafish TPR repeat protein Fleer/Ift70. Here we analyze the expression patterns of ccp deglutamylase genes during zebrafish development and the effects of ccp gene knockdown on cilia formation, morphology, and tubulin glutamylation. The deglutamylases ccp2, ccp5, and ccp6 are expressed in ciliated cells, whereas ccp1 expression is restricted to the nervous system. Only ccp5 knockdown increases cilia tubulin glutamylation, induces ciliopathy phenotypes, including axis curvature, hydrocephalus, and pronephric cysts, and disrupts multicilia motility, suggesting that Ccp5 is the principal tubulin deglutamylase that maintains functional levels of cilia tubulin glutamylation. The ability of ccp5 knockdown to restore cilia tubulin glutamylation in fleer/ift70 mutants and rescue pronephric multicilia formation in both fleer- and ift88-deficient zebrafish indicates that tubulin glutamylation is a key driver of ciliogenesis.     

Tubulin: An Integral Protein of Mammalian Synaptic Vesicle Membranes

Abstract: The major protein in isolated synaptic vesicles from bovine cerebral cortex has been compared to tubulin by sodium dodecyl sulphate-urea polyacrylamide gel electrophoresis, by two-dimensional gel electrophoresis and by peptide mapping following limited proteolysis of the protein by Staphylococcus aureus protease. The results establish in purified synaptic vesicles the presence of tubulin, which is composed of the α and β subunits. In the presence of ethyleneglycol bis (aminoethyl ether)- N, N' -tetraacetic acid (EGTA) or magnesium in the isolation buffers, the synaptic vesicles contained mainly the α-tubulin whereas the β subunit was less abundant. Similarly, synaptosomal plasma membranes that were prepared in the presence of EGTA also contained more of α-tubulin than of the β subunit. Non-ionic detergents such as Triton X-100 or Nonidet P-40 failed to solubilize the tubulin from the synaptic vesicles. Ionic detergents such as deoxycholate and sodium dodecyl sulphate solubilized all the vesicle proteins, including tubulin. The results indicate that α-tubulin is an integral vesicle membrane protein, whereas most of the β sub-unit is peripherally attached and can be easily dissociated from the vesicle membrane with EGTA.     

The relationship of the rat brain 68 kDa microtubule-associated protein with synaptosomal plasma membranes and with the Drosophila 70 kDa heat-shock protein.

A protein of molecular mass 68 kDa and pI5.6 is a major translation product of rat brain mRNA [Hall, Mahadevan, Whatley, Biswas & Lim (1984) Biochem. J. 219, 751-761]. In the rat brain this protein was associated with microtubule preparations and was present together with tubulin as a component of the synaptosomal plasma membranes, synaptic vesicles and post-synaptic structures. The brain mRNA for this protein was found to hybridize specifically to the Drosophila gene for the 70 kDa heat-shock protein, thus enabling its rapid isolation.     

Microtubule-membrane interactions in cilia. I. Isolation and characterization of ciliary membranes from Tetrahymena pyriformis

Tetrahymena ciliary membranes were prepared by four different techniques, and their protein composition was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), electron microscopy, and two-dimensional thin-layer peptide mapping. Extraction of the isolated cilia by nonionic detergent solubilized the ciliary membranes but left the axonemal microtubules and dyneine arms intact, as determined by quantitative electron microscopy. The proteins solubilized by detergent included a major 55,000-dalton protein, 1-3 high molecular weight proteins that comigrated, on SDS-PAGE, with the axonemal dynein, as well as several other proteins of 45,000-50,000 daltons. Each of the major proteins contained a small amount of carbohydrate, as determined by PAS-staining; no PAS-positive material was detected in the detergent-extracted axonemes. The major 55,000- dalton protein has proteins quite similar to those of tubulin, based on SDS-PAGE using three different buffer systems as well as two- dimensional maps of tryptic peptides from the isolated 55,000-dalton protein. To determine whether this tubulin-like protein was associated with the membrane or whether it was an axonemal or matrix protein released by detergent treatment, three different methods to isolate ciliary membrane vesicles were developed. The protein composition of each of these differetn vesicle preparations was the same as that of the detergent-solubilized material. These results suggest that a major ciliary membrane protein has properties similar to those of tubulin.     

The Fine Structure of Streptomyces coelicolor : I. The Cytoplasmic Membrane System

Colonies and spore suspensions of Streptomyces coelicolor were fixed by the method of Kellenberger, Ryter, and Séchaud (1958) and embedded in methacrylate or araldite. Thin sections were cut with an A. F. Huxley microtome and examined in a Siemens' Elmiskop I. At all stages of development the hyphae of Streptomyces coelicolor have an extensive membranous component in the cytoplasm. The membranes are continuous with the plasma membrane and have a variety of configurations at different places in the hyphae. Tubular structures, vesicles, and parallel stacks of membranes are seen. In some areas concentric layers of membranes form whorled structures which are particularly frequent in the region of developing cross-walls and within maturing spores. In the spores membranous structures often lie embedded in the nuclear material. In disintegrating hyphae the intracytoplasmic membranes round off into small vesicles and remain when the rest of the cytoplasmic structure has gone. In the absence of typical mitochondria and other cytoplasmic membranous structures it is possible that the membranous component of the cytoplasm of Streptomyces coelicolor may perform the functions of the endoplasmic reticulum and/or the mitochondria of higher cells.     

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.     

Ciliary membrane tubulin and associated proteins: a complex stable to Triton X-114 dissociation

When either membranes from scallop gill cilia or reconstituted membranes from the same source are solubilized with Triton X-114 and the detergent is condensed by warming, no significant fraction of any major membrane protein partitions into the micellar detergent. Rather, most of the membrane lipids condense with the detergent phase, forming mixed micelles from which nearly pure lipid vesicles may be produced by adsorption of detergent with polystyrene beads. One minor membrane protein, with a molecular weight of about 20 000, is associated consistently with these vesicles. The aqueous phase contains a fairly homogeneous protein-Triton X-114 micelle sedimenting at 2.6 S in the analytical ultracentrifuge. Sucrose gradient velocity analysis in a detergent-free gradient indicates moderate size polydispersity but constant polypeptide composition throughout the sedimenting protein zone. Sucrose gradient equilibrium analysis (also in a detergent-free gradient) results in a protein-detergent complex banding at a density of 1.245 g/cm3. Sedimentation of the protein-detergent complex in the ultracentrifuge, followed by fixation and normal processing for electron microscopy, reveals a fine, reticular material consisting of 5-10-nm granules. These data are consistent with previous evidence that membrane tubulin and most other membrane proteins exist together as a discrete lipid-protein complex in molluscan gill ciliary membranes.     

Characterization of a membrane-specific tubulin isoform by peptide mapping

A membrane-specific tubulin-like protein, found in preparations of synaptic plasma membranes and brain mitochondria, was analyzed by chemical and proteolytic peptide mapping to determine which part of the molecule was different from cytoplasmic tubulin. The membrane polypeptide was identical to alpha tubulin in the first two-thirds of the molecule containing the amino terminal, as found by peptide mapping. However, some differences were observed in the peptide maps of the carboxy terminal one third of the molecule which includes a domain that is important in the regulation of tubulin self-assembly.     

Cytochemical study of liposome and lipid vesicle phagocytosis

Electron microscopy cytochemistry has been used to study the cytoplasmic location of liposomes and lipid vesicles following specific antibody-dependent phagocytosis. The vesicle compositions were 94–99 mol% ‘fluid’ lipid (egg phosphatidylcholine or dimyristoylphosphatidylcholine at 37°C or ‘solid’ lipid (dipalmitoylphosphatidylcholine at 37°C). In some cases, 4 mol% phosphatidylserine was included in the vesicle membrane so as to vary the surface charge density. These vesicles undergo specific antibody-dependent phagocytosis by RAW264 macrophages when the lipid membranes contain 1–2 mol% dinitrophenyl lipid hapten in the presence of rabbit anti-dinitrophenyl IgG antibody. Internalized lipid vesicles can be visualized with the electron microscope when ferritin is trapped in the internal aqueous compartments prior to internalization. The lipid vesicles were demonstrated to be internal to the macrophage plasma membranes by selectively staining the plasma membranes with Ruthenium red. The cytoplasmic location of vesicles and liposomes was studied by electron microscopic staining for activities of the following enzymes: (1) acid phosphatase; (2) inorganic trimetaphosphatase; (3) adenosine triphosphatase; and (4) glucose-6-phosphatase. The first two enzymatic activities were found in association with ferritin-containing vesicles after antibody-dependent phagocytosis, showing the formation of vesicle-containing phagolysosomes. Adenosine triphosphatase and glucose-6-phosphatase were primary not associated with the vesicles, suggesting a minimal association of vesicles with plasma membrane, Golgi, endoplasmic reticulum and perinuclear cisternae. Phagosome-lysosome fusion did not appear to depend on the type of target lipid vesicle or liposome, on the ‘fluidity’ of the target membrane, or the presence of phosphatidylserine in the target membrane.     

The distribution of dopamine-like immunoreactivity in the thoracic and abdominal ganglia of the locust (Schistocerca gregaria)

Summary An indirect gold-labeling method utilizing the lectin from Limax flavus was employed to characterize the subcellular distribution of sialic acid in glycoconjugages of the salamander olfactory mucosa. The highest density of lectin binding sites was in secretory vesicles of sustentacular cells. Significantly lower densities of lectin binding sites were found in secretory granules of acinar cells of both Bowman's and respiratory glands. Lectin binding in acinar cells of Bowman's glands was confined primarily to electron-lucent regions and membranes of secretory granules. In the olfactory mucus, the density of lectin binding sites was greater in the region of mucus closest to the nasal cavity than in that closest to the epithelial surface. At the epithelial surface, the density of lectin binding sites associated with olfactory cilia was 2.4-fold greater than that associated with microvilli of sustentacular cells or non-ciliary plasma membranes of olfactory receptor neurons, and 7.9-fold greater than non-microvillar sustentacular cell plasma membranes. Lectin binding sites were primarily associated with the glycocalyx of olfactory receptor cilia. The cilia on cells in the respiratory epithelium contained few lectin binding sites. Thus, sialylated glycoconjugates secreted by sustentacular cells are preferentially localized in the glycocalyx of the cilia of olfactory receptor neurons.     

A role for Tctex-1 (DYNLT1) in controlling primary cilium length

The microtubule motor complex cytoplasmic dynein is known to be involved in multiple processes including endomembrane organization and trafficking, mitosis, and microtubule organization. The majority of studies of cytoplasmic dynein have focused on the form of the motor that is built around the dynein-1 heavy chain. A second isoform, dynein heavy chain-2, and its specifically associated light intermediate chain, LIC3 (D2LIC), are known to be involved in the formation and function of primary cilia. We have used RNAi in human epithelial cells to define the cytoplasmic dynein subunits that function with dynein heavy chain 2 in primary cilia. We identify the dynein light chain Tctex-1 as a key modulator of cilia length control; depletion of Tctex-1 results in longer cilia as defined by both acetylated tubulin labeling of the axoneme and Rab8a labeling of the cilia membrane. Suppression of dynein heavy chain-2 causes concomitant loss of Tctex-1 and this correlates with an increase in cilia length. Compared to individual depletions, double siRNA depletion of DHC2 and Tctex-1 causes an even greater increase in cilia length. Our data show that Tctex-1 is a key regulator of cilia length and most likely functions as part of dynein-2.     

Experimental model for studying the primary cilia in tissue culture cells.

In HeLa, PK, 3T3, PtK1 cells and rat embryo fibroblasts (REF), antibodies against acetylated tubulin stained centrioles, primary cilia, some cytoplasmic microtubules and microtubule bundles of the mid-body. The primary cilia were stained more intensively than cytoplasmic microtubules and could easily be distinguished. This makes it possible to detect the primary cilia in cultured cells and to estimate their number by light microscopy. The four cultures studied had 1/4 to 1/3 of interphase cells with detectable primary cilia, and only in HeLa cells the primary cilia were very rare. Comparison of electron microscopic and immunofluorescence data showed that the frequencies of occurrence of the primary cilia in four tissue cultures determined by these two methods were the same. Therefore, antibodies against acetylated tubulin can be used to study the primary cilia. In synchronized mitotic fibroblasts (3T3 and REF) the primary cilia appeared first 2 h after the cells had been plated on coverslips, which is 1 h after the cells had entered the interphase. Four hours after plating the number of ciliated cells reached the average level for nonsynchronous population. This model can be used for further studies of the expression of primary cilia.     

STUDIES ON THE DEPLETION AND ACCUMULATION OF MICROVILLI AND CHANGES IN THE TUBULOVESICULAR COMPARTMENT OF MOUSE PARIETAL CELLS IN RELATION TO GASTRIC ACID SECRETION

Gastric parietal cells in mice present a spectrum of microscopic appearances due mainly to variations in the abundance of the tubular and vesicular component of the cytoplasm and in the size and number of microvilli lining the intracellular canaliculi. Differences in the range of forms among parietal cells of fasting versus fed mice were not especially striking, but cells with very numerous tubules and vesicles were more common after fasting. However, in mice treated with drugs or hormones that induce acid secretion, parietal cells were more uniform in appearance. There was a marked reduction of these cytoplasmic membranes and a concomitant increase in both the number and size of microvilli. Measurements of acid secretion in control animals and in animals treated with acid secretagogues indicated hydrogen ion secretion contemporaneous with depletion of the cytoplasmic tubulovesicular membranes and with increase of the microvilli. In mice with inhibited acid secretion, parietal cells showed an accumulation of cytoplasmic tubules and vesicles and reduction in the numbers of microvilli. Stereological methods were used to quantitate 10 different parietal cell compartments. Tracer studies with lanthanum did not reveal continuity between the tubules and the plasma membrane. However, there were regions of close apposition between the tubulovesicular membranes and the cell membrane of the canaliculus, and instances where cytoplasmic tubules extended from the cell into the core of enlarged microvilli.