Plus-end motors override minus-end motors during transport of squid axon vesicles on microtubules.

Plus- and minus-end vesicle populations from squid axoplasm were isolated from each other by selective extraction of the minus-end vesicle motor followed by 5'-adenylyl imidodiphosphate (AMP-PNP)-induced microtubule affinity purification of the plus-end vesicles. In the presence of cytosol containing both plus- and minus-end motors, the isolated populations moved strictly in opposite directions along microtubules in vitro. Remarkably, when treated with trypsin before incubation with cytosol, purified plus-end vesicles moved exclusively to microtubule minus ends instead of moving in the normal plus-end direction. This reversal in the direction of movement of trypsinized plus-end vesicles, in light of further observation that cytosol promotes primarily minus-end movement of liposomes, suggests that the machinery for cytoplasmic dynein-driven, minus-end vesicle movement can establish a functional interaction with the lipid bilayers of both vesicle populations. The additional finding that kinesin overrides cytoplasmic dynein when both are bound to bead surfaces indicates that the direction of vesicle movement could be regulated simply by the presence or absence of a tightly bound, plus-end kinesin motor; being processive and tightly bound, the kinesin motor would override the activity of cytoplasmic dynein because the latter is weakly bound to vesicles and less processive. In support of this model, it was found that (a) only plus-end vesicles copurified with tightly bound kinesin motors; and (b) both plus- and minus-end vesicles bound cytoplasmic dynein from cytosol.

KIF3C and KIF3A form a novel neuronal heteromeric kinesin that associates with membrane vesicles.

We have cloned from rat brain the cDNA encoding an 89,828-Da kinesin-related polypeptide KIF3C that is enriched in brain, retina, and lung. Immunocytochemistry of hippocampal neurons in culture shows that KIF3C is localized to cell bodies, dendrites, and, in lesser amounts, to axons. In subcellular fractionation experiments, KIF3C cofractionates with a distinct population of membrane vesicles. Native KIF3C binds to microtubules in a kinesin-like, nucleotide-dependent manner. KIF3C is most similar to mouse KIF3B and KIF3A, two closely related kinesins that are normally present as a heteromer. In sucrose density gradients, KIF3C sediments at two distinct densities, suggesting that it may be part of two different multimolecular complexes. Immunoprecipitation experiments show that KIF3C is in part associated with KIF3A, but not with KIF3B. Unlike KIF3B, a significant portion of KIF3C is not associated with KIF3A. Consistent with these biochemical properties, the distribution of KIF3C in the CNS has both similarities and differences compared with KIF3A and KIF3B. These results suggest that KIF3C is a vesicle-associated motor that functions both independently and in association with KIF3A.

Distribution of sialoglycoconjugates on the luminal surface of the endothelial cell in the fenestrated capillaries of the pancreas.

Sialic acid-bearing molecules on the luminal surface of the vascular endothelium in mouse and rat pancreatic capillaries were detected electron microscopically by using a procedure with ferritin hydrazide (FH), after preferential oxidation of sialyl residues with sodium periodate. The distribution of FH on the endothelial surface demonstrated the existence of microdomains with various densities of sialoglycoconjugates oxidizable by sodium periodate and accessible to the tracer. On the plasmalemma proper, FH binding sites were heterogeneously distributed. Their concentration on various microdomains decreased as follows: plasmalemma proper greater than coated pits greater than stomal diaphragms of plasmalemmal vesicles and transendothelial channels, and fenestral diaphragms. The membrane of plasmalemmal vesicles and transendothelial channels was not labeled by FH. Nonspecific binding of FH to the nonoxidized endothelial surface or that oxidized after neuraminidase treatment was relatively low.

Distribution of sialoglycoconjugates on acinar cells of the mammalian pancreas.

Using the sialic acid-specific lectin, limulin (LPA; from Limulus polyphemus hemolymph), the distribution and nature of sialoglycoconjugates on the surface of rat pancreatic cells has been investigated. Binding of rhodaminated LPA (Rh-LPA) or horseradish peroxidase-conjugated LPA (HRP-LPA) to fixed-frozen sections of adult rat pancreas resulted in intense linear staining of the apical surface of acinar cells with fainter staining on the basal but not the lateral cell surfaces. LPA binding was specific in that it could be abolished by 1) pretreatment of tissue sections with neuraminidase or periodic acid; 2) competition with sialic acid; and 3) incubation in Ca2+ -free buffers. Pretreatment of sections with proteases abolished LPA binding to the apical surfaces of acinar cells and also enhanced LPA binding to the lateral cell surface. Lipid extraction of sections following protease treatment markedly reduced LPA binding to the acinar cell periphery. These results suggest that LPA binding sites on the acinar cell apical surface may be primarily sialoglycoproteins, while those on the basolateral surfaces may consist in part of gangliosides. Electron microscopy of collagenase-dispersed acini exposed to HRP-LPA confirmed binding of LPA to the basal plasmalemma and, in addition, revealed staining of basal lamina when present. LPA binding to the acinar cell surface was not affected by digestion of tissue sections with hyaluronidase, heparinase, collagenase, or 6 M guanidine-HCl. Control experiments indicated that rat pancreatic secretory proteins contain undetectable amounts of sialoglycoproteins and thus that the apical localization of LPA is not due to adherent secretory proteins. Islets of Langerhans were always uniformly and heavily stained with LPA conjugates; this staining was protease insensitive. Appearance of LPA binding sites was examined on embryonic pancreatic epithelia. At day 15 of gestation, Rh-LPA stained the entire periphery of the epithelial cells, including the lateral cell surface, although more intense staining was already noted on the apical surface. This pattern persisted through day 17 of gestation, but by day 19 an adult staining pattern was observed with loss of staining of the lateral cell surfaces.

Sialoglycoconjugates of a pancreatic tumor: markers for cell polarity, membrane fluidity, and possible role in exocytosis.

The distribution and nature of sialoglycoconjugates on the surface of cells of a pancreatic carcinoma and their behavior when interacting with the sialic acid-specific lectin, limulin (LPA; from Limulus polyphemus hemolymph) were compared to those of normal pancreatic acinar cells. Fluorescence microscopy of frozen sections, using rhodaminated LPA (Rh-LPA), revealed protease-resistant binding sites evenly distributed over the cell surface of neoplastic cells, contrasting with the asymmetric distribution of sialoglycoconjugates on normal acinar cells. An asymmetric staining pattern, resembling that of normal acinar cells, was occasionally observed in tumor cells that had regained their structural polarity when in contact with the basement membranes of blood vessels. Cytochemistry, using horseradish peroxidase-conjugated LPA (HRP-LPA), showed that the binding of limulin to neoplastic cells was less intense than that to any plasmalemmal domain of normal acinar cells. In tumor cells, local intensification of LPA binding was systematically observed on plasmalemmal regions adjacent to zymogen granules. Fixed dissociated cells, both tumor and normal, treated with Rh-LPA, retained the fluorescence distribution of Rh-LPA observed in situ. Nonfixed neoplastic cells showed lectin-induced patching of limulin binding sites and were more susceptible to agglutination by LPA than normal acinar cells.

Purification and use of limulin: a sialic acid-specific lectin.

A simple and rapid method for the isolation of the sialic acid-specific lectin, Limulus polyphemus hemagglutinin (LPA), from the hemolymph of Limulus polyphemus is described. Declotted hemolymph is adsorbed to an affinity chromatographic column consisting of hog gastric mucin glycopeptides coupled to agarose and LPA is eluted in a single step with a Ca2+-free buffer, giving an apparent purification of approximately 25,000-fold. The eluted material is homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing and consists of identical subunits each of 29,000 daltons. Hemagglutination inhibition studies with horse red blood cells indicate specificity of LPA for N-acetyl- and N-glycolylneuraminic acid; binding is Ca2+ -dependent and abolished by neuraminidase treatment. LPA was covalently coupled to rhodamine and to horseradish peroxidase for use in detection of sialoglycoconjugates on cells and tissues by light and electron microscopy. Examples of the use of LPA for detection of sialoglycoconjugates in rat renal tubules and glomeruli, blood vessels in rat pancreas, and on horse red blood cells are shown. The procedures described here should prove useful as a cytochemical probe for detection of sialoglycoconjugates in a variety of systems. An accompanying article utilizes these probes for the detection of sialoglycoconjugates on the plasmalemma of adult and differentiating rat pancreatic acinar cells.

A hemagglutination test for binding of hydrazide-derivatized cytochemical markers.

A sensitive hemagglutination test was developed in order to predict the ability of amine- or hydrazide-derivatized cytochemical markers to interact with cell surface aldehydes in various in vitro and in situ systems. Human red blood cells were modified by glutaraldehyde fixation and subsequent oxidation with sodium periodate, thus exposing free aldehyde residues on their surface, able to form Schiff bases with accessible hydrazide groups on the marker molecule. Fixed and oxidized erythrocytes could be stored for more than 1 year without losing their capacity to be agglutinated by polyhydrazides. The aggregating capacity of hydrazide-enriched molecules correlated well with their binding to oxidized cell surface moieties.

Differential distribution of sialic acid in exocrine pancreas and parotid gland.

The sialic acid-specific lectin limulin (LPA, from Limulus polyphemus hemolymph) was used for the investigation of the distribution of accessible sialoglycoconjugates on the surface of cells from rat and rabbit parotid gland and exocrine pancreas. Fluorescence microscopy with rhodamine-conjugated LPA on fresh-frozen and fixed-frozen sections of the parotid gland revealed lectin binding sites on acinar and ductular epithelial cells. The staining was localized only at the periphery of the acini and ducts and was absent from the apical and lateral surface of epithelial cells. This staining pattern contrasted with that found in epithelial cells of acini and ducts in exocrine pancreas, where the luminal surface was intensely labeled by the fluorescent lectin. The luminal content of ductular tract and acini in parotid gland and pancreas was devoid of lectin-reactive sialoglycoconjugates. Connective tissue surrounding ducts and blood vessels bound the lectin heavily in both glands. These results outline that cells with similar structure and function, but constituents of different exocrine glands, exhibit differential distribution of sialoglycoconjugates on their corresponding plasmalemmal domains.

Intracellular fate of a multivalent ligand covalently bound to cell surface components. An electron microscopic study.

The electron dense tracer ferritin hydrazide (FH) was covalently attached to sialoglycoconjugates of the luminal surface of capillary endothelium in rat pancreas, after chemical modification of sialyl residues. Mild oxidation of the vasculature by perfusion with 1 mM sodium periodate was followed by a 30-90 min incubation with the tracer, at 37 degrees C. More than 60% of coated pits and coated vesicles were marked by FH, although in only approximately 25% of these structures the label density was higher than on adjacent domains of plasmalemma proper. Intracellularly, even after prolonged (90 min) perfusion of the vasculature with FH, the tracer was detected only in coated vesicles and in a few smooth-surfaced vesicles, but not in multivesicular bodies and endosomes. Cationized ferritin (CF), pI approximately 8.4, which binds to cell surface acidic sites (including sialoglycoconjugates) via an electrostatic noncovalent interaction, was rapidly internalized by the oxidized endothelium in coated vesicles and vacuoles. Unlike FH, after 30 min CF was already found in some multivesicular bodies and large vacuoles. At 60 to 90 min, most of multivesicular bodies contained the tracer. Similarly, a conjugate of wheat germ agglutinin with ferritin, which binds noncovalently to cell surface glycoconjugates containing N-acetylneuraminyl and N-acetylglucosaminyl residues, was internalized by the capillary endothelium in coated vesicles and transported to multivesicular bodies. It is concluded that FH, being unable to dissociate from its binding sites, is stopped on the endocytotic route in a compartment prior to its delivery to multivesicular bodies, thus escaping lysosomal degradation.

One axon, many kinesins: What's the logic?

A large number of membrane-bounded organelles, protein complexes, and mRNAs are transported along microtubules to different locations within the neuronal axon. Axonal transport in the anterograde direction is carried out by members of a superfamily of specialized motor proteins, the kinesins. All kinesins contain a conserved motor domain that hydrolyses ATP to generate movement along microtubules. Regions outside the motor domain are responsible for cargo binding and regulation of motor activity. Present in a soluble, inactive form in the cytoplasm, kinesins are activated upon cargo binding. Selective targeting of different types of kinesin motors to specific cargoes is directed by amino acid sequences situated in their variable tails. Cargo proteins with specific function at their destination, bind directly to specific kinesins for transport. Whereas most kinesins move to microtubule plus-ends, a small number of them move to microtubule minus-ends, and may participate in retrograde axonal transport. Axonal transport by kinesins has a logic: Fully assembled, multisubunit, functional complexes (e.g., ion channel complexes, signaling complexes, RNA-protein complexes) are transported to their destination by kinesin motors that interact transiently (i.e., during transport only) with one of the complexes' subunits.

Pathways of transcytosis in the fenestrated endothelium of pancreatic capillaries.

A procedure which covalently attaches an electron dense tracer to cell surface sialoglycoconjugates was used in order to label the luminal surface of capillary endothelium in rat pancreas. Mild oxidation of the vasculature by perfusion with 1 mM sodium periodate, followed by a 30-90 min incubation with ferritin hydrazide (FH) at 37 degrees C, led to a characteristic decoration of the endothelial surface. FH was taken up by the endothelium in coated vesicles and, to a much lesser extent, in plasmalemmal vesicles or vacuoles. Though rarely, FH was found at the abluminal front of the endothelium in coated vesicles opened to the extracellular space either directly or via a smooth-surfaced vesicle, thus suggesting the involvement of coated structures in transendothelial transport (transcytosis). A conjugate of wheat germ agglutinin with ferritin, which binds noncovalently to cell surface glycoconjugates containing N-acetylneuraminyl and N-acetylglucosaminyl residues, was transported transcellularly in monomeric form, mainly through smooth-surfaced (plasmalemmal) vesicles and transendothelial channels. Cationized ferritin (CF), pI approximately 8.4, which binds to cell surface acidic sites via an electrostatic noncovalent interaction, was rapidly internalized by the oxidized endothelium in coated structures and in vacuoles. Big aggregates of CF were transported and delivered to the abluminal front of the endothelium via large vacuoles, vesicles, and through coated structures and multivesicular bodies. These results show that the capillary endothelial cell possesses multiple mechanisms for transporting macromolecules across the cell, involving vacuoles, smooth or coated vesicles, channels, and lysosomal-related compartments. They also indicate that, besides molecular weight and electric charge of macromolecules, other characteristics of the ligands (e.g., type of bond with cell surface components, state of aggregation) are factors influencing the transendothelial transport.

High and low molecular weight tracers for the electron microscopical detection of sialoglycoconjugates.

Hydrazide-derivative tracers of different molecular weights have been synthesized for use in the electron microscopical detection of sodium periodate-oxidized sialyl residues of glycoconjugates in various tissues and cells. Haemundecapeptide hydrazide, horseradish peroxidase hydrazide, and Limulus polyphemus haemocyanin hydrazide were obtained by coupling adipic acid dihydrazide to the tracers with the aid of water-soluble carbodiimide. The enzymatic tracers thus prepared retained their peroxidatic activity. On conversion to the hydrazide derivative, the haemocyanin molecule dissociated into its hexameric subunits. In order to test by transmission electron microscopy the ability of the conjugates to bind to the sialoglycoconjugates of endothelial cell surfaces, each tracer was perfused in situ into rat pancreatic vasculature previously oxidized with 1 mM sodium periodate. The three tracers characteristically labelled the various microdomains of the luminal cell coat of the capillary endothelial cell. The electron opacity of the haemocyanin subunits allowed their easy detection when bound to the cell surface or to components of the extracellular matrix. The bound markers were not displaced by a high ionic strength buffer, and did not label desialylated cell surfaces. These results indicate that the three hydrazide-derivative tracers may be useful tools for the electron microscopical detection of cellular and extracellular sialoglycoconjugates.

Far-Eastern interpretation of cellular pathology: yang-type components of a pancreatic acinar tumor.

Conjugates of a sialic acid-binding lectin, limulin, with rhodamine and horseradish peroxidase, were used to detect yang-type components at the surface of pancreatic carcinoma cells. Fluorescence microscopy of frozen sections revealed an even distribution of yang residues over the surface of neoplastic cells, in contrast to the polarized one on normal acinar cells. Electron microscopy indicated a lower concentration of yang-type molecules on the tumor cell surface than on its normal counterpart. It was concluded that the neoplastic process induces an alteration of yin/yang ratio at cellular level.

A mixed hemagglutination test for binding of glycosylated cytochemical markers.

A sensitive hemagglutination test which uses simultaneously erythrocytes from two different species was developed to test the ability of glycosylated cytochemical markers to interact with membrane-bound lectins. The interaction between galactose-terminated glycoconjugates and Ricinus communis agglutinin 120 was chosen for exemplification. The lectin was covalently attached to the surface of sheep red blood cells, thus rendering them highly agglutinating for human erythrocytes. Small amounts of a large variety of terminal galactose-containing molecules inhibited the hemagglutination. The assay proved to be particularly useful for testing colloidal gold-adsorbed glycoconjugates of low protein concentration.

Complex intermolecular interactions maintain a stable linkage between the photoreceptor connecting cilium axoneme and plasma membrane.

Microtubule-membrane cross-linkers in motile and nonmotile cilia are supramolecular structures, held together by strong interactions between the constituent molecules. We have characterized these interactions in the photoreceptor connecting cilium, where cross-linkers co-fractionate and maintain their in situ location after Triton X-100 extraction of axonemes. In bovine photoreceptor cells, the transmembrane assemblage that is cross-linked to the connecting cilium axoneme contains three high molecular mass glycoconjugates of 425, 600, and 700 kDa (Horst et al., 1987). The relative amounts of the three glycoconjugates, as judged from band intensity in electrophoretograms, depend strongly on sample treatment prior to electrophoresis. The electrophoretic pattern was reproducible after several weeks of storage of the axoneme fraction in extraction buffer containing 50% sucrose. Removal of sucrose from the buffer by dialysis eliminated the 600 kDa and 700 kDa, and decreased the detected amount of the 425 kDa glycoconjugate. When samples were incubated in Laemmli sample buffer at increasing temperatures (23 degrees, 60 degrees, 95 degrees C), a gradual reduction in the intensity of the three bands was observed. The quantitative reduction of high molecular mass glycoconjugates was accompanied by the appearance of novel protein species of lower molecular mass, as detected by lectin and antibody overlays of axonemal transblots. These results suggest that the previously characterized cross-linker glycoconjugates are complex, SDS-resistant multi-molecular conglomerates. We have further used fluorescent lectins to monitor the presence of glycoconjugates on whole-mounted axonemes, in conditions aimed to selectively solubilize the cross-linkers. The cross-linker complexes could not be dissociated from the axoneme by incubation with buffers containing 1 M of either Na2SO4 or NaI. The results indicate that the connecting cilium-specific cross-linker complexes are bound via high-affinity interactions to both axoneme and overlying plasma membrane.

Gamma-tubulin in differentiated cell types: localization in the vicinity of basal bodies in retinal photoreceptors and ciliated epithelia.

gamma-Tubulin, a newly discovered member of the tubulin superfamily required for microtubule nucleation, is associated with the centrosome(s) throughout the vertebrate cell cycle. We have used a polyclonal antibody, generated against a highly conserved segment of gamma-tubulin, to localize this protein in postmitotic, ciliated cells, in which the major microtubule organizing centers are the basal bodies. Single-cilium photoreceptor cells from bovine retina contained a strongly immunoreactive species, with molecular characteristics of gamma-tubulin, in association with a detergent-resistant, cytoskeletal fraction devoid of cytoplasmic microtubules. gamma-Tubulin was discretely localized throughout the basal body region, extending opposite to the axonemal shaft, in mechanically detached rod outer segments and whole-mounted, connecting cilium-derived axonemes. In multiciliated epithelia from bovine trachea and oviduct, gamma-tubulin immunoreactivity was detected at the base of the cilia, where basal bodies are located. These results suggest that this key centrosomal protein of mitotically active cells is also an integral component of microtubule organizing centers, required for the generation of the microtubule network in terminally differentiated cells.

The kinesin motor KIF3A is a component of the presynaptic ribbon in vertebrate photoreceptors.

Kinesin motors are presumed to transport various membrane compartments within neurons, but their specific in vivo functions, cargoes, and expression patterns in the brain are unclear. We have investigated the distribution of KIF3A, a member of the heteromeric family of kinesins, in the vertebrate retina. We find KIF3A at two distinct sites within photoreceptors: at the basal body of the connecting cilium axoneme and at the synaptic ribbon. Immunoelectron microscopy of the photoreceptor ribbon synapse shows KIF3A to be concentrated both at the ribbon matrix and on vesicles docked at the ribbon, a result that is consistent with the presence of both detergent-extractable and resistant KIF3A fractions at these synapses. KIF3A is also present in the inner plexiform layer, again at presynaptic ribbons. These findings suggest that within a single cell, the photoreceptor, one kinesin polypeptide, KIF3A, can serve two distinct functions, one specific for ribbon synapses.

Cell surface chemistry of arterial endothelium and blood monocytes in the normolipidemic rabbit.

Chemical mapping of the luminal surface of normal rabbit aortic and coronary endothelium was investigated cytochemically to establish a baseline for further comparison with the biochemical changes possibly induced by the experimental hypercholesterolemia. Morphometric analysis showed that in the aortic endothelium the plasma membrane exposes a large number of uniformly-distributed positively-charged groups of high pKa, and a heterogeneous pattern of dense anionic groups of low pKa. Among the latter, only a third was represented by neuraminidase-cleavable sialic acids. These are constituted by various classes of N-, and O-substituted sialyl residues in glycoconjugates, most frequent being those non-O-acetylated at C8 or C9. Among the oligosaccharides detected with lectins, very abundant were the glycoconjugates containing mannosyl and subterminal galactosyl, whereas N-acetyl-glucosamine, terminal galactosyl and N-acetyl-galactosaminyl moieties were rather poorly represented. The density of the latter two markedly increased after its unmasking by neuraminidase treatment. Coated pits contained both anionic and cationic sites, but only few sialic acids and saccharide residues in significantly lower amounts than plasma membrane. The membrane of plasmalemmal vesicles displayed a high number of cationic sites and mannosyl residues, but very few anionic groups, sialyl residues, and galactosyl and N-acetyl-galactosaminyl moieties. Coronary endothelium displayed a chemical pattern similar to aorta, with some differences, especially in the frequency of some oligosaccharides. Vena cava was low in acidic groups but rather rich in galactose. Plasmalemmal vesicles were only occasionally labeled by the probes used. Monocyte surface exhibited a high density of anionic sites, and binding sites for wheat germ agglutinin and Ricinus communis agglutinin. No mononuclear cells were observed adhering to endothelial surface.

Prelesional events in atherogenesis. Changes induced by hypercholesterolemia in the cell surface chemistry of arterial endothelium and blood monocytes, in rabbit.

We investigated the modifications that diet-induced hypercholesterolemia, in rabbit, can produce in the cell surface charge and chemistry of arterial endothelium (E) and blood monocytes (M). Weekly, up to 8 weeks, after blood samples were taken for lipid analysis and blood cell preparation, the vasculature was washed free of blood and the endothelial luminal surface (ES) exposed to cytochemical probes for detecting charged groups, sialoconjugates and oligosaccharides. After fixation in situ, specimens collected from lesion-prone regions (aortic arch and coronary artery) and vena cava, were processed for electron microscopy. Morphometric analysis of tracer distribution on endothelium of nonlesional and lesional areas occurring in various stages of structural alterations, showed a remarkable resistance of the cell coat to very high level of serum cholesterol. In nonlesional zones the E surface charge and glycoconjugates were not significantly changed. In lesional areas, including those with forming fatty streaks, while cationic sites, galactosyl-, and N-acetyl-galactosaminyl residues were not altered whereas mannosyl moieties increased in density. A reduction in anionic groups and sialoconjugates appeared only after advanced extracellular and intracellular accumulation of lipoprotein-derived material and stromal proliferation developed in the intima. Moreover, these ES changes were usually restricted to the relatively rare E cells heavily loaded with lipid inclusions. The modulations were generally paralleled by comparable variations in the M surface. Regardless the extent of surface charge reduction, monocytes continued to migrate and foam cells to egress from the vessel wall. The results suggest that the onset and progression of early intimal lesions are not preceded but followed by significant restricted alterations in cell surface charge and glycoconjugates of arterial endothelium and monocytes.