Showing papers in "Journal of Cell Biology in 1992"

Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation.

Abstract: Programmed cell death (PCD) plays a key role in developmental biology and in maintenance of the steady state in continuously renewing tissues. Currently, its existence is inferred mainly from gel electrophoresis of a pooled DNA extract as PCD was shown to be associated with DNA fragmentation. Based on this observation, we describe here the development of a method for the in situ visualization of PCD at the single-cell level, while preserving tissue architecture. Conventional histological sections, pretreated with protease, were nick end labeled with biotinylated poly dU, introduced by terminal deoxy-transferase, and then stained using avidin-conjugated peroxidase. The reaction is specific, only nuclei located at positions where PCD is expected are stained. The initial screening includes: small and large intestine, epidermis, lymphoid tissues, ovary, and other organs. A detailed analysis revealed that the process is initiated at the nuclear periphery, it is relatively short (1-3 h from initiation to cell elimination) and that PCD appears in tissues in clusters. The extent of tissue-PCD revealed by this method is considerably greater than apoptosis detected by nuclear morphology, and thus opens the way for a variety of studies.

Brefeldin A: insights into the control of membrane traffic and organelle structure.

Abstract: THE definition of cellular organelles has evolved over the last hundred years largely driven by morphologic observations, but more recently has been supplemented and complemented by functional and biochemical studies (Palade, 1975) . Thus, organelles are now identified both by their morphology and by the set ofcomponents that comprise them . Determining how organelle identity is established and maintained and how newly synthesized protein and membrane are sorted to different organelles are the central issues of organellogenesis . Essential to the many cellular functions that take place within the central vacuolar system (which consists ofthe ER, Golgi apparatus, secretory vesicles, endosomes, and lysosomes) is membrane traffic which mediates the exchange of components between different organelles . There are two critical characteristics of membrane traffic . First, only certain sets oforganelles exchange membrane and the patterns of this exchange define what are called membrane pathways . Second, multiple pathways intersect at specific points within the central vacuolar system . For specific components to "choose" the correct pathway at such points of crossing, mechanisms exist to impose choices on specific molecules . This process is called sorting . The characteristicsofeachorganelle within the central vacuolar system are likely to be intimately tied to the properties ofmembrane traffic . An imbalance in the magnitude ofmembrane input into and egress from an organelle would have profound effects on the size ofthat compartment . In addition, failures in sorting or aberrations in targeting pathways would be expected to profoundly affect the identity of individual organelles . Recently, the relationship between the control of membrane traffic and the maintenance of organelle structure has been investigated with the use ofa remarkable drug, brefeldin A (BFA).' In this review we will summarize recent findings with BFA and propose some speculative models concerning the mechanism and regulation ofmembrane traffic within the central vacuolar system .

Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth

Abstract: Hepatocyte Growth Factor (HGF, also known as Scatter Factor) is a powerful mitogen or motility factor in different cells, acting through the tyrosine kinase receptor encoded by the MET protooncogene Endothelial cells express the MET gene and expose at the cell surface the mature protein (p190MET) made of a 50 kD (alpha) subunit disulfide linked to a 145-kD (beta) subunit HGF binding to endothelial cells identifies two sites with different affinities The higher affinity binding site (Kd = 035 nM) corresponds to the p190MET receptor Sub-nanomolar concentrations of HGF, but not of a recombinant inactive precursor, stimulate the receptor kinase activity, cell proliferation and motility HGF induces repairs of a wound in endothelial cell monolayer HGF stimulates the scatter of endothelial cells grown on three-dimensional collagen gels, inducing an elongated phenotype In the rabbit cornea, highly purified HGF promotes neovascularization at sub-nanomolar concentrations HGF lacks activities related to hemostasis-thrombosis, inflammation and endothelial cells accessory functions These data show that HGF is an in vivo potent angiogenic factor and in vitro induces endothelial cells to proliferate and migrate

Tyrosine phosphorylation of paxillin and pp125FAK accompanies cell adhesion to extracellular matrix: a role in cytoskeletal assembly.

Abstract: Cells in culture reveal high levels of protein tyrosine phosphorylation in their focal adhesions, the regions where cells adhere to the underlying substratum. We have examined the tyrosine phosphorylation of proteins in response to plating cells on extracellular matrix substrata. Rat embryo fibroblasts, mouse Balb/c 3T3, and NIH 3T3 cells plated on fibronectin-coated surfaces revealed elevated phosphotyrosine levels in a cluster of proteins between 115 and 130 kD. This increase in tyrosine phosphorylation was also seen when rat embryo fibroblasts were plated on laminin or vitronectin, but not on polylysine or on uncoated plastic. Integrin mediation of this effect was suggested by finding the same pattern of elevated tyrosine phosphorylation in cells plated on the cell-binding fragment of fibronectin and in cells plated on a synthetic polymer containing multiple RGD sequences. We have identified one of the proteins of the 115-130-kD cluster as pp125FAK, a tyrosine kinase recently localized in focal adhesions (Schaller, M. D., C. A. Borgman, B. S. Cobb, R. R. Vines, A. B. Reynolds, and J. T. Parsons. 1992. Proc. Natl. Acad. Sci. USA. 89:5192). A second protein that becomes tyrosine phosphorylated in response to extracellular matrix adhesion is identified as paxillin, a 70-kD protein previously localized to focal adhesions. Treatment of cells with the tyrosine kinase inhibitor herbimycin A diminished the adhesion-induced tyrosine phosphorylation of these proteins and inhibited the formation of focal adhesions and stress fibers. These results suggest a role for integrin-mediated tyrosine phosphorylation in the organization of the cytoskeleton as cells adhere to the extracellular matrix.

Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction

Abstract: For determination of the physiological role and mechanism of vacuolar proteolysis in the yeast Saccharomyces cerevisiae, mutant cells lacking proteinase A, B, and carboxypeptidase Y were transferred from a nutrient medium to a synthetic medium devoid of various nutrients and morphological changes of their vacuoles were investigated. After incubation for 1 h in nutrient-deficient media, a few spherical bodies appeared in the vacuoles and moved actively by Brownian movement. These bodies gradually increased in number and after 3 h they filled the vacuoles almost completely. During their accumulation, the volume of the vacuolar compartment also increased. Electron microscopic examination showed that these bodies were surrounded by a unit membrane which appeared thinner than any other intracellular membrane. The contents of the bodies were morphologically indistinguishable from the cytosol; these bodies contained cytoplasmic ribosomes, RER, mitochondria, lipid granules and glycogen granules, and the density of the cytoplasmic ribosomes in the bodies was almost the same as that of ribosomes in the cytosol. The diameter of the bodies ranged from 400 to 900 nm. Vacuoles that had accumulated these bodies were prepared by a modification of the method of Ohsumi and Anraku (Ohsumi, Y., and Y. Anraku. 1981. J. Biol. Chem. 256:2079-2082). The isolated vacuoles contained ribosomes and showed latent activity of the cytosolic enzyme glucose-6-phosphate dehydrogenase. These results suggest that these bodies sequestered the cytosol in the vacuoles. We named these spherical bodies "autophagic bodies." Accumulation of autophagic bodies in the vacuoles was induced not only by nitrogen starvation, but also by depletion of nutrients such as carbon and single amino acids that caused cessation of the cell cycle. Genetic analysis revealed that the accumulation of autophagic bodies in the vacuoles was the result of lack of the PRB1 product proteinase B, and disruption of the PRB1 gene confirmed this result. In the presence of PMSF, wild-type cells accumulated autophagic bodies in the vacuoles under nutrient-deficient conditions in the same manner as did multiple protease-deficient mutants or cells with a disrupted PRB1 gene. As the autophagic bodies disappeared rapidly after removal of PMSF from cultures of normal cells, they must be an intermediate in the normal autophagic process. This is the first report that nutrient-deficient conditions induce extensive autophagic degradation of cytosolic components in the vacuoles of yeast cells.

Transforming growth factor-beta: recent progress and new challenges.

Abstract: I T is just 10 years since the peptide, transforming growth factor-/31 (TGF-~I) 1, was isolated from human platelets, human placenta, and bovine kidney and characterized as a discrete molecular entity, namely a 25-kD homodimer with a unique NH2-terminal sequence. Two years later this molecule was cloned, and subsequently four other closely related isoforms have been found in vertebrates; three isoforms (TGF-~s 1, 2, and 3) are known in man. TGF-~ can now be considered the prototype of a multifunctional cytokine, especially after the discovery that it could act both as an inhibitor and stimulator of cell replication, as well as control the synthesis of many of the components of the extracellular matrix (for reviews of the above see Roberts and Sporn, 1990; Massagu6, 1990; Moses et al., 1990; Sporn and Roberts, 1990). There has been immense progress in TGF-/~ research in the past two years. This mini-review will highlight some of these accomplishraents and indicate a few challenges for the future. We will confine this brief review to the TGF-/3s themselves and will not consider the extended TGF-B family, which includes the inhibins, activins, bone morphogenetic proteins, and related morphogenetic peptides, all of which are of increasing importance in many areas of cell biology, such as reproduction and development.

The three members of the selectin receptor family recognize a common carbohydrate epitope, the sialyl Lewis(x) oligosaccharide.

Abstract: The selectins (lectin-EGF-complement binding-cell adhesion molecules [LEC-CAMs]) are a family of mammalian receptors implicated in the initial interactions between leukocytes and vascular endothelia, leading to lymphocyte homing, platelet binding, and neutrophil extravasation. The three known selectins, L-selectin (leukocyte adhesion molecule-1 [LECAM-1]), E-selectin (endothelial-leukocyte adhesion molecule-1 [ELAM-1]), and P-selectin (GMP-140) share structural features that include a calcium-dependent lectin domain. The sialyl Lewis(x) carbohydrate epitope has been reported as a ligand for both E- and P-selectins. Although L-selectin has been demonstrated to bind to carbohydrates, structural features of potential mammalian carbohydrate ligand(s) have not been well defined. Using an ELISA developed with a sialyl Lewis(x)-containing glycolipid and an E-selectin-IgG chimera, we have demonstrated the direct binding of the L-selectin-IgG chimera to sialyl Lewis(x). This recognition was calcium dependent, and could be blocked by Mel-14 antibody but not by other antibodies. Recognition was confirmed by the ability of cells expressing the native L-selectin to adhere to immobilized sialyl Lewis(x). These data suggest that the sialyl Lewis(x) oligosaccharide may form the basis of a recognition domain common to all three selectins.

Enhanced synthesis of brain-derived neurotrophic factor in the lesioned peripheral nerve: different mechanisms are responsible for the regulation of BDNF and NGF mRNA.

Abstract: Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are molecules which regulate the development and maintenance of specific functions in different populations of peripheral and central neurons, amongst them sensory neurons of neural crest and placode origin. Under physiological conditions NGF is synthesized by peripheral target tissues, whereas BDNF synthesis is highest in the CNS. This situation changes dramatically after lesion of peripheral nerves. As previously shown, there is a marked rapid increase in NGF mRNA in the nonneuronal cells of the damaged nerve. The prolonged elevation of NGF mRNA levels is related to the immigration of activated macrophages, interleukin-1 being the most essential mediator of this effect. Here we show that transsection of the rat sciatic nerve also leads to a very marked increase in BDNF mRNA, the final levels being even ten times higher than those of NGF mRNA. However, the time-course and spatial pattern of BDNF mRNA expression are distinctly different. There is a continuous slow increase of BDNF mRNA starting after day 3 post-lesion and reaching maximal levels 3-4 wk later. These distinct differences suggest different mechanisms of regulation of NGF and BDNF synthesis in non-neuronal cells of the nerve. This was substantiated by the demonstration of differential regulation of these mRNAs in organ culture of rat sciatic nerve and Schwann cell culture. Furthermore, using bioassays and specific antibodies we showed that cultured Schwann cells are a rich source of BDNF- and ciliary neurotrophic factor (CNTF)-like neurotrophic activity in addition to NGF. Antisera raised against a BDNF-peptide demonstrated BDNF-immunoreactivity in pure cultured Schwann cells, but not in fibroblasts derived from sciatic nerve.

A novel endothelial-specific membrane protein is a marker of cell-cell contacts.

Abstract: mAbs were raised in mice against cultured human endothelial cells (EC) and screened by indirect immunofluorescence for their ability to stain intercellular contacts. One mAb denoted 7B4 was identified which, out of many cultured cell types, specifically decorated cultured human EC. The antigen recognized by mAb 7B4 is bound at the appositional surfaces of cultured EC only as they become confluent and is stably expressed at intercellular boundaries of confluent monolayers. EC recognition specificity was maintained when the antibody was assayed by immuno-histochemistry in tissue sections of many normal and malignant tissues and in blood vessels of different size and type. The antigen recognized by 7B4 was enriched at EC intercellular boundaries similarly in vitro and in situ. In vitro, addition of mAb 7B4 to confluent EC increased permeation of macromolecules across monolayers even without any obvious changes of cell morphology. In addition, when EC permeability was increased by agents such as thrombin, elastase, and TNF/gamma IFN, its distribution pattern at intercellular contact rims was severely altered. mAb 7B4 immunoprecipitated a major protein of 140 kD from metabolically and surface-labeled cultured EC extracts which appeared to be an integral membrane glycoprotein. On the basis of its distribution in cultured cells and in tissues in situ, 7B4 antigen is distinct from other described EC proteins enriched at intercellular contacts. NH2-terminal sequencing of the antigen, immunopurified from human placenta, and sequencing of peptides from tryptic peptide maps revealed identity to the cDNA deduced sequence of a recently identified new member of the cadherin family (Suzuki, S., K. Sano, and H. Tanihara. 1991. Cell Regul. 2:261-270.) These data indicate that 7B4 antigen is an endothelial-specific cadherin that plays a role in the organization of lateral endothelial junctions and in the control of permeability properties of vascular endothelium.

Integrin-dependent phosphorylation and activation of the protein tyrosine kinase pp125FAK in platelets.

Abstract: We have investigated mechanisms involved in integrin-mediated signal transduction in platelets by examining integrin-dependent phosphorylation and activation of a newly identified protein tyrosine kinase, pp125FAK (FAK, focal adhesion kinase). This kinase was previously shown to be localized in focal adhesions in fibroblasts, and to be phosphorylated on tyrosine in normal and Src-transformed fibroblasts. We show that thrombin and collagen activation of platelets causes an induction of tyrosine phosphorylation of pp125FAK and that pp125FAK molecules isolated from activated platelets display enhanced levels of phosphorylation in immune-complex kinase assays. pp125FAK was not phosphorylated on tyrosine after thrombin or collagen treatment of Glanzmann's thrombasthenic platelets deficient in the fibrinogen receptor GPIIb-IIIa, or of platelets pretreated with an inhibitory monoclonal antibody to GP IIb-IIIa. Fibrinogen binding to GP IIb-IIIa was not sufficient to induce pp125FAK phosphorylation because pp125FAK was not phosphorylated on tyrosine in thrombin-treated platelets that were not allowed to aggregate. These results indicate that tyrosine phosphorylation of pp125FAK is dependent on platelet aggregation mediated by fibrinogen binding to the integrin receptor GP IIb-IIIa. The induction of tyrosine phosphorylation of pp125FAK was inhibited in thrombin- and collagen-treated platelets preincubated with cytochalasin D, which prevents actin polymerization following activation. Under all of these conditions, there was a strong correlation between the induction of tyrosine phosphorylation of pp125FAK in vivo and stimulation of the phosphorylation of pp125FAK in vitro in immune-complex kinase assays. This study provides the first genetic evidence that tyrosine phosphorylation of pp125FAK is dependent on integrin-mediated events, and demonstrates that there is a strong correlation between tyrosine phosphorylation of pp125FAK in platelets, and the activation of pp125FAK-associated phosphorylating activity in vitro.

Cell surface receptors for herpes simplex virus are heparan sulfate proteoglycans.

Abstract: The role of cell surface heparan sulfate in herpes simplex virus (HSV) infection was investigated using CHO cell mutants defective in various aspects of glycosaminoglycan synthesis. Binding of radiolabeled virus to the cells and infection were assessed in mutant and wild-type cells. Virus bound efficiently to wild-type cells and initiated an abortive infection in which immediate-early or alpha viral genes were expressed, despite limited production of late viral proteins and progeny virus. Binding of virus to heparan sulfate-deficient mutant cells was severely impaired and mutant cells were resistant to HSV infection. Intermediate levels of binding and infection were observed for a CHO cell mutant that produced undersulfated heparan sulfate. These results show that heparan sulfate moieties of cell surface proteoglycans serve as receptors for HSV.

VIP21, a 21-kD membrane protein is an integral component of trans-Golgi-network-derived transport vesicles.

Abstract: In simple epithelial cells, apical and basolateral proteins are sorted into separate vesicular carriers before delivery to the appropriate plasma membrane domains. To dissect the putative sorting machinery, we have solubilized Golgi-derived transport vesicles with the detergent CHAPS and shown that an apical marker, influenza haemagglutinin (HA), formed a large complex together with several integral membrane proteins. Remarkably, a similar set of CHAPS-insoluble proteins was found after solubilization of a total cellular membrane fraction. This allowed the cloning of a cDNA encoding one protein of this complex, VIP21 (Vesicular Integral-membrane Protein of 21 kD). The transiently expressed protein appeared on the Golgi-apparatus, the plasma membrane and vesicular structures. We propose that VIP21 is a component of the molecular machinery of vesicular transport.

Lymphocyte CD44 binds the COOH-terminal heparin-binding domain of fibronectin.

Abstract: The lymphocyte-high endothelial venule (HEV) cell interaction is an essential element of the immune system, as it controls lymphocyte recirculation between blood and lymphoid organs in the body. This interaction involves an 85-95-kD class of lymphocyte surface glycoprotein(s), CD44. A subset of lymphocyte CD44 molecules is modified by covalent linkage to chondroitin sulfate (Jalkanen, S., M. Jalkanen, R. Bargatze, M. Tammi, and E. C. Butcher. 1988. J. Immunol. 141:1615-1623). In this work, we show that removal of chondroitin sulfate by chondroitinase treatment of lymphocytes or incubation of HEV with chondroitin sulfate does not significantly inhibit lymphocyte binding to HEV, suggesting that chondroitin sulfate is not involved in endothelial cell recognition of lymphocytes. Affinity-purified CD44 antigen was, on the other hand, observed to bind native Type I collagen fibrils, laminin, and fibronectin, but not gelatin. Binding to fibronectin was studied more closely, and it was found to be mediated through the chondroitin sulfate-containing form of the molecule. The binding site on fibronectin was the COOH-terminal heparin binding domain, because (a) the COOH-terminal heparin-binding fragment of fibronectin-bound isolated CD44 antigen; (b) chondroitin sulfate inhibited this binding; and (c) finally, the ectodomain of another cell surface proteoglycan, syndecan, which is known to bind the COOH-terminal heparin binding domain of fibronectin (Saunders, S., and M. Bernfield. 1988. J. Cell Biol. 106: 423-430), inhibited binding of CD44 both to intact fibronectin and to its heparin binding domain. Moreover, inhibition studies showed that binding of a lymphoblastoid cell line, KCA, to heparin binding peptides from COOH-terminal heparin binding fragment of fibronectin was mediated via CD44. These findings suggest that recirculating lymphocytes use the CD44 class of molecules not only for binding to HEV at the site of lymphocyte entry to lymphoid organs as reported earlier but also within the lymphatic tissue where CD44, especially the subset modified by chondroitin sulfate, is used for interaction with extracellular matrix molecules such as fibronectin.

Identification of a specific glycoprotein ligand for P-selectin (CD62) on myeloid cells.

Abstract: P-selectin (CD62, GMP-140, PADGEM), a Ca(2+)-dependent lectin on activated platelets and endothelium, functions as a receptor for myeloid cells by interacting with sialylated, fucosylated lactosaminoglycans. P-selectin binds to a limited number of protease-sensitive sites on myeloid cells, but the protein(s) that carry the glycans recognized by P-selectin are unknown. Blotting of neutrophil or HL-60 cell membrane extracts with [125I]P-selectin and affinity chromatography of [3H]glucosamine-labeled HL-60 cell extracts were used to identify P-selectin ligands. A major ligand was identified with an approximately 250,000 M(r) under nonreducing conditions and approximately 120,000 under reducing conditions. Binding of P-selectin to the ligand was Ca2+ dependent and was blocked by mAbs to P-selectin. Brief sialidase digestion of the ligand increased its apparent molecular weight; however, prolonged digestion abolished binding of P-selectin. Peptide:N-glycosidase F treatment reduced the apparent molecular weight of the ligand by approximately 3,000 but did not affect P-selectin binding. Western blot and immunodepletion experiments indicated that the ligand was not lamp-1, lamp-2, or L-selectin, which carry sialyl Le(x), nor was it leukosialin, a heavily sialylated glycoprotein of similar molecular weight. The preferential interaction of the ligand with P-selectin suggests that it may play a role in adhesion of myeloid cells to activated platelets and endothelial cells.

Cadherin-mediated cell-cell adhesion is perturbed by v-src tyrosine phosphorylation in metastatic fibroblasts.

Abstract: Rat 3Y1 cells acquire metastatic potential when transformed with v-src, and this potential is enhanced by double transformation with v-src and v-fos (Taniguchi, S., T. Kawano, T. Mitsudomi, G. Kimura, and T. Baba. 1986. Jpn. J. Cancer Res. 77:1193-1197). We compared the activity of cadherin cell adhesion molecules of normal 3Y1 cells with that of v-src transformed (SR3Y1) and v-src and v-fos double transformed (fosSR3Y1) 3Y1 cells. These cells expressed similar amounts of P-cadherin, and showed similar rates of cadherin-mediated aggregation under suspended conditions. However, the aggregates or colonies of these cells were morphologically distinct. Normal 3Y1 cells formed compacted aggregates in which cells are firmly connected with each other, whereas the transformed cells were more loosely associated, and could freely migrate out of the colonies. Overexpression of exogenous E-cadherin in these transformed cells had no significant effect on their adhesive properties. We then found that herbimycin A, a tyrosine kinase inhibitor, induced tighter cell-cell associations in the aggregates of the transformed cells. In contrast, vanadate, a tyrosine phosphatase inhibitor, inhibited the cadherin-mediated aggregation of SR3Y1 and fosSR3Y1 cells but had little effect on that of normal 3Y1 cells. These results suggest that v-src-mediated tyrosine phosphorylation perturbs cadherin function directly or indirectly, and the inhibition of tyrosine phosphorylation restores cadherin action to the normal state. We next studied tyrosine phosphorylation on cadherins and the cadherin-associated proteins, catenins. While similar amounts of catenins were expressed in all of these cells, the 98-kD catenin was strongly tyrosine phosphorylated only in SR3Y1 and fosSR3Y1 cells. Cadherins were also weakly tyrosine phosphorylated only in the transformed cells. The tyrosine phosphorylation of these proteins was enhanced by vanadate, and inhibited by herbimycin A. Thus, the tyrosine phosphorylation of the cadherin-catenin system itself might affect its function, causing instable cell-cell adhesion.

Divalent cation regulation of the function of the leukocyte integrin LFA-1.

Abstract: The integrin lymphocyte function-associated antigen-1 (LFA-1) expressed on T cells serves as a useful model for analysis of leukocyte integrin functional activity. We have assessed the role of divalent cations Mg2+, Ca2+, and Mn2+ in LFA-1 binding to ligand intercellular adhesion molecule-1 (ICAM-1) and induction of the divalent cation-dependent epitope recognized by mAb 24. Manganese strongly promoted both expression of the 24 epitope and T cell binding to ICAM-1 via LFA-1, suggesting that Mn2+ is able to directly alter the conformation of LFA-1 in a manner that favors ligand binding. Since Mn2+ also promotes functional activity of other integrins, parallels in mechanism of ligand binding may span the integrin family. In contrast, induction of 24 epitope expression by Mg2+ required removal of Ca2+ from T cell LFA-1 with EGTA. Furthermore, binding of mAb 24 to T cell LFA-1 in the presence of either Mn2+ or Mg2+ was found to be specifically inhibited by Ca2+, suggestive of a negative regulatory role for Ca2+ in the control of leukocyte integrin function. Analysis of T cell binding to ICAM-1 via LFA-1 in the presence of Mg2+ or Mn2+, confirmed that Ca2+ exerted inhibitory effects upon LFA-1 function. The implication of our findings is that Ca2+ bound with relatively high affinity to LFA-1 may serve to maintain an inactive state. Thus induction of function and 24 epitope expression may occur as a result of displacement of Ca2+ from leukocyte integrins or alternatively, such activators may be able to impose the required conformational change in the presence of bound Ca2+.

GTP-binding mutants of rab1 and rab2 are potent inhibitors of vesicular transport from the endoplasmic reticulum to the Golgi complex.

Abstract: We have examined the role of ras-related rab proteins in transport from the ER to the Golgi complex in vivo using a vaccinia recombinant T7 RNA polymerase virus to express site-directed rab mutants. These mutations are within highly conserved domains involved in guanine nucleotide binding and hydrolysis found in ras and all members of the ras superfamily. Substitutions in the GTP-binding domains of rab1a and rab1b (equivalent to the ras 17N and 116I mutants) resulted in proteins which were potent trans dominant inhibitors of vesicular stomatitis virus glycoprotein (VSV-G protein) transport between the ER and cis Golgi complex. Immunofluorescence analysis indicated that expression of rab1b121I prevented delivery of VSV-G protein to the Golgi stack, which resulted in VSV-G protein accumulation in pre-Golgi punctate structures. Mutants in guanine nucleotide exchange or hydrolysis of the rab2 protein were also strong trans dominant transport inhibitors. Analogous mutations in rab3a, rab5, rab6, and H-ras did not inhibit processing of VSV-G to the complex, sialic acid containing form diagnostic of transport to the trans Golgi compartment. We suggest that at least three members of the rab family (rab1a, rab1b, and rab2) use GTP hydrolysis to regulate components of the transport machinery involved in vesicle traffic between early compartments of the secretory pathway.

Surface structure and properties of plant seed oil bodies

Abstract: Storage triacylglycerols (TAG) in plant seeds are present in small discrete intracellular organelles called oil bodies. An oil body has a matrix of TAG, which is surrounded by phospholipids (PL) and alkaline proteins, termed oleosins. Oil bodies isolated from mature maize (Zea mays) embryos maintained their discreteness, but coalesced after treatment with trypsin but not with phospholipase A2 or C. Phospholipase A2 or C exerted its activity on oil bodies only after the exposed portion of oleosins had been removed by trypsin. Attempts were made to reconstitute oil bodies from their constituents. TAG, either extracted from oil bodies or of a 1:2 molar mixture of triolein and trilinolein, in a dilute buffer were sonicated to produce droplets of sizes similar to those of oil bodies; these droplets were unstable and coalesced rapidly. Addition of oil body PL or dioleoyl phosphatidylcholine, with or without charged stearylamine/stearic acid, or oleosins, to the medium before sonication provided limited stabilization effects to the TAG droplets. High stability was achieved only when the TAG were sonicated with both oil body PL (or dioleoyl phosphatidylcholine) and oleosins of proportions similar to or higher than those in the native oil bodies. These stabilized droplets were similar to the isolated oil bodies in chemical properties, and can be considered as reconstituted oil bodies. Reconstituted oil bodies were also produced from TAG of a 1:2 molar mixture of triolein and trilinolein, dioleoyl phosphatidylcholine, and oleosins from rice (Oryza sativa), wheat (Triticum aestivum), rapeseed (Brassica napus), soybean (Glycine max), or jojoba (Simmondsia chinensis). It is concluded that both oleosins and PL are required to stabilize the oil bodies and that oleosins prevent oil bodies from coalescing by providing steric hindrance. A structural model of an oil body is presented. The current findings on seed oil bodies could be extended to the intracellular storage lipid particles present in diverse organisms.

Transforming growth factor-beta 1 in the rat brain: increase after injury and inhibition of astrocyte proliferation.

Abstract: Transforming growth factor-beta 1 (TGF-beta 1) has been shown to up-regulate the synthesis of nerve growth factor (NGF) in cultured rat astrocytes and in neonatal brain in vivo (Lindholm, D., B. Hengerer, F. Zafra, and H. Thoenen. 1990. NeuroReport. 1:9-12). Here we show that mRNA encoding TGF-beta 1 increased in rat cerebral cortex after a penetrating brain injury. The level of NGF mRNA is also transiently increased after the brain trauma, whereas that of brain-derived neurotrophic factor remained unchanged. In situ hybridization experiments showed a strong expression of TGF-beta 1 4 d after the lesion in cells within and in the vicinity of the wound. Staining of adjacent sections with OX-42 antibodies, specific for macrophages and microglia/brain macrophages, revealed a similar pattern of positive cells, suggesting that invading macrophages, and perhaps reactive microglia, are the source of TGF-beta 1 in injured brain. Both astrocytes and microglia express TGF-beta 1 in culture, and TGF-beta 1 mRNA levels in astrocytes are increased by various growth factors, including FGF, EGF, and TGF-beta itself. TGF-beta 1 is a strong inhibitor of astrocyte proliferation and suppresses the mitotic effects of FGF and EGF on astrocytes. The present results indicate that TGF-beta 1 expressed in the lesioned brain plays a role in nerve regeneration by stimulating NGF production and by controlling the extent of astrocyte proliferation and scar formation.

Isometric contraction by fibroblasts and endothelial cells in tissue culture: a quantitative study.

Abstract: We have used an isometric force transducer to study contraction of two types of nonmuscle cells in tissue culture. This method permits the quantitative measurement of contractile force generated by cells of defined type under the influence of external agents while allowing detailed morphological observation. Chick embryo fibroblasts (CEF), which form a contractile network inside a collagen matrix, and human umbilical vein endothelial cells (HUVE), which are located in a monolayer on the surface of the collagen matrix, were studied. CEF and HUVE in 10% FCS produce a substantial tension of 4.5 +/- 0.2 x 10(4) dynes/cm2 and 6.1 x 10(4) dynes/cm2, respectively. Both cell types contract when stimulated with thrombin, generating a force per cell cross-sectional area of approximately 10(5) dynes/cm2, a value approximately an order of magnitude less than smooth muscle. The integrity of the actin cytoskeleton is essential for force generation, as disruption of actin microfilaments with cytochalasin D results in a rapid disappearance of force. Intact microtubules appear to reduce isometric force exerted by CEF, as microtubule-disrupting drugs result in increased tension. Contraction by HUVE precedes a dramatic rearrangement of actin microfilaments from a circumferential ring to stress fibers.

Dynamic organization of DNA replication in mammalian cell nuclei: spatially and temporally defined replication of chromosome-specific alpha-satellite DNA sequences.

Abstract: Five distinct patterns of DNA replication have been identified during S-phase in asynchronous and synchronous cultures of mammalian cells by conventional fluorescence microscopy, confocal laser scanning microscopy, and immunoelectron microscopy. During early S-phase, replicating DNA (as identified by 5-bromodeoxyuridine incorporation) appears to be distributed at sites throughout the nucleoplasm, excluding the nucleolus. In CHO cells, this pattern of replication peaks at 30 min into S-phase and is consistent with the localization of euchromatin. As S-phase continues, replication of euchromatin decreases and the peripheral regions of heterochromatin begin to replicate. This pattern of replication peaks at 2 h into S-phase. At 5 h, perinucleolar chromatin as well as peripheral areas of heterochromatin peak in replication. 7 h into S-phase interconnecting patches of electron-dense chromatin replicate. At the end of S-phase (9 h), replication occurs at a few large regions of electron-dense chromatin. Similar or identical patterns have been identified in a variety of mammalian cell types. The replication of specific chromosomal regions within the context of the BrdU-labeling patterns has been examined on an hourly basis in synchronized HeLa cells. Double labeling of DNA replication sites and chromosome-specific alpha-satellite DNA sequences indicates that the alpha-satellite DNA replicates during mid S-phase (characterized by the third pattern of replication) in a variety of human cell types. Our data demonstrates that specific DNA sequences replicate at spatially and temporally defined points during the cell cycle and supports a spatially dynamic model of DNA replication.

Alzheimer-like paired helical filaments and antiparallel dimers formed from microtubule-associated protein tau in vitro.

Abstract: Recent evidence from several laboratories shows that the paired helical filaments of Alzheimer's disease brains consist mainly of the protein tau in an abnormally phosphorylated form, but the mode of assembly is not understood. Here we use EM to study several constructs derived from human brain tau and expressed in Escherichia coli. All constructs or tau isoforms are rodlike molecules with a high tendency to dimerize in an antiparallel fashion, as shown by antibody labeling and chemical crosslinking. The length of the rods is largely determined by the region of internal repeats that is also responsible for microtubule binding. One unit length of the repeat domain (three or four repeats) is around 22-25 nm, comparable to the cross-section of Alzheimer PHF cores. Constructs corresponding roughly to the repeat region of tau can form synthetic paired helical filaments resembling those from Alzheimer brain tissue. A similar self-assembly occurs with the chemically cross-linked dimers. In both cases there is no need for phosphorylation of the protein.

Developmental changes in heparan sulfate expression: in situ detection with mAbs.

Abstract: Two mAbs that are specific for heparan sulfate-related epitopes have been raised and used to analyze the cellular and tissular distribution of this glycosaminoglycan during development. mAb 10E4 reacts with an epitope that occurs in native heparan sulfate chains and that is destroyed by N-desulfation of the glycosaminoglycan. The antibody does not react with hyaluronate, chondroitin sulfate, or DNA, and reacts only poorly with heparin. The reactivity of proteoglycan extracts or tissue sections with the 10E4 antibody is completely abolished by heparitinase, but is only partially affected by heparinase. mAb 3G10, in contrast, reacts only with heparitinase-treated heparan sulfate chains, proteoglycans, or tissue sections. The 3G10 epitope is destroyed by treatment with mercuric acetate, which indicates that the desaturated uronate generated by the lyase is essential for the reactivity of the antibody. The 3G10 epitope is not generated by treating heparan sulfate proteoglycans with heparinase or chondroitin sulfate proteoglycans with chondroitin sulfate lyases, which indicates that the 3G10 antibody recognizes desaturated uronates that occur in specific structural contexts. The antibody 10E4 and, after heparitinase treatment, the antibody 3G10 decorate the surfaces of many cell types and the extracellular matrix in proximity of the cells, in particular, the basement membranes. The analysis of embryonic and adult tissues reveals important temporal and regional differences in the abundance of the 10E4 and 3G10 epitopes at these sites. Moreover, the staining pattern of the two antibodies is not always superimposable, which is indicative of regional differences in the exposure or structure of the tissular heparan sulfates. As a whole the results suggest that heparan sulfate abounds at sites of active morphogenesis and that the expression of this glycosaminoglycan is developmentally regulated.

Tissue and cellular distribution of the extended family of protein kinase C isoenzymes

Abstract: Polyclonal isoenzyme-specific antisera were developed against four calcium-independent protein kinase C (PKC) isoenzymes (delta, epsilon, epsilon', and zeta) as well as the calcium-dependent isoforms (alpha, beta I, beta II, and gamma). These antisera showed high specificities, high titers, and high binding affinities (3-370 nM) for the peptide antigens to which they were raised. Each antiserum detected a species of the predicted molecular weight by Western blot that could be blocked with the immunizing peptide. PKC was sequentially purified from rat brain, and the calcium-dependent forms were finally resolved by hydroxyapatite chromatography. Peak I reacted exclusively with antisera to PKC gamma, peak II with PKC beta I and -beta II, and peak III with PKC alpha. These same fractions, however, were devoid of immunoreactivity for the calcium-independent isoenzymes. The PKC isoenzymes demonstrated a distinctive tissue distribution when evaluated by Western blot and immunocytochemistry. PCK delta was present in brain, heart, spleen, lung, liver, ovary, pancreas, and adrenal tissues. PKC epsilon was present in brain, kidney, and pancreas, whereas PKC epsilon' was present predominantly in brain. PKC zeta was present in most tissues, particularly the lung, brain, and liver. Both PKC delta and PKC zeta showed some heterogeneity of size among the different tissues. PKC alpha was present in all organs and tissues examined. PKC beta I and -beta II were present in greatest amount in brain and spleen. Although the brain contained the most PKC gamma immunoreactivity, some immunostaining was also seen in adrenal tissue. These studies provide the first evidence of selective organ and tissue distributions of the calcium-independent PKC isoenzymes.

Coordinated expression of extracellular matrix-degrading proteinases and their inhibitors regulates mammary epithelial function during involution

Abstract: Extracellular matrix (ECM) plays an important role in the maintenance of mammary epithelial differentiation in culture. We asked whether changes in mouse mammary specific function in vivo correlate with changes in the ECM. We showed, using expression of beta-casein as a marker, that the temporal expression of ECM-degrading proteinases and their inhibitors during lactation and involution are inversely related to functional differentiation. After a lactation period of 9 d, mammary epithelial cells maintained beta-casein expression up to 5 d of involution. Two metalloproteinases, 72-kD gelatinase (and its 62-kD active form), and stromelysin, and a serine proteinase tissue plasminogen activator were detected by day four of involution, and maintained expression until at least day 10. The expression of their inhibitors, the tissue inhibitor of metalloproteinases (TIMP) and plasminogen activator inhibitor-1, preceded the onset of ECM-degrading proteinase expression and was detected by day two of involution, and showed a sharp peak of expression centered on days 4-6 of involution. When involution was accelerated by decreasing lactation to 2 d, there was an accelerated loss of beta-casein expression evident by day four and a shift in expression of ECM-remodeling proteinases and inhibitors to a focus at 2-4 d of involution. To further extend the correlation between mammary-specific function and ECM remodeling we initiated involution by sealing just one gland in an otherwise hormonally sufficient lactating animal. Alveoli in the sealed gland contained casein for at least 7 d after sealing, and closely resembled those in a lactating gland. The relative expression of TIMP in the sealed gland increased, whereas the expression of stromelysin was much lower than that of a hormone-depleted involuting gland, indicating that the higher the ratio of TIMP to ECM-degrading proteinases the slower the process of involution. To test directly the functional role of ECM-degrading proteinases in the loss of tissue-specific function we artificially perturbed the ECM-degrading proteinase-inhibitor ratio in a normally involuting gland by maintaining high concentrations of TIMP protein with the use of surgically implanted slow-release pellets. In a concentration-dependent fashion, involuting mammary glands that received TIMP implants maintained high levels of casein and delayed alveolar regression. These data suggest that the balance of ECM-degrading proteinases and their inhibitors regulates the organization of the basement membrane and the tissue-specific function of the mammary gland.(ABSTRACT TRUNCATED AT 400 WORDS)

Soluble L-selectin is present in human plasma at high levels and retains functional activity.

Abstract: L-selectin expressed by granulocytes, lymphocytes, and monocytes is responsible for initial leukocyte attachment to inflamed endothelium and high endothelial venules of peripheral lymph nodes. After leukocyte activation in vitro, L-selectin is rapidly shed from the cell surface. In this study, shed L-selectin (sL-selectin) from both lymphocytes and neutrophils was demonstrated to be present in high levels in human plasma by Western blot analysis and using a quantitative ELISA. In serum from normal human blood donors, a mean sL-selectin level of 1.6 +/- 0.8 micrograms/ml (n = 63) was found by ELISA. In addition, semipurified sL-selectin from plasma inhibited L-selectin-specific attachment of lymphocytes to cytokine-activated endothelium in a dose-dependent manner. L-selectin-dependent leukocyte attachment was completely inhibited at sL-selectin concentrations of 8-15 micrograms/ml, while physiological concentrations of sL-selectin caused a small but consistent inhibition of lymphocyte attachment. sL-selectin in plasma also inhibited anti-L-selectin mAb (2-5 micrograms/ml) binding to the surface of leukocytes. Interestingly, one epitope present within the EGF-like domain of L-selectin was lost in sL-selectin, suggesting a conformational change in the structure of the receptor after shedding. The presence of serum sL-selectin with functional activity indicates a potential role for sL-selectin in the regulation of leukocyte attachment to endothelium.

Control of microtubule dynamics and length by cyclin A- and cyclin B-dependent kinases in Xenopus egg extracts.

Abstract: In eukaryotic cells, the onset of mitosis involves cyclin molecules which interact with proteins of the cdc2 family to produce active kinases. In vertebrate cells, cyclin A dependent kinases become active in S- and pro-phases, whereas a cyclin B-dependent kinase is mostly active in metaphase. It has recently been shown that, when added to Xenopus egg extracts, bacterially produced A- and B-type cyclins associate predominantly with the same kinase catalytic subunit, namely p34cdc2, and induce its histone H1 kinase activity with different kinetics. Here, we show that in the same cell free system, both the addition of cyclin A and cyclin B changes microtubule behavior. However, the cyclin A-dependent kinase does not induce a dramatic shortening of centrosome-nucleated microtubules whereas the cyclin B-dependent kinase does, as previously reported. Analysis of the parameters of microtubule dynamics by fluorescence video microscopy shows that the dramatic shortening induced by the cyclin B-dependent kinase is correlated with a several fold increase in catastrophe frequency, an effect not observed with the cyclin A-dependent kinase. Using a simple mathematical model, we show how the length distributions of centrosome-nucleated microtubules relate to the four parameters that describe microtubule dynamics. These four parameters define a threshold between unlimited microtubule growth and the establishment of steady-state dynamics, which implies that well defined steady-state length distributions can be produced by regulating precisely the respective values of the dynamical parameters. Moreover, the dynamical model predicts that increasing catastrophe frequency is more efficient than decreasing the rescue frequency to reduce the average steady state length of microtubules. These theoretical results are quantitatively confirmed by the experimental data.

Two Saccharomyces cerevisiae kinesin-related gene products required for mitotic spindle assembly.

Abstract: Two Saccharomyces cerevisiae genes, CIN8 and KIP1 (a.k.a. CIN9), were identified by their requirement for normal chromosome segregation. Both genes encode polypeptides related to the heavy chain of the microtubule-based force-generating enzyme kinesin. Cin8p was found to be required for pole separation during mitotic spindle assembly at 37 degrees C, although overproduced Kip1p could substitute. At lower temperatures, the activity of at least one of these proteins was required for cell viability, indicating that they perform an essential but redundant function. Cin8p was observed to be a component of the mitotic spindle, colocalizing with the microtubules that lie between the poles. Taken together, these findings suggest that these proteins interact with spindle microtubules to produce an outwardly directed force acting upon the poles.

The hyaluronan receptor (CD44) participates in the uptake and degradation of hyaluronan.

Abstract: The hyaluronan receptor belongs to the polymorphic family of CD44 glycoproteins, which have been implicated in a variety of cellular functions including adhesion to hyaluronan and collagen, the binding of lymphocytes to high endothelial cells during extravasation, and conferring metastatic potential to carcinoma cells. Here, we demonstrate that the receptor also participates in the uptake and degradation of hyaluronan by both transformed fibroblasts (SV-3T3 cells) and alveolar macrophages. These cells were incubated with isotopically labeled hyaluronan for various periods of time, and the extent of degradation was determined by either molecular-sieve chromatography or centrifugation through Centricon 30 microconcentrators. The macrophages degraded the hyaluronan at a faster rate than the SV-3T3 cells, which may reflect the fact that they contained a greater number of receptors. More importantly, in both cell types, the degradation of hyaluronan was specifically blocked by antibodies directed against the receptor. However, the receptor by itself did not have the ability to degrade hyaluronan, since preparations of SV-3T3 membranes containing the receptor did not break down hyaluronan. Subsequent experiments revealed that macrophages can internalize fluorescein-tagged hyaluronan, and this process was blocked by antibodies against the receptor. Furthermore, the subsequent degradation of hyaluronan was inhibited by agents that block the acidification of lysosomes (chloroquine and NH4Cl). Thus, the most likely explanation for these results is that the receptor mediates the uptake of hyaluronan into the cell where it can be degraded by acid hydrolases in lysosomes. The ability of cells expressing the receptor to degrade hyaluronan may be important during tissue morphogenesis and cell migration.

Mechanisms of actin rearrangements mediating platelet activation.

Abstract: The detergent-insoluble cytoskeleton of the resting human blood platelet contains approximately 2,000 actin filaments approximately 1 micron in length crosslinked at high angles by actin-binding protein and which bind to a spectrin-rich submembrane lamina (Fox, J., J. Boyles, M. Berndt, P. Steffen, and L. Anderson. 1988. J. Cell Biol. 106:1525-1538; Hartwig, J., and M. DeSisto. 1991. J. Cell Biol. 112:407-425). Activation of the platelets by contact with glass results within 30 s in a doubling of the polymerized actin content of the cytoskeleton and the appearance of two distinct new actin structures: bundles of long filaments within filopodia that end at the filopodial tips (filopodial bundles) and a circumferential zone of orthogonally arrayed short filaments within lamellipodia (lamellipodial network). Neither of these structures appears in cells exposed to glass with cytochalasin B present; instead the cytoskeletons have numerous 0.1-0.3-microns-long actin filament fragments attached to the membrane lamina. With the same time course as the glass-induced morphological changes, cytochalasin-sensitive actin nucleating activity, initially low in cytoskeletons of resting platelets, increases 10-fold in cytoskeletons of thrombin-activated platelets. This activity decays with a time course consistent with depolymerization of 0.1-0.3-microns-long actin filaments, and phalloidin inhibits this decay. Cytochalasin-insensitive and calcium-dependent nucleation activity also increases markedly in platelet extracts after thrombin activation of the cells. Prevention of the rise in cytosolic Ca2+ normally associated with platelet activation with the permeant Ca2+ chelator, Quin-2, inhibits formation of lamellipodial networks but not filopodial bundles after glass contact and reduces the cytochalasin B-sensitive nucleation activity by 60% after thrombin treatment. The filopodial bundles, however, are abnormal in that they do not end at the filopodial tips but form loops and return to the cell body. Addition of calcium to chelated cells restores lamellipodial networks, and calcium plus A23187 results in cytoskeletons with highly fragmented actin filaments within seconds. Immunogold labeling with antibodies against gelsolin reveals gelsolin molecules at the ends of filaments attached to the submembrane lamina of resting cytoskeletons and at the ends of some filaments in the lamellipodial networks and filopodial bundles of activated cytoskeletons. Addition of monomeric actin to myosin subfragment 1-labeled activated cytoskeletons leads to new (undecorated) filament growth off the ends of filaments in the filopodial bundles and the lamellipodial network. The simplest explanation for these findings is that gelsolin caps the barbed ends of the filaments in the resting platelet. Uncapping some of these filaments after activation leads to filopodial bundles.(ABSTRACT TRUNCATED AT 400 WORDS)