bluuebunny/crossref_metadata_2025 huggingface dataset:
Association of Rap1a and Rap1b proteins with late endocytic/phagocytic compartments and Rap2a with the Golgi complex
Authors: Pizon Véronique , Desjardins Michel , Bucci Cecilia , Parton Robert G. , Zerial Marino
Published: April 2021

Among the small GTPases of the Ras family, Rap proteins exhibit the highest homology with p21Ras. The four Rap proteins so far identified constitute two subgroups, comprising the Rap1(A,B) and the Rap2(A,B) proteins. The intracellular location of Rap1A, Rap1B and Rap2A proteins was investigated in mammalian cells by confocal immunofluorescence microscopy. Using a specific anti-Rap1 affinity-purified antibody, both Rap1A and Rap1B proteins were localized to late endocytic compartments (late endosomes/lysosomes) in fibroblasts. The localization of the Rap1A and B proteins transiently overexpressed with the vaccinia T7 system was identical to that observed for endogenous Rap1 proteins. In contrast, epitope-tagged Rap2A protein colocalized with several markers of the Golgi complex, thus indicating that its site of function was distinct from that of Rap1A. In addition, morphological and subcellular fractionation studies provided evidence for the association of Rap1 proteins with phagosomes displaying biochemical features of late endocytic structures in J774 macrophages. Thus, the localization of Rap1A and Rap1B implicates their involvement in late endocytic/phagocytic processes.
Comparison of human CAP and CAP2, homologs of the yeast adenylyl cyclase-associated proteins
Authors: Yu Gang , Swiston John , Young Dallan
Published: April 2021

We previously reported the identification of human CAP, a protein that is related to the Saccharomyces cerevisiae and Schizosaccharomyces pombe adenylyl cyclase-associated CAP proteins. The two yeast CAP proteins have similar functions: the N-terminal domains are required for the normal function of adenylyl cyclase, while loss of the C-terminal domains result in morphological and nutritional defects that are unrelated to the cAMP pathways. We have amplified and cloned cDNAs from a human glioblastoma library that encode a second CAP-related protein, CAP2. The human CAP and CAP2 proteins are 64% identical. Expression of either human CAP or CAP2 in S. cerevisiae cap− strains suppresses phenotypes associated with deletion of the C-terminal domain of CAP, but does not restore hyper-activation of adenylyl cyclase by RAS2val19. Similarly, expression of either human CAP or CAP2 in S. pombe cap− strains suppresses the morphological and temperature-sensitive phenotypes associated with deletion of the C-terminal domain of CAP in this yeast. In addition, expression of human CAP, but not CAP2, suppresses the propensity to sporulate due to deletion of the N-terminal domain of CAP in S. pombe. This latter observation suggests that human CAP restores normal adenylyl cyclase activity in S. pombe cap− cells. Thus, functional properties of both N-terminal and C-terminal domains are conserved between the human and S. pombe CAP proteins.
Effects of activators (SC-9 and OAG) and inhibitors (staurosporine and H-7) of protein kinase C on the proliferation of mouse epidermal melanoblasts in serum-free culture
Authors: Hirobe Tomohisa
Published: April 2021

Mouse epidermal melanoblasts preferentially proliferated from disaggregated epidermal cell suspensions derived from newborn mouse skin in a serum-free melanoblast proliferation medium containing dibutyryl adenosine 3′:5′cyclic monophosphate and basic fibroblast growth factor. After 12 days, almost all of the keratinocytes died and pure cultures of melanoblasts (∼80%) and melanocytes (∼20%) could be obtained. No further proliferation of melanoblasts was observed in the melanoblast proliferation medium. In order to clarify the role of protein kinase C, which is important for the regulation of cellular proliferation, activators or inhibitors of protein kinase C were added to the culture of the quiescent melanoblasts at 12 days. The proliferation of melanoblasts was induced by an activator of protein kinase C, N-(6-phenylhexyl)-5-chloro-1-naphthalene-sulfonamide or 1-oleoyl-2-acetyl-glycerol. It was also induced by an inhibitor of protein kinase C, staurosporine or 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine. However, the melanoblasts failed to proliferate in the melanoblast proliferation medium supplemented with both the activator and the inhibitor. These results suggest that the proliferation of mouse epidermal melanoblasts in culture is regulated by activating or inhibiting the activity of protein kinase C.
Characterization of cadherin-4 and cadherin-5 reveals new aspects of cadherins
Authors: Tanihara Hidenobu , Kido Mitsuru , Obata Shuichi , Heimark Ronald L. , Davidson Mari , John Tom St , Suzuki Shintaro
Published: April 2021

Several properties of cadherin-4 and cadherin-5 were char- acterized by using the cDNA transfection approach. The proteins of both cadherins had a relative molecular mass of about 130 kDa and were present at the cell periphery, especially at cell-cell contact sites. These cadherins were easily digested with trypsin, and Ca2+ protected cadherin- 4, but not cadherin-5, from the digestion. In immunopre- cipitation, cadherin-4 co-precipitated with two major proteins of 105 kDa and 95 kDa, respectively. The 105 kDa and the 95 kDa proteins are likely to correspond to α- and β-catenins. Cadherin-5 co-precipitated with only one major protein of 95 kDa, but seems to associate with the 105 kDa protein. On the other hand, plakoglobin or γ-catenin did not co-precipitate well with either cadherin-4 or cadherin- 5 in immunoprecipitation, but plakoglobin also appears to associate weakly with these cadherins. Cadherin-4 trans- fectants aggregated within 30 minutes in a cell aggregation assay, but cadherin-5 transfectants did not aggregate under the same conditions. Furthermore, the transfectants of chimeric cadherin-4 with cadherin-5 cytoplasmic domain showed cell aggregation activity comparable to that of wild- type cadherin-4 transfectants, whereas the transfectants of chimeric cadherin-5 with cadherin-4 cytoplasmic domain did not show appreciable cell aggregation, suggesting that the extracellular domains of cadherins, in conjunction with their cytoplasmic domains, play an important role in cell aggregation activity. These results show that cadherin-4 is very similar to the classical cadherins, whereas cadherin-5 is functionally as well as structurally distinct from classical cadherins.
Sites in human nuclei where damage induced by ultraviolet light is repaired: localization relative to transcription sites and concentrations of proliferating cell nuclear antigen and the tumour suppressor protein, p53
Authors: Jackson Dean A. , Hassan A. Bass , Errington Rachel J. , Cook Peter R.
Published: April 2021

The repair of damage induced in DNA by ultraviolet light involves excision of the damaged sequence and synthesis of new DNA to repair the gap. Sites of such repair synthesis were visualized by incubating permeabilized HeLa or MRC-5 cells with the DNA precursor, biotin-dUTP, in a physiological buffer; then incorporated biotin was immunolabelled with fluorescent antibodies. Repair did not take place at sites that reflected the DNA distribution; rather, sites were focally concentrated in a complex pattern. This pattern changed with time; initially intense repair took place at transcriptionally active sites but when transcription became inhibited it continued at sites with little transcription. Repair synthesis in vitro also occurred in the absence of transcription. Repair sites generally contained a high concentration of proliferating cell nuclear antigen but not the tumour-suppressor protein, p53.
Modulation of α-actinin levels affects cell motility and confers tumorigenicity on 3T3 cells
Authors: Glück Ursula , Ben-Ze’ev Avri
Published: April 2021

α-Actinin is an abundant actin crosslinking protein, also localized at adherens type junctions. In adhesion plaques, α-actinin can link the actin filaments to integrin via vinculin and talin, or directly by binding to the cytoplasmic domain of β1-integrin. The expression of α-actinin is rapidly elevated in growth-activated quiescent cells, and is reduced in SV40-transformed 3T3 cells and various differentiating cell types (reviewed by Glück, U., Kwiatkowski, D. J. and Ben-Ze’ev, A. Proc. Nat. Acad. Sci. USA 90, 383-387, 1993). To study the effect of changes in α-actinin levels on cell behavior, α-actinin expression was elevated in 3T3 cells by transfection with a full-length human nonmuscle α-actinin cDNA. To suppress α-actinin levels, 3T3 cells were transfected with an antisense α-actinin cDNA construct. Cells overexpressing α-actinin by 40-60% displayed a significant reduction in cell motility, as demonstrated by their slower locomotion into an artificial wound, and by forming shorter phagokinetic tracks on colloidal gold-coated substrata. 3T3 cells in which the expression of α-actinin was reduced to 25-60% of control levels, after antisense α-actinin transfection, had an increased cell motility. Moreover, such α-actinin-deficient 3T3 cells formed tumors upon injection into nude mice. The results demonstrate that modulations in α-actinin expression can affect, in a major way, the motile and tumorigenic properties of cells, and support the view that decreased α-actinin expression could be a common regulatory pathway to malignant transformation of 3T3 cells.
The mode of anchorage to the cell surface determines both the function and the membrane location of Thy-1 glycoprotein
Authors: Tiveron M.-C. , Nosten-Bertrand M. , Jani H. , Garnett D. , Hirst E. M. A. , Grosveld F. , Morris R. J.
Published: April 2021

The surface glycoprotein, Thy-1, when expressed by transfection in NG115/401L neural cells, inhibits their neurite outgrowth over astrocytes. We have investigated the role of the glycosylphosphatidylinositol anchor of Thy-1 in this inhibition. Hybrid molecules, in which the lipid anchor was replaced by polypeptide transmembrane domains, were expressed by transfection. Lines expressing Thy-1 with the transmembrane and full cytoplasmic domains of NCAM-140, or with the transmembrane and truncated cytoplasmic domain of CD8, were not inhibited in their ability to extend neurites over astrocytes. Truncation of the cyto-plasmic domain of NCAM-140 to just two amino acids, however, produced a transmembrane form of Thy-1 that, when expressed at high levels, inhibited neurite outgrowth. All forms of Thy-1 were concentrated in clusters that occurred primarily on fine filopodia. In double transfectants expressing normal Thy-1 and Thy-1 with the full NCAM cytoplasmic tail, the clusters of each form were separate, with no instances of the transmembrane form being found within the clusters of lipid-anchored Thy-1. Thy-1 with the two-amino-acid cytoplasmic domain of NCAM also occurred in clusters separate from those occupied by lipid-anchored Thy-1, but substantial ‘invasion’ of the clusters of normal Thy-1 by this trans-membrane construct occurred. We suggest that the ability of this hybrid protein to enter the lipid-anchored clusters enables it to activate the signalling pathways that normal Thy-1 uses. Thus the membrane anchor, in targetting Thy-1 to different microdomains on the cell surface, determines its ability to inhibit neurite outgrowth on astrocytes.
Mitotic arrest with anti-microtubule agents or okadaic acid is associated with increased glycoprotein terminal GlcNAc’s
Authors: Chou Chih-Fong , Omary M. Bishr
Published: April 2021

The two major intermediate filament glycoproteins in human simple epithelia are keratins 8 and 18 (K8/18). A dramatic increase in terminal N-acetylglucosamine (GlcNAc) residues in K8/18 was previously noted after arresting cells in G2/M using anti-microtubule agents. Here we use in vitro galactosylation to show that increased terminal GlcNAc’s is a general phenomenon that occurs in glycoproteins isolated from nuclear and plasma membrane fractions after cells are arrested in mitosis using colcemid, nocodazole, or okadaic acid. All three agents also resulted in a hyperphosphorylated form of K8 as determined by phosphatase treatment and tryptic phosphopeptide mapping. The altered glycosylation was found to be independent of microtubule disassembly, and was not directly related to the G2/M phase of the cell cycle after aphidicolin synchronization. Staurosporine (1 μM) inhibited K8/18 phosphorylation in okadaic acid- or nocodazole-treated cells, and inhibited the increase in K8/18 glycosylation without inhibiting the increase in terminal GlcNAc’s of membrane-associated glycoproteins. In contrast, brefeldin A resulted in a dramatic increase in terminal GlcNAc’s of membrane-associated but not intermediate filament proteins. Golgi complex-related staining using anti-β-COP antibody showed significant fragmentation under conditions associated with altered membrane protein glycosylation. Our results suggest that Golgi disruption may be involved in the observed increase in terminal GlcNAc’s of membrane but not intermediate filament glycoproteins. The mechanism of increased glycoprotein terminal GlcNAc’s in association with mitotic arrest appears to be distinct for intermediate filaments and membrane-associated proteins, and in the case of intermediate filament proteins, phosphorylation may play an important role. Some of the effects of agents that induce mitotic arrest may be mediated by glycosylation changes.
A homologue of the human regulator of mitotic spindle assembly protein (RMSA-1) is present in crane fly and is associated with meiotic chromosomes
Authors: Yeo Jing-Ping , Forer Arthur , Toh Ban-Hock
Published: April 2021

In a previous study, we have shown that a newly identified chromosomal protein, RMSA-1 (Regulator of Mitotic Spindle Assembly-1), identified and cloned using a human autoimmune serum, is essential for mitotic spindle assembly; we proposed that RMSA-1 was a previously unknown physiological substrate for cdc 2 kinase. In the present study, we show that this protein is present in crane fly and is associated with the chromosomes of spermatocytes.

A 31 kDa molecule in extracts from crane-fly nuclei, isolated from larvae, pupae and adults, reacts with affinity-purified anti-RMSA-1 autoantibody, shown by immunoblotting. The autoantibody reacts, as shown by immunofluorescence, with crane-fly spermatocyte chromosomes in prophase through anaphase of both meiosis-1 and meiosis-II but does not react with preprophase or telophase nuclei or with spermatid nuclei. In all meiotic stages, the crane-fly sex chromosomes stain more intensely than the autosomes. We conclude that, since RMSA-1 is present in insect and mammalian cells, it is conserved across a variety of animal species. Further, since RMSA-1 binds to chromosomes in meiotic cells, it also may be essential for assembly of the meiotic spindle.
Molecular phylogeny of the kinesin family of microtubule motor proteins
Authors: Goodson Holly V. , Kang Sang Joon , Endow Sharyn A.
Published: April 2021

The rapidly expanding kinesin family of microtubule motor proteins includes proteins that are involved in diverse microtubule-based functions in the cell. Phylogenetic analysis of the motor regions of the kinesin proteins reveals at least five clearly defined groups that are likely to identify kinesins with different roles in basic cellular processes. Two of the groups are consistent with overall sequence similarity, while two groups contain proteins that are related in overall structure or function but show no significant sequence similarity outside the motor domain. One of these groups consists only of kinesin proteins with predicted C-terminal motor domains; another includes only kinesins required for mitotic spindle bipolarity. Drosophila Nod, presently an ungrouped protein, may represent a class of kinesins that, like the myosin I proteins, function as monomers. The analysis indicates that many types of kinesin proteins exist in eukaryotic organisms. At least two of the five groups identified in this analysis are expected to be present in most, or all, eukaryotes.
Movement of membrane tubules along microtubules in vitro: evidence for specialised sites of motor attachment
Authors: Allan Viki , Vale Ronald
Published: April 2021

We have studied the microtubule-dependent formation of tubular membrane networks in vitro, using a heterologous system composed of Xenopus egg cytosol combined with rat liver membrane fractions enriched in either Golgi stacks or rough endoplasmic reticulum. The first step in membrane network construction involves the extension of membrane tubules along microtubules by the action of microtubule-based motor proteins. We have observed for both membrane fractions that 80-95% of moving tubule tips possess a distinct globular domain. These structures do not form simply as a consequence of motor protein activity, but are stable domains that appear to be enriched in active microtubule motors. Negative stain electron microscopy reveals that the motile globular domains associated with the RER networks are generally smaller than those observed in networks derived from a crude Golgi stack fraction. The globular domains from the Golgi fraction are often packed with very low density lipoprotein particles (the major secretory product of hepatocytes) and albumin, which suggests that motor proteins may be specifically enriched in organelle regions where proteins for export are accumulated. These data raise the possibility that the concentration of active motor proteins into specialised membrane domains may be an important feature of the secretory pathway.
Actin microfilaments are associated with the migrating nucleus and the cell cortex in the green alga Micrasterias Studies on living cells
Authors: Meindl Ursula , Zhang Dahong , Hepler Peter K.
Published: April 2021

Rhodamine-phalloidin or FITC-phalloidin has been injected in small amounts into living, developing cells of Micrasterias denticulata and the stained microfilaments visualized by confocal laser scanning microscopy. The results reveal that two different actin filament systems are present in a growing cell: a cortical actin network that covers the inner surface of the cell and is extended far into the tips of the lobes in both the growing and the non-growing semicell; it is also associated with the surface of the chloroplast. The second actin system ensheathes the nucleus at the isthmus-facing side during nuclear migration. Its arrangement corresponds to that of the microtubule system that has been described in earlier electron microscopic investigations. The spatial correspondence between the distribution of actin filaments and microtubules suggests a cooperation between both cytoskeleton elements in generating the motive force for nuclear migration. The function of the cortical actin network is not yet clear. It may be involved in processes like transport and fusion of secretory vesicles and may also function in shaping and anchoring the chloroplast.
Dephosphorylation of the largest neurofilament subunit protein influences the structure of crossbridges in reassembled neurofilaments
Authors: Gotow Takahiro , Tanaka Toshihisa , Nakamura Yu , Takeda Masatoshi
Published: April 2021

Phosphorylation-dependent change in electrophoretic mobility is the most unique characteristic of NF-H, the largest molecular mass subunit of the neurofilament. We dephosphorylated NF-H using Escherichia coli alkaline phosphatase, then reassembled it into neurofilaments with NF-M and NF-L, and into NF-H filaments with NF-H alone. We compared these dephosphorylated filaments with control: projections by low-angle rotary-shadow, crossbridges by quick-freeze deep-etch, and core filament packing density by thin-section electron microscopy. Projections in the dephosphorylated filaments were basically similar in structure to those in control, although there was a tendency for them to be wider and less dense, especially in NF-H filaments. Dephosphorylated filaments were still able to form crossbridges between core filaments, but their crossbridges were significantly wider, less dense, more branched and more irregular than crossbridges in control, and core filaments were more densely packed. These structural differences may be brought about by the removal of phosphate groups from NF-H tail and consequent reduction of electrostatic repulsion between adjacent cross-bridges extending from the same core filament. The results indicate that phosphorylation of NF-H is necessary for forming well developed crossbridges, straight and at constant intervals, like those of in vivo axonal neurofilaments.
Function of type I and type II keratin head domains: their role in dimer, tetramer and filament formation
Authors: Hatzfeld Mechthild , Burba Matthias
Published: April 2021

To examine the role of the keratin head region and its subdomains in filament assembly we constructed several deletion mutants of type I and type II keratins and analysed their in vitro IF forming capacity. The ΔK8 (1-74) and 6KIS (1-56) mutants formed only soluble oligomers, predominantly tetramers with their heterotypic partners. KS mutants that retained either the entire ΔK8 (1-64)) or nearly the entire ΔK8 (1-66)) Hl subdomain formed some short and irregular IF-like structures with K18. However, filaments never reached the normal length and more protofilamentous material was observed. Analysis of the soluble complexes in 2 M guanidine-HCI indicated that tetramer formation was impaired in the truncated molecules. The length of the deletion correlated with the degree of tetramer destabilization. These results suggest that the head domain - specifically the Hl subdomain of type II keratins - plays a direct role in IF assembly. Its functions include a stabilization of the tetramer molecule, suggesting a role in directing the alignment of dimers as well as in elongation.

We also analysed whether both head domains are required or if either type I or type II head domains alone are sufficient for IF formation. Hybrid molecules carrying their partner keratins head domains (K1S (8 head) and KS (18 head)) were combined with their wild-type partners and tested for IF-forming ability. Both combinations formed filaments distinct from normal IF. The effect of the ‘replaced’ head domains was not compensated when both hybrid molecules were combined.

Taken together, the results indicate that complete removal of the head domains of either KS or K 18 arrested IF assembly at the state of soluble oligomers. Replacement of the head domains by head domains of the complementary partner partly compensated for the effect. However, regular IF formation could not take place when either the head domain was missing or it was replaced by the partner’s keratin head.
The role of transcription factors, chromatin structure and DNA replication in 5 S RNA gene regulation
Authors: Wolffe Alan P.
Published: April 2021

Differential expression of the oocyte and somatic 5 S RNA genes during Xenopus development can be explained by changes in transcription factor and histone interactions with the two types of gene. Both factors and histones bind 5 S RNA genes with specificity. Protein-protein interactions determine the stability of potentially transcriptionally active or repressed nucleoprotein complexes. A decline in transcription factor abundance, differential binding of transcription factors to oocyte and somatic 5 S genes, and increased competition with the histones for association with DNA during early embryogenesis, can account for the developmental decision to selectively repress the oocyte genes, while retaining the somatic genes in the transcriptionally active state.

The 5 S ribosomal genes of Xenopus are perhaps the simplest eukaryotic genes to show regulated expression during development. A large multigene family (oocyte 5 S DNA) is transcriptionally active in oocytes but is repressed in somatic cells, whereas a small multigene family (somatic 5 S DNA) is active in both cell types. A potential molecular mechanism to explain the developmental switch that turns off oocyte 5 S DNA transcription has been experimentally reconstructed in vitro and more recently tested in vivo. Central to this mechanism is the specific association of both transcription factors and histones with 5 S RNA genes. How the interplay of histones and transcription factors is thought to affect transcription, and how their respective contributions might change during development from an oocyte, to an embryo and eventually to a somatic cell is the focus of this review.
Structural motifs for recognition and adhesion in members of the immunoglobulin superfamily
Authors: Holness Claire L. , Simmons David L.
Published: April 2021

The immunoglobulin superfamily is the most abundant family of cell surface molecules, accounting for 50% of leukocyte surface glycoproteins. This evolutionary success story is thought to be due to the stability of the Ig domain, which is able to resist the harsh proteolytic and oxidative environment of the extracellular world. By mutation and selection, the Ig domain has evolved to serve many different functions including: receptors for growth factors (CSF-1 receptor, PDGF receptor, FGF receptors); receptors for the Fc region of Ig (IgG receptors; CD 16, CD32, CD64; CD89 IgA receptor); and as adhesion molecules, which now seems to be the function of the majority (CD2/CD58, CD28 and CTLA4, which bind to B7 and B70; CD4/class II, CD8/class I, CD31/CD31, CD50 (ICAM-3)/LFA-1, CD54 (ICAM-1)/LFA-1, CD102 (ICAM-2)/LFA-1, CD106 (VCAM)/VLA-4, NCAM/NCAM. L1, MAG, TAG-1, CEA) (Springer, 1990).
Challenge with high concentrations of cyclic AMP induces transient changes in the cytosolic free calcium concentration in Dictyostelium discoideum
Authors: Schlatterer Christina , Gollnick Frank , Schmidt Eckhard , Meyer Rainer , Knoll Gerd
Published: April 2021

Dictyostelium discoideum cells use cyclic AMP (cAMP) for chemotactic signaling as well as for differentiation. The precise regulation of the cytosolic Ca2+ concentration ([Ca2+]i) seems to play a key role for both processes. We performed single cell measurements of [Ca2+]i in amoebae that were starved in suspension for various times and scrape-loaded with the Ca2+ indicator fura-2. Stimulation of cells with cAMP at the concentration required to induce gene expression (> 100 μM) elicited a global transient increase in [Ca2+]i that depended on the presence of external Ca2+. Both vegetative and aggregation-competent cells displayed a rise in [Ca2+]i, with aggregation-competent cells responding more often than vegetative cells. Basal [Ca2+]i in the presence of Ca2+ was high in vegetative cells and declined during development; the cAMP-induced rise in [Ca2+]i was higher and lasted longer in vegetative cells than in aggregative cells. The addition of 2′-deoxy-cAMP, which binds to the cAMP receptor, induced an increase in [Ca2+]i, whereas the membranepermeant analogue 8-bromo-cAMP that has a low affinity for the receptor but activates cAMP-dependent protein kinase had no effect. This indicates that the change in [Ca2+]i is mediated by the cell surface cAMP receptor. Since HC85 mutant cells, which lack the Gα2 subunit of the G-protein that couples the receptor to phospholipase C, also responded to stimulation with cAMP, the Ca2+ influx does not seem to be triggered by the phosphoinositide signaling cascade.

In order to find out whether such changes in [Ca2+]i could be part of the differentiation process in vivo we determined the volume of the extracellular space in aggregates of cells as a basis for the estimation of the physiological cAMP concentration in the multicellular organism. Electron microscopy revealed only narrow gaps between the cells, with the extracellular space comprising roughly 1% of the cell volume. From the estimation of the resulting concentration range of cAMP we consider it highly probable that cells in an aggregate encounter doses of cAMP that elicit a global rise in [Ca2+]i; this could be part of the signal transduction system involved in the processes of differentiation.
Changes in annexin I and II levels during the postnatal development of rat pancreatic islets
Authors: Ohnishi Makoto , Tokuda Masaaki , Masaki Tsutomu , Fujimura Takashi , Tai Yuji , Matsui Hideki , Itano Toshifumi , Ishida Toshihiko , Takahara Jiro , Konishi Ryoji , Hatase Osamu
Published: April 2021

The expression patterns and the dynamic changes in content of both annexin I and annexin II in the rat pancreatic islets during postnatal development were investigated by both western blot analysis and immunohistochemistry. Immunohistochemical methods clearly demonstrated the presence of annexins I and II exclusively in pancreatic islets, while exocrine tissues were not stained by anti-annexin antibodies. Pancreatic islets were diffusely stained with no specific differences in distribution between different cell types. The expression of annexin I in pancreatic islets gradually increased with postnatal development. A developmental study of annexins I and II by western blot analysis essentially supported the results obtained by immunohistochemistry. In addition, the increasing expression of two protein tyrosine kinases, epidermal growth factor-receptor/kinase and pp60sre, which phosphorylate annexin I and annexin II, respectively, and of protein kinase C, which phosphorylates both proteins, was also shown during postnatal development in rat pancreatic islets. Thus, a relationship between the expression of annexins I and II and the maturation of islet cell function is suggested.
Identification of a key integrin-binding sequence in VCAM-1 homologous to the LDV active site in fibronectin
Authors: Clements John M. , Newham Peter , Shepherd Maura , Gilbert Richard , Dudgeon Timothy J. , Needham Lindsey A. , Edwards R. Mark , Berry Linda , Brass Andrew , Humphries Martin J.
Published: April 2021

The integrin adhesion receptor α4β1 binds two ligands, the extracellular matrix glycoprotein fibronectin and the immunoglobulin superfamily member VCAM-1. Ligandbinding sites are contained with the HepII/IIICS domain of fibronectin, and within the homologous immunoglobulin domains 1 and 4 of VCAM-1. Previous studies have shown that the binding of each ligand to α4β1 is mutually exclusive, suggesting that they may employ similar mechanisms to bind receptor. Fibronectin contains at least three distinct peptide sequences that are active sites for α4β1 binding, two homologous sequences Leu-Asp-Val-Pro (LDVP) and Ile-Asp-Ala-Pro (IDAP), and a third related to Arg-Gly-Asp (RGD). Using a combination of site-directed mutagenesis and synthetic peptide approaches in conjunction with VCAM-1-dependent cell adhesion assays, we now report the identification of a key α4β1-binding sequence in both domains 1 and 4 of VCAM-1 as the tetrapeptide Ile-Asp-Ser-Pro (IDSP). Mutagenesis studies also suggest that an additional sequence in domain 1, KLEK, participates in receptor binding. Since IDSP is homologous to the LDVP and IDAP fibronectin peptides, this therefore provides a molecular explanation for the promiscuity of ligand binding by α4β1 and has implications for the design of synthetic VCAM-1 antagonists. The extrapolation of these findings to other integrin-binding immunoglobulin ligands is also discussed.
Mammalian cells have two functional RCC1 proteins produced by alternative splicing
Authors: Miyabashira Junko , Sekiguchi Takeshi , Nishimoto Takeharu
Published: April 2021

Previously we cloned two human RCC1 cDNAs that differed in their noncoding region. In this study, we have found new human and hamster RCC1 cDNAs, which have an even more different coding region from that of the previously cloned RCC1 cDNAs yet can complement the RCC1 mutation in the tsBN2 cell line. The newly found RCC1 cDNAs encode a protein (designated as RCC1-I) that has an insertion of 31 (human) and 13 (hamster) amino acids at valine25 in the N-terminal region outside the RCC1-seven repeat. The inserted nucleotide sequence was searched for, within the human RCC1 genomic sequence that had already been determined, and was found to be located between the 6th and 7th exons, designated as the 6′ exon. Both the 5′ and 3′ ends of the 6′ exon correspond to the GT-AG rules for splicing, indicating that human RCC1-I mRNAs are produced by alternative splicing. The finding that both humans and hamsters have the insertion at the same RCC1 site suggests that the pattern of alternative splicing in the RCC1 gene has been conserved through evolution.
Expression of prelamin A but not mature lamin A confers sensitivity of DNA biosynthesis to lovastatin on F9 teratocarcinoma cells
Authors: Sinensky M. , McLain T. , Fantle K.
Published: April 2021

The role of inhibition of prelamin A processing in the inhibition of DNA synthesis by lovastatin was examined by expressing prelamin A in F9 teratocarcinoma cells. These cells, normally lacking expression of the A/C lamins, were transfected with constructs expressing either prelamin A or mature lamin A and the effect of lovastatin on DNA biosynthesis was assessed. It was found that expression of prelamin A specifically conferred sensitivity to inhibition of DNA biosynthesis by lovastatin on F9 cells.
Isolation and characterization of a sea urchin zygote cortex that supports in vitro contraction and reactivation of furrowing
Authors: Walker Gary R. , Kane Robert , Burgess David R.
Published: April 2021

The isolation of the cortex of the sea urchin blastomere by detergent lysis was explored with the aim of analyzing components important in the structure and function of the cortical cytoskeleton, and their relationship to such phenomena as contraction. Buffered EGTA medium supplemented with isotonic glycerol and with magnesium, at a level close to the reported internal cellular concentration, yields stable cytoskeletal cortices that retain their spherical shape. Cortices prepared this way contain actin, myosin, fascin and spectrin, components normally associated with the cortical cytoskeleton in a similar distribution to that in intact zygotes. They retain the organized cortical filamentous structure, including the actin-fascin bundles that form cores of microvilli. ATP and NaCl caused changes in cortical shape, described as either contraction or expansion, respectively. Spectrin, but not myosin, was partially extracted by NaCl, resulting in expansion of the cortex that suggests a role for spectrin in maintenance of cortical structure. ATP (but not ADP nor ATPyS), which caused the partial removal of myosin and spectrin, led to the contraction of the cortex, consistent with a role for myosin in cortical tension. In cortices isolated from dividing eggs, the zygotes retained their cleavage furrows and ATP induced continuation of furrow progression. This preparation appears to be a useful in vitro model for cytokinesis.
Imaging subcellular structures of rat mammary carcinoma cells by scanning force microscopy
Authors: Pietrasanta L. I. , Schaper A. , Jovin T. M.
Published: April 2021

Scanning force microscopy (SFM) was used for imaging subcellular structures of cultured rat mammary carcinoma cells dried in air. Identification of cellular substructures was achieved by immunofluorescence and specific fluores-cence probes. Cells grown attached to a glass support exhibited submicrometer thickness in the dried state. Inside the nuclear domain the nucleoli appeared as prominent conical protrusions. Membrane extensions, microspikes and microvilli were well preserved at the cell periphery after fixation in glutaraldehyde vapor and air-drying and were distinguishable either as isolated elements or intercellular communications. The plasma membrane and soluble proteins were selectively removed with non-ionic detergent in a buffer system. The mitochondria were concentrated primarily in the perinuclear space and exhibited a well defined filamentous shape. Their identity was confirmed by specific fluorescence staining with rhodamine 123. In the membrane-free system achieved by dry-cleaving of the sample surface, the cytoskeletal network was resolved as a complex mesh of actin-containing fiber bundles interwoven with a filigree arrangement of thinner filaments. The smallest fibrous substructures revealed by SFM with the scanning tips used to date were ∼8 to 10 nm in height and 80 nm in width.
Estrogen-dependent expression of the cystic fibrosis transmembrane regulator gene in a novel uterine epithelial cell line
Authors: Rochwerger Leonora , Dho Sascha , Parker Linda , Foskett J. Kevin , Buchwald Manuel
Published: April 2021

We have demonstrated previously the modulation of CFTR expression by estrogen in vivo in the rat uterine epithelium. The purpose of this study was to establish a suitable in vitro system to investigate the regulation of CFTR by steroid hormones. Primary cultures of rat uterine epithelial cells, which showed high levels of CFTR expression in vitro, were infected with an adeno/SV40 virus. One clone, UIT 1.16, which retained the morphology of the primary epithelial cells yet proliferated beyond the life span of the primary culture, was isolated and characterized. Successful immortalization of UIT 1.16 cells was verified by the presence of a band corresponding to the SV40 large T-antigen in western blots, as well as by their ability to proliferate continuously. Transmission electron microscopy studies revealed that these cells maintained the characteristics of a polarized epithelium with well-established membrane domains and specialized intercellular junctions. A high transepithelial electrical resistance was also observed when cells were assayed in modified Ussing chambers. When the basolateral cellular membrane of cells grown in vitrogencoated filters was permeabilized with nystatin, a forskolinstimulated Cl− permeability was observed in the apical membrane, similar to that present in other CFTR-expressing epithelial cells. UIT 1.16 cells showed high levels of CFTR expression on northern blots. The expression of CFTR was dependent on the presence of estrogen in the culture medium, since almost undetectable levels of CFTR mRNA were observed when the cells were cultured in medium containing serum depleted of steroid hormones. However, addition of estrogen to this medium prevented the disappearance of CFTR mRNA, confirming estrogenregulated expression of CFTR in the UIT 1.16 cell line. The newly developed UIT 1.16 cell line provides a valuable model to analyze the regulation of CFTR expression by steroid hormones. Moreover, the cell line could also be used to investigate the role of CFTR in the uterus during the normal female cycle as well as for the study of other uterine epithelial functions and the agents that regulate them.
A novel role for E- and P-selectins: shape control of endothelial cell monolayers
Authors: Kaplanski Gilles , Farnarier Catherine , Benoliel Anne-Marie , Foa Colette , Kaplanski Solange , Bongrand Pierre
Published: April 2021

The migration of neutrophils from blood vessels to peripheral tissues is a key step of inflammation. This requires the formation of transient gaps between endothelial cells with concomitant leucocyte squeezing through these narrow apertures and immediate restoration of endothelium continuity. It is currently considered that the main role of selectins is to mediate the initial contact between flowing leucocytes and endothelial cells. We show here that the binding of E- or P-selectins by specific antibodies induces a marked ‘rounding up’ of interleukin-1- or thrombin-activated human endothelial cells, respectively. Also, anti-E-selectin antibodies trigger a transient increase in cytosolic calcium involving intracellular calcium stores. No such effect is observed when von Willebrand factor or intercellular adhesion molecule 1 are similarly bound. Thus, in addition to promoting the initial interaction between activated endothelium and moving leucocytes, selectins might play a role in the induction of subsequent endothelial deformation, which would facilitate leucocyte arrest and transmigration towards peripheral tissues, and enhance the diffusion of soluble molecules between intravascular and peripheral compartments. Our results are consistent with this hypothesis and demonstrate a new property of endothelial selectins.
Platelet-derived growth factor is an autocrine growth stimulator in retinal pigmented epithelial cells
Authors: Campochiaro Peter A. , Hackett Sean F. , Vinores Stanley A. , Freund John , Csaky Carl , LaRochelle William , Henderer Jeff , Johnson Mike , Rodriguez Ignacio R. , Friedman Zvi , Derevjanik Nancy , Dooner James
Published: April 2021

The retinal pigmented epithelium (RPE) plays a major role in normal and exaggerated retinal wound repair; the latter can result in epiretinal membrane formation and loss of vision. The RPE forms a stable monolayer of highly differentiated cells that proliferates only during wound repair. The mechanism underlying the change to the pro-liferating phenotype is unknown.

When grown on a plastic substratum, cultured RPE cells mimic the proliferating phenotype in situ; they escape density arrest and proliferate in serum-free medium. In this study, we have demonstrated that a platelet-derived growth factor (PDGF) autocrine loop is involved in RPE growth in serum-free medium, because: (1) RPE cells secrete PDGF into their media and express PDGF receptors; (2) the PDGF receptors on RPE cells are autophosphorylated in serum-free medium and suramin, an agent that displaces PDGF and other growth factors from their receptors, blocks the autophosphorylation; and (3) a neutralizing antibody to PDGF significantly decreases RPE growth in serum-free medium. When a linear scrape is made in an RPE monolayer, the cells migrate and pro-liferate to fill in the gap mimicking wound repair in situ. Cells along the edge of the scrape show increased expression of PDGF and PDGF-β receptors, and increased staining for proliferating cell nuclear antigen.

Immunohistochemistry and in situ hybridization demon-strate expression of PDGF in ganglion cells and cells of retinal blood vessels. PDGF is not detected in the outer retina or RPE in untreated eyes, but is detected in RPE participating in wound repair, either adjacent to laser burns or underlying retinal detachment. PDGF and PDGF receptors are also expressed in RPE in epiretinal membranes removed during vitreous surgery.

These data suggest that PDGF is an autocrine stimulator of growth in RPE that plays a role in retinal wound repair and epiretinal membrane formation.
Surface antigens of Leishmania mexicana amastigotes: characterization of glycoinositol phospholipids and a macrophage-derived glycosphingolipid
Authors: Winter Gerhard , Fuchs Manuela , McConville Malcolm J. , Stierhof York-Dieter , Overath Peter
Published: April 2021

Amastigotes of the protozoan parasite Leishmania prolif-erate in phagolysosomes of macrophages. They abundantly express glycoinositol phospholipids (GIPLs), which are considered necessary for parasite survival by providing a shield at the surface against lysosomal hydrolases and by serving as receptors for the interaction with host cells. The structures of four GIPLs of L. mexicana amastigotes were characterized by a combination of gas-liquid chromatog-raphy-mass spectrometry, methylation linkage analysis and enzymatic treatments. They contain the glycan struc-tures Manα1-3Manα1-4GlcN (iM2), Manα1-6(Manα1-3)Manα1-4GlcN (iM3), Manα1-2Manα1-6(Manα1-3)-Manα1-4GlcN (iM4) and (NH2-CH2CH2-PO4)Manα1-6(Manα1-3)Manα1-4GlcN (EPiM3), which are linked to alkylacyl-phosphatidylinositol. The predominant amastigote GIPL, EPiM3 (∼2×107 molecules/cell), is located at the parasite cell surface, in the flagellar pocket and in lysosomal membranes, but not on host cell structures as shown by immunofluorescence and immunoelectron microscopy. In addition, amastigotes in infected Balb/c mice contain a glycolipid with similar distribution as EPiM3, which has the same characteristics as the Forssman antigen of mammalian cells. In contrast to EPiM3, there is strong evidence that this glycosphingolipid is not synthe-sized by amastigotes but by macrophages in the lesion. This suggests a mechanism of lipid transfer from the macrophage to the parasite.
Ezrin has properties to self-associate at the plasma membrane
Authors: Andréoli Christophe , Martin Marianne , Borgne Roland Le , Reggio Hubert , Mangeat Paul
Published: April 2021

Ezrin, a member of a family of proteins involved in the interaction of the microfilament cytoskeleton with the plasma membrane, plays a role in membrane translocation in gastric parietal cells (Hanzel, D., Reggio, H., Bretscher, A., Forte, J. G. and Mangeat, P. (1991). EMBO J. 10, 2363-2373). Human ezrin was expressed in and purified from Escherichia coli. It possesses all the major biophysical, immunological and physiological properties of natural ezrin. Upon microinjection in live gastric HGT-1 cells, ezrin was incorporated into the dorsal microvilli, a site where the endogeneous protein is localized. By coimmuno-precipitation and ezrin-affinity assays, two HGT-1 cell proteins of 77 and 72 kDa behaved as ezrin-binding proteins. In enriched gastric apical membranes, 125I-ezrin labelled proteins of 80, 77 and 72 kDa by overlay assay. The 80 kDa protein was identified as ezrin and the 77 and 72 kDa proteins as gastric forms of proteins structurally related to ezrin, such as radixin and moesin. In insect cells infected with a recombinant baculovirus, one-third of over-expressed ezrin accumulated at the plasma membrane. Ezrin bound a 77 kDa endogenous peripheral membrane protein, behaving as an insect counterpart of the mammalian ezrin family. In addition to the respective role of the amino- and carboxyl-terminal domains of ezrin in linking the membrane and the cytoskeleton (Algrain, M., Turunen, O., Vaheri, A., Louvard, D. and Arpin, M. (1993). J. Cell Biol. 120, 129-139), both domains interacted syner-gistically in a salt-dependent manner to trigger self-association of ezrin. Ezrin’s self-association properties could represent another way of regulating the number of ezrin molecules bound at specific membrane sites.
Analysis of a cortical cytoskeletal structure: a role for ezrin-radixin-moesin (ERM proteins) in the marginal band of chicken erythrocytes
Authors: Winckler Bettina , González Agosti Charo , Magendantz Margaret , Solomon Frank
Published: April 2021

We are studying how the cytoskeleton determines cell shape, using a simple model system, the marginal band of chicken erythrocytes. We previously identified a minor component of the marginal band by a monoclonal antibody, called 13H9 (Birgbauer and Solomon (1989). J. Cell Biol. 109, 1609-1620; Goslin et al. (1989). J. Cell Biol. 109, 1621-1631). mAb 13H9 also binds to the leading edges of fibroblasts and to neuronal growth cones and recognizes the cytoskeletal protein ezrin. In recent years, two proteins with a high degree of homology to ezrin were identified: moesin and radixin, together comprising the ERM protein family. We now show that the contiguous epitope sufficient for mAb 13H9 binding is a sequence present in each of the ERM proteins, as well as the product of the gene associated with neurofibromatosis 2, merlin or schwannomin. We used biochemical and immunological techniques, as well as PCR to characterize the expression and localization of the ERM proteins in chicken erythrocytes. The results demonstrate that radixin is the major ERM protein associated with the cytoskeleton. Both ezrin and radixin localize to the position of the marginal band. Our results suggest that the ERM proteins play functionally conserved roles in quite diverse organelles.