bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein composition of cornified cell envelopes of epidermal keratinocytes
Authors: Steven Alasdair C. , Steinert Peter M.
Published: April 2021

Terminally differentiated mammalian epidermal cells are lined with a 15 nm thick layer of proteins cross-linked by isodipeptide and disulfide bonds, called the cornified cell envelope (CE). A number of proteins, including involucrin, loricrin, cystatin A, filaggrin, a cysteine-rich protein (CRP) and the ‘small proline-rich’ proteins (SPRRs) have been reported to be components of this complex, but little infor-mation has been obtained as to their relative abundances because the acute insolubility of the CEs has precluded direct methods of analysis. To address this question, we have determined the amino acid compositions of isolated CEs, and then modelled them in terms of linear combina-tions of the candidate proteins. The results show that stratum corneum CEs have a loricrin content of 65-70% (w/w) in human, and 80-85% in mouse. In human epidermal CEs, the secondary contributors are filaggrin and CRP (each ~10%), with smaller amounts of involucrin, SPRR and cystatin A (2-5% each) also present. Mouse epidermal CEs have about the same amount of filaggrin and somewhat more SPRR, but only trace amounts of the other proteins. In marked contrast, the major constituents of the CEs of cultured keratinocytes induced to terminal differentiation in vitro are cystatin A, involucrin and CRP (each ~30%). No significant amount of loricrin was detected except in sloughed mouse cells, which represent a more advanced state of terminal differentiation than attached cells. These results demonstrate that the CEs of cultured cells are quite different from mature CEs from native epidermis, and further suggest that generation of the CE in vivo is a multi-stage process in which involucrin, SPRR, CRP and cystatin A are initially attached to the cell membrane, and are subsequently overlaid with a heavy deposition of loricrin together with some filaggrin. Our data suggest that cultured CEs approximate only the first stage(s) of this process.
Cytolocation of prosome antigens on intermediate filament subnetworks of cytokeratin, vimentin and desmin type
Authors: Olink-Coux Marguerite , Arcangeletti Cristina , Pinardi Federica , Minisini Rosalba , Huesca Michèle , Chezzi Carlo , Scherrer Klaus
Published: April 2021

Analysis by double-label indirect immunofluorescence of PtK1 and HeLa cells had previously demonstrated that prosome* antigens form networks that superimpose on those of the intermediate filaments of the cytokeratin type. We show here that in PtK1 cells various prosomal antigens also reside to a variable extent on intermediate filaments subnetworks of the vimentin type. In proliferating human fibroblasts the prosome and vimentin networks were found to coincide, while in proliferating myoblasts of the C2.7 mouse myogenic cell line the prosomal antigens seem to superimpose on the intermediate filaments of the desmin type. Thus, the prosomes, which are RNP particles of variable composition and subcomplexes of untranslated mRNP, and carry a multicatalytic proteinase activity, seem to co-localize with the specific kind of cytoplasmic intermediate filament in relation to the cell type. These results, which generalize the previous data, are discussed in view of possible role(s) for prosomes in mRNA metabolism and/or intermediate filaments remodelling.
Species specificity and organ, cellular and subcellular localization of the 100 kDa Ras GTPase activating protein
Authors: Mollat Patrick , Fournier Agnès , Yang Chun Zhi , Alsat Eliane , Zhang Yuehe , Evain-Brion Danièle , Grassi Jacques , Thang Ming Nguy
Published: April 2021

A p100-GAP isoform, generated by an alternative splicing mechanism that eliminates the 180 hydrophobic amino acids at the amino terminus of p120-GAP, has been described in human placenta, in addition to the known p120GAP and neurofibromin. This p100-GAP possesses full Ras-GTPase stimulating activity. p120-GAP is ubiquitously localized in the cytosol while the localization of p100-GAP is unknown. Here we have explored the precise localization of p100-GAP and show that p100-GAP is present only in extracts of primate placenta. It is abundant in both human and Maccaca Rhesus placentae, where it is present in far larger amounts than p120-GAP. The p100-GAP is species-specific since it was not detected in the placenta of pig, sheep, mouse or rat. p100-GAP was also found to be organ-specific, since it was not detectable in organs other than the placenta. In this connection, we substantiated our previous finding that p100-GAP is mainly localized in the trophoblasts. Both subcellular trophoblast fractionation and immunofluorescence analyses showed that this protein was distributed between the cytosol, plasma membrane and a fraction bound to the nucleus, but not inside it. This highly restrictive specificity of p100-GAP localization in relation to species, organ and cell type, confirms the extreme singularity of this protein, and strongly suggests a particular specific function in the trophoblast.
Calponin is localised in both the contractile apparatus and the cytoskeleton of smooth muscle cells
Authors: North Alison J. , Gimona Mario , Cross Robert A. , Small J. Victor
Published: April 2021

Calponin and caldesmon are two thin filament-binding proteins found in smooth muscle that have both been attributed a role in modulating the interaction of actin and myosin. Using high-resolution dual-label immunocyto-chemistry we have determined the distribution of calponin relative to the contractile and cytoskeletal compartments of the smooth muscle cell. We show, using chicken gizzard smooth muscle, that calponin occurs in the cytoskeleton, with β-cytoplasmic actin, filamin and desmin, as well as in the contractile apparatus, with myosin and caldesmon. According to the observed labelling intensities, calponin was more concentrated in the cytoskeleton and it was additionally localised in the cytoplasmic dense bodies as well as in the adhesion plaques at the cell surface, which both harbour the β-cytoplasmic isoform of actin.

It is probable that these results explain earlier conflicting reports on the composition of smooth muscle thin filaments and suggest that calponin, together with a Ca2+-receptor protein, could just as likely serve a role in the cytoskeleton of smooth muscle as in the contractile apparatus.
Molecular analysis of the γ heavy chain of Chlamydomonas flagellar outerarm dynein
Authors: Wilkerson Curtis G. , King Stephen M. , Witman George B.
Published: April 2021

We report here the complete sequence of the γ dynein heavy chain of the outer arm of the Chlamydomonas flagellum, and partial sequences for six other dynein heavy chains. The γ dynein heavy chain sequence contains four P-loop motifs, one of which is the likely hydrolytic site based on its position relative to a previously mapped epitope. Comparison with available cytoplasmic and flagellar dynein heavy chain sequences reveals regions that are highly conserved in all dynein heavy chains sequenced to date, regions that are conserved only among axonemal dynein heavy chains, and regions that are unique to individual dynein heavy chains. The presumed hydrolytic site is absolutely conserved among dyneins, two other P loops are highly conserved among cytoplasmic dynein heavy chains but not in axonemal dynein heavy chains, and the fourth P loop is invariant in axonemal dynein heavy chains but not in cytoplasmic dynein. One region that is very highly conserved in all dynein heavy chains is similar to a portion of the ATP-sensitive microtubule-binding domain of kinesin. Two other regions present in all dynein heavy chains are predicted to have high α-helical content and have a high probability of forming coiled-coil structures. Overall, the central one-third of the γ dynein heavy chain is most conserved whereas the N-terminal one-third is least conserved; the fact that the latter region is divergent between the cytoplasmic dynein heavy chain and two different axonemal dynein heavy chains suggests that it is involved in chain-specific functions.
Association of cytoplasmic dynein with manchette microtubules and spermatid nuclear envelope during spermiogenesis in rats
Authors: Yoshida Toshimichi , Ioshii Sergio O. , Imanaka-Yoshida Kyoko , Izutsu Kosaku
Published: April 2021

During spermiogenesis, the shape of the spermatid nucleus, which is spherical, changes and it becomes the sperm head. A microtubular structure called a manchette is thought to be involved in this morphogenetic process. In this report, we demonstrate the localization of cytoplasmic dynein and manchette development by a double immunofluorescence technique using anti-bovine brain MAP 1C and antitubulin. Before step 6 of the Leblond and Clermont staging, the microtubules showed a fine reticular network, and the dynein staining was homogeneous. In step 6, the microtubular network was concentrated around the nucleus. The manchette developed in step 7 spermatids, and was fully formed, with a skirt-like appearance, covering the nuclear surface in step 8. Dynein fluorescence was associated with the microtubular manchette in steps 7-10. During these steps, the nucleus was protruded from the cytoplasm. In steps 11-13, the most active stages in nuclear shaping, the dynein was densely localized at the nuclear surface covered by the manchette. As the nucleus acquired a shape similar to the mature spermatozoon at step 14, the dynein fluorescence was localized only at the concave side of the nuclear caudal edge. The manchette became narrower and elongated. In step 15, the manchette extended into the elongated cytoplasm, diminishing during steps 16-18. The localization of the dynein was limited to the ventral aspect of the caudal head in these steps. There was little dynein fluorescence in mature spermatozoa. Immunoelectron microscopy showed positive reactions in the nuclear envelope and the inner region of the microtubular manchette. These observations suggest that cytoplasmic dynein, possibly bound to the nuclear envelope, and manchette microtubules are involved in the protrusion of the spermatid nucleus from the cytoplasm.
Sequence analysis of the Chlamydomonas alpha and beta dynein heavy chain genes
Authors: Mitchell David R. , Brown Kimberly S.
Published: April 2021

We have sequenced genomic clones spanning the complete coding region of one heavy chain (beta) and the catalytic domain of a second (alpha) of the Chlamydomonas reinhardtii flagellar outer arm dynein ATPase. The beta heavy chain gene (ODA-4 locus) spans 20 kb, is divided into at least 30 exons, and encodes a predicted 520 kDa protein. Comparison with sea urchin beta dynein sequences reveals homology that extends throughout both proteins. Over the most conserved central catalytic region, the Chlamydomonas alpha and beta chains are equally divergent from the sea urchin beta chain (64% and 65% similarity, respectively), whereas the Chlamydomonas gamma chain is more divergent from urchin beta (54% similarity). The four glycine-rich loops identified as potential nucleotide-binding sites in other dynein heavy chains are also present in Chlamydomonas alpha and beta dyneins. Two of these four nucleotide-binding motifs are highly conserved among flagellar dyneins, but only the motif previously identified as the catalytic site in sea urchin dynein is highly conserved between flagellar and cytoplasmic dynein heavy chains. Predictions of secondary structure suggest that all dynein heavy chains possess three large domains, with the four nucleotide-binding consensus sequences located in a central 185 kDa domain that is bounded on both sides by regions that form multiple, short alpha-helical coiled-coils.
The distribution of cytoplasmic bacteria in the early Drosophila embryo is mediated by astral microtubules
Authors: Callaini Giuliano , Riparbelli Maria Giovanna , Dallai Romano
Published: April 2021

Maternally inherited cytoplasmic bacteria have occasionally been observed in embryos and adults of different strains of several Drosophila species. While there is a considerable body of data on the relationship between bacteria and embryo viability, little is known about the behavior of these bacteria during the early development of Drosophila. In eggs laid by infected Drosophila melanogaster females we showed that cytoplasmic bacteria were initially concentrated in a thin cortical layer and scattered in the yolk region. During the following syncytial blastoderm mitoses the bacteria mainly accumulated towards the poles of the mitotic spindles, suggesting that astral microtubules play a role in localizing bacteria. This is supported by the observation that treatment of the infected embryos with the microtubule-disrupting drug colchicine led to the partial dissociation of the bacteria from the spindle poles, whereas cytochalasin treatment left almost all the bacterial clusters intact. Moreover, bacteria were not found near the polar bodies and yolk nuclei, which were without astral microtubules. In mitosis-defective embryos, with centrosomes dissociated from the nuclei, the bacteria were concentrated in association with the isolated astral microtubules, and in cold-treated embryos, in which microtubules regrew from isolated centrosomes after recovering, the bacteria clustered around the newly formed asters. These observations, also supported by electron microscope analysis, indicate a close relationship between cytoplasmic bacteria and astral microtubules, and suggest that the latter were able to build discrete cytoplasmic domains ensuring the proper distribution of cytoplasmic components during the blastoderm mitoses, despite the lack of cell membranes.
Cloning of Drosophila β-adaptin and its localization on expression in mammalian cells
Authors: Camidge D. Ross , Pearse Barbara M. F.
Published: April 2021

A Drosophila cDNA (BAD1) encoding a structural and assembly-competent homologue of the mammalian coated pit β-adaptins (β and β’ has been cloned and sequenced. In its amino-terminal region (residues 1-575), the BAD1 sequence appears intermediate between that of the mammalian β-adaptin and a predicted sequence, from cDNA 105a, which appears to code for a version of β’-adaptin. To test its functional characteristics, a ‘myc’-tagged version of BAD1 was expressed in Cos cells. The BAD1 protein was detected most clearly in plasma membrane coated pits, where it colocalized with α-adaptin, although other coated pits were noted which apparently did not contain α-adaptin. However, these are probably γ-adaptin containing pits, as BAD1 was also found colocalized with γ-adaptin in Golgi coated pits in which, typically, α-adaptin is absent. Immunoprecipitation experiments confirmed that the BAD1 protein was present in both types of adaptor complex, unlike β-adaptin which complexes with α-adaptin and β’-adaptin which partners γ-adaptin exclusively. In spite of this, BAD1 expression does not appear to mix α-adaptin and γ-adaptin distribution amongst all the coated pits: thus the location of these adaptor complexes in mammalian cells does not depend on the differences between β subunits but rather on membrane-specific interactions of other adaptor polypeptides. The differential interaction of β with α-adaptin and β’ with γ-adaptin in mammalian cells is likely to depend on the few non-conservative differences between their respective sequences and BAD1. Four of these (one with respect to β and three versus 105a) are clustered in a particular region (residues 155 to 305), which may therefore represent a domain that influences the choice of partner adaptin.
Genetic deficiency in low density lipoprotein receptor-related protein confers cellular resistance to Pseudomonas exotoxin A Evidence that this protein is required for uptake and degradation of multiple ligands
Authors: Willnow Thomas E. , Herz Joachim
Published: April 2021

The low density lipoprotein receptor-related protein (LRP) is a large multifunctional receptor implicated in the cellular uptake of functionally diverse ligands. Biochemical evidence suggests that LRP is a clearance receptor for apoE-rich remnant lipoproteins, lipoprotein lipase, α2-macroglobulin/protease complexes, plasminogen activator/inhibitor complexes, the active protease tissue-type plasminogen activator and exotoxin A from Pseudomonas aeruginosa. Mice genetically deficient in LRP die early during gestation, underscoring the essential physiological role of this gene in vivo. To study the effect of LRP deficiency at the cellular level, we have used Pseudomonas exotoxin A (PEA) to select murine embryonic fibroblasts that are genetically deficient in LRP. Our results demonstrate that this single gene defect is sufficient to confer resistance to PEA on cultured cells. In addition, embryonic fibroblasts lacking LRP are unable to bind, internalize and degrade methylamine-activated α2-macroglobulin and complexes of urokinase with plasminogen activator inhibitor-1. Furthermore, cellular uptake and degradation of receptor-associated protein, a 39 kDa accessory protein of LRP, is reduced by 90% in the absence of LRP. These results provide genetic evidence for the multifunctional nature of LRP and its crucial role in protease/inhibitor complex metabolism.
Immunolocalization of microfibril and microfibril-associated proteins in the subendothelial matrix of the developing mouse aorta
Authors: Davis Elaine C.
Published: April 2021

In the developing aorta, endothelial cell connecting filaments extend from the abluminal surface of the endothelial cell to the subjacent elastic lamina. The connecting filaments are in alignment with intracellular stress fibers and are oriented parallel to the direction of blood flow. In the present study, the composition of the endothelial cell connecting filaments was investigated by indirect immuno-gold labeling with antibodies to the microfibril proteins, MP340 (fibrillin) and MAGP, and to fibronectin and heparan sulfate proteoglycan (HSPG). In the subendothelial matrix of both 15-day gestational and 5-day post-natal mouse aortae, the connecting filaments showed moderate immunoreactivity with anti-MP340; however, no significant immunoreaction was seen with anti-MAGP. Antifibronectin strongly labeled the connecting filaments and a weak immunoreaction was seen with anti-HSPG. In contrast, the adjacent ‘elastin-associated microfibrils’ showed a very strong immunoreaction with anti-MP340 and a moderate reaction with anti-MAGP. Little or no reaction was seen with anti-fibronectin or anti-HSPG. The filaments that connect endothelial cells to the subjacent elastic lamina during aortic development are thus microfibrillar in nature and related to elastin-associated microfibrils as evidenced by their positive immunoreaction with anti-MP340. The absence of labeling with anti-MAGP, however, suggests that either these fibrillin-containing filaments do not contain MAGP or that the immunoreactive epitopes are blocked by the proteins that coat the connecting filaments such as fibronectin. These results suggest that microfibrils not in association with elastin may play a role in cell anchorage and, more specifically, in the aorta may be involved in maintaining the structural integrity of the endothelial cell layer during early development of the vessel wall. Furthermore, the absence of immunoreactivity with anti-MAGP on the fibrillin-containing endothelial cell connecting filaments raises the possibility that microfibrils may consist of a family of related filaments rather than a single structural entity.
Ductus arteriosus smooth muscle cell migration on collagen: dependence on laminin and its receptors
Authors: Clyman Ronald I. , Tannenbaum Jamie , Chen Yao Qi , Cooper Douglas , Yurchenco Peter D. , Kramer Randall H. , Waleh Nahid S.
Published: April 2021

During permanent closure of the ductus arteriosus, smooth muscle cells migrate through the extracellular matrix (ECM) to form intimal mounds that occlude the vessel’s lumen. Smooth muscle cells (SMC) migrate over surfaces coated with collagen in vitro. During the migration SMC also synthesize fibronectin (FN) and laminin (LN). Anti-bodies against FN and LN inhibit migration on collagen by 30% and 67%, respectively. Because of the apparent importance of LN in migration, we examined how SMC interact with LN and LN fragments (P1, E8, P1′, E1′, E3, E4, and G). Ductus SMC adhere to high concentrations of LN and to two fragments of the molecule: P1 and E8. They use a unique set of integrin receptors to bind to LN (α1β1, α6β1 and αVβ3), to P1 (α1β1, αVβ3), and to E8 (α6β1, αVβ3).

The αVβ3 integrin binds to the P1 fragment of LN in an RGD peptide-dependent manner, and to the E8 fragment in an RGD-independent manner; the RGD site on the P1 fragment probably is not available to the cell in intact LN. Antibodies against β1 integrins completely inhibit SMC adhesion to LN; antibodies against the αVβ3 integrin do not block SMC adhesion to LN, but do prevent cell spreading. LN is also capable of interfering with SMC adhesion to other ECM components. The antiadhesive effect of LN is located in the E1′ domain. Both exogenous and endogenous LN increase SMC motility on collagen I. The locomotion-promoting activity of LN resides in the E1′ antiadhesive domain, and not in its adhesive (P1, E8) domains. LN causes a decrease in the number of focal contacts on collagen I. This might enable SMC to alter their mobility as they move through the extracellular matrix to occlude the ductus arteriosus lumen.
Molecular cloning and characterization of a novel liver-specific transport protein
Authors: Simonson Gregg D. , Vincent Alexandra C. , Roberg Kevin J. , Huang Yue , Iwanij Victoria
Published: April 2021

Monoclonal antibodies that specifically recognize a membrane component located on the sinusoidal domain of the hepatocyte have been used to screen a rat liver cDNA expression library and a clone encoding a novel transporter (NLT) protein has been identified. Analysis of the deduced 535 amino acid protein sequence indicates that it is unique, but shares the twelve-transmembrane domain hydrophathicity profile as well as the presence of transporter-specific amino acid motifs with bacterial and mammalian transporters. Since overall homology of NLT to known transporter genes is low (20-25% identity) it may represent a new subgroup within the transporter family of proteins. The NLT was characterized further with respect to its tissue distribution and its expression during liver development. A 2.1 kb transcript has been found in liver and at lower levels in kidney, but not in several other tissues tested. Studies on the developing liver indicate that NLT transcripts are present at a very low level from 19 through 21 gestation days with a 4- to 5-fold increase within two weeks after birth. Overall, we have cloned a novel transporter that is preferentially expressed in liver, is located on the sinusoidal domain of the plasma membrane and represents a marker for the late stage of liver development.
Surface morphology and mechanical properties of MDCK monolayers by atomic force microscopy
Authors: Hoh Jan H. , Schoenenberger Cora-Ann
Published: April 2021

We describe the morphology and mechanical stability of the apical surface of MDCK monolayers by atomic force microscopy (AFM). Living cells could be imaged in physiological solution for several hours without noticeable deterioration. Cell boundaries appear as ridges that clearly demarcate neighboring cells. In some cases the nucleus of individual cells could be seen, though apparently only in very thin areas of the monolayer. Two types of protrusions on the surface could be visualized. Smooth bulges that varied in width from a few hundred nanometers to several micrometers, which appear to represent relatively rigid subapical structures. Another type of protrusion extended well above the membrane and was swept back and forth during the imaging. However, the microvilli that are typically present on the apical surface could not be resolved. For comparison, a transformed MDCK cell line expressing the K-rasoncogene was also examined. When cultured on solid substrata at low density, the R5 cells spread out and are less than 100 nm thick over large areas with both extensive processes and rounded edges. Many intracellular structures such as the nucleus, cytoskeletal elements and vesicles could be visualized. None of the intracellular structures seen in the AFM images could be seen by scanning electron microscopy. Both R5 cells and MDCK monolayers required imaging forces of >2 nN for good image contrast. Force measurements on the MDCK monolayers show that they are very soft, with an effective spring constant of ∼0.002 N/m for the apical plasma membrane, over the first micrometer of deformation, resulting in a height deformation of approximately 500 nm per nanoNewton of applied force. The mechanical properties of the cells could be manipulated by addition of glutaraldehyde. These changes were monitored in real time by collecting force curves during the fixation reaction. The curves show a stiffening of the apical plasma membrane that was completed in ∼1 minute. On the basis of these measurements and the imaging forces required, we conclude that deformation of the plasma membrane is an important component of the contrast mechanism, in effect ‘staining’ structures based on their relative rigidity.
Receptor-mediated endocytosis is sensitive to antibodies against the uncoating ATPase (hsc70)
Authors: Höning Stefan , Kreimer Georg , Robenek Horst , Jockusch Brigitte M.
Published: April 2021

We have investigated the functional role of the coated vesicle-uncoating ATPase (UA), a cognate heat shock protein (hsc70), in receptor-mediated endocytosis. A monoclonal antibody against bovine brain UA/hsc70 was generated that recognizes a 26 kDa proteolytic fragment harbouring the putative clathrin-binding site. In vitro, this antibody blocked the UA/hsc70-mediated release of clathrin from isolated coated vesicles (CVs). Upon microinjection into tissue culture cells, it specifically inhibited the heat shock-induced nuclear migration of UA/hsc70.

This antibody also interfered with endocytosis of ligand-receptor complexes in injected cells. Two different systems were studied: the uptake of aggregated human IgG by BHK cells transfected with a human Fc receptor (FcRII), and the internalization of LDL by human fibroblasts. Injection of the monoclonal antibody in concentrations yielding approximately equal molar ratios of antibody to enzyme resulted in a reduction of endocytosis to 20-30% of control values, as seen by conventional light and confocal laser scanning microscopy, and by electron microscopy. In the transfected BHK cells, the endocytosed ligand remained associated with the labeling for clathrin and was not delivered to the endosomal compartment within the period expected from control serum- or non-injected cells. Thin sections revealed an accumulation of coated structures in the antibody-injected cells as compared to controls. Thus, our data show that UA is essential for normal receptor-mediated endocytosis, and is presumably involved in the uncoating of CVs preceding their fusion with endosomes.
The kinetics of H1 histone kinase activation during the cell cycle of wild-type and wee mutants of the fission yeast Schizosaccharomyces pombe
Authors: Creanor J. , Mitchison J. M.
Published: April 2021

H1 histone kinase activity has been followed in selection-synchronised cultures of fission yeast wild-type and wee1mutant cells, and in induction-synchronised cells of the mutant cdc2-33. The main conclusions are: (1) in all three cases, the peak of activity is near mitosis. (2) The rise in activity is relatively slow starting in wild type at 0.4 of the cycle before mitosis. It is proposed that the beginning of the rise is the first identifiedevent in the mitotic control. (3) The rise is twice as fast in weeand starts nearer to mitosis. (4) In all cases the beginning of the rise is in G2. (5) The fall in activity is also slow, lasting for 0.25 of the cycle, in wild type. Exit from mitosis happens well before activity has fallen to baseline. (6) In a range of size mutants, activity is roughly proportional to cell size. It is suggested that the kinase may have a cytoplasmic function. (7) Estimates have been made of the timing of mitosis in the mutants. In wee, mitosis is 0.14 of the cycle earlier than in wild type because the cells have a longer septated period at the end of the cycle. (8) A novel method has been developed for eliminating the effects of the partial asynchrony in synchronous cultures, without which the kinetic analysis would have been inaccurate.
Differential regulation of C-CAM isoforms in epithelial cells
Authors: Hunter Irene , Lindh Monica , Öbrink Björn
Published: April 2021

C-CAM is a Ca2+-independent cell adhesion molecule (CAM) that mediates intercellular adhesion of isolated rat hepatocytes. It is widely distributed in epithelia, where its presence both at lateral cell borders and on apical cell surfaces suggests that it may have diverse biological functions. Two major isoforms, C-CAM1 and C-CAM2, which differ in the lengths of their cytoplasmic domains, have been identified. The lack of suitable in vitro systems has so far prevented a detailed study of the physiological role of C-CAM in epithelia. We now report on the identification, biochemical characterization and functional analysis of C-CAM isoforms in the established epithelial cell line NBT II, derived from a chemically induced carcinoma of rat bladder. C-CAM in NBT II cells is a 110-115 kDa cell surface glycoprotein located predominantly at sites of cell-cell contact but also present on the apical cell surface. Northern blotting analysis revealed the presence of both C-CAM1 and C-CAM2, with the major transcripts for both isoforms present within the 4.0 kb size range. The dissociation of NBT II cell colonies by anti-C-CAM antibodies indicated that at least one function of C-CAM in these cells is to mediate intercellular adhesion. The maintenance of extensive cell-cell contacts and the expression of C-CAM at the contact sites in cells grown in low Ca2+medium suggested that, like its counterpart in hepatocytes, C-CAM in NBT II cells may be a Ca2+-independent cell-cell adhesion molecule. The co-localization and coordinate reorganization of both C-CAM and actin by anti-C-CAM antibodies indicated that these two proteins were associated and suggested that interactions with the cytoskeleton may be important for the regulation of C-CAM function. The specific upregulation of C-CAM1 in cells induced to undergo epithelial to mesenchymal-like transitions (EMT) by the serum substitute Ultroser G suggested that C-CAM isoforms are important modulators of the adhesive properties of these cells.
Modulation of scatter factor/hepatocyte growth factor activity by cell-substratum adhesion
Authors: Clark Peter
Published: April 2021

Scatter factor/hepatocyte growth factor (SF/HGF) is a multifunctional growth and motility factor whose activities vary with cell type. Here, the composition of the substratum was found to profoundly alter the scattering activities of SF/HGF, but not its mitogenetic effects, in MDCK cells. Whereas enhancement of DNA synthesis and induction of cell flattening by SF/HGF were independent of substratum composition (i.e. occurred on both fibronectin and vitronectin surfaces), colony dispersion as a result of cell separation fails to occur or is markedly reduced on surfaces where vitronectin is the major adhesive ligand. Prolonged exposure of non-scattering cultures to SF/HGF resulted in cells at colony margins producing long protrusions, which indicate that the motility of these cells is stimulated but ‘frustrated’ by the lack of breakdown of cell-cell adhesion. Scattering therefore appears to comprise two major components: increased motility and breakdown of cell-cell adhesion. The pathway leading to the breakdown of cell-cell contacts is modulated by downstream signals from extracellular matrix receptors. When cultured on immobilised fibronectin, vitronectin or a surface containing both, colony dissociation correlates with the presence of fibronectin, suggesting that positive signals from fibronectin receptors are required for SF/HGF-induced cell separation. Comparison of the findings in this study with those of a recent report on the modulation of SF/HGF-induced tubulogenesis by ECM (Santos, O. F. P. and Nigam, S. K. (1993) Dev. Biol. 160, 293-302), where vitronectin in type-1 collagen gels alters the pattern of SF/HGF-induced MDCK tubule formation from highly branched to long and unbranched, suggests that cell motility enhancement leads to tubule formation whereas the breakdown of cell-cell adhesion is required for tubule branching.
Does transcription by RNA polymerase play a direct role in the initiation of replication?
Authors: Hassan A. Bassim , Cook Peter R.
Published: April 2021

RNA polymerases have been implicated in the initiation of replication in bacteria. The conflicting evidence for a role in initiation in eukaryotes is reviewed.
Nuclear mitotic apparatus protein (NuMA): spindle association, nuclear targeting and differential subcellular localization of various numa isoforms
Authors: Tang Tang K. , Tang Chieh-ju C. , Chao Yu-Jane , Wu Cheng-Wen
Published: April 2021

We have recently shown that the nuclear mitotic apparatus protein (NuMA) is composed of at least three isoforms that differ mainly at the carboxy terminus, and are generated by alternative splicing of a common mRNA precursor from a single NuMA gene (J. Cell Sci. (1993) 104, 249-260). Transient expression of human NuMA-l isoform (T33/p230) in Chinese hamster ovary polyoma (CHOP) cells showed that NuMA-l was present in interphase nuclei and was concentrated at the polar regions of the spindle apparatus in mitotic cells. However, expression of two other isoforms (NuMA-m and -s) revealed a distinct sub-cellular localization. NuMA-m (U4/p195) and NuMA-s (U6/p194) were present in the interphase cytosol and appeared to be mainly located at the centrosomal region. When cells entered into mitosis, however, NuMA-m and −s moved to the mitotic spindle pole. Analysis of a series of linker scanning-mutants and NuMA/β-galactosidase chimeric proteins showed that residues 1972-2007 of NuMA-l constitute a novel nuclear localization signal (NLS) and residues 1538-2115 are necessary and sufficient for spindle association. Further analysis of the NLS by site-specific mutagenesis indicated that Lys1988 is essential for nuclear targeting, whereas Arg1984 is not. These results have allowed us tentatively to assign specific biological activities to distinct structural domains of the NuMA polypeptide.
A monoclonal antibody identifies a novel GPI-anchored glycoprotein involved in epithelial intercellular adhesion
Authors: Rabino Mauro , Trusolino Livio , Prat Maria , Cremona Ottavio , Savoia Paola , Marchisio Pier Carlo
Published: April 2021

BD31 mAb, raised against a line of gastric carcinoma cells, reacts with intercellular boundaries of human transformed cells originating from carcinomas or sarcomas growing in epithelial-like clusters as well as in primary cultures of epithelial and endothelial cells. BD31 also reacts with inter- cellular rims of normal and transformed epithelial tissues and is particularly abundant in glands and fast-growing epithelia but absent in nervous and muscle tissues as well as in blood and in mesenchyme-derived cells. Confocal analysis indicates that BD31 is located in dots at cell-cell contacts but not in basal and apical domains of cultured and in situ epithelial cells. mAb BD31 precipitates a 100 kDa protein from cells labeled with [35S]methionine or [3H]glucosamine as well as from 125I-surface-labeled cells. This glycoprotein resists to trypsin in the presence of Ca2+, releases an 80 kDa fragment in the medium and does not react with antibodies to the conserved cytodomain of known cadherins or, specifically, to the ectodomain of E- cadherin in western blotting; moreover, the lack of cadherin cytodomain and protein removal by phospho- inositide-specific phospholipase indicate its membrane anchoring by a glycosyl-phosphatidylinositol (GPI) moiety. BD31 IgGs do not impair cell-matrix adhesion but induce inhibition of Ca2+-dependent aggregation, loss of cell-cell adhesion, scattering of confluent cells and appearance of migratory cell phenotypes in a dose- and time-dependent manner. This novel GPI-anchored glycoprotein may regulate intercellular adhesion by a mechanism involving membrane-associated phospholipases.
Cell cycle analysis and chromosomal localization of human Plk1, a putative homologue of the mitotic kinases Drosophila polo and Saccharomyces cerevisiae Cdc5
Authors: Golsteyn Roy M. , Schultz Sharon J. , Bartek Jiri , Ziemiecki Andrew , Ried Thomas , Nigg Erich A.
Published: April 2021

Polo and CDC5 are two genes required for passage through mitosis in Drosophila melanogaster and Saccharomyces cerevisiae, respectively. Both genes encode structurally related protein kinases that have been implicated in regu- lating the function of the mitotic spindle. Here, we report the characterization of a human protein kinase that displays extensive sequence similarity to Drosophila polo and S. cerevisiae Cdc5; we refer to this kinase as Plk1 (for polo-like kinase 1). The largest open reading frame of the Plk1 cDNA encodes a protein of 68,254 daltons, and a protein of this size is detected by immunoblotting of HeLa cell extracts with monoclonal antibodies raised against the C-terminal part of Plk1 expressed in Escherichia coli. Northern blot analysis of RNA isolated from human cells and mouse tissues shows that a single Plk1 mRNA of 2.3 kb is highly expressed in tissues with a high mitotic index, con- sistent with a possible function of Plk1 in cell proliferation. The Plk1 gene maps to position p12 on chromosome 16, a locus for which no associations with neoplastic malignan- cies are known. The Plk1 protein levels and its distribution change during the cell cycle, in a manner consistent with a role of Plk1 in mitosis. Thus, like Drosophila polo and S. cerevisiae Cdc5, human Plk1 is likely to function in cell cycle progression.
A cytoplasmic dynein motor in Drosophila: identification and localization during embryogenesis
Authors: Hays Tom S. , Porter Mary E. , McGrail Maura , Grissom Paula , Gosch Pam , Fuller Margaret T. , McIntosh J. Richard
Published: April 2021

We have characterized a cytoplasmic dynein motor isoform that is present in extracts of Drosophila embryos. A prominent high molecular weight (HMW) polypeptide (>400 kDa) is enriched in microtubules prepared from nucleotide-depleted embryonic extracts. Based on its ATP-sensitive microtubule binding activity, 20 S sedimentation coefficient, sensitivity to UV-vanadate and nucleotide specificity, the HMW polypeptide resembles cytoplasmic dyneins prepared from other organisms. The Drosophila cytoplasmic dynein acts as a minus-end motor that promotes microtubule translocation in vitro. A polyclonal antibody raised against the dynein heavy chain polypeptide was used to localize the dynein antigen in whole-mount preparations of embryos by immunofluorescence microscopy. These studies show that the dynein motor is associated with microtubules throughout embryogenesis, including mitotic spindle microtubules and microtubules of the embryonic nervous system.
The cytoplasmic domain of syndecan-1 is not required for association with Triton X-100-insoluble material
Authors: Miettinen Heini M. , Jalkanen Markku
Published: April 2021

Cell surface heparan sulfate proteoglycans such as syndecan-1 bind various extracellular matrix proteins and have been suggested to interact with the cytoskeleton. Such interactions are thought to be important for stabilizing cell morphology. Syndecan-1 resists extraction with Triton X-100. This insolubility was reported not to be affected by removal of the glycosaminoglycan chains, suggesting that the insolubility is not due to binding to the extracellular matrix, but rather to an association with the actin cytoskeleton (Rapraeger, A., Jalkanen, M. and Bernfield, M. (1986) J. Cell Biol. 103, 2683-2696). To examine further the interaction of syndecan-1 with the Triton X-100-insoluble residue, we expressed wild-type mouse syndecan-1 and a cytoplasmic deletion mutant (tail-less) in Chinese hamster ovary cells. We observed that both the wild-type and the tail-less syndecan-1 were partly insoluble in Triton X-100. The insolubility was not affected by increasing temperature (37°C or 50°C) or by cytochalasin D. Removal of the glycosaminoglycan chains from the ectodomain, however, resulted in complete Triton X-100 solubility, unlike previous reports. Syndecan-1 could also be released into the Triton X-100-soluble fraction by addition of heparin or heparan sulfate to the extraction medium. We conclude that the cytoplasmic domain of syndecan-1 is not responsible for Triton X-100 insolubility. Instead, our results indicate that Triton X-100 insolubility is caused by an interaction of syndecan-1 molecules with other cellular and/or extracellular molecules mediated by the heparan sulfate chains.
Vimentin’s tail interacts with actin-containing structures in vivo
Authors: Cary Robert B. , Klymkowsky Michael W. , Evans Robert M. , Domingo Alberto , Dent Joseph A. , Backhus Leilah E.
Published: April 2021

The tail domain of the intermediate filament (IF) protein vimentin is unnecessary for IF assembly in vitro. To study the role of vimentin’s tail in vivo, we constructed a plasmid that directs the synthesis of a ‘myc-tagged’ version of the Xenopus vimentin-1 tail domain in bacteria. This polypep- tide, mycVimTail, was purified to near homogeneity and injected into cultured Xenopus A6 cells. In these cells the tail polypeptide co-localized with actin even in the presence of cytochalasin. Two myc-tagged control polypeptides argue for the specificity of this interaction. First, a similarly myc-tagged lamin tail domain localizes to the nucleus, indi- cating that the presence of the myc tag did not itself confer the ability to co-localize with actin (Hennekes and Nigg (1994)J. Cell Sci. 107, 1019-1029). Second, a myc-tagged polypeptide with a molecular mass and net charge at phys- iological pH (i.e. −4) similar to that of the mycVimTail polypeptide, failed to show any tendency to associate with actin-containing structures, indicating that the interaction between mycVimTail and actin-containing structures was not due to a simple ionic association. Franke (1987; Cell Biol. Int. Rep. 11, 831) noted a similarity in the primary sequence between the tail of the type I keratin DG81A and vimentin. To test whether the DG81A tail interacted with actin-containing structures, we constructed and purified myc-tagged DG81A tail polypeptides. Unexpectedly, these keratin tail polypeptides were largely insoluble under phys- iological conditions and formed aggregates at the site of injection. While this insolubility made it difficult to determine if they associated with actin-containing struc- tures, it does provide direct evidence that the tails of vimentin and DG81A differ dramatically in their physical properties. Our data suggest that vimentin’s tail domain has a highly extended structure, binds to actin-containing structures and may mediate the interaction between vimentin filaments and microfilaments involved in the control of vimentin filament organization (Hollenbeck et al. (1989)J. Cell Sci. 92, 621; Tint et al. (1991)J. Cell Sci. 98, 375).
An inhibitor of neurite outgrowth produced by astrocytes
Authors: Smith-Thomas Linda C. , Fok-Seang Juin , Stevens James , Du Jian-Sheng , Muir Elizabeth , Faissner Andreas , Geller Herbert M. , Rogers John H. , Fawcett James W.
Published: April 2021

We have produced a number of astrocytic cell lines, some of which promote abundant neurite outgrowth, some of which are poor promoters of neurite outgrowth. The critical difference between these lines lies in the extracellular matrix, cell lines that are good promoters of axon growth producing a matrix that promotes axon growth, cell lines that are poor promoters of axon growth producing a non-permissive matrix. We were unable to find any consistent correlations between promotion of axon growth and production of proteases, protease inhibitors, N-cadherin, growth cone collapsing activity, and several extracellular matrix molecules. In the present study we have compared the least permissive of our cell lines, Neu7, with the most permissive, A7. Medium conditioned by the cell lines has the same properties as the matrix, since dorsal root ganglia (DRGs) grown in conditioned medium from the Neu7 line grow axons poorly, while DRGs grown in medium conditioned by A7 or primary astrocytes grow many long axons. Since matrix produced by all the cell lines contains large amounts of laminin, we looked to see whether the cells were producing laminin-blocking activity. Medium from the Neu7 line blocked laminin, while that from the A7 and primary astrocytes did not. However, when the conditioned media were heat-treated to remove neurite-promoting activity, they all had laminin-blocking activity: the blocking activity is heat stable. The neurite-promoting properties of the conditioned media therefore probably reflect a balance between promoting molecules and blockers. The laminin-blocking activity could be reduced by treatment of the heat-treated conditioned media with trypsin, keratanase, chondrointase ABC, but not chondroitinase AC or heparitinase. Fractionation of the conditioned medium on an ion-exchange column revealed that the laminin-blocking activity was found in the sulphatelabelled fractions, which are predominantly proteoglycan. Whole Neu7 extracellular matrix was treated with enzymes, and its neurite-promoting activity could be increased by chondroitinase ABC and to a lesser extent by keratinase, but not by heparitinase. We conclude that the critical difference between matrix produced by astrocytic cell lines that promote axon growth and those that do not lies in the level of production of a dermatan/keratan sulphate proteoglycan.
Perturbing cell surface β-(1,4)-galactosyltransferase on F9 embryonal carcinoma cells arrests cell growth and induces laminin synthesis
Authors: Maillet* Carol M. , Shur Barry D.
Published: April 2021

Cell growth and differentiation are influenced by intercellular contact, suggesting that cell adhesion molecules may be instrumental in triggering these events. F9 embryonal carcinoma cells are an ideal system in which to examine the function of cell adhesion molecules in growth and differentiation, since the relevant cell adhesion molecules and differentiation markers are well defined. Intercellular adhesion in F9 cells is mediated by uvomorulin, or E-cadherin, and cell surface β-(1,4)-galactosyltransferase. Since previous studies suggested that neither F9 cell growth nor differentiation is directly dependent on uvomorulin function, in this study we examined whether cell surface galactosyltransferase plays any role in F9 cell growth or differentiation.

A variety of galactosyltransferase perturbants, including anti-galactosyltransferase antibodies, UDPgalactose, and the substrate modifier protein α-lactalbumin, inhibited the growth of F9 cells, whereas control reagents did not. To examine this in more detail, we analyzed the effects of perturbing surface galactosyltransferase on progression through the F9 cell cycle. Anti-galactosyltransferase IgG treatment inhibited ornithine decarboxylase activity and lengthened the F9 cell cycle during G1 and G2, the latter mimicking the effects of retinoic acid, a reagent known to prolong the F9 cell cycle and induce differentiation. In contrast, anti-uvomorulin antibodies had no effect on F9 cell growth, ornithine decarboxylase activity, or progression through the cell cycle. Furthermore, perturbation of surface galactosyltransferase adhesions in F9 cell aggregates induced precocious F9 cell differentiation, as assayed by increased laminin synthesis, whereas control reagents had no effect. Thus, perturbing surface galactosyltransferase adhesions in F9 cells both decreases growth and stimulates synthesis of laminin. These results imply that interactions between surface galactosyltransferase and its oligosaccharide ligand during cell adhesion may affect the normal growth-regulatory and differentiation-inducing signals, as is seen, in part, during treatment with retinoic acid.