bluuebunny/crossref_metadata_2025 huggingface dataset:
Direct involvement of a lamin-B-related (54 kDa) protein in the association of intermediate filaments with the postsynaptic membrane of the Torpedo marmorata electrocyte
Authors: Cartaud Annie , Jasmin Bernard J. , Changeux Jean-Pierre , Cartaud Jean
Published: April 2021

Mechanisms by which motor innervation induces postsynaptic membrane differentiation and functional compart-mentalization of the subneural sarcoplasm in skeletal muscle fibres are still poorly understood. However, trans-membrane control of cytoskeletal activities by the nerve terminal may be considered. Here, we examine several properties of a 54 kDa protein, previously identified in the postsynaptic membrane of the Torpedo marmorata electrocyte with anti-lamin B antibodies, in order to study its role in the assembly of the subneural intermediate filament meshwork. Using a ligand blot assay, we show that this protein binds desmin, a type III intermediate filaments protein, at micromolar concentrations. Moreover, purified acetylcholine receptor-rich membrane fragments are able to generate arrays of desmin filaments in vitro. Immunofluorescence experiments indicate that the 54 kDa protein becomes associated with the acetylcholine receptor-rich membrane at an early stage of development of the electrocyte, and that a polarized desmin network develops concomitantly from the postsynaptic membrane. Taken together, these data show that, like karyoskeletal lamin B, the 54 kDa protein is involved in the organization of the subneural intermediate filament meshwork. Control of the assembly of the subneural cytoskeleton by components of the postsynaptic membrane may thus be a prerequisite for the functional compartmentalization of the muscle fibre triggered by motor innervation.
The A/B domain of truncated retinoic acid receptors can block differentiation and promote features of malignancy
Authors: Aneskievich Brian J. , Fuchs Elaine
Published: April 2021

Recently, we discovered that stable introduction of a carboxyl-terminally truncated retinoic acid receptor gamma (tRARγ) into an epidermal keratinocyte line blocked the ability of these cells to differentiate, as judged by their failure to express late markers of squamous differ-entiation. We now demonstrate a correlation between the level of residual endogenous RAR activity of tRARγ-expressing keratinocyte lines and degree of terminal differ-entiation. Mutagenesis studies localize the effects to the A/B subdomain of the truncated receptor. Despite tRARγ’s capacity to interfere with RAR-mediated transactivation of retinoic acid response elements (RAREs) in keratinocytes, the effects of the truncated receptor are independent of its ability to bind DNA and directly interact with endogenous RARs. tRARα also inhibits RARE-mediated gene expression in keratinocytes, even though its full-length counterpart enhances RARE activity in these cells. Intrigu-ingly, both tRARγ and RARγ suppress keratin promoter activity in epidermal cells, although for tRARγ, the effect is mediated through the A/B domain whereas for RARγ, the effects require DNA binding. Taken together, these findings suggest that the truncation allows for new and aberrant interactions with transcriptional proteins/co-factors that participate in governing RARE activity. This discovery may have relevance in tumorigenesis, where genetic lesions can result in mutant RARs or in loss of receptor expression.
Dermal fibroblasts convert to a myogenic lineage in mdx mouse muscle
Authors: Gibson Alexander J. , Karasinski Janusz , Relvas João , Moss Jill , Sherratt Tim G. , Strong Peter N. , Watt Diana J.
Published: April 2021

Duchenne muscular dystrophy is a primary muscle disease that manifests itself in young boys as a result of a defect in a gene located on the X-chromosome. This gene codes for dystrophin, a normal muscle protein that is located beneath the sarcolemma of muscle fibres. Therapies to alleviate this disease have centred on implanting normal muscle precursor cells into dystrophic fibres to compensate for the lack of this gene and its product. To date, donor cells for implantation in such therapy have been of myogenic origin, derived from paternal biopsies. Success in human muscle, however, has been limited and may reflect immune rejection problems. To overcome this problem the patient’s own myogenic cells, with the dystrophin gene inserted, could be used, but this could lead to other problems, since these cells are those that are functionally compromised by the disease. Here, we report the presence of high numbers of dystrophin-positive fibres after implanting dermal fibroblasts from normal mice into the muscle of the mdx mouse -the genetic homologue of Duchenne muscular dystrophy. Dystrophin-positive fibres were also abundant in mdx muscle following the implantation of cloned dermal fibroblasts from the normal mouse. Our results suggest the in vivo conversion of these non-myogenic cells to the myogenic pathway resulting in the formation of dystrophin-positive muscle fibres in the deficient host. The use of dermal fibroblasts may provide an alternative approach to the previously attempted myoblast transfer therapy, which in human trials has yielded disappointing results.
The role of protein phosphorylation in the assembly of a replication competent nucleus: investigations in Xenopus egg extracts using the cyanobacterial toxin microcystin-LR
Authors: Murphy Jacqueline , Crompton Catherine M. , Hainey Sandra , Codd Geoffrey A. , Hutchison Christopher J.
Published: April 2021

Cell-free extracts of Xenopus eggs support nuclear assembly and DNA replication in vitro. Extracts supple-mented with the protein phosphatase inhibitor micro-cystin-LR displayed various inhibitory effects at different concentrations of the toxin. In the presence of cycloheximide, additions of microcystin did not induce histone H1-kinase activity. Nevertheless, increasing concentrations of microcystin did sequentially prevent DNA replication, nuclear lamina assembly and nuclear envelope assembly. DNA replication was prevented when microcystin was added at 250 nM. Furthermore, this effect could be reversed after the addition of the catalytic sub-unit of protein phosphatase 2A to inhibited extracts. At a concentration of 250 nM microcystin, nuclear membrane assembly, nuclear lamina assembly and nuclear transport all occurred in egg extracts. In addition single-stranded M13 DNA replication was also permitted. However, it appeared that replicase assembly was not completed, since nuclei assembled in microcystin-treated extracts displayed an unusual distribution of proliferating cell nuclear antigen (PCNA). Although PCNA was located at sites that resembled pre-replication foci, this nuclear protein was readily solubilised when nuclei were isolated and extracted sequentially with Triton, nucleases and salts. Despite this, nuclei containing pre-assembled replication forks could synthesise DNA when transferred into microcystin-treated extracts.
Divergent modes of autophagy in the methylotrophic yeast Pichia pastoris
Authors: Tuttle Daniel L. , Dunn William A.
Published: April 2021

The budding yeast Pichia pastoris responds to methanolic media by synthesizing high levels of cytosolic enzymes (e.g. formate dehydrogenase) and peroxisomal enzymes (e.g. alcohol oxidase), which are necessary to assimilate this carbon source. Major alterations in cellular metabolism are initiated upon a shift in carbon source to ethanol or glucose. These alterations require the synthesis of new proteins and the rapid degradation of those enzymes no longer needed for methanol utilization. In this study, we have measured cytosolic and peroxisomal enzyme activities and examined the fate of morphologically distinct peroxisomes to assess the degradative response of this yeast during nutrient adaptation. Utilizing biochemical, morphological and genetic approaches, we have shown that there exist in P. pastoris at least two pathways for the sequestration of peroxisomes into the vacuole for degradation. The ethanol-induced pathway is independent of protein synthesis and includes an intermediate stage in which individual peroxisomes are sequestered into autophagosomes by wrapping membranes, which then fuse with the vacuole. This process is analogous to macroau-tophagy. The glucose-induced pathway invokes the engulfment of clusters of peroxisomes by finger-like protrusions of the vacuole by a process analogous to microautophagy. Unlike ethanol adaptation, glucose stimulated the degradation of formate dehydrogenase as well. Peroxisomes remained outside the vacuoles of glucose-adapted cycloheximide-treated normal cells, suggesting that protein synthesis is required for peroxisome entry into the yeast vacuole. Two complementary mutants (gsa1 and gsa2) that are unable to degrade peroxisomes or formate dehydrogenase during glucose adaptation were isolated. The mutated gene products appear to function in one or more events upstream of degradation within the vacuole, since ethanol-induced peroxisome degradation proceeded normally in these mutants and peroxisomes were found outside the vacuoles of glucoseadapted gsa2 cells. Mutants lacking vacuolar proteinases A and B were unable to degrade alcohol oxidase or formate dehydrogenase during ethanol or glucose adaptation. Peroxisomes were found to accumulate within the vacuoles of these proteinase mutants during adaptation. Combined, the results suggest that there exist in Pichia pastoris two independent pathways for the sequestration of peroxisomes into the vacuole, the site of degradation.
A yeast protein that bidirectionally affects nucleocytoplasmic transport
Authors: Singleton David R. , Chen Shaoping , Hitomi Midori , Kumagai Chino , Tartakoff Alan M.
Published: April 2021

We have identified a temperature-sensitive mutant of Sac-charomyces cerevisiae (npl3) that accumulates polyadeny-lated RNA in the nucleus at 37°C, as judged by in situ hybridization. The strong nuclear signal is not simply due to increased cytoplasmic turnover of mRNA, as reincuba-tion at 37°C with an RNA polymerase inhibitor shows no diminution in the in situ signal. Over several hours at 37°C, the average poly(A) tail length increases and a character-istic ultrastructural alteration of the nucleoplasm occurs. Cloning and sequencing indicate that the corresponding gene is NPL3/NOP3, which codes for a nucleolar/nuclear protein implicated in protein import into the nucleus (Bossie et al. (1992). Mol. Biol. Cell 3, 875-893) and in rRNA maturation (Russell and Tollervey (1992). J. Cell Biol. 119, 737-747). NPL3 includes bipartite RNA recognition motifs (RRM) and a Gly-Arg repeat domain, as in several nucleolar proteins. A point mutation adjacent to one of the RRM has been identified in the ts copy of the gene. Although this protein is not concentrated in nuclear pores, NPL3 is implicated in both import and export from the nucleus. Judging from the site of the npl3 mutation and since the block in RNA export can be detected prior to an obvious nuclear import defect in npl3, the defect in RNA export may be primary. Since other mutants that interrupt RNA export do not block protein import, the NPL3 protein itself appears to be implicated in protein import.
Lamin A precursor is localized to intranuclear foci
Authors: Sasseville A. Marie-Josée , Raymond Yves
Published: April 2021

Lamin A is synthesized in the cytoplasm as a precursor bearing a carboxyl-terminal CaaX box or isoprenylation signal. This precursor is post-translationally processed through multiple steps: isoprenylation with a farnesyl residue on the cysteine of the CaaX box, proteolytic removal of the last three amino acids, carboxymethylation of the cysteine residue and, finally, proteolytic removal of 15 amino acids from the carboxyl terminus. This last step gives rise to mature lamin A from which the isoprenylated terminus has been removed. Isoprenylation is a prerequisite for all other steps of processing. The subcellular location of these processing steps for lamin A is still a matter of debate. We have produced an antibody specific to the 18 amino acid carboxyl terminus of the lamin A precursor that does not recognize mature lamin A. This antibody detects intranuclear foci by immunofluorescence. Larger amounts of lamin A precursor were accumulated by treating cells with mevinolin (MVN), an inhibitor of iso-prenoid synthesis. In MVN-treated cells, the lamin A precursor accumulated most strikingly in the peripheral nuclear lamina where it was assembled, while intranuclear foci were maintained. The addition of an excess of meval-onate (MVA), which restores isoprenylation activity, to MVN-treated cells led to a progressive disappearance of the lamin A precursor from the peripheral lamina. This process was completed after 4 hours of MVA treatment, after which the lamin A precursor was restricted to intranuclear foci. We conclude from these results that the non-isoprenylated lamin A precursor appears competent for assembly into the peripheral nuclear lamina, and that all the processing steps leading to mature lamin A can occur within the nuclear space.
Interleukin-8 induces motile behavior and loss of focal adhesions in primary fibroblasts
Authors: Dunlevy Jane R. , Couchman John R.
Published: April 2021

Interleukin-8 (IL-8) is a proinflammatory cytokine that promotes neutrophil migration. Although fibroblasts are known to secrete IL-8, the actions of this cytokine on fibroblasts have not been previously reported. We have found that in subconfluent populations of cultured primary fibroblasts, IL-8 causes an increase in the percentage of cells lacking focal adhesions. Most of the IL-8-stimulated cells not only exhibit a lack of focal adhesions but also have a migratory phenotype that includes a protrusive leading edge and trailing tail. In addition, IL-8 was found to promote primary fibroblast chemotaxis in modified Boyden chambers as well as chemokinesis on serum-coated cover-slips. Human primary fibroblasts were also found to specif-ically bind to IL-8 with high affinity. We have previously shown that a lack of focal structures in primary fibroblasts can be used as an index of chemokinetic locomotion and have fully characterized this system using newborn rat heart conditioned medium. The main stimulus in heart con-ditioned medium that is responsible for the lack of focal adhesions in the majority of cells can be immunoprecipi-tated using a polyclonal antibody against recombinant human IL-8. Additionally, video microscopy assays using heart conditioned medium depleted with the IL-8 antibody show an increase in the percentage of stationary cells, a consequent decrease in the percentage of migrating cells, and a twofold increase in the mitotic rate.

Interleukin-1α and tumor necrosis factor-α, which are early inflammatory cytokines, have been previously shown to stimulate IL-8 production in macrophages, fibroblasts, endothelial and epithelial cells. Our findings indicate that these two cytokines also cause an increase in the percent-age of fibroblasts without focal adhesions. Additionally, this increase in cells lacking focal structures can be largely attributed to the production and subsequent autocrine action of a factor immunoprecipitated with an IL-8 antibody. Conversely, GRO-α, which has a high homology with IL-8, does not cause a similar increase in the percent-age of cells lacking focal adhesions, but was not antagonis-tic to the effects of IL-8.
Stabilization and bundling of subtilisin-treated microtubules induced by microtubule associated proteins
Authors: Saoudi Yasmina , Paintrand Isabelle , Multigner Luc , Job Didier
Published: April 2021

The acidic carboxy-terminal regions of α- and β-tubulin subunits are currently thought to be centrally involved in microtubule stability and in microtubule association with a variety of proteins (MAPs) such as MAP2 and tau proteins.

Here, pure tubulin microtubules were exposed to subtilisin to produce polymers composed of cleaved tubulin subunits lacking carboxy termini. Polymer exposure to subtilisin was achieved in buffer conditions compatible with further tests of microtubule stability. Microtubules composed of normal α-tubulin and cleaved β-tubulin were indistinguishable from control microtubules with regard to resistance to dilution-induced disassembly, to cold temperature-induced disassembly and to Ca2+-induced disassem-bly. Microtubules composed of cleaved α- and β-tubulins showed normal sensitivity to dilution-induced disassembly and to low temperature-induced disassembly, but marked resistance to Ca2+-induced disassembly. Polymers composed of normal α-tubulin and cleaved β-tubulin or of cleaved α- and β-tubulins were stabilized in the presence of added MAP2, myelin basic protein and histone H1. Cleavage of tubulin carboxy termini greatly potentiated microtubule stabilization by tau proteins. We show that this potentiation of polymer stabilization can be ascribed to tau-induced microtubule bundling. In our working con-ditions, such bundling upon association with tau proteins occurred only in the case of microtubules composed of cleaved α- and β-tubulins and triggered apparent micro-tubule cross-stabilization among the bundled polymers.

These results, as well as immunofluorescence analysis, which directly showed interactions between subtilisin-treated microtubules and MAPs, suggest that the carboxy termini of α- and β-tubulins are not primarily involved in the binding of MAPs onto microtubules. However, inter-actions between tubulin carboxy termini and MAPs remain possible and might be involved in the regulation of MAP-induced microtubule bundling.
Novel tools for the study of development, migration and turnover of nematocytes (cnidarian stinging cells)
Authors: Weber Jakob
Published: April 2021

The rhodamine derivatives tetramethyl-rhodamine-5/6-maleimide (TROMI) and tetramethyl-rhodamine-6-iso-thiocyanate (TRITC) were allowed to react with living Hydra vulgaris. The two fluorescent dyes stain the polyps to different degrees, apparently without impairing their viability and behaviour. Concerning nematocytes, TROMI preferentially couples to cytoskeletal elements only of mounted nematocytes whereas TRITC selectively reacts with structural components of cysts of late nematoblasts, which thereafter develop apparently normally into mature nematocytes. Hence TROMI-labelling indicates that nematocytes are mounted and ready for discharge; TRITC-labelling can be used as a tool to investigate the final mat-uration, migration and installation of nematocytes in Hydra.

Together with a new non-fixative method to dissociate Hydra polyps into single, identifiable cells, the two labelling methods allow direct quantitative dynamic studies of nematocyte turnover and open new possibilities of investigating the regulation and the mechanisms of nematocyte supply and migration.
The 190 kda centrosome-associated protein of Drosophila melanogaster contains four zinc finger motifs and binds to specific sites on polytene chromosomes
Authors: Whitfield W. G. F. , Chaplin M. A. , Oegema K. , Parry H. , Glover D. M.
Published: April 2021

Microinjection of a bacterially expressed, TRITC labelled fragment of the centrosome-associated protein CP190 of Drosophila melanogaster, into syncytial Drosophila embryos, shows it to associate with the centrosomes during mitosis, and to relocate to chromatin during interphase. Indirect immunofluorescence staining of salivary gland chromosomes of third instar Drosophila larvae, with anti-bodies specific to CP190, indicate that the protein is asso-ciated with a large number of loci on these interphase polytene chromosomes. The 190 kDa CP190 protein is encoded by a 4.1 kb transcript with a single, long open reading frame specifying a polypeptide of 1,096 amino acids, with a molecular mass of 120 kDa, and an isoelectric point of 4.5. The central region of the predicted amino acid sequence of the CP190 protein contains four CysX2CysX12HisX4His zinc-finger motifs which are similar to those described for several well characterised DNA binding proteins. The data suggest that the function of CP190 involves cell cycle dependent associations with both the centrosome, and with specific chromosomal loci.
Phalloidin unzips nebulin from thin filaments in skeletal myofibrils
Authors: Ao Xiaolei , Lehrer Sherwin S.
Published: April 2021

Fluorescent phallotoxins such as rhodamine-phalloidin take hours to bind uniformly to thin filaments of skeletal myofibrils, after fast initial binding to both ends of thin filaments. Observation of this process in skeletal and cardiac myofibrils and of the resulting re-distribution of nebulin using anti-nebulin antibody showed that: (1) rhodamine-phalloidin binds uniformly to actin in cardiac myofibrils within minutes, in contrast to skeletal myofib-rils; (2) overnight pre-incubation of skeletal myofibrils with phalloidin results in uniform initial binding of rhodamine-phalloidin and a changed nebulin localization; (3) pre-incu-bation of skeletal myofibrils with Ca2+-calmodulin results in uniform initial binding of rhodamine-phalloidin; (4) the binding of rhodamine-phalloidin to actin in skeletal myofibrils is unidirectional, i.e. the fluorescence of incor-porated rhodamine-phalloidin moves from the pointed ends where it is bound initially toward the barbed end at the Z-band; (5) the unidirectional binding of rhodamine-phalloidin results in redistribution of nebulin, i.e. the initial fluorescent bands associated with the epitopes of bound nebulin antibody change to a single band located close to Z-line. These results indicate that nebulin inhibits rhodamine-phalloidin binding to actin and suggests that the unidirectional rhodamine-phalloidin binding may be due to cooperative competitive binding, i.e. phalloidin ‘unzips’ nebulin starting from the pointed ends of the thin filaments.
The roles of Ca2+ and plasma membrane ion channels in hyphal tip growth of Neurospora crassa
Authors: Levina Natalia N. , Lew Roger R. , Hyde Geoffrey J. , Heath I. Brent
Published: April 2021

Growing hyphae of the ascomycete fungus Neurospora crassa contained a tip-high gradient of cytoplasmic Ca2+, which was absent in non-growing hyphae and was insen-sitive to Gd3+in the medium. Patch clamp recordings in the cell-attached mode, from the plasma membrane of these hyphae, showed two types of channel activities; spon-taneous and stretch activated. The spontaneous channels were identified as inward K+channels based on inhibition by tetraethylammonium. The stretch activated channels had increased amplitudes in response to elevated Ca2+in the pipette solution, and thus are permeable to Ca2+and mediate inward Ca2+movement. Gd3+, which is an inhibitor of some stretch activated channels, incompletely inhibited stretch activated channel activity. Both tetraethylammonium and Gd3+only transiently reduced the rates of tip growth without changing tip morphology, thus indicating that the channels are not absolutely essential for tip growth. Furthermore, in contrast to the hyphae of another tip growing organism, Saprolegnia ferax, tip-high gradients of neither spontaneous nor stretch activated channels were found. Voltage clamping of the apical plasma membrane potential in the range from −300 to +150 mV did not affect the rates of hyphal elongation. Collectively, these data suggest that ion transport across the plasma membrane at the growing tip in Neurospora is not obligatory for the maintainance of tip growth, but that a gradient of Ca2+, possibly generated from internal stores in an unknown way, is required.
Yeast ypt51p and mammalian rab5: Counterparts with similar function in the early endocytic pathway
Authors: Singer-Krüger Birgit , Stenmark Harald , Zerial Marino
Published: April 2021

Ypt51p, a small GTPase of Saccharomyces cerevisiae, has been previously identified as a structural homolog of mammalian Rab5. Although disruption analysis revealed that the protein is required for endocytic transport and for vacuolar protein sorting, the precise step controlled by Ypt51p was not determined. In this work we show that by heterologous expression in animal cells Ypt51p was targeted to Rab5-positive early endosomes and stimulated endocytosis. Furthermore, two Ypt51p mutants induced similar morphological alterations as the corresponding Rab5 mutants. Also in yeast cells Ypt51p was found to be required at an early step in endocytic membrane traffic, since α-factor accumulated in an early endocytic inter-mediate in the absence of Ypt51p. Cell fractionation analysis revealed cofractionation of Ypt51p with endocytic intermediates, while no association with the late Golgi com-partment could be detected. Indirect immunofluorescence microscopy allowed us to morphologically identify the Ypt51p-containing compartment. Similar to the mammalian system larger Ypt51p-positive structures were revealed upon expression of Ypt51p Q66L. These structures were also positive for α-factor receptor and for car-boxypeptidase Y, thus providing direct evidence for their endocytic nature and for the convergence of the vacuolar biosynthetic and endocytic pathways.
Fgf inhibits neurite outgrowth over monolayers of astrocytes and fibroblasts expressing transfected cell adhesion molecules
Authors: Williams Emma J. , Mittal Bina , Walsh Frank S. , Doherty Patrick
Published: April 2021

We have cultured cerebellar neurons on monolayers of cortical astrocytes in control medium or medium containing recombinant basic fibroblast growth factor (FGF). FGF was found to inhibit neurite outgrowth, with a significant effect seen at 0.5 ng/ml and a maximal effect at 10 ng/ml. FGF increased the production of arachidonic acid (AA) in cerebellar neurons, and when added directly to cultures or generated endogenously via activation of phospholipase A2 using melittin, this second messenger could mimic the inhibitory effect of FGF. FGF and AA could also specifically inhibit neurite outgrowth stimulated by three cell adhesion molecules (NCAM, N-cadherin and L1) expressed in transfected fibroblasts, or in the case of L1 bound to a tissue culture substratum. These data demonstrate that, in certain cellular contexts, FGF can act as an inhibitory cue for axonal growth and that arachidonic acid is the second messenger responsible for this activity. We discuss the possibility that arachidonic acid inhibits neurite outgrowth by desensitising the second messenger pathway underlying neuronal responsiveness to cell adhesion molecules.
Loss of mdck cell α2β1 integrin expression results in reduced cyst formation, failure of hepatocyte growth factor/scatter factor-induced branching morphogenesis, and increased apoptosis
Authors: Saelman Edwin U. M. , Keely Patricia J. , Santoro Samuel A.
Published: April 2021

Cellular interactions with collagen in a model of kidney tubulogenesis were investigated using Madin-Darby canine kidney (MDCK) cells in an in vitro morphogenetic system. MDCK cells adhered to collagen types I and IV in a Mg2+-dependent manner, typical of the α2β1 integrin. Collagen-Sepharose affinity chromatography and immunoblotting demonstrated the presence and collagen binding activity of the α2β1 integrin on MDCK cells. To assess the function of α2β1 integrin, MDCK cells were transfected with a plasmid pRSVα2′ which allowed the expression of α2-integrin subunit antisense RNA. Three G418-resistant clones showing reduced adhesion to collagen, stable genomic integration of the antisense construct, decreased α2-integrin subunit mRNA and decreased α2-integrin subunit protein expression were selected for analysis in morphogenetic experiments. MDCK cells and plasmid-only control transfectants, cultured in three-dimensional collagen type I gels, showed normal cyst formation, whereas the antisense RNA transfectants showed increased apoptosis and formed small rudimentary cysts. Stimulation with hepatocyte growth factor/scatter factor-containing 3T3 fibroblast-conditioned medium or recombinant hepatocyte growth factor/scatter factor resulted in extensive branching of the preformed control cysts whereas the surviving small cysts formed by antisense expressing cells increased in size but failed to elongate and branch upon stimulation. We conclude that α2β1 integrin collagen interactions play a crucial role in the hepatocyte growth factor/scatter factor-induced tubulogenesis and branching morphogenesis of MDCK cells in collagen gels as well as an important role in cell survival.
Calcium requirements during mitotic cdc2 kinase activation and cyclin degradation in Xenopus egg extracts
Authors: Lindsay H. D. , Whitaker M. J. , Ford C. C.
Published: April 2021

Activation of p34cdc2 kinase is essential for entry into mitosis while subsequent deactivation and cyclin degradation are associated with exit. In Xenopus embryos, both of these phases are regulated by post-translational modifications and occur spontaneously on incubation of extracts prepared late in the first cell cycle. Even though high levels of calcium buffer were initially used to prepare these extracts, we found that free calcium levels in them remained in the observed physiological range (200-500 nM). Further addition of calcium buffers only slightly reduced free calcium levels, but inhibited histone H1 (cdc2) kinase deactivation and cyclin degradation. Higher buffer concentrations slowed the kinase activation phase. Reducing the free buffer concentration by premixing with calcium reversed the effects of the buffer, indicating that the inhibitory effects arose from the calcium-chelating properties of the buffer rather than non-specific side effects. Furthermore, additions of calcium buffer at the end of the H1 kinase activation phase did not prevent deactivation. From these results, and the order of effectiveness of different calcium buffers in disrupting the H1 kinase cycle, we suggest that local transient increases in free calcium influence the rate of cdc2 kinase activation and are required to initiate the pathway leading to cyclin degradation and kinase inactivation in mitotic cell cycles.
Stratifin, a keratinocyte specific 14-3-3 protein, harbors a pleckstrin homology (ph) domain and enhances protein kinase c activity
Authors: Dellambra Elena , Patrone Mauro , Sparatore Bianca , Negri Armando , Ceciliani Fabrizio , Bondanza Sergio , Molina Francesco , Cancedda Fiorella Descalzi , Luca Michele De
Published: April 2021

The intrinsic signal(s) responsible for the onset of human keratinocyte terminal differentiation is not yet fully under-stood. Evidence has been recently accumulated linking the phospholipase-mediated activation of protein kinase C to the coordinate changes in gene expression occurring during keratinocyte terminal differentiation. Here we report the purification of a keratinocyte-derived protein enhancing protein kinase C enzymatic activity. The stimulator eluted as a peak with estimated molecular mass of approximately 70 kDa, while analysis by SDS-PAGE showed a 30 kDa protein migrating as a distinct doublet, suggesting the formation of a 30 kDa homodimer. The amino acid sequence analysis allowed the unambigous identification of the protein kinase C stimulator as a mixture of the highly homologous σ (stratifin) and ζ isoforms of 14-3-3 proteins, which are homodimers of identical 30 kDa subunits. Mono Q anion exchange chromatography and immunoblot analysis further confirmed that stratifin enhances protein kinase C activity. Stratifin was originally sequenced from a human keratinocyte protein database, but its function was unknown. The pleckstrin homology domain has been recently related to protein translocation to the cell membrane as well as to functional interactions of intracel-lular proteins involved in signal transduction. We show here that stratifin (and 14-3-3 ζ) harbors a pleckstrin homology domain, and the consequent functional implications will be discussed.
Neither erk nor jnk/sapk map kinase subtypes are essential for histone h3/hmg-14 phosphorylation or c-fos and c-jun induction
Authors: Cano Eva , Hazzalin Catherine A. , Kardalinou Efterpi , Buckle Robin S. , Mahadevan Louis C.
Published: April 2021

The effects of EGF, TPA, UV radiation, okadaic acid and anisomycin on ERK and JNK/SAPK MAP kinase cascades have been compared with their ability to elicit histone H3/HMG-14 phosphorylation and induce c-fos and c-jun in C3H 10TG cells. EGF and UV radiation activate both ERKs and JNK/SAPKs but to markedly different extents; EGF activates ERKs more strongly than JNK/SAPKs, whereas UV radiation activates JNK/SAPKs much more strongly than ERKs. Anisomycin and okadaic acid activate JNK/SAPKs but not ERKs, and conversely, TPA activates ERKs but not JNK/SAPKs. Nevertheless, all these agents elicit phosphorylation of ribosomal and pre-ribosomal S6, histone H3 and HMG-14, and the induction of c-fos and c-jun, showing that neither cascade is absolutely essential for these responses. We then analysed the relationship between ERKs, JNK/SAPKs and the transcription factors Elk-1 and c-Jun, implicated in controlling c-fos and c-jun, respec-tively. JNK/SAPKs bind to GST-cJun1-79, and ERKs, par-ticularly ERK-2, to GST-Elk1307-428; there is no cross-specificity of binding. Further, GST-Elk1307-428 binds preferentially to active rather than inactive ERK-2. In vitro, JNK/SAPKs phosphorylate both GST-cJun1-79 and GST-Elk1307-428, whereas ERKs phosphorylate GST-Elk1307-428 but not GST-cJun1-79. Thus, neither ERKs nor JNK/SAPKs are absolutely essential for nuclear signalling and c-fos and c-jun induction. The data suggest either that activation of a single MAP kinase subtype is sufficient to elicit a complete nuclear response, or that other uncharac-terised routes exist.
The distribution of Na+,K+-atpase and 5a11 antigen in apical microvilli of the retinal pigment epithelium is unrelated to α-spectrin
Authors: Rizzolo Lawrence J. , Zhou Shaoming
Published: April 2021

The retinal pigment epithelium was used to study the relationship between the cortical cytoskeleton and two plasma membrane proteins that associate with it. These proteins were the Na+,K+-ATPase, an ion pump, and the 5A11 antigen, a member of the immunoglobulin super-family of receptor proteins. The cytoskeleton was marked by two of its constituents, α-spectrin and ankyrin. Ankyrin links the Na+,K+-ATPase to spectrin in many cells. The RPE is of interest, because unlike most epithelia it distributes the Na+,K+-ATPase to the apical membrane. The development of polarity was studied during chick embryogenesis. On embryonic day 6 (E6), each of these proteins was observed in the apical and lateral plasma membranes. As development proceeded, only the Na+,K+-ATPase was removed from the lateral membranes. Beginning on E12, ankyrin, spectrin and 5A11 appeared together in patches along the basal plasma membrane. By E16, these patches coalesced into a uniform distribution along the basal membrane. At the apical pole, α-spectrin appeared near the base of the microvilli, but was undetected in the microvilli them-selves. This distribution resembled the distribution of α-spectrin in the intestine and proximal kidney tubule. By contrast, a pool of ankyrin and 5A11 and nearly all the Na+,K+-ATPase appeared in the microvilli. Despite its segregation from α-spectrin, the Na+,K+-ATPase appeared to associate with a macromolecular complex, as judged by extraction with Triton X-100. Changes in spectrin distribution could not be related to changes in isoform expression, as only one isoform of β-spectrin was detected by co-immunoprecipitation with α-spectrin. By contrast, multiple ankyrin-like peptides could be identified by immunoblotting. These data illustrate some of the unique properties of RPE microvilli. These properties prevent the Na+,K+-ATPase from complexing with the α-spectrin-based cytoskeleton by sequestering the enzyme into the compartment where its activity is required.
Elucidation of nucleus-cytoplasm interaction: change in ability of the nucleus to express sexuality according to clonal age in Paramecium
Authors: Haga Nobuyuki
Published: April 2021

The expression of sexual activity in Paramecium caudatum is repressed for about 50 fissions after conjugation. The ability of the macronucleus to affect the expression of sexual activity according to the age of clonal development was investigated using the macronuclear fusionreorganization method. When a mature macronucleus was transplanted into an immature cell and fused with a macronucleus in the immature cell, the clones derived from the recipient showed sexual immaturity. In a reverse experiment, an immature macronucleus was transplanted into a mature cell, and the clones also showed sexual immaturity. The ability of the macronucleus to transform mature cells to immature cells was clonal agedependent. The characteristics of the immaturemature hybrid macronucleus indicate that the immature macronucleus is dominant over the mature macronucleus with respect to the ability to express sexual activity. On the other hand, in cells of the early immaturity period, the micronucleus, known as the germ nucleus, shows the ability to undergo meiosis and eventually to produce progeny under control of the mature macronucleus. The expression of sexual activity is thought to be governed by the clonal age of the macronucleus and not by the clonal age of the micronucleus or cytoplasm. The macronucleus seems to determine the ability to express sexual activity by counting postconjugation divisions and keeping track of the age of the clone.
A 182 bp fragment of the mouse proα1(II) collagen gene is sufficient to direct chondrocyte expression in transgenic mice
Authors: Zhou Guang , Garofalo Silvio , Mukhopadhyay Krish , Lefebvre Véronique , Smith Chad N. , Eberspaecher Heidi , Crombrugghe Benoit de
Published: April 2021

Type II collagen is a major chondrocyte-specific component of the cartilage extracellular matrix and it represents a typical differentiation marker of mature chondrocytes. In order to delineate cis-acting elements of the mouse proα1(II) collagen gene that control chondrocyte-specific expression in intact mouse embryos, we generated transgenic mice harboring chimeric constructions in which varying lengths of the promoter and intron 1 sequences were linked to a β-galactosidase reporter gene. A construction containing a 3,000 bp promoter and a 3,020 bp intron 1 fragment directed high levels of β-galactosidase expression specifically to chondrocytes. Expression of the transgene coincided with the temporal expression of the endogenous gene at all stages of embryonic development. Successive deletions of intron 1 delineated a 182 bp fragment which targeted β-galactosidase expression to chondrocytes with the same specificity as the larger intron 1 fragment. Transgenic mice harboring a 309 bp Col2a1 promoter lacking intron 1 tester sequences showed no β-galactosidase expression in chondrocytes. Reduction of the 182 bp fragment to a 73 bp subfragment surrounding a decamer sequence previously reported to be involved in chondrocyte specificity, resulted in loss of transgene expression in chondrocytes. When the Col2a1 promoter was replaced with a minimal β-globin promoter, the 182 bp intron 1 sequence was still able to target expression of the transgene to chondrocytes. We conclude that a 182 bp intron 1 DNA segment of the mouse Col2a1 gene contains the necessary information to confer highlevel, temporally correct, chondrocyte expression on a reporter gene in intact mouse embryos and that Col2a1 promoter sequences are dispensable for chondrocyte expression.
Matrigel induces thymosin β4 gene in differentiating endothelial cells
Authors: Grant D. S. , Kinsella J. L. , Kibbey M. C. , LaFlamme S. , Burbelo P. D. , Goldstein A. L. , Kleinman H. K.
Published: April 2021

We performed differential cDNA hybridization using RNA from endothelial cells cultured for 4 hours on either plastic or basement membrane matrix (Matrigel), and identified early genes induced during the morphological differentiation into capillarylike tubes. The mRNA for one clone, thymosin β4, was increased 5-fold. Immunostaining localized thymosin β4 in vivo in both growing and mature vessels as well as in other tissues. Endothelial cells transfected with thymosin β4 showed an increased rate of attachment and spreading on matrix components, and an accelerated rate of tube formation on Matrigel. An antisense oligo to thymosin β4 inhibited tube formation on Matrigel. The results suggest that thymosin β4 is induced and likely involved in differentiating endothelial cells. Thymosin β4 may play a role in vessel formation in vivo.
Localization of the rat myosin I molecules myr 1 and myr 2 and in vivo targeting of their tail domains
Authors: Ruppert Christian , Godel Jasminca , Müller Rainer T. , Kroschewski Ruth , Reinhard Jutta , Bähler Martin
Published: April 2021

Myr 1 is a widely distributed mammalian myosin I molecule related to brush border myosin I. A second widely distributed myosin I molecule similar to myr 1 and brush border myosin I, called myr 2, has now been identified. Specific antibodies and expression of epitope-tagged molecules were used to determine the subcellular localization of myr 1 and myr 2 in NRK cells. Myr 1 was detected at the plasma membrane and was particularly enriched in cell protrusions like lamellipodia, membrane ruffles and filopodia. In dividing cells myr 1 localized to the cleavage furrow. Myr 2 was localized in a discrete punctate pattern in resting cells and in cells undergoing cytokinesis. In sub-cellular fractionation experiments myr 1 and myr 2 were both partly soluble and partly associated with smooth membranes of medium density.

The tail domains of myosin I molecules have been proposed to interact with a receptor and thereby determine the subcellular localization. To test this hypothesis we expressed the tail domains of myr 1 and myr 2 that lack the F-actin-binding myosin head domain in NRK cells. These tail domains also partly copurified with smooth membranes of medium density and immunolocalized similar to the respective endogenous myosin I; however, they exhibited a lower affinity for membranes and an increased diffuse cytosolic localization. These results suggest that the tail domains of myr 1 and myr 2 are suf-ficient for subcellular targeting but that their head domains also contribute significantly to maintaining a proper sub-cellular localization.
Expression of laminin isoforms in mouse myogenic cells in vitro and in vivo
Authors: Schuler Frank , Sorokin Lydia M.
Published: April 2021

The expression of laminin-1 (previously EHS laminin) and laminin-2 (previously merosin) isoforms by myogenic cells was examined in vitro and in vivo. No laminin α2 chain-specific antibodies react with mouse tissues, so rat mono-clonal antibodies were raised against the mouse laminin α2 chain: their characterization is described here. Myoblasts and myotubes from myogenic cell lines and primary myogenic cultures express laminin β1 and γ1 chains and form a complex with a 380 kDa α chain identified as laminin α2 by immunofluorescence, immunoprecipitation and PCR.

PCR from C2C12 myoblasts and myotubes for the laminin α2 chain gene (LamA2) provided cDNA sequences which were used to investigate the in vivo expression of mouse LamA2 mRNA in embryonic tissues by in situ hybridization. Comparisons were made with specific probes for the laminin α1 chain gene (LamA1). LamA2 but not LamA1 mRNA was expressed in myogenic tissues of 14-and 17-day-old mouse embryos, while the laminin α2 polypeptide was localized in adjacent basement membranes in the muscle fibres. In situ hybridization also revealed strong expression of the LamA2 mRNA in the dermis, indicating that laminin α2 is expressed other than by myogenic cells in vivo. Immunofluorescence studies localized laminin α2 in basement membranes of basal ker-atinocytes and the epithelial cells of hair follicles, providing new insight into basement membrane assembly during embryogenesis.

In vitro cell attachment assays revealed that C2C12 and primary myoblasts adhere to laminin-1 and -2 isoforms in a similar manner except that myoblast spreading was sig-nificantly faster on laminin-2. Taken together, the data suggest that laminins 1 and 2 play distinct roles in myo-genesis.
Receptor-mediated adhesive and anti-adhesive functions of chondroitin sulfate proteoglycan preparations from embryonic chicken brain
Authors: Ernst Heidemarie , Zanin Mary K. B. , Everman David , Hoffman Stanley
Published: April 2021

Chondroitin sulfate proteoglycans inhibit the adhesion of cells to extracellular matrix proteins that otherwise permit adhesion. Although proteoglycans are widely assumed to act by masking the other protein in a mixed substrate, recent studies suggest that proteoglycans inhibit adhesion through mechanisms initiated by their binding to specific cell surface receptors. To explore this issue, we developed a purification scheme to isolate proteoglycan aggregates, monomers, and core proteins. Two distinct adhesion assays were used to study the interaction of these proteoglycan preparations with human foreskin fibroblasts: the gravity assay in which cell attachment is stabilized by cell spreading, and the centrifugation assay in which spreading does not play a role. All proteoglycan preparations mediate adhesion in the centrifugation assay but not in the gravity assay. In the centrifugation assay, proteoglycan aggregates and monomers are considerably more active than other extracellular matrix proteins while proteoglycan core proteins are at least as active as other extracellular matrix proteins. Proteoglycan core proteins bind to cell-associated hyaluronic acid, but not to integrins. Using mixed sub-strates in the gravity assay, all proteoglycan preparations inhibited cell attachment to fibronectin and vitronectin but not to collagen I and laminin. Although proteoglycan aggregates and monomers are more active than core proteins in inhibiting adhesion in the gravity assay, core proteins are still clearly active. A variety of control exper-iments suggest that the inhibition of cell attachment by pro-teoglycans is mediated through the specific interactions of proteoglycans with cell surface receptors, resulting in the inhibition of cell spreading. These results suggest at least two molecular mechanisms for proteoglycan-fibroblast interactions, one involving the chondroitin sulfate on the proteoglycan and an as yet unidentified receptor, the other involving the proteoglycan core protein and cell-associated hyaluronic acid.
Differential targeting of recombinant fibronectins in AtT-20 cells based on their efficiency of aggregation
Authors: Castle Anna M. , Schwarzbauer Jean E. , Wright Rhonda L. , Castle J. David
Published: April 2021

In pituitary-derived AtT-20 cells, recombinant fibronectin containing the N-terminal matrix assembly domain and the C-terminal half of fibronectin does not follow the regulated secretory pathway but instead concentrates in distinct organelles prior to secretion. These organelles are larger than the dense-core granules and localize to the cell body at sites that differ from lysosomes, endosomes and endo-plasmic reticulum. Unlike the dense-core granules, their discharge is not stimulated by 8-bromo-cyclic-AMP or phorbol esters. The kinetics of intracellular transport and secretion of the recombinant fibronectin suggest that it is present in a post-Golgi pool that turns over more slowly than constitutive vesicles. Indeed, the fibronectin-contain-ing organelles disappear with a half-time of 3 hours after inhibiting protein synthesis. Presence of the organelles correlates with intracellular aggregation of dimeric fibronectin polypeptides. The organelles are absent in cells expressing monomeric recombinant fibronectin (lacking C-terminal dimerization sites) or the C-terminal half of fibronectin (which dimerizes but lacks the N-terminal matrix assembly domain), both of which aggregate less efficiently than dimeric fibronectin. Instead, the latter polypeptides enter the dense-core granules. Thus while the formation of the fibronectin-containing organelles may require efficient aggregation, it may not require a specific structural signal. Moreover, efficient aggregation is not necessarily a prerequisite for following the regulated pathway.