bluuebunny/crossref_metadata_2025 huggingface dataset:
Matrix metalloproteinase-9 (92 kDa gelatinase/type IV collagenase) from U937 monoblastoid cells: correlation with cellular invasion
Authors: Watanabe Hideto , Nakanishi Isao , Yamashita Kyoko , Hayakawa Taro , Okada Yasunori
Published: April 2021

The role of matrix metalloproteinase-9 (MMP-9, 92 kDa gelatinase/type IV collagenase) in invasion of mononuclear phagocytes was studied with U937 monoblastoid cells. 12-o-tetradecanoyl 13-phorbol acetate (TPA) differentiated them to macrophage-like cells with induction of MMP-9, and tumor necrosis factor TNF and interleukin-1α (IL-1α) stimulated the production of MMP-9 by TPA-treated cells. TNF also induced the production of MMP-9 by TPA-untreated U937 cells without morphological differentiation. Other agents including dimethyl sulfoxide (DMSO), all-trans-retinoic acid (all-trans-RA), platelet-derived growth factor and 3′ ;5′-cyclic monophosphate had no effects on MMP-9 production by TPA-treated or-untreated cells, but all-trans-RA and DMSO did have a morphological effect on the differentiation of the cells. These data suggest that MMP-9 production by U937 cells is regulated by a mechanism independent of the differentiation to macrophage-like cells. MMP-9 was purified to homogeneity as an inactive zymogen with Mr 92,000 (proMMP-9) from TPA-differentiated U937 cells treated with TNF. ProMMP-9 was activated by p-aminophenylmercuric acetate (APMA) generating an active species of Mr 67,000. Trypsin and cathepsin G also attained activation of the zymogen to its full activity obtained by APMA activation, but plasmin, leukocyte elastase, thrombin and plasma kallikrein had no ability to activate it. APMA-activated MMP-9 degraded type I gelatin readily and cleaved native collagen types III, IV and V. Invasion assays using reconstituted basement membrane coupled with a type IV collagenolysis assay showed good correlations between invasiveness, type IV collagenolysis and proMMP-9 production. Invasion was significantly inhibited by EDTA, 2-macroglobulin and tissue inhibitor of metalloproteinases-1, but not by inhibitors of cathepsin G and leukocyte elastase. These data suggest that MMP-9 plays an important role in the invasion of mononuclear phagocytes through basement membranes.
A methodology for the systematic and quantitative study of cell contact guidance in oriented collagen gels Correlation of fibroblast orientation and gel birefringence
Authors: Guido Stefano , Tranquillo Robert T.
Published: April 2021

Despite the likely role of contact guidance in every physiological process involving cell migration, its study in a three-dimensional tissue-equivalent environment has been precluded, heretofore, by inherent difficulties in systematically preparing well-defined contact guidance fields and quantifying the resultant contact guidance. Here, we describe a novel use of a magnetic field to orient collagen fibrils during fibrillogenesis, entrapping cells dispersed in the collagen solution. Using computer-controlled staging and image analysis, we show from automated birefringence measurements of the resultant slab of cell-populated gel contained in a specially designed observation chamber that the fibril orientation is biased along the long axis of the chamber uniformly throughout the chamber. Further, we show that the degree of fibril orientation, and consequently the elicited contact guidance, can be controlled by independently varying the magnetic field strength or temperature during fibrillogenesis. We characterize the contact guidance response to the imposed contact guidance field by measuring cell orientation relative to the axis of fibril orientation from still images obtained in time-lapse via automated image analysis. We present the first quantitative correlation of contact guidance (based on cell orientation) with collagen fibril orientation (based on birefringence) for human foreskin fibroblasts cultured in a collagen gel, by using gels of varying orientation resulting from different magnetic field strengths and temperatures during fibrillogenesis, and by using sufficiently low cell concentrations and early observation times.
Modulation of Muc-1 mucin expression in the mouse uterus during the estrus cycle, early pregnancy and placentation
Authors: Braga Vania M. M. , Gendler Sandra J.
Published: April 2021

The Muc-1 protein is an integral membrane protein that is expressed apically by simple secretory epithelia in many different organs. We present in this paper a study on Muc-1 protein expression in the mouse uterus during early pregnancy, placentation and the estrus cycle. Muc-1 immunopositive reaction is found in the decidua by day 8 of pregnancy onwards. The observed pattern was unusual, since Muc-1 protein was present in spherical cytoplasmic granules in granular metrial gland cells. Both the intracellular pattern of expression and the lymphoid origin of these cells were striking results. Muc-1 is thought to be an epithelial differentiation marker, and this is the first report of its expression by non-epithelial cells. Our results on Muc-1 expression in the uterus of cycling mice showed that higher levels of Muc-1 mRNA and protein correlate with higher levels of plasma estrogen in the estrus and proestrus phases. However, in ovariectomized mice without hormone replacement, the endometrium expressed high levels of this protein. These levels could not be substantially changed by estrogen, although progesterone reduced the levels of Muc-1 protein associated with the epithelium. These data together with the normal expression in the cycling mice suggest that progesterone might repress Muc-1 expression during the metestrus and diestrus phases. In cycling mice, when plasma progesterone is at its nadir and the estrogen level is elevated in estrus and proestrus phases, Muc-1 concentration would increase to its basal level, not because of estrogen stimulation, but due to lack of progesterone repression. The low level of expression is also observed in the endometrium during early pregnancy, where reduced levels of Muc-1 protein are seen at the time of implantation.
Controlled induction of focal adhesion disassembly and migration in primary fibroblasts
Authors: Dunlevy Jane R. , Couchman John R.
Published: April 2021

Fibroblast migration is an integral component of biological processes such as wound healing and embryogenesis. Previous experiments examining fibroblast locomotion from tissue explants have shown that migrating fibroblasts lack, or contain only transient, focal adhesions (focal contacts). Focal adhesions are specialized regions of tight cell-matrix interaction, assembled by a complex process of transmembrane signalling. Although the explant model has been used for studying several aspects of fibroblast locomotion, it is limited by the lack of control over migration, and only a small percentage of the cells actually locomoting. Therefore, we have developed an in vitro model for cultured fibroblast strains where the presence or absence of focal adhesions can be manipulated, and in the latter case 70% of these cells become locomotory.

The stimulus used to decrease the percentage of cells containing focal adhesions, and hence enhance locomotion, was newborn rat heart-conditioned medium (HCM). Addition of HCM to rat embryo fibroblasts induced both chemokinesis and chemotaxis. Cells disassembled focal adhesions on a variety of extracellular matrix substrates after approximately 6 h of stimulation with HCM; conversely, removal of HCM promoted reformation of focal adhesions within 12-24 h. HCM-stimulated fibroblasts which lacked focal adhesions concomitantly lacked F-actin stress fibers and focal concentrations of vinculin and talin. Therefore, fibroblast migration can be readily controlled in an on-off manner through conditioned medium, which influences the absence or presence of focal adhesions.
Topoisomerase II inhibition prevents anaphase chromatid segregation in mammalian cells independently of the generation of DNA strand breaks
Authors: Clarke Duncan J. , Johnson Robert T. , Downes C. Stephen
Published: April 2021

Yeast temperature-sensitive mutants of DNA topoisomerase II are incapable of chromosome condensation and anaphase chromatid segregation. In mammalian cells, topoisomerase II inhibitors such as etoposide (VP-16-123) have similar effects. Unfortunately, conclusions drawn from work with mammalian cells have been limited by the fact that the standard inhibitors of topoisomerase II also generate DNA strand breaks, which when produced by other agents (e.g. ionizing radiation) are known to affect progression into and through mitosis. Here we show that the anti-tumour agent ICRF-193, recently identified as a topoisomerase II inhibitor operating by a non-standard mechanism, generates neither covalent complexes between topoisomerase II and DNA, nor adjacent DNA strand breaks, in mitotic HeLa. However, the drug does prevent anaphase segregation in HeLa and PtK2 cells, with effects similar to those of etoposide. We therefore conclude that topoisomerase II function is required for anaphase chromosome segregation in mammalian cells, as it is in yeast.
Regulation of p53 protein expression in human breast cancer cell lines
Authors: Vojtěšek Bořivoj , Lane David P.
Published: April 2021

Mutation of the p53 gene is a common occurrence in human breast cancers but is by no means universal. However, even in tumours where the gene is not mutated altered levels of p53 protein are often detected. This is also observed in cell lines derived from human breast cancers. By transfecting such cell lines containing either wild type or mutant p53 genes with a temperature-sensitive mutant mouse p53 gene we have established that the cellular environment plays a critical role in the regulation of p53 protein expression. The results suggest that tumours that aberrantly express wild-type p53 may have lost the normal growth regulatory response to the protein and thus be functionally similar to those expressing the mutant protein.
Differential localization patterns of myristoylated and nonmyristoylated c-Src proteins in interphase and mitotic c-Src overexpresser cells
Authors: David-Pfeuty Thérèse , Bagrodia Shubha , Shalloway David
Published: April 2021

Myristoylation of pp60src is required for its membrane attachment and transforming activity. The mouse monoclonal antibody, mAb327, which recognizes both normal, myristoylated pp60c-src and a nonmyristoylated mutant, pp60c-src/myr−, has been used to compare the effects of preventing myristoylation on the localization of c-Src in NIH 3T3-derived overexpresser cells using immunofluorescence microscopy. During interphase, pp60c-src partitions between the plasma membrane and the centrosome, while pp60c-src/myr− is predominantly cytoplasmic but also partly nuclear. The cytoplasmic, but not the nuclear, staining can be readily washed out by brief pretritonization of the cells before fixation, indicating that the cytoplasmic pool of pp60c-src/myr−, in contrast with the nuclear one, does not associate tightly with structures that are insoluble in the presence of nonionic detergents. We have previously shown that during G2 phase, pp60c-src leaves the plasma membrane and is redistributed diffusely throughout the cytoplasm and to two clusters of patches surrounding the two separating centriole pairs. In contrast, we now find that pp60c-src/myr− translocates to the nucleus in late G2 or early prophase prior to there being any clear evidence of nuclear membrane breakdown or nuclear lamina disassembly. Similar nuclear translocation of pp60c-src/myr−, but not of pp60c-src, is also observed when cells are arrested in G0 or at the G1/S transition. Furthermore, during mitosis, pp60c-src is found primarily in diffuse and patchy structures dispersed throughout the cytoplasm while pp60c-src/myr− more specifically associates with the main components of the spindle apparatus (poles and fibers) and inside the interchromosomal space.

These results suggest that a possible role for myristoylation might be to prevent unregulated nuclear transport of proteins whose nonmyristoylated counterparts are readily moved into the nucleus. They also raise the possibility that a subfraction of wild-type pp60c-src may behave, at specific times, like its nonmyristoylated counterpart, and may translocate to the nucleus and exert specific functions in that location.
Binding of the α-fodrin SH3 domain to the leading lamellae of locomoting chicken fibroblasts
Authors: Meriläinen J. , Palovuori R. , Sormunen R. , Wasenius V.-M. , Lehto V.-P.
Published: April 2021

Fodrin (nonerythroid spectrin) is a membrane skeletal protein that plays an important role in the establishment and maintenance of the cell shape and polarity. We have identified in α-fodrin an src homology 3 (SH3)-related region, a small domain that is present in a large number of proteins that are involved in signal transduction, cell polarization and membrane-cytoskeleton interactions. In this study we have explored the function of the α-fodrin SH3 by incubating fixed and permeabilized cultured chicken fibroblasts with the αfodrin SH3 peptide, expressed in bacteria as a fusion protein with glutathione S-transferase. Immunofluorescence and immunoelectron microscopy showed that αfodrin SH3 binds to the cytoplasmic face of the plasma membrane in the leading lamellae and the pseudopodial lobes of the spreading and locomoting cells. No, or only minimal, binding was seen in immotile cells, or in the stationary trailing ends of the locomoting cells. SH3 binding was also seen in cytochalasin-D-treated cells, suggesting that actin filaments are not responsible for the binding. These findings suggest that α-fodrin SH3 interacts with plasma membrane components that are present in the leading lamellae exclusively or are modulated in a manner specific to the leading lamellae.
Distribution of acidic and basic fibroblast growth factors in ovine skin during follicle morphogenesis
Authors: Cros D. L. du , Isaacs K. , Moore G. P. M.
Published: April 2021

Acidic and basic fibroblast growth factors (aFGF and bFGF) have been localized by immunochemistry in ovine skin during wool follicle morphogenesis. At 40 days of gestation, prior to the appearance of follicle pri-mordia, bFGF immunoreactivity was detected in the intermediate and periderm layers of the epidermis and at the dermal-epidermal junction. Antibodies to aFGF did not bind to skin at this age. During early follicle formation, at 76 days of gestation, both FGFs were found in the epidermis and associated with the follicle primordia. Antibodies to aFGF, in particular, bound to the basal cells of the epidermis and the follicle cell aggregations. With the development of epidermal plugs, bFGF was confined to the intermediate layers of the epidermis and the dermal-epidermal junction, whereas aFGF staining was associated with the cells of the epidermis and the plugs. At 90 days, when many different stages of follicle development were in evidence, immunoreactivity for both FGFs was associated with the cells of the elongating epidermal column, particularly those adjacent to the dermal-epidermal junction. During follicle maturation, bFGF was found in the suprabasal layer of the epidermis, in the outer root sheath of the follicle and in the basement membrane zone surrounding the bulb matrix. Conversely, strong staining for aFGF was observed in the epidermis and pilary canal contiguous with the epidermis, and in cells of the upper bulb matrix of the follicle in the region of the keratogenous zone. Western blotting of extracts of mature follicles that had been isolated from the skin showed the presence of a major aFGF immunoreactive band with an apparent molecular mass of 27 kDa.

The distributions of aFGF and bFGF, particularly around the dermal-epidermal junction during follicle development, demonstrate that these growth factors may have related functions in local tissue remodelling during follicle morphogenesis. However, in adult skin, the presence of bFGF adjacent to the proliferative zone of the follicle suggests its involvement in regulating the mitotic activity in the follicle bulb. By contrast, the localization of aFGF to the cells of the upper follicle bulb, in the zone of keratinization, implicates this growth factor in cellular differentiation.
Redundant enhancer elements guide β1 tubulin gene expression in apodemes during Drosophila embryogenesis
Authors: Buttgereit Detlev
Published: April 2021

During Drosophila embryogenesis, the β1 tubulin gene ( Tub56D) is expressed in the CNS and PNS as well as in the apodemes. In this report we determine the regulation of β1 tubulin gene expression during formation of the attachment sites of the somatic muscles, the apodemes. The process was analysed in transgenic flies carrying β1 enhancer-hsp70 promoter-lacZ fusion constructs. Expression is first detected at late stage 13 and remains until hatching. By deletion analysis of the intron we identified a 14-bp element present in three copies. This element represents a classical enhancer, as it acts on a heterologous promoter. Separate fragments containing the respective elements yield nearly identical expression patterns, and no cooperativity was observed between the three copies. Thus, the expression of the β1 tubulin gene in the apodemes is under control of redundant enhancer elements. Double staining for β1 tubulin gene expression in apodemes and for β3 tubulin gene expression in muscles allowed us to correlate apodeme and muscle formation. Cells of the apodemes that are in contact with their corresponding muscles show expression of the reporter gene as monitored by antibody staining.
Expression of high molecular weight tau in the central and peripheral nervous systems
Authors: Georgieff Irene S. , Liem Ronald K. H. , Couchie Dominique , Mavilia Carmelo , Nunez Jacques , Shelanski Michael L.
Published: April 2021

Using a novel PCR approach, we have cloned a cDNA encoding the entire high molecular weight tau molecule from rat dorsal root ganglia. The resulting 2080 bp cDNA differs from low molecular weight rat brain tau by the insertion of a novel 762 bp region (exon 4a) between exons 4 and 5. This cDNA clone is identical in sequence with a high molecular weight tau (HMW) cDNA from rat PC12 tumor cells and is closely related to a HMW tau cDNA from mouse N115 tumor cells. In vitro transcription/translation produces a protein that migrates on SDS-PAGE with the same apparent molecular weight as HMW tau purified from rat sciatic nerve. The HMW tau protein is generated from an 8 kb mRNA, which can be detected by northern blots in peripheral ganglia, but not in brain. A more sensitive assay using PCR and Southern blot analysis demonstrates the presence of exon 4a in spinal cord and in retina. In combination with immunohistochemical studies of spinal cord, these data suggest that HMW tau, though primarily in the peripheral nervous system, is also expressed in limited areas of the central nervous system, although its presence cannot be detected in the cerebral cortices.
Endothelial cell attachment and spreading on human tenascin is mediated by α2β1 and αVβ 3 integrins
Authors: Sriramarao P. , Mendler Markus , Bourdon Mario A.
Published: April 2021

Human umbilical vein endothelial cells were found to attach and partially spread on human tenascin. The attachment of endothelial cells to tenascin results in elongated cells with interconnecting processes and is distinct from the flattened appearance of endothelial cells on fibronectin, collagen, vitronectin or laminin substrata, suggesting a role for tenascin in modulating cell adhesion and motility. Endothelial attachment to tenascin was partially inhibitable by the SRRGDMS peptide derived from human tenascin and completely inhibitable by anti-integrin antibodies to α2β1 and αvβ3. Endothelial cell attachment to tenascin could be inhibited up to 80% with anti-x2 and anti-β1 monoclonal antibodies P1E6 and P4C10, respectively, and this was associated with a complete loss in cell spreading. In contrast, pretreatment of endothelial cells with the anti-(XVβ3 monoclonal antibody LM609, resulted in a 35% inhibition in cell attachment but did not alter cell spreading. In combination the anti-α2 and anti-œVβ3 antibodies, could completely abrogate cell spreading and attachment to tenascin-coated surfaces. Affinity purification of 125I-labeled endothelial cell extract on a tenascin matrix column followed by immunoprecipitation with monoclonal antibodies to different integrin ex and β subunits resulted in the identification of α2β1 and (XVβ3 integrins, respectively, as tenascin binding receptors. Collagen affinity-purified ot2β1 receptor from endothelial cells bound not only to collagen and laminin but also to tenascin in a radio receptor binding assay. The results demonstrate that (X2β1 and αVβ3 mediate distinct endothelial cell interactions with tenascin; cell spreading and cell binding, respectively. Binding by cxVβ3 is mediated by the SRRGDMS site on tenascin, whereas the α2β1 binding site remains undefined. The interaction of oc2β1 and cxVβ3 with tenascin may be regulated in a cell typespecific manner as evidenced by the binding of endothelial cell oc2β1 and (XVβ3 to tenascin, and the lack of binding by the same receptors on osteosarcoma MG63 to tenascin.
Ezrin is concentrated in the apical microvilli of a wide variety of epithelial cells whereas moesin is found primarily in endothelial cells
Authors: Berryman Mark , Franck Zsofia , Bretscher Anthony
Published: April 2021

Ezrin and moesin are two cytoskeletal proteins originally purified from human placenta that are 74% identical in overall protein sequence. They are believed to be membrane-cytoskeletal linking proteins because they share sequence homology with erythrocyte band 4.1 and colocalize with actin specifically in microvilli and membrane ruffles in cultured cells. To determine if ezrin and moesin share similar distributions in vivo, we studied their localizations with respect to F-actin in tissue sections. Surprisingly, ezrin and moesin exhibited very different cellular distributions. Ezrin was highly concentrated and colocalized with actin on the apical surface of many epithelial cell types. During enterocyte differentiation, the pattern of expression and redistribution of ezrin was consistent with it performing a role in microvillus assembly. Immunoelectron microscopy in differentiated cells revealed that ezrin was restricted mainly to the plasma membrane of microvilli and other actin-rich surface projections. Moesin was found in endothelial cells and was also enriched in the apical microvilli of a restricted set of epithelial cells. All polarized cell types with abundant microvilli contained one or both proteins, suggesting that ezrin and moesin perform related functions. However, the differential expression of ezrin and moesin indicates that they have distinct properties, which are uniquely adapted to specific cell types.
Reversion analysis of dynein intermediate chain function
Authors: Mitchell David R. , Kang Yong
Published: April 2021

The ODA6 locus of Chlamydomonas reinhardtii encodes a 70 kDa intermediate chain protein of the flagellar outer row dynein ATPase, and mutations at this locus prevent assembly of the entire outer row dynein arm complex. To initiate a structure-function analysis of the 70 kDa protein, we used transformation with chimeric mutant/wild-type genes to localize the defect in one assembly mutation, oda6-95. Sequence analysis revealed a frame-shift mutation in codon 53, which is followed by a stop codon after 13 amino acids in the new reading frame. By selecting intragenic pseudorevertants of this mutation we obtained 11 new oda6 alleles. Many of these pseudorevertants encode intermediate chain proteins that permit assembly of outer row arms but do not restore full wild-type motility. Revertant strains fall into two phenotypic classes, one with average beat frequencies of 54 Hz (similar to wild type) and one with average frequencies of 27 Hz (compared with 24 Hz for oda6-95) during normal forward swimming. Low beat frequency strains also display abnormalities during photophobic reversal (symmetric waveform). Amplification and sequence analysis of revertant alleles indicated that each reversion caused a second frame-shift, within a 115 nt interval, which restored the original reading frame, and that phenotypic severity was related to both direction (5’ or 3’ and distance between the original mutation and the reversion event. On the basis of immunoblot analysis of outer arm proteins, we conclude that revertant motility defects do not correlate with deficits in assembly of a specific dynein heavy chain or intermediate chain polypeptide, and electron microscopy confirms that revertants have normal outer arm structures. These results suggest that the 70 kDa intermediate chain plays a direct role in outer arm function distinct from its role in the assembly process.
Molecular cloning and cellular localization of a bip homologue in Trypanosoma brucei: Divergent er retention signals in a lower eukaryote
Authors: Bangs James D. , Uyetake Lyle , Brickman Marla Jo , Balber Andrew E. , Boothroyd John C.
Published: April 2021

Using the polymerase chain reaction with degenerate primers, three new members of the hsp70 gene family of Trypanosoma brucei have been identified. A genomic clone of one of these, gA, has been fully sequenced and the corresponding gene product has been characterized using antibody to recombinant gA fusion protein. gA is the trypanosomal homologue of BiP, an endoplasmic reticulum resident hsp70 gene family member, based on four lines of evidence: (1) gA protein has 64% deduced amino acid identity with rat BiP; (2) the deduced amino acid sequence has a putative secretory signal peptide; (3) the gA gene product is a soluble luminal resident of a trypanosomal microsome fraction; (4) the gA polypeptide does not cofractionate with mitochondrial markers. Trypanosomes are the most primitive eukaryote yet in which BiP has been identified. The gA polypeptide has been used as a specific marker for the direct visualization of endoplasmic reticulum in trypanosomes by both indirect immunofluorescence and cryoimmuno electron microscopy. The endoplasmic reticulum is seen as a tubular network that extends throughout the cell excluding the flagellum. The C-terminal tetrapeptide of gA is MDDL, which, together with the C-terminal tetrapeptide (KQDL) of a trypanosome protein disulfide isomerase homologue (Hsu et al. (1989)Biochemistry 28, 6440-6446), indicates that endoplasmic reticulum retrieval signals in trypanosomes may be as divergent and heterogeneous as any seen in the other eukaryotes yet studied.
Evidence for two intracellular calcium pools in Dictyostelium: the campinduced calcium influx is directed into a nbd-cland 2,5-di-(tert-butyl)-1,4 hydroquinone-sensitive pool
Authors: Flaadt H. , Jaworski E. , Malchow D.
Published: April 2021

Signal transduction in Dictyostelium for oriented movement and differentiation involves a fine tuning of the cytosolic Ca2+ concentration. We have previously shown that cAMP binding to the cell surface receptor elicits two cellular events: (i) to enhance Ca2+ entry across the plasma membrane; (ii) to increase Ca2+ uptake into Ca2+-sequestering organelles. Here we used permeabilised cells to show that cAMP-induced Ca2+ uptake in these cells was sensitive to the Ca2+ transport ATPase blocker 2,5-di-(tert-butyl)-1,4-hydroquinone (BHQ) and the vacuolar H+-ATPase inhibitor NBD-Cl. By contrast, bafilomycin A1 and vanadate, inhibitors of Ca2+ uptake into acidosomes in Dictyostelium, did not reduce the cAMP-induced Ca2+ uptake of permeabilised cells. GTPγS served as a tool to measure Ins(l,4,5) P3-(InsP3)-sensitive Ca2+ release. Following NBD-Cl or BHQ treatment Ca2+ release was reversibly inhibited. We conclude that the cAMP-controlled Ca2+ influx is directed into a NBD-Cl and BHQ-sensitive compartment, which comprises the InsP3-releasable pool. The acidosomal Ca2+ store seems to provide for additional Ca2+ if required.
Association of the brain anion exchanger, AE3, with the repeat domain of ankyrin
Authors: Morgans Catherine W. , Kopito Ron R.
Published: April 2021

The 89 kDa NH2-terminal domain of erythrocyte ankyrin is composed almost entirely of 22 tandem repeats of a 33 amino acid sequence and constitutes the binding site for the cytoplasmic NH2-terminal domain of the erythrocyte anion exchanger, AE1. We have developed an assay to evaluate the in vivo interaction between a fragment of ankyrin corresponding to this domain (ANK90) and two non-erythroid anion exchangers, AE2 and AE3, that share considerable structural homology with AE1. Association was assessed by coimmunoprecipitation of ANK90-anion exchanger complexes from detergent extracts of cells cotransfected with plasmids encoding the ankyrin fragment and the anion exchanger or mutants thereof. ANK90 was coimmunoprecipitated with AE1 but not with an AE1 deletion mutant lacking the cytoplasmic NH2-terminal domain. Using this assay, we show that the brain anion exchanger AE3, but not the closely related homologue, AE2, is capable of binding to ankyrin.
Cell-to-cell transport via motile tubules in growing hyphae of a fungus
Authors: Shepherd V. A. , Orlovich D. A. , Ashford A. E.
Published: April 2021

The system of pleiomorphic, motile tubules and vacuoles in growing hyphal tips of Pisolithus tinctorius has been shown to play a role in intracellular transport. Here we show that the same system also exchanges material between adjacent cells. This exchange is most obvious between terminal and penultimate cells following nuclear division in the tip cell and just before dissolution of the cell wall between the clamp connection and penultimate cell. At this stage the two new dolipore septa are complete. The process was studied in living hyphae using confocal and conventional fluorescence microscopy. Tubules could move in either direction across the septum and often extended and retracted several times and penetrated for some distance (e.g. 40 µm) into the receiving cell. Movements appeared co-ordinated and during the exchange tubules transiently interconnected vacuoles in adjacent cells and by peristaltic movements appeared to transfer material between them.

The fluorescent tubules occupied a specific plane in the vicinity of the septum and remained in this plane for the duration of their movement, suggesting that their orientation and direction of movement is controlled. In freeze-substituted hyphae, tubular cisternae of similar dimensions to fluorescent tubules passed through the parenthesome pores perpendicular to the septum and in some cases entered the mouth of the septal pore. This indicates that the septal pore is of an appropriate dimension to accommodate the tubules and that they can cross the septal pore to exchange material between vacuole systems of adjacent cells. This is the first direct demonstration of such intercellular transport via a sub-cellular compartment.
The highly divergent α- and β-tubulins from Dictyostelium discoideum are encoded by single genes
Authors: Triviños-Lagos Leda , Ohmachi Tetsuo , Albrightson Caryn , Burns Roy G. , Ennis Herbert L. , Chisholm Rex L.
Published: April 2021

As a step in the characterization of the microtubule system of Dictyostelium discoideum, we have isolated and sequenced full-length cDNA clones that encode the Dic tyostelium a- and β-tubulins, as well as the Dictyostelium a -tubulin gene. Southern blot analysis suggests that Dic tyostelium is unusual in that its genome contains single α- and β-tubulin genes, rather than the multi-gene family common in most eukaryotic organisms. The complete α-tubulin cDNA contains 1558 nucleotides, with an open reading frame, that encode a protein of 457 amino acids. The complete β-tubulin cDNA contains 1572 nucleotides and encodes a protein of 456 amino acids. Analysis of the deduced protein sequences indicates that while there is a significant degree of sequence similarity between the Dictyostelium tubulins and other known tubulins, the Dictyostelium α-tubulin displays the greatest sequence divergence yet described. Single α- and β-tubulin transcripts are detected by northern blot analysis during all stages of Dictyostelium development. The highest levels of message accumulate late in germinating spores and vegetative amoebae. Despite changes in α - and β-tubulin mRNA levels, protein levels remain constant throughout development. We have expressed the carboxy-terminal two-thirds of the α- and β-tubulins as trpE fusions in Escherichia coli and used this protein to produce polyclonal antisera specific for the Dic tyostelium ơ- and β-tubulins. These antisera recognize one ex- and two β-tubulin spots on western blots of 2-D gels and, by indirect immunofluorescence, both recognize the interphase and mitotic microtubule arrays in vegetative amoebae.
The small gtp-binding protein, Rab6p, is associated with both golgi and post-golgi synaptophysin-containing membranes during synaptogenesis of hypothalamic neurons in culture
Authors: Tixier-Vidal Andrée , Barret Alain , Picart Renée , Mayau Véronique , Vogt Dorothée , Wiedenmann Bertram , Goud Bruno
Published: April 2021

We have recently localized a small GTP-binding protein (Rab6p) thought to be involved in vesicular membrane transport, to the medial and trans-cisternae of the Golgi apparatus in NRK (normal rat kidney) cells. Here, we have localized and quantified Rab6p during the development in culture of embryonic neurons, up to synapse formation, and compared its subcellular distribution and level of expression to that of synaptophysin, a major integral membrane protein of small synaptic vesicles. Using immunocytochemistry (laser scanning confocal microscopy, immunoelectron microscopy), fractionation and immunoisolation methods, we show that during the early phase of synaptogenesis, Rab6p is associated with synaptophysin-containing membranes of a trans-Golgi subcompartment, post-Golgi vesicles and small synaptic vesicles or their precursors. Concomitantly, Rab6p undergoes translocation from cytosol to membranes and its level of expression increases. However, at late stages, the association of Rab6p to small synaptic vesicles sharply decreases and its level of expression plateaus. These findings suggest a role for Rab6p in the post-Golgi transport of synaptophysin, at an early step of the biogenesis of small synaptic vesicles.
Implanted myoblasts not only fuse with myofibers but also survive as muscle precursor cells
Authors: Yao Shou-Nan , Kurachi Kotoku
Published: April 2021

Intramuscular implanted myoblasts can fuse with existing myofibers. Here we report that implanted primary myoblasts marked with retroviral transgenes can also persist as muscle precursor cells. These cells can be recovered as viable myoblasts from muscles of recipent mice even months after myoblast implantation, and they can fully resume expression of the transgenes in culture. Upon re-implantation into muscles, they again not only fuse with existing myofibers, but also survive as muscle precursor cells in the tissue. These reserve myogenic cells should be able to contribute to host myofibers in muscle regeneration when the recombinant myofibers are damaged, providing an additional mechanism to maintain a persistent expression of transgenes delivered by myoblast-mediated gene transfer.
The spatial distribution and relative abundance of gap-junctional connexin40 and connexin43 correlate to functional properties of components of the cardiac atrioventricular conduction system
Authors: Gourdie Robert G. , Severs Nicholas J. , Green Colin R. , Rothery Stephen , Germroth Patricia , Thompson Robert P.
Published: April 2021

Electrical coupling between heart muscle cells is mediated by specialised regions of sarcolemmal interaction termed gap junctions. In previous work, we have demonstrated that connexin42, a recently identified gapjunctional protein, is present in the specialised conduction tissues of the avian heart. In the present study, the spatial distribution of the mammalian homologue of this protein, connexin40, was examined using immunofluorescence, confocal scanning laser microscopy and quantitative digital image analysis in order to determine whether a parallel distribution occurs in rat. Con-nexin40 was detected by immunofluorescence in all main components of the atrioventricular conduction system including the atrioventricular node, atrioventricular bundle, and Purkinje fibres. Quantitation revealed that levels of connexin40 immunofluorescence increased along the axis of atrioventricular conduction, rising over 10-fold between atrioventricular node and atrioventricular bundle and a further 10-fold between atrioventricular bundle and Purkinje fibres. Connexin40 and con-nexin43, the principal gap-junctional protein of the mammalian heart, were co-localised within atrioventricular nodal tissues and Purkinje fibres. By applying a novel photobleach/double-labelling protocol, it was demonstrated that connexin40 and connexin43 are colocalised in precisely the same Purkinje fibre myocytes. A model, integrating data on the spatial distribution and relative abundance of connexin40 and connexin43 in the heart, proposes how myocyte-type-specific patterns of connexin isform expression account for the electrical continuity of cardiac atrioventricular conduction.
Organization of the double-stranded rna-activated protein kinase dai and virus-associated va rnai in adenovirus-2-infected hela cells
Authors: Jiménez-García Luis F. , Green Simon R. , Mathews Michael B. , Spector David L.
Published: April 2021

We have examined the cellular distribution of the double-stranded RNA-activated protein kinase DAI in adenovirus 2 (Ad2) -infected and uninfected HeLa cells. In uninfected cells DAI was found to be concentrated in the cytoplasm. In addition, DAI was localized in the nucleoli and diffusely distributed throughout the nucleoplasm. Cells treated with α-interferon displayed a similar pattern of distribution for DAI. When RNA polymerase I activity was inhibited by the drug actinomycin D, nucleoli segregated and DAI was found to colocalize with the dense fibrillar region of the nucleoli. During mitosis, the distribution of DAI paralleled that of rRNA. In adenovirus-infected cells the localization of DAI was similar to that in uninfected interphase cells. VA RNAI was detected in Ad2-infected cells by 10-14 hours postinfection as fine dots in the nucleoplasm. By 18-24 hours post-infection, VA RNAI appeared in bigger and more abundant dots in the nucleoplasm and the cytoplasm was intensively labeled. Transient expression of the VA RNAI gene in uninfected cells resulted in a similar localization of the RNA. Our results are consistent with a role for DAI and VA RNAI in protein synthesis and suggest that DAI may play an early role in ribosome biogenesis in the nucleolus in addition to its cytoplasmic role in translation.
Regional variation in distribution of egf receptor in developing and adult corneal epithelium
Authors: Zieske James D. , Wasson Michael
Published: April 2021

Epidermal growth factor receptor has been localized to the proliferative cell layers in a variety of stratified squamous epithelia. In the current study, the rat cornea was used as an experimental model to determine if epidermal growth factor receptor is concentrated in epithelial stem cells. Epidermal growth factor receptor was localized using immunofluorescence microscopy in adult and neonatal (1-day to 4-week) rat corneas. Antibody binding to epidermal growth factor receptor was present in basal cells across the adult cornea but was more intense in the limbal zone. In rats 1 day to 1 week of age, the corneal epithelium consisted of one or two layer of cells that were intensely labeled by anti-epidermal growth factor receptor. Following epithelial stratification, which occurred just prior to eyelid opening (∼12 days), expression of epidermal growth factor receptor was greatly reduced in central corneal epithelium and gained an adult pattern by 3 weeks of age. Expression of epidermal growth factor receptor was also examined by incubating 1 mm slices of adult corneas with 125I-epidermal growth factor (4 nM) for 90 minutes, followed by washing and autoradiography. Basal cells in the limbal zone contained 4.5-fold more silver grains per cell than did basal cells in the central cornea. These data suggest that cells with high potential for proliferation, i.e. limbal basal cells and all basal cells in developing rats, express high epidermal growth factor receptor levels. High levels of receptor may allow these cells to be rapidly stimulated by growth factors to undergo cell division during development and following wounding in adult corneas. High epidermal growth factor receptor levels may also provide a mechanism whereby limbal basal cells are maintained in an undifferentiated stem cell state rather than entering the pathway of terminal differentiation.
Regulation of keratinocyte terminal differentiation by integrin-extracellular matrix interactions
Authors: Watt Fiona M. , Kubler M.-Dominique , Hotchin Neil A. , Nicholson Linda J. , Adams Josephine C.
Published: April 2021

Suspension-induced terminal differentiation of human epidermal keratinocytes can be inhibited by fibronectin through binding to the α5β1 integrin. We have investigated the effect of fibronectin on expression of integrins and proteins of the actin cytoskeleton and have explored the nature of the differentiation stimulus by testing different combinations of anti-integrin monoclonal antibodies or extracellular matrix proteins in the suspension assay. Fibronectin prolonged cell surface expression of β1 integrins but did not overcome the inhibition of intracellular transport of integrins that occurs when keratinocytes are placed in suspension. Fibronectin did not prevent the suspension-induced decline in the level of mRNAs encoding the β1 integrin subunit, actin, filamin and α-actinin; furthermore, the inhibition of terminal differentiation did not depend on the state of assembly of microfilaments or microtubules. Terminal differentiation could be partially inhibited by an adhesion-blocking monoclonal antibody to the β1 integrin subunit or by a combination of adhesion blocking antibodies recognising the α subunits that associate with α1 (α2, α3 and α5). Although laminin and type IV collagen do not inhibit terminal differentiation individually, they were inhibitory when added to cells in combination with a low concentration of fibronectin. We conclude that the proportion of keratinocyte β1 integrins occupied by ligand can regulate the initiation of terminal differentiation independently of the state of assembly of the actin cytoskeleton.
Evidence that vault ribonucleoprotein particles localize to the nuclear pore complex
Authors: Chugani Diane C. , Rome Leonard H. , Kedersha Nancy L.
Published: April 2021

Vaults are cytoplasmic ribonucleoprotein organelles that are highly conserved among diverse eukaryotic species. Their mass (12.9 MDa), diameter (26-35 nm) and shape (two halves, each with eightfold radial symmetry) have recently been determined and are similar to those ascribed to the central plug (or transporter) of the nuclear pore complex (NPC). The size and eightfold symmetry of the vault particle make it conducive to interacting physically in a complementary manner with NPCs. The present study demonstrates that vaults specifically associate with nuclei by both immunoblotting and immunofluorescence. Immunogold EM confirmed that vaults associate with the nuclear envelope in tissue sections and with NPCs of isolated nuclei.
In vivo expression and molecular characterization of the porcine slow myosin heavy chain
Authors: Chang Kin-Chow , Fernandes Kenneth , Goldspink Geoffrey
Published: April 2021

We report on the molecular characterization of the porcine slow-myosin heavy chain (HC) β gene and the isolation of its 5’ end cDNA. In vivo expression study, by in situ hybridization and histochemistry, revealed a highly regular rosette pattern of fiber arrangement, with a slow fiber occupying the central core, in all the skeletal muscles examined. This feature can be advantageous in the distinction of primary and secondary fibers in myogenic lineage studies. In the neonatal heart, ß isoform expression is diffuse, with higher expression occurring in the ventricle than in the atrium. Transient transfection assays showed the porcine promoter functions in a muscle- and differentiation stage-specific manner. In the 5’ regulatory region are several putative positive and negative regulatory elements, including a positive and a negative element in close proximity to each other in intron 1.
Cell binding specificity of mouse r-cadherin and chromosomal mapping of the gene
Authors: Matsunami Hiroaki , Miyatani Seiji , Inoue Takayoshi , Copeland Neal G. , Gilbert Debra J. , Jenkins Nancy A. , Takeichi Masatoshi
Published: April 2021

R-cadherin was originally identified as a chicken cad- herin expressed by the retina. Here, we describe the identification of a mouse homologue of R-cadherin. We isolated mouse cDNAs encoding a cadherin with 94% identity in amino acid sequence to the chicken R-cad- herin, and defined this molecule as mouse R-cadherin. L cells transfected with the mouse R-cadherin cDNA acquired a cadherin-mediated cell-cell adhesiveness as found for other cadherins. To examine the binding specificity of mouse R-cadherin, L cells expressing this cadherin (mRL) were mixed with L cells expressing chicken R-cadherin (cRL), mouse N-cadherin (mNL), mouse E-cadherin (mEL) and mouse P-cadherin (mPL). While mRL cells randomly intermixed with cRL cells, those cells aggregated separately from mEL or mPL cells. Mixing of mRL with mNL cells gave an intermediate result; that is, they formed both separate and chimeric aggregates, suggesting that R- and N-cadherin can interact with each other although each has a preference to bind to its own type. Similar properties were previously found for chicken R-cadherin. Thus, the cell binding specificity of R-cadherin is entirely conserved between the two species, suggesting a conserved role for this protein in morphogenesis. We also located the mouse R-cadherin gene to chromosome 2.
Endocytosis of folate-protein conjugates: ultrastructural localization in kb cells
Authors: Turek John J. , Low Philip S.
Published: April 2021

It has been demonstrated that proteins covalently conjugated to folic acid may be taken up by cells via endo-cytosis after binding to a folate binding protein (FBP) in the cell membrane. The proteins taken up in this manner remain catalytically active and they may modify physiological processes occurring in the cytosol. Confocal fluorescence microscopy of KB cells incubated with FITC-bovine serum albumin-folic acid conjugates showed that after uptake, the conjugates resided in large vesicular structures. The purpose of the present study was to determine the subcellular localization of protein-folic acid conjugates in KB cells using folic acid-bovine serum albumin-colloidal gold (F-BSA-CG) as a tracer. F-BSA-CG conjugates were taken up via uncoated pits or caveolae, and resided primarily in multivesicular bodies (MVBs) and other tubular endosomes at early time points (15-60 min). At later time points (6 hours), conjugates were still contained in MVBs but some were also found in secondary lysosomes or free in the cytoplasm. Coincubation of KB cells with transferrin-colloidal gold (TF-CG) and F-BSA-CG resulted in colocalization of TF-CG and F-BSA-CG within endosomal elements at times later than 15 minutes, indicating that the caveolae-mediated F-BSA-CG endocytic pathway converged with a pathway utilized by clathrin-coated pits.