bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expression and localization of annexin VII (synexin) in muscle cells
Authors: Selbert Stefan , Fischer Petra , Pongratz Dieter , Stewart Murray , Noegel Angelika A. 
Published: April 2021

ABSTRACT
Annexin VII (synexin) is a member of the annexin family of proteins, which are characterized by Ca2+-dependent binding to phospholipids. We used PCR to isolate from a λgt11-mouse fibroblast library annexin VII cDNA fragments corresponding to the two isoforms found in both humans and Dictyostelium discoideum. The two isoforms of 47 kDa and 51 kDa differed by 22 amino acids inserted into the proximal third of the hydrophobic N terminus. Annexin VII-specific polypeptides expressed in Escherichia coli were used to generate isoform-specific monoclonal antibodies. Expression of the two isoforms during myogenesis was followed in the myogenic cell lines BC3H1 and L6. Only the 47 kDa isoform was present in undifferentiated L6 or BC3H1 myoblasts. The 51 kDa isoform appeared after myogenesis had been induced and in striated muscle only the 51 kDa isoform was observed. Immunofluorescence showed that annexin VII was located in the cytosol of mononucleated and fused polynucleated cultured cells, whereas in striated muscle, annexin VII was located preferentially at the plasma membrane and the transverse tubules. However, there was also some residual cytosolic staining, which was more abundant in type II (fast twitch) than in type I (slow twitch) fibers. Permeabilization of L6 cells with digitonin in the presence of 5 mM EGTA led to a release of annexin VII from the cells, which paralleled the loss of cytosolic lactate dehydrogenase (LDH) at low detergent concentrations (50 μM). In the presence of 100 μM extracellular Ca2+, annexin VII remained bound to the plasma membrane even in the presence of high digitonin concentrations. Incubation with the Ca2+-specific ionophore A23187 and 100 μM extracellular Ca2+ led to a redistribution of annexin VII from the cytosol to the plasma membrane after 30 minutes of incubation. The results obtained indicate a developmentally and Ca2+-regulated localization and expression of annexin VII and raise the possibility that annexin VII may play a role in excitation-contraction coupling in skeletal muscle.
β-Scruin, a homologue of the actin crosslinking protein scruin, is localized to the acrosomal vesicle of Limulus sperm
Authors: Way Michael , Sanders Mitchell , Chafel Mark , Tu Ya-Huei , Knight Alex , Matsudaira Paul 
Published: April 2021

ABSTRACT
Scruin (α-scruin) is an actin bundling protein found in the acrosomal process of Limulus polyhemus sperm. We have cloned and sequenced a second scruin isoform from Limulus, β-scruin, that is 67% identical to α-scruin. Northern and Southern analyses confirm that β-scruin and α-scruin are encoded by distinct genes. The sequence of βscruin, like α-scruin, is organized into N- and C-terminal superbarrel domains that are characterized by a six-fold repeat of a 50 residue motif. Western analysis using rabbit polyclonal antisera specific for αand β-scruin indicate that β-scruin, like α-scruin, is found in Limulus sperm but not blood or muscle. Both immunofluorescence microscopy and immunogold-EM localize β-scruin within the acrosomal vesicle at the anterior of sperm but not in the acrosomal process. The function of β-scruin in this membrane-bounded compartment that is devoid of actin is unknown. However, the location of β-scruin together with the fact that it contains two putative β-superbarrel structural folds, which are known to be catalytic domains in a number of proteins, suggests it may have a possible enzymatic role.
The pegs on the decorated tubules of the contractile vacuole complex of Paramecium are proton pumps
Authors: Fok Agnes K. , Aihara Marilynn S. , Ishida Masaki , Nolta Kathleen V. , Steck Theodore L. , Allen Richard D. 
Published: April 2021

ABSTRACT
Our previous study has shown that the decorated tubules (collectively known as the decorated spongiome) of the contractile vacuole complex (CVC) in Paramecium are the site of fluid segregation, as the binding of microinjected monoclonal antibody (mAb) DS-1 to the tubules reduced the CVC’s fluid output. In this study, we showed by immunogold labeling on cryosections that the antigenic sites for mAb DS-1 were located on the 15 nm ‘pegs’ protruding from the cytosolic surface of the decorated tubules. In immunofluorescence studies, both polyclonal antibodies against the subunits of the V-ATPase of Dictyostelium discoideum and against the 57 kDa B-subunit of the V-ATPase of chromaffin granules gave identical labeling patterns to that produced by mAb DS-1. On cryosections, all three antigens were located most consistently near or on the pegs of the decorated tubules. These data support the notion that the pegs on the membrane of the decorated tubules represent the V1 complex of a proton pump. Concanamycin B, a potent inhibitor of V-ATPase activity and of acidification of lysosomes and endosomes, strongly and reversibly inhibited fluid output from the CVC but had minimal effect on the integrity of the decorated spongiome as observed by immunofluorescence. Such inhibition suggests that a V-ATPase is intimately involved in fluid segregation. Exposing Paramecium to 12°C or 1°C for 30 minutes resulted in the dissociation of the decorated tubules from the smooth spongiome that borders the collecting canals; thus the DS-1-reactive A4 antigen, the 75 kDa and 66 kDa antigens were all found dispersed in the cytosol. Fluid output in these cells also ceased. In summary, the results obtained from proton pump antibodies, a V-ATPase inhibitor and cold treatment showed that the pegs on the decorated spongiome of Paramecium are V1 subunits of the V-ATPase and that these V1V0 complexes must remain active and in place for normal fluid segregation to occur.
Bordetella bronchiseptica dermonecrotizing toxin stimulates assembly of actin stress fibers and focal adhesions by modifying the small GTP-binding protein rho
Authors: Horiguchi Yasuhiko , Senda Takao , Sugimoto Nakaba , Katahira Jun , Matsuda Morihiro 
Published: April 2021

ABSTRACT
We studied the biochemical mechanism of morphological changes in cells treated with Bordetella dermonecrotizing toxin (DNT). DNT caused the morphological changes of serum-starved MC3T3-E1 cells from flat shapes to reflactile ones. These changes were accompanied by the assembly of actin stress fibers and focal adhesions, which is known to be regulated by the small GTP-binding protein rho. Clostridium botulinum C3 exoenzyme, which ADP-ribosylates and inactivates rho protein, ‘rounded’ the cells within 2 hours after addition to the extracellular fluid and their rounded shapes were maintained for at least 10 hours. However, when the cells were co-treated with C3 exoenzyme and DNT, they were rounded at 2 hours but recovered an apparently intact morphology after 3-8 hours of incubation. rho proteins in lysates from DNT-treated cells and untreated cells were radiolabeled by [32P]ADP-ribosylation with C3 exoenzyme and analyzed by SDS-polyacrylamide gel electrophoresis. Whereas the lysate from untreated cells showed a single band of [32P]ADP-ribosylated rho protein, the lysate from DNT-treated cells showed an additional two bands as well as the band identical to that of the lysate from untreated cells. Recombinant rhoA protein treated with DNT in vitro also showed a mobility shift in SDS-polyacrylamide gel electrophoresis. These results indicate that DNT causes the assembly of actin stress fibers and focal adhesions by directly modifying rho protein.
Alternatively spliced exons of the β tropomyosin gene exhibit different affinities for F-actin and effects with nonmuscle caldesmon
Authors: Pittenger Mark F. , Kistler Amy , Helfman David M. 
Published: April 2021

ABSTRACT
The rat β-tropomyosin (TM) gene expresses two isoforms via alternative RNA splicing, namely skeletal muscle β-TM and fibroblast TM-1. The latter is also expressed in smooth muscle where it corresponds to smooth muscle β-TM. Skeletal muscle β-TM contains exons 7 and 10, whereas exons 6 and 11 are used in fibroblasts and smooth muscle. In order to study the properties of the alternatively spliced proteins, recombinant TMs derived from bacterial and insect cell expression systems were produced, including the normal β gene products, fibroblast TM-1 and β skeletal muscle TM, two carboxy-terminal chimeric TMs, TM-6/10 and TM-7/11, as well as a carboxyl-truncated version of each, TM-6Cla and TM-7Cla. The purified TM isoforms were used in actin filament association studies. The apparent TM association constants (Ka) were taken as the free concentration at half saturation and were found to be 6 μM for β Sk TM, 8.5 for TM-6/10, 25 μM for TM-1, and 30 μM for TM-7/11 at an F-actin concentration of 42 μM. For the truncated TMs, the values determined were higher still but the binding was not carried out to full saturation. Isoforms were also produced using the baculovirus-insect cell system which produces proteins with an acetylated amino terminus as is normally found in vivo. This modification significantly enhanced the F-actin association of TM-1 but not the β skeletal TM or the other isoforms. Fibroblast TM-2 or TM-3, both products of the α gene, enhanced the affinity of TM-1 for F-actin, demonstrating different isoforms can act cooperatively on binding to actin. This effect was not detected with the other expressed β gene products. The presence of 83 kDa nonmuscle caldesmon was found to enhance the binding of TM-1 for F-actin. This effect was dependent on the presence of both exons 6 and 11, as caldesmon had little effect on the other β gene products. Collectively these results demonstrate TMs differ in their affinity for F-actin, which can be altered by other TMs or actin-binding proteins. The β tropomyosin isoforms were fluorescently-tagged and microinjected into cultured cells to study their in vivo localization where it was found that each of the full-length TMs bound to microfilaments but, at the light microscopy level, the isoforms were not differentially localized in these fibroblasts.
Mouse sperm-egg plasma membrane interactions: analysis of roles of egg integrins and the mouse sperm homologue of PH-30 (fertilin) β
Authors: Evans Janice P. , Schultz Richard M. , Kopf Gregory S. 
Published: April 2021

ABSTRACT
The guinea pig sperm protein, PH-30 (also known as fertilin), is postulated to participate in the interaction between the sperm and egg plasma membranes. The β subunit of guinea pig PH-30 (gpPH-30β) contains a domain with homology to disintegrins, snake venom proteins that bind to integrins via an integrin-binding domain containing the tripeptide RGD. This raises the question of whether an egg integrin serves as a receptor for PH-30. Although mouse eggs express integrin subunits, their role in mouse fertilization is unresolved. Therefore, we examined fertilization for two different hallmarks of integrin function, namely, dependence of ligand binding on divalent cations and the ability to inhibit ligand binding with RGD peptides. We demonstrate that sperm binding to zona pellucida-free eggs is supported by Ca2+, Mg2+, or Mn2+. Ca2+ was necessary and sufficient for sperm-egg fusion, with 2.5 mM Ca2+ being the most effective concentration. In addition, fertilization could be partially inhibited with various RGD peptides, which caused a decrease in sperm-egg fusion by 30-58%. This partial inhibition of fusion with RGD peptides prompted the cloning of the mouse homologue of gpPH-30β (hereafter referred to as mPH-30β) to determine if it possessed the tripeptide RGD or a different amino acid sequence in its disintegrin domain. mPH-30β, which is expressed during meiotic and post-meiotic phases of spermatogenesis, shares significant similarities to gpPH-30β throughout the length of the molecule, from the signal sequence to the cytoplasmic tail. The full-length deduced amino acid sequence of mPH-30β is 55% identical and 72% homologous to gpPH-30β. The disintegrin domain of mPH-30β has the tripeptide QDE (instead of RGD) in its cell recognition region. Peptides containing this QDE sequence decrease the binding and fusion of sperm with zona pellucida-free eggs by approximately 70%, suggesting that the disintegrin domain of mPH-30β participates in the interaction between sperm and egg membranes.
Phosphorylation of numa occurs during nuclear breakdown and not mitotic spindle assembly
Authors: Sparks C. A. , Fey E. G. , Vidair C. A. , Doxsey S. J. 
Published: April 2021

ABSTRACT
NuMA, the nuclear mitotic apparatus protein, is a component of the nuclear matrix at interphase that redis-tributes to the spindle poles at mitosis. While the function of NuMA is not known, it has been implicated in spindle organization during mitosis and nuclear reformation. Phosphorylation is thought to play a regulatory role in NuMA function. In this study, NuMA phosphorylation was examined through the cell cycle using highly synchronized cells. In intact cells labeled with 32P-orthophosphate, NuMA appeared as a 250 kDa phosphoprotein in inter-phase that shifted to a higher apparent molecular mass in mitosis. The shift was due to phosphorylation as shown by reduction of the shifted band to interphase mobility by phosphatase treatment. This phosphorylation event occurred roughly at the G2/M transition at the time of NuMA’s release from the nucleus and its redistribution to the mitotic spindle. However, mitotic phosphorylation did not require spindle formation since the phosphorylated species was detected in nocodazole-treated cells lacking microtubule spindles. Dephosphorylation of NuMA occurred in two distinct steps, after lamin B assembled into the nuclear lamina, in early G1 and at the end of G1. Based on the timing of the phosphorylation and dephosphoryla-tion observed in this study, we propose that they may play a role in nuclear events such as nuclear organization, tran-scription, or initiation of DNA replication at G1/S.
The three-dimensional organization of ribosomal genes and the architecture of the nucleoli vary with g1, s and g2 phases
Authors: Junéra H. R. , Masson C. , Géraud G. , Hernandez-Verdun D. 
Published: April 2021

ABSTRACT
The three-dimensional (3-D) organization of the nucleolus, a defined nuclear territory containing tandem repeats of the ribosomal genes (rDNA), was investigated in PtK1 cells. Identification of the interphase stages was performed in single cells using DNA quantification by cytometry for the G1 and G2 phases while the S phase was identified by immunolabelling of the proliferating cell nuclear antigen (PCNA). The 3-D organization of the rDNA in the nucleolus was analyzed by fluorescence in situ hybridization using confocal microscopy. All the rDNA was located inside the nucleolar structures during all stages and the two rDNA loci were orthogonal. The rDNA was heterogeneously distributed in each nucleolus during G1, S and G2, with alternate sites of clustered genes (spots) and of genes in more extended configurations. The number of spots (4 to 6 in G1) increased during S phase (7 to 12) and their 3-D organization was progressively relaxed from G1 to G2. Double spots in G2 could reflect a similar gene organization of two chromatids. During mid-S phase, PCNA co-localized with some clustered genes (spots), indicating that rDNA replication occurs inside nucleoli and at different sites of the same locus simultaneously. The evaluation of the rDNA transcription units in 5,6-dichloro-1-β-D-ribofuranosyl-benzimidazole (DRB)-treated cells indicated a mean of 16 units per G1 nucleus and 25 units per G2 nucleus. For G1 and G2, the fine 3-D structure of nucleoli was reconstructed using ultrathin serial sections after specific contrast of DNA and RNA, digitization of the serial section images and computer-assisted 3-D architecture. Fibrillar centers (FCs) formed discrete structures (about 10 in G1 and 20 in G2) connected by a network of the dense fibrillar component. The 3-D arrangement of the FCs in G1 and G2 are similar to the rDNA spots. In conclusion, the architecture of the nucleoli during interphase reflects the distribution of the rDNA that is characterized by alternation of clustered and extended genes.
Lamin proteins form an internal nucleoskeleton as well as a peripheral lamina in human cells
Authors: Hozák Pavel , Marie-Josée Sasseville A. , Raymond Yves , Cook Peter R. 
Published: April 2021

ABSTRACT
The nuclear lamina forms a protein mesh that underlies the nuclear membrane. In most mammalian cells it contains the intermediate filament proteins, lamins A, B and C. As their name indicates, lamins are generally thought to be confined to the nuclear periphery. We now show that they also form part of a diffuse skeleton that ramifies throughout the interior of the nucleus. Unlike their peripheral counterparts, these internal lamins are buried in dense chromatin and so are inaccessible to antibodies, but accessibility can be increased by removing chromatin. Knobs and nodes on an internal skeleton can then be immunola-belled using fluorescein- or gold-conjugated anti-lamin A antibodies. These results suggest that the lamins are misnamed as they are also found internally.
Extent of shock-induced membrane leakage in human and mouse myotubes depends on dystrophin
Authors: Menke Andre , Jockusch Harald 
Published: April 2021

ABSTRACT
A lack of the cytoskeletal protein dystrophin causes muscle fiber necrosis in Duchenne/Becker muscular dystrophies (DMD/BMD) and in murine X-linked muscular dystrophy (MDX). However, no overt disease symptoms are observed in dystrophin-less cultured myotubes, and the biological function of dystrophin in normal muscle cells is still unknown. In this work, we have extended our studies on a model system, using hypoosmotic shock to determine stress resistance of muscle cells. In frozen sections of control human and mouse myotubes, dystrophin was shown to be localized at the cell periphery as in mature muscle fibers. Dystrophin-less DMD and MDX myotubes were more susceptible to hypoosmotic shock than controls, as monitored by the uptake of external horseradish peroxidase and release of the soluble enzymes creatine kinase or pyruvate kinase and of radiolabelled proteins. Control experiments indicated that this difference is not due to differences in metabolism or ion fluxes. Treatment with cytochalasin D drastically increased the shock sensitivity of myotubes and abolished the difference between dystrophin-less and control cells. These results lend further support to the suggested stabilizing role of dystrophin in the context of the membrane-cytoskeletal complex.
Nucleoplasmin associates with and is phosphorylated by casein kinase ii
Authors: Vancurova Ivana , Paine Terry Maloney , Lou Willard , Paine Philip L. 
Published: April 2021

ABSTRACT
Nucleoplasmin is a phosphorylated nuclear-accumulating protein. We report herein that the kinetics of its cytoplasm r nucleus transport are affected by its degree of phosphorylation. Therefore, we sought to identify any protein kinase which specifically associates with nucleoplasmin. We discovered that nucleoplasmin co-isolates by two independent methods (immunoabsorption and chromatography) in a complex including a kinase which phosphorylates nucleoplasmin. The co-purifying kinase is casein kinase II-like because: (i) it phosphorylates casein; (ii) its phospho-transferase activity can be competed out by GTP; (iii) it is stimulated by polylysine; and (iv) it is inhibited by heparin. Moreover, a polyclonal antibody to the α (38 kDa) and α′ (36 kDa) catalytic subunits of casein kinase II specifically recognizes 38 and 36 kDa polypeptides in the nucleoplasmin-complex, and a specific inhibitor of casein kinase II inhibits nucleoplasmin’s nuclear transport. Additionally, we found that phosphorylation of nucleoplasmin by its associated casein kinase II is strongly inhibited by histones and that, in addition to nucleoplasmin, another protein (p100) in the nucleoplasmin-complex is phosphorylated by casein kinase II.
Polarized exocytosis in mdck cells is regulated by phosphorylation
Authors: Brewer Colleen B. , Roth Michael G. 
Published: April 2021

ABSTRACT
Protein phosphorylation and dephosphorylation systems modulate many cellular activities and have recently been implicated in the in vitro transport of newly synthesized proteins. Here we show that polarized transport from the Golgi to the plasma membrane in intact MDCK cells is regulated by phosphorylation-dephosphorylation. Transport is inhibited by the phosphatase inhibitor okadaic acid and is stimulated by the kinase inhibitor staurosporine. Stimulation of apical transport exceeds stimulation of basolateral transport by up to 5-fold. We also find that the G protein activator aluminum fluoride, which stimulates transport to the surface at low fluoride concentrations as previously reported, inhibits transport at higher concentrations. In the nonpolarized fibroblast cell line CV-1, neither staurosporine nor aluminum fluoride stimulates transport to the cell surface. Our results suggest that the phosphorylation-dephosphorylation system, like the G protein, may be involved in the specialized sorting process characteristic of polarized cells. We show some evidence that these two mechanisms of regulation may act through common intermediates.
Evidence that tenascin and thrombospondin-1 modulate sprouting of endothelial cells
Authors: Canfield Ann E. , Schor Ana M. 
Published: April 2021

ABSTRACT
Cultured endothelial cells undergo a reversible transition from a resting (cobblestone) phenotype to an angiogenic (sprouting) phenotype. This transition mimics the early events of angiogenesis. We have previously reported that the addition of exogenous xylosides inhibits endothelial cell sprouting and modifies the extracellular matrix (ECM) synthesised by the cells. We have now investigated whether endothelial sprouting is mediated by the nature of the extracellular matrix in contact with the cells. Accordingly, cell-free matrices deposited by bovine aortic endothelial cells (BAEC) were isolated. These matrices were produced under conditions in which the formation of the sprouting phenotype was permitted (controls) or inhibited (by the addition of exogenous xylosides). BAEC were then plated on these matrices and grown under conditions which promote sprouting. Sprouting proceeded normally on control matrices, whereas it was inhibited when the cells were grown on matrices deposited in the presence of xylosides. The composition of the permissive and inhibitory matrices was then analysed. Inhibitory matrices contained reduced levels of tenascin and increased levels of thrombospondin-1 by comparison to the permissive matrices. In contrast, no differences were detected in the relative levels of laminin. The roles of tenascin and thrombospondin-1 in endothelial sprouting were confirmed using specific antibodies. Immunolocalisation studies revealed the presence of both proteins in sprouting cells. Antibodies to tenascin inhibited the formation of sprouting cells on permissive matrices and on gelatin-coated dishes without affecting cell growth. Tenascin synthesis was increased when sprouting cells were present in the cultures. Antibodies to thrombospondin-1 stimulated sprouting on inhibitory matrices. These results suggest that the transition from a resting to a sprouting phenotype is promoted by tenascin and inhibited by thrombospondin-1.