bluuebunny/crossref_metadata_2025 huggingface dataset:
Inositol 1,4,5-trisphosphate receptor-like protein in plasmalemmal caveolae is linked to actin filaments
Authors: Fujimoto Toyoshi , Miyawaki Atsushi , Mikoshiba Katsuhiko
Published: April 2021

We reported that a plasmalemmal inositol 1,4,5-trisphos-phate receptor-like protein (PM InsP3R-L) is localized in caveolae of various non-neuronal cells in vivo (Fujimoto et al. (1992)J. Cell Biol. 119, 1507-1513). In the present study, we investigated the distribution of PM InsP3R-L in cultured cells. In mouse epidermal keratinocytes (Pam 212) cultured in standard Ca2+ (1.8 mM), PM InsP3R-L was dis-tributed densely in the vicinity of cell-to-cell contacts. In contrast, when Pam cells were cultured in low Ca2+ (0.06 mM) without making cell-to-cell contacts, PM InsP3R-L was observed randomly; by restoring the Ca2+ concentra-tion, the circumferential actin filaments became obvious and the density of PM InsP3R-L increased in the contact region. Treatment of Pam cells with cytochalasin D caused aggregation of caveolae where PM InsP3R-L as well as F-actin and fodrin were localized. In bovine aortic endothelial cells, PM InsP3R-L was aligned along actin filaments crossing the cytoplasm in various directions. PM InsP3R-L of Pam cells was hardly extracted by treatment with 0.5% Triton X-100 or 60 mM octyl-glucoside in a cytoskeleton-stabilizing buffer for 15 minutes at 4°C.

The results show that the distribution of caveolae bearing PM InsP3R-L changes when the actin cytoskeleton is modified. They also indicate that the association of PM InsP3R-L with actin filaments may mediate the redistribution of caveolae. Since caveolae are thought to be related to signal transduction, their location defined by the actin cytoskeleton may affect the site where cellular reaction is to occur in response to various stimuli.
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

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

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

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

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

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

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

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

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

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

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

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

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

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.
Gap junction regulation in the uterus and ovaries of immature rats by estrogen and progesterone
Authors: Risek Boris , Klier F. George , Phillips A. , Hahn D. W. , Gilula N. B.
Published: April 2021

The effects of estrogen (E2) and progesterone (P) were examined on the expression levels of multiple gap junction (GJ) gene products (α1=Cx43, β1=Cx32, β2=Cx26) in the uterus and ovaries of immature rats by immunohisto- chemistry, electron microscopy and northern blot analysis. E2 induced the expression of α1 connexin in the uterus (specifically in the myometrium and in endometrial stroma proximal to luminal epithelium) and ovaries. The E2- induced α1 expression was completely suppressed by P in the uterus, but only partly in ovaries. Steroid hormones also modulated the quantity, size, and distribution of β1 and β2 containing junctional plaques along lateral cell borders in polarized luminal and glandular uterine epithelia. Small GJs were detected at basolateral regions in proliferative luminal epithelium following administration of E2. In contrast, large GJs were localized at subapical- lateral cell borders of the secretory epithelium following P- treatment. The co-administration of E2+P had a synergis- tic effect on β1 and β2 expression in the luminal epithelium, but an inhibitory effect on β2 expression in glandular epithelium. Myometrial GJs were detected in freeze- fracture replicas as aggregates containing regularly arranged particles with particle free zones. In contrast, GJs in secretory epithelium contained particles which were arranged in a non-crystalline fashion. These GJs contained domains of mixed and segregated β1 and β2 antigens within a single plaque as revealed by laser scanning confocal microscopy analysis of immuno-double-labeled secretory epithelium. The demonstration of segregated antigens within a single GJ plaque indicates the possibility of multiple channel populations formed by homo-oligomeric connexons. These results suggest that different connexins can be differentially regulated by steroid hormones in different cell types, and that the same steroid hormone can have different effects on the same connexin in different cell types.
Mapping of adherens junction components using microscopic resonance energy transfer imaging
Authors: Kam Zvi , Volberg Tova , Geiger Benjamin
Published: April 2021

Quantitative microscopic imaging of resonance energy transfer (RET) was applied for immunological high resolution proximity mapping of several cytoskeletal components of cell adhesions. To conduct this analysis, a micro-scopic system was developed, consisting of a highly stable field illuminator, computer-controlled filter wheels for rapid multiple-color imaging and a sensitive, high resolu-tion CCD camera, enabling quantitative data recording and processing. Using this system, we have investigated the spatial inter-relationships and organization of four adhesion-associated proteins, namely vinculin, talin, α-actinin and actin. Cultured chick lens cells were double labeled for each of the junctional molecules, using fluores-cein- and rhodamine-conjugated antibodies or phalloidin. RET images were acquired with fluorescein excitation and rhodamine emission filter setting, corrected for fluorescein and rhodamine fluorescence, and normalized to the fluo-rescein image. The results pointed to high local densities of vinculin, talin and F-actin in focal adhesions, manifested by mean RET values of 15%, 12% and 10%, respectively. On the other hand, relatively low values (less than 1%) were observed following double immunofluorescence labeling of the same cells for α-actinin. Double indirect labeling for pairs of these four proteins (using fluorophore-conjugated antibodies or phalloidin) resulted in RET values of 5% or lower, except for the pair α-actinin and actin, which yielded significantly higher values (13-15%). These results suggest that despite their overlapping staining patterns, at the level of resolution of the light microscope, the plaque proteins vinculin and talin are not homogeneously interspersed at the molecular level but form segregated clusters. α-Actinin, on the other hand, does not appear to form such clusters but, rather, closely interacts with actin. We discuss here the conceptual and applicative aspects of RET measurements and the implications of the results on the subcellular molecular organization of adherens-type junctions.
Identification of intrinsic dimer and overexpressed monomeric forms of γ-tubulin in Sf9 cells infected with baculovirus containing the Chlamydomonas γ-tubulin sequence
Authors: Vassilev Alexei , Kimble Mary , Silflow Carolyn D. , LaVoie Matthew , Kuriyama Ryoko
Published: April 2021

A new member of the tubulin superfamily, γ-tubulin, is localized at microtubule-organizing centers (MTOCs) in a variety of organisms. Chlamydomonas cDNA coding for the full-length sequence of γ-tubulin was expressed in insect ovarian Sf9 cells using the baculovirus expression system. Approximately half of the induced 52 kDa γ-tubulin was recovered in the supernatant after centrifugation of Sf9 cell lysates at 18,000 g for 15 minutes. When the cell super-natant was analyzed by FPLC on a Superdex 200 sizing column, Chlamydomonas γ-tubulin separated into two major peaks. The lagging peak contained a monomeric form of γ-tubulin with a sedimentation coefficient of 2.5 S, which interacted with the Superdex column in a salt-dependent manner. The leading peak, with an apparent molecular mass of 900 kDa, corresponded to a molecular chaperonin complex, and TCP1 chaperonin released folded γ-tubulin polypeptide from the complex in the presence of MgATP. The released γ-tubulin monomers were capable of binding to microtubules in vitro and biochemical quanti-ties of active monomers were further purified using a com-bination of size-exclusion and ion-exchange column chro-matography. The endogenous Sf9 cell γ-tubulin migrated faster than Chlamydomonas γ-tubulin with an apparent molecular mass of 49 kDa on gels. Analyses on gel filtration and sucrose density gradient centrifugation showed that, while overexpressed Chlamydomonas γ-tubulin was present in a monomeric form, endogenous γ-tubulin from Sf9 and HeLa cells exists as a dimer. These results may suggest the possibility that γ-tubulin could form a heterodimer with hitherto unknown molecule(s).
Mutants lacking myosin II cannot resist forces generated during multicellular morphogenesis
Authors: Shelden Eric , Knecht David A.
Published: April 2021

We have used fluorescent labeling, confocal microscopy and computer-assisted motion analysis to observe and quantify individual wild-type and myosin II mutant cell behavior during early multicellular development in Dic-tyostelium discoideum. When cultured with an excess of unlabeled wild-type cells, labeled control cells are randomly distributed within aggregation streams, while myosin II mutant cells are found primarily at the lateral edges of streams. Wild-type cells move at average rates of 8.5±4.9 μm/min within aggregation streams and can exhibit regular periodic movement at 3.5 minute intervals; half as long as the 7 minute period reported previously for isolated cells. Myosin II mutants under the same conditions move at 5.0±4.8 μm/min, twice as fast as reported previously for isolated myosin II mutant cells, and fail to display regular periodic movement. When removed from aggregation streams myosin II mutant cells move at only 2.5±2.0 μm/min, while wild-type cells under these conditions move at 5.9±4.5 μm/min. Analysis of cell morphology further reveals that myosin II mutant cells are grossly and dynamically deformed within wild-type aggregation streams but not when removed from streams and examined in isolation. These data reveal that the loss of myosin II has dramatic consequences for cells undergoing multicellular develop-ment. The segregation of mutant cells to aggregation stream edges demonstrates that myosin II mutants are unable to penetrate a multicellular mass of wild-type cells, while the observed distortion of myosin II mutant cells suggests that the cortex of such cells is too flacid to resist forces generated during movement. The increased rate of mutant cell movement and distortion of mutant cell mor-phology seen within wild-type aggregation streams further argues both that movement of wild-type cells within a mul-ticellular mass can generate traction forces on neighboring cells and that mutant cell morphology and behavior can be altered by these forces. In addition, the distortion of myosin II mutant cells within wild-type aggregation streams indicates that myosin is not required for the formation of cell-cell contacts. Finally, the consequences of the loss of myosin II for cells during multicellular development are much more severe than has been previously revealed for isolated cells. The techniques used here to analyze the behavior of individual cells within multicellular aggregates provide a more sensitive assay of mutant cell phenotype than has been previously available and will be generally applicable to the study of motility and cytoskeletal mutants in Dictyostelium.
Stimulation of tyrosine phosphorylation of distinct proteins in response to antibody-mediated ligation and clustering of α3 and α6 integrins
Authors: Jewell Kevin , Kapron-Bras Carolyn , Jeevaratnam Premala , Dedhar Shoukat
Published: April 2021

The interaction of cells with components of the extracellular matrix through their integrin receptors results in the stimulation of tyrosine phosphorylation of several proteins, suggesting that these receptors play a key role in signal transduction. Here we report that antibody-mediated ligation and clustering of α3β1 and α6β1/α6β4 integrins resulted in the stimulation of tyrosine phosphorylation of proteins that are specific for each heterodimer. Thus, ligation and clustering of the α3β1 integrin on human prostate carcinoma cells (PC-3) and human umbilical vein endothelial cells (HUVEC) with anti-α3 antibodies resulted in the stimulation of tyrosine phosphorylation of a 55 kDa protein. In contrast, ligation and clustering of the α6β1 integrin on these cells with anti-α6 antibody resulted in the dramatic stimulation of tyrosine phosphorylation of a 90 kDa protein in addition to a 52 kDa protein, and ligation and clustering of α5β1 on HUVEC did not result in the apparent stimulation of tyrosine phosphorylation of any proteins. Clustering with anti-β1 antibodies triggered the tyrosine phosphorylation of all of these proteins, whereas ligation and clustering of PC-3 cells with an anti-β4 antibody resulted in the tyrosine phosphorylation of a distinct 62 kDa protein. Since the PC-3 cells express both α6β1 and α6β4, these data suggest that these two receptors can transduce distinct signals. All of the phosphorylations could be inhibited by treating the cells with Genistein, a tyrosine kinase inhibitor. Antibody-mediated ligation and clustering of integrins on the two types of cells did not result in the stimulation of tyrosine phosphorylation of pp125 focal adhesion kinase, although this was observed upon cell attachment and spreading on fibronectin, laminin and anti-α3 monoclonal antibody. Collectively, these data demonstrate that cross-linking of different integrin het-erodimers can stimulate tyrosine kinase activities, leading to the phosphorylation of distinct proteins, which are also different from those observed when cells are allowed to spread on a matrix.
The state of actin assembly regulates actin and vinculin expression by a feedback loop
Authors: Bershadsky Alexander D. , Glück Ursula , Denisenko Oleg N. , Sklyarova Tatyana V. , Spector Ilan , Ben-Ze’ev Avri
Published: April 2021

Actin filaments are major determinants of cell shape, motility and adhesion, which control important biological processes including embryonic development and wound healing. These processes are associated with changes in actin assembly, which is regulated by controlling the balance between polymerized and non-polymerized actin. To maintain a significant pool of non-polymerized actin, mechanism(s) linking actin synthesis to its state of poly-merization were proposed. We have studied this relation-ship between actin synthesis and organization by modulat-ing actin assembly using different drugs. Unassembled actin was increased in 3T3 cells using either the Clostrid-ium botulinum C2 toxin, which ADP-ribosylates actin, or by latrunculin A, a Red Sea sponge product, which binds monomeric actin. The synthesis of actin was dramatically reduced in these cells owing to a concomitant decrease in actin RNA level. Similar results were obtained with HeLa cells grown in both monolayer and in suspension, suggest-ing that cell shape changes associated with drug treatment are not the primary cause for the effect on actin synthesis. In contrast, the scrape-loading of 3T3 cells with phalloidin, a stabilizer of polymerized actin that increased the level of assembled actin, resulted in elevated actin synthesis and RNA content. The expression of vinculin, a major component of adhesion plaques and cell-cell junctions, which is involved in actin-membrane associations, was altered in parallel with that of actin in cells treated with these drugs. The decrease in actin RNA resulted from destabilization of actin mRNA in cells where unassembled actin level was elevated. This is suggested by the unchanged transcription of actin in isolated nuclei from drug-treated cells, and by demonstrating that actin mRNA was degraded faster in cells after C2 toxin treatment than in control cells. This feedback control mechanism is mainly confined to the cytoplasm, as it remained active in enucleated cells. The results suggest the existence of an autoregulatory pathway for the expression of actin and other microfilament-associ-ated proteins which is linked to the state of actin polymer-ization in the cell.
The levels of ubiquitinated histone H2A are highly upregulated in transformed human cells: Partial colocalization of uH2A clusters and PCNA/cyclin foci in a fraction of cells in S-phase
Authors: Vassilev Alex P. , Rasmussen Hanne H. , Christensen Erik I. , Nielsen Søren , Celis Julio E.
Published: April 2021

The levels and distribution of ubiquitinated histone H2A (uH2A) have been studied in normal and transformed human cells using a monoclonal antibody (mAb E6C5) that reacts specifically with this ubiquitin conjugate as determined by two-dimensional gel western blotting and microsequencing. Immunoblotting experiments demonstrated that the levels of the protein are highly upregulated in SV40-transformed human fibroblasts (WI-38 SV40) and keratinocytes (K14) relative to their normal counterparts, a finding that was further confirmed by indirect immuno-fluorescence studies of formaldehyde/Triton X-100-treated cells, which showed that about 97% of the transformed cells and 26% of the normal populations reacted with the antibody to yield a fine granular nuclear staining associ-ated with the chromatin. Transformed cells contained in addition clusters of uH2A that were quite abundant and that showed variable size, shape and distribution even within a single cell line. The clusters, which were rare in normal cells, did not colocalize with other known nuclear antigens and may correspond to novel nuclear domains where ubiquitination/deubiquitination takes place. Electron microscopic immunocytochemistry of K14 cells confirmed the existence of the clusters. Double immunoflu-orescence studies of K14 keratinocytes with proliferating cell nuclear antigen (PCNA)/cyclin antibodies, which react with the nuclei of cells engaged in DNA replication, showed partial colocalization of PCNA/cyclin foci and large uH2A clusters in about 14% of the S-phase cells, and these cor-responded mainly to late S-phase cells. Inhibition of DNA replication with hydroxyurea resulted in an overall increase in the intensity of the uH2A staining as well as in a more clear colocalization of uH2A clusters and PCNA/cyclin foci. Taken together, the results support the contention that uH2A plays a role at some stage of DNA replication.
αvβ1 is a receptor for vitronectin and fibrinogen, and acts with α5β1 to mediate spreading on fibronectin
Authors: Marshall John F. , Rutherford Deborah C. , McCartney Alison C. E. , Mitjans Francesc , Goodman Simon L. , Hart Ian R.
Published: April 2021

We have shown previously that VUP was the only line out of ten human melanoma lines that failed to express the vit-ronectin receptor αvβ3, but instead expressed αvβ1. Levels of αvβ1 expression were low on parental VUP cells so that iterative sorting by FACS, using an anti-αv antibody (13C2), was utilised to derive sublines with 8-to 10-fold higher amounts of cell surface αvβ1. There was little dif-ference between low (V−) and high (V+) αvβ1-expressing sublines with regard to adherence to collagen type I, collagen type IV or laminin substrata. However, adherence to vitronectin and fibrinogen correlated closely with αvβ1 expression (35-42% adhesion for V(+) lines versus 6-8% adhesion for V− lines on vitronectin, for example). Utilising a high αvβ1-expressing subline (V+B2) we have shown that binding to vitronectin and fibrinogen was inhibited specif-ically by function-blocking antibodies to αv (17E6 and 14D9) and β1 (A11B2). V(+) sublines spread more compared with V(−) sublines on both vitronectin and fibronectin. However, neither α5-nor αv-blocking anti-bodies had any effect on attachment or spreading of V+B2 on fibronectin whereas the combination of α5 (PID6)-and αv(17E6)-blocking antibodies abrogated binding to fibronectin almost completely. This is the first report of an αvβ1 integrin able to recognise vitronectin and fibrinogen, and also cooperate with α5β1 to mediate attachment to and spreading on fibronectin.
Dynamics of fibroblast spreading
Authors: Dunn Graham A. , Zicha Daniel
Published: April 2021

A new technique of microinterferometry permits cellular growth and motile dynamics to be studied simultaneously in living cells. In isolated chick heart fibroblasts, we have found that the non-aqueous mass of each cell tends to increase steadily, with minor fluctuations, throughout the cell cycle. The spread area of each cell also tends to increase during interphase but fluctuates between wide limits. These limits are dependent on the cell’s mass and the upper limit is particularly sharp and directly proportional to mass. From a dynamical point of view, the spread area of a cell is determined by the balance between the rates of two antagonistic processes: protrusion of cellular material into new territory and retraction of material from previously occupied territory. The spatial asymmetry of these processes determines the translocation of the cell. We have found with the chick fibroblasts that the rates of the two processes are generally closely matched to each other and appear to be dependent on the cell’s area of spreading. Both continue incessantly in well spread cells, even when there is no net translocation of the cell, and the lower limit of each activity is directly proportional to spread area. The two processes show different behaviour, however, during changes in the spread area of the cell. Both increases and decreases in area appear to be brought about by changes in the rate of retraction, the rate of protrusion remaining relatively constant. A simple stochastic model based on a limited supply of adhesion molecules can simulate all our observations including the mass-limited spreading, the strong correlation between protrusion and retraction and the retraction-dominated changes in area. We conclude that the spread area of the cell is actively regulated, possibly by a simple automatic mechanism that adjusts the area of spreading in relation to the mass of the cell and controls the rate of protrusion to compensate rapidly for spontaneous fluctuations in retraction.
Rapid bulk replacement of acceptor membrane by donor membrane during phagosome to phagoacidosome transformation in Paramecium
Authors: Allen Richard D. , Bala Nenita P. , Ali Rose F. , Nishida Dawn M. , Aihara Marilynn S. , Ishida Masaki , Fok Agnes K.
Published: April 2021

The extent to which a donor membrane will be retrieved, or if it is retrieved at all after it fuses with an acceptor membrane, is usually difficult to determine. We have studied the dynamics of membrane retrieval in the phagosome system of Paramecium multimicronucleatum using six monoclonal antibody markers. Our previous freeze-fracture and transmission electron microscopic studies have indicated that extensive changes take place in the membrane of the young phagosome as it progresses through its cycle. Using immunofluorescence and immu-noelectron microscopy to determine the times of entry and exit of these individual antigens into the digestive vacuole system, we showed that two hydrophilic antigens, one located on the cytosolic and one on the lumenal side of the discoidal membrane (phagosome membrane precursor), were completely retrieved from the phagosome by tubula-tion within the first three minutes. At the same time that this membrane was retrieved, membrane from a second population of vesicles, the acidosomes, fused with the phagosome to produce the phagoacidosome. On the basis of immunogold localization on cryosections of a total of six antigens, the two specific for phagosome/discoidal vesicle membrane as well as four specific for the acidosome/phagoacidosome membrane, this replacement is total. We also showed that in the presence of the actin-active drug cytochalasin B, this replacement was essentially prevented. However, when vacuole acidification was neu-tralized by ammonium chloride, this replacement process continued unaffected after a lag. Consequently, acidifica-tion, per se, is not required to trigger the replacement of the phagosome membrane. We conclude, on the basis of these studies as well as our previous freeze-fracture studies that during phagoacidosome formation most of the acceptor membrane is retrieved and is replaced by the donor membrane. This shows that at least one cell type possesses the mechanisms needed to substantially replace the membrane of a phagosomal compartment when radical and rapid changes are needed to modulate the digestive and absorptive processes.