bluuebunny/crossref_metadata_2025 huggingface dataset:
Behaviour of ejaculated spermatozoa from bull, boar and ram during thin-layer countercurrent partition in aqueous two-phase systems
Authors: Harrison R. A. P. , Jacques M. L. , Minguez M. L. Pascual , Miller N. G. A.
Published: April 2021

Ejaculated spermatozoa from bulls, boars and rams were subjected to thin-layer countercurrent partition in aqueous two-phase systems composed of dextran T500 and polyethylene glycol 6000 (PEG) in sucrose-based Hepes-buffered media. In the basal system, the great majority of spermatozoa tended to partition with the dextran-rich bottom phase; however, by including very low levels of phosphate, they could be made to partition increasingly with the PEG-rich top phase (complete at 10 mM phosphate). A procedure was developed for carrying out four separations simultaneously under identical conditions, whereby it could be shown that distribution varied with the number of spermatozoa in the sample. In the case of bull, the effect of cell number could be reduced considerably by inclusion of small quantities of seminal plasma in the phase system, but no such effect was found for ram or boar. Considerable variation in distribution pattern was seen between samples, which did not appear to be due to technical inconsistency. Livability in the phase systems was also variable, and we believe that PEG may exert a detergent-like effect on the sperm surface that is exacerbated in highly defined media free of protective proteins.
Identification of the intermediate filament-associated protein gyronemin as filamin: Implications for a novel mechanism of cytoskeletal interaction
Authors: Brown Kevin D. , Binder Lester I.
Published: April 2021

In a previous paper, a monoclonal antibody (designated Ml.4) that recognized a 240 kDa polypeptide was characterized. This antibody stained the intermediate filaments in several cell lines, and biochemical characteristics of the 240 kDa polypeptide led us to conclude that it was a novel intermediate filament-associated protein, which we termed gyronemin. Here we report that gyronemin is expressed in adult rat organs that contain a substantial smooth muscle component. Taking advantage of this observation, this protein was purified from bovine uterine tissue and, by biochemical, immunological and amino acid sequence analysis, found to be homologous to the actin-associated protein filamin. Three novel monoclonal antibodies raised using purified bovine gyronemin as the immunogen show this protein to be associated with actin-containing stress fibers, although our original Ml.4 antibody continued to be localized along vimentin filaments. Since two-dimensional electrophoretic analysis did not demonstrate a difference in either relative molecular mass or isoelectric point of this polypeptide when associated with either filamentous system, we conclude that filamin is a bifunctional protein capable of associating with both the intermediate filament and actin cytoskeletal systems.
Involvement of the consensus sequence motif at coil 2b in the assembly and stability of vimentin filaments
Authors: Kouklis Panos D. , Traub Peter , Georgatos Spyros D.
Published: April 2021

Nearly all intermediate filament (IF) proteins share two sequence motifs located at the N- and the C-terminal ends of their helical rod domain (‘coil la’ and ‘coil 2b’, respectively). To examine the structural role of the coil 2b motif, we have performed in vitro assembly studies and in vivo microinjection experiments employing two site-specific reagents: (a) a 20-residue synthetic peptide (C-2) representing the conserved motif itself and (b) a monoclonal antibody (anti-IFA) that recognises an epitope within the conserved coil 2b sequence. We demonstrate here that vimentin protofilaments, when induced to assemble in the presence of C-2 or anti-IFA, show a lower propensity to polymerise and yield various abberant structures. The few filaments that are formed under these conditions appear much shorter than normal IFs and are unravelled or aggregated. Furthermore, when preformed vimentin filaments are exposed to C-2 or anti-IFA, most of the normal IFs are converted into shorter filamentous forms that possess an abberant morphology. None of these effects is seen when vimentin subunits are coincubated with control peptides. Microinjection of anti-IFA into the cytoplasm of interphasic 3T3 cells provokes collapse of vimentin IFs into a juxtanuclear mass and formation of numerous amorphous aggregates distributed throughout the cytoplasm. These two effects are not seen when the anti-IFA is microinjected into the cell nucleus. Our results provide experimental evidence supporting previous suggestions for a role for the conserved coil 2b sequence in filament assembly. We propose that this region is interacting with other sites along the vimentin molecule and that these interactions are essential for proper protofilament-protofilament alignment and filament stability.
Association of cyclin-bound p34cdc2 with subcellular structures in Xenopus eggs
Authors: Leiss D. , Félix M.-A. , Karsenti E.
Published: April 2021

Cell cycle progression is controlled by changes in kinase activity of homologs of the fission yeast protein p34cdc2. The p34cdc2 kinase is activated by its association with a cyclin subunit, followed by post-translational modifications. Here, we show that in Xenopus eggs stimulated to enter the early embryonic cell cycle by an electric shock, part of the p34cdc2 becomes associated with subcellular fractions as the eggs progress towards mitosis. This occurs as a result of cyclin accumulation because most of the B-type cyclins and some of the A-type cyclins are found in the particulate fraction. Moreover, as soon as cyclins are degraded, p34cdc2 is released in the soluble fraction. The p34cdc2-cyclin complex can be solubilised by 80 mM β-glycerophos-phate (in the standard MPF extraction buffer) or by high salt concentrations. The post-translational modifications leading to cdc2 kinase activation by cyclin occur in the insoluble form. Following fractionation of egg extracts by sucrose gradient centrifugation, the p34cdc2 -cyclin B complex is found in several fractions, but especially in two discrete peaks. We present evidence that in the slow-sedimenting peak the p34cdt2-cyclin B complex is associated with the 60 S subunit of monoribosomes. It could be targeted in this fashion to substrates such as ribosomal proteins and maybe to cytoskeletal proteins, since ribosomes bind to microtubules and are present in the spindle. The p34cdc2-cyclin B complex is also found in a faster-migrating fraction containing various membranous structures, including Golgi stacks. Therefore, as observed by immunofluorescence in other systems, it seems that cyclin subunits target p34cdc2 to specific cellular sites and this is certainly important for its function. In addition, we present preliminary evidence suggesting that some component present in the ribosome-containing fraction is required for activation of the p34cdc2-cyclin B complex.
Adhesion of 8701-bc breast cancer cells to type v collagen and 67 kda receptor
Authors: Minafra Salvatore , Luparello Laudio , Pucci-Minafra Ida , Sobel Mark E. , Garbisa Spiridione
Published: April 2021

Ductal infiltration carcinomas (d.i.c.) of the breast are potentially highly metastatic tumours, associated with drastic alterations of the architecture and molecular composition of the extracellular matrix at the tumourhost interface. 8701-BC, a recently characterized cell line, isolated from primary d.i.c., was used to study different aspects of tumor cell-substratum interactions. Since type V collagen deposition is augmented in d.i.c. we have examined the ability of 8701-BC cells to interact with this collagen species. We have found that cell binding to type V collagen was mediated by protein homologous to the 67 kDa laminin receptor (67-R). This conclusion is substantied by the following observations: (a) a major band having an apparent molecular mass of 67 kDa and immunoreactive to the anti-67 R antibody was detectable by SDS-PAGE of the membrane proteins; (b) the antibody inhibited cellular adhesion to type V collagen in a dose-dependent way; (c) membrane proteins purified by affinity chromatography on type V collagen were immunoreactive to anti-67 R antibody, but not to anti-VL Al, VLA2 and VLA3 integrin antibodies. This receptor appears to have prominent carbohydrate-binding properties, since lactose competes with cell adhesion to type V collagen.
Differential lectin binding to presumptive cortical cells of the wool follicle bulb
Authors: Campbell Mary T. , Dollin Anne E. , Adelson David L.
Published: April 2021

An a-D-galactoside-specific lectin from Bandeiraea sim-plicifolia (BSLI) showed differential binding to cortical cells of the wool follicle bulb. The lectin bound to cells on one side only of the bulb and was completely blocked by a-D-galactose. The region of lectin binding extended from presumptive cortical cells at the base of the bulb to cortical cells at the top of the bulb, disappearing as cortical cells entered the fibre cortex. An orthocortexspecific monoclonal antibody was used to show that cortical cells recognised by the lectin lie directly below the fibre orthocortex and presumably give rise to the orthocortex. The results suggest that two distinct populations of presumptive cortical cells are present only two to three cell layers from the base of the bulb in a region where no morphological differences are detectable. The lectin-bound pre-cortical cells appear to give rise to orthocortical cells while cells not bound by lectin presumably give rise to paracortical cells.

Electron microscopy showed that the lectin bound to sites on the plasma membrane, probably on the extracellular surface. This suggests that the lectin target may be a membrane glycoprotein or glycolipid. Two polypeptides recognised by BSLI were separated from wool follicle extracts by SDS-gel electrophoresis. These polypeptides migrated at approximately 69 kDa and 17 kDa. However, only the 69 kDa molecule showed the expected loss of binding by BSLI in the presence of a-D-galactose. Further work will be required to determine if this glycoprotein is the bulb cell molecule recognised by BSLI. Dermal papilla cells were also bound by BSLI and may carry the same, or a similar, cell-surface molecule to presumptive orthocortical cells.

BSLI appears to be a useful marker for detecting patterns of cell differentiation in the bulb that correlate with patterns of cortical cell type in the wool fibre. The lectin may also be a useful tool for distinguishing cortical cells of different lineage in cell culture. Furthermore, the pattern of BSLI binding seen in small follicle bulbs appears to provide some support for a reaction-diffusion model of follicle differentiation.
Phosphorylated baculovirus p10 is a heat-stable microtubule-associated protein associated with process formation in Sf9 cells
Authors: Cheley Stephen , Kosik Kenneth S. , Paskevich Peter , Bakalis Shelley , Bayley Hagan
Published: April 2021

Insect ovarian Sf? cells extend processes with complex morphologies when infected with a recombinant baculovirus encoding the catalytic subunit of protein kinase A. Within the shafts of the processes are abundant microtubules, which, in contrast to those in Sf9 cells expressing the microtubule-associated protein tau, are generally not organized into parallel bundles. During infection the late viral polypeptide p10 becomes phosphorylated by the protein kinase A catalytic subunit at its penultimate residue, Ser92. The expression or phosphorylation of other major host cell or viral polypeptides does not change, compared with polypeptides from a wild-type viral infection. Once phosphorylated, p10 associates with microtubules in the infected cells and may thereby play a role in process formation.
A gene family consisting of ezrin, radixin and moesin Its specific localization at actin filament/plasma membrane association sites
Authors: Sato Naruki , Funayama Noriko , Nagafuchi Akira , Yonemura Sfugenobu , Tsukita Sachiko , Tsukita Shoichiro
Published: April 2021

Radixin is a barbed end-capping actin-modulating protein which was previously reported to be concentrated at cell-to-cell adherens junctions (AJ) and cleavage furrows. Recently, cDNA encoding mouse radixin was isolated, showing that radixin is highly homologous to but distinct from ezrin. From mouse teratocarcinoma cells we isolated and analyzed cDNA encoding another radixin-related protein. Sequence analysis has demonstrated that this protein is a mouse homologue of human moesin (98.3% identity) and that it shares 71.7% and 80.1% identity with ezrin and radixin, respectively. Translation experiments in vitro combined with immunoblot analyses led us to conclude that there is a gene family consisting of ezrin, radixin and moesin. These members are coexpressed in various types of cells. Then, by immunofluorescence microscopy, we closely analyzed their distribution using polyclonal and monoclonal antibodies, which could recognize all three members. In addition to cell-to-cell AJ and cleavage furrows, it was shown that they were concentrated at microvilli and ruffling membranes in various types of cells. Furthermore, the cell-to-substrate AJ (focal contacts) were clearly stained by anti-radixin pAb only after the apical/lateral membranes and cytoplasm were removed by the zinc method. We conclude that at least one of the members of the ezrinradixin-moesin family is concentrated at specific regions where actin filaments are densely associated with plasma membranes.
BN46/51, a new nucleolar protein, binds to the basal body region inNaegleria gruberiflagellates
Authors: Trimbur Gina M. , Walsh Charles J.
Published: April 2021

Indirect immunofluorescence with the monoclonal antibody, BN5.1, labels the nucleolus of amebae of the amebo-flagellate Naegleria gruberi. When amebae differentiate into flagellates, BN5.1 binds to nucleoli and to the basal body region. The BN5.1 antigen is not present in basal bodies when basal bodies form at about 60 min after initiation of the differentiation or when flagella form at about 70 min. The BN5.1 antigen is first detectable in the basal body region at 85 min after initiation, a time when the basal body region acts as a microtubule organizing center for the formation of the microtubule cytoskeleton (MTCS) of flagellates. When flagellates revert spontaneously to amebae, the BN5.1 antigen is lost from the basal body region coincident with the loss of the MTCS. The BN5.1 antigen, composed of approximately equal amounts of two subunits of 46 kDa and 51 kDa, both of which carry the BN5.1 epitope, has been named BN46/51. BN46/51 in the basal body region comigrates with the nucleolar antigen by two-dimensional gel electrophoresis. Approximately 75% of the nucleolar BN46/51 is solubilized by extracton with 0.4 M NaCl. However, the antigen associated with the basal body region is resistant to extraction, even with 2 M NaCl. Solubilized BN46/51 exists as a heterogeneous multimer that elutes on gel filtration with a peak at 400 to 500 kDa and sediments on sucrose gradients at 5.5 S. The multimers consist of only the 46 kDa and the 51 kDa subunits in equal amounts as judged by glutaraldehyde cross-linking and by chromatography on BN5.1 affinity columns. Nucleolar BN46/51 is associated with the dense fibrillar and granular components of the nucleolus. However, it does not resemble any previously described nucleolar protein. Neither BN5.1, nor three other mAbs that recognize additional epitopes on both the 46 kDa and 51 kDa subunits of BN46/51, binds to nucleoli from Saccharomyces cerevisiae or mammalian cells. BN5.1 does not bind to the nucleoli of Dictyostelium discoideum or Euglena gracilis. Thus BN46/51 is an unusual and perhaps unique nucleolar component whose presence in the basal body region presents a challenge to our understanding of the cytoskeleton.
Association of myosin with the connecting cilium of rod photoreceptors
Authors: Williams David S. , Hallett Mark A. , Arikawa Kentaro
Published: April 2021

The cilium of a vertebrate photoreceptor cell connects the phototransductive outer segment of the cell to the inner segment. Previous studies have shown that, within the connecting cilium, there is a small cluster of actin filaments, which play a critical role in the formation of new disk membranes. Here, we have detected a polypeptide in rat rod outer segments that is recognized by myosin heavy chain antibodies and was found to possess other characteristics of conventional non-muscle myosin heavy chain: it comigrates in SDS-PAGE with non-muscle myosin heavy chain; it associates with the cytoskeleton of rod outer segments in an ATP-sensitive manner; and it binds to purified actin filaments in the absence of ATP. Myosin ATPase activity was also detected in isolated rod outer segments. Electron immunomicroscopy revealed that myosin is present in the small actin-containing domain within the connecting cilium at the site of disk membrane morphogenesis. These results pose the possibility that an actin-myosin contractile mechanism functions in the formation of new photoreceptor disk membranes.
Endocytosis: what goes in and how?
Authors: Watts Colin , Marsh Mark
Published: April 2021

During the last decade the term ‘endocytosis’ has become virtually synonymous with the activity of clathrin-coated vesicles. These vesicles, which are derived from cell surface clathrin-coated pits, are transport vehicles responsible for the transfer of plasma membrane receptors and their ligands, between the first two stations of the endocytic pathway: namely, the plasma membrane and early endosomes (Goldstein et al., 1985; van Deurs et al., 1989; Griffiths and Gruenberg, 1991). Despite the irrefutable evidence that clathrin-coated vesicles mediate endocytosis, their contribution to the total endocytic activity of the cell and the composition of the membrane they internalise remains controversial. Here we discuss: (1) the evidence that non-clathrin-mediated endocytic mechanisms operate alongside the clathrin-mediated pathway; (2) the evidence that endocytosis occurs for surface molecules that are not enriched in clathrin-coated pits and; (3) the sorting activities of cell surface clathrincoated pits and the notion that plasma membrane proteins that show particularly slow rates of uptake are actively excluded from the endocytic pathway.
Characterization of early and late endocytic compartments of the transferrin cycle Transferrin receptor antibody blocks erythroid differentiation by trapping the receptor in the early endosome
Authors: Killisch Iris , Steinlein Peter , Römisch Karin , Hollinshead Ruth , Beug Hartmut , Griffiths Gareth
Published: April 2021

We describe a detailed morphological characterization of the endocytic pathway in differentiating chicken erythroblasts transformed by a temperature-sensitive mutant of avian erythroblastosis virus (AEV). These cells express high levels of transferrin receptors (TfR) when induced to differentiate at 42°C. Biochemical analysis showed that most (≈90%) of the internalized 125I-Tf recycled within ≈30 min while a smaller fraction of l25I-Tf required up to 2 h for recycling. By immunocytochemistry, the bulk of Tf and TfR was localized at the plasma membrane and in tubuloreticular early endosomes. This structure contained coated buds that labelled with an antibody specific for the clathrin light chain. Decreasing amounts of both Tf and TfR were detected in two distal compartments, spherical endosome vesicles resembling multivesicular bodies and the prelysosomal compartment (PLC) enriched in cation-independent mannose 6-phosphate receptor. As shown by fluorescent (F1TC-Tf) labelling of living cells, the movement of Tf/TfR complex into these late structures was accompanied by a significant drop in pH from about 6, the value displayed by early endosomes, to values below pH 5.0. Since no detectable 125I-Tf degradation was observed during a 4 h period we believe that the Tf/TfR detected in these late endocytic structures avoids degradation and recycles back to the cell surface.

The addition of an anti-TfR monoclonal antibody to the culture medium of these cells blocks their differentiation. Under this condition the antibody-TfR complex was trapped in an early endosome compartment that enlarged to more than twice its normal size. However, this condition did not affect the transport kinetics of horseradish peroxidase from the medium to the PLC.
Dimeric transcription factor families: it takes two to tango but who decides on partners and the venue?
Authors: Lee Kevin A. W.
Published: April 2021

Dimeric transcription factors that bind to DNA are often grouped into families on the basis of dimerization and DNA-binding specificities. cDNA cloning studies have established that members of the same family have structurally related dimerisation and DNA-binding domains but diverge in other regions that are important for transcriptional activation. These features lead to the straightforward suggestion that although all members of a family bind to similar DNA elements, individual members exhibit distinct transcriptional effector functions. This simple view is now supported by experimental evidence from those systems that have proved amenable to study. There are however some largely unaddressed questions that concern the mechanisms that allow family members to go about their business without interference from their highly related siblings. Here I will discuss some insights from studies of the bZIP class of transcription factors.
Intranuclear co-location of newly replicated DNA and PCNA by simultaneous immunofluorescent labelling and confocal microscopy in MCF-7 cells
Authors: Humbert C. , Santisteban M. S. , Usson Y. , Robert-Nicoud M.
Published: April 2021

The intranuclear distribution of newly replicated DNA and of the proliferating cell nuclear antigen (PCNA) was mapped by confocal laser scanning microscopy after simultaneous immunofluorescent labelling of incorporated bromodeoxyuridine (BrdUrd) and PCNA. A mild hydrolysis with HCI followed by an enzymic digestion of DNA was used to produce single-stranded DNA required for BrdUrd immunorevelation, since this procedure preserves PCNA antigenicity. Optical sections obtained with a laser scanning microscope clearly showed a similar distribution of PCNA and BrdUrd within the nuclei, thus confirming previous observations on parallel labelled synchronized cultures. The intranuclear distribution of PCNA and BrdUrd varies concomitantly during the S phase of MCF-7 cells.
The pebble gene is required for cytokinesis in Drosophila
Authors: Lehner Christian F.
Published: April 2021

Cytokinesis is developmentally controlled during Drosophila embryogenesis. It is omitted during the initial nuclear division cycles. The nuclei of the resulting syncytium are then cellularized at a defined stage, and cytokinesis starts in somatic cells with mitosis 14. However, cytokinesis never occurs in somatic cells of embryos homozygous or transheterozygous for mutations in the pebble gene. Interestingly, the process of cellularization, which involves steps mechanistically similar to cytokinesis, is not affected. Moreover, all the nuclear aspects of mitosis (nuclear envelope breakdown, chromosome condensation, spindle assembly and function) proceed normally in pebble mutant embryos, indicating that pebble is specifically required for the coordination of mitotic spindle and contractile ring functions.

The pebble phenotype is also observed, but only with very low penetrance, during the early divisions of the germ line progenitors (the pole cells). -Amanitin injection experiments indicate that these early pole cell divisions, the first cell divisions during embryogenesis, do not require zygotic gene expression. These divisions might therefore rely on maternally contributed pebble function. The maternal contribution from heterozygous mothers might be insufficient in rare cases for all the pole cell divisions.
Control of division plane in normal and griseofulvin-treated microsporocytes of Magnolia
Authors: Brown Roy C. , Lemmon Betty E.
Published: April 2021

Meiotic cytokinesis in microsporocytes of Magnolia is an unusual form of the simultaneous type; phragmoplast expansion is not accompanied by a cell plate, wall deposition is centripetal, and infurrowing of the cytoplasm after first meiosis results in semicells connected by an isthmus. Dyad domains are further defined by interaction of extensive radial systems of microtubules emanating from the daughter nuclei and by a band of organelles polarized in the equatorial region. After second meiosis, phragmoplasts are organized in the interzonal regions between the sister nuclei in each semicell and also at the interfaces of microtubules forming secondary interzonals between non-sister nuclei. Wall deposition is not initiated until after phragmoplasts expand to the cell periphery and fuse in the isthmus. Centripetal wall deposition in boundaries of spore domains marked by radial arrays of microtubules results in simultaneous quadripartitioning of the microsporocyte into a tetrad of microspores. Treatment of microsporocytes with griseofulvin resulted in atypically placed nuclei and supernumerary nuclei. Abnormalities could be traced to displaced spindles and to spindles with multiple poles. Drug-induced multinucleate coenocytes were able to organize microtubules and initiate cytokinesis in altered patterns. The data suggest that spindle alignment and aggregation of spindle poles are two components of spatial control that are operative in determining the normal arrangement of nuclei, and that the final placement of walls is a function of the postmeiotic nuclear-based radial arrays of microtubules which define spore domains.
Very low cadmium concentrations stimulate DNA synthesis and cell growth
Authors: Zglinicki T. Von , Edwall C. , Östlund E. , Lind B. , Nordberg M. , Ringertz N. R. , Wroblewski J.
Published: April 2021

Uptake of cadmium into cultured cells and its effects on cell growth and DNA synthesis are measured over a range of Cd concentrations of seven orders of magnitude. Cd uptake is found to be proportional to the external Cd concentration and to incubation time over a very broad range of concentrations. At least 200 mmol cadmium per kg dry weight of cells can be accumulated in this way, leading to exhaustion of the major intracellular Cd binding sites before cell death.

On the other hand, very low cadmium concentrations down to 100 pM stimulate cell growth and DNA synthesis significantly. Stimulation is found in all three mammalian cell types examined: namely L6J1, a rat permanent myoblast cell line, LLC-PK1 porcine renal epithelial cells, and a primary rat chondrocyte culture. Cd acts as a cofactor with serum in L6J1 cultures, but is stimulatory only in serum-free cultures of chondrocytes. Stimulation occurs at Cd concentrations too low to result in a measurable induction of metallothionein. This might implicate the action of response amplifiers in the chain of events leading to Cd-stimulated DNA replication and cell growth.
A monoclonal antibody study of protein distribution in the membrane skeleton of the ciliate Pseudomicrothorax
Authors: Curtenaz Sophie , Peck Robert K.
Published: April 2021

The membrane skeleton, or epiplasm, of the ciliated protozoon Pseudomicrothorax dubius is a chemically and structurally complex layer. It is responsible for the cell shape and the positioning of some cortical organelles. One may expect that its possible morphogenetic role can be achieved only via a regional differentiation of the protein distribution in the epiplasm. We have tried to demonstrate such differentiation by preparing an epiplasm extract, which consists predominantly of concanavalin A (ConA)-positive glycoproteins. This fraction, either untreated or deglycosylated, was used to raise monoclonal antibodies (mAbs), whose specificity was tested on western blots of either untreated or deglycosylated epiplasm. The recognized polypeptides were then localized in situ by fluorescence and electron microscopic immunocytochemistry. Six mAbs are presented here. Four of them are directed against ConA-positive glycoproteins and show a localization of the latter on the outer surface of the epiplasm. The two others are directed against other epiplasmic polypeptides: one is specific for a common epitope shared by most of the epiplasmic proteins, but not by the glycoproteins, and labels the entire membrane skeleton, whereas the other recognizes three minor polypeptides, which seem localized to the inner part of the epiplasm.
Redistribution of a Golgi glycoprotein in plant cells treated with Brefeldin A
Authors: Satiat-Jeunemaitre Béatrice , Hawes Chris
Published: April 2021

The fungal fatty acid derivative Brefeldin A (BFA), has been used to study the reversible distribution of a Golgi glycoprotein, the JIM 84 epitope, into the cytosol of higher plant cells. Treatment of both maize and onion root tip cells resulted in a rearrangement of the Golgi stacks into either circular formations or a perinuclear distribution. The Golgi cisternae became curved and vesiculated and in cells where the Golgi apparatus was totally dispersed the JIM 84 epitope was associated with large areas in the cytosol which were also vesiculated. On removal of the BFA the Golgi apparatus reformed and the JIM 84 epitope was again located in the cisternal stacks. This mode of BFA action is compared with that so far described for animal cells.
Altering the cellular mechanical force balance results in integrated changes in cell, cytoskeletal and nuclear shape
Authors: Sims John R. , Karp Seth , Ingber Donald E.
Published: April 2021

Studies were carried out with capillary endothelial cells cultured on fibronectin (FN)-coated dishes in order to analyze the mechanism of cell and nuclear shape control by extracellular matrix (ECM). To examine the role of the cytoskeleton in shape determination independent of changes in transmembrane osmotic pressure, membranes of adherent cells were permeabilized with saponin (25 g/ml) using a buffer that maintains the functional integrity of contractile microfilaments. Realtime videomicroscopic studies revealed that addition of 250 M ATP resulted in time-dependent retraction and rounding of permeabilized cells and nuclei in a manner similar to that observed in intact living cells following detachment using trypsin-EDTA. Computerized image analysis confirmed that permeabilized cells remained essentially rigid in the absence of ATP and that retraction was stimulated in a dose-dependent manner as the concentration of ATP was raised from 10 to 250 M. Maximal rounding occurred by 30 min with projected cell and nuclear areas being reduced by 69 and 41%, respectively. ATP-induced rounding was also accompanied by a redistribution of microfilaments resulting in formation of a dense net of F-actin surrounding retracted nuclei. Importantly, ATP-stimulated changes in cell, cytoskeletal, and nuclear form were prevented in permeabilized cells using a synthetic myosin peptide (IRICRKG) that has been previously shown to inhibit actomyosin filament sliding in muscle. In contrast, both the rate and extent of cell and nuclear rounding were increased in permeabilized cells exposed to ATP when the soluble FN peptide, GRGDSP, was used to dislodge immobilized FN from cell surface integrin receptors. GRGDSP had little effect on cell or nuclear shape in the absence of ATP and, hence, in the absence of cytoskeletal tension. These data suggest that large-scale changes in cell and nuclear shape result from the action of mechanical tension that is generated within the cytoskeleton via an actomyosin filament sliding mechanism, transmitted across integrin receptors and physically resisted by immobilized adhesion sites within the extracellular matrix. Rapid and coordinated changes of cell, cytoskeletal and nuclear form result when this cellular force balance is altered.
Co-localization and molecular association of dystrophin with laminin at the surface of mouse and human myotubes
Authors: Dickson George , Azad Ajith , Morris Glenn E. , Simon Horst , Noursadeghi Mahdad , Walsh Frank S.
Published: April 2021

In Duchenne muscular dystrophy (DMD), deficiency of the protein dystrophin results in necrosis of muscle myofibres, associated with lesions in the sarcolemma and surrounding basal lamina. Dystrophin has been proposed to be a major component of the sub-sar-colemmal cytoskeleton involved in maintaining the integrity of the myofibre plasma membrane, and is known to associate with a group of sarcolemmal glycoproteins, one of which exhibits high affinity binding to the basal lamina component laminin. However, a direct or indirect transmembrane association of dystrophin in muscle cells with the myofibre basal lamina has not been demonstrated. To address this question we have examined dystrophin immunostaining and immunoprecipitation patterns in cultured mouse and human myotubes in comparison with that of the basal lamina component, laminin. Dual-immunolabelling revealed virtually complete co-localization of dystrophin on the inside surface of the muscle cell sarcolemma with plaques and veined arrays of laminin accumulating on the extracellular face. This pattern of laminin and dystrophin distribution was distinct from that of other cell surface molecules expressed in myotubes such as the neural cell adhesion molecule, NCAM, and the 1 integrin receptor, and immunoprecipitation of dystrophin from solubilized myotube extracts resulted in co-purification of laminin B1 chain confirming an association between these two components. The results thus provide the first direct cellular evidence of a transmembrane linkage between dystrophin in the sarcolemmal cytoskeleton with laminin in the overlying basal lamina. While the immunocytochemical distribution of laminin was apparently normal in dystrophin-deficient muscle cells, elevated levels of soluble laminin were present in extracts of mdx compared with normal mouse skeletal muscle. The absence of dystrophin may thus result in disruption at the molecular level of the cytoskeletal-basal lamina superstructure surrounding the sarcolemma in dystrophic myofibres.
Localization of ahe 6 and 4 integrin subunits in normal human nonlymphoid tissues
Authors: Natali P. G. , Nicotra M. R. , Bigotti A. , Martino C. De
Published: April 2021

The 6/ 4 integrin, of undefined receptor activity, has been shown to be expressed in a variety of murine epithelial cells. To gain information on the role of this heterodimer in tissue architecture as well as in malignant transformation we have performed an extensive immunohistochemical analysis of normal human tissues using monoclonal antibodies to 6 and 4 subunits. Because 6 is known to associate also with the 1 subunit to form a non-promiscuous receptor for laminin, the expression of 1 chain was also evaluated. The results of this study have shown that the 6 chain has a wide distribution in tissues, including small vessels and peripheral nerves. 6 colocalizes with 4 and 1 in most epithelial cells at the basolateral or basal aspect abutting the basement membrane. In a minority of tissues lacking 4, the 6 chain is coexpressed with 1. These findings demonstrate that the expression of 6/ 1 laminin receptor and 6/ 4 heterodimer is phylogenetically conserved, suggesting that they are likely to play an important role in cellular scaffolding through binding to laminin and to still uncharacterized ligand/s present in basement membranes.
The efficiency and timing of plasmid DNA replication in Xenopus eggs: correlations to the extent of prior chromatin assembly
Authors: Sanchez J. Aquiles , Marek Doreen , Wangh Lawrence J.
Published: April 2021

Injection of the circular plasmid FV1 (derived from type I bovine papilloma virus) into Xenopus eggs before the start of the first cell cycle dramatically increases the efficiency of plasmid replication once eggs are chemically activated. We call this the preloading effect and report kinetic and quantitative characterization of this phenomenon here. The timing and the amount of FV1 synthesis were measured by both BrdUTP density labelling and an optimized method of selective enzymatic digestion of replicated and unreplicated molecules using the three methyladenosine-sensitive isoschizomers, DpnI, MboI and Sau3a. DpnI in 100 mM NaCl proved particularly useful for distinguishing and quantitating unreplicated, once-replicated, and repeatedly replicated molecules accumulated over several cell cycles. Our results reveal that both the amount of DNA replicated and the timing of synthesis during the first S-phase correlate with the length of the preloading period. Longer preloading leads to larger amounts of DNA being replicated sooner. In fact, up to 30-50% of 1 ng injected plasmid can replicate in a semiconservative cell cycle-dependent manner during the first S-phase. But such high levels of synthesis during the first cell cycle appear to limit the egg’s ability to rereplicate this material in subsequent cell cycles. The preloading effect does not depend on synthesis of either viral or egg proteins, but does appear to correlate with the extent of plasmid assembly into chromatin before the start of the cell cycle. We postulate that each plasmid molecule must achieve a critical degree of chromatin assembly before it can proceed along the replication pathway. These observations illuminate some of the difficulties inherent in building a vector for gene insertion into Xenopus embryos, but also suggest an experimental strategy toward this aim.
Selective interactions of cells with crystal surfaces Implications for the mechanism of cell adhesion
Authors: Hanein Dorit , Sabanay Helena , Addadi Lia , Geiger Benjamin
Published: April 2021

In this study we have characterized the mode of cell adhesion to calcite and calcium (R,R)-tartrate tetrahydrate crystals. The use of crystals as adhesion substrata was motivated by their well-established chemical nature and structurally defined surfaces. We show that calcite binds A6 Xenopus laevis epithelial cells rapidly and efficiently, most likely via surface-adsorbed proteins. Surface topology had only a limited effect on the adhesive interactions. Calcium (R,R)-tartrate tetrahydrate crystals exhibits two chemically equivalent, yet structurally distinct faces that differ mainly in the surface distribution of their lattice water molecules and charges. However, despite the gross similarity between the two faces striking differences were noted in their adhesive behavior. One of the faces was highly adhesive for cells, leading to protein-independent attachment and spreading followed by cell death. In contrast, cell adhesion to the other surface of tartrate was slow ( 24 h) and apparently mediated by RGD-containing protein(s). It was further shown that the latter face of tartrate crystals could be “conditioned” by long (24 h) incubation with serum-containing medium, after which it becomes highly adhesive.

The results presented here indicate that crystal surfaces may serve as excellent, structurally defined, substrata for cell adhesion, that cell binding may occur directly or via RGD-containing proteins and that cell adhesion may be dramatically modulated by variations in surface structure. The implications of the results to the mechanism of cell-substratum adhesion are discussed.
Internal lamin structures within G1 nuclei of human dermal fibroblasts
Authors: Bridger Joanna M. , Kill Ian R. , O’Farrell Minnie , Hutchison Christopher J.
Published: April 2021

The nuclear lamina is a mesh-like network of fibres subjacent to the inner nuclear membrane that is believed to be involved in the specific spatial reorganisation of chromatin after mitosis. To determine how the lamina might be involved in chromatin reorganisation, we have performed indirect immunofluorescence studies on quiescent and proliferating human dermal fibroblasts (HDF). Two monoclonal antibodies recognising human lamins A and C and three different fixation methods were employed. In indirect immunofluorescence studies, cultures of quiescent cells displayed a uniform perinuclear distribution of the antibodies. In proliferating cultures two distinct populations of cells were observed: one population displayed a typical perinuclear antibody distribution, while the second population displayed an unusual pattern consisting of a series of spots and fibres within the nucleus. By inducing cell-cycle synchrony in cultures we were able to determine that the unusual internal distribution of the lamin antibodies was restricted to cells in G1. Optical sectioning and 3-D reconstruction of the lamina structures in G1 nuclei was performed with a confocal laser scanning microscope (CLSM). This revealed that the internal lamin structures consisted of small foci and fibres proliferating throughout the nucleus. These structures were shown to be closely associated with areas of condensed chromatin but not nuclear membrane. As cells progress towards S phase the internal lamin foci disappear.
Cell wall oligogalacturonides increase cytosolic free calcium in carrot protoplasts
Authors: Messiaen J. , Read N. D. , Cutsem P. Van , Trewavas A. J.
Published: April 2021

The mode of action of cell wall elicitors in the induction of various plant cell responses, such as the activation of host defence mechanisms, is unknown, but signal transduction through cytosolic free calcium ([Ca2+]i) has been suggested. This paper shows that polygalacturonic acid or oligogalacturonides cause a prolonged increase in [Ca2+]i of carrot protoplasts within 20 min of induction. Our data support the view that a special conformation of the oligogalacturonides possessing >9 residues is necessary to induce an elevation in [Ca2+]i. The localization of [Ca2+]i elevation around the periphery of protoplast cytoplasm and the inhibition of the response with verapamil suggest that exogenous Ca2+ is the major source for the rise in [Ca2+]i.
Aα and Bβ chains of fibrinogen stimulate proliferation of human fibroblasts
Authors: Gray Andrew J. , Bishop Jill E. , Reeves John T. , Laurent Geoffrey J.
Published: April 2021

During blood coagulation and wound healing, fibrinogen polymerises to form a fibrin matrix, providing a substratum over which connective tissue cells migrate and proliferate. Although a number of growth factors have been implicated in this process, a possible role for the fibrin(ogen) molecules themselves has not been considered. In this study we have investigated the ability of the constituent chains of fibrin(ogen) to induce fibroblast replication. Fibrinogen chains (Aα1, Aα2, Bβandγ) were separated by cation exchange chromatography and their mitogenic activity was assessed before and after treatment with thrombin. The Aα1, Aβ2 and Bβchains where all found to stimulate fibroblast replication (23 2.9%, 29.2 5.3% and 31.4 5% stimulation above control, respectively) and on the addition of thrombin this activity was enhanced. No activity was observed in the γ chain before or after treatment with thrombin. These results indicate that growth promoting activity is inherent in fibrin(ogen) structure, suggesting a novel mechanism for fibroblast proliferation during wound healing.
Specific responses of axons and dendrites to cytoskeleton perturbations: an in vitro study
Authors: Lafont F. , Rouget M. , Rousselet A. , Valenza C. , Prochiantz A.
Published: April 2021

Several factors can influence the development of axons and dendrites in vitro. Some of these factors modify the adhesion of neurons to their substratum. We have previously shown that the threshold of neuron-substratum adhesion necessary for initiation and elongation of dendrites is higher than that required for axonal growth. To explain this difference we propose that, in order to antagonize actin-driven surface tension, axons primarily rely on the compression forces of microtubules whereas dendrites rely on adhesion. This model was tested by seeding the cells in conditions allowing the development either of axons or of axons and dendrites, then adding cytochalasin B or nocodazole 1 hour or 24 hours after plating. The addition of cytochalasin B, which depolymerizes actin filaments and thus reduces actin-tensile forces, increases the length of both axons and dendrites, indicating that both axons and dendrites have to antagonize surface tension in order to elongate. The addition of nocodazole, which acts primarily on microtubules, slightly reduces dendrite elongation and totally abolishes axonal growth. Similar results are obtained when the drugs are added 1 or 24 hours after plating, suggesting that the same mechanisms are at work both in initiation and in elongation. Finally, we find that in the presence of cytochalasin B axons adopt a curly morphology, a fact that could be explained by the importance of tensile forces in antagonizing the asymmetry created by polarized microtubules presenting a uniform minus/plus orientation.
A reversibly palmitoylated resident protein (p63) of an ER-Golgi intermediate compartment is related to a circulatory shock resuscitation protein
Authors: Schweizer Anja , Rohrer Jack , Jenö Paul , DeMaio Antonio , Buchman Timothy G. , Hauri Hans-Peter
Published: April 2021

The recently identified 63 kDa membrane protein, p63, is a resident protein of a membrane network interposed in between rough ER and Golgi apparatus. To characterize p63 at the molecular level a 2.91 kb cDNA encoding p63 has been isolated from a human placenta gt10 cDNA library. Sequence analysis of tryptic peptides prepared from isolated p63 confirmed the identity of the cloned gene. The translated amino acid sequence consists of 601 amino acids (65.8 kDa) with a single putative membrane-spanning region and a N-terminal cytoplasmic domain of 106 amino acids. The human p63 cDNA exhibits a high level of sequence identity to the pig hepatic cDNA 3AL (accession number M27092) whose expression is enhanced after resuscitation from circulatory shock. An additional remarkable feature of p63 is that it becomes reversibly palmitoylated when intracellular protein transport is blocked by the drug brefeldin A. Overexpression of p63 in COS cells led to the development of a striking tubular membrane network in the cytoplasm. This suggests that the protein may be determinant for the structure of the p63 compartment.
Elevated cAMP levels induce multilayering of MDCK cells without disrupting cell surface polarity
Authors: Ullrich Oliver , Haase Winfried , Koch-Brandt Claudia
Published: April 2021

The effect of hormones on the morphology and cell surface polarity of the epithelial cell line MDCK was examined. When MDCK cells were seeded in high densities in media containing FCS a regular monolayer was formed. However, in serum-free medium supplemented with insulin, transferrin, prostaglandin E1, hydrocortisone and triiodothyronine, the development of a multilayer with intercellular lumina was observed. In hormone-depletion studies we identified PGE1 as the inducer of these multilayers. Since dibutyryl cyclic AMP and the phosphodiesterase inhibitor isobutyl methylxanthine could substitute for PGE1, we conclude that an elevated intracellular cAMP level resulted in formation of the multilayer.

Further analysis by electron microscopy and immunocytochemistry revealed a polarized organization of the multilayered cells. Junctional complexes, enclosing microvilli-rich membrane domains, were found at the apices of adjacent cells facing the medium and those surrounding the intercellular lumina. Surprisingly, cells participitating in the formation of both the free surface and the surface of the intercellular lumen, exhibited two distinct membranes with microvilli, each separated by junctional complexes. Immunolocalization of membrane marker proteins demonstrated that an apical 114 kDa membrane protein was localized to the free cell surfaces, the same membrane domains where extensive microvilli were also observed. The distribution of a basolateral 58 kDa membrane protein was restricted to sites of cell contact. These results provided evidence that nontransformed epithelial MDCK cells form multilayers in response to elevated cAMP levels; however, they retain the potential of developing cell surface polarity.