bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of Myf-5 in somitogenesis and the development of skeletal muscles in vertebrates
Authors: Arnold Hans-Henning , Braun Thomas
Published: April 2021

Muscle research has become a paradigm for the investigation of developmental processes that trigger determination and differentiation of specialized cell types in vertebrates. The isolation of a family of muscle regulatory genes which includes MyoD, myogenin, Myf-5 and MRF4 (also called herculin and Myf-6) represented a breakthrough in our attempt to identify important genetic components required for myogenesis (for recent reviews see: Emerson, 1990; Olson, 1990; Weintraub et al., 1991). About twenty years ago, the first experimental evidence was provided in muscle cell culture systems that suggested the existence of a “master gene” that controls the myogenic fate of cells (O’Neill and Stockdale, 1974; Holtzer et al., 1975). This hypothesis has been revived recently by the identification of the myogenic regulatory genes. While MyoD was the first gene to be cloned that had the ability to induce the muscle phenotype in cultured fibroblasts (Davis et al., 1987), it is now clear that the other members of this family have similar properties and may serve overlapping biological functions.
Characterization of a putative Drosophila GTP-binding protein
Authors: Fredieu John R. , Mahowald Anthony P.
Published: April 2021

We generated a set of monoclonal antibodies raised against Drosophila antigen eluted from a lectin affinity column. One antibody, mAb 13D5 recognizes an anti-gen found in the most dorsal regions of the ventral mid-line of Drosophila embryos at stages prior to and during axonogenesis. 13D5 recognizes cells dorsal to the ecto-derm in the extended germ band beginning approx-imately 7 hours after fertilization and in the peripheral nervous system (PNS) at about 13 hours. In addition, 24-hour-old cultures of isolated embryonic neuroblasts possess a number of cells that express the 13D5 antigen and are not recognized by horseradish peroxidase anti-sera. These cells extend ramified processes with mult-iple growth cone-like structures or large individual processes with a broad growth cone structure. 13D5 immunoprecipitates a single band with an apparent molecular mass of 58 kDa and isolated a 1.9 kb EcoRI fragment from the Xgt11 expression libraries. In situ hybridization to staged embryos using the digoxygenin-labeled probe reveals a pattern of expression in cells just lateral to the dorsal-most regions (mesectoderm) of the ventral midline in 8-to 9-hour embryos. In situ hybrid-ization to cultured cells derived from whole embryos reveals several cell types with differing morphologies that express transcript recognized by the digoxygenin-labeled probe. These cells may possess either broad processes containing detectable transcript, or long thin processes with no detectable transcript. Northern analy-sis reveals a 2.1 kb RNA transcript detectable in all embryonic stages. Nucleotide sequence obtained from the 1.9 kb insert reveals homology with the GTP-bind-ing regions of two signal recognition particle receptors (SNRP) isolated from canine and human tissues.
Structural reorganization of the rough endoplasmic reticulum without size expansion accounts for dexamethasone-induced secretory activity in AR42J cells
Authors: Rajasekaran Ayyappan K. , Morimoto Takashi , Hanzel David K. , Rodriguez-Boulan Enrique , Kreibich Gert
Published: April 2021

A striking reorganization of the rough endoplasmic reticulum (RER) from a tubulovesicular (TV-RER) to a stacked cisternal (SC-RER) configuration was observed when the secretory activity of AR42J cells, a cell line derived from a rat pancreatic acinar carcinoma, was induced by dexamethasone. Treatment with 10 nM dexamethasone resulted in a 6.6-fold increase in the intracellular and a 4.6-fold increase in the secreted amylase activity, respectively. On the basis of the morphometric analysis of thin-section electron micrographs it has been previously reported that this increase in secretory activity is accompanied by a 2.4-fold or 30-fold increase in the size of the RER. We have developed a new biochemical method to determine the size of the RER by quantifying the membrane-bound ribosomes. Using this procedure we did not detect any change in the size of the RER after induction of an active secretory state in AR42J cells. Electron microscopic observation showed the predominance of SC-RER in dexamethasone-treated cells compared to the abundance of TV-RER in control cells. Laser scanning confocal microscopy showed a patchy distribution of ER staining in dexamethasone-treated cells compared to more basal localization in control cells. On the basis of our observations we conclude that in AR42J cells the increase in secretory activity induced by dexamethasone is accompanied by a reorganization of the RER rather than by an increase in ER surface area, as reported by others. Our results suggest that SC-RER is a biosynthetically more efficient form of the RER, which is found predominantly in actively secreting cells.
Transepithelial calcium transport in the chick chorioallantoic membrane I. Isolation and characterization of chorionic ectoderm cells
Authors: Akins Robert E. , Tuan Rocky S.
Published: April 2021

The chicken eggshell supplies approximately 80% of the calcium found in the hatchling chick. The mobilization of eggshell calcium into the developing embryo involves the transepithelial transport of large amounts of calcium in a development-specific manner. The cells responsible for the transport of eggshell calcium into the embryonic circulation are the ectodermal cells of the chorioallantoic membrane. In this report, we present a method for the isolation and culture of chorioallantoic membrane ectodermal cells, which are amenable to direct experimental manipulation. Cell preparations are characterized with respect to the expression of an ectoderm-specific cell surface marker (transcalcin, a calcium-binding protein), and a specific enzymatic activity (elevated Ca2+-activated ATPase). Functional assessment of in vitro cellular calcium uptake by 45Ca2+ tracer kinetics indicates the persistence of a temperature-sensitive, rapid-influx pathway similar to that observed in vivo. The preparations of primary ectodermal cells present an in vitro system applicable to the experimental analysis of calcium metabolism and transport by the chick chorioallantoic membrane.
Transepithelial calcium transport in the chick chorioallantoic membrane II. Compartmentalization of calcium during uptake
Authors: Akins Robert E. , Tuan Rocky S.
Published: April 2021

Calcium transport from the eggshell to the developing chick embryo is carried out by the ectoderm cells of the chick chorioallantoic membrane. Primary cells isolated from chick chorioallantoic membrane ectoderm were used to analyze the subcellular distribution of 45Ca2+ accumulated from the extracellular medium. We present evidence suggesting that calcium may be sequestered into endosome-like vesicles during the initial phase of uptake. A combination of techniques were utilized to monitor calcium fluxes and calcium compartmentalization in the cultured chorioallantoic membrane cells: (1) fura-2 fluorescence was used to indicate cytosolic free calcium concentrations, (2) 45Ca2+ tracer was used to follow calcium accumulation in all cellular compartments, and (3) digitonin was used to differentially permeabilize subcellular membranes in order to localize 45Ca2+ by following tracer release profiles.

Differences between cytosolic calcium flux and whole cell calcium accumulation suggested that the pathway of calcium uptake from the medium involves sequestration into an internal compartment separate from the cytosol. Kinetic analysis of the digitonin-mediated release of specific subcellular markers (lactate dehydrogenase, NAD-dependent isocitrate dehydrogenase, [3H]inulin, and [3H]-2-deoxyglucose) and preloaded 45Ca2+ indicated that calcium was localized in a compartment similar to endosomal vesicles. Our results are consistent with a transcytotic mechanism for chorioallantoic membrane calcium transport.
Xenopus nuclear factor 7 (xnf7) possesses an NLS that functions efficiently in both oocytes and embryos
Authors: Li Xiaoxia , Etkin Laurence D.
Published: April 2021

Xenopus nuclear factor 7 (xnf7) is a nuclear phosphoprotein that is encoded by a member of a novel zinc finger gene family and likely functions as a transcription factor. It possesses a nuclear localization signal (NLS) similar to the bipartite basic NLS of nucleoplasmin, but unlike nucleoplasmin, which re-enters nuclei immediately after fertilization, xnf7 remains cytoplasmic until the mid-blastula transition (MBT). We have measured the accumulation of injected labeled xnf7 protein or protein produced from synthetic xnf7 transcripts in the oocyte nuclei (GV). The data show that the NLS of xnf7 functions efficiently in oocytes. Mutations in either of the bipartite basic domains of the xnf7 NLS inhibit nuclear accumulation, while mutations in the spacer sequences have no effect. The xnf7 NLS linked to pyruvate kinase directs the efficient accumulation of this protein into nuclei of early embryos prior to the MBT. These data suggest that retention of the xnf7 protein during development is the result of a mechanism that interferes with the xnf7 NLS function.
An in vitro model for the analysis of intestinal brush border assembly II. Changes in expression and localization of brush border proteins during cell contact-induced brush border assembly in Caco-2BBe cells
Authors: Peterson Michelle D. , Bement William M. , Mooseker Mark S.
Published: April 2021

In the companion paper (M. D. Peterson and M. S. Mooseker (1993). J. Cell Sci. 105, 445-460) we describe a method for modeling brush border assembly in the Caco-2BBe clones. In this study we have examined the molecular changes accompanying cell contact-induced brush border assembly. A subset of brush border proteins was tracked throughout brush border assembly by immunoblotting and by immunofluorescent localization using laser scanning confocal microscopy. Actin, fodrin, villin and presumptive unconventional myosin immunogens were distributed at the periphery of depolarized cells. All proteins partitioned primarily with the membrane fraction upon differential sedimentation of depolarized cell lysates; the fractionation patterns were comparable to those of confluent cells. After a monolayer had formed, each protein showed a redistribution to the apical domain in a discrete sequence. Actin and villin began to shift apically at 2 d, while fodrin and the unconventional myosin immunogens did not redistribute until 3 d. Enterocyte-like localization was observed by 5 d for all proteins. Sucrase-isomaltase was not reliably detectable until 9 d by immunofluorescence, after brush border assembly was complete. Quantitative immunoblot analysis of total cell extracts demonstrated an average 10-fold increase in villin levels, while fodrin levels appeared to remain unchanged. Three putative unconventional myosin immunogens of 140 kDa, 130 kDa, and 110 kDa have been detected previously in the C2BBe cells with a head-specific monoclonal antibody to avian brush border myosin I (M. D. Peterson and M. S. Mooseker (1992) J. Cell Sci. 102, 581-600). Each of these immunogens displayed distinct expression patterns during brush border assembly. The 140 kDa species decreased by half, while the 130 kDa immunogen(s) did not change in any consistent fashion. The 110 kDa protein, presumed to be human brush border myosin I, rose on average 8-fold. A ribonuclease protection assay was also performed using a probe for human brush border myosin I. Equal amounts of total RNA from depolarized and confluent cells were assayed; the level of protected product was approximately 9-fold greater in the confluent cells. The expression patterns of the brush border proteins, coupled with the correlation to the ultrastructural features during brush border assembly in C2BBe cells, show that differentiation of the C2BBe cells closely resembles the changes that occur during human fetal intestinal differentiation.
Cell surface CD44-related chondroitin sulfate proteoglycan is required for transforming growth factor-β-stimulated mouse melanoma cell motility and invasive behavior on type I collagen
Authors: Faassen Anne E. , Mooradian Daniel L. , Tranquillo Robert T. , Dickinson Richard B. , Letourneau Paul C. , Oegema Theodore R. , McCarthy James B.
Published: April 2021

Tumor cell metastasis involves a complex series of events, including the adhesion, migration and invasive behavior of tumor cells on components of the extracellular matrix. Multiple cell surface receptors mediate interactions with the surrounding extracellular matrix and thereby influence cell adhesion, motility and invasion. We have previously described a cell surface CD44-related chondroitin sulfate proteoglycan on highly metastatic melanoma cells. CD44-chondroitin sulfate proteoglycan was shown to be important in melanoma cell motility and invasive behavior on type I collagen matrices. In our current studies, the role of cell surface CD44-chondroitin sulfate proteoglycan in collagen-mediated mouse melanoma cell migration and invasive behavior is further evaluated using transforming growth factor-1. We report that transforming growth factor-1 stimulates the migratory and invasive behavior of mouse melanoma cells on type I collagen. Transforming growth factor-1 stimulated cell surface CD44-chondroitin sulfate proteoglycan synthesis in mouse melanoma cells, specifically through an upregulation of chondroitin sulfate production, while the expression of CD44-chondroitin sulfate proteoglycan core protein was not affected. Furthermore, transforming growth factor-1-mediated enhancement of cell polarity, migration and invasive behavior on type I collagen gels was markedly inhibited in the presence of -D-xyloside, an agent that blocks chondroitin sulfate addition to the core protein. Collectively, our findings indicate that mouse melanoma cell surface CD44-chondroitin sulfate proteoglycan is required for transforming growth factor-1-enhanced cell motility and invasion, and that CD44-chondroitin sulfate proteoglycan may play a role in forming and/or maintaining a dominant leading lamella, which is required for efficient locomotion.
Antisera against a channel-forming 16 kDa protein inhibit dye-coupling and bind to cell membranes in Drosophila ovarian follicles
Authors: Bohrmann Johannes
Published: April 2021

In Drosophila ovarian follicles, communication via gap junctions can be observed between the oocyte and its surrounding follicular epithelium. In the present study, the intercellular exchange of the fluorescent tracer Lucifer Yellow was analysed following pressure-injections of five different sera or protein solutions into the oocyte of stage-10 follicles. Three of the tested sera are directed against a channel-forming 16 kDa protein, which is a component of the vacuolar H+-ATPase and of Nephrops norvegicus gap junctions. When one of these antisera was injected 5-10 min prior to the dye, the percentage of follicles showing dye-coupling between oocyte and follicle cells was extremely small. On the other hand, injections of non-immune serum or of bovine serum albumin solution had only minor inhibitory effects. With indirect immunofluorescence, the three Nephrops antisera revealed a discrete punctate pattern at the membranes between neighbouring follicle cells as well as between follicle cells and oocyte. Most likely, this fluorescent pattern represents the distribution of gap junctions in the follicular epithelium. On immunoblots, the Nephrops antisera recognized a 29 kDa Drosophila ovary protein with high specificity. Affinity purification of one of these antisera against the 29 kDa protein revealed that this protein of Drosophila and the 16 kDa membrane-channel protein of Nephrops are immuno-logically related. Thus, the Nephrops antisera might help to reveal, in future injection experiments, the functional role of gap-junction mediated communication in Drosophila.
CO2 production in cell-free extracts of fission yeast detects cell cycle changes
Authors: Novak B. , Sveiczer A. , Mitchison J. M.
Published: April 2021

CO2 production was followed by manometry in starved cell-free extracts of fission yeast stimulated by unstarved cell-free extracts from a synchronous culture. The degree of stimulus, measured by the lag time in CO2 production, varied markedly during the cell cycle, with a maximum for cells at about mitosis and a minimum for septated cells. Similar differences in lag time were found with unstarved extracts of cdc13.117 grown at 37°C and 35°C.
Ultraviolet microbeam irradiation of chromosomal spindle fibres in Haemanthus katherinae endosperm I. Behaviour of the irradiated region
Authors: Czaban B. Barbara , Forer Arthur , Bajer Andrew S.
Published: April 2021

We used an ultraviolet microbeam to irradiate chromosomal spindle fibres in metaphase Haemanthus endosperm cells. An area of reduced birefringence (ARB) was formed at the position of the focussed ultra-violet light with all wavelengths we used (260, 270, 280, and 290 nm). The chromosomal spindle fibre regions (kinetochore microtubules) poleward from the ARBs were unstable: they shortened (from the ARB to the pole) either too fast for us to measure or at rates of about 40 m per minute. The chromosomal spindle fibre regions (kinetochore microtubules) kinetochore-ward from the ARBs were stable: they did not change length for about 80 seconds, and then they increased in length at rates of about 0.7 m per minute. The lengthening chromosomal spindle fibres sometimes grew in a direction different from that of the original chromosomal spindle fibre. The chromosome associated with the irradiated spindle fibre sometimes moved off the equator a few micrometers, towards the non-irradiated half-spindle.

We discuss our results in relation to other results in the literature and conclude that kinetochores and poles influence the behaviour of kinetochore microtubules.
Regulation of the intracellular distribution of cytoplasmic dynein by serum factors and calcium
Authors: Lin Sharron X. H. , Collins Christine A.
Published: April 2021

Previous work has indicated that cytoplasmic dynein localizes primarily to lysosomes in cultured fibroblasts, consistent with a function for dynein in retrograde movement. We now show that dynein can be redistributed from a lysosome-associated pool to a more diffuse cytoplasmic pool upon shifting fibroblasts to culture medium lacking serum for several hours. This effect on dynein localization is readily reversed upon addition of serum, with a substantial return to a control appearance of punctate staining within 10 minutes. The serum effect appears to be selective for dynein, in that the localization of kinesin and the overall morphology of intracellular organelles does not change. However, the distribution of kinesin-positive vesicles and lysosomes does appear to be altered during serum starvation, in that these organelles are located to greater extents in the peripheral regions of the cell. Dynein is also associated with the mitotic apparatus, but this localization does not change in response to serum starvation. Removal of calcium from the extracellular medium also results in the loss of punctate dynein staining, which can be recovered upon addition of calcium to calcium-free medium. The redistribution of dynein observed under these experimental conditions may reflect the activity of a regulatory process controlling the association of dynein with organelles, thereby providing one means of modulating intracellular transport.
Modulation of pp60c-src activity and cellular localization during differentiation of human trophoblast cells in culture
Authors: Rebut-Bonneton C. , Boutemy-Roulier S. , Evain-Brion D.
Published: April 2021

The morphological and functional differentiation of human trophoblast cells ends with the formation of terminally differentiated multinucleated syncytial trophoblasts. This in vivo differentiation is mimicked in vitro during the primary culture of extravillous cytotrophoblasts: isolated mononuclear cytotrophoblasts aggregate and fuse to form syncytia. This in vitro differentiation is associated with an increase in epidermal growth factor receptor (EGF-R) expression and a transitory increase in E-cadherin expression during cell aggregation. In the present study, we investigated the expression of pp60c-src during morphological differentiation of trophoblast cells. Cultures were terminated at various time intervals and pp60c-src was analysed by immunocytochemistry using a specific antibody. In addition, pp60c-src was investigated by western blot analysis and its tyrosine kinase activity was measured concomitantly. In mononuclear cytotrophoblasts, pp60c-src was localized at cell-matrix contacts and during the aggregation of cytotrophoblasts, pp60c-src was distributed on the cell surface at points of cell-cell contact being colocalized with EGF-R and E-cadherin. The kinase activity of the pp60c-src protein increased significantly at day 2 when cells were completely aggregated and started to fuse, and remained elevated while cells underwent further differentiation. Inhibition of pp60c-src by herbimycin A at 0.25 to 1 g/ml during the first day of culture was associated with a decreased expression of tyrosine kinase activity of EGF-R and an increase in E-cadherin expression. These data suggest that pp60c-src is involved in the modulation of trophoblast cell aggregation and fusion leading to syncytial formation.
Tyrosine phosphorylation of the focal adhesion kinase pp125FAK during development: relation to paxillin
Authors: Turner Christopher E. , Schaller Michael D. , Parsons J. Thomas
Published: April 2021

Significant changes in the level of protein tyrosine phosphorylation accompany avian embryonic development. A comparison of different tissues reveals that a similar and remarkably restricted complement of proteins is modified in this manner. In each case the major proteins detected using anti-phosphotyrosine antibodies have molecular masses of approximately 170, 150, 125, 70 and 50 kDa. As a first step in determining the function of this protein modification in embryogenesis we have initiated a study to identify these phosphoproteins. We have previously reported that the 70 kDa band is paxillin, a component of actin-membrane attachment sites associated with regions of cell adhesion (Turner, C.E. (1991) J. Cell Biol. 115, 201-207). We report here that the 125 kDa phosphotyrosine-containing protein is the tyrosine kinase pp125FAK, a protein that co-localizes with paxillin at sites of adhesion (Schaller et al. (1992)Proc. Nat. Acad. Sci. USA 89, 5192-5196). Tyrosine phosphorylation of both pp125FAK and paxillin was detected at low levels as early as embryonic day 3 and increased steadily during the first half of development, reached a maximum between embryonic days eight and twelve, and declined to background levels prior to hatching. Paxillin protein expression also increased during the first half of embryogenesis, suggesting little change in the overall phosphorylation of this protein through embryonic day 8. In contrast, pp125FAK, following an initial increase, is expressed at a constant high level during these early embryonic stages, implying an increase in its overall phosphotyrosine content. In the second half of embryonic development pp125FAK expression decreased in parallel with the decrease in tyrosine phosphorylation of this and the other phosphopeptides. The alternatively-spliced 41/43 kDa form of pp125FAK is also present in the embryo, but at much reduced levels, and is not phosphorylated on tyrosine. At the stage of development corresponding to the highest level of phosphorylation of pp125FAK (day 12) approximately 54% of the pp125FAK was phosphorylated on tyrosine. pp125FAK and paxillin exhibit a similar tissue distribution with the exception of brain where only pp125FAK was detected. Immunoprecipitates of pp125FAK from embryonic smooth muscle extract exhibited tyrosine kinase activity that phosphorylated pp125FAK and a 60 kDa protein. This immune complex also catalyzed the tyrosine phosphorylation of purified paxillin. Our identification of intracellular proteins associated with cell attachment, namely pp125FAK and paxillin, as two of the major targets for tyrosine phosphorylation during embryogenesis suggests that this protein modification may contribute to the regulation of important cell adhesion events during embryonic morphogenesis.
Aggregation of band 3 in hereditary ovalocytic red blood cell membranes Electron microscopy and protein rotational diffusion studies
Authors: Che Alexis , Cherry Richard J. , Bannister L. H. , Dluzewski A. R.
Published: April 2021

Microaggregation of band 3 proteins in hereditary ovalocytic membranes was investigated by rotational diffusion measurements and by electron microscopy. It was previously shown that band 3 in ovalocytic membranes has decreased rotational mobility compared with band 3 in normal cells (Tilley, L., Nash, G. B., Jones, G. L. and Sawyer, W. L. (1991) J. Membr. Biol. 121, 59-66). This result could arise from either altered interactions with cytoskeletal proteins or from band 3 microaggregation. In the present study it was found that removal of spectrin and actin from the membrane had no effect on the rotational mobility of ovalocytic band 3. Additional removal of ankyrin and band 4.1, as well as cleavage of the cytoplasmic domain of band 3 with trypsin, did enhance band 3 mobility, as is the case in the membranes from normal cells. However, the rotational mobility of ovalocytic band 3 was always considerably less than that of normal band 3 under the same conditions. Scanning electron microscopy and low power electron micrographs of freeze-fracture replicas revealed that the surfaces of ovalocytes were more irregular than those of normal erythrocytes. At higher magnification, numerous linearly arranged intramembranous particles were observed on the P-faces of freeze-fractured ovalocytes but not on normal cells. These clusters consist of straight or slightly curved lines of 10-15 particles in single rows. From these results it is deduced that the reduced rotational mobility of band 3 in ovalocytes is a consequence of the formation of microaggregates, which are very probably induced by the mutation in the membrane-bound domain of ovalocytic band 3.
Interaction of the human autoantigen p150 with splicing snRNPs
Authors: Blencowe Benjamin J. , Carmo-Fonseca Maria , Behrens Sven-Erik , Lührmann Reinhard , Lamond Angus I.
Published: April 2021

An important goal of studies on pre-mRNA splicing is to identify factors that mediate the snRNP-snRNP and snRNP-pre-mRNA interactions that take place in the spliceosome. The U4/U6 snRNP is one of the four snRNPs that are subunits of spliceosomes. A rare patient autoimmune serum (MaS serum) has recently been identified that specifically immunoprecipitates U4/U6 snRNP from HeLa cell extracts through recognition of a 150 kDa autoantigen (p150) (Okano and Medsger, Journal of Immunology, 146, 535-542, 1991). Here we show that in addition to U4/U6 snRNP, p150 can also be detected associated with 20 S U5, U4/U6.U5 and 17 S U2 snRNPs, but not with U1 snRNP. In each particle p150 is present in sub-stoichiometric levels relative to the major snRNP proteins. We show that MaS serum selectively immunoprecipitates a sub-population of U4/U6 snRNPs in which the m3G-cap structure is masked and that p150 is preferentially associated with U6 snRNA in the U4/U6 particle. Anti-p150 antibodies show widespread nucleoplasmic staining, excluding nucleoli, with an elevated concentration in coiled bodies. This changes to a discrete punctate pattern when cells are treated with α-amanitin. Both the cytological and biochemical data indicate that the p150 autoantigen is a snRNP-associated factor in vivo. We also present biochemical evidence confirming that assembly of U4/U6 and U5 snRNPs into a U4/U6.U5 tri-snRNP particle is an integral step in the spliceosome assembly pathway. Addition of the purified U4/U6.U5 tri-snRNP restores splicing activity to inactivated HeLa nuclear extracts in which splicing had been inhibited by specific depletion of either the U4/U6 or U5 snRNPs.
Polarized distribution of Listeria monocytogenes surface protein ActA at the site of directional actin assembly
Authors: Kocks Christine , Hellio Raymond , Gounon Pierre , Ohayon Helene , Cossart Pascale
Published: April 2021

The facultative intracellular pathogen Listeria monocy -togenes can infect host tissues by using directional actin assembly to propel itself from one cell into another. The movement is generated by continuous actin assembly from one end of the bacterium into a tail, which is left behind in the cytoplasm. Bacterial actin assembly requires expression of the bacterial gene actA. We have used immunocytochemistry to show that the actA gene product, ActA, is distributed asymmetrically on the bacterial surface: it is not expressed at one pole and is increasingly concentrated towards the other. This polarized distribution of ActA was linked to bacterial division: ActA protein was not, or only faintly, expressed at the pole that had been formed during the previous division. On intracellular bacteria ActA was expressed at the site of actin assembly, suggesting that ActA may be involved in actin filament nucleation off the bacterial surface. We predict that the asymmetrical distribution of this protein is required for the ability of intracellular Listeria to move in the direction of the non-ActA expressing pole.
Heat shock results in cell cycle delay and synchronisation of mitotic domains in cellularised Drosophila melanogaster embryos
Authors: Maldonado-Codina Gabriela , Llamazares Salud , Glover David M.
Published: April 2021

Cells of Drosophila embryos that are subjected to a 37°C temperature shock whilst undergoing the S-phase of cell cycle 14 arrest with their microtubules in an interphaselike state, and with nuclei showing unusual chromatin condensation. They do not recover from this state within a 30 minute period even though extensive gastrulation movements can occur. Cells of embryos heat shocked in G2-phase are delayed in interphase with high levels of cyclins A and B. Within ten minutes recovery from heat shock, cells enter mitosis throughout the embryo. The degradation of the mitotic cyclins A and B in these synchronised mitotic domains does not follow the normal timing, but is delayed. These findings point to a need for caution when interpreting experiments that use the heat shock promoter to study the expression of cell cycle control genes in Drosophila.
The relaxed crossbridge pattern in isolated rabbit psoas muscle thick filaments
Authors: Kensler Robert W. , Stewart Murray
Published: April 2021

Rabbit muscle is a major source of material for biochemical experiments and spin labelling studies of contraction, and so it is important to establish how closely this material resembles the frog and fish muscles usually used for structural studies. Previous studies have shown that relaxed rabbit muscle thick filaments lose the characteristic order of their crossbridges when they are cooled below about 15-19°C, whereas the order of fish and frog muscles is retained above 0°C. The lack of order has frustrated attempts to examine rabbit thick filament structure and has raised questions about how closely they might resemble other thick filaments. We have therefore developed a procedure for preserving the crossbridge order in isolated filaments. Electron microscopy of these thick filaments after either negative staining or metal shadowing has shown that the crossbridge pattern has a 43 nm axial repeat and is based on three near-helical strands. Computed transforms of either type of image show a series of layer lines confirming that the native relaxed pattern has been preserved, and computer reconstructions show the individual crossbridges lying on a slightly perturbed 3-stranded lattice. These data indicate an unexpectedly high degree of similarity between the rabbit and frog patterns and indicate that, in fully preserved material, there is little structural difference between the two thick filaments at the temperature at which each normally functions. Overall, therefore, our results validate the integration of biochemical data from rabbit muscle with structural data from frog and indicate that the differences between the two muscle types do not result from fundamental structural differences in the arrangement of myosin molecules in their thick filaments.
Paracrine induction of angiogenesis in vitro by Swiss 3T3 fibroblasts
Authors: Montesano R. , Pepper M.S. , Orci L.
Published: April 2021

During angiogenesis, normally quiescent endothelial cells that line existing microvessels are induced to invade the surrounding extracellular matrix and to form capillary sprouts in response to paracrine factors released by neighboring cell types. In an attempt to mimic the physiological conditions under which angiogenesis is triggered in vivo, we have designed two co-culture systems that are suitable for the study of paracrine interactions between microvascular endothelial cells and cell types that might produce angiogenic factors. In the first model, cells to be co-cultured with endothelial cells were suspended within a collagen gel and overlaid with an additional collagen gel devoid of cells, onto which bovine microvascular endothelial cells were subsequently seeded and grown to confluence. In the second model, a small collagen gel disc containing a suspension of endothelial cells was embedded into a larger, cell-free collagen gel disc, which in turn was surrounded by an annular collagen gel containing other cell types. We show that Swiss 3T3 fibroblasts and their 3T3-L1 substrain induce the formation of capillary-like tubes by endothelial cells in these co-culture systems, whereas Balb 3T3 cells, as well as a number of other fibroblasts and epithelial cells, lack this ability. Thus, endothelial cells grown on a collagen gel containing Swiss 3T3 or 3T3-L1 cells invaded the underlying matrix to form a network of interconnecting tubules. In addition, Swiss 3T3 and 3T3-L1 cells stimulated extensive radial outgrowth of tubular sprouts from the periphery of endothelial-cell-containing collagen discs. Conditioned medium from Swiss 3T3 cells mimicked the effect of co-culture by inducing formation of capillary-like tubes, and also increased plasminogen activator activity in endothelial cells. Conditioned medium from Balb 3T3 cells, in contrast, lacked these activities. Preliminary evidence suggests that the factor(s) in Swiss 3T3 conditioned medium that induces tubule formation by endothelial cells may be different from a number of well-characterized angiogenic cytokines. The co-culture systems described here should prove to be useful for the identification of physiological regulators of angiogenesis produced by various cell types.
The bip protein and the endoplasmic reticulum of Schizosaccharomyces pombe: fate of the nuclear envelope during cell division
Authors: Pidoux Alison L. , Armstrong John
Published: April 2021

A polyclonal antibody was raised to the C-terminal region of fission yeast BiP. The use of this antibody for immunoprecipitation, western blotting and immunofluorescence has confirmed and extended the observations made previously with an epitope-tagged BiP molecule. A fraction of BiP protein is glycosylated in Sc h izo s a c charomyces pombe cells. Pulse-chase experiments showed that this modification occurs rapidly upon synthesis and that the extent of glycosylation does not then change with time. BiP protein is induced by elevated temperatures and by treatment with tunicamycin. The antibody cross-reacts with proteins of similar molecular weight in the yeasts Kluyveromyces lactis and Schizosaccharomyces japonicus. Immunofluorescence of BiP has been used to follow the behaviour of the ER and in particular the nuclear envelope through the cell cycle.
Spatial localization of agonist-induced Ca2+ entry in bovine adrenal chromaffin cells: Different patterns induced by histamine and angiotensin II, and relationship to catecholamine release
Authors: Cheek Timothy R. , Morgan Alan , O’Sullivan Antony J. , Moreton Roger B. , Berridge Michael J. , Burgoyne Robert D.
Published: April 2021

The spatial organization of agonist-induced Ca2+ entry in single bovine adrenal chromaffin cells has been investigated using video-imaging techniques to visualize fura2 quenching by the Ca2+ surrogate, Mn 2+.

The potent secretagogue histamine, in addition to releasing Ca2+ from intracellular stores, resulted in a large influx of external Mn2+ that occurred over the entire surface of the cell. The influx of Ca2+ that this mirrors was found to be an obligatory requirement for the triggering of catecholamine release by histamine, which suggests that such a global influx of Ca2+ into the cell probably underlies the ability of this agonist to stimulate a large secretory response. By contrast, the weaker secretagogue angiotensin II, which also acts through the second messenger inositol trisphosphate, produced a localized entry of external Mn2+ in 64% of cells. In these cells, localized Mn2+ entry always occurred at the pole of the cell in which the angiotensin II-induced rise in [Ca2+]i was largest. Since exocytosis in response to angiotensin II has previously been shown to be restricted to this same pole of the cell (Cheek et al. (1989). J. Cell Biol. 109, 1219-1227), these results suggest that localized influx of Ca2+ in response to angiotensin II could underlie the polarized exocytotic response observed with this stimulus. These results directly demonstrate that different agonists can induce different patterns of divalent cation influx in the same cells and, furthermore, suggest how these different patterns can have a direct influence on cellular function.
The control of chondrocyte differentiation during endochondral bone growth in vivo: changes in TGF-β and the proto-oncogene c-myc
Authors: Loveridge N. , Farquharson C. , Hesketh J. E. , Jakowlew S. B. , Whitehead C. C. , Thorp B. H.
Published: April 2021

The expression of transforming growth factor-β and the c-myc proto-oncogene was studied in situ in the chondrocytes of the tibial growth plate of normal chicks and those with avian tibial dyschondroplasia in which the chondrocytes are developmentally arrested in the transitional phase between proliferation and differentiation. This results in an accumulation of unmineralised and avascular cartilage. Dyschondroplastic chicks showed reduced c-myc expression in the transitional chondrocytes but unaltered levels in the proliferating chondrocytes. Transforming growth factor-β expression was reduced in the transitional chondrocytes of dyschondroplastic chicks. In areas where the lesion was being repaired there was evidence of increased expression of both c-myc protein and transforming growth factor-β. Addition of 1,25-dihydroxyvitamin D to the diet, which is known to reduce the incidence of dyschondroplasia, resulted in an increase in c-myc production. These results suggest that both transforming growth factor-β and the proto-oncogene c-myc may be important elements of the cascade of events that lead to chondrocyte differentiation, hypertrophy and mineralisation.
Quantification and immunolocalisation of porcine articular and growth plate cartilage collagens
Authors: Wardale R. John , Duance Victor C.
Published: April 2021

The collagens of growth plate and articular cartilage from 5-6 month old commercial pigs were characterised. Growth plate cartilage was found to contain less total collagen than articular cartilage as a proportion of the dry weight. Collagen types I, II, VI, IX and XI are present in both growth plate and articular cartilage whereas type X is found exclusively in growth plate cartilage. Types III and V collagen could not be detected in either cartilage.

Type I collagen makes up at least 10% of the collagenous component of both cartilages. There are significant differences in the ratios of the quantifiable collagen types between growth plate and articular cartilage. Collagen types I, II, and XI were less readily extracted from growth plate than from articular cartilage following pepsin treatment, although growth plate cartilage contains less of the mature collagen cross-links, hydroxylysyl-pyridinoline and lysyl-pyridinoline. Both cartilages contain significant amounts of the divalent reducible collagen cross-links, hydroxylysyl-ketonorleucine and dehydro-hydroxylysinonorleucine.

Immunofluorescent localisation indicated that type I collagen is located predominantly at the surface of articular cartilage but is distributed throughout the matrix in growth plate. Types II and XI are located in the matrix of both cartilages whereas type IX is predominantly pericellular in the calcifying region of articular cartilage and the hypertrophic region of the growth plate. Collagen type VI is located primarily as a diffuse area at the articular surface.
The mouse brown (b) locus protein has dopachrome tautomerase activity and is located in lysosomes in transfected fibroblasts
Authors: Winder Alison J. , Wittbjer Anna , Rosengren Evald , Rorsman Hans
Published: April 2021

Many genes mapping to pigmentation loci are involved in the regulation of melanin synthesis in the mouse. The brown (b) locus controls black/brown coat coloration, and its product has significant homology to the key melanogenic enzyme tyrosinase. This has led to suggestions that the b-protein is itself a melanogenic enzyme. In order to investigate its function, we have established lines of mouse fibroblasts stably expressing the b-protein by co-transfection of a b-protein expression vector and a plasmid conferring resistance to the antibiotic G418. The b-protein synthesised by these cells has the expected molecular mass of 75 kDa and reacts with three different anti-b-protein antibodies. We were unable to confirm previous reports that the b-protein has tyrosinase or catalase activity, but detected stereospecific dopachrome tautomerase activity in b-proteinexpressing fibroblasts. This dopachrome tautomerase binds to Concanavalin A-Sepharose, and the major product of its action on L-dopachrome is 5,6-di-hydroxyindole-2-carboxylic acid. Since this activity is not present in untransfected cells we conclude that the b-protein has dopachrome tautomerase activity. Fibroblasts do not contain melanosomes, the specialised organelles in which the b-protein is located in melanocytes. Nevertheless, indirect immunofluorescence localisation of the b-protein in transfected fibroblasts produces a distinctive pattern of intense juxtanuclear staining combined with punctate cytoplasmic staining. Double-labelling shows co-localisation of the b-protein with the late endosomal/lysosomal markers β-glucuronidase and LAMP-1, both in transfected fibroblasts and in mouse melanoma cells. These findings are consistent with the hypothesis that melanosomes are closely related to lysosomes.
Synthesis and assembly of fibrillin by fibroblasts and smooth muscle cells
Authors: Kielty Cay M. , Shuttleworth C. Adrian
Published: April 2021

The expression and assembly of the microfibrillar glycoprotein fibrillin has been investigated in cultures of nuchal ligament fibroblasts, skin fibroblasts and vascular smooth muscle cells. The level of fibrillin expression varied with the cell type and growth conditions. Higher levels of synthesis were recorded in quiescent post-confluent cells than in actively dividing subconfluent cultures. Nuchal ligament fibroblasts consistently synthesized the highest levels of fibrillin. Growth of cells in the presence of ascorbate resulted in an increased proportion of newly synthesized fibrillin retained within cell layers. Fibrillin was immunoprecipitated from medium and cell layer extracts in the form of monomers and high-Mr disulphide-bonded aggregates. Rotary shadowing electron microscopy of cell layer extracts and collagen gels provided direct evidence for the assembly of extensive intact microfibrils by smooth muscle cells and fibroblast cultures. Gel filtration chromatography of medium and cell layer extracts, in combination with immunoprecipitation of column fractions, provided a means of analysing the size distribution and assembly of newly synthesized fibrillin. This cell culture approach provides an opportunity to evaluate normal and aberrant synthesis and assembly of fibrillin in a wide range of cell types.
Retinoic acid mediates post-transcriptional regulation of keratin 19 mrna levels
Authors: Crowe David L.
Published: April 2021

Stratified squamous epithelia have been shown to preferentially express a site-specific pattern of keratin intermediate filaments. Retinoic acid (RA) is known to modulate expression of the basal cell keratins K19 and K5. Expression of these genes is dependent on extracellular RA concentration. We have found that K19 mRNA levels increase over time in cultured keratinocytes exposed to elevated concentrations of RA. K5 mRNA levels decrease in response to RA in a similar fashion. The observed changes in K5 message are primarily the result of RA-induced alterations in gene transcription. However, the RA-mediated induction of K19 mRNA is not the result of increased transcription but is primarily due to enhanced mRNA stability. These results suggest that an RA-dependent post-transcriptional mechanism modulates K19 intermediate filament expression in stratified squamous epithelia.
The origin of parasitophorous vacuole membrane lipids in malaria-infected erythrocytes
Authors: Ward Gary E. , Miller Louis H. , Dvorak James A.
Published: April 2021

During invasion of an erythrocyte by a malaria merozoite, an indentation develops in the erythrocyte surface at the point of contact between the two cells. This indentation deepens as invasion progresses, until the merozoite is completely surrounded by a membrane known as the parasitophorous vacuole membrane (PVM). We incorporated fluorescent lipophilic probes and phospholipid analogs into the erythrocyte membrane, and followed the fate of these probes during PVM formation with low-light-level video fluorescence microscopy. The concentration of probe in the forming PVM was indistinguishable from the concentration of probe in the erythrocyte membrane, suggesting that the lipids of the PVM are continuous with and derived from the host cell membrane during invasion. In contrast, fluorescently labeled erythrocyte surface proteins were largely excluded from the forming PVM. These data are consistent with a model for PVM formation in which the merozoite induces a localized invagination in the erythrocyte lipid bilayer, concomitant with a localized restructuring of the host cell cytoskeleton.
Prp2o, the Saccharomyces cerevisiae homolog of the regulator of chromosome condensation, rcc1, interacts with double-stranded dna through a multi-component complex containing gtp-binding proteins
Authors: Lee Arianna , Tam Rose , Belhumeur Pierre , DiPaolo Tiziano , Clark Michael W.
Published: April 2021

Prp20, a homolog to the mammalian negative regulator of chromosome condensation, RCC1, is retained on double-stranded (ds) DNA-cellulose when extracts are prepared from asynchronously growing wild-type yeast strains. Conversely, neither Prp20 from ts mutant cell extracts nor wt yeast Prp20 produced in Escherichia coli, bind to dsDNA-cellulose. In vitro reconstitution assays using E. coli-expressed Prp20 and inactivated ts mutant extracts of prp20-1 reveal that the Prp20 protein requires the assistance of other proteins in the cell extract to promote its binding to dsDNA. Immunoprecipitations and sizing-column-chromatography indicate that the Prp20 protein binds to the dsDNA column through a multicomponent complex composed of six to seven proteins, which has a collective molecular mass greater than 150,000 Da. At least three of the members of this Prp20 complex will bind GTP in vitro. Moreover, the Prp20 complex is shown to specifically lose its ability to bind dsDNA during the DNA replication phase of the cell cycle. This loss of dsDNA binding during the S phase of the cell cycle does not affect the proper organization of the nucleoplasm and appears to be reversed before the cell enters mitosis.