bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of dna in the nucleoids of chloroplasts and mitochondria in Euglena gracilis by immunoelectron microscopy
Authors: Hayashi-Isimaru Yasuko , Ueda Katsumi , Nonaka Mitsuko
Published: April 2021

DNA in the nucleoids of chloroplasts and of mitochondria in Euglena gracilis was detected with anti-DNA antibodies by immunoelectron microscopy. After treatment with the antibodies, DNA in these organelles combined with gold particles that had been coated with anti-IgM antibodies such that it was possible to trace the outlines of the nucleoids. Nucleoids in chloroplasts appeared to be composed of twisted threads 50-70 nm in diameter. The twisted threads were entangled to form thicker nodes of 100-200 nm diameter. Most nucleoids in mitochondria were spherical or ovoid, 70-130 nm in diameter. Nucleoids both in chloroplasts and in mitochondria contained cores with which DNA threads were in tight contact. The structure of the nucleoids was very different from those previously observed by conventional electron microscopy.
Identification and preliminary characterization of a 65 kDa higher-plant microtubule-associated protein
Authors: Chang-Jie Jiang , Sonobe Seiji
Published: April 2021

Microtubules in plant cells, as in animal cells, are dynamic structures. However, our lack of knowledge about the constituents of microtubules in plant cells has prevented us from understanding the mechanisms that control microtubule dynamics. To characterize some of these constituents, a cytoplasmic extract was prepared from evacuolated protoplasts (miniprotoplasts) of tobacco BY-2 cells, and microtubules were assembled in the presence of taxol and disassembled by cold treatment in the presence of Ca2+ and a high concentration of NaCl. SDS-PAGE analysis of triplecycled microtubule protein revealed the presence of 120 kDa, 110 kDa and a group of 60-65 kDa polypeptides in addition to tubulin. Since these polypeptides had copolymerized with tubulin, through the three cycles of assembly and disassembly, and they bundle microtubules, we tentatively identified the three polypeptides as microtubule-associated proteins (MAPs).

To characterize these factors further, triple-cycled microtubule protein was fractionated by Mono-Q anion-exchange chromatography and the microtubulebundling activity of each fraction was examined. Fractions having microtubule-bundling activity contained only the 65 kDa MAP, an indication that the 65 kDa MAP is responsible for the bundling of microtubules. Purified 65 kDa MAP formed cross-bridge structures between adjacent microtubules in vitro.

Polyclonal antibodies were raised in mice against the 65 kDa MAP. Immunofluorescence microscopy revealed that the 65 kDa MAP colocalized with microtubules in BY-2 cells throughout the cell cycle. Western blotting analysis of extracts from several species of plants suggested that the 65 kDa MAP and/or related peptides are widely distributed in the plant kingdom.
Vcam-1 is a cs1 peptide-inhibitable adhesion molecule expressed by lymph node high endothelium
Authors: May Michael J. , Entwistle Gillian , Humphries Martin J. , Ager Ann
Published: April 2021

Previous studies have shown that unactivated lymphocytes bind to CS1 peptide and that the adhesion of these cells to high endothelium is inhibited by CS1 peptide. These results suggest that lymphocyte binding occurs via recognition of the CS1-containing splice variant of fibronectin expressed on the high endothelial surface. We have now extended these studies by determining the role of the CS1 receptor, α4β1 (VLA-4) and the alternative VLA-4 ligand, VCAM-1 in a rat model of lymphocyte-high endothelial cell interaction. Anti-VLA-4 antibody, HP2/1, blocked lymphocyte adhesion to resting and IFN-γ (interferon-γ) pretreated cultured high endothelial cells (HEC) in a dose-dependent manner with maximal inhibition of 60%. HP2/1 completely blocked the adhesion of rat lymphocytes to immobilized CS1 peptide and to a recombinant soluble (rs) form of human VCAM-1. Lymphocyte binding to rsVCAM-1 was also completely blocked by CS1 peptide. Anti-rat VCAM-1 monoclonal antibody 5F10 inhibited adhesion to untreated and IFN-γ-treated HEC equally and its effect at 50% inhibition was slightly less than that of HP2/1. These findings suggest that a CS1 peptideinhibitable ligand expressed by high endothelium is VCAM-1. The majority of cultured HEC expressed significant levels of VCAM-1 under basal conditions, as did HEV in peripheral lymph nodes. VCAM-1 expression by HEC was upregulated by cytokine pretreatment and the effects were ordered: IFN-γ > TNF-α > IL-1 β.

The results described here demonstrate that rat peripheral lymph node HEC express VCAM-1, its expression is upregulated by cytokines, in particular IFN-γ, and it supports the adhesion of unactivated lymphocytes. They also suggest that the VLA-4/VCAM-1 adhesion pathway may operate during the constitutive migration of lymphocytes into lymphoid organs. Although the mechanism of CS1 peptide inhibition was not determined, these results show that VCAM-1 is a CS1 peptide-inhibitable ligand and therefore CS1, on its own, cannot be used as a specific indicator of fibronectin activity.
Internucleosomal dna fragmentation and programmed cell death (apoptosis) in the interdigital tissue of the embryonic chick leg bud
Authors: Garcia-Martinez V. , Macias D. , Gañan Y. , Garcia-Lobo J. M. , Francia M. V. , Fernandez-Teran M. A. , Hurle J. M.
Published: April 2021

In this work we have attempted to characterize the programmed cell death process in the chick embryonic interdigital tissue. Interdigital cell death is a prominent phenomenon during limb development and has the role of sculpturing the digits. Morphological changes in the regressing interdigital tissue studied by light, transmission and scanning electron microscopy were correlated with the occurrence of internucleosomal DNA fragmentation, evaluated using agarose gels. Programming of the cell death process was also analyzed by testing the chondrogenic potential of the interdigital mesenchyme, in high density cultures. Our results reveal a progressive loss of the chondrogenic potential of the interdigital mesenchyme, detectable 36 hours before the onset of the degenerative process. Internucleosomal DNA fragmentation was only detected concomitant with the appearance of cells dying with the morphology of apoptosis, but unspecific DNA fragmentation was also present at the same time. This unspecific DNA fragmentation was explained by a precocious activation of the phagocytic removal of the dying cells, confirmed in the tissue sections. From our observations it is suggested that programming of cell death involves changes before endonuclease activation. Further, cell surface changes involved in the phagocytic uptake of the dying cells appear to be as precocious as endonuclease activation.
The nuclear pore complex: three-dimensional surface structure revealed by field emission, in-lens scanning electron microscopy, with underlying structure uncovered by proteolysis
Authors: Goldberg M. W. , Allen T. D.
Published: April 2021

The structure of the nuclear pore complex (NPC) has been previously studied by many different electron microscopic techniques. Recently, scanning electron microscopes have been developed that can visualise biologically relevant structural detail at the same level of resolution as transmission electron microscopes and have been used to study NPC structure. We have used such an instrument to visualise directly the structure of both cytoplasmic and nucleoplasmic surfaces of the NPC of manually isolated amphibian oocyte nuclear envelopes that have been spread, fixed, critical point dried and coated with a thin fine-grained film of chromium or tantalum. We present images that directly show features of the NPC that are visible at each surface, including coaxial rings, cytoplasmic particles, plug/spoke complexes and the nucleoplasmic basket or fishtrap. Some cytoplasmic particles are rod-shaped or possibly “T”-shaped, can be quite long structures extending into the cytoplasm and may be joined to the coaxial ring at a position between each subunit. Both coaxial rings, which are proud of the membranes, can be exposed by light proteolytic digestion, revealing eight equal subunits each of which may be bipartite. We have determined that the nucleoplasmic filaments that make up the baskets are attached to the outer periphery of the coaxial ring at a position between each of its subunits. These filaments extend into the nucleoplasm and insert at the distal end to the smaller basket ring. The space left between adjacent basket filaments would exclude particles bigger than about 25 nm, which is consistent with the exclusion limit previously found for NPC-transported molecules.
Nuclei that lack a lamina accumulate karyophilic proteins and assemble a nuclear matrix
Authors: Jenkins Hazel , Hölman Thomas , Lyon Carol , Lane Birgitte , Stick Reimer , Hutchison Christopher
Published: April 2021

Xenopus egg extracts, which support nuclear assembly and DNA replication in vitro, were physically depleted of lamin B3 using monoclonal antibodies linked to magnetic beads. Depleted extracts were still able to support nuclear envelope assembly around demembranated sperm heads but the resulting pronuclei lacked a lamina and were unable to initiate semiconservative DNA replication or to assemble replicases, confirming previous data. Immunoblotting analysis of isolated nuclei and nuclear matrix fractions indicated that lamin-depleted nuclei still accumulated nucleoporins and PCNA. Furthermore, the rate of PCNA uptake was identical in lamin-depleted and control nuclei. However, neither the nucleoporins nor the PCNA was associated with nuclear matrix fractions. The major protein components of sperm pronuclear matrix fractions were characterized by two-dimensional gel electrophoresis. Of these proteins only three out of 22 species, other than the lamins, were significantly reduced in lamin-depleted nuclei, indicating that these nuclei do assemble a nuclear matrix.
Selective killing induced by an inhibitor of n-linked glycosylation
Authors: Larsson Olle , Carlberg Magdalena , Zetterberg Anders
Published: April 2021

Treatment with a low dose (0.5 μg/ml) of tunicamycin (an inhibitor of N-linked glycosylation) blocked the cell cycle progression of both normal Balb/c 3T3 cells (A31) and their SV40-transformed derivatives (SVA31) specifically in early G1 (0-3 h after mitosis). Upon release after an 8-h treatment the A31 cells returned to the cell cycle via a 9-h recovery phase, indicating that they were arrested in G0. The A31 cells were fully viable after this treatment. In contrast, the postmitotic SVA31 cells, which were unable to arrest in G0, did not divide after the removal of tunicamycin. Instead, these cells died but this did not occur until 22-34 h after release from the treatment. SVA31 cells that had passed the postmitotic phase of G1 survived during the parental generation and divided normally. However, a large portion of these cells died during the next cycle, and in total during a 48-h period approximately 50% of the cells were killed as a consequence of an 8-h exposure to tunicamycin. In contrast, treatment with inhibitors of protein synthesis and HMG CoA reductase activity as well as inhibitors of modification of N-linked oligosaccharide chains did not result in cell death.
Internalization of hyaluronan by chondrocytes occurs via receptor-mediated endocytosis
Authors: Hua Qiang , Knudson Cheryl B. , Knudson Warren
Published: April 2021

Several studies have suggested that chondrocytes must have the capacity to internalize and degrade extracellular hyaluronan. In the present study we show direct evidence that hyaluronan is, in fact, endocytosed by chondrocytes and that the endocytosis is mediated via cell surface CD44/hyaluronan receptors. Cultures of bovine articular chondrocytes as well as rat chondrosarcoma chondrocytes were incubated with either fluoresceinor 3H-labeled hyaluronan. Intense binding and accumulation of labeled hyaluronan was visualized by fluorescence microscopy or bright-field/dark-field microscopy following autoradiography. Cell surface hyaluronan was removed with either trypsin or Streptomyces hyaluronidase in order to distinguish and quantify intracellular endocytosed hyaluronan. Labeled hyaluronan was visualized within small discrete intracellular vesicles distributed throughout the cytoplasm. Binding and endocytosis of fluoresceinor 3H-labeled hyaluronan was totally blocked by the addition of excess unlabeled hyaluronan or hyaluronan hexasaccharides, competitive inhibitors of hyaluronan/hyaluronan receptor interactions. Binding and endocytosis was also blocked by the addition of anti-CD44 monoclonal antibodies. Characterization of endocytosed 3H-labeled hyaluronan demonstrated that a significant portion of the hyaluronan was degraded by both the bovine articular and rat chondrosarcoma chondrocytes. Interestingly, a higher proportion of bound hyaluronan was internalized by the bovine chondrocytes. Therefore, hyaluronan receptormediated endocytosis and degradation of hyaluronan may provide a critical link to the maintenance and homeostasis of cartilage tissue.
Endothelial cells adhere to the rgd domain and the fibrinogen-like terminal knob of tenascin
Authors: Joshi Paritosh , Chung Chang-Y. , Aukhil Ikramuddin , Erickson Harold P.
Published: April 2021

We have found that endothelial cells adhere much more strongly than fibroblasts to domains of tenascin and fibronectin. Endothelial cells adhered weakly, without spreading, to bacterial expression proteins corresponding to the tenth fibronectin type III (FN-III) domain of fibronectin, which contains the RGD. A larger fibronectin protein, containing this domain and the three amino-terminal ‘synergy’ domains gave strong adhesion and spreading. Two widely separated domains of tenascin gave adhesion. The third FN-III domain, TNfn3, which contains an RGD sequence in human and chicken tenascin, gave very strong adhesion and spreading of endothelial cells when tested as an isolated domain. Larger segments containing TNfn3 and the adjacent TNfn2 gave weaker adhesion, probably because the RGD sequence is partially blocked. Adhesion to this domain required divalent cations, was exquisitely sensitive to soluble GRGDSP peptide, and was blocked by antisera to the integrin αvβ3. The second tenascin adhesion domain was the fibrinogen-like C-terminal knob, TNfbg. Cells adhered to but did not spread on this domain. This adhesion required divalent cations and was also sensitive to GRGDSP peptide, so it may be mediated by an integrin receptor.

We have explored a range of conditions for preparing the adhesion substratum, and our results may resolve the controversy over whether tenascin can act as a substratum adhesion molecule. When coated for short times (1-2 hours) on plastic, tenascin had no adhesion activity, in contrast to fibronectin and the expression proteins, which gave strong adhesion under these conditions. When coated for longer times (12-24 hours) on plastic, the tenascin substratum supported good adhesion, but not spreading, of endothelial cells. Tenascin coated on nitrocellulose gave substantially stronger adhesion than on plastic, but still required long coating times for maximal activity. Adhesion of endothelial cells to native TN was inhibited by GRDGSP peptide. The cell adhesion activity demonstrates the presence on endothelial cells of tenascin receptors, which may play a supportive role in angiogenesis, in the structure of blood vessels, or in binding tenascin to the cell surface to elicit or enhance a signalling function.
Commitment of the teratocarcinoma-derived mesodermal clone c1 towards terminal osteogenic differentiation
Authors: Poliard A. , Lamblin D. , Marie P. J. , Buc-Caron M. H. , Kellermann O.
Published: April 2021

The mesodermal clone C1 was derived from the multipotent embryonal carcinoma 1003 cell line transformed with the plasmid pK4 carrying SV40 oncogenes under the control of the adenovirus E1A promoter. We have shown that the C1 clone becomes committed to the osteogenic pathway when cultured in aggregates in the presence of mediators of the osteogenic differentiation. To further validate C1 as a model with which to study osteogenesis in vitro the kinetics of its differentiation was studied, focusing on the histology of the aggregates and on the expression of a set of genes corresponding to representative bone matrix proteins. The presence of ascorbic acid and - glycerophosphate specifically leads to mineralization in almost 100% of the aggregates. Transcription of the above genes, silent in exponentially growing cells, specifically occurred with the establishment of cell-cell contacts independently of the presence of ascorbic acid and inorganic phosphate. The latter, however, were absolutely required for matrix deposition and mineralization. In their presence, one observed an overall decline in type I collagen and alkaline phos-phatase transcripts while osteocalcin and osteopontin transcripts preferentially accumulated in cells lining the mineralizing foci. Concomitantly, type I collagen and osteocalcin became extracellularly deposited. The osteogenic differentiation of C1 occurred while cells were still proliferating. The C1 clone thus behaves as a mesodermal stem cell, becoming committed to the osteogenic pathway upon: firstly, establishment of cel-lular contacts; and secondly, addition of ascorbate and β-glycerophosphate. It therefore appears to be a promis-ing in vitro system for deciphering the molecular basis of osteoblast ontogeny. More generally it emphasizes the potential of the pK4-immortalized cell lines for the study of lineage specification.
Cytoarchitecture of size-excluding compartments in living cells
Authors: Provance D. William , McDowall Alasdair , Marko Michael , Luby-Phelps Katherine
Published: April 2021

By fluorescence ratio imaging of large and small inert tracer particles in living cells, we have previously shown that particles 24 nm in radius are excluded from other-wise uncharacterized compartments in the distal and perinuclear cytoplasm (Luby-Phelps, K. and Taylor, D.L., 1988. Cell Motil. Cytoskel. 10, 28-37). In this study we examined the cytoarchitecture of these compartments. Whole-mount TEM showed that distal size-excluding compartments were devoid of membrane-bounded organelles and were filled with a dense cytomatrix consisting of numerous, long bundles of thin filaments interconnected by a more random meshwork of short thin filaments. The mean diameter of void spaces in the cytomatrix of distal excluding compartments was 31 nm, compared to 53 nm in adjacent nonexcluding domains. The height of the distal excluding compartments was generally ≤ 50% of the height in the adjacent non-excluding compartment. An electrondense structure having the same projected outline as the perinuclear size-excluding compartment was visible by whole-mount TEM, but the cells were too thick and osmiophilic in this region to resolve any detail. Immunofluorescence localization of cytoskeletal proteins in distal excluding compartments indicated the presence of filament bundles containing F-actin, nonmuscle filamin (ABP280) and α-actinin. F-actin and ABP280, but not α-actinin, were found also in between these filament bundles. Microtubules and vimentin generally were rare or absent from distal excluding domains. Staining of living cells with DMB-ceramide revealed that the perinuclear size-excluding compartment consisted of a compact, juxtanuclear domain coinciding with the transGolgi, surrounded by a more diffuse domain coinciding with a perinuclear concentration of endoplasmic reticulum. Intense immunofluorescence staining for vimentin was also observed in the perinuclear size-excluding compartment. We propose that the most likely mechanism for exclusion from distal compartments is molecular sieving by a meshwork of actin filament bundles interconnected by an F-actin/ABP280 gel network, while exclusion from the perinuclear compartment may be due to close apposition of cisternae in the trans-Golgi and a network or basket of vimentin filaments in the centrosomal region of the cell.
Recycling of a secretory granule membrane protein after stimulated secretion
Authors: Hurtley Stella M.
Published: April 2021

Recycling of a secretory granule membrane protein, dopamine-β-hydroxylase, was examined in primary cultures of bovine adrenal chromaffin cells. Cells were stimulated to secrete in the presence of antibodies directed against the luminal domain of dopamine-hydroxylase. The location of the antibodies after various times of reincubation and after a second secretory stimulus was assessed using immunofluorescence microscopy. Stimulation led to the exposure of dopamine-β-hydroxylase at the plasma membrane, which could be detected by a polyclonal antibody in living and fixed cells. The plasma membrane dopamine-β-hydroxylase, either alone or complexed with antibody, was rapidly internalized after removal of the secretagogue. Internalized protein-antibody complex remained stable for at least 24 hours of reculture. Twenty four hours after stimulation the cells with internalized anti-body could respond to further stimulation and some of the antibody was re-exposed at the plasma membrane. These findings were confirmed using FACS analysis. This suggests that the antibody-protein complex had returned to secretory granules that could respond to further secretagogue stimulation.
Keratocyte networks visualised in the living cornea using vital dyes
Authors: Poole C. Anthony , Brookes Nigel H. , Clover Gillian M.
Published: April 2021

Fluorescent viability probes have been used to visualise and investigate the viability, morphology and organisation of the keratocyte within the stroma of the intact living cornea. The live cell probe, calcien-AM, in combination with a dead cell probe, ethidium homodimer (Live/Dead Assay, Molecular Probes, U.S.A.) proved superior to earlier generation vital dyes such as fluorescein diacetate or 5,6-carboxyfluorescein diacetate, initially used in combination with ethidium bromide. The ubiquitous distribution of esterase enzymes that cleave calcien-AM within the keratocyte cytoplasm produced a high concentration of fluorescently active calcein throughout the cell, including fine cell processes. Epi-illuminated fluorescence microscopy on transparent corneal dissections subsequently revealed details of keratocyte microanatomy and three-dimensional network organisation in situ. Three morphologically discrete subpopulations of keratocytes were identified: two formed relatively small bands of cells, immediately subjacent to either Bowman’s or Descemet’s membranes, the third subpopulation constituting the majority of keratocytes typically located within the corneal stroma. The results indicate that calcein-AM is able to penetrate intact living cornea revealing cell viability, and it also has the capacity to ‘trace’ cellular elements and reveal fine structure within a dense connective tissue matrix.
A centrosome-associated antibody from Drosophila melanogaster reveals a new microtubule-dependent structure in the equatorial zone of Parascaris univalens embryos
Authors: Jiménez M. , Goday C.
Published: April 2021

The distribution of antigens to two antibodies (Bx63 and Rb188) that associate to Drosophila. melanogaster cen-trosomes has been investigated in the nematode Paras-caris. By western blot analysis both antibodies identify in Parascaris polypeptides of the same molecular mass as in Drosophila (Rb188 a 185 kDa antigen and Bx63 185 kDa and 66 kDa antigens). By immunocytochemistry we show that the centrosomes of Parascaris con-tain the 185 kDa antigen recognized by polyclonal Rb188 and monoclonal Bx63 antibodies. In addition, Bx63 reveals cytoplasmic midzone structures, not found in Drosophila, that display a cell cycle-dependent organization in embryos. These structures, which most prob-ably contain the 66 kDa antigen revealed by Bx63, appear at the onset of anaphase as fibrillar-like structures that during anaphase form a ring-like structure encircling the equatorial plane of the blastomere. Before furrowing, the antigen participates in the formation of the midbody and associates with convergent polar microtubules. After blastomere division, Bx63 signal persists as a single body between the daughter cells. The analysis of chilled and nocodazole-treated embryos suggests that the localization of the midzone Bx63 anti-gen is dependent on non-kinetochore microtubules. Inhibition of furrowing by cytochalasin B shows that the antigen persists after the disassembly of microfilaments. Cytological observations of contractile ring and Bx63 ring assembly indicate that both structures do not simul-taneously colocalize at the equatorial zone. The data suggest a spindle-dependent distribution of the Bx63 antigen during cytokinesis. We discuss the participation of this antigen in the organization of the midbody before furrowing, and consider the possible relevance of the midbody with respect to cell to cell communication during early development in nematodes.
Titin aggregates associated with intermediate filaments align along stress fiber-like structures during human skeletal muscle cell differentiation
Authors: Ven Peter F. M. van der , Schaart Gert , Croes Huib J. E. , Jap Paul H. K. , Ginsel Leo A. , Ramaekers Frans C. S.
Published: April 2021

Differentiating human skeletal muscle cell cultures were used to study the association of titin with other sarcomeric and cytoskeletal proteins during myofibrillo-genesis. Several developmental stages of these cultures were double stained with antibodies to titin in combination with antibodies to α-actin, α-actinin, myosin heavy chain (MHC), nebulin, desmin, and β-tubulin. The first indications of titin expression were found in postmitotic mononuclear myoblasts where it is located in a random, punctate fashion. At the light microscope level no evidence was found for an association of these titin spots with any of the other proteins studied, with the exception of MHC, which colocalized with titin in a small minority of the titin expressing cells. Subsequently the titin spots were found to be linked to longitudinally oriented stress fiber-like structures (SFLS), containing-actinin and sarcomeric α-actin, but not MHC, nebulin or desmin. Upon further maturation titin antibodies seemed to stain SFLS in a rather homogeneous fashion together with MHC, α-actin and α-actinin. Thereafter a more periodic localization of titin, MHC, α-actin andα-actinin on SFLS became obvious. From these structures myofibrils developed as a result of further differentiation. Initially only short stretches with a striated titin, MHC, F-actin and α-actinin organization were found. Nebulin was integrated in these young myofibrils at a later developmental stage. Desmin was not found to be incorporated in these myofibrils until complete alignment of the sarcomeres in mature myotubes had occurred. At the ultrastructural level titin antibodies recognized aggregates that were associated with intermediate filaments (IF) in postmitotic mononuclear myoblasts. At a later maturational stage, prior to the development of cross-striated myofibrils, the IF-associated titin aggregates were found in close association with subsarcolemmally located SFLS. We conclude that IF and SFLS play an important role in the very early stages of in vitro human myofibrillogenesis. On the basis of our results we assume that titin aggregates are targeted to SFLS through IF. The association of titin with SFLS might be crucial for the unwinding of titin necessary for the assembly of sarcomeres and the first association of titin with other sarcomeric proteins.
Rab6 is associated with a compartment that transports rhodopsin from the trans-Golgi to the site of rod outer segment disk formation in frog retinal photoreceptors
Authors: Deretic Dusanka , Papermaster David S.
Published: April 2021

The biogenesis of light sensitive membranes in retinal rod photoreceptors involves polarized sorting and tar-geting of newly synthesized rhodopsin to a specialized domain, the rod outer segment (ROS). We have isolated and characterized the population of post-Golgi mem-branes that mediate intracellular transport of rhodopsin. In the present study we have examined the association of small (20-25 kDa) GTP-binding (G) pro-teins with these membranes. We found that one of the small G proteins, rab6, behaves like an integral mem-brane protein of the post-Golgi vesicles, although ∼30% of rab6 is soluble. The distribution of the membrane-associated and the soluble forms is highly polarized. By confocal and EM immunocytochemistry it can be seen that most of rab6 is associated with the photoreceptor trans-Golgi cisternae, trans-Golgi network (TGN) and post-Golgi vesicles. The photoreceptor axon and synaptic terminal are unlabeled, but dendrites of deeper retinal layers are labeled. The distribution of rab6 across sucrose density gradient fractions parallels the distribution of sialyltransferase (a TGN marker) activity. About 9% of membrane-bound rab6 is associated, how-ever, with the rhodopsin-bearing sialyltransferase-free post-Golgi vesicles, which represent a very small fraction (<1%) of the total retinal membranes. Rab6 is absent from the mature ROS disk membranes but it is present at the sites of new ROS disk formation and in the ROS cytoplasm. This suggests that rab6 becomes soluble upon disk membrane formation. Therefore, rab6 may function not only as a component of the sorting machinery of photoreceptors that delivers rhodopsin to its appropriate subcellular domain but may also par-ticipate in some aspects of ROS disk morphogenesis.
Diffusible, retinal factors stimulate the barrier properties of junctional complexes in the retinal pigment epithelium
Authors: Rizzolo Lawrence J. , Li Zhi-Qiang
Published: April 2021

The retinal pigment epithelium lies at the interface between the neural retina and the choriocapillaris where it forms a blood-retinal barrier. Barrier function requires a polarized distribution of plasma membrane proteins and ‘tight’ tight junctions. During chicken embryogenesis, these features develop gradually. Although terminal junctional complexes are established by embryonic day 4, the distribution of the Na+/K+-ATPase is not polarized in all cells of the epithelium until embryonic day 11. Similarly, the tight junctions of early embryos are leaky, but become tight by hatching (embry-onic day 21). We used primary cell culture to examine the molecular basis of this gradual induction of polar-ized function. Pigment epithelium harvested from embryonic day 7, and cultured on filters, formed mono-layers coupled by junctional complexes. The distribution of the Na+/K+-ATPase was non-polarized and the tight junctions were leaky with a transepithelial electrical resistance of 20-30 cm2. To isolate diffusible factors that stimulate the transepithelial electrical resistance, neural retinas from embryonic day 7, 14 or 16 embryos were incubated at 37°C in base medium for 6 hours. The conditioned medium was added to the apical chamber of freshly cultured pigment epithelium. The distribution of the Na+/K+-ATPase became basolateral, and the electrical resistance gradually increased two to three times over 6 days. The increase in electrical resistance corresponded to a decrease in the rate of [3H]inulin diffusion across the monolayer. The effectiveness of the conditioned medium increased steadily with increasing age of the neural retina. Rather than increased production of an active factor, apparently different active factors were produced at different ages. The embryonic day 14 retinal-derived factors were heat-and alkali-labile and smaller than 10 kDa. Additionally, they were acid-stable and insensitive to proteolysis by cathepsin D. These studies provide evidence that diffusible factors from the neural retina regulate the gradual development of bar-rier function in the retinal pigment epithelium.
Transient effect of epidermal growth factor on the motility of an immortalized mammary epithelial cell line
Authors: Matthay Michael A. , Thiery Jean-Paul , Lafont Frank , Stampfer Martha F. , Boyer Brigitte
Published: April 2021

The effects of growth factors on epithelial cell motility and dispersion have been examined on an immortalized human mammary epithelial cell line, the 184A1 non-tumorigenic cell line. Among all the molecules tested, epidermal growth factor (EGF) and transforming growth factor-α (TGF-α)were demonstrated to stimulate an increase in mammary epithelial cell motility and wound closure that was associated with a morphological transformation of the cells and was accompanied by modifications in cell-cell and cell-substrate adhesion systems. The EGF-induced increase in cell motility and monolayer wound closure occurred over a 24 hour period and was not dependent on an increase in cell number. The effect of EGF was abolished by inhibiting2 integrins with specific antibodies, indicating that part of the mechanism for the increase in cell motility and accelerated wound closure depends on α2 integrin functional expression. After 72 hours of exposure to EGF, the EGF-induced alterations in cell morphology, motility and cell adhesion systems underwent a spontaneous reversion that was correlated with a 10-fold reduction in the number of EGF receptors. The ability to regulate the scattering response induced by growth factors might be an important feature distinguishing normal epithelial cells from their tumoral counterparts.
Novel developmentally regulated exon identified in the rat fast skeletal muscle troponin T gene
Authors: Morgan Matthew J. , Earnshaw John C. , Dhoot Gurtej K.
Published: April 2021

In theory, the rat fast skeletal muscle troponin T gene can generate 64 different isoforms. Here we report the identification of a novel alternative exon (exon y) that increases the potential isoform variation to 128. The inclusion of exon y in fast skeletal muscle troponin T mRNA occurs in perinatal, but not adult, skeletal muscle. Exon y is located between exons 8 and 9. This is the first time that a developmentally regulated exon located amongst a set of alternatively spliced exons has been described. Exon y is included in two mRNA iso-forms. The proteins that these mRNAs would encode have molecular masses greater than that of the largest fast skeletal muscle troponin T isoform lacking exon y. These two proteins correlate well in both size and pat-tern of expression with the two fast skeletal muscle troponin T isoforms expressed in perinatal skeletal muscle. These results indicate that there is coordinated regulation of the splicing of exon y with other alternative exons.
Repeated UVB irradiations do not have the same potential to promote stimulation of melanogenesis in cultured normal human melanocytes
Authors: Aberdam Edith , Roméro Christine , Ortonne Jean-Paul
Published: April 2021

The major stimulus for human melanin production is ultraviolet (UV) radiation. Little is known about the mechanisms underlying this response and the eventual enzyme regulation resulting from this activation. We treated normal human melanocytes in culture with daily UVB radiations. Cumulative increases in UVB doses resulted in proportional increases in tyrosinase activity over the first few days whereas an intermittent pattern of tyrosinase activation was observed after the fifth day of irradiation. This intermittent pattern consisted of latency periods where no melanogenic response was elicited despite exposure to UVB. Tyrosinase activity in cellular extracts increased shortly after an effective irra-diation, peaked at 3 hours and thereafter decreased to below basal levels. Increased tyrosinase activity was associated with increased amounts of both the newly synthesized and mature forms of the enzyme. Decreased tyrosinase activity following an activation period was correlated with decreases in both the expression of tyrosinase mRNA and the amount of the newly synthe-sized form of the enzyme present in the melanocytes 24 hours after six irradiations. This particular pattern of stimulation of tyrosinase was not observed in S-91 murine melanoma cells after repeated UVB irradiations. Taken together these results may suggest a photo-pro-tective mechanism developed by irradiated normal human melanocytes.
The Cdc25 homologue Twine is required for only some aspects of the entry into meiosis in Drosophila
Authors: White-Cooper Helen , Alphey Luke , Glover David M.
Published: April 2021

The twineHB5 mutation prevents spindle formation during the entry into meiosis in Drosophila males, but chromosome condensation and nuclear envelope break-down both still occur. This suggests the possibility that this particular cdc25 homologue is required to activate a p34cdc2 kinase required for only some of the events of this G2-M transition. In contrast, meiotic spindles do form in twineHB5 females, although these appear abnor-mal. However, the female meiotic divisions do not arrest at metaphase I as in wild type, but continue repeatedly, leading to gross non-disjunction. Small chromatin masses, corresponding in size to the fourth chromo-somes, often segregate properly to the spindle poles. These can persist into the embryos derived from twineHB5 females, where they appear to participate in mitotic divisions on thin spindles. In addition, these embryos contain a small number of large chromatin masses that are not associated with spindles.
Rous sarcoma virus-transformed cells develop peculiar adhesive structures along the cell periphery
Authors: Nakamura Norimasa , Tanaka Junya , Sobue Kenji
Published: April 2021

Alteration of the cell/substratum adhesive structures of rat fibroblasts (3Y1 cells) upon transformation by Rous sarcoma virus (RSV) was investigated by immunofluo-rescence microscopy. In serum-containing culture medium, 3Y1 cells developed focal adhesions as their main adhesive structures, while BY1 cells expressed peculiar close contacts along the cell periphery with the vitronectin receptor integrin, in addition to podosomes. These peripheral close contacts are referred to as the peripheral adhesions. The peripheral adhesions were observed as a darker region than podosomes by inter-ference reflection microscopy. They were more easily destroyed by incubating the cells with RGD-containing peptide than were the focal adhesions. In contrast to focal adhesions and podosomes, actin bundles were not detected within the peripheral adhesions, where pp60v-src and tyrosine-phosphorylated proteins accumu-lated. Expression of the integrin was determined by the substratum composition when BY1 cells were cultured in serum-free culture medium. Under such conditions, BY1 cells expressed the peripheral adhesions within 3 hours on adhesion molecule-coated glass. On the other hand, in serum-containing medium, they first developed focal adhesions transiently at their early stage of adhe-sion, and then the peripheral adhesions were predomi-nantly expressed within 12 hours. Podosomes were formed in a time course similar to that of the periph-eral adhesions. These findings suggest that the periph-eral adhesion is a class of stable adhesive structure dis-tinct from the focal adhesion or podosome of BY1 cells. Similar close contact-type peripheral adhesions with the integrin were also observed in a variety of cultured cells such as normal fibroblasts at their logarithmic growth phase, phorbol ester-treated fibroblasts, and several malignant tumor cells, with poorly organized focal adhe-sions and stress fibers. These findings further suggest that the peripheral adhesions may be widely involved in the adhesion of cells that inadequately develop stress fibers and focal adhesions.
(Pro)collagenase (matrix metalloproteinase-1) is present in rodent osteoclasts and in the underlying bone-resorbing compartment
Authors: Delaissé J. M. , Eeckhout Y. , Neff L. , François-Gillet Ch. , Henriet P. , Su Y. , Vaes G. , Baron R.
Published: April 2021

Osteoclasts resorb the extracellular matrix of bone by secreting enzymes and acid into a sealed-off compart-ment that they form upon attachment to the bone sur-face. Although the lysosomal cysteine proteinases can degrade collagen after the demineralization of bone at low pH, several lines of evidence suggest that collage-nase (matrix metalloproteinase-1, EC 3.4.24.7) may also be involved in this process. The question of whether col-lagenase is present in the osteoclast and/or in the bone-resorbing compartment has however not been resolved. We have prepared an antimouse collagenase antiserum and affinity-purified an IgG fraction that specifically immunoblots and immunoprecipitates (pro)collagenase. Using these antibodies, we demonstrate by immunolo-calization the presence of (pro)collagenase both in the osteoclasts and in the extracellular subosteoclastic bone-resorbing compartment. These specific localizations were observed not only in mice but also in rat and rabbit osteoclasts and using not only the antibody we have pre-pared but also antibodies raised in other laboratories against rat (Jeffrey et al., J. Cell. Physiol. 143, 396-403, 1990) and rabbit (Brinckerhoff et al., J. Biol. Chem. 265, 22262-22269, 1990) collagenase. Intracellular collage-nase was observed in the osteoclasts whether the cells were plated on bone or cultured on glass coverslips. It is proposed that osteoclastic collagenase is secreted in the resorbing compartment where it may cooperate with the lysosomal cysteine proteinases in the degradation of the collagen component of the matrix during the resorp-tion of bone.
Functional down-regulation of α5β1 integrin in keratinocytes is reversible but commitment to terminal differentiation is not
Authors: Hotchin Neil A. , Kovach Nicholas L. , Watt Fiona M.
Published: April 2021

Extracellular matrix receptors of the integrin family have a dual role in the epidermis, regulating both adhesion and differentiation. Loss of contact with the extra-cellular matrix causes keratinocytes to become commit-ted to terminal differentiation, and results in a decrease in the ability of the α5β1 integrin to bind fibronectin. We have investigated whether the decrease in ligand-binding ability is reversible and, if so, whether commitment to terminal differentiation can also be reversed. Keratinocytes that had been placed in suspension for 5 hours to induce commitment were compared with the starting population (0 hour cells) in the presence or absence of 8A2, an activating anti-β 1 antibody. 8A2 IgG or FAb fragments increased the amount of α5β1 in cell extracts that bound to fibronectin-Sepharose and in the presence of 8A2 the amount of bound α5β1 in 0 hour and 5 hour extracts was equal. 8A2 also restored α5β1function in adhesion assays of intact 5 hour cells. Ca2+, Mg2+ and Mn2+ alone, at concentrations of up to 1 mM, did not increase the adhesiveness of 5 hour cells relative to 0 hour cells; however, the effect of 8A2 on keratinocytes was dependent on Ca2+. Although 8A2 restored 5 1 ligand-binding ability it did not prevent committed cells from withdrawing from the cell cycle and expressing involucrin, a differentiation marker. The results suggest that loss of matrix contact triggers two distinct events in keratinocytes: a reversible change in α5β1 conformation and generation of an irreversible signal through the receptor that culminates in terminal differentiation.
Kinesin-like molecules involved in spindle formation
Authors: Rodionov Vladimir I. , Gelfand Vladimir I. , Borisy Gary G.
Published: April 2021

To study the possible involvement of kinesin-like mole-cules in mitosis a polyclonal antibody against the head domain of Drosophila kinesin heavy chain (HD anti-body) was microinjected into PtK1 cells at the prophase-prometaphase transition. Progress of the cell through mitosis was recorded for subsequent detailed analysis. Cells injected with pre-immune IgG progressed through mitosis at rates similar to those for noninjected cells. After HD antibody injections, chromosomes failed to congress to an equatorial plane and cells failed to form a bipolar spindle. Rather, the spindle poles came together, resulting in a monopolar-like configuration with chromosomes arranged about the poles in a rosette. Sometimes the monopolar array moved to the margin of the cell in a way similar to anaphase B movement in normal cells. Antibody-injected cells progressed into the next cell cycle as evidenced by chromosome decondensation and nuclear envelope reformation. Anti-tubulin immunofluorescence confirmed the presence of a radial monopolar array of microtubules in injected cells. HD antibody stained in a punctate pattern in interphase and the spindle region in mitotic PtK1 cells. The antibody also reacted with spindle fibers of isolated mitotic CHO spindles and with kinetochores of isolated CHO chro-mosomes. Immunoblotting indicated that the major component recognized by the antibody is the 120 kDa kinesin heavy chain. At higher protein loads the anti-body recognized also a 34 kDa polypeptide in PtK1 cell extracts, a 135 kDa polypeptide in a preparation of CHO spindles and a 300 kDa polypeptide in a preparation of CHO mitotic chromosomes. We conclude that a kinesin-like molecule is important for the formation and/or maintenance of the structure of mitotic spindle.
Molecular cloning and subcellular localization of three GTP-binding proteins of the rab subfamily
Authors: Olkkonen Vesa M. , Dupree Paul , Killisch Iris , Lütcke Anne , Zerial Marino , Simons Kai
Published: April 2021

Small GTPases of the rab subfamily are involved in reg-ulation of intracellular membrane transport events. We recently used a PCR approach to isolate short cDNA fragments of a number of novel rab sequences. These PCR fragments have now been used with cDNA library screening and PCR-based techniques to clone the cDNAs encoding three of these proteins, rab12, rab22, and rab24. By northern blot analysis, the messages were found to be present in a wide variety of mouse tissues. However, quantitative differences in the mRNA levels between the tissues were detected. We determined the subcellular localization of the GTPases by expressing the c-myc epitope-tagged proteins with the Semliki Forest virus and the vaccinia T7 vector systems. Transiently expressed rab12 was localized to the Golgi complex. This localization was confirmed using a polyclonal anti-peptide antibody detecting the endogenous protein in BHK cells. rab22 expressed from the cDNA was local-ized to endosomal compartments and to the plasma membrane. After longer periods of expression, the protein was found on abnormally large perinuclear endo-somal structures, suggesting that it is a potent regulator of events in the endocytic pathway. Finally, rab24 was found in the endoplasmic reticulum/cis-Golgi region and on late endosomal structures. The localization of rab24 may indicate its involvement in autophagy-related processes.
Immunological properties and cDNA sequence analysis of an intermediate-filament-like protein from squid neuronal tissue
Authors: Adjaye James , Marsh Philip J. , Eagles Peter A. M.
Published: April 2021

A cDNA library has been constructed in the expression vector gt11 from mRNA isolated from squid (Loligo forbesi) optic lobes. The library was screened with anti-bodies generated against purified squid neurofilaments. A positive clone was isolated, which harboured a gt11 recombinant having an insert size of 3.5 kb. Hybridiz-ation analysis by Southern and northern blotting showed that the corresponding protein is encoded by a single gene that gives rise to a transcript of 2.6 kb. Translation of the full nucleotide sequence of the gene revealed an open reading frame covering 557 amino acids. This squid-neurofilament-like protein, SNLK, bears the characteristic N-terminal head, rod and C-ter-minal tail domains present in all intermediate filament (IF) proteins. The rod has the classical heptad repeats indicating coiled-coil-forming ability, and the predicted lengths of the coils are similar to coils 1a, 1b and 2 of intermediate filaments. At the C-terminal end of the rod there is a strongly conserved IF epitope, and a fusion protein containing SNLK is recognised by the pan-specific intermediate filament antibody, IFA. A poly-clonal antibody raised against SNLK has been used to show that the protein is present only in neuronal tissues and that it is immunologically related to neurofilaments from Myxicola but not from mammals.