bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of integrin receptors and their role in adhesion, spreading and migration of normal human melanocytes
Authors: Zambruno Giovanna , Marchisio Pier Carlo , Melchiori Antonella , Bondanza Sergio , Cancedda Ranieri , Luca Michele De
Published: April 2021

Integrin receptors of human melanocytes in vivo and of melanocytes isolated and cultured from in vitro recon-stituted normal human epidermis were investigated. Melanocytes were studied by high-resolution immuno-cytochemistry of in situ epidermis and were found to expose only the integrin subunits α3, α6, αv and β1 on their plasma membrane surface. Instead, cultured normal melanocytes expressed α3β1, α5β1, α6β1 and αvβ3, which were immunoprecipitated from both meta-bolically and surface-labeled cells. Betα1 integrins were diffused on the adhesion surface, while αvβ3 was clus-tered in focal contacts both in control cells and upon dendrite induction with phorbol 12-myristate 13-acetate (PMA). The functional roles of integrins were studied in vitro by cell adhesion, spreading and migration assays. The sum of the data indicated that, in normal human melanocytes: (i) adhesion to defined substrata is mainly mediated by specific β1 integrins; (ii) spreading is mainly modulated by αvβ3; (iii) the β1 and β3 het-erodimers cooperate in regulating migration. The in vitro expression of two integrins (αvβ3 and α5β1) that are not exposed in situ, and their role in the spreading and migratory properties of melanocytes, strongly suggest that they are involved in regenerating a nor-mally pigmented epidermis during wound healing by controlling melanocyte spreading and migration over a provisional matrix. Tumor promoters, such as PMA, selectively increased the expression of α3β1. We suggest that this integrin might be involved in melanocyte migration on the newly formed basement membrane during wound healing as well as in intercellular recognition of adjacent keratinocytes.
Cyclic AMP- and Ins(1,4,5)P3-induced ca2+ fluxes in permeabilised cells of Dictyostelium discoideum: cGMP regulates ca2+ entry across the plasma membrane
Authors: Flaadt Heidrun , Jaworski Elke , Schlatterer Christina , Malchow Dieter
Published: April 2021

Transduction of chemotactic signals in Dictyostelium dis -coideum apparently involves a precise regulation of the cytosolic ca2+ concentration. Cyclic AMP stimulation causes ca2+ influx followed by ca2+ extrusion, the mag-nitude of extrusion depending on the state of differen-tiation. Here, we show that the cAMP receptor controls ca2+ influx both at the level of entry across the plasma membrane and at the level of transport into ca2+-sequestering organelles. The use of permeabilised cells allowed us to discriminate between both fluxes. Perme-abilised cells still showed the cAMP-induced ca2+ uptake. The flux across the plasma membrane was more sensitive to Bα2+ and Mn2+, respectively, than ca2+ sequestration. We have shown previously, using stmF mutants, that cGMP regulates ca2+ influx. We confirmed this result with the membrane-permeant cGMP-analogue, Sp-8-Br-cGMPS, which enhanced the cAMP-induced ca2+ influx in intact cells but not the uptake in permeabilised cells, indicating that cGMP regulates ca2+ influx across the plasma membrane. Occasionally, a fast transient ca2+ efflux, preceding the influx, occurred in intact cells. A small cAMP-induced ca2+ release was also found in permeabilised cells. A simi-larly sized ca2+ release was elicited by Ins(1,4,5)P3 and could be substituted for by GTP or GTPyS. This result suggests that rapid ca2+ release can be mediated by Ins(1,4,5)P3.
Non-enzymatic access to the plasma membrane of Medicago root hairs by laser microsurgery
Authors: Kurkdjian Armen , Leitz Guenther , Manigault Pierre , Harim Abdellah , Greulich Karl Otto
Published: April 2021

Using UV laser microsurgery, the cell walls of root hairs from Medicago sativa (alfalfa) were perforated under plasmolysing conditions, giving direct access to the plasma membrane without enzyme treatment. The opening in the cell wall of a few m in diameter results in immediate movement of the protoplasm and partial or complete extrusion of the cell contents. The move-ment of the protoplasm is retarded by increases in cal-cium concentration. The calcium-dependency of the movement of the protoplasm allows us to obtain pref-erentially the extrusion of protoplasm, or to gain access to a small area of plasma membrane in situ. The com-plete protoplasm can be expelled, to form a protoplast. Fluorescein diacetate staining indicated esterase activity and membrane integrity of the protoplasts. Microscopic examination revealed organelle movement and the pres-ence of a nucleus. The plasma membrane was free from cell wall fragments, as shown by Tinopal staining. Con-ditions for obtaining plasmolysis without disturbing the physiology of the root hairs too much were achieved by slow, stepwise and reversible plasmolysis. Cytoplasmic streaming in root hairs was maintained during plas-molysis and laser microperforation. This laser technique should be suitable for the performance of electrophysi-ological studies using the patch-clamp technique on plasma membrane from non-enzyme-treated cells.
Integrin receptor-mediated mobilisation of intranuclear calcium in rat osteoclasts
Authors: Shankar Geetha , Davison Ian , Helfrich Miep H. , Mason William T. , Horton Michael A.
Published: April 2021

Cell-matrix interactions have been shown to play an important role in regulating cell function and behav-iour. In bone, where calcified matrix formation and resorption events are required to be in dynamic equi-librium, regulation of adhesive interactions between bone cells and their matrix is critical. The present study focuses on the osteoclast, the bone resorbing cell, as well as integrins, which are cell surface adhesion receptors that mediate osteoclast attachment to bone matrix. In osteoclasts, the most abundant integrin receptor is the vitronectin receptor (VNR, αv3). The objective of the study was to investigate changes in intracellular cal-cium, a regulator of osteoclast function, following addition of peptides that bind integrins, in particular the αv3 form of the vitronectin receptor (VNR), which is highly expressed in osteoclasts.

The study demonstrated a unique spatial localisation of the calcium signal in response to cell membrane receptor occupancy by integrin ligands in rat osteo-clasts. Addition of peptides with the Arg-Gly-Asp (RGD) sequence such as BSP-IIA, GRGDSP and GRGDS to rat osteoclasts evoked an immediate increase in free calcium ion concentration [Ca2+]i, localised to the nuclei and to the thin cytoplasmic skirt. These responses were inhibited by F11, a monoclonal antibody to the rat integrin 3 chain, as well as echistatin, a snake venom shown to colocalise with the αv chain in osteoclasts, suggesting that the calcium signal is mediated by the αv3 form of VNR. In contrast, a uniform increase in [Ca2+]i through-out the osteoclast was observed with the calcium-regu-lating hormone calcitonin, as a consequence of calcium entry from extracellular sources. Addition of 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ), a non-nuclear calcium ATPase inhibitor, resulted in a calcium signal with spatial characteristics distinct from that evoked by calcitonin or RGD-containing peptides. Thus, an increase in intranuclear calcium to the exclusion of a concomitant rise in cytoplasmic [Ca2+]i was restricted to responses induced by RGD-containing peptides.

The present findings demonstrate that, in rat osteo-clasts, there is a signalling pathway linked to the inte-grin VNR, which can rapidly modulate the concentration of nuclear [Ca2+]i, which in turn may regulate nuclear calcium-dependent processes.
Demonstration of cell cycle kinetics in thyroid primary culture by immunostaining of proliferating cell nuclear antigen: differences in cyclic AMP-dependent and -independent mitogenic stimulations
Authors: Baptist Mireille , Dumont Jacques E. , Roger Pierre P.
Published: April 2021

In this study, experimental conditions are described that allowed us to follow the fate of the DNA polymerase 3-associated proliferating cell nuclear antigen (PCNA), by immunolabeling during the overall cell cycle. Differences in subcellular localization or the presence of PCNA allowed us to identify each phase of the cell cycle. Using these cell cycle markers in dog thyroid epithelial cells in primary culture, we found unexpected differences in cell cycle kinetics, in response to stimulations through cAMP-dependent and cAMP-independent pathways. These provide a new dimension to the view that the two pathways are largely separate, but co-operate on DNA synthesis initiation. More precisely, thyrotropin (TSH), acting via cAMP, exerts a potent triggering effect on DNA synthesis, associated with a precocious induction of PCNA appearance. This constitutes the major influence of TSH (cAMP) in determining cell cycle progression, which is only partly moderated by TSH-dependent lengthening of S- and G2-phases.
immunolocalisation of collagenase in rabbit periosteal tissue explants and extraction of the enzyme: the effect of the cytokines IL-1α and EGF
Authors: van der Zee Erwin , Everts Vincent , Hoeben Kees , Beertsen Wouter
Published: April 2021

The effect of interleukin-1α (IL-1α) and murine epidermal growth factor (EGF) on incorporation of endogenously produced collagenase in the extracellular matrix of soft connective tissue was studied in an in vitro model system using periosteal explants obtained from rabbit calvariae. Immunohistochemical analysis indicated the highest level of collagenase in explants cultured for 72 hours with IL-1α in combination with EGF. Most enzyme appeared to be associated with the extracellular matrix, but labeling was also found in numerous fibroblast-like cells. Explants cultured in the presence of IL-1α alone contained less enzyme and in periostea treated without cytokines, or with EGF alone, only a faint label, if any, was seen. Freshly isolated, non-cultured periostea contained no detectable enzyme.

Extraction of collagenase from periostea revealed that: (1) non-cultured periosteum did not contain detectable levels of enzyme. (2) The amount of total activatable enzyme synergistically increased (10-fold) under the influence of IL-1α and EGF, whereas IL-1α alone showed a 4-fold enhancement compared to control or EGF-incubated explants. (3) The latent fraction of the enzyme was synergistically increased (up to 100-fold or more) in periostea cultured in the presence of IL-1α+EGF (21.17 mU/explant versus 0.05 mU/explant in controls). (4) Active collagenase, on the other hand, appeared to be present in a relatively high concentration in explants cultured without cytokines (2.45 mU/explant versus 0.36 mU/explant in IL-1α+EGF-treated explants). (5) No enzyme could be extracted from devitalized explants that were incubated in media containing high levels of latent collagenase. It was concluded that collagenase had been incorporated somehow in the extracellular matrix during culturing. In an attempt to analyze breakdown of collagen the amount of hydroxyproline in culture media was assessed. The level of this imino acid released by periostea treated without cytokines or with EGF alone was significantly higher than by those incubated with IL-1α and EGF.

In conclusion, incorporation of collagenase in periosteal connective tissue seems to occur during culturing; the level of the incorporated latent enzyme being strongly enhanced by a combination of the cytokines IL-1α and EGF. However, as the amount of collagen breakdown had decreased, these findings indicate that IL-1α in combination with EGF is able to induce deposition of a large reservoir of latent collagenase in the extracellular matrix, but does not stimulate adequate activation mechanisms to result in enhanced degradation.
A 28 kDa sarcolemmal antigen in kidney principal cell basolateral membranes: relationship to orthogonal arrays and MIP26
Authors: Verbavatz Jean-Marc , Van Hoek Alfred N. , Ma Tonghui , Sabolic Ivan , Valenti Giovanna , Ellisman Mark H. , Ausiello Dennis A. , Verkman A. S. , Brown Dennis
Published: April 2021

Two recently cloned water channels, CHIP28 and WCH-CD, are homologous to MIP26, an integral membrane channel-forming protein found in lens fiber plasma membranes. CHIP28 is found in basolateral and apical plasma membranes of kidney proximal tubules and thin descending limbs of Henle, whereas WCH-CD is apically located in collecting duct principal cells. So far, the putative water channel that may be responsible for the high constitutive permeability of principal cell basolateral membranes has not been identified. Interestingly, freeze-fracture electron microscopy has shown that characteristic orthogonal arrays of intramembrane particles (OAPs) are found on the basolateral plasma membranes of collecting duct principal cells, and that morphologically identical OAPs present in lens fiber cell plasma membranes contain the protein MIP26. Similar OAPs have also been detected on plasma membranes of other cell types including gastric parietal cells, astroglial cells and skeletal muscle fibers. By indirect immunofluorescence, western blotting and northern blotting, MIP26 was found only in lens fibers. In addition, functional studies on reconstituted and oocyte-expressed MIP26 excluded the possibility that MIP26 might be a basolateral water channel in the kidney. However, a polyclonal antibody raised against skeletal muscle sarcolemmal vesicles, which are enriched in OAPs, produced an intense staining of principal cell basolateral plasma membranes in kidney collecting duct and immunoprecipitated a 28 kDa protein from kidney papilla. The immunoprecipitated protein from papilla was not recognized by anti-CHIP28 or anti-MIP26 antibodies, indicating that principal cell basolateral membranes contain a novel member of the CHIP/MIP family. Because this antibody also stained brain astrocyte end feet, which are enriched in OAPs, it is possible that the 28 kDa protein is related to these structures. We conclude that OAPs probably contain related but distinct proteins that may have different membrane channel functions in different cell types.
Cytoskeleton-dependent transport of cytoplasmic particles in previtellogenic to mid-vitellogenic ovarian follicles of Drosophila: time-lapse analysis using video-enhanced contrast microscopy
Authors: Bohrmann Johannes , Biber Knut
Published: April 2021

In Drosophila oogenesis, several morphogenetic determinants and other developmental factors synthesized in the nurse cells have been shown to accumulate in the oocyte during pre- to mid-vitellogenic stages. However, the mechanisms of the involved intercellular transport processes that seem to be rather selective have not been revealed so far. We have investigated in vitro, by means of video-enhanced contrast time-lapse microscopy, the transport of cytoplasmic particles from the nurse cells through ring canals into the oocyte during oogenesis stages 6-10A. At stage 7, we first observed single particles moving into the previtellogenic oocyte. The particle transfer was strictly unidirectional and seemed to be selective, since only some individual particles moved whereas other particles lying in the vicinity of the ring canals were not transported. The observed transport processes were inhibitable with 2,4-dinitrophenol, cytochalasin B or N-ethylmaleimide, but not with microtubule inhibitors. At the beginning of vitellogenesis (stage 8), the selective translocation of particles through the ring canals became faster (up to 130 nm/second) and more frequent (about 1 particle/minute), whereas during mid-vitellogenesis (stages 9-10A) the velocity and the frequency of particle transport decreased again. Following their more or less rectilinear passage through the ring canals, the particles joined a circular stream of cytoplasmic particles in the oocyte. This ooplasmic particle streaming started at stage 6/7 with velocities of about 80 nm/second and some reversals of direction at the beginning. The particle stream in the oocyte was sensitive to colchicine and vinblastine, but not to cytochalasin B, and we presume that it reflects the rearrangement of ooplasmic microtubules described recently by other authors. We propose that during stages 7-10A, a selective transport of particles into the oocyte occurs through the ring canal along a polarized scaffold of cytoskeletal elements in which microfilaments are involved. This transport might be driven by a myosin-like motor molecule. Either attached to, or organized into, such larger particles or organelles, specific mRNAs and proteins might become selectively transported into the oocyte.
Sense and antisense transfection analysis of tau function: tau influences net microtubule assembly, neurite outgrowth and neuritic stability
Authors: Esmaeli-Azad Babak , McCarty Joseph H. , Feinstein Stuart C.
Published: April 2021

Microtubules are fundamental elements participating in many aspects of cell behavior and maintenance, yet the factors regulating microtubule behavior in vivo remain poorly understood. Employing the nerve growth factor (NGF)-responsive cell line, PC12, we have used sense and antisense DNA transfection strategies to examine the role of the microtubule-associated protein (MAP) tau in several aspects of neuronal cell behavior. Stable transfectants over-expressing tau accumulate more microtubule mass and extend neurites more rapidly than control cells, while transfectants under-expressing tau exhibit reduced micro-tubule levels and slower neurite outgrowth. Further, tau over-expressing cells are markedly more resistant to nocodazole-induced neuritic degeneration when compared to wild-type or tau under-expressing cells. These observations provide direct support for the model that tau is capable of influencing: (i) net microtubule assembly, (ii) the rate of neurite elongation and (iii) neuritic stability. These capabilities suggest that tau plays crucial roles in the development and maintenance of neuronal cells.
Quantitative determination of the proportion of microtubule polymer present during the mitosis-interphase transition
Authors: Zhai Ye , Borisy Gary G.
Published: April 2021

We have developed a new method for determining levels of tubulin polymer, based on quantitative fluorescence detection of x-rhodamine tubulin microinjected into living cells and we have applied this method to analysis of the mitosis-interphase transition. LLC-PK cells in interphase and mitosis were microinjected, then cooled and rewarmed to drive tubulin incorporation. Total tubulin fluorescence in individual, living cells was quantified using a cooled, scientific grade CCD image sensor. Cells were then washed and lysed into a microtubule-stabilizing buffer to extract the soluble pool. Total tubulin polymer fluorescence was determined for the extracted cells in the same way as for living cells. Fluorescence images were corrected by flatfielding and background subtraction. The ratio of extracted cell fluorescence/living cell fluorescence for individual cells, was taken as the proportion of tubulin as polymer. Cells in M-phase, G1 and random interphase were analyzed. G1 cells had almost the same proportion as random interphase cells. Mitotic cells gave a value of 90±5% of G1 cells at 37°C. Within M-phase, levels of tubulin as polymer in metaphase and early anaphase were not significantly different. In contrast to the general expectation of micro-tubule depolymerization at anaphase onset, these results indicate that as cells exit mitosis, the overall proportion of tubulin as polymer does not change dramatically even though the mitotic spindle disassembles. We conclude that the mitosis-interphase transition is accompanied by a redistribution of tubulin at an essentially constant polymer level. Therefore, a global shift to depolymerization conditions is not the driving force for anaphase chromosome movement.
Identification of a beta-type adaptin in plant clathrin-coated vesicles
Authors: Holstein S.E. , Drucker M. , Robinson D.G.
Published: April 2021

Plant clathrin-coated vesicles (CCV), suitably protected against proteolysis, were isolated from zucchini hypocotyls, and screened for the presence of adaptin-like polypeptides using monoclonal antibodies prepared against alpha, beta(beta') and gamma-adaptins of bovine brain. An immunoreactive polypeptide in plant CCV was only detected in the case of the beta(beta')-adaptin antibody. This polypeptide has a molecular mass of 108 kDa in SDS-PAGE, and gives rise to a major cleavage product of 70 kDa after proteolysis with trypsin. Gel filtration of 0.75 M MgCl2-dissociated coat proteins showed that the plant beta(beta')-type adaptin eluted with other polypeptides in a manner similar to the adaptor complexes of brain CCV. Upon subsequent hydroxyapatite chromatography the immunoreactive polypeptide eluted in fractions corresponding to Golgi (HA-I) rather than plasma membrane (HA-II) brain adaptor complexes. In addition, this polypeptide did not shift to a higher molecular mass when subjected to urea-SDS-PAGE. Confirmation of the presence of a beta-type adaptin in plants was provided by dot and Southern blotting experiments using genomic DNA from zucchini hypocotyls and a beta-adaptin cDNA clone from human fibroblasts.
Development of a spontaneous permanent cell line of rabbit corneal epithelial cells that undergoes sequential stages of differentiation in cell culture
Authors: Castro-Muñozledo Federico
Published: April 2021

Established epithelial cell lines that retain their differen-tiation potential and growth regulatory characteristics can provide valuable tools for studying gene regulation, extra-cellular matrix synthesis or growth factor response. They are also useful for drug development and toxicity testing. Experiments were therefore carried out to optimize culture conditions for the long-term, serial transfer of corneal epithelial cells in the presence of 3T3 feeder layers; and to establish a permanent cell line. In such experiments, rabbit corneal epithelial cells were seeded at low inoculation densities, and transferred every 5 days. After 80 popula-tion doublings, an epithelial cell line, RCE1, emerged. The cell line is heteroploid, with an average population doubling time of 15.5 hours (vs 18 hours for primary cultures). When RCE1 cells reached confluence, they strat-ified to form a three-to five-layered epithelium and expressed the differentiation-related keratin pair K3/K12 as shown by immunoblot and immunostaining. Biosyn-thetic labeling of proliferating, confluent and stratified cultures further showed that RCE1 cells expressed keratin pairs K5/K14, K6/K16 and K3/K12, thus mimicking faith-fully the stage-dependent differentiation of primary cultures of rabbit corneal keratinocytes. The results demonstrated that RCE1 cells provide a useful model for studying corneal cell growth and differentiation.
Ca2+ Signaling in the Chlamydomonas flagellar regeneration system: cellular and molecular responses
Authors: Cheshire J. L. , Evans J. H. , Keller L. R.
Published: April 2021

In response to certain extracellular stimuli, Chlamydomonas reinhardtii cells excise their flagella, induce expression of more than 200 different flagellar mRNAs, and assemble a new flagellar pair. Normally, flagellar excision, gene induction and outgrowth are tightly coupled temporally. Our previous studies showed that uncoupling the cellular response of flagellar excision from flagellar outgrowth resulted in submaximal flagellar gene induction, and led us to propose that normal flagellar gene induction is a composite response. The present study extends these observations by measuring flagellar gene induction in Chlamydomonas cells stimulated under conditions where both flagellar excision and flagellar outgrowth are blocked. We find that the flagellar genes are induced in a Ca2+-dependent manner in response to stimulation in the absence of flagellar excision and outgrowth. Flagellar gene induction is therefore independent of flagellar excision and outgrowth but sensitive to extracellular Ca2+ levels. Thus, flagellar excision, flagellar outgrowth and flagellar gene induction are three responses to a common stimulus that are related by their requirement for extracellular Ca2+
Astrocytes modulate retinal vasculogenesis: effects on fibronectin expression
Authors: Jiang B. , Liou G. I. , Behzadian M. A. , Caldwell R. B.
Published: April 2021

Vasculogenesis is the formation of blood-vessels by differentiation of vascular precursor cells. Experiments using retinal models were designed to test the hypothesis that astrocytes influence this process by effects on the composition of the extracellular matrix. Retinal vasculogenesis was studied in relation to the migration of astrocytes and expression of the extracellular matrix proteins laminin and fibronectin by in vivo experiments in neonatal rats. The results show that astrocytes spread into the retina just ahead of the newly formed vessels, where they probably initiate vasculogenesis. They also establish that fibronectin, but not laminin, is expressed in the zone of vasculogenesis immediately prior to vessel formation. Increased amounts of fibronectin mRNA indicate that fibronectin is synthesized by cells within this same region during this same time period. Later, as the new vessels form, differentiation of endothelial cells is correlated with the appearance of pericytes in the vessel wall and laminin in the vascular basement membrane. In vitro experiments using conditioned medium approaches showed that astrocytes stimulate endothelial cell fibronectin expression. Taken together with the in vivo observations these in vitro results suggest that fibronectin expression is an essential component in the initiation of retinal vasculogenesis. This study is the first indication that astrocytes influence the fibronectin component of the extracellular matrix during retinal vasculogenesis and that expression of fibronectin precedes that of laminin in this process.
The effects of diazepam on mitosis and the microtubule cytoskeleton I. Observations on the diatoms Hantzschia amphioxys and Surirella robusta
Authors: Spurck Timothy P. , Pickett-Heaps Jeremy D.
Published: April 2021

The effects of diazepam (DZP) on mitosis and the micro-tubule (MT) cytoskeleton in the live diatoms Hantzschia amphioxys and Surirella robusta were followed using time-lapse video microscopy. Similarly treated cells were fixed and later examined for immunoflouresence staining of MTs or for transmission electron microscopy.

DZP treatment (250 μM) had no effect on interphase cells but affected mitosis, resulting in the majority of prometaphase and metaphase chromosomes releasing from one or both spindle poles and collecting irregularly along the central spindle. Chromosomes remaining attached to one pole continued to display slight prometaphase oscillations; however, this activity was never observed in metaphase spindles. Following removal of DZP, some chromosomes still bipolarly attached, immediately released elastically from one pole. Within the first 2 minutes of recovery, all chromosomes recommenced spindle attachment, exhibiting normal prometaphase oscillations and proceeded through mitosis. DZP treatment during anaphase had no detectable effect on chromosome motion or cell cleavage.

These results suggest that DZP acts as an anti-MT agent, selectively affecting polar MTs at prophase, prometaphase and metaphase, and thereby weakening kinetochore connection to the poles. From these and other results (unpub-lished), its mode of action is different to that of most anti-MT agents.
Cell-to-cell adherens junction formation and actin filament organization: similarities and differences between non-polarized fibroblasts and polarized epithelial cells
Authors: Yonemura Shigenobu , Itoh Masahiko , Nagafuchi Akira , Tsukita Shoichiro
Published: April 2021

Cadherin has an intimate spatial relationship with actin filaments (AF) in various types of cells, forming the cell-to-cell adherens junction (AJ). We compared the AJ/AF rela-tionship between non-polarized fibroblasts (NRK cells) and polarized epithelial cells (MTD-1A cells). E/P-cadherin, α-catenin, ZO-1 and vinculin were localized with reference to AF in these cells using laser scan microscopy as well as conventional light and electron microscopy. NRK cells adhered to each other at the tips of thin cellular processes, where spot-like AJ were formed, where P-cadherin, α-catenin, ZO-1 and vinculin were concentrated. Some stress-fiber-like AF bundles ran axially in these processes and terminated at spot-like AJ on their tips. At the electron micro-scopic level these spot-like AJ were seen as aggregates of small ‘units’ of AJ, where AF were densely and perpendicularly associated with the plasma membrane. In MTD-1A cells, the AJ/AF relationship was investigated during the cell polarization process after replating or wounding. At the early stage, the AJ/AF relationship was quite similar to that in NRK cells. As polarization proceeded, the spot-like AJs were gradually fused side by side with the concomitant shortening of the associated stress-fiber-like AF bundles. Finally, the belt-like AJ was established, which was lined with circumferential AF bundles. The similarities and differences in the AJ/AF relationship between non-polarized fibroblasts and polarized epithelial cells are discussed.
The exit of mouse oocytes from meiotic M-phase requires an intact spindle during intracellular calcium release
Authors: Winston Nicola J. , McGuinness Orla , Johnson Martin H. , Maro Bernard
Published: April 2021

To study the role of the metaphase spindle during the period of oocyte activation, mouse oocytes were fertilised or activated parthenogenetically in the presence or absence of the microtubule inhibitor nocodazole. In both cases, nocodazole caused the disappearance of the spindle and prevented the passage of the oocytes into interphase. However, the calcium spiking responses of the oocytes were not affected by nocodazole, being repetitive after fertilisation and a single spike after activation. If, after their activation or fertilisation in nocodazole, oocytes were later removed from the drug, only those that had been fertilised progressed into interphase. This progress was associated with continuing calcium spiking. Moreover, both the spiking and the progress to interphase could be blocked or reduced in incidence by removal of external calcium or addition of 5,5′-dimethyl BAPTA-AM. Oocytes that had been activated by ethanol in the presence of nocodazole and then removed from it, to allow reformation of the spindle, only progressed into interphase if given a second exposure to ethanol, thereby eliciting a second calcium transient. These results show that exit from meiotic M-phase requires the simultaneous presence of a fully intact spindle during the release of calcium and that those factors leading to the degradation of cyclin B are only activated transiently. Since cyclin is being degraded continuously in the metaphase-II-arrested mouse oocyte and since this degradation is micro-tubule-dependent, these data suggest that the superimposition of a high concentration of intracellular calcium is required to tilt the equilibrium further in favour of cyclin degradation if exit from M-phase is to occur.
Characterization of moesin in the sea urchin Lytechinus variegatus: redistribution to the plasma membrane following fertilization is inhibited by cytochalasin B
Authors: Bachman Eric Scott , McClay David R.
Published: April 2021

We have investigated the distribution and function of an ezrin-radixin-moesin-like (ERM) molecule in the sea urchin. A sea urchin homologue of moesin was cloned that shares 75% amino acid similarity in the conserved N-terminal region to other moesin molecules. A 6.3 kb message is transcribed late in embryogenesis and is present in adult tissues. Polyclonal antibodies were generated to proteins expressed by a bacterial expression vector, and affinity purified. These antibodies recognize a single 75 kDa protein that is present throughout development in approximately equal abundance, and specifically they immuno-precipitate a single protein. We show by immunolocalization that SUmoesin has two predominant patterns during development. First, SUmoesin is rapidly redistributed after fertilization from a location throughout the egg cytoplasm to a location in the egg cortex. Later in embryogenesis, SUmoesin is localized to the apical ends of cells in the regions of cell-cell junctions. We show that SUmoesin is present in actin-rich regions of the embryo. Finally, we show that the location of SUmoesin requires an intact actin-based cytoskeleton. SUmoesin fails to localize to the plasma membrane after fertilization in the presence of cytocha-lasin B. Furthermore, SUmoesin loses its apical position in the region of cell-cell junctions in the presence of cytocha-lasin B in later stages of embryogenesis. This effect is reversible, and the microtubule inhibitor colchicine has no effect. These results show that SUmoesin becomes associated with apical plasma membrane structures early in development, and that SUmoesin is both coincident with actin and requires the assembly of actin filmaments to maintain its localization.
Molecular analysis of a cytoplasmic dynein light intermediate chain reveals homology to a family of ATPases
Authors: Hughes Sharon M. , Vaughan Kevin T. , Herskovits Jonathan S. , Vallee Richard B.
Published: April 2021

Cytoplasmic dynein is a multi-subunit complex involved in retrograde organelle transport and some aspects of mitosis. In previous work we have cloned and sequenced cDNAs encoding the rat cytoplasmic dynein heavy and intermedi-ate chains. Here we report the cloning of the remaining class of cytoplasmic dynein subunits, which we refer to as the light intermediate chains (LICs: 53-59 kDa). Four LIC electrophoretic bands were resolved in purified bovine cytoplasmic dynein preparations by one-dimensional gel electrophoresis. These four bands were simplified to two bands (LIC53/55 and LIC57/59) by alkaline phosphatase treatment. N-terminal amino acid sequence was obtained from a total of 11 proteolytic peptides generated from both LIC53/55 and LIC57/59. Overlapping cDNA clones encoding LIC53/55 were isolated by oligonucleotide screening using probes based on the LIC53/55 peptide sequence. The cDNA sequence contained a 497 codon open reading frame encoding a polypeptide with a molecular mass of ∼55 kDa. Each of the LIC53/55 peptides was found within the deduced amino acid sequence, as well as four of the LIC57/59 peptides. Analysis of the LIC53/55 primary sequence revealed homology with the ABC transporter family of ATPases in the region surrounding the P-loop sequence element. Together these data identify the LICs as a novel family of dynein subunits with potential ATPase activity. They also reveal that the complexity of the LICs is due to both post-translational modification and the existence of at least two LIC polypeptides for which we propose the names LIC-1a and LIC-2.
Interaction of chromosome-6-encoded dystrophin related protein with the extracellular matrix
Authors: Khurana Tejvir S. , Kunkel Louis M. , Frederickson Alan D. , Carbonetto Salvatore , Watkins Simon C.
Published: April 2021

Dystrophin-related protein/utrophin is a large, cytoskeletal protein that shares significant sequence similarity with dys-trophin. Dystrophin-related protein is known to be enriched where cell-extracellular matrix contacts are well defined; however, the mechanism of dystrophin-related protein enrichment and its functional role(s) at these sites are yet to be defined. Here, we demonstrate that dystrophin-related protein is concentrated in patches of astrocyte membrane in apposition with the extracellular matrix and that the distribution of dystrophin-related protein is temporally modulated by the extracellular matrix constituent laminin. Furthermore, we demonstrate the existence of a specific biochemical association between dystrophin-related protein and laminin in astrocytes. In these astrocytes, the depletion of dystrophin-related protein by the use of antisense dystrophin-related protein oligonucleotides causes marked reduction in the formation of functional substratum-membrane attachments. Taken together, these data suggest that dystrophin-related protein may function in the generation and maintenance of regional substratum-associated membrane specializations, such as those found at the blood-brain barrier.
rab8 in retinal photoreceptors may participate in rhodopsin transport and in rod outer segment disk morphogenesis
Authors: Deretic Dusanka , Huber Lukas A. , Ransom Nancy , Mancini Michael , Simons Kai , Papermaster David S.
Published: April 2021

Small GTP-binding protein rab8 regulates transport from the TGN to the basolateral plasma membrane in epithelial cells and to the dendritic plasma membrane in cultured hippocampal neurons. In our approach to identify proteins involved in rhodopsin transport and sorting in retinal pho-toreceptors, we have found, using [32P]GTP overlays of 2D gel blots, that six small GTP-binding proteins are tightly bound to the post-Golgi membranes immunoisolated with a mAb to the cytoplasmic domain of frog rhodopsin. We report here that one of these proteins is rab8. About 50% of photoreceptor rab8 is membrane associated and ∼13% is tightly bound to the post-Golgi vesicles. By confocal microscopy, antibody to rab8 specifically labels calycal processes and the actin bundles of the photoreceptor inner segment that extend inward to the junctional complexes that comprise the outer limiting membrane. Anti-rab8 shows a striking periodicity of high density labeling at 1±0.12 μm intervals along the actin bundles. Rhodopsin-bearing post-Golgi membranes cluster around the base of the cilium where rab8 and actin are also co-localized, as revealed by confocal microscopy of retinal sections double labeled with anti-rab8 and phalloidin. Microfilaments have been implicated in rod outer segment (ROS) disk morpho-genesis. Our data suggest that rab6, which we have previ-ously localized to the post-Golgi compartment, and rab8 associate with the post-Golgi membranes sequentially at different stages of transport. rab8 may mediate later steps that involve interaction of transport membranes with actin filaments and may participate in microfilament-dependent ROS disk morphogenesis.
Protein storage vacuoles form de novo during pea cotyledon development
Authors: Hoh Birgit , Hinz Giselbert , Jeong Byung-Kap , Robinson David G.
Published: April 2021

We have investigated the formation of protein storage vacuoles in peas (Pisum sativum L.) in order to determine whether this organelle arises de novo during cotyledon development. A comparison of different stages in cotyledon development indicates that soluble protease activities decline and the amounts of storage proteins and the integral membrane protein of the protein body, α-TIP, increase during seed maturation. On linear sucrose density gradients we have been able to distinguish between two separate vesicle populations: one enriched in α-TIP, and one in TIP-Ma 27, a membrane protein characteristic of vegetative vacuoles. Both vesicle populations possess, however, PPase and V-ATPase activities. Conventionally fixed cotyledonary tissue at an intermediate stage in cotyledon development reveals the presence of a complex tubular-cisternal membrane system that seems to surround the pre-existing vacuoles. The latter gradually become compressed as a result of dilation of the former membrane system. This was confirmed immunocytochemically with the TIP-Ma 27 antiserum. Deposits of the storage proteins vicilin and legumin in the lumen, and the presence of α-TIP in the membranes of the expanding membrane system provide evidence of its identity as a precursor to the protein storage vacuole.
The isolation and long-term culture of normal human endometrial epithelium and stroma: Expression of mRNAs for angiogenic polypeptides basally and on oestrogen and progesterone challenges
Authors: Zhang Lyna , Rees Margaret C. P. , Bicknell Roy
Published: April 2021

A highly reproducible and technically straightforward technique for the isolation and long-term culture of normal human endometrial epithelial cells is described. The essential conditions for long-term culture are that the cells be seeded onto a gelatin matrix and that ‘endothelial cell growth supplement’ be present in the culture medium. Normal endometrial epithelial cells express cytokeratins and oestrogen receptors. They may be passaged five to six times without change in properties. Growth of normal endometrial epithelial cells was stimulated by 17-β;-oestradiol and epidermal growth factor. Expression of the mRNA coding for seven polypeptide angiogenic factors, by normal endometrial epithelial, stromal and three endometrial carcinoma lines, was examined. The endometrial epithelial and stromal cells express mRNA for the polypeptide angio-genic factors, basic fibroblast growth factor, vascular endothelial cell growth factor, transforming growth factor-β;1 and pleiotrophin, as well as the cytokine midkine. Expression of the mRNA for both vascular endothelial growth factor and midkine by normal endometrial epithelial cells showed a 2-fold increase on treatment with a physiological dose of 17-β;-oestradiol (1010 M) while, in contrast, the mRNA of transforming growth factor-β;1 decreased 4-fold on treatment with 17-β;-oestradiol (1010 M) and was abolished by exposure to progesterone (5×109 M). Expression of the mRNAs for angiogenic polypeptides by the endometrial carcinoma lines was more restricted.
Angiotensin II stimulation of rapid paxillin tyrosine phosphorylation correlates with the formation of focal adhesions in rat aortic smooth muscle cells
Authors: Turner C.E. , Pietras K.M. , Taylor D.S. , Molloy C.J.
Published: April 2021

Angiotensin II is a potent vasoconstrictor that has been also implicated in vascular hyperproliferative diseases, including atherosclerosis and restenosis following angioplasty. Treatment of cultured, serum-starved rat aortic smooth muscle cells with angiotensin II causes rapid protein tyrosine phosphorylation that precedes cell mitogenesis. We have identified two of the phosphoproteins as paxillin (75 kilodaltons) and the tyrosine kinase pp125Fak, both components of actin-associated focal adhesion sites. Angiotensin II stimulated a 5-fold increase in the tyrosine phosphorylation of paxillin and a smaller (1.5-fold) increase in pp125Fak tyrosine phosphorylation. Paxillin tyrosine phosphorylation was evident within 1 minute, and was maximal after 10 minutes. Similar elevated protein tyrosine phosphorylation levels of paxillin were obtained with exposure of the rat aortic smooth muscle cells to peptides endothelin-1 and alpha-thrombin that function, as angiotensin II, through binding to members of the seven transmembrane domain G protein coupled receptors. Angiotensin II treatment also stimulated the production of a well-ordered actin-containing stress fiber network and prominent paxillin-containing focal adhesions. The focal adhesions stained intensely with anti-phosphotyrosine antibody suggesting the tyrosine phosphorylation of paxillin and cytoskeletal reorganization were tightly coupled. Angiotensin II receptor occupancy has been shown previously to lead to protein kinase C activation. However, compared to angiotensin II stimulation, a smaller, delayed increase in paxillin tyrosine phosphorylation was observed following direct protein kinase C activation by the phorbol ester phorbol 12-myristate-13-acetate. Paxillin tyrosine phosphorylation was selective for certain agonists since no increase in tyrosine phosphorylation of this protein was observed following exposure to the potent mitogen PDGF. Thus, actin-based cytoskeletal changes involving sites of cell adhesion to the extracellular matrix may play an important role in normal and pathophysiologic smooth muscle cell growth regulation in response to certain angiotensin II-type vasoactive agonists.
Association of vimentin intermediate filaments with the centrosome
Authors: Trevor Katrina T. , McGuire Jacqueline G. , Leonova Elena V.
Published: April 2021

SW-13 cells that lack cytoplasmic intermediate filaments (IFs) were stably transfected with a human vimentin cDNA expression vector. Isolated subclones displayed two prevalent patterns of vimentin distribution as observed by indirect immuno-localization: (1) cytoplasmic filaments characteristic of a vimentin IF network; and (2) a distinct, juxtanuclear focus with limited filamentous extensions. Comparative analysis of two subclones that uniquely seg-regated these patterns of vimentin organization indicated that vimentin accumulated as a perinuclear focus in cells that expressed a 4-fold lower level of the protein. The observed variation in cellular organization was not due to detectable differences in vimentin protein modification, as determined by two-dimensional gel analysis. Increasing the amount of vimentin in a low expressing clone by a secondary transfection with human or mouse vimentin cDNA resulted in well-dispersed, cytoplasmic filaments, suggesting that the distinct juxtanuclear organization of vimentin arose due to lower cellular vimentin levels.

Employing anti-γ -tubulin and anti-vimentin antibodies, dual immunofluorescence together with confocal microscopy revealed that the juxtanuclear focus of vimentin was located in the centrosomal region. Electron microscopy showed a spheroidal, filamentous structure with at least some filaments closely associated with the peri-centriolar material (PCM). Because vimentin IF organiz-ation is at least partially dependent on microtubules, the effects of nocodazole and taxol on perinuclear vimentin foci were examined. Neither drug affected the juxtanuclear localization of foci, although taxol (10 μM, 5 hours) caused a release of pericentriolar γ -tubulin from the nuclear region in 50-60% of the cells. These studies indicate that lower, in vivo, levels of vimentin fail to form extended IFs but rather are organized as a perinuclear aggregate. Moreover, the PCM of the centrosome appears to possess attachment sites for vimentin IFs.