bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of the β6 integrin subunit in development, neoplasia and tissue repair suggests a role in epithelial remodeling
Authors: Breuss J. M. , Gallo J. , DeLisser H. M. , Klimanskaya I. V. , Folkesson H. G. , Pittet J. F. , Nishimura S. L. , Aldape K. , Landers D. V. , Carpenter W. , Gillett N. , Sheppard D. , Matthay M. A. , Albelda S. M. , Kramer R. H. , Pytela R.
Published: April 2021

The αvβ6 integrin was identified in cultured epithelial cells and functions as a fibronectin receptor. We have now used monoclonal antibodies to determine in vivo expression patterns of the β6 subunit in normal and pathological human or primate tissues, and during experimental wound healing or induced lung injury. The results indicate that β6 expression is restricted to epithelia and is up-regulated in parallel with morphogenetic events, tumorigenesis, and epithelial repair. During development of the kidney, lung, and skin, we found that β6 is expressed by specific types of epithelial cells, whereas it is mostly undetectable in normal adult kidney, lung and skin. In contrast, we detected high-level expression in several types of carcinoma. For example, β6 is almost invariably neo-expressed in squamous cell carcinomas derived from the oral mucosa, often focally localized at the infiltrating edges of tumor islands.

Expression of β6 is also upregulated in migrating keratinocytes at the wound edge during experimental epidermal wound healing. Similarly, β6 expression is induced in type II alveolar epithelial cells during lung injury caused by injection of live bacteria. We also observed β6 expression in adult lungs and kidneys at focal sites of subclinical inflammation, as well as in a variety of clinical specimens from patients with chronic or acute inflammation of the lungs or kidneys. From these findings and earlier results, we hypothesize that αvβ6 affects cell spreading, migration and growth during reorganization of epithelia in development, tissue repair, and neoplasia.
Visualization of exocytosis during sea urchin egg fertilization using confocal microscopy
Authors: Terasaki Mark
Published: April 2021

A Ca2+ wave at fertilization triggers cortical granule exocytosis in sea urchin eggs. New methods for visualizing exocytosis of individual cortical granules were developed using fluorescent probes and confocal microscopy. Electron microscopy previously provided evidence that cortical granule exocytosis results in the formation of long-lived depressions in the cell surface. Fluorescent dextran or ovalbumin in the sea water seemed to label these depressions and appeared by confocal microscopy as disks. FM 1-43, a water-soluble fluorescent dye which labels membranes in contact with the sea water, seemed to label the membrane of these depressions and appeared as rings. In double-labeling experiments, the disk and ring labeling by the two types of fluorescent dyes were coincident to within 0.5 second. The fluorescent labeling is coincident with the disappearance of cortical granules by transmitted light microscopy, demonstrating that the labeling corresponds to cortical granule exocytosis. Fluorescent labeling was simultaneous with an expansion of the space occupied by the cortical granule, and labeling by the fluorescent dextran was found to take 0.1-0.2 second. These results are consistent with, and reinforce the previous electron microscopic evidence for, long-lived depressions formed by exocytosis; in addition, the new methods provide new ways to investigate cortical granule exocytosis in living eggs.

The fluorescence labeling methods were used with the Ca2+ indicator Ca Green-dextran to test if Ca2+ and cortical granule exocytosis are closely related spatially and temporally. In any given region of the cortex, Ca2+ increased relatively slowly. Furthermore, Ca2+ began to rise about 8 seconds before the first fluorescent labeling of exocytosis, suggesting that it takes a relatively long time for the Ca2+ concentration to reach a threshold value that triggers fusion. No evidence was found for highly localized Ca2+ increases that could account for the pattern of individual exocytic events. These results indicate that there is a long lag period between Ca2+ rise and cortical granule exocytosis, and that Ca2+ and cortical granule exocytosis are not tightly coupled spatially and temporally.
Modulation of melanogenic protein expression during the switch from eu-to pheomelanogenesis
Authors: Kobayashi Takeshi , Vieira Wilfred D. , Potterf Brian , Sakai Chie , Imokawa Genji , Hearing Vincent J.
Published: April 2021

Mammalian melanocytes can produce two basic types of melanin, eumelanin and pheomelanin, within discrete organelles termed melanosomes. The physiological signals that regulate this switch are extrinsic to the melanocyte, and include α-melanocyte stimulating hormone and the agouti protein. Tyrosinase, encoded at the albino locus, is the enzyme essential for the synthesis of both types of melanin, but other tyrosinase-related proteins (e.g. TRP1 encoded at the brown locus and TRP2 encoded at the slaty locus) regulate eumelanogenesis catalytically at steps distal to tyrosinase (as 5,6-dihydroxyindole-2-carboxylic acid oxidase and DOPAchrome tautomerase, respectively). The silver protein is another melanosomal protein, and although it has some limited homology to the tyrosinase-related proteins, it does not have any known enzymatic function and probably serves as a structural matrix protein. The role of each of those melanosomal proteins in pheomelanogenesis, however, is still unclear. In this study, we have compared the expression and catalytic functions of those proteins in pheomelanic and eumelanic hair bulb melanocytes. There was no detectable expression of TRP1 or TRP2, or either of their enzymatic activities, in hair bulbs of lethal yellow (Ay/a) newborn mice, and tyrosinase activity was present at a reduced level compared to that found in hair bulbs of black (a/a) newborn mice. Similar results were observed in regenerating hair bulbs of adult lethal yellow mice and in hair bulbs of 5-to 7-day-old agouti mice (A/A), an age where pheomelanin is produced predominantly. Expression of the silver protein was similarly not observed in hair bulbs of the pheomelanic mice. These results support the hypothesis that TRP1 and TRP2 may have only eumelanogenesis-specific functions and further suggest that the silver protein has a structural and/or unknown catalytic function that is similarly eumelanogenesis-specific. Expression of TRP1, TRP2 and/or the silver protein is not required for the switch to pheomelanogenesis, since their functions are specifically down-regulated by the action of the agouti protein, the physiological regulator of the switch to produce pheomelanin.
Cytoskeletal filament assembly and the control of cell spreading and function by extracellular matrix
Authors: Mooney David J. , Langer Robert , Ingber Donald E.
Published: April 2021

This study was undertaken to analyze how cell binding to extracellular matrix produces changes in cell shape. We focused on the initial process of cell spreading that follows cell attachment to matrix and, thus, cell ‘shape’ changes are defined here in terms of alterations in projected cell areas, as determined by computerized image analysis. Cell spreading kinetics and changes in microtubule and actin microfilament mass were simultaneously quantitated in hepatocytes plated on different extracellular matrix substrata. The initial rate of cell spreading was highly dependent on the matrix coating density and decreased from 740 μm2/h to 50 μm2/h as the coating density was lowered from 1000 to 1 ng/cm2. At approximately 4 to 6 hours after plating, this initial rapid spreading rate slowed and became independent of the matrix density regardless of whether laminin, fibronectin, type I collagen or type IV collagen was used for cell attachment. Analysis of F-actin mass revealed that cell adhesion to extracellular matrix resulted in a 20-fold increase in polymerized actin within 30 minutes after plating, before any significant change in cell shape was observed. This was followed by a phase of actin microfilament disassembly which correlated with the most rapid phase of cell extension and ended at about 6 hours; F-actin mass remained relatively constant during the slow matrix-independent spreading phase. Microtubule mass increased more slowly in spreading cells, peaking at 4 hours, the time at which the transition between rapid and slow spreading rates was observed. However, inhibition of this early rise in microtubule mass using either nocodazole or cycloheximide did not prevent this transition. Use of cytochalasin D revealed that microfilament integrity was absolutely required for hepatocyte spreading whereas interference with microtubule assembly (using nocodazole or taxol) or protein synthesis (using cycloheximide) only partially suppressed cell extension. In contrast, cell spreading could be completely inhibited by combining sub-optimal doses of cytochalasin D and nocodazole, suggesting that intact microtubules can stabilize cell form when the microfilament lattice is partially compromised. The physiological relevance of the cytoskeleton and cell shape in hepatocyte physiology was highlighted by the finding that a short exposure (6 hour) of cells to nocodazole resulted in production of smaller cells 42 hours later that exhibited enhanced production of a liver-specific product (albumin). These data demonstrate that spreading and flattening of the entire cell body is not driven directly by net polymerization of either microfilaments or microtubules. Instead, extracellular matrix appears to control cell shape and function by producing global changes in a structurally integrated cytoskeletal network.
gp330 on type ii pneumocytes mediates endocytosis leading to degradation of pro-urokinase, plasminogen activator inhibitor-1 and urokinase-plasminogen activator inhibitor-1 complex
Authors: Stefansson Steingrimur , Kounnas Maria Z. , Henkin Jack , Mallampalli Rama K. , Chappell David A. , Strickland Dudley K. , Argraves W. Scott
Published: April 2021

Glycoprotein 330 (gp330) is a member of a family of receptors related to the low density lipoprotein receptor (LDLR). Although several ligands have been shown to bind gp330 in solid-phase assays, the ability of gp330 to mediate ligand endocytosis has not been demonstrated. To develop a cellular model for gp330 function we screened a variety of cultured cell lines and identified several that expressed this protein, including immortalized rat type II pneumocytes and a human and two rodent tumor cell lines. Using type II pneumocytes, endocytosis of a previously described gp330 ligand, urokinase (uPA) complexed with plasminogen activator inhibitor-1 (uPA:PAI-1) and two new ligands, PAI-1 and pro-uPA, was demonstrated. RAP, the 39 kDa receptor-associated protein known to antagonize ligand binding to gp330 in solid-phase binding assays, completely inhibited both internalization and degradation of the radiolabeled ligands by type II pneumocytes. This suggested that the clearance of these ligands was dependent on either gp330 or the LDLR-related protein (LRP), which shares several ligand-binding characteristics with gp330. By using polyclonal antibodies to gp330, the cellular internalization and degradation of the ligands were inhibited by 30-50%; remaining ligand internalization and degradation activity could be partially inhibited by polyclonal antibodies against LRP. These findings indicate that gp330, like other LDLR family members, mediates endocytosis of its ligands. In addition, gp330 acts in concert with LRP in type II pneumocytes to mediate clearance of a variety of proteins involved in plasminogen activation, including uPA:PAI-1 complexes PAI-1 and pro-uPA. The evidence that gp330 can mediate clearance of these proteins in cultured cells implicates it as having a role in extracellular protease regulation in the many specialized epithelia where it is expressed in vivo.
Increased microvascular permeability and endothelial fenestration induced by vascular endothelial growth factor
Authors: Roberts W.G. , Palade G.E.
Published: April 2021

The vascular endothelial growth factor (VEGF) was originally described as vascular permeability factor due to its ability to increase microvascular permeability to plasma proteins. However, the vessel types (arteriolar, venular, and capillary) affected by VEGF and the modification of endothelial morphology in response to increased permeability induced by VEGF in vivo have not been precisely documented. By topical application or intradermal injection of recombinant human VEGF-165 we find that VEGF increases the permeability of postcapillary venules as well as muscular venules and capillaries. Surprisingly, we also find that endothelia of small venules and capillaries become fenestrated within 10 minutes of VEGF application. Fenestrations appeared in vascular beds which do not normally have fenestrated endothelium, namely the cremaster muscle and skin. Histamine, saline, and heat-inactivated VEGF do not cause fenestrations. Increased permeability is completely inhibited when VEGF is cleared by immunoprecipitation with anti-VEGF monoclonal antibodies. The VEGF effect on permeability is unlike that of any other mediator described to date since both muscular venules and capillaries are affected.
Chromatin diminution is strictly correlated to somatic cell behavior in early development of the nematode Parascaris univalens
Authors: Esteban M. R. , Giovinazzo G. , Goday C.
Published: April 2021

We have studied the relationship between the occurrence of chromatin diminution and the developmental behavior of somatic blastomeres in early development of the nematode Parascaris univalens. A cytological and immuno-cytochemical analysis of chromatin diminution was performed in P. univalens embryos exposed to ‘vegetalizing’ (LiCl) and ‘animalizing’ (NaSCN) substances during early developmental stages. We have also analyzed chromatin diminution in embryos displaying only symmetrical ‘somatic-like’ divisions due to a brief cytochalasin B treatment at the pronuclear stage. The results show that LiCl treatment induces chromatin diminution in P0-P4 pregerminal blastomeres while NaSCN treatment prevents it. Pregerminal cells undergoing chromatin diminution in ‘vegetalized’ embryos behaved like somatic cells with respect to division and cleavage patterns. NaSCN treatment results in undiminuted polynucleated embryos that are not capable of cleavage. In cytochalasin B-pulsed embryos, chromatin diminution occurs in all blastomeres. From our results we conclude that chromatin diminution and somatic cell behavior are not separable and constitute strictly correlated events in Parascaris. Moreover, the results indicate that the segregation of the cytoplasmic factors involved in chromatin diminution in early Parascaris development are microfilament-mediated. Here, we also report the formation of a latter pregerminal cell precursor (P5) not susceptible to LiCl-induced chromatin diminution.
caenorhabditis elegans cyclin a- and b-type genes: A cyclin a multigene family, an ancestral cyclin b3 and differential germline expression
Authors: Kreutzer Monique A. , Richards James P. , Silva-Udawatta Mihiri N. De , Temenak Joseph J. , Knoblich Jürgen A. , Lehner Christian F. , Bennett Karen L.
Published: April 2021

We have cloned cDNAs for Caenorhabditis elegans cyclins A1, B and B3. While cyclins A1 and B are most closely related to either A- or B-type cyclins of other species, cyclin B3 is less related to these cyclins. However, this cyclin is most similar to the recently identified chicken cyclin B3. Our identification of a Caenorhabditis homolog demonstrates that cyclin B3 has been conserved in evolution. cyclin A1 is a member of an A-type multigene family; however the cyclin A1 cDNA only recognizes a single band on northern blots. A single-sized RNA is also observed for the cyclin B3 cDNA. In contrast, three different transcripts are observed for the cyclin B cDNA. Based on our analyses using RNAs from germline-defective mutants and from populations enriched for males, one cyclin B transcript is specific to the paternal germline. The two other cyclin B transcripts, as well as the cyclin A1 and cyclin B3 transcripts, are most abundant in the maternal germline and are only present at low levels in other tissues. Moreover, the 3′ untranslated regions of each Caenorhabditis cyclin cDNA possess several copies of potential translational control elements shown in Xenopus and Drosophila maternal cyclin mRNAs to function during oogenesis and early embryogenesis.
Invasion of Toxoplasma gondii occurs by active penetration of the host cell
Authors: Morisaki J. Hiroshi , Heuser John E. , Sibley L. David
Published: April 2021

Toxoplasma gondii is an obligate intracellular parasite that infects a wide variety of vertebrate cells including macrophages. We have used a combination of video microscopy and fluorescence localization to examine the entry of Toxoplasma into macrophages and nonphagocytic host cells. Toxoplasma actively invaded host cells without inducing host cell membrane ruffling, actin microfilament reorganization, or tyrosine phosphorylation of host proteins. Invasion occurred rapidly and within 25-40 seconds the parasite penetrated into a tight-fitting vacuole formed by invagination of the plasma membrane. In contrast, during phagocytosis of Toxoplasma, extensive membrane ruffling captured the parasite in a loose-fitting phagosome that formed over a period of 2-4 minutes. Phagocytosis involved both reorganization of the host cytoskeleton and tyrosine phosphorylation of host proteins. In some cases, parasites that were first internalized by phagocytosis, were able to escape from the phagosome by a process analogous to invasion. These studies reveal that active penetration of the host cell by Toxoplasma is fundamentally different from phagocytosis or induced endocytic uptake. The novel ability to penetrate the host cell likely contributes to the capability of Toxoplasma-containing vacuoles to avoid endocytic processing.
The α2β1 integrin regulates collagen-mediated mdck epithelial membrane remodeling and tubule formation
Authors: Schwimmer Randi , Ojakian George K.
Published: April 2021

Previous studies have demonstrated that incubation of MDCK cell epithelial cysts in collagen gel induced a reversal in cell surface polarity that was regulated by β1 integrins. Further experiments were done to identify the specific collagen binding integrin involved by applying collagen gel overlays to the apical membrane of subconfluent MDCK monolayers. Cell surface levels of the apical membrane glycoprotein gp135 were monitored by ELISA to quantitate the extent of collagen-mediated membrane remodeling. After an 8 hour incubation with collagen, there was a 35% reduction in gp135 while the cell surface levels of the α2, α3 and β1 integrin subunits were not affected. Immunofluorescence microscopy confirmed the loss of gp135 from selected regions of the apical cell surface while the α2 and β1 integrin subunits were distributed in small clusters over the entire apical membrane in both control and collagen-treated monolayers. Collagen-mediated loss of gp135 was inhibited by monoclonal antibodies which recognize either the α2 or β1 integrin subunits but not by a monoclonal antibody against the α6β1 integrin. These results demonstrated that remodeling of the apical membrane had occurred, allowing the selective retention of β1 integrins but not gp135. They were supported by the observation that collagen-mediated loss of apical membrane microvilli was inhibited by the monoclonal antibody against the α2 integrin subunit. Incubation of confluent monolayers with collagen gel induced the formation of polarized epithelial tubules within 16 hours. Epithelial tubule biogenesis was completely inhibited by monoclonal antibodies against either the α2 or β1 integrin subunits, providing strong evidence that the α2β1 integrin is essential for collagen-mediated epithelial membrane remodeling and tubule formation.
Mapping of nucleoporins to the center of the nuclear pore complex by post-embedding immunogold electron microscopy
Authors: Grote Monika , Kubitscheck Ulrich , Reichelt Rudolf , Peters Reiner
Published: April 2021

Ultrathin sections of Lowicryl K4M embedded cultured 3T3 cells, human keratinocytes and mouse/rat liver tissue were incubated with polyspecific primary antibodies against p62 and other nucleoporins followed by 10 nm gold labeled secondary antibodies. By quantitatively evaluating both cross sections and tangential sections of the NPC, we found that irrespective of the cell type antibodies predominantly bound within a radius of 25 nm around the central axis of the nuclear pore complex (NPC). Superposition of a current structural model of the NPC with the nucleoporin distribution observed by us showed that nucleoporins mapped predominantly to the controversely discussed ‘central granule’. Our experimental approach was verified by mapping gp210, another nuclear pore protein, at or very close to the NPC in the perinuclear cisterna thus establishing a distribution pattern completely different from that of the nucleoporins.
Monoclonal and polyclonal antibodies detect a new type of post-translational modification of axonemal tubulin
Authors: Levilliers Nicolette , Fleury Anne , Hill Anne-Marie
Published: April 2021

Polyclonal (PAT) and monoclonal (AXO 49) antibodies against Paramecium axonemal tubulin were used as probes to reveal tubulin heterogeneity. The location, the nature and the subcellular distribution of the epitopes recognized by these antibodies were, respectively, determined by means of: (i) immunoblotting on peptide maps of Paramecium, sea urchin and quail axonemal tubulins; (ii) immunoblotting on ciliate tubulin fusion peptides generated in E. coli to discriminate antibodies directed against sequential epitopes (reactive) from post-translational ones (non reactive); and (iii) immunofluorescence on Paramecium cells, using throughout an array of antibodies directed against tubulin sequences and post-translational modifications as references.

AXO 49 monoclonal antibody and PAT serum were both shown to recognize epitopes located near the carboxylterminal end of both subunits of Paramecium axonemal tubulin, whereas the latter recognized additional epitopes in α-tubulin; AXO 49 and a fraction of the PAT serum proved to be unreactive over fusion proteins; both PAT and AXO 49 labelled a restricted population of very stable microtubules in Paramecium, consisting of axonemal and cortical ones, and their reactivity was sequentially detected following microtubule assembly; finally, both antibodies stained two upward spread bands in Paramecium axonemal tubulin separated by SDS-PAGE, indicating the recognition of various α- and β-tubulin isoforms displaying different apparent molecular masses.

These data, taken as a whole, definitely establish that PAT and AXO 49 recognize a post-translational modification occurring in axonemal microtubules of protozoa as of metazoa. This modification appears to be distinct from the previously known ones, and all the presently available evidence indicates that it corresponds to the very recently discovered polyglycylation of Paramecium axonemal α- and β-tubulin.
Osteoclast ATP receptor activation leads to a transient decrease in intracellular pH
Authors: Yu Hesheng , Ferrier Jack
Published: April 2021

Application of extracellular adenosine triphosphate (ATP) induces a pulsed decrease in osteoclast intracellular pH (pHi), as measured with seminaphthofluorescein (SNAFL)-calcein on a laser scanning confocal microscope. Adenosine diphosphate also produces a pHi decrease, but adenosine monophosphate, uridine triphosphate, 2-methylthio-ATP, and β,γ-methylene-ATP have little effect on pHi. The ATP-induced pHi decrease is largely inhibited by suramin, a P2 purinergic receptor blocker. Clamping intracellular free [Ca2+] ([Ca2+]i) with BAPTA/AM does not affect the ATP-induced pHi change, showing that this pHi decrease is not caused by the increased intracellular [Ca2+]i that is produced by activation of osteoclast purinergic receptors. We show that an increase in [Ca2+]i by itself will produce a pHi increase. The ATP effect is not blocked by inhibition of Na+/H+ exchange by either Na+-free bathing medium or amiloride. Two inhibitors of the osteoclast cell membrane proton pump, N-ethylmaleimide and vanadate, produce partial inhibition of the ATP-induced pHi decrease. Two other proton pump inhibitors, bafilomycin and N,N′-dicyclohexylcarbodiimide, have no influence on the ATP effect. None of the proton pump inhibitors but vanadate has a direct effect on pHi. Vanadate produces a transient pHi increase upon application to the bathing medium, possibly as a result of its known effect of stimulating the Na+/H+ exchanger. Inhibition of Cl−/HCO3− exchange by decreasing extracellular Cl− gives a pronounced long-term pHi increase, supporting the hypothesis that this exchange has an important role in osteoclast pHi homeostasis. In Cl−-free extracellular medium, there is a greatly reduced effect of extracellular ATP on pHi. The ATP effect is partially inhibited by diisothiocyanatostilbene sulfonic acid, an inhibitor of the Cl−/HCO3− exchanger. These data provide evidence that ATP binding to a P2 purinergic receptor results in a transient enhancement of Cl− /HCO3− exchange across the osteoclast cell membrane.
Cyclic AMP modifies the cellular distribution of connexin43 and induces a persistent increase in the junctional permeability of mouse mammary tumor cells
Authors: Atkinson Michael M. , Lamper Paul D. , Lin Helen H. , Kollander Rahn , Li Xen-Ren , Kiang David T.
Published: April 2021

Direct communication between cells via gap junctions is thought to be an important component of homeostasis and coordinated cellular responses to external signals. We investigated how the second messenger cAMP exerts its effects on junctional communication in a mouse mammary tumor cell line, MMT22. Junctional permeance was quantitatively assessed using dye microinjection and video microscopy. An increase of permeance was found after exposure to 8-bromo-cAMP, being detectable after 30 minutes of treatment and attaining a fourfold higher level of permeance by 24 hours. This elevated level was maintained with continuous exposure to 8-bromo-cAMP for seven days. The permeability change was accompanied by an increase in gap junctions as shown by freeze-fracture electron microscopy and by confocal microscopy using antibodies directed against the gap junction protein, connexin43. The amount of detergent-insoluble connexin43 also increased with 8-bromo-cAMP treatment, and most of the increase could be attributed to an increase of slower migrating (i.e. phosphorylated) species of connexin43. However, connexin43 mRNA and the total cellular content of connexin43 did not change over this period of exposure to 8-bromo-cAMP, as shown by densitometric analyses of northern and western blots. We conclude that 8-bromo-cAMP affects the distribution of connexin43 such that a greater proportion of the protein is utilized for channel formation. Since these changes were relatively slow to develop and persisted with prolonged exposure to 8-bromo-cAMP, it is possible that the junctional permeability of these mammary tumor cells is linked to the ‘basal’ level of cAMP, i.e. levels maintained by the cells in accordance with a particular cell state.
DNA polymerase alpha, a component of the replication initiation complex, is essential for the checkpoint coupling S phase to mitosis in fission yeast
Authors: D’Urso Gennaro , Grallert Beáta , Nurse Paul
Published: April 2021

Genetic analysis in the yeast Schizosaccharomyces pombe has shown that three genes cdc18, cut5, and cdtl, are essential for DNA synthesis and also for the checkpoint control that couples completion of DNA replication to the onset of mitosis. To test whether assembly of the replication initiation complex is an important element in the checkpoint control pathway we have investigated if DNA polymerase alpha (poll), a component of the initiation complex, is essential for the S-phase checkpoint control. We show that germinating S. pombe spores disrupted for the pol1 gene enter mitosis despite defects in DNA synthesis. This is shown by monitoring septation index, DNA content, and by direct immunofluorescence of mitotic spindles using antibodies to alpha-tubulin. In addition we have isolated six temperature sensitive mutants in the pol1 gene that cause cell cycle arrest when grown at the non-permissive temperature. Our experiments support a model in which DNA polymerase alpha, in addition to being part of the initiation complex, is required for a checkpoint signal that is activated as cells traverse START, and is essential to prevent mitosis until S phase has been completed. In contrast, proteins responsible for the elongation of DNA may not be necessary for this checkpoint signal.
The carboxyl terminus of myosin binding protein C (MyBP-C, C-protein) specifies incorporation into the A-band of striated muscle
Authors: Gilbert Rénald , Kelly Michael G. , Mikawa Takashi , Fischman Donald A.
Published: April 2021

Myosin binding protein-C (MyBP-C), also known as C-protein, is a major constituent of the thick filaments of vertebrate striated muscles. The protein, ∼130 kDa, consists of a series of 10 globular motifs (numbered I to X) each of ∼90-100 amino acids, bearing resemblance to the C2-set of immunoglobins (Ig C2) and to the fibronectin type III (FnIII) motifs. Using pure preparations of myosin and MyBP-C, it has been demonstrated that the major myosin binding domain of MyBP-C resides within the C-terminal Ig C2 motif (motif X). However, in the context of the in vivo thick filament, it is uncertain if the latter domain is sufficient to target MyBP-C correctly to the A-band or if other regions of the molecule are required for this process. To answer this question, cultures of skeletal muscle myoblasts were transfected with expression plasmids encoding seven truncation mutants of MyBP-C, and their targeting to the A-band investigated by immunofluorescence microscopy. To distinguish the recombinant proteins from endogenous MyBP-C, a myc epitope was inserted at each amino terminus. Recombinant MyBP-C exhibited an identical distribution in the sarcomere to that of native MyBP-C; i.e. it was found exclusively in the C-zone of the A-band. A mutant encoding the C-terminal 372 amino acids, but lacking motifs I-VI (termed Δ1-6), also targeted correctly to the A-band. This fragment, which is composed of two Ig C2 and two FnIII motifs, was the minimal protein fragment required for correct A-band incorporation. Larger aminoterminal deletions or deletion of motif X, the myosin binding domain, abolished all localization to the A-band. One construct (Δ10) lacking only motif X strongly inhibited myofibril assembly. We conclude that the myosin binding domain of MyBP-C, although essential, is not sufficient for correct incorporation into the A-band and that motifs VII to IX are required for this process. The data suggest a topological model in which MyBP-C is associated with the thick filament through its C terminus.
Isolation and characterization of fission yeast mutants defective in the assembly and placement of the contractile actin ring
Authors: Chang Fred , Woollard Alison , Nurse Paul
Published: April 2021

Fission yeast cells divide by medial cleavage using an actin-based contractile ring. We have conducted a genetic screen for temperature-sensitive mutants defective in the assembly and placement of this actin ring. Six genes necessary for actin ring formation and one gene necessary for placement of the actin ring have now been identified. The genes can be further organized into different phenotypic groups, suggesting that the gene products may have different functions in actin ring formation. Mutants of cdc3 and cdc8, which encode profilin and tropomyosin respectively, display disorganized actin patches in all cells. cdc12 and cdc15 mutants display disorganized actin patches during mitosis, but normal interphase actin patterns. cdc4 and rng2 mutants display disorganized actin cables during mitosis, but normal interphase actin patterns. In mid1 mutants, the actin ring and septum are positioned at random locations and angles on the cell surface, although the nucleus is positioned normally, indicating that the mid1 gene product is required to couple the division site to the position of the nucleus. mid1 mutant cells may reveal a new cell cycle checkpoint in telophase that coordinates cell division and the proper distribution of nuclei. The actin ring forms medially in a β-tubulin mutant, showing that actin ring formation and placement are not dependent on the mitotic spindle.
Coordinated movements between autosomal half-bivalents in crane-fly spermatocytes: evidence that ‘stop’ signals are sent between partner half-bivalents
Authors: Yin Beihong , Forer Arthur
Published: April 2021

During anaphase-I in crane-fly spermatocytes, sister half-bivalents separate and move to opposite poles. When we irradiate a kinetochore spindle fibre with an ultraviolet microbeam, the associated half-bivalent temporarily stops moving and so does the partner half-bivalent with which it was paired during metaphase. To test whether a ‘signal’ is transmitted between partner half-bivalents we irradiated the spindle twice, once in the interzone (the region between separating partner half-bivalents) and once in a kinetochore fibre. For both irradiations we used light of wavelength 290 μm and a dose that, after irradiating a spindle fibre only, altered movement in 63% of irradiations (12/19); in 11 of the 12 cells both partner half-bivalents stopped moving after the irradiation. In control experiments we irradiated the interzone only: these irradiations generally did not stop chromosomal poleward motion but sometimes (14/29) caused poleward movement to each pole to be abruptly reduced to about half the velocity prior to irradiation.

In double irradiation experiments we varied the order of the irradiations. In some double irradiation experiments we irradiated the interzonal region first and the spindle fibre second; in 75% (9/12) of the cells the half-bivalent associated with the irradiated fibre stopped moving while the partner half-bivalent moved normally, i.e. in 9/12 cells the interzonal irradiations uncoupled the movements of the partner half-bivalents. In other double irradiation experiments we irradiated the spindle fibre first and the interzone second: in 80% (4/5) of the cells the half-bivalents not associated with the irradiated spindle fibre resumed movement immediately after the irradiation while the other half-bivalent remained stopped. Interzonal irradiations therefore uncouple the poleward movements of sister half-bivalents and the uncoupling does not depend on the order of the irradiation. Our experiments suggest therefore that the irradiation of a spindle fibre causes negative (‘stop’) signals to be transmitted across the interzone and that irradiation of the interzone blocks the transmission of the stop signal.
Human keratinocytes migrate to the negative pole in direct current electric fields comparable to those measured in mammalian wounds
Authors: Nishimura Karen Y. , Isseroff R. Rivkah , Nuccitelli Richard
Published: April 2021

Previous measurements of the lateral electric fields near skin wounds in guinea pigs have detected DC fields between 100-200 mV/mm near the edge of the wound. We have studied the translocation response of motile primary human keratinocytes migrating on a collagen substrate while exposed to similar physiological DC electric fields. We find that keratinocytes migrate randomly on collagen in fields of 5 mV/mm or less, but in larger fields they migrate towards the negative pole of the field, exhibiting galvanotaxis. Since these cells have an average cell length of 50 µm, this implies that they are able to detect a voltage gradient as low as 0.5 mV along their length. This cath-odally-directed movement exhibits increased directedness with increasing field strengths between 10 and 100 mV/mm. We observe a maximally directed response at 100 mV/mm with half of the cells responding to the field within 14 minutes. The average speed of migration tended to be greater in fields above 50 mV/mm than in smaller fields. We conclude that human keratinocytes migrate towards the negative pole in DC electric fields that are of the same magnitude as measured in vivo near wounds in mammalian skin.
Three-dimensional organization of rat hepatocyte cytoskeleton: relation to the asialoglycoprotein endocytosis pathway
Authors: Novikoff P. M. , Cammer M. , Tao L. , Oda H. , Stockert R. J. , Wolkoff A. W. , Satir P.
Published: April 2021

Analysis by confocal microscopy has revealed features of the microtubule network of rat hepatocytes in culture, establishing the three-dimensional disposition of the microtubule-based cytoskeleton, its relation to the actin-based cytoskeleton and to ligand-containing endosomes during receptor-mediated endocytosis and the alterations in its structure and disposition by the microtubule pertubant, Taxol. By co-localization studies, we have been able to demonstrate that the microtubules have a significant role in receptor-mediated endocytosis of asialoglycoproteins in this cell. Asialoorosomucoid-containing endosomes attach to widely spaced arrays of microtubules running under the baso-lateral surface of the hepatocytes 5-15 minutes after the initiation of endocytosis and then travel along microtubule paths to become concentrated with microtubules near the centrosome and at bile canaliculi after 30-60 minutes of receptor-mediated endocytosis. Receptor-mediated endocytosis is affected, but not abolished by Taxol, which inhibits the rate of asialoorosomucoid degradation at the same concentrations as those that disrupt microtubule and cytoplasmic dynein distribution, and that prevent the concentration of endosomes centrally. The results support suggestions that asialoorosomucoid-containing endosomes are captured by microtubules just below the actin layer at the cell periphery and these are actively transported centrally along microtubules, possibly by cytoplasmic dynein, so that the concentration of endosomes near the centrosome, and the subsequent efficient lysosomal degradation of ligand, are consequences of the confluence of microtubules in this region.
Degradation of nuclear matrix and DNA cleavage in apoptotic thymocytes
Authors: Weaver Valerie M. , Carson Christine E. , Walker P. Roy , Chaly Nathalie , Lach Boleslaw , Raymond Yves , Brown David L. , Sikorska Marianna
Published: April 2021

In dexamethasone-treated thymocyte cultures an increase in nuclear proteolytic activity paralleled chromatin fragmentation and the appearance of small apoptotic cells. The elevation of nuclear proteolytic activity was accompanied by site-specific degradation of nuclear mitotic apparatus protein and lamin B, two essential components of the nuclear matrix. Nuclear mitotic apparatus protein phosphorylation and cleavage into 200 and 48 kDa fragments occured within 30 minutes of dexamethasone treatment. Cleavage of lamin B, which generated a fragment of 46 kDa consistent with the central rod domain of the protein, was also detected after 30 minutes of exposure to the steroid hormone. The level of lamin B phosphorylation did not change as a result of the dexamethasone treatment and the lamina did not solubilize until the later stages of apoptosis. Initial DNA breaks, detected by the terminal transferasemediated dUTP-biotin nick end labeling assay, occurred throughout the nuclei and solubilization of lamina was not required for this process to commence. The data presented in this paper support a model of apoptotic nuclear destruction brought about by the site-specific proteolysis of key structural proteins. Both the nuclear mitotic apparatus protein and lamin B were specifically targeted by protease(s) at early stages of the cell death pathway, which possibly initiate the cascade of degradative events in apoptosis.
Plant and animal profilins are functionally equivalent and stabilize microfilaments in living animal cells
Authors: Rothkegel Martin , Mayboroda Oleg , Rohde Manfred , Wucherpfennig Carmen , Valenta Rudolf , Jockusch Brigitte M.
Published: April 2021

We have analyzed the degree of functional similarity between birch and mammalian profilins, two members of the profilin family which show only a moderate sequence homology (22%) in living animal cells. The plant profilin, derived from birch pollen, was stably expressed in BHK-21 cells. Plant and endogenous profilin synthesis and cellular distribution were monitored by specific monoclonal antibodies. Quantitation of profilin and actin on calibrated immunoblots showed that two stable clones contained in total 1.4 and 2.0 times as much profilin as the parental cells. Using double fluorescence and confocal laser scanning microscopy, it was seen that the endogenous and the plant profilin colocalized with dynamic microfilaments, in particular with F-actin-rich foci and cortical microfilament webs of spreading cells, with dynamic microfilament bundles induced by serum deprival, and with cytochalasin D- and latrunculininduced transient F-actin aggregates. The increase in the overall profilin concentration correlated with a significantly higher resistance of actin filaments to these drugs. Our data indicate that even profilins of highly distant evolutionary origin can functionally substitute for each other and support the hypothesis that in animal cells, profilins are engaged in regulating either the stability or the kinetic properties of actin filaments.
A single gamma-tubulin gene and mRNA, but two gamma-tubulin polypeptides differing by their binding to the spindle pole organizing centres
Authors: Lajoie-Mazenc Isabelle , Détraves Claire , Rotaru Victor , Garès Michèle , Tollon Yvette , Jean Catherine , Julian Monique , Wright Michel , Raynaud-Messina Brigitte
Published: April 2021

Cells of eukaryotic organisms exhibit microtubules with various functions during the different developmental stages. The identification of multiple forms of α- and β-tubulins had raised the question of their possible physiological roles. In the myxomycete Physarum polycephalum a complex polymorphism for α- and β-tubulins has been correlated with a specific developmental expression pattern. Here, we have investigated the potential heterogeneity of γ-tubulin in this organism. A single gene, with 3 introns and 4 exons, and a single mRNA coding for γ-tubulin were detected. They coded for a polypeptide of 454 amino acids, with a predicted molecular mass of 50,674, which presented 64-76% identity with other γ-tubulins. However, immunological studies identified two γ-tubulin polypeptides, both present in the two developmental stages of the organism, uninucleate amoebae and multinucleate plasmodia. The two γ-tubulins, called γs- and γf-tubulin for slow and fast electrophoretic mobility, exhibited apparent molecular masses of 52,000 and 50,000, respectively. They were recognized by two antibodies (R70 and JH46) raised against two distinct conserved sequences of γ-tubulins. They were present both in the preparations of amoebal centrosomes possessing two centrioles and in the preparations of plasmodial nuclear metaphases devoid of structurally distinct polar structures. These two γ-tubulins exhibited different sedimentation properties as shown by ultracentrifugation and sedimentation in sucrose gradients. Moreover, γs-tubulin was tightly bound to microtubule organizing centers (MTOCs) while γf-tubulin was loosely associated with these structures. This first demonstration of the presence of two γ-tubulins with distinct properties in the same MTOC suggests a more complex physiological role than previously assumed.
Three homologs of rds/peripherin in Xenopus laevis photoreceptors that exhibit covalent and non-covalent interactions
Authors: Kedzierski Wojciech , Moghrabi Walid N. , Allen Ann C. , Jablonski-Stiemke Monica M. , Azarian Sassan M. , Bok Dean , Travis Gabriel H.
Published: April 2021

We have isolated and characterized three homologs of mammalian rds/peripherin from Xenopus retinae. One (xrds38) is likely the Xenopus ortholog, while the other two (xrds36 and -35) are more distant relatives. By immunocytochemical analysis of retinal sections, xrds38 is distributed in both rod and cone photoreceptors, while xrds36 and xrds35 are present in rods only. At the EM level, xrds38 is present specifically in the rims and incisures of rod and cone outer segment discs. All are N-glycosylated and form covalent dimers. Immunoprecipitation analysis showed that in rods, these three proteins interact to form heterotetrameric or higher-order complexes. The pattern of sequence conservation among the xrds proteins, mammalian rds/peripherin, and mammalian rom-1 suggest that the central portion of the intradiscal D2 loop contains the interacting structural elements.
Elongation factor-1α is an overexpressed actin binding protein in metastatic rat mammary adenocarcinoma
Authors: Edmonds Brian T. , Wyckoff Jeffrey , Yeung Yee-Guide , Wang Yun , Stanley E. Richard , Jones Joan , Segall Jeffrey , Condeelis John
Published: April 2021

Overexpression of elongation factor-1α (EF1α) mRNA has been correlated with increased metastatic potential in mammary adenocarcinoma; however, this relationship was not explored at the level of protein expression. As EF1α has been shown in other cell types to be a component of the actin cytoskeleton, a likely effector in metastasis, the actin binding activity of EF1α from metastatic and nonmetastatic rat breast tumors and cell lines was investigated. We have shown that EF1α protein is overexpressed in metastatic compared to nonmetastatic cells and whole tumors. Similarly to other EF1αs, both types of tumor EF1α bind to F-actin, but EF1α from metastatic cells has a reduced affinity for actin. In addition, there is a high correlation between the intracellular distribution of filamentous actin and EF1α in those cytoskeletal structures thought to be important for supporting the cellular motility required for metastasis. Following stimulation with EGF, there is a parallel increase in the amount of F-actin and EF1α associated with the cytoskeleton. The response to EGF can be blocked with cytochalasin D indicating that the binding of EF1α to the cytoskeleton is mediated by F-actin. We propose that a weakened association of EF1α with actin may be related to the metastatic process via an altered organization of the actin cytoskeleton and the differential translation of mRNAs associated with the cytoskeleton.
NRD convertase: a putative processing endoprotease associated with the axoneme and the manchette in late spermatids
Authors: Chesneau Valérie , Prat Annik , Segretain Dominique , Hospital Véronique , Dupaix Alain , Foulon Thierry , Jégou Bernard , Cohen Paul
Published: April 2021

N-arginine dibasic convertase is a novel metalloendopeptidase which selectively cleaves at the N terminus of arginine residues in paired basic amino acids. Although present in brain and several other tissues, NRD convertase is particularly abundant in testis, where its expression appeared to be restricted to germ cells. Low levels of both mRNA and its corresponding protein were detected early in spermatogenesis. However, a marked accumulation of the protein was observed during late steps (14 to 19) of spermiogenesis. By electron microscopy, the NRD convertase immunoreactivity was localized in the cytoplasm of elongating and elongated spermatids, with a noticeable concentration at the level of two microtubular structures, i.e. the manchette and the axoneme. These observations strongly support the hypothesis that NRD convertase is involved in processing events potentially associated with the morphological transformations occurring during spermiogenesis.