bluuebunny/crossref_metadata_2025 huggingface dataset:
Transcription-dependent redistribution of nuclear protein 4.1 to SC35-enriched nuclear domains
Authors: Lallena María-José , Correas Isabel
Published: April 2021

Protein 4.1, originally identified as a component of the membrane-skeleton of the red blood cell, has also been localized in the nucleus of mammalian cells. To learn more about nuclear 4.1 protein, we have analyzed the nature of its association with the nuclear structure in comparison with SC35 and snRNP antigens, splicing proteins of the nuclear speckle domains. When MDCK or HeLa cells were digested with DNase I and washed in the presence of high salt (2 M NaCl), snRNP antigens were extracted whereas protein 4.1 and SC35 remained colocalizing in nuclear speckles. In cells treated with RNase A or heat shocked, nuclear 4.1 distribution also resembled that of SC35. Experiments carried out in transcriptionally active nuclei showed that protein 4.1 distributed in irregularly shaped speckles which appeared to be interconnected. During transcriptional inhibition, protein 4.1 accumulated in rounded speckles lacking interconnections. When cells were released from transcriptional inhibition, protein 4.1 redistributed back to the interconnected speckle pattern of transcriptionally active cells, as it was also observed for SC35. Finally, coprecipitation of 4.1 and SC35 proteins from RNase A digested HeLa nuclei further indicates that these two proteins are associated, forming part of the nuclear speckle domains to which they attach more tightly than snRNP antigens.
Localisation of the Schizosaccharomyces pombe rho1p GTPase and its involvement in the organisation of the actin cytoskeleton
Authors: Arellano Manuel , Duran Angel , Perez Pilar
Published: April 2021

The Schizosaccharomyces pombe rholp GTPase directly activates the (1-3)β-D-glucan synthase and participates in the regulation of cell wall growth and morphogenesis in this fission yeast. Indirect immunofluorescence experiments using rho1p tagged with hemagglutinin have revealed that rho1p was located at the growing tips during interphase and at the septum prior to cytokinesis, localising to the same areas as actin patches. In S. pombe cdc10-129 mutant cells, arrested in G1, HA-rho1p accumulates at one tip whereas in cdc25-22 mutants, arrested in G2, HA-rho?p accumulates at both tips. In tea1-1 and tea2-1 cdc11-119 mutant cells, HA-rho?p is localised to the new growing tips. Overexpression of different rhol mutant alleles caused different effects on cortical actin patch distribution, (1-3)β-D-glucan synthase activation, and sensitivity to cell wall specific antifungal drugs. These results indicate that multiple cellular components are activated by rholp. Overexpression of the dominant negative rhol?20N allele was lethal as was the rho1+ deletion. Moreover, when rho1+ expression was repressed in actively growing S. pombe, cells died in about 10 to 12 hours. Under these conditions, normal cell morphology was maintained but the level of (1-3)β-D-glucan synthase activity decreased and the actin patches disappeared. Most cells lysed after cytokinesis during the process of separation, and lysis was not prevented by an osmotic stabiliser. We conclude that rho1p localisation is restricted to growth areas and regulated during the cell cycle and that rho1p is involved in cell wall growth and actin cytoskeleton organisation in S. pombe.
Extinction of immunoglobulin gene expression in B cells upon fusion with HeLa cells is preceded by rapid nuclear depletion of essential transcription factors and is accompanied by widespread inactivation of genes expressed in a B cell-specific manner
Authors: Junker Steffen , Lamm Martin , Nielsen Viggo , Matthias Patrick
Published: April 2021

When immunoglobulin (Ig) expressing B cells are fused with non-B cells, Ig expression is rapidly suppressed at the level of transcription, a phenomenon termed extinction. Here we demonstrate that fusion of HeLa cells with either diploid or tetraploid B cells (Daudi) results in widespread extinction of several other B cell-encoded genes that are expressed in a B cell-specific manner. In contrast, expression of B cell-expressed genes that are not dependent on cell-specific controls is unaffected. We show that the molecular mechanism(s) underlying Ig gene extinction can be explained, at least in part, by a lack of transcription factors that are essential for Ig gene transcription. These transcription factors are either not produced due to block of transcription of their respective genes (Oct-2, OBF-1, PU.1), or are rendered inactive posttranslationally (NF-κB, E47). By isolating Daudi × HeLa heterokaryons a few hours after fusion, we have studied the initial fate of two B cellspecific transcription factors involved in Ig gene transcription, Oct-2 and NF-κB. This report provides the first demonstration that upon fusion with HeLa cells, the nuclear contents of B cell-expressed transcription factors are depleted within a few hours with kinetics that are as fast or faster than that of Ig gene extinction. Thus, the extinguishing mechanism is effective very early after fusion. We suggest that extinction of Ig genes is part of a global mechanism that suppresses the differentiation program foreign to the HeLa phenotype.
Commitment to differentiation and cell cycle re-entry are coincident but separable events in the transformation of African trypanosomes from their bloodstream to their insect form
Authors: Matthews Keith R. , Gull Keith
Published: April 2021

African trypanosomes undergo extensive changes in cellular morphology, biochemistry and surface antigen expression as they differentiate from their bloodstream form to those forms that colonise the midgut of their tsetse fly vector. If initiated with stumpy-form cells, a nondividing sub-type of the bloodstream parasite, differentiation and cell cycle re-entry occur synchronously in the population and provide a means to dissect the respective controls of proliferation and transformation. We have exploited this synchrony to determine the respective importance and hierarchy of the known triggers for differen-tiation (cis aconitate, temperature drop) for individual components of both differentiation and the cell cycle. This has revealed the pre-eminence of cis aconitate as a primary trigger for parasite differentiation, and has allowed us to determine that the cellular commitment to both differentiation and cell-cycle re-entry are precisely co-incident processes
To bead or not to bead? Lens-specific intermediate filaments revisited
Authors: Georgatos Spyros D. , Gounari Fotini , Goulielmos George , Aebi Ueli
Published: April 2021

For nearly three decades cytoplasmic intermediate filaments (IFs) have been described as 10 nm thick, unbranched ropes radiating from the cell nucleus and extending to the plasma membrane. This stereotype is now being challenged by the discovery and molecular characterization of the beaded filaments (BFs), a novel class of IFs composed of the lens-specific proteins filensin and phakinin. In contrast to ‘mainstream’ IFs, BFs have a distinctly nodular appearance and form a meshwork underneath the plasma membrane of the lens fiber cells. In vitro assembly studies, expression of filensin and phakinin in cultured cells, and analysis of the corresponding genes reveal that these proteins have evolved from two different subfamilies of IF proteins, thus yielding a unique structure. The new information provides a basis for understanding how the various forms of tissue-specific IF proteins might have developed adopting to the constraints of a specialized environment.
Distinct mechanisms of nuclear accumulation regulate the functional consequence of E2F transcription factors
Authors: Allen K. Elizabeth , Luna Susana de la , Kerkhoven Ron M. , Bernards René , Thangue Nicholas B. La
Published: April 2021

Transcription factor E2F plays an important role in co-ordinating and integrating early cell cycle progression with the transcription apparatus. It is known that physiological E2F arises when a member of two families of proteins, E2F and DP, interact as E2F/DP heterodimers and that transcriptional activity is regulated through the physical association of pocket proteins such as pRb. However, little information is available regarding the mechanisms which control the levels of functional E2F. In this study, we have characterised one such mechanism which regulates the nuclear accumulation and activity of E2F. Specifically, we show that E2F proteins fall into two distinct categories according to their ability to accumulate in nuclei, one being exemplified by E2F-1 and the other by E2F-4 and -5. Thus, E2F-1 possesses an intrinsic nuclear localization signal whereas E2F-4 and -5 are devoid of such a signal. Furthermore, we find for E2F-4 and -5 that two distinct processes govern their nuclear accumulation whereby the nuclear localization signal is supplied in trans from either a DP heterodimer partner or a physically associated pocket protein. It is consistent with the role of pocket proteins in regulating nuclear accumulation that we find E2F-5 to be nuclear during early cell cycle progression with an increased cytoplasmic concentration in cycling cells. Our data show that the mechanism of nuclear accumulation determines the functional consequence of E2F on cell cycle progression: pocket protein-mediated accumulation impedes cell cycle progression, whereas DP-regulated nuclear accumulation promotes cell cycle progression. Moreover, the inactivation of pocket proteins by the adenovirus E1a protein, and subsequent release of E2F, failed to displace nuclear E2F. Our study identifies a new level of regulation in the control of E2F activity exerted at the level of nuclear accumulation where subunit composition and interaction with pocket proteins dictates the functional consequence on cell cycle progression.
Nuclear export signal of IκBα interferes with the Rev-dependent posttranscriptional regulation of human immunodeficiency virus type I
Authors: Bachelerie Françoise , Rodriguez Manuel S. , Dargemont Catherine , Rousset Dominique , Thomas Dominique , Virelizier Jean-Louis , Arenzana-Seisdedos Fernando
Published: April 2021

De novo synthesized IκBα accumulates transiently in the nucleus where it inhibits NF-κB-dependent transcription and reduces nuclear NF-κB content. A sequence present in the C-terminal domain of IκBα and homologous to the HIV-1 Rev nuclear export signal (NES) has been recently defined as a functional NES conferring on IκBα the ability to export IκBα/NF-κB complexes. Rev utilises its RNA-binding activity and NES sequence to promote specifically the transport of unspliced and monospliced viral RNAs to the cytoplasm. The object of this work was to determine if nuclear IκBα could interfere with Revdependent transport of viral RNA from the nucleus to the cytoplasm. We report that accumulation of IκBα in the cell nucleus blocks viral replication. This effect could be dissociated from the capacity of IκBα to inhibit NF-κB-DNA-binding activity and required a functional IκBα NES motif. Indeed, mutation of the NES abrogated the capacity of IκBα to inhibit Rev-dependent mechanisms involved in the replication of either wild-type or NF-κB-mutated HIV-1 molecular clones. Nuclear accumulation of a reporter protein tagged with a nuclear localization signal (NLS) and fused to the IκBα NES motif (NLS-PK-NES) was sufficient to inhibit HIV-1 replication at a post-transcriptional level by specifically blocking the expression of a Rev-dependent gene. Furthermore, in cells pulsed with TNF, a treatment which favors nuclear accumulation of newly synthesized IκBα, NLS-PK-NES expression promoted sustained accumulation of nuclear NF-κB lacking DNA-binding activity. This NES-mediated accumulation of inactive nuclear NF-κB is likely the consequence of interference in the IκBα-mediated export of NF-κB. These findings indicate that IκBα and Rev compete for the same nuclear export pathway and suggest that nuclear accumulation of IκBα, which would occur during normal physiological cell activation process, may interfere with the Rev-NES-mediated export pathway of viral RNAs, thus inhibiting HIV-1 replication.
Contact guidance of CNS neurites on grooved quartz: influence of groove dimensions, neuronal age and cell type
Authors: Rajnicek Ann M. , Britland Stephen , McCaig Colin D.
Published: April 2021

We used an in vitro system that eliminates competing guidance cues found in embryos to determine whether sub-stratum topography alone provides important neurite guidance information. Dissociated embryonic Xenopus spinal cord neurons and rat hippocampal neurons were grown on quartz etched with a series of parallel grooves. Xenopus neurites grew parallel to grooves as shallow as 14 nm and as narrow as 1 μm. Hippocampal neurites grew parallel to deep, wide grooves but perpendicular to shallow, narrow ones. Grooved substrata determined the sites at which neurites emerged from somas: Xenopus neurites sprouted from regions parallel to grooves but presumptive axons on rat hippocampal neurons emerged perpendicular to grooves and presumptive dendrites emerged parallel to them. Neurites grew faster in the favored direction of orientation and turned through large angles to align on grooves. The frequency of perpendicular alignment of hippocampal neurites depended on the age of the embryos from which neurons were isolated, suggesting that contact guidance is regulated in development. Collectively, the data indicate that substratum topography is a potent morphogenetic factor for developing CNS neurons and suggest that in addition to a role in pathfinding the geometry of the embryo assists in establishing neuronal polarity. In the companion paper (A. M. Rajnicek and C. D. McCaig (1997) J. Cell Sci. 110, 2915-2924) we explore the cellular mechanism for contact guidance of growth cones.
Guidance of CNS growth cones by substratum grooves and ridges: effects of inhibitors of the cytoskeleton, calcium channels and signal transduction pathways
Authors: Rajnicek Ann M. , McCaig Colin D.
Published: April 2021

We exploited our observation that embryonic Xenopus spinal neurites align parallel to grooves in a quartz surface and that embryonic rat hippocampal neurites align perpendicular to shallow, narrow grooves (see companion paper: A. M. Rajnicek, S. Britland and C. D. McCaig, 1997) (J. Cell Sci. 110, 2905-2913) to investigate the mechanism of growth cone contact guidance. Substratum topography affected the pattern of growth cone filopodia and microtubules but parallel orientation of Xenopus neurites and perpendicular orientation of hippocampal neurites were unperturbed by cytochalasin B, which virtually eliminated filopodia. Hippocampal growth cone orientation and turning in response to grooves was unaffected by disruption of microtubules using taxol or nocodazole. Gross cytoskeletal reorganization on grooved substrata was therefore not required for growth cone steering.

Inhibitors were used to identify the signal transduction pathway for perpendicular alignment of hippocampal neurites. Alignment persisted in the presence of gadolinium chloride, a blocker of stretch-activated calcium channels, the G protein inhibitor pertussis toxin, the protein tyrosine kinase inhibitor genistein, the protein kinase A and G inhibitor HA1004, the protein kinase A inhibitor KT5720and the protein kinase G inhibitor KT5823. Low concentrations of the protein kinase C inhibitors staurosporine, bisindolylmaleimide or H-7 did not affect perpendicular orientation but higher concentrations inhibited it. The calcium channel blockers flunarizine, nifedipine and diltiazem also inhibited perpendicular orientation. Influx of calcium and protein kinase C activity therefore appear to be involved in perpendicular contact guidance.
A heterotrimeric G protein-phospholipase A2 signaling cascade is involved in the regulation of peroxisomal motility in CHO cells
Authors: Huber Christoph M. , Saffrich Rainer , Anton Markus , Paßreiter Michael , Ansorge Wilhelm , Gorgas Karin , Just Wilhelm W.
Published: April 2021

Peroxisomal motility was studied in vivo in CHO cells following transfection with a green fluorescent protein construct containing the C-terminal peroxisomal targeting signal 1 (GFP-PTS1). Time-lapse imaging and evaluation of difference images revealed that peroxisomes attach to microtubules in a Ca2+ requiring step and are transported in an ATP-dependent manner.

Following microinjection of guanosine-5′-O-(3-thiotriphosphate) (GTPγS), peroxisomal movements were arrested, indicating regulation by GTP-binding proteins. The effect of GTPγS was mimicked by AlF4− and mastoparan, two drugs which are known to activate heterotrimeric G proteins. Pertussis toxin which prevents Gi/Go protein activation completely abolished the effect of GTPγS and mastoparan on peroxisomal motility suggesting that the G protein belongs to the Gi/Go class. At least one effector of the G protein is phospholipase A2 as demonstrated by the observation that the phospholipase A2 activating protein peptide efficiently blocks peroxisomal motility, and that the effect of mastoparan and AlF4− is largely abolished by various phospholipase A2 inhibitors.

In summary, these data provide evidence for a new type of regulation of organelle motility mediated by a Gi/Go- phospholipase A2 signaling pathway. This type of regulation has not been observed so far with other cell organelles such as mitochondria, the endoplasmic reticulum or axonal vesicles. Thus, motility is regulated individually for each cell organelle by distinct mechanisms enabling the cell to fulfill its vital functions.
2′,3′-Cyclic nucleotide 3′-phosphodiesterase is associated with mitochondria in diverse adrenal cell types
Authors: McFerran Brian W. , Burgoyne Robert D.
Published: April 2021

In this study, we have examined the expression and intracellular localisation of the myelin protein 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP) in bovine adrenal medullary chromaffin cell cultures. By immunoblotting, using two distinct anti-CNP monoclonal antibodies, CNP was detected in medullary cell cultures and expression of CNP was confirmed by reverse transcription and PCR amplification. CNP did not leak from digitonin-permeabilised chromaffin cells, suggesting that there is no cytosolic pool of this protein. Immunofluorescence studies with both antibodies showed that all cells in the medullary chromaffin cell culture were stained with a punctate appearance consistent with an intracellular localisation for CNP. More specifically it was demonstrated that CNP is colocalised with mitochondria. Various cell types in chromaffin cell cultures were stained with a mitochondrial pattern and CNP staining was co-localised with mitochondrial staining. These results show that CNP is a widely expressed protein that is associated with mitochondria and provides new clues as to its cellular function outside of myelin structures.
Ezrin: A protein requiring conformational activation to link microfilaments to the plasma membrane in the assembly of cell surface structures
Authors: Bretscher Anthony , Reczek David , Berryman Mark
Published: April 2021

The cortical cytoskeleton of eucaryotic cells provides structural support to the plasma membrane and also contributes to dynamic processes such as endocytosis, exocytosis, and transmembrane signaling pathways. The ERM (ezrinradixin-moesin) family of proteins, of which ezrin is the best studied member, play structural and regulatory roles in the assembly and stabilization of specialized plasma membrane domains. Ezrin and related molecules are concentrated in surface projections such as microvilli and membrane ruffles where they link the microfilaments to the membrane.

The present knowledge about ezrin is discussed from an historical perspective. Both biochemical and cell biological studies have revealed that ezrin can exist in a dormant con-formation that requires activation to expose otherwise masked association sites. Current results indicate that activated ezrin monomers or head-to-tail oligomers associate directly with F-actin through a domain in its C terminus, and with the membrane through its N-terminal domain. The association of ezrin with transmembrane proteins can be direct, as in the case of CD44, or indirect through EBP50. Other binding partners, including the regulatory subunit of protein kinase A and rho-GDI, suggest that ezrin is an integral component of these signaling pathways. Although the membrane-cytoskeletal linking function is clear, further studies are necessary to reveal how the activation of ezrin and its association with different binding partners is regulated.
Dynamics of connexins, E-cadherin and α-catenin on cell membranes during gap junction formation
Authors: Fujimoto Kazushi , Nagafuchi Akira , Tsukita Shoichiro , Kuraoka Akio , Ohokuma Akiko , Shibata Yosaburo
Published: April 2021

We examined the dynamics of connexins, E-cadherin and α-catenin during gap-junction disassembly and assembly in regenerating hepatocytes by immunofluorescence microscopy, and immunogold-electron microscopy using SDS-digested freeze-fracture replicas. The present findings suggest that during the disappearance of gap junctions most of the gap junction plaques are broken up into smaller aggregates, and then the gap junction proteins may be removed from the cell membrane, but some of the connexons or connexins remain dispersed in the plane of membrane as pure morphologically indistinguishable intramembrane proteins. Double-immunogold electron microscopy using a polyclonal antibody for connexins and a monoclonal antibody for E-cadherin or α-catenin revealed co-localization of these molecules at cell-to-cell contact sites during the reappearance of gap junction plaques. This implies that, at least in regenerating hepatocytes, the cadherin-catenin complex-mediated cell-to-cell contact sites act as foci for gap junction formation. In addition, connexin-immunoreactivity was also observed along tight junctional strands, suggesting that the gap junction may also form along the tight junctions.
Differentiation of U-937 promonocytic cells by etoposide and ICRF-193, two antitumour DNA topoisomerase II inhibitors with different mechanisms of action
Authors: Pérez Concepción , Vilaboa Nuria E. , García-Bermejo Laura , Blas Elena de , Creighton Andrew M. , Aller Patricio
Published: April 2021

We have compared the action on U-937 human promonocytic leukemia cells of two DNA topoisomerase II inhibitors, namely the epipodophyllotoxin etoposide and the bisdioxopiperazine ICRF-193. One hour pulsetreatment with 3 µM etoposide caused topoisomerase-associated, primary DNA breakage, which was rapidly followed by apoptosis. By contrast, these effects were not observed upon pulse-treatment with 6 µM ICRF-193. However, continuous treatments with subcytotoxic concentrations of etoposide (0.15 µM) and ICRF-193 (0.3 µM) produced several similar effects, namely decreased cell proliferation, accumulation of cells at G2, increase in cell mass, and induction of differentiation. Under these conditions, etoposide produced a biphasic activation of protein kinase C, which consisted in an early transient activation (from hours 1 to 6) of the membrane-bound enzyme followed by a later activation (hour 48) of the total, membrane-bound and cytosolic enzyme. By contrast, ICRF-193 only provoked a late activation (from hours 72 to 96) of the total enzyme. When used at differentiation-inducing concentrations, both topoisomerase inhibitors caused a great stimulation of AP-1 binding activity, with maximum value at hour 12 in etoposide-treated cells and at hour 48 in ICRF-193-treated cells. By contrast, the binding activity of the NF-κB and EGR-1 transcription factors was little affected. It is concluded that topoisomerase II inhibitors may induce the differentiation of promonocytic cells, independently of their capacity to cause DNA strand breaks. However, there are other effects, such as the early activation of protein kinase C, which are probably derived from the production of primary DNA breakage by some anti-topoisomerase drugs.
Focal adhesion kinase: at the crossroads of signal transduction
Authors: Ilic Duško , Damsky Caroline H. , Yamamoto Tadashi
Published: April 2021

Morphogenetic processes during development, including cell migration, depend on signals from both the extracellular matrix (ECM) and soluble signaling factors. Extensive evidence has shown that the nonreceptor tyrosine kinase, focal adhesion kinase (FAK), is activated in response to both kind of signal. The most definitive evidence that FAK is directly downstream of signals initiated by the ECM comes from comparing the phenotypes of mice deficient for FAK and the ECM molecule, fibronectin: in both cases embryos die at about E8.5 and display almost identical severe vascular and other mesodermal defects. It is now clear that there are additional FAK-like proteins, indicating the existence of a FAK family. Furthermore, FAK is not located at adhesive sites in all cells where it is expressed. This, plus extensive data indicating that FAK becomes activated in response to several soluble signaling factors, suggests that the FAK family may be at the crossroads of multiple signaling pathways that affect cell and developmental processes.
Subpellicular and flagellar microtubules of Trypanosoma brucei are extensively glutamylated
Authors: Schneider André , Plessmann Uwe , Weber Klaus
Published: April 2021

To determine the spectrum of tubulin variants in cytoskeletons of Trypanosoma brucei carboxy-terminal fragments of α- and β-tubulin were isolated and characterized by sequencing and mass spectrometry. All variants arise by posttranslational modifications. We confirm the presence of tyrosinated and detyrosinated α-tubulin. Unexpectedly, but in line with its sequence, β-tubulin also occurs with and without its carboxy-terminal tyrosine. Both tyrosinated and detyrosinated α- and β-tubulins are extensively glutamylated. Unglutamylated tubulins are only trace components of the cytoskeletal microtubules. The maximal numbers of glutamyl residues in the lateral chain are 15 and 6 for α- and β-tubulin, respectively. The oligoglutamyl side chain is linked via an isopeptide bond to glutamic acid residues 445 of α- and 435 of β-tubulin. The same sites are used in glutamylated tubulins of mammalian brain. No tubulin variants based on polyglycylation are detected in cytoskeletal preparations or in isolated flagella. Tubulin specific incorporation of radioactive glutamate but not of glycine is observed when protein biosynthesis is completely inhibited in Trypanosoma cells. Possible reasons for the absence of polyglycylated tubulins from the trypanosomal axoneme are discussed. Finally we show that lysine 40 of the flagellar α-tubulin is completely acetylated.
How do injured cells communicate with the surviving cell monolayer?
Authors: Sammak Paul J. , Hinman Lee E. , Tran Phuong Oanh T. , Sjaastad Michael D. , Machen Terry E.
Published: April 2021

Mechanically scratching cell monolayers relieves contact inhibition and induces surviving cells near the wound edge to move and proliferate. The present work was designed to test whether surviving cells passively respond to newly available space, or whether cells are actively stimulated by signals from injured cells nearby. We monitored intracellular free Ca2+ ([Ca2+]i) while scratching confluent monolayers of bovine pulmonary endothelial cells and mouse mammary epithelial cells. Within seconds after wounding, a transient elevation of [Ca2+]ɪ was observed in surviving cells. In endothelial cells, the [Ca2+]ɪ elevation propagated into the monolayer for a distance of 10 to 12 cell rows at a speed of 20 to 28 µm/second. The amplitude of the wave of [Ca2+]i was reduced as it propagated into the monolayer, but the velocity of the wave was nearly constant. Cells that experienced the [Ca2+]i elevation had intact plasma membranes, and survived for over 24 hours post wounding. Removing extracellular Ca2+ decreased the amplitude by two-thirds and reduced the propagation rate by half, suggesting that Ca2+ influx contributed to the increased [Ca2+]i. To determine how [Ca2+]| waves were stimulated, we blocked extracellular communication by fluid perfusion or intercellular communication by breaks in the monolayer.

In bovine pulmonary artery endothelial cultures, the [Ca2+]i wave passed over breaks in the monolayer, and was prevented from traveling upstream in a perfusion chamber. Conditioned media from injured cells also elevated [Ca2+]i in unwounded reporter cultures. In mouse mammary epithelial monolayers with established cell-cell contacts, the [Ca2+]i wave passed over breaks in the monolayer, but was only partially prevented from traveling upstream during perfusion. These experiments showed that mechanical wounds lead to long distance, [Ca2+]i-dependent communication between the injured cells and the surviving cell monolayer through at least two mechanisms: first, extracellular release of a chemical stimulus from wounded cells that diffused to neighboring cells (present in both monolayers); second, transmission of an intercellular signal through cell-cell junctions (present in the mammary epithelial monolayers). Thus, mechanical injury provided a direct, chemical stimulus to nearby cells which have not themselves been damaged.
Splicing factors associate with nuclear HCMV-IE transcripts after transcriptional activation of the gene, but dissociate upon transcription inhibition: evidence for a dynamic organization of splicing factors
Authors: Dirks Roeland W. , Pauw Elmar S. D. de , Raap Anton K.
Published: April 2021

Before being transported to the cytoplasm, intron-containing pre-mRNAs have to be spliced somewhere in the cell nucleus. Efficient splicing requires an ordered assembly of splicing factors onto the pre-mRNAs. To accomplish this, intron containing genes may be preferentially localized at nuclear sites enriched for splicing factors or alternatively, splicing factors may circulate throughout the nucleus and have the ability to associate with randomly positioned nascent transcripts. Combined detection of HCMV-IE mRNA/DNA and splicing factors in rat 9G cells that can be induced for IE gene expression shows that IE genes are not associated with speckled regions enriched for splicing factors when transcriptionally inactive, but ‘attract’ splicing factors when transcriptionally activated. This process proved reversible after transcription inhibition. IE transcripts appeared to be retained near the transcription site in tracklike domains by splicing factors associated with them until splicing has been completed. Double-hybridization experiments revealed that a substantial part of the accumulated transcripts contain a poly(A) tail suggesting that most, if not all, IE transcripts are polyadenylated at the site of transcription. These results indicate that RNA processing may occur independent of the position of the gene in the cell nucleus relative to speckle domains.
Zona pellucida glycoprotein mZP3 bioactivity is not dependent on the extent of glycosylation of its polypeptide or on sulfation and sialylation of its oligosaccharides
Authors: Liu C. , Litscher E.S. , Wassarman P.M.
Published: April 2021

During fertilization in mice, free-swimming sperm bind to mZP3, one of three egg zona pellucida glycoproteins. Sperm recognize and bind to specific serine/threonine-linked (O-linked) oligosaccharides located at the mZP3 combining site for sperm. Shortly after binding to mZP3, sperm undergo the acrosome reaction, a form of cellular exocytosis. Here, we examined the influence of extent of glycosylation, sulfation, and sialylation of mZP3 (M(r) approximately 65,000-100,000) on its bioactivity; i.e. its ability to inhibit binding of sperm to eggs and to induce the acrosome reaction in vitro. Low (av. M(r) approximately 70,000), medium (av. M(r) approximately 82,000), and high (av. M(r) approximately 94,000) M(r) fractions of mZP3 were purified and shown to vary in extent of asparagine-linked (N-linked) glycosylation. All three size-fractions exhibited bioactivity, suggesting that the ability of mZP3 to inhibit binding of sperm to eggs is not related to the extent of glycosylation of its polypeptide (M(r) approximately 44,000). Digestion of mZP3 by neuraminidase decreased its average M(r) from approximately 83,000 to approximately 77,000 and increased its average pI from approximately 4.7 to approximately 6.0, but did not significantly affect mZP3 bioactivity. Terminal sialic acid largely accounts for the glycoprotein's acidic nature, but is not an essential element of the mZP3 combining site for sperm. Experiments with stably transfected embryonal carcinoma (EC) cells that secrete bioactive EC-mZP3 revealed that, of the sulfate present, approximately 70–75% was located on N-linked and approximately 25–30% on O-linked oligosaccharides. EC-mZP3 devoid of sulfate inhibited binding of sperm to eggs and induced the acrosome reaction to the same extent as sulfated EC-mZP3. These results suggest that sulfation of EC-mZP3 oligosaccharides is not essential for bioactivity. Overall, these findings contrast with those reported for certain other glycoproteins involved in cellular adhesion that require sulfate and/or sialic acid for bioactivity.
The amphipathic alpha helices of the Toxoplasma protein GRA2 mediate postsecretory membrane association
Authors: Mercier Corinne , Cesbron-Delauw Marie-France , Sibley L. David
Published: April 2021

The Toxoplasma gondii protein GRA2 is secreted into the parasite-containing vacuole where it is rapidly and specifically targeted to a network of membranous tubules that connect with the vacuolar membrane. To examine the molecular basis of this association, we expressed an HA9 epitope-tagged form of GRA2 by stable transformation of Toxoplasma. GRA2-HA9 was correctly packaged inside the dense granules, secreted into the PV and targeted to the network, as shown by immunoelectron microscopy, immunofluorescence and cell fractionation. Expression of deletion mutants of GRA2-HA9 lacking either of two amphipathic alpha helices resulted in the production and secretion of soluble proteins which were unable to stably associate with the network. A mutant in which the amino acids of the first alpha helix were rearranged to a nonamphipathic pattern localized correctly to the network but failed to remained stably associated with the membrane. Collectively, these results demonstrate that targeting and membrane association occur by separate mechanisms and that the combination of both alpha helices is essential for stable localization of GRA2 to the network.
The integrin αvβ6 is critical for keratinocyte migration on both its known ligand, fibronectin, and on vitronectin
Authors: Huang Xiaozhu , Wu Jianfeng , Spong Suzanne , Sheppard Dean
Published: April 2021

The integrin αvβ6 is expressed on a variety of epithelial cells during dynamic processes including organogenesis, tissue injury and malignant transformation. However, because of the lack of tools to specifically inhibit the function of this integrin, little is known about its effects on cell behavior. To directly examine the role of this integrin in cell migration, we used keratinocytes derived from wildtype mice or mice expressing a null mutation in the β6 subunit (β6−/−) to perform migration assays in vitro. Migration on the known αvβ6 ligand, fibronectin was reduced in keratinocytes from β6−/−mice. Interestingly, keratinocytes from β6−/− mice also demonstrated markedly reduced migration on vitronectin, a protein not previously known to be a ligand for αvβ6. An anti-αvβ6 monoclonal antibody 10D5, generated by immunization of β6−/− mice with murine keratinocytes, inhibited adhesion and migration of wild-type keratinocyte on both vitronectin and fibronectin to levels similar to those seen with keratinocytes from β6−/−mice. αvβ6-mediated migration on both ligands was dramatically augmented by treatment with phorbol myrisate acetate (PMA) or with hepatocyte growth factor, and augmentation of migration by either stimulus could be abolished by the PKC inhibitor GF109203X, suggesting a critical role for PKC in enhancement of αvβ6-mediated cell migration.
Localization of endogenous ARF6 to sites of cortical actin rearrangement and involvement of ARF6 in cell spreading
Authors: Song Jia , Khachikian Zareh , Radhakrishna Harish , Donaldson Julie G.
Published: April 2021

To study the function of the endogenous ARF6 GTP binding protein in cells, we generated an antibody which specifically recognizes ARF6, and not the other ARF proteins. Using this antibody, ARF6 was detected in all mouse organs tested and in a variety of cultured cell lines including RBL, MDCK, NRK, BHK, COS, and HeLa cells. In NRK cells, by immunofluorescence, ARF6 localized to the plasma membrane, especially at regions exhibiting membrane ruffling, and was also concentrated in a fine punctate distribution in the juxtanuclear region. This pattern of localization of the endogenous protein was similar to the localization of ARF6 when overexpressed in NRK, or HeLa, cells. Treatments which perturb cortical actin in NRK cells, such as replating of cells after trypsinization or treatment with phorbol ester, resulted in the recruitment of endogenous ARF6 to the regions of cortical actin rearrangement. ARF6 activation and subsequent membrane recycling was required for cell spreading activity since expression of the dominantnegative, GTP-binding defective mutant of ARF6, T27N, previously shown to inhibit ARF6-regulated membrane recycling, inhibited cell attachment and spreading in HeLa cells. Furthermore, phorbol ester treatment enhanced the cell spreading activities in NRK cells, and in HeLa cells, but was not observed in cells expressing T27N. Taken together, these observations support a role for endogenous ARF6 in modeling the plasma membrane and cortical actin cytoskeleton.
Subcellular targeting and cytoskeletal attachment of SAP97 to the epithelial lateral membrane
Authors: Wu Hongju , Reuver Susanne M. , Kuhlendahl Sven , Chung Wook Joon , Garner Craig C.
Published: April 2021

The synapse-associated protein SAP97 is a member of a novel family of cortical cytoskeletal proteins involved in the localization of ion channels at such membrane specializations as synaptic junctions. These multidomain proteins have binding sites for protein 4.1, GKAPs/SAPAPs, voltage- and ligand-gated ion channels and cell-adhesion molecules containing C-terminal T/SXV motifs. In this study, we evaluated the contribution of individual domains in SAP97 to its selective recruitment and attachment to the cortical cytoskeleton in epithelial cells. We find that the PDZ, SH3 and GK domains, as well as the I3 insert in SAP97, are not essential for subcellular targeting, though both PDZ1-2 domains and the I3 insert affect the efficiency of localization. Instead, we show that the first 65 amino acid residues in SAP97, which are absent from SAP90/PSD-95 and SAP102, direct the selective subcellular localization and can mediate at least one point of attachment of SAP97 to the cytoskeleton assembled at sites of cell-cell contact. Our data demonstrate that it is the sequences unique to SAP97 that direct its subcellular targeting to the epithelial lateral membrane.
Desmoglein 3 anchors telogen hair in the follicle
Authors: Koch Peter J. , Mahoney M. G. , Cotsarelis George , Rothenberger Kyle , Lavker Robert M. , Stanley John R.
Published: April 2021

Little is known about the function of desmosomes in the normal structure and function of hair. Therefore, it was surprising that mice without desmoglein 3 (the autoantigen in pemphigus vulgaris) not only developed mucous membrane and skin lesions like pemphigus patients, but also developed hair loss. Analysis of this phenotype indicated that hair was normal through the first growth phase (‘follicular neogenesis’). Around day 20, however, when the hair follicles entered the resting phase of the hair growth cycle (telogen), mice with a targeted disruption of the desmoglein 3 gene (DSG3−/−) lost hair in a wave-like pattern from the head to the tail. Hair then regrew and was lost again in the same pattern with the next synchronous hair cycle. In adults, hair was lost in patches. Gentle hair pulls with adhesive tape showed that anagen (growing) hairs were firmly anchored in DSG3−/− mice, but telogen hairs came out in clumps compared to that of DSG3+/− and +/+ littermates in which telogen hairs were firmly anchored. Histology of bald skin areas in DSG3−/− mice showed cystic telogen hair follicles without hair shafts. Histology of hair follicles in early telogen, just before clinical hair loss occurred, showed loss of cell adhesion (acantholysis) between the cells surrounding the telogen club and the basal layer of the outer root sheath epithelium. Electron microscopy revealed ‘half-desmosomes’ at the plasma membranes of acantholytic cells. Similar acantholytic histology and ultrastructural findings have been previously reported in skin and mucous membrane lesions of DSG3−/− mice and pemphigus vulgaris patients. Immunoperoxidase staining with an antibody raised against mouse desmoglein 3 showed intense staining on the cell surface of keratinocytes surrounding the telogen hair club in normal mice. Similar staining was seen in human telogen hair with an anti-human desmoglein 3 antibody. Finally, a scalp biopsy from a pemphigus vulgaris patient showed empty telogen hair follicles. These data demonstrate that desmoglein 3 is not only critical for cell adhesion in the deep stratified squamous epithelium, but also for anchoring the telogen hair to the outer root sheath of the follicle and underscore the importance of desmosomes in maintaining the normal structure and function of hair.
Sensory nerves affect the recruitment and differentiation of rat periovarian brown adipocytes during cold acclimation
Authors: Giordano Antonio , Morroni Manrico , Carle Flavia , Gesuita Rosaria , Marchesi Gian Franco , Cinti Saverio
Published: April 2021

Rat periovarian adipose tissue contains a low number of uncoupling protein-expressing brown adipocytes scattered into lobules of white fat. Their increase following cold acclimation is matched by a major increase in noradrenergic and neuropeptide Y-, substance P- and calcitonin gene-related peptide-containing nerves. To ascertain whether periovarian fat is provided with sensory nerves, and whether any relationship exists between such nerves (in particular the calcitonin gene-related peptide-containing fibers found in cold-acclimated rats in close association with brown adipocytes) and brown fat recruitment, the effects of capsaicin desensitization on neuropeptide-containing nerves and brown adipocyte density were studied in the periovarian tissue of rats kept at 20°C and on a group acclimated to 4°C for 14 days. In both groups, systemic capsaicin administration considerably reduced the expression of substance P and calcitonin gene-related peptide in vascular-nerve bundles and parenchyma. In cold-acclimated rats, the increase in brown adipocyte density was significantly checked by capsaicin administration (21.11 versus 7.96 brown adipocytes/mm2, P<0.05). Finally, ultrastructural investigation showed the occurrence of brown adipocyte precursors filled with aggregates of glycogen and poorly differentiated multilocular adipocytes in capsaicin-treated cold-acclimated rats. These data suggest that periovarian adipose tissue is indeed provided with sensory neuropeptide-containing nerves and that they play a role in the recruitment and differentiation of brown adipocytes.
The putative sponge aggregation receptor: Isolation and characterization of a molecule composed of scavenger receptor cysteine-rich domains and short consensus repeats
Authors: Blumbach Barbara , Pancer Zeev , Diehl-Seifert Bärbel , Steffen Renate , Münkner Jessica , Müller Isabel , Müller Werner E. G.
Published: April 2021

Porifera (sponges) are the oldest extant metazoan phylum. Dissociated sponge cells serve as a classic system to study processes of cell reaggregation. The reaggregation of dissociated cells is mediated by an extracellularly localized aggregation factor (AF), based on heterophilic interactions of the third order; the AF bridges two cells by ligating a cell-surface-bound aggregation receptor (AR). In the present study we report cloning, expression and immunohistochemical localization of a polypeptide from the marine sponge Geodia cydonium, which very likely represents the AR. The presumed AR gene gives rise to at least three forms of alternatively spliced transcripts of 6.5, 4.9 and 3.9 kb, as detected by northern blotting. Two cDNA clones corresponding to the shorter forms were already reported earlier; here we present an analysis of the largest. All three putative polypeptides feature scavenger receptor cysteine-rich (SRCR) domains. The largest form, SRCR-SCR-Car, is a cell-surface receptor of molecular mass 220 kDa, which is assumed to be the cell-adhesion receptor AR; the second form, SRCR-Re, is also a putative receptor of 166 kDa, while the third form, SRCR-Mo, is a soluble molecule of 129 kDa. The SRCR-SCR-Car molecule consists of fourteen SRCR domains, six short consensus repeats (SCRs), a C-terminal transmembrane domain and a cytoplasmic tail; its fourteenth SRCR domain features an Arg-Gly-Asp tripeptide. To obtain monoclonal antibodies, a 170-amino-acid-long polypeptide that is found in all three forms of the SRCR-containing proteins was expressed in E. coli. In a western blot of sponge cells lysate the monoclonal antibody raised against the recombinant polypeptide recognized two major immuno-reacting polypeptides (220 and 117 kDa) and two minor bands (36 and 32 kDa). The antibody was found to react with antigen(s) predominantly localized on the plasma membranes of cells, especially those of spherulous cells. In a functional assay Fab′ fragments of the antibodies suppressed AF-mediated cell-cell reaggregation. Additionally, a recombinant SRCR-soluble fragment effectively inhibited AF-mediated cell-cell reaggregation. We conclude that the 220 kDa SRCR-containing protein of the sponge G. cydonium is very likely the AR.
Expression of Mel-CAM in implantation site intermediate trophoblastic cell line, IST-1, limits its migration on uterine smooth muscle cells
Authors: Shih I.-M. , Wang T.-L. , Wu T.-C. , Kurman R. J. , Gearhart J. D.
Published: April 2021

An immortalized implantation site intermediate trophoblastic cell line, IST-1, was established from a human placenta of 7 weeks gestation. IST-1 cells phenotypically resembled the implantation site intermediate trophoblastic cells in situ and expressed Mel-CAM (MUC 18 or CD146). Mel-CAM is a cell adhesion molecule belonging to the immunoglobulin gene superfamily. It is involved in heterophilic cell-cell adhesion and plays a role in several biological processes including tumor progression. We have previously shown that Mel-CAM was highly expressed in the intermediate (extravillous) trophoblast in the human implantation site. In this study we determined the function of Mel-CAM in the interaction of trophoblast and uterine smooth muscle in the implantation site. IST-1 cells failed to adhere to immobilized recombinant Mel-CAM in solid phase whereas the uterine smooth muscle cells did. The presence of the putative Mel-CAM ligand in smooth muscle cells was further supported by the finding that Mel-CAM-transfected but not the mock-transfected U937 leukemia cells bind to the confluent monolayer of uterine smooth muscle cells. IST-1 cells attached efficiently to the monolayer of the uterine smooth muscle cells and acquired a spindle-shaped morphology simulating smooth muscle cells. The cell binding was only marginally affected by Mel-CAM blocking antibodies. However, Mel-CAM blocking antibodies and recombinant Mel-CAM promoted cell migration from IST-1 cell spheroids on the smooth muscle monolayer. Taken together, our results suggest that IST-1 cells express Mel-CAM but not the putative Mel-CAM ligand. In contrast, the uterine smooth muscle cells express the putative Mel-CAM ligand which binds to Mel-CAM on the surface of the IST-1 cells. The interaction between Mel-CAM and its putative ligand confers a stationary phenotype for trophoblastic cells. These observations are consistent with an important role for Mel-CAM in limiting trophoblastic migration within the myometrium in the implantation site.