bluuebunny/crossref_metadata_2025 huggingface dataset:
Positive and negative regulation of TGF-β signaling
Authors: Miyazono Kohei
Published: April 2021

Cytokines of the transforming growth factor β (TGF-β) superfamily, including TGF-βs, activins and bone morphogenetic proteins (BMPs), bind to specific serine/threonine kinase receptors and transmit intracellular signals through Smad proteins. Upon ligand stimulation, Smads move into the nucleus and function as components of transcription complexes. TGF-β and BMP signaling is regulated positively and negatively through various mechanisms. Positive regulation amplifies signals to a level sufficient for biological activity. Negative regulation occurs at the extracellular, membrane, cytoplasmic and nuclear levels. TGF-β and BMP signaling is often regulated through negative feedback mechanisms, which limit the magnitude of signals and terminate signaling. Negative regulation is also important for formation of gradients of morphogens, which is crucial in developmental processes. In addition, other signaling pathways regulate TGF-β and BMP signaling through cross-talk. Nearly 20 BMP isoforms have been identified, and their activities are regulated by various extracellular antagonists. Regulation of TGF-β signaling might be tightly linked to tumor progression, since TGF-β is a potent growth inhibitor in most cell types.
Unscheduled re-entry into the cell cycle induced by NGF precedes cell death in nascent retinal neurones
Authors: María Frade José
Published: April 2021

During their early postmitotic life, a proportion of the nascent retinal ganglion cells (RGCs) are induced to die as a result of the interaction of nerve growth factor (NGF) with the neurotrophin receptor p75. To analyse the mechanisms by which NGF promotes apoptosis, an in vitro culture system consisting of dissociated E5 retinal cells was established. In this system, NGF-induced apoptosis was only observed in the presence of insulin and neurotrophin-3, conditions that favour the birth of RGCs and other neurones expressing the glycoprotein G4. The pro-apoptotic effect of NGF on the G4-positive neurones was evident after 10 hours in vitro and was preceded by a significant upregulation of cyclin B2, but not cyclin D1, and the presence of mitotic nuclei in these cells. Brain-derived neurotrophic factor prevented both the increase of cyclin B2 expression in the G4-positive neurones and the NGF-induced cell death. Finally, pharmacologically blocking cell-cycle progression using the cyclin-dependent kinase inhibitor roscovitine prevented NGF-induced cell death in a dose-dependent manner. These results strongly suggest that the apoptotic signalling initiated by NGF requires a driving stimulus manifested by the neuronal birth and is preceded by the unscheduled re-entry of postmitotic neurones into the cell cycle.
Chromosome condensation induced by geminivirus infection of mature plant cells
Authors: Bass Hank W. , Nagar Steven , Hanley-Bowdoin Linda , Robertson Dominique
Published: April 2021

Tomato golden mosaic virus (TGMV) is a geminivirus that replicates its single-stranded DNA genome through double-stranded DNA intermediates in nuclei of differentiated plant cells using host replication machinery. We analyzed the distribution of viral and plant DNA in nuclei of infected leaves using fluorescence in situ hybridization (FISH). TGMV-infected nuclei showed up to a sixfold increase in total volume and displayed a variety of viral DNA accumulation patterns. The most striking viral DNA patterns were bright, discrete intranuclear compartments, but diffuse nuclear localization was also observed. Quantitative and spatial measurements of high resolution 3-dimensional image data revealed that these compartments accounted for 1-18% of the total nuclear volume or 2-45% of the total nuclear FISH signals. In contrast, plant DNA was concentrated around the nuclear periphery. In a significant number of nuclei, the peripheral chromatin was organized as condensed prophase-like fibers. A combination of FISH analysis and indirect immunofluorescence with viral coat protein antibodies revealed that TGMV virions are associated with the viral DNA compartments. However, the coat protein antibodies failed to cross react with some large viral DNA inclusions, suggesting that encapsidation may occur after significant viral DNA accumulation. Infection by a TGMV mutant with a defective coat protein open reading frame resulted in fewer and smaller viral DNA-containing compartments. Nevertheless, nuclei infected with the mutant virus increased in size and in some cases showed chromosome condensation. Together, these results established that geminivirus infection alters nuclear architecture and can induce plant chromatin condensation characteristic of cells arrested in early mitosis.
Interaction between the cytodomains of the α3 and β1 integrin subunits regulates remodelling of adhesion complexes on laminin
Authors: Laplantine Emmanuel , Vallar Laurent , Mann Karlheinz , Kieffer Nelly , Aumailley Monique
Published: April 2021

The first step of laminin 1-induced signal transduction is initiated by the formation of α6β1 integrin-specific adhesion complexes. In contrast, on other laminin isoforms the adhesion complexes are α3β1 integrin-specific due to a transdominant regulation of the α6β1 integrin by the α3β1 integrin. To determine the mechanism of this regulation, peptides representing the cytoplasmic domain of the α3 or α6 integrin subunits were microinjected together with recombinant enhanced green fluorescence protein into live fibroblasts. Microinjection of the α3 integrin peptide to laminin 1-adherent cells displaying α6β1 integrin-specific adhesion complexes resulted in the disengagement of the α6β1 integrin, while microinjection of green fluorescence protein alone or in combination with the α6 integrin cytodomain had no effect. Further surface plasmon resonance studies revealed that the cytodomain of the β1 integrin subunit interacts with low affinity with the cytoplasmic tail of the α3 integrin subunit, but not with that of several other α subunits including α6. These results imply that the cytoplasmic tails of the integrin α subunits play a critical role in the regulation of integrin-induced signal transduction. In particular, the intracellular tail of the α3 integrin subunit controls the formation of adhesion complexes in cells adhering to laminins.
The Saccharomyces cerevisiae SDA1gene is required for actin cytoskeleton organization and cell cycle progression
Authors: Buscemi Giacomo , Saracino Francesca , Masnada Debora , Agostoni Carbone Maria Luisa
Published: April 2021

The organization of the actin cytoskeleton is essential for several cellular processes. Here we report the characterization of a Saccharomyces cerevisiaenovel gene, SDA1, encoding a highly conserved protein, which is essential for cell viability and is localized in the nucleus. Depletion or inactivation of Sda1 cause cell cycle arrest in G1by blocking both budding and DNA replication, without loss of viability. Furthermore, sda1-1temperature-sensitive mutant cells arrest at the non-permissive temperature mostly without detectable structures of polymerized actin, although a normal actin protein level is maintained, indicating that Sda1 is required for proper organization of the actin cytoskeleton. To our knowledge, this is the first mutation shown to cause such a phenotype. Recovery of Sda1 activity restores proper assembly of actin structures, as well as budding and DNA replication. Furthermore we show that direct actin perturbation, either in sda1-1or in cdc28-13cells released from G1block, prevents recovery of budding and DNA replication. We also show that the block in G1caused by loss of Sda1 function is independent of Swe1. Altogether our results suggest that disruption of F-actin structure can block cell cycle progression in G1and that Sda1 is involved in the control of the actin cytoskeleton.
Apical endocytosis of ricin in MDCK cells is regulated by the cyclooxygenase pathway
Authors: Llorente Alicia , Deurs Bo van , Garred Øystein , Eker Per , Sandvig Kirsten
Published: April 2021

Addition of arachidonic acid or stimulation of arachidonic acid production by secretory phospholipase A2 selectively upregulated apical endocytosis of ricin in MDCK cells without affecting basolateral endocytosis. Electron microscopic studies revealed that MDCK cells treated with secretory phospholipase A2 and incubated with horseradish peroxidase had an increased number of normal appearing peroxidase-labeled endosomes and no sign of membrane ruffling. Moreover, inhibition of basal arachidonic acid release, either by decreasing the cytosolic phospholipase A2activity or the diacylglycerol lipase activity, reduced the rate of apical endocytosis. Furthermore, indomethacin, an inhibitor of the cyclooxygenase pathway, counteracted the stimulation of endocytosis seen with both secretory phospholipase A2 and arachidonic acid, suggesting that formation of eicosanoids such as prostaglandins could be essential for the regulation. This idea was supported by the finding that prostaglandin E2, the predominant prostaglandin formed in kidney, also upregulated ricin uptake. The regulatory effect of the cyclooxygenase pathway on apical endocytosis of ricin was found to be independent of protein kinases A and C, which are known to selectively control apical clathrin-independent endocytosis in polarized cells.
Morphology and behaviour of dinoflagellate chromosomes during the cell cycle and mitosis
Authors: Bhaud Yvonne , Guillebault Delphine , Lennon Jean-François , Defacque Hélène , Soyer-Gobillard Marie-Odile , Moreau Hervé
Published: April 2021

The morphology and behaviour of the chromosomes of dinoflagellates during the cell cycle appear to be unique among eukaryotes. We used synchronized and aphidicolin-blocked cultures of the dinoflagellate Crypthecodinium cohniito describe the successive morphological changes that chromosomes undergo during the cell cycle. The chromosomes in early G1phase appeared to be loosely condensed with numerous structures protruding toward the nucleoplasm. They condensed in late G1, before unwinding in S phase. The chromosomes in cells in G2phase were tightly condensed and had a double number of arches, as visualised by electron microscopy. During prophase, chromosomes elongated and split longitudinally, into characteristic V or Y shapes.

We also used confocal microscopy to show a metaphase-like alignment of the chromosomes, which has never been described in dinoflagellates. The metaphase-like nucleus appeared flattened and enlarged, and continued to do so into anaphase. Chromosome segregation occurred via binding to the nuclear envelope surrounding the cytoplasmic channels and microtubule bundles. Our findings are summarized in a model of chromosome behaviour during the cell cycle.
Actin filaments and microtubules play different roles during bristle elongation in Drosophila
Authors: Tilney Lewis G. , Connelly Patricia S. , Vranich Kelly A. , Shaw Michael K. , Guild Gregory M.
Published: April 2021

Developing bristles in Drosophilapupae contain 7-11 bundles of crosslinked actin filaments and a large population of microtubules. During bristle growth the rate of cell elongation increases with bristle length. Thin section EM shows that bundle size is correlated with the amount of cytoplasm at all points along the bristle. Thus, as the bristle elongates and tapers, fewer actin filaments are used. To ensure penetration of inhibitors we isolated thoraces and cultured them in vitro; bristles elongate at rates identical to bristles growing in situ. Interestingly, inhibitors of actin filament assembly (cytochalasin D and latrunculin A) dramatically curtailed bristle elongation while a filament stabilizer (jasplakinolide) accelerated elongation. In contrast, inhibitors of microtubule dynamics (nocodazole, vinblastine, colchicine and taxol) did not affect bristle elongation. Surprisingly, the bristle microtubules are stable and do not turn over. Furthermore, the density of microtubules decreases as the bristle elongates. These two facts coupled with calculations and kinetics of elongation and the fact that the microtubules are short indicate that the microtubules are assembled early in development and then transported distally as the bristle grows. We conclude that actin assembly is crucial for bristle cell elongation and that microtubules must furnish other functions such as to provide bulk to the bristle cytoplasm as well as playing a role in vesicle transport.
Ca2+-dependent myosin II activation is required for uropod retraction during neutrophil migration
Authors: Eddy Robert J. , Pierini Lynda M. , Matsumura Fumio , Maxfield Frederick R.
Published: April 2021

Buffering of intracellular Ca2+transients in human neutrophils leads to reduced motility due to defective uropod detachment on fibronectin and vitronectin-coated surfaces. Since one potential target of a rise in [Ca2+]iis the activation of myosin II, we characterized the role of myosin II during motility. Treatment of neutrophils with a myosin inhibitor (2,3-butanedione monoxime), or myosin light chain kinase inhibitors (ML-7, ML-9, or KT5926) resulted in impaired uropod retraction and a dose-dependent decrease in chemokinesis following stimulation with N-formyl-Met-Leu-Phe (fMLP). Treatment with ML-9 resulted in a redistribution of F-actin and talin to the non-retracted uropods, mimicking the redistribution observed during [Ca2+]ibuffering. Impairment of uropod retraction and redistribution of F-actin and talin by myosin II inhibition was only observed on adhesive substrates such as fibronectin and not on poorly adhesive substrates such as human serum-coated glass. At higher concentrations of ML-9, cell polarization was inhibited and pseudopod extension occurred radially. Using an antibody specific for serine 19-phosphorylated regulatory light chain of myosin II, regions of activated myosin II were found at the leading edge as well as the uropod in motile fMLP-stimulated cells. [Ca2+]idepletion caused a 50% decrease in the level of serine 19-phosphorylated myosin II suggesting that activation of myosin II by intracellular Ca2+transients may be an essential step in establishing a polarized pseudopod and providing the force required for uropod retraction during PMN motility on adhesive surfaces.
A reference guide for referee phrases
Authors: A Caveman
Published: April 2021

I realize that I have given the appearance of having a rather cynical view of some of the wording in research papers and their alternative meanings (see previous Sticky Wickets on ‘Data not shown…
Upregulation of the secretory pathway in cysteine protease inhibitor-resistant Trypanosoma cruzi
Authors: Engel Juan C. , García Cristina Torres , Hsieh Ivy , Doyle Patricia S. , McKerrow James H.
Published: April 2021

A novel chemotherapy in development for Chagas’ disease targets cruzain, the major cysteine protease of Trypanosoma cruzi. Peptidomimetic inhibitors disrupt the intracellular cycle of the parasite and rescue animals from a lethal infection. Inhibitor killing of parasites results from interruption of autocatalytic cruzain processing and transport to lysosomes, and massive accumulation of precursor protein in the Golgi complex. To further understand the mechanisms of protease processing and transport in this primitive eukaryote, and uncover potential mechanisms for resistance to these drugs, we generated cysteine-protease inhibitor (CPI)-resistant epimastigotes in vitro and investigated the mechanisms involved at the biochemical and structural levels. Resistance to 20-fold the lethal CPI concentration, achieved after a year of gradual drug increase, was accompanied by a modest decrease in growth rate. A marked increase in the number of vesicles trafficking from the Golgi complex to the flagellar pocket occurs in resistant cells. No mature protease reaches lysosomes though accumulation of endocytosed gold particles in lysosomes appears to be normal. Higher molecular mass cruzain species, consistentwith complexes of cruzain precursors and inhibitor, are secreted by CPI-resistant parasites into the culture supernatant. Release of these cruzain precursors may be facilitated by an enhanced acidification of trans-Golgi cisternae in resistant parasites. The pH within Golgi cisternae is higher in control epimastigotes and most mature cruzain is lysosomal. Cruzain activity is negligible in CPI-resistant epimastigote extracts compared to the parental clone. Activity is restored following withdrawal of the inhibitor. No cross-resistance to the therapeutic drugs nifurtimox and benznidazole occurred and, conversely, parasites resistant to these drugs were sensitive to CPI. Protease inhibitors are thus potential therapeutical alternatives in cases of nifurtimox/benznidazole resistance. Cumulatively, these results suggest that CPI-resistance induces upregulation of Golgi complex function and post- Golgi secretory pathway, and release of precursors before the enzyme reaches its site of biologic activity.
Differential expression and cellular distribution of centrin isoforms during human ciliated cell differentiation in vitro
Authors: Laoukili Jamila , Perret Eric , Middendorp Sandrine , Houcine Odile , Guennou Christiane , Marano Francelyne , Bornens Michel , Tournier Frédéric
Published: April 2021

Centrin protein is an ubiquitously expressed cytoskeletal component and is a member of the EF-hand superfamily of calcium-binding proteins. It was first discovered in the flagellar apparatus of unicellular green algae where it is involved in contraction of Ca2+-sensitive structures. Centrin protein is associated with centrosome-related structures such as spindle pole body in yeast, and centriole/basal bodies in flagellar and ciliated cells. Three centrin genes have been cloned in human cells. In this work, we have performed a comparative biochemical and functional analysis of centrin isoforms using a primary culture of human nasal epithelial cells which provides an efficient way to obtain a complete ciliated cell differentiation process. RT-PCR experiments show that the expression of the three human centrin genes increases during cell differentiation, and that only centrin 2 and 3 are expressed during cell proliferation. Using polyclonal antibodies raised against recombinant human centrin 2 and 3, we show a specific pattern of protein expression. Ultrastructural immunolocalization suggests that centrin proteins are involved in the early process of centriole assembly, as they are concentrated within the precursor structures of centriole/basal bodies. It also shows a differential localisation of centrin proteins in mature centriole/basal bodies, suggesting different functions for centrins 1/2 and centrin 3. This is also supported by functional analyses showing that centrin 1 and/or centrin 2 are involved in ciliary beating.
Selective permeability of different connexin channels to the second messenger inositol 1,4,5-trisphosphate
Authors: Niessen Heiner , Harz Hartmann , Bedner Peter , Krämer Karsten , Willecke Klaus
Published: April 2021

Intercellular propagation of signals through connexin32- containing gap junctions is of major importance in physiological processes like nerve activity-dependent glucose mobilization in liver parenchymal cells and enzyme secretion from pancreatic acinar cells. In these cells, as in other organs, more than one type of connexin is expressed. We hypothesized that different permeabilities towards second messenger molecules could be one of the reasons for connexin diversity. In order to investigate this, we analyzed transmission of inositol 1,4,5-trisphosphate-mediated calcium waves in FURA-2-loaded monolayers of human HeLa cells expressing murine connexin26, -32 or -43. Gap junction-mediated cell coupling in different connexin- transfected HeLa cells was standardized by measuring the spreading of microinjected Mn2+ that led to local quenching of FURA-2 fluorescence. Microinjection of inositol 1,4,5-trisphosphate into confluently growing HeLa connexin32 transfectants induced propagation of a Ca2+ wave from the injected cell to neighboring cells that was at least three- to fourfold more efficient than in HeLa Cx26 cells and about 2.5-fold more efficient than in HeLa Cx43 transfectants. Our results support the notion that diffusion of inositol 1,4,5-trisphosphate through connexin32- containing gap junctions is essential for the optimal physiological response, for example by recruiting liver parenchymal cells that contain subthreshold levels of this short lived second messenger.
Targeting of synaptotagmin to neurite terminals in neuronally differentiated PC12 cells
Authors: Krasnov Peter A. , Enikolopov Grigori
Published: April 2021

We have investigated structural elements that determine the accumulation of synaptotagmin, a major synaptic vesicle protein, in neurite terminals of neuronally differentiated neuroendocrine pheochromocytoma PC12 cells. We performed extensive deletion and point mutagenesis of rat synaptotagmin II, expressed mutant proteins in PC12 cells differentiated by nerve growth factor (NGF) and monitored their intracellular distribution by immunofluorescence. We found a structural element located at the carboxy-terminal domain of the synaptotagmin molecule, which is necessary for its accumulation at the terminal. Using alanine-scanning mutagenesis, we have identified two amino acids in this element, tryptophan W405 and leucine L408, that are critical for correct targeting of synaptotagmin II to neurite terminals. Changing either one of them to alanine prevents the accumulation of the protein at the terminals. These amino acids are evolutionarily conserved throughout the entire synaptotagmin family and also among synaptotagmin-related proteins, suggesting that different synaptotagmins may have similar mechanisms of targeting to neuronal cell terminals.
Accumulation of profilin II at the surface of Listeria is concomitant with the onset of motility and correlates with bacterial speed
Authors: Geese Marcus , Schlüter Kathrin , Rothkegel Martin , Jockusch Brigitte M. , Wehland Jürgen , Sechi Antonio S.
Published: April 2021

The spatial and temporal activity of the actin cytoskeleton is precisely regulated during cell motility by several microfilament-associated proteins of which profilin plays an essential role. We have analysed the distribution of green fluorescent protein (GFP)-tagged profilins in cultured and in Listeria-infected cells. Among the different GFP-profilin fusion proteins studied, only the construct in which the GFP moiety was fused to the carboxy terminus of profilin II (profilin II-GFP) was recruited by intracellular Listeria. The in vitro ligand-binding properties of this construct, e.g. the binding to monomeric actin, poly-L-proline and phosphatidylinositol 4,5-bisphosphate (PIP2), were unaffected by GFP. Profilin II-GFP co-localised with vinculin and Mena to the focal adhesions in REF-52 fibroblasts and was distributed as a thin line at the front of protruding lamellipodia in B16-F1 mouse melanoma cells. In Listeria-infected cells, profilin II-GFP was recruited, in an asymmetric fashion, to the surface of Listeria at the onset of motility whereas it was not detectable on non-motile bacteria. In contrast to the vasodilator-stimulated phosphoprotein (VASP), profilin II-GFP localised at the bacterial surface only on motile Listeria. Moreover, the fluorescence intensity of profilin II-GFP directly correlated with the speed of the bacteria. Thus, the use of GFP-tagged profilin II provides new insights into the role of profilins in cellular motility.
Functional overlap of the Dictyostelium RasG, RasD and RasB proteins
Authors: Khosla Meenal , Spiegelman George B. , Insall Rob , Weeks Gerald
Published: April 2021

Disruption of the rasG gene in Dictyostelium discoideum results in several distinct phenotypes: a defect in cytokinesis, reduced motility and reduced growth. Reintroduction of the rasG gene restores all of the properties of the rasG− cells to those of the wild type. To determine whether the defects are due to impaired interactions with a single or multiple downstream effectors, we tested the ability of the highly related but non identical Dictyostelium ras genes, rasD and rasB, to rescue the defects.

Introduction of the rasD gene under the control of the rasG promoter into rasG null (rasG−) cells corrected all phenotypes except the motility defect, suggesting that motility is regulated by a RasG mediated pathway that is different to those regulating growth or cytokinesis. Western blot analysis of RasD protein levels revealed that vegetative rasG− cells contained considerably more protein than the parental AX-3 cells, suggesting that RasD protein levels are negatively regulated in vegetative cells by RasG. The level of RasD was enhanced when the rasD gene was introduced under the control of the rasG promoter, and this increase in protein is presumably responsible for the reversal of the growth and cytokinesis defects of the rasG− cells. Thus, RasD protein levels are controlled by the level of RasG, but not by the level of RasD.

Introduction of the rasB gene under the control of the rasG promoter into rasG− cells produced a complex phenotype. The transformants were extremely small and mononucleate and exhibited enhanced motility. However, the growth of these cells was considerably slower than the growth of the rasG− cells, suggesting the possibility that high levels of RasB inhibit an essential process. This was confirmed by expressing rasB in wild-type cells; the resulting transformants exhibited severely impaired growth. When RasB protein levels were determined by western blot analysis, it was found that levels were higher in the rasG− cells than they were in the wild-type parental, suggesting that RasG also negatively regulates rasB expression in vegetative cells. Overexpression of rasB in the rasG− cells also reduced the level of RasD protein.

In view of the fact that alternate Ras proteins correct some, but not all, of the defects exhibited by the rasG− cells, we propose that RasG interacts with more than one downstream effector. In addition, it is clear that the levels of the various Ras proteins are tightly regulated in vegetative cells and that overexpression can be deleterious.
Regulation of cytokinesis by the Elm1 protein kinase in Saccharomyces cerevisiae
Authors: Bouquin Nicolas , Barral Yves , Courbeyrette Régis , Blondel Marc , Snyder Mike , Mann Carl
Published: April 2021

A Saccharomyces cerevisiae mutant unable to grow in a cdc28-1N background was isolated and shown to be affected in the ELM1 gene. Elm1 is a protein kinase, thought to be a negative regulator of pseudo-hyphal growth. We show that Cdc11, one of the septins, is delocalised in the mutant, indicating that septin localisation is partly controlled by Elm1. Moreover, we show that cytokinesis is delayed in an elm1Δ mutant. Elm1 levels peak at the end of the cell cycle and Elm1 is localised at the bud neck in a septin-dependent fashion from bud emergence until the completion of anaphase, at about the time of cell division. Genetic and biochemical evidence suggest that Elm1 and the three other septin-localised protein kinases, Hsl1, Gin4 and Kcc4, work in parallel pathways to regulate septin behaviour and cytokinesis. In addition, the elm1Δ morphological defects can be suppressed by deletion of the SWE1 gene, but not the cytokinesis defect nor the septin mislocalisation. Our results indicate that cytokinesis in budding yeast is regulated by Elm1.
Contributions of the extracellular and cytoplasmic domains of platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31) in regulating cell-cell localization
Authors: Sun Jing , Paddock Cathy , Shubert Julie , Zhang Hong-Bing , Amin Kunjlata , Newman Peter J. , Albelda Steven M.
Published: April 2021

PECAM-1/CD31, a vascular cell adhesion/signaling molecule that has been implicated in a number of vascular functions (including angiogenesis and the transmigration of leukocytes through endothelium) is highly enriched at the cell-cell borders of adjacent endothelial cells. To identify the mechanisms responsible for this localization, a series of PECAM-1 mutants and chimeric PECAM-1 molecules were transfected into non-PECAM-expressing cells and the ability of the constructs to move to cell-cell borders of adjacent cells was determined using immunohistochemistry and confocal microscopy. Although neither the extracellular domain, by itself, nor the cytoplasmic domain, by itself, was sufficient to direct cell-cell localization, the combination of the extracellular and transmembrane domains with a small group of highly charged amino acids in a membrane proximal region of the cytoplasmic domain was sufficient to direct efficient localization of the molecule to cell-cell borders. Importantly, only constructs that supported PECAM-1 mediated adhesion localized to cell-cell borders. Our data are consistent with a ‘diffusion trapping’ model in which movement of PECAM-1 in the cell membrane occurs relatively freely until the ‘stablized’ extracellular domain of the molecule encounters its ligand on an adjacent cell. When this occurs, the complex is ‘captured’ at the cell-cell interface leading to localization at cell-cell borders.
Nup2p is located on the nuclear side of the nuclear pore complex and coordinates Srp1p/importin-α export
Authors: Hood Jennifer K. , Casolari Jason M. , Silver Pamela A.
Published: April 2021

Proteins bearing canonical nuclear localization sequences are imported into the nucleus by the importin/karyopherin-α/β heterodimer. Recycling of the importin-α subunit to the cytoplasm requires the action of Cas, a member of the importin-β superfamily. In the yeast Saccharomyces ceresivisiae, the essential gene CSE1 encodes a Cas homologue that exports the yeast importin-α protein Srp1p/Kap60p from the nucleus. In this report, we describe a role for the FXFG nucleoporin Nup2p, and possibly the related Nup1p, in the Cse1p-mediated nuclear export pathway. Yeast cells lacking Nup2p or containing a particular temperature-sensitive mutation in NUP1 accumulate Srp1p in the nucleus. Similarly, Cse1p is displaced from the nuclear rim to the nuclear interior in Δnup2 cells. We do not observe any biochemical interaction between Cse1p and Nup2p. Instead, we find that Nup2p binds directly to Srp1p. We have localized Nup2p to the interior face of the nuclear pore complex, and have shown that its N terminus is sufficient for targeting Nup2p to the pore, as well as for binding to Srp1p. Taken together, these data suggest that Nup2p is an important NPC docking site in the Srp1p export pathway.
Cell cycle-dependent localization of the CDK2-cyclin E complex in Cajal (coiled) bodies
Authors: Liu Juinn-Lin , Hebert Michael D. , Ye Ying , Templeton Dennis J. , Kung Hsing-Jien , Matera A. Gregory
Published: April 2021

We have found that CDK2 and cyclin E, but not cyclin A, accumulates within Cajal bodies (CBs) in a cell cycle-dependent fashion. In the absence of cyclin E, CDK2 is not enriched in the CB compartment, suggesting that the translocation of CDK2 to CBs is dependent on cyclin E. CDK2 and cyclin E could be recruited to CBs as a functional complex or CBs may serve as ‘docking stations’ for CDK2-cyclin E activation by CAKs during the G1/S transition. Notably, CDK7-cyclin H-Mat1 complexes are known to accumulate in CBs. Treatment of cells with inhibitors of either CDKs (olomoucine, 200 μM) or RNA polymerase I (actinomycin D, 0.05 μg/ml), results in a striking reorganization of CDK2 and p80 coilin to the nucleolar periphery. Furthermore, we demonstrate that p80 coilin can be phosphorylated by purified CDK2-cyclin E complexes in vitro. Thus coilin and other CB proteins appear to be downstream targets of CDK2-cyclin E complex-mediated signaling pathways regulating cell cycle progression and controlling aspects of CB function. Possible roles for CDK2 and cyclin E in the well-documented association of CBs, histone gene clusters and RNA 3′ end processing factors are discussed.
Glycocalyx modulation is a physiological means of regulating cell adhesion
Authors: Sabri Siham , Soler Mireille , Foa Colette , Pierres Anne , Benoliel Anne-Marie , Bongrand Pierre
Published: April 2021

Here we present experimental evidence that phagocytic cells use modulation of specific components of their glycocalyx to regulate their binding capacity.

Particles coated with antibodies specific for the CD32 medium affinity IgG receptor were driven along human monocytic THP-1 cells (expressing CD32) in a flow chamber operated at low shear rate. Surprisingly, only minimal adhesion was observed. However, when cells were activated by exposure to fibronectin-coated surfaces and/or soluble gamma interferon, adhesion efficiency was dramatically increased, whereas the apparent glycocalyx thickness displayed 20% decrease, and the surface density of CD43/leukosialin carbohydrate epitopes displayed 30-40% decrease on activated cells.

The existence of a causal link between adhesion increase and glycocalyx alteration was strongly supported by the finding that (i) both phenomena displayed similar kinetics, (ii) an inverse relationship between THP-1 cell binding capacity and glycocalyx density was demonstrated at the individual cell level, and (iii) adhesion enhancement could not be ascribed to an increased binding site density or improved functional capacity of activated cells.

Additional experiments revealed that cell-to-particle adhesion resulted in delayed (i.e. more than a few minutes) egress of CD43/leukosialin from contact areas. Since the time scale of particle attachment was less than a second, surface mobility should not affect the potential of CD43 to impair the initial step of adhesion.

Finally, studies performed with fluorescent lectins suggested that THP-1 cell activation and increased adhesive potential were related to a decrease of O-glysosylation rather than N-glycosylation of surface glycoproteins.
Activation of TrkA tyrosine kinase in embryonal carcinoma cells promotes cell compaction, independently of tyrosine phosphorylation of catenins
Authors: Cozzolino Mauro , Giovannone Barbara , Serafino Annalucia , Knudsen Karen , Levi Andrea , Alemà Stefano , Salvatore AnnaMaria
Published: April 2021

Cadherins are transmembrane receptors whose extracellular domain mediates homophilic cell-cell interactions, while their cytoplasmic domain associates with a family of proteins known as catenins. Although the mechanisms that regulate the assembly and functional state of cadherin-catenin complexes are poorly understood, current evidence supports a role for protein tyrosine kinase activity in regulating cell adhesion and migration. Tyrosine phosphorylation of catenins is thought to mediate loss of intercellular adhesion promoted by activation of receptor tyrosine kinases in epithelial cells. Here, we show that activation of ectopically expressed TrkA, the tyrosine kinase receptor for nerve growth factor (NGF), stimulates embryonal carcinoma P19 cells to develop extensive intercellular contacts and to assemble into closely packed clusters. Thus, activation of receptor tyrosine kinases can differentially regulate adhesiveness by cell-type-specific mechanisms. Furthermore, activation of TrkA in P19 and epithelial MDCK cells induces tyrosine phosphorylation of p120ctn and of β-catenin, irrespective of the elicited cellular response. The selective Src tyrosine kinase inhibitor PP2, however, suppresses NGF- or HGF-induced tyrosine phosphorylation of catenins in both P19 and MDCK cells without interfering with the acquisition of a compacted or scattered phenotype. These findings provide a cogent argument for considering that tyrosine phosphorylation of catenins is dispensable for their interaction with cadherins and, ultimately, for the modulation of cadherin-based cell adhesion by receptor tyrosine kinases.
Molecular characterization of the major membrane skeletal protein in the ciliate Tetrahymena pyriformis suggests n-plication of an early evolutionary intermediate filament protein subdomain
Authors: Bouchard Philippe , Chomilier Jacques , Ravet Viviane , Mornon Jean-Paul , Viguès Bernard
Published: April 2021

Epiplasmin C is the major protein component of the membrane skeleton in the ciliate Tetrahymena pyriformis. Cloning and analysis of the gene encoding epiplasmin C showed this protein to be a previously unrecognized protein. In particular, epiplasmin C was shown to lack the canonical features of already known epiplasmic proteins in ciliates and flagellates. By means of hydrophobic cluster analysis (HCA), it has been shown that epiplasmin C is constituted of a repeat of 25 domains of 40 residues each. These domains are related and can be grouped in two families called types I and types II. Connections between types I and types II present rules that can be evidenced in the sequence itself, thus enforcing the validity of the splitting of the domains. Using these repeated domains as queries, significant structural similarities were demonstrated with an extra six heptads shared by nuclear lamins and invertebrate cytoplasmic intermediate filament proteins and deleted in the cytoplasmic intermediate filament protein lineage at the protostome-deuterostome branching in the eukaryotic phylogenetic tree.
Targeting of cytoskeletal proteins to the flagellum of Trypanosoma brucei
Authors: Ersfeld Klaus , Gull Keith
Published: April 2021

The eukaryotic flagellum represents one of the most complex macromolecular structures found in any organism and contains more than 250 proteins. Due to the relative ease of genetic manipulation the flagellum of Trypanosoma brucei has emerged as an accessible model system to study the morphogenesis and dynamics of this organelle. We have recently started to characterise the mechanisms by which components of the cytoskeletal fraction of the flagellum, such as the axoneme, the paraflagellar rod and the flagellar attachment zone, are targeted by proteins synthesised in the cytoplasm and assembled. Here, we present the identification of a novel actin-related protein as a component of the axoneme. We show that this protein shares the tripeptid motif histidine-leucine-alanine (HLA) with one of the major proteins of the paraflagellar rod, PFRA. Building on previous work from this lab which showed that a deletion comprising this motif abolished targeting of PFRA to the flagellum we demonstrate in this study that the deletion of the tripeptid motif is sufficient to achieve mistargeting both of the PFRA and the actin- related protein. We propose that this motif represents an essential part of a flagellar targeting machinery in trypanosomes and possibly in other flagellated organisms.
Glycosaminoglycan synthesis and secretion by the retinal pigment epithelium: polarized delivery of hyaluronan from the apical surface
Authors: Senanayake Preenie deS , Calabro Anthony , Nishiyama Kazutoshi , Hu Jane G. , Bok Dean , Hollyfield Joe G.
Published: April 2021

Hyaluronan and chondroitin sulfate glycosaminoglycan secretion from retinal pigment epithelial cells was established in confluent cultures with high transepithelial resistance. Cell cultures were maintained on Millicell-PCF culture plates, which allow separation of culture medium exposed to apical and basal epithelial surfaces. Following various times in culture, apical and basal culture media were sampled at three day intervals and the glycosaminoglycan content was quantified. Samples were digested with proteinase K to free the glycosaminoglycans from their core proteins, the glycosaminoglycans were ethanol precipitated, and subjected to hyaluronidase SD and chondroitinase ABC digestion to release hyaluronan and chondroitin sulfate disaccharides. Disaccharides were fluorotagged with 2-aminoacridone, separated on polyacrylamide gels and the molar fluorescence in each disaccharide band quantitated. Hyaluronan in the apical medium was significantly higher than in the basal medium (5-12 times) at all recovery intervals (P<0.0001). In contrast, the distribution of unsulfated chondroitin, 4-sulfated chondroitin and 6-sulfated chondroitin disaccharides in apical and basal media was non-polar. Confocal microscopy of cultures probed with a hyaluronan- specific fluorotag established that the HA evident in these cultures is restricted to the apical border of the RPE cultures. Collectively, these data indicate that hyaluronan synthesized by the retinal pigment epithelium is secreted preferentially from the apical surface, suggesting that this tissue is an important source of hyaluronan present in the interphotoreceptor matrix.
Immunolocalization of cytoplasmic dynein and dynactin subunits in cultured macrophages: enrichment on early endocytic organelles
Authors: Habermann Anja , Schroer Trina A. , Griffiths Gareth , Burkhardt Janis K.
Published: April 2021

Cytoplasmic dyneins and their cofactor, dynactin, work together to mediate the movement of numerous cargo organelles toward the minus-ends of microtubules. In many cases, there is compelling evidence that dynactin functions in part to attach dyneins to cargo organelles, but this may not always be the case. We have localized three dynactin subunits (Arp1, p62 and p150Glued) and two subunits of conventional cytoplasmic dynein (dynein intermediate chain and dynein heavy chain 1) in murine macrophages using immunogold labeling of thawed cryosections. Using stereological techniques, we have quantified the relative distributions of each of these subunits on specific membrane organelles to generate a comprehensive analysis of the distribution of these proteins in a single cell type. Our results show that each of the subunits tested exhibits the same distribution with respect to different membrane organelles, with highest levels present on early endosomes, and lower levels present on later endocytic organelles, the mitochondrial outer membrane, the plasma membrane and vesicles in the Golgi region. An additional pool of punctate dynactin labeling was detected in the cell periphery, in the absence of dynein labeling. Even when examined closely, membrane organelles could not be detected in association with these dynactin-positive sites; however, double labeling with anti-tubulin antibody revealed that at least some of these sites represent the ends of microtubules. The similarities among the labeling profiles with respect to membrane organelles suggest that dynein and dynactin bind to membrane organelles as an obligate unit. In contrast, our results show that dynactin can associate with microtubule ends in the absence of dynein, perhaps providing sites for subsequent organelle and dynein association to form a functional motility complex.
Df31 is a novel nuclear protein involved in chromatin structure in Drosophila melanogaster
Authors: Crevel Gilles , Huikeshoven Hella , Cotterill* Sue
Published: April 2021

We originally isolated the Df31 protein from Drosophila embryo extracts as a factor which could decondense Xenopus sperm, by removing the sperm specific proteins and interacting with histones to facilitate their loading onto DNA. We now believe that this protein has a more general function in cellular DNA metabolism.

The Df31 gene encodes a very hydrophilic protein with a predicted molecular mass of 18.5 kDa. Immunostaining showed that Df31 was present in a wide range of cell types throughout differentiation and in both dividing and non- dividing cells. In all cases the protein is present in large amounts, comparable with the level of nucleosomes. Injection of antisense oligonucleotides to lower the level of Df31 in embryos caused severe disruption of the nuclear structure. Large irregular clumps of DNA were formed, and in most cases the amount of DNA associated with each clump was more than that found in a normal nucleus. Immunofluorescence, cell fractionation, and formaldehyde cross-linking show that Df31 is associated with chromatin and that a significant fraction of it binds very tightly. It also shows the same binding characteristics when loaded onto chromatin in vitro. Chromatin fractionation shows that Df31 is tightly associated with nucleosomes, preferentially with oligonucleosomes. Despite this no differences were observed in the properties of nucleosomes loaded in the in vitro system in the presence and absence of Df31.

These results suggest that Df31 has a role in chromosomal structure, most likely acting as a structural protein at levels of folding higher than that of nucleosomes.
Two type V myosins with non-overlapping functions in the fission yeast Schizosaccharomyces pombe: Myo52 is concerned with growth polarity and cytokinesis, Myo51 is a component of the cytokinetic actin ring
Authors: Win Thein Z. , Gachet Yannick , Mulvihill Daniel P. , May Karen M. , Hyams Jeremy S.
Published: April 2021

The fission yeast genome project has identified five myosin genes: one type I myosin, myo1+, two type II myosins, myo2+ and myp2+, and two type V myosins, myo51+ and myo52+. Cells deleted for myo51+ show normal morphology and growth rates whereas deletion of myo52+ results in a partial loss of cell polarity, slow growth and cytokinetic defects. Combining both deletions in a single strain is phenotypically non-additive, myo52Δ being epistatic to myo51Δ. Overproduction of Myo51 gives rise to elongated cells which fail to form functional septa whereas overproduction of Myo52 results in branched cells with aberrant septa that fail to cleave. Myo52 localises to the poles of growing cells but during cell division it relocalises to the cell equator as a bar that is bisected by the cytokinetic septum. Myo51 shows no obvious localisation during interphase but at cytokinesis it is associated with the contractile cytokinetic actin ring (CAR). Both myosins are dependent upon an intact actin cytoskeleton for localisation. Myo52 partially colocalises with the α-glucan synthase Mok1 at the cell tips and to a lesser extent at the septum. Mok1 is delocalised and upregulated in myo52Δ and myo52Δ cell walls are resistant to digestion by the cell wall degrading enzyme zymolyase. Thus myo52+ appears to be involved in the local delivery or positioning of vesicles containing cell wall precursors at the cell tips and has a role in the maturation or cleavage of the septum. Myo51 has a non-essential role in cytokinesis as a component of the cytokinetic actin ring.
A nuclear tale of two yeasts: The Yeast Nucleus
Authors: Fantes P. , Beggs J.
Published: April 2021

Without question, numerous studies in yeast and mammals have revealed a striking commonality of underlying mechanisms that govern basic biological operations. Perhaps the most famous example from recent years has been the recognition that genes required for maintaining the yeast genome play a critical role in preventing cancer in humans. However, examining the molecular differences -the variations on a common theme, so to speak -can also be useful for understanding core biological processes. These ideas are the foundation for The Yeast Nucleus, a valuable contribution to Oxford University Press’s ‘Frontiers in Molecular Biology’ series. The textbook compares and contrasts various nuclear processes in budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe), pointing out the similarities - and differences -that make these two somewhat unrelated yeasts the dominant model systems for studying fundamental eukaryotic processes.