bluuebunny/crossref_metadata_2025 huggingface dataset:
The fission yeast origin recognition complex is constitutively associated with chromatin and is differentially modified through the cell cycle
Authors: Lygerou Zoi , Nurse Paul
Published: April 2021

The origin recognition complex (ORC) binds to the well defined origins of DNA replication in budding yeast. Homologous proteins in other eukaryotes have been identified but are less well characterised. We report here the characterisation of a fission yeast ORC complex (SpORC). Database searches identified a fission yeast Orc5 homologue. SpOrc5 is essential for cell viability and its deletion phenotype is identical to that of two previously identified ORC subunit homologues, SpOrc1 (Orp1/Cdc30) and SpOrc2 (Orp2). Co-immunoprecipitation experiments demonstrate that SpOrc1 forms a complex with SpOrc2 and SpOrc5 and gel filtration chromatography shows that SpOrc1 and SpOrc5 fractionate as high molecular mass complexes. SpORC subunits localise to the nucleus in a punctate distribution which persists throughout interphase and mitosis. We developed a chromatin isolation protocol and show that SpOrc1, 2 and 5 are associated with chromatin at all phases of the cell cycle. While the levels, nuclear localisation and chromatin association of SpORC remain constant through the cell cycle, one of its subunits, SpOrc2, is differentially modified. We show that SpOrc2 is a phosphoprotein which is hypermodified in mitosis and is rapidly converted to a faster migrating isoform as cells proceed into G1 in preparation for S-phase.
Decreasing oncoprotein 18/stathmin levels reduces microtubule catastrophes and increases microtubule polymer in vivo
Authors: Howell Bonnie , Deacon Heather , Cassimeris Lynne
Published: April 2021

Oncoprotein 18/stathmin (Op18) has been identified recently as a protein which destabilizes microtubules. To characterize the function of Op18 in living cells, we used microinjection of anti-Op18 antibodies or antisense oligonucleotides to block either Op18 activity or expression in interphase newt lung cells. Anti-tubulin staining of cells microinjected with anti-Op18 and fixed 1-2 hours after injection showed an increase in total microtubule polymer. In contrast, microinjection of either non-immune IgG or anti-Op18 preincubated with bacterially-expressed Op18 had little effect on microtubule polymer level. Cells treated with Op18 antisense oligonucleotides for 4 days had ≥50% reduced levels of Op18 with no change in the soluble tubulin level. Measurement of MT polymer level in untreated, antisense or nonsense oligonucleotide treated cells demonstrated that reduced Op18 levels resulted in a 2.5-fold increase in microtubule polymer. Next, the assembly dynamics of individual microtubules at the peripheral regions of living cells were examined using video-enhanced contrast DIC microscopy. Microinjection of antibodies against oncoprotein 18 resulted in a 2.2-fold reduction in catastrophe frequency and a slight reduction in plus end elongation velocity compared to uninjected cells or cells microinjected with non-immune IgG. Preincubation of anti-Op18 antibody with recombinant Op18 greatly diminished the effects of the antibody. Similarly, treatment of cells with antisense oligonucleotides reduced catastrophes 2.5- to 3-fold compared to nonsense oligonucleotide treated or untreated cells. The other parameters of dynamic instability were unchanged after reducing Op18 with antisense oligonucleotides. These studies are consistent with Op18 functioning to regulate microtubule catastrophes during interphase in vivo.
Profilin is predominantly associated with monomeric actin in Acanthamoeba
Authors: Kaiser Donald A. , Vinson Valda K. , Murphy Douglas B. , Pollard Thomas D.
Published: April 2021

We used biochemical fractionation, immunoassays and microscopy of live and fixed Acanthamoeba to determine how much profilin is bound to its known ligands: actin, membrane PIP2, Arp2/3 complex and polyproline sequences. Virtually all profilin is soluble after gentle homogenization of cells. During gel filtration of extracts on Sephadex G75, approximately 60% of profilin chromatographs with monomeric actin, 40% is free and none voids with Arp2/3 complex or other large particles. Selective monoclonal antibodies confirm that most of the profilin is bound to actin: 65% in extract immunoadsorption assays and 74-89% by fluorescent antibody staining. Other than monomeric actin, no major profilin ligands are detected in crude extracts. Profilin-II labeled with rhodamine on cysteine at position 58 retains its affinity for actin, PIP2 and poly-L-proline. When syringe-loaded into live cells, it distributes throughout the cytoplasm, is excluded from membrane-bounded organelles, and concentrates in lamellapodia and sites of endocytosis but not directly on the plasma membrane. Some profilin fluorescence appears punctate, but since no particulate profilin is detected biochemically, these spots may be soluble profilin between organelles that exclude profilin. The distribution of profilin in fixed human A431 cells is similar to that in amoebas. Our results show that the major pool of polymerizable actin monomers is complexed with profilin and spread throughout the cytoplasm.
Kinesin-mediated transport of neurofilament protein oligomers in growing axons
Authors: Yabe Jason T. , Pimenta Aurea , Shea Thomas B.
Published: April 2021

We examined cytoskeleton-associated forms of NF proteins during axonal neuritogenesis in cultured dorsal root ganglion (DRG) neurons and NB2a/d1 neuroblastoma. In addition to filamentous immunoreactivity, we observed punctate NF immunoreactivity throughout perikarya and neurites. Immuno-electron microscopy revealed this punctate immunoreactivity to consist of non-membrane-bound 75 nm round/ovoid structures consisting of amorphous, fibrous material. Endogenous and microinjected NF subunits incorporated into dots prior to their accumulation within filaments. A transfected GFP-conjugated NF-M incorporated into dots and translocated at a rate consistent with slow axonal transport in real-time video analyses. Some dots converted into a filamentous form or exuded filamentous material during transport. Dots contained conventional kinesin immunoreactivity, associated with microtubules, and their transport into axons was blocked by anti-kinesin antibodies and nocodazole. These oligomeric structures apparently represent one form in which NF subunits are transported in growing axons and may utilize kinesin as a transport motor.
The contractile vacuole network of Dictyostelium as a distinct organelle: its dynamics visualized by a GFP marker protein
Authors: Gabriel Daniela , Hacker Ulrike , Köhler Jana , Müller-Taubenberger Annette , Schwartz Jean-Marc , Westphal Monika , Gerisch Günther
Published: April 2021

The contractile vacuole system is an osmoregulatory organelle composed of cisternae and interconnecting ducts. Large cisternae act as bladders that periodically fuse with the plasma membrane, forming pores to expel water. To visualize the entire network in vivo and to identify constituents of the vacuolar complex in cell fractions, we introduced a specific marker into Dictyostelium cells, GFP-tagged dajumin. The C-terminal, GFP-tagged region of this transmembrane protein is responsible for sorting to the contractile vacuole complex. Dajumin-GFP negligibly associates with the plasma membrane, indicating its retention during discharge of the bladder. Fluorescent labeled cell-surface constituents are efficiently internalized by endocytosis, while no significant cycling through the contractile vacuole is observed. Endosomes loaded with yeast particles or a fluid-phase marker indicate sharp separation of the endocytic pathway from the contractile vacuole compartment. Even after dispersion of the contractile vacuole system during mitosis, dajumin-GFP distinguishes the vesicles from endosomes, and visualizes post-mitotic re-organization of the network around the nucleus. Highly discriminative sorting and membrane fusion mechanisms are proposed to account for the sharp separation of the contractile vacuole and endosomal compartments. Evidence for a similar compartment in other eukaryotic cells is discussed.
Activation of microglia reveals a non-proteolytic cytokine function for tissue plasminogen activator in the central nervous system
Authors: Rogove Andrew D. , Siao Chia-Jen , Keyt Bruce , Strickland Sidney , Tsirka Stella E.
Published: April 2021

Tissue plasminogen activator mediates excitotoxin-induced neurodegeneration and microglial activation in the mouse hippocampus. Here we show that tissue plasminogen activator (tPA) acts in a protease-independent manner to modulate the activation of microglia, the cells of the central nervous system with macrophage properties. Cultured microglia from tPA-deficient mice can phagocytose as efficiently as wild-type microglia. However, tPA-deficient microglia in mixed cortical cultures exhibit attenuated activation in response to lipopolysaccharide, as judged by morphological changes, increased expression of the activation marker F4/80 and the release of the pro-inflammatory cytokine tumor necrosis factor-α. When tPA is added to tPA deficient cortical cultures prior to endotoxin stimulation, microglial activation is restored to levels comparable to that observed in wild-type cells. Proteolytically-inactive tPA can also restore activation of tPA-deficient microglia in culture and in vivo. However, this inactive enzyme does not restore susceptibility of tPA-deficient hippocampal neurons to excitotoxin-mediated cell death. These results dissociate two different functions of tPA: inactive enzyme can mediate microglial activation, whereas proteolytically-competent protein also promotes neuronal degeneration. Thus tPA is identified as a new cytokine in the central nervous system.
Differential subcellular localization of DNA-dependent protein kinase components Ku and DNA-PKcs during mitosis
Authors: Koike Manabu , Awaji Takeo , Kataoka Masakazu , Tsujimoto Gozoh , Kartasova Tonja , Koike Aki , Shiomi Tadahiro
Published: April 2021

The Ku protein is a complex of two subunits, Ku70 and Ku80. Ku plays an important role in DNA-PKcs-dependent double-strand break repair and V(D)J recombination, and in growth regulation, which is DNA-PKcs-independent. We studied the expression and the subcellular localization of Ku and DNA-PKcs throughout the cell cycle in several established human cell lines. Using immunofluorescence analysis and confocal laser scanning microscopy, we detected Ku70 and Ku80 in the nuclei in interphase cells. In mitotic cells (1) most of Ku protein was found diffused in the cytoplasm, (2) a fraction was detected at the periphery of condensed chromosomes, (3) no Ku protein was present in the chromosome interior. Association of Ku with isolated chromosomes was also observed. On the other hand, DNA-PKcs was detected in the nucleus in interphase cells and not at the periphery of condensed chromosomes during mitosis. Using indirect immunoprecipitation, we found that throughout the cell cycle, Ku70 and Ku80 were present as heterodimers, some in complex with DNA-PKcs. Our findings suggest that the localization of Ku at the periphery of metaphase chromosomes might be imperative for a novel function of Ku in the G2/M phase, which does not require DNA-PKcs.
Impaired integrin-mediated signal transduction, altered cytoskeletal structure and reduced motility in Hck/Fgr deficient macrophages
Authors: Suen Patrick W. , Ilic Dusko , Caveggion Elena , Berton Giorgio , Damsky Caroline H. , Lowell Clifford A.
Published: April 2021

Integrin-mediated adhesion of monocytes and macrophages initiates a signal transduction pathway that leads to actin cytoskeletal reorganization, cell migration and immunologic activation. This signaling pathway is critically dependent on tyrosine kinases. To investigate the role of the Src-family of tyrosine kinases in integrin signal transduction, we have examined the adhesive properties of macrophages isolated from hck-/-fgr-/- double knockout mice which lack two of the three predominant Src-family kinases expressed in myeloid cells. Previous examination of polymorphonuclear leukocytes from these animals indicated that these kinases were critical in initiating the actin cytoskeletal rearrangements that lead to respiratory burst and granule secretion following integrin ligation. Double mutant peritoneal exudate macrophages demonstrated markedly reduced tyrosine phosphorylation responses compared to wild-type cells following plating on fibronectin, collagen or vitronectin-coated surfaces. Tyrosine phosphorylation of several actin-associated proteins (cortactin, paxillin, and tensin), as well as the Syk and Pyk2 tyrosine kinases, were all significantly reduced in double mutant cells. The subcellular localization of focal-adhesion associated proteins was also dramatically altered in mutant macrophages cultured on fibronectin-coated surfaces. In wild-type cells, filamentous actin, paxillin, and talin were concentrated along leading edges of the plasma membrane, suggesting that these proteins contribute to cellular polarization during migration in culture. Double mutant cells failed to show the polarized subcellular localization of these proteins. Likewise, double mutant macrophages failed to form normal filopodia under standard culture conditions. Together, these signaling and cytoskeletal defects may contribute to the reduced motility observed in in vitro assays. These data provide biochemical and morphological evidence that the Src-family kinases Hck and Fgr are required for normal integrin-mediated signal transduction in murine macrophages.
Motif Trap: a rapid method to clone motifs that can target proteins to defined subcellular localisations
Authors: Bejarano Luis A. , González Cayetano
Published: April 2021

We have developed a rapid procedure termed Motif Trap (MT) to identify protein motifs that are able to target proteins to a distinct subcellular localisation in eukaryotic cells. By expressing random DNA fragments fused to green fluorescent protein (GFP), individual cells with the GFP localisation of interest are readily isolated allowing for the expressed DNA fragments to be cloned by RT-PCR. These can then be used to identify the corresponding full-length cDNAs. Using MT, we have identified patterns of GFP localisation which correspond to every major organelle and compartment. We have shown that MT is useful to identify new sequences that determine subcellular localisation as well as known targeting motifs.
Mussel defensins are synthesised and processed in granulocytes then released into the plasma after bacterial challenge
Authors: Mitta Guillaume , Vandenbulcke Franck , Hubert Florence , Roch Philippe
Published: April 2021

MGD1 (Mytilus galloprovincialis defensin 1), a new member of the arthropod defensin family, is a 4 kDa antibacterial peptide previously isolated from the plasma of Mediterranean mussels. We report here the presence of MGD1 in the organelle-rich fraction of hemocytes and the cDNA sequence corresponding to MGD1 and one new isoform mRNA: MGD2. Sequence analysis indicated that MGDs are synthesised as precursors consisting of a putative signal peptide of 21 residues, the active peptide of 39 amino acids and a 21 residue carboxyl-terminal extension, rich in acidic amino acids. Localisation of the transcripts by northern blot revealed that the precursors are abundantly expressed in hemocytes.

Immunocytochemistry at both the optical and ultrastructural levels showed that defensins (i) are predominantly located in vesicles of a granulocyte subclass of hemocytes containing small granules, (ii) are also found in large clear granules of another granulocyte subclass, and (iii) that MGD immune reactivity existed in granular structures of enterocytes. Finally, we revealed that bacterial challenge triggered a plasmatic increase of MGD1 concentration and gave evidence of the simultaneous release of the peptides from the hemocytes.
Distinct roles for astrocyte αvβ5 and αvβ8 integrins in adhesion and migration
Authors: Milner Richard , Huang Xiaozhu , Wu Jianfeng , Nishimura Stephen , Pytela Robert , Sheppard Dean , ffrench-Constant Charles
Published: April 2021

The Αv integrins are likely to be an important group of molecules for regulating astrocyte behaviour within the central nervous system. Together with their ligand vitronectin, they are expressed by astrocytes in vivo and are further upregulated during neurological disease. Here we have characterised the expression of Αv integrins on primary astrocytes from both rat and mouse, and shown that they express just two members, ΑvΒ5 and ΑvΒ8. By using RGD peptides and function-blocking antibodies against the Β1 integrins and ΑvΒ5, we find that both αvβ5 and αvβ8 can act as functional receptors for vitronectin. However, while αvβ5 is largely responsible for astrocyte adhesion to vitronectin this integrin appears to play no role in migration on vitronectin, with αvβ8 playing the dominant role in promoting migration on this substrate. β1 integrins are not involved in mediating interactions between astrocytes and vitronectin. These results were confirmed in experiments with astrocytes derived from mice in which the β5 gene had been deleted by homologous recombination. β5 null astrocytes attached to vitronectin at a reduced rate, but showed no defect in migration on vitronectin relative to wild-type astrocytes. These data provide the first evidence that αvβ8 regulates migration and show that astrocyte αvβ5 and αvβ8 integrins have distinct functions.
Tubulin polyglutamylase: isozymic variants and regulation during the cell cycle in HeLa cells
Authors: Regnard C. , Desbruyères E. , Denoulet P. , Eddé B.
Published: April 2021

Polyglutamylation is a posttranslational modification of tubulin that is very common in neurons and ciliated or flagellated cells. It was proposed to regulate the binding of microtubule associated proteins (MAPs) and molecular motors as a function of the length of the polyglutamyl side-chain. Though much less common, this modification of tubulin also occurs in proliferating cells like HeLa cells where it is associated with centrioles and with the mitotic spindle. Recently, we partially purified tubulin polyglutamylase from mouse brain and described its enzymatic properties. In this work, we focused on tubulin polyglutamylase activity from HeLa cells. Our results support the existence of a tubulin polyglutamylase family composed of several isozymic variants specific for α orβ-tubulin subunits. In the latter case, the specificity probably also concerns the differentβ-tubulin isotypes. Interestingly, we found that tubulin polyglutamylase activity is regulated in a cell cycle dependent manner and peaks in G2-phase while the level of glutamylated tubulin peaks in mitosis. Consistent results were obtained by treating the cells with hydroxyurea, nocodazole or taxotere. In particular, in mitotic cells, tubulin polyglutamylase activity was always low while glutamylation level was high. Finally, tubulin polyglutamylase activity and the level of glutamylated tubulin appeared to be inversely related. This paradox suggests a complex regulation of both tubulin polyglutamylase and the reverse deglutamylase activity.
Differential expression of allograft inflammatory factor 1 and of glutathione peroxidase during auto- and allograft response in marine sponges
Authors: Kruse Michael , Steffen Renate , Batel Renato , Müller Isabel M. , Müller Werner E. G.
Published: April 2021

Very recently, Porifera (sponges) have been proven to be suitable model systems to study auto- and allograft recognition at the molecular level. Several potential immune molecules have been isolated from the marine sponges Suberites domuncula and Geodia cydonium, among them those which comprise Ig-like domains in their extracellular part. Here we report on the isolation of two cDNAs from S. domuncula that code for molecules involved in mammals in cytokine-mediated graft response; a putative allograft inflammatory factor 1 (AIF-1) and a non-selenium glutathione peroxidase (GPX). Both polypeptides share high similarity with the corresponding mammalian proteins. The expression of the two genes during auto- and allograft recognition in S. domuncula and G. cydonium was determined. It is shown that the expression of the AIF-1-related gene is upregulated only in allografts, while the GPX-related gene is expressed in the fusion zones formed between auto-as well as allografts. Taken together, these findings suggest that besides cell-mediated defense reactions a cytokine-dependent immune response is also elicited during graft recognition in sponges.
Sec61β, a subunit of the protein translocation channel, is required during: Drosophila development
Authors: Valcárcel Rafael , Weber Ursula , Jackson David B. , Benes VladimÍr , Ansorge Wilhelm , Bohmann Dirk , Mlodzik Marek
Published: April 2021

We have identified and isolated mutations in the first Drosophila gene encoding a subunit of the Sec61 protein translocation channel, DSec61β. While neither the Saccharomyces cerevisiae Sec61β nor its functional Escherichia coli homologue are essential for viability or for protein translocation, we show that DSec61β is essential for embryonic development. Homozygous mutant embryos die at the end of embryogenesis and are impaired in the secretion of cuticle proteins from the epidermis. DSec61β germ line clones, result in defects in dorso-ventral patterning of the egg and are consistent with affected secretion of the protein Gurken from the oocyte to the follicle cells. Clonal analyses in the imaginal discs reveal defects in adult structures, including rhabdomere morphogenesis and a reduction of the size of tarsal segments in the leg. This is the first in vivo study of a component of the protein translocation machinery in higher eukaryotes, and illustrates how a protein that has an inessential, kinetic function in single-cell organisms can become critical for the complex development of a multicellular organism.
Detection of cytokeratin dynamics by time-lapse fluorescence microscopy in living cells
Authors: Windoffer Reinhard , Leube Rudolf E.
Published: April 2021

To monitor the desmosome-anchored cytokeratin network in living cells fusion protein HK13-EGFP consisting of human cytokeratin 13 and the enhanced green fluorescent protein was stably expressed in vulvar carcinoma-derived A-431 cells. It is shown for A-431 subclone AK13-1 that HK13-EGFP emits strong fluorescence in fixed and living cells, being part of an extended cytoplasmic intermediate filament network that is indistinguishable from that of parent A-431 cells. Biochemical, immunological and ultrastructural analyses demonstrate that HK13-EGFP behaves identically to the endogenous cytokeratin 13 and is therefore a reliable in vivo tag for this polypeptide and the structures formed by it. Time-lapse fluorescence microscopy reveals that the cytokeratin 13-containing network is in constant motion, resulting in continuous restructuring occurring in single and migratory cells, as well as in desmosome-anchored cells. Two major types of movement are distinguished: (i) oscillations of mostly long filaments, and (ii) an inward-directed flow of fluorescence originating as diffuse material at the cell periphery and moving in the form of dots and thin filaments toward the deeper cytoplasm where it coalesces with other filaments and filament bundles. Both movements are energy dependent and can be inhibited by nocodazole, but not by cytochalasin D. Finally, disassembly and reformation of cytokeratin filament networks are documented in dividing cells revealing distinct and rapidly occurring stages of cytokeratin organisation and distribution.

Movies available on-line: http://www.biologists.com/JCS/movies/jcs0841.html http://www.uni-mainz.de/FB/Medizin/Anatomie/Leube/
Inhibition of cadherin function differentially affects markers of terminal differentiation in cultured human keratinocytes
Authors: Hines Michelle D. , Jin Hong C. , Wheelock Margaret J. , Jensen Pamela J.
Published: April 2021

Cadherin function is required for normal keratinocyte intercellular adhesion and stratification. In the present study, we have investigated whether cadherin-cadherin interactions may also modulate keratinocyte differentiation, as evidenced by alterations in the levels of several differentiation markers. Confluent keratinocyte cultures, propagated in low Ca2+medium in which cadherins are not active, were pre-incubated with antibodies that block the function of E-cadherin and/or P-cadherin; Ca2+was then elevated to 1 mM to activate the cadherins and induce differentiation. In control cultures (incubated with no antibody or with antibodies to other cell surface molecules), Ca2+elevation induced an increase in type 1 transglutaminase, profilaggrin, and loricrin, as measured by western blotting and in agreement with previous results. However, the concurrent addition of antibodies against both E- and P-cadherin prevented this increase in transglutaminase 1 protein. Incubation with either antibody alone had no consistent effect. Profilaggrin and loricrin, which are later markers of keratinocyte differentiation, responded differently from transglutaminase 1 to addition of antibodies. In the presence of anti-E-cadherin antibody, both loricrin and profilaggrin levels were dramatically enhanced compared to the high Ca2+control cells, while addition of antibody to P-cadherin slightly attenuated the Ca2+-induced increase. In the presence of both antibodies, loricrin and profilaggrin protein levels were intermediate between those observed in the presence of either antibody alone. The expression of involucrin, however, was unaffected by addition of antibodies. In addition, effects of the anti-cadherin antibodies were not secondary to alterations in proliferation or programmed cell death, as determined by several independent assays of these processes. Thus, the consequences of cadherin inhibition depend upon both the particular cadherin and the differentiation marker under study. Taken together, these data suggest that E-cadherin and P-cadherin contribute to the orderly progression of terminal differentiation in the epidermis in multiple ways.
Identification of PA2.26 antigen as a novel cell-surface mucin-type glycoprotein that induces plasma membrane extensions and increased motility in keratinocytes
Authors: Scholl Francisco G. , Gamallo Carlos , Vilaró Senén , Quintanilla Miguel
Published: April 2021

PA2.26 antigen was identified as a cell-surface protein induced in epidermal carcinogenesis and skin remodeling processes. PA2.26 is expressed in carcinoma cell lines and cultured fibroblasts but absent in nontumorigenic keratinocytes. In tissues, PA2.26 is present in epithelial cells of the choroid plexus, ependyma, glomerulus and alveolus, in mesothelial cells, and in endothelia of lymphatic vessels. Biochemical characterization of PA2.26 protein and sequence analysis of the isolated cDNA demonstrate that PA2.26 antigen is a mucin-like transmembrane glycoprotein. Confocal and immunoelectron microscopy analysis in cultured cells reveal that PA2.26 is concentrated in actin-rich microvilli and plasma membrane projections, such as filopodia, lamellipodia and ruffles, where it colocalizes with members of the ERM (ezrin, radixin, moesin) family protein. Ezrin and moesin, but not radixin, can be coimmunoprecipitated together with PA2.26 from cell lysates. Ectopic expression of PA2.26 in immortalized, nontumorigenic, keratinocytes induces an epithelial-fibroblastoid morphological conversion with increased plasma membrane extensions, concomitantly to a major reorganization of the actin cytoskeleton, redistribution of ezrin to cell-surface projections, and enhanced motility. These findings suggest an involvement of PA2.26 in cell migration.
Vimentin contributes to human mammary epithelial cell migration
Authors: Gilles Christine , Polette Myriam , Zahm Jean-Marie , Tournier Jean-Marie , Volders Laure , Foidart Jean-Michel , Birembaut Philippe
Published: April 2021

Vimentin expression in human mammary epithelial MCF10A cells was examined as a function of their migratory status using an in vitro wound-healing model. Analysis of the trajectories of the cells and their migratory speeds by time lapse-video microscopy revealed that vimentin mRNA and protein expression were exclusively induced in cells at the wound’s edge which were actively migrating towards the center of the lesion. Actin labeling showed the reorganization of actin filaments in cells at the wound’s edge which confirmed the migratory phenotype of this cell subpopulation. Moreover, the vimentin protein disappeared when the cells became stationary after wound closure. Using cells transfected with the vimentin promoter controlling the green fluorescent protein gene, we also demonstrated the specific activation of the vimentin promoter in the migratory cells at the wound’s edge. Transfection of the antisense vimentin cDNA into MCF10A cells clearly reduced both their ability to express vimentin and their migratory speed. Taken together, these observations demonstrate that vimentin is transiently associated with, and could be functionally involved in, the migratory status of human epithelial cells.
Characterization of a coiled coil protein present in the basal body of Trypanosoma brucei
Authors: Dilbeck Vincent , Berberof Magali , Cauwenberge Anne Van , Alexandre Henri , Pays Etienne
Published: April 2021

TBBC(for Trypanosoma bruceibasal body component) is a unique gene transcribed in a 4.8 kb mRNA encoding a 1,410 amino acid protein that consists almost entirely of a coiled coil structure. This protein appeared to localize in the basal body, with an accessory presence at the posterior end of the cell, the nucleus and over the flagellum. Since the two other known components of the trypanosome basal body are γ-tubulin and an uncharacterized component termed BBA4 we performed double immunofluorescence experiments with anti-TBBC and either anti-BBA4 or anti-γ-tubulin antibodies. These three components did not colocalize but were very closely associated, BBA4 being the most proximal to the kinetoplast DNA. Anti-TBBC antibodies detected a 170 kDa protein in western blots of total HeLa cell extracts. Moreover, these antibodies stained the centriole of HeLa and COS cells as well as the centriole of mouse spermatozoa, indicating that a TBBC-like centriolar component has been conserved during the evolution of eukaryotes.
Adhesion and migration of avian neural crest cells on fibronectin require the cooperating activities of multiple integrins of the β1 and β3 families
Authors: Testaz Sandrine , Delannet Muriel , Duband Jean-Loup
Published: April 2021

Based on genetic, functional and histological studies, the extracellular matrix molecule fibronectin has been proposed to play a key role in the migration of neural crest cells in the vertebrate embryo. In the present study, we have analyzed in vitro the repertoire and function of integrin receptors involved in the adhesive and locomotory responses of avian truncal neural crest cells to fibronectin. Immunoprecipitation experiments showed that neural crest cells express multiple integrins, namely α3β1, α4β1, α5β1, α8β1, αvβ1, αvβ3 and a β8 integrin, as potential fibronectin receptors, and flow cytometry analyses revealed no major heterogeneity among the cell population for expression of integrin subunits. In addition, the integrin repertoire expressed by neural crest cells was found not to change dramatically during migration. At the cellular level, only αvβ1 and αvβ3 were concentrated in focal adhesion sites in connection with the actin microfilaments, whereas the other integrins were predominantly diffuse over the cell surface. In inhibition assays with function-perturbing antibodies, it appeared that complete abolition of cell spreading and migration could be achieved only by blocking multiple integrins of the β1 and β3 families, suggesting possible functional compensations between different integrins. In addition, these studies provided evidence for functional partitioning of integrins in cell adhesion and migration. While spreading was essentially mediated by αvβ1 and α8β1, migration involved primarily α4β1, αvβ3 and α8β1 and, more indirectly, α3β1. α5β1 and the β8 integrin were not found to play any major role in either adhesion or migration. Finally, consistent with the results of inhibition experiments, recruitment of α4β1 and αvβ3, individually or in combination using antibodies or recombinant VCAM-1 and PECAM-1 molecules as a substratum, was required for migration but was not sufficient to produce migration of the cell population as efficiently as with fibronectin. In conclusion, our study indicates that neural crest cells express a multiplicity of fibronectin-binding integrins and suggests that dispersion of the cell population requires cooperation between distinct integrins regulating different events of cell adhesion, locomotion and, possibly, proliferation and survival.
Integrin subunits β1C-1 and β1C-2 expressed in GD25T cells are retained and degraded intracellularly rather than localised to the cell surface
Authors: Svineng Gunbjørg , Johansson Staffan
Published: April 2021

We have previously identified the integrin β1C-2 and characterised the distribution of β1C-1 and β1C-2 transcripts in various cell lines and normal cells. In this study we have investigated the expression of the two β1C-variants in integrin β1 deficient mouse GD25T cells. After stable transfection of the GD25T cells with cDNAs coding for β1A, β1C-1 and β1C-2, the cell surface expression of the β1C-1 and β1C-2 variants was found to be very low while the β1A variant was expressed at high levels. Northern blot analysis showed that the level of β1-transcript in the β1C-1 and β1C-2 clones was equal or higher than in the β1A clones. Metabolic labelling and deglycosylation by endoglycosidase H treatment clearly demonstrated that the majority of the β1C-1 and β1C-2 chains did not become maturely glycosylated, nor did they dimerise with α subunits. After 20 hours of chase, the labelled β1C-1 and β1C-2 chains had been gradually degraded, whereas immature β1A was converted into the maturely glycosylated form during the same period of time. Immunostaining showed intracellular β1 localisation in the β1C-1 and β1C-2 expressing clones, while in the β1A expressing clones the β1 chains were mainly localised to focal adhesion sites and along fibronectin fibres. Taken together, we have shown that expression of both integrin β1C-1 and β1C-2 in GD25T cells result in very low cell surface expression compared with the normal β1A isoform. Instead, both β1C-1 and β1C-2 chains remain in the endoplasmic reticulum until they are intracellularly degraded.
A contractile activity that closes phagosomes in macrophages
Authors: Swanson Joel A. , Johnson Melissa T. , Beningo Karen , Post Penny , Mooseker Mark , Araki Nobukazu
Published: April 2021

Studies of Fc-mediated phagocytosis by mouse macrophages identified a contractile activity at the distal margins of forming phagosomes. Time-lapse video microscopic analysis of macrophages containing rhodamine-labeled actin and fluorescein dextran showed that actin was concentrated at the distal margins of closing phagosomes. Phagocytosis-related contractile activities were observed when one IgG-opsonized erythrocyte was engaged by two macrophages. Both cells extended pseudopodia until they met midway around the erythrocyte. It was then constricted and pulled into two phagosomes, which remained interconnected by a string of erythrocyte membrane. Butanedione monoxime, an uncompetitive inhibitor of class II and perhaps other myosins, and wortmannin and LY294002, inhibitors of phosphoinositide 3-kinase, prevented the constrictions without inhibiting the initial pseudopod extension. Immunofluorescence microscopy showed the presence of myosins IC, II, V and IXb in phagosomes. Of these, only myosin IC was concentrated around the strings connecting shared erythrocytes, suggesting that myosin IC mediates the purse-string-like contraction that closes phagosomes. The sequential processes of pseudopod extension and contraction can explain how macropinosomes and spacious phagosomes form without guidance from a particle surface.
Clathrin, adaptors and eps15 in endosomes containing activated epidermal growth factor receptors
Authors: Sorkina Tatiana , Bild Andrea , Tebar Francesc , Sorkin Alexander
Published: April 2021

Activation of the epidermal growth factor receptor (EGFR) by EGF results in binding of clathrin adaptor protein complex AP-2 to the receptor cytoplasmic tail. The transient interaction with AP-2 is thought to be responsible for the selective recruitment of the EGFR into coated pits during endocytosis. In this study we found that EGF-induced EGFR/AP-2 association, measured by co-immunoprecipitation, persists after receptor internalization. Double-label immunofluorescence of EGF-treated A-431 and COS-1 cells revealed the presence of AP-2, clathrin and eps15, another component of the plasma membrane coated pits, in the large perinuclear endosomes loaded with EGFRs. By optical sectioning and image deconvolution, the immunoreactivities were seen to be distributed within vesicular and tubular elements of these endosomes. In addition, these compartments contained the transferrin receptors and a EEA.1 protein, markers of early endosomes. Furthermore, Golgi clathrin adaptor complex AP-1 was found in EGFR-containing endosomes and EGFR immunoprecipitates in A-431 cells. The direct interaction of the EGFR with µ1 as well as µ2 subunits of AP-1 and AP-2, correspondingly, was shown using the yeast two-hybrid assay. Brefeldin A, a drug that releases AP-1 from the trans-Golgi membranes, had no effect on AP-1 association with endosomes and its co-precipitation with EGFR. Taken together, the data suggest that endosomal EGFR-AP complexes make up a significant portion of the total amount of these complexes detectable by coimmunoprecipitation. It can be proposed that APs are capable of binding to the endosomal membrane via a mechanism that requires AP interaction with the intracellular tails of multimeric receptors like activated EGFR, which in turn allows recruitment of clathrin and eps15. The hypothesis that the competition between adaptor complexes for binding to the receptor tails in endosomes may regulate of the sorting of receptors is discussed.
Antagonistic effects of NES and NLS motifs determine S. cerevisiae Rna1p subcellular distribution
Authors: Feng Wenqin , Benko Ann L. , Lee Jia-Hai , Stanford David R. , Hopper Anita K.
Published: April 2021

Nucleus/cytosol exchange requires a GTPase, Ran. In yeast Rna1p is the GTPase activating protein for Ran (RanGAP) and Prp20p is the Ran GDP/GTP exchange factor (GEF). RanGAP is primarily cytosolic and GEF is nuclear. Their subcellular distributions led to the prediction that Ran-GTP hydrolysis takes place solely in the cytosol and GDP/GTP exchange solely in the nucleus. Current models propose that the Ran-GTP/Ran-GDP gradient across the nuclear membrane determines the direction of exchange. We provide three lines of evidence that Rna1p enters and leaves the nuclear interior. (1) Rna1p possesses leucine-rich nuclear export sequences (NES) that are able to relocate a passenger karyophilic protein to the cytosol; alterations of consensus residues re-establish nuclear location. (2) Rna1p possesses other sequences that function as a novel nuclear localization sequence able to deliver a passenger cytosolic protein to the nucleus. (3) Endogenous Rna1p location is dependent upon Xpo1p/Crm1p, the yeast exportin for leucine-rich NES-containing proteins. The data support the hypothesis that Rna1p exists on both sides of the nuclear membrane, perhaps regulating the Ran-GTP/Ran-GDP gradient, participating in a complete RanGTPase nuclear cycle or serving a novel function.
Intracellular processing of endocytosed triglyceride-rich lipoproteins comprises both recycling and degradation
Authors: Heeren Jörg , Weber Wilfried , Beisiegel Ulrike
Published: April 2021

The current study was performed to investigate the intracellular fate of triglyceride-rich lipoproteins. Triglyceride-rich lipoproteins are responsible for the delivery of lipids to various tissues, however, their intracellular pathway has not yet been elucidated. Here radiolabeled triglyceride-rich lipoproteins, associated with lipoprotein lipase, were used for the quantitative evaluation of the intracellular metabolism. Pulse chase experiments showed that after 90 minutes approximately 60% of the labeled protein, mainly apoproteins E and C, was released intact into the medium, where it re-associates with lipoproteins. Apoprotein B, in contrast, was degraded, following the same pathway as the apoprotein B from low density lipoproteins. In kinetic experiments uptake and intracellular fate of triglyceride-rich lipoproteins was compared to that of transferrin and low density lipoproteins. These experiments revealed that apoproteins were retained inside the cell much longer than transferrin, and unlike low density lipoproteins were not degraded. Using immunofluorescence it was shown that apoprotein E and lipoprotein lipase follow a distinct route from the sorting compartment to the surface, which is clearly distinguishable from the perinuclear transferrin recycling compartment. In contrast, the fluorescence labeled lipids were delivered to lysosomal compartments. The data presented here show that surface proteins of triglyceride-rich lipoproteins, such as apoproteins E and C and lipoprotein lipase follow a recycling pathway, whereas lipids and high molecular mass core proteins are degraded.
Involvement of the protein kinase C substrate, SSeCKS, in the actin-based stellate morphology of mesangial cells
Authors: Nelson Peter J. , Moissoglu Konstadinos , Vargas Jesus , Klotman Paul E. , Gelman Irwin H.
Published: April 2021

Activation of protein kinase C is a key signal transduction event in mesangial cell dedifferentiation and proliferation, yet little is known about downstream substrates or their roles in normal or diseased glomeruli. SSeCKS, a novel protein kinase C substrate originally isolated as a srcsuppressed negative mitogenic regulator in fibroblasts, controls actin-based cytoskeletal architecture and scaffolds key signaling kinases such as protein kinase C and protein kinase A. Based on the morphologic similarity between SSeCKS-overexpressing fibroblasts and stellate mesangial cells, we hypothesized that SSeCKS might play a role in mesangial cell morphology in a protein kinase C-dependent manner. Immunoblotting, in situ staining and northern blotting detected abundant expression of SSeCKS in human and rodent mesangial cells and glomerular parietal cells but not in renal tubular epithelia. Immunofluorescence analysis showed enrichment of SSeCKS in mesangial cell podosomes and along a cytoskeletal network distinct from F-actin. Activation of protein kinase C by phorbol ester resulted in a rapid serine phosphorylation of SSeCKS and its subsequent translocation to perinuclear sites, coincident with the retraction of stellate processes. These effects were blocked by concentrations of bis-indolylmaleimide that selectively inhibit protein kinase C. Finally, ablation of SSeCKS expression using retroviral anti-sense vectors induced (1) an elongated, fibroblastic cell morphology, (2) production of thick, longitudinal stress fibers and (3) repositioning of vinculin-associated focal complexes away from the cell edges. These data suggest a role for SSeCKS as a downstream mediator of protein kinase C-controlled, actin-based mesangial cell cytoskeletal architecture.
MEC-12, an α-tubulin required for touch sensitivity in C. elegans
Authors: Fukushige Tetsunari , Siddiqui Zeba K. , Chou Michael , Culotti Joseph G. , Gogonea Camelia B. , Siddiqui Shahid S. , Hamelin Michel
Published: April 2021

mec-12 is one of a dozen genes required for touch receptor neuron function in Caenorhabditis elegans. Some mec-12 mutants (mechanosensory-defective) lack the large-diameter microtubules that are characteristic of these neurons (15 protofilaments, as opposed to 11). Mutants of mec-7, a β-tubulin encoding gene, have a similar phenotype. We have identified the nature of mec-12 by germline transformation rescue and characterization of a point mutation. Sequence analysis of the mec-12 encoded product (MEC-12) indicates that it corresponds to a novel C. elegans α-tubulin. MEC-12 is the only identified C. elegans α-tubulin that contains a lysine at position 40, a known site of post-translational acetylation. Some mec-12 mutations eliminate microtubule acetylation as assayed immunocytochemically; phenotypic rescue using a MEC-12 variant lacking the lysine-40 showed that acetylation is not required for MEC-12 activity. Although functionally needed only in the touch neurons, mec-12 is expressed in several other neuron types. These results support the notion that tubulin isotype diversity contributes to the formation of distinct classes of microtubules; 15protofilament microtubule assembly requires MEC-12 α-tubulin and MEC-7 β-tubulin, which are both highly expressed in the touch receptor neurons. MEC-12 is the first reported α-tubulin isotype that appears to be required in a single class of neuronal microtubules.
The nucleolar phosphoprotein B23 redistributes in part to the spindle poles during mitosis
Authors: Zatsepina Olga V. , Rousselet Annie , Chan Pui K. , Olson Mark O. J. , Jordan E. G. , Bornens Michel
Published: April 2021

B23 is a major phosphoprotein in the interphasic nucleolus where it is involved in the assembly of pre-ribosomes. Using several cultured animal cells, we report that, in addition to the known redistribution of the protein during mitosis, B23 also becomes associated with mitotic spindle poles starting from early prometaphase onwards. Colocalization of B23 with the protein NuMA (Nuclear Mitotic Apparatus protein) was studied in mitotic cells and taxol-arrested cells. During the onset of mitosis, we observed that a fraction of B23 associates with, and dissociates from, the poles later than NuMA. At metaphase, both proteins are colocalized at the poles. The polar redistribution of both B23 and NuMA is mediated by microtubules. In taxol-treated cells, B23 is associated with the microtubule minus ends in the center of mitotic asters together with NuMA. Association of B23 with microtubule minus ends of mitotic asters was further confirmed with an in vitro assay, where B23 was found by western blotting to co-sediment with taxol-induced microtubule asters formed in a mitotic cell extract. Immunolabeling demonstrated that B23 and NuMA were both present at the center of the asters. Furthermore, an additional hyperphosphorylated form of B23 appeared when microtubule asters formed and associated with the asters. Immunodepletion of B23 from the mitotic extract revealed that taxol-induced microtubule asters were still observed in B23-immunodepleted mitotic extract, indicating that the presence of B23 at the poles is unlikely to be essential for spindle formation or stabilisation.