bluuebunny/crossref_metadata_2025_split huggingface dataset:
Microtubule minus-end anchorage at centrosomal and non-centrosomal sites: the role of ninein
Authors: Mogensen Mette M. , Malik Azer , Piel Matthieu , Bouckson-Castaing Veronique , Bornens Michel 
Published: April 2021

ABSTRACT
The novel concept of a centrosomal anchoring complex, which is distinct from the γ-tubulin nucleating complex, has previously been proposed following studies on cochlear epithelial cells. In this investigation we present evidence from two different cell systems which suggests that the centrosomal protein ninein is a strong candidate for the proposed anchoring complex.
Ninein has recently been observed in cultured fibroblast cells to localise primarily to the post-mitotic mother centriole, which is the focus for a classic radial microtubule array. We show here by immunoelectron microscopical analyses of centrosomes from mouse L929 cells that ninein concentrates at the appendages surrounding the mother centriole and at the microtubule minus-ends. We further show that localisation of ninein in the cochlear supporting epithelial cells, where the vast majority of the microtubule minus-ends are associated with apical non-centrosomal sites, suggests that it is not directly involved in microtubule nucleation. Ninein seems to play an important role in the positioning and anchorage of the microtubule minus-ends in these epithelial cells. Evidence is presented which suggests that ninein is released from the centrosome, translocated with the microtubules, and is responsible for the anchorage of microtubule minus-ends to the apical sites. We propose that ninein is a non-nucleating microtubule minus-end associated protein which may have a dual role as a minus-end capping and anchoring protein.
α3β1 and α6β4 integrin receptors for laminin-5 are not essential for epidermal morphogenesis and homeostasis during skin development
Authors: DiPersio C. Michael , Neut Ronald van der , Georges-Labouesse Elisabeth , Kreidberg Jordan A. , Sonnenberg Arnoud , Hynes Richard O. 
Published: April 2021

ABSTRACT
Continuous regeneration and homeostasis of the stratified epidermis requires coordinated regulation of cell proliferation, cell differentiation, and cell survival. Integrin-mediated cell adhesion to the extracellular matrix has important roles in regulating each of these processes. Integrins α3β1 and α6β4 are both receptors on epidermal keratinocytes for the basement membrane protein laminin-5, the major ligand for epidermal adhesion in mature skin. Ablation in mice of either α3β1 or α6β4, through null mutation of the gene encoding the α3, α6, or β4 integrin subunit, results in epidermal blistering of varying severity. Our previous studies showed that, despite blistering, differentiation and stratification of the epidermis appeared essentially normal in mice that lacked either α3β1 or α6β4. However, these studies did not definitively address the specific developmental importance of each integrin, since they may have overlapping and/or compensatory functions. Given the individual importance of α3β1 or α6β4 in maintaining the dermo-epidermal junction in mature skin, we sought to determine the importance of these integrins for embryonic skin development and epidermal morphogenesis. In the current study, we analyzed skin development in mutant embryos that completely lack both integrins α3β1 and α6β4. Although α3β1/α6β4-deficient embryos displayed epidermal blistering by stage E15.5 of development, they also retained regions of extensive epidermal adhesion to the basement membrane through stage E16.5, indicating alternative adhesion mechanisms. Apoptosis was induced in detached epidermis of α3β1/α6β4-deficient embryos, exemplifying vividly the importance of epithelial attachment to the basement membrane for cell survival. However, apoptotic cells were completely absent from attached epidermis of α3β1/α6β4-deficient embryos, showing that epithelial adhesion that occurred independently of α3β1 and α6β4 also protected cells from apoptosis. Remarkably, in the absence of the known laminin-5 binding integrins (α3β1, α6β4, and α6β1), keratinocytes retained the capacity to proliferate in the epidermis, and epidermal stratification and skin morphogenesis appeared normal prior to blister formation. These findings show that while α3β1 and α6β4 are both required for integrity of the dermo-epidermal junction, neither one is essential for epidermal morphogenesis during skin development.
Involvement of Iba1 in membrane ruffling and phagocytosis of macrophages/microglia
Authors: Ohsawa Keiko , Imai Yoshinori , Kanazawa Hiroko , Sasaki Yo , Kohsaka Shinichi 
Published: April 2021

ABSTRACT
Ionized calcium binding adaptor molecule 1, Iba1, is an EF hand calcium binding protein whose expression is restricted to macrophages/microglia. In this study, Iba1 was shown to colocalize with F-actin in membrane ruffles induced by macrophage colony-stimulating factor and in phagocytic cups formed during zymosan phagocytosis. Expression of mutant Iba1 carrying either N-or C-terminal deletions or carrying a substitution in the calcium binding domain, suppressed the membrane ruffling and the phagocytosis. These results indicate that Iba1 is a key molecule in membrane ruffling and the phagocytosis of macrophages/microglia. Furthermore, Iba1 colocalized with a small GTPase Rac in the membrane ruffles and the phagocytic cups. The Iba1 mutants also suppressed membrane ruffling induced by dominant active Rac1V12, but do not affect microspikes by Cdc42V12 and stress fibers by RhoAV14. These observations suggest that Iba1 is involved in Rac and calcium signaling pathways.
Plakoglobin and β-catenin: Protein interactions, regulation and biological roles
Authors: Zhurinsky Jacob , Shtutman Michael , Ben-Ze′ev Avri 
Published: April 2021

ABSTRACT
β-Catenin can play different roles in the cell, including one as a structural protein at cell-cell adherens junctions and another as a transcriptional activator mediating Wnt signal transduction. Plakoglobin (γ-catenin), a close homolog of β-catenin, shares with β-catenin common protein partners and can fulfill some of the same functions. The complexing of catenins with various protein partners is regulated by phosphorylation and by intramolecular interactions. The competition between different catenin partners for binding to catenins mediates the cross-talk between cadherin-based adhesion, catenin-dependent transcription and Wnt signaling. Although plakoglobin differs from β-catenin in its functions and is unable to compensate for defects in Wnt signaling resulting from lack of β-catenin, recent evidence suggests that plakoglobin plays a unique role in Wnt signaling that is different from that of β-catenin. The functional difference between catenins is reflected in their differential involvement in embryonic development and cancer progression.
Transient suppression of cortactin ectopically induces large telencephalic neurons towards a GABAergic phenotype
Authors: Cheng Yu , Leung Soyan , Mangoura Dimitra 
Published: April 2021

ABSTRACT
Excitatory and inhibitory neuronal cell fates require specific expression of both neurotransmitter and morphological phenotypes. The role of the F-actin cytoskeleton in morphological phenotypes has been well documented, but its role in neurotransmitter phenotype expression remains unknown. Here we present evidence that the F-actin binding protein cortactin participates in determining both aspects of cell fate in large telencephalic neurons. We show that the expression of cortactin was upregulated early in development just prior to appearance of GABAergic neurons in the chick telencephalon at embryonic day 6. This program was faithfully maintained in primary neuronal cultures derived from E6 telencephalon, where immature neurons differentiate either to large pyramidal and large stellate excitatory neurons or to small inhibitory GABAergic neurons. Immunostaining revealed that cortactin was enriched in areas of membrane budding, growth cones, and in the cell cortex of immature neurons. With differentiation, intense punctate staining was also observed in an extraction-resistant cytosolic compartment of the soma and processes.
More importantly, suppression of cortactin by inhibition of cortactin mRNA translation with antisense oligonucleotides caused permanent phenotypic changes. Specifically, a transient suppression of cortactin was achieved in immature neurons with a single exposure to antisense oligonucleotides. This inhibition first induced both the expression of mRNA and the enzymatic activity of GAD significantly earlier than in control neurons. Second, cortactin-suppressed large projectional neurons exhibited significantly shorter processes and growth cones with protrusive filopodia and an enlarged lamellipodia veil. Most importantly, this remodeling of neuritic outgrowth in projectional somata was accompanied by the ectopic induction of GABA (γ-aminobutyric acid) expression. Considering this data altogether, it appears that cortactin may function to suppress concurrently several parameters of the GABAergic program in large developing neurons.
Protein kinase C activation leads to dephosphorylation of occludin and tight junction permeability increase in LLC-PK1 epithelial cell sheets
Authors: Clarke Hilary , Soler Alejandro Peralta , Mullin James M. 
Published: April 2021

ABSTRACT
Activation of protein kinase C by exposure of LLC-PK1 renal epithelial cells to 10−7 M TPA, a tumor promoting phorbol ester, results in a rapid and sustained increase in paracellular permeability as evidenced by a decrease in transepithelial electrical resistance. Occludin, the first identified transmembrane protein to be localized to the tight junction of both epithelial and endothelial cells is thought play an important role in tight junction barriers. Although transepithelial electrical resistance fell to less than 20% of initial values within 1 hour of TPA exposure, transmission electron microscopy showed no change in the gross morphology of the tight junction of cells treated with 10−7 M TPA for up to 2 hours. Immunofluorescence microscopy revealed a more rapid change in the membrane distribution of ZO-1 compared to occludin in the TPA-treated cells. Immunoblot analysis indicated that occludin levels in total cell lysates as well as cytosolic, membrane (Triton-X soluble) and cytoskeletal (Triton-X insoluble) fractions remained unchanged for at least 2 hours in cells treated with 10−7 M TPA compared to their corresponding control cells. As the phosphorylation state of occludin is thought to be important in both tight junction assembly and regulation, the effect of phorbol ester treatment on the phosphorylation of occludin was investigated. Surprisingly, activation of protein kinase C with 10−7 M TPA resulted in a time-dependent decrease in threonine phosphorylation of occludin which correlated closely with the rapid decrease in transepithelial electrical resistance. This dephosphorylation of occludin, occuring after activation of a serine/threonine kinase by TPA, suggested that protein kinase C was not acting directly on this tight junction target protein. If occludin dephosphorylation is involved in increasing tight junction premeability, then protein kinase C is apparently further upstream in the signaling pathway regulating epithelial barrier function, with a downstream serine/threonine phosphatase acting upon occludin.
Membrane localization and biological activity of SNAP-25 cysteine mutants in insulin-secreting cells
Authors: Gonelle-Gispert Carmen , Molinete Miguel , Halban Philippe A. , Sadoul Karin 
Published: April 2021

ABSTRACT
The tSNARE SNAP-25 is expressed in pancreatic β-cells and is involved in the regulated release of insulin. It has been shown previously that SNAP-25 associates with the plasma membrane consequent to palmitoylation of one or more cysteines in the central region of the molecule. The importance of palmitolyation in the biological function of SNAP-25 in exocytosis was not addressed. Furthermore, studies on both SNAP-25 and its non-palmitoylated homologues SNAP-29 and sec9, have suggested an alternative or complementary mechanism for membrane association involving interaction with syntaxin. To address these issues, we have now studied the behavior and biological activity of cysteine mutant SNAP-25 in insulin-secreting (HIT) cells. While 91% of native SNAP-25 was associated with the membrane, this value decreased to 56% for the single cysteine mutant C85/A and to 10% for the double (C85,88/A) and quadruple (C85,88,90,92/A) mutants. The mutant SNAP-25 forms were all found to bind syntaxin 1A with equal efficacy. Over-expression of syntaxin 1A in HIT cells allowed for partial relocalization of both the double and quadruple SNAP-25 cys mutants to the membrane. By introducing a further mutation to the SNAP-25 molecules to render them resistant to botulinum neurotoxin E, it was possible to study their ability to reconstitute regulated insulin secretion in toxin-treated HIT cells. Native SNAP-25 was able to fully reconstitute secretory activity in such cells. Despite the fact that the single cysteine mutant was significantly displaced to the cytosol, it still displayed 82% activity in the secretion reconstitution assay, and a similar discrepancy was seen for the double mutant. Even the quadruple mutant with no remaining cysteines was able to support a minimal level of secretion. It is concluded that both palmitoylation and binding to syntaxin are implicated in membrane association of SNAP-25. This as well as the discrepancy between membrane localization and biological activity of the cysteine mutants, suggests a complex, multi-component process for association of SNAP-25 with the membrane and its recruitment to a biologically productive state.
Clathrin-mediated endocytosis and recycling of autocrine motility factor receptor to fibronectin fibrils is a limiting factor for NIH-3T3 cell motility
Authors: Le Phuong U. , Benlimame Naciba , Lagana Annick , Raz Avraham , Nabi Ivan R. 
Published: April 2021

ABSTRACT
Autocrine motility factor receptor (AMF-R) is internalized via a clathrin-independent pathway to smooth endoplasmic reticulum tubules. This endocytic pathway is shown here to be inhibited by methyl-β-cyclodextrin (mβCD) implicating caveolae or caveolae-like structures in AMF internalization to smooth ER. AMF-R is also internalized via a clathrin-dependent pathway to a transferrin receptor-negative, LAMP-1/lgpA-negative endocytic compartment identified by electron microscopy as a multivesicular body (MVB). Endocytosed AMF recycles to cell surface fibrillar structures which colocalize with fibronectin; AMF-R recycling is inhibited at 20°C, which blocks endocytosis past the early endosome, but not by mβCD demonstrating that AMF-R recycling to fibronectin fibrils is mediated by clathrin-dependent endocytosis to MVBs. Microtubule disruption with nocodazole did not affect delivery of bAMF to cell surface fibrils indicating that recycling bAMF traverses the MVB but not a later endocytic compartment. Plating NIH-3T3 cells on an AMF coated substrate did not specifically affect cell adhesion but prevented bAMF delivery to cell surface fibronectin fibrils and reduced cell motility. AMF-R internalization and recycling via the clathrin-mediated pathway are therefore rate-limiting for cell motility. This recycling pathway to the site of deposition of fibronectin may be implicated in the de novo formation of cellular attachments or the remodeling of the extracellular matrix during cell movement.
Biochemical characterization and localization of the dual specificity kinase CLK1
Authors: Menegay Harry J. , Myers Michael P. , Moeslein Fred M. , Landreth Gary E. 
Published: April 2021

ABSTRACT
CLK1 was one of the first identified dual specificity kinases and is the founding member of the ‘LAMMER’ family of kinases. We have established the substrate site specificity of CLK1. We report here that truncation of the N terminus of CLK1 resulted in a dramatic increase in CLK1 enzymatic activity, indicating that the N terminus acts as a negative regulatory domain. The N-terminal truncation resulted in a 45-fold increase in Vmax, suggesting that this domain does not contain a pseudo-substrate motif, but may act to conformationally constrain the catalytic activity of CLK1. Tyrosine phosphorylation has been proposed to be critical for CLK1 activity, however, CLK1 activity was unaffected by exposure to tyrosine phosphatases. Treatment of CLK1 with the serine/threonine specific phosphatase PP2A, resulted in a 2-to 6-fold increase in enzymatic activity. Incubation of CLK1 with tyrosine phosphatases in combination with PP2A abolished CLK1 activity. These data suggest that CLK1 is regulated by three distinct mechanisms that serve to both positively and negatively regulate CLK1 activity. CLK1 activity is positively regulated by phosphorylation on either tyrosine residues or serine/threonine residues, and is negatively regulated by steric constraints mediated by the N-terminal domain, as well as, by phosphorylation on a subset of serine/threonine residues within the catalytic domain. CLK1 mRNA is expressed at low levels in all tissues and cell lines examined. The full-length and truncated splice forms are expressed at roughly equivalent levels in most tissues. The ratio of the two splice variants of CLK1 can be altered by treatment with cycloheximide. CLK1 protein expression is limited to a small subset of highly localized neuronal populations in the rat brain. Contrary to previous studies using overexpression systems, we show that CLK1 protein is primarily found in the cytoplasm of these cells, with only a small fraction localized to the nucleus.
A myosin family tree
Authors: Hodge Tony , Cope M. Jamie T. V. 
Published: April 2021

ABSTRACT
This unrooted phylogenetic tree of the myosin superfamily (see insert) is derived from an alignment of 139 members of the myosin superfamily. The alignment compared the core motor domains (equivalent to residues 88-780 of chicken skeletal myosin II) of each myosin, using distance matrix analysis performed with the Clustal-W package. The exceptions, shown with a dotted line, are SsVIIa, which is a partial sequence as reported in the databases, and Hs MysPDZ. The latter is reported as a complete coding sequence but has a truncated N terminus starting some 52 residues into the core motor region (i.e. residue 140 of chicken skeletal myosin II). These shorter sequences have no significant effect on the branching order of the rest of the tree.
Positive and negative regulation of telomerase access to the telomere
Authors: Evans Sara K. , Lundblad Victoria 
Published: April 2021

ABSTRACT
The protective caps on chromosome ends – known as telomeres – consist of DNA and associated proteins that are essential for chromosome integrity. A fundamental part of ensuring proper telomere function is maintaining adequate length of the telomeric DNA tract. Telomeric repeat sequences are synthesized by the telomerase reverse transcriptase, and, as such, telomerase is a central player in the maintenance of steady-state telomere length. Evidence from both yeast and mammals suggests that telomere-associated proteins positively or negatively control access of telomerase to the chromosome terminus. In yeast, positive regulation of telomerase access appears to be achieved through recruitment of the enzyme by the end-binding protein Cdc13p. In contrast, duplex-DNA-binding proteins assembled along the telomeric tract exert a feedback system that negatively modulates telomere length by limiting the action of telomerase. In mammalian cells, and perhaps also in yeast, binding of these proteins probably promotes a higher-order structure that renders the telomere inaccessible to the telomerase enzyme.
Exosome: from internal vesicle of the multivesicular body to intercellular signaling device
Authors: Denzer Kristin , Kleijmeer Monique J. , Heijnen Harry F. G. , Stoorvogel Willem , Geuze Hans J. 
Published: April 2021

ABSTRACT
Exosomes are small membrane vesicles that are secreted by a multitude of cell types as a consequence of fusion of multivesicular late endosomes/lysosomes with the plasma membrane. Depending on their origin, exosomes can play roles in different physiological processes. Maturing reticulocytes externalize obsolete membrane proteins such as the transferrin receptor by means of exosomes, whereas activated platelets release exosomes whose function is not yet known. Exosomes are also secreted by cytotoxic T cells, and these might ensure specific and efficient targeting of cytolytic substances to target cells. Antigen presenting cells, such as B lymphocytes and dendritic cells, secrete MHC class-I- and class-II-carrying exosomes that stimulate T cell proliferation in vitro. In addition, dendritic-cell-derived exosomes, when used as a cell-free vaccine, can eradicate established murine tumors. Although the precise physiological target(s) and functions of exosomes remain largely to be resolved, follicular dendritic cells (accessory cells in the germinal centers of secondary lymphoid organs) have recently been shown to bind B-lymphocyte-derived exosomes at their cell surface, which supports the notion that exosomes play an immunoregulatory role. Finally, since exosomes are derived from multivesicular bodies, their molecular composition might provide clues to the mechanism of protein and lipid sorting in endosomes.
Myosin light chain kinase plays an essential role in S. flexneri dissemination
Authors: Rathman Michelle , Lanerolle Primal de , Ohayon Hélène , Gounon Pierre , Sansonetti Philippe 
Published: April 2021

ABSTRACT
Shigella flexneri, the causitive agent of bacillary dysentery, has been shown to disseminate in colonic epithelial cells via protrusions that extend from infected cells and are endocytosed by adjacent cells. This phenomenon occurs in the region of the eukaryotic cell’s adherens junctions and is inhibited by pharmacological reagents or host cell mutations that completely disrupt the junctional complex. In this study, inhibitors of the myosin light chain kinase (MLCK) were shown to dramatically decrease intercellular spread of S. flexneri but to have no inhibitory effect on bacterial entry, multiplication or actin-based motility within the host cell. Furthermore, cell-to-cell spread of Listeria monocytogenes, another bacterial pathogen that uses an actin-based mechanism to move within the eukaryotic cytoplasm and to spread from cell to cell, was not affected by the MLCK inhibitors, indicating that (1) the inhibition of S. flexneri cell-to-cell spread in treated cells is not due to a complete break down of cell-cell contacts, which was subsequently confirmed by confocal microscopy, and (2) MLCK plays a role in a S. flexneri-specific mechanism of dissemination. Myosin has been shown to play a role in a variety of membrane-based phenomena. The work presented here suggests that activation of this molecule via phosphorylation by MLCK, at the very least participates in the formation of the bacteria-containing protrusion, and could also contribute to the endocytosis of this structure by neighboring cells.