bluuebunny/crossref_metadata_2025 huggingface dataset:
The ’ ligand-induced conformational change’ of α5βl integrin: Relocation of α5 subunit to uncover the β1 stalk region
Authors: Tsuchida Jun , Ueki Shoko , Takada Yoshikazu , Saito Yuji , Takagi Junichi
Published: April 2021

Integrin heterodimers undergo a conformational change upon the binding of ligand to their extracellular domains. An anti-β1 integrin monoclonal antibody AG89 can detect such a conformational change since it recognizes a ligand-inducible epitope in the stalk-like region of β1 subunits. The binding of a 125I-labeled AG89 Fab fragment to α5β1 integrins on K562 cells was assessed and analyzed by the Scatchard method. High affinity binding sites for AG89 are present on cells treated with ligand peptide. In addition, results revealed that cells treated with EDTA also express AG89 binding sites with the same affinity although the number of binding sites is 4-fold lower. AG89 immunoprecipitated α5β1 complexes from surface-labeled K562 cells treated with ligand peptide. By contrast, it immunoprecipitated only β1 chains when the ligand peptide was absent, suggesting that high affinity binding sites on EDTA-treated cells are associated with non-functional β1 monomer. Additional studies show that the epitope for AG89 is constitutively exposed on mutant β1 that cannot complex with α5. These data suggest that the AG89 epitope is masked by the α5 subunit. Ligand binding and integrin activation may uncover the β1 stalk region by triggering a conformational shift of α5 relative to β1.
The role of nucleation in patterning microtubule networks
Authors: Hyman A. , Karsenti E.
Published: April 2021

Control of microtubule nucleation is important for many microtubule dependent processes in cells. Traditionally, research has focused on nucleation of microtubules from centrosomes. However, it is clear that microtubules can nucleate from non-centrosome dependent sites. In this review we discuss the consequences of non-centrosome dependent microtubule nucleation for formation of microtubule patterns, concentrating on the assembly of mitotic spindles.
Localization and processing from a polycistronic precursor of novel snoRNAs in maize
Authors: Shaw Peter J. , Beven Alison F. , Leader David J. , Brown John W. S.
Published: April 2021

We have shown previously that groups of U14 snoRNA genes are clustered with other, novel snoRNAs in maize. These genes are transcribed polycistronically from an upstream promoter to give a precursor snoRNA, which is processed by a splicing-independent mechanism. The clusters contain both box C/D snoRNAs, thought to guide rRNA O-ribose methylations, and the first plant box H/ACA snoRNA so far identified, thought to guide an rRNA pseudo-uridylation. Here we show that four novel snoRNAs identified as members of U14-containing gene clusters each show distinct sub-nucleolar localizations. Two of the snoRNAs (snoR2, a box H/ACA snoRNA, and snoR3, a box C/D snoRNA) colocalise closely with nucleolar rDNA transcription sites. A third box C/D snoRNA, U49, is localised to a more extended region which includes the transcription sites. On the other hand snoR1, another box C/D snoRNA, is located in a quite different region of the nucleolus, and shows a similar distribution to that of 7–2/MRP, a snoRNA involved in the later pre-rRNA cleavage reactions. This may indicate that this snoRNA is involved at later stages of processing, whereas the other snoRNAs are involved early or cotranscriptionally. Probes to intergenic spacer regions of the precursor snoRNA have been used to determine the location of the precursor. This shows a clear labelling of both the dense fibrillar component of the nucleolus, and of coiled bodies. This distribution implies that the polycistronic precursor is imported into the nucleolus for processing to the mature snoRNAs, and that the import or processing pathway involves coiled bodies.
Advillin (p92): a new member of the gelsolin/villin family of actin regulatory proteins
Authors: Marks Peter W. , Arai Maya , Bandura Jennifer L. , Kwiatkowski David J.
Published: April 2021

A new member of the gelsolin/villin family of actin regulatory proteins was initially identified by screening an adult murine brain cDNA library with a probe for bovine adseverin. The predicted amino acid sequence of the 92 kDa murine protein p92 (advillin) is 75% homologous to villin and 65% homologous to gelsolin and adseverin. It shares a six domain structure with other gelsolin family members and has a carboxy-terminal headpiece, similar to, yet distinct from, villin. Northern blot analysis shows a high level of mRNA expression in murine uterus and human intestine. In situ mRNA analysis of adult murine tissues demonstrates that the message is most highly expressed in the endometrium of the uterus, the intestinal lining, and at the surface of the tongue. In murine embryonic development, strong expression of the message is observed by day 14.5 in dorsal root ganglia and trigeminal ganglia. Expression is also noted at day 16.5 in cerebral cortex. We propose that p92 (advillin) has unique functions in the morphogenesis of neuronal cells which form ganglia, and that it may compensate to explain the near normal phenotype observed in villin-deficient mice.
YEB3/VAC8 encodes a myristylated armadillo protein of the Saccharomyces cerevisiae vacuolar membrane that functions in vacuole fusion and inheritance
Authors: Pan X. , Goldfarb D.S.
Published: April 2021

Armadillo (Arm) repeat proteins such as beta-catenin and alpha-karyopherin (importin) are thought to mediate the docking of cargo at membrane-associated cytoskeletal elements. YEB3 encodes an uncharacterized Saccharomyces cerevisiae protein that contains eleven tandem Arm repeats. While YEB3 is nonessential for growth, yeb3delta cells accumulated numerous small vacuoles and are defective in vacuolar inheritance. A functional Yeb3p-green fluorescent protein (GFP) chimera localized to vacuolar membranes. Confocal microscopy revealed that Yeb3p-GFP is localized over the surface of the vacuole, but is concentrated approximately 5- to 7-fold in bands located between clustered vacuoles. N-terminal myristylation of Yeb3p is required for vacuolar localization. The first 69 amino acids of Yeb3p were sufficient to target a GFP reporter protein to the vacuolar membrane; however, this fusion protein also localized to the plasma membrane, indicating that additional sequence is required for exclusive steady state vacuolar localization. By analogy to the function of beta-catenin in cell-cell adhesion, alpha-karyopherin in nuclear transport, and smgGDS in the control of ras-like GTPases, Yeb3p may provide a link between vacuoles and the actin cytoskeleton during vacuolar inheritance and fusion and perhaps mediate the assembly of a GTPase regulated docking complex.
Antisense oligonucleotides against ‘cardiac’ and ‘skeletal’ DHP-receptors reveal a dual role for the ‘skeletal’ isoform in EC coupling of skeletal muscle cells in primary culture
Authors: Bulteau Laurence , Raymond Guy , Cognard Christian
Published: April 2021

Two dihydropyridine receptor mRNA isoforms (cardiac and skeletal) are expressed in rat skeletal muscle cells in primary culture. The progressive changes in excitationcontraction coupling mode from dual mode (‘skeletal’ and ‘cardiac’) to predominant ‘skeletal’ one during in vitro myogenesis are thought to be linked to the developmental changes in the relative expression of the two types of molecular entity previously observed in this preparation. In order to test this hypothesis, myotube cultures (5-to 7-day-old) were treated with antisense phosphorothioated oligodeoxynucleotides against cardiac or skeletal α1 subunit of L-type calcium channel. The oligodeoxynucleotide uptake by cells was checked by means of imaging of fluorescent oligodeoxynucleotide derivatives within the cells. Optimum concentration used (10 μM in the extracellular medium) and incubation time (70 hours) were empirically determined. Antisense directed against the cardiac type led to a 54% decrease in the averaged L-type calcium current peak density at −10 mV. The same type of experiment was performed with antisense against the skeletal isoform and led to a same order of inihibition (46%). This result clearly shows that the two isoforms can work as a calcium channel. Conversely, analysis of the shape of T–V (relative contractile amplitude versus membrane potential) curves shows that the treatment with ‘skeletal’ antisense depressed the contractile response in the medium membrane potential range whereas treatment with ‘cardiac’ antisense had no effect. This and other results taken together suggest that the skeletal isoform of dihydropyridine receptor is involved in both ‘cardiac’ and ‘skeletal’ types of EC coupling mechanisms at work in early stages of myotubes in vitro development. The type of coupling probably depends on the proximity of the skeletal dihydropyridine receptor and the ryanodine receptor.
Altered stem cell regeneration in irradiated intestinal crypts of senescent mice
Authors: Martin K. , Potten C. S. , Roberts S. A. , Kirkwood T. B. L.
Published: April 2021

Ageing is associated with a progressive deterioration in the functions of many organs within the body. In tissue with high cell turnover, the maintenance of the stem cells is of particular importance. Any accumulation of damage in stem cells may affect their function and hence threaten the homeostasis and regenerative capacity of the tissue. The small intestine represents a good model for the study of stem cells because of its spatial and hierarchical organisation. We have examined the effect of age on stem cell regenerative capacity after irradiation, using the microcolony assay. Crypt survival levels, the growth rate of surviving crypts, and the number of cells able to repopulate a crypt have been investigated by irradiating groups of 67 month old and 28-30 month old ICRFa male mice. After high doses of irradiation, the surviving crypts in old mice were both smaller and fewer in number than in young mice. The growth rate of surviving crypts was determined by measuring the crypt area and the number of cells/crypt at various times after 14 Gy irradiation. There was a growth delay of between about one half and one day in the older mice. Surprisingly, the number of clonogenic cells per crypt was estimated to be greater in the older mice. These studies indicate important age-related alterations in the capacity to regenerate the crypts after radiation damage.
Splenic T lymphocytes die preferentially during heat-induced apoptosis: NuMA reorganization as a marker
Authors: Sodja Caroline , Brown David L. , Walker P. R. , Chaly Nathalie
Published: April 2021

We are investigating nuclear events during apoptosis in mouse splenic lymphocytes cultured immediately after isolation (controls) or after heat treatment (42°C, 30 minutes), and have found that hyperthermia increased the level of apoptosis to double that of spontaneous apoptosis in controls within 6 hours. Immunolabelling for Nuclear Mitotic Apparatus Protein (NuMA) suggested that splenocytes were responding heterogeneously to the heat treatment. Whereas all nuclei in controls and about half of nuclei in heat-treated samples showed the usual diffuse nucleoplasmic labelling, 40-60% of nuclei in heated samples also contained numerous bright spots. We then examined whether the heterogeneity in NuMA organization might be an indication of a differential response of B and T lymphocytes to hyperthermia, and whether the presence of NuMA spots is related to the apoptotic process. NuMA labelling of heated fractionated splenocyte populations showed that 90% of nuclei in T-enriched cultures (≤4% IgG+ cells), but only 25% of nuclei in B-enriched samples (≤80% IgG+ cells), contained spots. As well, 2 hours after heat treatment of unfractionated cultures, ≥90% of nuclei that were accumulating DNA strand breaks, as detected by TUNEL, exhibited NuMA spots. These data indicate that cells with NuMA spots are targetted for, or have initiated, the death program. Since most T cells, but few or no B cells, were spotty after heating, we conclude further that hyperthermia induces apoptosis preferentially in splenic T lymphocytes. The observation that the proportion of T cells was, on average, threefold greater in control than in heated samples after 24 hours in culture reinforces this conclusion.
Regulation of macropinocytosis in v-Src-transformed fibroblasts: cyclic AMP selectively promotes regurgitation of macropinosomes
Authors: Veithen Alex , Amyere Mustapha , Smissen Patrick Van Der , Cupers Philippe , Courtoy Pierre J.
Published: April 2021

Stable transformation of Rat-1 fibroblasts by the v-Src oncoprotein results into the constitutive formation of macropinosomes. In the present report, we found that macropinosomes do not fuse with transferrin-containing endosomes and investigated the effects of cyclic AMP as a regulator of macropinocytosis in this cell system. The permeant analogs dibutyryl cyclic AMP and 8-bromo-cyclic AMP, as well as the pharmacological activator of adenylate cyclase forskolin, similarly decreased by about 35% the net endocytic accumulation of the fluid-phase tracer horseradish peroxidase at intervals >5 minutes in v-Src-transformed cells but not in the non-transformed parental Rat-1 cell line. However, and in contrast to the phospholipase C inhibitor 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate or the phosphatidylinositol 3-kinase inhibitor wortmannin, dibutyryl cyclic AMP neither returned the peroxidase accumulation rate of v-Src-transformed cells to that of parental Rat-1/control cells, nor prevented macropinosome formation, as shown by confocal microscopy. Detailed analysis of the kinetics of tracer entry and efflux in transformed cells revealed that dibutyryl cyclic AMP inhibited peroxidase accumulation only after intervals >5 minutes, due to accelerated peroxidase regurgitation, but did not alter the rate of transferrin recycling. Taken together, these data indicate that, in v-Src-transformed fibroblasts, macropinocytosis and micropinocytosis serve different pathways and that cyclic AMP affects neither micropinocytosis nor the formation of macropinosomes, but selectively promotes regurgitation therefrom.
Characterization of novel nuclear targeting and apoptosis-inducing domains in FAS associated factor 1
Authors: Fröhlich Thomas , Risau Werner , Flamme Ingo
Published: April 2021

FAS associated factor 1 (FAF1) has been described as an unusual protein that binds to the intracellular portion of the apoptosis signal transducing receptor FAS/Apo-1 and potentiates apoptosis in L-cells. By means of mRNA differential display we have identified the avian homologue (qFAF) as a fibroblast growth factor-inducible gene in pluripotent cells from E0 quail embryos during mesoderm induction in vitro. Later during embryonic development, qFAF expression is ubiquitous. We confirm that qFAF is associated with FAS, and show that it is phosphorylated on serine residues and localized to the nucleus. By in vitro mutagenesis we have delimited a novel nuclear targeting domain to a short 35 amino acid alpha-helical region in the amino-terminal half of the protein. The nuclear function of qFAF remains unclear. However, a probably dominant negative deletion mutant of qFAF causes apoptosis of transfected cells. This function resides in the carboxyterminal domain of qFAF which shares remarkable sequence homologies with a putative ubiquitin conjugating enzyme from Caenorhabditis elegans. Our data indicate a complex function for FAF, which may be executed during FAS signalling and/or in the ubiquitination pathway, and may be essential for cell differentiation and survival.
Are caspases involved in the death of cells with a transcriptionally inactive nucleus? Sperm and chicken erythrocytes
Authors: Weil Miguel , Jacobson Michael D. , Raff Martin C.
Published: April 2021

We show that mouse sperm die spontaneously within 1-2 days in culture and that treatment with either staurosporine (STS) and cycloheximide (CHX) or a peptide caspase inhibitor does not accelerate or delay the cell death. Chicken erythrocytes, by contrast, are induced to die by either serum deprivation or treatment with STS and CHX, and embryonic erythrocytes are more sensitive than adult erythrocytes to both treatments. Although these erythrocyte deaths display a number of features that are characteristic of apoptosis, they are not blocked, or even delayed, by peptide caspase inhibitors, and most of the cells die without apparently activating caspases. A small proportion of the dying erythrocytes do activate caspase-3, but even these cells, which seem to be the least mature erythrocytes, die just as quickly in the presence of caspase inhibitors. Our findings raise the possibility that both mouse sperm and chicken erythrocytes have a death programme that may not depend on caspases and that chicken erythrocytes lose caspases as they mature. Chicken erythrocytes may provide a useful ‘stripped down’ cell system to try to identify the protein components of such a death programme, which may serve to back-up the conventional caspase-dependent suicide mechanism in many cell types.
Endothelial cells assemble two distinct α6β4-containing vimentin-associated structures: roles for ligand binding and the β4 cytoplasmic tail
Authors: Homan Suzanne M. , Mercurio Arthur M. , LaFlamme Susan E.
Published: April 2021

The α6β4 laminin binding integrin functions in the assembly of type I hemidesmosomes, which are specialized cell-matrix adhesion sites found in stratified epithelial cells. Although endothelial cells do not express all the components of type I hemidesmosomes, endothelial cells can express the α6β4 integrin. Because endothelial cells lose expression of α6β4 in culture, we expressed recombinant α6β4 in the dermal microvascular endothelial cell line, HMEC-1, to test whether endothelial cells can assemble adhesion structures containing α6β4. Using immunofluorescence microscopy, we found that recombinant α6β4 concentrates specifically in a novel fibrillar structure on the basal surface of endothelial cells in the absence of an exogenous laminin substrate. This localization is regulated by an intracellular mechanism, because the β4 cytoplasmic domain is sufficient to direct a reporter domain (IL-2R) to the fibrillar structures independently of recombinant α6β4. In addition, this IL-2R-β4 chimera is sufficient to recruit the intermediate filament-associated protein HD1/plectin to these fibrillar structures and this also occurs in the absence of recombinant α6β4. The fibrillar localization pattern, as well as the recruitment of HD1/plectin, requires the first and second fibronectin type III repeats and the connecting segment of the β4 tail. In addition, when endothelial cells are provided a laminin 5-rich matrix, recombinant α6β4 redistributes from the fibrillar structure to type I hemidesmosome-like structures. The β4 cytoplasmic domain can also direct a reporter domain to these type I hemidesmosome-like structures; however, this process is dependent upon the expression of recombinant α6β4. Biochemical analysis indicates that both the fibrillar and the type I hemidesmosome-like structures are associated with the vimentin intermediate filament cytoskeleton. Thus, the results illustrate that endothelial cells have the essential components necessary to assemble at least two distinct α6β4-containing and vimentin-associated structures on their basal surface and that the β4 cytoplasmic tail and the availability of specific α6β4 ligands regulate receptor localization to these structures.
The vitronectin receptor associates with clathrin-coated membrane domains via the cytoplasmic domain of its β5 subunit
Authors: Deyne Patrick G. De , O’Neill Andrea , Resneck Wendy G. , Dmytrenko George M. , Pumplin David W. , Bloch Robert J.
Published: April 2021

Rat myotubes cultured in fetal calf serum adhere to vitronectin-coated substrates through two distinct structures, focal contacts and clathrin-coated membrane domains. We studied the integrins in myotubes to learn how they associate with these two domains. Double label immunofluorescence studies with antibodies specific for clathrin, vinculin and several forms of integrin showed that focal contacts and clathrin-coated membrane domains contain both vitronectin receptors (VnR, containing β3- and β5-integrins) and fibronectin receptors (FnR, containing β1-integrin). VnR but not FnR associates tightly with the substrate in both domains, as the VnR alone remains attached to the coverslip when the lipid bilayer and other membrane proteins are removed by detergent. Ultrastructural studies confirmed the localization of the β5 subunit of the VnR at both domains.

We used intracellular injection and affinity chromatography to test the possibility that clathrin at coated membrane domains associates with the cytoplasmic sequence of the β5 subunit of the VnR.

Injection of a synthetic peptide containing the NPXY motif from the cytoplasmic domain of the human β5 subunit, SRARYEMASNPLYRKPIST, depleted clathrin from coated membrane domains without affecting clathrin in perinuclear structures or vinculin at focal contacts. Injection of the homologous β1 peptide, MNAKWDTGENPIYKSAVITT, also containing an NPXY motif, had no significant effect on any of these structures. Affinity matrices containing the β5 but not the β1 peptide selectively retained clathrin from myotube extract, and bound clathrin could be selectively eluted by soluble forms of the β5 but not the β1 peptide. Thus, a sequence including the NPXY motif in the integrin β5 subunit is involved in the specific anchoring of the VnR, but not the FnR, to clathrin-coated membrane.
Schwann cells use a novel collagen-dependent mechanism for fibronectin fibril assembly
Authors: Chernousov Michael A. , Stahl Richard C. , Carey David J.
Published: April 2021

Cultured rat Schwann cells were stimulated to deposit fibrillar extracellular matrix by treatment with ascorbic acid in the absence of nerve cells. Immunofluoresence staining of the matrix showed that it contains collagens types I and IV, fibronectin and perlecan but not laminin. Collagen type IV, fibronectin and perlecan co-distributed completely in the matrix fibrils, whereas collagen type I was present in only a subset of these fibrils. Time course studies indicated that collagen type I fibrils appear at late stages of matrix formation. Digestion of Schwann cell extracellular matrix with collagenase effectively disrupted most of the matrix including fibronectin fibrils. This was in contrast with fibroblasts, where collagenase treatment removed collagen with no visible effect on fibronectin fibrils. α5 integrin was expressed on the cell surface of Schwann cells and partially codistributed with fibronectin-containing fibrils. This suggests that the inability of Schwann cells to deposit fibronectin-containing matrix through a conventional, collagen-independent mechanism was not due to the lack of fibronectin-binding integrins on their cell surface. Polyclonal anti-fibronectin antibodies inhibited the deposition of fibronectin into the matrix fibrils, whereas collagen type IV fibrils were generally unaffected. Growth of Schwann cells on collagen type IV-coated substrate in the absence of ascorbate induced deposition of fine fibronectin fibrils. These results suggest that Schwann cells use an apparently novel, collagen type IV-dependent mechanism for the deposition of fibronectin into their extracellular matrix.
Targeting of the myosin-I myr 3 to intercellular adherens type junctions induced by dominant active Cdc42 in HeLa cells
Authors: Stöffler Hanns-Eugen , Honnert Ulrike , Bauer Christian A. , Höfer Dirk , Schwarz Heinz , Müller Rainer T. , Drenckhahn Detlev , Bähler Martin
Published: April 2021

Myr 3, a member of the myosin-I family from rat, is shown in this study to be localized at adherens-type intercellular junctions in epithelial and nonepithelial tissues. Formation of intercellular junctions and the accompanying recruitment of myr 3 to these junctions involves signaling by the Rho subfamily of small GTP-binding proteins. This conclusion is based on studies with HtTA-1 HeLa cells that were induced by overexpression of constitutively active Cdc42Hs to form typical adherens-type intercellular junctions enriched in cadherins (N-cadherin), β-catenin, filamentous actin and myr 3. Recruitement of myr 3 to Cdc42-induced adherens junctions in HeLa cells was dependent on a short region of the tail domain and a functional myosin motor domain, but was independent of its myosin-I tail homology and SH3 regions. Overexpression of constitutively active Rac1 induced a distinct type of adherens junction in HeLa cells that was characterized by elaborate intercellular interdigitations enriched in N-cadherin, β-catenin and F-actin. Myr 3 was often present, but not specifically enriched in the intercellular junctions induced by constitutively active Rac1.
The activation of ProMMP-2 (gelatinase A) by HT1080 fibrosarcoma cells is promoted by culture on a fibronectin substrate and is concomitant with an increase in processing of MT1-MMP (MMP-14) to a 45 kDa form
Authors: Stanton H. , Gavrilovic J. , Atkinson S.J. , d'Ortho M.P. , Yamada K.M. , Zardi L. , Murphy G.
Published: April 2021

We have assessed the effect of fibronectin and laminin-1 on the expression of molecules involved in the activation pathway of MMP-2, a key proteinase in tissue remodelling. HT1080 fibrosarcoma cells cultured on fibronectin were shown to activate endogenous MMP-2, to a level comparable with that elicited by treatment with phorbol ester. In contrast, the MMP-2 expressed by HT1080 cells cultured on laminin-1 was mainly in the pro- (inactive form). Culture of the cells on peptide fragments of fibronectin derived from the central cell binding domain also promoted MMP-2 activation, indicating that signals via fibronectin binding to integrin receptors may be involved. HT1080 cells cultured on immobilised antibodies to the alpha5 and beta1 integrin subunits secreted levels of active MMP-2 similar to those observed for full length fibronectin, whereas cells cultured on an antibody to the alpha6 integrin subunit secreted mainly proMMP-2. The data demonstrate that the activation of MMP-2 by HT1080 cells is regulated by the nature of the extracellular matrix, and that signals via the alpha5beta1 integrin receptor may be involved in the fibronectin induced up-regulation of MMP-2 activation. We then assessed the effect of fibronectin on the components of the putative MT1-MMP/TIMP-2 ‘receptor’ complex implicated in MMP-2 activation. Levels of TIMP-2 protein expressed by HT1080 cells did not vary detectably between cells cultured on fibronectin or laminin-1. However, the expression of MT1-MMP protein was up-regulated when the cells were cultured on fibronectin, which could be attributed to an increase in levels of a truncated 45 kDa form. Parallel studies using gelatin zymography demonstrated that the up-regulation of the production of the 45 kDa band was concomitant with MMP-2 activation. Inhibitor studies revealed that the truncation of MT1-MMP to a 45 kDa form is MMP mediated, although not inhibited by TIMP-1. In vitro, the 45 kDa form could be generated by cleavage of membrane-bound native MT1-MMP with several recombinant MMPs, including both active MT1-MMP and MMP-2. The implication that either MMP-2 or MT1-MMP can process MT1-MMP to 45 kDa, raises the possibility that truncation of MT1-MMP represents a self-regulatory end-point in the activation pathway of MMP-2.
Differential regulation of CXCR4 and CCR5 endocytosis
Authors: Signoret N. , Rosenkilde M.M. , Klasse P.J. , Schwartz T.W. , Malim M.H. , Hoxie J.A. , Marsh M.
Published: April 2021

The chemokine receptors CCR5 and CXCR4 are major co-receptors/receptors for the CD4-dependent and CD4-independent entry of human and simian immunodeficiency viruses. The chemokines that bind and activate these receptors can inhibit the entry of viruses that use the respective co-receptor molecules. Chemokine-induced co-receptor internalisation is a significant component of the mechanism through which chemokines inhibit virus entry. CXCR4 internalisation is induced by the CXCR4 ligand stromal cell derived factor-1 (SDF-1), phorbol esters and, in T cells, cellular activation. Here we show that CXCR4 endocytosis can be mediated through either one of two distinct internalisation signals. A COOH-terminal serine rich domain is required for ligand- but not phorbol ester- induced CXCR4 internalisation. However, a Ser/IleLeu motif, similar to that required for the endocytosis of CD4 and the T cell receptor/CD3 complex, is required for phorbol ester-induced, but not ligand-induced, CXCR4 endocytosis. By contrast, CCR5 internalisation is induced by the beta-chemokine RANTES but not by phorbol esters. CCR5 lacks the Ser/IleLeu sequence required for phorbol ester-induced uptake of CXCR4. Together these results indicate that distinct mechanisms can regulate CXCR4 and CCR5 endocytosis and trafficking.
Strategies of epithelial repair: Modulation of stem cell and transit amplifying cell proliferation
Authors: Lehrer Michael S. , Sun Tung-Tien , Lavker Robert M.
Published: April 2021

Using double labeling techniques, we studied the replication of corneal epithelial stem cells that reside exclusively in the limbal zone, and their progeny transit amplifying cells. We show that corneal epithelial stem cells can be induced to enter DNA synthesis by wounding and by TPA. We demonstrate the existence of a hierarchy of TA cells; those of peripheral cornea undergo at least two rounds of DNA synthesis before they become post-mitotic, whereas those of central cornea are capable of only one round of division. However, the cell cycle time of these TA cells can be shortened and the number of times these TA cells can replicate is increased in response to wounding. These results thus demonstrate three strategies of epithelial repair: (i) stem cell replication, (ii) the unleashing of additional rounds of cell proliferation that remain as an untapped reserve under normal circumstances, and (iii) enhancement of TA cell proliferation via a shortening of the cycling time.
High levels of actin tyrosine phosphorylation: Correlation with the dormant state of Dictyostelium spores
Authors: Kishi Yoshiro , Clements Chris , Mahadeo Dana C. , Cotter David A. , Sameshima Masazumi
Published: April 2021

Upon removal of nutrients, the amoebae of the cellular slime mold Dictyostelium discoideum differentiate into dormant spores which survive starvation stress. In this study, we demonstrate that half of the actin molecules in the spores are tyrosine-phosphorylated. The phosphorylated actin is distributed around immobile crenate mitochondria and vesicles, as well as in the cytoplasm of the spores. The actin isolated from spore lysates contains phosphorylated and unphosphorylated forms at the same molar ratio as that of the original whole spore lysate. Under actin polymerizing conditions they form actin filaments and then they are completely depolymerized under actin depolymerizing conditions, indicating that tyrosine phosphorylation of actin may not prohibit actin polymerization nor stimulate depolymerization. The phosphorylation levels increase at the end of the culmination stage when spores have matured morphologically and physiologically, and reach maximum levels after an additional 12 hours of development. The levels are stable for 20 days following spore maturation, and decline to undetectable levels within the next 10 days.

Spores having high levels of phosphorylation show high viability, and vice versa. Following activation of spores with nutrient medium containing spore germination promoters, the phosphorylation levels quickly decrease with a half-life of about 5 minutes. After 20 minutes spores begin to swell. At this later time, most of the phosphorylated actin already has been dephosphorylated. Also, in heat-activated spores actin dephosphorylation occurs prior to spore swelling. However, addition of phosphatase inhibitors following heat-activation, prevented spore swelling and dephosphorylation of actin. Our data indicate that the high levels of actin tyrosine phosphorylation, specific to the spore stage, may be required for maintaining dormancy to withstand starvation stress. The rapid dephosphorylation of actin leads to a reactivated dynamic actin system which participates in spore swelling, vesicle movement, and mitochondrial shape changes during the spore germination process.
Anosmin-1 underlying the X chromosome-linked Kallmann syndrome is an adhesion molecule that can modulate neurite growth in a cell-type specific manner
Authors: Soussi-Yanicostas Nadia , Faivre-Sarrailh Catherine , Hardelin Jean-Pierre , Levilliers Jacqueline , Rougon Geneviève , Petit Christine
Published: April 2021

Anosmin-1 is an extracellular matrix glycoprotein which underlies the X chromosome-linked form of Kallmann syndrome. This disease is characterized by hypogonadism due to GnRH deficiency, and a defective sense of smell related to the underdevelopment of the olfactory bulbs. This study reports that anosmin-1 is an adhesion molecule for a variety of neuronal and non-neuronal cell types in vitro. We show that cell adhesion to anosmin-1 is dependent on the presence of heparan sulfate and chondroitin sulfate glycosaminoglycans at the cell surface. A major cell adhesion site of anosmin-1 was identified in a 32 amino acid (32R1) sequence located within the first fibronectin-like type III repeat of the protein. The role of anosmin-1 as a substrate for neurite growth was tested on either coated culture dishes or monolayers of anosmin-1-producing CHO cells. In both experimental systems, anosmin-1 was shown to be a permissive substrate for the neurite growth of different types of neurons. Mouse P5 cerebellar neurons cultured on anosmin-1 coated wells developed long neurites; the 32R1 peptide was found to underly part of this neurite growth activity. When the cerebellar neurons were cultured on anosmin-1-producing CHO cells, neurite growth was reduced as compared to wild-type CHO cells; in contrast, no difference was observed for E18 hippocampal and P1 dorsal root ganglion neurons in the same experimental system. These results indicate that anosmin-1 can modulate neurite growth in a cell-type specific manner. Finally, anosmin-1 induced neurite fasciculation of P5 cerebellar neuron aggregates cultured on anosmin-1-producing CHO cells. The pathogenesis of the olfactory defect in the X-linked Kallmann syndrome is discussed in the light of the present results and the recent data reporting the immunohistochemical localisation of anosmin-1 during early embryonic development.
Messenger ribonucleoprotein complexes containing human ELAV proteins: interactions with cytoskeleton and translational apparatus
Authors: Antic Dragana , Keene Jack D.
Published: April 2021

Mammalian ELAV proteins bind to polyadenylated messenger RNAs and have specificity for AU-rich sequences. Preferred binding sites in vitro include the AUUUA pentamer and related sequences present in the 3′ untranslated regions of many growth regulatory mRNAs. Human ELAV (hELAV) proteins have been implicated in post-transcriptional regulation of gene expression by their effects on the stability and translatability of growth regulatory mRNAs.

We have examined the intracellular localization of ELAV proteins in neurons and in tumor cells of neuronal origin using indirect immunofluorescence, confocal microscopy and biochemical separation. Mammalian neuronal ELAV proteins are found predominantly in the cytoplasm of cells in mRNP complexes termed α complexes which, when associated with polysomes, form large and high density β complexes, as assayed by glycerol and accudenz gradients, respectively. Puromycin, cytochalasin or EDTA treatments disrupt β complexes causing the release of α complexes, which then appear, by confocal microscopy, as large hELAV mRNP granules associated with microtubules. Association of partially purified hELAV mRNP α complexes with microtubules was confirmed by in vitro reconstitution assays. Furthermore, colchicine treatment of cells suggested that association of hELAV mRNP α complexes with microtubules is also necessary for the formation of β complexes.

Our data suggest a model in which a subset of mRNAs is associated with microtubules as ELAV mRNP particles (α complexes) which, in turn, associate with polysomes to form a translational apparatus (β complex) that is, through polysomes, associated with the microfilament cytoskeletal network. hELAV proteins in these mRNP granules may affect post-transcriptional regulation of gene expression via the intracellular transport, localization and/or translation of growth regulatory mRNAs.
Active nuclear pore complexes in Chironomus: visualization of transporter configurations related to mRNP export
Authors: Kiseleva Elena , Goldberg Martin W. , Allen Terence D. , Akey Christopher W.
Published: April 2021

The Nuclear Pore Complex (NPC) regulates nucleocytoplasmic transport by providing small channels for passive diffusion and multiple docking surfaces that lead to a central translocation channel for active transport. In this study we have investigated by high resolution scanning and transmission electron microscopy the dynamics of NPC structure in salivary gland nuclei from Chironomus during Balbiani ring (BR) mRNP translocation, and present evidence of rearrangement of the transporter related to mRNP export. Analysis of the individual NPC components verified a strong evolutionary conservation of NPC structure between vertebrates and invertebrates. The transporter is an integral part of the NPC and is composed of a central short double cylinder that is retained within the inner spoke ring, and two peripheral globular assemblies which are tethered to the cytoplasmic and nucleoplasmic coaxial rings by eight conserved internal ring filaments.

Distinct stages of BR mRNP nuclear export through the individual NPC components were directly visualized and placed in a linear transport sequence. The BR mRNP first binds to the NPC basket, which forms an expanded distal basket ring. In this communication we present stages of BR mRNP transport through the nucleoplasmic, central and cytoplasmic transporter subunits, which change their conformation during mRNP translocation, and the emegence of mRNP into the cytoplasm. We propose that the reorganization of the basket may be driven, in part, by an active translocation process at the transporter. Furthermore, the images provide dramatic evidence that the transporter functions as a central translocation channel with transiently open discrete gates in its globular assemblies. A model of NPC transporter reorganization accompanied with mRNP translocation is discussed.
Truncations of the C-terminal cytoplasmic domain of MG160, a medial Golgi sialoglycoprotein, result in its partial transport to the plasma membrane and filopodia
Authors: Gonatas Jacqueline O. , Chen You-Jun , Stieber Anna , Mourelatos Zisimos , Gonatas Nicholas K.
Published: April 2021

MG160, a type I cysteine-rich membrane sialoglycoprotein residing in the medial cisternae of the rat Golgi apparatus, is highly homologous to CFR, a fibroblast growth factor receptor, and ESL-1, an E-selectin ligand located at the cell surface of mouse myeloid cells and recently detected in the Golgi apparatus as well. The mechanism for the transport of MG160 from the Golgi apparatus to the cell surface is unknown.

In this study we found that differential processing of the carboxy-terminal cytoplasmic domain (CD), consisting of amino acids Arg1159 Ile Thr Lys Arg Val Thr Arg Glu Leu Lys Asp Arg1171, resulted in the partial transport of the protein to the plasma membrane and filopodia.

In Chinese hamster ovary cells (CHO), stably transfected with the entire cDNA encoding MG160, the protein was localized in the Golgi apparatus. However, when the terminal Arg1171 or up to nine distal amino acids were deleted, the protein was distributed to the plasma membrane and filopodia as well as the Golgi apparatus.

This report shows that the CD of an endogenous type I Golgi protein is important for its efficient retention and identifies a unique residue preference in this process. Cleavage within the CD of MG160 may constitute a regulatory mechanism for the partial export of the protein from the Golgi apparatus to the plasma membrane and filopodia.
Annexin VI defines an apical endocytic compartment in rat liver hepatocytes
Authors: Ortega David , Pol Albert , Biermer Michael , Jäckle Stefan , Enrich Carlos
Published: April 2021

Annexin VI has been demonstrated previously to be a marker for hepatic endosomes. By western blotting with an affinity purified anti-annexin VI antibody it was shown that annexin VI was present in the three morphologically and functionally different endosomal fractions from rat liver. We have quantified the gold-labeled endosomes by immunoelectron microscopy in ultrathin Lowicryl sections of rat liver and now demonstrate that 80% of the total labeling with anti-annexin VI was associated with endocytic structures surrounding the bile canaliculus, the apical domain of hepatocytes, whereas only 20% was found in the subsinusoidal endosomes. In double immuno-gold labeling experiments 80% of the Rab5 positive apical endosomes were also labeled with anti-annexin VI antibodies. However, there was no significant colocalization with antibodies to the polymeric immunoglobulin receptor. Finally, we demonstrate that 50% of endosomes containing internalized gold-labeled transferrin were double labeled with anti-annexin VI antibodies. Thus, annexin VI becomes the first known structural protein at the apical ‘early’ endocytic compartment of the hepatocyte that may be involved in the receptor recycling and transport to late endocytic/lysosomal compartment pathways.
An image correlation analysis of the distribution of clathrin associated adaptor protein (AP-2) at the plasma membrane
Authors: Brown Claire M. , Petersen Nils O.
Published: April 2021

Clathrin associated adaptor protein is involved in endocytosis at the plasma membrane (AP-2) and protein sorting at the Golgi membrane (AP-1). There is a great deal of information available on the structure, function and binding characteristics of AP-2, however, there is little quantitative data on the AP-2 distribution at the membrane. Image correlation spectroscopy is a technique which yields number counts from an autocorrelation analysis of intensity fluctuations within confocal microscopy images. Image correlation spectroscopy analysis of the indirect immunofluorescence from AP-2 at the plasma membrane of CV-1 cells shows that AP-2 is in a bimodal distribution consisting of large coated pit associated aggregates of ∼60 AP-2 molecules, and smaller aggregates containing ∼20 AP-2 molecules, which we propose are coated pit nucleation sites. Following hypertonic treatment 25% of the AP-2 molecules dissociate from the large AP-2 aggregates and form AP-2 dimers, leaving the remaining AP-2 as large aggregates with ∼45 molecules. The smaller AP-2 aggregates completely dissociate forming AP-2 dimers. Dispersion of AP-2 with hypertonic treatment is not seen qualitatively because the number of large AP-2 aggregates is unchanged, the aggregates are just 25% smaller. Change in temperature from 37°C to 4°C has no affect on the number of AP-2 aggregates or the AP-2 distribution between the two populations. These data and estimates of the coated pit size suggest that coated pits cover ∼0.9% of the cell membrane. Combination of image correlation spectroscopy analysis and measurements of the CV-1 cell surface area show that there are ∼6×105 AP-2 molecules per CV-1 cell with ∼2×105 AP-2 molecules within coated pit structures.
Polarized localizations of annexins I, II, VI and XIII in epithelial cells of intestinal, hepatic and pancreatic tissues
Authors: Massey-Harroche Dominique , Mayran Nathalie , Maroux Suzanne
Published: April 2021

The cellular and subcellular localizations of annexins I, II, VI and XIII in the rabbit intestine, liver and pancreas were studied by performing immunofluorescence labeling on thin frozen tissue sections using specific monoclonal antibodies. The expression of annexins was found to be finely regulated. Annexins XIII and I were expressed exclusively in the small intestine and the colon, respectively, whereas annexin II was present in all the tissues tested and annexin VI specifically in the liver and pancreas. These different annexins were concentrated in the basolateral domain of polarized cells, and some of them had an extra-apical localization: annexin XIII was concentrated in the lower 3/4 of enterocyte brush border microvilli; annexin II was present in the upper part of the terminal web in intestinal absorbent cells as well as in the bile canalicular area in hepatocytes, whereas annexin VI was detected on some apical vesicles concentrated around the bile canaliculi. In pancreatic acinar cells, the presence of annexin II on some zymogen granules provides further evidence that annexin II may be involved in exocytic events. In conclusion, this study shows that the basolateral domain of polarized cells appears to be the main site where annexins are located, and they may therefore be involved in the important cellular events occurring at this level.
Centrosome dynamics in early embryos of Caenorhabditis elegans
Authors: Keating Heather H. , White John G.
Published: April 2021

The early Caenorhabditis elegans embryo divides with a stereotyped pattern of cleavages to produce cells that vary in developmental potential. Differences in cleavage plane orientation arise between the anterior and posterior cells of the 2-cell embryo as a result of asymmetries in centrosome positioning. Mechanisms that position centrosomes are thought to involve interactions between microtubules and the cortex, however, these mechanisms remain poorly defined. Interestingly, in the early embryo the shape of the centrosome predicts its subsequent movement. We have used rhodamine-tubulin and live imaging techniques to study the development of asymmetries in centrosome morphology and positioning. In contrast to studies using fixed embryos, our images provide a detailed characterization of the dynamics of centrosome flattening. In addition, our observations of centrosome behavior in vivo challenge previous assumptions regarding centrosome separation by illustrating that centrosome flattening and daughter centrosome separation are distinct processes, and by revealing that nascent daughter centrosomes may become separated from the nucleus. Finally, we provide evidence that the midbody specifies a region of the cortex that directs rotational alignment of the centrosome-nucleus complex and that the process is likely to involve multiple interactions between microtubules and the cortex; the process of alignment involves oscillations and overshoots, suggesting a multiplicity of cortical sites that interact with microtubules.