bluuebunny/crossref_metadata_2025 huggingface dataset:
Live dynamics of Dictyostelium cofilin suggests a role in remodeling actin latticework into bundles
Authors: Aizawa Hiroyuki , Fukui Yoshio , Yahara Ichiro
Published: April 2021

Cofilin, an indispensable, actin-regulating protein represents the ‘cofilin family’ of actin-binding proteins existing in a wide variety of organisms. Our previous and other in vitro studies have implied that cofilin can accelerate transformation of filamentous (F)-actin and α-actinin latticework into bundles, and overexpression of cofilin induces formation of F-actin bundles in Dictyostelium. Here we expressed an Aequorea green fluorescent protein (GFP)-Dictyostelium cofilin fusion protein in Dictyostelium, and observed the live dynamics to examine the physiological function of cofilin. We show that purified GFP-cofilin binds to actin filaments and decreases the apparent viscosity of actin solution in a similar manner to authentic Dictyostelium cofilin. Expressed GFP-cofilin exhibits normal actin-binding activities in the cytoplasm as represented by incorporation into the actin rods induced with dimethyl sulfoxide. Free moving cells form a crown-like cortical structure on the dorsal surface, and GFP-cofilin exhibits dynamic assembly into actin bundles being formed beneath the cortex. During phagocytosis, GFP-cofilin accumulates into actin bundles formed in the region underlying the phagocytic cups. In cells chemotactically activated with cyclic AMP, GFP-cofilin exhibits a high level of accumulation in projecting leading edges. When the chemoattraction is experimentally changed, the redistribution of GFP-cofilin towards the new pseudopod occurs in a matter of 30-60 seconds. These results demonstrate that cofilin plays a crucial role in vivo in rapid remodeling of the cortical actin meshwork into bundles.
Internalization and recycling of the C5a anaphylatoxin receptor: evidence that the agonist-mediated internalization is modulated by phosphorylation of the C-terminal domain
Authors: Naik Nishigandha , Giannini Eric , Brouchon Laurence , Boulay François
Published: April 2021

The C5a anaphylatoxin receptor is a member of the G protein-coupled receptor family involved in chemoattraction and activation of myeloid cells, as well as in host defence against infection by Pseudomonas aeruginosa. Upon challenge by C5a, the C5a receptor undergoes a rapid phosphorylation on serine residues in the carboxyl-terminal region. In this study, we used cells stably transfected with either the wild-type C5a receptor, or mutants affected in their capacity to be phosphorylated, to examine the role played by phosphorylation in the intracellular trafficking of the C5a receptor. Upon agonist binding, the wildtype receptor was rapidly internalized into endosomes that cluster near the nucleus after 10 minutes. Internalization of a non-phosphorylable mutant was severely impaired relative to wild-type receptor, whereas a mutant phosphorylated on serine 327 and/or serine 338, showed a rate of internalization intermediate between that of wild-type receptor and that of the non-phosphorylable mutant. Under continuous exposure to C5a and in the absence of protein synthesis, the C5a receptor was maintained in a highly phosphorylated state but was not degraded. Confocal microscopy and ligand-binding studies indicated that internalized receptors were recycled to the plasma membrane. During this process, receptors were dephosphorylated with kinetics that correlated with the kinetics of receptor recovery on the cell surface. Altogether, our data suggest that phosphorylation plays a key role in the intracellular trafficking of the C5a receptor. Phosphorylated receptors might be recognized by an adaptor protein that interacts with the endocytic machinery.
Non-centrosomal microtubule formation and measurement of minus end microtubule dynamics in A498 cells
Authors: Yvon Anne-Marie C. , Wadsworth Patricia
Published: April 2021

Experiments performed on a cell line (A498) derived from a human kidney carcinoma revealed non-centrosomal microtubules in the peripheral lamella of many cells. These short microtubules were observed in glutaraldehyde-fixed cells by indirect immunofluorescence, and in live cells injected with rhodamine-labeled tubulin. The non-centrosomal microtubules were observed to form de novo in living cells, and their complete disassembly was also observed. Low-light-level fluorescence microscopy, coupled to imaging software, was utilized to record and measure the dynamic behavior of both ends of the non-centrosomal microtubules in these cells. For each, the plus end was differentiated from the minus end using the ratio of their transition frequencies and by measuring total assembly at each end. For comparative purposes, dynamics of the plus ends of centrosomally nucleated microtubules were also analyzed in this cell line. Our data reveal several striking differences between the plus and minus ends. The average pause duration was nearly 4-fold higher at the minus ends; the percentage of time spent in pause was 92% at the minus ends, compared to 55% at plus ends. Dynamicity was decreased 4-fold at the minus ends, and the average number of events per minute was reduced from 7.0 at the plus end to 1.5 at the minus ends. The minus ends also showed a 6-fold decrease in frequency of catastrophe over the plus ends. These data demonstrate that in living cells, microtubules can form at sites distant from the perinuclear microtubule organizing center, and once formed, non-centrosomal microtubules can persist for relatively long periods.
Ultrastructural localization of cPLA2 in unstimulated and EGF/A23187-stimulated fibroblasts
Authors: Bunt Gertrude , de Wit Joris , van den Bosch Henk , Verkleij Arie J. , Boonstra Johannes
Published: April 2021

The 85 kDa cytosolic phospholipase A2 is the key enzyme in the release of arachidonic acid. To gain insight into cytosolic phospholipase A2 action in mitogen-activated cells, the localization of the phospholipase was investigated in fibroblasts upon stimulation with epidermal growth factor and the calcium ionophore A23187. By the use of indirect immunofluorescence microscopy, staining of endogenous cytosolic phospholipase A2 resulted in a punctate labeling pattern randomly distributed throughout the cytoplasm of the cell. Immunogold electron microscopy revealed that this punctate labeling pattern exhibited the presence of the 85 kDa phospholipase A2 in small clusters. These clusters were found in the cytosol in the vicinity of all organellar membranes, except for the Golgi system. The enzyme showed no preference for the nuclear envelope, the endoplasmic reticulum or the plasma membrane. Stimula tion of cells with epidermal growth factor or A23187 or both did not change the punctate immunofluorescence labeling pattern. Furthermore, a similar labeling pattern was observed by the artificial introduction of extremely low or high intracellular calcium concentrations. Even by electron microscopy, translocation of cytosolic phospholipase A2 to membranes was not observed after stimulation of cells with epidermal growth factor and A23187. From these results it is concluded that cytosolic phospholipase A2 is localized in clusters close to membranes in stimulated as well as unstimulated fibroblasts, without preference for a specific organellar membrane.
Matrix metalloproteinase-2 activation modulates glioma cell migration
Authors: Deryugina Elena I. , Bourdon Mario A. , Luo Guang-Xiang , Reisfeld Ralph A. , Strongin Alex
Published: April 2021

Stable transfection of U251.3 glioma cells with cDNA encoding MT-MMP-1 resulted in increased cell surface expression of MT-MMP-1 and TIMP-2, constitutive activation of MMP-2 proenzyme and increased collagen degradation. In tumor spheroid outgrowth assays, cell migration of MT-MMP-1 transfectants relative to control was enhanced on collagen and decreased on vitronectin and fibronectin. These effects were reversed by TIMP-2 and were not associated with any substantial changes in cell adhesion. Binding of U251.3 cells to the C-terminal domain of MMP-2 was specifically inhibited by anti-αvβ3 integrin blocking antibody indicating that MMP-2 interacts with αvβ3 through the enzyme’s C-terminal portion at or near the integrin’s matrix adhesion sites. We propose that these mechanisms could govern directed matrix degradation in the tumor cells’ microenvironment by sequestration of active MMP-2 on the cell surface. Our data suggest that activation of MMP-2 and its proteolytic activity localized to the cell surface could differentially modulate tumor cell migration in response to particular matrix proteins by altering both composition of the extracellular matrix and expression of adhesion receptors on the cell surface.
Distinct nuclear assembly pathways for lamins A and C lead to their increase during quiescence in Swiss 3T3 cells
Authors: Pugh Gareth E. , Coates Philip J. , Lane E. Birgit , Raymond Yves , Quinlan Roy A.
Published: April 2021

The expression of A-type lamins coincides with cell differentiation and as A-type lamins specifically interact with chromatin, a role in the regulation of differential gene expression has been suggested for A-type lamins. Using the mouse Swiss 3T3 cell line as a model, the change in two A-type lamins, lamins A and C, during cellular quiescence has been investigated. This well established model system mimics the first stages of differentiation when cells exit the cell cycle. In fact, quiescence in Swiss 3T3 cells was accompanied by a significant increase (2.6-fold) in lamin A protein levels and a smaller but reproducible increase (1.4-fold) in lamin C. These effects were fully reversible upon restimulation of the cells with serum. No effect upon lamin B levels was observed. Conversely, levels of A-type lamin mRNA decreased markedly as a result of quiescence suggesting transcriptional mechanisms are involved in the change in levels of lamins A and C. No difference in the incorporation of microinjected human lamin A into nuclei of quiescent or proliferating cells was observed. These data suggest A-type lamin binding sites were not limiting and indicated little difference between A-type lamin assembly mechanisms in quiescent and proliferating cells. The data did demonstrate lamin A and lamin C incorporation into the nuclear lamina proceeded by different pathways when microinjected in Swiss 3T3 cells. The incorporation of recombinant lamin C into the nuclear lamina was delayed compared to lamin A and proceeded via intranuclear foci. Such foci were not seen with microinjected lamin A. Instead, recombinant lamin A was rapidly (<20 minutes) incorporated into the nuclear lamina. Comicroinjection of lamin A with lamin C did not prevent foci formation but assisted in the rapid clearing (t1/2=30 minutes) of these structures and the incorporation of both lamins A and C into the lamina. These data suggest that the incorporation of lamin C into the lamina is facilitated by lamin A. They demonstrate a distinct difference in the nuclear assembly pathways of lamins A and C and show for the first time a functional distinction for these two splice variants of the A-type lamin gene. From the differences in assembly pathways and changes in protein levels accompanying quiescence in 3T3 cells, we suggest distinct roles for lamin A and lamin C in proliferating and quiescent states of the cell cycle.
Myosin II-independent processes in mitotic cells of Dictyostelium discoideum: redistribution of the nuclei, re-arrangement of the actin system and formation of the cleavage furrow
Authors: Neujahr Ralph , Heizer Christina , Gerisch Günther
Published: April 2021

Mitosis was studied in multinucleated and mononucleated mutant cells of Dictyostelium discoideum that lack myosin II (Manstein et al. (1989)EMBO J. 8, 923-932). Multinucleated cells were produced by culture in suspension, mononucleated cells were enriched by growth on a solid surface (DeLozanne and Spudich (1987)Science 236, 10861091). The multinucleated cells disclosed interactions of mitotic complexes with the cell cortex that were not apparent in normal, mononucleated cells. During the anaphase stage, entire mitotic complexes consisting of spindle, microtubule asters, and separated sets of chromosomes were translocated to the periphery of the cells. These complexes were appended at a distance of about 3 µm from the cell surface, in a way that the spindle became orientated in parallel to the surface. Subsequently, lobes of the cell surface were formed around the asters of microtubules. These lobes were covered with tapered protrusions rich in coronin, an actin associated protein that typically accumulates in dynamic cell-surface projections (DeHostos et al. (1991)EMBO J. 10, 4097-4104). During their growth on a solid surface, mononucleated myosin II-null cells passed through the mitotic cleavage stages with a speed comparable to wild-type cells. Cytokinesis as linked to mitosis is distinguishable by several parameters from traction mediated cytofission, which results in the pinching off of pieces of a multinucleated cell in the interphase. The possibility is discussed that cells can cleave during mitosis without forming a contractile ring at the site of the cleavage furrow.
γ-Tubulin in trypanosomes: molecular characterisation and localisation to multiple and diverse microtubule organising centres
Authors: Scott V. , Sherwin T. , Gull K.
Published: April 2021

A genomic clone from Trypanosoma brucei, which contains a full length γ-tubulin gene, was isolated using degenerate oligonucleotide primers. The sequence of this clone predicts a protein of 447 amino acids having a high degree of homology with γ-tubulins from human and Xenopus laevis (67.2% amino acid identity) and only 57.7% identity with the Plasmodium falciparum γ-tubulin. Northern blot analysis of poly(A)+ selected RNA from a procyclic culture detects a major transcript of approximately 2.2 kb plus a minor transcript of approximately 3.6 kb.

A fusion protein comprising almost the full length γ-tubulin gene product (amino acids 8-447) plus an aminoterminal histidine tag has been expressed and purified from Escherichia coli and used to raise a polyclonal antibody. Immunofluorescence, using this antibody, shows classical centrosomal localisation in mammalian cells. In T. brucei γ-tubulin is present in the basal bodies which subtend the flagellum and also at the anterior tip of the cell body where many minus ends of microtubules are located. Furthermore the antibody reveals a small subset of the sub-pellicular microtubules and a discrete dot within the nucleus which alters form with progression through the mitotic cycle. Evidence is also presented for discrete punctate staining within the microtubules of the cell body which may represent the presence of γ-tubulin on the ends of individual microtubules. Our results indicate that γ-tubulin is associated with diverse microtubule organising centres and structures in trypanosomes.
Overexpression of full- or partial-length MAP4 stabilizes microtubules and alters cell growth
Authors: Nguyen Hoang-Lan , Chari Sripriya , Gruber Dorota , Lue Chia-Man , Chapin Steven J. , Bulinski Jeannette Chloë
Published: April 2021

To investigate the in vivo functions of MAP4, a microtubule-associated protein expressed almost ubiquitously in vertebrate cells, we prepared stably transfected clonal mouse Ltk- cell lines expressing full-length MAP4 (L-MAP4 cells) or its MT-binding domain (L-MTB cells). Although transfectants showed no dramatic defect in morphology, organellar distribution, or level of MT polymer, as compared to naive Ltk- cells or L-MOCK cells (transfected with vector alone), MTs in L-MAP4 and L-MTB cells showed greater stability than those in control cells, as monitored by the level of post-translationally detyrosinated α-tubulin and by a quantitative nocodazole-resistance assay. In vivo, the MT-binding domain of MAP4 stabilized MTs less potently than full-length MAP4, in contrast to the equivalent efficacy demonstrated in studies of in vitro MT polymerization (Aizawa et al. (1991)J. Biol. Chem. 266, 9841-9846). L-MAP4 and L-MTB cells grew significantly more slowly than control cells; this growth inhibition was not due to mitotic arrest or cell death. L-MAP4 and L-MTB cells also exhibited greater tolerance to the MT-depoly-merizing agent, nocodazole, but not to the MT-polymeriz-ing agent, Taxol. Our results demonstrate that MAP4 and its MT-binding domain are capable of MT stabilization in vivo, and that increasing the intracellular level of MAP4 affects cell growth parameters.
The centrosomal protein centrosomin A and the nuclear protein centrosomin B derive from one gene by post-transcriptional processes involving RNA editing
Authors: Petzelt Christian , Joswig Gaby , Mincheva Antonaeta , Lichter Peter , Stammer Hermann , Werner Dieter
Published: April 2021

The identification of a gene encoding concomitantly a nuclear protein and an intrinsic centrosomal protein further emphasizes the close and presumably developmental relationship between the cell nucleus and the centro-some. Screening of a murine RNA-based cDNA library with an antiserum to a centrosomal protein and rescreening with the insert of an initial clone released two complete cDNAs (1.2 kbp and 2.2 kbp) coding for proteins with notable characteristics. The amino-terminal sections of centrosomin A (276 amino acid residues, molecular mass 34.5 kDa) and of centrosomin B (447 amino acid residues, molecular mass 54.8 kDa) are identical over 272 amino acid residues. The carboxy-terminal section of the larger protein comprises additional 175 amino acid residues including nuclear location signals. The mRNAs encoding centrosomin A and B derive from a single gene. Chromosomal mapping (FISH) shows only one significantly hybridizing gene locus on chromosome 19D. PCR with genomic DNA as template and primer pairs complementary to the sequence which is identical in centrosomin A and B cDNAs results in amplification of only one DNA fragment. Moreover, one exon of the genomic sequence and the centrosomin B-encoding cDNA sequence include a G which is deleted in the centrosomin A-encoding cDNA. Accordingly, the two mRNAs are the products of either alternative splicing or alternative polyadenylation in combination with RNA editing. The recombinantly expressed chimeric protein consisting of centrosomin A and the green fluorescent protein from Aequorea victoria accumulates in centrosomes while the corresponding fusion protein with the centrosomin B sequence is transported into nuclei.
Epitopes of adhesion-perturbing monoclonal antibodies map within a predicted α-helical domain of the integrin β1 subunit
Authors: Shih Daw-Tsun , Boettiger David , Buck Clayton A.
Published: April 2021

Several recent studies have demonstrated the involvement of various domains of the β1 integrin subunit in ligand binding. Thus, specific amino acids have been shown to be important in divalent cation binding, and others have been implicated by peptide crosslinking to play an intimate role in integrin-ligand interactions. Added to these data are previous observations that a group of adhesion-blocking anti-chicken β1 antibodies mapped within the first 160 amino acid residues of the subunit. These observations suggested that this region plays a critical role in integrin ligand recognition. In order to further define the domain in which the epitopes for these antibodies are clustered, a series of mouse/chicken chimeric β1 constructs were examined for their reactivity with each of these antibodies. Most of the antibodies recognize a region between residues 124 to 160 of the chicken β1 subunit. Computer modeling predicted a possible amphipathic α-helical configuration for the region between residues 141 to 160. Consistent with this prediction, circular dichroism and NMR analysis revealed a tendency for a synthetic peptide containing these residues to form an α-helix. The significance of this structural characteristic was demonstrated by a mutation at residue 149 that disrupted the α-helix formation and resulted in a loss of the ability to form heterodimers with α subunits, localize to focal contacts, or be transported to the cell surface. The direct involvement of residues 141 to 160 in ligand binding was supported by the ability of a peptide with this sequence to elute integrins from a fibronectin affinity column. Thus, our data suggest that residues 141 to 160 of the integrin β1 subunit, when arranged in an α-helix configuration, participate in ligand binding.
Specific requirements for the ER to Golgi transport of GPI-anchored proteins in yeast
Authors: Sütterlin Christine , Doering Tamara L. , Schimmöller Frauke , Schröder Stephan , Riezman Howard
Published: April 2021

GPI-anchored proteins are attached to the membrane via a glycosylphosphatidylinositol-(GPI) anchor whose carbohydrate core is conserved in all eukaryotes. Apart from membrane attachment, the precise role of the GPI-anchor is not known, but it has been proposed to play a role in protein sorting. We have investigated the transport of the yeast GPI-anchored protein Gas1p. We identified two mutant strains involved in very different cellular processes that are blocked selectively in the transport of GPI-anchored proteins before arrival to the Golgi. The end8-1/ lcb1-100 mutant is defective in ceramide synthesis. In vitro data suggest a requirement for ceramides after the exit from the ER. We therefore propose that ceramides might function in the fusion of a GPI-containing vesicle with the Golgi, but we cannot exclude a role in the ER. The second mutant that blocks the transport of GPI-anchored proteins to the Golgi is ret1-1, a mutant in the α-subunit of coatomer. In both mutants, GPI-anchor attachment is normal and in ret1-1 cells, the GPI-anchors are remodeled with ceramide to the same extent as in wild-type cells.
Avian neural crest cell migration on laminin: interaction of the α1β1 integrin with distinct laminin-1 domains mediates different adhesive responses
Authors: Desban Nathalie , Duband Jean-Loup
Published: April 2021

In the present study, to further elucidate the molecular events that control neural crest cell migration, we have analyzed in vitro the adhesive and locomotory response of avian trunk neural crest cells to laminin-1 and searched for the integrin receptors involved in this process. Adhesion of crest cells on laminin-1 was comparable to that found on fibronectin or vitronectin. By contrast, migration was significantly greater on laminin-1 than on the other substrate molecules. Interaction of crest cells with laminin-1 involved two major cell-binding domains situated in different portions of the molecule, namely the E1′ and E8 fragments, which elicited different cellular responses. Cells were poorly spread on the E1′ fragment whereas, on E8, they were extremely flattened and cohesive. Either fragment supported cell locomotion, albeit not as efficiently as laminin-1. Immunoprecipitation and immunocytochemistry analyses revealed that crest cells expressed the α1β1, α3β1, α6β1 and αvβ3 integrins, as well as β8 integrins, as presumptive laminin-1 receptors, but not α6β4 and α2β1. Immunofluorescence labeling of cultured cells showed that the α1, αv, β1 and β3 subunits were diffuse on the cell surface and in focal contacts. In contrast, α3 and β8 were diffuse, while α6 was mostly intracytoplasmic and, secondarily, in focal contacts. Inhibition assays of cell adhesion and migration with function-perturbing antibodies demonstrated that α1β1 played a predominant role in both adhesion and migration on laminin-1 and interacted with either binding sites in the E1′ and E8 fragments. αvβ3 was also implicated in neural crest cell migration. In contrast, α3β1, α6β1 and the β8 integrins appeared to play only subsidiary roles in cell adhesion and migration. Finally, the ability of neural crest cells to interact with laminin-1 was found to increase with time in culture, possibly in correlation with changes in α3 distribution on the cell surface. In conclusion, our study indicates that (1) the preferential migration of neural crest cells along basal laminae can be accounted for by the ability of laminin-1 to promote migration with great efficiency; (2) interaction with laminin-1 involves two major cell binding domains that are both recognized by the α1β1 integrin; (3) α1β1 integrin can elicit different cellular responses depending on the laminin-1 domains with which it interacts; and (4) changes in the repertoire of integrins expressed by neural crest cells are consistent with the modulations of cell-substratum adhesion occurring throughout migration.
Mechanisms for de novo biogenesis of an apical membrane compartment in groups of simple epithelial cells surrounded by extracellular matrix
Authors: Ojakian George K. , Nelson W. James , Beck Kenneth A.
Published: April 2021

In open monolayers of epithelial cells grown in vitro, the apical membrane domain forms on the free cell surface that faces the culture medium. However, in vivo, the apical lumenal compartment arises within groups of cells that do not have a free cell surface. We designed in vitro culture conditions, using small colonies of MDCK cells overlaid with collagen, in which formation of the apical membrane must occur de novo by remodeling existing membrane domains that are contacted by other cells or extracellular matrix. Within 12 hours of collagen overlay, the apical membrane glycoprotein gp135 is removed from the free cell surface, while lateral membrane proteins (e.g. Na+,K+-ATPase) remain at sites of cell-cell contacts. Subsequently, lumenal structures, containing gp135 and the apically secreted protein gp81, formed within these cell-cell contacts. Na+,K+-ATPase, adherens junction (E-cadherin, α- and β-catenins) and tight junction (ZO-1) proteins were localized on the lateral membrane adjacent to, but excluded from the gp135-positive lumenal compartment.

Therefore, each lumen represents a newly formed apical compartment on the lateral membrane. The Golgi complex (α-mannosidase II and Golgi β-spectrin), centrosomes (γ-tubulin) and microtubules reorient to a cytoplasmic position adjacent to the newly-forming apical lumenal compartments. Significantly, addition of colchicine, nocodazole or brefeldin A inhibits apical lumen formation. These results demonstrate that simple epithelial cells form an apical lumenal compartment de novo through initial intermixing, and then sorting of apical and basal-lateral membrane proteins at sites of cell-cell contacts. In addition, apical lumen formation requires an intact microtubule network, microtubule-dependent reorientation of the Golgi complex and secretory apparatus, and fully functional protein delivery from the Golgi complex to the forming apical cell surface.
The composition and dynamics of cell-substratum adhesions in locomoting fish keratocytes
Authors: Lee Juliet , Jacobson Ken
Published: April 2021

Close contacts are the predominant type of cell-substratum adhesion in rapidly moving cells yet little is known about their composition and dynamics. To address these issues we have attempted to identify the molecular components of close contacts formed in rapidly moving fish epithelial keratocytes. In addition we have utilized the simple shape of keratocytes to explore the relationship between close contact formation and rapid locomotion. β1 Integrin and talin molecules were found to be localized within a narrow rim of very close contact along the leading edge. These molecules together with vinculin were also found within small foci distributed evenly throughout the lamella, corresponding to regions of variable close contact. α-Actinin was found in foci within older, more posteriorly located regions of the lamella and along stress fibers. In addition to close contacts, small focal adhesion-like structures which stained positively for all antibodies tested were found at the tips of stress fibers within retracting cell margins.

Interference reflection and total internal reflection microscopy of moving keratocytes showed cell-substratum contacts to be organized into distinct patterns that appear to move forwards, in concert with the leading edge. A feature common to all cells is a rim of very close contact at the leading edge. This region is specialized for the formation of new cell-substratum adhesions and is the site where patterns of close contact are generated. We have found that cell locomotion is most rapid when a uniform contact pattern is present but cell speed is progressively reduced as the contact pattern becomes more irregular. Furthermore, the local rate of lamellar extension is most rapid when underlain by regions of intermediate closeness to the substratum, but is reduced or ceases if the underlying contact is either very close or more distant, respectively.

Our results suggest that close contacts and focal adhesions are related structures formed from a common hierarchy of molecular interactions. In addition the relationship between close contact formation and lamellar extension indicates a direct coupling between these two processes at the leading edge. Furthermore, we can explain the dynamic behaviour of close contacts in terms of the relative rates of trapping and release of component molecules that is initiated at the leading edge. We suggest that regulation of the molecular dynamics involved in leading edge specialization determines both the pattern of cellsubstratum contacts and the net rate of actin filament assembly.
Absence of orthogonal arrays in kidney, brain and muscle from transgenic knockout mice lacking water channel aquaporin-4
Authors: Verbavatz Jean-Marc , Ma Tonghui , Gobin Renée , Verkman A. S.
Published: April 2021

Freeze-fracture electron microscopy (FFEM) of kidney collecting duct, muscle, astrocytes in brain, and other mammalian tissues has revealed regular square arrays of intramembrane particles called orthogonal arrays of particles (OAPs). Their possible role in membrane structure and transport have been proposed, and their absence or decrease has been noted in a variety of hereditary and acquired diseases. A transgenic mouse lacking water channel AQP4 was used to show that AQP4 is the OAP protein. FFEM was done on kidney, skeletal muscle, and brain from AQP4 wild-type [+/+], heterozygous [+/−] and knockout [−/−] mice. The [−/−] mice did not express detectable AQP4 protein, but were grossly indistinguish-able from [+/+] mice. FFEM was done on blinded samples of kidney, brain and muscle from 9 mice. In all 6 kidney samples from [+/+] and [+/−] mice, OAPs similar to those in AQP4-transfected CHO cells were found in basolateral membranes of collecting duct principal cells. In all muscle and brain samples from [+/+] and [+/−] mice, OAPs of identical ultrastructure to those in kidney were seen, but in smaller patch sizes. OAPs were not seen in any sample from [−/−] mice. Label-fracture analysis using a peptide-derived AQP4 polyclonal antibody showed immunogold labeling of OAPs in AQP4-expressing CHO cells. These studies provide direct evidence that AQP4 is required for formation of OAPs and is a component of OAPs, thus establishing the identity and function of OAPs.
HSF1 granules: a novel stress-induced nuclear compartment of human cells
Authors: Cotto José J. , Fox Susan G. , Morimoto Richard I.
Published: April 2021

Heat shock factor 1 (HSF1) is the ubiquitous stress-responsive transcriptional activator which is essential for the inducible transcription of genes encoding heat shock proteins and molecular chaperones. HSF1 localizes within the nucleus of cells exposed to heat shock, heavy metals, and amino acid analogues, to form large, irregularly shaped, brightly staining granules which are not detected during attenuation of the heat shock response or when cells are returned to their normal growth conditions. The kinetics of detection of HSF1 granules parallels the transient induction of heat shock gene transcription. HSF1 granules are also detected using an HSF1-Flag epitope tagged protein or a chimeric HSF1-green fluorescent protein which reveals that these nuclear structures are stress-induced and can be detected in living cells. The spatial organization of HSF1 granules in nuclei of stressed cells reveals that they are novel nuclear structures which are stress-dependent and provides evidence that the nucleus undergoes dynamic reorganization in response to stress.
Cytosolic Phox proteins interact with and regulate the assembly of coronin in neutrophils
Authors: Grogan Ann , Reeves Emer , Keep Nicholas , Wientjes Frans , Totty Nicholas F. , Burlingame Alma L. , Hsuan J. Justin , Segal Anthony W.
Published: April 2021

The NADPH oxidase generates microbicidal superoxide in phagocytes, and when defective it leads to chronic granu-lomatous disease (CGD). Oxidase specific proteins in the cytosol, p47phox and p67phox, as well as the small GTP binding protein p21rac are important for activation of superoxide production. Because the activity of this oxidase is normally tightly restricted to the phagocytic vacuole, and its temporal and spatial organisation might be regulated by cytoskeletal proteins, we examined the cytosolic phox proteins for interactions with cytoskeletal elements. p67phox copurified with a 57 kDa protein, identified as coronin, an actin binding protein that is important for movement and phagocytosis in Dictyostelium. Binding studies revealed that coronin attaches to the C-terminal half of p40phox, a binding partner of p67phox. The phox proteins and coronin had a similar distribution in the cell, and both accumulated around the phagocytic vacuole. PMA activation of adherent neutrophils resulted in a major rearrangement of these proteins, and of actin, which were lost from the periphery of the cell and condensed around the nucleus. The rearrangement of F-actin and coronin in adherent cells, were absent, or markedly diminished, in cells from patients lacking p47phox or p67phox in which an abnormally large proportion of the coronin was present as part of a large complex. The cytosolic phox proteins might play a regulatory role in the reorganisation of the cytoskeleton accompanying superoxide generation.
Occludin dephosphorylation in early development of Xenopus laevis
Authors: Cordenonsi Michelangelo , Mazzon Emanuela , Rigo Lidia De , Baraldo Simonetta , Meggio Flavio , Citi Sandra
Published: April 2021

Using immunobot and immunofluorescence analysis with a cross-reacting antiserum, we identified Xenopus laevis occludin as a 57-61 kDa antigen colocalized with cingulin in epithelial junctions of embryos. Occludin was completely extracted from unfertilized eggs and embryos with a solution containing 0.1% Triton X-100 and 1% NP40. Maternal occludin in unfertilized eggs migrated by SDS-PAGE as a 61 kDa protein. In fertilized eggs and in early cleavages up to blastula stage 8 it migrated as a series of polypeptides with 57-60 kDa. In gastrulae, neurulae and tailbud stage embryos, it migrated as a 57 kDa polypeptide. The electrophoretic mobility downshift was specifically reproduced by treatment of extracts with acid phosphatase, indicating that it is due to dephosphorylation. The correlation of occludin dephosphorylation with the de novo assembly of tight junction in native epithelia of Xenopus embryos suggests a possible role of occludin dephosphory-lation in the events leading to tight junction assembly. To identify kinases which can phosphorylate occludin, recom-binant chicken occludin (cytoplasmic domain) was subjected to in vitro phosphorylation. Occludin was phos-phorylated on serine and threonine residues by protein kinase CK2 and p34cdc2/cyclin B complex, but was not sig-nificantly phosphorylated by mitogen-activated protein kinase, protein kinase CK1 and p38Syk tyrosine kinase. We noted that occludin sequences contain a motif matching the activation loop of the cytoplasmic domain of insulin receptor kinase.
Differential fate of glycoproteins carrying a monoglucosylated form of truncated N-glycan in a new CHO line, MadIA214, selected for a thermosensitive secretory defect
Authors: Ermonval Myriam , Cacan René , Gorgas Karin , Haas Ingrid G. , Verbert André , Buttin Gérard
Published: April 2021

A temperature sensitive secretory line, MadIA214, was selected from mutagenized Chinese hamster ovary cells that express two heterologous export marker proteins: a secretory form of the human placental alkaline phosphatase (SeAP), and the Kd heavy chain of mouse MHC class I. SeAP secretion in MadIA214 was extremely reduced at elevated temperature (40°C), while the export of functional H-2Kd molecules to the plasma membrane was only slightly affected. This mutant constitutively transferred onto newly synthesized proteins a truncated oligosaccharide core, Man5GlcNAc2, which was monoglucosylated in the protein-bound form. Nevertheless, the final oligosaccharide-structures associated to mature SeAP and H-2Kd were similar in mutant and wild-type glycoproteins. The inaccessibility in MadIA214 endoplasmic reticulum (ER) of one or more components required for oligosaccharide chain elongation is supported by the reconstitution of a correct core structure, obtained after disruption of cellular compartments, but not after cell permeabilisation or blocking ER-to-Golgi transport. The increased association of the ER-chaperone BiP with immature SeAP correlated with the thermodependent decrease in SeAP secretion. The retention of incompletely folded polypeptides in MadIA214 parallels both a marked ER-dilation and an important glycoprotein degradation documented by the formation of soluble oligomannosides with one GlcNAc residue. Our data provide the first in vivo evidence that the initial step in N-glycosylation differentially governs glycoprotein maturation, transport and degradation.
Nuclear localization of IκBα promotes active transport of NF-κB from the nucleus to the cytoplasm
Authors: Arenzana-Seisdedos Fernando , Turpin Pierre , Rodriguez Manuel , Thomas Dominique , Hay Ronald T. , Virelizier Jean-Louis , Dargemont Catherine
Published: April 2021

IκBα tightly regulates the transcriptional activity of NF-κB by retaining it in the cytoplasm in an inactive form. In the present work, we report that IκBα, when expressed in the nuclear compartment, not only abrogates NF-κB/DNA interactions and NF-κB-dependent transcription, but also transports NF-κB back to the cytoplasm. This function of IκBα is insured by a nuclear export sequence located in the C-terminal domain of IκBα and homologous to the previously described export signal found in HIV-1 Rev protein as well as in PKI (the inhibitor of the catalytic subunit of protein kinase A). Thus, inhibition of NF-κB/DNA binding and the consecutive efficient nuclear export of the transcription factor by IκBα could represent an important mechanism for the control of the expression of NF-κB-dependent genes.
v-Crk-induced cell transformation: changes in focal adhesion composition and signaling
Authors: Nievers Mirjam G. , Birge Raymond B. , Greulich Heidi , Verkleij Arie J. , Hanafusa Hidesaburo , Henegouwen Paul M. P. van Bergen en
Published: April 2021

v-Crk is an oncogene product in which a viral Gag sequence is fused to a cellular Crk sequence. It contains one SH2 and one SH3 domain. To gain insight into the molecular mechanisms underlying v-Crk-induced cell transformation, we studied the subcellular localization and molecular interactions of v-Crk in v-Crk-transformed NIH-3T3 cells. Our results show that v-Crk specifically localizes to focal adhesions where it induces protein tyrosine phosphorylation. Subcellular fractionation studies indicated that a significant amount of v-Crk is present in the cytoskeletal cell fraction, a fraction that includes focal adhesions. Tyrosine phosphorylated proteins, including p130CAS, were also predominantly found in the cytoskeletal fraction. We show that v-Crk induces a translocation of p130CAS to the cytoskeleton, which is accompanied by hyperphosphorylation of this protein. Mutational analyses showed that a functional v-Crk SH2 domain is required for the localization of v-Crk in focal adhesions. Functional v-Crk SH2 and SH3 domains were both found to be required for the observed increase in tyrosine phosphorylation of focal adhesion proteins and for the translocation and hyperphosphorylation of p130CAS. v-Crk immunoprecipitation studies revealed that cytoskeleton-associated v-Crk interacts with both p130CAS and an unidentified tyrosine kinase. These findings suggest the formation of a focal adhesion-located complex consisting of v-Crk, a tyrosine kinase and p130CAS, which may lead to the hyperphosphorylation of p130CAS. These specific and localized signaling events may represent initial steps in the process of v-Crk-induced cell transformation.
Subcellular localization of myosin V in nerve growth cones and outgrowth from dilute-lethal neurons
Authors: Evans L. L. , Hammer J. , Bridgman P. C.
Published: April 2021

Myosin V-null mice (dilute-lethal mutants) exhibit apparent neurological defects that worsen from birth until death in the third postnatal week. Although myosin V is enriched in brain, the neuronal function of myosin V is unclear and the underlying cause of the neurological defects in these mice is unknown. To aide in understanding myosin V function, we examined the distribution of myosin V in the rodent superior cervical ganglion (SCG) growth cone, a well characterized neuronal structure in which myosin V is concentrated. Using affinity purified, myosin V-specific antibodies in immunofluorescence and immunoelectron microscopy, we observed that myosin V is concentrated in organellerich regions of the growth cone. Myosin V is present on a distinct population of small (50-100 nm) organelles, and on actin filaments and the plasma membrane. Myosin V-associated organelles are present on both microtubules and actin filaments. These results indicate that myosin V may be carried as a passenger on organelles that are transported along microtubules, and that these organelles may also be capable of movement along actin filaments. In addition, we found no abnormalities in outgrowth, morphology, or cytoskeletal organization of SCG growth cones from dilute-lethal mice. These results indicate that myosin V is not necessary for the traction force needed for growth cone locomotion, for organization of the actin cytoskeleton, or for filopodial dynamics.
Chromatin modifications during oogenesis in the mouse: removal of somatic subtypes of histone H1 from oocyte chromatin occurs post-natally through a post-transcriptional mechanism
Authors: Clarke Hugh J. , Bustin Michael , Oblin Colette
Published: April 2021

We examined the distribution of the somatic subtypes of histone H1 and the variant subtype, H10, and their encoding mRNAs during oogenesis and early embryogenesis in the mouse. As detected using immunocytochemistry, somatic H1 was present in the nuclei of oocytes of 18-day embryos. Following birth, however, somatic H1 became less abundant in both growing and non-growing oocytes, beginning as early as 4 days of age in the growing oocytes, and was scarcely detectable by 19 days. Together with previous results, this defines a period of time when somatic H1 is depleted in oocytes, namely, from shortly after birth when the oocytes are at prophase I until the 4-cell stage following fertilization. At the stages when somatic H1 was undetectable, oocyte nuclei could be stained using an antibody raised against histone H10, which suggests that this may be a major linker histone in these cells. In contrast to the post-natal loss of somatic H1 protein, mRNAs encoding four (H1a, H1b, H1d, H1e) of the five somatic subtypes were present, as detected using RT-PCR in growing oocytes of 9-day pups, and all five subtypes including H1c were present in fully grown oocytes of adults. All five subtypes were also present in embryos, both before and after activation of the embryonic genome. mRNA encoding H10 was also detected in oocytes and early embryos. Whole-mount in situ hybridization using cloned H1c and H1e cDNAs revealed that the mRNAs were present in the cytoplasm of oocytes and 1-cell embryos, in contrast to the sea urchin early embryo where they are sequestered in the cell nucleus. We suggest that, as in many somatic cell types, the chromatin of mouse oocytes becomes depleted of somatic H1 and relatively enriched in histone H10 postnatally, and that somatic H1 is reassembled onto chromatin in cleavage-stage embryos. The post-natal loss of somatic H1 appears to be regulated post-transcriptionally by a mechanism not involving nuclear localization.
Mechanisms for the coordination of intercellular calcium signaling in insulin secreting cells
Authors: Cao Dongrong , Lin George , Westphale Eileen M. , Beyer Eric C. , Steinberg Thomas H.
Published: April 2021

Insulin-mediated increases in cytosolic calcium are synchronized among the cells in a pancreatic islet, and result in pulsatile secretion of insulin. Pancreatic beta cells express the gap junction protein connexin43 and are functionally coupled, making gap junctional communication a likely mechanism for the synchronization of calcium transients among islet cells. To define the mechanism by which pancreatic islet cells coordinate calcium responses, we studied mechanically-induced intercellular calcium waves in the communication-deficient rat insulinoma cell line RINm5f, and in RINm5f cells transfected with the gap junction protein connexin43. Both RINm5f and RINm5f cells transfected with connexin43 propagated calcium waves that required release of calcium from intracellular stores, did not involve gap junctional communication, and appeared to be mediated by autocrine activity of secreted ATP acting on P2U purinergic receptors. Connexin43 transfectants also propagated calcium waves that required gap junctional communication and influx of extracellular calcium through voltage-gated calcium channels. Gap junction-dependent intercellular calcium waves were inhibited by preventing plasma membrane depolarization. These studies demonstrate two distinct pathways by which insulin-secreting cells can coordinate cytosolic calcium rises, and show that it is by ionic traffic that gap junctions synchronize calcium-dependent events in these cells.
Syndet is a novel SNAP-25 related protein expressed in many tissues
Authors: Wang Guangyi , Witkin Joan W. , Hao Gianming , Bankaitis Vytas A. , Scherer Philipp E. , Baldini Giulia
Published: April 2021

SNAP-25 is a synaptosomal associated protein localized at the plasma membrane of nerve terminals. SNAP-25 associates with syntaxin 1 and vesicle-associated membrane protein-2 (VAMP-2) and is thought to form a complex essential for neurotransmitter release. We have identified syndet, a novel protein related to the family of SNAP-25 isoforms. Like SNAP-25, syndet has regions with high probability of forming coiled coils, a cysteine rich-domain, and lacks a signal sequence or transmembrane domains. Syndet is tightly bound to membranes, possibly by acylation within the cysteine-rich domain. Syndet is expressed in non-neuronal tissues. In adipocytes, syndet is found at the plasma membrane and in an intracellular compartment. The identification of syndet supports the hypothesis that multiple SNAP-25 related proteins ensure specificity of vesicle fusion at the cell surface.