bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

ABSTRACT
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 (&lt;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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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.