bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dissection of the translocation and chaperoning functions of yeast BiP/Kar2p in vivo
Authors: Holkeri Heidi , Paunola Eija , Jämsä Eija , Makarow Marja 
Published: April 2021

ABSTRACT
We used the rat nerve growth factor receptor ectodomain (NGFRe) and Escherichia coli β-lactamase to dissect the functions of Saccharomyces cerevisiae BiP/Kar2p in vivo. Both were fused to the Hsp150Δ-polypeptide, which promotes proper folding of heterologous proteins which otherwise are misfolded in the yeast ER. Hsp150Δ-NGFRe and Hsp150Δ-β-lactamase acquired disulfides and were properly folded and secreted to the culture medium. When disulfide formation was prevented by incubating cells with dithiothreitol (DTT), Hsp150Δ-NGFRe remained in the endoplasmic reticulum (ER). The occupancy of an otherwise partially used N-glycosylation site of reduced NGFRe was complete suggesting that, normally, folding and disulfide formation occurred as rapidly as N-glycosylation. Removal of DTT resulted in remarkably rapid disulfide formation and secretion, suggesting only mild conformational distortion of reduced NGFRe. In contrast, reduced Hsp150Δ-β-lactamase was severely misfolded and attained a secretion competent conformation more slowly after reoxidation. When kar2-159 cells were incubated at permissive temperature 24°C with DTT, the reporter proteins were retained in the ER. After shift of the cells to 34°C to inactivate BiP/Kar2p irreversibly, and subsequent removal of DTT, most pre-accumulated Hsp150Δ-NGFRe was rapidly secreted, whereas Hsp150Δ-β-lactamase was secretion incompetent. Thus, Hsp150Δ-NGFRe did not require BiP/Kar2p for conformational maturation, though translocation was dependent on BiP/Kar2p. Apparently proteins differ in their post-translocational requirements for BiP/Kar2p, indicating that translocation and chaperoning are distinct functions.
Psoralen photoactivation promotes morphological and functional changes in fibroblasts in vitro reminiscent of cellular senescence
Authors: Herrmann Gernot , Brenneisen Peter , Wlaschek Meinhard , Wenk Jutta , Faisst Katrin , Quel Gudrun , Hommel Christina , Goerz Günter , Ruzicka Thomas , Krieg Thomas , Sies Helmut , Scharffetter-Kochanek Karin 
Published: April 2021

ABSTRACT
Premature aging of the skin is a prominent side effect of psoralen photoactivation, a treatment used widely for various skin disorders. The molecular mechanisms underlying premature aging upon psoralen photoactivation are as yet unknown. Here we show that treatment of fibroblasts with 8-methoxypsoralen (8-MOP) and subsequent ultraviolet A (UVA) irradiation resulted in a permanent switch of mitotic to stably postmitotic fibroblasts which acquired a high level of de novo expression of SA-β-galactosidase, a marker for fibroblast senescence in vitro and in vivo. A single exposure of fibroblasts to 8-MOP/UVA resulted in a 5.8-fold up-regulation of two matrix-degrading enzymes, interstitial collagenase (MMP-1) and stromelysin-1 (MMP-3), over a period of &gt;120 days, while TIMP-1, the major inhibitor of MMP-1 and MMP-3, was only slightly induced. This imbalance between matrix-degrading metalloproteases and their inhibitor may lead to connective tissue damage, a hallmark of premature aging. Superoxide anion and hydrogen peroxide, but not singlet oxygen, were identified as important intermediates in the downstream signaling pathway leading to these complex fibroblast responses upon psoralen photoactivation. Collectively, the end phenotype induced upon psoralen photoactivation shares several criteria of senescent cells. In the absence of detailed molecular data on what constitutes normal aging, it is difficult to decide whether the changes reported here reflect mechanisms underlying normal cellular aging/senescence or rather produce a mimic of cellular aging/senescence by quite different pathways.
Cell heterogeneity upon myogenic differentiation: down-regulation of MyoD and Myf-5 generates ‘reserve cells’
Authors: Yoshida Naoko , Yoshida Shosei , Koishi Kyoko , Masuda Kanjiro , Nabeshima Yo-ichi 
Published: April 2021

ABSTRACT
When a proliferating myoblast culture is induced to differentiate by deprivation of serum in the medium, a significant proportion of cells escape from terminal differentiation, while the rest of the cells differentiate. Using C2C12 mouse myoblast cells, this heterogeneity observed upon differentiation was investigated with an emphasis on the myogenic regulatory factors.
The differentiating part of the cell population followed a series of well-described events, including expression of myogenin, p21WAF1, and contractile proteins, permanent withdrawal from the cell cycle and cell fusion, whereas the rest of the cells did not initiate any of these events. Interestingly, the latter cells showed an undetectable or greatly reduced level of MyoD and Myf-5 expression, which had been originally expressed in the undifferentiated proliferating myoblasts. When these undifferentiated cells were isolated and returned to the growth conditions, they progressed through the cell cycle and regained MyoD expression. These cells demonstrated identical features with the original culture on the deprivation of serum. They produced both MyoD-positive differentiating and MyoD-negative undifferentiated populations once again. Thus the undifferentiated cells in the serum-deprived culture were designated ‘reserve cells’.
Upon serum deprivation, MyoD expression rapidly decreased as a result of down-regulation in approximately 50% of the cells. After this heterogenization, MyoD positive cells expressed myogenin, which is the earliest known event of terminal differentiation and marks irreversible commitment to this, while MyoD-negative cells did not differentiate and became the reserve cells. We also demonstrated that ectopic expression of MyoD converted the reserve cells to differentiating cells, indicating that down-regulation of MyoD is a causal event in the formation of reserve cells.
Expression of caveolin-1 and polarized formation of invaginated caveolae in Caco-2 and MDCK II cells
Authors: Vogel Ulla , Sandvig Kirsten , Deurs Bo van 
Published: April 2021

ABSTRACT
We have studied caveolin-1 expression and the frequency and distribution of typical invaginated caveolae as they are identified by electron microscopy in the polarized epithelial cell lines MDCK II and Caco-2. In wild-type MDCK II cells caveolin expression is high and more than 400 caveolae/mm filter were observed at the basolateral membrane. No caveolae were found at the apical surface. By contrast, wild-type Caco-2 cells do not express caveolin-1 and have extremely few, if any caveolae. Caco-2 cells were stably transfected with the gene for caveolin-1 in order to investigate if the formation of caveolae is polarized also in these cells. We have isolated Caco-2 clones expressing different levels of caveolin-1, where the level of expression varies from 10-100% of the endogenous level in MDCK II cells. Caveolin-1 expression in Caco-2 cells gives rise to a marked immunofluorescense labeling mainly at the lateral plasma membrane. By electron microscopy an increase from less than 4 caveolae/mm filter in wild-type Caco-2 cells to 21-76 caveolae/mm filter in Caco-2 clones transfected with caveolin-1 was revealed and these caveolae were exclusively localized to the basolateral membrane. Thus expression of heterologous caveolin-1 in Caco-2 cells leads to polarized formation of caveolae, but there is a lack of correlation between the amount of caveolin expressed in the cells and the number of caveolae, suggesting that factors in addition to caveolin are required for generation of caveolae.
Formation of a distinct connexin43 phosphoisoform in mitotic cells is dependent upon p34cdc2 kinase
Authors: Lampe Paul D. , Kurata Wendy E. , Warn-Cramer Bonnie J. , Lau Alan F. 
Published: April 2021

ABSTRACT
The gap junction protein connexin43 is a phosphoprotein that typically migrates as three bands (nonphosphorylated, P1 and P2) during polyacrylamide gel electrophoresis. The electrophoretic mobility of connexin43 from mitotic cells was distinctly reduced to a form (P3) that migrated slower than P2 from Rat1 cells prepared by shakeoff of nocodazole-treated and untreated cultures. Mitotic FT210 cells, which contain a temperature-sensitive mutation in the p34cdc2 kinase, showed abundant levels of the P3 connexin43 when maintained at the permissive temperature where p34cdc2 is active. In contrast, nocodozole-treated FT210 cells grown at the nonpermissive temperature did not contain P3 connexin43. These results indicated that generation of the P3 connexin43 was dependent upon active p34cdc2/cyclin B kinase. Although the p34cdc2 kinase phosphorylated connexin43 in vitro on peptides containing serine 255, the major phosphotryptic peptides in P3 connexin43 from mitotic cells appeared to be the consequence of another protein kinase(s), which may be activated by the p34cdc2/cyclin B kinase. The P3 connexin43 exhibited a marked redistribution from cell-cell plasma membrane interfaces to multiple, distinctly stained cytoplasmic structures. These events may be part of the dramatic structural changes observed in mitotic cells undergoing cell rounding and cytokinesis. Results of initial studies using inhibitors of protein degradative and synthetic pathways suggested the likelihood that protein degradation and synthesis participate in the disappearance of the P3 connexin43 and restoration of the pattern of connexin43 isoforms observed in nonmitotic cells.
The Cdk inhibitors p25rum1 and p40SIC1 are functional homologues that play similar roles in the regulation of the cell cycle in fission and budding yeast
Authors: Sánchez-Díaz Alberto , González Isabel , Arellano Manuel , Moreno Sergio 
Published: April 2021

ABSTRACT
p25rum1 and p40SIC1 are specific inhibitors of p34cdc2/CDC28 kinase complexes with B-type cyclins that play a central role in the regulation of the G1 phase of the cell cycle. We show here that low levels of expression of SIC1 in Schizosaccharomyces pombe rescues all the phenotypes of cells lacking the rum1+ gene. In addition, high level expression of SIC1 in S. pombe induces extra rounds of DNA replication without mitosis, a phenotype very similar to the overexpression of rum1+. Transient expression of rum1+ in S. cerevisiae restores the G1 arrest phenotype of cdc4 sic1Δ double mutants. Overproduction of rum1+ in Saccharomyces cerevisiae causes a cell cycle block in G1with a phenotype similar to inactivation of all the Clb cyclins. Finally, we have mapped the cyclin interacting domain and Cdk inhibitory domain to a region of about 80 amino acids in p25rum1 that has significant homology to the C-terminal domain of p40SIC1. All these observations suggest that fission yeast p25rum1 and budding yeast p40SIC1 define a family of Cdk inhibitors that specifically down regulate cyclin B/Cdk1 during the G1 phase of the cell cycle.
Analysis of CD44 interactions with hyaluronan in murine L cell fibroblasts deficient in glycosaminoglycan synthesis: a role for chondroitin sulfate
Authors: Esford Lesley E. , Maiti Arpita , Bader Sharon A. , Tufaro Frank , Johnson Pauline 
Published: April 2021

ABSTRACT
CD44 is a widely expressed cell adhesion molecule that binds the extracellular matrix component, hyaluronan, in a tightly regulated manner. Previous studies have shown that the CD44-hyaluronan interaction is affected by changes in the glycosylation state of CD44. In this study, we take advantage of several well-characterized murine L cell mutants defective in heparan sulfate synthesis (gro2C cells), heparan sulfate and chondroitin sulfate synthesis (sog9 cells), and glycosaminoglycan and oligosaccharide processing (sog8 cells) to assess the effects of these defects on the hyaluronan binding ability of CD44. In parental L cells and gro2C cells, CD44 was induced to bind hyaluronan after addition of the activating, anti-CD44 monoclonal antibody, IRAWB 14. By contrast, no inducible binding was observed in sog9 cells. Treatment of L cells with sodium chlorate, an inhibitor of sulfation, also abolished inducible hyaluronan binding. However, inducible and some constitutive hyaluronan binding was observed in sog8 cells. This indicates that sulfation and, in particular, the addition of chondroitin sulfate are required for inducible hyaluronan binding by CD44 in L cells. However, in the absence of fully processed oligosaccharides, chondroitin sulfate is not essential for hyaluronan binding, indicating that the effect of chondroitin sulfate is dependent upon the glycosylation state of the cell. Thus, in addition to glycosylation, chondroitin sulfate biosynthesis is an important post-translational modification that can affect the hyaluronan binding ability of CD44.
Physical and biochemical regulation of integrin release during rear detachment of migrating cells
Authors: Palecek Sean P. , Huttenlocher Anna , Horwitz Alan F. , Lauffenburger Douglas A. 
Published: April 2021

ABSTRACT
Cell migration can be considered as a repeated cycle of membrane protrusion and attachment, cytoskeletal contraction and rear detachment. At intermediate and high levels of cell-substratum adhesiveness, cell speed appears to be rate-limited by rear detachment, specifically by the disruption of cytoskeleton-adhesion receptor-extracellular matrix (ECM) linkages. Often, cytoskeletal linkages fracture to release integrin adhesion receptors from the cell. Cell-extracellular matrix bonds may also dissociate, allowing the integrins to remain with the cell. To investigate molecular mechanisms involved in fracturing these linkages and regulating cell speed, we have developed an experimental system to track integrins during the process of rear retraction in Chinese hamster ovary (CHO) cells. Integrin expression level was varied by transfecting CHO B2 cells, which express very little endogenous α5 integrin, with a plasmid containing human α5 integrin cDNA and sorting the cells into three populations with different α5 expression levels. Receptor/ligand affinity was varied using CHO cells transfected with either αIIbβ3 or αIIbβ3(β1-2), a high affinity variant. αIIbβ3(β1-2) is activated to a higher affinity state with an anti-LIBS2 antibody. Fluorescent probes were conjugated to non-adhesion perturbing anti-integrin antibodies, which label integrins in CHO cells migrating on a matrix-coated glass coverslip. The rear retraction area was determined using phase contrast microscopy and integrins initially in this area were tracked by fluorescence microscopy and a cooled CCD camera.
We find that rear retraction rate appears to limit cell speed at intermediate and high adhesiveness, but not at low adhesiveness. Upon rear retraction, the amount of integrin released from the cell increases as extracellular matrix concentration, receptor level and receptor-ligand affinity increase. In fact, integrin release is a constant function of cell-substratum adhesiveness and the number of cell-substratum bonds. In the adhesive regime where rear detachment limits the rate of cell migration, cell speed has an inverse relationship to the amount of integrin released at the rear of the cell. At high cell-substratum adhesiveness, calpain, a Ca2+-dependent protease, is also involved in release of cytoskeletal linkages during rear retraction. Inhibition of calpain results in decreased integrin release from the cell membrane, and consequently a decrease in cell speed, during migration. These observations suggest a model for rear retraction in which applied tension and calpain-mediated cytoskeletal linkage cleavage are required at high adhesiveness, but only applied tension is required at low adhesiveness.
Cyclic AMP-dependent protein kinases and human trophoblast cell differentiation in vitro
Authors: Keryer Guy , Alsat Eliane , Taskén Kjetil , Evain-Brion Danièle 
Published: April 2021

ABSTRACT
Human trophoblast cells offer a unique in vitro model for the study of aspects of the dynamic processes occurring during cell fusion and syncytium formation. In the human placenta, mononuclear cytotrophoblasts aggregate and fuse to form a multinucleated syncytiotrophoblast. In vitro, the addition of cyclic AMP analogs, 8-bromo-cyclic-AMP or Sp-8-bromo-cyclic AMPS, promotes syncytiotrophoblast formation, as shown by the disappearance of immunostained E-cadherin and desmoplakin, and increased numbers of nuclei per syncytium. An antagonist of cyclic AMP, Rp-8-bromo-cyclic AMPS, and an inhibitor of the cyclic AMP-dependent protein kinase catalytic subunit, H-89, impair cell fusion. This led us to study the pattern of expression and subcellular localization of cyclic-AMP-dependent protein kinase subunits during syncytium formation. Cytotrophoblasts expressed the RIα and RIIα regulatory subunits and the Cα and Cβ catalytic subunits. RIα was down-regulated during syncytium formation. No change in RIIα protein levels was observed, but there was a drastic subcellular redistribution. RIIα located in the Golgi-centrosomal area of cytotrophoblasts was scattered throughout the cytoplasm of the syncytiotrophoblast. Interestingly, an accumulation of RIIα was observed underneath the apical membrane of syncytiotrophoblast in vitro and in situ. This suggests a key role of cyclic AMPdependent protein kinase type IIα during cell fusion and microvilli formation, both of which are essential for the secretory and transfer functions of the syncytiotrophoblast.
Endocytic internalization in yeast and animal cells: similar and different
Authors: Geli M. Isabel , Riezman Howard 
Published: April 2021

ABSTRACT
The internalization step of endocytosis has been the focus of several laboratories during the last forty years. Unlike some other budding events in the cell, many fundamental questions regarding the molecular machinery involved in the mechanism of budding itself still remain unsolved. Over the last few years the general picture of the field has quickly evolved from the originally simplistic view which postulated that clathrin polymerization is the major force driving budding at the plasma membrane. Refinement of the assays and molecular markers to measure endocytosis in animal cells has shown that other factors in addition to the clathrin coat are required and that endocytosis can also take place through clathrin-independent mechanisms. At the same time, recent introduction of genetic approaches to study endocytosis has accelerated the identification of molecules required for this process. The isolation of endocytosis mutants in budding yeast has been especially fruitful in this respect. Preliminary comparison of the results obtained in yeast and animal cells did not seem to coincide, but further progress in both systems now suggests that part of the divergence originally seen may be due to the particular experimental approaches used rather than fundamental differences in endocytic mechanisms. In this review we present a short historical overview on the advances made in yeast and animal cells regarding the study of endocytosis, underlining both emerging similarities and still interesting differences.
Cornified cell envelope assembly: a model based on electron microscopic determinations of thickness and projected density
Authors: Jarnik Michal , Simon Martha N. , Steven Alasdair C. 
Published: April 2021

ABSTRACT
In stratifying squamous epithelia, the cornified cell envelope (CE), a peripheral layer of crosslinked protein, is assembled sequentially from precursor proteins initially dispersed in the cytoplasm. Its major component is loricrin (37 kDa in mouse), which contributes from approx. 60% to &gt;80% of the protein mass in different tissues. Despite its importance to the mechanical resilience and impenetrability of these tissues, detailed information has not been obtained on CE structure, even on such basic properties as its thickness or uniformity across a given CE or from tissue to tissue. To address this issue, we have studied CEs isolated from three murine epithelia, namely epidermis, forestomach and footpad, by electron microscopy of metal-shadowed specimens and scanning transmission electron microscopy (STEM) of unstained specimens. The former data reveal that the cytoplasmic surface is smoothly textured whereas the extracellular surface is corrugated, and that the average thickness is 15.3±1.2 nm, and strikingly uniform. Measurements of mass-per-unit-area from the STEM images yielded values of approx. 7.0±0.8 kDa/nm2, which were remarkably consistent over all three tissues. These data imply that the mature CE has a uniquely defined thickness. To explain its uniformity, we postulate that loricrin forms a molecular monolayer, not a variable number of multiple layers. In this scenario, the packing density is one loricrin monomer per 7 nm2, and loricrin should have an elongated shape, 2.5-3.0 nm wide by approx. 11 nm long. Moreover, we anticipate that any inter-tissue variations in the mechanical properties of CEs should depend more on protein composition and cross-linking pattern than on the thickness of the protein layer deposited.
Mutational analysis of the potential phosphorylation sites in the cytoplasmic domain of integrin β1A: Requirement for threonines 788-789 in receptor activation
Authors: Wennerberg Krister , Fässler Reinhard , Wärmegård Birgitta , Johansson Staffan 
Published: April 2021

ABSTRACT
To investigate the role of the potential phosphorylation sites in the cytoplasmic domain of integrin β1A, point mutated variants of the protein were stably expressed in the β1-deficient cell line GD25. Mutants T777A, Y783F, S785A, and Y795F were fully active in promoting cell adhesion, de novo formation of focal contacts, formation of fibronectin fibrils, and activation of focal adhesion kinase. Thus, phosphorylation of these residues is not required for several basic functions of integrin β1A. On the other hand, the TT788-9AA mutant, was defective in mediating cell attachment and did not contribute to fibronectin fibril formation. The conformation of the extracellular domain was shifted towards an inactive state as measured by binding of the monoclonal antibody 9EG7. Antibody induced clustering of β1ATT788-9AA demonstrated that the mutant cytoplasmic part was functional in mediating activation of focal adhesion kinase. Therefore, we conclude that threonines 788-789, which are conserved among most integrin β subunits, are of critical importance for integrin function due to effects on the extracellular conformation of the receptor.
Effects of collagenase-cleavage of type I collagen on α2β1 integrin-mediated cell adhesion
Authors: Messent Anthea J. , Tuckwell Danny S. , Knäuper Vera , Humphries Martin J. , Murphy Gillian , Gavrilovic Jelena 
Published: April 2021

ABSTRACT
In this paper we show that collagenase-3 cleavage of type I collagen has a marked effect on α2β1 integrin-mediated interactions with the collagen fragments generated. Isolated α2β1 integrin and α2 integrin A-domain were found to bind to both native collagen and native fragment and, to a lesser degree, native fragment. Whole integrin and integrin A-domain binding were lost after heat denaturation of the collagen fragments. At physiological temperature, cell adhesion to triple-helical fragment via α2β1 integrin was still possible; however, no α2β1 integrin-mediated adhesion to the fragment was observed.
Unwinding of the collagen fragment triple helices by heating to physiological temperatures prior to adsorption to plastic tissue culture plates resulted in total abrogation of HT1080 cell attachment to either fragment. These results provide significant evidence in support of a role for matrixmetalloproteinase cleavage of the extracellular matrix in modifying cell-matrix interactions.