bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dynamic distribution and formation of a para-sarcomeric banding pattern of prosomes during myogenic differentiation of satellite cells in vitro
Authors: Foucrier J. , Grand M. C. , Conto F. De , Bassaglia Y. , Géraud G. , Scherrer K. , Martelly I. 
Published: April 2021

ABSTRACT
Myogenesis proceeds by fusion of proliferating myoblasts into myotubes under the control of various transcription factors. In adult skeletal muscle, myogenic stem cells are represented by the satellite cells which can be cultured and differentiate in vitro. This system was used to investigate the subcellular distribution of a particular type of prosomes at different steps of the myogenic process. Prosomes constitute the MCP core of the 26S proteasomes but were first observed as subcomplexes of the untranslated mRNPs; recently, their RNase activity was discovered. A monoclonal antibody raised against the p27K subunit showed that the p27K subunit-specific prosomes move transiently into the nucleus prior to the onset of myoblast fusion into myotubes; this represents possibly one of the first signs of myoblast switching into the differentiation pathway. Prior to fusion, the prosomes containing the p27K subunit return to the cytoplasm, where they align with the gradually formed lengthwise-running desmin-type intermediate filaments and the microfilaments, co-localizing finally with the actin bundles. The prosomes progressively form discontinuous punctate structures which eventually develop a pseudo-sarcomeric banding pattern. In myotubes just formed in vitro, the formation of this pattern seems to preceed that produced by the muscle-specific sarcomeric α-actin. Interestingly, this pattern of prosomes of myotubes in terminal in vitro differentiation was very similar to that of prosomes observed in vivo in foetal and adult muscle. These observations are discussed in relation to molecular myogenesis and prosome/proteasome function.
Tropomodulin assembles early in myofibrillogenesis in chick skeletal muscle: evidence that thin filaments rearrange to form striated myofibrils
Authors: Almenar-Queralt Angels , Gregorio Carol C. , Fowler Velia M. 
Published: April 2021

ABSTRACT
Actin filament lengths in muscle and nonmuscle cells are believed to depend on the regulated activity of capping proteins at both the fast growing (barbed) and slow growing (pointed) filament ends. In striated muscle, the pointed end capping protein, tropomodulin, has been shown to maintain the lengths of thin filaments in mature myofibrils. To determine whether tropomodulin might also be involved in thin filament assembly, we investigated the assembly of tropomodulin into myofibrils during differentiation of primary cultures of chick skeletal muscle cells. Our results show that tropomodulin is expressed early in differentiation and is associated with the earliest premyofibrils which contain overlapping and misaligned actin filaments. In addition, tropomodulin can be found in actin filament bundles at the distal tips of growing myotubes, where sarcomeric α-actinin is not always detected, suggesting that tropomodulin caps actin filament pointed ends even before the filaments are cross-linked into Z bodies by α-actinin. Tropomodulin staining exhibits an irregular punctate pattern along the length of premyofibrils that demonstrate a smooth phalloidin staining pattern for F-actin. Strikingly, the tropomodulin dots often appear to be located between the closely spaced, dot-like Z bodies that are stained for α-actinin. Thus, in the earliest premyofibrils, the pointed ends of the thin filaments are clustered and partially aligned with respect to the Z bodies (the location of the barbed filament ends). At later stages of differentiation, the tropomodulin dots become aligned into regular periodic striations concurrently with the appearance of striated phalloidin staining for F-actin and alignment of Z bodies into Z lines. Tropomodulin, together with the barbed end capping protein, CapZ, may function from the earliest stages of myofibrillogenesis to restrict the lengths of newly assembled thin filaments by capping their ends; thus, transitions from nonstriated to striated myofibrils in skeletal muscle are likely due principally to filament rearrangements rather than to filament polymerization or depolymerization. Rearrangements of actin filaments capped at their pointed and barbed ends may be a general mechanism by which cells restructure their actin cytoskeletal networks during cell growth and differentiation.
Cloning, sequencing, and nucleolar targeting of the basal-body-binding nucleolar protein BN46/51
Authors: Trimbur Gina M. , Goeckeler Jennifer L. , Brodsky Jeffrey L. , Walsh Charles J. 
Published: April 2021

ABSTRACT
BN46/51 is an acidic protein found in the granular component of the nucleolus of the amebo-flagellate Naegleria gruberi. When Naegleria amebae differentiate into swimming flagellates, BN46/51 is found associated with the basal body complex at the base of the flagella. In order to determine the factors responsible for targeting BN46/51 to a specific subnucleolar region, cDNAs coding for both subunits were isolated and sequenced. Two clones, JG4.1 and JG12.1 representing the 46 kDa and 51 kDa subunits, respectively, were investigated in detail. JG12.1 encoded a polypeptide of 263 amino acids with a predicted size of 30.1 kDa that co-migrated with the 51 kDa subunit of BN46/51 when expressed in yeast. JG4.1 encoded a polypeptide of 249 amino acids with a predicted size of 28.8 kDa that co-migrated with the 46 kDa subunit of BN46/51. JG4.1 was identical to JG12.1 except for the addition of an aspartic acid between positions 94 and 95 of the JG12.1 sequence and the absence of 45 amino acids beginning at position 113. The predicted amino acid sequences were not closely related to any previously reported. However, the sequences did have 26-31% identity to a group of FKPBs (FK506 binding proteins) but lacked the peptidyl-prolyl cis-trans isomerase domain of the FKBPs. Both subunits contained two KKE and three KKX repeats found in other nucleolar proteins and in some microtubule binding proteins. Using ‘Far Western’ blots of nucleolar proteins, BN46/51 bound to polypeptides of 44 kDa and 74 kDa. The 44 kDa component was identified as the Naegleria homologue of fibrillarin. BN46/51 bound specifically to the nucleoli of fixed mammalian cells, cells which lack a BN46/51 related polypeptide. When the JG4.1 and JG12.1 cDNAs were expressed in yeast, each subunit was independently targeted to the yeast nucleolus. We conclude that BN46/51 represents a unique nucleolar protein that can form specific complexes with fibrillarin and other nucleolar proteins. We suggest that the association of BN46/51 with the MTOC of basal bodies may reflect its role in connecting the nucleolus with the MTOC activity for the mitotic spindle. This would provide a mechanism for nucleolar segregation during the closed mitosis of Naegleria amebae.
Polarized targeting of epithelial cell proteins in thyrocytes and MDCK cells
Authors: Prabakaran Daniel , Ahima Rexford S. , Harney John W. , Berry Marla J. , Larsen P. Reed , Arvan Peter 
Published: April 2021

ABSTRACT
Polarized trafficking signals may be interpreted differently in different cell types. In this study, we have compared the polarized trafficking of different proteins expressed endogenously in primary porcine thyroid epithelial cells to similar proteins expressed in MDCK cells. As in MDCK cells, NH4Cl treatment of filter-grown thyrocytes caused mis-sorted soluble proteins to exhibit enhanced secretion to the apical medium. In independent studies, thrombospondin 1 (a thyroid basolaterally secreted protein) was secreted basolaterally from MDCK cells. Likewise, the 5′-deiodinase (a thyroid basolateral membrane protein) encoded by the DIO1 gene was also distributed basolaterally in transfected MDCK cells. Consistent with previous reports, when the secretion of human growth hormone (an unglycosylated regulated secretory protein) was examined from transfected MDCK cells, the release was nonpolarized. However, transfected thyrocytes secreted growth hormone apically in a manner dependent upon zinc addition. Moreover, two additional regulated secretory proteins expressed in thyrocytes, thyroglobulin (the major endogenous glycoprotein) and parathyroid hormone (an unglycosylated protein expressed transiently), were secreted apically even in the absence of zinc. We hypothesize that while cellular mechanisms for interpreting polarity signals are generally similar between thyrocytes and MDCK cells, thyrocytes allow for specialized packaging of regulated secretory proteins for apical delivery, which does not require glycosylation but may involve availability of certain ions as well as appropriate intracellular compartmentation.
Mutational analysis of cysteine-string protein function in insulin exocytosis
Authors: Zhang Hui , Kelley William L. , Chamberlain Luke H. , Burgoyne Robert D. , Lang Jochen 
Published: April 2021

ABSTRACT
Cysteine-string proteins (Csps) are vesicle proteins involved in neurotransmission. They contain at least four domains: an N-terminal J-domain which can interact with the chaperone Hsc70, an adjacent linker region, the defining cysteine rich domain and a variable C terminus. As the relevance of these domains for the function of Csps in exocytosis is unknown, we have performed a mutational analysis of Csp domains using insulin release by large dense core vesicles (LDCVs) as a model of regulated exocytosis. All mutants were apparently palmitoylated and their subcellular distribution was similar to endogenous Csp. Point mutations within the highly conserved HPD motif of the J-domain abolished activation of Hsc70. However, these mutations altered the effect of Csp on exocytosis only after additional truncation of the extreme C terminus as found in the Csp splice variant Csp2. Furthermore, the strikingly conserved linker region adjacent to the J-domain was important for Csp function in exocytosis, but not for the activation of Hsc70 ATPase. The effects of Csp wild-type or mutants were preserved in permeabilized cells excluding an effect on transmembrane ion fluxes. These observations demonstrate a functional difference between the two isoforms and suggest a role for the J-domain co-chaperone function as well as for the newly defined linker region in LDCV exocytosis.
Activation of pp60src is critical for stretch-induced orienting response in fibroblasts
Authors: Sai Xiaorui , Naruse Keiji , Sokabe Masahiro 
Published: April 2021

ABSTRACT
When subjected to uni-axial cyclic stretch (120% in length, 1 Hz), fibroblasts (3Y1) aligned perpendicular to the stretch axis in a couple of hours. Concomitantly with this orienting response, protein tyrosine phosphorylation of cellular proteins (molecular masses of approximately 70 kDa and 120-130 kDa) increased and peaked at 30 minutes. Immuno-precipitation experiments revealed that paxillin, pp125FAK, and pp130CAS were included in the 70 kDa, and 120-130 kDa bands, respectively. Treatment of the cells with herbimycin A, a tyrosine kinase inhibitor, suppressed the stretch induced tyrosine phosphorylation and the orienting response suggesting that certain tyrosine kinases are activated by stretch. We focused on pp60src, the most abundant tyrosine kinase in fibroblasts. The kinase activity of pp60src increased and peaked at 20 minutes after the onset of cyclic stretch. Treatment of the cells with an anti-sense S-oligodeoxynucleotide (S-ODN) against pp60src, but not the sense S-ODN, inhibited the stretch induced tyrosine phosphorylation and the orienting response. To further confirm the involvement of pp60src, we performed the same sets of experiments using c-src-transformed 3Y1 (c-src-3Y1) fibroblasts. Cyclic stretch induced a similar orienting response in c-src-3Y1 to that in wild-type 3Y1, but with a significantly faster rate. The time course of the stretch-induced tyrosine phosphorylation was also much faster in c-src-3Y1 than in 3Y1 fibroblasts. These results strongly suggest that cyclic stretch induces the activation of pp60src and that pp60src is indispensable for the tyrosine phosphorylation of pp130CAS, pp125FAK and paxillin followed by the orienting response in 3Y1 fibroblasts.
RA regulation of keratin expression and myogenesis suggests different ways of regenerating muscle in adult amphibian limbs
Authors: Corcoran Jonathan P. , Ferretti Patrizia 
Published: April 2021

ABSTRACT
Formation of a regeneration blastema following limb amputation is believed to occur through a process of dedifferentiation. It has been suggested, however, that the cells contributed to the blastema by the stump muscle are satellite-like cells, rather than cells originated by dedifferentiation. We have previously shown that simple epithelial keratins 8 and 18 are expressed in the mesenchymal progenitor cells of the regenerating amphibian limb and in cultured cells with myogenic potential, and that their expression appears to be causally related to changes in proliferation and differentiation. We show here that retinoic acid (RA) affects the expression of these keratins differently in myogenic cells originated from normal limb and limb blastema. Furthermore, we find that the effects of RA on proliferation, myogenic differentiation and adhesion of these cells also differ. In fact, whereas RA does not affect keratin expression, proliferation or myogenic differentiation in blastemal cells, it does decrease keratin levels and thymidine incorporation and increase myogenesis in cells from normal limb. Conversely, RA increases cell adhesion only in blastemal cells. Significantly, these effects of RA on cultured cells are consistent with those observed in vivo. Overall the results presented here suggest that in the urodele limb there are two distinct cell populations with myogenic potential, one originating from dedifferentiation and one equivalent to the satellite cells of the mammalian muscle, which are likely to be primarily involved in blastema formation and muscle repair, respectively.
Influence of transforming growth factor β1 (TGF-β1) on the behaviour of porcine thyroid epithelial cells in primary culture through thrombospondin-1 synthesis
Authors: Claisse D. , Martiny I. , Chaqour B. , Wegrowski Y. , Petitfrere E. , Schneider C. , Haye B. , Bellon G. 
Published: April 2021

ABSTRACT
Transforming growth factor β1 (TGF-β1) is a secreted polypeptide that is thought to play a major role in the regulation of folliculogenesis and differentiation of thyroid cells. On porcine thyroid follicular cells cultured on plastic substratum, TGF-β1, in a concentration-dependent way, promoted the disruption of follicles, cell spreading, migration and confluency by a mechanism that did not involve cell proliferation. TGF-β1 strongly activated the production of thrombospondin-1 and αvβ3 integrin in a concentration-dependent manner whereas the expression of thyroglobulin was unaffected. Anisomycin, an inhibitor of protein synthesis, inhibited the effect of TGF-β1 on cell organization. Thrombospondin-1 reproduced the effect of TGF-β1. In the presence of thrombospondin-1 cells did not organize in follicle-like structures but, in contrast, spreaded and reached confluency independently of cell proliferation. This effect is suppressed by an RGD-containing peptide. The adhesive properties of thrombospondin-1 for thyroid cells were shown to be mediated by both the aminoterminal heparin-binding domain and the RGD domain of thrombospondin-1. Adhesion was shown to involve αvβ3 integrin. The results show that TGF-β1 exerted an influence upon function and behaviour of follicle cells partly mediated by the synthesis of thrombospondin-1 and of its receptor αvβ3 integrin.
Extracellular signal-regulated kinase functions in the urokinase receptor-dependent pathway by which neutralization of low density lipoprotein receptor-related protein promotes fibrosarcoma cell migration and Matrigel invasion
Authors: Webb Donna J. , Nguyen Diem H. D. , Gonias Steven L. 
Published: April 2021

ABSTRACT
The low density lipoprotein receptor-related protein (LRP) has been reported to regulate cellular migration. In this study, an antisense RNA expression strategy was used to reduce LRP to undetectable levels in HT 1080 fibrosarcoma cells. The LRP-deficient cells demonstrated increased levels of cell-surface uPAR, higher levels of uPA in conditioned medium, increased migration on vitronectin-coated surfaces, and increased invasion of Matrigel. LRP-deficient cells also demonstrated increased levels of phosphorylated extracellular signal-regulated kinase (ERK) in the absence of exogenous stimulants. Antibodies which block binding of endogenously produced uPA to uPAR reduced ERK phosphorylation and migration of LRP-deficient cells to the levels observed with control cells. Inhibitors of ERK activation, including PD098059 and dominant-negative MEK1, also decreased the migration of LRP-deficient but not control cells. By contrast, constitutively active MEK1 stimulated the migration of control but not LRP-deficient cells. Although Matrigel invasion by LRP-deficient cells was inhibited by the proteinase inhibitor, aprotinin, PD098059 in combination with aprotinin was necessary for an optimal effect. Expression of the VLDL receptor in LRP-deficient cells reversed the changes in cellular migration and invasion. These studies demonstrate that binding of endogenously produced uPA to uPAR may serve as a major determinant of basal levels of activated ERK and, by this mechanism, regulate cellular migration and invasion. By regulating the uPA/uPAR system, LRP may also regulate ERK activation, cellular migration, and invasion.
COPI vesicles accumulating in the presence of a GTP restricted Arf1 mutant are depleted of anterograde and retrograde cargo
Authors: Pepperkok Rainer , Whitney J. Andrew , Gomez Marie , Kreis Thomas E. 
Published: April 2021

ABSTRACT
Microinjection of the slowly hydrolyzable GTP analogue GTPγS or the ectopic expression of a GTP restricted mutant of the small GTPase arf1 (arf1[Q71L]) leads to the rapid accumulation of COPI coated vesicles and buds in living cells. This effect is blocked at 15°C and by microinjection of antibodies against β-COP. Anterograde and retrograde membrane protein transport markers, which have been previously shown to be incorporated into COPI vesicles between the endoplasmic reticulum and Golgi complex, are depleted from the GTPγS or arf1[Q71L] induced COPI coated vesicles and buds. In contrast, in control cells 30 to 60% of the COPI carriers co-localize with these markers. These in vivo data corroborate recent in vitro work, suggesting that GTPγS and arf1[Q71L] interfere with the sorting of membrane proteins into Golgi derived COPI vesicles, and provide the first in vivo evidence for a role of GTP hydrolysis by arf1 in the sorting of cargo into COPI coated vesicles and buds.
Skh1, the MEK component of the Mkh1 signaling pathway in Schizosaccharomyces pombe
Authors: Loewith Robbie , Hubberstey Andrew , Young Dallan 
Published: April 2021

ABSTRACT
We previously reported the identification of Mkh1, a MEK kinase in Schizosaccharomyces pombe that is required for cell wall integrity, and we presented genetic evidence that Pmk1/Spm1, a MAP kinase, functions downstream from Mkh1 in the same pathway. Here, we report the identification of Skh1, a MEK (MAP kinase kinase) in S. pombe. The sequence of Skh1 is nearly identical to that of the recently reported Pek1 sequence. We present biochemical and genetic evidence that Skh1 is the MEK component of the Mkh1-Spm1 MAP kinase cascade. Our yeast two-hybrid results indicate that Mkh1, Skh1, and Spm1 physically interact to form a ternary complex. Deletion of mkh1, skh1 or spm1 results in identical phenotypes, including sensitivity to β-glucanase treatment, growth inhibition on media containing KCl, and filamentous growth on medium containing caffeine. Double mutant strains exhibit phenotypes that are identical to the single mutant strains. Furthermore, expression of an activated HA-Skh1DD protein suppressed these defects in mkh1Δ cells, and overexpression of Spm1 suppressed these defects in skh1Δ cells. We also show that HA-Spm1 is hyper-phosphorylated on tyrosine residues in cells coexpressing the activated HA-Skh1DD protein. Furthermore, we found the phosphorylated/activated form of GFP-HA-Spm1 at detectable levels in wild-type cells, but not at appreciable levels in mkh1Δ or skh1Δ cells expressing this fusion protein. Together, our results indicate that Mkh1, Skh1 and Spm1 constitute a MAPK cascade in fission yeast.
Clathrin plays a novel role in the regulation of cell polarity, pseudopod formation, uropod stability and motility in Dictyostelium
Authors: Wessels Deborah , Reynolds Josh , Johnson Olof , Voss Edward , Burns Royce , Daniels Karla , Garrard Elizabeth , O’Halloran Teresa J. , Soll David R. 
Published: April 2021

ABSTRACT
Although the traditional role of clathrin has been in vesicle trafficking and the internalization of receptors, a novel role in cytokinesis was recently revealed in an analysis of a clathrin-minus Dictyostelium mutant (chc−). chc− cells grown in suspension were demonstrated to be defective in assembling myosin II into a normal contractile ring. To test whether this defect reflected a more general one of cytoskeletal dysfunction, chc− cells were analyzed for cell polarity, pseudopod formation, uropod stability, cell locomotion, chemotaxis, cytoskeletal organization and vesicle movement. chc− cells crawled, chemotaxed, localized F-actin in pseudopods, organized their microtubule cytoskeleton in a relatively normal fashion and exhibited normal vesicle dynamics. Although chc− cells extended pseudopods from the anterior half of the cell with the same frequency as normal chc+ cells, they extended pseudopods at twice the normal frequency from the posterior half of the cell. The uropods of chc− cells also exhibited spatial instability. These defects resulted in an increase in roundness, a reduction in polarity, a reduction in velocity, a dramatic increase in turning, a high frequency of 180° direction reversals and a decrease in the efficiency of chemotaxis. All defects were reversed in a rescued strain. These results are the first to suggest a novel role for clathrin in cell polarity, pseudopod formation, uropod stability and locomotion. It is hypothesized that clathrin functions to suppress pseudopod formation and to stabilize the uropod in the posterior half of a crawling cell, two behavioral characteristics that are essential for the maintenance of cellular polarity, efficient locomotion and efficient chemotaxis.
Src tyrosine kinase activity down-regulates Rho-dependent responses during Shigella entry into epithelial cells and stress fibre formation
Authors: Duménil Guillaume , Sansonetti Philippe , Nhieu Guy Tran Van 
Published: April 2021

ABSTRACT
Invasion of epithelial cells by Shigella, the causative agent of bacillary dysentery, is dependent upon the formation of characteristic membrane ruffles that engulf the bacteria in a macropinocytic-like process. We show here that Cdc42 and Rac GTPases, but not Rho, are critical for actin polymerisation, whereas Rho is necessary for the recruitment of ezrin and Src at the site of entry. Remarkably, cells expressing constitutively active Src did not show ezrin recruitment at Shigella entry foci. In these cells, formation of stress fibres induced by LPA stimulation, or microinjection of activated Rho (V14Rho), was inhibited. Src-mediated tyrosyl-phosphorylation of p190RhoGAP correlated with changes in the ability of p190RhoGAP to interact with Rho, suggesting that Src regulates Rho function via p190RhoGAP. We propose that Rho activation is required for proper organisation of Shigella entry foci and for Src recruitment, and that Src tyrosine kinase activity, in turn, down-regulates the function of Rho at the site of Shigella entry. The significance of this negative regulatory loop on Rhodependent responses is discussed.