bluuebunny/crossref_metadata_2025_split huggingface dataset:
Differentiation and integrity of cardiac muscle cells are impaired in the absence of βi integrin
Authors: Fässler Reinhard , Rohwedel Jürgen , Maltsev Victor , Bloch Wilhelm , Lentini Silvia , Guan Kaomei , Gullberg Donald , Hescheler Jürgen , Addicks Klaus , Wobus Anna M. 
Published: April 2021

ABSTRACT
Cellular interactions with substrata of the microenvironment are one of the major mechanisms for differentiation and morphogenesis. Many of these interactions are mediated via the β1 integrin subfamily of cell surface receptors, which are believed to transduce signals upon cell adhesion. We have used β1 integrin-deficient embryonic stem cells to test their ability to differentiate into cardiac muscle cells. We show here by several approaches that β1 integrin is important for normal cardiogenesis. First, the in vitro differentiation of β1 integrin-deficient embryonic stem cells into cardiac muscle cells is retarded. This is demonstrated by the delayed expression of cardiac muscle-specific genes and action potentials. Second, the specification of cardiac precursor cells into pacemaker-, atrial- and ventricular-like cells is significantly impaired in β1 integrin-deficient cells. The occurrence of atrial- and ventricular-like cells is reduced and transient. Only cells exhibiting pacemaker-like action potentials of high frequency and arrhythmias survive. Third, the sarcomeric architecture is incomplete and disarranged in the absence of β1 integrin. Fourth, β1-deficient embryonic stem cells can contribute to the developing heart in chimaeric mice but many areas with β1-null cells contain cell debris. The number of β1-null cells decreases from prenatal to postnatal stages and is lost completely in 6-month-old hearts.
Thus, we conclude that interactions with the extracellular matrix via β1 integrin is necessary for differentiation and the maintenance of a specialized phenotype of cardiac muscle cells.
Disruption of the vimentin intermediate filament system during adipose conversion of 3T3-L1 cells inhibits lipid droplet accumulation
Authors: Lieber Jonathan G. , Evans Robert M. 
Published: April 2021

ABSTRACT
During the differentiation of 3T3-L1 pre-adipocytes, vimentin intermediate filaments are reorganized to form cage-like structures around the nascent lipid droplets. Initial studies with 3T3-L1 cells indicated that aggregation of vimentin filaments by nocodazole treatment during or shortly after induction of adipose conversion dramatically reduced the lipid droplet content of 3T3-L1 cells 96-120 hours after induction. Specific but transient disruption of vimentin following anti-IFA antibody injection also resulted in a decrease in lipid droplet formation in differentiating cells. To specifically and stably affect filament organization, 3T3-L1 cells lines were established by transfection with a glucocorticoid-regulatable, dominant negative mutant vimentin cDNA expression plasmid. Treatment of these cells (83∆C) with dexamethasone resulted in expression of vimentin with a carboxyl-terminal deletion, which led to the disruption of the endogenous filament network. Induction of adipose conversion in 83∆C cells lead to the formation of lipid droplets comparable to those seen in untransfected 3T3-L1 cells. Addition of dexamethasone during the adipose conversion of 83∆C cells did not affect the induction of the marker enzyme glycerol-3-phosphate dehydrogenase or the incorporation of [14C]palmitate into triglycerides during a 10 minute pulse label. There was, however, a failure to form prominent lipid droplets and to accumulate [14C]palmitate-labeled triglycerides. Pulse-chase experiments indicated that the failure of these cells to accumulate triglyceride was associated with an increased rate of turnover. These studies indicate that vimentin filaments provide a function that influences lipid stability during adipose conversion of 3T3-L1 cells.
Cellulose microfibrils, cell motility, and plasma membrane protein organization change in parallel during culmination in Dictyostelium discoideum
Authors: Grimson Mark J. , Haigler Candace H. , Blanton Richard L. 
Published: April 2021

ABSTRACT
Prestalk cells of Dictyostelium discoideum contribute cellulose to two distinct structures, the stalk tube and the stalk cell wall, during culmination. This paper demonstrates by freeze fracture electron microscopy that two distinct types of intramembrane particle aggregates, which can be characterized as cellulose microfibril terminal complexes, occur in the plasma membranes of cells synthesizing these different forms of cellulose. The same terminal complexes were observed in situ in developing culminants and in vitro in monolayer cells induced to synthesize the two types of cellulose. We propose that cessation of cell motility is associated with a change in packing and intramembrane mobility of the particle aggregates, which causes a change in the nature of the cellulose synthesized. The terminal complexes are compared to those described in other organisms and related to the previous hypothesis of two modes of cellulose synthesis in Dictyostelium.
A novel structural component of the Dictyostelium centrosome
Authors: Kalt Astrid , Schliwa Manfred 
Published: April 2021

ABSTRACT
The microtubule-organizing center of D. discoideum is a nucleus-associated body (NAB) that consists of a multilayered, box-shaped core embedded in an amorphous corona from which the microtubules emerge. The composition of the NAB is still largely unresolved. Here we have examined a high molecular mass component of the NAB which was identified by a monoclonal antibody raised against isolated nucleus/NAB complexes. This antibody recognized a 350 kDa component which is immunologically related to the D. discoideum heavy chain of myosin II. The 350 kDa antigen was localized only at the NAB in interphase cells, while in mitotic cells it may also be found in the vicinity of the NAB as well as in association with the mitotic spindle. Immunogold labeling experiments showed that the protein is part of the NAB corona. This association was not destroyed by treatment with 2 M urea or 0.6 M KCl. The 350 kDa antigen was part of the thiabendazole-induced cytoplasmic microtubule-organizing centers. A direct role in the polymerization of tubulin could not be determined in an in vitro microtubule nucleation assay, whereas antibody electroporation of live cells appeared to interfere with the generation of a normal microtubule system in a subset of cells. Our observations suggest that the 350 kDa antigen is a structural component of the NAB corona which could be involved in its stabilization.
Dynamic changes of NuMA during the cell cycle and possible appearance of a truncated form of NuMA during apoptosis
Authors: Hsu Hsin-Ling , Yeh Ning-Hsing 
Published: April 2021

ABSTRACT
We have demonstrated that dynamic redistribution of nuclear-mitotic apparatus (NuMA) protein in the cell cycle is correlated temporally and spatially with its biochemical modifications. In interphase, NuMA behaves solely as a 220 kDa nuclear matrix-associated protein. After initiation of DNA condensation during mitosis, NuMA is phosphorylated by Cdc2 kinase into a 240 kDa form which is transported quickly to the centrosomal region. Once cells have passed the metaphase-anaphase transition, the 240 kDa form of NuMA either becomes a 180 kDa truncated form which is fated to be degraded completely before mitotic exit, or returns to the 220 kDa form that relocates to the daughter nuclei and remains throughout interphase. Apparently, a proteolytic enzyme is activated during the late stages of mitosis. After induction of a 180 kDa form of NuMA in interphase HeLa cells by 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole, nuclear apoptotic phenomena including chromatin condensation, DNA fragmentation, and micronucleation were observed. However, the same treatment did not induce apoptosis in mitotic phasearrested HeLa cells. The 180 kDa form of NuMA was demonstrated to be a truncated product, at least lacking the tail domain. When HL60 cells were stimulated by diverse apoptosis inducers such as camptothecin, staurosporine, cycloheximide, and A23187, the extent of NuMA cleavage to produce a 180 kDa product was comparable with the degree of oligonucleosomal laddering. NuMA cleavage is likely to be a consequence of the onset of apoptosis. The intact 220 kDa NuMA functions in interphase cells to retain the nuclear structural integrity. Additionally, NuMA appears to act as a nuclear structural target for a death protease during apoptosis.
Conditional differentiation of heartand smooth muscle-derived cells transformed by a temperature-sensitive mutant of SV40 T antigen
Authors: Jahn Lothar , Sadoshima Jun-ichi , Greene Adam , Parker Chris , Morgan Kathleen G. , Izumo Seigo 
Published: April 2021

ABSTRACT
To create muscle cell lines that conditionally differentiate in vitro we introduced a temperature-sensitive SV40 T antigen by retroviral infection into rat aortic smooth muscle cells (SMCs) and neonatal heart-derived cells. After G418 selection cell lines isolated were characterized at permissive (33°C) and non-permissive (39°C) temperatures. [3H]Thymidine uptake showed that progression through the cell cycle is greatly reduced at 39°C. Cytoskeletal proteins, such as actins and vimentin did not change significantly after temperature shift, while the number of desmin-positive SMCs significantly increased when cells were switched to 39°C. Heart-derived muscle cells showed sarcomeric myosin heavy chain reactivity only when grown at 39°C. After thrombin stimulation intracellular calcium in both cell types increased severalfold in 39°C-cells but not in 33°C-cells. Whole cell patch-clamp recordings of SMCs and heart-derived cells revealed a strong increase in nicardipine-sensitive Ca2+ current when cells were switched to 39°C. Nicardipine-insensitive Ca2+ current also increased in both cell types at the non-permissive temperature. Na+ current in SMCs was large at 33°C and small or not detectable at 39°C and absent in heart-derived cells. Using a cDNA probe specific for the α1 subunit of the dihydropyridine-sensitive Ca2+ channel we demonstrate a temperature-sensitive expression of the dihydropyridine receptor mRNA in smooth muscle-derived cells but not in heart-derived H10 cells. Our results suggest that upon downregulation of SV40 T antigen these cells become quiescent and exhibit a more differentiated phenotype. These cell lines may provide a useful tool to investigate ion channeland receptor signal transduction, as well as cell cycle control in smooth and possibly cardiac muscle cell differentiation.
Hysteretic behavior and differential apparent stability properties of microtubule species emerge from the regulation of post-translational modifications of microtubules
Authors: Laurent Michel , Fleury Anne 
Published: April 2021

ABSTRACT
At the epigenetic level, microtubule diversity is generated by several mechanisms of reversible post-translational modifications of tubulin subunits. In most cases, modification enzymes preferentially act on the tubulin subunits of microtubules, whereas the substrate of the enzymes which ensure the reverse reaction is preferentially the αβ-dimer of nonpolymerized tubulin. Most modifications identified to date appear to be nearly ubiquitous within the animal kingdom. Moreover, modifications are generally not mutually exclusive, so that cellular microtubules often bear several distinct biochemical alterations whose biological role is yet unknown.
Post-translational modifications often (but not always) occur on microtubule species with low turnover rate. However, in vitro comparison of the polymerization and depolymerization rates of modified or unmodified forms of tubulin did not reveal any significant difference between molecular species. Thus, post-translational modifications are thought to be the result rather than the cause of microtubule stability.
We re-examine this contention in the light of a regulated kinetic scheme for multiple and non-exclusive enzymatic modifications of microtubules. This study shows that different apparent stability properties of microtubule species emerge under such a kinetic regulation, although all the species were assumed to have identical intrinsic stability properties. This model can be used to reinterpret the results of well-known studies bearing on the relationship between microtubule stability and post-translational modifications. Another important finding is that the existence of a regulation loop in one of the multiple pathways of enzymatic differentiation of microtubules endows the system with hysteretic properties. These properties may be viewed, under restrictive conditions, as a buffering mechanism for the transitions between microtubule growing and shrinking phases during fluctuations in the regulation of centrosomal nucleating activity.
Identification of αvβ3 as a heterotypic ligand for CD31/PECAM-1
Authors: Buckley C. D. , Doyonnas R. , Newton J. P. , Blystone S. D. , Brown E. J. , Watt S. M. , Simmons D. L. 
Published: April 2021

ABSTRACT
CD31 (PECAM-1) is a member of the immunoglobulin gene superfamily (IgSF) and has an important role in a number of endothelial cell functions including angiogenesis, inflammation, integrin activation and cell-cell adhesion. CD31 has both homotypic and heterotypic adhesive properties and in common with other IgSF members contains multiple functional domains. Using chimaeric fusion proteins of CD31 and a panel of haematopoietic cell lines we show that CD31 can bind cells in a predominantly homotypic or heterotypic manner depending on the cell line used. Heterotypic binding was found to be cation and temperature dependent and enhanced by Mn2+: all features of integrin mediated binding. Using a panel of anti-CD31 and anti-integrin antibodies we show that αvβ3 is a ligand for CD31 on the monocytic cell line U937. The specificity of the interaction between αvβ3 and CD31 was further confirmed by solid phase binding assays and the use of αvβ3 transfected cells which bound CD31 specifically. Furthermore, we have mapped the binding site for αvβ3 to domains 1 and 2 of CD31. The interaction of CD31 with αvβ3 may be important in many aspects of endothelial function including leukocyteendothelial transmigration and angiogenesis.
Contributions of the structural domains of filensin in polymer formation and filament distribution
Authors: Goulielmos George , Remington Susann , Schwesinger Falk , Georgatos Spyros D. , Gounari Fotini 
Published: April 2021

ABSTRACT
Filensin and phakinin constitute the subunits of a heteropolymeric, lens-specific intermediate filament (IF) system known as the beaded-chain filaments (BFs). Since the rod of filensin is four heptads shorter than the rods of all other IF proteins, we decided to examine the specific contribution of this protein in filament assembly. For these purposes, we constructed chimeric proteins in which regions of filensin were exchanged with the equivalent ones of vimentin, a self-polymerizing IF protein. Our in vitro studies show that the filensin rod domain does not allow homopolymeric filament elongation. However, the filensin rod is necessary for co-polymerization of filensin with phakinin and seems to counteract the inherent tendency of the latter protein to homopolymerize into large, laterally associated filament bundles. Apart from the rod domain, the presence of an authentic or substituted tail domain in filensin is also essential for co-assembly with the naturally tail-less phakinin and formation of extended filaments in vitro. Finally, transfection experiments in CHO and MCF-7 cells show that the rod domain of filensin plays an important role in de novo filament formation and distribution. The same type of analysis further suggests that the end-domains of filensin interact with cell-specific, assembly-modulating factors.
MAP-1B/TAU functional redundancy during laminin-enhanced axonal growth
Authors: DiTella M. C. , Feiguin F. , Carri N. , Kosik K. S. , Cáceres A. 
Published: April 2021

ABSTRACT
When cultured cerebellar macroneurons develop attached to a laminin-containing substrate or after the acute addition of laminin to the tissue culture medium, there is an acceleration in the rate and extent of axonal elongation. Furthermore, laminin is capable of inducing axonal formation and microtubule stabilization in neurons arrested at stage II of neuritic development by tau suppression (Caceres and Kosik, 1990; Caceres et al., 1991). Laminin-enhanced or induced axonal extension is paralleled by a selective and dramatic incorporation of phosphorylated MAP-1b into axonal microtubules. Axonal formation in neurons growing in the presence of laminin is prevented by treatment of the cultures with a mixture of MAP-1b and tau antisense oligonucleotides, but not by the single suppression of any one of these MAPs. However, suppression of MAP-1b, but not of tau, greatly reduces the increase in the rate and extent of axonal elongation induced by laminin. No such effects are elicited by MAP-1b antisense oligonucleotides in neurons growing in the absence of laminin, e.g. polylysine alone, where most of the MAP-1b present in the cells is dephosphorylated and not associated with the cytoskeleton. Taken collectively, these data suggest that, with regard to axonal elongation, MAP-1b and tau can be functionally substituted, and that extracellular matrix molecules, such as laminin, affect axonal extension by promoting the in vivo utilization of MAP-1b.
Cortical actin movements during the first cell cycle of the Caenorhabditis elegans embryo
Authors: Hird Steven 
Published: April 2021

ABSTRACT
The first division of the Caenorhabditis elegans embryo is unequal, generating daughter cells with distinct fates. The differences between the cells are believed to result from the partitioning of cytoplasmic determinants during the first cell cycle. Actin microfilaments play a critical, but poorly defined, role in this event. In this paper, the actin cortex in live embryos is studied during cytoplasmic localisation by fluorescently labelling microfilaments in oocytes and then using in vivo fluorescence microscopy to observe their behaviour. This reveals that there is a concerted movement of cortical actin to the anterior of the embryo at the time cytoplasmic localisation takes place. Furthermore, it is demonstrated that endogenous foci of F-actin are asymmetrically distributed following this event; these structures have previously been seen in fixed cortices. A model for the participation of the actin cytoskeleton in cytoplasmic localisation is presented based on these results.
Autonomous differentiation in the mouse myogenic cell line, C2, involves a mutual positive control between insulin-like growth factor II and MyoD, operating as early as at the myoblast stage
Authors: Montarras Didier , Aurade Frédéric , Johnson Thomas , Ilan Joseph , Gros François , Pinset Christian 
Published: April 2021

ABSTRACT
We have studied the contribution of the endogenous production of insulin-like growth factor II (IGFII) and of the muscle regulatory factor, MyoD, to the autonomy of differentiation in isolated skeletal myoblasts. Inhibition of MyoD and IGFII gene expression in myoblasts of the mouse myogenic cell line, C2, was achieved by transfection and selection of stably transfected cells (anti-MyoD and anti-IGFII cells) with vectors producing MyoD or IGFII antisense RNA. We observed that inhibiting either MyoD or IGFII has multiple and similar consequences. In addition to the inhibition of the target gene, expression of MyoD transcripts in anti-IGFII myoblasts and expression of IGFII in anti-MyoD myoblasts were also abolished, whereas accumulation of transcripts for the muscle regulatory factor, Myf5, was markedly increased in both cell types. However, despite this Myf5 up-regulation, both anti-IGFII and anti-MyoD myoblasts lost the ability to undergo autonomous differentiation (differentiation in the absence of added IGF), further indicating that Myf5 and MyoD are not strictly interchangeable. Additional evidence of a link between MyoD and IGFII was obtained: (1) forced expression of the MyoD cDNA stimulated IGFII gene expression, and (2) treatment of C2 myoblasts with fibroblast growth factor, not only diminished MyoD expression and compromised differentiation as previously shown by others, but also abolished IGFII expression. These experiments showing loss or gain of function argue in favor of a mutual positive control between IGFII and MyoD operating as early as the myoblast stage.
Tenascin mediates human glioma cell migration and modulates cell migration on fibronectin
Authors: Deryugina Elena I. , Bourdon Mario A. 
Published: April 2021

ABSTRACT
The role of tenascin in mediating tumor cell migration was studied using two cell migration models. In migration/invasion Transwell assays U251.3 glioma cells rapidly migrated through the 8 µm pore size membranes onto tenascinand fibronectin-coated surfaces. In this assay the number of cells migrating onto tenascin was 52.2±9.6% greater than on fibronectin within 4 hours. To assess cell migration rates and cell morphology, U251.3 migration was examined in a two-dimension spheroid outgrowth assay. The radial distance migrated by U251.3 cells from tumor spheroids was found to be 53.8±4.9% greater on tenascin than on fibronectin. Cells migrating on tenascin display a very motile appearance, while cells migrating on fibronectin spread and maintain close intercellular contacts. Cell migration in the presence of integrin blocking antibodies demonstrated that migration on tenascin and fibronectin is mediated by distinct integrins, α2β1 and αvβ5/αvβ3, respectively. Since tenascin is coexpressed in malignant tumor matrices with fibronectin, we assessed the effects of tenascin on U251.3 cell migration mediated by fibronectin. Tenascin was found to provide a positive effect on fibronectin-mediated migration by altering cell morphology and enhancing cell motility. These effects of tenascin on fibronectin-mediated cell migration were inhibited by blocking β1 and α2β1integrins. The results suggest that tenascin may play a significant role in promoting tumor cell migration and invasiveness by modulating cell responses to normal matrix components.
Mechanism of endothelial cell shape change and cytoskeletal remodeling in response to fluid shear stress
Authors: Malek Adel M. , Izumo Seigo 
Published: April 2021

ABSTRACT
Endothelium exposed to fluid shear stress (FSS) undergoes cell shape change, alignment and microfilament network remodeling in the direction of flow by an unknown mechanism. In this study we explore the role of tyrosine kinase (TK) activity, intracellular calcium ([Ca2+]i), mechanosensitive channels and cytoskeleton in the mechanism of cell shape change and actin stress fiber induction in bovine aortic endothelium (BAE). We report that FSS induces β-actin mRNA in a time- and magnitudedependent fashion. Treatment with quin2-AM to chelate intracellular calcium release and herbimycin A to inhibit TK activity abolished BAE shape change and actin stress fiber induction by FSS, while inhibition of protein kinase C with chelerythrine had no effect. Altering intermediate filament structure with acrylamide did not affect alignment or F-actin induction by FSS. Examining the role of the BAE cytoskeleton revealed a critical role for microtubules (MT). MT disruption with nocodazole blocked both FSS-induced morphological change and actin stress fiber induction. In contrast, MT hyperpolymerization with taxol attenuated the cell shape change but did not prevent actin stress fiber induction under flow. Mechanosensitive channels were found not to be involved in the FSS-induced shape change. Blocking the shear-activated current (IK.S) with barium and the stretch-activated cation channels (ISA) with gadolinium had no effect on the shear-induced changes in morphology and cytoskeleton. In summary, FSS has a profound effect on endothelial shape and F-actin network by a mechanism which depends on TK activity, intracellular calcium, and an intact microtubule network, but is independent of protein kinase C, intermediate filaments and shear- and stretch-activated mechanosensitive channels.