bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular motors − a paradigm for mutant analysis
Authors: Endow Sharyn A. 
Published: April 2021

ABSTRACT
Molecular motors perform essential functions in the cell and have the potential to provide insights into the basis of many important processes. A unique property of molecular motors is their ability to convert energy from ATP hydrolysis into work, enabling the motors to bind to and move along cytoskeletal filaments. The mechanism of energy conversion by molecular motors is not yet understood and may lead to the discovery of new biophysical principles. Mutant analysis could provide valuable information, but it is not obvious how to obtain mutants that are informative for study. The analysis presented here points out several strategies for obtaining mutants by selection from molecular or genetic screens, or by rational design. Mutants that are expected to provide important information about the motor mechanism include ATPase mutants, which interfere with the nucleotide hydrolysis cycle, and uncoupling mutants, which unlink basic motor activities and reveal their interdependence. Natural variants can also be exploited to provide unexpected information about motor function. This general approach to uncovering protein function by analysis of informative mutants is applicable not only to molecular motors, but to other proteins of interest.
A functional knock-out of titin results in defective myofibril assembly
Authors: van der Ven Peter F. M. , Bartsch Jörg W. , Gautel Mathias , Jockusch Harald , Fürst Dieter O. 
Published: April 2021

ABSTRACT
Titin, also called connectin, is a giant muscle protein that spans the distance from the sarcomeric Z-disc to the M-band. Titin is thought to direct the assembly of sarcomeres and to maintain sarcomeric integrity by interacting with numerous sarcomeric proteins and providing a mechanical linkage. Since severe defects of such an important molecule are likely to result in embryonic lethality, a cell culture model should offer the best practicable tool to probe the cellular functions of titin. The myofibroblast cell line BHK-21/C13 was described to assemble myofibrils in culture. We have now characterized the sub-line BHK-21-Bi, which bears a small deletion within the titin gene. RNA analysis revealed that in this mutant cell line only a small internal portion of the titin mRNA is deleted. However, western blots, immunofluorescence microscopy and immunoprecipitation experiments showed that only the N-terminal, approx. 100 kDa central Z-disc portion of the 3 MDa titin protein is expressed, due to the homozygous deletion in the gene. Most importantly, in BHK-21-Bi cells the formation of thick myosin filaments and the assembly of myofibrils are impaired, although sarcomeric proteins are expressed. Lack of thick filament formation and of ordered actin-myosin arrays was confirmed by electron microscopy. Myogenisation induced by transfection with MyoD yielded myofibrils only in myotubes formed from wild type and not from mutant cells, ruling out that a principal failure in myogenic commitment of the BHK-21-Bi cells might cause the observed effects. These experiments provide the first direct evidence for the crucial role of titin in both thick filament formation as a molecular ruler and in the coordination of myofibrillogenesis.
A fission yeast general translation factor reveals links between protein synthesis and cell cycle controls
Authors: Grallert Beáta , Kearsey Stephen E. , Lenhard Michael , Carlson Cathrine Rein , Nurse Paul , Boye Erik , Labib Karim 
Published: April 2021

ABSTRACT
In two independent screens we isolated fission yeast mutations with phenotypes suggesting defects in B-cyclin function or expression. These mutations define a single gene which we call ded1. We show that ded1 encodes a general translation factor that is related in sequence and function to RNA helicases required for translation in other species. Levels of the B-cyclins Cig2 and Cdc13 are dramatically reduced upon inactivation of Ded1, and this reduction is independent of degradation by the anaphase promoting complex. When a ded1 mutant is grown under semi-restrictive conditions, the translation of Cig2 (and to a lesser extent Cdc13), is impaired relative to other proteins. We show that B-cyclin translation is specifically inhibited upon nitrogen starvation of wild-type cells, when B-cyclin/Cdc2 inactivation is a prerequisite for G1 arrest and subsequent mating. Our data suggest that translational inhibition of B-cyclin expression represents a third mechanism, in addition to cyclin degradation and Rum1 inhibition, that contributes to Cdc2 inactivation as cells exit from the mitotic cell cycle and prepare for meiosis.
A novel RGD-independent fibronectin assembly pathway initiated by α4β1 integrin binding to the alternatively spliced V region
Authors: Sechler Jan L. , Cumiskey Anne Marie , Gazzola Deana M. , Schwarzbauer Jean E. 
Published: April 2021

ABSTRACT
Fibronectin (FN) matrix assembly is a multi-step process that involves binding to integrin receptors, FN-FN interactions and connections to the actin cytoskeleton. Ultimately, FN is converted into stable matrix fibrils that are detergent-insoluble. RGD-binding integrins such as α5β1 play a major role in the assembly of fibrillar FN. Here we show that α4β1 binding to the alternatively spliced V (IIICS) region of FN initiates an alternative assembly pathway. Activation of α4β1 with exogenous agents such as Mn2+ or a β1-stimulatory antibody TS2/16 was sufficient to induce initiation of FN fibrillogenesis by Ramos B lymphoma cells and by CHO(B2)α4 cells. Using recombinant FNs lacking specific sequences, we show that assembly is independent of the RGD sequence but requires the V25/CS-1 segment. Previously, we have characterized an activated recombinant FN (FNΔIII1-7) that rapidly forms detergent-insoluble multimers upon binding to α5β1 integrin. α4β1 also formed FNΔIII1-7 multimers without the aid of exogenous stimulants, suggesting that an activated form of FN can override the need for activation of the integrin. In contrast to assembly by α5β1, actin filaments remained largely cortical and no change in cell growth rate was observed with α4β1-mediated assembly. These results show that binding sites on FN other than the RGD sequence/synergy site and distant from the cell binding domain can promote FN assembly. Thus, there appear to be multiple, integrin-specific mechanisms for assembly of FN matrix.
Determination of cell polarity in germinated spores and hyphal tips of the filamentous ascomycete Ashbya gossypii requires a rhoGAP homolog
Authors: Wendland J. , Philippsen P. 
Published: April 2021

ABSTRACT
In the filamentous ascomycete Ashbya gossypii, like in other filamentous fungi onset of growth in dormant spores occurs as an isotropic growth phase generating spherical germ cells. Thereafter, a switch to polarized growth results in the formation of the first hyphal tip. The initial steps of hyphal tip formation in filamentous fungi, therefore, resemble processes taking place prior to and during bud emergence of unicellular yeast-like fungi. We investigated whether phenotypic similarities between these distinct events extended to the molecular level. To this end we isolated and characterized the A. gossypii homolog of the Saccharomyces cerevisiae BEM2 gene which is part of a network of rho-GTPases and their regulators required for bud emergence and bud growth in yeast. Here we show that the AgBem2 protein contains a GAP-(GTPase activating protein) domain for rho-like GTPases at its carboxy terminus, and that this part of AgBem2p is required for complementation of an Agbem2 null strain. Germination of spores resulted in enlarged Agbem2 germ cells that were unable to generate the bipolar branching pattern found in wild-type germ cells. In addition, mutant hyphae were swollen due to defects in polarized cell growth indicated by the delocalized distribution of chitin and cortical actin patches. Surprisingly, the complete loss of cell polarity which lead to spherical hyphal tips was overcome by the establishment of new cell polarities and the formation of multiple new hyphal tips. In conclusion these results and other findings demonstrate that establishment of cell polarity, maintenance of cell polarity, and polarized hyphal growth in filamentous fungi require members of rho-GTPase modules.
During both interphase and mitosis, DNA topoisomerase II interacts with DNA as well as RNA through the protein’s C-terminal domain
Authors: Rzepecki Ryszard , Fisher Paul A. 
Published: April 2021

ABSTRACT
DNA topoisomerase II (topo II) is thought to be a nuclear enzyme; during interphase most was insoluble and could be recovered in the pellet after centrifugation of cell homogenates at 10,000 g (P-10). Upon entry into mitosis, the majority of topo II did not associate with condensed chromosomes but was apparently solubilized and redistributed throughout the cell. Although two non-chromosomal subfractions of mitotic topo II were defined by centrifugation at 130,000 g, the vast majority (&gt;90%) was recovered in the pellet (P-130). In vivo nucleic acid interactions with topo II were monitored by a recently developed approach of UV-photo-crosslinking, immunoprecipitation and 32P-labeling. P-10 (interphase) topo II was largely associated with DNA. P-130 (mitotic non-chromosomal) topo II was primarily associated with RNA. These nucleic acid interactions with both interphase and mitotic topo II occurred through the catalytically inert and as yet, poorly understood C-terminal domain of the protein. P-10 topo II was highly active enzymatically. Activity, measured by the ability of topo II to decatenate kDNA minicircles, was reduced by treatment with phosphatase. In contrast, P-130 topo II was relatively inactive but activity could be increased by phosphatase treatment. In vivo, P-130 topo II was more heavily phosphorylated than P-10 topo II; in both, only the C-terminal domain of topo II was detectably modified. Our observations suggest that cell cycle-dependent changes in the distribution, nucleic acid interactions and enzymatic activity of topo II are regulated, at least in part, by phosphorylation/dephosphorylation.
Release of an invasion promoter E-cadherin fragment by matrilysin and stromelysin-1
Authors: Noë Veerle , Fingleton Barbara , Jacobs Kathleen , Crawford Howard C. , Vermeulen Stefan , Steelant Wim , Bruyneel Erik , Matrisian Lynn M. , Mareel Marc 
Published: April 2021

ABSTRACT
The function of many transmembrane molecules can be altered by cleavage and subsequent release of their ectodomains. We have investigated ectodomain cleavage of the cell-cell adhesion and signal-transducing molecule E- cadherin. The E-cadherin ectodomain is constitutively shed from the surface of MCF-7 and MDCKts.srcC12 cells in culture. Release of the 80 kDa soluble E-cadherin fragment is stimulated by phorbol-12-myristate-13-acetate and is inhibited by overexpression of the tissue inhibitor of metalloproteinases-2. The metalloproteinases matrilysin and stromelysin-1 both cleave E-cadherin at the cell surface and release sE-CAD into the medium. The soluble E- cadherin fragment thus released inhibits E-cadherin functions in a paracrine way, as indicated by induction of invasion into collagen type I and inhibition of E-cadherin- dependent cell aggregation. Our results, therefore, suggest a novel mechanism by which metalloproteinases can influence invasion.
Subversion of integrins by enteropathogenic Yersinia
Authors: Isberg Ralph R. , Barnes Penelope 
Published: April 2021

ABSTRACT
Enteropathogenic Yersinia are gram-negative bacterial species that translocate from the lumen of the intestine and are able to grow within deep tissue sites. During the earliest stages of disease, the organism is able to bind integrin receptors that are presented on the apical surface of M cells in the intestine, which allows its internalization and subsequent translocation into regional lymph nodes. The primary integrin substrate is the outer-membrane protein invasin, which binds with extraordinarily high affinity to at least five different integrins that have the β1 chain. Bacterial uptake into host cells is modulated by the affinity of receptor-substrate interaction, receptor concentration and the ability of the substrate to aggregate target receptors.
Overexpression of Akt inhibits NGF-induced growth arrest and neuronal differentiation of PC12 cells
Authors: Bang Ok-Sun , Park Eui Kyun , Yang Sung-Il , Lee Sun-Ryung , Franke Thomas F. , Kang Shin-Sung 
Published: April 2021

ABSTRACTTo investigate the role of Akt in nerve growth factor (NGF)-induced neuronal differentiation, PC12 cells ectopically expressing wild-type or dominant-inhibitory forms of Akt were analyzed. NGF-induced neurite outgrowth was greatly accelerated in cells expressing dominant-inhibitory Akt, compared to parental PC12 cells, but was almost completely blocked in cells expressing wild-type Akt. Since neuronal differentiation requires an arrest of cell growth, several aspects of cell growth of the different cell lines were compared. Cells expressing wild-type Akt were not susceptible to the growth-arresting effect of NGF, whereas parental PC12 cells and notably cells expressing mutant Akt were so affected. Accompanying this, the expressions of CDKs and p21WAF1 were down- and up-regulated, respectively, in both parental PC12 cells and cells expressing mutant Akt. When treated with some growth arrest-inducing agents such as sodium nitroprusside, forskolin and butyrolactone I, cells expressing wild-type Akt regained their responsiveness to the effects of NGF on differentiation. In summary, our results indicate that Akt overrides the growth-arresting effect of NGF and thereby, negatively regulates neuronal differentiation.
Nuclear localization of Ku antigen is promoted independently by basic motifs in the Ku70 and Ku80 subunits
Authors: Bertinato Jesse , Schild-Poulter Caroline , Haché Robert J. G. 
Published: April 2021

ABSTRACT
The Ku antigen is a heteromeric (Ku70/Ku80), mostly nuclear protein. Ku participates in multiple nuclear processes from DNA repair to V(D)J recombination to telomere maintenance to transcriptional regulation and serves as a DNA binding subunit and allosteric regulator of DNA-dependent protein kinase. While some evidence suggests that subcellular localization of Ku may be subject to regulation, how Ku gains access to the nucleus is poorly understood. In this work, using a combination of indirect immunofluorescence and direct fluorescence, we have demonstrated that transfer of the Ku heterodimer to the nucleus is determined by basic nuclear localization signals in each of the Ku subunits that function independently. A bipartite basic nuclear localization signal between amino acids 539-556 of Ku70 was observed to be required for nuclear import of full-length Ku70 monomer, while a short Ku80 motif of four amino acids from 565-568 containing three lysines was required for the nuclear import of full- length Ku80. Ku heterodimers containing only one nuclear localization signal accumulated in the nucleus as efficiently as wild-type Ku, while site directed mutagenesis inactivating the basic motifs in each subunit, resulted in a Ku heterodimer that was completely localized to the cytoplasm. Lastly, our results indicate that mutations in Ku previously proposed to abrogate Ku70/Ku80 heterodimerization, markedly reduced the accumulation of Ku70 without affecting heterodimer formation in mammalian cells.
Lamins in disease: why do ubiquitously expressed nuclear envelope proteins give rise to tissue-specific disease phenotypes?
Authors: Hutchison Christopher J. , Alvarez-Reyes Mauricio , Vaughan Owen A. 
Published: April 2021

ABSTRACT
The nuclear lamina is a filamentous structure composed of lamins that supports the inner nuclear membrane. Several integral membrane proteins including emerin, LBR, LAP1 and LAP2 bind to nuclear lamins in vitro and can influence lamin function and dynamics in vivo. Results from various studies suggest that lamins function in DNA replication and nuclear envelope assembly and determine the size and shape of the nuclear envelope. In addition, lamins also bind chromatin and certain DNA sequences, and might influence chromosome position. Recent evidence has revealed that mutations in A-type lamins give rise to a range of rare, but dominant, genetic disorders, including Emery-Dreifuss muscular dystrophy, dilated cardiomyopathy with conduction-system disease and Dunnigan-type familial partial lipodystrophy. An examination of how lamins A/C, emerin and other integral membrane proteins interact at the INM provides the basis for a novel model for how mutations that promote disease phenotypes are likely to influence these interactions and therefore cause cellular pathology through a combination of weakness of the lamina or altered gene expression.
Can’t clone, won’t clone: Essential Molecular Biology, Vol. 1, 2nd edn edited by T. A. Brown
Authors: Evans Sara K. , Lundblad Victoria 
Published: April 2021

ABSTRACT
I have heard it said (though not to my face) that practical molecular biology is somewhat akin to cookery, and I have to admit (though not to my fellow Molecular Biologists) that there is an element of truth in this. Of course, our ovens are cooler, and our pie dishes smaller, but often it is a case of mixing ingredients in the right proportions and baking at 37°C for an hour.
Cyclin specificity: how many wheels do you need on a unicycle?
Authors: Miller Mary E. , Cross Frederick R. 
Published: April 2021

ABSTRACT
Cyclin-dependent kinase (CDK) activity is essential for eukaryotic cell cycle events. Multiple cyclins activate CDKs in all eukaryotes, but it is unclear whether multiple cyclins are really required for cell cycle progression. It has been argued that cyclins may predominantly act as simple enzymatic activators of CDKs; in opposition to this idea, it has been argued that cyclins might target the activated CDK to particular substrates or inhibitors. Such targeting might occur through a combination of factors, including temporal expression, protein associations, and subcellular localization.
HEMCAM/CD146 downregulates cell surface expression of β1 integrins
Authors: Alais Sandrine , Allioli Nathalie , Pujades Cristina , Duband Jean-Loup , Vainio Olli , Imhof Beat A. , Dunon Dominique 
Published: April 2021

ABSTRACT
HEMCAM/gicerin, an immunoglobulin superfamily protein, is involved in homophilic and heterophilic adhesion. It interacts with NOF (neurite outgrowth factor), a molecule of the laminin family. Alternative splicing leads to mRNAs coding for HEMCAM with a short (HEMCAM-s) or a long cytoplasmic tail (HEMCAM-l). To investigate the cellular function of these two variants, we stably transfected murine fibroblasts with either form of HEMCAM. Expression of each isoform of this protein in L cells delayed proliferation and modified their adhesion properties to purified extracellular matrix proteins. Expression of either HEMCAM-s or HEMCAM-l inhibited integrin-dependent adhesion and spreading of fibroblasts to laminin 1, showing that this phenomenon did not depend on the cytoplasmic region. By contrast, L-cell adhesion and spreading to fibronectin depended on the HEMCAM isoform expressed. Flow cytometry and immunoprecipitation studies revealed that the expression of HEMCAM downregulated expression of the laminin-binding integrins α3β1, α6β1 and α7β1, and fibronectin receptor α5β1 from the cell surface. Semi-quantitative PCR and northern blot experiments showed that the expression of α6β1 integrin modified by HEMCAM occurred at a translation or maturation level. Thus, our data demonstrate that HEMCAM regulates fibroblast adhesion by controlling β1 integrin expression.
Movies available on-line: http://www.biologists.com/JCS/movies/jcs1886.html, movie 1A, movie 1B, movie 2A, movie 2B
Prolactin signalling to milk protein secretion but not to gene expression depends on the integrity of the Golgi region
Authors: Lkhider Mustapha , Pétridou Barbara , Aubourg Alain , Ollivier-Bousquet Michèle 
Published: April 2021

ABSTRACT
Prolactin added to the incubation medium of lactating mammary epithelial cells is transported from the basal to the apical region of cells through the Golgi region and concomitantly stimulates arachidonic acid release and protein milk secretion. We report that when PRL is added after disorganisation of the Golgi apparatus by brefeldin A treatment, prolactin signalling to expression of genes for milk proteins and prolactin endocytosis are not affected. However, prolactin transport to the apical region of cells (transcytosis), as well as prolactin-induced arachidonic acid release and subsequent stimulation of the secretion of caseins, which are located in a post-Golgi compartment, are inhibited. This inhibition was not a consequence of damage to the secretory machinery, as under the same conditions, protein secretion could be stimulated by the addition of arachidonic acid to the incubation medium. Thus, it is possible to discriminate between prolactin-induced actions that are dependent (signalling to milk protein secretion) or independent (signalling to milk gene expression) on the integrity of the Golgi apparatus. These results suggest that these two biological actions may be transduced via distinct intracellular pathways, and support the hypothesis that prolactin signals may be emitted at various cellular sites.
Depletion of rafts in late endocytic membranes is controlled by NPC1-dependent recycling of cholesterol to the plasma membrane
Authors: Lusa Sari , Blom Titta S. , Eskelinen Eeva-Liisa , Kuismanen Esa , Månsson Jan-Eric , Simons Kai , Ikonen Elina 
Published: April 2021

ABSTRACT
In mammalian cells, cholesterol is thought to associate with sphingolipids to form lateral membrane domains termed rafts. Increasing evidence suggests that rafts regulate protein interactions, for example, during signalling, intracellular transport and host-pathogen interactions. Rafts are present in cholesterol-sphingolipid-enriched membranes, including early and recycling endosomes, but whether rafts are found in late endocytic organelles has not been analyzed. In this study, we analyzed the association of cholesterol and late endosomal proteins with low-density detergent-resistant membranes (DRMs) in normal cells and in cells with lysosomal cholesterol-sphingolipid accumulation. In normal cells, the majority of [3H]cholesterol released from [3H]cholesterol ester-LDL associated with detergent-soluble membranes, was rapidly transported to the plasma membrane and became increasingly insoluble with time. In Niemann-Pick C1 (NPC1) protein-deficient lipidosis cells, the association of LDL-cholesterol with DRMs was enhanced and its transport to the plasma membrane was inhibited. In addition, the NPC1 protein was normally recovered in detergent-soluble membranes and its association with DRMs was enhanced by lysosomal cholesterol loading. Moreover, lysosomal cholesterol deposition was kinetically paralleled by the sequestration of sphingolipids and formation of multilamellar bodies in late endocytic organelles. These results suggest that late endocytic organelles are normally raft-poor and that endocytosed LDL-cholesterol is efficiently recycled to the plasma membrane in an NPC1-dependent process. The cholesterol-sphingolipid accumulation characteristic to NPC disease, and potentially to other sphingolipidoses, causes an overcrowding of rafts forming lamellar bodies in the degradative compartments.