Tension-sensitive kinetochore phosphorylation in vitro

1998 ◽  
Vol 111 (21) ◽  
pp. 3189-3196 ◽  
Author(s):  
R.B. Nicklas ◽  
M.S. Campbell ◽  
S.C. Ward ◽  
G.J. Gorbsky
Keyword(s):  
Mammalian Cells ◽  
Chemical Change ◽  
Protein S ◽  
Living Cells ◽  
Mechanical Tension ◽  
In Vitro System ◽  
Substrate Protein ◽  
Meiotic Chromosomes ◽  
System A

Many cells have a checkpoint that detects a single misattached chromosome and delays anaphase, allowing time for error correction. Detection probably depends on tension-sensitive kinetochore protein phosphorylation. Somehow, mechanical tension, or some consequence of tension, produces a chemical change, dephosphorylation. The mechanism of tension-mediated dephosphorylation can be approached using an in vitro system. Earlier work showed that the kinetochores of washed chromosomes from a mammalian cell line can be phosphorylated in vitro simply by incubation with ATP and a phosphatase inhibitor. We confirm this for chromosomes from insect meiotic cells. Thus, kinetochores of washed chromosomes from diverse sources contain a complete phosphorylation system: a kinase, a phosphatase and the substrate protein(s). We show that phosphorylation in vitro is sensitive to tension, as it is in living cells. This makes the conditions required for phosphorylation in vitro relevant to the process in living cells. The phosphatase is ruled out as the tension-sensitive component in vitro, leaving either the kinase or the substrate as the sensitive component. We show that a kinase extracted from mammalian cells in mitosis phosphorylates the kinetochores of insect meiotic chromosomes very effectively. The mammalian kinase under-phosphorylates the kinetochore of the insect's X-chromosome, just as the native insect kinase does. This provides a clue to the evolution of a chromosome that is not detected by the checkpoint. The mammalian kinase is not tightly bound to the chromosome and thus functions primarily in solution. This suggests that the substrate's phosphorylatable groups are freely available to outside constituents, e.g. regulators, as well as to the kinetochore's own kinase and phosphatase.

Effect of double-strand breaks on homologous recombination in mammalian cells and extracts

1985 ◽  
Vol 5 (12) ◽  
pp. 3331-3336
Author(s):  
K Y Song ◽  
L Chekuri ◽  
S Rauth ◽  
S Ehrlich ◽  
R Kucherlapati
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Recombination Frequency ◽  
Double Strand Breaks ◽  
Base Pairs ◽  
In Vitro System ◽  
Strand Breaks ◽  
Cell Extracts ◽  
Nuclear Extracts

We examined the effect of double-strand breaks on homologous recombination between two plasmids in human cells and in nuclear extracts prepared from human and rodent cells. Two pSV2neo plasmids containing nonreverting, nonoverlapping deletions were cotransfected into cells or incubated with cell extracts. Generation of intact neo genes was monitored by the ability of the DNA to confer G418r to cells or Neor to bacteria. We show that double-strand breaks at the sites of the deletions enhanced recombination frequency, whereas breaks outside the neo gene had no effect. Examination of the plasmids obtained from experiments involving the cell extracts revealed that gene conversion events play an important role in the generation of plasmids containing intact neo genes. Studies with plasmids carrying multiple polymorphic genetic markers revealed that markers located within 1,000 base pairs could be readily coconverted. The frequency of coconversion decreased with increasing distance between the markers. The plasmids we constructed along with the in vitro system should permit a detailed analysis of homologous recombinational events mediated by mammalian enzymes.

scholarly journals Loss of Heterochromatin Protein 1 (HP1) chromodomain function in mammalian cells by intracellular antibodies causes cell death

2002 ◽  
Vol 115 (9) ◽  
pp. 1803-1813 ◽  
Author(s):  
Ilaria Filesi ◽  
Alessio Cardinale ◽  
Sjaak van der Sar ◽  
Ian G. Cowell ◽  
Prim B. Singh ◽  
...  
Keyword(s):  
Cell Death ◽  
Mammalian Cells ◽  
Wide Spectrum ◽  
Human Fibroblasts ◽  
Protein S ◽  
Single Chain ◽  
Heterochromatin Protein 1 ◽  
Apoptotic Pathway

The chromodomain (CD) is a highly conserved motif present in a variety of animal and plant proteins, and its probable role is to assemble a variety of macromolecular complexes in chromatin. The importance of the CD to the survival of mammalian cells has been tested. Accordingly, we have ablated CD function using two single-chain intracellular Fv (scFv) fragments directed against non-overlapping epitopes within the HP1 CD motif. The scFv fragments can recognize both CD motifs of HP1 and Polycomb (Pc) in vitro and, when expressed intracellularly, interact with and dislodge the HP1 protein(s) from their heterochromatin localization in vivo. Mouse and human fibroblasts expressing anti-chromodomain scFv fragments show a cell-lethal phenotype and an apoptotic morphology becomes apparent soon after transfection. The mechanism of cell death appears to be p53 independent, and the cells are only partly rescued by incubation with the wide spectrum caspase inhibitor Z-VAD fmk. We conclude that expression of anti-chromodomain intracellular antibodies is sufficient to trigger a p53-independent apoptotic pathway that is only partly dependent on the known Z-VAD-inhibitable caspases, suggesting that CD function is essential for cell survival.

scholarly journals Function of Protein S-Palmitoylation in Immunity and Immune-Related Diseases

2021 ◽  
Vol 12 ◽  
Author(s):  
Yuqi Zhang ◽  
Ziran Qin ◽  
Wenhuan Sun ◽  
Feng Chu ◽  
Fangfang Zhou
Keyword(s):  
Protein Interactions ◽  
Protein Function ◽  
Mammalian Cells ◽  
Large Body ◽  
Protein S ◽  
Cellular Membrane ◽  
Immune Signaling ◽  
Substrate Protein ◽  
Associated Proteins ◽  
Immunologic Diseases

Protein S-palmitoylation is a covalent and reversible lipid modification that specifically targets cysteine residues within many eukaryotic proteins. In mammalian cells, the ubiquitous palmitoyltransferases (PATs) and serine hydrolases, including acyl protein thioesterases (APTs), catalyze the addition and removal of palmitate, respectively. The attachment of palmitoyl groups alters the membrane affinity of the substrate protein changing its subcellular localization, stability, and protein-protein interactions. Forty years of research has led to the understanding of the role of protein palmitoylation in significantly regulating protein function in a variety of biological processes. Recent global profiling of immune cells has identified a large body of S-palmitoylated immunity-associated proteins. Localization of many immune molecules to the cellular membrane is required for the proper activation of innate and adaptive immune signaling. Emerging evidence has unveiled the crucial roles that palmitoylation plays to immune function, especially in partitioning immune signaling proteins to the membrane as well as to lipid rafts. More importantly, aberrant PAT activity and fluctuations in palmitoylation levels are strongly correlated with human immunologic diseases, such as sensory incompetence or over-response to pathogens. Therefore, targeting palmitoylation is a novel therapeutic approach for treating human immunologic diseases. In this review, we discuss the role that palmitoylation plays in both immunity and immunologic diseases as well as the significant potential of targeting palmitoylation in disease treatment.

scholarly journals Tension-driven multi-scale self-organisation in human iPSC-derived muscle fibers

2021 ◽  
Author(s):  
Qiyan Mao ◽  
Achyuth Acharya ◽  
Alejandra Rodriguez-delaRosa ◽  
Fabio Marchiano ◽  
Benoit Dehapiot ◽  
...  
Keyword(s):  
Induced Pluripotent Stem Cell ◽  
Human Muscle ◽  
Mechanical Tension ◽  
In Vitro System ◽  
Multi Scale ◽  
Self Organisation ◽  
Induced Pluripotent ◽  
Sarcomere Assembly ◽  
Human Ipsc

Human muscle is a hierarchically organised tissue with its contractile cells called myofibers packed into large myofiber bundles. Each myofiber contains periodic myofibrils built by hundreds of contractile sarcomeres that generate large mechanical forces. To better understand the mechanisms that coordinate human muscle morphogenesis from tissue to molecular scales, we adopted a simple in vitro system using induced pluripotent stem cell-derived human myogenic precursors. When grown on an unrestricted two-dimensional substrate, developing myofibers spontaneously align and self-organise into higher-order myofiber bundles, which grow and consolidate to stable sizes. Following a transcriptional boost of sarcomeric components, myofibrils assemble into chains of periodic sarcomeres that emerge across the entire myofiber. By directly probing tension we found that tension build-up precedes sarcomere assembly and increases within each assembling myofibril. Furthermore, we found that myofiber ends stably attach to other myofibers using integrin-based attachments and thus myofiber bundling coincides with stable myofiber bundle attachment in vitro. A failure in stable myofiber attachment results in a collapse followed by a disassembly of the myofibrils. Overall, our results strongly suggest that mechanical tension across sarcomeric components as well as between differentiating myofibers is key to coordinate the multi-scale self-organisation of muscle morphogenesis.

Disruption of Golgi structure and function in mammalian cells expressing a mutant dynamin

2000 ◽  
Vol 113 (11) ◽  
pp. 1993-2002 ◽  
Author(s):  
H. Cao ◽  
H.M. Thompson ◽  
E.W. Krueger ◽  
M.A. McNiven
Keyword(s):  
Mammalian Cells ◽  
Living Cells ◽  
Dominant Negative ◽  
Coated Vesicle ◽  
Large Numbers ◽  
Golgi Structure ◽  
And Function ◽  
Golgi Network ◽  
Mutant Cells

The large GTPase dynamin is a mechanoenzyme that participates in the scission of nascent vesicles from the plasma membrane. Recently, dynamin has been demonstrated to associate with the Golgi apparatus in mammalian cells by morphological and biochemical methods. Additional studies using a well characterized, cell-free assay have supported these findings by demonstrating a requirement for dynamin function in the formation of clathrin-coated, and non-clathrin-coated vesicles from the trans-Golgi network (TGN). In this study, we tested if dynamin participates in Golgi function in living cells through the expression of a dominant negative dynamin construct (K44A). Cells co-transfected to express this mutant dynamin and a GFP-tagged Golgi resident protein (TGN38) exhibit Golgi structures that are either compacted, vesiculated, or tubulated. Electron microscopy of these mutant cells revealed large numbers of Golgi stacks comprised of highly tubulated cisternae and an extraordinary number of coated vesicle buds. Cells expressing mutant dynamin and GFP-tagged VSVG demonstrated a marked retention (8- to 11-fold) of the nascent viral G-protein in the Golgi compared to control cells. These observations in living cells are consistent with previous morphological and in vitro studies demonstrating a role for dynamin in the formation of secretory vesicles from the TGN.

scholarly journals 70-kD heat shock-related protein is one of at least two distinct cytosolic factors stimulating protein import into mitochondria.

1988 ◽  
Vol 107 (6) ◽  
pp. 2051-2057 ◽  
Author(s):  
H Murakami ◽  
D Pain ◽  
G Blobel
Keyword(s):  
Heat Shock ◽  
Matrix Protein ◽  
Protein Import ◽  
Protein S ◽  
Yeast Mitochondria ◽  
Related Protein ◽  
Free System ◽  
In Vitro System ◽  
Cytosolic Factors

We have developed an in vitro system in which the posttranslational import of Put2 (delta-pyrroline-5-carboxylate dehydrogenase), into yeast mitochondria is dependent on the addition of yeast postribosomal supernatant (PRS). When mRNA for a nuclear-encoded yeast mitochondrial matrix protein, Put2, was translated in a wheat germ cell-free system, import into posttranslationally added yeast mitochondria was negligible. However, when a yeast PRS was added, significant import was observed. The import stimulating activity of the yeast PRS was shown to consist of at least two distinct factors. One of these is the recently purified 70-kD heat shock-related protein Ssalp/Ssa2p, two proteins that are 98% homologous. The other factor is an N-ethylmaleimide-sensitive protein(s). Both factors act synergistically.

scholarly journals Effect of double-strand breaks on homologous recombination in mammalian cells and extracts.

1985 ◽  
Vol 5 (12) ◽  
pp. 3331-3336 ◽  
Author(s):  
K Y Song ◽  
L Chekuri ◽  
S Rauth ◽  
S Ehrlich ◽  
R Kucherlapati
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Recombination Frequency ◽  
Double Strand Breaks ◽  
Base Pairs ◽  
In Vitro System ◽  
Strand Breaks ◽  
Cell Extracts ◽  
Nuclear Extracts

We examined the effect of double-strand breaks on homologous recombination between two plasmids in human cells and in nuclear extracts prepared from human and rodent cells. Two pSV2neo plasmids containing nonreverting, nonoverlapping deletions were cotransfected into cells or incubated with cell extracts. Generation of intact neo genes was monitored by the ability of the DNA to confer G418r to cells or Neor to bacteria. We show that double-strand breaks at the sites of the deletions enhanced recombination frequency, whereas breaks outside the neo gene had no effect. Examination of the plasmids obtained from experiments involving the cell extracts revealed that gene conversion events play an important role in the generation of plasmids containing intact neo genes. Studies with plasmids carrying multiple polymorphic genetic markers revealed that markers located within 1,000 base pairs could be readily coconverted. The frequency of coconversion decreased with increasing distance between the markers. The plasmids we constructed along with the in vitro system should permit a detailed analysis of homologous recombinational events mediated by mammalian enzymes.

In Vitro Characterisation of Two Naturally Occurring Mutations in the Thrombin-sensitive Region of Anticoagulant Protein S

1999 ◽  
Vol 82 (12) ◽  
pp. 1627-1633 ◽  
Author(s):  
Tusar Giri ◽  
Pablo García de Frutos ◽  
Tomio Yamazaki ◽  
Bruno Villoutreix ◽  
Björn Dahlbäck
Keyword(s):  
Molecular Modelling ◽  
Mammalian Cells ◽  
Recombinant Protein Expression ◽  
3D Structure ◽  
Protein S ◽  
Site Directed Mutagenesis ◽  
Type I ◽  
Sensitive Region ◽  
Naturally Occurring

SummaryThe molecular consequences of two naturally occurring mutations in the thrombin-sensitive region of protein S were investigated using a combination of recombinant protein expression, functional analysis and molecular modelling. Both mutations (R49H and R70S) have been found in thrombosis patients diagnosed as having type I protein S deficiency. Molecular modelling analysis suggested the R49H substitution not to disrupt the structure of thrombin-sensitive region, whereas the R70S substitution could affect the 3D structure mildly. To elucidate the molecular consequences of these substitutions experimentally, site directed mutagenesis of protein S cDNA and expression in mammalian cells created the two mutants. The secretion profiles and functional anticoagulant activities of the protein S mutants were characterised. Secretion of the R49H mutant was similar to that of wild type protein S, whereas the R70S mutant showed moderately decreased expression. Neither of the mutants showed any major functional defects as cofactors to activated protein C (APC) in an APTT-based assay or in degradation of factor Va. However, both mutants demonstrated decreased activity in a factor VIIIa degradation assay, which in addition to APC and protein S also included factor V as synergistic APC cofactor. In conclusion, the R49H substitution did not produce a quantitative abnormality in vitro, raising doubts as to whether it caused the type I deficiency. In contrast, the experimental data obtained for the R70S mutant agrees well with the observed type I deficiency. Our study illustrates that in vitro experimental characterisation together with computer-based structural analysis are useful tools in the analysis of the relationship between naturally occurring mutations and clinical phenotypes.

scholarly journals A Fusion Peptide in the Spike Protein of MERS Coronavirus

Viruses ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 825 ◽  
Author(s):  
Entedar A. J. Alsaadi ◽  
Benjamin W. Neuman ◽  
Ian M. Jones
Keyword(s):  
Mammalian Cells ◽  
Middle Eastern ◽  
Membrane Binding ◽  
Protein S ◽  
Syncytium Formation ◽  
Fusion Peptide ◽  
Amino Acid Replacement ◽  
Spike Protein ◽  
S Protein

Coronaviruses represent current and emerging threats for many species, including humans. Middle East respiratory syndrome-related coronavirus (MERS-CoV) is responsible for sporadic infections in mostly Middle Eastern countries, with occasional transfer elsewhere. A key step in the MERS-CoV replication cycle is the fusion of the virus and host cell membranes mediated by the virus spike protein, S. The location of the fusion peptide within the MERS S protein has not been precisely mapped. We used isolated peptides and giant unilamellar vesicles (GUV) to demonstrate membrane binding for a peptide located near the N-terminus of the S2 domain. Key residues required for activity were mapped by amino acid replacement and their relevance in vitro tested by their introduction into recombinant MERS S protein expressed in mammalian cells. Mutations preventing membrane binding in vitro also abolished S-mediated syncytium formation consistent with the identified peptide acting as the fusion peptide for the S protein of MERS-CoV.

Protein S Thr103Asn Mutation Associated with Type II Deficiency Reproduced In Vitro and Functionally Characterised

2000 ◽  
Vol 84 (09) ◽  
pp. 413-419 ◽  
Author(s):  
Tusar Giri ◽  
Pablo de Frutos ◽  
Björn Dahlbäck
Keyword(s):  
Mammalian Cells ◽  
Anticoagulant Activity ◽  
Protein S ◽  
Mutant Protein ◽  
Type Ii ◽  
Wild Type ◽  
Type Protein ◽  
Wild Type Protein ◽  
Functional Defect

SummaryProtein S functions as a cofactor to activated protein C (APC) in the degradation of FVa and FVIIIa. In protein S, the thrombin sensitive region (TSR) and the first EGF-like domain are important for expression of the APC cofactor activity. A naturally occurring Thr103Asn (T103N) mutation in the first EGF-like domain of protein S has been associated with functional (type II) protein S deficiency. To elucidate the functional consequences of the T103N mutation, recombinant protein S mutant was expressed in mammalian cells and functionally characterised. The expression level of protein S T103N from transiently transfected COS 1 cells was equal to that of wild type protein S. The mutant protein S and wild type protein S were also expressed in 293 cells after stable transfection, and the recombinant proteins purified. In APTT-and PT-based coagulation assays, the mutant protein demonstrated approximately 50% lower anticoagulant activity as compared to wild type protein S. The functional defect was further investigated in FVa-and FVIIIa-degradation assays. The functional defect of mutant protein S was attenuated at increasing concentrations of APC. The results demonstrate the region around residue 103 of protein S to be of functional importance, possibly through a direct interaction with APC.

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