Abstract：
:Actins are major eukaryotic cytoskeletal proteins, and they are involved in many important cell functions, including cell division, cell polarity, wound healing and muscle contraction. Despite obvious drawbacks, muscle actin, which is easily purified, is used extensively for biochemical studies of the non-muscle actin cytoskeleton. Here, we report a rapid and cost-effective method to purify heterologous actins expressed in the yeast Pichia pastoris Actin is expressed as a fusion with the actin-binding protein thymosin β4 and purified by means of an affinity tag introduced in the fusion. Following cleavage of thymosin β4 and the affinity tag, highly purified functional full-length actin is liberated. We purify actins from Saccharomycescerevisiae and Schizosaccharomycespombe, and the β- and γ-isoforms of human actin. We also report a modification of the method that facilitates expression and purification of arginylated actin, a form of actin thought to regulate dendritic actin networks in mammalian cells. The methods we describe can be performed in all laboratories equipped for molecular biology, and should greatly facilitate biochemical and cell biological studies of the actin cytoskeleton.
journal_name
J Cell Scijournal_title
Journal of cell scienceauthors
Hatano T,Alioto S,Roscioli E,Palani S,Clarke ST,Kamnev A,Hernandez-Fernaud JR,Sivashanmugam L,Chapa-Y-Lazo B,Jones AME,Robinson RC,Sampath K,Mishima M,McAinsh AD,Goode BL,Balasubramanian MKdoi
10.1242/jcs.213827subject
Has Abstractpub_date
2018-04-23 00:00:00issue
8eissn
0021-9533issn
1477-9137pii
jcs.213827journal_volume
131pub_type
杂志文章abstract：:The coordinated trafficking and tethering of membrane cargo within cells relies on the function of distinct cytoskeletal motors that are targeted to specific subcellular compartments through interactions with protein adaptors and phospholipids. The unique actin motor myosin VI functions at distinct steps during clathr...
journal_title：Journal of cell science
pub_type： 杂志文章,评审
doi：10.1242/jcs.095554
更新日期：2013-06-15 00:00:00
abstract：:Mutations within connexin50 (Cx50) have been linked to various cataract phenotypes. To determine the mechanism behind cataract formation we used the paired Xenopus oocyte system in conjunction with transfected HeLa cells and genetically engineered mouse models to examine the functional characteristics of gap junctions...
journal_title：Journal of cell science
pub_type： 杂志文章
doi：10.1242/jcs.012237
更新日期：2007-12-01 00:00:00
abstract：:Deubiquitylating (or deubiquitinating) enzymes (DUBs) are proteases that reverse protein ubiquitylation and therefore modulate the outcome of this post-translational modification. DUBs regulate a variety of intracellular processes, including protein turnover, signalling pathways and the DNA damage response. They have ...
journal_title：Journal of cell science
pub_type： 杂志文章,评审
doi：10.1242/jcs.201855
更新日期：2017-06-15 00:00:00
abstract：:Macroautophagy is a dynamic process whereby portions of the cytosol are encapsulated in double-membrane vesicles and delivered to the lysosome for degradation. Phosphatidylinositol-3-phosphate (PtdIns3P) is concentrated on autophagic vesicles and recruits effector proteins that are crucial for this process. The produc...
journal_title：Journal of cell science
pub_type： 杂志文章
doi：10.1242/jcs.080341
更新日期：2011-03-01 00:00:00
abstract：:Survivin, a human inhibitor of apoptosis protein (IAP), plays an important role in both cell cycle regulation and inhibition of apoptosis. Survivin is expressed in cells during the G(2)/M phase of the cell cycle, followed by rapid decline of both mRNA and protein levels at the G(1) phase. It has been suggested that ce...
journal_title：Journal of cell science
pub_type： 杂志文章
doi：
更新日期：2000-12-01 00:00:00
abstract：:Nematocysts are explosive organelles found in all Cnidaria. Explosion of nematocyst capsules is driven by the high pressure within the capsule formed by the high concentration of poly-gamma-glutamate in the capsule matrix. Poly-gamma-glutamate is a polyanion that binds cations tightly, including the fluorescent cation...
journal_title：Journal of cell science
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doi：
更新日期：2002-02-15 00:00:00
abstract：:Endothelial cell-based angiogenesis requires activation of survival signals that generate resistance to external apoptotic stimuli, such as tumor necrosis factor-alpha (TNF-alpha), during pathobiologic settings. Mechanisms by which this is achieved are not fully defined. Here, we use a model in which the multifunction...
journal_title：Journal of cell science
pub_type： 杂志文章
doi：10.1242/jcs.03361
更新日期：2007-02-01 00:00:00
abstract：:In somatic cells, the heterodimeric Structural Maintenance of Chromosomes (SMC) proteins are involved in chromosome condensation and gene dosage compensation (SMC2 and 4), and sister chromatid cohesion and DNA recombination (SMC1 and 3). We report here evidence for an involvement of mammalian SMC1 and SMC3 proteins in...
journal_title：Journal of cell science
pub_type： 杂志文章
doi：
更新日期：2000-02-01 00:00:00
abstract：:Microinjected GTP gamma S revealed three distinct steps in the exocytic transport of the temperature sensitive glycoprotein of vesicular stomatitis virus (ts-O45-G) from the ER to the cell surface in intact Vero cells. While COPII dependent export of ts-O45-G from the ER is blocked in cells injected with recombinant p...
journal_title：Journal of cell science
pub_type： 杂志文章
doi：
更新日期：1998-07-01 00:00:00
abstract：:We have shown previously, by fluorescent antibody staining, that desmosomal antigens are widely distributed in the tissues of vertebrate animals. Furthermore, we have demonstrated mutual desmosome formation between cells derived from man, cow, dog, chicken and frog. In this paper we have studied the components of desm...
journal_title：Journal of cell science
pub_type： 杂志文章
doi：
更新日期：1986-03-01 00:00:00
abstract：:The repair of point mutations can be directed by modified single-stranded DNA oligonucleotides and regulated by cellular activities including homologous recombination, mismatch repair and transcription. Now, we report that DNA replication modulates the gene repair process by influencing the frequency with which either...
journal_title：Journal of cell science
pub_type： 杂志文章
doi：10.1242/jcs.01250
更新日期：2004-08-01 00:00:00
abstract：:How cell fate becomes restricted during somatic cell differentiation is a long-lasting question in biology. Epigenetic mechanisms not present in pluripotent cells and acquired during embryonic development are expected to stabilize the differentiated state of somatic cells and thereby restrict their ability to convert ...
journal_title：Journal of cell science
pub_type： 杂志文章
doi：10.1242/jcs.113019
更新日期：2012-12-15 00:00:00
abstract：:Direct-current electric fields mediate motility (galvanotaxis) of many cell types. In 3T3 fibroblasts, electric fields increased the proportion, speed and cathodal directionality of motile cells. Analogous to fibroblasts' spontaneous migration, we initially hypothesized that reorientation of microtubule components mod...
journal_title：Journal of cell science
pub_type： 杂志文章
doi：10.1242/jcs.00986
更新日期：2004-03-15 00:00:00
abstract：:Drosophila abnormal spindle (asp) mutants exhibit a mitotic metaphase checkpoint arrest with abnormal spindle poles, which reflects a requirement for Asp for the integrity of microtubule organising centres (MTOCs). In male meiosis, the absence of a strong spindle integrity checkpoint enables asp mutant cells to procee...
journal_title：Journal of cell science
pub_type： 杂志文章
doi：
更新日期：2002-03-01 00:00:00
abstract：:The alpha6beta4 integrin is located at the basal surface of keratinocytes, in hemidesmosomal structures that mediate stable adhesion of epidermal cells to the underlying basement membrane component laminin-5. The absence of alpha6beta4 integrin causes junctional epidermolysis bullosa, a severe blistering disease of th...
journal_title：Journal of cell science
pub_type： 杂志文章
doi：10.1242/jcs.01689
更新日期：2005-03-01 00:00:00
abstract：:During muscle development myosin molecules form symmetrical thick filaments, which integrate with the thin filaments to produce the regular sarcomeric lattice. In Drosophila indirect flight muscles (IFMs) the details of this process can be studied using genetic approaches. The weeP26 transgenic line has a GFP-encoding...
journal_title：Journal of cell science
pub_type： 杂志文章
doi：10.1242/jcs.110361
更新日期：2013-01-01 00:00:00
abstract：:Feedback mechanisms preventing M phase occurrence before S phase completion are assumed to depend on inhibition of cyclin B-cdc2 kinase activation by unreplicated DNA. In sea urchin, fertilization stimulates protein synthesis and releases eggs from G1 arrest. We found that in the one-cell sea urchin embryo cyclin B-cd...
journal_title：Journal of cell science
pub_type： 杂志文章
doi：
更新日期：1995-07-01 00:00:00
abstract：:We have previously shown that Xenopus oocytes, eggs, and early embryos contain lamins LII and LIII, and that portions of each are associated with distinct egg vesicle populations. We now report that a lamin similar or identical to the B-type lamin LI is also present in oocyte nuclei and in egg extracts. We have quanti...
journal_title：Journal of cell science
pub_type： 杂志文章
doi：
更新日期：1996-07-01 00:00:00
abstract：:The inclusion of lymphocytes in high endothelial venules and their migration to the lymph nodes are critical steps in the immune response. Cell migration is regulated by the actin cytoskeleton and myosins. Myo1e is a long-tailed class I myosin and is highly expressed in B cells, which have not been studied in the cont...
journal_title：Journal of cell science
pub_type： 杂志文章
doi：10.1242/jcs.235275
更新日期：2020-03-02 00:00:00
abstract：:Epidermal growth factor receptor (EGFR) ligands undergo a complex series of processing events during their maturation to active signaling proteins. Like its mammalian homologs, the predominant Drosophila EGFR ligand Spitz is produced as a transmembrane pro-protein. In the secretory pathway, Spitz is cleaved within its...
journal_title：Journal of cell science
pub_type： 杂志文章
doi：10.1242/jcs.131169
更新日期：2013-10-01 00:00:00
abstract：:Single cell clones were isolated from the human teratoma line, Tera-2. The cells of three of these clones were studied. The progressively growing cells were shown to be tumorigenic, and they were characterized by the lack of expression of beta 2-microglobulin and HLA-A,B,C determinants on the cell surface. The majorit...
journal_title：Journal of cell science
pub_type： 杂志文章
doi：
更新日期：1984-12-01 00:00:00
abstract：:During vertebrate gastrulation, a well-orchestrated series of morphogenetic changes leads to the formation of the three germ layers: the ectoderm, mesoderm and endoderm. The analysis of gene expression patterns during gastrulation has been central to the identification of genes involved in germ layer formation. Howeve...
journal_title：Journal of cell science
pub_type： 杂志文章
doi：10.1242/jcs.02928
更新日期：2006-05-15 00:00:00
abstract：:Human procathepsin D carries two N-linked glycosylation sites at asparagine residues 70 and 199, widely separated on the surface of the folded protein. We created monoglycosylated procathepsin D molecules by site-directed mutagenesis in vitro of the individual glycosylation sites. With only two exceptions, all 12 muta...
journal_title：Journal of cell science
pub_type： 杂志文章
doi：
更新日期：1995-05-01 00:00:00
abstract：:The neuroprotective role of carbon monoxide (CO) has been studied in a cell-autonomous mode. Herein, a new concept is disclosed - CO affects astrocyte-neuron communication in a paracrine manner to promote neuroprotection. Neuronal survival was assessed when co-cultured with astrocytes that had been pre-treated or not ...
journal_title：Journal of cell science
pub_type： 杂志文章
doi：10.1242/jcs.187260
更新日期：2016-08-15 00:00:00
abstract：:The cornified cell envelope (CE), a structure formed in the outermost layers of stratified squamous epithelia, provides a physical barrier against environmental insults. It is composed of several structural proteins, which are irreversibly crosslinked by calcium-activated transglutaminases. The small proline rich prot...
journal_title：Journal of cell science
pub_type： 杂志文章
doi：
更新日期：2001-11-01 00:00:00
abstract：:Small latent TGF-beta consists of latency associated peptide (LAP) bound to the 25 kDa TGF-beta by noncovalent interactions. Small latent TGF-beta is secreted from cells and deposited into the extracellular matrix as covalent complexes with its binding proteins, LTBPs. Four LTBPs have been molecularly cloned and their...
journal_title：Journal of cell science
pub_type： 杂志文章
doi：
更新日期：2001-08-01 00:00:00
abstract：:The 5T4 oncofoetal antigen is a 72 kDa glycoprotein defined by a monoclonal antibody raised against human placental trophoblast and is expressed in many different carcinomas but detected only at low levels in some normal epithelia. Immunohistochemical analysis of the patterns of expression in colorectal carcinomas has...
journal_title：Journal of cell science
pub_type： 杂志文章
doi：
更新日期：1995-08-01 00:00:00
abstract：:The secreted Frizzled-related proteins (SFRPs) are a family of soluble proteins that are structurally related to Frizzled (Fz) proteins, the serpentine receptors that mediate the extensively used cell-cell communication pathway involving Wnt signalling. Because of their homology with the Wnt-binding domain on the Fz r...
journal_title：Journal of cell science
pub_type： 杂志文章,评审
doi：10.1242/jcs.026096
更新日期：2008-03-15 00:00:00
abstract：:Sister chromatid cohesion is essential for proper segregation of the genome in mitosis and meiosis. Central to this process is cohesin, a multi-protein complex conserved from yeast to human. Previous genetic studies in fungi have identified Pds5/BimD/Spo76 as an additional factor implicated in cohesion. Here we descri...
journal_title：Journal of cell science
pub_type： 杂志文章
doi：10.1242/jcs.02355
更新日期：2005-05-15 00:00:00
abstract：:Cells that are treated long-term with TNFalpha or short-term with TGFalpha together with cycloheximide (CHX) undergo apoptosis. Cell shrinkage and detachment during apoptosis is dependent on actomyosin contractility. Myosin II heavy chain (MHCII) isoforms have shared and distinct functions. Here, we investigated wheth...
journal_title：Journal of cell science
pub_type： 杂志文章
doi：10.1242/jcs.022640
更新日期：2008-05-15 00:00:00
