scholarly journals Re-expression of ABP-120 rescues cytoskeletal, motility, and phagocytosis defects of ABP-120- Dictyostelium mutants.

1996 ◽  
Vol 7 (5) ◽  
pp. 803-823 ◽  
Author(s):  
D Cox ◽  
D Wessels ◽  
D R Soll ◽  
J Hartwig ◽  
J Condeelis
Keyword(s):  
Homologous Recombination ◽  
Cell Line ◽  
Cell Lines ◽  
Binding Proteins ◽  
Chemical Mutagenesis ◽  
Actin Binding ◽  
Direct Result ◽  
Cross Linking ◽  
Wild Type ◽  
Actin Binding Proteins

The actin binding protein ABP-120 has been proposed to cross-link actin filaments in nascent pseudopods, in a step required for normal pseudopod extension in motile Dictyostelium amoebae. To test this hypothesis, cell lines that lack ABP-120 were created independently either by chemical mutagenesis or homologous recombination. Different phenotypes were reported in these two studies. The chemical mutant shows only a subtle defect in actin cross-linking, while the homologous recombinant mutants show profound defects in actin cross-linking, cytoskeletal structure, pseudopod number and size, cell motility and chemotaxis and, as shown here, phagocytosis. To resolve the controversy as to what the ABP-120- phenotype is, ABP-120 was re-expressed in an ABP-120- cell line created by homologous recombination. Two independently "rescued" cell lines that express wild-type levels of ABP-120 were analyzed. In both rescued cell lines, actin incorporation into the cytoskeleton, pseudopod formation, cell morphology, instantaneous velocity, phagocytosis, and chemotaxis were restored to wild-type levels. There is no alteration in the expression levels of several related actin binding proteins in either the original ABP-120- cell line or in the rescued cell lines, leading to the conclusion that neither the aberrant phenotype observed in ABP-120- cells nor the normal phenotype reasserted in rescued cells can be attributed to alterations in the levels of other abundant and related actin binding proteins. Re-expression of ABP-120 in ABP-120- cells reestablishes normal structural and behavioral parameters, demonstrating that the severity and properties of the structural and behavioral defects of ABP-120- cell lines produced by homologous recombination are the direct result of the absence of ABP-120.

scholarly journals Actin Cross-Linking Toxin Is a Universal Inhibitor of Tandem-Organized and Oligomeric G-Actin Binding Proteins

2018 ◽  
Vol 28 (10) ◽  
pp. 1536-1547.e9 ◽  
Author(s):  
Elena Kudryashova ◽  
David B. Heisler ◽  
Blake Williams ◽  
Alyssa J. Harker ◽  
Kyle Shafer ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Actin Binding ◽  
Cross Linking ◽  
Actin Binding Proteins

AcornHRD: A novel tool to identify homologous recombination deficiency events in whole genome sequencing data.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. e15078-e15078
Author(s):  
Kai Liu ◽  
Xueyu Hao ◽  
Mengmeng Zhang ◽  
Mingwei Li ◽  
Wang Wang ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Ovarian Cancer ◽  
Homologous Recombination ◽  
Whole Genome Sequencing ◽  
Cell Line ◽  
Cell Lines ◽  
Genome Sequencing ◽  
Whole Genome ◽  
Wild Type ◽  
Homologous Recombination Deficiency

e15078 Background: Recently, homologous recombination deficiency (HRD) scores are associated with the efficacy of Poly‐(ADP‐Ribose)‐Polymerase (PARP) inhibition and platinum-based chemotherapy in a variety of cancers. Evaluating HRD level in patients with cancers is becoming far more important and influential, so far, there is no standard method to be used in clinical. In this study, we developed an algorithm to detect HRD from next-generation sequencing (NGS) for finding additional patients may potentially benefit from target therapy. Methods: Forty-eight patients were enrolled, including breast cancer, ovarian cancer, prostatic cancer. Fifteen cell lines with breast cancer and endometrial carcinoma were collected from Cobioer biosciences co., LTD. Forty-eight Formalin-fixed, paraffin embedded (FFPE) samples and 15 cell lines were performed by DNA extracting. We developed an HRD score algorithm, termed as AcornHRD algorithm. HRD score was analyzed by whole-genome sequencing, and GATK mutect2 software was used to detect BRCA1/2mutation by deep sequencing. Results: BRCA1/2 deleterious mutations were observed in 20 patients (41.7%). HRD was explained by deficiencies in 17 patients (85.0%) with BRCA mutation, whereas eight HRD-high tumors were non- BRCA related (28.6%). Among BRCA wild-type patients, the corresponding percentage of HRD positive patients in breast cancer, ovarian cancer and prostate cancer were 36.3%, 37.5% and 11.1%, respectively. Similar results were also verified in the cell line datasets. The findings showed that 100% (3/3) BRCA1/2 deficient cell lines are also HRD-high. Furthermore, HRD scores were highly correlated with standard results in the cell line datasets. Conclusions: We here report the NGS-based HRD scores to distinguish similarly well between BRCA mutant and BRCA wild-type cases in a cohort of Chinese population. AcornHRD scores were highly associated with BRCA1/2 deficiency. AcornHRD algorithm can be a useful tool to detect HRD events in clinical settings.

Loss of PIP5KIβ Causes a Defect in Lamellipodia Formation and Shear Resistant Adhesion.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 141-141
Author(s):  
Xinsheng Chen ◽  
Yanfeng Wang ◽  
Edward K. Williamson ◽  
Timothy J. Stalker ◽  
Lawrence F. Brass ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Ex Vivo ◽  
Intracellular Localization ◽  
Second Messenger ◽  
Arterial System ◽  
Actin Binding ◽  
Wild Type ◽  
Differential Interference Contrast Microscopy ◽  
Actin Binding Proteins ◽  
Lamellipodia Formation

Abstract Phosphatidylinositol 4,5-bisphosphate (PIP2) is widely known for the production of lipid second messengers after its hydrolysis by phospholipase C or phosphorylation by phosphatidylinositol 3-kinase. PIP2 also regulates cytoskeletal dynamics by directly interacting with actin-binding proteins. Three isoforms of PIP5KI (α, β, and γ) are all capable of phosphorylating PI4P to synthesize PIP2. However, these isoforms have different primary structures, expression levels in various tissues, and intracellular localization. Our previous studies have demonstrated that PIP5KIβ and PIP5KIγ are the dominant isoforms present in platelets. We generated and bred mice heterozygous for a null mutation into the murine PIP5KIβ gene, and crossed these mice to determine the phenotype of mice lacking this protein. PIP5KIβ-null mice were born, appeared developmentally normal, had normal platelet counts, and exhibited no spontaneous hemorrhage. Compared to platelets derived from wild type littermates, platelets lacking PIP5KIβ had PIP2 concentrations that were 61% of normal under basal conditions (p<0.01), and 51% of normal 45 seconds following thrombin stimulation (p<0.01). Similarly, maximum IP3 levels were only 65% of normal in the knockout platelets (p<0.01). Consistent with this second messenger defect, PIP5KIβ −/− platelets had impaired aggregation in response to submaximal doses of thrombin, ADP, collagen, and a thromboxane analogue (U46619). PIP5KIβ-null platelets exhibited disaggregation suggesting that sustained second messenger formation is critical for a sustained aggregation response. Since PIP2 can directly associate with, and thereby regulate actin-binding proteins, we analyzed platelet spreading upon fibrinogen. PIP5KIβ knockout platelets start to spread, but ultimately spread less well than platelets derived from wild type littermates. Imaging this process with real time differential interference contrast microscopy, we found that PIP5KIβ-null platelets extend filopodia as efficiently as wild type platelets, but have difficulty anchoring down these extended membranes. When a filopod on a PIP5KIβ −/− platelet does ultimately adhere to the matrix, a normal lamellipod is rapidly formed. The cytoskeletal organization of PIP5KIβ knockout platelets spread upon fibrinogen was further studied in the electron microscope. This higher resolution analysis verified the profound defect in lamellipodia formation. We speculated that this process of lamellipodia formation is critical for adhesion under the shear conditions found within the arterial system. To test this hypothesis, we analyzed the ability of PIP5KIβ knockout platelets to adhere to collagen in a flow chamber. At all shear conditions between 200 and 1100/s, platelets lacking PIP5KIβ consistently adhered less than wild type platelets. To further analyze the necessity of PIP5KIβ in adhesion of platelets under conditions of arterial shear, we compared PIP5KIβ −/− and PIP5KIβ +/+ mice in a ferric chloride carotid injury model. Under conditions that induced thrombosis in 75% of wild type mice (n=4), we only detected thrombi in 20% of PIP5KIβ-null mice (n=5). Together, these data demonstrate that PIP5KIβ is required for sustained PIP2 and second messenger synthesis, the formation of actin-rich lamellipodia, and stable ex vivo and in vivo platelet adhesion under shear.

scholarly journals A Dictyostelium mutant lacking an F-actin cross-linking protein, the 120-kD gelation factor.

1990 ◽  
Vol 111 (4) ◽  
pp. 1477-1489 ◽  
Author(s):  
M Brink ◽  
G Gerisch ◽  
G Isenberg ◽  
A A Noegel ◽  
J E Segall ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Mutant Cell ◽  
Actin Binding ◽  
In Vitro Assays ◽  
Cell Cortex ◽  
Cross Linking ◽  
Actin Binding Proteins ◽  
Cell Extracts ◽  
Nonmuscle Cells

Actin-binding proteins are known to regulate in vitro the assembly of actin into supramolecular structures, but evidence for their activities in living nonmuscle cells is scarce. Amebae of Dictyostelium discoideum are nonmuscle cells in which mutants defective in several actin-binding proteins have been described. Here we characterize a mutant deficient in the 120-kD gelation factor, one of the most abundant F-actin cross-linking proteins of D. discoideum cells. No F-actin cross-linking activity attributable to the 120-kD protein was detected in mutant cell extracts, and antibodies recognizing different epitopes on the polypeptide showed the entire protein was lacking. Under the conditions used, elimination of the gelation factor did not substantially alter growth, shape, motility, or chemotactic orientation of the cells towards a cAMP source. Aggregates of the mutant developed into fruiting bodies consisting of normally differentiated spores and stalk cells. In cytoskeleton preparations a dense network of actin filaments as typical of the cell cortex, and bundles as they extend along the axis of filopods, were recognized. A significant alteration found was an enhanced accumulation of actin in cytoskeletons of the mutant when cells were stimulated with cyclic AMP. Our results indicate that control of cell shape and motility does not require the fine-tuned interactions of all proteins that have been identified as actin-binding proteins by in vitro assays.

Regulation of the cardiac L-type Ca2+ channel by the actin-binding proteins α-actinin and dystrophin

2002 ◽  
Vol 282 (6) ◽  
pp. C1502-C1511 ◽  
Author(s):  
Abbas Sadeghi ◽  
Andrew D. Doyle ◽  
Barry D. Johnson
Keyword(s):  
Cardiac Myocytes ◽  
Binding Proteins ◽  
Voltage Dependence ◽  
Actin Binding ◽  
Mdx Mice ◽  
Potential Mechanism ◽  
Channel Activity ◽  
Wild Type ◽  
Actin Binding Proteins ◽  
Channel Inactivation

The actin-binding proteins dystrophin and α-actinin are members of a family of actin-binding proteins that may link the cytoskeleton to membrane proteins such as ion channels. Previous work demonstrated that the activity of Ca2+ channels can be regulated by agents that disrupt or stabilize the cytoskeleton. In the present study, we employed immunohistochemical and electrophysiological techniques to investigate the potential regulation of cardiac L-type Ca2+channel activity by dystrophin and α-actinin in cardiac myocytes and in heterologous cells. Both actin-binding proteins were found to colocalize with the Ca2+ channel in mouse cardiac myocytes and to modulate channel function. Inactivation of the Ca2+channel in cardiac myocytes from mice lacking dystrophin ( mdx mice) was reduced compared with that in wild-type myocytes, voltage dependence of activation was shifted by 5 mV to more positive potentials, and stimulation by the β-adrenergic pathway and the dihydropyridine agonist BAY K 8644 was increased. Furthermore, heterologous coexpression of the Ca2+ channel with muscle, but not nonmuscle, forms of α-actinin was also found to reduce inactivation. As might be predicted from a reduction of Ca2+ channel inactivation, a prolonging of the mouse electrocardiogram QT was observed in mdx mice. These results suggest a combined role for dystrophin and α-actinin in regulating the activity of the cardiac L-type Ca2+ channel and a potential mechanism for cardiac dysfunction in Duchenne and Becker muscular dystrophies.

Actin-binding proteins sensitively mediate actin bundle stiffness

2006 ◽  
Vol 39 ◽  
pp. S240
Author(s):  
M. Bathe ◽  
M. Claessens ◽  
E. Frey ◽  
A. Bausch
Keyword(s):  
Binding Proteins ◽  
Actin Binding ◽  
Actin Binding Proteins ◽  
Actin Bundle

A review of actin binding proteins: new perspectives

2007 ◽  
Vol 36 (1) ◽  
pp. 121-125 ◽  
Author(s):  
Ricardo Uribe ◽  
David Jay
Keyword(s):  
Binding Proteins ◽  
Actin Binding ◽  
Actin Binding Proteins

scholarly journals Molecular analysis of ethyl methanesulfonate-induced reversion of a chromosomally integrated mutant shuttle vector gene in mammalian cells.

1988 ◽  
Vol 8 (10) ◽  
pp. 4185-4189 ◽  
Author(s):  
J A Greenspan ◽  
F M Xu ◽  
R L Davidson
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Shuttle Vector ◽  
Ethyl Methanesulfonate ◽  
Wild Type ◽  
Single Base ◽  
Type Sequence

The molecular mechanisms of ethyl methanesulfonate-induced reversion in mammalian cells were studied by using as a target a gpt gene that was integrated chromosomally as part of a shuttle vector. Murine cells containing mutant gpt genes with single base changes were mutagenized with ethyl methanesulfonate, and revertant colonies were isolated. Ethyl methanesulfonate failed to increase the frequency of revertants for cell lines with mutant gpt genes carrying GC----AT transitions or AT----TA transversions, whereas it increased the frequency 50-fold to greater than 800-fold for cell lines with mutant gpt genes carrying AT----GC transitions and for one cell line with a GC----CG transversion. The gpt genes of 15 independent revertants derived from the ethyl methanesulfonate-revertible cell lines were recovered and sequenced. All revertants derived from cell lines with AT----GC transitions had mutated back to the wild-type gpt sequence via GC----AT transitions at their original sites of mutation. Five of six revertants derived from the cell line carrying a gpt gene with a GC----CG transversion had mutated via GC----AT transition at the site of the original mutation or at the adjacent base in the same triplet; these changes generated non-wild-type DNA sequences that code for non-wild-type amino acids that are apparently compatible with xanthine-guanine phosphoribosyltransferase activity. The sixth revertant had mutated via CG----GC transversion back to the wild-type sequence. The results of this study define certain amino acid substitutions in the xanthine-guanine phosphoribosyltransferase polypeptide that are compatible with enzyme activity. These results also establish mutagen-induced reversion analysis as a sensitive and specific assay for mutagenesis in mammalian cells.

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