The synthesis and biological evaluation of a kabiramide C fragment modified with a WH2 consensus actin-binding motif as a potential disruptor of the actin cytoskeleton

2016 ◽  
Vol 52 (4) ◽  
pp. 807-810 ◽  
Author(s):  
Daniel J. Tetlow ◽  
Steve J. Winder ◽  
Christophe Aïssa
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Filaments ◽  
Biological Evaluation ◽  
Actin Binding ◽  
Binding Motif ◽  
Synthesis And Biological Evaluation ◽  
In Cells

Despite its low affinity for actin monomers, a fragment of kabiramide C disrupts actin filamentsin vitroand in cells.

scholarly journals Actin-depolymerizing Factor and Cofilin-1 Play Overlapping Roles in Promoting Rapid F-Actin Depolymerization in Mammalian Nonmuscle Cells

2005 ◽  
Vol 16 (2) ◽  
pp. 649-664 ◽  
Author(s):  
Pirta Hotulainen ◽  
Eija Paunola ◽  
Maria K. Vartiainen ◽  
Pekka Lappalainen
Keyword(s):  
Cell Motility ◽  
Actin Filament ◽  
Actin Filaments ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Actin Depolymerizing Factor ◽  
Cytoskeletal Dynamics ◽  
Nonmuscle Cells ◽  
In Cells

Actin-depolymerizing factor (ADF)/cofilins are small actin-binding proteins found in all eukaryotes. In vitro, ADF/cofilins promote actin dynamics by depolymerizing and severing actin filaments. However, whether ADF/cofilins contribute to actin dynamics in cells by disassembling “old” actin filaments or by promoting actin filament assembly through their severing activity is a matter of controversy. Analysis of mammalian ADF/cofilins is further complicated by the presence of multiple isoforms, which may contribute to actin dynamics by different mechanisms. We show that two isoforms, ADF and cofilin-1, are expressed in mouse NIH 3T3, B16F1, and Neuro 2A cells. Depleting cofilin-1 and/or ADF by siRNA leads to an accumulation of F-actin and to an increase in cell size. Cofilin-1 and ADF seem to play overlapping roles in cells, because the knockdown phenotype of either protein could be rescued by overexpression of the other one. Cofilin-1 and ADF knockdown cells also had defects in cell motility and cytokinesis, and these defects were most pronounced when both ADF and cofilin-1 were depleted. Fluorescence recovery after photobleaching analysis and studies with an actin monomer-sequestering drug, latrunculin-A, demonstrated that these phenotypes arose from diminished actin filament depolymerization rates. These data suggest that mammalian ADF and cofilin-1 promote cytoskeletal dynamics by depolymerizing actin filaments and that this activity is critical for several processes such as cytokinesis and cell motility.

Role of Pleckstrin 2 in Improved Erythropoiesis of Transferrin-Treated Beta-Thalassemic Mice

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 755-755 ◽  
Author(s):  
Maria Feola ◽  
Andrea Zamperone ◽  
Weili Bao ◽  
Tenzin Choesang ◽  
Huihui Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Actin Cytoskeleton ◽  
Actin Filaments ◽  
Conflicts Of Interest ◽  
Erythroid Differentiation ◽  
Actin Binding ◽  
Hematopoietic Stem ◽  
Cytoskeleton Reorganization ◽  
Erythroid Precursors

Abstract Erythropoiesis is a process during which multipotent hematopoietic stem cells proliferate, differentiate and ultimately produce enucleated reticulocytes. Terminal erythroid differentiation begins at the morphologically recognizable pro-erythroblast (pro-E) stage and is completed when orthochromatic erythroblasts (ortho-E) expel their nuclei to produce reticulocytes. Progressive differentiation between these stages occurs in homologous cell division progressively doubling proportions of pro-E, basophilic (baso-E), polychromatophilic (poly-E), and ortho-E, and multiple signaling pathways are involved in the generation of enucleated erythroid cells, including multiple steps requiring actin cytoskeleton reorganization. We have previously shown that β-thalassemic mice (th1/th1) demonstrate a disordered progression from pro-E to baso-E and that exogenous transferrin therapy restores normal proportion of early stage erythroid precursors in th1/th1 mice (Liu Blood 2013). To identify genes that play novel function in different stages of terminal erythropoiesis, we performed RNA seq analysis of sorted bone marrow pro-E from WT, th1/th1, and transferrin-treated th1/th1 mice. We identify pleckstrin-2 (plek2) as a gene of interest with a 15-fold increase in plek2 mRNA expression in th1/th1 relative to WT mice, normalized in transferrin-treated th1/th1 mice. Plek2 is an actin binding protein, like pleckstrin-1, contains a central DEP domain known to bind RacGTPase, is Epo dependent, and is expressed in all stages of terminal erythropoiesis. We evaluate plek2 mRNA and protein expression in sorted bone marrow erythroid precursors from WT, th1/th1, and transferrin-treated th1/th1 mice. Our data demonstrates a statistically significant increase in plek2 mRNA in th1/th1 relative to WT mice, with the highest expression of plek2 in poly-E, normalized in transferrin-treated th1/th1 mice (Figure 1A). A similar pattern of increased protein concentration in th1/th1 relative to WT mice and normalization in transferrin-treated th1/th1 mice is evident in sorted bone marrow samples (Figure 1B). Prior in vitro studies demonstrate that membrane localization of plek2 is required for erythroid differentiation. Thus, we performed sub-cellular fractionation in bone marrow erythroid precursors and determined for the first time that in sorted erythroblasts from WT bone marrow, plek2 is found exclusively in the cytoplasm in pro-E and in both cytoplasm and membrane from baso-E to ortho-E (Figure 2), co-localized with actin filaments in the membrane (data not shown). In contrast, sorted erythroblasts from th1/th1 bone marrow reveal membrane-associated plek2 starting from pro-E, demonstrating earlier co-localization with actin filaments (data not shown) and suggesting an earlier activation of plek2 and consequent actin cytoskeleton reorganization during erythroid differentiation in th1/th1 mice, normalized in transferrin-treated th1/th1 mice (Figure 2). Erythropoiesis involves a complicated and incompletely understood set of potentially related molecular signals influencing cell survival, differentiation, enucleation, and release into the circulation. For example, although Epo increases survival, Epo signaling also activates RacGTPases, inhibiting enucleation. Recent in vitro data demonstrates that knockdown of plek2 affected enucleation with significantly lower reticulocyte count. Although the involvement of RacGTPase in plek2-mediated erythroid differentiation has not been explored, we hypothesize that plek2 activation triggers RacGTPase and prevents enucleation in th1/th1 mice. Our data demonstrates that RacGTPase concentration is increased in sorted bone marrow erythroid precursors from th1/th1 relative to WT mice and normalized in transferrin-treated th1/th1 mice (Figure 1B). These results suggest that plek2 plays an important role in erythropoiesis likely as a key factor in the improved enucleation of transferrin-treated th1/th1 mice. Disclosures No relevant conflicts of interest to declare.

scholarly journals Mechanisms of actin disassembly

2013 ◽  
Vol 24 (15) ◽  
pp. 2299-2302 ◽  
Author(s):  
William Brieher
Keyword(s):  
Actin Cytoskeleton ◽  
Cell Motility ◽  
Actin Filament ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Actin Depolymerization ◽  
Filament Dynamics ◽  
Physiological Demands ◽  
In Cells

The actin cytoskeleton is constantly assembling and disassembling. Cells harness the energy of these turnover dynamics to drive cell motility and organize cytoplasm. Although much is known about how cells control actin polymerization, we do not understand how actin filaments depolymerize inside cells. I briefly describe how the combination of imaging actin filament dynamics in cells and using in vitro biochemistry progressively altered our views of actin depolymerization. I describe why I do not think that the prevailing model of actin filament turnover—cofilin-mediated actin filament severing—can account for actin filament disassembly detected in cells. Finally, I speculate that cells might be able to tune the mechanism of actin depolymerization to meet physiological demands and selectively control the stabilities of different actin arrays.

scholarly journals Distribution of actin-binding protein and myosin in macrophages during spreading and phagocytosis.

1980 ◽  
Vol 84 (2) ◽  
pp. 215-224 ◽  
Author(s):  
O I Stendahl ◽  
J H Hartwig ◽  
E A Brotschi ◽  
T P Stossel
Keyword(s):  
Actin Filaments ◽  
Structural Unit ◽  
Binding Protein ◽  
Actin Binding ◽  
Rabbit Lung ◽  
Actin Binding Protein ◽  
Lung Macrophages ◽  
In Cells

Actin-binding protein (ABP) and myosin are proteins that influence the rigidity and movement, respectively, of actin filaments in vitro. We examined the distribution of ABP and myosin molecules in acetone-fixed rabbit lung macrophages by means of immunofluorescence. The staining for both of these proteins in unspread cells was quite uniform, but was reduced in the nucleus and concentrated slightly in the periphery. The peripheral accumulation of staining attenuated in uniformly spread cells, although filopodia and hyaline veils definitely stained. In cells fixed during ingestion of yeast particles, the brightest staining correlated with the disposition of organelle-excluding pseudopodia initially surrounding the yeast. After phagocytosis was complete and the yeasts resided in intracellular vacuoles, no concentration of staining around the ingested yeasts was detectable. We conclude that ABP and myosin molecules are components of the structural unit of the cell responsible for spreading and phagocytosis, the hyaline cortex, a region known to be rich in actin filaments. The findings are consistent with the theory that these molecules control the rigidity and movement of filaments in the periphery of the living macrophage.

Villin-induced growth of microvilli is reversibly inhibited by cytochalasin D

1993 ◽  
Vol 105 (3) ◽  
pp. 765-775 ◽  
Author(s):  
E. Friederich ◽  
T.E. Kreis ◽  
D. Louvard
Keyword(s):  
Plasma Membrane ◽  
Actin Cytoskeleton ◽  
Binding Site ◽  
Actin Filaments ◽  
Living Cells ◽  
Cytochalasin D ◽  
Actin Binding ◽  
Fast Growing ◽  
Actin Binding Site

Villin is an actin-binding protein that is associated with the cytoskeleton of brush border microvilli. In vitro, villin nucleates, caps or severs actin filaments in a Ca(2+)-dependent manner. In the absence of Ca2+, villin organizes microfilaments into bundles. Transfection of a villin-specific cDNA into cultured cells that do not produce this protein results in the growth of long surface microvilli and the reorganization of the underlying actin cytoskeleton. Here we studied the effects of low concentrations of cytochalasin D on the induction of these plasma membrane-actin cytoskeleton specializations. Transfected cells were treated with concentrations of cytochalasin D that prevent the association of actin monomers with the fast-growing end of microfilaments in vitro. In villin-positive cells, cytochalasin D inhibited the growth of microvilli and promoted the formation of rodlet-like actin structures, which were randomly distributed throughout the cytoplasm. The formation of these structures was dependent on large amounts of villin and on the integrity of an actin-binding site located at the carboxy terminus of villin, which is required for microfilament bundling in vitro and for the growth of microvilli in vivo. The effect of cytochalasin D was reversible. The observation of living cells by video-imaging revealed that when cytochalasin D was removed, rapid disassembly of actin rodlets occurred after a lag phase. The present data stress the important role of the plasma membrane in the organization of the actin cytoskeleton and suggest that the extension of the microvillar plasma membrane is dependent on the elongation of microfilaments at their fast-growing end. Inhibition of microfilament elongation near the plasma membrane by cytochalasin D may result in the ‘random’ nucleation of actin filaments throughout the cytoplasm. On the basis of the present data, we propose that villin is involved in the assembly of the microvillar actin bundle by a mechanism that does not prevent monomer association with the preferred end of microfilaments. For instance, villin may stabilize actin filaments by lateral interactions. The functional importance of the carboxy-terminal F-actin binding site in such a mechanism is stressed by the fact that it is required for the formation of F-actin rodlets in cytochalasin D-treated cells. Finally, our data further emphasize the observations that the effects of cytochalasin D in living cells can be modulated by actin-binding proteins.

scholarly journals Pseudomonas aeruginosa exoenzyme Y directly bundles actin filaments

2020 ◽  
Vol 295 (11) ◽  
pp. 3506-3517 ◽  
Author(s):  
Jordan M. Mancl ◽  
Cristian Suarez ◽  
Wenguang G. Liang ◽  
David R. Kovar ◽  
Wei-Jen Tang
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Actin Cytoskeleton ◽  
Actin Filaments ◽  
Structural Model ◽  
Effector Proteins ◽  
Host Cells ◽  
Cyclase Activity ◽  
Actin Dynamics ◽  
Actin Binding

Pseudomonas aeruginosa uses a type III secretion system (T3SS) to inject cytotoxic effector proteins into host cells. The promiscuous nucleotidyl cyclase, exoenzyme Y (ExoY), is one of the most common effectors found in clinical P. aeruginosa isolates. Recent studies have revealed that the nucleotidyl cyclase activity of ExoY is stimulated by actin filaments (F-actin) and that ExoY alters actin cytoskeleton dynamics in vitro, via an unknown mechanism. The actin cytoskeleton plays an important role in numerous key biological processes and is targeted by many pathogens to gain competitive advantages. We utilized total internal reflection fluorescence microscopy, bulk actin assays, and EM to investigate how ExoY impacts actin dynamics. We found that ExoY can directly bundle actin filaments with high affinity, comparable with eukaryotic F-actin–bundling proteins, such as fimbrin. Of note, ExoY enzymatic activity was not required for F-actin bundling. Bundling is known to require multiple actin-binding sites, yet small-angle X-ray scattering experiments revealed that ExoY is a monomer in solution, and previous data suggested that ExoY possesses only one actin-binding site. We therefore hypothesized that ExoY oligomerizes in response to F-actin binding and have used the ExoY structure to construct a dimer-based structural model for the ExoY–F-actin complex. Subsequent mutational analyses suggested that the ExoY oligomerization interface plays a crucial role in mediating F-actin bundling. Our results indicate that ExoY represents a new class of actin-binding proteins that modulate the actin cytoskeleton both directly, via F-actin bundling, and indirectly, via actin-activated nucleotidyl cyclase activity.

Disruption of the actin cytoskeleton of mammalian cells by the capping complex actin-fragmin is inhibited by actin phosphorylation and regulated by Ca2+ ions

1998 ◽  
Vol 111 (12) ◽  
pp. 1695-1706 ◽  
Author(s):  
B. Constantin ◽  
K. Meerschaert ◽  
J. Vandekerckhove ◽  
J. Gettemans
Keyword(s):  
Actin Cytoskeleton ◽  
Cell Morphology ◽  
Actin Filaments ◽  
Mammalian Cells ◽  
Actin Binding ◽  
General Mechanism ◽  
Resting Cells ◽  
Dependent Manner

Fragmin from Physarum polycephalum is a gelsolin-like actin-binding protein and interferes with the growth of actin filaments in vitro by severing actin filaments and capping their barbed ends through formation of an actin-fragmin dimer in a Ca2+-dependent manner. The actin-fragmin dimer is phosphorylated in vivo and in vitro on the actin subunit by the actin-fragmin kinase. We have studied the properties of these capping proteins and their regulation by actin phosphorylation and Ca2+ ions in living PtK2, CV1 and NIH3T3 cultured cells by microinjection or by expression in conjunction with immunostaining and fluorescence microscopy. Microinjection of the actin-fragmin dimer disintegrated the actin cytoskeleton and altered cell morphology. This in vivo effect could be blocked by phosphorylation of the actin subunit by the actin-fragmin kinase in low Ca2+ conditions, and the capping activity could be recovered by high Ca2+ concentration, probably through activation of the second actin-binding site in fragmin. This suggests that in Physarum microplasmodia, actin polymerization can be controlled in a Ca2+-dependent manner through the phosphorylation of actin. Microinjected or overexpressed recombinant fragmin did not affect the actin-based cytoskeleton or cell morphology of resting cells, unless the cytosolic free Ca2+ concentration was increased by microinjection of a Ca2+-containing buffer. The cells were able to revert to their normal phenotype which indicates that endogenous regulatory mechanisms counteracted fragmin activity, probably by uncapping fragmin from the barbed ends of filaments. Fragmin also antagonized formation of stress fibers induced by lysophosphatidic acid. Our findings demonstrate that the interactions between actin and fragmin are tightly regulated by the cytosolic Ca2+ concentration and this provides a basis for a more general mechanism in higher organisms to regulate microfilament organization.

Hybrid Design of Isonicotinic Acid Hydrazide Derivatives: Machine Learning Studies, Synthesis and Biological Evaluation of their Antituberculosis Activity

2020 ◽  
Vol 17 (3) ◽  
pp. 365-375
Author(s):  
Vasyl Kovalishyn ◽  
Diana Hodyna ◽  
Vitaliy O. Sinenko ◽  
Volodymyr Blagodatny ◽  
Ivan Semenyuta ◽  
...  
Keyword(s):  
Machine Learning ◽  
Isonicotinic Acid ◽  
Acid Hydrazide ◽  
Predictive Ability ◽  
Antitubercular Activity ◽  
Biological Evaluation ◽  
Starting Point ◽  
Binary Classifiers ◽  
Synthesis And Biological Evaluation

Background: Tuberculosis (TB) is an infection disease caused by Mycobacterium tuberculosis (Mtb) bacteria. One of the main causes of mortality from TB is the problem of Mtb resistance to known drugs. Objective: The goal of this work is to identify potent small molecule anti-TB agents by machine learning, synthesis and biological evaluation. Methods: The On-line Chemical Database and Modeling Environment (OCHEM) was used to build predictive machine learning models. Seven compounds were synthesized and tested in vitro for their antitubercular activity against H37Rv and resistant Mtb strains. Results: A set of predictive models was built with OCHEM based on a set of previously synthesized isoniazid (INH) derivatives containing a thiazole core and tested against Mtb. The predictive ability of the models was tested by a 5-fold cross-validation, and resulted in balanced accuracies (BA) of 61–78% for the binary classifiers. Test set validation showed that the models could be instrumental in predicting anti- TB activity with a reasonable accuracy (with BA = 67–79 %) within the applicability domain. Seven designed compounds were synthesized and demonstrated activity against both the H37Rv and multidrugresistant (MDR) Mtb strains resistant to rifampicin and isoniazid. According to the acute toxicity evaluation in Daphnia magna neonates, six compounds were classified as moderately toxic (LD50 in the range of 10−100 mg/L) and one as practically harmless (LD50 in the range of 100−1000 mg/L). Conclusion: The newly identified compounds may represent a starting point for further development of therapies against Mtb. The developed models are available online at OCHEM http://ochem.eu/article/11 1066 and can be used to virtually screen for potential compounds with anti-TB activity.

scholarly journals Villin Severing Activity Enhances Actin-based Motility In Vivo

2007 ◽  
Vol 18 (3) ◽  
pp. 827-838 ◽  
Author(s):  
Céline Revenu ◽  
Matthieu Courtois ◽  
Alphée Michelot ◽  
Cécile Sykes ◽  
Daniel Louvard ◽  
...  
Keyword(s):  
Shigella Flexneri ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Loss Of Function ◽  
Mesenchymal Transition ◽  
Capping Protein ◽  
Function Strategy ◽  
In Cells

Villin, an actin-binding protein associated with the actin bundles that support microvilli, bundles, caps, nucleates, and severs actin in a calcium-dependant manner in vitro. We hypothesized that the severing activity of villin is responsible for its reported role in enhancing cell plasticity and motility. To test this hypothesis, we chose a loss of function strategy and introduced mutations in villin based on sequence comparison with CapG. By pyrene-actin assays, we demonstrate that this mutant has a strongly reduced severing activity, whereas nucleation and capping remain unaffected. The bundling activity and the morphogenic effects of villin in cells are also preserved in this mutant. We thus succeeded in dissociating the severing from the three other activities of villin. The contribution of villin severing to actin dynamics is analyzed in vivo through the actin-based movement of the intracellular bacteria Shigella flexneri in cells expressing villin and its severing variant. The severing mutations abolish the gain of velocity induced by villin. To further analyze this effect, we reconstituted an in vitro actin-based bead movement in which the usual capping protein is replaced by either the wild type or the severing mutant of villin. Confirming the in vivo results, villin-severing activity enhances the velocity of beads by more than two-fold and reduces the density of actin in the comets. We propose a model in which, by severing actin filaments and capping their barbed ends, villin increases the concentration of actin monomers available for polymerization, a mechanism that might be paralleled in vivo when an enterocyte undergoes an epithelio-mesenchymal transition.

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