Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 5 de 5
Filter
Add more filters










Database
Language
Publication year range
1.
Nat Commun ; 13(1): 628, 2022 02 02.
Article in English | MEDLINE | ID: mdl-35110533

ABSTRACT

Positive feedback loops involving signaling and actin assembly factors mediate the formation and remodeling of branched actin networks in processes ranging from cell and organelle motility to mechanosensation. The Arp2/3 complex inhibitor Arpin controls the directional persistence of cell migration by interrupting a feedback loop involving Rac-WAVE-Arp2/3 complex, but Arpin's mechanism of inhibition is unknown. Here, we describe the cryo-EM structure of Arpin bound to Arp2/3 complex at 3.24-Å resolution. Unexpectedly, Arpin binds Arp2/3 complex similarly to WASP-family nucleation-promoting factors (NPFs) that activate the complex. However, whereas NPFs bind to two sites on Arp2/3 complex, on Arp2-ArpC1 and Arp3, Arpin only binds to the site on Arp3. Like NPFs, Arpin has a C-helix that binds at the barbed end of Arp3. Mutagenesis studies in vitro and in cells reveal how sequence differences within the C-helix define the molecular basis for inhibition by Arpin vs. activation by NPFs.


Subject(s)
Actin-Related Protein 2-3 Complex/chemistry , Actin-Related Protein 2-3 Complex/drug effects , Carrier Proteins/pharmacology , Actin-Related Protein 2-3 Complex/metabolism , Actins/metabolism , Binding Sites , Cell Movement/drug effects , Cryoelectron Microscopy , Cytoskeletal Proteins , Humans , Models, Molecular , Protein Binding , Pseudopodia , Signal Transduction
2.
Neuroendocrinology ; 110(6): 535-551, 2020.
Article in English | MEDLINE | ID: mdl-31509830

ABSTRACT

BACKGROUND: Synaptic plasticity is the neuronal capacity to modify the function and structure of dendritic spines (DS) in response to neuromodulators. Sex steroids, particularly 17ß-estradiol (E2) and progesterone (P4), are key regulators in the control of DS formation through multiprotein complexes including WAVE1 protein, and are thus fundamental for the development of learning and memory. OBJECTIVES: The aim of this work was to evaluate the molecular switch Cdk5 kinase/protein phosphatase 2A (PP2A) in the control of WAVE1 protein (phosphorylation/dephosphorylation) and the regulation of WAVE1 and cortactin to the Arp2/3 complex, in response to rapid treatments with E2 and P4 in cortical neuronal cells. RESULTS: Rapid treatment with E2 and P4 modified neuronal morphology and significantly increased the number of DS. This effect was reduced by the use of a Cdk5 inhibitor (Roscovitine). In contrast, inhibition of PP2A with PP2A dominant negative construct significantly increased DS formation, evidencing the participation of kinase/phosphatase in the regulation of WAVE1 in DS formation induced by E2 and P4. Cortactin regulates DS formation via Src and PAK1 kinase induced by E2 and P4. Both cortactin and WAVE1 signal to Arp2/3 complex to synergistically promote actin nucleation. CONCLUSION: These results suggest that E2 and P4 dynamically regulate neuron morphology through nongenomic signaling via cortactin/WAVE1-Arp2/3 complex. The control of these proteins is tightly orchestrated by phosphorylation, where kinases and phosphatases are essential for actin nucleation and, finally, DS formation. This work provides a deeper understanding of the biological actions of sex steroids in the regulation of DS turnover and neuronal plasticity processes.


Subject(s)
Actin-Related Protein 2-3 Complex/metabolism , Cerebral Cortex/physiology , Dendritic Spines/physiology , Estradiol/physiology , Progesterone/physiology , Protein Phosphatase 2/metabolism , Signal Transduction/physiology , Wiskott-Aldrich Syndrome Protein Family/metabolism , Actin-Related Protein 2-3 Complex/drug effects , Animals , Cerebral Cortex/drug effects , Cortactin , Cyclin-Dependent Kinase 5/antagonists & inhibitors , Dendritic Spines/drug effects , Embryo, Mammalian , Estradiol/pharmacology , Progesterone/pharmacology , Protein Kinase Inhibitors/pharmacology , Protein Phosphatase 2/drug effects , Rats , Roscovitine/pharmacology , Signal Transduction/drug effects , Wiskott-Aldrich Syndrome Protein Family/drug effects
3.
PLoS Genet ; 12(11): e1006452, 2016 Nov.
Article in English | MEDLINE | ID: mdl-27870871

ABSTRACT

Fungal biofilms are complex, structured communities that can form on surfaces such as catheters and other indwelling medical devices. Biofilms are of particular concern with Candida albicans, one of the leading opportunistic fungal pathogens of humans. C. albicans biofilms include yeast and filamentous cells that are surrounded by an extracellular matrix, and they are intrinsically resistant to antifungal drugs such that resolving biofilm infections often requires surgery to remove the contaminated device. C. albicans biofilms form through a regulated process of adhesion to surfaces, filamentation, maturation, and ultimately dispersion. To uncover new strategies to block the initial stages of biofilm formation, we utilized a functional genomic approach to identify genes that modulate C. albicans adherence. We screened a library of 1,481 double barcoded doxycycline-repressible conditional gene expression strains covering ~25% of the C. albicans genome. We identified five genes for which transcriptional repression impaired adherence, including: ARC18, PMT1, MNN9, SPT7, and orf19.831. The most severe adherence defect was observed upon transcriptional repression of ARC18, which encodes a member of the Arp2/3 complex that is involved in regulation of the actin cytoskeleton and endocytosis. Depletion of components of the Arp2/3 complex not only impaired adherence, but also caused reduced biofilm formation, increased cell surface hydrophobicity, and increased exposure of cell wall chitin and ß-glucans. Reduced function of the Arp2/3 complex led to impaired cell wall integrity and activation of Rho1-mediated cell wall stress responses, thereby causing cell wall remodelling and reduced adherence. Thus, we identify important functional relationships between cell wall stress responses and a novel mechanism that controls adherence and biofilm formation, thereby illuminating novel strategies to cripple a leading fungal pathogen of humans.


Subject(s)
Actin-Related Protein 2-3 Complex/genetics , Biofilms/growth & development , Candida albicans/genetics , Drug Resistance, Fungal/genetics , Fungal Proteins/biosynthesis , Actin Cytoskeleton/genetics , Actin-Related Protein 2-3 Complex/drug effects , Biofilms/drug effects , Candida albicans/drug effects , Candida albicans/growth & development , Candida albicans/pathogenicity , Candidiasis/drug therapy , Candidiasis/genetics , Candidiasis/microbiology , Cell Adhesion/genetics , Cell Wall/genetics , Endocytosis/genetics , Fungal Proteins/genetics , Gene Expression Regulation, Fungal , Genome, Fungal , Genomics , Humans , Metabolic Networks and Pathways/genetics , Stress, Physiological/genetics
4.
Lipids Health Dis ; 14: 4, 2015 Feb 14.
Article in English | MEDLINE | ID: mdl-25971815

ABSTRACT

BACKGROUND: Omega-3 polyunsaturated fatty acids (n-3 PUFAs) show beneficial effects on cardiovascular health and cognitive functions, but the underlying molecular mechanisms are not completely understood. Because of the fact that cytoskeleton dynamics affect almost every cellular process, the regulation of cytoskeletal dynamics could be a new pathway by which n-3 PUFAs exert their effects on cellular level. METHODS: A 12-week open-label intervention study with 12 healthy men was conducted to determine the effects of 2.7 g/d n-3 PUFA on changes in mRNA expression of cytoskeleton-associated genes by quantitative real-time PCR in whole blood. Furthermore, the actin content in red blood cells was analyzed by immunofluorescence imaging. RESULTS: N-3 PUFA supplementation resulted in a significant down-regulation of cytoskeleton-associated genes, in particular three GTPases (RAC1, RHOA, CDC42), three kinases (ROCK1, PAK2, LIMK), two Wiskott-Aldrich syndrome proteins (WASL, WASF2) as well as actin related protein 2/3 complex (ARPC2, ARPC3) and cofilin (CFL1). Variability in F-actin content between subjects was high; reduced actin content was only reduced within group evaluation. CONCLUSIONS: Reduced cytoskeleton-associated gene expression after n-3 PUFA supplementation suggests that regulation of cytoskeleton dynamics might be an additional way by which n-3 PUFAs exert their cellular effects. Concerning F-actin, this analysis did not reveal unmistakable results impeding a generalized conclusion.


Subject(s)
Cytoskeleton/drug effects , Fatty Acids, Omega-3/pharmacology , Actin-Related Protein 2-3 Complex/drug effects , Adult , Cofilin 1/drug effects , Down-Regulation/drug effects , Fluorescent Antibody Technique , Gene Expression/drug effects , Humans , Lim Kinases/drug effects , Male , Real-Time Polymerase Chain Reaction , Wiskott-Aldrich Syndrome Protein Family/drug effects , Wiskott-Aldrich Syndrome Protein, Neuronal/drug effects , cdc42 GTP-Binding Protein/drug effects , p21-Activated Kinases/drug effects , rac1 GTP-Binding Protein/drug effects , rho-Associated Kinases/drug effects , rhoA GTP-Binding Protein/drug effects
5.
BMC Cell Biol ; 9: 42, 2008 Jul 30.
Article in English | MEDLINE | ID: mdl-18667055

ABSTRACT

BACKGROUND: Cytokinesis is the final step of cell division taking place at the end of mitosis during which the cytoplasmic content and replicated chromosomes of a cell are equally partitioned between the two daughter cells. This process is achieved by the formation and the ingression of an actomyosin contractile ring under the control of equatorial microtubules. The mechanisms of contractile ring formation are not fully understood but involve recruitment of preexisting actin filaments and de novo actin polymerisation. RESULTS: In this study, we evaluated the role of the actin nucleation factor, Arp2/3 complex, during cytokinesis. We found that the Arp2/3 complex is recruited late to the cleavage furrow suggesting a potential involvement of Arp2/3 complex during this process. Furthermore, wiskostatin a potent inhibitor of N-WASP activity towards the Arp2/3 complex blocked cytokinesis without affecting mitosis. Nonetheless, this inhibition could not be reproduced using alternative approaches targeting the N-WASP/Arp2/3 complex pathway. CONCLUSION: We conclude that the wiskostatin induced defective cytokinesis does not occur through the inhibition of the N-WASP/Arp2/3 pathway. Wiskostatin is likely to either directly target other proteins required for cytokinesis progression or alternately wiskostatin bound to N-WASP could affect the activity of other factors involved in cytokinesis.


Subject(s)
Actin-Related Protein 2-3 Complex/physiology , Carbazoles/pharmacology , Cytokinesis/physiology , Mitosis/physiology , Propanolamines/pharmacology , Wiskott-Aldrich Syndrome Protein, Neuronal/physiology , Actin-Related Protein 2-3 Complex/drug effects , Cytokinesis/drug effects , HeLa Cells/drug effects , HeLa Cells/physiology , Humans , Mitosis/drug effects , Nocodazole/pharmacology , Wiskott-Aldrich Syndrome Protein, Neuronal/drug effects
SELECTION OF CITATIONS
SEARCH DETAIL
...