scholarly journals Presence of WH2 like domain in VgrG-1 toxin of Vibrio cholerae reveals the molecular mechanism of actin cross-linking

2017 ◽  
Author(s):  
Priyanka Dutta ◽  
A.S. Jijumon ◽  
Mohit Mazumder ◽  
Drisya Dileep ◽  
Asish K. Mukhopadhyay ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Host Cell ◽  
Vibrio Cholerae ◽  
Actin Binding ◽  
Cross Linking ◽  
Secretion Systems ◽  
Type Vi Secretion ◽  
Wh2 Domain ◽  
Cytoskeleton Disruption

AbstractType VI secretion systems (T6SS) plays a crucial role in Vibrio cholerae mediated pathogenicity and predation. Tip of T6SS is homologous to gp27/gp5 complex or tail spike of T4 bacteriophage. VgrG-1 of V. cholerae T6SS is unusual among other VgrG because its effector domain is trans-located into the cytosol of eukaryotic cells with an additional actin cross-linking domain (ACD) at its C terminal end. ACD of VgrG-1 (VgrG-1-ACD) causes T6SS dependent host cell cytotoxicity through actin cytoskeleton disruption to prevent bacterial engulfment by macrophages. ACD mediated actin cross-linking promotes survival of the bacteria in the small intestine of humans, along with other virulence factors; establishes successful infection with the onset of diarrhoea in humans. Our studies demonstrated VgrG-1-ACD can bind to actin besides actin cross-linking activity. Computational analysis of ACD revealed the presence of WH2 domain through which it binds actin. Mutations in WH2 domain lead to loss of actin binding in vitro. VgrG-1-ACD having the mutated WH2 domain cannot cross-link actin efficiently in vitro and manifests less actin cytoskeleton disruption when transfected in HeLa cells.Summary statementActin cross-linking (ACD) domain of VgrG-1 toxin of Type VI secretion in Vibrio cholera has WASP Homology domain 2 (WH2) domain. ACD interact with actin through WH2 domain, WH2 is essential for ACD mediated cross-linking and disruption of actin cytoskeleton in the host cell.

scholarly journals Interactions among a Fimbrin, a Capping Protein, and an Actin-depolymerizing Factor in Organization of the Fission Yeast Actin Cytoskeleton

2001 ◽  
Vol 12 (11) ◽  
pp. 3515-3526 ◽  
Author(s):  
Kentaro Nakano ◽  
Kazuomi Satoh ◽  
Akeshi Morimatsu ◽  
Masaaki Ohnuma ◽  
Issei Mabuchi
Keyword(s):  
Actin Cytoskeleton ◽  
Fission Yeast ◽  
Actin Binding ◽  
Cell Cortex ◽  
Cross Linking ◽  
Actin Depolymerizing Factor ◽  
Binding Domains ◽  
Ef Hand ◽  
Actin Cables

We report studies of the fission yeast fimbrin-like protein Fim1, which contains two EF-hand domains and two actin-binding domains (ABD1 and ABD2). Fim1 is a component of both F-actin patches and the F-actin ring, but not of F-actin cables. Fim1 cross-links F-actin in vitro, but a Fim1 protein lacking either EF-hand domains (Fim1A12) or both the EF-hand domains and ABD1 (Fim1A2) has no actin cross-linking activity. Overexpression of Fim1 induced the formation of F-actin patches throughout the cell cortex, whereas the F-actin patches disappear in cells overexpressing Fim1A12 or Fim1A2. Thus, the actin cross-linking activity of Fim1 is probably important for the formation of F-actin patches. The overexpression of Fim1 also excluded the actin-depolymerizing factor Adf1 from the F-actin patches and inhibited the turnover of actin in these structures. Thus, Fim1 may function in stabilizing the F-actin patches. We also isolated the gene encoding Acp1, a subunit of the heterodimeric F-actin capping protein.fim1 acp1 double null cells showed more severe defects in the organization of the actin cytoskeleton than those seen in each single mutant. Thus, Fim1 and Acp1 may function in a similar manner in the organization of the actin cytoskeleton. Finally, genetic studies suggested that Fim1 may function in cytokinesis in cooperation with Cdc15 (PSTPIP) and Rng2 (IQGAP), respectively.

scholarly journals Microtubule Actin Cross-Linking Factor (Macf)

1999 ◽  
Vol 147 (6) ◽  
pp. 1275-1286 ◽  
Author(s):  
Conrad L. Leung ◽  
Dongming Sun ◽  
Min Zheng ◽  
David R. Knowles ◽  
Ronald K.H. Liem
Keyword(s):  
Calcium Binding ◽  
Coiled Coil ◽  
Specific Protein ◽  
Binding Domain ◽  
Full Length ◽  
Actin Binding ◽  
Cross Linking ◽  
Actin Binding Domain

We cloned and characterized a full-length cDNA of mouse actin cross-linking family 7 (mACF7) by sequential rapid amplification of cDNA ends–PCR. The completed mACF7 cDNA is 17 kb and codes for a 608-kD protein. The closest relative of mACF7 is the Drosophila protein Kakapo, which shares similar architecture with mACF7. mACF7 contains a putative actin-binding domain and a plakin-like domain that are highly homologous to dystonin (BPAG1-n) at its NH2 terminus. However, unlike dystonin, mACF7 does not contain a coiled–coil rod domain; instead, the rod domain of mACF7 is made up of 23 dystrophin-like spectrin repeats. At its COOH terminus, mACF7 contains two putative EF-hand calcium-binding motifs and a segment homologous to the growth arrest–specific protein, Gas2. In this paper, we demonstrate that the NH2-terminal actin-binding domain of mACF7 is functional both in vivo and in vitro. More importantly, we found that the COOH-terminal domain of mACF7 interacts with and stabilizes microtubules. In transfected cells full-length mACF7 can associate not only with actin but also with microtubules. Hence, we suggest a modified name: MACF (microtubule actin cross-linking factor). The properties of MACF are consistent with the observation that mutations in kakapo cause disorganization of microtubules in epidermal muscle attachment cells and some sensory neurons.

NUANCE, a giant protein connecting the nucleus and actin cytoskeleton

2002 ◽  
Vol 115 (15) ◽  
pp. 3207-3222 ◽  
Author(s):  
Yen-Yi Zhen ◽  
Thorsten Libotte ◽  
Martina Munck ◽  
Angelika A. Noegel ◽  
Elena Korenbaum
Keyword(s):  
Actin Cytoskeleton ◽  
Transmembrane Domain ◽  
Coiled Coil ◽  
Peripheral Blood Lymphocytes ◽  
Binding Domain ◽  
Actin Binding ◽  
Actin Binding Domain ◽  
Microfilament System

NUANCE (NUcleus and ActiN Connecting Element) was identified as a novel protein with an α-actinin-like actin-binding domain. A human 21.8 kb cDNA of NUANCE spreads over 373 kb on chromosome 14q22.1-q22.3. The cDNA sequence predicts a 796 kDa protein with an N-terminal actin-binding domain, a central coiled-coil rod domain and a predicted C-terminal transmembrane domain. High levels of NUANCE mRNA were detected in the kidney, liver,stomach, placenta, spleen, lymphatic nodes and peripheral blood lymphocytes. At the subcellular level NUANCE is present predominantly at the outer nuclear membrane and in the nucleoplasm. Domain analysis shows that the actin-binding domain binds to Factin in vitro and colocalizes with the actin cytoskeleton in vivo as a GFP-fusion protein. The C-terminal transmembrane domain is responsible for the targeting the nuclear envelope. Thus, NUANCE is the firstα-actinin-related protein that has the potential to link the microfilament system with the nucleus.

scholarly journals Tissue Expression and Actin Binding of a Novel N-Terminal Utrophin Isoform

2011 ◽  
Vol 2011 ◽  
pp. 1-18
Author(s):  
Richard A. Zuellig ◽  
Beat C. Bornhauser ◽  
Ralf Amstutz ◽  
Bruno Constantin ◽  
Marcus C. Schaub
Keyword(s):  
Actin Cytoskeleton ◽  
Tissue Expression ◽  
Protein Product ◽  
Binding Domain ◽  
Actin Binding ◽  
Rat Tissues ◽  
Cortical Actin ◽  
Actin Binding Domain ◽  
Spectrin Repeats

Utrophin and dystrophin present two large proteins that link the intracellular actin cytoskeleton to the extracellular matrix via the C-terminal-associated protein complex. Here we describe a novel short N-terminal isoform of utrophin and its protein product in various rat tissues (N-utro, 62 kDa, amino acids 1–539, comprising the actin-binding domain plus the first two spectrin repeats). Using different N-terminal recombinant utrophin fragments, we show that actin binding exhibits pronounced negative cooperativity (affinity constantsK1=∼5×106andK2=∼1×105 M-1) and is Ca2+-insensitive. Expression of the different fragments in COS7 cells and in myotubes indicates that the actin-binding domain alone binds exlusively to actin filaments. The recombinant N-utro analogue binds in vitro to actin and in the cells associates to the membranes. The results indicate that N-utro may be responsible for the anchoring of the cortical actin cytoskeleton to the membranes in muscle and other tissues.

scholarly journals Evolution of a cis-acting SNP that controls Type VI Secretion in Vibrio cholerae

2022 ◽  
Author(s):  
Siu Lung Ng ◽  
Sophia A. Kammann ◽  
Gabi Steinbach ◽  
Tobias Hoffmann ◽  
Peter J. Yunker ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Phenotypic Diversity ◽  
Constitutive Expression ◽  
Regulatory Elements ◽  
Regulatory Control ◽  
Type Vi Secretion System ◽  
Type Vi Secretion ◽  
Intergenic Regions

Mutations in regulatory mechanisms that control gene expression contribute to phenotypic diversity and thus facilitate the adaptation of microbes to new niches. Regulatory architecture is often inferred from transcription factor identification and genome analysis using purely computational approaches. However, there are few examples of phenotypic divergence that arise from the rewiring of bacterial regulatory circuity by mutations in intergenic regions, because locating regulatory elements within regions of DNA that do not code for protein requires genomic and experimental data. We identify a single cis-acting single nucleotide polymorphism (SNP) dramatically alters control of the type VI secretion system (T6), a common weapon for inter-bacterial competition. Tight T6 regulatory control is necessary for adaptation of the waterborne pathogen Vibrio cholerae to in vivo conditions within the human gut, which we show can be altered by this single non-coding SNP that results in constitutive expression in vitro. Our results support a model of pathogen evolution through cis-regulatory mutation and preexisting, active transcription factors, thus conferring different fitness advantages to tightly regulated strains inside a human host and unfettered strains adapted to environmental niches.

scholarly journals Mechanosensing through direct binding of tensed F-actin by LIM domains

2020 ◽  
Author(s):  
Xiaoyu Sun ◽  
Donovan Y. Z. Phua ◽  
Lucas Axiotakis ◽  
Mark A. Smith ◽  
Elizabeth Blankman ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Point Mutations ◽  
Cytoskeletal Proteins ◽  
Actin Binding ◽  
General Mechanism ◽  
Protein Protein Interaction ◽  
Cytoplasmic Actin ◽  
Lim Domains ◽  
Lim Proteins

SummaryMechanical signals transmitted through the cytoplasmic actin cytoskeleton must be relayed to the nucleus to control gene expression. LIM domains are protein-protein interaction modules found in cytoskeletal proteins and transcriptional regulators; however, it is unclear if there is a direct link between these two functions. Here we identify three LIM protein families (zyxin, paxillin, and FHL) whose members preferentially localize to the actin cytoskeleton in mechanically-stimulated cells through their tandem LIM domains. A minimal actin-myosin reconstitution system reveals that representatives of all three families directly bind F-actin only in the presence of mechanical force. Point mutations at a site conserved in each LIM domain of these proteins selectively disrupt tensed F-actin binding in vitro and cytoskeletal localization in cells, demonstrating a common, avidity-based mechanism. Finally, we find that binding to tensed F-actin in the cytoplasm excludes the cancer-associated transcriptional co-activator FHL2 from the nucleus in stiff microenvironments. This establishes direct force-activated F-actin binding by FHL2 as a mechanosensing mechanism. Our studies suggest that force-dependent sequestration of LIM proteins on the actin cytoskeleton could be a general mechanism for controlling nuclear localization to effect mechanical signaling.

scholarly journals Actin Cytoskeleton Manipulation by Effector Proteins Secreted by DiarrheagenicEscherichia coliPathotypes

2013 ◽  
Vol 2013 ◽  
pp. 1-22 ◽  
Author(s):  
Fernando Navarro-Garcia ◽  
Antonio Serapio-Palacios ◽  
Paul Ugalde-Silva ◽  
Gabriela Tapia-Pastrana ◽  
Lucia Chavez-Dueñas
Keyword(s):  
Actin Cytoskeleton ◽  
Host Cell ◽  
Virulence Factors ◽  
Cell Biology ◽  
Bacterial Species ◽  
Effector Proteins ◽  
Actin Binding ◽  
E Coli ◽  
Tissue Organization ◽  
Epithelial Barriers

The actin cytoskeleton is a dynamic structure necessary for cell and tissue organization, including the maintenance of epithelial barriers. Disruption of the epithelial barrier coincides with alterations of the actin cytoskeleton in several disease states. These disruptions primarily affect the paracellular space, which is normally regulated by tight junctions. Thereby, the actin cytoskeleton is a common and recurring target of bacterial virulence factors. In order to manipulate the actin cytoskeleton, bacteria secrete and inject toxins and effectors to hijack the host cell machinery, which interferes with host-cell pathways and with a number of actin binding proteins. An interesting model to study actin manipulation by bacterial effectors isEscherichia colisince due to its genome plasticity it has acquired diverse genetic mobile elements, which allow having differentE. colivarieties in one bacterial species. TheseE. colipathotypes, including intracellular and extracellular bacteria, interact with epithelial cells, and their interactions depend on a specific combination of virulence factors. In this paper we focus onE. colieffectors that mimic host cell proteins to manipulate the actin cytoskeleton. The study of bacterial effector-cytoskeleton interaction will contribute not only to the comprehension of the molecular causes of infectious diseases but also to increase our knowledge of cell biology.

scholarly journals Paraburkholderia phymatum STM815 σ54 Controls Utilization of Dicarboxylates, Motility, and T6SS-b Expression

Nitrogen ◽  
2020 ◽  
Vol 1 (2) ◽  
pp. 81-98
Author(s):  
Martina Lardi ◽  
Yilei Liu ◽  
Sebastian Hug ◽  
Samanta Bolzan de Campos ◽  
Leo Eberl ◽  
...  
Keyword(s):  
Wild Type Strain ◽  
Nitrogenase Activity ◽  
Secretion Systems ◽  
Free Living ◽  
Major Life ◽  
Life Styles ◽  
Type Vi Secretion ◽  
Flagellar Biosynthesis ◽  
Free Living Conditions

Rhizobia have two major life styles, one as free-living bacteria in the soil, and the other as bacteroids within the root/stem nodules of host legumes where they convert atmospheric nitrogen into ammonia. In the soil, rhizobia have to cope with changing and sometimes stressful environmental conditions, such as nitrogen limitation. In the beta-rhizobial strain Paraburkholderia phymatum STM815, the alternative sigma factor σ54 (or RpoN) has recently been shown to control nitrogenase activity during symbiosis with Phaseolus vulgaris. In this study, we determined P. phymatum’s σ54 regulon under nitrogen-limited free-living conditions. Among the genes significantly downregulated in the absence of σ54, we found a C4-dicarboxylate carrier protein (Bphy_0225), a flagellar biosynthesis cluster (Bphy_2926-64), and one of the two type VI secretion systems (T6SS-b) present in the P. phymatum STM815 genome (Bphy_5978-97). A defined σ54 mutant was unable to grow on C4 dicarboxylates as sole carbon source and was less motile compared to the wild-type strain. Both defects could be complemented by introducing rpoNin trans. Using promoter reporter gene fusions, we also confirmed that the expression of the T6SS-b cluster is regulated by σ54. Accordingly, we show that σ54 affects in vitro competitiveness of P. phymatum STM815 against Paraburkholderia diazotrophica.

scholarly journals STIP1/HOP Regulates the Actin Cytoskeleton through Interactions with Actin and Changes in Actin-Binding Proteins Cofilin and Profilin

2020 ◽  
Vol 21 (9) ◽  
pp. 3152 ◽  
Author(s):  
Samantha Joy Beckley ◽  
Morgan Campbell Hunter ◽  
Sarah Naulikha Kituyi ◽  
Ianthe Wingate ◽  
Abantika Chakraborty ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Binding Proteins ◽  
Major Effect ◽  
Vital Role ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Nuclear Accumulation ◽  
Actin Binding Proteins ◽  
Hsp90 Chaperone

Cell migration plays a vital role in both health and disease. It is driven by reorganization of the actin cytoskeleton, which is regulated by actin-binding proteins cofilin and profilin. Stress-inducible phosphoprotein 1 (STIP1) is a well-described co-chaperone of the Hsp90 chaperone system, and our findings identify a potential regulatory role of STIP1 in actin dynamics. We show that STIP1 can be isolated in complex with actin and Hsp90 from HEK293T cells and directly interacts with actin in vitro via the C-terminal TPR2AB-DP2 domain of STIP1, potentially due to a region spanning two putative actin-binding motifs. We found that STIP1 could stimulate the in vitro ATPase activity of actin, suggesting a potential role in the modulation of F-actin formation. Interestingly, while STIP1 depletion in HEK293T cells had no major effect on total actin levels, it led to increased nuclear accumulation of actin, disorganization of F-actin structures, and an increase and decrease in cofilin and profilin levels, respectively. This study suggests that STIP1 regulates the cytoskeleton by interacting with actin, or via regulating the ratio of proteins known to affect actin dynamics.

scholarly journals Structure of artificial and natural VE-cadherinbased adherens junctions

2008 ◽  
Vol 36 (2) ◽  
pp. 189-193 ◽  
Author(s):  
Jean-Christophe Taveau ◽  
Mathilde Dubois ◽  
Olivier Le Bihan ◽  
Sylvain Trépout ◽  
Sébastien Almagro ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Actin Cytoskeleton ◽  
Self Assembly ◽  
Adherens Junctions ◽  
Actin Binding ◽  
Vascular Endothelial ◽  
Vascular Integrity ◽  
Trans Orientation ◽  
Annexin 2

In vascular endothelium, adherens junctions between endothelial cells are composed of VE-cadherin (vascular endothelial cadherin), an adhesive receptor that is crucial for the proper assembly of vascular structures and the maintenance of vascular integrity. As a classical cadherin, VE-cadherin links endothelial cells together by homophilic interactions mediated by its extracellular part and associates intracellularly with the actin cytoskeleton via catenins. Although, from structural crystallographic data, a dimeric structure arranged in a trans orientation has emerged as a potential mechanism of cell–cell adhesion, the cadherin organization within adherens junctions remains controversial. Concerning VE-cadherin, its extracellular part possesses the capacity to self-associate in solution as hexamers consisting of three antiparallel cadherin dimers. VE-cadherin-based adherens junctions were reconstituted in vitro by assembly of a VE-cadherin EC (extracellular repeat) 1–EC4 hexamer at the surfaces of liposomes. The artificial adherens junctions revealed by cryoelectron microscopy appear as a two-dimensional self-assembly of hexameric structures. This cadherin organization is reminiscent of that found in native desmosomal junctions. Further structural studies performed on native VE-cadherin junctions would provide a better understanding of the cadherin organization within adherens junctions. Homophilic interactions between cadherins are strengthened intracellularly by connection to the actin cytoskeleton. Recently, we have discovered that annexin 2, an actin-binding protein connects the VE-cadherin–catenin complex to the actin cytoskeleton. This novel link is labile and promotes the endothelial cell switch from a quiescent to an angiogenic state.

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