2024-03-28T17:23:58Zhttp://repository.helmholtz-hzi.de/oai/requestoai:repository.helmholtz-hzi.de:10033/3385552019-08-30T11:28:23Zcom_10033_338554col_10033_338544
The EHEC-host interactome reveals novel targets for the translocated intimin receptor.
Blasche, Sonja
Arens, Stefan
Ceol, Arnaud
Siszler, Gabriella
Schmidt, M Alexander
Häuser, Roman
Schwarz, Frank
Wuchty, Stefan
Aloy, Patrick
Uetz, Peter
Stradal, Theresia
Koegl, Manfred
Helmholtz Centre for infection research; Inhooffenstr. 7; D-38124 Braunschweig; Germany.
Enterohemorrhagic E. coli (EHEC) manipulate their human host through at least 39 effector proteins which hijack host processes through direct protein-protein interactions (PPIs). To identify their protein targets in the host cells, we performed yeast two-hybrid screens, allowing us to find 48 high-confidence protein-protein interactions between 15 EHEC effectors and 47 human host proteins. In comparison to other bacteria and viruses we found that EHEC effectors bind more frequently to hub proteins as well as to proteins that participate in a higher number of protein complexes. The data set includes six new interactions that involve the translocated intimin receptor (TIR), namely HPCAL1, HPCAL4, NCALD, ARRB1, PDE6D, and STK16. We compared these TIR interactions in EHEC and enteropathogenic E. coli (EPEC) and found that five interactions were conserved. Notably, the conserved interactions included those of serine/threonine kinase 16 (STK16), hippocalcin-like 1 (HPCAL1) as well as neurocalcin-delta (NCALD). These proteins co-localize with the infection sites of EPEC. Furthermore, our results suggest putative functions of poorly characterized effectors (EspJ, EspY1). In particular, we observed that EspJ is connected to the microtubule system while EspY1 appears to be involved in apoptosis/cell cycle regulation.
2015-01-19T15:02:33Z
2015-01-19T15:02:33Z
2014
Article
The EHEC-host interactome reveals novel targets for the translocated intimin receptor. 2014, 4:7531 Sci Rep
2045-2322
25519916
10.1038/srep07531
http://hdl.handle.net/10033/338555
Scientific reports
en
oai:repository.helmholtz-hzi.de:10033/5963752019-08-30T11:36:05Zcom_10033_338554col_10033_338544
How distinct Arp2/3 complex variants regulate actin filament assembly.
Rottner, Klemens
Stradal, Theresia E B
Helmholtz Centre for infection research, Inhoffenstr. 7, D-38124 Braunschweig, Germany.
The heptameric Arp2/3 complex generates branched actin filament networks that drive lamellipodium protrusion, vesicle trafficking and pathogen motility. Distinct variants of the Arp2/3 complex are now shown to have different roles in tuning actin assembly and disassembly, in concert with the prominent actin regulators cortactin and coronin.
2016-02-16T15:53:36Z
2016-02-16T15:53:36Z
2015-12-23
Article
How distinct Arp2/3 complex variants regulate actin filament assembly. 2015, 18 (1):1-3 Nat. Cell Biol.
1476-4679
26693915
10.1038/ncb3293
http://hdl.handle.net/10033/596375
Nature cell biology
en
oai:repository.helmholtz-hzi.de:10033/6077392019-08-30T11:26:13Zcom_10033_338554col_10033_338544
Perinuclear Arp2/3-driven actin polymerization enables nuclear deformation to facilitate cell migration through complex environments.
Thiam, Hawa-Racine
Vargas, Pablo
Carpi, Nicolas
Crespo, Carolina Lage
Raab, Matthew
Terriac, Emmanuel
King, Megan C
Jacobelli, Jordan
Alberts, Arthur S
Stradal, Theresia
Lennon-Dumenil, Ana-Maria
Piel, Matthieu
Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany.
Cell migration has two opposite faces: although necessary for physiological processes such as immune responses, it can also have detrimental effects by enabling metastatic cells to invade new organs. In vivo, migration occurs in complex environments and often requires a high cellular deformability, a property limited by the cell nucleus. Here we show that dendritic cells, the sentinels of the immune system, possess a mechanism to pass through micrometric constrictions. This mechanism is based on a rapid Arp2/3-dependent actin nucleation around the nucleus that disrupts the nuclear lamina, the main structure limiting nuclear deformability. The cells' requirement for Arp2/3 to pass through constrictions can be relieved when nuclear stiffness is decreased by suppressing lamin A/C expression. We propose a new role for Arp2/3 in three-dimensional cell migration, allowing fast-moving cells such as leukocytes to rapidly and efficiently migrate through narrow gaps, a process probably important for their function.
2016-05-03T13:57:47Z
2016-05-03T13:57:47Z
2016
Article
Perinuclear Arp2/3-driven actin polymerization enables nuclear deformation to facilitate cell migration through complex environments. 2016, 7:10997 Nat Commun
2041-1723
26975831
10.1038/ncomms10997
http://hdl.handle.net/10033/607739
Nature communications
en
oai:repository.helmholtz-hzi.de:10033/6208932019-08-30T11:37:23Zcom_10033_338554col_10033_338544col_10033_620574
FMNL formins boost lamellipodial force generation.
Kage, Frieda
Winterhoff, Moritz
Dimchev, Vanessa
Mueller, Jan
Thalheim, Tobias
Freise, Anika
Brühmann, Stefan
Kollasser, Jana
Block, Jennifer
Dimchev, Georgi
Geyer, Matthias
Schnittler, Hans-Joachim
Brakebusch, Cord
Stradal, Theresia E B
Carlier, Marie-France
Sixt, Michael
Käs, Josef
Faix, Jan
Rottner, Klemens
Helmholtz Centre for infection research, Inhoffenstr.7, 38124 Braunschweig, Germany.
Migration frequently involves Rac-mediated protrusion of lamellipodia, formed by Arp2/3 complex-dependent branching thought to be crucial for force generation and stability of these networks. The formins FMNL2 and FMNL3 are Cdc42 effectors targeting to the lamellipodium tip and shown here to nucleate and elongate actin filaments with complementary activities in vitro. In migrating B16-F1 melanoma cells, both formins contribute to the velocity of lamellipodium protrusion. Loss of FMNL2/3 function in melanoma cells and fibroblasts reduces lamellipodial width, actin filament density and -bundling, without changing patterns of Arp2/3 complex incorporation. Strikingly, in melanoma cells, FMNL2/3 gene inactivation almost completely abolishes protrusion forces exerted by lamellipodia and modifies their ultrastructural organization. Consistently, CRISPR/Cas-mediated depletion of FMNL2/3 in fibroblasts reduces both migration and capability of cells to move against viscous media. Together, we conclude that force generation in lamellipodia strongly depends on FMNL formin activity, operating in addition to Arp2/3 complex-dependent filament branching.
2017-04-07T14:44:37Z
2017-04-07T14:44:37Z
2017-03-22
Article
FMNL formins boost lamellipodial force generation. 2017, 8:14832 Nat Commun
2041-1723
28327544
10.1038/ncomms14832
http://hdl.handle.net/10033/620893
Nature communications
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
oai:repository.helmholtz-hzi.de:10033/6211222019-12-03T01:57:49Zcom_10033_338554col_10033_338544col_10033_620574
FMNL2 and -3 regulate Golgi architecture and anterograde transport downstream of Cdc42.
Kage, Frieda
Steffen, Anika
Ellinger, Adolf
Ranftler, Carmen
Gehre, Christian
Brakebusch, Cord
Pavelka, Margit
Stradal, Theresia
Rottner, Klemens
Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr.7, 38124 Braunschweig, Germany.
The Rho-family small GTPase Cdc42 localizes at plasma membrane and Golgi complex and aside from protrusion and migration operates in vesicle trafficking, endo- and exocytosis as well as establishment and/or maintenance of cell polarity. The formin family members FMNL2 and -3 are actin assembly factors established to regulate cell edge protrusion during migration and invasion. Here we report these formins to additionally accumulate and function at the Golgi apparatus. As opposed to lamellipodia, Golgi targeting of these proteins required both their N-terminal myristoylation and the interaction with Cdc42. Moreover, Golgi association of FMNL2 or -3 induced a phalloidin-detectable actin meshwork around the Golgi. Importantly, functional interference with FMNL2/3 formins by RNAi or CRISPR/Cas9-mediated gene deletion invariably induced Golgi fragmentation in different cell lines. Furthermore, absence of these proteins led to enlargement of endosomes as well as defective maturation and/or sorting into late endosomes and lysosomes. In line with Cdc42 - recently established to regulate anterograde transport through the Golgi by cargo sorting and carrier formation - FMNL2/3 depletion also affected anterograde trafficking of VSV-G from the Golgi to the plasma membrane. Our data thus link FMNL2/3 formins to actin assembly-dependent functions of Cdc42 in anterograde transport through the Golgi apparatus.
2017-09-26T12:48:35Z
2017-09-26T12:48:35Z
2017-08-29
Article
FMNL2 and -3 regulate Golgi architecture and anterograde transport downstream of Cdc42. 2017, 7 (1):9791 Sci Rep
2045-2322
28852060
10.1038/s41598-017-09952-1
http://hdl.handle.net/10033/621122
Scientific reports
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
oai:repository.helmholtz-hzi.de:10033/6214572019-08-30T11:29:14Zcom_10033_338554col_10033_338544
Actin dynamics in host-pathogen interaction.
Stradal, Theresia E B
Schelhaas, Mario
Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany.
actin dynamics
bacterial invasion
host-pathogen interaction
viral entry
virulence factors
The actin cytoskeleton and Rho GTPase signaling to actin assembly are prime targets of bacterial and viral pathogens, simply because actin is involved in all motile and membrane remodeling processes, such as phagocytosis, macropinocytosis, endocytosis, exocytosis, vesicular trafficking and membrane fusion events, motility, and last but not least, autophagy. This article aims at providing an overview of the most prominent pathogen-induced or -hijacked actin structures, and an outlook on how future research might uncover additional, equally sophisticated interactions.
2018-08-28T14:11:31Z
2018-08-28T14:11:31Z
2018-06-23
Article
1873-3468
29935019
10.1002/1873-3468.13173
http://hdl.handle.net/10033/621457
http://creativecommons.org/licenses/by-nc-sa/3.0/us/
Attribution-NonCommercial-ShareAlike 3.0 United States
FEBS letters
oai:repository.helmholtz-hzi.de:10033/6215752019-08-30T11:29:42Zcom_10033_338554com_10033_311308col_10033_559591col_10033_338544col_10033_620574
Distinct Interaction Sites of Rac GTPase with WAVE Regulatory Complex Have Non-redundant Functions in Vivo.
Schaks, Matthias
Singh, Shashi P
Kage, Frieda
Thomason, Peter
Klünemann, Thomas
Steffen, Anika
Blankenfeldt, Wulf
Stradal, Theresia E
Insall, Robert H
Rottner, Klemens
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Arp2/3 complex
CRISPR/CAS9
Rho-GTPase
filopodium
lamellipodium
migration
protrusion
Cell migration often involves the formation of sheet-like lamellipodia generated by branched actin filaments. The branches are initiated when Arp2/3 complex [1] is activated by WAVE regulatory complex (WRC) downstream of small GTPases of the Rac family [2]. Recent structural studies defined two independent Rac binding sites on WRC within the Sra-1/PIR121 subunit of the pentameric WRC [3, 4], but the functions of these sites in vivo have remained unknown. Here we dissect the mechanism of WRC activation and the in vivo relevance of distinct Rac binding sites on Sra-1, using CRISPR/Cas9-mediated gene disruption of Sra-1 and its paralog PIR121 in murine B16-F1 cells combined with Sra-1 mutant rescue. We show that the A site, positioned adjacent to the binding region of WAVE-WCA mediating actin and Arp2/3 complex binding, is the main site for allosteric activation of WRC. In contrast, the D site toward the C terminus is dispensable for WRC activation but required for optimal lamellipodium morphology and function. These results were confirmed in evolutionarily distant Dictyostelium cells. Moreover, the phenotype seen in D site mutants was recapitulated in Rac1 E31 and F37 mutants; we conclude these residues are important for Rac-D site interaction. Finally, constitutively activated WRC was able to induce lamellipodia even after both Rac interaction sites were lost, showing that Rac interaction is not essential for membrane recruitment. Our data establish that physical interaction with Rac is required for WRC activation, in particular through the A site, but is not mandatory for WRC accumulation in the lamellipodium.
2018-11-20T13:29:37Z
2018-11-20T13:29:37Z
2018-10-25
Article
1879-0445
30393033
10.1016/j.cub.2018.10.002
http://hdl.handle.net/10033/621575
http://creativecommons.org/licenses/by-nc-sa/3.0/us/
Attribution-NonCommercial-ShareAlike 3.0 United States
Current biology : CB
oai:repository.helmholtz-hzi.de:10033/6215992019-08-30T11:30:27Zcom_10033_338554col_10033_338544
Regulation of MRTF-A by JMY via a nucleation-independent mechanism.
Kluge, Franziska
Weissbach, Julia
Weber, Anja
Stradal, Theresia
Posern, Guido
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Actin
JMY
Mrtf
Nucleators
Transcription
WH2 domains
MRTF-A (myocardin-related transcription factor A) is a coactivator for SRF-mediated gene expression. The activity of MRTF-A is critically dependent on the dissociation of G-actin from N-terminal RPEL motifs. MRTF-SRF induction often correlates with enhanced polymerization of F-actin. Here we investigate MRTF regulation by the multifunctional JMY protein, which contains three WASP/verprolin homology 2 (WH2/V) domains and facilitates Arp2/3-dependent and -independent actin nucleation. Co-immunoprecipitation experiments, immunofluorescence and luciferase reporter assays were combined with selective inhibitors to investigate the effect of JMY and its domains on MRTF-A in NIH 3 T3 mouse fibroblasts. JMY induced MRTF-A transcriptional activity and enhanced its nuclear translocation. Unexpectedly, MRTF-A was hyperactivated when the Arp2/3-recruiting CA region of JMY was deleted or mutated, suggesting an autoinhibitory mechanism for full-length JMY. Moreover, isolated WH2/V domains which are unable to nucleate actin were sufficient for nuclear accumulation and SRF activation. Recombinant WH2/V regions of JMY biochemically competed with MRTF-A for actin binding. Activation of MRTF-A by JMY was unaffected by Arp3 knockdown, by an Arp2/3 inhibitor, and by latrunculin which disassembles cellular F-actin. Restriction of JMY to the nucleus abrogated its MRTF-A activation. Finally, JMY RNAi reduced basal and stimulated transcriptional activation via MRTF-A. Our results suggest that JMY activates MRTF-SRF independently of F-actin via WH2/V-mediated competition with the RPEL region for G-actin binding in the cytoplasm. Furthermore, the C-terminal region facilitates an autoinhibitory effect on full-length JMY, possibly by intramolecular folding.
2018-12-04T12:10:56Z
2018-12-04T12:10:56Z
2018-11-21
Article
1478-811X
30463620
10.1186/s12964-018-0299-x
http://hdl.handle.net/10033/621599
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
Springer-Nature
Cell communication and signaling : CCS
oai:repository.helmholtz-hzi.de:10033/6216762019-08-30T11:29:14Zcom_10033_338554col_10033_338544
Visualization of translocons in Yersinia type III protein secretion machines during host cell infection.
Nauth, Theresa
Huschka, Franziska
Schweizer, Michaela
Bosse, Jens B
Diepold, Andreas
Failla, Antonio Virgilio
Steffen, Anika
Stradal, Theresia E B
Wolters, Manuel
Aepfelbacher, Martin
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Type III secretion systems (T3SSs) are essential virulence factors of numerous bacterial pathogens. Upon host cell contact the T3SS machinery-also named injectisome-assembles a pore complex/translocon within host cell membranes that serves as an entry gate for the bacterial effectors. Whether and how translocons are physically connected to injectisome needles, whether their phenotype is related to the level of effector translocation and which target cell factors trigger their formation have remained unclear. We employed the superresolution fluorescence microscopy techniques Stimulated Emission Depletion (STED) and Structured Illumination Microscopy (SIM) as well as immunogold electron microscopy to visualize Y. enterocolitica translocons during infection of different target cell types. Thereby we were able to resolve translocon and needle complex proteins within the same injectisomes and demonstrate that these fully assembled injectisomes are generated in a prevacuole, a PI(4,5)P2 enriched host cell compartment inaccessible to large extracellular proteins like antibodies. Furthermore, the operable translocons were produced by the yersiniae to a much larger degree in macrophages (up to 25% of bacteria) than in HeLa cells (2% of bacteria). However, when the Rho GTPase Rac1 was activated in the HeLa cells, uptake of the yersiniae into the prevacuole, translocon formation and effector translocation were strongly enhanced reaching the same levels as in macrophages. Our findings indicate that operable T3SS translocons can be visualized as part of fully assembled injectisomes with superresolution fluorescence microscopy techniques. By using this technology, we provide novel information about the spatiotemporal organization of T3SS translocons and their regulation by host cell factors.
2019-02-01T14:58:16Z
2019-02-01T14:58:16Z
2018-12-01
Article
1553-7374
30586431
10.1371/journal.ppat.1007527
http://hdl.handle.net/10033/621676
Plospathogens
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
PLOS
PLoS pathogens
oai:repository.helmholtz-hzi.de:10033/6216832019-08-30T11:33:04Zcom_10033_620533com_10033_338554com_10033_311308col_10033_620534col_10033_620721col_10033_338544
xCELLanalyzer: A Framework for the Analysis of Cellular Impedance Measurements for Mode of Action Discovery
Franke, Raimo
Hinkelmann, Bettina
Fetz, Verena
Stradal, Theresia
Sasse, Florenz
Klawonn, Frank
Brönstrup, Mark
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
mode of action
impedance spectroscopy
target identification
natural products
actinomycin D
Mode of action (MoA) identification of bioactive compounds is very often a challenging and time-consuming task. We used a label-free kinetic profiling method based on an impedance readout to monitor the time-dependent cellular response profiles for the interaction of bioactive natural products and other small molecules with mammalian cells. Such approaches have been rarely used so far due to the lack of data mining tools to properly capture the characteristics of the impedance curves. We developed a data analysis pipeline for the xCELLigence Real-Time Cell Analysis detection platform to process the data, assess and score their reproducibility, and provide rank-based MoA predictions for a reference set of 60 bioactive compounds. The method can reveal additional, previously unknown targets, as exemplified by the identification of tubulin-destabilizing activities of the RNA synthesis inhibitor actinomycin D and the effects on DNA replication of vioprolide A. The data analysis pipeline is based on the statistical programming language R and is available to the scientific community through a GitHub repository.
2019-02-12T08:42:10Z
2019-02-12T08:42:10Z
2019-01-25
Article
2472-5552
2472-5560
30681906
10.1177/2472555218819459
http://hdl.handle.net/10033/621683
SLAS Discovery
http://journals.sagepub.com/doi/10.1177/2472555218819459
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
Sage
247255521881945
SLAS DISCOVERY: Advancing Life Sciences R&D
oai:repository.helmholtz-hzi.de:10033/6217162019-11-21T12:00:01Zcom_10033_620857com_10033_338554col_10033_620858col_10033_338544
The Effect of Cytochalasans on the Actin Cytoskeleton of Eukaryotic Cells and Preliminary Structure⁻Activity Relationships.
Kretz, Robin
Wendt, Lucile
Wongkanoun, Sarunyou
Luangsa-Ard, J Jennifer
Surup, Frank
Helaly, Soleiman E
Noumeur, Sara R
Stadler, Marc
Stradal, Theresia E B
HZI, Helmholtz Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig Germany.
Ascomycota
Xylariales
actin cytoskeleton
chromatography
secondary metabolites
structure elucidation
In our ongoing search for new bioactive fungal metabolites, two new cytochalasans were isolated from stromata of the hypoxylaceous ascomycete Hypoxylon fragiforme. Their structures were elucidated via high-resolution mass spectrometry (HR-MS) and nuclear magnetic resonance (NMR) spectroscopy. Together with 23 additional cytochalasans isolated from ascomata and mycelial cultures of different Ascomycota, they were tested on their ability to disrupt the actin cytoskeleton of mammal cells in a preliminary structure–activity relationship study. Out of all structural features, the presence of hydroxyl group at the C7 and C18 residues, as well as their stereochemistry, were determined as important factors affecting the potential to disrupt the actin cytoskeleton. Moreover, reversibility of the actin disrupting effects was tested, revealing no direct correlations between potency and reversibility in the tested compound group. Since the diverse bioactivity of cytochalasans is interesting for various applications in eukaryotes, the exact effect on eukaryotic cells will need to be determined, e.g., by follow-up studies involving medicinal chemistry and by inclusion of additional natural cytochalasans. The results are also discussed in relation to previous studies in the literature, including a recent report on the anti-Biofilm activities of essentially the same panel of compounds against the pathogenic bacterium, Staphylococcus aureus. View Full-Text
2019-03-06T15:27:16Z
2019-03-06T15:27:16Z
2019-02-19
Article
Biomolecules. 2019 Feb 19;9(2). pii: biom9020073. doi: 10.3390/biom9020073
2218-273X
30791504
10.3390/biom9020073
http://hdl.handle.net/10033/621716
Biomolecules
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
MPDI
Biomolecules
oai:repository.helmholtz-hzi.de:10033/6217392019-08-30T11:30:26Zcom_10033_620857com_10033_338554col_10033_620858col_10033_338544
The Effect of Cytochalasans on the Actin Cytoskeleton of Eukaryotic Cells and Preliminary Structure⁻Activity Relationships.
Kretz, Robin
Wendt, Lucile
Wongkanoun, Sarunyou
Luangsa-Ard, J Jennifer
Surup, Frank
Helaly, Soleiman E
Noumeur, Sara R
Stadler, Marc
Stradal, Theresia E B
HZI, Helmholtz Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig Germany.
Ascomycota
Xylariales
actin cytoskeleton
chromatography
secondary metabolites
structure elucidation
In our ongoing search for new bioactive fungal metabolites, two new cytochalasans were isolated from stromata of the hypoxylaceous ascomycete Hypoxylon fragiforme. Their structures were elucidated via high-resolution mass spectrometry (HR-MS) and nuclear magnetic resonance (NMR) spectroscopy. Together with 23 additional cytochalasans isolated from ascomata and mycelial cultures of different Ascomycota, they were tested on their ability to disrupt the actin cytoskeleton of mammal cells in a preliminary structure⁻activity relationship study. Out of all structural features, the presence of hydroxyl group at the C7 and C18 residues, as well as their stereochemistry, were determined as important factors affecting the potential to disrupt the actin cytoskeleton. Moreover, reversibility of the actin disrupting effects was tested, revealing no direct correlations between potency and reversibility in the tested compound group. Since the diverse bioactivity of cytochalasans is interesting for various applications in eukaryotes, the exact effect on eukaryotic cells will need to be determined, e.g., by follow-up studies involving medicinal chemistry and by inclusion of additional natural cytochalasans. The results are also discussed in relation to previous studies in the literature, including a recent report on the anti-Biofilm activities of essentially the same panel of compounds against the pathogenic bacterium, Staphylococcus aureus.
2019-04-04T13:54:21Z
2019-04-04T13:54:21Z
2019-02-19
Article
2218-273X
30791504
10.3390/biom9020073
http://hdl.handle.net/10033/621739
Biomolecules
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
MDPI
Biomolecules
oai:repository.helmholtz-hzi.de:10033/6217632019-08-30T11:35:08Zcom_10033_620636com_10033_338554col_10033_620637col_10033_338544
Spatiotemporal control of FlgZ activity impacts Pseudomonas aeruginosa flagellar motility.
Bense, Sarina
Bruchmann, Sebastian
Steffen, Anika
Stradal, Theresia E B
Häussler, Susanne
Düvel, Juliane
HZI, Helmholtz Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig Germany.
The c-di-GMP-binding effector protein FlgZ has been demonstrated to control motility in the opportunistic pathogen Pseudomonas aeruginosa and it was suggested that c-di-GMP-bound FlgZ impedes motility via its interaction with the MotCD stator. To further understand how motility is downregulated in P. aeruginosa and to elucidate the general control mechanisms operating during bacterial growth, we examined the spatiotemporal activity of FlgZ. We re-annotated the P. aeruginosaflgZ open reading frame and demonstrated that FlgZ-mediated downregulation of motility is fine-tuned via three independent mechanisms. First, we found that flgZ gene is transcribed independently from flgMN in stationary growth phase to increase FlgZ protein levels in the cell. Second, FlgZ localizes to the cell pole upon c-di-GMP binding and third, we describe that FimV, a cell pole anchor protein, is involved in increasing the polar localized c-di-GMP bound FlgZ to inhibit both, swimming and swarming motility. Our results shed light on the complex dynamics and spatiotemporal control of c-di-GMP-dependent bacterial motility phenotypes and on how the polar anchor protein FimV, the motor brake FlgZ and the stator proteins function to repress flagella-driven swimming and swarming motility.
2019-05-07T14:03:56Z
2019-05-07T14:03:56Z
2019-03-12
Article
Mol Microbiol. 2019 Mar 12. doi: 10.1111/mmi.14236.
1365-2958
30864240
10.1111/mmi.14236
http://hdl.handle.net/10033/621763
Molecular microbiology
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
Wiley-Blackwell
Molecular microbiology
oai:repository.helmholtz-hzi.de:10033/6221892020-04-07T05:14:41Zcom_10033_338554col_10033_338544
Lamellipodin tunes cell migration by stabilizing protrusions and promoting adhesion formation.
Dimchev, Georgi
Amiri, Behnam
Humphries, Ashley C
Schaks, Matthias
Dimchev, Vanessa
Stradal, Theresia E B
Faix, Jan
Krause, Matthias
Way, Michael
Falcke, Martin
Rottner, Klemens
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Arp2/3
Lamellipodium
VASP
WAVE regulatory complex
Efficient migration on adhesive surfaces involves the protrusion of lamellipodial actin networks and their subsequent stabilization by nascent adhesions. The actin binding protein lamellipodin (Lpd) is thought to play a critical role in lamellipodium protrusion, by delivering Ena/VASP proteins onto the growing plus ends of actin filaments and by interacting with the WAVE regulatory complex (WRC), an activator of the Arp2/3 complex, at the leading edge. Using B16-F1 melanoma cell lines, we demonstrate that genetic ablation of Lpd compromises protrusion efficiency and coincident cell migration without altering essential parameters of lamellipodia, including their maximal rate of forward advancement and actin polymerization. We also confirmed lamellipodia and migration phenotypes with CRISPR/Cas9-mediated Lpd knockout Rat2 fibroblasts, excluding cell type-specific effects. Moreover, computer-aided analysis of cell edge morphodynamics on B16-F1 cell lamellipodia revealed that loss of Lpd correlates with reduced temporal protrusion maintenance as a prerequisite of nascent adhesion formation. We conclude that Lpd optimizes protrusion and nascent adhesion formation by counteracting frequent, chaotic retraction and membrane ruffling.
2020-03-06T09:52:03Z
2020-03-06T09:52:03Z
2020-02-24
Article
J Cell Sci. 2020 Feb 24. pii: jcs.239020. doi: 10.1242/jcs.239020.
1477-9137
32094266
10.1242/jcs.239020
http://hdl.handle.net/10033/622189
Journal of cell science
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
The company of biologists
Journal of cell science
oai:repository.helmholtz-hzi.de:10033/6223322020-07-02T01:31:18Zcom_10033_620857com_10033_338554col_10033_620858col_10033_338544
New Peptaibiotics and a Cyclodepsipeptide from : Isolation, Identification, Cytotoxic and Nematicidal Activities.
Moussa, Ashaimaa Y
Lambert, Christopher
Stradal, Theresia E B
Ashrafi, Samad
Maier, Wolfgang
Stadler, Marc
Helaly, Soleiman E
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Sordariomycetes
cyclodepsipeptides
nematicidal activity
peptaibiotics
structure elucidation
Fungal associations with nematodes have attracted scientific attention because of the need to develop new biocontrol agents. In this context, Ijuhya vitellina, an antagonistic fungus previously isolated from the plant parasitic cyst nematode Heterodera filipjevi, was selected to carry out an in-depth metabolomic study for its active metabolites. Herein, three new nonapeptide peptaibols with leucinostatin based sequences were isolated and identified by 1, 2D NMR, and HR-ESI-MS-MS. The absolute configuration was assigned based on Marfay's analysis and Mosher ester formation. The new leucinostatins manifested moderate nematicidal effect against the plant pathogenic nematode Pratylenchus penetrans with LD90 values ranging from 5 to 7 µg/mL. Furthermore, a cyclodepsipeptide, named arthrichitin D, with five amino acid residues attached to a 3-hydroxy-2,4-dimethylhexadeca-4,6-dienoic fatty acid chain was discovered and showed weak nematicidal effect against Caenorhabditis elegans. Chaetoglobosin B and its 19-O-acetyl derivative were also obtained as minor metabolites, and the activity of chaetoglobosin B on the actin cytoskeleton of mammalian cells was assessed.
2020-07-01T13:12:45Z
2020-07-01T13:12:45Z
2020-03-22
Article
Antibiotics (Basel). 2020;9(3):132. Published 2020 Mar 22. doi:10.3390/antibiotics9030132.
2079-6382
32235703
10.3390/antibiotics9030132
http://hdl.handle.net/10033/622332
Antibiotics (Basel, Switzerland)
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
MDPI
9
3
Antibiotics (Basel, Switzerland)
Switzerland
oai:repository.helmholtz-hzi.de:10033/6224182020-08-26T01:38:27Zcom_10033_338554col_10033_338544col_10033_620574
The Small GTPase Rac1 Increases Cell Surface Stiffness and Enhances 3D Migration Into Extracellular Matrices.
Kunschmann, Tom
Puder, Stefanie
Fischer, Tony
Steffen, Anika
Rottner, Klemens
Mierke, Claudia Tanja
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Membrane ruffling and lamellipodia formation promote the motility of adherent cells in two-dimensional motility assays by mechano-sensing of the microenvironment and initiation of focal adhesions towards their surroundings. Lamellipodium formation is stimulated by small Rho GTPases of the Rac subfamily, since genetic removal of these GTPases abolishes lamellipodium assembly. The relevance of lamellipodial or invadopodial structures for facilitating cellular mechanics and 3D cell motility is still unclear. Here, we hypothesized that Rac1 affects cell mechanics and facilitates 3D invasion. Thus, we explored whether fibroblasts that are genetically deficient for Rac1 (lacking Rac2 and Rac3) harbor altered mechanical properties, such as cellular deformability, intercellular adhesion forces and force exertion, and exhibit alterations in 3D motility. Rac1 knockout and control cells were analyzed for changes in deformability by applying an external force using an optical stretcher. Five Rac1 knockout cell lines were pronouncedly more deformable than Rac1 control cells upon stress application. Using AFM, we found that cell-cell adhesion forces are increased in Rac1 knockout compared to Rac1-expressing fibroblasts. Since mechanical deformability, cell-cell adhesion strength and 3D motility may be functionally connected, we investigated whether increased deformability of Rac1 knockout cells correlates with changes in 3D motility. All five Rac1 knockout clones displayed much lower 3D motility than Rac1-expressing controls. Moreover, force exertion was reduced in Rac1 knockout cells, as assessed by 3D fiber displacement analysis. Interference with cellular stiffness through blocking of actin polymerization by Latrunculin A could not further reduce invasion of Rac1 knockout cells. In contrast, Rac1-expressing controls treated with Latrunculin A were again more deformable and less invasive, suggesting actin polymerization is a major determinant of observed Rac1-dependent effects. Together, we propose that regulation of 3D motility by Rac1 partly involves cellular mechanics such as deformability and exertion of forces.
2020-08-25T08:56:11Z
2020-08-25T08:56:11Z
2019-05-22
Article
Other
Sci Rep. 2019;9(1):7675. Published 2019 May 22. doi:10.1038/s41598-019-43975-0.
31118438
10.1038/s41598-019-43975-0
http://hdl.handle.net/10033/622418
2045-2322
Scientific reports
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
Nature research
9
1
7675
Scientific reports
England
oai:repository.helmholtz-hzi.de:10033/6225462020-10-29T01:35:50Zcom_10033_620857com_10033_338554col_10033_620858col_10033_338544col_10033_620574
Diversely Functionalised Cytochalasins through Mutasynthesis and Semi-Synthesis.
Wang, Chongqing
Lambert, Christopher
Hauser, Maurice
Deuschmann, Adrian
Zeilinger, Carsten
Rottner, Klemens
Stradal, Theresia E B
Stadler, Marc
Skellam, Elizabeth J
Cox, Russell J
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
cytochalasins
molecular tools
mutasynthesis
semi-synthesis
Mutasynthesis of pyrichalasin H from Magnaporthe grisea NI980 yielded a series of unprecedented 4'-substituted cytochalasin analogues in titres as high as the wild-type system (≈60 mg L-1 ). Halogenated, O-alkyl, O-allyl and O-propargyl examples were formed, as well as a 4'-azido analogue. 4'-O-Propargyl and 4'-azido analogues reacted smoothly in Huisgen cycloaddition reactions, whereas p-Br and p-I compounds reacted in Pd-catalysed cross-coupling reactions. A series of examples of biotin-linked, dye-linked and dimeric cytochalasins was rapidly created. In vitro and in vivo bioassays of these compounds showed that the 4'-halogenated and azido derivatives retained their cytotoxicity and antifungal activities; but a unique 4'-amino analogue was inactive. Attachment of larger substituents attenuated the bioactivities. In vivo actin-binding studies with adherent mammalian cells showed that actin remains the likely intracellular target. Dye-linked compounds revealed visualisation of intracellular actin structures even in the absence of phalloidin, thus constituting a potential new class of actin-visualisation tools with filament-barbed end-binding specificity.
2020-10-28T14:15:11Z
2020-10-28T14:15:11Z
2020-06-02
Article
Chemistry. 2020 Jun 2. doi: 10.1002/chem.202002241.
32484589
10.1002/chem.202002241
http://hdl.handle.net/10033/622546
1521-3765
Chemistry (Weinheim an der Bergstrasse, Germany)
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
Wiley-VCH
Chemistry (Weinheim an der Bergstrasse, Germany)
Germany
oai:repository.helmholtz-hzi.de:10033/6225502020-11-03T01:31:23Zcom_10033_620533com_10033_338554com_10033_620636col_10033_620534col_10033_620637col_10033_338544
Host-induced spermidine production in motile triggers phagocytic uptake.
Felgner, Sebastian
Preusse, Matthias
Beutling, Ulrike
Stahnke, Stephanie
Pawar, Vinay
Rohde, Manfred
Brönstrup, Mark
Stradal, Theresia
Häussler, Susanne
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Pseudomonas aeruginosa
dual-sequencing
flagella
host-pathogen interaction
infectious disease
microbiology
motility
spermidine
Exploring the complexity of host-pathogen communication is vital to understand why microbes persist within a host, while others are cleared. Here, we employed a dual-sequencing approach to unravel conversational turn-taking of dynamic host-pathogen communications. We demonstrate that upon hitting a host cell, motile Pseudomonas aeruginosa induce a specific gene expression program. This results in the expression of spermidine on the surface, which specifically activates the PIP3-pathway to induce phagocytic uptake into primary or immortalized murine cells. Non-motile bacteria are more immunogenic due to a lower expression of arnT upon host-cell contact, but do not produce spermidine and are phagocytosed less. We demonstrate that not only the presence of pathogen inherent molecular patterns induces immune responses, but that bacterial motility is linked to a host-cell-induced expression of additional immune modulators. Our results emphasize on the value of integrating microbiological and immunological findings to unravel complex and dynamic host-pathogen interactions.
2020-11-02T11:14:23Z
2020-11-02T11:14:23Z
2020-09-22
Article
Elife. 2020 Sep 22;9:e55744. doi: 10.7554/eLife.55744.
32960172
10.7554/eLife.55744
http://hdl.handle.net/10033/622550
2050-084X
eLife
en
info:eu-repo/grantAgreement/EC//H2020724290
http://creativecommons.org/licenses/by-nc-sa/4.0/
openAccess
Attribution-NonCommercial-ShareAlike 4.0 International
elifeSciences
9
eLife
International
International
International
International
England
oai:repository.helmholtz-hzi.de:10033/6227422021-02-16T03:17:51Zcom_10033_311624com_10033_6839com_10033_311308com_10033_338554col_10033_311625col_10033_620721col_10033_559591col_10033_338544
Crystal structure of bacterial cytotoxic necrotizing factor CNFy reveals molecular building blocks for intoxication.
Chaoprasid, Paweena
Lukat, Peer
Mühlen, Sabrina
Heidler, Thomas
Gazdag, Emerich-Mihai
Dong, Shuangshuang
Bi, Wenjie
Rüter, Christian
Kirchenwitz, Marco
Steffen, Anika
Jänsch, Lothar
Stradal, Theresia E B
Dersch, Petra
Blankenfeldt, Wulf
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Yersinia
AB-toxin
ADP-ribosyl transferase
CNF
DUF4765
Cytotoxic necrotizing factors (CNFs) are bacterial single-chain exotoxins that modulate cytokinetic/oncogenic and inflammatory processes through activation of host cell Rho GTPases. To achieve this, they are secreted, bind surface receptors to induce endocytosis and translocate a catalytic unit into the cytosol to intoxicate host cells. A three-dimensional structure that provides insight into the underlying mechanisms is still lacking. Here, we determined the crystal structure of full-length Yersinia pseudotuberculosis CNFY . CNFY consists of five domains (D1-D5), and by integrating structural and functional data, we demonstrate that D1-3 act as export and translocation module for the catalytic unit (D4-5) and for a fused β-lactamase reporter protein. We further found that D4, which possesses structural similarity to ADP-ribosyl transferases, but had no equivalent catalytic activity, changed its position to interact extensively with D5 in the crystal structure of the free D4-5 fragment. This liberates D5 from a semi-blocked conformation in full-length CNFY , leading to higher deamidation activity. Finally, we identify CNF translocation modules in several uncharacterized fusion proteins, which suggests their usability as a broad-specificity protein delivery tool.
2021-02-15T14:54:01Z
2021-02-15T14:54:01Z
2021-01-07
Article
EMBO J. 2021 Jan 7:e105202. doi: 10.15252/embj.2020105202. Epub ahead of print.
33410511
10.15252/embj.2020105202
http://hdl.handle.net/10033/622742
1460-2075
The EMBO journal
en
http://creativecommons.org/licenses/by/4.0/
Attribution 4.0 International
Springer
e105202
The EMBO journal
England
oai:repository.helmholtz-hzi.de:10033/6227842021-03-23T01:30:51Zcom_10033_338554col_10033_338544col_10033_620574
Induced Arp2/3 Complex Depletion Increases FMNL2/3 Formin Expression and Filopodia Formation.
Dimchev, Vanessa
Lahmann, Ines
Koestler, Stefan A
Kage, Frieda
Dimchev, Georgi
Steffen, Anika
Stradal, Theresia E B
Vauti, Franz
Arnold, Hans-Henning
Rottner, Klemens
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
F-actin branching
F-actin turnover
cell division
chemotaxis
filopodium
lamellipodium
migration
nuclear envelope breakdown
The Arp2/3 complex generates branched actin filament networks operating in cell edge protrusion and vesicle trafficking. Here we employ a conditional knockout mouse model permitting tissue- or cell-type specific deletion of the murine Actr3 gene (encoding Arp3). A functional Actr3 gene appeared essential for fibroblast viability and growth. Thus, we developed cell lines for exploring the consequences of acute, tamoxifen-induced Actr3 deletion causing near-complete loss of functional Arp2/3 complex expression as well as abolished lamellipodia formation and membrane ruffling, as expected. Interestingly, Arp3-depleted cells displayed enhanced rather than reduced cell spreading, employing numerous filopodia, and showed little defects in the rates of random cell migration. However, both exploration of new space by individual cells and collective migration were clearly compromised by the incapability to efficiently maintain directionality of migration, while the principal ability to chemotax was only moderately affected. Examination of actin remodeling at the cell periphery revealed reduced actin turnover rates in Arp2/3-deficient cells, clearly deviating from previous sequestration approaches. Most surprisingly, induced removal of Arp2/3 complexes reproducibly increased FMNL formin expression, which correlated with the explosive induction of filopodia formation. Our results thus highlight both direct and indirect effects of acute Arp2/3 complex removal on actin cytoskeleton regulation.
2021-03-22T15:52:43Z
2021-03-22T15:52:43Z
2021-02-01
Article
Front Cell Dev Biol. 2021 Feb 1;9:634708. doi: 10.3389/fcell.2021.634708.
2296-634X
33598464
10.3389/fcell.2021.634708
http://hdl.handle.net/10033/622784
Frontiers in cell and developmental biology
en
http://creativecommons.org/licenses/by/4.0/
Attribution 4.0 International
Frontiers
9
634708
Frontiers in cell and developmental biology
Switzerland
oai:repository.helmholtz-hzi.de:10033/6229512021-09-24T01:49:42Zcom_10033_338554com_10033_620636col_10033_621787col_10033_620638col_10033_338544col_10033_620574
Loss of Hem1 disrupts macrophage function and impacts migration, phagocytosis, and integrin-mediated adhesion.
Stahnke, Stephanie
Döring, Hermann
Kusch, Charly
de Gorter, David J J
Dütting, Sebastian
Guledani, Aleks
Pleines, Irina
Schnoor, Michael
Sixt, Michael
Geffers, Robert
Rohde, Manfred
Müsken, Mathias
Kage, Frieda
Steffen, Anika
Faix, Jan
Nieswandt, Bernhard
Rottner, Klemens
Stradal, Theresia E B
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Hem1
WAVE regulatory complex
WRC
adhesion
integrin
knockout
macrophage
phagocytosis
protrusion
rescue
Hematopoietic-specific protein 1 (Hem1) is an essential subunit of the WAVE regulatory complex (WRC) in immune cells. WRC is crucial for Arp2/3 complex activation and the protrusion of branched actin filament networks. Moreover, Hem1 loss of function in immune cells causes autoimmune diseases in humans. Here, we show that genetic removal of Hem1 in macrophages diminishes frequency and efficacy of phagocytosis as well as phagocytic cup formation in addition to defects in lamellipodial protrusion and migration. Moreover, Hem1-null macrophages displayed strong defects in cell adhesion despite unaltered podosome formation and concomitant extracellular matrix degradation. Specifically, dynamics of both adhesion and de-adhesion as well as concomitant phosphorylation of paxillin and focal adhesion kinase (FAK) were significantly compromised. Accordingly, disruption of WRC function in non-hematopoietic cells coincided with both defects in adhesion turnover and altered FAK and paxillin phosphorylation. Consistently, platelets exhibited reduced adhesion and diminished integrin αIIbβ3 activation upon WRC removal. Interestingly, adhesion phenotypes, but not lamellipodia formation, were partially rescued by small molecule activation of FAK. A full rescue of the phenotype, including lamellipodia formation, required not only the presence of WRCs but also their binding to and activation by Rac. Collectively, our results uncover that WRC impacts on integrin-dependent processes in a FAK-dependent manner, controlling formation and dismantling of adhesions, relevant for properly grabbing onto extracellular surfaces and particles during cell edge expansion, like in migration or phagocytosis.
2021-07-22T09:05:42Z
2021-07-22T09:05:42Z
2021-03-11
Article
Curr Biol. 2021 May 24;31(10):2051-2064.e8. doi: 10.1016/j.cub.2021.02.043. Epub 2021 Mar 11.
33711252
10.1016/j.cub.2021.02.043
http://hdl.handle.net/10033/622951
1879-0445
Current biology : CB
en
http://creativecommons.org/licenses/by/4.0/
Attribution 4.0 International
Wiley-VCH
31
10
2051
2064.e8
Current biology : CB
England
oai:repository.helmholtz-hzi.de:10033/6232352022-06-17T02:02:32Zcom_10033_338554col_10033_338544
Methylation of Salmonella Typhimurium flagella promotes bacterial adhesion and host cell invasion.
Horstmann, Julia A
Lunelli, Michele
Cazzola, Hélène
Heidemann, Johannes
Kühne, Caroline
Steffen, Pascal
Szefs, Sandra
Rossi, Claire
Lokareddy, Ravi K
Wang, Chu
Lemaire, Laurine
Hughes, Kelly T
Uetrecht, Charlotte
Schlüter, Hartmut
Grassl, Guntram A
Stradal, Theresia E B
Rossez, Yannick
Kolbe, Michael
Erhardt, Marc
The long external filament of bacterial flagella is composed of several thousand copies of a
single protein, flagellin. Here, we explore the role played by lysine methylation of flagellin in
Salmonella, which requires the methylase FliB. We show that both flagellins of Salmonella
enterica serovar Typhimurium, FliC and FljB, are methylated at surface-exposed lysine residues by FliB. A Salmonella Typhimurium mutant deficient in flagellin methylation is outcompeted for gut colonization in a gastroenteritis mouse model, and methylation of flagellin
promotes bacterial invasion of epithelial cells in vitro. Lysine methylation increases the
surface hydrophobicity of flagellin, and enhances flagella-dependent adhesion of Salmonella to
phosphatidylcholine vesicles and epithelial cells. Therefore, posttranslational methylation of
flagellin facilitates adhesion of Salmonella Typhimurium to hydrophobic host cell surfaces, and
contributes to efficient gut colonization and host infection.
2022-06-16T13:27:15Z
2022-06-16T13:27:15Z
2020-04-24
2019-09-09
Article
32332720
10.1038/s41467-020-15738-3
http://hdl.handle.net/10033/623235
2041-1723
Nature communications
en
http://creativecommons.org/licenses/by/4.0/
Attribution 4.0 International
11
1
2013
Nature communications
England