Publications by authors named "Jan Faix"

73 Publications

Loss of Hem1 disrupts macrophage function and impacts migration, phagocytosis, and integrin-mediated adhesion.

Curr Biol 2021 May 11;31(10):2051-2064.e8. Epub 2021 Mar 11.

Department of Cell Biology, Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany. Electronic address:

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.
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http://dx.doi.org/10.1016/j.cub.2021.02.043DOI Listing
May 2021

Capping protein is dispensable for polarized actin network growth and actin-based motility.

J Biol Chem 2020 11 31;295(45):15366-15375. Epub 2020 Aug 31.

Laboratoire Physico-Chimie Curie, Institut Curie, PSL Research University, CNRS, Paris, France; Sorbonne Université, Paris, France. Electronic address:

Heterodimeric capping protein (CP) binds the rapidly growing barbed ends of actin filaments and prevents the addition (or loss) of subunits. Capping activity is generally considered to be essential for actin-based motility induced by Arp2/3 complex nucleation. By stopping barbed end growth, CP favors nucleation of daughter filaments at the functionalized surface where the Arp2/3 complex is activated, thus creating polarized network growth, which is necessary for movement. However, here using an assay where Arp2/3 complex-based actin polymerization is induced on bead surfaces in the absence of CP, we produce robust polarized actin growth and motility. This is achieved either by adding the actin polymerase Ena/VASP or by boosting Arp2/3 complex activity at the surface. Another actin polymerase, the formin FMNL2, cannot substitute for CP, showing that polymerase activity alone is not enough to override the need for CP. Interfering with the polymerase activity of Ena/VASP, its surface recruitment or its bundling activity all reduce Ena/VASP's ability to maintain polarized network growth in the absence of CP. Taken together, our findings show that CP is dispensable for polarized actin growth and motility in situations where surface-directed polymerization is favored by whatever means over the growth of barbed ends in the network.
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http://dx.doi.org/10.1074/jbc.RA120.015009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7650241PMC
November 2020

Loss of Ena/VASP interferes with lamellipodium architecture, motility and integrin-dependent adhesion.

Elife 2020 05 11;9. Epub 2020 May 11.

Institute for Biophysical Chemistry, Hannover Medical School, Hannover, Germany.

Cell migration entails networks and bundles of actin filaments termed lamellipodia and microspikes or filopodia, respectively, as well as focal adhesions, all of which recruit Ena/VASP family members hitherto thought to antagonize efficient cell motility. However, we find these proteins to act as positive regulators of migration in different murine cell lines. CRISPR/Cas9-mediated loss of Ena/VASP proteins reduced lamellipodial actin assembly and perturbed lamellipodial architecture, as evidenced by changed network geometry as well as reduction of filament length and number that was accompanied by abnormal Arp2/3 complex and heterodimeric capping protein accumulation. Loss of Ena/VASP function also abolished the formation of microspikes normally embedded in lamellipodia, but not of filopodia capable of emanating without lamellipodia. Ena/VASP-deficiency also impaired integrin-mediated adhesion accompanied by reduced traction forces exerted through these structures. Our data thus uncover novel Ena/VASP functions of these actin polymerases that are fully consistent with their promotion of cell migration.
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http://dx.doi.org/10.7554/eLife.55351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7239657PMC
May 2020

Lamellipodin tunes cell migration by stabilizing protrusions and promoting adhesion formation.

J Cell Sci 2020 04 9;133(7). Epub 2020 Apr 9.

Division of Molecular Cell Biology, Zoological Institute, Technische Universität Braunschweig, Spielmannstrasse 7, 38106 Braunschweig, Germany

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, 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.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/jcs.239020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7157940PMC
April 2020

Formins specify membrane patterns generated by propagating actin waves.

Mol Biol Cell 2020 03 15;31(5):373-385. Epub 2020 Jan 15.

Max Planck Institute of Biochemistry, D-82152 Martinsried, Munich, Germany.

Circular actin waves separate two distinct areas on the substrate-attached cell surface from each other: an external area from an inner territory that is circumscribed by the wave. These areas differ in composition of actin-associated proteins and of phosphoinositides in the membrane. At the propagating wave, one area is converted into the other. By photo-conversion of Eos-actin and analysis of actin network structures we show that both in the inner territory and the external area the actin network is subject to continuous turnover. To address the question of whether areas in the wave pattern are specified by particular actin polymerizing machines, we locate five members of the formin family to specific regions of the wave landscape using TIRF microscopy and constitutively active formin constructs tagged with fluorescent protein. Formin ForB favors the actin wave and ForG the inner territory, whereas ForA, ForE, and ForH are more strongly recruited to the external area. Fluctuations of membrane binding peculiar to ForB indicate transient states in the specification of membrane domains before differentiation into ForB decorated and depleted ones. Annihilation of the patterns by 1 µM of the formin inhibitor SMIFH2 supports the implication of formins in their generation.
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http://dx.doi.org/10.1091/mbc.E19-08-0460DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7183788PMC
March 2020

Functional integrity of the contractile actin cortex is safeguarded by multiple Diaphanous-related formins.

Proc Natl Acad Sci U S A 2019 02 11;116(9):3594-3603. Epub 2019 Feb 11.

Institute for Biophysical Chemistry, Hannover Medical School, 30625 Hannover, Germany;

The contractile actin cortex is a thin layer of filamentous actin, myosin motors, and regulatory proteins beneath the plasma membrane crucial to cytokinesis, morphogenesis, and cell migration. However, the factors regulating actin assembly in this compartment are not well understood. Using the model system, we show that the three Diaphanous-related formins (DRFs) ForA, ForE, and ForH are regulated by the RhoA-like GTPase RacE and synergize in the assembly of filaments in the actin cortex. Single or double formin-null mutants displayed only moderate defects in cortex function whereas the concurrent elimination of all three formins or of RacE caused massive defects in cortical rigidity and architecture as assessed by aspiration assays and electron microscopy. Consistently, the triple formin and RacE mutants encompassed large peripheral patches devoid of cortical F-actin and exhibited severe defects in cytokinesis and multicellular development. Unexpectedly, many / / and mutants protruded efficiently, formed multiple exaggerated fronts, and migrated with morphologies reminiscent of rapidly moving fish keratocytes. In 2D-confinement, however, these mutants failed to properly polarize and recruit myosin II to the cell rear essential for migration. Cells arrested in these conditions displayed dramatically amplified flow of cortical actin filaments, as revealed by total internal reflection fluorescence (TIRF) imaging and iterative particle image velocimetry (PIV). Consistently, individual and combined, CRISPR/Cas9-mediated disruption of genes encoding mDia1 and -3 formins in B16-F1 mouse melanoma cells revealed enhanced frequency of cells displaying multiple fronts, again accompanied by defects in cell polarization and migration. These results suggest evolutionarily conserved functions for formin-mediated actin assembly in actin cortex mechanics.
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http://dx.doi.org/10.1073/pnas.1821638116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6397521PMC
February 2019

Functional Characterization of Ubiquitin-Like Core Autophagy Protein ATG12 in .

Cells 2019 01 19;8(1). Epub 2019 Jan 19.

Institute of Biochemistry I, Medical Faculty, University Hospital Cologne, 50931 Cologne, Germany.

Autophagy is a highly conserved intracellular degradative pathway that is crucial for cellular homeostasis. During autophagy, the core autophagy protein ATG12 plays, together with ATG5 and ATG16, an essential role in the expansion of the autophagosomal membrane. In this study we analyzed gene replacement mutants of in AX2 wild-type and ATG16‾ cells. RNA analysis revealed a strong enrichment of, firstly, autophagy genes among the up-regulated genes and, secondly, genes implicated in cell motility and phagocytosis among the down-regulated genes in the generated ATG12‾, ATG16‾ and ATG12‾/16‾ cells. The mutant strains showed similar defects in fruiting body formation, autolysosome maturation, and cellular viability, implying that ATG12 and ATG16 act as a functional unit in canonical autophagy. In contrast, ablation of ATG16 or of ATG12 and ATG16 resulted in slightly more severe defects in axenic growth, macropinocytosis, and protein homeostasis than ablation of only ATG12, suggesting that ATG16 fulfils an additional function in these processes. Phagocytosis of yeast, spore viability, and maximal cell density were much more affected in ATG12‾/16‾ cells, indicating that both proteins also have cellular functions independent of each other. In summary, we show that ATG12 and ATG16 fulfil autophagy-independent functions in addition to their role in canonical autophagy.
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http://dx.doi.org/10.3390/cells8010072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6356199PMC
January 2019

IQGAP-related protein IqgC suppresses Ras signaling during large-scale endocytosis.

Proc Natl Acad Sci U S A 2019 01 8;116(4):1289-1298. Epub 2019 Jan 8.

Division of Molecular Biology, Ruđer Bošković Institute, 10000 Zagreb, Croatia;

Macropinocytosis and phagocytosis are evolutionarily conserved forms of bulk endocytosis used by cells to ingest large volumes of fluid and solid particles, respectively. Both processes are regulated by Ras signaling, which is precisely controlled by mechanisms involving Ras GTPase activating proteins (RasGAPs) responsible for terminating Ras activity on early endosomes. While regulation of Ras signaling during large-scale endocytosis in WT has been, for the most part, attributed to the ortholog of human RasGAP NF1, in commonly used axenic laboratory strains, this gene is mutated and inactive. Moreover, none of the RasGAPs characterized so far have been implicated in the regulation of Ras signaling in large-scale endocytosis in axenic strains. In this study, we establish, using biochemical approaches and complementation assays in live cells, that IQGAP-related protein IqgC interacts with active RasG and exhibits RasGAP activity toward this GTPase. Analyses of and IqgC-overexpressing cells further revealed participation of this GAP in the regulation of both types of large-scale endocytosis and in cytokinesis. Moreover, given the localization of IqgC to phagosomes and, most prominently, to macropinosomes, we propose IqgC acting as a RasG-specific GAP in large-scale endocytosis. The data presented here functionally distinguish IqgC from other members of the IQGAP family and call for repositioning of this genuine RasGAP outside of the IQGAP group.
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http://dx.doi.org/10.1073/pnas.1810268116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6347711PMC
January 2019

Capping protein-controlled actin polymerization shapes lipid membranes.

Nat Commun 2018 04 24;9(1):1630. Epub 2018 Apr 24.

Lehrstuhl für Zellbiophysik, Technische Universität München, James-Franck-Str. 1, D-85748, Garching, Germany.

Arp2/3 complex-mediated actin assembly at cell membranes drives the formation of protrusions or endocytic vesicles. To identify the mechanism by which different membrane deformations can be achieved, we reconstitute the basic membrane deformation modes of inward and outward bending in a confined geometry by encapsulating a minimal set of cytoskeletal proteins into giant unilamellar vesicles. Formation of membrane protrusions is favoured at low capping protein (CP) concentrations, whereas the formation of negatively bent domains is promoted at high CP concentrations. Addition of non-muscle myosin II results in full fission events in the vesicle system. The different deformation modes are rationalized by simulations of the underlying transient nature of the reaction kinetics. The relevance of the regulatory mechanism is supported by CP overexpression in mouse melanoma B16-F1 cells and therefore demonstrates the importance of the quantitative understanding of microscopic kinetic balances to address the diverse functionality of the cytoskeleton.
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http://dx.doi.org/10.1038/s41467-018-03918-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5915599PMC
April 2018

Analysis of Random Migration of Dictyostelium Amoeba in Confined and Unconfined Environments.

Methods Mol Biol 2018 ;1749:341-350

Institute for Biophysical Chemistry, Hannover Medical School, Hannover, Germany.

Dictyostelium discoideum has proven to be an excellent model to study amoeboid cell migration. During their life cycle, Dictyostelium cells exhibit distinct modes of motility. Individual growth-phase cells explore new territories by random cell migration using the core cell motility machinery, but they can also hunt bacteria by detection and chemotaxis toward the by-product folate. After depletion of nutrients, the cells initiate a developmental program allowing streaming of the cells into aggregation centers by chemotaxis toward cAMP and by cell-to-cell adhesion. Subsequent development is associated with complex rotational movement of the compacted aggregates to drive cell type specific sorting, which in turn is necessary for terminal culmination and formation of fruiting bodies. Here we describe a protocol for the analyses of cell motility of vegetative Dictyostelium cells in unconfined and mechanically confined settings.
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http://dx.doi.org/10.1007/978-1-4939-7701-7_24DOI Listing
January 2019

Actin assembly mechanisms at a glance.

J Cell Sci 2017 Oct;130(20):3427-3435

Department of Neurology, University Hospital Regensburg, 93053 Regensburg, Germany

The actin cytoskeleton and associated motor proteins provide the driving forces for establishing the astonishing morphological diversity and dynamics of mammalian cells. Aside from functions in protruding and contracting cell membranes for motility, differentiation or cell division, the actin cytoskeleton provides forces to shape and move intracellular membranes of organelles and vesicles. To establish the many different actin assembly functions required in time and space, actin nucleators are targeted to specific subcellular compartments, thereby restricting the generation of specific actin filament structures to those sites. Recent research has revealed that targeting and activation of actin filament nucleators, elongators and myosin motors are tightly coordinated by conserved protein complexes to orchestrate force generation. In this Cell Science at a Glance article and the accompanying poster, we summarize and discuss the current knowledge on the corresponding protein complexes and their modes of action in actin nucleation, elongation and force generation.
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http://dx.doi.org/10.1242/jcs.206433DOI Listing
October 2017

EMC10 (Endoplasmic Reticulum Membrane Protein Complex Subunit 10) Is a Bone Marrow-Derived Angiogenic Growth Factor Promoting Tissue Repair After Myocardial Infarction.

Circulation 2017 Nov 20;136(19):1809-1823. Epub 2017 Sep 20.

From Division of Molecular and Translational Cardiology, Department of Cardiology and Angiology (M.R.R., M.K.-K., S.K., E.B., I.R., Y.W., K.C.W.), Core Unit Proteomics, Institute of Toxicology (F.P., A.P.), Department of Biophysical Chemistry (J.F.), Leibniz Research Laboratories for Biotechnology and Artificial Organs, Department of Cardiothoracic, Transplantation, and Vascular Surgery (G.K., I.G.), Institute of Legal Medicine (M.K.), Institute of Physiological Chemistry (M.G.), and Department of Cardiology and Angiology (J.B.), Hannover Medical School, Germany; F. Hoffmann-La Roche, Pharma Research and Early Development, Basel, Switzerland (H.-J.S., M.B.); Department of Pathology and Department of Cardiac Surgery, Institute for Cardiovascular Research, Vrije Universiteit University Medical Center, Amsterdam, The Netherlands (H.W.N.); and Department of Physiology and Cellular Biophysics and Department of Neuroscience, College of Physicians and Surgeons, Columbia University, New York, NY (J.A.G.).

Background: Clinical trials of bone marrow cell-based therapies after acute myocardial infarction (MI) have produced mostly neutral results. Treatment with specific bone marrow cell-derived secreted proteins may provide an alternative biological approach to improving tissue repair and heart function after MI. We recently performed a bioinformatic secretome analysis in bone marrow cells from patients with acute MI and discovered a poorly characterized secreted protein, EMC10 (endoplasmic reticulum membrane protein complex subunit 10), showing activity in an angiogenic screen.

Methods: We investigated the angiogenic potential of EMC10 and its mouse homolog (Emc10) in cultured endothelial cells and infarcted heart explants. We defined the cellular sources and function of Emc10 after MI using wild-type, -deficient, and bone marrow-chimeric mice subjected to transient coronary artery ligation. Furthermore, we explored the therapeutic potential of recombinant Emc10 delivered by osmotic minipumps after MI in heart failure-prone FVB/N mice.

Results: Emc10 signaled through small GTPases, p21-activated kinase, and the p38 mitogen-activated protein kinase (MAPK)-MAPK-activated protein kinase 2 (MK2) pathway to promote actin polymerization and endothelial cell migration. Confirming the importance of these signaling events in the context of acute MI, Emc10 stimulated endothelial cell outgrowth from infarcted mouse heart explants via p38 MAPK-MK2. Emc10 protein abundance was increased in the infarcted region of the left ventricle and in the circulation of wild-type mice after MI. Emc10 expression was also increased in left ventricular tissue samples from patients with acute MI. Bone marrow-derived monocytes and macrophages were the predominant sources of Emc10 in the infarcted murine heart. KO mice showed no cardiovascular phenotype at baseline. After MI, however, capillarization of the infarct border zone was impaired in KO mice, and the animals developed larger infarct scars and more pronounced left ventricular remodeling compared with wild-type mice. Transplanting KO mice with wild-type bone marrow cells rescued the angiogenic defect and ameliorated left ventricular remodeling. Treating FVB/N mice with recombinant Emc10 enhanced infarct border-zone capillarization and exerted a sustained beneficial effect on left ventricular remodeling.

Conclusions: We have identified Emc10 as a previously unknown angiogenic growth factor that is produced by bone marrow-derived monocytes and macrophages as part of an endogenous adaptive response that can be enhanced therapeutically to repair the heart after MI.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.117.029980DOI Listing
November 2017

Kindlin-2 recruits paxillin and Arp2/3 to promote membrane protrusions during initial cell spreading.

J Cell Biol 2017 11 14;216(11):3785-3798. Epub 2017 Sep 14.

Department of Molecular Medicine, Max Planck Institute of Biochemistry, Martinsried, Germany.

Cell spreading requires the coupling of actin-driven membrane protrusion and integrin-mediated adhesion to the extracellular matrix. The integrin-activating adaptor protein kindlin-2 plays a central role for cell adhesion and membrane protrusion by directly binding and recruiting paxillin to nascent adhesions. Here, we report that kindlin-2 has a dual role during initial cell spreading: it binds paxillin via the pleckstrin homology and F0 domains to activate Rac1, and it directly associates with the Arp2/3 complex to induce Rac1-mediated membrane protrusions. Consistently, abrogation of kindlin-2 binding to Arp2/3 impairs lamellipodia formation and cell spreading. Our findings identify kindlin-2 as a key protein that couples cell adhesion by activating integrins and the induction of membrane protrusions by activating Rac1 and supplying Rac1 with the Arp2/3 complex.
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http://dx.doi.org/10.1083/jcb.201701176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5674885PMC
November 2017

Bin1 directly remodels actin dynamics through its BAR domain.

EMBO Rep 2017 11 11;18(11):2051-2066. Epub 2017 Sep 11.

Proteostasis in Neurodegenerative Disease (B180), Schaller Research Group at the University of Heidelberg and DKFZ, Heidelberg, Germany

Endocytic processes are facilitated by both curvature-generating BAR-domain proteins and the coordinated polymerization of actin filaments. Under physiological conditions, the N-BAR protein Bin1 has been shown to sense and curve membranes in a variety of cellular processes. Recent studies have identified Bin1 as a risk factor for Alzheimer's disease, although its possible pathological function in neurodegeneration is currently unknown. Here, we report that Bin1 not only shapes membranes, but is also directly involved in actin binding through its BAR domain. We observed a moderate actin bundling activity by human Bin1 and describe its ability to stabilize actin filaments against depolymerization. Moreover, Bin1 is also involved in stabilizing tau-induced actin bundles, which are neuropathological hallmarks of Alzheimer's disease. We also provide evidence for this effect , where we observed that downregulation of Bin1 in a model of tauopathy significantly reduces the appearance of tau-induced actin inclusions. Together, these findings reveal the ability of Bin1 to modify actin dynamics and provide a possible mechanistic connection between Bin1 and tau-induced pathobiological changes of the actin cytoskeleton.
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http://dx.doi.org/10.15252/embr.201744137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5666605PMC
November 2017

Differential functions of WAVE regulatory complex subunits in the regulation of actin-driven processes.

Eur J Cell Biol 2017 Dec 4;96(8):715-727. Epub 2017 Sep 4.

Institute for Biophysical Chemistry, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. Electronic address:

The WAVE regulatory complex (WRC) links upstream Rho-family GTPase signaling to the activation of the ARP2/3 complex in different organisms. WRC-induced and ARP2/3 complex-mediated actin nucleation beneath the plasma membrane is critical for actin assembly in the leading edge to drive efficient cell migration. The WRC is a stable heteropentamer composed of SCAR/WAVE, Abi, Nap, Pir and the small polypeptide Brk1/Hspc300. Functional interference with individual subunits of the complex frequently results in diminished amounts of the remaining polypeptides of the WRC complex, implying the complex to act as molecular entity. However, Abi was also found to associate with mammalian N-WASP, formins, Eps8/SOS1 or VASP, indicating additional functions of individual WRC subunits in eukaryotic cells. To address this issue systematically, we inactivated all WRC subunits, either alone or in combination with VASP in Dictyostelium cells and quantified the protein content of the remaining subunits in respective WRC knockouts. The individual mutants displayed highly differential phenotypes concerning various parameters, including cell morphology, motility, cytokinesis or multicellular development, corroborating the view of additional roles for individual subunits, beyond their established function in WRC-mediated Arp2/3 complex activation. Finally, our data uncover the interaction of the actin polymerase VASP with WRC-embedded Abi to mediate VASP accumulation in cell protrusions, driving efficient cell migration.
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http://dx.doi.org/10.1016/j.ejcb.2017.08.003DOI Listing
December 2017

Distinct VASP tetramers synergize in the processive elongation of individual actin filaments from clustered arrays.

Proc Natl Acad Sci U S A 2017 07 30;114(29):E5815-E5824. Epub 2017 Jun 30.

Institute for Biophysical Chemistry, Hannover Medical School, 30625 Hannover, Germany;

Ena/VASP proteins act as actin polymerases that drive the processive elongation of filament barbed ends in membrane protrusions or at the surface of bacterial pathogens. Based on previous analyses of fast and slow elongating VASP proteins by in vitro total internal reflection fluorescence microscopy (TIRFM) and kinetic and thermodynamic measurements, we established a kinetic model of Ena/VASP-mediated actin filament elongation. At steady state, it entails that tetrameric VASP uses one of its arms to processively track growing filament barbed ends while three G-actin-binding sites (GABs) on other arms are available to recruit and deliver monomers to the filament tip, suggesting that VASP operates as a single tetramer in solution or when clustered on a surface, albeit processivity and resistance toward capping protein (CP) differ dramatically between both conditions. Here, we tested the model by variation of the oligomerization state and by increase of the number of GABs on individual polypeptide chains. In excellent agreement with model predictions, we show that in solution the rates of filament elongation directly correlate with the number of free GABs. Strikingly, however, irrespective of the oligomerization state or presence of additional GABs, filament elongation on a surface invariably proceeded with the same rate as with the VASP tetramer, demonstrating that adjacent VASP molecules synergize in the elongation of a single filament. Additionally, we reveal that actin ATP hydrolysis is not required for VASP-mediated filament assembly. Finally, we show evidence for the requirement of VASP to form tetramers and provide an amended model of processive VASP-mediated actin assembly in clustered arrays.
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http://dx.doi.org/10.1073/pnas.1703145114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5530675PMC
July 2017

FMNL formins boost lamellipodial force generation.

Nat Commun 2017 03 22;8:14832. Epub 2017 Mar 22.

Division of Molecular Cell Biology, Zoological Institute, Technische Universität Braunschweig, Spielmannstrasse 7, 38106 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.
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http://dx.doi.org/10.1038/ncomms14832DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5364437PMC
March 2017

The Arp2/3 inhibitory protein Arpin is dispensable for chemotaxis.

Biol Cell 2017 Apr 7;109(4):162-166. Epub 2017 Mar 7.

Ecole Polytechnique, Université Paris-Saclay, CNRS UMR7654, Palaiseau, 91120, France.

Arpin is an Arp2/3 inhibitory protein, which decreases the protrusion lifetime and hence directional persistence in the migration of diverse cells. Arpin is activated by the small GTPase Rac, which controls cell protrusion, thus closing a negative feedback loop that renders the protrusion intrinsically unstable. Because of these properties, it was proposed that Arpin might play a role in directed migration, where directional persistence has to be fine-tuned. We report here, however, that Arpin-depleted tumour cells and Arpin knock-out Dictyostelium amoeba display no obvious defect in chemotaxis. These results do not rule out a potential role of Arpin in other systems, but argue against a general role of Arpin in chemotaxis.
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http://dx.doi.org/10.1111/boc.201600064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5506374PMC
April 2017

A Diaphanous-related formin links Ras signaling directly to actin assembly in macropinocytosis and phagocytosis.

Proc Natl Acad Sci U S A 2016 11 7;113(47):E7464-E7473. Epub 2016 Nov 7.

Institute for Biophysical Chemistry, Hannover Medical School, 30625 Hannover, Germany;

Phagocytosis and macropinocytosis are Ras-regulated and actin-driven processes that depend on the dynamic rearrangements of the plasma membrane that protrudes and internalizes extracellular material by cup-shaped structures. However, the regulatory mechanisms underlying actin assembly in large-scale endocytosis remain elusive. Here, we show that the Diaphanous-related formin G (ForG) from the professional phagocyte Dictyostelium discoideum localizes to endocytic cups. Biochemical analyses revealed that ForG is a rather weak nucleator but efficiently elongates actin filaments in the presence of profilin. Notably, genetic inactivation of ForG is associated with a strongly impaired endocytosis and a markedly diminished F-actin content at the base of the cups. By contrast, ablation of the Arp2/3 (actin-related protein-2/3) complex activator SCAR (suppressor of cAMP receptor) diminishes F-actin mainly at the cup rim, being consistent with its known localization. These data therefore suggest that ForG acts as an actin polymerase of Arp2/3-nucleated filaments to allow for efficient membrane expansion and engulfment of extracellular material. Finally, we show that ForG is directly regulated in large-scale endocytosis by RasB and RasG, which are highly related to the human proto-oncogene KRas.
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http://dx.doi.org/10.1073/pnas.1611024113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5127317PMC
November 2016

Visualization of Actin Assembly and Filament Turnover by In Vitro Multicolor TIRF Microscopy.

Methods Mol Biol 2016 ;1407:287-306

Institute for Biophysical Chemistry, Hannover Medical School, Carl-Neuberg-Str. 1, 30625, Hannover, Germany.

In response to chemotactic signals, motile cells develop a single protruding front to persistently migrate in direction of the chemotactic gradient. The highly dynamic reorganization of the actin cytoskeleton is an essential part during this process and requires the precise interplay of various actin filament assembly factors and actin-binding proteins (ABPs). Although many ABPs have been implicated in cell migration, as yet only a few of them have been well characterized concerning their specific functions during actin network assembly and disassembly. In this chapter, we describe a versatile method that allows the direct visualization of the assembly of single actin filaments and higher structures in real time by in vitro total internal reflection fluorescence microscopy (TIRF-M) using purified and fluorescently labeled actin and ABPs.
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http://dx.doi.org/10.1007/978-1-4939-3480-5_21DOI Listing
December 2017

WHAMY is a novel actin polymerase promoting myoblast fusion, macrophage cell motility and sensory organ development in Drosophila.

J Cell Sci 2016 Feb 16;129(3):604-20. Epub 2015 Dec 16.

Institut für Neurobiologie, Universität Münster, Badestr. 9, Münster 48149, Germany

Wiskott-Aldrich syndrome proteins (WASPs) are nucleation-promoting factors (NPF) that differentially control the Arp2/3 complex. In Drosophila, three different family members, SCAR (also known as WAVE), WASP and WASH (also known as CG13176), have been analyzed so far. Here, we characterized WHAMY, the fourth Drosophila WASP family member. whamy originated from a wasp gene duplication and underwent a sub-neofunctionalization. Unlike WASP, we found that WHAMY specifically interacted with activated Rac1 through its two CRIB domains, which were sufficient for targeting WHAMY to lamellipodial and filopodial tips. Biochemical analyses showed that WHAMY promoted exceptionally fast actin filament elongation, although it did not activate the Arp2/3 complex. Loss- and gain-of-function studies revealed an important function of WHAMY in membrane protrusions and cell migration in macrophages. Genetic data further implied synergistic functions between WHAMY and WASP during morphogenesis. Double mutants were late-embryonic lethal and showed severe defects in myoblast fusion. Trans-heterozygous mutant animals showed strongly increased defects in sensory cell fate specification. Thus, WHAMY is a novel actin polymerase with an initial partitioning of ancestral WASP functions in development and subsequent acquisition of a new function in cell motility during evolution.
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http://dx.doi.org/10.1242/jcs.179325DOI Listing
February 2016

A resilient formin-derived cortical actin meshwork in the rear drives actomyosin-based motility in 2D confinement.

Nat Commun 2015 Sep 29;6:8496. Epub 2015 Sep 29.

Institute for Biophysical Chemistry, Hannover Medical School, Carl-Neuberg-Strasse 1, Hannover 30625, Germany.

Cell migration is driven by the establishment of disparity between the cortical properties of the softer front and the more rigid rear allowing front extension and actomyosin-based rear contraction. However, how the cortical actin meshwork in the rear is generated remains elusive. Here we identify the mDia1-like formin A (ForA) from Dictyostelium discoideum that generates a subset of filaments as the basis of a resilient cortical actin sheath in the rear. Mechanical resistance of this actin compartment is accomplished by actin crosslinkers and IQGAP-related proteins, and is mandatory to withstand the increased contractile forces in response to mechanical stress by impeding unproductive blebbing in the rear, allowing efficient cell migration in two-dimensional-confined environments. Consistently, ForA supresses the formation of lateral protrusions, rapidly relocalizes to new prospective ends in repolarizing cells and is required for cortical integrity. Finally, we show that ForA utilizes the phosphoinositide gradients in polarized cells for subcellular targeting.
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http://dx.doi.org/10.1038/ncomms9496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4598863PMC
September 2015

Coronin7 regulates WASP and SCAR through CRIB mediated interaction with Rac proteins.

Sci Rep 2015 Sep 28;5:14437. Epub 2015 Sep 28.

Center for Biochemistry, Medical Faculty, Center for Molecular Medicine Cologne (CMMC) and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, 50931 Köln, Germany.

Coronin7 (CRN7) stabilizes F-actin and is a regulator of processes associated with the actin cytoskeleton. Its loss leads to defects in phagocytosis, motility and development. It harbors a CRIB (Cdc42- and Rac-interactive binding) domain in each of its WD repeat domains which bind to Rac GTPases preferably in their GDP-loaded forms. Expression of wild type CRN7 in CRN7 deficient cells rescued these defects, whereas proteins with mutations in the CRIB motifs which were associated with altered Rac binding were effective to varying degrees. The presence of one functional CRIB was sufficient to reestablish phagocytosis, cell motility and development. Furthermore, by molecular modeling and mutational analysis we identified the contact regions between CRN7 and the GTPases. We also identified WASP, SCAR and PAKa as downstream effectors in phagocytosis, development and cell surface adhesion, respectively, since ectopic expression rescued these functions.
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http://dx.doi.org/10.1038/srep14437DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4585930PMC
September 2015

Single-molecule microscopy of molecules tagged with GFP or RFP derivatives in mammalian cells using nanobody binders.

Methods 2015 Oct 27;88:89-97. Epub 2015 Jun 27.

Randall Division of Cell and Molecular Biophysics, King's College London, SE1 1UL, United Kingdom; Institute of Biochemistry, ETH Zurich, Zurich, Switzerland; Institut für Biochemie und Chemie, Freie Universität Berlin, Thielallee 63, 14195 Berlin, Germany. Electronic address:

With the recent development of single-molecule localization-based superresolution microscopy, the imaging of cellular structures at a resolution below the diffraction-limit of light has become a widespread technique. While single fluorescent molecules can be resolved in the nanometer range, the delivery of these molecules to the authentic structure in the cell via traditional antibody-mediated techniques can add substantial error due to the size of the antibodies. Accurate and quantitative labeling of cellular molecules has thus become one of the bottlenecks in the race for highest resolution of target structures. Here we illustrate in detail how to use small, high affinity nanobody binders against GFP and RFP family proteins for highly generic labeling of fusion constructs with bright organic dyes. We provide detailed protocols and examples for their application in superresolution imaging and single particle tracking and demonstrate advantages over conventional labeling approaches.
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http://dx.doi.org/10.1016/j.ymeth.2015.06.018DOI Listing
October 2015

Actin-filament disassembly: it takes two to shrink them fast.

Curr Biol 2015 Jun;25(11):R450-2

Institute for Biophysical Chemistry, Hannover Medical School, Carl-Neuberg Str. 1, 30625 Hannover, Germany. Electronic address:

Actin-filament disassembly is indispensable for replenishing the pool of polymerizable actin and allows continuous dynamic remodelling of the actin cytoskeleton. A new study now reveals that ADF/cofilin preferentially dismantles branched networks and provides new insights into the collaborative work of ADF/cofilin and Aip1 on filament disassembly at the molecular level.
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http://dx.doi.org/10.1016/j.cub.2015.04.050DOI Listing
June 2015

The IQGAP-related protein DGAP1 mediates signaling to the actin cytoskeleton as an effector and a sequestrator of Rac1 GTPases.

Cell Mol Life Sci 2014 Aug 25;71(15):2775-85. Epub 2014 Mar 25.

Division of Molecular Biology, Ruđer Bošković Institute, Bijenička 54, 10000, Zagreb, Croatia.

Proteins are typically categorized into protein families based on their domain organization. Yet, evolutionarily unrelated proteins can also be grouped together according to their common functional roles. Sequestering proteins constitute one such functional class, acting as macromolecular buffers and serving as an intracellular reservoir ready to release large quantities of bound proteins or other molecules upon appropriate stimulation. Another functional protein class comprises effector proteins, which constitute essential components of many intracellular signal transduction pathways. For instance, effectors of small GTP-hydrolases are activated upon binding a GTP-bound GTPase and thereupon participate in downstream interactions. Here we describe a member of the IQGAP family of scaffolding proteins, DGAP1 from Dictyostelium, which unifies the roles of an effector and a sequestrator in regard to the small GTPase Rac1. Unlike classical effectors, which bind their activators transiently leading to short-lived signaling complexes, interaction between DGAP1 and Rac1-GTP is stable and induces formation of a complex with actin-bundling proteins cortexillins at the back end of the cell. An oppositely localized Rac1 effector, the Scar/WAVE complex, promotes actin polymerization at the cell front. Competition between DGAP1 and Scar/WAVE for the common activator Rac1-GTP might provide the basis for the oscillatory re-polarization typically seen in randomly migrating Dictyostelium cells. We discuss the consequences of the dual roles exerted by DGAP1 and Rac1 in the regulation of cell motility and polarity, and propose that similar signaling mechanisms may be of general importance in regulating spatiotemporal dynamics of the actin cytoskeleton by small GTPases.
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http://dx.doi.org/10.1007/s00018-014-1606-3DOI Listing
August 2014

The inverse BAR domain protein IBARa drives membrane remodeling to control osmoregulation, phagocytosis and cytokinesis.

J Cell Sci 2014 Mar 24;127(Pt 6):1279-92. Epub 2014 Jan 24.

Institute for Biophysical Chemistry, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany.

Here, we analyzed the single inverse Bin/Amphiphysin/Rvs (I-BAR) family member IBARa from Dictyostelium discoideum. The X-ray structure of the N-terminal I-BAR domain solved at 2.2 Å resolution revealed an all-α-helical structure that self-associates into a 165-Å zeppelin-shaped antiparallel dimer. The structural data are consistent with its shape in solution obtained by small-angle X-ray scattering. Cosedimentation, fluorescence anisotropy, and fluorescence and electron microscopy revealed that the I-BAR domain bound preferentially to phosphoinositide-containing vesicles and drove the formation of negatively curved tubules. Immunofluorescence labeling further showed accumulation of endogenous IBARa at the tips of filopodia, the rim of constricting phagocytic cups, in foci connecting dividing cells during the final stage of cytokinesis and most prominently at the osmoregulatory contractile vacuole (CV). Consistently, IBARa-null mutants displayed defects in CV formation and discharge, growth, phagocytosis and mitotic cell division, whereas filopodia formation was not compromised. Of note, IBARa-null mutants were also strongly impaired in cell spreading. Taken together, these data suggest that IBARa constitutes an important regulator of numerous cellular processes intimately linked with the dynamic rearrangement of cellular membranes.
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http://dx.doi.org/10.1242/jcs.140756DOI Listing
March 2014

A Cdc42- and Rac-interactive binding (CRIB) domain mediates functions of coronin.

Proc Natl Acad Sci U S A 2014 Jan 17;111(1):E25-33. Epub 2013 Dec 17.

Center for Biochemistry, Medical Faculty, Center for Molecular Medicine Cologne and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, 50931 Köln, Germany.

The Cdc42- and Rac-interactive binding motif (CRIB) of coronin binds to Rho GTPases with a preference for GDP-loaded Rac. Mutation of the Cdc42- and Rac-interactive binding motif abrogates Rac binding. This results in increased 1evels of activated Rac in coronin-deficient Dictyostelium cells (corA(-)), which impacts myosin II assembly. corA(-) cells show increased accumulation of myosin II in the cortex of growth-phase cells. Myosin II assembly is regulated by myosin heavy chain kinase-mediated phosphorylation of its tail. Kinase activity depends on the activation state of the p21-activated kinase a. The myosin II defect of corA(-) mutant is alleviated by dominant-negative p21-activated kinase a. It is rescued by wild-type coronin, whereas coronin carrying a mutated Cdc42- and Rac-interactive binding motif failed to rescue the myosin defect in corA(-) mutant cells. Ectopically expressed myosin heavy chain kinases affinity purified from corA(-) cells show reduced kinase activity. We propose that coronin through its affinity for GDP-Rac regulates the availability of GTP-Rac for activation of downstream effectors.
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http://dx.doi.org/10.1073/pnas.1315368111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3890859PMC
January 2014

Enhanced fear expression in Spir-1 actin organizer mutant mice.

Eur J Cell Biol 2014 May-Jun;93(5-6):225-37. Epub 2013 Nov 20.

University Hospital Regensburg, Department of Neurology, Molecular Cell Biology Laboratory, Franz-Josef-Strauß-Allee 11, 93053 Regensburg, Germany. Electronic address:

Spir proteins nucleate actin filaments at vesicle membranes and facilitate intracellular transport processes. The mammalian genome encodes two Spir proteins, namely Spir-1 and Spir-2. While the mouse spir-2 gene has a rather broad expression pattern, high levels of spir-1 expression are restricted to the nervous system, oocytes, and testis. Spir-1 mutant mice generated by a gene trap method have been employed to address Spir-1 function during mouse development and in adult mouse tissues, with a specific emphasis on viability, reproduction, and the nervous system. The gene trap cassette disrupts Spir-1 expression between the N-terminal KIND domain and the WH2 domain cluster. Spir-1 mutant mice are viable and were born in a Mendelian ratio. In accordance with the redundant function of Spir-1 and Spir-2 in oocyte maturation, spir-1 mutant mice are fertile. The overall brain anatomy of spir-1 mutant mice is not altered and visual and motor functions of the mice remain normal. Microscopic analysis shows a slight reduction in the number of dendritic spines on cortical neurons. Detailed behavioral studies of the spir-1 mutant mice, however, unveiled a very specific and highly significant phenotype in terms of fear learning in male mice. In contextual and cued fear conditioning experiments the male spir-1 mutant mice display increased fear memory when compared to their control littermates. Our data point toward a particular function of the vesicle associated Spir-1 actin organizer in neuronal circuits determining fear behavior.
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http://dx.doi.org/10.1016/j.ejcb.2013.11.001DOI Listing
May 2015
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