TY - JOUR AB - Regulation of the Arp2/3 complex is required for productive nucleation of branched actin networks. An emerging aspect of regulation is the incorporation of subunit isoforms into the Arp2/3 complex. Specifically, both ArpC5 subunit isoforms, ArpC5 and ArpC5L, have been reported to fine-tune nucleation activity and branch junction stability. We have combined reverse genetics and cellular structural biology to describe how ArpC5 and ArpC5L differentially affect cell migration. Both define the structural stability of ArpC1 in branch junctions and, in turn, by determining protrusion characteristics, affect protein dynamics and actin network ultrastructure. ArpC5 isoforms also affect the positioning of members of the Ena/Vasodilator-stimulated phosphoprotein (VASP) family of actin filament elongators, which mediate ArpC5 isoform–specific effects on the actin assembly level. Our results suggest that ArpC5 and Ena/VASP proteins are part of a signaling pathway enhancing cell migration. AU - Fäßler, Florian AU - Javoor, Manjunath AU - Datler, Julia AU - Döring, Hermann AU - Hofer, Florian AU - Dimchev, Georgi A AU - Hodirnau, Victor-Valentin AU - Faix, Jan AU - Rottner, Klemens AU - Schur, Florian KM ID - 12334 IS - 3 JF - Science Advances KW - Multidisciplinary SN - 2375-2548 TI - ArpC5 isoforms regulate Arp2/3 complex–dependent protrusion through differential Ena/VASP positioning VL - 9 ER - TY - COMP AB - A precise quantitative description of the ultrastructural characteristics underlying biological mechanisms is often key to their understanding. This is particularly true for dynamic extra- and intracellular filamentous assemblies, playing a role in cell motility, cell integrity, cytokinesis, tissue formation and maintenance. For example, genetic manipulation or modulation of actin regulatory proteins frequently manifests in changes of the morphology, dynamics, and ultrastructural architecture of actin filament-rich cell peripheral structures, such as lamellipodia or filopodia. However, the observed ultrastructural effects often remain subtle and require sufficiently large datasets for appropriate quantitative analysis. The acquisition of such large datasets has been enabled by recent advances in high-throughput cryo-electron tomography (cryo-ET) methods. This also necessitates the development of complementary approaches to maximize the extraction of relevant biological information. We have developed a computational toolbox for the semi-automatic quantification of segmented and vectorized fila- mentous networks from pre-processed cryo-electron tomograms, facilitating the analysis and cross-comparison of multiple experimental conditions. GUI-based components simplify the processing of data and allow users to obtain a large number of ultrastructural parameters describing filamentous assemblies. We demonstrate the feasibility of this workflow by analyzing cryo-ET data of untreated and chemically perturbed branched actin filament networks and that of parallel actin filament arrays. In principle, the computational toolbox presented here is applicable for data analysis comprising any type of filaments in regular (i.e. parallel) or random arrangement. We show that it can ease the identification of key differences between experimental groups and facilitate the in-depth analysis of ultrastructural data in a time-efficient manner. AU - Dimchev, Georgi A AU - Amiri, Behnam AU - Fäßler, Florian AU - Falcke, Martin AU - Schur, Florian KM ID - 14502 KW - cryo-electron tomography KW - actin cytoskeleton KW - toolbox TI - Computational toolbox for ultrastructural quantitative analysis of filament networks in cryo-ET data ER - TY - JOUR AB - A precise quantitative description of the ultrastructural characteristics underlying biological mechanisms is often key to their understanding. This is particularly true for dynamic extra- and intracellular filamentous assemblies, playing a role in cell motility, cell integrity, cytokinesis, tissue formation and maintenance. For example, genetic manipulation or modulation of actin regulatory proteins frequently manifests in changes of the morphology, dynamics, and ultrastructural architecture of actin filament-rich cell peripheral structures, such as lamellipodia or filopodia. However, the observed ultrastructural effects often remain subtle and require sufficiently large datasets for appropriate quantitative analysis. The acquisition of such large datasets has been enabled by recent advances in high-throughput cryo-electron tomography (cryo-ET) methods. This also necessitates the development of complementary approaches to maximize the extraction of relevant biological information. We have developed a computational toolbox for the semi-automatic quantification of segmented and vectorized filamentous networks from pre-processed cryo-electron tomograms, facilitating the analysis and cross-comparison of multiple experimental conditions. GUI-based components simplify the processing of data and allow users to obtain a large number of ultrastructural parameters describing filamentous assemblies. We demonstrate the feasibility of this workflow by analyzing cryo-ET data of untreated and chemically perturbed branched actin filament networks and that of parallel actin filament arrays. In principle, the computational toolbox presented here is applicable for data analysis comprising any type of filaments in regular (i.e. parallel) or random arrangement. We show that it can ease the identification of key differences between experimental groups and facilitate the in-depth analysis of ultrastructural data in a time-efficient manner. AU - Dimchev, Georgi A AU - Amiri, Behnam AU - Fäßler, Florian AU - Falcke, Martin AU - Schur, Florian KM ID - 10290 IS - 4 JF - Journal of Structural Biology KW - Structural Biology SN - 1047-8477 TI - Computational toolbox for ultrastructural quantitative analysis of filament networks in cryo-ET data VL - 213 ER - TY - JOUR AB - De novo loss of function mutations in the ubiquitin ligase-encoding gene Cullin3 lead to autism spectrum disorder (ASD). In mouse, constitutive haploinsufficiency leads to motor coordination deficits as well as ASD-relevant social and cognitive impairments. However, induction of Cul3 haploinsufficiency later in life does not lead to ASD-relevant behaviors, pointing to an important role of Cul3 during a critical developmental window. Here we show that Cul3 is essential to regulate neuronal migration and, therefore, constitutive Cul3 heterozygous mutant mice display cortical lamination abnormalities. At the molecular level, we found that Cul3 controls neuronal migration by tightly regulating the amount of Plastin3 (Pls3), a previously unrecognized player of neural migration. Furthermore, we found that Pls3 cell-autonomously regulates cell migration by regulating actin cytoskeleton organization, and its levels are inversely proportional to neural migration speed. Finally, we provide evidence that cellular phenotypes associated with autism-linked gene haploinsufficiency can be rescued by transcriptional activation of the intact allele in vitro, offering a proof of concept for a potential therapeutic approach for ASDs. AU - Morandell, Jasmin AU - Schwarz, Lena A AU - Basilico, Bernadette AU - Tasciyan, Saren AU - Dimchev, Georgi A AU - Nicolas, Armel AU - Sommer, Christoph M AU - Kreuzinger, Caroline AU - Dotter, Christoph AU - Knaus, Lisa AU - Dobler, Zoe AU - Cacci, Emanuele AU - Schur, Florian KM AU - Danzl, Johann G AU - Novarino, Gaia ID - 9429 IS - 1 JF - Nature Communications KW - General Biochemistry KW - Genetics and Molecular Biology TI - Cul3 regulates cytoskeleton protein homeostasis and cell migration during a critical window of brain development VL - 12 ER - TY - JOUR AB - 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. AU - Damiano-Guercio, Julia AU - Kurzawa, Laëtitia AU - Müller, Jan AU - Dimchev, Georgi A AU - Schaks, Matthias AU - Nemethova, Maria AU - Pokrant, Thomas AU - Brühmann, Stefan AU - Linkner, Joern AU - Blanchoin, Laurent AU - Sixt, Michael K AU - Rottner, Klemens AU - Faix, Jan ID - 7909 JF - eLife TI - Loss of Ena/VASP interferes with lamellipodium architecture, motility and integrin-dependent adhesion VL - 9 ER - TY - JOUR AB - The actin-related protein (Arp)2/3 complex nucleates branched actin filament networks pivotal for cell migration, endocytosis and pathogen infection. Its activation is tightly regulated and involves complex structural rearrangements and actin filament binding, which are yet to be understood. Here, we report a 9.0 Å resolution structure of the actin filament Arp2/3 complex branch junction in cells using cryo-electron tomography and subtomogram averaging. This allows us to generate an accurate model of the active Arp2/3 complex in the branch junction and its interaction with actin filaments. Notably, our model reveals a previously undescribed set of interactions of the Arp2/3 complex with the mother filament, significantly different to the previous branch junction model. Our structure also indicates a central role for the ArpC3 subunit in stabilizing the active conformation. AU - Fäßler, Florian AU - Dimchev, Georgi A AU - Hodirnau, Victor-Valentin AU - Wan, William AU - Schur, Florian KM ID - 8971 JF - Nature Communications KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Physics and Astronomy KW - General Chemistry SN - 2041-1723 TI - Cryo-electron tomography structure of Arp2/3 complex in cells reveals new insights into the branch junction VL - 11 ER - TY - JOUR AB - 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. AU - Dimchev, Georgi A AU - Amiri, Behnam AU - Humphries, Ashley C. AU - Schaks, Matthias AU - Dimchev, Vanessa AU - Stradal, Theresia E. B. AU - Faix, Jan AU - Krause, Matthias AU - Way, Michael AU - Falcke, Martin AU - Rottner, Klemens ID - 8434 IS - 7 JF - Journal of Cell Science KW - Cell Biology SN - 0021-9533 TI - Lamellipodin tunes cell migration by stabilizing protrusions and promoting adhesion formation VL - 133 ER - TY - JOUR AB - Cell migration is commonly accompanied by protrusion of membrane ruffles and lamellipodia. In two-dimensional migration, protrusion of these thin sheets of cytoplasm is considered relevant to both exploration of new space and initiation of nascent adhesion to the substratum. Lamellipodium formation can be potently stimulated by Rho GTPases of the Rac subfamily, but alsoby RhoG or Cdc42. Here we describe viable fibroblast cell lines geneticallydeficient for Rac1 that lack detectable levels of Rac2 and Rac3. Rac-deficient cells were devoid of apparent lamellipodia, but these structures were restored by expression of either Rac subfamily member, but not by Cdc42 or RhoG. Cells deficient in Rac showed strong reduction in wound closure and random cell migration and a notable loss of sensitivity to a chemotactic gradient. Despite these defects, Rac-deficient cells were able to spread, formed filopodia and established focal adhesions. Spreading in these cells was achieved by the extension of filopodia followed by the advancement of cytoplasmic veils between them. The number and size of focal adhesions as well as their intensity were largely unaffected by genetic removal of Rac1. However, Rac deficiency increased the mobility of different components in focal adhesions, potentially explaining how Rac - although not essential - can contribute to focal adhesion assembly. Together, our data demonstrate that Rac signaling is essential for lamellipodium protrusion and for efficient cell migration, but not for spreading or filopodium formation. Our findings also suggest that Rac GTPases are crucial to the establishment or maintenance of polarity in chemotactic migration. AU - Steffen, Anika AU - Ladwein, Markus AU - Georgi Dimchev AU - Hein, Anke AU - Schwenkmezger, Lisa AU - Arens, Stefan AU - Ladwein, Kathrin I AU - Holleboom, J. Margit AU - Florian Schur AU - Small, John V AU - Schwarz, Janett AU - Gerhard, Ralf AU - Faix, Jan AU - Stradal, Theresia E AU - Brakebusch, Cord H AU - Rottner, Klemens ID - 811 IS - 20 JF - Journal of Cell Science TI - Rac function is crucial for cell migration but is not required for spreading and focal adhesion formation VL - 126 ER -