Tadamoto Isogai

Initiation of lamellipodia and ruffles involves cooperation between mDia1 and the Arp2/3 complex

ABSTRACT Protrusion of lamellipodia and ruffles requires polymerization of branched actin filaments by the Arp2/3 complex. Although regulation of Arp2/3 complex activity has been extensively investigated, the mechanism of initiation of lamellipodia and ruffles remains poorly understood. Here, we show that mDia1 acts in concert with the Arp2/3 complex to promote initiation of lamellipodia and ruffles. We find that mDia1 is an epidermal growth factor (EGF)-regulated actin nucleator involved in membrane ruffling using a combination of knockdown and rescue experiments. At the molecular level, mDia1 polymerizes linear actin filaments, activating the Arp2/3 complex, and localizes within nascent and mature membrane ruffles. We employ functional complementation experiments and optogenetics to show that mDia1 cooperates with the Arp2/3 complex in initiating lamellipodia and ruffles. Finally, we show that genetic and pharmacological interference with this cooperation hampers ruffling and cell migration. Thus, we propose that the lamellipodium- and ruffle-initiating machinery consists of two actin nucleators that act sequentially to regulate membrane protrusion and cell migration. Highlighted Article: The formin mDia1 nucleates linear actin filaments that serve as mother filaments for initial activation of the Arp2/3 complex and formation of lamellipodia and ruffles.

SMIFH2 has effects on Formins and p53 that perturb the cell cytoskeleton

Formin proteins are key regulators of the cytoskeleton involved in developmental and homeostatic programs, and human disease. For these reasons, small molecules interfering with Formins’ activity have gained increasing attention. Among them, small molecule inhibitor of Formin Homology 2 domains (SMIFH2) is often used as a pharmacological Formin blocker. Although SMIFH2 inhibits actin polymerization by Formins and affects the actin cytoskeleton, its cellular mechanism of action and target specificity remain unclear. Here we show that SMIFH2 induces remodelling of actin filaments, microtubules and the Golgi complex as a result of its effects on Formins and p53. We found that SMIFH2 triggers alternated depolymerization-repolymerization cycles of actin and tubulin, increases cell migration, causes scattering of the Golgi complex, and also cytotoxicity at high dose. Moreover, SMIFH2 reduces expression and activity of p53 through a post-transcriptional, proteasome-independent mechanism that influences remodelling of the cytoskeleton. As the action of SMIFH2 may go beyond Formin inhibition, only short-term and low-dose SMIFH2 treatments minimize confounding effects induced by loss of p53 and cytotoxicity.

Flat clathrin lattices are dynamic actin-controlled hubs for clathrin-mediated endocytosis and signalling of specific receptors

Clathrin lattices at the plasma membrane coat both invaginated and flat regions forming clathrin-coated pits and clathrin plaques, respectively. The function and regulation of clathrin-coated pits in endocytosis are well understood but clathrin plaques remain enigmatic nanodomains. Here we use super-resolution microscopy, molecular genetics and cell biology to show that clathrin plaques contain the machinery for clathrin-mediated endocytosis and cell adhesion, and associate with both clathrin-coated pits and filamentous actin. We also find that actin polymerization promoted by N-WASP through the Arp2/3 complex is crucial for the regulation of plaques but not pits. Clathrin plaques oppose cell migration and undergo actin- and N-WASP-dependent disassembly upon activation of LPA receptor 1, but not EGF receptor. Most importantly, plaque disassembly correlates with the endocytosis of LPA receptor 1 and down-modulation of AKT activity. Thus, clathrin plaques serve as dynamic actin-controlled hubs for clathrin-mediated endocytosis and signalling that exhibit receptor specificity.

Proteomic Analyses Uncover a New Function and Mode of Action for Mouse Homolog of Diaphanous 2 (mDia2)

mDia2 is an auto-inhibited Formin influencing actin dynamics upon conversion to the active conformation. mDia2 regulates actin-based protrusions and cell invasion, cell differentiation, vesicle trafficking, and cytokinesis. However, whether mDia2 has additional functions and how its action is functionally specified remain unknown. Here we draw the interactome of auto-inhibited and constitutively active mDia2 to address these issues. We embed mDia2 in protein networks accounting for its attributed functions and unexpectedly link it to the Ubiquitin Proteasome System. Taking FBXO3 as a test case, we show that mDia2 binds FBXO3 and p53, and regulates p53 transcriptional activity in an actin-nucleation-independent and conformation-insensitive manner. Increased mDia2 and FBXO3 levels elevate p53 activity and expression thereby sensitizing cells to p53-dependent apoptosis, whereas their decrease produces opposite effects. Thus, we discover a new role of mDia2 in p53 regulation suggesting that the closed conformation is biologically active and an FBXO3-based mechanism to functionally specify mDia2s activity.

PFA fixation enables artifact-free super-resolution imaging of the actin cytoskeleton and associated proteins

ABSTRACT Super-resolution microscopy (SRM) allows precise localization of proteins in cellular organelles and structures, including the actin cytoskeleton. Yet sample preparation protocols for SRM are rather anecdotal and still being optimized. Thus, SRM-based imaging of the actin cytoskeleton and associated proteins often remains challenging and poorly reproducible. Here, we show that proper paraformaldehyde (PFA)-based sample preparation preserves the architecture of the actin cytoskeleton almost as faithfully as gold-standard glutaraldehyde fixation. We show that this fixation is essential for proper immuno-based localization of actin-binding and actin-regulatory proteins involved in the formation of lamellipodia and ruffles, such as mDia1, WAVE2 and clathrin heavy chain, and provide detailed guidelines for the execution of our method. In summary, proper PFA-based sample preparation increases the multi-color possibilities and the reproducibility of SRM of the actin cytoskeleton and its associated proteins. Summary: We show that proper PFA fixation allows high-quality super-resolution imaging of the actin cytoskeleton and can outperform gold-standard glutaraldehyde fixation for imaging of actin-binding proteins.

New nuclear and perinuclear functions of formins

Formin family proteins (formins) represent an evolutionary conserved protein family encoded in the genome of a wide range of eukaryotes. Formins are hallmarked by a formin homology 1 (FH1) domain juxtaposed to an FH2 domain whereby they control actin and microtubule dynamics. Not surprisingly, formins are best known as key regulators of the cytoskeleton in a variety of morphogenetic processes. However, mounting evidence implicates several formins in the assembly and organization of actin within and around the nucleus. In addition, actin-independent roles for formins have recently been discovered. In this mini-review, we summarize these findings and highlight the novel nuclear and perinulcear functions of formins. In light of the emerging new biology of formins, we also discuss the fundamental principles governing the versatile activity and multimodal regulation of these proteins.

Invadosomes - shaping actin networks to follow mechanical cues.

Invadosomes are actin-based protrusions formed by cells in response to obstacles in their microenvironment, especially basement membranes and dense interstitial matrices. A versatile set of proteins controls assembly and dynamics of the actin networks at invadosomes and adhesive molecules link them with the extracellular matrix. Furthermore, polarized delivery of proteases makes invadosomes degradative. Therefore, invadosomes have been classically viewed as specialized protrusions involved in cell migration and remodeling of the microenvironment. Recent discoveries have considerably broadened this picture by showing that invadosomes respond to traction forces and can self-organize into dynamic arrays capable of following the topography of the substrate. Although these findings suggest that invadosomes may function as mechanosensors, this possibility has not been critically evaluated. In this review, we first summarize the organization and dynamics of actin in invadosomes and their superstructures with emphasis on force-production mechanisms. Next, we outline our current understanding of how mechanical cues impinge on invadosomes and modify their behavior. From this perspective, we provide an outlook of the outstanding open questions and the main challenges in the field.

CLIC4 is regulated by RhoA-mDia2 signaling through Profilin-1 binding to modulate filopodia length

CLIC4 is a cytosolic protein implicated in diverse actin-based processes, including integrin trafficking, cell adhesion and tubulogenesis. CLIC4 is rapidly recruited to the plasma membrane by G12/13-coupled receptor agonists and then partly co-localizes with β1 integrins. Receptor-mediated CLIC4 translocation depends on actin polymerization, but the mechanism and functional significance of CLIC4 trafficking are unknown. Here we show that RhoA activation by either LPA or EGF is necessary and sufficient for CLIC4 translocation, with a regulatory role for the RhoA effector mDia2, an inducer of actin polymerization. We find that CLIC4 directly interacts with the G-actin-binding protein Profilin-1 via conserved residues that are required for CLIC4 trafficking and lie in a concave surface. Consistently, silencing of Profilin-1 impaired CLIC4 trafficking induced by either LPA or EGF. CLIC4 knockdown promoted the formation of long integrin-dependent filopodia, a phenotype rescued by wild-type CLIC4 but not by trafficking-incompetent CLIC4(C35A). Our results establish CLIC4 as a Profilin-1-binding protein and suggest that CLIC4 translocation provides a feedback mechanism to modulate mDia2/Profilin-1-driven cortical actin assembly and membrane protrusion.

PO-274 Tumour subtype-specific cells of origin of malignant mesothelioma

Introduction Malignant Mesothelioma (MM) is an aggressive malignancy of the lining of the thoracic and peritoneal cavity. The primary cause is previous asbestos exposure. The pathological diagnosis of MM is rather complex due to a lack of useful biological markers and because multiple cell types are involved in the development of MM. We aimed to investigate to what extent the tumour subtype is determined by the cell of origin rather than the somatically acquired driver mutations. Material and methods Co-deletion of the conditional tumour suppressors, NF2, p53 and Cdkn2a in freshly isolated mesothelial cells using Cre viruses allowed us to establish clonal cell lines with epithelial, sacomatoid and biphasic morphology as also observed in human MM. Cells were analysed for clinical relevant protein marker profiles, tumorigenic potential and RNA expression. Results were compared to (poly)clonal cell lines obtained from conditional mice injected intra-thoracically with lentiviruses expressing Cre-recombinase driven by tissue-specific promoters. Results and discussions Using the ex vivo approach we were able to obtain clonal cell lines that upon transplantation gave rise to the main three MM tumour epithelial, sarcomatoid and biphasic subtypes. The epithelial and sarcomatoid phenotypes observed in vitro retained in the tumours, also after serial transplantation of the cell lines. Clonal biphasic cells co-expressed both epithelial and sarcomatoid markers and external factors could skew these biphasic cells towards a more epithelial or sarcomatoid phenotype. Transplantation of clonal biphasic cells only gave rise to tumours when the cells were grafted in immune-deficient mice, this in contrast to the epithelial and sarcomatoid cell lines that effectively gave tumours in syngeneic immunocompetent recipients. Analysis of tumour cell populations and derived clonal cell lines induced by lentiviral Cre-mediated switching of the same tumour suppressors in the mesothelial lining of conditional mice showed a high level of heterogeneity that included the same tumour subtypes also obtained using the ex vivo procedure. Conclusion A set of the same genetic mutations is able to drive heterogeneous MM development suggesting that the specific phenotype of the resulting tumour depends on the specific cell-of-origin. Therefore, epigenetic differences rather than acquired mutations appear a determining factor for the subtype of MM that arises. This epigenetic state appears relative stability as interconversion of the tumour subtypes was not observed.