Cancer cells utilize filopodia to explore, adhere to, and invade their surrounding microenvironment, yet the protein networks that organize these protrusions remain incompletely defined. To uncover the molecular machinery underlying MYO10-positive filopodia, we targeted the fast biotin ligase TurboID to the motor protein MYO10. Proximity biotinylation in two cell types revealed hundreds of potential MYO10 interactors. A targeted microscopy and siRNA screen identified MINK1, SCRIB, CSNK1A1, and CCT8 as new regulators of MYO10 filopodia formation. Focusing on one of the common interactors between cell lines, CCT8, known as a subunit of the chaperonin TRiC (TCP1 Ring Complex), we found that CCT8 associates with the MYO10 motor domain and regulates MYO10 filopodia independently of TRiC. Depleting CCT8 affected filopodia dynamics and impaired cell spreading, migration, and invasion in breast cancer cells. These findings establish CCT8 as a TRiC-independent regulator of MYO10 filopodia across diverse cancer cell types.

Cancer cells utilize filopodia to explore, adhere to, and invade their surrounding microenvironment, yet the protein networks that organize these protrusions remain incompletely defined. To uncover the molecular machinery underlying MYO10-positive filopodia, we targeted the fast biotin ligase TurboID to the filopo-dia tip-localized motor protein MYO10. Proximity biotinylation in two cell types revealed hundreds of potential MYO10 interactors. Surprisingly, there was limited overlap between the cell lines, indicating a previously unknown level of cell-type specificity in filopodia composition. A targeted microscopy and siRNA screen identified MINK1, SCRIB, CSNK1A1, and CCT8 as new regulators of filopodia formation. Focusing on one common interactor between cell lines, CCT8, known as a subunit of the chaperonin TRiC (TCP1 Ring Complex), we found that CCT8 associates with the MYO10 motor domain and regulates MYO10 filopodia independently of TRiC. Depleting CCT8 affected filopodia dynamics and impaired cell spreading, migration, and invasion in breast cancer cells. These findings establish CCT8 as a TRiC-independent regulator of MYO10 filopodia across different cancer cell types, highlight the surprising cell-type-specificity of filopodia composition, and provide a strategy and resource for studying filopodia in various biological contexts.

Myosin-X (MYO10), a molecular motor localizing to filopodia, is thought to transport various cargo to filopodia tips, modulating filopodia function. Yet, only a few MYO10 cargoes have been described. Here, using GFP-Trap and BioID approaches combined with mass spectrometry, we identified lamellipodin (RAPH1) as a novel MYO10 cargo. We report that the FERM domain of MYO10 is required for RAPH1 localization and accumulation at filopodia tips. Previous studies have mapped RAPH1 interaction with adhesome components to its talinbinding and Ras-association domains. Surprisingly, we find that the RAPH1 MYO10-binding site is not within these domains. Instead, it comprises an area with previously unknown functions. Functionally, RAPH1 supports MYO10 filopodia formation and stability but is not involved in regulating integrin activity in filopodia tips. Taken together, our data indicate a feed-forward mechanism whereby MYO10 filopodia are positively regulated by MYO10-mediated transport of RAPH1 to the filopodium tip.

Summary: Filopodia assemble unique integrin-adhesion complexes to sense the extracellular matrix. However, the mechanisms of integrin regulation in filopodia are poorly defined. Here, we report that active integrins accumulate at the tip of myosin-X (MYO10)-positive filopodia, while inactive integrins are uniformly distributed. We identify talin and MYO10 as the principal integrin activators in filopodia. In addition, deletion of MYO10’s FERM domain, or mutation of its β1-integrin-binding residues, reveals MYO10 as facilitating integrin activation, but not transport, in filopodia. However, MYO10’s isolated FERM domain alone cannot activate integrins, potentially because of binding to both integrin tails. Finally, because a chimera construct generated by swapping MYO10-FERM by talin-FERM enables integrin activation in filopodia, our data indicate that an integrin-binding FERM domain coupled to a myosin motor is a core requirement for integrin activation in filopodia. Therefore, we propose a two-step integrin activation model in filopodia: receptor tethering by MYO10 followed by talin-mediated integrin activation.

Actin-rich cellular protrusions direct versatile biological processes from cancer cell invasion to dendritic spine development. The stability, morphology, and specific biological functions of these protrusions are regulated by crosstalk between three main signaling axes: integrins, actin regulators, and small guanosine triphosphatases (GTPases). SHANK3 is a multifunctional scaffold protein, interacting with several actin-binding proteins and a well-established autism risk gene. Recently, SHANK3 was demonstrated to sequester integrin-activating small GTPases Rap1 and R-Ras to inhibit integrin activity via its Shank/ProSAP N-terminal (SPN) domain. Here, we demonstrate that, in addition to scaffolding actin regulators and actin-binding proteins, SHANK3 interacts directly with actin through its SPN domain. Molecular simulations and targeted mutagenesis of the SPN-ankyrin repeat region (ARR) interface reveal that actin binding is inhibited by an intramolecular closed conformation of SHANK3, where the adjacent ARR domain covers the actin-binding interface of the SPN domain. Actin and Rap1 compete with each other for binding to SHANK3, and mutation of SHANK3, resulting in reduced actin binding, augments inhibition of Rap1-mediated integrin activity. This dynamic crosstalk has functional implications for cell morphology and integrin activity in cancer cells. In addition, SHANK3-actin interaction regulates dendritic spine morphology in neurons and autism-linked phenotypes in vivo.

In this study, direct and effective intracellular delivery of CRISPR/Cas9 plasmids for homology-directed repair is achieved by functionalized mesoporous silica nanoparticles (MSNs). The functionalized MSNs (Cy5.5-MSNs-NLS) are synthesized by in situ labeling of a fluorescent dye (Cy5.5) and surface conjugation of nuclear localization sequence (NLS, PKKKRKV), showing a high loading efficiency (50%) toward the plasmids (PXN cutdown plasmid: GFP-Cas9-paxillin_gRNA and repair plasmid: AICSDP-1: PXN-EGFP). Subsequently, a polymeric coating of the poly(dimethyldiallylammonium chloride) (PDDA) is electrostatically deposited onto the plasmid-loaded Cy5.5-MSNs-NLS by microfluidic nanoprecipitation. The coating layer offers effective protection against the denaturation of plasmids by EcoRV restriction enzymes, and is shown to prevent premature release. Moreover, owing to the positive charge and pH-responsive disaggregation of PDDA, enhanced cellular internalization (16 h) and endosomal escape (4 h) of the nanocarrier are observed. After escape of nanocarrier system into the cytoplasm, the NLS on the surface of MSNs facilitates nuclear transport of the CRISPR/Cas9 plasmids, achieving successful GFP-tag knock-in of the PXN genomic sequence in U2OS cells. This intracellular delivery system thus offers an attractive method to overcome physiological barriers for CRISPR/Cas9 delivery, showing considerable promise for paxillin-associated focal adhesion and signaling regulator investigation.

Cellular mechanics play a crucial role in tissue homeostasis and are often misregulated in disease. Traction force microscopy is one of the key methods that has enabled researchers to study fundamental aspects of mechanobiology; however, traction force microscopy is limited by poor resolution. Here, we propose a simplified protocol and imaging strategy that enhances the output of traction force microscopy by increasing i) achievable bead density and ii) the accuracy of bead tracking. Our approach relies on super-resolution microscopy, enabled by fluorescence fluctuation analysis. Our pipeline can be used on spinning-disk confocal or widefield microscopes and is compatible with available analysis software. In addition, we demonstrate that our workflow can be used to gain biologically relevant information and is suitable for fast long-term live measurement of traction forces even in light-sensitive cells. Finally, using fluctuation-based traction force microscopy, we observe that filopodia align to the force field generated by focal adhesions.

Abstract RAS proteins play a direct causal role in human cancers, with activating mutations in RAS occurring in ∼30% of tumors. Despite comprehensive efforts, efficient targeting of RAS has not yet reached the clinic. Here, we report SHANK3, a synaptic scaffolding protein and a regulator of integrin activity, as a novel tumor suppressor targeting RAS proto-oncogenes, especially mutant KRAS. We previously found the N-terminal part of SHANK3 as an unexpected RAS-association domain with high affinity for active RAS proteins. Now we show that the direct binding of SHANK3 to active RAS abrogates the MAPK/ERK signaling and thereby inhibits the RAS-stimulated transformation and slows the growth of RAS-dependent tumor cells. SHANK3 expression is downregulated in RAS-driven human cancers, and high expression of SHANK3 correlates with improved patient survival in KRAS-driven pancreatic and lung adenocarcinoma. These data support that targeting the MAPK/ERK pathway by SHANK3 represents a novel promising therapeutic approach for RAS-driven cancers. Citation Format: Johanna Lilja, Guillaume Jcaquement, Itziar Martinez D. Posada, Petra Laasola, Ella-Maria Vesilahti, Mitro Miihkinen, Siiri Salomaa, Hans-Juergen Kreienkamp, Igor Barsukov, Daniel Abankwa, Johanna Ivaska. SHANK3 in oncogenic RAS signaling [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B29.

Actin-rich cellular protrusions direct versatile biological processes from cancer cell invasion to dendritic spine development. The stability, morphology and specific biological function of these protrusions are regulated by crosstalk between three main signaling axes: integrins, actin regulators and small GTPases. SHANK3 is a multifunctional scaffold protein, interacting with several actin-binding proteins, and a well-established autism risk gene. Recently, SHANK3 was demonstrated to sequester integrin-activating small GTPases Rap1 and R-Ras to inhibit integrin activity via its N-terminal SPN domain. Here, we demonstrate that SHANK3 interacts directly with actin using its SPN domain. Actin binding can be inhibited by an intramolecular closed conformation of SHANK3, where the adjacent ARR domain covers the actin-binding interface of the SPN domain. Actin and Rap1 compete with each other for binding to SHANK3 and loss of SHANK3-actin binding augments inhibition of Rap1-mediated integrin activity. This dynamic crosstalk has functional implications for filopodia formation in cancer cells, dendritic spine morphology in neurons and autism-linked phenotypes in vivo .

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Mitro Miihkinen is co-first author on ‘ProLIF – quantitative integrin protein–protein interactions and synergistic membrane effects on proteoliposomes’, published in the ‘Reconstituting Cell Biology’ special issue of Journal of Cell Science. Mitro is a PhD student in the lab of Johanna Ivaska at Turku Centre for Biotechnology, Turku, Finland, investigating the role of integrins and filopodia in cancer cell invasion.

While it is clear that key transcriptional programmes are important for maintaining pluripotency, the requirement for cell adhesion to the extracellular matrix remains poorly defined. Human pluripotent stem cells (hPSCs) form colonies encircled by an actin ring and large stable cornerstone focal adhesions (FA). Using superresolution two-colour interferometric photo-activated localisation microscopy, we examine the three-dimensional architecture of cornerstone adhesions and report vertical lamination of FA proteins with three main structural features distinct from previously studied focal adhesions: 1) integrin β5 and talin are present at high density, at the edges of cornerstone FA, adjacent to a vertical kank-rich protein wall, 2) vinculin localises higher than previously reported, displaying a head-above-tail orientation, and 3) surprisingly, actin and α-actinin are present in two discrete z-layers. Finally, we report that depletion of kanks diminishes FA patterning, and actin organisation within the colony, indicating a role for kanks in hPSC colony architecture.

Filopodia are adhesive cellular protrusions specialised in the detection of extracellular matrix (ECM)-derived cues. While ECM engagement at focal adhesions is known to trigger the recruitment of hundreds of proteins (“adhesome”) to fine-tune cellular behaviour, the components of the filopodia adhesions remain undefined. Here, we performed a structured illumination microscopy-based screen to map the localisation of 80 target proteins, linked to cell adhesion and migration, within filopodia. We demonstrate preferential enrichment of several adhesion proteins to either filopodia tips, filopodia shafts, or shaft subdomains suggesting divergent, spatially restricted functions for these proteins. Moreover, proteins with phospho-inositide (PI) binding sites are particularly enriched in filopodia. This, together with the strong localisation of PI(3,4)P2 in filopodia tips, predicts critical roles for PIs in regulating filopodia ultra-structure and function. Our mapping further reveals that filopodia adhesions consist of a unique set of proteins, the filopodome, that are distinct from classical nascent adhesions, focal adhesions and fibrillar adhesions. Using live imaging, we observe that filopodia adhesions can give rise to nascent adhesions, which, in turn, form focal adhesions. Finally, we demonstrate that p130Cas (BCAR1) is recruited to filopodia tips via its CCHD domain and acts as a mechanosensitive regulator of filopodia stability.

Integrin transmembrane heterodimeric receptors control a wide range of biological interactions by triggering the assembly of large multiprotein complexes at their cytoplasmic interface. A diverse set of methods have been used to investigate cytoplasmic interactions between integrins and intracellular proteins. These predominantly consist of peptide-based pull-downs and biochemical immuno-isolations from detergent-solubilized cell lysates. However, quantitative methods to probe integrin-protein interactions in a more biologically relevant context where the integrin is embedded within a lipid bilayer have been lacking. Here we describe a technique called ProLIF (Protein-Liposome Iinenteractions by Flow cytometry) to reconstitute recombinant integrin transmembrane domain (TMD) and cytoplasmic tail (CT) fragments on liposomes as individual α or β subunits or as αβ heterodimers and, using flow cytometry, to rapidly and quantitatively measure protein interactions with these membrane-embedded integrins. Importantly, the assay can analyse binding of fluorescent proteins directly from cell lysates without further purification steps. By combining integrins with membrane lipids to generate proteoliposomes, the effects of membrane composition such as PI(4,5)P2 presence on protein recruitment to the integrin CTs can be analyzed. ProLIF requires no specific instrumentation, apart from a standard flow cytometer and can be applied to measure a broad range of membrane-dependent protein-protein interactions with the potential for high-throughput/multiplex analyses.

Human pluripotent stem cells (hPSC) can generate almost all adult cell lineages. While it is clear that key transcriptional programmes are important elements for maintaining pluripotency, the equally essential requirement for cell adhesion to specific extracellular matrix components remains poorly defined. Our recent observation that hPSC colonies form unusually large cornerstone focal adhesions (FA), distinct from parental somatic cells, that are lost following differentiation, emphasises the potential of these atypical FA as gatekeepers of pluripotency. Here, using nanopatterns, we further demonstrate that physical restriction of adhesion size, in hPSC colonies, is sufficient to trigger differentiation. Using superresolution two-colour interferometric photo-activated localization microscopy (iPALM), we examined the three-dimensional architecture of these cornerstone adhesions and report vertical lamination of FA proteins with three main structural peculiarities: 1) integrin β5 and talin are present at high density, at the edges of cornerstone FA, adjacent to a vertical kank-rich protein wall. 2) Vinculin localises higher than expected with respect to the substrata and displays a head-above-tail orientation, and 3) surprisingly, actin and α-actinin are present in two discrete layers, a previously undescribed localisation for these proteins. Finally, we report that depletion of kanks diminishes FA patterning, and actin organisation within the colony, indicating a key role for kanks in hPSC colony architecture.

Sharpin, a multifunctional adaptor protein, regulates several signalling pathways. For example, Sharpin enhances signal-induced NF-κB signalling as part of the linear ubiquitin assembly complex (LUBAC) and inhibits integrins, the T cell receptor, caspase 1 and PTEN. However, despite recent insights into Sharpin and LUBAC function, a systematic approach to identify the signalling pathways regulated by Sharpin has not been reported. Here, we present the first 'Sharpin interactome', which identifies a large number of novel potential Sharpin interactors in addition to several known ones. These data suggest that Sharpin and LUBAC might regulate a larger number of biological processes than previously identified, such as endosomal trafficking, RNA processing, metabolism and cytoskeleton regulation. Importantly, using the Sharpin interactome, we have identified a novel role for Sharpin in lamellipodium formation. We demonstrate that Sharpin interacts with Arp2/3, a protein complex that catalyses actin filament branching. We have identified the Arp2/3-binding site in Sharpin and demonstrate using a specific Arp2/3-binding deficient mutant that the Sharpin-Arp2/3 interaction promotes lamellipodium formation in a LUBAC-independent fashion.This article has an associated First Person interview with the first author of the paper.