Novel insights into filopodia function – A focus on integrin and F-actin regulation

Filopodia are actin-rich cell protrusions that are extended from the plasma membrane by different cell types in order to sense the surrounding environment. In cancer cells, the emergence of filopodia supports metastasis, while in neuronal cells filopodia help to form synapses by probing adjacent cells for suitable presynaptic partners. Filopodia can make contact with the extracellular matrix with small, integrinmediated adhesions located at their tips. During cancer invasion, recognition of the extracellular matrix by filopodia not only supplies migrating cells with guidance cues but has also been linked with increased colony growth at the metastatic site. In neuronal cells, inhibition of integrin activity has been shown to negatively impact synapse formation. Although both filopodia and integrin-positive adhesions have roles in metastasis or in synaptogenesis, filopodial adhesions remain under-studied cellular structures and there exists minimal literature on how these adhesions are regulated or how they function. The objective of this study was to reveal novel regulatory mechanisms of filopodia by studying two integrin and filopodia linked proteins: myosin-X and SHANK3. The work presented here provides fundamental information on how 1) integrins are activated at filopodia tips, 2) which integrinlinked proteins are recruited to adhesions at filopodia providing a road-map to classify these adhesions and 3) how SHANK3, an F-actin network organizer and filopodia regulator, modulates the crosstalk between integrin and F-actin via direct and conformationally regulated binding with F-actin. The thesis also provides novel methodology in the form of a high-end biochemical binding assay (4) where protein binding to integrin tails can be interrogated in the presence of a lipidic membrane.Solun filopodien toiminta Filopodit ovat solukalvon pullistumia, joita täyttää sisältäpäin solun aktiinitukiranka. Filopodit syntyvät solukalvon pullistuessa ulospäin aktiinitukirangan vaikutuksesta ja solut käyttävät niitä ympäristönsä aistimiseen käyttämällä filopodien kärjissä sijaitsevia tarttumisreseptoreja - integriinejä. Syövän leviämisen aikana solujen kyky tunnistella ympäröivää soluväliainetta tarjoaa solulle tarkkaa tietoa sen ympäristöstä, ohjaten solun kulkua vaikeassa 3-ulotteisessa soluväliaineen verkostossa. Toisaalta hermosolut käyttävät filopodeja naapurisolujensa (tai ympäröivän kudoksen) tarkasteluun hermoliitosten muodostusprosessissa. Vaikka filopodit ja integriinireseptorit ohjaavat sekä syövän leviämistä ja aivojen normaalia toimintaa, filopodien välittämien integriini-positiivisten soluadheesioiden synty tunnetaan huonosti. Lisäksi filopodien soluadheesioiden säätely sekä vaikutukset solun toiminnalle ovat heikosti ymmärrettyjä tapahtumasarjoja. Tämän väitöskirjatyön tarkoituksena oli ymmärtää filopodien solullisia säätelyketjuja paremmin tutkimalla kahta integriineihin ja filopodeihin aiemmin liitettyä proteiinia: myosiini-X ja SHANK3. Tässä väitöskirjatyössä esitetyt tulokset tuovat täysin uutta tietoa seuraavista tapahtumista: 1) miten filopodit muodostavat adheesioita soluväliaineen kanssa 2) mitkä solun adheesioproteiinit sijoittuvat filopodien kärkiin adheesiomuodostusprosessin aikana sekä 3) kuinka SHANK3 proteiini säätelee samanaikaisesti solun aktiinitukirankaa ja integriiniaktiivisuutta. Lisäksi väitöskirja sisältää menetelmällisen julkaisun uudesta biokemiallisesta koeasetelmasta, jonka avulla voidaan paremmin tutkia solun proteiinien sitoutumista integriinireseptoreihin solukalvon läheisyydessä

Myosin-X and talin modulate integrin activity at filopodia tips

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 b1-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.Peer reviewe

Filopodome Mapping Identifies p130Cas as a Mechanosensitive Regulator of Filopodia Stability

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. We demonstrate that p130Cas (BCAR1) is recruited to filopodia tips via its C-terminal Cas family homology domain (CCHD) and acts as a mechanosensitive regulator of filopodia stability. Finally, we demonstrate that our map based on myosin-X-induced filopodia can be translated to endogenous filopodia and fascin- and IRSp53-mediated filopodia

Fluctuation-Based Super-Resolution Traction Force Microscopy

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

Myosin-X and talin modulate integrin activity at filopodia tips

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 b1-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

Superresolution architecture of cornerstone focal adhesions in human pluripotent stem cells

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.</p

The Sharpin interactome reveals a role for Sharpin in lamellipodium formation via the Arp2/3 complex

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, caspase1 and PTEN. However, despite recent insights into Sharpin and LUBAC function, a systematic approach to identify signalling pathways regulated by Sharpin has not been reported. Here, we present the first ‘Sharpin interactome’, which identifies a large amount 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 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.</p

Effective Delivery of the CRISPR/Cas9 System Enabled by Functionalized Mesoporous Silica Nanoparticles for GFP-Tagged Paxillin Knock-In

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

SHANK3 conformation regulates direct actin binding and crosstalk with Rap1 signaling

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.Peer reviewe

SHANK3 conformation regulates direct actin binding and crosstalk with Rap1 signaling

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