Positive and negative durotaxis - mechanisms and emerging concepts.

Cell migration is controlled by the coordinated action of cell adhesion, cytoskeletal dynamics, contractility and cell extrinsic cues. Integrins are the main adhesion receptors to ligands of the extracellular matrix (ECM), linking the actin cytoskeleton to the ECM and enabling cells to sense matrix rigidity and mount a directional cell migration response to stiffness gradients. Most models studied show preferred migration of single cells or cell clusters towards increasing rigidity. This is referred to as durotaxis, and since its initial discovery in 2000, technical advances and elegant computational models have provided molecular level details of stiffness sensing in cell migration. However, modeling has long predicted that, depending on cell intrinsic factors, such as the balance of cell adhesion molecules (clutches) and the motor proteins pulling on them, cells might also prefer adhesion to intermediate rigidity. Recently, experimental evidence has supported this notion and demonstrated the ability of cells to migrate towards lower rigidity, in a process called negative durotaxis. In this Review, we discuss the significant conceptual advances that have been made in our appreciation of cell plasticity and context dependency in stiffness-guided directional cell migration.

Novel tools to study cell-ECM interactions, cell adhesion dynamics and migration.

Integrin-mediated cell adhesion is essential for cell migration, mechanotransduction and tissue integrity. In vivo, these processes are regulated by complex physicochemical signals from the extracellular matrix (ECM). These nuanced cues, including molecular composition, rigidity and topology, call for sophisticated systems to faithfully explore cell behaviour. Here, we discuss recent methodological advances in cell-ECM adhesion research and compile a toolbox of techniques that we expect to shape this field in future. We outline methodological breakthroughs facilitating the transition from rigid 2D substrates to more complex and dynamic 3D systems, as well as advances in super-resolution imaging for an in-depth understanding of adhesion nanostructure. Selected methods are exemplified with relevant biological findings to underscore their applicability in cell adhesion research. We expect this new "toolbox" of methods will allow for a closer approximation of in vitro experimental setups to in vivo conditions, providing deeper insights into physiological and pathophysiological processes associated with cell-ECM adhesion.

Focal adhesions contain three specialized actin nanoscale layers.

Focal adhesions (FAs) connect inner workings of cell to the extracellular matrix to control cell adhesion, migration and mechanosensing. Previous studies demonstrated that FAs contain three vertical layers, which connect extracellular matrix to the cytoskeleton. By using super-resolution iPALM microscopy, we identify two additional nanoscale layers within FAs, specified by actin filaments bound to tropomyosin isoforms Tpm1.6 and Tpm3.2. The Tpm1.6-actin filaments, beneath the previously identified α-actinin cross-linked actin filaments, appear critical for adhesion maturation and controlled cell motility, whereas the adjacent Tpm3.2-actin filament layer beneath seems to facilitate adhesion disassembly. Mechanistically, Tpm3.2 stabilizes ACF-7/MACF1 and KANK-family proteins at adhesions, and hence targets microtubule plus-ends to FAs to catalyse their disassembly. Tpm3.2 depletion leads to disorganized microtubule network, abnormally stable FAs, and defects in tail retraction during migration. Thus, FAs are composed of distinct actin filament layers, and each may have specific roles in coupling adhesions to the cytoskeleton, or in controlling adhesion dynamics.

Dynamic Micropatterning Reveals Substrate-Dependent Differences in the Geometric Control of Cell Polarization and Migration.

Cells are highly dynamic and adopt variable shapes and sizes. These variations are biologically important but challenging to investigate in a spatiotemporally controlled manner. Micropatterning, confining cells on microfabricated substrates with defined geometries and molecular compositions, is a powerful tool for controlling cell shape and interactions. However, conventional binary micropatterns are static and fail to address dynamic changes in cell polarity, spreading, and migration. Here, a method for dynamic micropatterning is reported, where the non-adhesive surface surrounding adhesive micropatterns is rapidly converted to support specific cell-matrix interactions while allowing simultaneous imaging of the cells. The technique is based on ultraviolet photopatterning of biotinylated polyethylene glycol-grafted poly-L-lysine, and it is simple, inexpensive, and compatible with a wide range of streptavidin-conjugated ligands. Experiments using biotinylation-based dynamic micropatterns reveal that distinct extracellular matrix ligands and bivalent integrin-clustering antibodies support different degrees of front-rear polarity in human glioblastoma cells, which correlates to altered directionality and persistence upon release and migration on fibronectin. Unexpectedly, however, neither an asymmetric cell shape nor centrosome orientation can fully predict the future direction of migration. Taken together, biotinylation-based dynamic micropatterns allow easily accessible and highly customizable control over cell morphology and motility.

Fibrillar adhesion dynamics govern the timescales of nuclear mechano-response via the vimentin cytoskeleton.

The cell nucleus is continuously exposed to external signals, of both chemical and mechanical nature. To ensure proper cellular response, cells need to regulate not only the transmission of these signals, but also their timing and duration. Such timescale regulation is well described for fluctuating chemical signals, but if and how it applies to mechanical signals reaching the nucleus is still unknown. Here we demonstrate that the formation of fibrillar adhesions locks the nucleus in a mechanically deformed conformation, setting the mechanical response timescale to that of fibrillar adhesion remodelling (~1 hour). This process encompasses both mechanical deformation and associated mechanotransduction (such as via YAP), in response to both increased and decreased mechanical stimulation. The underlying mechanism is the anchoring of the vimentin cytoskeleton to fibrillar adhesions and the extracellular matrix through plectin 1f, which maintains nuclear deformation. Our results reveal a mechanism to regulate the timescale of mechanical adaptation, effectively setting a low pass filter to mechanotransduction.

Defined extracellular matrix compositions support stiffness-insensitive cell spreading and adhesion signaling.

Integrin-dependent adhesion to the extracellular matrix (ECM) mediates mechanosensing and signaling in response to altered microenvironmental conditions. In order to provide tissue- and organ-specific cues, the ECM is composed of many different proteins that temper the mechanical properties and provide the necessary structural diversity. Despite most human tissues being soft, the prevailing view from predominantly in vitro studies is that increased stiffness triggers effective cell spreading and activation of mechanosensitive signaling pathways. To address the functional coupling of ECM composition and matrix rigidity on compliant substrates, we developed a matrix spot array system to screen cell phenotypes against different ECM mixtures on defined substrate stiffnesses at high resolution. We applied this system to both cancer and normal cells and surprisingly identified ECM mixtures that support stiffness-insensitive cell spreading on soft substrates. Employing the motor-clutch model to simulate cell adhesion on biochemically distinct soft substrates, with varying numbers of available ECM-integrin-cytoskeleton (clutch) connections, we identified conditions in which spreading would be supported on soft matrices. Combining simulations and experiments, we show that cell spreading on soft is supported by increased clutch engagement on specific ECM mixtures and even augmented by the partial inhibition of actomyosin contractility. Thus, "stiff-like" spreading on soft is determined by a balance of a cell's contractile and adhesive machinery. This provides a fundamental perspective for in vitro mechanobiology studies, identifying a mechanism through which cells spread, function, and signal effectively on soft substrates.

IGFBP2 secretion by mammary adipocytes limits breast cancer invasion.

The progression of noninvasive ductal carcinoma in situ to invasive ductal carcinoma for patients with breast cancer results in a significantly poorer prognosis and is the precursor to metastatic disease. In this work, we have identified insulin-like growth factor-binding protein 2 (IGFBP2) as a potent adipocrine factor secreted by healthy breast adipocytes that acts as a barrier against invasive progression. In line with this role, adipocytes differentiated from patient-derived stromal cells were found to secrete IGFBP2, which significantly inhibited breast cancer invasion. This occurred through binding and sequestration of cancer-derived IGF-II. Moreover, depletion of IGF-II in invading cancer cells using small interfering RNAs or an IGF-II-neutralizing antibody ablated breast cancer invasion, highlighting the importance of IGF-II autocrine signaling for breast cancer invasive progression. Given the abundance of adipocytes in the healthy breast, this work exposes the important role they play in suppressing cancer progression and may help expound upon the link between increased mammary density and poorer prognosis.

Myosin-X recruits lamellipodin to filopodia tips.

Myosin-X (MYO10), a molecular motor localizing to filopodia, is thought to transport various cargo to filopodia tips, modulating filopodia function. However, 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 the RAPH1 interaction domain for adhesome components to its talin-binding and Ras-association domains. Surprisingly, we find that the RAPH1 MYO10-binding site is not within these domains. Instead, it comprises a conserved helix located just after the RAPH1 pleckstrin homology domain with previously unknown functions. Functionally, RAPH1 supports MYO10 filopodia formation and stability but is not required to activate integrins at 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.

PP2A methylesterase PME-1 suppresses anoikis and is associated with therapy relapse of PTEN-deficient prostate cancers.

While organ-confined prostate cancer (PCa) is mostly therapeutically manageable, metastatic progression of PCa remains an unmet clinical challenge. Resistance to anoikis, a form of cell death initiated by cell detachment from the surrounding extracellular matrix, is one of the cellular processes critical for PCa progression towards aggressive disease. Therefore, further understanding of anoikis regulation in PCa might provide therapeutic opportunities. Here, we discover that PCa tumours with concomitant inhibition of two tumour suppressor phosphatases, PP2A and PTEN, are particularly aggressive, having < 50% 5-year secondary-therapy-free patient survival. Functionally, overexpression of PME-1, a methylesterase for the catalytic PP2A-C subunit, inhibits anoikis in PTEN-deficient PCa cells. In vivo, PME-1 inhibition increased apoptosis in in ovo PCa tumour xenografts, and attenuated PCa cell survival in zebrafish circulation. Molecularly, PME-1-deficient PC3 cells display increased trimethylation at lysines 9 and 27 of histone H3 (H3K9me3 and H3K27me3), a phenotype known to correlate with increased apoptosis sensitivity. In summary, our results demonstrate that PME-1 supports anoikis resistance in PTEN-deficient PCa cells. Clinically, these results identify PME-1 as a candidate biomarker for a subset of particularly aggressive PTEN-deficient PCa.

An extracellular receptor tyrosine kinase motif orchestrating intracellular STAT activation.

The ErbB4 receptor isoforms JM-a and JM-b differ within their extracellular juxtamembrane (eJM) domains. Here, ErbB4 isoforms are used as a model to address the effect of structural variation in the eJM domain of receptor tyrosine kinases (RTK) on downstream signaling. A specific JM-a-like sequence motif is discovered, and its presence or absence (in JM-b-like RTKs) in the eJM domains of several RTKs is demonstrated to dictate selective STAT activation. STAT5a activation by RTKs including the JM-a like motif is shown to involve interaction with oligosaccharides of N-glycosylated cell surface proteins such as ß1 integrin, whereas STAT5b activation by JM-b is dependent on TYK2. ErbB4 JM-a- and JM-b-like RTKs are shown to associate with specific signaling complexes at different cell surface compartments using analyses of RTK interactomes and super-resolution imaging. These findings provide evidence for a conserved mechanism linking a ubiquitous extracellular motif in RTKs with selective intracellular STAT signaling.

MYO10-filopodia support basement membranes at pre-invasive tumor boundaries.

Ductal carcinoma in situ (DCIS) is a pre-invasive stage of breast cancer. During invasion, the encapsulating DCIS basement membrane (BM) is compromised, and tumor cells invade the surrounding stroma. The mechanisms that regulate functional epithelial BMs in vivo are poorly understood. Myosin-X (MYO10) is a filopodia-inducing protein associated with metastasis and poor clinical outcome in invasive breast cancer (IBC). We identify elevated MYO10 expression in human DCIS and IBC, and this suggests links with disease progression. MYO10 promotes filopodia formation and cell invasion in vitro and cancer-cell dissemination from progressively invasive human DCIS xenografts. However, MYO10-depleted xenografts are more invasive. These lesions exhibit compromised BMs, poorly defined borders, and increased cancer-cell dispersal and EMT-marker-positive cells. In addition, cancer spheroids are dependent on MYO10-filopodia to generate a near-continuous extracellular matrix boundary. Thus, MYO10 is protective in early-stage breast cancer, correlating with tumor-limiting BMs, and pro-invasive at later stages, facilitating cancer-cell dissemination.

Directed cell migration towards softer environments.

How cells sense tissue stiffness to guide cell migration is a fundamental question in development, fibrosis and cancer. Although durotaxis-cell migration towards increasing substrate stiffness-is well established, it remains unknown whether individual cells can migrate towards softer environments. Here, using microfabricated stiffness gradients, we describe the directed migration of U-251MG glioma cells towards less stiff regions. This 'negative durotaxis' does not coincide with changes in canonical mechanosensitive signalling or actomyosin contractility. Instead, as predicted by the motor-clutch-based model, migration occurs towards areas of 'optimal stiffness', where cells can generate maximal traction. In agreement with this model, negative durotaxis is selectively disrupted and even reversed by the partial inhibition of actomyosin contractility. Conversely, positive durotaxis can be switched to negative by lowering the optimal stiffness by the downregulation of talin-a key clutch component. Our results identify the molecular mechanism driving context-dependent positive or negative durotaxis, determined by a cell's contractile and adhesive machinery.

MASTL is enriched in cancerous and pluripotent stem cells and influences OCT1/OCT4 levels.

MASTL is a mitotic accelerator with an emerging role in breast cancer progression. However, the mechanisms behind its oncogenicity remain largely unknown. Here, we identify a previously unknown role and eminent expression of MASTL in stem cells. MASTL staining from a large breast cancer patient cohort indicated a significant association with ß3 integrin, an established mediator of breast cancer stemness. MASTL silencing reduced OCT4 levels in human pluripotent stem cells and OCT1 in breast cancer cells. Analysis of the cell-surface proteome indicated a strong link between MASTL and the regulation of TGF-ß receptor II (TGFBR2), a key modulator of TGF-ß signaling. Overexpression of wild-type and kinase-dead MASTL in normal mammary epithelial cells elevated TGFBR2 levels. Conversely, MASTL depletion in breast cancer cells attenuated TGFBR2 levels and downstream signaling through SMAD3 and AKT pathways. Taken together, these results indicate that MASTL supports stemness regulators in pluripotent and cancerous stem cells.

Pressure Drives Rapid Burst-Like Coordinated Cellular Motion from 3D Cancer Aggregates.

A key behavior observed during morphogenesis, wound healing, and cancer invasion is that of collective and coordinated cellular motion. Hence, understanding the different aspects of such coordinated migration is fundamental for describing and treating cancer and other pathological defects. In general, individual cells exert forces on their environment in order to move, and collective motion is coordinated by cell-cell adhesion-based forces. However, this notion ignores other mechanisms that encourage cellular movement, such as pressure differences. Here, using model tumors, it is found that increased pressure drove coordinated cellular motion independent of cell-cell adhesion by triggering cell swelling in a soft extracellular matrix (ECM). In the resulting phenotype, a rapid burst-like stream of cervical cancer cells emerged from 3D aggregates embedded in soft collagen matrices (0.5 mg mL-1 ). This fluid-like pushing mechanism, recorded within 8 h after embedding, shows high cell velocities and super-diffusive motion. Because the swelling in this model system critically depends on integrin-mediated cell-ECM adhesions and cellular contractility, the swelling is likely triggered by unsustained mechanotransduction, providing new evidence that pressure-driven effects must be considered to more completely understand the mechanical forces involved in cell and tissue movement as well as invasion.

Platelet SHARPIN regulates platelet adhesion and inflammatory responses through associations with αIIbß3 and LUBAC.

Platelets form hemostatic plugs to prevent blood loss, and they modulate immunity and inflammation in several ways. A key event during hemostasis is activation of integrin αIIbß3 through direct interactions of the ß3 cytoplasmic tail with talin and kindlin-3. Recently, we showed that human platelets express the adapter molecule Shank-associated RH domain interacting protein (SHARPIN), which can associate directly with the αIIb cytoplasmic tail and separately promote NF-κB pathway activation as a member of the Met-1 linear ubiquitination activation complex (LUBAC). Here we investigated the role of SHARPIN in platelets after crossing Sharpin flox/flox (fl/fl) mice with PF4-Cre or GPIbα-Cre mice to selectively delete SHARPIN in platelets. SHARPIN-null platelets adhered to immobilized fibrinogen through αIIbß3, and they spread more extensively than littermate control platelets in a manner dependent on feedback stimulation by platelet adenosine diphosphate (ADP) (P < .01). SHARPIN-null platelets showed increased colocalization of αIIbß3 with talin as assessed by super-resolution microscopy and increased binding of soluble fibrinogen in response to submaximal concentrations of ADP (P < .05). However, mice with SHARPIN-null platelets showed compromised thrombus growth on collagen and slightly prolonged tail bleeding times. Platelets lacking SHARPIN also showed reduced NF-κB activation and linear ubiquitination of protein substrates upon challenge with classic platelet agonists. Furthermore, the loss of platelet SHARPIN resulted in significant reduction in inflammation in murine models of colitis and peritonitis (P < .01). Thus, SHARPIN plays differential and context-dependent roles in platelets to regulate important inflammatory and integrin adhesive functions of these anucleate cells.

Sortilin-related receptor is a druggable therapeutic target in breast cancer.

In breast cancer, the currently approved anti-receptor tyrosine-protein kinase erbB-2 (HER2) therapies do not fully meet the expected clinical goals due to therapy resistance. Identifying alternative HER2-related therapeutic targets could offer a means to overcome these resistance mechanisms. We have previously demonstrated that an endosomal sorting protein, sortilin-related receptor (SorLA), regulates the traffic and signaling of HER2 and HER3, thus promoting resistance to HER2-targeted therapy in breast cancer. This study aims to assess the feasibility of targeting SorLA using a monoclonal antibody. Our results demonstrate that anti-SorLA antibody (SorLA ab) alters the resistance of breast cancer cells to HER2 monoclonal antibody trastuzumab in vitro and in ovo. We found that SorLA ab and trastuzumab combination therapy also inhibits tumor cell proliferation and tumor cell density in a mouse xenograft model of HER2-positive breast cancer. In addition, SorLA ab inhibits the proliferation of breast cancer patient-derived explant three-dimensional cultures. These results provide, for the first time, proof of principle that SorLA is a druggable target in breast cancer.

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.

Cargo-specific recruitment in clathrin- and dynamin-independent endocytosis.

Spatially controlled, cargo-specific endocytosis is essential for development, tissue homeostasis and cancer invasion. Unlike cargo-specific clathrin-mediated endocytosis, the clathrin- and dynamin-independent endocytic pathway (CLIC-GEEC, CG pathway) is considered a bulk internalization route for the fluid phase, glycosylated membrane proteins and lipids. While the core molecular players of CG-endocytosis have been recently defined, evidence of cargo-specific adaptors or selective uptake of proteins for the pathway are lacking. Here we identify the actin-binding protein Swiprosin-1 (Swip1, EFHD2) as a cargo-specific adaptor for CG-endocytosis. Swip1 couples active Rab21-associated integrins with key components of the CG-endocytic machinery-Arf1, IRSp53 and actin-and is critical for integrin endocytosis. Through this function, Swip1 supports integrin-dependent cancer-cell migration and invasion, and is a negative prognostic marker in breast cancer. Our results demonstrate a previously unknown cargo selectivity for the CG pathway and a role for specific adaptors in recruitment into this endocytic route.

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 ß1-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.