Tumor Suppressor DAPK1 Catalyzes Adhesion Assembly on Rigid but Anoikis on Soft Matrices

Cancer cells normally grow on soft surfaces due to impaired mechanosensing of the extracellular matrix rigidity. Upon restoration of proper mechanosensing, cancer cells undergo apoptosis on soft surfaces (anoikis) like most normal cells. However, the link between mechanosensing and activation of anoikis is not clear. Here we show that death associated protein kinase 1 (DAPK1), a tumor suppressor that activates cell death, is directly linked to anoikis activation through rigidity sensing. We find that when rigidity sensing is decreased through inhibition of DAPK1 activity, cells are transformed for growth on soft matrices. Further, DAPK1 catalyzes matrix adhesion assembly and is part of adhesions on rigid surfaces. This pathway involves DAPK1 phosphorylation of tropomyosin1.1, the talin1 head domain, and tyrosine phosphorylation of DAPK1 by Src. On soft surfaces, DAPK1 rapidly dissociates from the adhesion complexes and activates apoptosis as catalyzed by PTPN12 activity and talin1 head. Thus, DAPK1 is important for adhesion assembly on rigid surfaces and the activation of anoikis on soft surfaces through its binding to rigidity-sensing modules

Force-dependent focal adhesion assembly and disassembly: A computational study

Cells interact with the extracellular matrix (ECM) via cell–ECM adhesions. These physical interactions are transduced into biochemical signals inside the cell which influence cell behaviour. Although cell–ECM interactions have been studied extensively, it is not completely understood how immature (nascent) adhesions develop into mature (focal) adhesions and how mechanical forces influence this process. Given the small size, dynamic nature and short lifetimes of nascent adhesions, studying them using conventional microscopic and experimental techniques is challenging. Computational modelling provides a valuable resource for simulating and exploring various “what if?” scenarios in silico and identifying key molecular components and mechanisms for further investigation. Here, we present a simplified mechano-chemical model based on ordinary differential equations with three major proteins involved in adhesions: integrins, talin and vinculin. Additionally, we incorporate a hypothetical signal molecule that influences adhesion (dis)assembly rates. We find that assembly and disassembly rates need to vary dynamically to limit maturation of nascent adhesions. The model predicts biphasic variation of actin retrograde velocity and maturation fraction with substrate stiffness, with maturation fractions between 18–35%, optimal stiffness of ∼1 pN/nm, and a mechanosensitive range of 1-100 pN/nm, all corresponding to key experimental findings. Sensitivity analyses show robustness of outcomes to small changes in parameter values, allowing model tuning to reflect specific cell types and signaling cascades. The model proposes that signal-dependent disassembly rate variations play an underappreciated role in maturation fraction regulation, which should be investigated further. We also provide predictions on the changes in traction force generation under increased/decreased vinculin concentrations, complementing previous vinculin overexpression/knockout experiments in different cell types. In summary, this work proposes a model framework to robustly simulate the mechanochemical processes underlying adhesion maturation and maintenance, thereby enhancing our fundamental knowledge of cell–ECM interactions

Breast Cancer-Derived Microparticles Reduce Cancer Cell Adhesion, an Effect Augmented by Chemotherapy

Tumor cell heterogeneity is primarily dictated by mutational changes, sometimes leading to clones that undergo a metastatic switch. However, little is known about tumor heterogeneity following chemotherapy perturbation. Here we studied the possible involvement of tumor-derived extracellular vesicles, often referred to as tumor-derived microparticles (TMPs), as mediators of the metastatic switch in the tumor microenvironment by hindering cell adhesion properties. Specifically, we show that highly metastatic or chemotherapy-treated breast cancer cells shed an increased number of TMPs compared to their respective controls. We found that these TMPs substantially reduce cell adhesion and disrupt actin filament structure, therefore increasing their biomechanical force pace, further implicating tumor cell dissemination as part of the metastatic cascade. Our results demonstrate that these pro-metastatic effects are mediated in part by CD44 which is highly expressed in TMPs obtained from highly metastatic cells or cells exposed to chemotherapy when compared to cells with low metastatic potential. Consequently, when we inhibited CD44 expression on TMPs by a pharmacological or a genetic approach, increased tumor cell adhesion and re-organized actin filament structure were observed. We also demonstrated that breast cancer patients treated with paclitaxel chemotherapy exhibited increased CD44-expressing TMPs. Overall, our study provides further insights into the role of TMPs in promoting metastasis, an effect which is augmented when tumor cells are exposed to chemotherapy

Tropomyosin controls sarcomere-like contractions for rigidity sensing and suppressing growth on soft matrices

Cells test the rigidity of the extracellular matrix by applying forces to it through integrin adhesions. Recent measurements show that these forces are applied by local micrometre-scale contractions, but how contraction force is regulated by rigidity is unknown. Here we performed high temporal- and spatial-resolution tracking of contractile forces by plating cells on sub-micrometre elastomeric pillars. We found that actomyosin-based sarcomere-like contractile units (CUs) simultaneously moved opposing pillars in net steps of ∼2.5 nm, independent of rigidity. What correlated with rigidity was the number of steps taken to reach a force level that activated recruitment of α-actinin to the CUs. When we removed actomyosin restriction by depleting tropomyosin 2.1, we observed larger steps and higher forces that resulted in aberrant rigidity sensing and growth of non-transformed cells on soft matrices. Thus, we conclude that tropomyosin 2.1 acts as a suppressor of growth on soft matrices by supporting proper rigidity sensing

Perturbed actin cap as a new personalized biomarker in primary fibroblasts of Huntington’s disease patients

Primary fibroblasts from patient’s skin biopsies are directly isolated without any alteration in the genome, retaining in culture conditions their endogenous cellular characteristics and biochemical properties. The aim of this study was to identify a distinctive cell phenotype for potential drug evaluation in fibroblasts from Huntington’s Disease (HD) patients, using image-based high content analysis. We show that HD fibroblasts have a distinctive nuclear morphology associated with a nuclear actin cap deficiency. This in turn affects cell motility in a similar manner to fibroblasts from Hutchinson-Gilford progeria syndrome (HGPS) patients used as known actin cap deficient cells. Moreover, treatment of the HD cells with either Latrunculin B, used to disrupt actin cap formation, or the antioxidant agent Mitoquinone, used to improve mitochondrial activity, show expected opposite effects on actin cap associated morphological features and cell motility. Deep data analysis allows strong cluster classification within HD cells according to patients’ disease severity score which is distinct from HGPS and matching controls supporting that actin cap is a biomarker in HD patients’ cells correlated with HD severity status that could be modulated by pharmacological agents as tool for personalized drug evaluation

A Role for the Juxtamembrane Cytoplasm in the Molecular Dynamics of Focal Adhesions

Focal adhesions (FAs) are specialized membrane-associated multi-protein complexes that link the cell to the extracellular matrix and play crucial roles in cell-matrix sensing. Considerable information is available on the complex molecular composition of these sites, yet the regulation of FA dynamics is largely unknown. Based on a combination of FRAP studies in live cells, with in silico simulations and mathematical modeling, we show that the FA plaque proteins paxillin and vinculin exist in four dynamic states: an immobile FA-bound fraction, an FA-associated fraction undergoing exchange, a juxtamembrane fraction experiencing attenuated diffusion, and a fast-diffusing cytoplasmic pool. The juxtamembrane region surrounding FAs displays a gradient of FA plaque proteins with respect to both concentration and dynamics. Based on these findings, we propose a new model for the regulation of FA dynamics in which this juxtamembrane domain acts as an intermediary layer, enabling an efficient regulation of FA formation and reorganization

Tropomyosin controls sarcomere-like contractions for rigidity sensing and suppressing growth on soft matrices

Cells test the rigidity of the extracellular matrix by applying forces to it through integrin adhesions. Recent measurements show that these forces are applied via local micrometre-scale contractions, but how contraction force is regulated by rigidity is unknown. Here we performed high temporal- and spatial-resolution tracking of contractile forces by plating cells on sub-micron elastomeric pillars. We found that actomyosin-based sarcomere-like contractile units (CUs) simultaneously moved opposing pillars in net steps of ~2.5 nm, independent of rigidity. What correlated with rigidity was the number of steps taken to reach a force level that activated recruitment of α-actinin to the CUs. When we removed actomyosin restriction by depleting tropomyosin 2.1, we observed larger steps and higher forces that resulted in aberrant rigidity sensing and growth of non-transformed cells on soft matrices. Thus, we conclude that tropomyosin 2.1 acts as a suppressor of growth on soft matrices by supporting proper rigidity sensing

YAP mediates apoptosis through failed integrin adhesion reinforcement

Summary: Extracellular matrix (ECM) rigidity is a major effector of cell fate decisions. Whereas cell proliferation on stiff matrices, wherein Yes-associated protein (YAP) plays a pivotal role, is well documented, activation of apoptosis in response to soft matrices is poorly understood. Here, we show that YAP drives the apoptotic decision as well. We find that in cells on soft matrices, YAP is recruited to small adhesions, phosphorylated at the Y357 residue, and translocated into the nucleus, ultimately leading to apoptosis. In contrast, Y357 phosphorylation levels are dramatically low in large adhesions on stiff matrices. Furthermore, mild attenuation of actomyosin contractility allows adhesion growth on soft matrices, leading to reduced Y357 phosphorylation levels and resulting in cell growth. These findings indicate that failed adhesion reinforcement drives rigidity-dependent apoptosis through YAP and that this decision is not determined solely by ECM rigidity but rather by the balance between cellular forces and ECM rigidity