Caveolae: Mechanosensing and mechanotransduction devices linking membrane trafficking to mechanoadaptation.

Mechanical forces (extracellular matrix stiffness, vascular shear stress, and muscle stretching) reaching the plasma membrane (PM) determine cell behavior. Caveolae are PM-invaginated nanodomains with specific lipid and protein composition. Being highly abundant in mechanically challenged tissues (muscles, lungs, vessels, and adipose tissues), they protect cells from mechanical stress damage. Caveolae flatten upon increased PM tension, enabling both force sensing and accommodation, critical for cell mechanoprotection and homeostasis. Thus, caveolae are highly plastic, ranging in complexity from flattened membranes to vacuolar invaginations surrounded by caveolae-rosettes-which also contribute to mechanoprotection. Caveolar components crosstalk with mechanotransduction pathways and recent studies show that they translocate from the PM to the nucleus to convey stress information. Furthermore, caveolae components can regulate membrane traffic from/to the PM to adapt to environmental mechanical forces. The interdependence between lipids and caveolae starts to be understood, and the relevance of caveolae-dependent membrane trafficking linked to mechanoadaption to different physiopathological processes is emerging.This study was supported by grants from the Spanish Ministry of Science and Innovation(MICIIN)/Agencia Estatal de Investigación (AEI)/European Regional Development Fund (ERDF/FEDER) “A way to make Europe” – (SAF2014-51876-R, SAF2017-83130-R, IGP-SO grant MINSEV1512-07-2016, CSD2009-0016 and BFU2016-81912-REDC), Comunidad Autónoma de Madrid (Tec4Bio-CM, S2018/NMT¬4443), Fundació La Marató de TV3 (385/C/2019) and the Worldwide Cancer Research Foundation (15-0404), all to M.A.d.P. We received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 641639. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MICIIN) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505).S

Caveolin-1 dolines form a distinct and rapid caveolae-independent mechanoadaptation system.

In response to different types and intensities of mechanical force, cells modulate their physical properties and adapt their plasma membrane (PM). Caveolae are PM nano-invaginations that contribute to mechanoadaptation, buffering tension changes. However, whether core caveolar proteins contribute to PM tension accommodation independently from the caveolar assembly is unknown. Here we provide experimental and computational evidence supporting that caveolin-1 confers deformability and mechanoprotection independently from caveolae, through modulation of PM curvature. Freeze-fracture electron microscopy reveals that caveolin-1 stabilizes non-caveolar invaginations-dolines-capable of responding to low-medium mechanical forces, impacting downstream mechanotransduction and conferring mechanoprotection to cells devoid of caveolae. Upon cavin-1/PTRF binding, doline size is restricted and membrane buffering is limited to relatively high forces, capable of flattening caveolae. Thus, caveolae and dolines constitute two distinct albeit complementary components of a buffering system that allows cells to adapt efficiently to a broad range of mechanical stimuli.We thank R. Parton (Institute for Molecular Biosciences, Queensland), P. Pilch (Boston University School of Medicine) and L. Liu (Boston University School of Medicine) for kindly providing PTRFKO cells and reagents, S. Casas Tintó for kindly providing SH-Sy5y cells, P. Bassereau (Curie Institute, Paris) for kindly providing OT setup, V. Labrador Cantarero from CNIC microscopy Unit for helping with ImageJ analysis, O. Otto and M. Herbig for providing help with RTDC experiments, S. Berr and K. Gluth for technical assistance in cell culture, F. Steiniger for support in electron tomography, and A. Norczyk Simón for providing pCMV-FLAG-PTRF construct. This project received funding from the European Union Horizon 2020 Research and Innovation Programme through Marie Sklodowska-Curie grant 641639; grants from the Spanish Ministry of Science and Innovation (MCIN/AEI/10.13039/501100011033): SAF2014-51876-R, SAF2017-83130-R co-funded by ‘ERDF A way of making Europe’, PID2020-118658RB-I00, PDC2021-121572-100 co-funded by ‘European Union NextGenerationEU/PRTR’, CSD2009- 0016 and BFU2016-81912-REDC; and the Asociación Española Contra el Cáncer foundation (PROYE20089DELP) all to M.A.d.P. M.A.d.P. is member of the Tec4Bio consortium (ref. S2018/NMT¬4443; Comunidad Autónoma de Madrid/FEDER, Spain), co-recipient with P.R.-C. of grants from Fundació La Marató de TV3 (674/C/2013 and 201936- 30-31), and coordinator of a Health Research consortium grant from Fundación Obra Social La Caixa (AtheroConvergence, HR20-00075). M.S.-A. is recipient of a Ramón y Cajal research contract from MCIN (RYC2020-029690-I). The CNIC Unit of Microscopy and Dynamic Imaging is supported by FEDER ‘Una manera de hacer Europa’ (ReDIB ICTS infrastructure TRIMA@CNIC, MCIN). We acknowledge the support from Deutsche Forschungsgemeinschaft through grants to M.M.K. (KE685/7-1) and B.Q. (QU116/6-2 and QU116/9-1). Work in D.N. laboratory was supported by grants from the European Union Horizon 2020 Research and Innovation Programme through Marie Sklodowska-Curie grant 812772 and MCIN (DPI2017-83721-P). Work in C.L. laboratory was supported by grants from Curie, INSERM, CNRS, Agence Nationale de la Recherche (ANR-17-CE13-0020-01) and Fondation ARC pour la Recherche (PGA1-RF20170205456). Work in P.R.-C. lab is funded by the MCIN (PID2019-110298GB-I00), the EC (H20 20-FETPROACT-01-2016-731957). Work in X.T. lab is funded by the MICIN (PID2021-128635NB-I00), ERC (Adv-883739) and La Caixa Foundation (LCF/PR/HR20/52400004; co-recipient with P.R.-C.). IBEC is recipient of a Severo Ochoa Award of Excellence from the MINECO. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the MCIN and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033).S

Los hemocitos, atraídos por la secreción de dAatsTyr, son los responsables de la eliminación de las células perdedoras durante la competición celular en "Drosophila melanogaster"

La competición celular es un fenómeno, descrito por primera vez en Drosophila melanogaster, que surge como consecuencia de la aparición de mutaciones que producen una población celular metabólicamente diferente del resto de células del tejido. Como resultado de esta diferencia se genera una competición entre ambos tipos celulares que conduce a la muerte por apoptosis de las células "menos aptas" (en el sentido Darwiniano), denominadas perdedoras y, la proliferación de las células "más aptas", denominadas ganadoras. Al final del proceso, los restos apoptóticos de las células perdedoras son fagocitados y eliminados del tejido mientras que las células ganadoras se dividen y se extienden ocupando el espacio dejado por las perdedoras, de forma que el número total del células no varía. En este sentido, la competición celular podría servir como mecanismo para entender fenómenos clínicos como el de la "cancerización de campo", que supone la expansión clonal de una célula mutante inicial que genera así un "campo mutante" susceptible de transformarse en un tumor al acumular nuevas mutaciones. Tal y como se ha esbozado anteriormente, un aspecto importante de la competición celular es la eliminación de los restos apoptóticos de las células perdedoras que, dada la posible implicación de la competición en el desarrollo pretumoral, supone un aspecto importante en la expansión de las células ganadoras (tumorales en el contexto oncogénico). En contra de lo que se pensaba hasta el momento, nuestros resultados muestran que no las células ganadoras las encargadas de fagocitar a las células perdedoras. De hecho, nuestras observaciones indican que son los hemocitos (macrófagos circulantes en la hemolinfa de Drosophila melanogaster) los encargados de llevar a cabo la mayor parte de esta tarea. Asimismo, hemos reunido diferentes evidencias que revelan la secreción, por parte de las células perdedoras, de una señal capaz de atraer hemocitos circulantes a los lugares de competición celular. Esta señal de atracción es la dTirosil-tRNA sintetasa (dAatsTyr), que forma parte de la maquinaria de traducción celular, pero que en condiciones apoptóticas es secretada al exterior celular, de la misma forma que se ha descrito previamente para AatsTyr (el homólogo humano) en células humanas en cultivo. Diferentes estudios ponen de manifiesto la implicación de los macrófagos en la progresión de algunos tumores humanos. Sin embargo, se desconocen los mecanismos implicados en esta contribución. En este sentido, nuestros resultados podrían ayudar a profundizar en la relación entre la fagocitosis, la competición celular y la actividad pro-oncogénica de algunas células inmunes, como los macrófagos

Active JNK-dependent secretion of Drosophila Tyrosyl-tRNA synthetase by loser cells recruits haemocytes during cell competition

Cell competition is a process by which the slow dividing cells (losers) are recognized and eliminated from growing tissues. Loser cells are extruded from the epithelium and engulfed by the haemocytes, the Drosophila macrophages. However, how macrophages identify the dying loser cells is unclear. Here we show that apoptotic loser cells secrete Tyrosyl-tRNA synthetase (TyrRS), which is best known as a core component of the translational machinery. Secreted TyrRS is cleaved by matrix metalloproteinases generating MiniTyr and EMAP fragments. EMAP acts as a guiding cue for macrophage migration in the Drosophila larvae, as it attracts the haemocytes to the apoptotic loser cells. JNK signalling and Kish, a component of the secretory pathway, are autonomously required for the active secretion of TyrRS by the loser cells. Altogether, this mechanism guarantees effective removal of unfit cells from the growing tissue.Work is funded by the European Research Council, Swiss National Science Foundation, Josef Steiner Cancer Research Foundation, and the Swiss Cancer League and Eurobioimaging. We specially thank Federico Halpern Blasco and ‘Jesus Serra foundation’ for funding part of the research. S.C.-T. was a recipient of a Ramon y Cajal contract from the Spanish MICINN

Elimination of classically-activated macrophages in tumor-conditioned medium by alternatively-activated macrophages

Cellular interactions are critical during development, tissue fitness and epithelial tumor development. The expression levels of specific genes confer to tumoral cells a survival advantage versus the normal neighboring cells. As a consequence, cells surrounding tumors are eliminated and engulfed by macrophages. We propose a novel scenario in which circulating cells facing a tumor can reproduce these cellular interactions. In vitro cultured macrophages from murine bone marrow were used to investigate this hypothesis. M1 macrophages in tumoral medium upregulated markers of a suboptimal condition, such as Sparc and TyrRS, and undergo apoptosis. However, M2 macrophages display higher Myc expression levels and proliferate at the expense of M1. Resulting M1 apoptotic debris is engulfed by M2 in a Sparc- and TyrRS-dependent manner. These findings suggest that tumor-dependent macrophage elimination could deplete immune response against tumors. This possibility could be relevant for macrophage based anti-tumoral strategies.This work was supported by Ministerio de Economía y Competitividad (RyC-2012-11410 and BFU2015-65685P to S.C.-T.) and Fundación Jesús Serra

Autophagy and the Lysosomal System in Cancer

Autophagy and the lysosomal system, together referred to as the autophagolysosomal system, is a cellular quality control network which maintains cellular health and homeostasis by removing cellular waste including protein aggregates, damaged organelles, and invading pathogens. As such, the autophagolysosomal system has roles in a variety of pathophysiological disorders, including cancer, neurological disorders, immune- and inflammation-related diseases, and metabolic alterations, among others. The autophagolysosomal system is controlled by TFEB, a master transcriptional regulator driving the expression of multiple genes, including autophagoly sosomal components. Importantly, Reactive Oxygen Species (ROS) production and control are key aspects of the physiopathological roles of the autophagolysosomal system, and may hold a key for synergistic therapeutic interventions. In this study, we reviewed our current knowledge on the biology and physiopathology of the autophagolysosomal system, and its potential for therapeutic intervention in cancer

Caveolae couple mechanical stress to integrin recycling and activation

Cells are subjected to multiple mechanical inputs throughout their lives. Their ability to detect these environmental cues is called mechanosensing, a process in which integrins play an important role. During cellular mechanosensing, plasma membrane (PM) tension is adjusted to mechanical stress through the buffering action of caveolae; however, little is known about the role of caveolae in early integrin mechanosensing regulation. Here, we show that Cav1KO fibroblasts increase adhesion to FN-coated beads when pulled with magnetic tweezers, as compared to wild type fibroblasts. This phenotype is Rho-independent and mainly derived from increased active β1-integrin content on the surface of Cav1KO fibroblasts. Florescence recovery after photobleaching analysis and endocytosis/recycling assays revealed that active β1-integrin is mostly endocytosed through the clathrin independent carrier/glycosylphosphatidyl inositol (GPI)-enriched endocytic compartment pathway and is more rapidly recycled to the PM in Cav1KO fibroblasts, in a Rab4 and PM tension-dependent manner. Moreover, the threshold for PM tension-driven β1-integrin activation is lower in Cav1KO mouse embryonic fibroblasts (MEFs) than in wild type MEFs, through a mechanism dependent on talin activity. Our findings suggest that caveolae couple mechanical stress to integrin cycling and activation, thereby regulating the early steps of the cellular mechanosensing response

Caveolin-1 dolines form a distinct and rapid caveolae-independent mechanoadaptation system

In response to diferent types and intensities of mechanical force, cells modulate their physical properties and adapt their plasma membrane (PM). Caveolae are PM nano-invaginations that contribute to mechanoadaptation, bufering tension changes. However, whether core caveolar proteins contribute to PM tension accommodation independently from the caveolar assembly is unknown. Here we provide experimental and computational evidence supporting that caveolin-1 confers deformability and mechanoprotection independently from caveolae, through modulation of PM curvature. Freeze-fracture electron microscopy reveals that caveolin-1 stabilizes non-caveolar invaginations—dolines—capable of responding to low-medium mechanical forces, impacting downstream mechanotransduction and conferring mechanoprotection to cells devoid of caveolae. Upon cavin-1/PTRF binding, doline size is restricted and membrane bufering is limited to relatively high forces, capable of fattening caveolae. Thus, caveolae and dolines constitute two distinct albeit complementary components of a bufering system that allows cells to adapt efciently to a broad range of mechanical stimuli.Peer ReviewedPostprint (published version