Macrophage mechanosensing during their pro-inflammatory response

Macrophages are innate immune cells responsible for engulfing microbes and cell debris through phagocytosis and orchestrating immune responses to maintain homeostasis. While conducting immune surveillance over all types of organs and tissues, macrophages face inherently heterogeneous microenvironments with unique biophysical features. For instance, microglia residing in the brain, Kupffer cells living in the skin and bone osteoclasts are exposed to very distinct tissue stiffnesses. Despite the research done in the last decade clearly indicates that macrophages are sensitive to physical factors, how mechanical cues modulate their inflammatory response remains poorly understood. The present study aims at investigating how microenvironment stiffness influences the pro-inflammatory behaviour of macrophages. Besides characterising the regulatory effect on pro-inflammatory gene expression and cytokine production, this work examines the impact of stiffness on the inflammasome, one of the main macrophage signalling platforms. For this, an in vitro system based in 2D polyacrylamide hydrogels whose stiffness can be independently tuned was established. Using substrates with an elastic moduli between 0.2 and 33.1 kPa, bone marrow-derived macrophages adopted a less spread and rounder morphology on compliant compared to stiff polyacrylamide. Upon priming with lipopolysaccharide, the expression levels of the gene encoding for TNF-α were higher on more compliant hydrogels, yet there were no significant differences in the expression of other major pro-inflammatory genes. Additionally stimulating macrophages with the ionophore nigericin revealed higher secreted protein levels of IL-1β and IL-6 on compliant substrates. Interestingly, macrophages challenged on compliant polyacrylamide also displayed an enhanced formation of the NLRP3 inflammasome as well as increased levels of pyroptotic cell death. The upregulation of inflammasome assembly on compliant hydrogels was not primarily attributed to the reduced cell spreading, since spatially confining cells on micropatterns led to a decrease of inflammasome-positive cells compared to well-spread cells. Finally, interfering with actomyosin contractility diminished the differences in inflammasome formation between compliant and stiff substrates. In summary, these results show that substrate stiffness affects the pro-inflammatory response of macrophages and for the first time describe that the NLRP3 inflammasome is one of the signalling components affected by stiffness mechanosensing. The work presented here expands our understanding of how microenvironment stiffness affects macrophage behaviour and which elements of their machinery might contribute to integrate mechanical cues into the regulation of their inflammatory functions. The onset of pathological processes or the implant of foreign bodies represent immune challenges in which macrophages can face a mechanically changing environment. Therefore, a better insight on how macrophages detect and process biophysical signals could potentially provide a basis for new strategies to modulate inflammatory responses.:INTRODUCTION 1.1 Macrophage cell biology 1.1.1 The origin of macrophages 1.1.2 The macrophage: a swiss army knife 1.1.3 The macrophage pro-inflammatory response 1.2 Immunobiophysics: the force of the immune system 1.2.1 Exertion of immune cell forces 1.2.2 Immune cell mechanosensing 1.3 Cellular mechanosensing and mechanotransduction 1.3.1 Cell adhesions to the extracellular matrix 1.3.2 Nuclear mechanotransduction 1.3.3 Membrane mechanosensing elements 1.4 Macrophage mechanosensing AIMS AND SCOPE OF THE THESIS RESULTS 3.1 Morphol. characterisation of macrophages cultured on substrates of varying stiffness 3.1.1 BMDMs adhere and can be cultured on polyacrylamide hydrogels 3.1.2 Macrophage morphology is influenced by substrate stiffness 3.1.3 PEG-Hep hydrogels induce similar morphological differences as PAA substrates but do not constitute a suitable macrophage culture platform 3.1.4 Substrate stiffness affects membrane architecture 3.2 Impact of substrate stiffness on the pro-inflammatory response of macrophages 3.2.1 The morphol. differences induced by different stiffness persist after macrophage priming 3.2.2 Tuning substrate stiffness does not cause major changes in the expression of pro-inflammatory genes 3.2.3 Lower substrate stiffness upregulates the secretion of the cytokines IL-6 and IL-1β 3.2.4 Stiffer substrates diminish macrophage pyroptotic cell death 3.2.5 Compliant substrates enhance NLRP3 inflammasome formation 3.3 Investigation of macrophage mechanotransducing elements 3.3.1 Limiting cell spreading alone does not recapitulate the effects induced by stiffness on inflammasome formation 3.3.2 Actomyosin contractility may play a role in transducing the mechanical cues given by substrate stiffness DISCUSSION AND CONCLUSIONS 4.1 Compliant substrates enhance the macrophage pro-inflammatory response 4.2 Substrate stiffness influences the formation of the NLRP3 inflammasome 4.3 Exclusively altering cell spreading does not explain the differences induced by substrate stiffness 4.4 Actomyosin contractility as a potential macrophage mechanotransducer element 4.5 Potential impact of the study in the context of cancer 4.6 Potential impact of the study in the context of implant design 4.7 Conclusions of the study MATERIALS AND METHODS 5.1 Production of polyacrylamide (PAA) hydrogels 5.2 Production of polyethylenglycol-heparin (PEG-Hep) hydrogels 5.3 Mechanical characterisation of hydrogels and macrophages 5.4 Isolation and culture of bone marrow-derived macrophages (BMDMs) 5.5 Fluorescence confocal microscopy 5.6 Scanning electron microscopy (SEM) 5.7 Gene expression analysis using quantitative real-time PCR (qRT-PCR) 5.8 Cytokine quantification assays 5.9 Cell viability assay 5.10 Culture of BMDMs on micropatterns 5.11 Optical diffraction tomography (ODT) 5.12 Statistical analysis and data visualisation APPENDIX LIST OF ACRONYMS AND ABBREVIATIONS LIST OF FIGURES BIBLIOGRAPHY ACKNOWLEDGEMENTSAls Teil des angeborenen Immunsystems sind Makrophagen dafür verantwortlich Pathogene und Zellrückstände durch Phagozytose zu beseitigen. Sie orchestrieren Immunantworten um homöostatische Bedingungen von Organen und Geweben aufrechtzuerhalten. Dabei sind sie extrem heterogenen Mikroumgebungen ausgesetzt, welche sich jeweils durch eine einzigartige Kombination von (bio)chemischen und mechanischen Eigenschaften, vor allem Gewebesteifigkeiten, auszeichnen. Dies veranschaulichen beispielsweise im Gehirn residierende Mikroglia, Kupffer-Zellen in der Haut und Osteoklasten in Knochen. Obwohl diverse Studien aus dem letzten Jahrzehnt eindeutig zeigen, dass Makrophagen auf mechanische Signale reagieren, ist der zugrunde liegende Mechanismus, wie diese Signale eine Entzündungsreaktion modulieren, noch immer unzureichend verstanden. Die vorliegende Studie beinhaltet die systematische Untersuchung, wie die Steifigkeit der Mikroumgebung das proinflammatorische Verhalten von Makrophagen beeinflusst. Neben der Charakterisierung der regulatorischen Wirkung auf die proinflammatorische Genexpression und Zytokinproduktion untersucht diese Arbeit auch den Einfluss der Steifigkeit auf das Inflammasom; eine der wichtigsten Signalplattformen für Makrophagen. Zu diesem Zweck wurde zunächst ein Zellkultursystem mit 2D-Polyacrylamid-Hydrogelen als Zellsubstrat entwickelt, bei dem das Elastizitätsmodul der Gelsubstrate gezielt eingestellt werden kann. Unter Verwendung von Substraten mit einem Elastizitätsmodul zwischen 0,2 kPa und 33,1 kPa zeigt die mikroskopische Analyse, dass aus Knochenmark stammende Makrophagen im Vergleich zu steifem Polyacrylamid eine weniger ausgebreitete und rundere Morphologie annehmen. Nach dem Primen mit Lipopolysaccharid waren die Expressionsniveaus des Gens, das für TNF-α kodiert, auf deformierbareren Hydrogelen höher, jedoch gab es keine signifikanten Unterschiede in der Expression anderer wichtiger pro-inflammatorischer Gene. Eine zusätzliche Stimulierung von Makrophagen mit dem Ionophor Nigericin bewirkte höhere sekretierte Proteinspiegel von IL-1β und IL-6 auf deformierbaren Substraten. Makrophagen, die deformierbarem Polyacrylamid ausgesetzt waren, zeigten auch eine verstärkte Bildung des NLRP3-Inflammasoms sowie ein erhöhtes Ausmaß an pyroptotischem Zelltod. Die Hochregulierung der Inflammasom-Assemblierung auf deformierbaren Hydrogelen wurde nicht primär auf die reduzierte Zellausbreitung zurückgeführt, da räumlich begrenzte Zellen auf Mikromustern zu einer Abnahme von Inflammasom-positiven Zellen im Vergleich zu stark ausgebreiteten Zellen führten. Schließlich verringerte eine Störung der Aktomyosin-Kontraktilität die Unterschiede in der Inflammasombildung zwischen deformierbaren und steifen Substraten. Zusammenfassend zeigen diese Ergebnisse, dass die Substratsteifigkeit die proinflammatorische Reaktion von Makrophagen beeinflusst und beschreiben erstmalig, dass das NLRP3-Inflammasom eine der Signalkomponenten ist, die von der zellulären Steifheitswahrnehmung beeinflusst werden. Die hier vorgestellte Arbeit erweitert unser Verständnis davon, wie die Steifigkeit der Mikroumgebung das Verhalten von Makrophagen beeinflusst und welche Elemente ihrer Maschinerie dazu beitragen könnten mechanische Signale in die Regulierung ihrer Entzündungsfunktionen zu integrieren. Das Einsetzen pathologischer Prozesse oder die Implantation von Fremdkörpern stellen Immunherausforderungen dar, bei denen Makrophagen einer sich mechanisch verändernden Umgebung ausgesetzt sein können. Daher könnte ein besserer Einblick in die Art und Weise, wie Makrophagen biophysikalische Signale erkennen und verarbeiten, möglicherweise eine Grundlage für neue Strategien zur Modulation von Entzündungsreaktionen bieten.:INTRODUCTION 1.1 Macrophage cell biology 1.1.1 The origin of macrophages 1.1.2 The macrophage: a swiss army knife 1.1.3 The macrophage pro-inflammatory response 1.2 Immunobiophysics: the force of the immune system 1.2.1 Exertion of immune cell forces 1.2.2 Immune cell mechanosensing 1.3 Cellular mechanosensing and mechanotransduction 1.3.1 Cell adhesions to the extracellular matrix 1.3.2 Nuclear mechanotransduction 1.3.3 Membrane mechanosensing elements 1.4 Macrophage mechanosensing AIMS AND SCOPE OF THE THESIS RESULTS 3.1 Morphol. characterisation of macrophages cultured on substrates of varying stiffness 3.1.1 BMDMs adhere and can be cultured on polyacrylamide hydrogels 3.1.2 Macrophage morphology is influenced by substrate stiffness 3.1.3 PEG-Hep hydrogels induce similar morphological differences as PAA substrates but do not constitute a suitable macrophage culture platform 3.1.4 Substrate stiffness affects membrane architecture 3.2 Impact of substrate stiffness on the pro-inflammatory response of macrophages 3.2.1 The morphol. differences induced by different stiffness persist after macrophage priming 3.2.2 Tuning substrate stiffness does not cause major changes in the expression of pro-inflammatory genes 3.2.3 Lower substrate stiffness upregulates the secretion of the cytokines IL-6 and IL-1β 3.2.4 Stiffer substrates diminish macrophage pyroptotic cell death 3.2.5 Compliant substrates enhance NLRP3 inflammasome formation 3.3 Investigation of macrophage mechanotransducing elements 3.3.1 Limiting cell spreading alone does not recapitulate the effects induced by stiffness on inflammasome formation 3.3.2 Actomyosin contractility may play a role in transducing the mechanical cues given by substrate stiffness DISCUSSION AND CONCLUSIONS 4.1 Compliant substrates enhance the macrophage pro-inflammatory response 4.2 Substrate stiffness influences the formation of the NLRP3 inflammasome 4.3 Exclusively altering cell spreading does not explain the differences induced by substrate stiffness 4.4 Actomyosin contractility as a potential macrophage mechanotransducer element 4.5 Potential impact of the study in the context of cancer 4.6 Potential impact of the study in the context of implant design 4.7 Conclusions of the study MATERIALS AND METHODS 5.1 Production of polyacrylamide (PAA) hydrogels 5.2 Production of polyethylenglycol-heparin (PEG-Hep) hydrogels 5.3 Mechanical characterisation of hydrogels and macrophages 5.4 Isolation and culture of bone marrow-derived macrophages (BMDMs) 5.5 Fluorescence confocal microscopy 5.6 Scanning electron microscopy (SEM) 5.7 Gene expression analysis using quantitative real-time PCR (qRT-PCR) 5.8 Cytokine quantification assays 5.9 Cell viability assay 5.10 Culture of BMDMs on micropatterns 5.11 Optical diffraction tomography (ODT) 5.12 Statistical analysis and data visualisation APPENDIX LIST OF ACRONYMS AND ABBREVIATIONS LIST OF FIGURES BIBLIOGRAPHY ACKNOWLEDGEMENT

Compliant Substrates Enhance Macrophage Cytokine Release and NLRP3 Inflammasome Formation During Their Pro-Inflammatory Response.

Immune cells process a myriad of biochemical signals but their function and behavior are also determined by mechanical cues. Macrophages are no exception to this. Being present in all types of tissues, macrophages are exposed to environments of varying stiffness, which can be further altered under pathological conditions. While it is becoming increasingly clear that macrophages are mechanosensitive, it remains poorly understood how mechanical cues modulate their inflammatory response. Here we report that substrate stiffness influences the expression of pro-inflammatory genes and the formation of the NLRP3 inflammasome, leading to changes in the secreted protein levels of the cytokines IL-1β and IL-6. Using polyacrylamide hydrogels of tunable elastic moduli between 0.2 and 33.1 kPa, we found that bone marrow-derived macrophages adopted a less spread and rounder morphology on compliant compared to stiff substrates. Upon LPS priming, the expression levels of the gene encoding for TNF-α were higher on more compliant hydrogels. When additionally stimulating macrophages with the ionophore nigericin, we observed an enhanced formation of the NLRP3 inflammasome, increased levels of cell death, and higher secreted protein levels of IL-1β and IL-6 on compliant substrates. The upregulation of inflammasome formation on compliant substrates was not primarily attributed to the decreased cell spreading, since spatially confining cells on micropatterns led to a reduction of inflammasome-positive cells compared to well-spread cells. Finally, interfering with actomyosin contractility diminished the differences in inflammasome formation between compliant and stiff substrates. In summary, we show that substrate stiffness modulates the pro-inflammatory response of macrophages, that the NLRP3 inflammasome is one of the components affected by macrophage mechanosensing, and a role for actomyosin contractility in this mechanosensory response. Thus, our results contribute to a better understanding of how microenvironment stiffness affects macrophage behavior, which might be relevant in diseases where tissue stiffness is altered and might potentially provide a basis for new strategies to modulate inflammatory responses

Compliant Substrates Enhance Macrophage Cytokine Release and NLRP3 Inflammasome Formation During Their Pro-Inflammatory Response.

Immune cells process a myriad of biochemical signals but their function and behavior are also determined by mechanical cues. Macrophages are no exception to this. Being present in all types of tissues, macrophages are exposed to environments of varying stiffness, which can be further altered under pathological conditions. While it is becoming increasingly clear that macrophages are mechanosensitive, it remains poorly understood how mechanical cues modulate their inflammatory response. Here we report that substrate stiffness influences the expression of pro-inflammatory genes and the formation of the NLRP3 inflammasome, leading to changes in the secreted protein levels of the cytokines IL-1β and IL-6. Using polyacrylamide hydrogels of tunable elastic moduli between 0.2 and 33.1 kPa, we found that bone marrow-derived macrophages adopted a less spread and rounder morphology on compliant compared to stiff substrates. Upon LPS priming, the expression levels of the gene encoding for TNF-α were higher on more compliant hydrogels. When additionally stimulating macrophages with the ionophore nigericin, we observed an enhanced formation of the NLRP3 inflammasome, increased levels of cell death, and higher secreted protein levels of IL-1β and IL-6 on compliant substrates. The upregulation of inflammasome formation on compliant substrates was not primarily attributed to the decreased cell spreading, since spatially confining cells on micropatterns led to a reduction of inflammasome-positive cells compared to well-spread cells. Finally, interfering with actomyosin contractility diminished the differences in inflammasome formation between compliant and stiff substrates. In summary, we show that substrate stiffness modulates the pro-inflammatory response of macrophages, that the NLRP3 inflammasome is one of the components affected by macrophage mechanosensing, and a role for actomyosin contractility in this mechanosensory response. Thus, our results contribute to a better understanding of how microenvironment stiffness affects macrophage behavior, which might be relevant in diseases where tissue stiffness is altered and might potentially provide a basis for new strategies to modulate inflammatory responses

Adipose cells and tissues soften with lipid accumulation while in diabetes adipose tissue stiffens

Adipose tissue expansion involves both differentiation of new precursors and size increase of mature adipocytes. While the two processes are well balanced in healthy tissues, obesity and diabetes type II are associated with abnormally enlarged adipocytes and excess lipid accumulation. Previous studies suggested a link between cell stiffness, volume and stem cell differentiation, although in the context of preadipocytes, there have been contradictory results regarding stiffness changes with differentiation. Thus, we set out to quantitatively monitor adipocyte shape and size changes with differentiation and lipid accumulation. We quantified by optical diffraction tomography that differentiating preadipocytes increased their volumes drastically. Atomic force microscopy (AFM)-indentation and -microrheology revealed that during the early phase of differentiation, human preadipocytes became more compliant and more fluid-like, concomitant with ROCK-mediated F-actin remodelling. Adipocytes that had accumulated large lipid droplets were more compliant, and further promoting lipid accumulation led to an even more compliant phenotype. In line with that, high fat diet-induced obesity was associated with more compliant adipose tissue compared to lean animals, both for drosophila fat bodies and murine gonadal adipose tissue. In contrast, adipose tissue of diabetic mice became significantly stiffer as shown not only by AFM but also magnetic resonance elastography. Altogether, we dissect relative contributions of the cytoskeleton and lipid droplets to cell and tissue mechanical changes across different functional states, such as differentiation, nutritional state and disease. Our work therefore sets the basis for future explorations on how tissue mechanical changes influence the behaviour of mechanosensitive tissue-resident cells in metabolic disorders

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

Compliant Substrates Enhance Macrophage Cytokine Release and NLRP3 Inflammasome Formation During Their Pro-Inflammatory Response

Immune cells process a myriad of biochemical signals but their function and behavior are also determined by mechanical cues. Macrophages are no exception to this. Being present in all types of tissues, macrophages are exposed to environments of varying stiffness, which can be further altered under pathological conditions. While it is becoming increasingly clear that macrophages are mechanosensitive, it remains poorly understood how mechanical cues modulate their inflammatory response. Here we report that substrate stiffness influences the expression of pro-inflammatory genes and the formation of the NLRP3 inflammasome, leading to changes in the secreted protein levels of the cytokines IL-1b and IL-6. Using polyacrylamide hydrogels of tunable elastic moduli between 0.2 and 33.1 kPa, we found that bone marrow-derived macrophages adopted a less spread and rounder morphology on compliant compared to stiff substrates. Upon LPS priming, the expression levels of the gene encoding for TNF-a were higher on more compliant hydrogels. When additionally stimulating macrophages with the ionophore nigericin, we observed an enhanced formation of the NLRP3 inflammasome, increased levels of cell death, and higher secreted protein levels of IL-1b and IL-6 on compliant substrates. The upregulation of inflammasome formation on compliant substrates was not primarily attributed to the decreased cell spreading, since spatially confining cells on micropatterns led to a reduction of inflammasome-positive cells compared to well-spread cells. Finally, interfering with actomyosin contractility diminished the differences in inflammasome formation between compliant and stiff substrates. In summary, we show that substrate stiffness modulates the pro-inflammatory response of macrophages, that the NLRP3 inflammasome is one of the components affected by macrophage mechanosensing, and a role for actomyosin contractility in this mechanosensory response. Thus, our results contribute to a better understanding of how microenvironment stiffness affects macrophage behavior, which might be relevant in diseases where tissue stiffness is altered and might potentially provide a basis for new strategies to modulate inflammatory responses

Adipose cells and tissues soften with lipid accumulation while in diabetes adipose tissue stiffens

Adipose tissue expansion involves both differentiation of new precursors and size increase of mature adipocytes. While the two processes are well balanced in healthy tissues, obesity and diabetes type II are associated with abnormally enlarged adipocytes and excess lipid accumulation. Previous studies suggested a link between cell stiffness, volume and stem cell differentiation, although in the context of preadipocytes, there have been contradictory results regarding stiffness changes with differentiation. Thus, we set out to quantitatively monitor adipocyte shape and size changes with differentiation and lipid accumulation. We quantified by optical diffraction tomography that differentiating preadipocytes increased their volumes drastically. Atomic force microscopy (AFM)-indentation and -microrheology revealed that during the early phase of differentiation, human preadipocytes became more compliant and more fluid-like, concomitant with ROCK-mediated F-actin remodelling. Adipocytes that had accumulated large lipid droplets were more compliant, and further promoting lipid accumulation led to an even more compliant phenotype. In line with that, high fat diet-induced obesity was associated with more compliant adipose tissue compared to lean animals, both for drosophila fat bodies and murine gonadal adipose tissue. In contrast, adipose tissue of diabetic mice became significantly stiffer as shown not only by AFM but also magnetic resonance elastography. Altogether, we dissect relative contributions of the cytoskeleton and lipid droplets to cell and tissue mechanical changes across different functional states, such as differentiation, nutritional state and disease. Our work therefore sets the basis for future explorations on how tissue mechanical changes influence the behaviour of mechanosensitive tissue-resident cells in metabolic disorders

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