Molecular and Cellular Pathophysiology 20211 Mechanobiology: where biology and biophysics meet Jaromír Gumulec j.gumulec@med.muni.cz @jarogumulec 2010 Patapoutian 10.1126/science.11932702 2010 Patapoutian 10.1126/science.11932703 Mechanically activated cationt channel for touch sensation Suppression of mechanically activated currents by means of Piezo1 siRNA. representative currents (averaged traces) induced by means of negative pipette pressure in a N2A cell transfected with (left) scrambled siRNA or (right) Piezo1 siRNA. Define footer – presentation title / department4 Mechanical forces in human tissues Human elbow actuation. Simulation of an elbow composed of three bones performing a complete flexion. Simulations for active and passive force normalized with peak force (𝐹m/𝐹max) during the isometric exercise 5 Mechanical forces in human tissues Hemodynamic shear stress. Cross-section of blood vessels illustrating shear stress, 𝛕s, the frictional force per unit area acting on inner vessel wall and endothelium as result of flow of viscous blood, diagram of shear magnitues in vessels. 1 dyne/cm2 = 0.1 Pa = 0.1 N/m2 Define footer – presentation title / department6 Cell mechanics sub-field of biophysics that focuses on the mechanical properties and behavior of living cells and how it relates to cell function PC-3 cells, 10x, quantitative phase imaging Define footer – presentation title / department7 Cells in environment, the Tumor Microenvironment Rodrigues 2020 https://doi.org/10.1016/j.trecan.2020.10. 009 8 ECM stiffness regulates tumor metabolism mechanics of the cellular microenvironment continuously modulates cell functions such as growth, survival, apoptosis, differentiation and morphogenesis via cytoskeletal remodelling and actomyosin contractility Transfer of human bronchial epithelial cells from stiff to soft substrates causes a downregulation of glycolysis via proteasomal degradation of the rate-limiting metabolic enzyme phosphofructokinase cancer cells maintain high glycolytic rates regardless of environmental mechanics Park et al https://www.nature.com/articles/s41586-020-1998-1 https://twitter.com/Marta_Shahbazi/status/1234532810154823685 2019 Mohamed https://doi.org/10.3389/fbioe.2019.001629 Cell functions affected by surrounding microenvironment • via signalling factors, extracellular matrix ligands • also via biomechanical cues: viscoelasticity, topography of extracellular matrix Define footer – presentation title / department10 Outline ̶ mechanobiology, mechanotransduction ̶ factors involved in mechanotransduction ̶ celluar consequences following mechanotransduction ̶ examples in physiology ̶ cell mechanics and pathology Define footer – presentation title / department11 Mechanotransduction ̶ the molecular mechanisms by which cells sense and respond to mechanical signals genetic and biochemical basis of disease X changes in cell mechanics, extracellular matrix, or mechanotransduction contribute to the development diseases: atherosclerosis, fibrosis, asthma, osteoporosis, heart failure cancer Define footer – presentation title / department12 Signaling and mechanics ̶ mechanotransduction contributors ̶ stretch-activated ion channels, ̶ caveolae, ̶ integrins, ̶ cadherins, ̶ growth factor receptors, ̶ myosin motors, ̶ cytoskeletal filaments, ̶ nuclei, ̶ extracellular matrix, ̶ and numerous other signaling molecules Define footer – presentation title / department13 Parameters involved in choosing the mechanical measurement tool. The choice of experimental tool requires consideration of (a) the lengthscale, (b) the timescale of the measurement and (c) the level of forces (or elasticity of the sample). Moeendarbary https://doi.org/10.1002/wsbm.127 Define footer – presentation title / department14 ̶ Whole-body scale: ̶ bodies exposed to forces, e.g. gravity ̶ body source of forces: locomotion permitted by tensile muscular forces ̶ Tissue scale ̶ blood pressure and shear stress on vessels due flow ̶ Single cell scale ̶ mechanical forces on cells regulate functions ̶ cell movement 2005 Wang15 Mechanotransduction Load-sensitive cells: fibroblasts, chondrocytes, osteoblasts, endothelial cells, smooth muscle cells,... 1 cell components in mechanotransduction: Extracellular matrix, cytoskeleton, integrins, Gproteins, receptor tyrosine kinases, MAPK, stretchactivated protein channels 2 mechanical forces on cells regulate functions: gene expression, protein synthesis, cell growth, differentiation. 3 excessive/abnormal mechanical load – tilts cell equilibrium to catabolism, tissue pathophysiogy 4 2015 Jansen16 Cell inside a three dimensional fibrous ECM network. (i) Integrins : α and β subunit - clustered in focal adhesions (FAs) together with other FA proteins (triangle, square and circle). - The adhesions connect the extracellular matrix (ECM) and the (actin) cytoskeleton. (ii)ECM provides multiple cues to the cell, specifically pore size, stiffness, nanotopography and dimensionality. (iii)Cytoskeleton: actin, intermediate filaments and microtubules (iv)Signalling pathways. Define footer – presentation title / department17 Cytoskeleton Define footer – presentation title / department18 Define footer – presentation title / department19 Define footer – presentation title / department20 Define footer – presentation title / department21 Define footer – presentation title / department22 Define footer – presentation title / department23 cytoskeleton involved in • cell shape and cell mechanic properties (no cell wall in animal cell) • providing mechanical strength • cell movement • chromosome separation • intracellular transport of organelles • enable cell communication • cytoskeletal fibers + motor proteins • dynamic instability, • self-assembly http://media.cellsignal.com/www/html/science/landscap es/adhesion Cytoskeleton in eukaryotic cells 24 Microfilaments Intermediary filaments Microtubules build of G-actin/F-actin various a-tubulin/ b-tubulin diameter 7 nm 10-12 nm 25 nm molecular motors myosins none kinesin / dynein polymeration fuel ATP none GTP properties most flexible, fast assembly very flexible, more permanent stiff function structure stabilisation, muscle contraction, cytokinesis, cell movement mechanical stability (bearing tension, retaining shape). structure, securing organelles . cell-specific resist compression, intracel. transport, mitotic spindle 2018 Raudenská https://www.lekarskeknihy.cz/produkt/109803-vybrane-kapitoly-z-bunecne-fyziologie/25 Structure of intermediate filaments actin microfilaments and microtubules are formed from globular subunits Intermediate filaments formed from fibrous protein units, globular parts localized at the ends monomers round around themselves, forming polymers. tetramers are basic organisation unit filament unit intermediate filament 2018 Raudenská https://www.lekarskeknihy.cz/produkt/109803-vybrane-kapitoly-z-bunecne-fyziologie/26 microvilli adhesion belt cell skeleton contractille ring stress fibers myosin filaments actin filaments contraction actin fillaments Actin filaments location in cells. actin shown green muscle contraction: motor molecule of myosin interacts with actin, resulting in contraction. by hydrolysis of ATP and resulting morphology changes 2018 Raudenská https://www.lekarskeknihy.cz/produkt/109803- vybrane-kapitoly-z-bunecne-fyziologie/ 2018 Raudenská https://www.lekarskeknihy.cz/produkt/109803-vybrane-kapitoly-z-bunecne-fyziologie/27 end endmicrotubulegrowth polymeration depolymeration GTP-binding tubulin dimer GDP-binding tubulin dimer Microtubule structure tubulin dimer composed of two subunits: alpha-tubulin and beta- tubulin dynamic structure always changing: dynamic instability: polymeration – tubulin elongation, depolymeration tubulin shortening GTP as energy source 2018 Raudenská https://www.lekarskeknihy.cz/produkt/109803-vybrane-kapitoly-z-bunecne-fyziologie/28 end transported molecule light chain heavy chain head microtubule filament end Molecular motors transport of membrane vesicles and organelles along microtubules driven by molecular motors: kinesins and dyneins: Kinesin to + end (to cell periphery) Dynein to - end (to cell centre) ATP as energy source Define footer – presentation title / department30 Measuring kinesin motor velocities. Fluorescently labeled In vivo measurements of kinesin molecules fused to GFP. The kymograph shown on the right shows that the motors move roughly 2 microns in roughly 4 seconds. Histogram of motor speeds from the measurements of ten cells like those made in (B). Adapted from S. M. Block et al., Nature 348:348, 1990, B, C Adapted from M. E. Tanenbaum et al., Cell 159:635, 2014.) 0.7 μm/s = 42 μm/min = 2.5 mm/hour Define footer – presentation title / department31 Define footer – presentation title / department32 Caveolin-1 is ubiquitously distributed throuhhout cells, although originally described as mebrane protein. FaDu primary oropharyngeal cancer, untreated control, confocal microcopy, 64x. magenta goes for tubulin. 2015 Janssen33 Cytoskeleton and mechanical role ̶ actin and intermediate filaments: main source of cell stiffness ̶ microtubules: resistence to compression force Paul, 2017, https://www.nature.com/articles/nrc.2016.12334 Physical limits for migration ̶ Nuclear size and stiffness control confined migration ̶ as confinment increases, deformation and squeezing is challenging ̶ knockdown of lamin A, (component of the nuclear lamina) decreases nuclear stiffness and enhances the transmigration ̶ progerin (a mutant form of lamin A) increases nuclear stiffness and suppresses confined cell migration 35 Nucleus stiffness. lamin A/C as limiting factor in migration We use combined AFM and side-view SPIM to study how forces correlate with nuclear shape change under compression in live cells. https://twitter.com/C_M_Hobson/status/1227278696798539777 Hobson 2020 https://doi.org/10.1091/mbc.E20-01-0073 Define footer – presentation title / department36 Extracellular matrix 2006 Wang37 Extracellular matrix ̶ preciselly organised structural and functional to biomechanical function of tissues ̶ proteins (collagen, glycoprotien, elastin), ̶ proteoglycans ̶ adhesive glycoproteins (fibronectin, laminin,...) ̶ ECM provides ̶ structural support, ̶ mechanical strength, ̶ attachement sites for cell receptors = ligand for integrins ̶ reservoir for signalling molecules 2015 Janssen38 Basement membrane classess of ECM connective tissue ̶ thin ̶ 2D substrate for adhesion of epithelial and endothelial cells ̶ laminin, collagen IV, nidogen, heparan sulphate ̶ fibrous 3D scaffold ̶ fibrillar colagens (type I, II mixed with III and V), proteoglycans, glycosaminoglycans 2006 Wang39 Extracellular matrix ̶ ECM component synthesis regulated by various factors and cytokines ̶ ECM degradated by matrix metalloproteases (MMPs) ̶ selectively digest ECM components collagen, fibronectin ̶ functional regulation of non ECM molecules (growth factors, cytokines,...) van Helvert https://doi.org/10.1038/s41556-017-0012-040 Dynamic ECM remodelling by cells - mechanoreciprocity ̶ Moving cells sense and respond to tissue mechanics and induce transient or permanent tissue modifications: ̶ extracellular matrix stiffening, ̶ compression and deformation, ̶ protein unfolding, ̶ proteolytic remodelling ̶ jamming transitions. van Helvert https://doi.org/10.1038/s41556-017-0012-041 migration of fibroblasts and endothelial cells into wound - realign and degrade provisional ECM - synthesize collagen and basement membrane proteins - undergo a transition of engaged integrin systems. - Outcome: tissue alignment, density and stiffness are reciprocally linked to fibroblast function Mechanoreciprocity in cancer invasion. Dual function of ECM deposition and stiffening by myofibroblasts in subregions, leading to tumour encapsulation or invasion along collagen interfaces. van Helvert https://doi.org/10.1038/s41556-017-0012-042 Mechanoreciprocity: Spiral concept of mechanical cell–tissue interactions Cells impose ‘mediators’ (pulling, pushing, ECM deposition and ECM degradation) and thereby alter tissue modules. Through iterative reinforcement and positive or negative feedback (indicated by the spiral), both cell and tissue modules undergo coevolution towards altered morphology and function. 2018 Raudenská https://www.lekarskeknihy.cz/produkt/109803-vybrane-kapitoly-z-bunecne-fyziologie/ 2015 Janssen 43 Strain-stiffening: Cell reaction to substrate stiffness Cells adapt their structure to substrate they grown on by cytoskeletal remodelling actin and intermediate fillaments increase stiffness under influence of force: cells strain-stiffen on harder substrates substrate stiffness degreeeofactinfilamentorganisation desorganised actin filaments completely polarized local organisation 2021 De Pieri https://doi.org/10.1002/adbi.20200016844 Profibrotic matrix stiffness and mechanotransduction feedback loop Mechanotransduction pathways mediate matrix stiffness-induced myofibroblast activation. Stiffness-mediated traction forces are transmitted across integrins, which induce actomyosin cell contractility signals activate the effectors YAP (Yes-associated protein), TAZ (transcriptional coactivator with PDZ-binding motif), which increase the expression of profibrotic αSMA and collagen type I. Increased collagen deposition and crosslinking further increases ECM stiffening, focal adhesion kinase (FAK) RHO-associated kinase (ROCK). 2015 Jansen45 Unclear: cells sense environment by: - by applying constant stress (force) and reading out strain (deformation) - OR vice versa Define footer – presentation title / department46 ̶ ability of cancer cells to invade via ̶ MMP-independent amoeboidal mode versus ̶ an MMP-dependent mesenchymal mode ̶ may not solely be attributed to cell-intrinsic properties ̶ but also to the 3D architecture of the local microenvironment. ̶ mouse mammary gland:significantly less fibrous tissue than the corresponding human Comparison of human and mouse mammary glands. (A) Hematoxylin & eosin (H&E) stained section of human breast tissue showing a terminal ductal lobular unit comprised of ducts and acini embedded in a fibrous connective tissue stroma. (B) Schematic representation of a human terminal ductal lobular unit, emphasizing the intimate association of epithelial structures with interstitial fibrous connective tissue stroma and the more distant adipose tissue. (C) H&E stained section of the mouse mammary gland, showing ducts imbedded in a stroma composed of adipose tissue. (D) Schematic representation of the mouse mammary gland, displaying ducts in intimate contact with fibroblasts and adipocyte Parmar et al 2004 10.1677/erc.1.00659 Define footer – presentation title / department47 Integrins 2019 Isomursu, 2015 Jansen48 ̶ heterodimeric transmembrane proteins connecting ECM to cytoskeleton ̶ act as bi-directional signalling receptor ̶ outside-in signalling: ligand binding > conformational changes modulating signalling cascades ̶ inside-out signalling: intracell. proteins > increase integrin affinity for extracel. ligands Integrin-based signaling49 Integrins Define footer – presentation title / department50 Mechanosensing in soft and stiff substrates 1. substrate rigidity can modulate the composition and dimensions of the FAs. 2. FAs promote cytoskeleton reorganization 3. thereby mediates tension (FA angle) cells on soft substrates have small FA complexes, with low degree of assembly, which experience low levels of tension, cells plated on stiff substrates present a higher degree of FA assembly and experience greater levels of tension. the most common elements of the mechanical response to substrate rigidity including sensing and transduction modules. (integrin or some cytoskeleton elements, can exhibit both functions). Espina 2021 https://doi.org/10.1111/febs.15862 Define footer – presentation title / department51 Interaction between the cell’s cytoskeletal mechanics, nuclear mechanics and the matrix mechanics impacts cell migration and invasion through environmental confinements 2019 Isomursu52 van Helvert https://doi.org/10.1038/s41556-017-0012-053 Cell migration modes in 3D environments, including single-cell and collective migration. Define footer – presentation title / department59 Mechanics in pathology 2006 Wang60 Effect of mechanical force ̶ Physiologically cells adapted to force: ̶ blood elements x compression and shear stress ̶ muscle cells X tensile and compression forces ̶ bone change shape, density and stiffness after altered mechanical loads ̶ blood vessels remodel in response to altered blood pressure ̶ cells responsible for tissue remodeling Raudenska 2021 10.48095/ccko202120261 ECM stiffness in disease ̶ excessive ECM fibrotisation: ̶ remodelation via release of proteolytic enzymes + demosition of components ̶ collagen elongation and crosslinking ̶ lysyloxidase activity (lysine – aldehydes – forming crosslinks in ECM proteins ̶ Stiff ECM increase metastasing probability: stromal stiffening modulation as therapy target? Define footer – presentation title / department62 Cell response to forces related to pathology ̶ abnormal mechanical loading ̶ alter cell function ̶ alter extracellular matrix ̶ lead to pathologies ̶ osteoporosis, osteoarthritis, tendinopathy, fibrosis, .... Define footer – presentation title / department63 ECM stiffness in cancerogenesis ECM affected by ̶ desmoplastic reaction in tumors ̶ compression forces – tumor expansion ̶ tensile forces of rigid ECM Cancer cell actively response to ECM stiffness via mechanotransduction ̶ YAP/TAZ signalling ̶ FAK signaling „mechanosensitive“ cancer cell pro-survival adhesion, migration, gene expression, cell-cell interactions, stem cell differentiation Define footer – presentation title / department64 ̶ cells exposed to mechanical stimuli: compression, tensile forces between cells and ECM, compression of interstitial fluid, shear stress ̶ tumor ECM change during progression ̶ tumor ECM may support cancer cell aggresiveness 65 Late stages of the metastatic cascade and biomechanical interrogation. During their metastatic journey, cancer cells are exposed to a number of biophysical challenges. Their adaptation to overcome these threats can be explored using different tools. Each one of the phenotyping techniques relies on the application of a force of known magnitude and tracking of the resulting cell deformation. 10.1091/mbc.E18-08-0545 blood circulation shear forces (red arrows around the cell) collision extravasation Nuclear size and compressive stiffness (as measured using AFM, red arrow in cell sitting on the endothelium) target organ tensile stresses caused by tissue deformations target organ adhering cells exert contractile forces • parallel to the surface • out-of-plane ones Tumor primary stroma variable X selection during metastasis stereotypic Shear stress Tomas Vicar BUT Eldridge et al. 2019, Biophysical Journal PC-3 cells on fibronectin-coated plastic, 12,8 Pa 12x time compression 22Rv1 cells on fibronectin-coated plastic, 6.4 Pa cells Define footer – presentation title / department67 Mechanical stimuli on tumor cells • non-tumor tissue ECM x cell forces in equilibrium • in response to tensile force (increased ECM stiffness in tumor stroma) → mechanosensitive signalling activation • YAP/TAZ: proliferation, dedifferentiation, invasion, resistence • focal adhesion kinase (FAK): increased migration (FA maturation, actomyosin contractility in stress fibers), • tumor cell expansion limited by stroma: ↑ interstitial pressure • increased ECM stiffness → ↑ mechanical stress → gradient supporting metastatic spread → migration along collagen fibers • vessels: shear stress Raudenska 2021 10.48095/ccko2021202. Define footer – presentation title / department68 Mechanotransduction affects cancer cells • stiff ECM activates mechanotransduction driving proliferation, invasion, migration • increased deposition and crosslinking of collagen stiffens ECM → ↑ integrin clustering → FAK activation → Rac1/Cdc42 GTPases activation → migration • RhoA > Rac1/Cdc42 → stress fiber formation + FA maturation • stiff ECM → ↑ YAP/TAZ sctivation via PI3K Raudenska 2021 10.48095/ccko2021202. 2018 Raudenská https://www.lekarskeknihy.cz/produkt/109803-vybrane-kapitoly-z-bunecne-fyziologie/69 Location of movement structures in the mesenchymal type of movement. Formation of structures enabling cell movementis significantly regulated by the activity of small GTPase from the Rho family • Rac1 = Ras-related C3 botulinum toxin substrate 1, • Cdc42 = Celldivision control protein 42 homolog • RhoA= Transforming protein RhoA 2018 Raudenská https://www.lekarskeknihy.cz/produkt/109803- vybrane-kapitoly-z-bunecne-fyziologie/ stress fibers actin filaments movement growth factors + various signals Define footer – presentation title / department70 Rho GTPases in the cell motility cycle. 1. A migratory cell enters the cell motility cycle in response to a chemoattractant signal. 2. Cdc42 determines the direction of motion. 3. Rac induces the formation of actin-rich lamellopodial protrusion at the leading edge. 4. New protrusion is stabilized by the formation of new adhesions to the underlying substratum, a process controlled mainly by Rac and RhoA . 5. Rho acts at the rear end leading to the formation of stress fibers and actin–myosin contractility providing tension for the cell to retract its tail and move forward. 2013 Hanna http://dx.doi.org/10.1016/j.cellsig.2013.04.009 Cell deformability prof. Jochen Guck MPL Erlangen Kyoohun Kim MPL Erlangen YOUNG MODULUS scales with Young modulus (AFM) … and with zinc resistance Cells AFM, sharp tip Young modulus (kPa) AFM, 5 um sphere Young modulus (kPa) RTDC, 30um Young modulus (kPa) PC-3, wild type 1.20 0.99 0.89 PC-3, Zn-resist. 1.70 2.00 1.23 1.2e-05 0 1 2 3 4 wild.type resistant Cell line Youngmodulus(kPa) 1.34 1.34 1.35 1.35 1.36 MeanRIvalue AFM, qp-SCONT (0,01 N/m) 5 µm silica sphere, AtomicJ (DMT model) N=18 PC-3 cells, wild-type vs zinc-resistant Define footer – presentation title / department72 Stiff matrix increase glycolysis, cancer cells regardless stifness New research out today from @gdanuser1 & @RJDLab in @nature found mechanical forces regulate epithelial cell metabolism. Stiff matrix ↑glycolysis, soft matrix ↓glycolysis, and cancer cells were glycolytic regardless – they ignore mechanical cues. https://nature.com/articles/s41586-020-1998-1nn https://twitter.com/CRI_UTSW/status/122767256977766400 5?s=20 (Park study) j.gumulec@med.muni.cz | @jarogumulec | www.med.muni.cz/masariklab