Optogenetic control of cellular forces and mechanotransduction

Contractile forces are the end effectors of cell migration, division, morphogenesis, wound healing and cancer invasion. Here we report optogenetic tools to upregulate and downregulate such forces with high spatiotemporal accuracy. The technology relies on controlling the subcellular activation of RhoA using the CRY2/CIBN light-gated dimerizer system. We fused the catalytic domain (DHPH domain) of the RhoA activator ARHGEF11 to CRY2-mCherry (optoGEF-RhoA) and engineered its binding partner CIBN to bind either to the plasma membrane or to the mitochondrial membrane. Translocation of optoGEF-RhoA to the plasma membrane causes a rapid and local increase in cellular traction, intercellular tension and tissue compaction. By contrast, translocation of optoGEF-RhoA to mitochondria results in opposite changes in these physical properties. Cellular changes in contractility are paralleled by modifications in the nuclear localization of the transcriptional regulator YAP, thus showing the ability of our approach to control mechanotransductory signalling pathways in time and space

Mapping mechanical stress in curved epithelia of designed size and shape

We thank C. Pérez-González, N. Castro, and all of the members of the Roca-Cusachs, Arroyo, and Trepat laboratories for their discussions and support. This work was supported by: Generalitat de Catalunya (Agaur, SGR-2021-01425 to X.T., SGR-2021-00523 to R.S., the CERCA Programme, and “ICREA Academia” award to M.A. and P.R-C.); Spanish Ministry for Science and Innovation MICCINN/FEDER (PID2021- 128635NB-I00, MCIN/AEI/ 10.13039/501100011033 and “ERDF-EU A way of making Europe” to X.T., PID2019-110949GB-I00 to M.A., PID2019- 110298GB-I00 to P.R.-C., PID2021-128674OB-I00, RTI2018-101256-J-I00, and RYC2019-026721-I to R.S.); European Research Council (Adv883739 to X.T., CoG-681434 to M.A.); Fundació la Marató de TV3 (project 201903-30-31-32 to X.T.); Deutsche Forschungsgemeinschaft (DFG GO3403/1-1 to T.G.); IBEC, IRB, and CIMNE are recipients of a Severo Ochoa Award of Excellence from the MINECO; European Commission (H2020-FETPROACT-01-2016-731957 to P.R-C.); La Caixa Foundation (LCF/PR/HR20/52400004 and ID 100010434 under the agreement LCF/ PR/HR20/52400004 to P.R-C. and X.T.). R.S. is a Serra Húnter fellow.The function of organs such as lungs, kidneys and mammary glands relies on the three-dimensional geometry of their epithelium. To adopt shapes such as spheres, tubes and ellipsoids, epithelia generate mechanical stresses that are generally unknown. Here we engineer curved epithelial monolayers of controlled size and shape and map their state of stress. We design pressurized epithelia with circular, rectangular and ellipsoidal footprints. We develop a computational method, called curved monolayer stress microscopy, to map the stress tensor in these epithelia. This method establishes a correspondence between epithelial shape and mechanical stress without assumptions of material properties. In epithelia with spherical geometry we show that stress weakly increases with areal strain in a size-independent manner. In epithelia with rectangular and ellipsoidal cross-section we find pronounced stress anisotropies that impact cell alignment. Our approach enables a systematic study of how geometry and stress influence epithelial fate and function in three-dimensions.Peer ReviewedPostprint (published version

Mechanical stress in curved epithelia of designed size and shape

[eng] GENERAL AIM: The general aim of this thesis is to understand how mechanical stress depends on pressure, size and shape in fluid-filled curved epithelial sheets. SPECIFIC AIMS: This general aim can be divided in the following specific objectives: 1. To develop a TFM-compatible protocol to generate curved epithelial monolayers with any desired size and shape. 2. To study the effect of size on the mechanical properties of spherical-cap epithelial monolayers. 3. To validate an inference method to map the stress tensor anywhere in the monolayer without assumptions of mechanical properties. 4. To study the effect of anisotropy on the mechanical properties of curved epithelial monolayers with rectangular and ellipsoidal footprints. 5. To study the relationship between stress anisotropy and cellular geometry and alignment in curved epithelial monolayers. 6. To study the relationship between stress anisotropy and nuclear geometry and alignment in curved epithelial monolayers

Plasmid expression in stressed bacteria

Treball de fi de grau en BiomèdicaTutors: Jordi García-Ojalvo, Letícia Galera LaportaPlasmids are small double-stranded DNA molecules that are present in a high variety of bacteria. They usually encode non-essential proteins, such as antibiotic resistances or toxins, which can become an advantage during the course of infection. Even though the behaviour of bacteria under stress has been widely studied, the dynamics underlying the expression and copy number of plasmids under these conditions have yet to be characterized. We have cloned into two plasmids with different nominal copy numbers a construct that contains a fluorescent protein gene (YFP) regulated by an inducible promoter. After transformation of these plasmids into two different Bacillus subtilis strains, one with a chromosomal integration of the same construct and a wild-type one, we have studied the changes in fluorescence levels produced by variations in stress conditions. Flow cytometry results show an increased level of YFP expression from the high-copy plasmids in cells under stress, in comparison to those that grow in common LB medium. Transformed cells give rise to two populations with different fluorescence values; the first one presents similar expression to that of cells with only a chromosomal copy of the construct while the second shows higher fluorescence levels

ERK-Mediated Mechanochemical Waves Direct Collective Cell Polarization

分子活性の波が細胞集団に伝わる制御機構を解明 --細胞同士の綱引きが情報を遠くに伝える--. 京都大学プレスリリース. 2020-06-04.Cells communicate by doing the 'wave'. 京都大学プレスリリース. 2020-07-22.During collective migration of epithelial cells, the migration direction is aligned over a tissue-scale expanse. Although the collective cell migration is known to be directed by mechanical forces transmitted via cell-cell junctions, it remains elusive how the intercellular force transmission is coordinated with intracellular biochemical signaling to achieve collective movements. Here, we show that intercellular coupling of extracellular signal-regulated kinase (ERK)-mediated mechanochemical feedback yields long-distance transmission of guidance cues. Mechanical stretch activates ERK through epidermal growth factor receptor (EGFR) activation, and ERK activation triggers cell contraction. The contraction of the activated cell pulls neighboring cells, evoking another round of ERK activation and contraction in the neighbors. Furthermore, anisotropic contraction based on front-rear polarization guarantees unidirectional propagation of ERK activation, and in turn, the ERK activation waves direct multicellular alignment of the polarity, leading to long-range ordered migration. Our findings reveal that mechanical forces mediate intercellular signaling underlying sustained transmission of guidance cues for collective cell migration

Optogenetic control of cellular forces and mechanotransduction

Contractile forces are the end effectors of cell migration, division, morphogenesis, wound healing and cancer invasion. Here we report optogenetic tools to upregulate and downregulate such forces with high spatiotemporal accuracy. The technology relies on controlling the subcellular activation of RhoA using the CRY2/CIBN light-gated dimerizer system. We fused the catalytic domain (DHPH domain) of the RhoA activator ARHGEF11 to CRY2-mCherry (optoGEF-RhoA) and engineered its binding partner CIBN to bind either to the plasma membrane or to the mitochondrial membrane. Translocation of optoGEF-RhoA to the plasma membrane causes a rapid and local increase in cellular traction, intercellular tension and tissue compaction. By contrast, translocation of optoGEF-RhoA to mitochondria results in opposite changes in these physical properties. Cellular changes in contractility are paralleled by modifications in the nuclear localization of the transcriptional regulator YAP, thus showing the ability of our approach to control mechanotransductory signalling pathways in time and space

Traction forces at the cytokinetic ring regulate cell division and polyploidy in the migrating zebrafish epicardium

Epithelial repair and regeneration are driven by collective cell migration and division. Both cellular functions involve tightly controlled mechanical events, but how physical forces regulate cell division in migrating epithelia is largely unknown. Here we show that cells dividing in the migrating zebrafish epicardium exert large cell–extracellular matrix (ECM) forces during cytokinesis. These forces point towards the division axis and are exerted through focal adhesions that connect the cytokinetic ring to the underlying ECM. When subjected to high loading rates, these cytokinetic focal adhesions prevent closure of the contractile ring, leading to multi-nucleation through cytokinetic failure. By combining a clutch model with experiments on substrates of different rigidity, ECM composition and ligand density, we show that failed cytokinesis is triggered by adhesion reinforcement downstream of increased myosin density. The mechanical interaction between the cytokinetic ring and the ECM thus provides a mechanism for the regulation of cell division and polyploidy that may have implications in regeneration and cancer

Traction forces at the cytokinetic ring regulate cell division and polyploidy in the migrating zebrafish epicardium

Epithelial repair and regeneration are driven by collective cell migration and division. Both cellular functions involve tightly controlled mechanical events, but how physical forces regulate cell division in migrating epithelia is largely unknown. Here we show that cells dividing in the migrating zebrafish epicardium exert large cell–extracellular matrix (ECM) forces during cytokinesis. These forces point towards the division axis and are exerted through focal adhesions that connect the cytokinetic ring to the underlying ECM. When subjected to high loading rates, these cytokinetic focal adhesions prevent closure of the contractile ring, leading to multi-nucleation through cytokinetic failure. By combining a clutch model with experiments on substrates of different rigidity, ECM composition and ligand density, we show that failed cytokinesis is triggered by adhesion reinforcement downstream of increased myosin density. The mechanical interaction between the cytokinetic ring and the ECM thus provides a mechanism for the regulation of cell division and polyploidy that may have implications in regeneration and cancer