Mechanobiology: Proceedings of the 4th International Symposium on Mechanobiology. 6th - 9th November 2022. Sydney, Australia. 9783031453793, 3031453794

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Springer Series in Biophysics  25

Boris Martinac · Charles D. Cox Kate Poole · Sara Baratchi Daryan Kempe   Editors

Mechanobiology Proceedings of the 4th International Symposium on Mechanobiology. 6th 9th November 2022. Sydney, Australia.

Springer Series in Biophysics

Volume 25

Series Editor Boris Martinac, Molecular Cardiology & Biophysics, Victor Chang Cardiac Research Institute, Darlinghurst, NSW, Australia

The “Springer Series in Biophysics” spans all areas of modern biophysics, such as molecular, membrane, cellular or single molecule biophysics. More than that, it is one of the few series of its kind to present biophysical research material from a biological perspective. All postgraduates, researchers and scientists working in biophysical research will benefit from the comprehensive and timely volumes of this well-structured series.

Boris Martinac  •  Charles D. Cox Kate Poole • Sara Baratchi • Daryan Kempe Editors

Mechanobiology Proceedings of the 4th International Symposium on Mechanobiology. 6th - 9th November 2022. Sydney, Australia.

Editors Boris Martinac Lowry Packer Building Victor Chang Cardiac Research Institute Darlinghurst, NSW, Australia Kate Poole School of Biomedical Sciences UNSW Sydney Sydney, NSW, Australia

Charles D. Cox Lowy Packer Building, Level 6 Victor Chang Cardiac Research Institute Darlinghurst, NSW, Australia Sara Baratchi Baker Heart and Diabetes Institute Melbourne, VIC, Australia

Daryan Kempe EMBL Australia, Single Molecule Science node, School of Biomedical Sciences UNSW Sydney Sydney, NSW, Australia

ISSN 0932-2353     ISSN 1868-2561 (electronic) Springer Series in Biophysics ISBN 978-3-031-53904-6    ISBN 978-3-031-45379-3 (eBook) https://doi.org/10.1007/978-3-031-45379-3 © Australian Society for Mechanobiology 2024 Chapter 4 is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/). For further details see license information in the chapter. This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland Paper in this product is recyclable.

Preface

The 4th International Symposium of Mechanobiology, held in Sydney, Australia, in November 2022, marked the occasion where pioneering minds from across the globe converged to explore the intricate interplay between mechanics and biology. This volume presents a collection of several studies serving as a testament to the current knowledge, innovation, and international collaboration in the mechanobiology research field. Mechanobiology, a cutting-edge multidisciplinary field combining physics, biology, bioengineering, and medicine, has witnessed remarkable growth in recent years. The symposium provided an ideal platform for over 200 scientists from 14 countries and regions around the world to share their groundbreaking work and unravel how mechanical forces shape biological phenomena at multiple scales. It offered common ground to researchers from various cultures in their pursuit of advancing mechanobiology, fostering a global network of like-minded scientists united by a shared vision for the future of their research. The several chapters contained within this volume showcase a selected cross section of research, each contributing to a deeper understanding of mechanobiology’s multifaceted complexities. With contributions spanning from fundamental discoveries to innovative applications, this volume is intended to capture the breadth and depth of the symposium’s discussions. The chapters presented here highlight several representative topics, including ion channel signalling in cellular mechanotransduction (Chaps. 1 and 2), biomaterials for tissue engineering (Chap. 3), intramolecular fluorescence-­based force measurement in protein unfolding (Chap. 4), mechanical forces in cancer development (Chap. 5), and cardiac pressure-volume relationship known as the Frank-Starling Law (Chap. 6). The symposium’s agenda sought to address both fundamental questions and practical implications of mechanobiological research, highlighting the potential to transform medical diagnostics, therapeutic interventions, and regenerative medicine. We extend our gratitude to the symposium organizers, all the speakers and attendees, as well as numerous sponsors whose contributions and support made this event a resounding success. Their collective efforts have enriched the field of mechanobiology and paved the way for future discoveries that hold the promise of transforming basic and clinical science and reshaping our understanding of life itself. In closing, this volume stands as a lasting testament to the remarkable research and discussions that took place during the 4th International Symposium of Mechanobiology. As the world continues to witness rapid advancements in the captivating mechanobiol-

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ogy field, we hope that the findings presented here will inspire future generations of mechanobiologists to continue unravelling the mysteries of life’s mechanics. Sydney, NSW, Australia  Kate Poole Melbourne, VIC, Australia   Sara Baratchi Darlinghurst, NSW, Australia   Charles Cox Sydney, NSW, Australia   Daryan Kempe

Contents

1 M  echanically-Evoked TRPV4-Mediated Currents Are Modulated by Activated Integrin β1������������������������������������������������������������������������������������  1 Jessica Richardson, Lioba Schroeter, and Kate Poole 2 Shear  Force Activation of Epithelial Na+ Channel (ENaC) Is Modulated by N-Glycans of the β ENaC Subunit������������������������������������������ 21 Jan-Peter Baldin, Daniel Barth, Fenja Knoepp, and Martin Fronius 3 Fueling  Biologically Relevant Next-Generation Microvasculature-on-a-Chip Platforms with Mechanobiology ������������������������ 35 Monique Bax and Valentin Romanov 4 Using  FRET to Determine How Myo10 Responds to Force in Filopodia�������� 67 Francine Parker, Eulashini Chuntharpursat-Bon, Justin E. Molloy, and Michelle Peckham 5 The  Physical Factors Involved in Cancer Progression�������������������������������������� 79 Che-Tien Lee, Chieh-Sen Hu, and Tzyy Yue Wong 6 The  Molecular Basis of the Frank-Starling Law of the Heart: A Possible Role for PIEZO1?���������������������������������������������������������������������������� 99 C. G. dos Remedios, K. Y. C. Law, J. W. McNamara, T. Kraft, M. Peckham, J. van der Velden, W. A. Linke, M. Ackerman, V. Sequeira, S. Lal, R. Cooke, M. Grosser, K. S. Campbell, B. Martinac, and A. Li

This montage of 18 images shows the phases of the total lunar eclipse on November 8, 2022, over Sydney Harbor, Australia. The lucky amateur astrophotographer who took the images was Professor Jeffrey R Holt from Harvard Medical School, a keynote speaker at the 4th ISMB Symposium. (An article about the event that was published in Sky & Telescope can be accessed at https://skyandtelescope.org/observing/fortune-­favors-­the-­prepared-­astrophotographer/#.Y_ fvoUADOO4.twitter)

Group Photo to wrap up the 2nd day of the 4th International Symposium on Mechanobiology, Sydney, Australia #ISMB2022@AuSMBSoc

Symposium Program

4th International Symposium on Mechanobiology Sydney Nanoscience Hub, The University of Sydney, Australia PROGRAM OVERVIEW Day 1: Sunday 6 November, 2022 (All times in AEDT, GMT+11) @ Messel Lecture Theatre, Sydney Nanoscience Hub 2:00pm – 2:15pm 2:15pm – 2:30pm 2:30pm – 3:20pm 3:20pm – 3:55pm 3:55pm – 4:20pm

WELCOME TO COUNTRY WELCOME TO CONFERENCE PLENARY TALK: Martin Chalfie KEYNOTE TALK: David Beech Afternoon break with refreshments

Boris Martinac Chair: Boris Martinac

MECHANICS IN CANCER session Chair: Yu Suk Choi 4:20pm – 4:45pm 4:45pm – 5:10pm 5:10pm – 5:35pm 5:35pm – 6:00pm

INVITED TALK: Chwee Teck Lim INVITED TALK: Geraldine O’Neill INVITED TALK: Adam Engler INVITED TALK: Dennis Discher

Day 2: Monday 7 November, 2022 9:00am – 9:05am 9:05am – 9:55am 9:55am – 10:30am 10:30am – 10:55am 10:55am – 11:25am

ACKNOWLEDGEMENT OF COUNTRY @ Messel Lecture Theatre, Sydney Nanoscience Hub PLENARY TALK: Rong Li Chair: Sara Baratchi KEYNOTE TALK: Satyajit Mayor INVITED TALK: Samantha Stehbens Morning break with refreshments

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Symposium Program

NON-MAMMALIAN MECHANOBIOLOGY session @ Messel Lecture Theatre, Sydney Nanoscience Hub

Chair: Boris Martinac

MECHANOSENSITIVE CHANNELS session @ Lecture Theatre 2, Room 424, Physics Building 11:25am – 11:50am 11:50am – 12:15pm 12:15pm – 12:30pm 12:30pm – 12:45pm 12:45pm – 1:45pm

INVITED TALK: Joshua Coomey INVITED TALK: JeanMarie Frachisse SELECTED TALK: Linda Kenney SELECTED TALK: Kenjiro Yoshimura Lunch break

Chair: Charles Cox INVITED TALK: Eric Honoré INVITED TALK: Ruhma Syeda SELECTED TALK: Sarah Boyle SELECTED TALK: Jonathan A. Garcia Contreras

MECHANO-IMAGING I session @ Messel Lecture Theatre, Sydney Nanoscience Hub

Chair: Daryan Kempe

MECHANO-IMAGING II session @ Lecture Theatre 2, Room 424, Physics Building

1:45pm – 2:10pm 2:10pm – 2:35pm 2:35pm – 2:50pm 2:50pm – 3:05pm 3:05pm – 3:20pm

INVITED TALK: Medha Pathak

Chair: Peter Su

INVITED TALK: Anne Lagendijk

INVITED TALK: Delphine INVITED TALK: Kathryn Stok Delacour SELECTED TALK: Keng-hui Lin SELECTED TALK: Barry Thompson SELECTED TALK: Andrew SELECTED TALK: Vidhyalakshmi Holle Acharya SELECTED TALK: Thomas Cox SELECTED TALK: Qiutan Yang

Symposium Program 3:20pm – Afternoon break with refreshments 3:50pm 3:50pm – KEYNOTE TALK: Gary Lewin @ Messel 4:25pm Lecture Theatre 4:25pm – FLASH TALKS (11 x 3 min) @ Messel 5:05pm Lecture Theatre 5:05pm – GROUP PHOTO 5:15pm 5:05pm – POSTER Session with drinks/refreshments 8:00pm Board meeting 5:15pm – 6:15pm @ ROOM 4001

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Chair: Jessica Richardson Chair: Jessica Richardson

International Society of Mechanobiology

Day 3: Tuesday 8 November, 2022 9:00am – 9:05am

ACKNOWLEDGEMENT OF COUNTRY

@ Messel Lecture Theatre, Sydney Nanoscience Hub

THEORY session  Chair: Maté Biro 9:05am – 9:55am 9:55am – 10:20am 10:20am – 10:45am 10:45am – 11:15am

PLENARY LECTURE: Stephan Grill INVITED TALK: Richard Morris INVITED TALK: Madan Rao Morning break with refreshments

SENSORY TRANSDUCTION session @ Messel Lecture Theatre

Chair: Charles Cox

TISSUE MECHANICS session @ Lecture Theatre 2, Chair: Charles Cox Room 424, Physics 11:15am – 11:40am INVITED TALK: Valeria Vásquez 11:40am – 12:05pm 12:05pm – 12:20pm 12:20pm – 12:35pm 12:35pm – 12:45pm 12:45pm – 1:45pm

 Yu Suk Choi

INVITED TALK: Misato Iwashita INVITED TALK: Uhtaek Oh INVITED TALK: Masahiro Sokabe SELECTED TALK: Martin Fronius SELECTED TALK: Amy Gelmi SPONSOR TALK: MAWA 10 min SELECTED TALK: X. Frank Zhang SPONSOR TALK: TAIHOYA SPONSOR TALK: LUMICKS Sponsor Video 10 min 10 min Lunch break AuSMB Annual General Meeting @ ROOM 4001

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Symposium Program

CELLULAR MECHANOSENSING session EXTRACELLULAR MECHANICAL SIGNALS session @ Messel Lecture Chair: Kate Poole @ Lecture Theatre 2, Room Chair: Jan Theatre 424, Physics Lauko 1:45pm – 2:00pm SELECTED TALK: SELECTED TALK: Jennifer Benjamin Dalton Young 2:00pm – 2:15pm SELECTED TALK: SELECTED TALK: Marco Massimo Vassalli Enriquez 2:15pm – 2:30pm SELECTED TALK: SELECTED TALK: Iris Michelle Peckham Doolaar 2:30pm – 2:45pm 2:45pm – 3:00pm 3:00pm – 3:15pm 3:15pm – 3:45pm 3:45pm – 4:20pm 4:20pm – 4:55pm 4:55pm – 5:20pm 5:45pm – 6:00pm 6:30pm – 11:30pm

SELECTED TALK: Arnd Pralle SELECTED TALK: Yuanyuan Zhang SELECTED TALK: Julio F. Cordero-Morales Afternoon break with refreshments KEYNOTE TALK: Peter Kohl

SELECTED TALK: Remi Peyronnet SELECTED TALK: Woei Ming Lee SELECTED TALK: Dietmar Oelz

Chair: Sara Baratchi

KEYNOTE TALK: Peter Yingxiao Wang INVITED TALK: Rob Parton

Coaches will depart at 6:00pm from Fisher Library (5 min walk from Sydney Nano) to conference dinner site CONFERENCE DINNER at the Harbourside Room, Museum of Contemporary Art, The Rocks, Sydney

Day 4: Wednesday 9 November, 2022

9:00am – 9:05am 9:05am – 9:55am 9:55am – 10:30am 10:30am – 10:55am 10:55am – 11:20am

ACKNOWLEDGEMENT OF COUNTRY PLENARY TALK DAY 4: Michael Sheetz KEYNOTE TALK: Jeffrey Holt INVITED TALK: Eduardo Perozo Morning break with refreshments

@ Messel Lecture Theatre, Sydney Nanoscience Hub Chair: Kate Poole

Symposium Program TISSUE ENGINEERING session 11:55am – 12:20pm 12:20pm – 12:45pm 12:45pm – 1:00pm 12:45pm – 1:45pm

@ Messel Lecture Theatre, Sydney Nanoscience Hub INVITED TALK: Song Li INVITED TALK: Chun-Xia Zhao CLOSING REMARKS and AWARDS PRESENTATIONS Lunch break

Chair: Arnold Ju

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Chapter 1 Mechanically-Evoked TRPV4-Mediated Currents Are Modulated by Activated Integrin β1 Jessica Richardson1, Lioba Schroeter1, and Kate Poole1 (*) 1 

School of Biomedical Sciences, Faculty of Medicine and Health, University of New South Wales, Sydney, NSW, Australia [email protected]

Abstract.  TRPV4 is a polymodal, non-selective cation channel which can be activated by mechanical stimuli only when they are applied directly at the cell-substrate interface. This restricted profile of mechanical activation suggests that mechanical gating of TRPV4 is highly dependent on either the specific physical context or molecular interaction partners within this cellular compartment. However, it is presently unknown what aspects of the cell-substrate interface confer mechanosensitivity to TRPV4. Integrin β1 has previously been indirectly linked to the mechanical activation of TRPV4 but this link has not yet been investigated in assays where it is clear that the gating stimulus was the mechanical input. We therefore aimed to test whether increasing integrin β1 activation could sensitise TRPV4-mediated, mechanically-­ evoked currents using whole-cell patch-clamp electrophysiology combined with a technique to apply mechanical stimuli directly at cell-substrate connections. We found that TRPV4-mediated currents were sensitised when integrin β1 was activated using the functional anti-integrin β1 [12G10] antibody. However, activation of integrin β1 using fibronectin-coated substrates only lead to minor increases in TRPV4 sensitivity. These data suggest that mechanical signalling via TRPV4 in the cell-­ substrate interface can be tuned by the activation state of integrin β1. Keywords:  TRPV4 · Ion channels · Mechanotransduction · Cell-substrate interface · Integrin beta1

Introduction TRPV4 is a non-selective cation channel activated by multiple gating stimuli including extracellular hypotonicity, moderate changes in ambient temperature, several specific ligands, low pH and mechanical stimuli [7, 22, 38, 39]. The mechanical gating of TRPV4 was first proposed when TRPV4 was shown to be activated in response to osmotic stress [35]. The hypothesis that TRPV4 is mechanosensitive was originally postulated because resultant cell swelling leads to membrane stretch. However, when © The Author(s), under exclusive license to Springer Nature Switzerland AG 2024 B. Martinac et al. (eds.), Mechanobiology, Springer Series in Biophysics 25, https://doi.org/10.1007/978-3-031-45379-3_1

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this hypothesis was tested by multiple teams, it was found that TRPV4 is not responsive to membrane stretch applied via the patch pipette in physiological conditions [21, 29, 35], though moderate changes in open probability are noted under potentiating conditions [14]. Additional studies have revealed that activation of TRPV4 by hypo-osmotic stimuli is dependent on a second messenger cascade, with 5,6,-EET, an endogenous ligand of TRPV4 acting as the gating stimulus, rather than direct mechanical activation of the channel due to membrane stretch [34, 38]. More recent studies, however, have demonstrated that TRPV4 can be directly activated by mechanical inputs, but only when forces are applied directly at the cell-­ substrate interface [29, 30, 33]. In these experiments, cells are cultured on the surface of deformable pillar arrays and mechanical stimuli are applied by deflecting an individual pilus subjacent to the cell. Simultaneously, whole-cell patch-clamp electrophysiology is used to monitor mechanically-evoked currents [27, 32]. TRPV4-dependent currents have been measured in both primary chondrocytes [29] and HEK-293T cells [29, 33] using this technique. The resulting, mechanically-evoked currents are sensitive to TRPV4 specific antagonists and exhibit rapid kinetics suggestive of direct mechanical activation of the channel [6]. These observations suggest that mechanical activation of TRPV4 is highly context-­ dependent and that either a specific mechanical environment or specific molecular interaction partners within the cell-substrate interface are required for channel activation in response to externally applied forces. It is presently unknown what aspects of the cell-substrate interface are required for mechanical activation of TRPV4. Calcium imaging analysis has suggested that integrin β1 may be involved in mechanical activation of TRPV4. Magnetic beads coated with anti-integrin β1 and adhered to the surface of a cell produced a localised increase in Ca++ ions when a force was applied via manipulation of the bead [17]. Co-immunoprecipitation studies suggest a direct interaction between integrin β1 and TRPV4 [1, 2, 28] and it has been postulated that the cytoplasmic tail of integrin β1, CD98hc and TRPV4 form an axis required for activation of TRPV4 by mechanical inputs [28]. However, the relatively low temporal resolution of the techniques (in comparison to the temporal resolution afforded by patch-clamp electrophysiology) used to measure TRPV4 activation via engagement of integrin β1 and the promiscuity of TRPV4 activation mean that it is possible that this axis involves generation of a second messenger that activates TRPV4. This question is particularly pertinent as deleting integrin α1β1 from chondrocytes inhibited TRPV4 activation by hypo-osmotic stimuli and the potent and specific TRPV4 agonist GSK1016790A [12], suggesting a more general role of integrins in TRPV4 activation than a specific influence on mechanical gating of the channel. Here, we have created a new cell line to investigate whether integrin β1 can modulate TRPV4 activation by mechanical inputs applied directly at the cell-substrate interface. Previous studies have investigated mechanical activation of TRPV4 in HEK-293T cells, expressing a low level of endogenous PIEZO1, which may confound the study of potential mechanically activated ion channels [8] and the activation of integrins [18]. Thus, we have used the HEK-293T PIEZO1 (HEK-P1KO) knockout cell line [15] as a background to create cells in which controlled levels of TRPV4 can be induced by the application of tetracycline. We have used these cells to show that TRPV4 can be acti-

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vated by mechanical inputs, in the absence of PIEZO1, and that the activation of integrin β1 potentiates TRPV4-dependent, mechanically-evoked currents at the cell-substrate interface.

Materials and Methods Cell Culture The HEK-P1KO cell line [15] was kindly provided by Ardem Patapoutian. All HEK-­ P1KO cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (SigmaAldrich) supplemented with 10% foetal bovine serum (FBS) (Gibco) and 1% penicillin/ streptomycin (Sigma-Aldrich). In order to create stable cell lines, the Flp-In T-REx kit (Invitrogen) was used to modify cells, as per manufacturer’s instructions. Briefly, first an FRT site was introduced into the genome, by transfecting cells with the pFRT/LacZeo plasmid, provided by manufacturer and integrants were selected in the presence of Zeocin (500 μg/mL, Thermo Fisher Scientific). A clonal population was then isolated and characterised to confirm integration of a single FRT site and sufficient gene expression from the FRT site, assessed using the LacZ reporter gene. Candidate cells were then stably transfected with the pcDNA™6/TR plasmid to introduce the Tet-­repressor, randomly integrated into the genome. The resulting cells were cultured in complete media containing both blasticidin (4  μg/mL, Sigma-Aldrich) and Zeocin (as above). Finally, cells were co-transfected with a plasmid encoding TRPV4 (pcDNA™5/FRT/ TO/TRPV4) and p0G44 encoding FLP, to allow Flp-mediated recombination and the integration of TRPV4 into the FRT site. The resulting cells were selected and maintained in media containing hygromycin (100 μg/mL) and blasticidin (4 μg/mL) (Thermo Fisher Scientific). TRPV4 levels were determined using western blot and qPCR to select the cell line with the lowest TRPV4 expression prior to induction (HEK-P1KO-­ TR-TRPV4). The parental clone of this cell line containing the FRT site and Tet-­ repressor was used as a control for experiments (HEK-P1KO-FRT-TR). This clone was also used to generate a second Flp-In TREx cell line expressing TRPV4 and cytosolic mEmerald as a reporter of expression levels (HEK-293T-P1KO-TR-TRPV4IRES-mEm). For cryogenic storage, cells were frozen in complete DMEM containing 10% dimethyl sulfoxide (DMSO, Sigma Aldrich). When thawed, cells were centrifuged at 200 g for 5 min and resuspended in media lacking DMSO or any other antibiotics. One passage after thaw, media was replaced with complete media containing all appropriate antibiotics for selection. Cells were used between passages 4–20 before discarding and were routinely screened for mycoplasma contamination by the UNSW Mycoplasma Testing Facility. To induce expression of TRPV4 or TRPV4 and mEmerald, cells were incubated in DMEM containing 1 μg/mL tetracycline-hydrochloride (Sigma-Aldrich) for 4 h at 37 °C prior to experimentation. To increase integrin β1 activation, cells were incubated with anti-integrin β1 antibody, [12G10] clone (ab30394, Abcam), (10  μg/ mL) for 4 h at 37 °C, at the same time as tetracycline induction.

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qPCR Quantitative-PCR (qPCR) was used to verify expression of TRPV4, post-induction, in the cell lines created for use in this study. RNA was extracted from cells using the RNeasy Mini-Kit (Qiagen) according to manufacturer’s instructions and RNA concentration and quality assessed using spectrophotometry. RNA was reverse transcribed to cDNA using M-MuLV reverse transcriptase (200  U/μL, New England Biolabs). The cDNA was subsequently utilised for probe-based qPCR using predesigned and validated PrimeTime primers and probes from Integrated DNA Technologies, with ACTB and TBP used as reference, house-keeping genes. Western Blot Analysis Samples for Western blot analyses were prepared by mechanically disrupting cells in cold lysis buffer (RIPA buffer containing protease and phosphatase inhibitors). Protein concentrations were determined using a Pierce™ BCA Assay (Thermo Fisher Scientific) as per manufacturer’s instructions. Protein samples were prepared for electrophoresis in reducing sample buffer and heated at 70 °C for 10 min before separation on a Bolt™ Bis-Tris gel (Thermo Fisher Scientific) in MOPS buffer, containing 0.25% NuPageTM antioxidant using a Mini Gel Module (Life Technologies). Electrophoresed proteins were then transferred to a PVDF membrane via wet transfer in an X-Cell II™ Blot Module (Thermo Fisher Scientific). Post transfer, membranes were blocked using 5% skim milk in TBST for 1 h at room temperature, labelled with 1 μg/mL anti-TRPV4 antibody (ab191580, Abcam), then an HRP-conjugated anti-IgG antibody (1:1000, 7074, Cell Signaling Technology). Labelled bands were detected using enhanced chemiluminescence and visualised using a ChemiDoc™ MP Imaging System. Pillar Arrays Pillar arrays were fabricated as previously described [27, 31, 32]. Briefly, positive masters were silanised with vapour phase trichloro(1H,1H,2H,2H-perfluorooctyl) silane (Sigma-Aldrich). Negative masters were cast from silanised positive masters in polydimethylsiloxane (PDMS) (Sylgard 184, Dow Corning), mixed at a ratio of 1:10 (curing agent:bulk elastomer) and cured for 15 min at 110 °C. These negative masters were subsequently silanised (as above) and used to cast pillar arrays. For pillar array casting, silanised negative masters were coated with degassed PDMS (1:10 ratio of curing agent:bulk elastomer) and left for 30 min before a thickness 2 coverslip was placed over the liquid PDMS. The PDMS was then cured for 1 h at 110 °C, before the master was pulled gently away from the array, bonded to the coverglass. For imaging experiments, 35 mm FluoroDishes (World Precision Instruments) were placed, upside down, over the liquid PDMS in place of the coverslip and cured for 16 h at 60 °C. Pillar arrays were activated with oxygen plasma (Zepto plasma system, Diener) for 90 s, resulting in both sterilisation and activation of the array surface. Cells were then seeded on the array within 30 min of plasma activation. Cells were left to adhere for at least 16 h before any experiments were conducted to allow strong attachment between cells and substrate.

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Arrays were functionalised by coating with either 0.01% poly-L-lysine (PLL) (Sigma-­ Aldrich) or 5 μg/mL fibronectin (FN) (Gibco) and incubated for 1 h before rinsing with 1× PBS and subsequent plating of cells. Using Pillar Arrays to Apply Mechanical Stimuli Fire-polished micropipettes driven by a LS-MM3A micromanipulator (Kleindiek Nanotechnik) were used to deflect individual pili underneath a cell to apply mechanical stimuli. Serial deflections (ranging from 1 to 1000 nm) were applied at a single pilus to produce a range of stimulus magnitudes. To determine the size of the stimulus, a bright-­ field image was taken before and after pillar deflection using a CMOS camera (Nikon DS-Qi2, Zeiss AxioCam HR R3 or ThorLabs DCC1545M-GL, depending on the specific rig used for the experiment). A custom MATLAB script was then used to determine the centre of the pilus in each image and track displacement between subsequent images [4]. Electrophysiology The direct measurement of mechanically-evoked currents was conducted using whole-­ cell patch-clamp electrophysiology, with all recordings performed at room temperature. Patching pipettes were pulled from filamented glass capillaries (1.5 mm outer diameter, 0.86  mm inner diameter, Harvard Apparatus) using a pipette puller (P-1000, Sutter Instruments Co) to create pipettes with a resistance of between 3 and 6  MΩ. Electrophysiology data were collected on two distinct patching rigs, with recordings sampled at 5 kHz using either an Axon Digidata 1440A digitizer (Molecular Devices, LLC) and an Axopatch 1D amplifier filtering at 3 kHz (Molecular Devices, LLC), or an Axon Digidata 1550B digitiser and an Axopatch 200B amplifier. All recordings were made with the Clampex Version 10.7.03 software (Molecular Devices, LLC). The extracellular bath solution contained a buffer with the following composition: 140 mM NaCl, 4  mM KCl, 2  mM CaCl2, 1  mM MgCl2, 4  mM glucose and 10  mM HEPES, adjusted to a pH of 7.4 with NaOH. The intracellular solution contained 110 mM KCl, 10 mM NaCl, 1 mM EGTA, 10 mM HEPES and 1 mM MgCl2, adjusted to a pH of 7.3 using KOH. Appropriate osmolarity of solutions was confirmed using an osmometer (Fiske® Micro-Osmometer, Model 210). After formation of a high resistance seal between glass pipette and cell membrane (>1 GΩ), the membrane within the pipette was disrupted with a short application of pressure, the holding potential set at – 60 mV and series resistance compensated by at least 60%. For details of how mechanical stimuli were applied to cells, see section “Using pillar arrays to apply mechanical stimuli”. Calcium Imaging To confirm induction of functional TRPV4, cells were loaded with 1 μM fluorometric calcium dye, CalBryte 520 AM (AAT Bioquest) in serum free DMEM. Cells were then incubated for 1  h at 37  °C before media was replaced with phenol red free DMEM

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(Fluorobrite, Gibco). Samples were mounted on a wide-field, epifluorescence microscope (Olympus IX710) and imaged using a 20× objective and appropriate filters using a ThorLabs DCC1545M-GL CMOS camera. An image was obtained before and 1 min after application of 100 nM GSK1016790A (Sigma-Aldrich), the resulting F/F0 value was calculated for each cell (with any cell that did not subsequently respond to application of ATP excluded from the data set). To visualise the localisation of TRPV4 activation by pillar deflection, cells cultured on pillar arrays in FluoroDishes were loaded with 1  μM CalBryte 520  AM (AAT Bioquest) in serum free DMEM.  Cells were then incubated for 1  h at 37  °C before media was replaced with phenol red free DMEM (Fluorobrite, Gibco). Changes in Ca++ were determined from alterations in the intensity of CalBryte 520 AM, as measured using wide field epifluorescence, with a 40× objective and appropriate filters from images captured over 5 s at 10 fps following the deflection of a single pilus. Assessment of Integrin β1 Activation and Localisation Cells were cultured on either flat glass substrates or on pillar arrays cast directly onto FluoroDishes. Substrates were activated with oxygen plasma and then coated with either FN or PLL before seeding cells. For visualising activated or total integrin β1, cells were fixed using 4% PFA and labelled with mouse monoclonal anti-integrin β1 [12G10] antibody (ab30394, Abcam) or mouse monoclonal AlexaFlour488 conjugated anti-integrin β1 [K20] antibody (NBP2-52708AF488, Novus Biologicals) respectively. For dual staining, cells were first labelled with the [12G10] antibody, then stained with goat anti-mouse IgG H&L AlexaFluor 594 (ab150116, Abcam). Samples were then washed and treated with 4% PFA for 15 min before thorough washing and subsequent overnight incubation with the conjugated AlexaFluor488 anti-integrin β1 [K20] antibody. Samples were imaged with confocal microscopy (Zeiss LSM 900) using a 40× objective (numerical aperture = 1.3). Statistical Analysis Prior to applying statistical analysis for hypothesis testing, the datasets were assessed using a Shapiro-Wilk test to determine if they were normally distributed, with the exception of qPCR data where the sample number was low (n