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      Fluorescence microscopy of piezo1 in droplet hydrogel bilayers

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          ABSTRACT

          Mechanosensitive ion channels are membrane gated pores which are activated by mechanical stimuli. The focus of this study is on Piezo1, a newly discovered, large, mammalian, mechanosensitive ion channel, which has been linked to diseases such as dehydrated hereditary stomatocytosis ( Xerocytosis) and lymphatic dysplasia. Here we utilize an established in-vitro artificial bilayer system to interrogate single Piezo1 channel activity. The droplet-hydrogel bilayer (DHB) system uniquely allows the simultaneous recording of electrical activity and fluorescence imaging of labelled protein. We successfully reconstituted fluorescently labelled Piezo1 ion channels in DHBs and verified activity using electrophysiology in the same system. We demonstrate successful insertion and activation of hPiezo1-GFP in bilayers of varying composition. Furthermore, we compare the Piezo1 bilayer reconstitution with measurements of insertion and activation of KcsA channels to reproduce the channel conductances reported in the literature. Together, our results showcase the use of DHBs for future experiments allowing simultaneous measurements of ion channel gating while visualising the channel proteins using fluorescence.

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          Most cited references 19

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          Mechanical stretch triggers rapid epithelial cell division through Piezo1

          Despite acting as a barrier for the organs they encase, epithelial cells turnover at some of the fastest rates in the body. Yet, epithelial cell division must be tightly linked to cell death to preserve barrier function and prevent tumour formation. How do the number of dying cells match those dividing to maintain constant numbers? We previously found that when epithelial cells become too crowded, they activate the stretch-activated channel Piezo1 to trigger extrusion of cells that later die 1 . Conversely, what controls epithelial cell division to balance cell death at steady state? Here, we find that cell division occurs in regions of low cell density, where epithelial cells are stretched. By experimentally stretching epithelia, we find that mechanical stretch itself rapidly stimulates cell division through activation of the same Piezo1 channel. To do so, stretch triggers cells paused in early G2 to activate calcium-dependent ERK1/2 phosphorylation that activates cyclin B transcription necessary to drive cells into mitosis. Although both epithelial cell division and cell extrusion require Piezo1 at steady state, the type of mechanical force controls the outcome: stretch induces cell division whereas crowding induces extrusion. How Piezo1-dependent calcium transients activate two opposing processes may depend on where and how Piezo1 is activated since it accumulates in different subcellular sites with increasing cell density. In sparse epithelial regions where cells divide, Piezo1 localizes to the plasma membrane and cytoplasm whereas in dense regions where cells extrude, it forms large cytoplasmic aggregates. Because Piezo1 senses both mechanical crowding and stretch, it may act as a homeostatic sensor to control epithelial cell numbers, triggering extrusion/apoptosis in crowded regions and cell division in sparse regions.
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            Removal of the mechanoprotective influence of the cytoskeleton reveals PIEZO1 is gated by bilayer tension

            Mechanosensitive ion channels are force-transducing enzymes that couple mechanical stimuli to ion flux. Understanding the gating mechanism of mechanosensitive channels is challenging because the stimulus seen by the channel reflects forces shared between the membrane, cytoskeleton and extracellular matrix. Here we examine whether the mechanosensitive channel PIEZO1 is activated by force-transmission through the bilayer. To achieve this, we generate HEK293 cell membrane blebs largely free of cytoskeleton. Using the bacterial channel MscL, we calibrate the bilayer tension demonstrating that activation of MscL in blebs is identical to that in reconstituted bilayers. Utilizing a novel PIEZO1–GFP fusion, we then show PIEZO1 is activated by bilayer tension in bleb membranes, gating at lower pressures indicative of removal of the cortical cytoskeleton and the mechanoprotection it provides. Thus, PIEZO1 channels must sense force directly transmitted through the bilayer.
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              Architecture of the mammalian mechanosensitive Piezo1 channel.

              Piezo proteins are evolutionarily conserved and functionally diverse mechanosensitive cation channels. However, the overall structural architecture and gating mechanisms of Piezo channels have remained unknown. Here we determine the cryo-electron microscopy structure of the full-length (2,547 amino acids) mouse Piezo1 (Piezo1) at a resolution of 4.8 Å. Piezo1 forms a trimeric propeller-like structure (about 900 kilodalton), with the extracellular domains resembling three distal blades and a central cap. The transmembrane region has 14 apparently resolved segments per subunit. These segments form three peripheral wings and a central pore module that encloses a potential ion-conducting pore. The rather flexible extracellular blade domains are connected to the central intracellular domain by three long beam-like structures. This trimeric architecture suggests that Piezo1 may use its peripheral regions as force sensors to gate the central ion-conducting pore.
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                Author and article information

                Journal
                Channels (Austin)
                Channels (Austin)
                KCHL
                kchl20
                Channels
                Taylor & Francis
                1933-6950
                1933-6969
                2019
                18 March 2019
                18 March 2019
                : 13
                : 1
                : 102-109
                Affiliations
                [a ]School of Biotechnology and Biomolecular Science, UNSW , Kensington, Australia
                [b ]Victor Chang Cardiac Research Institute , Darlinghurst, Australia
                Author notes
                CONTACT Matthew A. B. Baker matthew.baker@ 123456unsw.edu.au
                Article
                1586046
                10.1080/19336950.2019.1586046
                6527062
                30885080
                © 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                Page count
                Figures: 6, References: 24, Pages: 8
                Product
                Funding
                Funded by: Australian Research Council 10.13039/501100000923
                Award ID: DP160103993
                Funded by: Australian Research Council 10.13039/501100000923
                Award ID: DP190100497
                Funded by: Commonwealth Scientific and Industrial Research Organisation 10.13039/501100000943
                Award ID: Future Science Platform Synthetic Biology Project Grant
                Funded by: University of New South Wales 10.13039/501100001773
                Funded by: University of New South Wales 10.13039/501100001773
                Award ID: Scientia Research Fellowship
                BM and MABB were supported by a UNSW-Tsinghua Seed Grant. BM was supported by an Australian Research Council Discovery Project Grant (DP160103993) and a Principal Research Fellowship from the National Health and Medical Research Council of Australia. MABB was supported by an Australian Research Council [Discovery Project  DP190100497]; Commonwealth Scientific and Industrial Research Organisation [Future Science Platform Synthetic Biology Project Grant]; University of New South Wales [UNSW-Tsinghua Seed Fund]; University of New South Wales [Scientia Research Fellowship].
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