Red blood cells (RBCs) experience significant mechanical forces in their lifetime as they traverse the mammalian circulatory system, squeezing through capillaries of smaller diameters than themselves (Chien, 1987). Central to the ability of RBCs to deform in response to shear stress is their biconcave disk shape (Lister, 1827; Mohandas and Evans, 1994). RBCs lack organelles and a transcytosolic network and their biconcave shape is instead imparted by the plasma membrane and the membrane skeleton, a 2D quasihexagonal network of actin filament (F-actin) nodes connected by (α₁β₁)₂-spectrin tetramers bound to transmembrane proteins (Fowler, 2013; Gratzer, 1981). In response to shear stress a Ca²⁺ influx is activated in RBCs (Larsen et al., 1981; Dyrda et al., 2010). Ca²⁺ entry into RBCs modulates junctional protein interactions in the membrane skeleton (Nunomura, 2006) in addition to activating the Gardos channel (Gardos, 1958), a K⁺ channel, leading to K⁺ efflux and subsequent cell volume decrease (Danielczok et al., 2017), which together facilitate flexibility of the cell membrane.

Piezo1 was first suggested as the mechanosensitive Ca²⁺ channel in RBCs after identification of gain-of-function mutations in patients with dehydrated xerocytosis (Bae et al., 2013; Albuisson et al., 2013; Andolfo et al., 2013; Zarychanski et al., 2012). Subsequent studies using a hematopoietic PIEZO1 knockout mouse confirmed that the channel is responsible for stretch-induced calcium influx in RBCs (Cahalan et al., 2015). How Piezo1 is organized in the RBC membrane, however, is not known. The unique biconcave shape of the RBC poses interesting questions about how membrane curvature, which locally is known to be important for Piezo1 structure (Guo and MacKinnon, 2017; Lin et al., 2019; Haselwandter and MacKinnon, 2018), might globally impact Piezo1 distribution and function. It has been proposed that due to its intrinsic curvature, Piezo1 might tend to concentrate in the dimple region of RBCs (Svetina et al., 2019), but direct experimental evidence is lacking. More broadly, how Piezo channels are organized in cell membranes is poorly understood. Based on electrophysiological recordings in overexpressing cells (Gottlieb et al., 2012) or fluorescence microscopy of a stable cell line (Ridone et al., 2020), some authors have reported clustering of Piezo1, but channel densities in both cases are likely non-physiological. Extensive patch-clamp studies and simulation of Piezo1 densities in neuro2Acells, which express Piezo1 natively, led others to the conclusion that Piezo1 channels are homogenously expressed across cell membranes and function as independent mechanotransducers (Lewis and Grandl, 2021). Additionally, it is unclear what role the actin cytoskeleton plays in organizing and gating the Piezo1 channel in cells. Piezo1 can be pressure activated in membrane blebs lacking cytoskeleton (Cox et al., 2016) and exhibits spontaneous openings in asymmetric lipid droplet bilayers (Syeda et al., 2016), consistent with a ‘force-from-lipids’ model of activation (Martinac et al., 1990). Others claim that Piezo1 is tethered to the actin cytoskeleton and is gated by a ‘force-from-filament’ mechanism (Wang et al., 2022).

Here, we set out to image and analyze the organization of Piezo1 channels in RBC membranes. The small size of RBCs and their inherent autofluorescence makes them refractory to conventional fluorescence microscopy, so we employed more sensitive super-resolution microscopic methods. Fluorescent Piezo1 spots can be resolved into single Piezo1 channels which, along with electron microscopy studies, confirm that they do not cluster in RBC membranes. Although not clustering, we find a non-uniform distribution of Piezo1 in the RBC membrane with enrichment at the dimple, which can be rationalized through the energetic coupling of the intrinsic curvature of Piezo1 to the curvature of the surrounding membrane. Single particle tracking (SPT) studies of Piezo1 demonstrate its lateral mobility in the plasma membrane. This is consistent with co-immunostaining analysis, suggesting that Piezo1 is not tethered to the membrane skeleton. Mobility of Piezo1 in the RBC membrane permits Piezo1 to explore the curvature landscape of the RBC surface and may allow Piezo1 to respond to local and global changes in membrane curvature as well as membrane tension.

Since its discovery (Coste et al., 2010), Piezo1 has been identified as a primary mechanosensitive molecule in a wide range of force-sensing physiological processes from cell and organ development (Sun et al., 2019; Li et al., 2014; Ranade et al., 2014) to blood pressure regulation (Zeng et al., 2018) and stem cell lineage choice (Pathak et al., 2014). How Piezo1 is organized in different cell types and how this might pertain to the sensing of different types of physiological force is currently not well understood. Here we studied Piezo1 organization in RBCs, both because of the clear importance of Piezo1 in RBC physiology (Bae et al., 2013; Zarychanski et al., 2012; Cahalan et al., 2015) and because of the unique cellular structure of RBCs, particularly their biconcave disk shape.

At the smallest level of Piezo1 organization, that is with itself, we find that Piezo1 does not cluster in the RBC membrane. By 3D-SIM microscopy (Figure 1) we observe diffraction-limited fluorescent spots that could technically represent multiple, closely located, Piezo1 channels. To distinguish these spots as individual channels, we needed to employ 2D STED microscopy (Figure 2). As the power of the depletion laser was increased to improve resolution, the size of each spot decreased correspondingly, but never resolved into multiple spots, even when the achieved resolution was near the size of the Piezo1 channel. Further, after Huygens deconvolution post-processing of our 2D STED data we could resolve triple-antibody-labeled Piezo1 channels, with an inter-subunit diameter consistent with the channel size. Thus, we conclude that each fluorescent spot, whether viewed by 3D-SIM or 2D STED, corresponds to a single Piezo1 channel. The average distance between Piezo1 channels is approximately 550 nm, although occasionally we observe two to three channels within 100-nm distances. This is in good agreement with studies of neuro2A cells, which natively express Piezo1, that also found that Piezo1 channels do not tend to cluster (Lewis and Grandl, 2021). While clustering could affect Piezo1 gating through Piezo’s membrane footprint, the precise coupling of Piezo1 gating to clustering would depend on the specific scenario considered, and clustering does not necessarily facilitate gating (Haselwandter and MacKinnon, 2018). A relatively homogenous membrane distribution of Piezo1 might be well suited to RBCs that are tumbling through solution, experiencing shear forces from all angles, and may contribute to the remarkable plasticity of RBC shapes under stress (Dupire et al., 2012). Of course, our data on RBCs do not rule out the possibility of Piezo1 clustering in other cell types.

Because of the intrinsic curvature of Piezo1 we were curious about its distribution as a function of the curvature of the RBC membrane. Experimentally we were able to ask this question through 3D-SIM microscopy, which yielded sufficiently high-resolution spatial information on Piezo1 channels while enabling easy assignment of the RBC dimple and rim membrane regions (Figure 3). Per μm² area of membrane we find an almost twofold enrichment of Piezo1 in the dimple relative to the rim. Our experimental data can be rationalized by a physical model of curvature coupling between the intrinsic curvature of the Piezo1 dome and the surrounding membrane curvature of the RBC along its biconcave surface (see Methods). Whilst the curved biconcave shape of RBCs is unique, the good agreement between our experimental data and theoretical prediction based on energetic curvature coupling would suggest an inherent relationship between Piezo’s intrinsic curvature and the surrounding membrane that is broadly relevant to other cell types. This is because the curvature coupling theory, which was developed with experiments on lipid bilayer vesicles, depends only on the bending elasticity of membranes and the intrinsic curvature of Piezo1, not on the unique shape of RBCs. It will be interesting to explore whether Piezo1, for example, is enriched in membrane invaginations or other curvature-matched membrane regions in different cell types. Recently, a study analyzing overexpressed Piezo1 in HeLa cells showed depletion of the channel from membrane filopodia, consistent with a mechanism of curvature coupling whereby the channel unfavorably distributes into membrane regions of high opposing curvature (Yang et al., 2022).

Curvature sensing of membrane proteins by curvature matching has been well documented for other intrinsically curved proteins such as Bin/Amphiphysin/Rvs (BAR) (Simunovic et al., 2015; Tsai et al., 2021) proteins. More recently, it has been shown that glycosylation can determine the membrane curvature sensing properties of some proteins (Lu et al., 2022). It is unclear from our current data whether the enrichment of Piezo1 in the RBC dimple is simply a corollary of the curved structure of Piezo1, which determines the channel’s tension sensitivity as described by the membrane dome model (Guo and MacKinnon, 2017; Haselwandter and MacKinnon, 2018), or whether it also has some important consequences for physiological force sensation of RBCs. In this context, it is noteworthy that the force-generating protein myosin IIA also shows an enrichment in the RBC dimple relative to the RBC rim (Alimohamadi et al., 2020).

Consistent with its ability to equilibrate along the RBC surface as a function of membrane curvature, we find that Piezo1 exhibits lateral mobility within the RBC plasma membrane. We used gold nanoparticle tracking under DIC (Figure 4) so we could record for minutes to an hour without being limited by photobleaching and at fast time resolution (100 Hz). This enabled us to record both the confined (<500 nm) diffusion of Piezo1 as well as Piezo1’s ‘hopping’ into more freely diffusing behavior. The spatial scales associated with the confined diffusion regime most likely reflect the compartment sizes of the actin–spectrin meshwork we observe by 2D STED. Such a model of confined diffusion set by actin–spectrin is well documented in RBCs (Tsuji et al., 1988) and other cell types (Fujiwara et al., 2016). Similar to observations by others using STORM microscopy (Pan et al., 2018), we observe ~200–500 nm voids that are deficient in actin or spectrin signal by 2D STED. Similar nanoscale ‘defects’ in the actin–spectrin meshwork have also been observed by atomic force microscopy imaging of RBCs (Nowakowski et al., 2001) and may serve as weak points in the meshwork that facilitate fast RBC remodeling during shear stress. Such defects may also permit faster diffusion of transmembrane proteins such as Piezo1.

Our double labeling experiments suggest that Piezo1 does not form a complex with actin or spectrin, which is consistent with Piezo1’s confined diffusion within the actin–spectrin meshwork compartments, and should be contrasted with the near-immobilization observed for proteins such as CFTR and Band3 (Kodippili et al., 2009, 3; Haggie et al., 2006), which both form complexes with actin. Additionally, our data are not consistent with Piezo1 being tethered to the underlying actin and activated by a ‘force-from-filament’ mechanism as has been traditionally described (Cox et al., 2019; Zhang et al., 2015). Of course, the actin–spectrin meshwork organizes the RBC membrane into compartments whose boundary conditions could impact how changes in local curvature and lateral tension of the membrane gate Piezo1. This could also explain how chemical agents that disrupt the actin cytoskeleton affect Piezo1 currents in other cell types (Gottlieb et al., 2012; Wang et al., 2022), that is through changes in organization of the membrane rather than disruption of a direct tether to the channel itself.

Additionally, we asked whether Piezo1 might be colocalized with the Ca²⁺-activated K⁺ (Gardos) channel. Ca²⁺ flux into the RBC through Piezo1 and subsequent activation of the Gardos channel is thought to be the initial step in mechanically activated RBC volume regulation (Danielczok et al., 2017; Cahalan et al., 2015). We do not, however, observe significant spatial proximity between Piezo1 and Gardos channels. Given the small size of RBCs, approximately 1 μm thick in the dimple and 2–3 μm thick in the rim, and the Piezo1 density of around 0.5 per μm², it seems that colocalization is probably not necessary for rapid opening of the Gardos channel following Piezo activation. For diffusion in three dimensions, the mean dispersion time can be approximated by ${\displaystyle \tau =\frac{x^2}{6D}}$ , ${\displaystyle x}$ the mean distance and $D$ the diffusion coefficient. The apparent diffusion coefficient for intracellular Ca²⁺ is in the range 13–65 μm²/s (Nakatani et al., 2002), meaning that Ca²⁺ should equilibrate within tens of ms within a RBC. In larger cell types, colocalization of Piezo1 with other channels might be necessary to elicit a functional coupling of ion fluxes. In rat ventricular myocytes, for example, Piezo1 has recently been shown, by diffraction-limited confocal microscopy, to colocalize with transient receptor melastatin (TRPM4) channels (Yu et al., 2022).

The experimentally determined values of the intrinsic curvature and the bending stiffness of Piezo1 in lipid bilayers enable us to estimate changes in Piezo1 curvature at the dimple versus the rim of a RBC membrane (Haselwandter et al., 2022a). We estimate a radius of curvature of 41.4 nm at the dimple and 42.7 nm at the rim and thus the curvature differences across the unperturbed RBC membrane are, alone, insufficient to substantially change the shape of Piezo1, and thus are not expected to open its pore. However, that Piezo1 distributes on the surface of RBCs by sensing the membrane curvature, by corollary, means that it must have the capacity to sense lateral membrane tension by a force-through-membrane mechanism. To see this, consider that Piezo1 has been shown to flex, that is, change its curvature, as a function of local membrane curvature (Haselwandter et al., 2022b). Consider also that membrane tension changes membrane curvature by flattening it. In this manner, curvature sensing and lateral membrane tension sensing are inextricably linked. This linkage lies at the heart of the membrane dome model of Piezo gating (Guo and MacKinnon, 2017; Haselwandter and MacKinnon, 2018; Haselwandter et al., 2022b; Haselwandter et al., 2022a). When RBCs squeeze through small capillaries, the membrane shape as well as the transmembrane pressure change transiently. The resulting changes in membrane curvature and lateral membrane tension can both, according to the membrane dome model of Piezo gating, open Piezo1’s pore to trigger Ca²⁺ entry.

Data generated and analyzed are included in main figures and figure supplements of manuscript. Source files for raw data used for tracking analysis in Figure 4 and Figure 4—figure supplement 1 have been deposited to Dryad: https://doi.org/10.5061/dryad.c2fqz61c7. Source files for raw data used for optimal transport colocalization analysis in Figure 5 have been deposited to Dryad: https://doi.org/10.5061/dryad.1ns1rn8x6.

1. 1. Mackinnon R

   (2022) Dryad Digital Repository

   Data from: Piezo1 as a force-through-membrane sensor in red blood cells.

   https://doi.org/10.5061/dryad.c2fqz61c7
2. 1. Mackinnon R

   (2022) Dryad Digital Repository

   Data from: Piezo1 as a force-through-membrane sensor in red blood cells.

   https://doi.org/10.5061/dryad.1ns1rn8x6

1. 1. Albuisson J
   2. Murthy SE
   3. Bandell M
   4. Coste B
   5. Louis-Dit-Picard H
   6. Mathur J
   7. Fénéant-Thibault M
   8. Tertian G
   9. de Jaureguiberry J-P
   10. Syfuss P-Y
   11. Cahalan S
   12. Garçon L
   13. Toutain F
   14. Simon Rohrlich P
   15. Delaunay J
   16. Picard V
   17. Jeunemaitre X
   18. Patapoutian A

   (2013) Dehydrated hereditary stomatocytosis linked to gain-of-function mutations in mechanically activated Piezo1 ion channels

   Nature Communications 4:1884.

   https://doi.org/10.1038/ncomms2899

   • PubMed
   • Google Scholar
2. 1. Alimohamadi H
   2. Smith AS
   3. Nowak RB
   4. Fowler VM
   5. Rangamani P

   (2020) Non-Uniform distribution of myosin-mediated forces governs red blood cell membrane curvature through tension modulation

   PLOS Computational Biology 16:e1007890.

   https://doi.org/10.1371/journal.pcbi.1007890

   • PubMed
   • Google Scholar
3. 1. Andolfo I
   2. Alper SL
   3. De Franceschi L
   4. Auriemma C
   5. Russo R
   6. De Falco L
   7. Vallefuoco F
   8. Esposito MR
   9. Vandorpe DH
   10. Shmukler BE
   11. Narayan R
   12. Montanaro D
   13. D’Armiento M
   14. Vetro A
   15. Limongelli I
   16. Zuffardi O
   17. Glader BE
   18. Schrier SL
   19. Brugnara C
   20. Stewart GW
   21. Delaunay J
   22. Iolascon A

   (2013) Multiple clinical forms of dehydrated hereditary stomatocytosis arise from mutations in Piezo1

   Blood 121:3925–3935.

   https://doi.org/10.1182/blood-2013-02-482489

   • PubMed
   • Google Scholar
4. 1. Andrey P
   2. Kiêu K
   3. Kress C
   4. Lehmann G
   5. Tirichine L
   6. Liu Z
   7. Biot E
   8. Adenot PG
   9. Hue-Beauvais C
   10. Houba-Hérin N
   11. Duranthon V
   12. Devinoy E
   13. Beaujean N
   14. Gaudin V
   15. Maurin Y
   16. Debey P

   (2010) Statistical analysis of 3D images detects regular spatial distributions of centromeres and chromocenters in animal and plant nuclei

   PLOS Computational Biology 6:e1000853.

   https://doi.org/10.1371/journal.pcbi.1000853

   • PubMed
   • Google Scholar
5. 1. Arakawa T
   2. Kobayashi-Yurugi T
   3. Alguel Y
   4. Iwanari H
   5. Hatae H
   6. Iwata M
   7. Abe Y
   8. Hino T
   9. Ikeda-Suno C
   10. Kuma H
   11. Kang D
   12. Murata T
   13. Hamakubo T
   14. Cameron AD
   15. Kobayashi T
   16. Hamasaki N
   17. Iwata S

   (2015) Crystal structure of the anion exchanger domain of human erythrocyte band 3

   Science 350:680–684.

   https://doi.org/10.1126/science.aaa4335

   • PubMed
   • Google Scholar
6. 1. Arganda-Carreras I
   2. Kaynig V
   3. Rueden C
   4. Eliceiri KW
   5. Schindelin J
   6. Cardona A
   7. Sebastian Seung H

   (2017) Trainable weka segmentation: a machine learning tool for microscopy pixel classification

   Bioinformatics 33:2424–2426.

   https://doi.org/10.1093/bioinformatics/btx180

   • PubMed
   • Google Scholar
7. 1. Bae C
   2. Gnanasambandam R
   3. Nicolai C
   4. Sachs F
   5. Gottlieb PA

   (2013) Xerocytosis is caused by mutations that alter the kinetics of the mechanosensitive channel piezo1

   PNAS 110:E1162–E1168.

   https://doi.org/10.1073/pnas.1219777110

   • PubMed
   • Google Scholar
8. 1. Ballas SK
   2. Krasnow SH

   (1980)

   Structure of erythrocyte membrane and its transport functions

   Annals of Clinical and Laboratory Science 10:209–219.

   • PubMed
   • Google Scholar
9. 1. Beck JS

   (1978) Relations between membrane monolayers in some red cell shape transformations

   Journal of Theoretical Biology 75:487–501.

   https://doi.org/10.1016/0022-5193(78)90358-2

   • PubMed
   • Google Scholar
10. 1. Brugnara C
    2. De Franceschi L
    3. Alper SL

    (1993) Ca (2+) -activated K+ transport in erythrocytes. Comparison of binding and transport inhibition by scorpion toxins

    The Journal of Biological Chemistry 268:8760–8768.

    https://doi.org/10.1016/S0021-9258(18)52939-2

    • PubMed
    • Google Scholar
11. 1. Bryk AH
    2. Wiśniewski JR

    (2017) Quantitative analysis of human red blood cell proteome

    Journal of Proteome Research 16:2752–2761.

    https://doi.org/10.1021/acs.jproteome.7b00025

    • PubMed
    • Google Scholar
12. 1. Cahalan SM
    2. Lukacs V
    3. Ranade SS
    4. Chien S
    5. Bandell M
    6. Patapoutian A

    (2015) Piezo1 links mechanical forces to red blood cell volume

    eLife 4:e07370.

    https://doi.org/10.7554/eLife.07370

    • PubMed
    • Google Scholar
13. Book

    1. Carmo M

    (2016)

    Differential Geometry of Curves & Surfaces

    Dover Publications, INC.

    • Google Scholar
14. 1. Chien S

    (1987) Red cell deformability and its relevance to blood flow

    Annual Review of Physiology 49:177–192.

    https://doi.org/10.1146/annurev.ph.49.030187.001141

    • PubMed
    • Google Scholar
15. 1. Coste B
    2. Mathur J
    3. Schmidt M
    4. Earley TJ
    5. Ranade S
    6. Petrus MJ
    7. Dubin AE
    8. Patapoutian A

    (2010) Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels

    Science 330:55–60.

    https://doi.org/10.1126/science.1193270

    • PubMed
    • Google Scholar
16. 1. Cox CD
    2. Bae C
    3. Ziegler L
    4. Hartley S
    5. Nikolova-Krstevski V
    6. Rohde PR
    7. Ng C-A
    8. Sachs F
    9. Gottlieb PA
    10. Martinac B

    (2016) Removal of the mechanoprotective influence of the cytoskeleton reveals Piezo1 is gated by bilayer tension

    Nature Communications 7:10366.

    https://doi.org/10.1038/ncomms10366

    • PubMed
    • Google Scholar
17. 1. Cox CD
    2. Bavi N
    3. Martinac B

    (2019) Biophysical principles of ion-channel-mediated mechanosensory transduction

    Cell Reports 29:1–12.

    https://doi.org/10.1016/j.celrep.2019.08.075

    • PubMed
    • Google Scholar
18. 1. Danielczok JG
    2. Terriac E
    3. Hertz L
    4. Petkova-Kirova P
    5. Lautenschläger F
    6. Laschke MW
    7. Kaestner L

    (2017) Red blood cell passage of small capillaries is associated with transient Ca2+-mediated adaptations

    Frontiers in Physiology 8:979.

    https://doi.org/10.3389/fphys.2017.00979

    • PubMed
    • Google Scholar
19. 1. Daumas F
    2. Destainville N
    3. Millot C
    4. Lopez A
    5. Dean D
    6. Salomé L

    (2003) Confined diffusion without fences of a G-protein-coupled receptor as revealed by single particle tracking

    Biophysical Journal 84:356–366.

    https://doi.org/10.1016/S0006-3495(03)74856-5

    • PubMed
    • Google Scholar
20. 1. Del Mármol JI
    2. Touhara KK
    3. Croft G
    4. MacKinnon R

    (2018) Piezo1 forms a slowly-inactivating mechanosensory channel in mouse embryonic stem cells

    eLife 7:e33149.

    https://doi.org/10.7554/eLife.33149

    • PubMed
    • Google Scholar
21. 1. Dumitru AC
    2. Stommen A
    3. Koehler M
    4. Cloos A-S
    5. Yang J
    6. Leclercqz A
    7. Tyteca D
    8. Alsteens D

    (2021) Probing Piezo1 localization upon activation using high-resolution atomic force and confocal microscopy

    Nano Letters 21:4950–4958.

    https://doi.org/10.1021/acs.nanolett.1c00599

    • PubMed
    • Google Scholar
22. 1. Dupire J
    2. Socol M
    3. Viallat A

    (2012) Full dynamics of a red blood cell in shear flow

    PNAS 109:20808–20813.

    https://doi.org/10.1073/pnas.1210236109

    • PubMed
    • Google Scholar
23. 1. Dyrda A
    2. Cytlak U
    3. Ciuraszkiewicz A
    4. Lipinska A
    5. Cueff A
    6. Bouyer G
    7. Egée S
    8. Bennekou P
    9. Lew VL
    10. Thomas SLY

    (2010) Local membrane deformations activate Ca2+-dependent K+ and anionic currents in intact human red blood cells

    PLOS ONE 5:e9447.

    https://doi.org/10.1371/journal.pone.0009447

    • PubMed
    • Google Scholar
24. 1. Ellefsen KL
    2. Holt JR
    3. Chang AC
    4. Nourse JL
    5. Arulmoli J
    6. Mekhdjian AH
    7. Abuwarda H
    8. Tombola F
    9. Flanagan LA
    10. Dunn AR
    11. Parker I
    12. Pathak MM

    (2019) Myosin-ii mediated traction forces evoke localized piezo1-dependent ca2+ flickers

    Communications Biology 2:298.

    https://doi.org/10.1038/s42003-019-0514-3

    • PubMed
    • Google Scholar
25. Preprint

    1. Ershov D
    2. Phan MS
    3. Pylvänäinen JW
    4. Rigaud SU
    5. Le Blanc L
    6. Charles-Orszag A
    7. Conway JRW
    8. Laine RF
    9. Roy NH
    10. Bonazzi D
    11. Duménil G
    12. Jacquemet G
    13. Tinevez JY

    (2021) Bringing TrackMate into the Era of Machine-Learning and Deep-Learning

    bioRxiv.

    https://doi.org/10.1101/2021.09.03.458852

    • Google Scholar
26. 1. Fairbanks G
    2. Steck TL
    3. Wallach DF

    (1971) Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane

    Biochemistry 10:2606–2617.

    https://doi.org/10.1021/bi00789a030

    • PubMed
    • Google Scholar
27. 1. Fowler VM

    (2013) The human erythrocyte plasma membrane

    Current Topics in Membranes 72:39–88.

    https://doi.org/10.1016/B978-0-12-417027-8.00002-7

    • Google Scholar
28. 1. Fujiwara TK
    2. Iwasawa K
    3. Kalay Z
    4. Tsunoyama TA
    5. Watanabe Y
    6. Umemura YM
    7. Murakoshi H
    8. Suzuki KGN
    9. Nemoto YL
    10. Morone N
    11. Kusumi A

    (2016) Confined diffusion of transmembrane proteins and lipids induced by the same actin meshwork lining the plasma membrane

    Molecular Biology of the Cell 27:1101–1119.

    https://doi.org/10.1091/mbc.E15-04-0186

    • PubMed
    • Google Scholar
29. 1. Gardos G

    (1958) The function of calcium in the potassium permeability of human erythrocytes

    Biochimica et Biophysica Acta 30:653–654.

    https://doi.org/10.1016/0006-3002(58)90124-0

    • PubMed
    • Google Scholar
30. 1. Gautier E-F
    2. Leduc M
    3. Cochet S
    4. Bailly K
    5. Lacombe C
    6. Mohandas N
    7. Guillonneau F
    8. El Nemer W
    9. Mayeux P

    (2018) Absolute proteome quantification of highly purified populations of circulating reticulocytes and mature erythrocytes

    Blood Advances 2:2646–2657.

    https://doi.org/10.1182/bloodadvances.2018023515

    • PubMed
    • Google Scholar
31. 1. Giorgi F
    2. Coglitore D
    3. Curran JM
    4. Gilliland D
    5. Macko P
    6. Whelan M
    7. Worth A
    8. Patterson EA

    (2019) The influence of inter-particle forces on diffusion at the nanoscale

    Scientific Reports 9:12689.

    https://doi.org/10.1038/s41598-019-48754-5

    • PubMed
    • Google Scholar
32. 1. Gottlieb PA
    2. Bae C
    3. Sachs F

    (2012) Gating the mechanical channel Piezo1: a comparison between whole-cell and patch recording

    Channels 6:282–289.

    https://doi.org/10.4161/chan.21064

    • PubMed
    • Google Scholar
33. 1. Gratzer WB

    (1981) The red cell membrane and its cytoskeleton

    The Biochemical Journal 198:1–8.

    https://doi.org/10.1042/bj1980001

    • PubMed
    • Google Scholar
34. 1. Guo YR
    2. MacKinnon R

    (2017) Structure-Based membrane dome mechanism for Piezo mechanosensitivity

    eLife 6:e33660.

    https://doi.org/10.7554/eLife.33660

    • PubMed
    • Google Scholar
35. 1. Gustafsson MGL

    (2000) Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy

    Journal of Microscopy 198:82–87.

    https://doi.org/10.1046/j.1365-2818.2000.00710.x

    • PubMed
    • Google Scholar
36. 1. Haggie PM
    2. Kim JK
    3. Lukacs GL
    4. Verkman AS

    (2006) Tracking of quantum dot-labeled CFTR shows near immobilization by C-terminal PDZ interactions

    Molecular Biology of the Cell 17:4937–4945.

    https://doi.org/10.1091/mbc.e06-08-0670

    • PubMed
    • Google Scholar
37. 1. Harraz OF
    2. Klug NR
    3. Senatore AJ
    4. Hill-Eubanks DC
    5. Nelson MT

    (2022) Piezo1 is a mechanosensor channel in central nervous system capillaries

    Circulation Research 130:1531–1546.

    https://doi.org/10.1161/CIRCRESAHA.122.320827

    • PubMed
    • Google Scholar
38. 1. Haselwandter CA
    2. MacKinnon R

    (2018) Piezo’s membrane footprint and its contribution to mechanosensitivity

    eLife 7:e41968.

    https://doi.org/10.7554/eLife.41968

    • PubMed
    • Google Scholar
39. 1. Haselwandter CA
    2. Guo YR
    3. Fu Z
    4. MacKinnon R

    (2022a) Elastic properties and shape of the Piezo dome underlying its mechanosensory function

    PNAS 119:e2208034119.

    https://doi.org/10.1073/pnas.2208034119

    • PubMed
    • Google Scholar
40. 1. Haselwandter CA
    2. Guo YR
    3. Fu Z
    4. MacKinnon R

    (2022b) Quantitative prediction and measurement of piezo’s membrane footprint

    PNAS 119:e2208027119.

    https://doi.org/10.1073/pnas.2208027119

    • PubMed
    • Google Scholar
41. 1. Heine J
    2. Reuss M
    3. Harke B
    4. D’Este E
    5. Sahl SJ
    6. Hell SW

    (2017) Adaptive-illumination STED nanoscopy

    PNAS 114:9797–9802.

    https://doi.org/10.1073/pnas.1708304114

    • PubMed
    • Google Scholar
42. 1. Heintzmann R
    2. Huser T

    (2017) Super-Resolution structured illumination microscopy

    Chemical Reviews 117:13890–13908.

    https://doi.org/10.1021/acs.chemrev.7b00218

    • PubMed
    • Google Scholar
43. 1. Helfrich W

    (1973) Elastic properties of lipid bilayers: theory and possible experiments

    Zeitschrift für Naturforschung C 28:693–703.

    https://doi.org/10.1515/znc-1973-11-1209

    • Google Scholar
44. 1. Hell SW
    2. Wichmann J

    (1994) Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy

    Opt Lett 19:780–782.

    https://doi.org/10.1364/ol.19.000780

    • PubMed
    • Google Scholar
45. 1. Kodippili GC
    2. Spector J
    3. Sullivan C
    4. Kuypers FA
    5. Labotka R
    6. Gallagher PG
    7. Ritchie K
    8. Low PS

    (2009) Imaging of the diffusion of single band 3 molecules on normal and mutant erythrocytes

    Blood 113:6237–6245.

    https://doi.org/10.1182/blood-2009-02-205450

    • PubMed
    • Google Scholar
46. 1. Larsen FL
    2. Katz S
    3. Roufogalis BD
    4. Brooks DE

    (1981) Physiological shear stresses enhance the Ca2+ permeability of human erythrocytes

    Nature 294:667–668.

    https://doi.org/10.1038/294667a0

    • PubMed
    • Google Scholar
47. 1. Lee C-H
    2. MacKinnon R

    (2018) Activation mechanism of a human SK-calmodulin channel complex elucidated by cryo-EM structures

    Science 360:508–513.

    https://doi.org/10.1126/science.aas9466

    • PubMed
    • Google Scholar
48. 1. Lew VL
    2. Muallem S
    3. Seymour CA

    (1982) Properties of the Ca2+-activated K+ channel in one-step inside-out vesicles from human red cell membranes

    Nature 296:742–744.

    https://doi.org/10.1038/296742a0

    • PubMed
    • Google Scholar
49. 1. Lewis SM
    2. Osborn JS
    3. Stuart PR

    (1968) Demonstration of an internal structure within the red blood cell by ion etching and scanning electronmicroscopy

    Nature 220:614–616.

    https://doi.org/10.1038/220614a0

    • PubMed
    • Google Scholar
50. 1. Lewis AH
    2. Grandl J

    (2021) Piezo1 ion channels inherently function as independent mechanotransducers

    eLife 10:e70988.

    https://doi.org/10.7554/eLife.70988

    • PubMed
    • Google Scholar
51. 1. Li A
    2. Seipelt H
    3. Müller C
    4. Shi Y
    5. Artmann GM

    (1999) Effects of salicylic acid derivatives on red blood cell membranes

    Pharmacology & Toxicology 85:206–211.

    https://doi.org/10.1111/j.1600-0773.1999.tb02010.x

    • PubMed
    • Google Scholar
52. 1. Li J
    2. Hou B
    3. Tumova S
    4. Muraki K
    5. Bruns A
    6. Ludlow MJ
    7. Sedo A
    8. Hyman AJ
    9. McKeown L
    10. Young RS
    11. Yuldasheva NY
    12. Majeed Y
    13. Wilson LA
    14. Rode B
    15. Bailey MA
    16. Kim HR
    17. Fu Z
    18. Carter DA
    19. Bilton J
    20. Imrie H
    21. Ajuh P
    22. Dear TN
    23. Cubbon RM
    24. Kearney MT
    25. Prasad RK
    26. Evans PC
    27. Ainscough JF
    28. Beech DJ

    (2014) Piezo1 integration of vascular architecture with physiological force

    Nature 515:279–282.

    https://doi.org/10.1038/nature13701

    • PubMed
    • Google Scholar
53. 1. Lin Y-C
    2. Guo YR
    3. Miyagi A
    4. Levring J
    5. MacKinnon R
    6. Scheuring S

    (2019) Force-Induced conformational changes in Piezo1

    Nature 573:230–234.

    https://doi.org/10.1038/s41586-019-1499-2

    • PubMed
    • Google Scholar
54. 1. Lister JJ

    (1827) Xxvi. Notice of some miscroscopic observations of the blood and animal tissues

    The Philosophical Magazine 2:130–138.

    https://doi.org/10.1080/14786442708674422

    • Google Scholar
55. 1. Lu C-H
    2. Pedram K
    3. Tsai C-T
    4. Jones T
    5. Li X
    6. Nakamoto ML
    7. Bertozzi CR
    8. Cui B

    (2022) Membrane curvature regulates the spatial distribution of bulky glycoproteins

    Nature Communications 13:3093.

    https://doi.org/10.1038/s41467-022-30610-2

    • PubMed
    • Google Scholar
56. 1. Lux SE

    (2016) Anatomy of the red cell membrane skeleton: unanswered questions

    Blood 127:187–199.

    https://doi.org/10.1182/blood-2014-12-512772

    • PubMed
    • Google Scholar
57. 1. Marikovsky Y
    2. Danon D

    (1969) Electron microscope analysis of young and old red blood cells stained with colloidal iron for surface charge evaluation

    The Journal of Cell Biology 43:1–7.

    https://doi.org/10.1083/jcb.43.1.1

    • PubMed
    • Google Scholar
58. 1. Martinac B
    2. Adler J
    3. Kung C

    (1990) Mechanosensitive ion channels of E. coli activated by amphipaths

    Nature 348:261–263.

    https://doi.org/10.1038/348261a0

    • PubMed
    • Google Scholar
59. 1. Mohandas N
    2. Evans E

    (1994) Mechanical properties of the red cell membrane in relation to molecular structure and genetic defects

    Annual Review of Biophysics and Biomolecular Structure 23:787–818.

    https://doi.org/10.1146/annurev.bb.23.060194.004035

    • PubMed
    • Google Scholar
60. 1. Nakatani K
    2. Chen C
    3. Koutalos Y

    (2002) Calcium diffusion coefficient in rod photoreceptor outer segments

    Biophysical Journal 82:728–739.

    https://doi.org/10.1016/S0006-3495(02)75435-0

    • PubMed
    • Google Scholar
61. 1. Nowakowski R
    2. Luckham P
    3. Winlove P

    (2001) Imaging erythrocytes under physiological conditions by atomic force microscopy

    Biochimica et Biophysica Acta (BBA) - Biomembranes 1514:170–176.

    https://doi.org/10.1016/S0005-2736(01)00365-0

    • Google Scholar
62. 1. Nunomura W

    (2006) Regulation of protein 4.1R interactions with membrane proteins by ca2+ and calmodulin

    Frontiers in Bioscience 11:1522.

    https://doi.org/10.2741/1901

    • Google Scholar
63. 1. Pan L
    2. Yan R
    3. Li W
    4. Xu K

    (2018) Super-resolution microscopy reveals the native ultrastructure of the erythrocyte cytoskeleton

    Cell Reports 22:1151–1158.

    https://doi.org/10.1016/j.celrep.2017.12.107

    • Google Scholar
64. 1. Pathak M
    2. Nourse JL
    3. Tran T
    4. Hwe J
    5. Arulmoli J
    6. Le DTT
    7. Bernardis E
    8. Flanagan LA
    9. Tombola F

    (2014) Stretch-activated ion channel Piezo1 directs lineage choice in human neural stem cells

    PNAS 111:16148–16153.

    https://doi.org/10.1073/pnas.1409802111

    • Google Scholar
65. 1. Ranade SS
    2. Qiu Z
    3. Woo SH
    4. Hur SS
    5. Murthy SE
    6. Cahalan SM
    7. Xu J

    (2014) Piezo1, a mechanically activated ion channel, is required for vascular development in mice

    PNAS 111:10347–10352.

    https://doi.org/10.1073/pnas.1409233111

    • Google Scholar
66. 1. Ravenhill BJ
    2. Kanjee U
    3. Ahouidi A
    4. Nobre L
    5. Williamson J
    6. Goldberg JM
    7. Antrobus R
    8. Dieye T
    9. Duraisingh MT
    10. Weekes MP

    (2019) Quantitative comparative analysis of human erythrocyte surface proteins between individuals from two genetically distinct populations

    Communications Biology 2:350.

    https://doi.org/10.1038/s42003-019-0596-y

    • PubMed
    • Google Scholar
67. 1. Ridone P
    2. Pandzic E
    3. Vassalli M
    4. Cox CD
    5. Macmillan A
    6. Gottlieb PA
    7. Martinac B

    (2020) Disruption of membrane cholesterol organization impairs the activity of Piezo1 channel clusters

    The Journal of General Physiology 152:e201912515.

    https://doi.org/10.1085/jgp.201912515

    • PubMed
    • Google Scholar
68. 1. Saotome K
    2. Murthy SE
    3. Kefauver JM
    4. Whitwam T
    5. Patapoutian A
    6. Ward AB

    (2018) Structure of the mechanically activated ion channel Piezo1

    Nature 554:481–486.

    https://doi.org/10.1038/nature25453

    • PubMed
    • Google Scholar
69. 1. Schloetel J-G
    2. Heine J
    3. Cowman AF
    4. Pasternak M

    (2019) Guided STED nanoscopy enables super-resolution imaging of blood stage malaria parasites

    Scientific Reports 9:4674.

    https://doi.org/10.1038/s41598-019-40718-z

    • PubMed
    • Google Scholar
70. 1. Schoonderwoert V
    2. Dijkstra R
    3. Luckinavicius G
    4. Kobler O
    5. van der Voort H

    (2013) Huygens STED deconvolution increases signal-to-noise and image resolution towards 22 nm

    Microscopy Today 21:38–44.

    https://doi.org/10.1017/S1551929513001089

    • Google Scholar
71. 1. Sheetz MP

    (1983)

    Membrane skeletal dynamics: role in modulation of red cell deformability, mobility of transmembrane proteins, and shape

    Seminars in Hematology 20:175–188.

    • PubMed
    • Google Scholar
72. 1. Simunovic M
    2. Voth GA
    3. Callan-Jones A
    4. Bassereau P

    (2015) When physics takes over: BAR proteins and membrane curvature

    Trends in Cell Biology 25:780–792.

    https://doi.org/10.1016/j.tcb.2015.09.005

    • PubMed
    • Google Scholar
73. 1. Staudt T
    2. Engler A
    3. Rittweger E
    4. Harke B
    5. Engelhardt J
    6. Hell SW

    (2011) Far-Field optical nanoscopy with reduced number of state transition cycles

    Optics Express 19:5644–5657.

    https://doi.org/10.1364/OE.19.005644

    • PubMed
    • Google Scholar
74. 1. Sun W
    2. Chi S
    3. Li Y
    4. Ling S
    5. Tan Y
    6. Xu Y
    7. Jiang F
    8. Li J
    9. Liu C
    10. Zhong G
    11. Cao D
    12. Jin X
    13. Zhao D
    14. Gao X
    15. Liu Z
    16. Xiao B
    17. Li Y

    (2019) The mechanosensitive Piezo1 channel is required for bone formation

    eLife 8:e47454.

    https://doi.org/10.7554/eLife.47454

    • PubMed
    • Google Scholar
75. 1. Svetina S
    2. Švelc Kebe T
    3. Božič B

    (2019) A model of Piezo1-based regulation of red blood cell volume

    Biophysical Journal 116:151–164.

    https://doi.org/10.1016/j.bpj.2018.11.3130

    • PubMed
    • Google Scholar
76. 1. Swihart AH
    2. Mikrut JM
    3. Ketterson JB
    4. Macdonald RC

    (2001) Atomic force microscopy of the erythrocyte membrane skeleton

    Journal of Microscopy 204:212–225.

    https://doi.org/10.1046/j.1365-2818.2001.00960.x

    • PubMed
    • Google Scholar
77. 1. Syeda R
    2. Florendo MN
    3. Cox CD
    4. Kefauver JM
    5. Santos JS
    6. Martinac B
    7. Patapoutian A

    (2016) Piezo1 channels are inherently mechanosensitive

    Cell Reports 17:1739–1746.

    https://doi.org/10.1016/j.celrep.2016.10.033

    • PubMed
    • Google Scholar
78. Software

    1. Tameling C
    2. Naas J

    (2021) Ctameling/OTC: optimal transport colocalization, version v1.0

    Zenodo.

    https://doi.org/10.5281/zenodo.4553632
79. 1. Tameling C
    2. Stoldt S
    3. Stephan T
    4. Naas J
    5. Jakobs S
    6. Munk A

    (2021) Colocalization for super-resolution microscopy via optimal transport

    Nature Computational Science 1:199–211.

    https://doi.org/10.1038/s43588-021-00050-x

    • PubMed
    • Google Scholar
80. 1. Tarantino N
    2. Tinevez J-Y
    3. Crowell EF
    4. Boisson B
    5. Henriques R
    6. Mhlanga M
    7. Agou F
    8. Israël A
    9. Laplantine E

    (2014) Tnf and IL-1 exhibit distinct ubiquitin requirements for inducing NEMO-IKK supramolecular structures

    The Journal of Cell Biology 204:231–245.

    https://doi.org/10.1083/jcb.201307172

    • PubMed
    • Google Scholar
81. 1. Tinevez J-Y
    2. Perry N
    3. Schindelin J
    4. Hoopes GM
    5. Reynolds GD
    6. Laplantine E
    7. Bednarek SY
    8. Shorte SL
    9. Eliceiri KW

    (2017) TrackMate: an open and extensible platform for single-particle tracking

    Methods 115:80–90.

    https://doi.org/10.1016/j.ymeth.2016.09.016

    • PubMed
    • Google Scholar
82. 1. Tomishige M
    2. Sako Y
    3. Kusumi A

    (1998) Regulation mechanism of the lateral diffusion of band 3 in erythrocyte membranes by the membrane skeleton

    The Journal of Cell Biology 142:989–1000.

    https://doi.org/10.1083/jcb.142.4.989

    • PubMed
    • Google Scholar
83. 1. Tsai F-C
    2. Simunovic M
    3. Sorre B
    4. Bertin A
    5. Manzi J
    6. Callan-Jones A
    7. Bassereau P

    (2021) Comparing physical mechanisms for membrane curvature-driven sorting of BAR-domain proteins

    Soft Matter 17:4254–4265.

    https://doi.org/10.1039/d0sm01573c

    • PubMed
    • Google Scholar
84. 1. Tsuji A
    2. Kawasaki K
    3. Ohnishi S
    4. Merkle H
    5. Kusumi A

    (1988) Regulation of band 3 mobilities in erythrocyte ghost membranes by protein association and cytoskeletal meshwork

    Biochemistry 27:7447–7452.

    https://doi.org/10.1021/bi00419a041

    • PubMed
    • Google Scholar
85. 1. Vallese F
    2. Kim K
    3. Yen LY
    4. Johnston JD
    5. Noble AJ
    6. Calì T
    7. Clarke OB

    (2022) Architecture of the human erythrocyte ankyrin-1 complex

    Nature Structural & Molecular Biology 29:706–718.

    https://doi.org/10.1038/s41594-022-00792-w

    • PubMed
    • Google Scholar
86. 1. Wang J
    2. Jiang J
    3. Yang X
    4. Zhou G
    5. Wang L
    6. Xiao B

    (2022) Tethering piezo channels to the actin cytoskeleton for mechanogating via the cadherin-β-catenin mechanotransduction complex

    Cell Reports 38:110342.

    https://doi.org/10.1016/j.celrep.2022.110342

    • PubMed
    • Google Scholar
87. 1. Wu Y
    2. Ali MRK
    3. Chen K
    4. Fang N
    5. El-Sayed MA

    (2019) Gold nanoparticles in biological optical imaging

    Nano Today 24:120–140.

    https://doi.org/10.1016/j.nantod.2018.12.006

    • Google Scholar
88. 1. Xia X
    2. Liu S
    3. Zhou ZH

    (2022) Structure, dynamics and assembly of the ankyrin complex on human red blood cell membrane

    Nature Structural & Molecular Biology 29:698–705.

    https://doi.org/10.1038/s41594-022-00779-7

    • PubMed
    • Google Scholar
89. Preprint

    1. Yang S
    2. Miao X
    3. Arnold S
    4. Li B
    5. Ly AT
    6. Wang H
    7. Wang M
    8. Guo X
    9. Pathak MM
    10. Zhao W
    11. Cox CD
    12. Shi Z

    (2022) Membrane Curvature Governs the Distribution of Piezo1 in Live Cells

    bioRxiv.

    https://doi.org/10.1101/2022.06.22.497259

    • Google Scholar
90. 1. Yu Z-Y
    2. Gong H
    3. Kesteven S
    4. Guo Y
    5. Wu J
    6. Li JV
    7. Cheng D
    8. Zhou Z
    9. Iismaa SE
    10. Kaidonis X
    11. Graham RM
    12. Cox CD
    13. Feneley MP
    14. Martinac B

    (2022) Piezo1 is the cardiac mechanosensor that initiates the cardiomyocyte hypertrophic response to pressure overload in adult mice

    Nature Cardiovascular Research 1:577–591.

    https://doi.org/10.1038/s44161-022-00082-0

    • Google Scholar
91. 1. Zarychanski R
    2. Schulz VP
    3. Houston BL
    4. Maksimova Y
    5. Houston DS
    6. Smith B
    7. Rinehart J
    8. Gallagher PG

    (2012) Mutations in the mechanotransduction protein Piezo1 are associated with hereditary xerocytosis

    Blood 120:1908–1915.

    https://doi.org/10.1182/blood-2012-04-422253

    • PubMed
    • Google Scholar
92. 1. Zeng W-Z
    2. Marshall KL
    3. Min S
    4. Daou I
    5. Chapleau MW
    6. Abboud FM
    7. Liberles SD
    8. Patapoutian A

    (2018) PIEZOs mediate neuronal sensing of blood pressure and the baroreceptor reflex

    Science 362:464–467.

    https://doi.org/10.1126/science.aau6324

    • PubMed
    • Google Scholar
93. 1. Zhang W
    2. Cheng LE
    3. Kittelmann M
    4. Li J
    5. Petkovic M
    6. Cheng T
    7. Jin P
    8. Guo Z
    9. Göpfert MC
    10. Jan LY
    11. Jan YN

    (2015) Ankyrin repeats convey force to gate the NOMPC mechanotransduction channel

    Cell 162:1391–1403.

    https://doi.org/10.1016/j.cell.2015.08.024

    • PubMed
    • Google Scholar

Author details

1. George Vaisey

   Laboratory of Molecular Neurobiology and Biophysics, Howard Hughes Medical Institute, The Rockefeller University, New York, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8359-1314

2. Priyam Banerjee

   Bio-Imaging Resource Center, The Rockefeller University, New York, United States

   Contribution

   Resources, Methodology, Writing – review and editing, Design of imaging experiments

   Competing interests

   No competing interests declared

3. Alison J North

   Bio-Imaging Resource Center, The Rockefeller University, New York, United States

   Contribution

   Resources, Methodology, Writing – review and editing, Design of imaging experiments

   Competing interests

   No competing interests declared

4. Christoph A Haselwandter

   Department of Physics and Astronomy and Department of Quantitative and Computational Biology, University of Southern California, Los Angeles, United States

   Contribution

   Formal analysis, Writing – review and editing, Developed calculations of probability distribution along RBC membrane

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5012-5640

5. Roderick MacKinnon

   Laboratory of Molecular Neurobiology and Biophysics, Howard Hughes Medical Institute, The Rockefeller University, New York, United States

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Writing – original draft, Writing – review and editing, Developed calculations of probability distribution along RBC membrane

   For correspondence

   mackinn@rockefeller.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7605-4679

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