Close

GenEPi: Piezo1-based fluorescent reporter for visualizing mechanical stimuli with high spatiotemporal resolution

Sine Yaganoglu, Nordine Helassa, Benjamin M. Gaub, Maaike Welling, Jian Shi, Daniel J. Müller, Katalin Török, Periklis Pantazis

Preprint posted on 15 July 2019 https://www.biorxiv.org/content/10.1101/702423v1

Article now published in Nature Communications at http://dx.doi.org/10.1038/s41467-023-40134-y

No irrational number π! GenEPi is a genetically-encoded non-invasive reporter to measure cellular mechanosensing.

Selected by Ramona Jühlen

Updated 21 August 2023 with a postLight by Ramona Jühlen

This bioRxiv preprint from 2019 has now (!) been published in Nature Communications with a whole new set of additional data and authors. Ten (!) more authors have been added to the study which underlines how much the original preprint has been extended. These changes were not necessarily a result of the peer review process (a Peer Review File is available here), but were mostly made by the authors before submission to the journal.

The original preprint relied on in vitro data and the most striking update is that the authors also generated a GenEPi zebrafish transgenic line and successfully tested the application of GenEPi in vivo. The in vitro data has also been updated by visualizing Piezo1-dependent activity during autonomous cellular processes in HEK293T, HFF and HeLa cells. Moreover, the initial systematic screen for an optical reporter of Piezo1 activity has been extended by a huge amount of data which clearly shows that GenEPi behaves similar to the wild-type Piezo1 channel.

The experiments in cardiomyocytes were supplemented by the use of norepipherine to show that GenEPi is also able to resolve higher-frequency cardiomyocyte contractions and by the use of nifedipine to confirm the specificity of GenEPi. These data were indeed added during the review process.

Overall, the preprint has been supplemented with a lot of new and intriguing data highlighting the broad range of applications of mechanosensing using GenEPi. No wonder that the authors chose an updated title which infers all this: Highly specific and non-invasive imaging of Piezo1-dependent activity across scales using GenEPi.

Background

Cellular mechanosensing, defined as the cell’s ability to respond to mechanical stimuli like tension or compression, is essential for a variety of cellular developmental and physiological processes, like embryogenesis and wound-healing. Mechanosensing has been an active field of study which employs several methodological tools. Some of these tools, however, need the tissue to be dissociated (e.g. atomic force microscopy (AFM)) while others can damage the tissue (e.g. optical or magnetic tweezers). Recently, improvements have been made by using genetically-encoded tools and Förster resonance transfer-based fluorescent tension sensors, which measure mechanical forces across distinct cytoskeletal proteins (e.g. vinculin) (preLight written by Amanda Haage: https://prelights.biologists.com/highlights/tunable-molecular-tension-sensors-reveal-extension-based-control-vinculin-loading/). However, their use is restricted by their specificity and force sensitivity to a distinct biological context and force range.

Thus, the authors set out to develop a non-invasive, genetically-encoded mechanosensor that could be used across a wide range of biological questions. For this purpose they generated a reporter for Piezo1 activity. Piezo (borrowed from Greek: to squeeze) proteins are capable of responding to mechanical stimuli and are stretch-gated ion channels. Piezo1 contributes to mechanosensing in many different organs (e.g. lung, bladder or skin). The C-terminus of Piezo1 resides in the cytoplasm and contains the ion channel which has a preference for divalent cations, like Ca2+. Mechanical stimuli induce Piezo1-channel opening and an influx of Ca2+. By targeting a genetically-encoded Ca2+-indicator to Piezo1, the authors developed an optical tool to measure Piezo1-mechanosensing activity.

Key findings

First, the authors reasoned that since Ca2+ is a crucial second messenger in several cellular processes, they would need a genetically-encoded Ca2+-indicator with low affinity plus a wide dynamic range. They decided to investigate low-affinity GCaMPs fused to the C-terminus of Piezo1. GCaMPs are genetically-encoded Ca2+-indicators consisting of a single circularly permutated EGFP molecule (cpEGFP), N-terminally linked to the M13 fragment of myosin light chain kinase (M13) and C-terminally linked to calmodulin (CaM) (1). M13 is a target sequence of CaM, and thus, by binding of Ca2+ to CaM the fluorescence activity of cpEGFP is enhanced due to the Ca2+–CaM–M13 interaction (Figure 1). By systematically screening different variants of GCaMP-Piezo1 for their response to mechanical stimuli and cytosolic Ca2+-fluctuations dependent and independent of Piezo1, the authors identified one GCaMP-Piezo1 variant (GenEPi) which fulfilled their requirements.

Figure 1. Schematic representation of GenEPi mechanosensing. GCaMP consists of the M13 fragment from myosin light chain kinase (M13), a circularly permutated EGFP (cpEGFP) and calmodulin (CaM) located N- to C-terminus, and GCaMP is fused to the C-terminus of Piezo1. Mechanical stimuli induce Piezo1-channel opening and an influx of Ca2+. Binding of Ca2+ to CaM induces a conformational change in cpEGFP due to Ca2+–CaM–M13 interaction, and thereby enhances fluorescent intensity (modified from preprint).

They showed that GenEPi did not affect cell viability in HEK 293T and cellular localisation resembled wild-type Piezo1. GenEPi retained low affinity for Ca2+: the signal induced by ionophore ionomycin, raising the intracellular level of Ca2+, was comparable to that of the control fusion protein Piezo1-eGFP. The activity of GenEPi was analysed using Yoda1, a Piezo1-specific antagonist, which significantly increased the signal of GenEPi. However, GenEPi did not respond to physiological increases in cytosolic Ca2+-signalling.

Next, the authors tested the response of GenEPi to mechanical stimuli other than fluid shear stress. By using an AFM-based approach they applied precisely-timed compressive forces to HEK 293T cells expressing GenEPi. GenEPi responded to short compressive forces (250 ms) with fast kinetics, whereby the mechanical sensitivity of Piezo1 in the GenEPi fusion protein was not compromised. Additionally, electrochemical response, ion selectivity and channel kinetics of Piezo1 were not altered.

Finally, the authors generated doxycycline-inducable GenEPi mouse embryonic stem cells (mESCs) and differentiated them to cardiomyocytes to analyse the performance of GenEPi in a 3D and multicellular environment. Beating patches of cardiomyocytes showed noticeable GenEPi activity. When using blebbistatin, a myosin inhibitor, GenEPi signal decreased revealing that the source of GenEPi response was indeed cardiomyocyte contraction.
In summary, GenEPi provides a Piezo1-specific and non-invasive intensiometric tool for studying mechanical stimuli in cells, as well in small microenvironments. GenEPi has a broad range of applicability with high spatiotemporal resolution as compared to other genetically-encoded mechanosensors.

What I like about this work and open questions

The authors provide an GenEPi-inducible mESC cell line which can be differentiated into skeletal myoblasts and further into myotubes. GenEPi-expressing myotubes would be an excellent read-out for the mechanosensing abilities of especially pathological cells and give insights into distinct muscular dystrophies.
Interestingly, Piezo1 activity has been recently linked to myotube fusion by promoting the assembly of RhoA-mediated actomyosin modules at the lateral cortex of myotubes (2). Maybe the authors can comment on how and if GenEPi is applicable to study proper myotube formation.

Additional references

  1. J. Nakai, M. Ohkura, K. Imoto, Nature Biotechnology. 19, 137–141 (2001).
  2. M. Tsuchiya et al., Nature Communications. 9, 1–15 (2018).

Tags: gcamps, mechanosensing, piezo1

Posted on: 7 August 2019 , updated on: 22 August 2023

doi: https://doi.org/10.1242/prelights.12979

Read preprint (No Ratings Yet)

Author's response

Periklis Pantazis shared

Thank you for promoting our work Ramona!

We are truly excited to see the warm welcome our study has received by the community. In that regard, we appreciate highlighting our study.

We are aware that our fluorescent Piezo1 sensor GenEPi – short for genetically encoded Piezo1 indicator… inspired by the traditional herbal liqueur or aperitif popularised in the French and Italian Alpine regions (https://en.m.wikipedia.org/wiki/Génépi) – will allow a plethora of mechanobiological investigations in physiology and homeostasis.

Interestingly, our primary motivation to explore a mechanosensitive force sensor was based on the need to visualise directly the emergence and evolution of mechanical forces such as membrane tension during embryo development, be it asymmetry generation or pattern formation. Driven by this desire, we designed GenEPi to be broadly applicable in various cellular contexts and flexible enough to be used in combination with other fluorescent signals. The resulting sensor is therefore based on an intensiometric readout occupying a single optical channel (green visible spectrum) and can be used with carefully selected promoters for various applications.

Not surprisingly, we anticipate that GenEPi will be a powerful tool beyond our initial research focus. The various requests we have received from groups working in different scientific areas are a testament of GenEPi‘s appeal.

As for your particular question, using GenEPi for myotube fusion is a very attractive research endeavour. GenEPi will allow to assess whether its spatiotemporal activity is correlated to important steps during the fusion process. Importantly, the GenEPi readout will allow to causally link its activity to downstream molecular factors such as cytoskeleton modulators and other process-relevant factors. Please note that we are in the process of generating transgenic lines to understand such mechanical forces also in an in vivo context. Exciting times ahead!

Thank you,

Periklis (Laki) Pantazis

Have your say

Your email address will not be published. Required fields are marked *

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Register here

preLists in the biochemistry category:

Preprint Peer Review – Biochemistry Course at UFRJ, Brazil

Communication of scientific knowledge has changed dramatically in recent decades and the public perception of scientific discoveries depends on the peer review process of articles published in scientific journals. Preprints are key vehicles for the dissemination of scientific discoveries, but they are still not properly recognized by the scientific community since peer review is very limited. On the other hand, peer review is very heterogeneous and a fundamental aspect to improve it is to train young scientists on how to think critically and how to evaluate scientific knowledge in a professional way. Thus, this course aims to: i) train students on how to perform peer review of scientific manuscripts in a professional manner; ii) develop students' critical thinking; iii) contribute to the appreciation of preprints as important vehicles for the dissemination of scientific knowledge without restrictions; iv) contribute to the development of students' curricula, as their opinions will be published and indexed on the preLights platform. The evaluations will be based on qualitative analyses of the oral presentations of preprints in the field of biochemistry deposited in the bioRxiv server, of the critical reports written by the students, as well as of the participation of the students during the preprints discussions.

 



List by Marcus Oliveira

CellBio 2022 – An ASCB/EMBO Meeting

This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.

 



List by Nadja Hümpfer et al.

20th “Genetics Workshops in Hungary”, Szeged (25th, September)

In this annual conference, Hungarian geneticists, biochemists and biotechnologists presented their works. Link: http://group.szbk.u-szeged.hu/minikonf/archive/prg2021.pdf

 



List by Nándor Lipták

Fibroblasts

The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!

 



List by Osvaldo Contreras

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)

 



List by Madhuja Samaddar et al.

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019

 



List by Dey Lab

Cellular metabolism

A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.

 



List by Pablo Ranea Robles

MitoList

This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.

 



List by Sandra Franco Iborra

Also in the cell biology category:

‘In preprints’ from Development 2022-2023

A list of the preprints featured in Development's 'In preprints' articles between 2022-2023

 



List by Alex Eve, Katherine Brown

preLights peer support – preprints of interest

This is a preprint repository to organise the preprints and preLights covered through the 'preLights peer support' initiative.

 



List by preLights peer support

The Society for Developmental Biology 82nd Annual Meeting

This preList is made up of the preprints discussed during the Society for Developmental Biology 82nd Annual Meeting that took place in Chicago in July 2023.

 



List by Joyce Yu, Katherine Brown

CSHL 87th Symposium: Stem Cells

Preprints mentioned by speakers at the #CSHLsymp23

 



List by Alex Eve

Journal of Cell Science meeting ‘Imaging Cell Dynamics’

This preList highlights the preprints discussed at the JCS meeting 'Imaging Cell Dynamics'. The meeting was held from 14 - 17 May 2023 in Lisbon, Portugal and was organised by Erika Holzbaur, Jennifer Lippincott-Schwartz, Rob Parton and Michael Way.

 



List by Helen Zenner

9th International Symposium on the Biology of Vertebrate Sex Determination

This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.

 



List by Martin Estermann

Alumni picks – preLights 5th Birthday

This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.

 



List by Sergio Menchero et al.

CellBio 2022 – An ASCB/EMBO Meeting

This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.

 



List by Nadja Hümpfer et al.

Fibroblasts

The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!

 



List by Osvaldo Contreras

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.

 



List by Alex Eve

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020

 



List by Ana Dorrego-Rivas

Planar Cell Polarity – PCP

This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.

 



List by Ana Dorrego-Rivas

BioMalPar XVI: Biology and Pathology of the Malaria Parasite

[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria

 



List by Dey Lab, Samantha Seah

1

Cell Polarity

Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.

 



List by Yamini Ravichandran

TAGC 2020

Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20

 



List by Maiko Kitaoka et al.

3D Gastruloids

A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.

 



List by Paul Gerald L. Sanchez and Stefano Vianello

ECFG15 – Fungal biology

Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome

 



List by Hiral Shah

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)

 



List by Madhuja Samaddar et al.

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019

 



List by Dey Lab

Autophagy

Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.

 



List by Sandra Malmgren Hill

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.

 



List by Rob Hynds

Cellular metabolism

A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.

 



List by Pablo Ranea Robles

BSCB/BSDB Annual Meeting 2019

Preprints presented at the BSCB/BSDB Annual Meeting 2019

 



List by Dey Lab

MitoList

This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.

 



List by Sandra Franco Iborra

Biophysical Society Annual Meeting 2019

Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA

 



List by Joseph Jose Thottacherry

ASCB/EMBO Annual Meeting 2018

This list relates to preprints that were discussed at the recent ASCB conference.

 



List by Dey Lab, Amanda Haage

Also in the genetics category:

9th International Symposium on the Biology of Vertebrate Sex Determination

This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.

 



List by Martin Estermann

Alumni picks – preLights 5th Birthday

This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.

 



List by Sergio Menchero et al.

Semmelweis Symposium 2022: 40th anniversary of international medical education at Semmelweis University

This preList contains preprints discussed during the 'Semmelweis Symposium 2022' (7-9 November), organised around the 40th anniversary of international medical education at Semmelweis University covering a wide range of topics.

 



List by Nándor Lipták

20th “Genetics Workshops in Hungary”, Szeged (25th, September)

In this annual conference, Hungarian geneticists, biochemists and biotechnologists presented their works. Link: http://group.szbk.u-szeged.hu/minikonf/archive/prg2021.pdf

 



List by Nándor Lipták

2nd Conference of the Visegrád Group Society for Developmental Biology

Preprints from the 2nd Conference of the Visegrád Group Society for Developmental Biology (2-5 September, 2021, Szeged, Hungary)

 



List by Nándor Lipták

EMBL Conference: From functional genomics to systems biology

Preprints presented at the virtual EMBL conference "from functional genomics and systems biology", 16-19 November 2020

 



List by Jesus Victorino

TAGC 2020

Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20

 



List by Maiko Kitaoka et al.

ECFG15 – Fungal biology

Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome

 



List by Hiral Shah

Autophagy

Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.

 



List by Sandra Malmgren Hill

Zebrafish immunology

A compilation of cutting-edge research that uses the zebrafish as a model system to elucidate novel immunological mechanisms in health and disease.

 



List by Shikha Nayar

Also in the molecular biology category:

‘In preprints’ from Development 2022-2023

A list of the preprints featured in Development's 'In preprints' articles between 2022-2023

 



List by Alex Eve, Katherine Brown

CSHL 87th Symposium: Stem Cells

Preprints mentioned by speakers at the #CSHLsymp23

 



List by Alex Eve

9th International Symposium on the Biology of Vertebrate Sex Determination

This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.

 



List by Martin Estermann

Alumni picks – preLights 5th Birthday

This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.

 



List by Sergio Menchero et al.

CellBio 2022 – An ASCB/EMBO Meeting

This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.

 



List by Nadja Hümpfer et al.

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.

 



List by Alex Eve

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020

 



List by Ana Dorrego-Rivas

ECFG15 – Fungal biology

Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome

 



List by Hiral Shah

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)

 



List by Madhuja Samaddar et al.

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.

 



List by Rob Hynds

MitoList

This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.

 



List by Sandra Franco Iborra
Close