GenEPi: Piezo1-based fluorescent reporter for visualizing mechanical stimuli with high spatiotemporal resolution
Preprint posted on 15 July 2019 https://www.biorxiv.org/content/10.1101/702423v1
No irrational number π! GenEPi is a genetically-encoded non-invasive reporter to measure cellular mechanosensing.
Selected by Ramona Jühlen
Categories: biochemistry, bioengineering, cell biology, genetics, molecular biology
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
- J. Nakai, M. Ohkura, K. Imoto, Nature Biotechnology. 19, 137–141 (2001).
- M. Tsuchiya et al., Nature Communications. 9, 1–15 (2018).
Posted on: 7 August 2019 , updated on: 4 October 2019
doi: https://doi.org/10.1242/prelights.12979
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