Difference between revisions of "Team:Slovenia/Basic Part"

 
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<b>Project</b>
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<b>All parts</b>
 
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<b>New basic part</b>
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<div class="main ui citing justified container"><h1 class = "ui left dividing header"><span class="section">&nbsp;</span>New Basic part</h1>
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<div class="main ui citing justified container"><h1 class = "ui left dividing header"><span class="section colorize">&nbsp;</span>Candidate for best new basic part</h1>
 
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<p>The BioBrick nominated for best part is MscS (<a href="http://parts.igem.org/Part:BBa_K1965000">BBa_K1965000</a>) from our <a href="https://2016.igem.org/Team:Slovenia/Part_Collection#Mechanosensing">Mechanosensing Collection</a>. This part
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<p>The BioBrick nominated for best part is MscS (<a href="http://parts.igem.org/Part:BBa_K1965000">BBa_K1965000</a>) from  
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contains the coding sequence of the bacterial mechanosensitive channel of small conductance (MscS). It has been previously described as an important receptor involved
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our <a href="https://2016.igem.org/Team:Slovenia/Part_Collection#Mechanosensing">Mechanosensing Collection</a>. It contains the coding sequence for the E.coli small-conductance  
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in bacterial sensing of mechanical stress. <br/>
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mechanosensitive channel, MscS. Its role is to mediate turgor regulation in bacteria and it is activated by changes in the osmotic pressure <x-ref>Perozo2003</x-ref>. It has been  
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We expressed and characterized it in mammalian cells, where it served as the sensor that translates the ultrasound stimulation
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previously shown that MscS forms a homoheptamer. Each subunit is 31kDa in size and contains three transmembrane helices <ref>2</ref> with the N-terminus facing the periplasm and
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into calcium ion influx, thereby playing a central role our design of ultrasound responsive cells as well as cells responsive to the touch (Touchpaint).</p>
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the C-terminus embedded in the cytoplasm <x-ref>Pivetti2003</x-ref>.</p>
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<div style="float:left; width:100%">
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<figure data-ref="2">
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<p>MscS can be described as an important receptor, involved in the response to ultrasound stimulation. We used the MscS channel as a source of Ca<sup>2+</sup> influx
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<img src="https://static.igem.org/mediawiki/2016/e/ec/T--Slovenia--BBa_K1965000.PNG">
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when stimulated with ultrasound <ref>1</ref>. </p>
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<figcaption><b>Localization and expression of mechanosensitive ion channel MscS. </b><br/>
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<div style="margin-left:auto; margin-right:auto; width:50%">
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<p style="text-align:justify">HEK293 cells were transfected with MscS. Expression by Western blot and localization by confocal microscopy were analyzed using anti-HA antibodies.
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<figure data-ref="1">
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(A) Scheme of bacterial ion channel MscS
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<img onclick="resize(this)" src="https://static.igem.org/mediawiki/2016/f/f2/T--Slovenia--S.3.1.1.png">
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(B) Ion channel MscS is localized on plasma membrane. HEK293 cells were transfected with the MscS:HA plasmid. 24 h after transfection cells were fixed with paraformaldehyde, permeabilized and stained with anti-HA antibodies. Localization was confirmed with confocal microscopy.
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<figcaption><b> Schematic presentation of MscS channel function in our system.</b><br/><p style="text-align:justify"> Cells in resting stage with closed MscS channels in the plasma membrane
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(D) Ion channels expressed in HEK293 cells. HEK293T cells were transfected with plasmids, encoding gas vesicle-forming proteins GvpA and GvpC. 48 h after transfection cells were collected, lysed and total protein concentration was measured. 50ng of proteins were loaded on SDS page gels. After separation by size, proteins were transferred to nitrocellulose membrane by western blot. Membranes were then immunoblotted with anti-FLAG or anti-AU1 antibodies.  
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(left). Upon ultrasound stimulation MscS channels open, leading to Ca <sup>2+</sup> influx (right). </p>
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</figcaption>
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</div>
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<div style="clear:both">
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<h5>Characterization</h5>
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<p>Expression and subcellular localization of MscS channels in HEK293T cells was inspected. HEK293T cells were transfected with plasmids encoding HA-tagged MscS channel
 +
and protein expression was confirmed by western blot analysis, while protein localization was investigated by confocal microscopy.</p>
 +
 
 +
<div style="float:right; width:60%">
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<figure data-ref="2">
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<img src="https://static.igem.org/mediawiki/2016/e/ec/T--Slovenia--BBa_K1965000.PNG">
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<figcaption><b>Localization and expression of mechanosensitive ion channel MscS. </b><br/>
 +
<p style="text-align:justify">HEK293 cells were transfected with MscS. Expression by Western blot and localization by confocal microscopy were analyzed using anti-HA antibodies.
 +
(A) Scheme of bacterial ion channel MscS
 +
(B) Ion channel MscS is localized on plasma membrane. HEK293 cells were transfected with the MscS:HA plasmid. 24 h after transfection cells were fixed with paraformaldehyde,
 +
permeabilized and stained with anti-HA antibodies. Localization was confirmed with confocal microscopy.
 +
(D) Ion channels expressed in HEK293 cells. HEK293T cells were transfected with plasmids, encoding gas vesicle-forming proteins GvpA and GvpC. 48 h after  
 +
transfection cells were collected, lysed and total protein concentration was measured. 50ng of proteins were loaded on SDS page gels. After separation by
 +
size, proteins were transferred to nitrocellulose membrane by western blot. Membranes were then immunoblotted with anti-FLAG or anti-AU1 antibodies. </p>
 +
 
 +
</figcaption>
 +
</figure>
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</div>
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 +
 
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<p>After confirming MscS expression in HEK293 cells, we stimulated the transfected cells with ultrasound to verify and characterize channel activity.
 +
Our experimental setup included an in-house built hardware MODUSON connected to unfocused transducer Olympus V318-SU. To monitor cell response in situ and
 +
in real time we used standard ratiometric fluorescent calcium indicators Fura Red, AM and Fluo-4, AM, which can be easily detected with confocal microscopy.
 +
When activated, mechanosensitive channels open, leading to calcium influx, which in turn binds the fluorescent calcium indicators. The indicator conformation
 +
changes upon calcium binding, resulting in an increase or a decrease of fluorescence.</p>
 +
 
 +
<p>When cells transfected with a mock plasmid were stimulated with ultrasound, we did not observe calcium influx. On the contrary, when cells
 +
transfected with the plasmid, encoding the MscS channel were stimulated with ultrasound, we detected a significant increase in calcium influx <ref>3</ref>. </p>
 +
 
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<div style="float:left; width:100%">
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<figure data-ref="3">
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<img src="https://static.igem.org/mediawiki/2016/8/8a/T--Slovenia--3.2.4.png">
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<figcaption><b> MscS channel improves sensitivity of cells for ultrasound. </b><br/>
 +
<p style="text-align:justify">(A) Schematic presentation of the stimulation sequence and (B) signal parameters used for stimulation.
 +
(C, D )Cells expressing MscS showed increased sensitivity to ultrasound stimulation in comparison to the cells transfected with a mock plasmid. HEK293 cells
 +
either expressing MscS or transfected with a mock plasmid were stimulated with ultrasound for 10 s and calcium influx was recorded in real time (D) using confocal
 +
microscopy. Changes in fluorescence intensity of calcium indicators Fluo-4, AM (green line) and Fura Red, AM (red line) are shown. The blue line represents the
 +
ratio of Fluo-4 and Fura Red intensities, indicating the increase in intracellular free calcium ions after ultrasound stimulation. </p>
 +
 
 +
</figcaption>
 +
</figure>
 +
</div>
 +
 
 +
 
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<p style="clear:both">In an attempt to improve calcium influx, we co-transfected HEK293 cells with the MscS channel and gas vesicle-forming proteins GvpC (BBa_K1965003)
 +
and GvpA (BBa_K1965004). The voltage of ultrasound stimulation was decreased to 450 Vpp as higher voltage also causes calcium influx in cells expressing
 +
only gas vesicle-forming proteins <a href="https://2016.igem.org/Team:Slovenia/Mechanosensing/Gas_vesicles(">Read more</a>)
 +
. By decreasing the voltage of ultrasound stimulation we successfully showed that only cells expressing both the MscS channel and the gas vesicle-forming
 +
proteins were activated as a result of ultrasound stimulation <ref>4</ref>. </p>
 +
 
 +
<div style="float:left; width:100%">
 +
<figure data-ref="4">
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<img src="https://static.igem.org/mediawiki/2016/9/99/T--Slovenia--3.3.7.png">
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<figcaption><b> 4 MscS with Gvps improve sensitivity of cells to ultrasound even at lower voltages.</b><br/>
 +
<p style="text-align:justify">(A) Schematic presentation of the stimulation sequence and (B) signal parameters used for stimulation.
 +
(C,D) Co-expression of mechanosensitive channels and gas vesicle-forming proteins increased sensitivity to ultrasound stimulation in comparison to the cells
 +
without exogenous mechanosensitive channels.
 +
HEK293 cells expressing gas vesicle-forming proteins GvpA and GvpC with or without MscS were stimulated with ultrasound for 10 s and calcium influx was
 +
recorded in real time (D) using confocal microscopy. Changes in fluorescence intensity of calcium indicators Fluo-4, AM (green line) and Fura Red, AM (red line)
 +
are shown. The blue line represents the ratio of Fluo-4 and Fura Red intensities, indicating the increase in intracellular free calcium ions after ultrasound
 +
stimulation. </p>
 +
 
 +
</figcaption>
 +
</figure>
 +
</div>
 +
 
 +
 
 +
<p>To verify that calcium influx was indeed a result of mechanosensitive channel activity , we used gadolinium (Gd3+), an inhibitor of ion channels, which
 +
is a trivalent ion of the lanthanide series. Due to its high charge density and similar ionic radius to Ca2+ <x-ref>Bourne1982</x-ref> it blocks the pore of
 +
the channel and therefore acts as an inhibitor of calcium ion channels. As shown in <ref>5</ref>, the addition of the inhibitor prevented calcium influx after
 +
ultrasound stimulation, confirming that cell response was indeed dependent on the activity of mechanosensitive channels.</p>
  
 +
<div style="float:left;width:100%">
 +
<figure data-ref="5">
 +
<img src="https://static.igem.org/mediawiki/2016/4/4c/T--Slovenia--3.3.8.png">
 +
<figcaption><b> Calcium influx after ultrasound stimulation is mediated by activation of mechanosensitive channels.</b><br/>
 +
<p style="text-align:justify">(A) Schematic presentation of the stimulation sequence and (B) signal parameters used for stimulation.
 +
(C,D) Gadolinium inhibits activation of mechanosensitive ion channels after ultrasound treatment.
 +
HEK293 cells expressing gas vesicle-forming proteins GvpA and GvpC with or without MscS were untreated (grey line) or treated with gadolinium (red line)
 +
and stimulated with ultrasound for 10 s.  Calcium influx was recorded in real time (D) using confocal microscopy. Changes in fluorescence intensity of calcium
 +
indicators Fluo-4, AM (green line) and Fura Red, AM (red line) are shown. The blue line represents the ratio of Fluo-4 and Fura Red intensities, indicating the
 +
increase in intracellular free calcium ions after ultrasound stimulation.
  
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                                        <figcaption><b>INSERT!!!</b><br/></figcaption>
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<p style="clear:both">The construct was further caracterized under <a href="https://2016.igem.org/Team:Slovenia/Mechanosensing/Mechanosensitive_channels">Mechanosensitive
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                                    <figure data-ref="6">
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channels</a>, <a href="https://2016.igem.org/Team:Slovenia/Mechanosensing/Gas_vesicles#vesicles">Gas vesicles</a> and <a href="https://2016.igem.org/Team:Slovenia/Proof">Touch
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                                        <img class="ui big centered image"
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painitng</a>.</p>
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                                                <!-- experiment: 20161002 2 MscS + Gvp 200V 90s -->
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                                                <!-- experiment: 20161002 2 MscS + Gvp 200V 90s -->
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                                        <figcaption><b> MscS channel improves sensitivity of cells for
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                                            ultrasound.</b><br/>
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                                            <p style="text-align:justify">(A) Schematic representation of a stimulation sequence and (B) signal
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                                            parameters used for stimulation.
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                                            (C) and (D)Cells expressing MscS showed increased sensitivity to ultrasound
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                                            stimulation in comparison to the cells without exogenous mechanosensitive
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                                            channel. HEK293 cells expressing MscS channels or control cells transfected
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                                            with vector were stimulated with ultrasound for 10 s and calcium influx
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                                            was recorded in real time (D) using a confocal microscope. For comparison
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                                            cells without ectopic MscS were used. Fluo-4 (D, green line) and Fura Red
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                                            dyes (D, red line) were used for ratiometric calcium imaging. (D) Ratio
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                                            (blue line) was calculated from fluorescence intensities of Fura Red and
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                                            Fluo-4
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                                            using <a href="https://2016.igem.org/Team:Slovenia/Software">CaPTURE</a>.
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Latest revision as of 13:25, 19 October 2016

New Basic part

All parts New basic part Part Collections

 Candidate for best new basic part

The BioBrick nominated for best part is MscS (BBa_K1965000) from our Mechanosensing Collection. It contains the coding sequence for the E.coli small-conductance mechanosensitive channel, MscS. Its role is to mediate turgor regulation in bacteria and it is activated by changes in the osmotic pressure Perozo2003. It has been previously shown that MscS forms a homoheptamer. Each subunit is 31kDa in size and contains three transmembrane helices 2 with the N-terminus facing the periplasm and the C-terminus embedded in the cytoplasm Pivetti2003.

MscS can be described as an important receptor, involved in the response to ultrasound stimulation. We used the MscS channel as a source of Ca2+ influx when stimulated with ultrasound 1.

Schematic presentation of MscS channel function in our system.

Cells in resting stage with closed MscS channels in the plasma membrane (left). Upon ultrasound stimulation MscS channels open, leading to Ca 2+ influx (right).

Characterization

Expression and subcellular localization of MscS channels in HEK293T cells was inspected. HEK293T cells were transfected with plasmids encoding HA-tagged MscS channel and protein expression was confirmed by western blot analysis, while protein localization was investigated by confocal microscopy.

Localization and expression of mechanosensitive ion channel MscS.

HEK293 cells were transfected with MscS. Expression by Western blot and localization by confocal microscopy were analyzed using anti-HA antibodies. (A) Scheme of bacterial ion channel MscS (B) Ion channel MscS is localized on plasma membrane. HEK293 cells were transfected with the MscS:HA plasmid. 24 h after transfection cells were fixed with paraformaldehyde, permeabilized and stained with anti-HA antibodies. Localization was confirmed with confocal microscopy. (D) Ion channels expressed in HEK293 cells. HEK293T cells were transfected with plasmids, encoding gas vesicle-forming proteins GvpA and GvpC. 48 h after transfection cells were collected, lysed and total protein concentration was measured. 50ng of proteins were loaded on SDS page gels. After separation by size, proteins were transferred to nitrocellulose membrane by western blot. Membranes were then immunoblotted with anti-FLAG or anti-AU1 antibodies.

After confirming MscS expression in HEK293 cells, we stimulated the transfected cells with ultrasound to verify and characterize channel activity. Our experimental setup included an in-house built hardware MODUSON connected to unfocused transducer Olympus V318-SU. To monitor cell response in situ and in real time we used standard ratiometric fluorescent calcium indicators Fura Red, AM and Fluo-4, AM, which can be easily detected with confocal microscopy. When activated, mechanosensitive channels open, leading to calcium influx, which in turn binds the fluorescent calcium indicators. The indicator conformation changes upon calcium binding, resulting in an increase or a decrease of fluorescence.

When cells transfected with a mock plasmid were stimulated with ultrasound, we did not observe calcium influx. On the contrary, when cells transfected with the plasmid, encoding the MscS channel were stimulated with ultrasound, we detected a significant increase in calcium influx 3.

MscS channel improves sensitivity of cells for ultrasound.

(A) Schematic presentation of the stimulation sequence and (B) signal parameters used for stimulation. (C, D )Cells expressing MscS showed increased sensitivity to ultrasound stimulation in comparison to the cells transfected with a mock plasmid. HEK293 cells either expressing MscS or transfected with a mock plasmid were stimulated with ultrasound for 10 s and calcium influx was recorded in real time (D) using confocal microscopy. Changes in fluorescence intensity of calcium indicators Fluo-4, AM (green line) and Fura Red, AM (red line) are shown. The blue line represents the ratio of Fluo-4 and Fura Red intensities, indicating the increase in intracellular free calcium ions after ultrasound stimulation.

In an attempt to improve calcium influx, we co-transfected HEK293 cells with the MscS channel and gas vesicle-forming proteins GvpC (BBa_K1965003) and GvpA (BBa_K1965004). The voltage of ultrasound stimulation was decreased to 450 Vpp as higher voltage also causes calcium influx in cells expressing only gas vesicle-forming proteins Read more) . By decreasing the voltage of ultrasound stimulation we successfully showed that only cells expressing both the MscS channel and the gas vesicle-forming proteins were activated as a result of ultrasound stimulation 4.

4 MscS with Gvps improve sensitivity of cells to ultrasound even at lower voltages.

(A) Schematic presentation of the stimulation sequence and (B) signal parameters used for stimulation. (C,D) Co-expression of mechanosensitive channels and gas vesicle-forming proteins increased sensitivity to ultrasound stimulation in comparison to the cells without exogenous mechanosensitive channels. HEK293 cells expressing gas vesicle-forming proteins GvpA and GvpC with or without MscS were stimulated with ultrasound for 10 s and calcium influx was recorded in real time (D) using confocal microscopy. Changes in fluorescence intensity of calcium indicators Fluo-4, AM (green line) and Fura Red, AM (red line) are shown. The blue line represents the ratio of Fluo-4 and Fura Red intensities, indicating the increase in intracellular free calcium ions after ultrasound stimulation.

To verify that calcium influx was indeed a result of mechanosensitive channel activity , we used gadolinium (Gd3+), an inhibitor of ion channels, which is a trivalent ion of the lanthanide series. Due to its high charge density and similar ionic radius to Ca2+ Bourne1982 it blocks the pore of the channel and therefore acts as an inhibitor of calcium ion channels. As shown in 5, the addition of the inhibitor prevented calcium influx after ultrasound stimulation, confirming that cell response was indeed dependent on the activity of mechanosensitive channels.

Calcium influx after ultrasound stimulation is mediated by activation of mechanosensitive channels.

(A) Schematic presentation of the stimulation sequence and (B) signal parameters used for stimulation. (C,D) Gadolinium inhibits activation of mechanosensitive ion channels after ultrasound treatment. HEK293 cells expressing gas vesicle-forming proteins GvpA and GvpC with or without MscS were untreated (grey line) or treated with gadolinium (red line) and stimulated with ultrasound for 10 s. Calcium influx was recorded in real time (D) using confocal microscopy. Changes in fluorescence intensity of calcium indicators Fluo-4, AM (green line) and Fura Red, AM (red line) are shown. The blue line represents the ratio of Fluo-4 and Fura Red intensities, indicating the increase in intracellular free calcium ions after ultrasound stimulation.

The construct was further caracterized under Mechanosensitive channels, Gas vesicles and Touch painitng.