Difference between revisions of "Part:BBa K1965000"

 
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<partinfo>BBa_K1965000 short</partinfo>
 
<partinfo>BBa_K1965000 short</partinfo>
  
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This part contains the coding sequence of bacterial mechanosensitive channel of small conductance, MscS. Its role is to mediate turgor regulation in bacteria and it is activated by changes in the osmotic pressure (Perozo and Rees 2003). It has been previously shown that MscS is a homoheptamer, each subunit is 31kDa in size and contains three transmembrane helices (Figure 3.3.2 A upper) with the N-terminal facing the periplasm and the C-terminal embedded in the cytoplasm(Pivetti et al. 2003).
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This part was used as a mechanosensitive ion channel, since it has been previously described as important receptors involved in the response to ultrasound stimulation. In our system it served as a source of Ca2+ influx when stimulated with ultrasound (figure 1).  
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<h3>Introduction </h3>
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<p>This part was nominated for best new part award by iGEM Team Slovenia 2016. 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 <sup>[1]</sup>. It has been 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 the C-terminus embedded in the cytoplasm <sup>[2]</sup>.</p>
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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 when stimulated with ultrasound <ref>1</ref>. </p>
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<div>
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    <figure data-ref="1">
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        <img class="ui medium image" src="https://static.igem.org/mediawiki/2016/f/f2/T--Slovenia--S.3.1.1.png">
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        <figcaption><b> Schematic presentation of MscS channel function in our system.</b><br/> Cells in resting stage with closed MscS channels in the plasma membrane (left). Upon ultrasound stimulation MscS channels open, leading to Ca <sup>2+</sup> influx (right).  
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        </figcaption>
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    </figure>
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</div>
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<h3>Characterization</h3>
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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>
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<div>
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    <figure data-ref="2">
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        <img class="ui medium image" 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/>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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(A) Scheme of bacterial ion channel MscS
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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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(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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        </figcaption>
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    </figure>
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</div>
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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>
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 +
<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>
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    <figure data-ref="3">
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        <img class="ui medium image" src="https://static.igem.org/mediawiki/2016/8/8a/T--Slovenia--3.2.4.png">
 +
        <figcaption><b> MscS channel improves sensitivity of cells for ultrasound. </b><br/>(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.
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        </figcaption>
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    </figure>
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</div>
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 +
 
 +
<p>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="http://2016.igem.org/Team:Slovenia/Mechanosensing/Gas_vesicles(">Read more</a>)
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. 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>
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    <figure data-ref="4">
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        <img class="ui medium image" 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/>(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.
 +
 
 +
        </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+<sup>[3]</sup> 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>
 +
    <figure data-ref="5">
 +
        <img class="ui medium image" 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/>(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.
 +
 
 +
        </figcaption>
 +
    </figure>
 +
</div>
 +
 
 +
 
 +
<h3>References</h3>
 +
 
 +
<sup>[1]</sup>Perozo, Eduardo, and Douglas C. Rees. 2003. “Structure and Mechanism in Prokaryotic Mechanosensitive Channels.” Current Opinion in Structural Biology 13(4): 432–42. <br>
 +
<sup>[2]</sup>Pivetti, Christopher D et al. 2003. “Two Families of Mechanosensitive Channel Proteins.” Microbiology and molecular biology reviews : MMBR 67(1): 66–85, table of contents. <br>
 +
<sup>[3]</sup>Bourne, G. W., & Trifaró, J. M. (1982). The gadolinium ion: A potent blocker of calcium channels and catecholamine release from cultured chromaffin cells. Neuroscience, 7(7), 1615–1622. https://doi.org/10.1016/0306-4522(82)90019-7.<br>
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</body>
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</html>
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Revision as of 15:57, 18 October 2016


MscS

Introduction

This part was nominated for best new part award by iGEM Team Slovenia 2016. 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 [1]. 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 [2].

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+[3] 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.

References

[1]Perozo, Eduardo, and Douglas C. Rees. 2003. “Structure and Mechanism in Prokaryotic Mechanosensitive Channels.” Current Opinion in Structural Biology 13(4): 432–42.
[2]Pivetti, Christopher D et al. 2003. “Two Families of Mechanosensitive Channel Proteins.” Microbiology and molecular biology reviews : MMBR 67(1): 66–85, table of contents.
[3]Bourne, G. W., & Trifaró, J. M. (1982). The gadolinium ion: A potent blocker of calcium channels and catecholamine release from cultured chromaffin cells. Neuroscience, 7(7), 1615–1622. https://doi.org/10.1016/0306-4522(82)90019-7.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]