Difference between revisions of "Team:Slovenia/Mechanosensing/Mechanosensitive channels"

 
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                         <b>Overview</b>
 
                         <b>Overview</b>
 
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                     </a>
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                    <a class="item" href="//2016.igem.org/Team:Slovenia/Mechanosensing/Mechanosensitive_channels" style="color:#DB2828;">
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                        <i class="selected radio icon"></i>
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                        <b>Mechanosensitive channels</b>
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                    </a>
 
                     <a class="item" href="#intro" style="margin-left: 10%">
 
                     <a class="item" href="#intro" style="margin-left: 10%">
 
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                         <b>Motivation</b>
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                         <b>Introduction</b>
 
                     </a>
 
                     </a>
 
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                         <div>
 
                         <div>
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                             <h1 class="ui left dividing header"><span id="intro" class="section">&nbsp;</span>Enhanced
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                             <h1 class="ui left dividing header"><span id="intro" class="section colorize">&nbsp;</span>Enhanced
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                                 Mechanosensitivity by overexpressed <br/>Mechanosensitive Channels</h1>
+
                                 mechanosensitivity by overexpressed <br/>mechanosensitive channels</h1>
 
                             <div class="ui segment" style="background-color: #ebc7c7; ">
 
                             <div class="ui segment" style="background-color: #ebc7c7; ">
 
                                 <ul>
 
                                 <ul>
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                         </div>
 
                         </div>
 
                         <div class="ui segment">
 
                         <div class="ui segment">
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                             <h4><span id="mot" class="section">&nbsp;</span></h4>
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                             <h3><span id="mot" class="section colorize">&nbsp;</span></h3>
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                             <p>We chose to test two mechanosensitive channels, human nonspecific cation channel TRPC1
+
 
 +
                            <div style="float:right; width:40%">
 +
                                <figure data-ref="shema">
 +
                                    <img src="https://static.igem.org/mediawiki/2016/8/88/T--Slovenia--3.2.1.png">
 +
                                    <figcaption><b>Proposed gating mechanisms of mechanosensitive channels (adapted from
 +
                                        Christensen & Corey
 +
                                        <x-ref>Christensen2007</x-ref>
 +
                                        ).</b></figcaption>
 +
                                </figure>
 +
                            </div>
 +
 
 +
 
 +
                            <p> The detailed mechanism of mechanosensing is not known, however most mechanosensitive
 +
                                receptors respond to mechanical stimuli through
 +
                                opening of the channel pore and allowing calcium ions to enter the cell
 +
                                <x-ref>Zheng2013</x-ref>
 +
                                . Membrane composition has been shown to play
 +
                                an important role in bacterial channel activation, however cytoskeleton apparently also
 +
                                mediates mechanosensing as several mechanosensitive channels
 +
                                comprise domains that can interact with the cytoskeleton (<ref>shema</ref>).
 +
                            </p>
 +
 
 +
                             <p>We chose to test two mechanosensitive channels: human nonspecific cation channel TRPC1
 
                                 and bacterial channel MscS, previously described as important receptors involved
 
                                 and bacterial channel MscS, previously described as important receptors involved
−
                                 in the response to mechanical stimulation in human and bacteria
+
                                 in the response to mechanical stimulation in humans and bacteria
 
                                 <x-ref>Haswell2011, Ye2013</x-ref>
 
                                 <x-ref>Haswell2011, Ye2013</x-ref>
 
                                 .
 
                                 .
 
                             </p>
 
                             </p>
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                             <div class="ui styled fluid accordion">
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                             <div style="width:60%" class="ui styled fluid accordion">
 
                                 <div class="title">
 
                                 <div class="title">
 
                                     <i class="dropdown icon"></i>
 
                                     <i class="dropdown icon"></i>
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                                         formation of functional homo- or hetero-tetramers
 
                                         formation of functional homo- or hetero-tetramers
 
                                         <x-ref>Bianchi2007</x-ref>
 
                                         <x-ref>Bianchi2007</x-ref>
−
                                         .
+
                                         (<ref>1a</ref>).
 
                                     </p>
 
                                     </p>
 
                                     <div style="clear:left; width:50%">
 
                                     <div style="clear:left; width:50%">
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                                         <figure data-ref="1">
+
                                         <figure data-ref="1a">
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                                             <img onclick="resize(this);"  
+
                                             <img onclick="resize(this);"
 
                                                 src="https://static.igem.org/mediawiki/2016/7/7a/T--Slovenia--3.2.2.PNG">
 
                                                 src="https://static.igem.org/mediawiki/2016/7/7a/T--Slovenia--3.2.2.PNG">
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                                             <figcaption><b>Structure of a tetrameric homologous TRPv6 channel (<a
+
                                             <figcaption><b>Structure of a tetrameric homologous TRPV6 channel (<a
 
                                                     href="http://www.rcsb.org/pdb/explore.do?structureId=5IRX">PDB
 
                                                     href="http://www.rcsb.org/pdb/explore.do?structureId=5IRX">PDB
 
                                                 5IRX</a>) presented from side and top with seen ion pore.</b>
 
                                                 5IRX</a>) presented from side and top with seen ion pore.</b>
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                                     </div>
 
                                     </div>
 
                                     <p>The second channel that we selected is the bacterial mechanosensitive channel
 
                                     <p>The second channel that we selected is the bacterial mechanosensitive channel
−
                                         MscS. Its role is to mediate turgor regulation in bacteria and it is
+
                                         MscS. Its role is to mediate turgor regulation in bacteria and is
 
                                         activated by changes in osmotic pressure
 
                                         activated by changes in osmotic pressure
 
                                         <x-ref>Perozo2003</x-ref>
 
                                         <x-ref>Perozo2003</x-ref>
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                                         periplasm and the C-terminus embedded in the cytoplasm
 
                                         periplasm and the C-terminus embedded in the cytoplasm
 
                                         <x-ref>Pivetti2003</x-ref>
 
                                         <x-ref>Pivetti2003</x-ref>
 +
                                        (<ref>2a</ref>)
 
                                         .
 
                                         .
 
                                     </p>
 
                                     </p>
 
                                     <div style="clear:left; width:50%">
 
                                     <div style="clear:left; width:50%">
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                                         <figure data-ref="2">
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                                         <figure data-ref="2a">
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                                             <img onclick="resize(this);"  
+
                                             <img onclick="resize(this);"
 
                                                 src="https://static.igem.org/mediawiki/2016/d/df/T--Slovenia--3.2.3.PNG">
 
                                                 src="https://static.igem.org/mediawiki/2016/d/df/T--Slovenia--3.2.3.PNG">
 
                                             <figcaption><b>. Crystal structure of the MscS channel (<a
 
                                             <figcaption><b>. Crystal structure of the MscS channel (<a
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                                 </div>
 
                                 </div>
 
                             </div>
 
                             </div>
 +
                            <p style="clear:both"></p>
 
                         </div>
 
                         </div>
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                         <h1><span class="section">&nbsp;</span>Results</h1>
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                         <h1><span class="section colorize">&nbsp;</span>Results</h1>
 
                         <div class="ui segment">
 
                         <div class="ui segment">
 
                             <div>
 
                             <div>
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                                 <h4><span id="loc" class="section">&nbsp;</span>Localization and expression</h4>
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                                 <h3><span id="loc" class="section colorize">&nbsp;</span>Localization and expression</h3>
 
                                 <p>The mechanosensitive TRPC1 channel with each subunit comprising six transmembrane
 
                                 <p>The mechanosensitive TRPC1 channel with each subunit comprising six transmembrane
−
                                     helices (
+
                                     helices (<ref>3a</ref>A lower) and the MscS channel with three transmembrane
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                                    <ref>3</ref>
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                                     helices (<ref>3a</ref>A upper) were expressed in HEK293T cells (<ref>3a</ref>D). MscS was detected as the 31 kDa band. TRPC1 was observed at 60 kDa, which was
−
                                    A) and the MscS channel with three transmembrane
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                                     lower than expected. We observed that the membrane localization in HEK293 was more
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                                     helices (
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                                    evident
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                                    <ref>3</ref>
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                                     for MscS (<ref>3a</ref>B) rather than TRPC1 (<ref>3a</ref>C).
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                                    A) were expressed in HEK293T cells (
+
−
                                    <ref>3</ref>
+
−
                                    D). MscS was detected as the 31 kDa band. TRPC1 was observed at 60 kDa, which was
+
−
                                     lower t
+
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                                    han expected. We observed that the membrane localization in HEK293 was more evident
+
−
                                     for MscS (
+
−
                                    <ref>3</ref>
+
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                                    B) rather than TRPC1 (
+
−
                                    <ref>3</ref>
+
−
                                    C).
+
 
                                 </p>
 
                                 </p>
 
                                 <div style="clear:left; width:100%">
 
                                 <div style="clear:left; width:100%">
−
                                     <figure data-ref="3">
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                                     <figure data-ref="3a">
 
                                         <img src="https://static.igem.org/mediawiki/2016/1/1e/T--Slovenia--3.2.2.png">
 
                                         <img src="https://static.igem.org/mediawiki/2016/1/1e/T--Slovenia--3.2.2.png">
 
                                         <figcaption><b>Localization and expression of mechanosensitive ion channels MscS
 
                                         <figcaption><b>Localization and expression of mechanosensitive ion channels MscS
 
                                             and TRPC1. </b><br/>
 
                                             and TRPC1. </b><br/>
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                                             <p style="text-align:justify">(A) Scheme of bacterial ion channel MscS (upper) and human ion channel TRPC1
+
                                             <p style="text-align:justify">(A) Scheme of bacterial ion channel MscS
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                                            (lower).
+
                                                (upper) and human ion channel TRPC1
−
                                            (B) Ion channel MscS localized to plasma membrane. (C) TRPC1 predominantly
+
                                                (lower).
−
                                            localized in the ER.
+
                                                (B) Ion channel MscS localized to plasma membrane. (C) TRPC1
−
                                            (D) Ion channels MscS and TRPC1 were expressed in HEK293 cells. HEK293 cells
+
                                                predominantly
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                                            were transfected with plasmids encoding HA tagged MscS or Myc-tagged TRPC1.
+
                                                localized in the ER.
−
                                            Expression by Western blot and localization by confocal microscopy were
+
                                                (D) Ion channels MscS and TRPC1 were expressed in HEK293 cells. HEK293
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                                            analyzed using anti-HA and anti-Myc antibodies, respectively.
+
                                                cells
−
</p>
+
                                                were transfected with plasmids encoding HA tagged MscS or Myc-tagged
−
</figcaption>
+
                                                TRPC1.
 +
                                                Expression by Western blot and localization by confocal microscopy were
 +
                                                analyzed using anti-HA and anti-Myc antibodies, respectively.
 +
                                            </p>
 +
                                        </figcaption>
 
                                     </figure>
 
                                     </figure>
 
                                 </div>
 
                                 </div>
  
 
                                 <p style="clear:both">To improve membrane localization of TRPC1 we fused a FAS
 
                                 <p style="clear:both">To improve membrane localization of TRPC1 we fused a FAS
−
                                     transmembrane domain to TRPC1 (
+
                                     transmembrane domain to TRPC1 (<ref>4a</ref>A), since the
−
                                    <ref>4</ref>
+
−
                                    A), since the
+
 
                                     transmembrane FAS domain has been very efficient in Jerala lab for the membrane
 
                                     transmembrane FAS domain has been very efficient in Jerala lab for the membrane
 
                                     localization
 
                                     localization
 
                                     <x-ref>Majerle2015</x-ref>
 
                                     <x-ref>Majerle2015</x-ref>
 
                                     . The strategy of adding an
 
                                     . The strategy of adding an
−
                                     additional transmembrane domain has to own knowledge not been applied before for the
+
                                     additional transmembrane domain has to our knowledge not been applied before for the
 
                                     ion channels. The addition of FAS transmembrane domain to the
 
                                     ion channels. The addition of FAS transmembrane domain to the
 
                                     N-terminus of TRPC1 improved localization of the protein to plasma membrane in
 
                                     N-terminus of TRPC1 improved localization of the protein to plasma membrane in
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                                     of non-permeabilized cells we verified that the modified channel was inserted into
 
                                     of non-permeabilized cells we verified that the modified channel was inserted into
 
                                     plasma membrane as predicted, since in non-permeabilized cells antibodies
 
                                     plasma membrane as predicted, since in non-permeabilized cells antibodies
−
                                     stained the exposed extracellular HA-tag but not the intracellular Myc-tag (
+
                                     stained the exposed extracellular HA-tag but not the intracellular Myc-tag (<ref>4a</ref>B).
−
                                    <ref>4</ref>
+
−
                                    B).
+
 
                                 </p>
 
                                 </p>
 
                                 <div style="float:left; width:100%">
 
                                 <div style="float:left; width:100%">
−
                                     <figure data-ref="4">
+
                                     <figure data-ref="4a">
 
                                         <img src="https://static.igem.org/mediawiki/2016/1/13/T--Slovenia--3.2.3.png">
 
                                         <img src="https://static.igem.org/mediawiki/2016/1/13/T--Slovenia--3.2.3.png">
 
                                         <figcaption><b>Localization of fusion protein P3:FAStm:TRPC1.</b><br/>
 
                                         <figcaption><b>Localization of fusion protein P3:FAStm:TRPC1.</b><br/>
−
                                             <p style="text-align:justify">(A) Scheme of ion channel P3:FAStm:TRPC1. (B) Ion channel P3:FAStm:TRPC1 was
+
                                             <p style="text-align:justify">(A) Scheme of ion channel P3:FAStm:TRPC1. (B)
−
                                            localized to plasma membrane. HEK293 cells were transfected with
+
                                                Ion channel P3:FAStm:TRPC1 was
−
                                            P3:FAStm:TRPC1 plasmid. 24 h after transfection cells were permeabilized
+
                                                localized to plasma membrane. HEK293 cells were transfected with
−
                                            (upper) or non-permeabilized (lower) and stained with antibodies against
+
                                                P3:FAStm:TRPC1 plasmid. 24 h after transfection cells were permeabilized
−
                                            HA and Myc-tag. Localization on plasma membrane is shown with arrows.
+
                                                (upper) or non-permeabilized (lower) and stained with antibodies against
−
</p>
+
                                                HA and Myc-tag. Localization on plasma membrane is shown with arrows.
 +
                                            </p>
 
                                         </figcaption>
 
                                         </figcaption>
 
                                     </figure>
 
                                     </figure>
Line 213: Line 232:
 
                                     the protein are orientated towards the interior of the cell. By addition
 
                                     the protein are orientated towards the interior of the cell. By addition
 
                                     of the FAS transmembrane domain, the N-terminus of P3:FAStm:TRPC1 chimera (where P3
 
                                     of the FAS transmembrane domain, the N-terminus of P3:FAStm:TRPC1 chimera (where P3
−
                                     stands for coiled coil, hyperlink to CC page) is exposed in the extracellular
+
                                     stands for <a
 +
                                            href="https://2016.igem.org/Team:Slovenia/Protease_signaling/Logic#ant">coiled
 +
                                        coil</a>) is exposed in the extracellular
 
                                     space and could interact with different proteins from outside the cell via the
 
                                     space and could interact with different proteins from outside the cell via the
 
                                     N-terminal tag. We reasoned that this interaction could be used to achieve a higher
 
                                     N-terminal tag. We reasoned that this interaction could be used to achieve a higher
Line 220: Line 241:
 
                                 <p>After we showed that the selected ion channels MscS, TRPC1 and P3:FAStm:TRPC1 are
 
                                 <p>After we showed that the selected ion channels MscS, TRPC1 and P3:FAStm:TRPC1 are
 
                                     expressed in HEK293 and localized at the plasma membrane, we further tested their
 
                                     expressed in HEK293 and localized at the plasma membrane, we further tested their
−
                                     function as mechanosensors by exposing them to the ultrasound stimulation.
+
                                     function as mechanosensors by exposing them to ultrasound stimulation.
 
                                 </p>
 
                                 </p>
 
                             </div>
 
                             </div>
 
                             <div>
 
                             <div>
−
                                 <h4><span id="us" class="section">&nbsp;</span>Ultrasound stimulation</h4><br/>
+
                                 <h3><span id="us" class="section colorize">&nbsp;</span>Ultrasound stimulation</h3><br/>
 
                                 <div style="clear:both" class="ui styled fluid accordion">
 
                                 <div style="clear:both" class="ui styled fluid accordion">
 
                                     <div class="title">
 
                                     <div class="title">
Line 234: Line 255:
 
                                             Ultrasound stimulation offers potentially remarkable advantages over the
 
                                             Ultrasound stimulation offers potentially remarkable advantages over the
 
                                             majority of external stimuli used for targeted cell stimulation.
 
                                             majority of external stimuli used for targeted cell stimulation.
−
                                             Optogenetics as another
+
                                             Optogenetics, as another
−
                                             promising approach to cell stimulation requires invasive surgery to
+
                                             promising approach to cell stimulation, requires invasive surgery to
 
                                             implement optical fibers connected to the source of light – LED or laser
 
                                             implement optical fibers connected to the source of light – LED or laser
 
                                             <x-ref>Warden2014</x-ref>
 
                                             <x-ref>Warden2014</x-ref>
Line 241: Line 262:
 
                                             hand, ultrasound offers a non-invasive approach to overcome the problems
 
                                             hand, ultrasound offers a non-invasive approach to overcome the problems
 
                                             which appear in
 
                                             which appear in
−
                                             the abovementioned method. Its use has been demonstrated potentially even
+
                                             the above mentioned method. Its use has been demonstrated potentially even
 
                                             for noninvasive ultrasound therapy through an intact skull
 
                                             for noninvasive ultrasound therapy through an intact skull
 
                                             <x-ref>Hynynen1998</x-ref>
 
                                             <x-ref>Hynynen1998</x-ref>
Line 247: Line 268:
 
                                             Previously, ultrasound had been used in several in vitro studies to directly
 
                                             Previously, ultrasound had been used in several in vitro studies to directly
 
                                             stimulate clusters of neurons but also in few model organisms (among others
 
                                             stimulate clusters of neurons but also in few model organisms (among others
−
                                             <x-ref>King2013</x-ref>
+
                                             <x-ref>King2013</x-ref>)
 
                                             .
 
                                             .
 
                                         </p>
 
                                         </p>
Line 254: Line 275:
 
                                 <br/>
 
                                 <br/>
 
                                 <p>For mechanical stimulation of cells with ultrasound we designed our own unique
 
                                 <p>For mechanical stimulation of cells with ultrasound we designed our own unique
−
                                     experimental setup, which included the
+
                                     <a href="https://2016.igem.org/Team:Slovenia/Hardware#set">experimental setup</a>,
 +
                                    which included the
 
                                     <a href="https://2016.igem.org/Team:Slovenia/Hardware">ultrasound device MODUSON</a>
 
                                     <a href="https://2016.igem.org/Team:Slovenia/Hardware">ultrasound device MODUSON</a>
−
                                     that we constructed connected to the unfocused transducer Olympus
+
                                     that we constructed and connected to the unfocused transducer Olympus
 
                                     V318-SU and a 3D printed support for a transducer to fix it at a defined position
 
                                     V318-SU and a 3D printed support for a transducer to fix it at a defined position
 
                                     relative to the cells. Stimulation conditions were optimized for our cell
 
                                     relative to the cells. Stimulation conditions were optimized for our cell
Line 264: Line 286:
 
                                     concentration as a ratio of the fluorescence intensity at two wavelengths,
 
                                     concentration as a ratio of the fluorescence intensity at two wavelengths,
 
                                     which was superior to the intensity based measurements, since it is independent of
 
                                     which was superior to the intensity based measurements, since it is independent of
−
                                     photobleaching and dye sequestration
+
                                     photobleaching and dye sequestration.
 
                                 </p>
 
                                 </p>
  
Line 296: Line 318:
 
                                 </p>
 
                                 </p>
  
−
                                 <p>We showed that by expressing the MscS channel, cells gained sensitivity for
+
                                 <p>We showed that by expressing the MscS channel cells gained sensitivity for
−
                                     ultrasound stimulation in comparison to non-transfected cells (
+
                                     ultrasound stimulation in comparison to non-transfected cells (<ref>5a</ref> and <ref>6a</ref>).
−
                                    <ref>6</ref>
+
−
                                    ).
+
 
                                     Influx of calcium ions was observed at a lower rate in the case of ectopically
 
                                     Influx of calcium ions was observed at a lower rate in the case of ectopically
 
                                     expressed TRPC1 (data not shown), probably due to its poor membrane localization.
 
                                     expressed TRPC1 (data not shown), probably due to its poor membrane localization.
 
                                 </p>
 
                                 </p>
  
−
                                 <div style="clear:both; width:60%; margin-left:auto; margin-right:auto;">
+
                                 <div style="clear:both; width:70%; margin-left:auto; margin-right:auto;">
−
                                     <figure data-ref="5">
+
                                     <figure data-ref="5a">
 
                                         <img src=" https://static.igem.org/mediawiki/2016/f/f2/T--Slovenia--S.3.1.1.png ">
 
                                         <img src=" https://static.igem.org/mediawiki/2016/f/f2/T--Slovenia--S.3.1.1.png ">
−
                                         <figcaption><b>INSERT!!!</b><br/></figcaption>
+
                                         <figcaption><b>Cells expressing exogenous MscS channels gained sensitivity
 +
                                            for ultrasound stimulation.</b><br/>
 +
<p style="text-align:justify">
 +
Scheme of cells expressing MscS. Upon stimulation with ultrasound channels open and calcium ions enter the cells.
 +
</p>
 +
</figcaption>
 
                                     </figure>
 
                                     </figure>
 
                                 </div>
 
                                 </div>
  
 
                                 <div style="clear:both;" align="center">
 
                                 <div style="clear:both;" align="center">
−
                                     <figure data-ref="6">
+
                                     <figure data-ref="6a">
 
                                         <img class="ui big centered image"
 
                                         <img class="ui big centered image"
 
                                             src="https://static.igem.org/mediawiki/2016/2/27/T--Slovenia--3.2.5.png">
 
                                             src="https://static.igem.org/mediawiki/2016/2/27/T--Slovenia--3.2.5.png">
Line 347: Line 372:
 
                                         <figcaption><b> MscS channel improves sensitivity of cells for
 
                                         <figcaption><b> MscS channel improves sensitivity of cells for
 
                                             ultrasound.</b><br/>
 
                                             ultrasound.</b><br/>
−
                                             <p style="text-align:justify">(A) Schematic representation of a stimulation sequence and (B) signal
+
                                             <p style="text-align:justify">(A) Schematic representation of a stimulation
−
                                            parameters used for stimulation.
+
                                                sequence and (B) signal
−
                                            (C) and (D )Cells expressing MscS showed increased sensitivity to ultrasound
+
                                                parameters used for stimulation.
−
                                            stimulation in comparison to the cells without exogenous mechanosensitive
+
                                                (C) and (D)Cells expressing MscS showed increased sensitivity to
−
                                            channel. HEK293 cells expressing MscS channels or control cells transfected
+
                                                ultrasound
−
                                            with vector were stimulated with ultrasound for 10 s and calcium influx
+
                                                stimulation in comparison to the cells without exogenous
−
                                            was recorded in real time (D) using a confocal microscope. For comparison
+
                                                mechanosensitive
−
                                            cells without ectopic MscS were used. Fluo-4 (D, green line) and Fura Red
+
                                                channel. HEK293 cells expressing MscS channels or control cells
−
                                            dyes (D, red line) were used for ratiometric calcium imaging. (D) Ratio
+
                                                transfected
−
                                            (blue line) was calculated from fluorescence intensities of Fura Red and
+
                                                with vector were stimulated with ultrasound for 10 s and calcium influx
−
                                            Fluo-4
+
                                                was recorded in real time (D) using a confocal microscope. For
−
                                            using <a href="https://2016.igem.org/Team:Slovenia/Software">CaPTURE</a>.
+
                                                comparison
−
</p>
+
                                                cells without ectopic MscS were used. Fluo-4 (D, green line) and Fura
 +
                                                Red
 +
                                                dyes (D, red line) were used for ratiometric calcium imaging. (D) Ratio
 +
                                                (blue line) was calculated from fluorescence intensities of Fura Red and
 +
                                                Fluo-4
 +
                                                using <a href="https://2016.igem.org/Team:Slovenia/Software">CaPTURE</a>.
 +
                                            </p>
 
                                         </figcaption>
 
                                         </figcaption>
 
                                     </figure>
 
                                     </figure>
Line 366: Line 397:
 
                                     improve its membrane localization, but also significantly enhanced its sensitivity
 
                                     improve its membrane localization, but also significantly enhanced its sensitivity
 
                                     to ultrasound
 
                                     to ultrasound
−
                                     stimulation (
+
                                     stimulation (<ref>7a</ref> and <ref>8a</ref>), suggesting the importance of membrane localization in the function of
−
                                    <ref>8</ref>
+
−
                                    C), suggesting the importance of membrane localization in the function of
+
 
                                     mechanosensors.
 
                                     mechanosensors.
 
                                 </p>
 
                                 </p>
  
−
                                 <div style="clear:left;">
+
                                 <div style="clear:left; width:70%; margin-left:auto; margin-right:auto;">
−
                                     <figure data-ref="7">
+
                                     <figure id="krneki" data-ref="7a">
−
                                         <img onclick="resize(this);" class="ui medium image"
+
                                         <img
 
                                             src=" https://static.igem.org/mediawiki/2016/f/fd/T--Slovenia--S.3.1.2.png">
 
                                             src=" https://static.igem.org/mediawiki/2016/f/fd/T--Slovenia--S.3.1.2.png">
−
                                         <figcaption><b>INSERT!!!</b><br/></figcaption>
+
                                         <figcaption><b>With fusion of FAS transmembrane domain to TRPC1 we enhanced
 +
                                            sensitivity of cells to ultrasound stimulation</b><br/>
 +
<p style="text-align:justify">
 +
Scheme of cells expressing modified TRPC1. Upon stimulation with ultrasound channels open and calcium ions enter the cells.
 +
</p>
 +
</figcaption>
 
                                     </figure>
 
                                     </figure>
 
                                 </div>
 
                                 </div>
  
 
                                 <div style="clear:both;" align="center">
 
                                 <div style="clear:both;" align="center">
−
                                     <figure data-ref="8">
+
                                     <figure data-ref="8a">
 
                                         <img class="ui big centered image"
 
                                         <img class="ui big centered image"
 
                                             src="https://static.igem.org/mediawiki/2016/8/8a/T--Slovenia--3.2.4.png">
 
                                             src="https://static.igem.org/mediawiki/2016/8/8a/T--Slovenia--3.2.4.png">
Line 416: Line 450:
 
                                         <figcaption><b>P3:FAS:TRPC1 channel improves sensitivity of cells for
 
                                         <figcaption><b>P3:FAS:TRPC1 channel improves sensitivity of cells for
 
                                             ultrasound.</b><br/>
 
                                             ultrasound.</b><br/>
−
                                             <p style="text-align:justify">(A) Schematic presentation of a stimulation sequence and (B) signal
+
                                             <p style="text-align:justify">(A) Schematic presentation of a stimulation
−
                                            parameters used for stimulation.
+
                                                sequence and (B) signal
−
                                            (C) and (D ) Cells expressing P3:FAS:TRPC1 showed increased sensitivity to
+
                                                parameters used for stimulation.
−
                                            ultrasound stimulation in comparison to the cells without exogenous
+
                                                (C) and (D) Cells expressing P3:FAS:TRPC1 showed increased sensitivity
−
                                            mechanosensitive channel.
+
                                                to
−
                                            HEK293 cells expressing P3:FAS:TRPC1 were stimulated with ultrasound for 10
+
                                                ultrasound stimulation in comparison to the cells without exogenous
−
                                            s and calcium influx was recorded in real time (D) using a confocal
+
                                                mechanosensitive channel.
−
                                            microscope. For comparison
+
                                                HEK293 cells expressing P3:FAS:TRPC1 were stimulated with ultrasound for
−
                                            cells without ectopic MscS were used. Fluo-4 (D, green line) and Fura Red
+
                                                10
−
                                            dyes (D, red line) were used for ratiometric calcium imaging. (D) Ratio
+
                                                s and calcium influx was recorded in real time (D) using a confocal
−
                                            (blue line) was calculated
+
                                                microscope. For comparison
−
                                            from fluorescence intensities of Fura Red and Fluo-4 using <a
+
                                                cells without ectopic MscS were used. Fluo-4 (D, green line) and Fura
−
                                            href="https://2016.igem.org/Team:Slovenia/Software">CaPTURE</a>.
+
                                                Red
−
</p>
+
                                                dyes (D, red line) were used for ratiometric calcium imaging. (D) Ratio
 +
                                                (blue line) was calculated
 +
                                                from fluorescence intensities of Fura Red and Fluo-4 using <a
 +
                                                        href="https://2016.igem.org/Team:Slovenia/Software">CaPTURE</a>.
 +
                                            </p>
 
                                         </figcaption>
 
                                         </figcaption>
 
                                     </figure>
 
                                     </figure>
 
                                 </div>
 
                                 </div>
 
                                 <p style="clear:both">In order to observe mechanostimulation of cells with ectopically
 
                                 <p style="clear:both">In order to observe mechanostimulation of cells with ectopically
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                                     expressed mechanoreceptors we had to use high-power ultrasound, however we tested
+
                                     expressed mechanoreceptors we had to use high-power ultrasound. However, we tested
 
                                     that the cells nevertheless
 
                                     that the cells nevertheless
 
                                     did not lose the viability by ultrasound stimulation. Our next challenge was to
 
                                     did not lose the viability by ultrasound stimulation. Our next challenge was to
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                         </div>
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                        <h3 class="ui left dividing header"><span id="ref-title" class="section colorize">&nbsp;</span>References
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                        </h3>
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                        <div class="ui segment citing" id="references"></div>
 
                     </div>
 
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        <img border="0" alt="iGEM" src="//2016.igem.org/wiki/images/8/84/T--Slovenia--logo_250x250.png" width="5%"
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Latest revision as of 13:25, 19 October 2016

Mechanosensitive channels

Overview Mechanosensitive channels Achievements Introduction Localization and
Expression Ultrasound stimulation Gas Vesicles

 Enhanced mechanosensitivity by overexpressed
mechanosensitive channels

  • Ectopically expressed mechanosensitive ion channels MscS and P3:FAStm:TRPC1 were used to enhance sensitivity of mammalian cells to ultrasound stimulation.
  • Membrane localization of the mechanosensitive channel TRPC1 was improved by fusing it with a FAS transmembrane domain, which also led to increased sensitivity to ultrasound stimulation.

 

Proposed gating mechanisms of mechanosensitive channels (adapted from Christensen & Corey Christensen2007 ).

The detailed mechanism of mechanosensing is not known, however most mechanosensitive receptors respond to mechanical stimuli through opening of the channel pore and allowing calcium ions to enter the cell Zheng2013 . Membrane composition has been shown to play an important role in bacterial channel activation, however cytoskeleton apparently also mediates mechanosensing as several mechanosensitive channels comprise domains that can interact with the cytoskeleton (shema).

We chose to test two mechanosensitive channels: human nonspecific cation channel TRPC1 and bacterial channel MscS, previously described as important receptors involved in the response to mechanical stimulation in humans and bacteria Haswell2011, Ye2013 .

Further explanation ...

Transient receptor potential channel 1 (TRPC1) is a human non-specific cation channel located at the plasma membrane. It has been previously reported as broadly expressed in human tissues where it functions as a store-operating calcium channel Xu2001 . It belongs to the TRP superfamily that act as tetrameric transmembrane proteins consisting of a domain formed by six transmembrane helices, with a pore between S5 and S6 Nilius2007 . These helices present the N- and C-termini to the cytoplasm, promoting the formation of functional homo- or hetero-tetramers Bianchi2007 (1a).

Structure of a tetrameric homologous TRPV6 channel (PDB 5IRX) presented from side and top with seen ion pore.

The second channel that we selected is the bacterial mechanosensitive channel MscS. Its role is to mediate turgor regulation in bacteria and is activated by changes in osmotic pressure Perozo2003 . It has been previously shown that MscS is a homoheptamer, each subunit is 31kDa in size and contains three transmembrane helices with the N-terminus facing the periplasm and the C-terminus embedded in the cytoplasm Pivetti2003 (2a) .

. Crystal structure of the MscS channel (PDB 5AJI) presented from the side view with presented membrane lipids and from the top with seen ion pore

 Results

 Localization and expression

The mechanosensitive TRPC1 channel with each subunit comprising six transmembrane helices (3aA lower) and the MscS channel with three transmembrane helices (3aA upper) were expressed in HEK293T cells (3aD). MscS was detected as the 31 kDa band. TRPC1 was observed at 60 kDa, which was lower than expected. We observed that the membrane localization in HEK293 was more evident for MscS (3aB) rather than TRPC1 (3aC).

Localization and expression of mechanosensitive ion channels MscS and TRPC1.

(A) Scheme of bacterial ion channel MscS (upper) and human ion channel TRPC1 (lower). (B) Ion channel MscS localized to plasma membrane. (C) TRPC1 predominantly localized in the ER. (D) Ion channels MscS and TRPC1 were expressed in HEK293 cells. HEK293 cells were transfected with plasmids encoding HA tagged MscS or Myc-tagged TRPC1. Expression by Western blot and localization by confocal microscopy were analyzed using anti-HA and anti-Myc antibodies, respectively.

To improve membrane localization of TRPC1 we fused a FAS transmembrane domain to TRPC1 (4aA), since the transmembrane FAS domain has been very efficient in Jerala lab for the membrane localization Majerle2015 . The strategy of adding an additional transmembrane domain has to our knowledge not been applied before for the ion channels. The addition of FAS transmembrane domain to the N-terminus of TRPC1 improved localization of the protein to plasma membrane in comparison to the unmodified TRPC1. From the confocal microscopy images of non-permeabilized cells we verified that the modified channel was inserted into plasma membrane as predicted, since in non-permeabilized cells antibodies stained the exposed extracellular HA-tag but not the intracellular Myc-tag (4aB).

Localization of fusion protein P3:FAStm:TRPC1.

(A) Scheme of ion channel P3:FAStm:TRPC1. (B) Ion channel P3:FAStm:TRPC1 was localized to plasma membrane. HEK293 cells were transfected with P3:FAStm:TRPC1 plasmid. 24 h after transfection cells were permeabilized (upper) or non-permeabilized (lower) and stained with antibodies against HA and Myc-tag. Localization on plasma membrane is shown with arrows.

In addition to the improved membrane localization, the FAS transmembrane domain linked to the TRPC1 presents another advantage. The TRPC1 is an ion channel with six transmembrane helices, therefore both the N- and the C-terminus of the protein are orientated towards the interior of the cell. By addition of the FAS transmembrane domain, the N-terminus of P3:FAStm:TRPC1 chimera (where P3 stands for coiled coil) is exposed in the extracellular space and could interact with different proteins from outside the cell via the N-terminal tag. We reasoned that this interaction could be used to achieve a higher sensitivity to mechanical stimuli.

After we showed that the selected ion channels MscS, TRPC1 and P3:FAStm:TRPC1 are expressed in HEK293 and localized at the plasma membrane, we further tested their function as mechanosensors by exposing them to ultrasound stimulation.

 Ultrasound stimulation


Further explanation ...

Ultrasound stimulation offers potentially remarkable advantages over the majority of external stimuli used for targeted cell stimulation. Optogenetics, as another promising approach to cell stimulation, requires invasive surgery to implement optical fibers connected to the source of light – LED or laser Warden2014 in order to target cells in tissue to activate or silence them. On the other hand, ultrasound offers a non-invasive approach to overcome the problems which appear in the above mentioned method. Its use has been demonstrated potentially even for noninvasive ultrasound therapy through an intact skull Hynynen1998 . Previously, ultrasound had been used in several in vitro studies to directly stimulate clusters of neurons but also in few model organisms (among others King2013) .


For mechanical stimulation of cells with ultrasound we designed our own unique experimental setup, which included the ultrasound device MODUSON that we constructed and connected to the unfocused transducer Olympus V318-SU and a 3D printed support for a transducer to fix it at a defined position relative to the cells. Stimulation conditions were optimized for our cell line and experimental setup. To measure the changes of free calcium ion concentration we stained cells with two fluorescent dyes Fura Red and Fluo-4. The combination of these two dyes enabled us to present changes in the calcium ion concentration as a ratio of the fluorescence intensity at two wavelengths, which was superior to the intensity based measurements, since it is independent of photobleaching and dye sequestration.

Further explanation ...

Fura Red and Fluo-4 are visible light-excitable dyes used for ratiometric measurement of calcium ions which excitation maximum is at 488 nm. While Fluo-4 exhibits an increase in fluorescence emission at 515 nm upon binding of calcium ions, fluorescence emission at 655 nm of Fura Red decreases once the indicator binds calcium ions. By calculating the ratio of fluorescence emission intensities captured at 488 nm exaction (where the difference of fluorescence between the bound and free indicator is at its maximum), we could observe changes in intracellular calcium concentrations in real time.


We followed changes of calcium concentration after ultrasound stimulation in real time using ratiometric confocal microscopy. For processing of data we developed our software CaPTURE, which automatically calculated the ratio between fluorescence intensities of FuraRed and Fluo-4 and presented the data as image and calculated values.

We showed that by expressing the MscS channel cells gained sensitivity for ultrasound stimulation in comparison to non-transfected cells (5a and 6a). Influx of calcium ions was observed at a lower rate in the case of ectopically expressed TRPC1 (data not shown), probably due to its poor membrane localization.

Cells expressing exogenous MscS channels gained sensitivity for ultrasound stimulation.

Scheme of cells expressing MscS. Upon stimulation with ultrasound channels open and calcium ions enter the cells.

MscS channel improves sensitivity of cells for ultrasound.

(A) Schematic representation of a stimulation sequence and (B) signal parameters used for stimulation. (C) and (D)Cells expressing MscS showed increased sensitivity to ultrasound stimulation in comparison to the cells without exogenous mechanosensitive channel. HEK293 cells expressing MscS channels or control cells transfected with vector were stimulated with ultrasound for 10 s and calcium influx was recorded in real time (D) using a confocal microscope. For comparison cells without ectopic MscS were used. Fluo-4 (D, green line) and Fura Red dyes (D, red line) were used for ratiometric calcium imaging. (D) Ratio (blue line) was calculated from fluorescence intensities of Fura Red and Fluo-4 using CaPTURE.

Fusion of the FAS transmembrane domain to TRPC1 did not only improve its membrane localization, but also significantly enhanced its sensitivity to ultrasound stimulation (7a and 8a), suggesting the importance of membrane localization in the function of mechanosensors.

With fusion of FAS transmembrane domain to TRPC1 we enhanced sensitivity of cells to ultrasound stimulation

Scheme of cells expressing modified TRPC1. Upon stimulation with ultrasound channels open and calcium ions enter the cells.

P3:FAS:TRPC1 channel improves sensitivity of cells for ultrasound.

(A) Schematic presentation of a stimulation sequence and (B) signal parameters used for stimulation. (C) and (D) Cells expressing P3:FAS:TRPC1 showed increased sensitivity to ultrasound stimulation in comparison to the cells without exogenous mechanosensitive channel. HEK293 cells expressing P3:FAS:TRPC1 were stimulated with ultrasound for 10 s and calcium influx was recorded in real time (D) using a confocal microscope. For comparison cells without ectopic MscS were used. Fluo-4 (D, green line) and Fura Red dyes (D, red line) were used for ratiometric calcium imaging. (D) Ratio (blue line) was calculated from fluorescence intensities of Fura Red and Fluo-4 using CaPTURE.

In order to observe mechanostimulation of cells with ectopically expressed mechanoreceptors we had to use high-power ultrasound. However, we tested that the cells nevertheless did not lose the viability by ultrasound stimulation. Our next challenge was to further improve sensitivity of cells to respond to lower power ultrasound as this would avoid stimulation of any endogenous channels and limit stimulation only to the engineered.

 References