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

 
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                        <b>Overview</b>
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<b>Project</b>
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                    <a class="item" href="//2016.igem.org/Team:Slovenia/Mechanosensing/Mechanosensitive_channels" style="color:#DB2828;">
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                        <b>Mechanosensitive channels</b>
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<b>Achievements</b>
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                        <b>Achievements</b>
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<b>Medal requirements</b>
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                    <a class="item" href="#mot" style="margin-left: 10%">
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                        <b>Introduction</b>
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                        <b>Localization and</b>
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                        <br/>
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                        <b style="margin-left: 12%">Expression</b>
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                        <b>Ultrasound stimulation</b>
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<div class="main ui citing justified container"><h1 class = "ui centered dividing header"><span class="section">&nbsp;</span></h1>
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                    <a class="item" href="//2016.igem.org/Team:Slovenia/Mechanosensing/Gas_vesicles">
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<p><b><ul><li>Ectopically expressed mechanosensitive ion channels MscS and P3:FAStm:TRPC1 in mammalian cells enhance sensitivity of cells to ultrasound stimulation.
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                        <b>Gas Vesicles</b>
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<li>Membrane localization of the mechanosensitive channel TRPC1 can be improved by fusing it with a FAS transmembrane domain, which also led to increased sensitivity
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                    </a>
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to ultrasound stimulation.
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<p>We chose to test two mechanosensitive channels, human nonspecific cation channel TRPC1 and bacterial channel MscS, previously described as important receptors involved
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in the response to mechanical stimulation in human and bacteria <x-ref>Haswell2011, Ye2013</x-ref>.
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Further explanation ...
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<p>Transient receptor potential channel 1 (TRPC1) is a human non-specific cation channel located at the plasma membrane. It has been previously reported as
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broadly expressed in human tissues where it functions as a store-operating calcium channel <x-ref>Xu2001</x-ref>. It belongs to the TRP superfamily that
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act as tetrameric transmembrane proteins consisting of a domain formed by six transmembrane helices, with a pore between S5 and S6 <x-ref>Nilius2007</x-ref>.
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These helices present the N- and C-termini to the cytoplasm, promoting the formation of functional homo- or hetero-tetramers <x-ref>Bianchi2007</x-ref>.
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<figure data-ref="1">
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<img onclick="resize(this);" class="ui medium image" 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 href="http://www.rcsb.org/pdb/explore.do?structureId=5IRX">PDB 5IRX</a>) presented from side and top with seen ion pore.</figcaption>
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</figure>
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<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
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activated by changes in osmotic pressure <x-ref>Perozo2003</x-ref>. It has been previously shown that MscS is a homoheptamer, each subunit is 31kDa in
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size and contains three transmembrane helices with the N-terminus facing the periplasm and the C-terminus embedded in the cytoplasm <x-ref>Pivetti2003</x-ref>.
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<img onclick="resize(this);" class="ui medium image" src="https://static.igem.org/mediawiki/2016/d/df/T--Slovenia--3.2.3.PNG" >
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<figcaption><b>. Crystal structure of the MscS channel (<a href="http://www.rcsb.org/pdb/explore.do?structureId=5AJI">PDB 5AJI</a>) presented from the side view with presented membrane lipids and from the top with seen ion pore</figcaption>
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<h1><span class="section">&nbsp;</span>Results</h1>
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                            <h1 class="ui left dividing header"><span id="intro" class="section colorize">&nbsp;</span>Enhanced
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<p>The mechanosensitive TRPC1 channel with each subunit comprising six transmembrane helices (<ref>3</ref>A) and the MscS channel with three transmembrane
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                                mechanosensitivity by overexpressed <br/>mechanosensitive channels</h1>
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helices (<ref>3</ref>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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                            <div class="ui segment" style="background-color: #ebc7c7; ">
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han expected. We observed that the membrane localization in HEK293 was more evident for MscS (<ref>3</ref>B) rather than TRPC1 (<ref>3</ref>C).
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                                <ul>
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</p>
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                                    <li><b>Ectopically expressed mechanosensitive ion channels MscS and P3:FAStm:TRPC1
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<div style = "clear:left;">  
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                                        were used to enhance sensitivity of mammalian cells to ultrasound
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<figure data-ref="3">
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                                        stimulation.</b>
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<img onclick="resize(this);" class="ui medium image" src="https://static.igem.org/mediawiki/2016/1/1e/T--Slovenia--3.2.2.png" >
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                                    <li><b>Membrane localization of the mechanosensitive channel TRPC1 was improved by
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<figcaption><b>Localization and expression of mechanosensitive ion channels MscS and TRPC1. </b><br/>
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                                        fusing it with a FAS transmembrane domain, which also led to increased
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(A) Scheme of bacterial ion channel MscS (upper) and human ion channel TRPC1 (lower).
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                                        sensitivity to ultrasound stimulation.</b>
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(B) Ion channel MscS localized to plasma membrane. (C) TRPC1 predominantly localized in the ER.
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                                </ul>
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(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.</figcaption> 
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                            <h3><span id="mot" class="section colorize">&nbsp;</span></h3>
  
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<p style="clear:both">To improve membrane localization of TRPC1 we fused a FAS transmembrane domain to TRPC1 (<ref>4</ref>A), since the
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                            <div style="float:right; width:40%">
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transmembrane FAS domain has been very efficient in Jerala lab for the membrane localization <x-ref>Majerle2015</x-ref>. The strategy of adding an
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                                <figure data-ref="shema">
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additional transmembrane domain has to own knowledge not been applied before for the ion channels. The addition of FAS transmembrane domain to the
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                                    <img src="https://static.igem.org/mediawiki/2016/8/88/T--Slovenia--3.2.1.png">
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N-terminus of TRPC1 improved localization of the protein to plasma membrane in comparison to the unmodified TRPC1. From the confocal microscopy images
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                                    <figcaption><b>Proposed gating mechanisms of mechanosensitive channels (adapted from
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of non-permeabilized cells we verified that the modified channel was inserted into plasma membrane as predicted, since in non-permeabilized cells antibodies
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                                        Christensen & Corey
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stained the exposed extracellular HA-tag but not the intracellular Myc-tag (<ref>4</ref>B).
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                                        <x-ref>Christensen2007</x-ref>
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</p>
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                                        ).</b></figcaption>
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<div style = "float:left;">  
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                                </figure>
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<figure data-ref="4">
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                            </div>
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<img onclick="resize(this);" class="ui medium image" src="https://static.igem.org/mediawiki/2016/1/13/T--Slovenia--3.2.3.png" >
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<figcaption><b>Localization of fusion protein P3:FAStm:TRPC1.</b><br/>
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(A) Scheme of ion channel P3:FAStm:TRPC1. (B) Ion channel P3:FAStm:TRPC1 was localized to plasma membrane. HEK293 cells were transfected with
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P3:FAStm:TRPC1 plasmid. 24 h after transfection cells were permeabilized (upper) or non-permeabilized (lower) and stained with antibodies against
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HA and Myc-tag. Localization on plasma membrane is shown with arrows.</figcaption>
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  </figure>
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</div>  
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<p>In addition to the improved membrane localization, the FAS transmembrane domain linked to the TRPC1 presents another advantage. The TRPC1 is an ion
 
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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
 
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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
 
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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.</p>
 
  
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<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
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                            <p> The detailed mechanism of mechanosensing is not known, however most mechanosensitive
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function as mechanosensors by exposing them to the ultrasound stimulation.
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                                receptors respond to mechanical stimuli through
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</p>
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                                opening of the channel pore and allowing calcium ions to enter the cell
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                                <x-ref>Zheng2013</x-ref>
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                                . Membrane composition has been shown to play
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                                an important role in bacterial channel activation, however cytoskeleton apparently also
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                                mediates mechanosensing as several mechanosensitive channels
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                                comprise domains that can interact with the cytoskeleton (<ref>shema</ref>).
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                            </p>
  
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<div style="clear:both" class="ui styled fluid accordion">
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                            <p>We chose to test two mechanosensitive channels: human nonspecific cation channel TRPC1
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<div class="title">
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                                and bacterial channel MscS, previously described as important receptors involved
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<i class="dropdown icon"></i>
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                                in the response to mechanical stimulation in humans and bacteria
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Further explanation ...
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                                <x-ref>Haswell2011, Ye2013</x-ref>
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</div>
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                                .
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<div class="content">
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                            </p>
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<p>
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                            <div style="width:60%" class="ui styled fluid accordion">
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Ultrasound stimulation offers potentially remarkable advantages over the majority of external stimuli used for targeted cell stimulation. Optogenetics as another
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                                <div class="title">
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promising approach to cell stimulation requires invasive surgery to implement optical fibers connected to the source of light – LED or laser <x-ref>Warden2014</x-ref>
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                                    <i class="dropdown icon"></i>
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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
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                                    Further explanation ...
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the abovementioned method. Its use has been demonstrated potentially even for noninvasive ultrasound therapy through an intact skull <x-ref>Hynynen1998</x-ref>.  
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                                </div>
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Previously, ultrasound had been used in several in vitro studies to directly stimulate clusters of neurons but also in few model organisms (among others
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                                <div class="content">
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<x-ref>King2013</x-ref>.
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                                    <p>Transient receptor potential channel 1 (TRPC1) is a human non-specific cation
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</p>
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                                        channel located at the plasma membrane. It has been previously reported as
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</div>
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                                        broadly expressed in human tissues where it functions as a store-operating
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</div><br/>
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                                        calcium channel
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                                        <x-ref>Xu2001</x-ref>
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<p>For mechanical stimulation of cells with ultrasound we designed our own unique experimental setup, which included the
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                                        . It belongs to the TRP superfamily that
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<a href="https://2016.igem.org/Team:Slovenia/Hardware">ultrasound device MODUSON</a> that we constructed connected to the unfocused transducer Olympus
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                                        act as tetrameric transmembrane proteins consisting of a domain formed by six
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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
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                                        transmembrane helices, with a pore between S5 and S6
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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.  
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                                        <x-ref>Nilius2007</x-ref>
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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,
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                                        .
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which was superior to the intensity based measurements, since it is independent of photobleaching and dye sequestration
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                                        These helices present the N- and C-termini to the cytoplasm, promoting the
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</p>
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                                        formation of functional homo- or hetero-tetramers
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                                        <x-ref>Bianchi2007</x-ref>
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<div class="ui styled fluid accordion">
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                                        (<ref>1a</ref>).
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Further explanation ...
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                                        <figure data-ref="1a">
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<p>Fura Red and Fluo-4 are visible light-excitable dyes used for ratiometric measurement of calcium ions which excitation maximum is at 488 nm.
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                                            <figcaption><b>Structure of a tetrameric homologous TRPV6 channel (<a
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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
+
                                                    href="http://www.rcsb.org/pdb/explore.do?structureId=5IRX">PDB
−
decreases once the indicator binds calcium ions. By calculating the ratio of fluorescence emission intensities captured at 488 nm exaction
+
                                                5IRX</a>) presented from side and top with seen ion pore.</b>
−
(where the difference of fluorescence between the bound and free indicator is at its maximum), we could observe changes in intracellular
+
                                            </figcaption>
−
calcium concentrations in real time.
+
                                        </figure>
−
</p>
+
                                    </div>
−
</div>
+
                                    <p>The second channel that we selected is the bacterial mechanosensitive channel
−
</div><br/>
+
                                        MscS. Its role is to mediate turgor regulation in bacteria and is
−
+
                                        activated by changes in osmotic pressure
−
<p>We followed changes of calcium concentration after ultrasound stimulation in real time using ratiometric confocal microscopy. For processing of data we
+
                                        <x-ref>Perozo2003</x-ref>
−
developed our software <a href="https://2016.igem.org/Team:Slovenia/Software">CaPTURE</a>, which automatically calculated the ratio between fluorescence
+
                                        . It has been previously shown that MscS is a homoheptamer, each subunit is
−
intensities of FuraRed and Fluo-4 and presented the data as image and calculated values.
+
                                        31kDa in
−
</p>
+
                                        size and contains three transmembrane helices with the N-terminus facing the
 +
                                        periplasm and the C-terminus embedded in the cytoplasm
 +
                                        <x-ref>Pivetti2003</x-ref>
 +
                                        (<ref>2a</ref>)
 +
                                        .
 +
                                    </p>
 +
                                    <div style="clear:left; width:50%">
 +
                                        <figure data-ref="2a">
 +
                                            <img onclick="resize(this);"
 +
                                                src="https://static.igem.org/mediawiki/2016/d/df/T--Slovenia--3.2.3.PNG">
 +
                                            <figcaption><b>. Crystal structure of the MscS channel (<a
 +
                                                    href="http://www.rcsb.org/pdb/explore.do?structureId=5AJI">PDB
 +
                                                5AJI</a>) presented from the side view with presented membrane lipids
 +
                                                and from the top with seen ion pore</b></figcaption>
 +
                                        </figure>
 +
                                    </div>
 +
                                </div>
 +
                            </div>
 +
                            <p style="clear:both"></p>
 +
                        </div>
 +
                        <h1><span class="section colorize">&nbsp;</span>Results</h1>
 +
                        <div class="ui segment">
 +
                            <div>
 +
                                <h3><span id="loc" class="section colorize">&nbsp;</span>Localization and expression</h3>
 +
                                <p>The mechanosensitive TRPC1 channel with each subunit comprising six transmembrane
 +
                                    helices (<ref>3a</ref>A lower) and the MscS channel with three transmembrane
 +
                                    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
 +
                                    lower than expected. We observed that the membrane localization in HEK293 was more
 +
                                    evident
 +
                                    for MscS (<ref>3a</ref>B) rather than TRPC1 (<ref>3a</ref>C).
 +
                                </p>
 +
                                <div style="clear:left; width:100%">
 +
                                    <figure data-ref="3a">
 +
                                        <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
 +
                                            and TRPC1. </b><br/>
 +
                                            <p style="text-align:justify">(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.
 +
                                            </p>
 +
                                        </figcaption>
 +
                                    </figure>
 +
                                </div>
  
−
<p>We showed that by expressing the MscS channel, cells gained sensitivity for ultrasound stimulation in comparison to non-transfected cells (<ref>6</ref>4).
+
                                <p style="clear:both">To improve membrane localization of TRPC1 we fused a FAS
−
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.
+
                                    transmembrane domain to TRPC1 (<ref>4a</ref>A), since the
−
</p>
+
                                    transmembrane FAS domain has been very efficient in Jerala lab for the membrane
−
+
                                    localization
−
<div style = "clear:left;">
+
                                    <x-ref>Majerle2015</x-ref>
−
<figure data-ref="5">
+
                                    . The strategy of adding an
−
<img onclick="resize(this);" class="ui medium image" src=" https://static.igem.org/mediawiki/2016/f/f2/T--Slovenia--S.3.1.1.png " >
+
                                    additional transmembrane domain has to our knowledge not been applied before for the
−
<figcaption><b>INSERT!!!</b><br/></figcaption>
+
                                    ion channels. The addition of FAS transmembrane domain to the
−
<!--ŽIGA, vstavi GIF-->
+
                                    N-terminus of TRPC1 improved localization of the protein to plasma membrane in
−
</figure>
+
                                    comparison to the unmodified TRPC1. From the confocal microscopy images
−
</div>
+
                                    of non-permeabilized cells we verified that the modified channel was inserted into
−
+
                                    plasma membrane as predicted, since in non-permeabilized cells antibodies
−
<div style = "clear:left;">
+
                                    stained the exposed extracellular HA-tag but not the intracellular Myc-tag (<ref>4a</ref>B).
−
<figure data-ref="6">
+
                                </p>
−
<img onclick="resize(this);" class="ui medium image" src="https://static.igem.org/mediawiki/2016/2/27/T--Slovenia--3.2.5.png" >
+
                                <div style="float:left; width:100%">
−
<figcaption><b> MscS channel improves sensitivity of cells for ultrasound.</b><br/>
+
                                    <figure data-ref="4a">
−
(A) Schematic representation of a stimulation sequence and (B) signal parameters used for stimulation.
+
                                        <img src="https://static.igem.org/mediawiki/2016/1/13/T--Slovenia--3.2.3.png">
−
(C) and (D )Cells expressing MscS showed increased sensitivity to ultrasound stimulation in comparison to the cells without exogenous mechanosensitive
+
                                        <figcaption><b>Localization of fusion protein P3:FAStm:TRPC1.</b><br/>
−
channel. HEK293 cells expressing MscS channels or control cells transfected with vector were stimulated with ultrasound for 10 s and calcium influx
+
                                            <p style="text-align:justify">(A) Scheme of ion channel P3:FAStm:TRPC1. (B)
−
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
+
                                                Ion channel P3:FAStm:TRPC1 was
−
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
+
                                                localized to plasma membrane. HEK293 cells were transfected with
−
using <a href="https://2016.igem.org/Team:Slovenia/Software">CaPTURE</a>.</figcaption>
+
                                                P3:FAStm:TRPC1 plasmid. 24 h after transfection cells were permeabilized
−
</figure>
+
                                                (upper) or non-permeabilized (lower) and stained with antibodies against
−
</div>
+
                                                HA and Myc-tag. Localization on plasma membrane is shown with arrows.
−
<p style="clear:both">Fusion of the FAS transmembrane domain to TRPC1 did not only improve its membrane localization, but also significantly enhanced its sensitivity to ultrasound
+
                                            </p>
−
stimulation (<ref>8</ref>C), suggesting the importance of membrane localization in the function of mechanosensors.
+
                                        </figcaption>
−
</p>
+
                                    </figure>
−
+
                                </div>
−
<div style = "clear:left;">  
+
−
<figure data-ref="7">
+
−
<img onclick="resize(this);" class="ui medium image" src=" https://static.igem.org/mediawiki/2016/f/fd/T--Slovenia--S.3.1.2.png" >
+
−
<figcaption><b>INSERT!!!</b><br/></figcaption>
+
−
</figure>
+
−
</div>
+
−
+
−
<div style = "clear:left;">  
+
−
<figure data-ref="8">
+
−
<img onclick="resize(this);" class="ui medium image" src="https://static.igem.org/mediawiki/2016/8/8a/T--Slovenia--3.2.4.png" >
+
−
<!--ŽIGA, vstavi GIF-->
+
−
<figcaption><b>P3:FAS:TRPC1 channel improves sensitivity of cells for ultrasound.</b><br/> 
+
−
(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 <a href="https://2016.igem.org/Team:Slovenia/Software">CaPTURE</a>. </figcaption>
+
−
</figure>
+
−
</div>
+
−
<p style="clear:both">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.
+
−
</p>
+
  
 +
                                <p style="clear:both">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 <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
 +
                                    N-terminal tag. We reasoned that this interaction could be used to achieve a higher
 +
                                    sensitivity to mechanical stimuli.</p>
  
 +
                                <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
 +
                                    function as mechanosensors by exposing them to ultrasound stimulation.
 +
                                </p>
 +
                            </div>
 +
                            <div>
 +
                                <h3><span id="us" class="section colorize">&nbsp;</span>Ultrasound stimulation</h3><br/>
 +
                                <div style="clear:both" class="ui styled fluid accordion">
 +
                                    <div class="title">
 +
                                        <i class="dropdown icon"></i>
 +
                                        Further explanation ...
 +
                                    </div>
 +
                                    <div class="content">
 +
                                        <p>
 +
                                            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
 +
                                            <x-ref>Warden2014</x-ref>
 +
                                            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
 +
                                            <x-ref>Hynynen1998</x-ref>
 +
                                            .
 +
                                            Previously, ultrasound had been used in several in vitro studies to directly
 +
                                            stimulate clusters of neurons but also in few model organisms (among others
 +
                                            <x-ref>King2013</x-ref>)
 +
                                            .
 +
                                        </p>
 +
                                    </div>
 +
                                </div>
 +
                                <br/>
 +
                                <p>For mechanical stimulation of cells with ultrasound we designed our own unique
 +
                                    <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>
 +
                                    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.
 +
                                </p>
  
 +
                                <div class="ui styled fluid accordion">
 +
                                    <div class="title">
 +
                                        <i class="dropdown icon"></i>
 +
                                        Further explanation ...
 +
                                    </div>
 +
                                    <div class="content">
 +
                                        <p>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.
 +
                                        </p>
 +
                                    </div>
 +
                                </div>
 +
                                <br/>
  
−
+
                                <p>We followed changes of calcium concentration after ultrasound stimulation in real
−
+
                                    time using ratiometric confocal microscopy. For processing of data we
−
+
                                    developed our software <a
−
+
                                            href="https://2016.igem.org/Team:Slovenia/Software">CaPTURE</a>, which
−
</div>
+
                                    automatically calculated the ratio between fluorescence
−
</div>
+
                                    intensities of FuraRed and Fluo-4 and presented the data as image and calculated
−
</div>
+
                                    values.
−
</div>
+
                                </p>
−
</div>
+
 
−
</div>
+
                                <p>We showed that by expressing the MscS channel cells gained sensitivity for
−
</div>
+
                                    ultrasound stimulation in comparison to non-transfected cells (<ref>5a</ref> and <ref>6a</ref>).
 +
                                    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.
 +
                                </p>
 +
 
 +
                                <div style="clear:both; width:70%; margin-left:auto; margin-right:auto;">
 +
                                    <figure data-ref="5a">
 +
                                        <img src=" https://static.igem.org/mediawiki/2016/f/f2/T--Slovenia--S.3.1.1.png ">
 +
                                        <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>
 +
                                </div>
 +
 
 +
                                <div style="clear:both;" align="center">
 +
                                    <figure data-ref="6a">
 +
                                        <img class="ui big centered image"
 +
                                            src="https://static.igem.org/mediawiki/2016/2/27/T--Slovenia--3.2.5.png">
 +
                                        <div style="clear:both; display:block; width: 90%; margin-right: auto; margin-left: auto">
 +
                                            <div style="display: block; float: left; width: 80%;">
 +
                                                <!-- experiment: 20161002 2 MscS + Gvp 200V 90s -->
 +
                                                <img class="playme" id="gifGroup8"
 +
                                                    src="//2016.igem.org/wiki/images/a/ad/T--Slovenia--Group71.png"
 +
                                                    width="100%"
 +
                                                    data-alt="//2016.igem.org/wiki/images/b/be/T--Slovenia--20160929_2_MscS_200V_110s_graf.gif">
 +
                                            </div>
 +
                                            <div>
 +
                                                <!-- experiment: 20161002 2 MscS + Gvp 200V 90s -->
 +
                                                <div style="display: block; float: left; width: 20%;">
 +
                                                    <img class="gifGroup8"
 +
                                                        src="//2016.igem.org/wiki/images/7/70/T--Slovenia--Group72.png"
 +
                                                        width="100%"
 +
                                                        data-alt="//2016.igem.org/wiki/images/8/84/T--Slovenia--20160929_2_MscS_200V_110s_Regions.gif">
 +
                                                </div>
 +
                                                <div style="display: block; float: left; width: 20%;">
 +
                                                    <img class="gifGroup8"
 +
                                                        src="//2016.igem.org/wiki/images/8/83/T--Slovenia--Group73.png"
 +
                                                        width="100%"
 +
                                                        data-alt="//2016.igem.org/wiki/images/8/8f/T--Slovenia--20160929_2_MscS_200V_110s_Controls.gif">
 +
                                                </div>
 +
                                                <div style="display: block; float: left; width: 20%;">
 +
                                                    <img class="gifGroup8"
 +
                                                        src="//2016.igem.org/wiki/images/0/0e/T--Slovenia--Group74.png"
 +
                                                        width="100%"
 +
                                                        data-alt="//2016.igem.org/wiki/images/e/e4/T--Slovenia--20160929_2_MscS_200V_110s_HeatMap.gif">
 +
                                                </div>
 +
                                            </div>
 +
                                        </div>
 +
                                        <figcaption><b> MscS channel improves sensitivity of cells for
 +
                                            ultrasound.</b><br/>
 +
                                            <p style="text-align:justify">(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 <a href="https://2016.igem.org/Team:Slovenia/Software">CaPTURE</a>.
 +
                                            </p>
 +
                                        </figcaption>
 +
                                    </figure>
 +
                                </div>
 +
                                <p style="clear:both">Fusion of the FAS transmembrane domain to TRPC1 did not only
 +
                                    improve its membrane localization, but also significantly enhanced its sensitivity
 +
                                    to ultrasound
 +
                                    stimulation (<ref>7a</ref> and <ref>8a</ref>), suggesting the importance of membrane localization in the function of
 +
                                    mechanosensors.
 +
                                </p>
 +
 
 +
                                <div style="clear:left; width:70%; margin-left:auto; margin-right:auto;">
 +
                                    <figure id="krneki" data-ref="7a">
 +
                                        <img
 +
                                            src=" https://static.igem.org/mediawiki/2016/f/fd/T--Slovenia--S.3.1.2.png">
 +
                                        <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>
 +
                                </div>
 +
 
 +
                                <div style="clear:both;" align="center">
 +
                                    <figure data-ref="8a">
 +
                                        <img class="ui big centered image"
 +
                                            src="https://static.igem.org/mediawiki/2016/8/8a/T--Slovenia--3.2.4.png">
 +
                                        <div style="clear:both; display:block; width: 90%; margin-right: auto; margin-left: auto">
 +
                                            <div style="display: block; float: left; width: 80%;">
 +
                                                <!-- experiment: 20161002 2 MscS + Gvp 200V 90s -->
 +
                                                <img class="playme" id="gifGroup9"
 +
                                                    src="//2016.igem.org/wiki/images/1/10/T--Slovenia--Group51.png"
 +
                                                    width="100%"
 +
                                                    data-alt="//2016.igem.org/wiki/images/4/41/T--Slovenia--201607192_P3_FAS_TRPC1200V.gif">
 +
                                            </div>
 +
                                            <div>
 +
                                                <!-- experiment: 20161002 2 MscS + Gvp 200V 90s -->
 +
                                                <div style="display: block; float: left; width: 20%;">
 +
                                                    <img class="gifGroup9"
 +
                                                        src="//2016.igem.org/wiki/images/c/c7/T--Slovenia--Group52.png"
 +
                                                        width="100%"
 +
                                                        data-alt="//2016.igem.org/wiki/images/0/00/T--Slovenia--201607192_P3_FAS_TRPC1200Vregion.gif">
 +
                                                </div>
 +
                                                <div style="display: block; float: left; width: 20%;">
 +
                                                    <img class="gifGroup9"
 +
                                                        src="//2016.igem.org/wiki/images/b/be/T--Slovenia--Group53.png"
 +
                                                        width="100%"
 +
                                                        data-alt="//2016.igem.org/wiki/images/6/60/T--Slovenia--201607192_P3_FAS_TRPC1200Vcontrol.gif">
 +
                                                </div>
 +
                                                <div style="display: block; float: left; width: 20%;">
 +
                                                    <img class="gifGroup9"
 +
                                                        src="//2016.igem.org/wiki/images/c/cb/T--Slovenia--Group54.png"
 +
                                                        width="100%"
 +
                                                        data-alt="//2016.igem.org/wiki/images/b/b9/T--Slovenia--201607192_P3_FAS_TRPC1200Vheatmap.gif">
 +
                                                </div>
 +
                                            </div>
 +
                                        </div>
 +
                                        <figcaption><b>P3:FAS:TRPC1 channel improves sensitivity of cells for
 +
                                            ultrasound.</b><br/>
 +
                                            <p style="text-align:justify">(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 <a
 +
                                                        href="https://2016.igem.org/Team:Slovenia/Software">CaPTURE</a>.
 +
                                            </p>
 +
                                        </figcaption>
 +
                                    </figure>
 +
                                </div>
 +
                                <p style="clear:both">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.
 +
                                </p>
 +
                            </div>
 +
                        </div>
 +
                        <h3 class="ui left dividing header"><span id="ref-title" class="section colorize">&nbsp;</span>References
 +
                        </h3>
 +
                        <div class="ui segment citing" id="references"></div>
 +
                    </div>
 +
                </div>
 +
            </div>
 +
        </div>
 +
    </div>
 +
</div>
 +
 
 +
<script>
 +
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 +
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 +
</script>
 +
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 +
    <a href="//igem.org/Main_Page">
 +
        <img border="0" alt="iGEM" src="//2016.igem.org/wiki/images/8/84/T--Slovenia--logo_250x250.png" width="5%"
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 +
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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