Difference between revisions of "Team:Slovenia/Mechanosensing/Gas vesicles"

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<h1 class = "ui left dividing header"><span class="section">&nbsp;</span>Enhanced Mechanosensitivity by Gas Vesicles expressed in Mammalian Cells</h1>
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<div class="main ui citing justified container"><h1 class = "ui left dividing header"><span class="section">&nbsp;</span>Enhanced Mechanosensitivity by overexpressed <br/>Mechanosensitive Channels</h1>
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<p><b><ul><li>Addition of synthetic lipid microbubbles improved the responsiveness of cells to low-power ultrasound.
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<p><b><ul><li>Ectopically expressed mechanosensitive ion channels MscS and P3:FAStm:TRPC1 were used to enhance sensitivity of mammalian cells to ultrasound stimulation.
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<li>Gas vesicle-forming proteins were expressed in mammalian cells where they improved sensitivity of cells to ultrasound.
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<li>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.
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<li>Combination of the ectopic expression of the mechanosensing bacterial channel MscS and gas vesicle-forming proteins sensitized cells to mechanical stimulation.</ul></b>
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<h3><span id = "motivation" class="section"> &nbsp; </span></h3>
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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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<p>For activation of mechanoreceptors TRPC1 or MscS, a high-power ultrasound wave (900 Vpp) is required. Our aim was to improve responsiveness of cells to respond to the lower power of ultrasound as this would increase the selectivity, avoiding stimulation of endogenous channels and prevent cell damage. We decided to test gas-filled lipid microbubbles, since it has been reported that microbubbles can amplify the ultrasonic signal <x-ref> Ibsen2015 </x-ref>.</p>
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in the response to mechanical stimulation in human and bacteria <x-ref>Haswell2011, Ye2013</x-ref>.
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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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Microbubbles are small gas-filled lipid vesicles which are used as contrast agents in medicine. Their size is in the range of micrometers. They work by resonating in an ultrasound beam, rapidly contracting and expanding in response to the pressure changes of the sound wave<x-ref>Blomley2001</x-ref>.  
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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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Ibsen et al. <x-ref>Ibsen2015</x-ref> have used microbubbles for transduction of the ultrasonic wave in order to make neurons of <i>C.elegans</i> sensitive to ultrasound.
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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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<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.</b></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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<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>
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<h1><span id = "results" class="section"> &nbsp; </span>Results</h1>
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<h1><span class="section">&nbsp;</span>Results</h1>
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<h4><span id = "results" class="section"> &nbsp; </span>Microbubbles</h4>
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<h4><span class="section">&nbsp;</span>Localization and expression</h4>
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<div style = "float:right;">  
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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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<figure data-ref="3.3.1.">
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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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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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<figcaption><b>Synthetic lipid microbubbles.</b><br>(A)Schematic of a cell with an increased sensitivity to ultrasound stimulation in the presence of microbubbles. When exposed to mechanical stimuli the microbubbles contract and expand, resulting in activation of mechanosensitive channels on the cell membrane. (B) Mixed size distribution of lipid microbubbles was obtained. Stabilized lipid microbubbles were prepared by sonication and detected by microscopy.
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<figcaption><b>Localization and expression of mechanosensitive ion channels MscS and TRPC1. </b><br/>
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(A) Scheme of bacterial ion channel MscS (upper) and human ion channel TRPC1 (lower).
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(B) Ion channel MscS localized to plasma membrane. (C) TRPC1 predominantly localized in the ER.
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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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<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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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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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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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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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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stained the exposed extracellular HA-tag but not the intracellular Myc-tag (<ref>4</ref>B).
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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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<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
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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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function as mechanosensors by exposing them to the ultrasound stimulation.
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<h4><span class="section">&nbsp;</span>Ultrasound stimulation</h4><br/>
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Further explanation ...
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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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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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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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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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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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<x-ref>King2013</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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<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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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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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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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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which was superior to the intensity based measurements, since it is independent of photobleaching and dye sequestration
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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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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
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decreases once the indicator binds calcium ions. By calculating the ratio of fluorescence emission intensities captured at 488 nm exaction
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(where the difference of fluorescence between the bound and free indicator is at its maximum), we could observe changes in intracellular
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calcium concentrations in real time.
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<p>We followed changes of calcium concentration after ultrasound stimulation in real time using ratiometric confocal microscopy. For processing of data we
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developed our software <a href="https://2016.igem.org/Team:Slovenia/Software">CaPTURE</a>, which automatically calculated the ratio between fluorescence
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intensities of FuraRed and Fluo-4 and presented the data as image and calculated values.
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<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).
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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.
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<p>Properties of microbubbles for example rigidity, are affected by the composition of the lipid membrane and the gas core. We prepared our lipid microbubbles from a mixture of DSPC:DSPE. Before sonication we added gas perfluorohexane (as described in the Protocols section), which facilitates compression and expansion of the microbubbles upon ultrasound stimulation (<ref>3.3.1.</ref>A). A heterogeneous mixture of microbubbles in the range from 5 to 100 <span>&#181;</span>m in size were generated by this procedure (<ref>3.3.1.</ref>B). Microbubbles are most effective in the size range corresponding to the resonance frequency of the ultrasound. However care has to be taken in the applied energy to prevent cavitation, that can sonoporate cell membranes.</p>
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<p style="clear:both">Application of microbubbles to cells expressing mechanosensitive channel MscS significantly improved calcium influx after mechanical stimulation using low-power ultrasound wave (450 Vpp) (<ref>3.3.2.</ref>).</p>
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<figcaption><b> MscS channel improves sensitivity of cells for ultrasound.</b><br/>  
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(A) Schematic representation of a stimulation sequence and (B) signal parameters used for stimulation.  
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(C) and (D )Cells expressing MscS showed increased sensitivity to ultrasound stimulation in comparison to the cells without exogenous mechanosensitive
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channel. HEK293 cells expressing MscS channels or control cells transfected with vector were stimulated with ultrasound for 10 s and calcium influx  
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was recorded in real time (D) using a confocal microscope. For comparison cells without ectopic MscS were used. Fluo-4 (D, green line) and Fura Red  
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dyes (D, red line) were used for ratiometric calcium imaging. (D) Ratio (blue line) was calculated from fluorescence intensities of Fura Red and Fluo-4  
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<figcaption><b> Ultrasound response of cells is amplified by the use of lipid microbubbles. </b><br/>(A) Presentation of the ultrasound stimulation sequence and (B) signal parameters used for the stimulation.  
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using <a href="https://2016.igem.org/Team:Slovenia/Software">CaPTURE</a>.</figcaption>
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(C, D) Lipid microbubbles strongly enhanced response of cells expressing MscS channels. HEK293 cells expressing MscS channels 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.
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<p>However, there are some drawbacks related to the use of lipid microbubbles, as their delivery requires injection into the selected tissue. Additionally lifetime of lipid microbubbles is limited in the tissue to tens of minutes and they need to be prepared freshly at least once a week.</p>
 
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<p>To overcome the described drawback we thought of alternative options. One idea that initially looked too crazy to work was to use genetically encoded gas vesicles that are produced in bacteria. Bacterial gas vesicles have been used as contrasting agents for ultrasonography in animals <x-ref> Shapiro2014</x-ref>. This demonstrated that ultrasound can have effect on gas vesicles. However adding bacterial gas vesicles instead of microbubbles would not solve the problem. The best solution would be if the gas vesicles could be produced in the functional form in mammalian cells. iGEM team OUC_China 2012 demonstrated that only two protein components were sufficient to prepare functional gas vesicles in <i>E.coli</i>. Since gas vesicles are compressible their size should vary with pressure variations in the ultrasound or by mechanical stimulus, which would strongly influence the cytoskeleton or cell membrane therefore they would likely amplify activation of mechano-channels.</p>
 
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Gas vesicles are stable gas-filled structures, which provide buoyancy in a wide variety of planktonic prokaryotes <x-ref> Buchholz1993</x-ref> Two structural proteins have been identified in the gas vesicles of cyanobacteria and the halophilic archaea. The major protein, GvpA, is small hydrophobic protein that forms the ribs of the gas vesicle wall, while the minor gas vesicle protein, GvpC, is larger, hydrophilic protein that stabilizes the structure<x-ref>Hayes1986</x-ref>.
 
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<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
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stimulation (<ref>8</ref>C), suggesting the importance of membrane localization in the function of mechanosensors.
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  <figcaption><b>Expression of gas vesicle forming proteins in mammalian cells. </b><br/> Expression of (A) GvpC and (B) GvpA protein was determined by Western blot using (A) anti-FLAG and (B) anti-AU1, respectively. Expected sizes (marked with arrow) are 22,5 kDa and 8,5 kDa for GvpC and GvpA, respectively.
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<p>HEK293 cells were transfected with plasmids expressing both gas vesicle forming proteins, GvpA and GvpC. We could obtain GvpC from the Registry and added to its characterization, while the plasmid for GvpA could not be recovered and its coding sequence was synthesized using mammalian codon usage. Expression of both proteins was confirmed by the western blot (<ref>3.3.3.</ref>) and colocalization was observed by confocal microscopy (<ref>3.3.4.</ref>).</p>
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<figcaption><b> GvpA and GvpC were both expressed in the cytosol and colocalized.</b><br/><p  style="text-align:justify;"> HEK293T cells were transfected with plasmids encoding gas vesicle proteins, GvpA and GvpC. 24 h after transfection cells were fixed, permeabilized and immunostained with anti-FLAG (upper row) and anti-AU1 (lower row). Both GvpA and GvpC are located in cytosol as expected. Colocalization is presented in the overlay picture.</p></figcaption>
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<figcaption><b> Cell viability was not affected by expression of gas vesicle forming proteins.</b><br/> HEK293 cells were transfected with GvpA and/or GvpC. 24 h after the transfection viability of cells was measured using trypan blue.
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<p>A toxicity test was performed in order to ensure that gas vesicles were not toxic to mammalian cells. <ref>3.3.5.</ref> shows that the viability of cells was not altered when expressing gas vesicle forming proteins.</p>
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<p style = "clear:both;">HEK293 cells expressing gas vesicle-forming proteins exhibited increased sensitivity to ultrasound stimulation, even in the absence of exogenous mechanosensitive channels (<ref>3.3.6.</ref>), which was most likely due to activation of the endogenous mechanosensitive channels in mammalian cells.</p>
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<figcaption><b>P3:FAS:TRPC1 channel improves sensitivity of cells for ultrasound.</b><br/>
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(A) Schematic presentation of a stimulation sequence and (B) signal parameters used for stimulation.  
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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
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<figcaption><b> Gas protein vesicles improve the sensitivity of cells to ultrasound.</b><br/>(A) Presentation of the ultrasound stimulation sequences for 900 Vpp (red) and 450 Vpp (grey) Vpp ultrasound waves and (B) signal parameters used for stimulation. (C, D) Genetically encoded gas vesicle-forming proteins greatly increased cell response at high-power (900 Vpp) in comparison to low-power ultrasound (450 Vpp). HEK293 cells expressing Gvps were stimulated with ultrasound for 10 s and calcium influx was recorded in real time (D). 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.
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<p>In order to maximize the sensitivity of cells to ultrasound, we transfected cells with a combination of mechanosensitive channel MscS and gas vesicle-forming proteins. By decreasing the power of ultrasound stimulation we demonstrated that cells expressing a combination of both ectopic channels and gas vesicles were activated as a result of the ultrasound stimulation (<ref>3.3.7.</ref>).</p>
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<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
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<figcaption><b> Coexpression MscS with Gvps in mammalian cells improves sensitivity of cells to ultrasound even at lower voltages.</b><br/>(A) Presentation of the ultrasound stimulation sequence and (B) signal parameters used for stimulation.
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(C, D) Co-expression of mechanosensitive channels and gas vesicle-forming proteins increased sensitivity to ultrasound stimulation in comparison to the cells without exogenous mechanosensitive channels.
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HEK293 cells expressing gas vesicle-forming proteins GvpA and GvpC with or without MscS were stimulated with ultrasound for 10 s and calcium influx was recorded in real time (D) using a confocal microscope. Changes in fluorescence intensity of calcium indicators Fluo-4 (green line) and Fura Red (red line) are shown. (D) Ratio (blue line) was calculated from fluorescence intensities of Fura Red and Fluo-4 using CaPTURE.</figcaption>
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<p style="clear:both;">To prove that calcium influx was the mediator of activation of mechanosensitive channels, we used an inhibitor of ion channels gadolinium (Gd3+), which has high charge density and similar ionic radius to Ca2+ .By blocking the pore of the channel it acts as an general inhibitor of calcium ion channels <x-ref>Bourne1982</x-ref> . As shown on <ref>3.3.8.</ref>, the addition of gadolinium prevented calcium influx triggered by ultrasound stimulation.</p>
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<figcaption><b> Calcium influx after ultrasound stimulation is mediated by activation of mechanosensitive channels.</b><br/>(A) Presentation of the ultrasound stimulation sequence and (B) signal parameters used for stimulation. (C, D) Gadolinium inhibits activation of mechanosensitive ion channels after ultrasound stimulation. HEK293 cells expressing gas vesicle-forming proteins GvpA and GvpC with or without MscS were treated with gadolinium (red line) or not (grey line) and stimulated with ultrasound for 10 s and calcium influx was recorded in real time (D). Changes in fluorescence intensity of calcium indicators Fluo-4 (green line) and Fura Red (red line) are shown. (D) Ratio (blue line) was calculated from fluorescence intensities of Fura Red and Fluo-4 using CaPTURE.</figcaption>
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<p><br /><br />This is the first time demonstration that that gas vesicle-forming proteins can be expressed in human cells and that they improve the sensitivity of mechanosensitive channels for ultrasound. Moreover when the gas vesicle-forming proteins were co-expressed with ectopic ion channels the calcium influx could be achieved with low-power ultrasound.</p>
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<p>Ultrasound is a type of mechanical stimulus, therefore we reasoned that cells might exhibit response also to other types of stimulus such as the touch. The next task was to couple the response of mechanosensitive channels to the mediator of signaling or to the genetically encoded reporter. Mediator of this activation are Ca2+ ions, which are sensed by different cellular proteins and which have been in the literature detected by several designed sensors. The mechanosensing device was then coupled to the calcium split luciferase reporter based on <a href="//2016.igem.org/Team:Slovenia/Mechanosensing/CaDependent_mediator">M13 and calmodulin</a> or can be coupled to the <a href="//2016.igem.org/Team:Slovenia/Protease_signaling/Split_proteases">protease split system</a> and response of cells was determined against mechanical stimulus in a <a href="//2016.igem.org/Team:Slovenia/Implementation/Touch_painting">Touchpaint implementation</a>.</p>
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Revision as of 16:23, 17 October 2016

Mechanosensitive channels

Project Achievements Medal requirements Idea

 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.

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 human 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.

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 it 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.

. 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 (3A) and the MscS channel with three transmembrane helices (3A) were expressed in HEK293T cells (3D). MscS was detected as the 31 kDa band. TRPC1 was observed at 60 kDa, which was lower t han expected. We observed that the membrane localization in HEK293 was more evident for MscS (3B) rather than TRPC1 (3C).

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 (4A), 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 own 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 (4B).

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, hyperlink to CC page) 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 the 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 abovementioned 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 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 (64). 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.

INSERT!!!
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 (8C), suggesting the importance of membrane localization in the function of mechanosensors.

INSERT!!!
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.