Part:BBa_K1965000
MscS
Introduction
This part was nominated for best new part award by iGEM Team Slovenia 2016. It contains the coding sequence for the E.coli small-conductance mechanosensitive channel, MscS. Its role is to mediate turgor regulation in bacteria and it is activated by changes in the osmotic pressure [1]. It has been previously shown that MscS forms a homoheptamer. Each subunit is 31kDa in size and contains three transmembrane helices (2A) with the N-terminus facing the periplasm and the C-terminus embedded in the cytoplasm [2].
MscS can be described as an important receptor, involved in the response to ultrasound stimulation. We used the MscS channel as a source of Ca2+ influx when stimulated with ultrasound (1).
Cells in resting stage with closed MscS channels in the plasma membrane (left). Upon ultrasound stimulation MscS channels open, leading to Ca 2+ influx (right).
Characterization
Expression and subcellular localization of MscS channels in HEK293T cells was inspected. HEK293T cells were transfected with plasmids encoding HA-tagged MscS channel and protein expression was confirmed by western blot analysis, while protein localization was investigated by confocal microscopy.
HEK293 cells were transfected with MscS. Expression by Western blot and localization by confocal microscopy were analyzed using anti-HA antibodies. (A) Scheme of bacterial ion channel MscS (B) Ion channel MscS is localized on plasma membrane. HEK293 cells were transfected with the MscS:HA plasmid. 24 h after transfection cells were fixed with paraformaldehyde, permeabilized and stained with anti-HA antibodies. Localization was confirmed with confocal microscopy. (D) Ion channels expressed in HEK293 cells. HEK293T cells were transfected with plasmids, encoding gas vesicle-forming proteins GvpA and GvpC. 48 h after transfection cells were collected, lysed and total protein concentration was measured. 50ng of proteins were loaded on SDS page gels. After separation by size, proteins were transferred to nitrocellulose membrane by western blot. Membranes were then immunoblotted with anti-FLAG or anti-AU1 antibodies.
After confirming MscS expression in HEK293 cells, we stimulated the transfected cells with ultrasound to verify and characterize channel activity. Our experimental setup included an in-house built hardware MODUSON connected to unfocused transducer Olympus V318-SU. To monitor cell response in situ and in real time we used standard ratiometric fluorescent calcium indicators Fura Red, AM and Fluo-4, AM, which can be easily detected with confocal microscopy. When activated, mechanosensitive channels open, leading to calcium influx, which in turn binds the fluorescent calcium indicators. The indicator conformation changes upon calcium binding, resulting in an increase or a decrease of fluorescence.
When cells transfected with a mock plasmid were stimulated with ultrasound, we did not observe calcium influx. On the contrary, when cells transfected with the plasmid, encoding the MscS channel were stimulated with ultrasound, we detected a significant increase in calcium influx (3).
(A) Schematic presentation of the stimulation sequence and (B) signal parameters used for stimulation. (C, D )Cells expressing MscS showed increased sensitivity to ultrasound stimulation in comparison to the cells transfected with a mock plasmid. HEK293 cells either expressing MscS or transfected with a mock plasmid were stimulated with ultrasound for 10 s and calcium influx was recorded in real time (D) using confocal microscopy. Changes in fluorescence intensity of calcium indicators Fluo-4, AM (green line) and Fura Red, AM (red line) are shown. The blue line represents the ratio of Fluo-4 and Fura Red intensities, indicating the increase in intracellular free calcium ions after ultrasound stimulation.
In an attempt to improve calcium influx, we co-transfected HEK293 cells with the MscS channel and gas vesicle-forming proteins GvpC (BBa_K1965003) and GvpA (BBa_K1965004). The voltage of ultrasound stimulation was decreased to 450 Vpp as higher voltage also causes calcium influx in cells expressing only gas vesicle-forming proteins (Read more) . By decreasing the voltage of ultrasound stimulation we successfully showed that only cells expressing both the MscS channel and the gas vesicle-forming proteins were activated as a result of ultrasound stimulation (4).
(A) Schematic presentation of the stimulation sequence and (B) signal parameters used for stimulation. (C,D) Co-expression of mechanosensitive channels and gas vesicle-forming proteins increased sensitivity to ultrasound stimulation in comparison to the cells without exogenous mechanosensitive channels. HEK293 cells expressing gas vesicle-forming proteins GvpA and GvpC with or without MscS were stimulated with ultrasound for 10 s and calcium influx was recorded in real time (D) using confocal microscopy. Changes in fluorescence intensity of calcium indicators Fluo-4, AM (green line) and Fura Red, AM (red line) are shown. The blue line represents the ratio of Fluo-4 and Fura Red intensities, indicating the increase in intracellular free calcium ions after ultrasound stimulation.
To verify that calcium influx was indeed a result of mechanosensitive channel activity , we used gadolinium (Gd3+), an inhibitor of ion channels, which is a trivalent ion of the lanthanide series. Due to its high charge density and similar ionic radius to Ca2+[3] it blocks the pore of the channel and therefore acts as an inhibitor of calcium ion channels. As shown in 5, the addition of the inhibitor prevented calcium influx after ultrasound stimulation, confirming that cell response was indeed dependent on the activity of mechanosensitive channels.
(A) Schematic presentation of the stimulation sequence and (B) signal parameters used for stimulation. (C,D) Gadolinium inhibits activation of mechanosensitive ion channels after ultrasound treatment. HEK293 cells expressing gas vesicle-forming proteins GvpA and GvpC with or without MscS were untreated (grey line) or treated with gadolinium (red line) and stimulated with ultrasound for 10 s. Calcium influx was recorded in real time (D) using confocal microscopy. Changes in fluorescence intensity of calcium indicators Fluo-4, AM (green line) and Fura Red, AM (red line) are shown. The blue line represents the ratio of Fluo-4 and Fura Red intensities, indicating the increase in intracellular free calcium ions after ultrasound stimulation.
References
[1]Perozo, Eduardo, and Douglas C. Rees. 2003. “Structure and Mechanism in Prokaryotic Mechanosensitive Channels.” Current Opinion in Structural Biology 13(4): 432–42.[2]Pivetti, Christopher D et al. 2003. “Two Families of Mechanosensitive Channel Proteins.” Microbiology and molecular biology reviews : MMBR 67(1): 66–85, table of contents.
[3]Bourne, G. W., & Trifaró, J. M. (1982). The gadolinium ion: A potent blocker of calcium channels and catecholamine release from cultured chromaffin cells. Neuroscience, 7(7), 1615–1622. https://doi.org/10.1016/0306-4522(82)90019-7.
Team UM-Macau 2024: Contribution on BBa_K1965000
In contrast to the previous project using MscS for environmental issue, this year we integrated MscS with Lamp2B, into a plasmid for eukaryotic use, generated exosomes from HEK293T, and transferred them to eukaryotic cancer cells, where ultrasound was used to induce calcium overload in the cells, which in turn mediated cancer cell death. Unlike this 2016 project, this year UM-Macau has taken the use of MscS beyond prokaryotes and used it for the first time to induce eukaryotic calcium influx - we have used a new plasmid that is suitable for eukaryotes and successfully validated its potential for inducing calcium overload in cancer cells, undoubtedly expanding the scope of application of MscS exponentially from BBa_K1965000.
Our project focuses on developing a novel approach by engineering exosomes to deliver essential proteins to cancer cells, stimulating calcium overloading and inducing cancer cell death. By introducing the MscS channels into cancer cells, we aim to generate calcium overload upon ultrasound stimulation. The ultrasound stimulation could precisely control cell death in tumors by opening the channels and leading to calcium ion influx. We utilized the exosomal transmembrane protein lamp2b, fused with our target proteins, to enhance the loading of MscS into the exosomes.
We used the supernatant of the successfully infected HEK293T cell to isolate the exosomes. And we confirm the production of exosomes by TEM. The halo-like structure is the exosomes, and the light dots shown on the background are salts.
We used DLS to provide valuable insights into the size distribution and polydispersity of exosomes, which typically range in size from 200 to 300 nanometers.
In our Western blot experiments, we could detect the CD81 exosome marker in the samples 1833-1 (1833: LentiV-Lamp2b/MscS/HA: EGFP/Neo).
The A2780 ovarian cancer cells treated with the exosome (1833: LentiV-Lamp2b/MscS/HA: EGFP/Neo) have an increase in necrosis, but there is no difference in apoptosis.
The NCI-H1299 lung cancer cells treated with the exosome (1833: LentiV-Lamp2b/MscS/HA: EGFP/Neo) have an increase in apoptosis (although not significant due to a single experiment), but there is no change in necrosis.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |