project

design

Introduction

Sense of Hearing

As founded in human’s ears, mechanosensitive (MS) channels taking responsibility for the sense of hearing. [6] In plants, cell membrane-associated proteins (also belonging to the MS channels) are proposed to be associated with sound sensing. [7] They are founded in response to sound of specific frequency and intensity. [8]

Some researches have been made in discovering the relationship between MS channels and sound, but these experiments have been done on cell level. Most of researchers employed ultrasound, which might cause cell damage during experiments.[10] [9] It is hard to explain how the ears can hear sound by sensing the weak and low-frequented vibration. In our project, we chose a kind of MS channel, TRPC5 (found in the central nervous system) as researching object. Ultrasound as well as normal sound was used to test the response of MS channel. We hoped we could make some tiny contributions in solving this problem and help the hearing impaired. Additionally, we expected to select some mutated MS channels that sensitive to distinctive sound frequency.

Gene regulation

Gene expression is a complex and stochastic process involving numerous progresses and various reaction steps.[1] With continuing development in synthesis biology, a large amount of methods are developed to regulate gene expression artificially, including the most common two regulation systems, chemical genetics [2] and optogenetics [3, 4]. In chemical genetics small molecular drugs such as aTc are used to induce the gene expression, while it lacks spatial specificity and the concentration of small chemical in the cell is not stable. Optogenetics employs light as an input signal to induce gene expression. However, there are the side effect from laser-induced heating and the abnormal ion distribution caused by over-expressed pumps or channels. What’s more, undesired network homeostasis can make experimental interpretation very difficult [5].

Based on the previous study, we hope to find out a mechanical channel which can response to specific soundwaves and to realize the audio-genetics. Compared to the methods above, sound signal is easier to generate and less harmful to research objects.

Study Overview

In our study, audio-genetics is defined as a method of gene regulation by sound frequency precisely and safely. The response to the external signal is executed through a mechanosensitive channel (TRPC5 [11-13] or piezo channel[14, 15]). Different from the other MS channels, they open the gate to selectively permeate calcium when the force applied. The influx of calcium promotes the activation of TRPC5 channel and more calcium will influx into cytoplasm [4]. Because of the less complex structure of TRPC5, we applied the directed mutation within selective region, hoping a definite response could establish.

Piezo channel has been proved more sensitive to mechanical force, so we considered it more probable used in audio-genetic. Since the structure of it is still unsolved yet, directed mutation could not be operated. It was involved in our study as a test component.

To measure the activation level (quantify the intracellular calcium), we employed a fluorescent indicator, R-GECO. It is an artificial calcium indicated deriving from GCaMP. After it binds with calcium, it emits red fluorescent of about 600nm wavelength [16].

Calcium in cytoplasm regulates a series of phosphorylation. We chose the specific promoter (PNFAT) as an inductive element to regulate the downstream transgene expression. For the convenience, green fluorescent protein (GFP) was employed as the output signal to quantitatively analyze the regulatory ability of our audio-genetic system [4].

We tested the system above in CHO-K1 cell line. It can be cultured suspended and grows fast. It is convenient for us to test the channel response to sound wave in different cases. Moreover, to quantify the sensibility of those MS channels to mechanical stress, we firstly active those MS channels by microfluidics [17] and hypoosmolarity [18] to observe the calcium influx level. Then, we applied sounds with various frequencies and intensities by using commercial sound generators such as a buzzer, balanced armature, horn, ultrasound and atomizer.

The successful audio-genetics will reshape the landscape of current methods of gene regulation and this novel technology will open a new door to audio control of gene expression, neuronal activities and other biological process in cells or organisms.

Plasmid construction562x723px

1. R-GECO is derived from GCaMP, a genetically encoded calcium indicator. After it binding with calcium ion, it can emit fluorescence with wavelength of approximately 600nm [1].

We use it to quantify the intracellular calcium after mechanical stimulation on CHO-k1 cell by using live cell imaging. Due to the immediate expression of R-GECO, it can only be used in short-term test and observation.

2. pBX087-NFAT-GFP

Piggybac is a transposon which could promote the integration of PNFAT -GFP to genome.

P-NFAT is a promoter that could be activated by NFAT(nuclear factor of activated T cells).

After Ca2+ entering cell, it will activate calmodulin. The serine/threonine phosphatase calcineurin will then be activated, which dephosphorylates the serine-rich region and Serine-proline repeats in the amino terminus of NFAT. This activates a conformational change that exposes a nuclear localization signal, results in nuclear import, and enables binding of NFAT to specific promoters (PNFAT) and cooperation with resident transcription factors to induce transgene expression [2].

In the lab, we introduced GFP269x312px as a indicator of PNFAT downstream expression. After Ca2+ entering cell, the cascades could be activated, which could finally generate green fluorescence and demonstrate the result. Since the green fluorescent protein could express continuously, it was used for long-term test and observation.

3. Original Piezo1 plasmid was provided by Professor Maojun Yang from Tsinghua University. We used restriction enzyme digestion to insert it into a new backbone.

It is a mammalian mechanosensitive channel. Piezo1 activity triggered by traction forces elicited influx of Ca2+ [3]. Piezo1 has broad roles in multiple physiological processes, including sensing shear stress of blood flow for proper blood vessel development [4,5].

We employed various mechanical forces to stimulate the CHO-k1 cells transfected with Piezo1 plasmid, adding doxycycline to promote TetON expression.

4. pBX097-NeoLoxp was constructed by connecting the backbone(pBX097) which has piggybac sequence with the NeoLoxp fragment.

Piggybac is a traditional transposon system that can help to integrate gene into multiple sites of cell’s chromosome.

NeoLoxp is the combination of neomycin antibotic gene with Loxp sequence.

Neomycin gene could be used in later cell screening to get successfully transfected cell.

Loxp is a key component in Cre-loxp system. This figure below elucidates it’s principle briefly.

In the lab, we firstly used Piggybac system to integrate this plasmid containing Loxp into chromosome. After that, quantitative PCR was used to select cells with single copy of Loxp. This228x265px159x277px Loxp was then used to transfect TRPC5 into cell’s chromosome through Cre-Loxp recombination system. Thus, we could compare the sensitivity of TRPC5 toward mechanical force quantitatively.

5. One part of TRPC5 was synthesized by IDT, another part was synthesized by Wuxi Qinglan Biotech Cooperation. We use polymerase chain reaction and restriction enzyme digestion to construct a complete and useful TRPC5 plasmid.

Transient receptor potential channel 5(TRPC5) is also a mechanosensitive channel. It’s able to sense the mechanical force and open the channel gate to selectively permeate calcium ion. Also,553x259px the influx of calcium ion can promote the activation of TRPC5 channel, thus more calcium will influx into cytosolic [2].

Similar to the experiment of Piezo1 plasmid, we used mechanical force including shear force, ultrasound vibration to stimulate the cells transfected with wild-type TRPC5 channel. Since it’s not so sensitive compared with Piezo1, we’ve done random mutagenesis in the ankyrin repeats of TRPC5, trying to screen the mutated plasmids which are quite sensitive to mechanical force.

References:

[1] Zhao, Y., et al., An expanded palette of genetically encoded Ca(2)(+) indicators. Science, 2011. 333(6051): p. 1888-91.

[2] Ye, H., et al., A synthetic optogenetic transcription device enhances blood-glucose homeostasis in mice. Science, 2011. 332(6037): p. 1565-8.

[3] Pathak, M. M., Nourse, J. L., & Tran, T., Stretch-activated ion channel Piezo1 directs lineage choice in human neural stem cells. PNAS, 2014. 111(45): p. 16148-16153.

[4] Ranade, S. S. et al. Piezo1, a mechanically activated ion channel, is required for
vascular development in mice. Proc. Natl Acad. Sci. USA , 2014. 111: p.10347–10352.

[5] Li, J. et al. Piezo1 integration of vascular architecture with physiological force.
Nature, 2014. 515: p. 279–282.

Measurement

Test of R-GECO

GCaMPs consists of a circularly permuted enhanced green fluorescent protein (such as EGFP), which is flanked on one side by the calcium-binding protein calmodulin and on the other side by the calmodulin-binding peptide M13. In the presence of calcium, calmodulin-M13 interactions elicit conformational changes in the fluorophore environment that leads to an increase in the emitted fluorescence. (Fig. 1a). With a series of artificial modification, a group of calcium indicators, GECO family, was created. They are useful in the study of neuronal activity and cell image. We could use histamine to induce calcium influx in HeLa cell and the test the efficiency of R-GECO[19] (Fig. 1b) However, there is no H1 receptor in CHO-K1 cell membrane so we employed the following methods.

553x394px

Fig. the mechanism and high sensitivity of calcium indicator. a) EGFP is flanked on one side by the calcium-binding protein calmodulin and on the other side by the calmodulin-binding peptide M13. In the presence of calcium, calmodulin-M13 interactions elicit conformational changes in the fluorophore environment that lead to an increase in the emitted fluorescence. b) Intensity versus time traces for transfected HeLa cells. [21]

1. Hyperpolarization

Potassium ion is involved to make a hyperpolarization and lead the increases in cytosolic free calcium [20].

553x258px

Need modified

Fig. the result of test of R-GECO. a) No KCl added. b) Add 200mM KCl at t=240s

2. Ionomycin

Ionomycin is an ionophore produced by the bacterium Streptomyces conglobatus. It is used in research to raise the intracellular level of calcium (Ca2+) by stimulating store-regulated cation entry.[22]

Fig. result (10 μM ionomycin in HHBSS)

(to be continued)

Test of TRPC5 and Piezo

Transient receptor potential (TRP) channels are from a diverse family of cation channels that respond to a variety of signals [23, 24]. For example, some are involved in sensory perception and are directly activated by chemical ligands and/or physical sensory stimuli such as temperature, mechanical and osmotic stresses. Others are activated downstream of receptor stimulation through a phospholipase C (PLC)-dependent pathway. TRPC5 is a calcium permeable cation channel predominantly expressed in the CNS, There is an impressive array of other activators of TRPC5 channels, such as nitric oxide, lysophospholipids, sphingosine-1-phosphate, reduced thioredoxin, protons, lanthanides, and calcium, and many can cause its direct activation. Moreover, TRPC5 shows constitutive activity, and it is responsive to membrane stretch and cold. Thus, TRPC5 channels have significant potential for synergistic activation and may serve as an important focal point in calcium signaling and electrogenesis. The range of biological functions of TRPC5 channels is also impressive, from neurotransmission to control of axon guidance and vascular smooth muscle cell migration and contractility [11].

Piezo protein is a functionally diverse mechanosensitive cation ion channel that in humans is encoded by the gene PIEZO1. It is expressed in the lungs, bladder and skin, where mechanosensation play an important biological roles. It also has broad roles in multiple physiological processes, include including sensing shear stress of blood flow for proper blood vessel development, regulating red blood cell function and controlling cell migration and differentiation.[14]

Directed evolution of TRPC5

TRPC5 structure

Here we randomly mutated TRPC5 directly. To get a good mutation of TRPC5 with high sensitivity to mechanical stress, we need find out the specific fragment of TRPC5 coding sequence which related to stress sensing. Many researches had revealed the structure of TRPC5 [11, 25, 26]. (Fig. 6) Based on their study, we knew the Ankyrin repeats
[27] and pore region [25] S4-S5 linker were the crucial positions for our directed evolution.

However, mutation at S4-S5 linker or pore helix would cause continuously Ca2+ influx and cell death, so it might not be appropriate to mutate at this transmembrane protein area [25]. Crystallographic studies have shown that multiple Ankyrin repeats could form a helical structure, which might act as a gating spring connected to cytoskeletal elements [18, 27], so we finally chose the Ankyrin repeating region as the mutated region.553x383px

Fig. 6 Structure of TRPC channels. Structural features of TRPC channels. See text for description of structural features. ANK, ankyrin-like repeats; CC-N and CC-C are N-terminal and C-terminal coiled-coil domains, respectively. The region shaded in gray at the extreme C-terminus is specific for TRPC 4 and 5 [12].

Construct library

We used GeneMorph II Random Mutagenesis Kit to make a library of Ankyrin repeats by error-prone PCR. (Fig. 7a) ( provided by Agilent Technologies Company) The GeneMorph II kit has been used to mutagenize plasmid DNA targets up to 6 kb in length. Its mutation rate can be controlled simply by varying the initial amount of target DNA in the reaction or the number of amplification cycles performed. This kit also allows researchers to choose the mutation frequency that is most appropriate for a particular application.

In directed evolution studies, mutation frequencies of 1~4 amino acid changes (2~7 nucleotide changes) per gene are commonly employed. Proteins with improved activities have also been isolated from highly mutagenized libraries exhibiting 20 mutations per gene.

Using the mutated (Ankyrin repeats) fragments, we compared the efficiency of three different ways (whole plasmid mutagenesis, Gibson assembly, ligation) to construct the mutated Ankyrin repeats fragments into intact plasmids.

Whole plasmid mutagenesis to construct the mutated Ankyrin repeats fragments into intact plasmids. It uses a pair of complementary mutagenic primers (the mutated Ankyrin repeats fragments) to amplify the entire plasmid in a thermocycling reaction using a high-fidelity non-strand-displacing DNA polymerase such as KOD polymerase, and the reaction generates a nicked, circular DNA. Gibson assembly is a method to combine more than ten DNA fragments based on sequence identity. It requires that the DNA fragments contain ~20-40 base pairs overlap with adjacent DNA fragments. The way of ligation is a method use T4 ligase to join dsDNA fragments with the same sticky end.

By experiments in different groups, we found out ligation method is the most efficient way to construct the mutated Ankyrin repeats fragments into intact plasmid s( about 0.8%).

Screening

To screen out the desire mutation of TRPC5 with high sensitive to mechanical stress, we first transfected the CHO-k1 cells with a great library mixture of TRPC5 mutation cloned in the same plasmid backbone. Then we use fluorescence activated cell sorting (FACS) to select the cells with high intensity of GFP, (Fig. 7b) because high sensitivity TRPC5 channel with induce more calcium influx into cytosolic under the same sound wave signal, and the high concentration of free calcium in cytosolic will induce more GFP expression in the downstream of specific promoter (PNFAT). To make sure each transfected cell only contain one mutated TRPC5. Otherwise, cells contain two or more copy number will be high sensitive the to mechanical stress because of overlapping effect.

We use Cre-Loxp system to firstly select the single cell colony only with one copy number of loxp after transfected by PiggyBac mediated multiplex gene transfer method[28], which can be done by with qPCR. Then transfect the cells with mutated TRPC5 that add a loxp sequence ahead, therefore, all cells will contain one copy of mutated TRPC5 and it is in the same position of genome of CHO-K1. We use vibration to stimulate transfected CHO-K1 cell that cultured in flask.

189x151px 361x149px

Fig. 7 the main experimental steps of an in vitro directed evolution process. a) Sequence diversity is created through a random mutagenesis step, (the symbol * represents point mutation). The library of DNA sequences is ligated into an expression vector. b) Fluorescence-activated cell sorting (FACS) enables the fluorescence measurement of individual cells and the separation of distinct subpopulations by electrostatic deflection.

Reference

1. Ozbudak, E.M., et al., Regulation of noise in the expression of a single gene. Nat Genet, 2002. 31(1): p. 69-73.

2. Nevozhay, D., et al., Negative autoregulation linearizes the dose-response and suppresses the heterogeneity of gene expression. Proc Natl Acad Sci U S A, 2009. 106(13): p. 5123-8.

3. Leifer, A.M., et al., Optogenetic manipulation of neural activity in freely moving Caenorhabditis elegans. Nat Methods, 2011. 8(2): p. 147-52.

4. Ye, H., et al., A synthetic optogenetic transcription device enhances blood-glucose homeostasis in mice. Science, 2011. 332(6037): p. 1565-8.

5. Hausser, M., Optogenetics: the age of light. Nat Methods, 2014. 11(10): p. 1012-4.

6. Muller, U., Cadherins and mechanotransduction by hair cells. Curr Opin Cell Biol, 2008. 20(5): p. 557-66.

7. Telewski, F.W., A unified hypothesis of mechanoperception in plants. Am J Bot, 2006. 93(10): p. 1466-76.

8. Qin, Y.C., et al., Biochemical and physiological changes in plants as a result of different sonic exposures. Ultrasonics, 2003. 41(5): p. 407-11.

9. Haswell, E.S., R. Phillips, and D.C. Rees, Mechanosensitive channels: what can they do and how do they do it? Structure, 2011. 19(10): p. 1356-69.

10. Mishra, R.C., R. Ghosh, and H. Bae, Plant acoustics: in the search of a sound mechanism for sound signaling in plants. J Exp Bot, 2016. 67(15): p. 4483-94.

11. Zholos, A.V., Trpc5. Handb Exp Pharmacol, 2014. 222: p. 129-56.

12. Vazquez, G., et al., The mammalian TRPC cation channels. Biochim Biophys Acta, 2004. 1742(1-3): p. 21-36.

13. Song, M.Y. and J.X. Yuan, Introduction to TRP channels: structure, function, and regulation. Adv Exp Med Biol, 2010. 661: p. 99-108.

14. Ge, J., et al., Architecture of the mammalian mechanosensitive Piezo1 channel. Nature, 2015. 527(7576): p. 64-9.

15. Pathak, M.M., et al., Stretch-activated ion channel Piezo1 directs lineage choice in human neural stem cells. Proc Natl Acad Sci U S A, 2014. 111(45): p. 16148-53.

16. Zhao, Y., et al., An expanded palette of genetically encoded Ca(2)(+) indicators. Science, 2011. 333(6051): p. 1888-91.

17. Li, J., et al., Piezo1 integration of vascular architecture with physiological force. Nature, 2014. 515(7526): p. 279-82.

18. Shen, B., et al., Plasma membrane mechanical stress activates TRPC5 channels. PLoS One, 2015. 10(4): p. e0122227.

19. Sauve, R., et al., Oscillatory activation of calcium-dependent potassium channels in HeLa cells induced by histamine H1 receptor stimulation: a single-channel study. J Membr Biol, 1987. 96(3): p. 199-208.

20. Ratz, P.H., et al., Regulation of smooth muscle calcium sensitivity: KCl as a calcium-sensitizing stimulus. Am J Physiol Cell Physiol, 2005. 288(4): p. C769-83.

21. Tian, L., et al., Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat Methods, 2009. 6(12): p. 875-81.

22. Morgan, A.J. and R. Jacob, Ionomycin enhances Ca2+ influx by stimulating store-regulated cation entry and not by a direct action at the plasma membrane. Biochem J, 1994. 300 ( Pt 3): p. 665-72.

23. Flockerzi, V., An introduction on TRP channels. Handb Exp Pharmacol, 2007(179): p. 1-19.

24. Ramsey, I.S., M. Delling, and D.E. Clapham, An introduction to TRP channels. Annu Rev Physiol, 2006. 68: p. 619-47.

25. Beck, A., et al., Conserved gating elements in TRPC4 and TRPC5 channels. J Biol Chem, 2013. 288(27): p. 19471-83.

26. Gaudet, R., TRP channels entering the structural era. J Physiol, 2008. 586(15): p. 3565-75.

27. Owsianik, G., et al., Structure-function relationship of the TRP channel superfamily. Rev Physiol Biochem Pharmacol, 2006. 156: p. 61-90.

28. Lu, X. and W. Huang, PiggyBac mediated multiplex gene transfer in mouse embryonic stem cell. PLoS One, 2014. 9(12): p. e115072.