WO2018069788A1

WO2018069788A1 - The method for improvement of responsiveness of cells to ultrasound and mechanical stimuli with gas vesicles and sensitised mechanosensors

Info

Publication number: WO2018069788A1
Application number: PCT/IB2017/056064
Authority: WO
Inventors: Roman Jerala; Mojca Bencina; Maja MESKO; Tina LEBAR; Jan LONZARIC; Tina FINK; Ziga STRMSEK; Fabio LAPENTA; Tjasa PLAPER; Katja LEBEN; Kosta CEROVIC; Estera MERLJAK; Nik FRANKO; Rok KRESE; Miha GRADISEK; Arne PRAZNIK; Nina JERALA; Lidija MAGDEVSKA; Samo ROSKAR; Ziga PUSNIK
Original assignee: EN-FIST CENTER ODLICNOSTI; Kemijski Institut
Current assignee: EN-FIST CENTER ODLICNOSTI; Kemijski Institut
Priority date: 2016-10-10
Filing date: 2017-10-02
Publication date: 2018-04-19
Anticipated expiration: 2019-04-10
Also published as: SI25290A; EP3523317A1

Abstract

The invention relates to the method of enhancement of cells' sensitivity to ultrasound or other, direct or indirect, mechanical stimulus. The method includes spontaneously assembled genetically encoded gas vesicle forming proteins expressed by cells coexpressing or not additional mechanosensory ion channels. Gas vesicles boost the effect of a mechanical perturbance inside the cell that affects mechanosensory channels and activates intracellular protin cascades. The invention relates to cells expressing gas vesicles with or without additionally expressing mechanosensnsitive ion channels.

Description

The method for improvement of responsiveness of cells to ultrasound and mechanical stimuli with gas vesicles and sensitised mechanosensors

Field of the Invention

The Invention relates to improvement of cells' responsiveness to ultrasound stimuli, direct and indirect mechanical stimuli. Improved sensitivity of cells is achieved by genetically encoded bacterial gas vesicles, which are expressed and self-assembled within mammalian cells. The Invention also relates to eukaryote cells containing spontaneously reconstituted gas vesicles. Due to expression of proteins which form gas vesicles, sensitivity of cells' mechanoreceptors increases.

Spontaneous formation of protein gas vesicles in eukaryote cells increases sensitivity of endo- or exogenous mechanoreceptors - membrane channels allowing for an influx of ions in response to mechanical stimuli. Opening of the channels triggers transcription of target genes, secretion of molecules or activation of membrane action potential of cells such as neurons, which is useful for treatment of neural diseases, hormonal disorders, metabolism and flow disorders and other diseases and disorders. The Invention is useful for regulation of human, animal or plant cells, signal transfer to different cells, secretion of peptides, proteins and other molecules and mechanical signal detection.

State of the Art

Time and space defined activation of target cells is an important technical challenge. It can be performed with chemical activators, change of temperature or pH, use of electrodes or light stimuli. Cell activation is can be performed with direct or indirect mechanical stimuli such as touch, shear forces, liquid flow, hypo- and hyper-osmotic stress and ultrasound.

Sensory perception of mechanical signals within organisms is not fully explained, it is however mostly performed by mechanosensitive receptors, which are ionic channels opening as a result of mechanic forces acting on cell membranes, opening cell's channels and allowing an influx of ions, such as calcium ions, into the cell. Ion influx triggers cell responses such as neuron activation, transcription of target genes, secretion of target metabolites, proteins etc.

It has been reported that secretion of insulin from pancreatic beta cells can be stimulated by ultrasound - patent no. US2016/0236012. However, due to the high intensity of this ultrasound stimulus negatively influenced survival of the cells after longer exposures and could due to the high intensity activate other body cells. It has been shown that TRP4 expression in C.elegans cells made recipient cells ultrasound responsive - patent no. US2016/0220672. In that case, changed behavior of the animal with inserted TRP4; however, it could not have been precisely controlled. Measurable response required synthetic lipid microbubbles to be added to the cell suspension, which increased the response and made it detectable. C. elegans worms were surrounded by lipid microbubbles filled with air, which enables targeted stimulation of microscopic organisms such as C. elegans. In case of a human animal or plant administration of gas filled microbubbles would require injection into the target tissue. Addition of lipid microbubbles significantly improved the response of cell mechanosensors to ultrasound, but the microbubbles are complicated to use due to short life expectancy, which would require daily preparation, quick removal and a need to inject them into the target tissue.

A more suitable option would be air filled bubbles selectively expressed only in the target cells. Natural protein gas vesicles have been discovered in several unicellular prokaryotes, such as cyanobacteria B. megatherium and archaea living in water, where their function is to control buoyancy (Walsby, 1994 Microbiological Reviews, 58 (1): 94-144). Gas vesicles are spontaneously assembled units of proteins, where gas of the same composition and partial pressure as gases of the environment is gathered inside the vesicle due to its hydrophobicity of the inner side.

It has been discovered that the main components of gas vesicles in halophilic bacteria Planktotrix rubescens are proteins GvpA and GvpC. Functional reconstitution of gas vesicles in E. coli bacteria composed of expressed proteins GvpA and GvpC from Planktotrix rubescens was reported (Hayes and Powel, Arch.Micorbiol. 1995, 164:50-57.; Wang et al., J.Ocean Univ.China 2015, 14:84-88)). Gas vesicles were used as contrast agent for ultrasound in such a manner that they were inserted in the organism after isolation from bacteria and reflection of ultrasound waves on gas vesicles was detected. The response was further enhanced by application of large pressure which led the vesicles to collapse and improved the contrast (Patent no. US2014/0288421 Al, Shaipro et al, Nature Nanotechnology 2014, 9,311-316).

Technical problem

It is desired to stimulate only target cells within the organism for defined amount of time. Important parameters for selecting the manner of stimulation are spatial resolution, speed of stimulation and response, operation at distance (if possible even without direct contact with responding cells). Stimulation of the cells with chemical substances does not provide high spatial and time resolution, due to fast decrease of concentration of reagent and chemical signals affecting on the entire organism. Light enables very quick alteration between on and off states of the signal, but the tissue is not sufficiently permeable for visible light, therefore a source of light needs to be delivered in close proximity of target tissue. In case of stimulation of cells in tissues of multicellular organisms, surgical procedure is needed. Ultrasound enables not only spatial but also time resolution, because it can act organism-wide as the sound is conducted through the matter. Time course of ultrasound stimulation can be very well controlled and can be focused on chosen spatial segment by combining several sources of ultrasound waves. However, spatial and time resolution of activation of cells with ultrasound does not allow differentiation of individual types of cells within groups of cells, tissues and organisms.

One of previously described improvements are protein and lipid gas bubbles, which significantly improve mechanical stimulus (including ultrasound), but do not ensure spatial resolution. Moreover, the bubbles require invasive application in case of activation of a tissue in an organism.

It is desirable to improve responsiveness of the target cells to ultrasound and direct or indirect mechanical stimuli in such a way that certain degree, intensity and duration of stimulation solely or predominantly activate target cells.

Problem Solution

The Invention resoles the above mentioned problems. The Invention increases sensitivity of the target cells to stimulation by ultrasound and direct or indirect mechanical stimuli via expression of spontaneously assembled protein gas vesicles inside target cells.

It is the expression of genetically encoded protein gas vesicles, which are assembled in the cytosol that enables resolution on the cellular level. So far, there have been no reports of protein gas vesicles expression in mammalian cells and form functional gas vesicles without harmful effects on said mammalian cells.

Application of mechanical stimulus, such as ultrasound, causes the gas within gas vesicles to compress and expand changing the volume and position of the vesicles, resulting in an effect on the cytoskeleton, cell membrane and other cell components and increases activation of mechanosensitive channels, native or introduced to the cell. The channels let an influx of ions such as calcium, which activates the cell - Figure 1. Cell activation can result in mechanically stimulated exocytosis of proteins and cell metabolites and gene transcription. Not only the Invention solves the problem of selective activation of cells on distance by using ultrasound in deep in the tissue, but also the problem to detect direct and indirect mechanical stimuli acting on target cells, such as pressure on the cells due to pressure in the tissue.

Figure legends

Figure 1. Schematic display of increased responsiveness of mechanosensors by protein gas vesicles. A

- Components of bacterial protein gas vesicles spontaneously assemble into vesicles filled with air from surroundings. B - Application of mechanical stimulus, such as ultrasound, touch, fluid flow, osmotic stress and other direct or indirect mechanical forces, causes the vesicles to oscillate, contract and expand due to compressibility of gas consequently acting on cytoskeleton and cell membrane. By acting on the cell membrane, mechanosensitive ion channels are activated and opened, which lets the ions through the cannels. Ions serve as secondary messenger molecules and change in concentration of free ions, primarily calcium ions, triggers signal cascade, which results in increased expression of genes and secretion of cell metabolites, which may endogenous or of recombinant origin.

ure 2. Expression and localization of protein gas vesicles in cells. A - Western Blot of cell lysate of cells transfected with genes encoding components of protein gas vesicles, marked with anti-AUl antibodies and B - Western Blot marked with anti-FLAG antibodies. The arrows show signals of adequate size. C - Localization of protein gas vesicles. Cells transfected with genes, encoding components of protein gas vesicles, were fixated and stained with anti-AUl and anti-FLAG antibodies.

ure 3. Localization of recombinant ion channels in human cells. A - Localization of bacterial MscS ion channel. B - Localization of human TRP ion channels.

ure 4. A - Ultrasound activation of cells, which contain gas vesicles and mechanosensitive MscS ion channels. Legend: (a) Cells expressing mechanosensitive MscS channel without gas vesicle proteins; (b) Cells expressing mechanosensitive MscS channel and gas vesicle proteins; (c) Cells expressing mechanosensitive MscS channel and lipid bubbles. B - Ultrasound activation of cells. Ultrasound selectively activates cells, which contain Gvp protein gas vesicles and does not activate cells transfected only with the vector (negative control).

ure 5. Mechanical cell activation. A - Activation of cells expressing protein gas vesicles with 10 second ultrasound stimulation. B - Activation of cells with touch, achieved by adding liquid into wells with cells. Cells were grown in microtiter plate and were stimulated with injection of 5-20μ1 of liquid and registered activation by measuring luciferase activity.

ure 6. Gene expression as a result of cell activation by ultrasound and gas vesicles. A - Expression of ion channels TrpCl and MscS. Western blot of cell lysate of transfected cells expressing ion channels stained by anti-Myc and anti-HA antibodies. B - Calcium dependent luciferase reporter expression. Cells were activated by ultrasound 24h after transfection with protein gas vesicles components and with or without additional ion channel. Luciferase activation was measured after 6h. Detailed description of invention with examples

The invention relates to a method which increases cells' sensibility to mechanical stimuli, preferentially ultrasound or touch and involves the use of protein gas vesicles, which express and fold in cells interior. Method presented by the Invention includes proteins which spontaneously form protein gas vesicles. Proteins are chosen amongst those expressed in floating unicellular microorganisms like plankton, cyanobacteria, B. megatherium and archaea. The invention refers preferentially to proteins from Planktotrix rubescens, especially to GvpA and GvpC and homologous proteins from formerly listed organisms. This includes proteins with SEQ ID 2 and SEQ ID 4 and similar proteins with at least 30% homology. Besides protein gas vesicles the method includes expression of mechanosensitive nonselective ion channels for additional increase of cells sensibility to mechanical stimuli. Mechanosensitive ion channels involved in the invention originate primarily from either bacteria or eukaryotes. Those from bacteria are preferentially MscS and MscL type, or other with SEQ ID 6 and SEQ ID 8. Mechanosensitive ion channels from eukaryotes are TRP type, namely TRPC, TRPV and TRPA, especially TRPC1, TRPC3, TRPV1 and TRPV1 or other with SEQ ID 10, SEQ ID 12 and SEQ ID 14. Mechanosensitive ion channels can be linked to protein gas vesicles directly or indirectly by use of dimerization domains.

The invention relates to a method which increases calls' sensibility to mechanical stimuli, preferentially ultrasound, fluid flow and touch and includes use of protein gas vesicles, which are spontaneously formed in cytosol and can be linked on mechanosensitive nonselective ion channels.

The invention relates to cells containing protein gas vesicles and optionally mechanosensitive ion channels for detection of mechanical stimuli. The cells can sense and respond to mechanical stimuli not only such as sound but also osmotic changes, mechanical pressure and flow of fluids surrounding the cells.

The invention relates to cells expressing gas vesicles and optionally mechanosensitive ion channels, which can be used for activation with ultrasound and other mechanical stimuli. Stimulation with above mentioned mechanical stimuli leads to formation of activation potential, which can be presented in a variety of ways such as (i) release of calcium in and out of the cells, essential for activation of muscle cells and neurons; (ii) release of endogenous neurotransmitters, cell metabolites (hormones); (iii) regulation of gene expression.

The invention relates to cells chosen amongst eukaryotic or plant cells, human cell lines or cells from fungi. Invention refers especially to cells, chosen from mammalian cells and human cell lines, preferentially neuronal or other cells from neural system, beta cells or pancreatic beta cells, antigen presenting cells T-lymphocytes or other cells from immune response, that include protein gas vesicles and optionally mechanosensitive ion channels.

Above described cells containing spontaneously formed protein gas vesicles with or without non-specific mechanosensitive ion channels exhibit increased sensibility to sound waves like ultrasound and other mechanical stimuli such as direct or indirect pressure, change in air pressure or fluid flow.

The invention relates to cells expressing protein gas vesicles and optionally mechanosensitive receptors that can be activated by mechanical stimulation, resulting in changing the state of proteins, preferentially calcium binding proteins, preferentially proteins that impact the state of the cell, preferentially signalling the state by changes in fluorophore characteristics or light emission, including genes for transcriptional activation or excreting peptides and other hormones.

Definitions:

The term "protein gas vesicles" refers to gas vesicles that are spontaneously formed by specific proteins inside the cell. In regards to this inventors refer preferentially to protein gas vesicles from unicellular proteins, which regulate organisms' buoyancy. It involves proteins from five families of bacteria, two groups of archaea, cyanobacteria, limited on plankton microorganisms. The size of formed protein gas vesicles ranges from 45 to 200 nm. Formation of protein vesicles involves several proteins spontaneously creating structures that capture cellular gasses.

The term "mechanical stimuli" used here refers to all kinds of mechanical disruptions/forces such as ultrasound, touch, osmotic stress, friction due to fluid flow and shear stress. The result of mechanical stimulation leads to changes in speed of fluids that affects cells or cellular membrane. Mechanical stimuli can occur due to pressure of specific hard object, fluid or other cells on cellular membrane, due to gravitational, centrifugal, shear and other direct force or indirect forces such as osmosis, expansion or contraction due to temperature or other factors, which cause effects on cellular membrane.

The term "ultrasound" used here denotes sound waves with frequencies between approximately 20 kHz and 15 MHz. Sound waves are generated from ultrasound generator which allows us to control intensity or amplitude of sound wave, frequency in combination with adequate converter and plan for time dependent ultrasound pulses.

The term "cell" used here refers to eukaryotic cell on unicellular or multicellular organism (cell line) cultivated like unicellular entity which is or was used as a recipient of nucleic acid. It also refers to daughter cells of original cell, which was genetically modified with nucleic acid. The term refers to cells from higher developed eukaryotic organisms such as vertebrates, preferentially mammals. The term "cells" refers also to human cell lines and plant cells. Daughter cells are not necessarily completely identical parental cells in morphologic shape and in total DNA complement, due to natural, coincidental or designed mutations. "Genetically modified host cell" (also "recombinant host cell") is host cell in which the nucleic acid was introduced. Eukaryotic genetically modified host cell is produced by insertion of nucleic acid or recombinant nucleic acid in appropriate eukaryotic host cell. The invention further includes host cells and organisms that contain nucleic acid of the Invention (transient or stable), that hold the sequence for operons of the present invention. Appropriate host cells are presented in state of technique and include eukaryotic cells. It is known that protein expressed in mammalian cells can originate from following organisms: human, rodent, cattle, pork, poultry, rabbits and similar. Host cells can be grown as cell lines from primary or immortalised cell lines.

The term "nucleic acid" used here refers to polymeric form of nucleotides of variable lengths, ribonucleotides or deoxy ribonucleotides.

The term "polypeptide", "protein" and "peptide" used here refer to polymeric form of amino acids of variable lengths, which include coding amino acids. The term "functional polypeptide" used here refers to polypeptide form of amino acids of variable lengths, which express any kind of function such as: structure formation, guiding on specific localization in the cell, targeting of specific organelles, ease or activation of chemical reactions and binding to other functional polypeptides.

The term "chimeric protein" used here has a general meaning and refers to polymeric form of amino acids of variable lengths, composed of more than one protein/domain/segment, optionally one connected with the other by linker of variable length, preferentially composed from 1 to 40 amino acids.

The term "heterologous" used here in the context of genetically modified host cells, refers to polypeptide for which applies at least one from the following statements: (a) polypeptide is foreign for host cell (host cell in nature doesn't contain the polypeptide - "exogenous"); (b) polypeptide is present in nature in specific host organism or host cell ("endogenous"), but is expressed in unusual amounts in the cell (more than expected or in higher amounts that is found in nature) or differs in nucleic sequence from endogenous nucleic sequence so that remains the same protein as endogenous (has the same or considerably similar amino acid sequence), produced in unusual amounts in the cell (more than expected or in higher amounts that is found in nature).

The term "homologous" used here refers to proteins or nucleic acids with preserved amino acid or nucleic sequence, preferentially at least 50% similarity, with minimal 20% conservation, determined by protein or nucleic acids comparison techniques that are known to experts on related scientific fields. Homologous proteins are characterized by carrying out same function in the cell. Homologous nucleic acids are coding for homologous proteins.

The term "recombinant" used here means that specific nucleic acid (DNA or RNA) is a product of various combinations of cloning, restrictions and/or ligations, which lead to a construct that has the structure of a coding or non-coding sequence, different from endogenous nucleic acids in natural systems. Generally, the DNA sequence, that codes structurally coding sequence, can be comprised from cDNA fragments, short oligonucleotide linkers or a series of synthetic oligonucleotides, from which we gain synthetic nucleic acid that, can be expressed by transcriptional machinery inside the cells or from non-cellular transcription and translation system. So acquired sequence can be used in form of open reading frame without disruption of transcription and translation due to internal non-translated sequences or introns, which are naturally present in eukaryotic genes. Genomic DNA with important sequences can also be used for formation of recombinant gene or transcriptional unit. Sequences of non-translated DNA can be located on 5' or 3' end of open reading frame where these sequences don't affect the manipulation or expression of coding regions and can serve as production modulators of target products by various mechanisms.

The introduction of vectors in host cells is achieved by conventional methods known from state of technology, referring to those of transformation and transfection that include: chemical introduction, electroporation, microinjection, DNA lipofection, cellular sonication, gene bombarding, viral DNA delivery and others. The introduction of DNA can be transient or stable. Transient DNA insertion refers to introduction of DNA with vector, which doesn't insert the DNA of the present invention into host genome. Stable introduction is achieved by insertion of DNA of the present invention into host genome. The introduction of DNA of the present invention for production of host organism with stably inserted DNA of the present invention can be controlled with the presence of markers. DNA sequence for markers refers to genes coding for proteins involved in resistance to antibiotics or chemicals and can be included on the same vector as DNA of the present invention or on separate vector.

The use of method and cells originating from invention is extremely diverse. Activation of cells (such as neurons, antigen presenting cells, B-cells, T-cells) with ultrasound or other mechanical stimuli leads to activation potential expressed in ways like (i) release of calcium in and out of the cells, essential for activation of muscle cells and neurons (ii) release of endogenous neurotransmitters, cell metabolites (for example hormones), (iii) regulation of gene expression. Cells originating from invention detect besides ultrasound also osmotic changes and movement of surrounding fluids. Examples of implementation which we will further describe are designed to present the invention to the best extend. These descriptions don't have a purpose to limit the scientific field of invention and its usability, however carry a purpose to increase understanding of invention and its use.

Examples

Example 1. Preparation of cells, expressing protein gas vesicles

Preparation of DNA constructs for protein gas vesicles and mechanosensitive ion channels

For DNA constructs preparation the inventors used methods of molecular biology including: chemical transformation of competent E.coli bacterial cells, plasmid DNA isolation, DNA amplification with polymerase chain reaction (PCR), reverse transcription - PCR, ligation with PCR, determination of nucleic acid concentration, DNA electrophoresis with agarose gel, isolation of DNA fragments from agarose gel, chemical synthesis of DNA, DNA restriction with restriction enzymes, restriction of plasmid vectors, ligation of DNA fragments, cleaning of plasmid DNA in higher amounts. Precise procedures of experimental techniques and methods are well known to experts on these scientific fields and are described in manuals for molecular biology.

For all experimental work inventors used sterile work techniques, which are also well known to experts on these scientific fields. All plasmids, full and partial were transformed in E.coli bacteria with chemical transformation. Plasmids for transfection in cell lines (mammalian, plant or human) were isolated using DNA isolation kit that eliminates endotoxins.

Final gene constructs and proteins of the present invention are listed in table 1. All operons were prepared with techniques by methods known to experts. Operons were inserted into suitable plasmids, appropriate for eukaryotic systems. Suitability of operons nucleotide sequences were confirmed by inventors with DNA sequencing and restriction analysis. Table 1 : Fusion proteins used for demonstration of the invention

Seq name plasmid construct description

ID. vector

1, 2 AUlrGvpA pcDNA3 Protein GvpA with N-terminal peptide tag AU1.

3,4 3FLAG:GvpC pcDNA3 Protein GvpC with N-terminal FLAG peptide tag.

5,6 MscS:HA pcDNA3 Mechanosensitive ion channel MscS with C-terminal

HA peptide tag.

7,8 TrpCl :Myc pcDNA3 Mechanosensitive ion channel TrpCl with C-terminal

Myc peptide tag.

9,10 HA:Fas:GS30:TrpCl:Myc pcDNA3 Mechanosensitive ion channel TrpCl with N-terminal

HA peptide tag and Fas transmembrane domain and C- terminal Myc peptide tag. Fas domain and TrpCl channel are linked by flexible 30 amino acids long peptide linker (GS30)

11,12 Myc:nLuc:GS10:M13 pcDNA3 N-terminal part of split luciferase fused with Ml 3 peptide on C-terminal and N-terminal Myc protein tag.

NLuc and M13 are linked by flexible 10 amino acids long peptide linker (GS10).

13,14 Calmodulin(E104Q):GS10:cLuc:HA pcDNA3 C-terminal part of split luciferase linked to Calmodulin

(E104Q) on N-terminal and C-terminal HA peptide tag.

CLuc and Calmodulin (E104Q) are linked by flexible

10 amino acids long peptide linker (GS10).

15,16 Myc:NFAT pcDNA3 Transcriptional factor NFAT with N-terminal Myc peptide tag.

17,18 3xnfat_Pmin_Fluc pGL4.16 Reporter plasmid with three repeats of binding site for transcriptional factor NFAT upstream from minimal promoter and gene for reporter protein firefly luciferase.

Preparation of cells expressing protein gas vesicles and mechanosensitive channels Methods and techniques of cell cultivation are well known to experts on the field, therefore they are briefly described here only for the purpose of illustrating an example. We cultured HEK293 cell line at 37°C and 5% CO₂. DMEM culture medium with added 10% FBS, which contains all the necessary nutrients and growth factors, was used for cell cultivation. When the cell culture reached the appropriate density, cells were transferred to a new culture flask and/or diluted. For the use of cells in experiments, we counted the cells with a haemocytometer and seeded them at the density 2.5xl0⁴ of cells per well in a microliter plate (6, 12 or 96 wells) 18-24 hours before transfection. We incubated the seeded plates at 37°C and 5% CO₂, until the cells reached 50-70% confluency. Next the cells were transfected with a transfection reagent. We carried out the transfection according to the protocol from the manufacturer of the transfection reagent (for example JetPei, Lipofectamin 2000) and adjusted the volumes to the used microtiter plate.

Immunodetection of the protein

The protocols for the preparation of SDS-PAGE gels, the transfer of proteins to the membrane and the analysis with Western Blot are commonly known to experts; therefore they are described here only for the purpose of illustration. We added 4x reducing loading buffer with SDS to the samples (supernatant or partially purified proteins) and denatured them by heating them to 100°C for 5 min. Afterwards, we loaded the samples on the gel. We used SeeBluePlus (Fermentas) as the protein size standard. For the electrophoresis, we used the vertical Mini-Protean II system and a 10% polyacrylamide gel. The electrophoresis was performed in a lx SDS electrophoresis buffer for 45-60 min at constant voltage 200V. After the electrophoresis was done we removed the loading gel and used the separation gel for the Western Blot. We soaked the PAGE gel, filter papers and the nitrocellulose membrane in the buffer for wet transfer and assembled the apparatus for the wet transfer. The transfer was performed for 1 hour at a constant electric current 350 mA. We blocked the unspecific binding site on the membrane with 0,2% I- Block reagent dissolved in lxPBS/0,1% Tween-20. Blocking was performed either for 1,5 hours with shaking at room temperature or overnight at 4°C and gentle shaking. We incubated the nitrocellulose membrane in the blocking solution (0,2% I-Block reagent/lxPBS/0,1% Tween-20) with mouse monoclonal AntiHA, AntiMyc, AntiFLAG or AntiAUl primary antibodies (Qiagen). The antibodies were diluted in a ratio 1: 2000 and incubated either for 1,5 hours with gentle shaking at room temperature or overnight at 4°C and gentle shaking. After incubation, we washed the membrane (4x5min) with a wash buffer (lxPBS/0,1% Tween-20). Next, we incubated the membrane for 45 minutes at room temperature and gentle shaking in the blocking solution with secondary goat anti mouse antibodies, conjugated with horseradish peroxidase, diluted in ratio 1 : 3000. After washing the membrane with wash buffer (3x5 min) we incubated the membrane in Super Signal West Pico chemilummescent reagent for 5 minutes. The substrate contains luminol, which is oxidized by the horseradish peroxidase on the secondary antibodies. The oxidized luminol enters the excited state and when it passes to the ground state light is released and detected on the film.

Results: Figures 2 A and 2B show the expression of the gas vesicle protein and Figure 6 A shows the expression of the ion channel proteins. The results confirm the presence of mechanosensitive channels and the proteins necessary for the formation of gas vesicles in the lysate of mammalian cells, which were transiently transfected with plasmids with the sequences for their expression.

Localization of fusion proteins within the cells

The internalization of fusion proteins into cells was determined by confocal microscopy. The methods and techniques of work with a confocal microscope, fixation, staining of cellular proteins with antibodies for the display of proteins and the use of use of dyes for tagging cellular organelles are commonly known to experts. Here we describe only the details necessary for the demonstration of the invention.

The purpose of the experiment was to determine whether the expression and location of the fusion proteins was correct. We determined the localization with immunodetection with the use of antibodies against peptide tags and secondary antibodies conjugated with dyes.

We examined the tagged live or fixed cells under the confocal microscope Leica TCS SP5 on the Leica DMI 6000 CS stand. This microscope is intended for laser scanning of fluorescently labelled live or fixed cells. We used the 63x oil immersion objective and acquired the images with the LAS AF 1.8.0. Leica Microsystems program. Which laser is used depends on the wavelengths we wanted to use for exiting.

In order to check, whether the proteins necessary for the formation of gas vesicles and the mechanosensitive channels are expressed in mammalian cells, we transiently transfected the HEK293 cells, seeded in 6-well plates, with plasmids encoding gas vesicle proteins Au GvpA or 3FLAG:GvpC or the sequence for the mechanosensitive channels MscS:HA or TrpC Myc. Two days after transfection we collected and lysed the cells and performed immunodetection on a nitrocellulose membrane according to the protocol described above. For detection of the GvpA protein we resuspended the cell lysates in formic acid and dialyzed them against PBS, before preparing the samples for the polyacrylamide gel electrophoresis.

In another experiment, we transiently transfected HEK293T cells, seeded in microscope plates with 8 wells, with plasmids which contained the sequences for the proteins GvpA or GvpC or HA:Fas:HA:TrpCl:Myc or TRPV1 and TRPA1. One day after transfection we checked the localization of the proteins with confocal microscopy, according to the protocol described above.

Results of the spontaneously assembled gas vesicles (Figure 2) clearly show that all mentioned proteins are expressed in the mammalian cell line HEK293T.

The same is true for the expression of mechanosensitive ion channels. The mechanosensitive channel MscS:HA is localized on the membrane of cells (Figure 3A), other mechanosensitive channels TrpCl, TRPV1 and TRPA1 are present on the membrane in lower amounts (Figure 3B). We achieved the translocation to the membrane with the addition of the transmembrane Fas domain to the N terminal end of the protein TrpCl (fusion protein HA:Fas:HA:TrpCl:Myc). We also showed the localization of TRPV1 and TRPA1 (Figure 3B).

Example 2. Measuring responsiveness of cells to ultrasound

Sensing the activation of cells stimulated with ultrasound through calcium mediated luciferase activation

For sensing the activation of cells after ultrasound stimulation we developed a reporter system, based on split firefly luciferase Flue. The system is made of two fusion proteins nLuc:GS10:M13-P and Calmodulin-P:GS10:cLuc. In the absence of activation at physiological intracellular calcium concentrations, there is no binding of the M13 peptide on Calmodulin. Calcium influx after cell activation causes a conformational change in Calmodulin and the binding to the Ml 3 peptide. This results in reassembly of the split Flue and it regains its function. We transiently transfected HEK293 cells, seeded in 6 well plates, one day before the experiment with plasmids encoding for the proteins AUl:GvpA and 3FLAG:GvpC and with the reporter system described above. Before stimulation, both 4mM CaC12 and ImM luciferin were added to the medium. We stimulated the cells for 10s with ultrasound generator Moduson and immersion probe Olympus Panametrics V318-SU. The activity of assembled split Flue was monitored by a fluorescence monitoring device G:BOX (Syngene).

Results: From Figure 4B it is evident, that after ultrasound activation, the luciferase activity is higher in the eukaryotic cells, which express gas vesicles. This indicates their presence increases the sensitivity of cells to ultrasound stimulation through activation of endogenous ion channels.

»In situ« detection of cell activation through measuring intracellular calcium with dyes

We tested cell responsiveness to ultrasound by monitoring the changes in the intracellular calcium concentrations. Changes in calcium concentration were detected by ratiometric fluorescent dyes for calcium imaging Fura Red (Setareh Biotech) and Fluo-4 AM (Biotium) on the confocal microscope Leica TCS SP5, with a 40x oil immersion objective. One day after transfection we incubated the previously transfected HEK293 cells, which were transfected with plasmids encoding for mechanosensitive channel MscS:HA (lOOng) and gas vesicle proteins AUl :GvpA (250ng) and 3FLAG:GvpC (50ng), with a mixture of both dyes at 37°C for 45 min. After, 4mM CaCl₂ was added to the medium and cells were stimulated for 10s by the ultrasound generator Moduson and the immersion probe Olympus Panametrics V318-SU on 6 well plates. We determined the percentage of cells which responded to ultrasound stimulation with the program CaPTURE.

Results: Figure 4A shows the percentage of cells, which responded to the ultrasound stimulation and confirm the increased sensitivity of cells expressing the mechanosensitive channel MscS and the proteins AUl:GvpA and 3FLAG:GvpC which form gas vesicles. Compared to the alternative increase of mechanoreceptor sensitivity by addition of synthesized lipid bubbles, the intracellular gas vesicles show comparable increase of responsiveness to ultrasound stimulation.

Luciferase activity.

A test with two reporters was used for monitoring luciferase activity: (a) firefly luciferase Flue or split Flue and (b) Renilla luciferase Rluc or SEAP (Secreted Alkaline Phosphatase). Firefly luciferase Flue, which uses CoA, ATP, and luciferin as a substrate, is functionally linked to the NFAT promoter which senses the concentration increase of free calcium ions. The other reporter is transfected into the cells simultaneously with the plasmid encoding Flue and the plasmids encoding the investigated fusion proteins serves as a reporter for the efficiency of transfection. The reporter plasmid encodes Renilla luciferase Rluc, which uses coelenterazine as substrate or SEAP (Secreted Alkaline Phosphatase). Rluc or SEAP are expressed in cells independently of the conditions.

To analyze the expression of reporter proteins we lysed the cells with a buffer according to manufacturer's instructions (Promega). We first measured the activity of firefly luciferase, and then we measured the activity of the Renilla luciferase (Rluc - http://www.promega.com/vectors/prltk.txt). The activity of Rluc indicates the percentage of transfected cells, while the activity of Flue indicates the activation of the reporter gene. The ratio Fluc/Rluc (RLA - relative luciferase activity) gives us the normalized value of stimulated cells in reference to the transfected cells.

The split firefly luciferase is designed in a way, that the N terminal end of Flue (Seq ID 11, 12) is connected with the protein Ml 3 and the C terminal end of Flue (Seq ID 13,14) is connected with the protein Calmodulin. In the presence of calcium, Ml 3 binds to Calmodulin. This reassembles the split Flue to an active enzyme, which is able to cleave its substrate. We detected the activation of Flue through the emitted light.

We used the method to detect the activation of mechanosensitive channels when they were exposed to simulation of a mechanical stimulus, for example, touch (Figure 5 A and 5B -Example 3).

Example 3. Measuring the response of cells to mechanical stress

We tested the responsiveness of the cells to a mechanical stimulus as described above through the reporter system with split luciferase. One day after transfection we added 4mM CaC¾ and luciferin, to HEK293 cells, which we previously transfected with plasmids encoding for mechanosensitive channel MscS:HA (lOOng) and gas vesicle proteins AUl:GvpA (250ng) and 3FLAG:GvpC (50ng). The luciferase activity was measured before the mechanical stimulus. Then we shook the plate vigorously with our hands and measured the luciferase activity again.

Results: Figure 5A shows the difference of luciferase activity before and after mechanical stimulation of cells.

In another experiment we transfected HEK293 cells, seeded in 96 well plates, with plasmids encoding for mechanosensitive channel MscS:HA ( ng) and gas vesicle proteins AUl:GvpA (50ng) and

3FLAG:GvpC (lOng). With the injection of different volumes of PBS buffer into the wells we simulated mechanical stress and measured luciferase activity before and after the stimulation.

Results: Figure 5B shows the activity of luciferase in cells transfected with gas vesicles. It is evident from the graph that the activation depends on the volume of the injected fluid. This indicates the activation of cells increases proportionally with the increase in mechanical stress.

Example 4. Transcriptional activation of cells

Stimulation with ultrasound in an ultrasonic bath

One day after transfection we added 4mM CaCl₂ to the medium and stimulated one plate of transfected cells for 30 min with ultrasound in an ultrasonic bath, while the other plate transfected with the same transfection mix was not stimulated. We lysed the cells on both plates 60 minutes after the end of stimulation on the first plate and measured the luciferase activity as described above.

To show a more general use of the system we activated the expression of a reporter gene on a plasmid with ultrasound stimulation. Calcium influx to the cell causes a conformational change of the protein Calmodulin, which in turn interacts with the protein Calcineurin. When Calcineurin interacts with the transcription factor NFAT it catalyzes the dephosphorylation of NFAT. The dephosphorylated NFAT is then translocated to the cell nucleus, where it binds to its DNA binding site and activates gene transcription. We transiently transfected HEK293T cells, seeded in 96 well plates, with plasmids encoding mechanosensitive channel TrpCl:Myc (25ng), transcription factor Myc:NFAT (lng), gas vesicle proteins Aul:GvpA (50ng) and GvpC (lOng) and with the reporter plasmid 3nfat_Pmin_fLuc (150ng). After stimulation, which was done according to the protocol described above, we measured the luciferase activity in the lysates of stimulated and unstimulated cells.

Results: Results, depicted on Figure 6B show the luciferase activity of transiently transfected HEK293T cells, stimulated with ultrasound in an ultrasound bath. From the graph it is evident, that ultrasound does not activate the transcription of the reporter gene, if cells are not transfected with plasmids encoding the mechanosensitive channel and plasmids encoding two gas vesicles forming proteins. After ultrasound stimulation, luciferase activity is significantly higher when plasmids encoding gas vesicle proteins (Gvp) are present. This indicates the activity of gas vesicles and the activation of endogenous mechanosensitive channels. When only the plasmid encoding mechanosensitive channel TrpCl:Myc is present, the luciferase activity does not change after stimulation with ultrasound. However, when the plasmids encoding vesicle proteins GvpA and GvpC are present in addition the plasmid encoding mechanosensitive channel TrpCl:Myc the transcription of the reporter gene is significantly increased. The change in the expression of the reporter gene is bigger in cells expressing the mechanosensitive channel TrpC 1 :Myc in comparison to cells which do not express the mechanosensitive channel TrpCl:Myc. This indicates the activity of gas vesicles and the activation of the above-mentioned channel additionally to the endogenous channels in the cell.

Claims

1. A method for improving eukaryote cells' sensitivity to ultrasound and mechanic stimuli affecting cells containing genetically encoded protein gas vesicles which are synthesized and self- assembled inside the cell.

2. The method according to claim 1, wherein protein components of protein gas vesicles originate in planktonic microorganisms, preferentially bacteria and archaea.

3. The method according to claims 1 or 2, wherein proteins GvpA and GvpC have protein sequences SEQ ID 2 and SEQ ID. 4 or homologs of said sequences with at least 30% similarity in amino acid sequence.

4. The method according to claims 1 to 3, wherein cells contain mechanosensitive nonspecific ion channels, possibly bacterial ion channels chosen from MscL or MscS, beside gas vesicle forming proteins.

5. The method according to claims 1 to 3, wherein cells contain beside gas vesicle forming proteins mechanosensitive nonspecific ion channels, optionally eukaryotic ion channels chosen from TRP family of ion channels, preferentially TRPC, TRPV and TRPA, preferentially TRPC1, TRPC3, TRPV1 and TRPA 1.

6. The method according to claims 1 to 5, wherein proteins GvpA and GvpC are connected to nonspecific ion channels directly or via dimerization domains.

7. Cells containing protein gas vesicle forming components and expressing stronger mechanosensory response to ultrasound stimulation or other mechanic stimuli.

8. Cells according to claim 7, wherein cells originate in human, animal, fungus or plant.

9. Cells according to any one of claims 7 or 8, wherein cells are T-lymphocites or other cells of the immune response, neural cells or other cells of nervous system, beta cells or pancreatic beta cells.

10. Cells according to any one of claims 7 to 9, wherein said cells detect mechanic action directed towards cells, preferentially mechanic action is direct or indirect pressure, change in air or liquid pressure or sound undulation such as ultrasound.

11. Cells according to any one of claims 7 to 10, wherein cells express additional mechanosensitive receptors of claims 4 and 5 in addition to endogenous mechanosensitive proteins.

12. Cells according to any one of claims 7 to 11, wherein cells contain calcium sensitive proteins, which change their state when activated, preferentially calcium binding proteins, preferentially proteins which change cell state, preferentially proteins which signal the change of state via change in fluorophore characteristics or light emission.

13. Cells according to any one of claims 7 to 12, wherein cells contain information encoding transcription activating proteins.

14. Cells according to any one of claims 7 to 13, wherein cells secrete peptide or other hormones upon activation via ultrasound or other mechanic stimulus.