WO2019012414A1

WO2019012414A1 - Identification of interactions between subcellular organelles using split gfp

Info

Publication number: WO2019012414A1
Application number: PCT/IB2018/055063
Authority: WO
Inventors: Tito CALI'; Marisa Brini
Original assignee: Universita' Degli Studi Di Padova
Priority date: 2017-07-10
Filing date: 2018-07-10
Publication date: 2019-01-17
Also published as: IT201700077259A1

Abstract

The object of the present invention is a method for determining the interactions between sub-cellular organelles in a cell comprising: a) expressing in said cell or providing said cell with a fusion protein comprising a localization signal X for a first sub-cellular organelle and a complementary fragment F of a fluorophore and a fusion protein comprising a localization signal Y for a second sub-cellular organelle and a complementary fragment F' of the same fluorophore, wherein said fragments F and F' are self- complementary to each other and, once self- assembled, reconstitute the fluorophore; b) displaying the fluorescent signal and therefore the interaction points between said first and second sub-cellular organelles; wherein said fluorophore is selected from GFP or variants thereof.

Description

"Identification of interactions between subcellular organelles using split GFP"

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[001] The object of the present invention is a method for determining the interactions between sub-cellular organelles in a cell comprising:

a) expressing in said cell or providing said cell with a fusion protein comprising a localization signal X for a first sub-cellular organelle and a complementary fragment F of a fluorophore and a fusion protein comprising a localization signal Y for a second sub-cellular organelle and a complementary fragment F' of the same fluorophore, wherein said fragments F and F' are self- complementary to each other and, once self- assembled, reconstitute the fluorophore;

b) displaying the fluorescent signal and therefore the interaction points between said first and second sub-cellular organelles;

wherein said fluorophore is selected from GFP or variants thereof.

Background art

[002] The proximity between the organelles is an important mechanism used by eukaryotic cells to ensure a fine coordination of the subcellular activities. A network of contact sites between membranes of different organelles ensures their mutual communication, creating microdomains that favor different signaling and metabolic pathways (Prinz et al . J Cell Biol 2014; 205, 759-769) . Because of the centrality in many fundamental cellular processes, such as in autophagy, in mitochondrial dynamics, in lipid biosynthesis, in mitochondrial bioenergetics, in calcium signaling and in apoptosis, the contact sites between mitochondria and the endoplasmic reticulum (ER) so far are the most characterized (Filadi et al . , Proc Natl Acad Sci USA 2015; 112, E2174-2181; Murley and Nunnari, Mol Cell 2016; 61, 648-653) .

[003]Among the techniques used today to evaluate the contact between subcellular organelles there is electron microscopy (EM) , which offers the necessary resolution to quantify the number of contacts and their distance. It is a costly approach which is necessarily to be conducted on fixed samples. Alternatively, it is possible to work with fluorescent proteins selectively targeted to the mitochondrial matrix and to the ER lumen

(Rizzuto et al . Science 1998; 280, 1763-1766), analyzing the co-localization of the signal. This methodology has the advantage of better respecting the physiology of the sample but has an undisputed limit since confocal microscopy, necessary for this type of measurement, has an optical resolution of about 200 nm, where the distances under examination are between 10 and 100 nm. A further methodology recently made available is based on the binding induced by rapamycin between a fluorescent protein directed to the mitochondria and another one directed to ER (Csordas et al . , Mol Cell 2010; 39, 121- 132) . The applicability of the latter, however, is strongly limited by the use of rapamycin which can artificially force the proximity of the organelles.

[004] The need for a methodology capable of overcoming the limits of the current ones and of giving an accurate, reproducible feedback that respects the physiology of the system is therefore strongly felt.

Description of the invention

[005] The object of the present invention are fluorescent probe systems and methods for measuring contacts between subcellular organelles, in a preferred embodiment contacts between the endoplasmic reticulum (ER) and mitochondria .

[006] Description of the figures

Figure 1: A) scheme of a cell and constructs in embodiment a) and b) with localization signal for ER and mitochondrion, respectively. B) diagram of the same cell with highlighted the ER-mitochondrial contact points that emit fluorescence following the expression and correct complementation of the probes coded by constructs a) and b) .

Figure 2: schematic representation of the constructs according to an embodiment and their expression.

Figure 3: diagram of a cell and of a further embodiment c) of the construct.

Figure 4: embodiments of vectors according to the present invention: A) ERs-βιι—OMM-GFPi-io, B) ER_L- n—

OMM-GFPi-io, C) DsRed-UAS- ER_s- n^~O M-GFPi-io .

Figure 5: fluorescence emitted in Hela cells after transfection with the indicated constructs.

Figure 6: fluorescence emitted in HeLa cells after transfection with the indicated constructs, colocalization with endogenous markers (mtHSp60 for mitochondria and CRT, calreticulin, for ER) .

Figure 7: mean value of the number of ER contacts/mitochondria per single cell.

Figure 8: immunogold assay in HeLa cells. Gold particles indicate the reconstituted probe at the contact points between ER-mitochondria .

Figure 9: A) variation of the mitochondrial Ca²⁺ concentration, mean of three independent experiments, after cell stimulation with histamine; B) quantification of the mitochondrial Ca²⁺ values at the peak reached following stimulation with histamine.

Figure 10: fluorescence in Hela cells after transfection with the indicated constructs and incubation with tunicamycin or exposure to HBSS (nutrient-free medium) . A) , C) fluorescence images obtained with the ERs-βιι and ERL-βιι constructs, respectively; B) , D) quantification. Figure 11: fluorescence in Hek293 cells after transfection with the indicated constructs.

Figure 12: in vivo fluorescence in Zebrafish embryos after transfection with the indicated constructs. Colocalization with exogenous markers, pDsRed2-ER for the endoplasmic reticulum and pTagRFP-mito for mitochondria .

Figure 13: in vivo fluorescence in Zebrafish embryos after transfection with the indicated constructs and quantization of the ER-mitochondria contacts.

Definitions

[007]Unless otherwise specified, all terms, notations and other scientific terminologies used in this document are intended to have the meanings commonly understood by those skilled in the art to which the present invention relates. In some cases, terms with commonly understood meanings are defined here for clarity and/or for ready reference and the inclusion of such definitions is not necessarily to be interpreted as representing a substantial difference compared to what is generally understood in the field. The techniques and procedures described or cited herein are generally well understood and commonly used using conventional methodologies by those skilled in the art, such as molecular cloning methodologies, widely described in Sambrook et al . Molecular Cloning: A Laboratory Manual 2001 Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and the standard protocols in molecular biology (Ausbel et al. Ed. John Wiley & Sons, Inc. 2001) .

[008] Procedures involving the use of commercially available kits and reagents are generally performed according to the protocols defined by the manufacturer, unless otherwise indicated.

[009]A "fluorescent protein", as used herein, is a fluorescent protein (GFP) of Aequorea victoria, or variants thereof, where the expression "variants thereof" is to be understood as structural variants thereof, folding versions of GFP (i.e., more soluble versions, superfolder versions), spectral variants of GFP (for example, YFP, CFP) and GFP-like fluorescent proteins (such as DsRed) . The term "GFP-like fluorescent protein" is used to refer to fluorescent proteins that share the structure βΐΐ of the GFP, as well as structural, folding and spectral variants thereof. GFP- like fluorescent proteins share structural and functional features such as, without limitation, the ability to form internal chromophores without requiring cofactors, external enzymatic catalysis or substrates, with the exception of molecular oxygen.

[0010]A "variant" of a fluorescent protein is a protein derived from a fluorescent protein and preserves the structure βΐΐ thereof, as well as the intrinsic fluorescence and includes structures with substitutions, deletions or insertions of amino acids that can confer new or modified biological properties (for example, greater stability, better solubility, improved folding, displacement of the emission or excitation spectrum, reduced or eliminated ability to form multimers, etc.) or structures that have modifications at the N and/or C terminus .

[0011] The term "complementary fragments" indicates individually inactive fragments which do not express the reporter phenotype, capable of complementing each other to restore the reporter activity. The terms "auto- complementary", "self-assembly" and "spontaneous association", when used to describe two or more protein fragments herein, mean that the fragments, not fluorescent per se, can be reconstituted into a fluorophore .

[0012] The terms "subcellular compartment", "subcellular element" and "subcellular localization" are used to refer to various distinctive parts, components or organelles of a cell including, without limitation, nucleus, cytoplasm, plasma membrane, endoplasmic reticulum, Golgi apparatus, filaments of actin and tubulin, endosomes, lysosomes, peroxisomes and mitochondria .

[0013] The terms "identity" or "percentage of identity", in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences that are the same or have a specified percentage of amino acid or nucleotide residues, e.g., about 70% identity, preferably 75%, 80%, 85%, 90% or 95% identity in a given region, compared and aligned for maximum match in a comparison window or determined region measured by sequence comparison algorithms, preferably BLAST, or with manual alignment and which are therefore considered "substantially identical".

[0014] For sequence comparison, generally a sequence acts as a reference sequence and test sequences are compared thereto. When using a sequence comparison algorithm, the test and reference sequences are entered into a computer and sequence algorithm program parameters are designated. Predefined program parameters may be used or alternative parameters may be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences related to the reference sequence, based on the program parameters.

[0015]A "comparison window" includes a reference to a segment of any of the number of contiguous positions selected from the group consisting of 20 to 600, usually from about 50 to about 200, typically from about 100 to about 150 where a sequence can be compared with a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Sequence alignment methods for comparison are well known in the art. The optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman, 1981, Adv. Appl . Matematica. 2: 482, by the homologous alignment algorithm of Needleman & Wunsch, 1970, J. Mol. Biol. 48: 443, with the research of the similarity method of Pearson & Lipman, 1988, Proc. Nat ' 1. Acad. Sci. USA 85: 2444, through computerized implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software package, Genetics Computer Group, 575 Science Dr., Madison, WI) or by manual alignment and visual inspection.

[0016]A preferred example of an algorithm that is suitable for determining the sequence identity percentage and sequence similarity are the BLAST and BLAST 2.0 algorithms, described in Altschul et al . , 1977, Nuc. Acid Res. 25: 3389-3402 and Altschul et al . , 1990, J. Mol. Biol. 215: 403-410, respectively. The BLAST analysis software is publicly available through the National Center for Biotechnology Information.

[0017] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin & Altschul, 1993, Proc Natl Acad Sci USA 90: 5873-5787) . A measure of similarity provided by the BLAST algorithm is the minimum sum probability (P

(N) ) , which gives an indication of the probability that a coincidence would occur between two sequences of nucleotides or amino acids. For example, a nucleic acid is considered to be similar to a reference sequence if the minimum sum probability in a comparison of the test nucleic acid with the reference nucleic acid is less than about 0.2, more preferably less than about 0.01 and more preferably less than about 0.001.

[0018] The term "bond", as used herein, refers to a physical bond, as well as to the bond that occurs due to the coexistence within a biological particle, for example phage, bacteria, yeasts or other eukaryotic cells .

[0019] "Physical link" refers to any method known in the art to functionally connect two molecules (which are defined as "physically bound"), such as for example recombinant fusion with or without intervening domains, non-covalent association, covalent bonding, hydrogen bonding; electrostatic bonding, conformational adhesion, for example antigen-antibody and biotin-avidin associations .

[0020]As used here, "linker" refers to a molecule or group of molecules that links two molecules and serves to put the two molecules in a preferred configuration.

[0021] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to amino acid residues .

[0022] The term "amino acid" refers to natural and synthetic amino acids, as well as analogous amino acids and mimetic amino acids that work similarly to natural amino acids. Natural amino acids are those encoded by the genetic code, as well as those amino acids which are subsequently modified, for example hydroxyproline, γ- carboxylglutamate and O-phosphoserine . Amino acid analogs refer to compounds having the same basic chemical structure as a natural amino acid, i.e. a carbon a which is bound to a hydrogen, a carboxyl group, an amine group and an R group, e.g. homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs maintain the same basic chemical structure as a natural amino acid. Amino acid mimetics refer to chemical compounds that have a different structure from the general chemical structure of an amino acid but that work similarly to a natural amino acid .

[0023] The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single or double form. The term includes nucleic acids containing known analogs of natural nucleotides that have similar binding properties such as the reference nucleic acid and are metabolized in a manner similar to natural nucleotides.

[0024]With regards to amino acid sequences, single substitutions, deletions or additions to a nucleic acid, peptide, polypeptide or sequence of proteins that alter, add or eliminate a single amino acid or a small percentage of amino acids in the coding sequence are to be understood as "conservatively modified variant" where the alteration causes the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. These conservatively modified variants are in addition and do not exclude polymorphic, interspecies homologous and alleles of the invention .

[0025] The following eight groups each contain amino acids that are conservative substitutions: 1) Alanine (A), Glycine (G) ; 2) aspartic acid (D) , glutamic acid (E) ; 3) asparagine (N) , glutamine (Q) ; 4) arginine (R) , lysine (K) ; 5) isoleucine (I), leucine (L) , methionine (M) , valine (V); 6) phenylalanine (F), tyrosine (Y) , tryptophan (W) ; 7) Serine (S), threonine (T) ; E 8)

Cysteine (C) , Methionine (M) .

[0026]Macromolecular structures such as polypeptide structures can be described in terms of different levels of organization. "Primary structure" refers to the amino acid sequence of a given peptide. "Secondary structure" refers to three-dimensional structures ordered locally within a polypeptide. These structures are commonly known as domains. The domains are portions of a polypeptide that form a compact unit of the polypeptide and are typically from 25 to about 500 amino acids. Typical domains are made up of minor organization sections, such as β-sheets and -helices. "Tertiary structure" refers to the complete three-dimensional structure of a polypeptide monomer. "Quaternary structure" refers to the three-dimensional structure formed by the non-covalent association of independent tertiary units.

Detailed description of the invention:

[0027] Split systems of fluorescent proteins and their use for the study of subcellular organelles interactions, in particular for the study of the contact points between ER and mitochondria, are described herein .

[0028] The preferred fluorescent protein systems used in the present invention are those derived from proteins similar to GFP and GFP. GFP-like proteins are an expanding family of homogeneous polypeptides of 25 to 30 kDa . The family of GFP-like proteins currently comprises about 100 members and includes red, yellow and green fluorescent proteins and a variety of non-fluorescent chromoproteins .

[0029] The split systems of fluorescent proteins according to the present invention, when expressed in cells, preferably in eukaryotic cells, lead to the expression of two complementary fragments, a complementary fragment on the endoplasmic reticulum and a second one on the outer membrane of the mitochondria. Said fragments are self-complementary, i.e. when they are expressed in sufficient proximity, they reconstitute the fluorophore, thus producing a fluorescent signal only where said two organelles expressing specifically one or the other of said complementary fragments are sufficiently close (Figure 1 and Figure 2) .

[0030] In one embodiment, said system comprises two constructs, a) and b) , encoded by two independent vectors. Each of said constructs comprises a complementary fragment, F and F', respectively, linked to a localization signal X for ER or to a localization signal Y for the outer mitochondrial membrane (OMM) . Said construct a) comprises a localization signal X for ER linked to the complementary fragment F. Said construct b) comprises a localization signal Y for OMM linked to the complementary fragment F' (Figure 1) .

[0031] The two complementary fragments F and F' self- complement each other, thus constituting the fluorescent protein when expressed sufficiently close to each other, as shown schematically in figure 1, panel B and in figure 2.

[0032] Said fluorescent protein is selected from GFP or variants thereof, such as YFP, CFP, BFP, or it is a GFP- like fluorescent protein. In a preferred embodiment, the fluorescent protein is GFP. Even more preferably, the system according to the present invention uses a very stable and intense fluorescence variant of the Green Fluorescent Protein (GFP) called superfolderGFP . In this embodiment, the complementary fragment F corresponds to the GFP β-strand 11 (βιι) and the complementary fragment F' corresponds to the GFP 1-10 (GFPi-io) portion.

[0033] In one embodiment, the localization signal X is bound to the β-strand 11 fragment of the GFP, amino acids 215-230, and the localization signal Y is bound to the GFPi-io portion which comprises the amino acids 1 to 214. The spontaneous association of the two complementary fragments determines the complementation and the concomitant reconstitution of the GFP fluorescence. Where a fragment is anchored to the subcellular element of interest, such as ER, and the fragment complementary to the second subcellular element of interest, for example the mitochondrion, the fragments self-complement each other where the two organelles come into contact, thus generating detectable fluorescence .

[0034] In one embodiment, said GFPi-io portion has at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90% identity with SEQ ID no. 1:

ATGTCCAAAGGAGAAGAACTGTTTACCGGCGTGGTGCCAATTCTCGTGGAACTGGA TGGCGATGTGAATGGCCACAAATTTTCTGTCAGAGGAGAGGGTGAAGGTGATGCCA CAATCGGAAAGCTCACCCTGAAATTCATCTGCACCACTGGAAAGCTCCCTGTGCCA TGGCCAACACTGGTCACTACCCTGACCTACGGCGTGCAGTGCTTTTCCAGATACCC AGACCATATGAAGAGGCATGACτTTTTCAAGAGCGCCATGCCCGAGGGCTATGTGC AGGAGAGAACCATCTCTTTCAAAGATGACGGGAAATACAAGACCCGCGCTGTGGTC AAGTTCGAAGGAGACACACTGGTGAATAGAATCGAGTTGAAGGGCACAGACTTTAA GGAAGATGGAAACATTCTCGGCCACAAGCTGGAATACAACTTTAACTCCCACAATG TGTACATCACAGCCGACAAGCAAAAGAATGGCATCAAGGCTAACTTCACAGTCAGA CACAACGTCGAGGATGGAAGCGTGCAGCTGGCCGACCATTATCAACAGAACACTCC AATCGGCGACGGCCCTGTGCTCCTCCCAGACAACCATTACCTGTCCACCCAGACAG TCCTGAGCAAAGATCCAAATGAAAAAGGAACATAA

[0035] Alternatively, said GFPi-io portion has at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90% identity with SEQ ID no. 15:

ATGAGCAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAG ATGGTGATGTTAATGGGCACAAATTTTCTGTCAGAGGAGAGGGTGAAGGTGATG CTACAATCGGAAAACTCACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGT TCCATGGCCAACACTTGTCACTACTCTGACCTATGGTGTTCAATGCTTTTCCCGT TATCCGGATCACATGAAAAGGCATGACτTTTTCAAGAGTGCCATGCCCGAAGGT TATGTACAGGAACGCACTATATCTTTCAAAGATGACGGGAAATACAAGACGCGT GCTGTAGTCAAGTTTGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAGGGTA CTGATTTTAAAGAAGATGGAAACATTCTCGGACACAAACTCGAGTACAACTTTAA CTCACACAATGTATACATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAA CTTCACAGTTCGCCACAACGTTGAAGATGGTTCCGTTCAACTAGCAGACCATTAT CAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACC TGTCGACACAAACTGTCCTTTCGAAAGATCCCAACGAAAAGGGTACCTAA

[0036] Alternatively, said GFPi-io portion has at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90% identity with SEQ ID no. 16:

ATGAGCAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAG ATGGAGATGTTAATGGGCACAAATTTTCTGTCAGAGGAGAGGGTGAAGGTGATG CTACAAACGGAAAACTCACCCTTAAATTCATTTGCACTACTGGAAAACTACCTGT TCCATGGCCAACGCTTGTCACTACTCTGACCTATGGTGTTCAATGCTTTTCCCGT TATCCGGATCACATGAAACAGCATGACτTTTTCAAGAGTGCCATGCCCGAAGGT TATGTACAGGAACGCACTATATATTTCAAAGATGACGGGAACTACAAGACGCGT GCTGTAGTCAAGTTTGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAGGGTA CTGATTTTAAAGAAGATGGAAACATTCTCGGACACAAACTCGAGTACAACTTTAA CTCACACAATGTATATATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAAC TTCACAATTCGCCACAACGTTGTAGATGGTTCCGTTCAACTAGCAGACCATTATC AACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACTT GTCGACACAAACTGTCCTTTCGAAAGATCCCAACGAAAAGGGTACCTAA

[0037] In a preferred embodiment, said GFPi-io portion has SEQ ID no. 1.

[0038] In one embodiment, said GFP β has at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90% identity with sequence SEQ ID no. 2:

CGGGACCACATGGTGCTGCACGAGTACGTGAACGCCGCTGGCATCACA.

[0039]Alternatively, said GFP βη has at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90% identity with sequence SEQ ID no. 17:

AAGCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACAGGT ACCTAA

[0040]Alternatively, said GFP βη has at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90% identity with sequence SEQ ID no. 18:

AAGCGTGACCACATGGTCCTTCATGAGTTTGTAACTGCTGCTGGGATTACAGGT ACCTAA

[0041] In a preferred embodiment, said GFP β has SEQ ID no . 2.

[0042]Said localization signal X which selectively directs to ER is selected from the localization signals described, for example, in Hedge RS and Keenan RJ Nature Reviews 2011, 12: 787-798. Preferably, it is a portion of the protein Sacl, a phosphatase which is an integral membrane protein of ER, and said sequence has at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90% identity with SEQ ID no. 3:

AGAGTGTTCCTGGCCCTGCCCATCATCATGGTGGTGGCCTTCAGCATGTGCATCAT CTGCCTGCTGATGGCCGGCGACACCTGGACAGAGACACTGGCCTACGTGCTGTTCT GGGGCGTGGCCAGCATCGGCACCTTTTTCATCATCCTGTACAACGGCAAGGACTTC GTGGACGCCCCCAGACTGGTGCAGAAAGAGAAGATCGAC .

[0043] Said localization signal X is separated from said complementary fragment F by a linker. Said linker is functional to define the distance between the protein encoded by said localization signal and the fluorescent protein portion. The length of said linker therefore determines where said complementary fragment is positioned in space with respect to said localization signal when expressed and located at the ER level.

[0044] The authors of the present invention have developed ER-βη constructs capable of originating a fluorescent signal as a function of the distance at which said ERs and said mitochondria expressing OMM- GFPi-io are located. In fact, constructs defined ERs-βιι have been obtained which when expressed in the cell that co-expresses OMM-GFPi-io originate a fluorescent signal where ER and mitochondria come into contact at a distance of about 7-20 nm, preferably 9-12 nm and constructs, defined ERL-βιι, which when expressed in the cell that co-expresses the same OMM-GFPi-io, originate a fluorescent signal where ER and mitochondria come into contact at a distance of about 40-100 nm, preferably 45- 50 nm.

[0045] Said "short" linker consists of about 15 - 50 AA, or 20 - 40 AA, or 25 - 35 AA, or 29 AA. In a preferred embodiment, said "short" linker has a sequence of at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90% identity with SEQ ID no. 5:

ATGCGGGACCACATGGTGCTGCACGAGTACGTGAACGCCGCTGGCATCACAGGCGG AGATGGCGGATCTGGCGGCGGAAGC .

[0046]Said "long" linker consists of 100-180 AA, or 120 - 160 AA, or 130 - 155 AA, or 146 AA, said "long" linker has at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90% identity with SEQ ID no. 6:

ATGCGGGACCACATGGTGCTGCACGAGTACGTGAACGCCGCTGGCATCACAGGCGG AGATGGCGGATCTGGCGGCGGAAGCAAACTGATGTGGCACGAGGGACTGGAAGAGG CCAGCAGACTGTACTTCGGCGAGCGGAACGTGAAGGGCATGTTCGAGGTGCTGGAA CCCCTGCACGCCATGATGGAAAGAGGCCCCCAGACCCTGAAAGAGACAAGCTTCAA CCAGGCCTACGGCCGGGACCTGATGGAAGCCCAGGAATGGTGCCGGAAGTACATGA AGTCCGGCAATGTGAAGGACCTGACACAGGCCTGGGACCTGTACTACCACGTGTTC CGGCGGATCAGCAAGCAGGGCTCTGAAGCCGCCGCTAGAGAAGCTGCTGCTAGAGG CGGAGCTTCTGGCGCTGGCGCAGGCGCTGGGGCCATCCTGAATAGC

[0047]Said localization signal Y which selectively directs to OMM is selected from the localization signals described, for example, in Walther DM and Rapaport D Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 2009, 1793: 42-51. Preferably, it is a sequence of the TOM20 protein, translocase of the outer mitochondrial membrane, and has at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90% identity with SEQ ID no. 4:

GTGGGCCGGAACAGCGCCATCGCCGCGGGCGTGTGCGGTGCCCTCTTCATAGGGTA CTGCATCTACTTTGACCGCAAAAGGCGGAGTGACCCCAAC.

[0048] In one embodiment, said cleavage sequence is P2A (self-cleaving 2A peptide) and has the sequence SEQ ID no . 7 :

GGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAA CCCTGGACCT.

[0049] In one embodiment, said constructs are conveyed by the plasmid pSYC-181 (Kim JH et al . Plos One 2011 6(4)) .

[0050] Figure 4 exemplifies some embodiments according to the present invention. In panel A, the vector which encodes the fusion proteins X-F and Y-F' is schematized, linked by a cleavage sequence that is P2A, in the "short" version, that is, suitable for detecting close interactions between ER and mitochondria. Panel B schematizes the same vector that encodes the fusion proteins X-F and Y-F' linked by the P2A cleavage sequence, in the "long" version. Panel C schematizes a vector for the expression of said fragments in vivo, in particular in Zebrafish. Also in this embodiment, X-F and Y-F' are linked by a cleavage sequence which is P2A. It detects that said reporters are under the control of the UAS bidirectional promoter, a promoter that also controls the expression of DsRed, ensuring an internal transfection control.

[0051] In a preferred embodiment, the fusion proteins X-F and Y-F', linked by a cleavage sequence which is P2A, are encoded by SEQ ID no. 13 for the short version, or by SEQ ID no. 14 for the long version, where SEQ ID no. 13 is:

ATGCGGGACCACATGGTGCTGCACGAGTACGTGAACGCCGCTGGCATCACA GGCGGAGATGGCGGATCTGGCGGCGGAAGCAAGCTGAGAGTGTTCCTGGCC CTGCCCATCATCATGGTGGTGGCCTTCAGCATGTGCATCATCTGCCTGCTG ATGGCCGGCGACACCTGGACAGAGACACTGGCCTACGTGCTGTTCTGGGGC GTGGCCAGCATCGGCACCTTTTTCATCATCCTGTACAACGGCAAGGACTTC GTGGACGCCCCCAGACTGGTGCAGAAAGAGAAGATCGACCCCGGGCTTAAG GAGCTCGCATGCGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCT GGAGACGTGGAGGAGAACCCTGGACCTAGATCTGAATTCATGGTGGGCCGG AACAGCGCCATCGCCGCGGGCGTGTGCGGTGCCCTCTTCATAGGGTACTGC ATCTACTTTGACCGCAAAAGGCGGAGTGACCCCAACTCCAAAGGAGAAGAA CTGTTTACCGGCGTGGTGCCAATTCTCGTGGAACTGGATGGCGATGTGAAT GGCCACAAATTTTCTGTCAGAGGAGAGGGTGAAGGTGATGCCACAATCGGA AAGCTCACCCTGAAATTCATCTGCACCACTGGAAAGCTCCCTGTGCCATGG CCAACACTGGTCACTACCCTGACCTACGGCGTGCAGTGCTTTTCCAGATAC CCAGACCATATGAAGAGGCATGACτTTTTCAAGAGCGCCATGCCCGAGGGC TATGTGCAGGAGAGAACCATCTCTTTCAAAGATGACGGGAAATACAAGACC CGCGCTGTGGTCAAGTTCGAAGGAGACACACTGGTGAATAGAATCGAGTTG AAGGGCACAGACTTTAAGGAAGATGGAAACATTCTCGGCCACAAGCTGGAA

TACAACTTTAACTCCCACAATGTGTACATCACAGCCGACAAGCAAAAGAAT

GGCATCAAGGCTAACTTCACAGTCAGACACAACGTCGAGGATGGAAGCGTG

CAGCTGGCCGACCATTATCAACAGAACACTCCAATCGGCGACGGCCCTGTG

CTCCTCCCAGACAACCATTACCTGTCCACCCAGACAGTCCTGAGCAAAGAT

CCAAATGAAAAAGGAACATAA; detta SEQ ID no. 14 is:

ATGCGGGACCACATGGTGCTGCACGAGTACGTGAACGCCGCTGGCATCACA

GGCGGAGATGGCGGATCTGGCGGCGGAAGCAAACTGATGTGGCACGAGGGA

CTGGAAGAGGCCAGCAGACTGTACTTCGGCGAGCGGAACGTGAAGGGCATG

TTCGAGGTGCTGGAACCCCTGCACGCCATGATGGAAAGAGGCCCCCAGACC

CTGAAAGAGACAAGCTTCAACCAGGCCTACGGCCGGGACCTGATGGAAGCC

CAGGAATGGTGCCGGAAGTACATGAAGTCCGGCAATGTGAAGGACCTGACA

CAGGCCTGGGACCTGTACTACCACGTGTTCCGGCGGATCAGCAAGCAGGGC

TCTGAAGCCGCCGCTAGAGAAGCTGCTGCTAGAGGCGGAGCTTCTGGCGCT

GGCGCAGGCGCTGGGGCCATCCTGAATAGCAGAGTGTTCCTGGCCCTGCCC

ATCATCATGGTGGTGGCCTTCAGCATGTGCATCATCTGCCTGCTGATGGCC

GGCGACACCTGGACAGAGACACTGGCCTACGTGCTGTTCTGGGGCGTGGCC

AGCATCGGCACCTTTTTCATCATCCTGTACAACGGCAAGGACTTCGTGGAC

GCCCCCAGGCTGGTGCAGAAAGAGAAGATCGACCCCGGGCTTAAGGAGCTC

GCATGCGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGAC

GTGGAGGAGAACCCTGGACCTAGATCTGAATTCATGGTGGGCCGGAACAGC

GCCATCGCCGCGGGCGTGTGCGGTGCCCTCTTCATAGGGTACTGCATCTAC

TTTGACCGCAAAAGGCGGAGTGACCCCAACTCCAAAGGAGAAGAACTGTTT

ACCGGCGTGGTGCCAATTCTCGTGGAACTGGATGGCGATGTGAATGGCCAC

AAATTTTCTGTCAGAGGAGAGGGTGAAGGTGATGCCACAATCGGAAAGCTC

ACCCTGAAATTCATCTGCACCACTGGAAAGCTCCCTGTGCCATGGCCAACA CTGGTCACTACCCTGACCTACGGCGTGCAGTGCTTTTCCAGATACCCAGAC CATATGAAGAGGCATGACτTTTTCAAGAGCGCCATGCCCGAGGGCTATGTG CAGGAGAGAACCATCTCTTTCAAAGATGACGGGAAATACAAGACCCGCGCT GTGGTCAAGTTCGAAGGAGACACACTGGTGAATAGAATCGAGTTGAAGGGC ACAGACTTTAAGGAAGATGGAAACATTCTCGGCCACAAGCTGGAATACAAC TTTAACTCCCACAATGTGTACATCACAGCCGACAAGCAAAAGAATGGCATC AAGGCTAACTTCACAGTCAGACACAACGTCGAGGATGGAAGCGTGCAGCTG GCCGACCATTATCAACAGAACACTCCAATCGGCGACGGCCCTGTGCTCCTC CCAGACAACCATTACCTGTCCACCCAGACAGTCCTGAGCAAAGATCCAAAT GAAAAAGGAACATAA

[0052] The embodiments which consist in the use of a single vector comprising the two portions of fluorescent protein controlled by the two different localization signals, linked together by a cleavage sequence and regulated by a single promoter, have proved particularly advantageous where they allow an equimolar expression of the two constructs.

[0053] Advantageously, said system also works in in vivo systems. As exemplified in the following examples, the solution according to the present invention allows displaying interactions between in vivo subcellular organelles clearly and without background noise.

[0054]A further object of the present invention is a method for detecting the contact points between endoplasmic reticulum (ER) (3) and mitochondria (4) in a cell (1), comprising, with reference to figure 1: i) expressing in the cell or providing the cell with a fusion protein comprising a localization signal X which selectively directs to ER and a first complementary fragment F of a fluorescent protein;

ii) expressing in the cell or providing the cell with a fusion protein comprising a localization signal Y that selectively directs to the outer mitochondrial membrane (OMM) and a second complementary analysis fragment of the fluorescent protein, capable of spontaneous association with the first complementary fragment F when said two complementary fragments are found to be sufficiently close; and

iii) detecting fluorescence, thus detecting the contact points between ER and OMM.

[0055] In a further embodiment, with reference to figure 3, said steps i) and ii) take place in a single step, where a fusion protein comprising a localization signal X which selectively directs to the endoplasmic reticule

(ER) and a first complementary fragment F of a fluorescent protein is expressed in the cell or provided to the cell, said fusion protein being linked by a cleavage sequence to a second fusion protein comprising a localization signal Y which selectively directs to the outer mitochondrial membrane (OMM) and a complementary analysis fragment F' of the fluorescent protein, capable of self-complementing with the first complementary fragment F when expressed sufficiently close.

[0056]A further aspect of the present invention is an isolated polypeptide having an amino acid sequence selected from the group consisting of SEQ ID no. 8, 9,

10, 11, 12.

SEQ ID no. 8

M R D H M V L H E Y V N A A G I T G G D G G S G G G S K L R V F L A L P I I M V V A F S M C I I C L L M A G D T W T E T L A Y V L F W G V A S I G T F F I I L Y N G K D F V D A P R L V Q K E K I D SEQ ID no. 9

M R D H M V L H E Y V N A A G I T G G D G G S G G G S K

L M W H E G L E E A S R L Y F G E R N V K G M F E V L E

P L H A M M E R G P Q T L K E T S F N Q A Y G R D L M E

A Q E W C R K Y M K S G N V K D L T Q A W D L Y Y H V F

R R I S K Q G S E A A A R E A A A R G G A S G A G A G A

G A I L N S R V F L A L P I I M V V A F S M C I I C L L

M A G D T W T E T L A Y V L F W G V A S I G T F F I I L

Y N G K D F V D A P R L V Q K E K I D

SEQ ID no. 10

M V G R N S A I A A G V C G A L F I G Y C I Y F D R K R R S D P N S K G E E L F T G V V P I L V E L D G D V N G H K F S V R G E G E G D A T I G K L T L K F I C T T G K L P V P W P T L V T T L T Y G V Q C F S R Y P D H M K R H D F F K S A M P E G Y V Q E R T I S F K D D G K Y K T R A V V K F E G D T L V N R I E L K G T D F K E D G N I L G H K L E Y N F N S H N V Y I T A D K Q K N G I K A N

F T V R H N V E D G S V Q L A D H Y Q Q N T P I G D G P

V L L P D N H V L S T Q T V L S K D P N E K G T SEQ ID no. 11

M R D H M V L H E Y V N A A G I T G G D G G S G G G S K L R V F L A L P I I M V V A F S M C I I C L L M A G D T W T E T L A Y V L F W G V A S I G T F F I I L Y N G K D F V D A P R L V Q K E K I D P G L K E L A C G S G A T N F S L L K Q A G D V E E N P G P R S E F M V G R N S A I A A G V C G A L F I G Y C I Y F D R K R R S D P N S K G E E L F T G V V P I L V E L D G D V N G H K F S V R G E G E G D A T I G K L T L K F I C T T G K L P V P W P T L

V T T L T Y G V Q C F S R Y P D H M K R H D F F K S A M P E G Y V Q E R T I S F K D D G K Y K T R A V V K F E G D T L V N R I E L K G T D F K E D G N I L G H K L E Y N F N S H N V Y I T A D K Q K N G I K A N F T V R H N V E D G S V Q L A D H Y Q Q N T P I G D G P V L L P D N H Y L S T Q T V L S K D P N E K G T SEQ ID no. 12

M R D H M V L H E Y V N A A G I T G G D G G S G G G S K L M W H E G L E E A S R L Y F G E R N V K G M F E V L E P L H A M M E R G P Q T L K E T S F N Q A Y G R D L M E A Q E W C R K V M K S G N V K D L T Q A W D L Y Y H V F R R I S K Q G S E A A A R E A A A R G G A S G A G A G A G A I L N S R V F L A L P I I M V V A F S M C I I C L L M A G D T W T E T L A Y V L F W G V A S I G T F F I I L Y N G K D F V D A P R L V Q K E K I D P G L K E L A C G S G A T N F S L L K Q A G D V E E N P G P R S E F M V G R N S A I A A G V C G A L F I G Y C I Y F D R K R R S D P N S K G E E L F T G V V P I L V E L D G D V N G H K F S V R G E G E G D A T I G K L T L K F I C T T G K L P V P W P T L V T T L T Y G V Q C F S R Y P D H M K R H D F F K S A M P E G Y V Q E R T I S F K D D G K Y K T R A V V K F E G D T L V N R I E L K G T D F K E D G N I L G H K L E Y N F N S H N V Y I T A D K Q K N G I K A N F T V R H N V E D G S V Q L A D H Y Q Q N T P I G D G P V L L P D N H Y L S T Q T V L S K D P N E K G T

[0057]A further aspect of the present invention is a kit comprising one or more of said polypeptides, in one embodiment said kit comprises SEQ ID no. 8 and SEQ ID no. 10, or SEQ ID no. 9 and SEQ ID no. 10; or SEQ ID no. 11, or SEQ ID no. 12.

[0058]A further aspect of the present invention is an expression vector comprising a nucleic acid molecule selected from the group consisting of SEQ ID no. 1, 2, 3, 4, 5, 6, 7, 13, 14. Preferably, said expression vector is pSyc-181, or is pT2. In a further embodiment, said vector is pT2 and comprises a bidirectional promoter which controls the expression of the selected nucleic acid molecule in the group consisting of SEQ ID no. 1, 2, 3, 4, 5, 6, 7, 13, 14 and a marker.

[0059] The sub-cellular localization tests of the invention are simple and only require the use of fluorescence-based tools and methods, such as fluorescence microscopy, confocal microscopy, and the like. The method does not require the addition of reagents and can be carried out in living cells, allowing images in real time in the cells.

[ 0060]A specific advantage of the present invention is the absence of background fluorescence before the complementation. Only if a complementation occurs at a particular point of contact, this becomes fluorescent.

[ 0061 ] Furthermore, as shown by the following examples, the constructs according to the present invention are successfully used also in vivo, overcoming the current limits linked to the imaging of subcellular structures in live animals, particularly when the imaging is applied to structures present in neurons or axons, where the in vivo verification of contact points between organelles is still technically difficult.

Examples

[ 0062 ] Example 1: Characterization of the probes in HeLa cells .

[ 0063]With the aim of monitoring the "short" and "long" ER-mitochondria interactions, i.e. in the range of about 10 or about 50 nm, two constructs were developed, ER- Short β-11 ( ERs-βιι) and ER-Long β-11 ( ERL-βιι) , respectively. The two constructs differ in the linkers present between the localization sequence X and the fragment F β-11.

[ 0064]As controls, the Kate β-ll construct, a red fluorescent protein fused with β-ll and the GFPi-io construct, i.e. a complementary fragment of the soluble GFP protein, were used without localization signal.

[ 0065 ] Figure 5 shows the results obtained. As a control, the coexpression of OMM-GFPi-io with Kate β-ll (first column on the left) leads to a homogeneously distributed fluorescent signal in the mitochondria. The expression of ERs-βιι o of ERL-βιι with GFPi-io (second and third column) leads to a general staining of ER . By co- expressing ERs-βιι or ERL-βιι or OMM-GFPi-io (last two columns on the right) , a dotted fluorescent signal is observed, a clear indication of a selective reconstitution at the points of contact between the two organelles. These data indicate the correct localization, expression and spontaneous association ability of the constructs used. In addition, the phenotype observed with ERs-βιι , compared with that obtained with ERL-βιι , only differs in terms of number of points, suggesting that probably the points are the sites where the two organelles are found in the specific distance covered by the probes (i.e. about 15 nm for the short probe and about 50 nm for the long probe) , excluding the possibility of a random effect obtained with the self-complementary fragments. The results obtained indicate that the OMM-GFPi-io and ERs/L-βιι constructs are efficiently expressed and localized in the correct cellular compartment, i.e. OMM and ER, respectively, and that they maintain the ability to spontaneously associate only in those regions that fall within the range of distance imposed by the linker region .

[0066] To better characterize the observed point expression, an immunofluorescence analysis was performed using endogenous markers of ER and mitochondria. In figure 6, the fluorescent points fall within both the mitochondrial signal of the mtHSP60 marker, and within the signal of the lattice marker, Calreticulin (CRT) , thus indicating that said points well represent the ER/mitochondria interface. From the overlapping photo in figure 6 (last column), it is noted that, as expected, the mitochondria and ER network is not fully involved in the formation of the contact points.

[0067] The number of points per cell was quantified by 3D reconstruction of the image with a plugin of the ImageJ software called VolumeJ. The results are shown in the graph in figure 7 and indicate, as expected, a greater number of "long distance" contacts than "short distance" ones .

[0068] To further confirm the correct localization, anti- GFP antibodies and gold nanoparticles directed against the same were used. The analysis shows a preferential labeling of the mitochondria and ER membranes surrounding the mitochondria, as indicated by the arrows in figure 8.

[0069] To exclude that the contact points were artificially created following the expression of the constructs and the self-assembly of the two complementary fragments, measurements of accumulation of mitochondrial Ca²⁺ induced by cell stimulation with histamine were performed, using the recombinant probe aequorin directed selectively to the mitochondrial matrix (Rizzuto et al . , Nature 1992; 358, 325-328) . As shown by the traces in figure 9 A, which represent each the mean of the results obtained from three independent experiments, and the quantification shown in the graph in figure 9B, no differences in the height of the mitochondrial Ca²⁺ peak reached with the histamine stimulus are observed between controls and cells expressing the OMM-GFPi-io and ERs/L-βιι constructs. These results show that the expression of the generated reporters does not artificially increase the ER/mitochondria contacts, and support the conclusion that the fluorescence points observed by confocal fluorescence microscopy analysis are actually representative of the ER/mitochondria contact points present in the experimental conditions considered and not artificially induced by the expression of the constructs themselves.

[0070] Example 2: characterization of the probes in HeK293 cells.

[0071] The transfection experiments with the constructs of example 1 were repeated in HeK293 cells. The data obtained from the confocal fluorescence microscopy analysis shown in figure 11 show that also in this cellular model a well-defined pattern for the reconstitution of the fluorescent point signal is obtained, an indication of a correct self-assembly of the fluorescent probe in the contact points of interest.

[0072 ] Example 3: pharmacological modulation of the ER/mitochondria interface.

[0073] The constitutive passage of Ca²⁺ from ER to the mitochondria is essential for the correct maintenance of cellular bioenergetics (Cardenas C et al . , Cell 2010; 142, 270-283) and the same is modulated to meet the metabolic demands of the cell. Under ER stress and activation of autophagy, an increased coupling between ER/mitochondria is observed (Bravo R. et al . , J Cell Sci 2011; 124, 2143-2152; Bravo-Sagua R. et al . , Scientific reports 2016; 6, 36394; Csordas G. et al . , J Cell Biol 2006; 174, 915-921) .

[0074] The experiment was conducted by inducing ER stress by treating cells with tunicamycin or by keeping the cells in HBSS to activate the autophagy process. HeLa cells were cotransfected with OMM-GFPi-io and ERs-βιι or ERi-βιι and incubated for 4 hours with tunicamycin (10 μg/ml) or with HBSS or untreated for the same time interval. Both treatments induce a significant increase in short-distance contact sites, Figure 10 A and B. It is interesting to note that the increase in short- distance contact points is accompanied by a statistically significant reduction in long-distance ER/mitochondria interactions in cells treated with tunicamycin, but not in cells exposed to HBSS (Figure 10 C, D) . These results show that the constructs according to the present invention find application in monitoring in living cells the variation of the ER/mitochondria interactions both in the short and long distance.

[ 0075 ] Example 4: in vivo characterization of constructs.

[ 0076 ] Zebrafish embryos were used. The constructs according to the present invention were injected into fertilized Zebrafish eggs. In particular, ERs-βιι with cytosolic GFPi-io , and OMM-GFPi-io with DJ-Ιβη, a cytosolic protein fused with the sequence βιι, were injected. The fluorescence signal observed co-expressing OMM-GFPi-io and DJ-1 β is perfectly localized at the mitochondrial level, co-expressing ERs-βιι and GFPi-io at the ER level, such signals co-localize perfectly with those of two specific proteins coexpressed as markers of the two organelles, i.e. pTagRFP-mito and DsRed2 ER, respectively (figure 12) .

[0077]A construct was then used that encodes both the complementary fragments of the reporter developed herein, fused to two distinct localization signals. In particular, a pT2-DsRed-UAS-spGFP2A construct was generated (figure 4 C) . In the construct, the UAS bidirectional promoter was used, which controls the expression of the probe and of the cytosolic DsRed, which acts as a transfection marker. The probe includes ERs-βιι and OMM-GFPi-io linked by a cleavage sequence that is P2A. The UAS promoter is activated in the presence of the transcription factor GAL4. In this regard, a transgenic Zebrafish line was used, the sl002t:GAL4, which constitutively expresses GAL4 in a specific neuronal population, i.e. in Rohon-Beard (RB) neurons

(Sagasti A et al . , Curr Biol 2005; 15: 804-814), neurons characterized by a very long axon and therefore preferred for cell imaging analysis. Injection of the pT2-DsRed-UAS-spGFP2A vector in sl002t:GAL4 Zebrafish generated the selective and specific expression of spGFP2A and DsRed in RB neurons, suggesting that the vector is a valid tool for imaging of ER-mitochondria contacts in vivo.

[0078] Therefore, the presence of GFP signal was evaluated in the DsRed positive neurons, i.e. the presence of a point-like signal indicating the ER- mitochondria contacts. The data are shown in figure 13, A and B. There are many interactions in the soma of RB neurons, whose number is comparable to that observed in HeLa cells. The interactions are also present in the axons, where some areas are particularly enriched, figure 13 C. Probably, these areas in which the ER- mitochondria interactions are particularly present represent axonal areas responsible for specific functions, as described for example in Bannai H. et al . , Cell Sci 2004; 117, 163-175; Cui-Wang T. et al . , Cell 2012; 148, 309-321; Villegas R. et al . , J Neurosci 2014; 34, 7179-7189. The number of interactions was quantified and normalized as a function of the reference area and it was found that the number of ER-mitochondria contacts did not vary considerably between axon and soma (figure 13 D) .

Claims

1. A method for determining the interactions between sub-cellular organelles in a cell comprising:

b) displaying the fluorescent signal and therefore the contact points between said first and second sub-cellular organelles;

wherein said fluorophore is selected from GFP or variants thereof.

2. A method according to claim 1, wherein said first sub-cellular organelle is the endoplasmic reticulum (ER) and said localization signal X is selected from the localization signals selectively targeting proteins to ER or represents a portion of a known protein responsible for anchorage thereof to ER membrane, preferably it is a portion of the Sacl protein and said second sub-cellular organelle is the mitochondrion and said localization signal Y is selected from the localization signals selectively targeting the outer mitochondrial membrane (OMM) .

3. A method according to claim 1 or 2, wherein said cell is an in vitro cell or an in vivo cell.

4. A method according to any one of claims 1 to 3, wherein said localization signal X has at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90% identity with SEQ ID no. 3: AGAGTGTTCCTGGCCCTGCCCATCATCATGGTGGTGGCCTTCAGCATGTGC ATCATCTGCCTGCTGATGGCCGGCGACACCTGGACAGAGACACTGGCCTAC GTGCTGTTCTGGGGCGTGGCCAGCATCGGCACCTTTTTCATCATCCTGTAC AACGGCAAGGACTTCGTGGACGCCCCCAGACTGGTGCAGAAAGAGAAGATC GAC.

5. A method according to any one of claims 1 to 3, wherein said localization signal Y has at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90% identity with SEQ ID no. 4: GTGGGCCGGAACAGCGCCATCGCCGCGGGCGTGTGCGGTGCCCTCTTCATA GGGTACTGCATCTACTTTGACCGCAAAAGGCGGAGTGACCCCAAC .

6. A method according to any one of claims 1 to 5, wherein said fluorophore is GFP and said complementary fragment F is GFP β-strand 11 (βΐΐ) and is selected from the group having at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90% identity with SEQ ID no. 2, 17 or 18 and the complementary fragment F' corresponds to the portion GFP 1-10 (GFPl-10) and is selected from the group having at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90% identity with SEQ ID no. 1, 15 or 16.

7. A method according to any one of claims 1 to 6, wherein said complementary fragment F βΐΐ has the sequence SEQ ID no. 2:

CGGGACCACATGGTGCTGCACGAGTACGTGAACGCCGCTGGCATCACA

and said complementary fragment F' GFPl-10 has SEQ ID no . 1 :

ATGTCCAAAGGAGAAGAACTGTTTACCGGCGTGGTGCCAATTCTCGTGGAA CTGGATGGCGATGTGAATGGCCACAAATTTTCTGTCAGAGGAGAGGGTGAA GGTGATGCCACAATCGGAAAGCTCACCCTGAAATTCATCTGCACCACTGGA AAGCTCCCTGTGCCATGGCCAACACTGGTCACTACCCTGACCTACGGCGTG CAGTGCTTTTCCAGATACCCAGACCATATGAAGAGGCATGACTTTTTCAAG AGCGCCATGCCCGAGGGCTATGTGCAGGAGAGAACCATCTCTTTCAAAGAT GACGGGAAATACAAGACCCGCGCTGTGGTCAAGTTCGAAGGAGACACACTG GTGAATAGAATCGAGTTGAAGGGCACAGACTTTAAGGAAGATGGAAACATT CTCGGCCACAAGCTGGAATACAACTTTAACTCCCACAATGTGTACATCACA GCCGACAAGCAAAAGAATGGCATCAAGGCTAACTTCACAGTCAGACACAAC GTCGAGGATGGAAGCGTGCAGCTGGCCGACCATTATCAACAGAACACTCCA ATCGGCGACGGCCCTGTGCTCCTCCCAGACAACCATTACCTGTCCACCCAG ACAGTCCTGAGCAAAGATCCAAATGAAAAAGGAACATAA.

8. A method according to any one of claims 1 to 7, wherein said localization signal X is SEQ ID no. 3, said localization signal Y is SEQ ID no. 4, said complementary fragment F is SEQ ID no. 2 and said complementary fragment F' is SEQ ID no. 1.

9. A method according to any one of claims 1 to 8, wherein said method is adapted to display the interactions between organelles within the range of 7-20 nm and said localization signal X is separated from said complementary fragment F by a linker consisting of about 15-50 AA, preferably said linker has a sequence having at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90% identity with SEQ ID no. 5:

ATGCGGGACCACATGGTGCTGCACGAGTACGTGAACGCCGCTGGCATCACA GGCGGAGATGGCGGATCTGGCGGCGGAAGC .

10. A method according to any one of claims 1 to 8, wherein said method is adapted to display the interactions between organelles within the range of 40-100 nm and said localization signal X is separated from said complementary fragment F by a linker consisting of about 100-180 AA, preferably said linker has a sequence having at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90% identity with SEQ ID no. 6:

ATGCGGGACCACATGGTGCTGCACGAGTACGTGAACGCCGCTGGCATCACA GGCGGAGATGGCGGATCTGGCGGCGGAAGCAAACTGATGTGGCACGAGGGA CTGGAAGAGGCCAGCAGACTGTACTTCGGCGAGCGGAACGTGAAGGGCATG TTCGAGGTGCTGGAACCCCTGCACGCCATGATGGAAAGAGGCCCCCAGACC CTGAAAGAGACAAGCTTCAACCAGGCCTACGGCCGGGACCTGATGGAAGCC CAGGAATGGTGCCGGAAGTACATGAAGTCCGGCAATGTGAAGGACCTGACA CAGGCCTGGGACCTGTACTACCACGTGTTCCGGCGGATCAGCAAGCAGGGC TCTGAAGCCGCCGCTAGAGAAGCTGCTGCTAGAGGCGGAGCTTCTGGCGCT GGCGCAGGCGCTGGGGCCATCCTGAATAGC .

11. A method according to any one of claims 1 to 10, wherein said fusion protein X-F and said fusion protein Y-F' are coded by a single vector, under the control of a single promoter, joined together by a cleavage sequence which is preferably P2A, SEQ ID no . 7 :

GGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAG GAGAACCCTGGACCT .

12. An isolated polypeptide having an amino acid sequence selected from the group consisting of SEQ ID no . 8, 9, 10, 11, 12.

13. A kit comprising one or more polypeptides according to claim 12, wherein said kit comprises SEQ ID: no. 8 and SEQ ID no. 10, or SEQ ID no. 9 and SEQ ID no. 10, or SEQ ID no. 11, or SEQ ID no. 12.

14. An expression vector comprising a nucleic acid molecule selected from the group consisting of SEQ ID no. 1, 2, 3, 4, 5, 6, 7, 13, 14.