Colocalisation experiments in Phaeodactylum tricornutum

GFP and CFP expression provides a good tool to study the intracellular localisation of proteins. However, sometimes a clear differentiation between closely associated com-ponents is difficult. In order to confirm intracellular protein localisations it might be useful to express two different reporter proteins within one cell to ensure that targeting signals direct a protein into the same compartment. It has been described before that co-transformation of an unselected gene with the Phaeodactylum tricornutum trans-formation vector pPha-T1 may also promote its stable integration into the genome [42]. First we tested whether we can direct GFP and the cyan variant CFP simulta-neously to different subcellular locations in the diatomP. tricornutum and whether it is possible to distinguish the two fluorophores microscopically. Therefore we used two different vectors, one containing the GFP gene fused to the presequence of the oxygen evolving enhancer 1 (PtOEE1; PtOEE1pre:GFP) and another containing the CFP gene only. Sequence analyses of the presequence of the P. tricornutum oxygen evolv-ing enhancer 1 protein (PtOEE1) showed a high prediction probability for a signal peptide and a weaker for a transit peptide (Table A.3). An “ASAFAP”-motif is also present in PtOEE1 but they do not coincide with the predicted signal peptide cleavage site (Figure 4.1A). As this protein is located in the thylakoids (Ammon and Kroth, un-published), for a better visualisation of GFP fluorescence, the third targeting domain responsible for thylakoid targeting has been deleted [82]. PtOEE1pre has previously been characterised intensively [81, 82, 53] and it was shown that it is targeted to the plastid stroma. When a mixture of the PtOEE1 containing plasmid and the CFP containing plasmid was then used to transformP. tricornutum, we were able to detect GFP fluorescence within the plastid and CFP in the cytosol of the cell (Figure 4.1B).

This indicates that discrimination between GFP and CFP fluorescence within the same cell is possible. Another control experiment was the simultaneous detection of GFP and CFP within the same compartment. Expression of PtOEE1∆F:GFP and PtOEE1∆F:CFP (lacking the phenylalanine at the +1 position of the transit peptide-like domain) in P. tricornutum, leads to congruent fluorescence of GFP and CFP within the same compartment (Figure 4.1C) in a structure previously described as “blob”-like structure [82]. In previous studies it has been demonstrated that the unmodified TpNTT3 and PtPGDH [7, 54] are targeted preferrably to the periplas-tidic space [53, 51, 141] and show the same phenotype as described by Gruber et al.

PtOEE1pre:GFP + CFP

(B)

DIC Chlorophyll GFP ChlGFPCFP

PtOEE1 Fpre:GFP + PtOEE1 Fpre:CFPΔ Δ

(C)

DIC Chlorophyll GFP ChlGFPCFP

(A)

: signal peptide predicted by SignalP´s hidden Markov models : estimated transit peptide domain

: mature protein

lower case: artificial sequence

GREY: conserved motif at signal peptide cleavage site

enhanced green fluorescent protein / enhanced cyan fluorescent protein MKFTAACSLALVASASAF

Figure 4.1.: Co-localisation control constructs in P. tricornutum. (A) PtOEE1pre:GFP, PtOEE1∆Fpre:GFP and PtOEE1∆F:CFP fusion constructs; (B) expression of PtOEE1pre:GFP and CFP inP. tricornutum;(C) expression of PtOEE1∆Fpre:GFP and PtOEE1∆F:CFP inP. tricornutum, GFP and CFP accumulates in a small reticular structure tightly associated to the plastid but outside the stroma, previously described as “blob”-like structure (BLS) [82]; Nomarski’s differential interfer-ence contrast (DIC), Chlorophyll autofluorescinterfer-ence, GFP fluorescinterfer-ence, CFP fluorescinterfer-ence and merged image showing the respective channels in the indicated colours are shown from left to right; scale bars represent10µm.

(2007). To check whether there is a difference in localisation between nBLS and aBLS, we fused the presequence from TpNTT3 and PtPGDH to GFP (native presequences leading to accumulation of GFP in BLS when expressed as a GFP fusion construct inP. tricornutum) and used these two constructs to genetically transform the diatom P. tricornutum simultaneously with the fusion protein PtOEE1∆Fpre:CFP (modified

presequence leading to accumulation of GFP in BLS when expressed as a GFP fusion construct in P. tricornutum) (Figure 4.2B). No differences between GFP and CFP localisation were found, we observed that images of both fluorophores within the cell were congruent (Figure 4.2C and D). We therefore conclude that nBLSs and aBLSs actually represent the same cellular compartment, presumably the periplastidic space.

This also suggests that presequences mediating transport into aBLS and nBLS are recognised by the same protein import pathway.

To check whether it is also possible to transform and co-transform, respectively, T. pseudonana cells and whether there is a difference between aBLS and nBLS locali-sation, respectively, we used again the fluorophores GFP and CFP. For that we fused the OEE1 protein from P. tricornutum (with the deleted F at signal peptides cleav-age site) to GFP and NADPH thioredoxin reductase C (NTRC) fromT. pseudonana to CFP. NTRC is exclusively found in oxygenic photosynthetical organisms including some, but not all, cyanobacteria, green algae, and plants, where it is localised in chloro-plasts [112, 67, 19]. From the diatomP. tricornutum it is known that a PtNTRC:GFP fusion protein lead to a BLS, which might mean that it is in the periplastidic space located [157].

Because it was unclear, whether a co-transformation with three plasmids (an ad-ditional seperate plasmid including the cloNat resistance cassette: pTpNR:nat have to be transformed) or with two plasmids (cloNat cassette is already in one fusion construct included, but resulting in one big plasmid, here: TpNTRCpre:CFP:nat) is more problematic, we tested both approaches (Figure 4.3B). Therefore we con-structed TpNTRCpre:CFP and TpNTRC:CFP:nat fusion proteins, respectively, and expressed them each with PtOEE1pre∆F:GFP in the diatom T. pseudonana. In the case of the approach where we co-transformed TpNTRCpre:CFP together with PtOEE1pre∆F:GFP we added an additional seperate plasmid including the cloNat resistance cassette to the transformation mixture. Unfortunately in both cases we observed only GFP accumulation in a BLS but no CFP fluorescence (Figure 4.3C and D). This means that in both cases the transformation was partly successful, because of fluorescing (GFP) transformants, but it is thinkable that in one case (Tp-NTRCpre:CFP:nat + PtOEE1∆Fpre:GFP) the CFP expression or fluorescence is too weak, or in the other case (TpNTRCpre:CFP + PtOEE1∆Fpre:GFP + pTpNR:nat) the plasmid containing the CFP gene was not integrated into the genome. At the mo-ment it is not possible to make an evidence whether there is a difference in localisation of aBLS and nBLS in T. pseudonana or not.

(A)

: signal peptide predicted by SignalP´s hidden Markov models : estimated transit peptide domain

: mature protein

lower case: artificial sequence

GREY: conserved motif at signal peptide cleavage site

enhanced green fluorescent protein / enhanced cyan fluorescent protein MRAPTTSVATTLALLAAASTTEA

TpNTT3pre:GFP + PtOEE1 Fpre:CFPΔ (C)

DIC Chlorophyll GFP ChlGFPCFP

PtPGDHpre:GFP + PtOEE1 Fpre:CFPΔ (D)

DIC Chlorophyll GFP CFP ChlGFPCFP

CFP

or

+

TpNTT3pre:GFP PtPGDHpre:GFP

(B)

Figure 4.2.: Co-localisation experiment in Phaeodactylum tricornutum. (A) TpNTT3pre:GFP, PtPGDHpre:GFP and PtOEE1∆Fpre:CFP fusion constructs; (B) used vectors for co-transformation ofP. tricornutum;(C)expression of TpNTT3pre:GFP and PtOEE1∆Fpre:CFP inP. tricornutum, GFP and CFP accumulates in a small reticular structure tightly associated to the plastid but outside the stroma, previously described as “blob”-like structure (BLS) [82]; (D)expression of PtPGDHpre:GFP and PtOEE1∆Fpre:CFP in P. tricornutum, GFP and CFP accumulates in a small reticular structure tightly associated to the plastid but outside the stroma, previously described as “blob”-like structure (BLS) [82]; Nomarski’s differential interference contrast (DIC), Chlorophyll autofluorescence, GFP fluo-rescence, CFP fluorescence and merged image showing the respective channels in the indicated colours are shown from left to right; scale bars represent10µm.

TpNTRCpre:CFP:nat + PtOEE1ΔFpre:GFP (C)

DIC Chlorophyll GFP ChlGFPCFP

(A)

: signal peptide predicted by SignalP´s hidden Markov models : estimated transit peptide domain

: mature protein

lower case: artificial sequence

GREY: conserved motif at signal peptide cleavage site

enhanced green fluorescent protein / enhanced cyan fluorescent protein MSMLRTVLLLLCVAFTT

TpNTRCpre:CFP + PtOEE1 Fpre:GFP + natΔ (D)

DIC Chlorophyll GFP CFP ChlGFPCFP

TpNTRCpre:CFP:nat

Figure 4.3.: Co-localisation in Thalassiosira pseudonana. (A) TpNTRCpre:CFP and PtOEE1∆Fpre:GFP fusion constructs; (B) used vectors and vector-combination, respectively, for co-transformation ofT. pseudonana;(C) expression of TpNTRCpre:CFP:nat and PtOEE1∆Fpre:GFP in T. pseudonana, we could observe only a GFP fluorescence in a BLS and no CFP fluorescence;(D) expression of TpNTRCpre:CFP and PtOEE1∆Fpre:GFP in T. pseudonana, we could observe only a GFP fluorescence in a BLS and no CFP fluorescence; Nomarski’s differential interference contrast (DIC), Chlorophyll autofluorescence, GFP fluorescence, CFP fluorescence and merged image showing the respective channels in the indicated colours are shown from left to right; scale bars represent 10µm.