CemOrange2 fusions facilitate multifluorophore subcellular imaging in C. elegans

Abstract

Due to its ease of genetic manipulation and transparency, Caenorhabditis elegans (C. elegans) has become a preferred model system to study gene function by microscopy. The use of Aequorea victoria green fluorescent protein (GFP) fused to proteins or targeting sequences of interest, further expanded upon the utility of C. elegans by labeling subcellular structures, which enables following their disposition during development or in the presence of genetic mutations. Fluorescent proteins with excitation and emission spectra different from that of GFP accelerated the use of multifluorophore imaging in real time. We have expanded the repertoire of fluorescent proteins for use in C. elegans by developing a codon-optimized version of Orange2 (CemOrange2). Proteins or targeting motifs fused to CemOrange2 were distinguishable from the more common fluorophores used in the nematode; such as GFP, YFP, and mKate2. We generated a panel of CemOrange2 fusion constructs, and confirmed they were targeted to their correct subcellular addresses by colocalization with independent markers. To demonstrate the potential usefulness of this new panel of fluorescent protein markers, we showed that CemOrange2 fusion proteins could be used to: 1) monitor biological pathways, 2) multiplex with other fluorescent proteins to determine colocalization and 3) gain phenotypic knowledge of a human ABCA3 orthologue, ABT-4, trafficking variant in the C. elegans model organism.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1. Excitation and emission spectra and cytosolic expression patterns of fluorescent proteins in C. elegans.

Fig 2. CemOrange2 subcellular expression patterns within C. elegans.

Representative confocal images of transgenic C. elegans strains expressing CemOrange2 fused with: the N-terminal SV-40 and C-terminal egl-13 nuclear localization signals (A, B); LMN-1, a nuclear lamin (C, D); TRAM-1, a rough ER protein (E, F); PISY-1, ER and Golgi protein (G, H); AMAN-2, a Golgi protein (I, J); RAB-5, an early endosomal protein (K, L); RAB-7, a late endosomal protein (M, N); LMP-1 and CUP-5, lysosomal membrane proteins (O-R); GLO-1, lysosomal related organelle protein (LRO) (S, T); SKL, a C-terminal peroxisomal targeting peptide (U, V); mt, an N-terminal mitochondrial target peptide (W, X); SQST-1 and LGG-1, autophagosome-related proteins (Y-BB); and AQP-1, an apical and basolateral plasma membrane protein (CC, DD). All transgenes were expressed using the intestinal specific promoter, Pnhx-2. Images were collected over >20 z-planes using a 40x PlanApo oil immersion objective (N.A. 1.3) at wavelengths Ex555 nm/Em565-590 nm. Either a maximum intensity projection (A—D) or a single XY-plane (E-DD) of the CemOrange2 expression are shown. The dashed box demarks a single intestinal cell to highlight the subcellular expression pattern shown in the right column (B, D, F, H, J, L, N, P, R, T, V, X, Z, BB, DD). Scale bars = 25 μm (left column) or 5 μm (right column). See text for description of arrowheads and asterisk.

Fig 3. Colocalization of CemOrange2 probes with differentially labeled subcellular markers.

Transgenic animals expressing the LMP-1, CUP-5, RAB-5, RAB-7, SKL, mt, TRAM-1, AMAN-2 or AQP-1 markers fused to CemOrange2 were labeled with previously described subcellular organelle markers (colocalization marker) and examined by confocal microscopy. Images were acquired over >20 z-planes using a 40x PlanApo oil immersion objective (N.A. 1.3) at wavelengths Ex555 nm/Em565-590 nm and a single XY-plane of representative images of animals are shown. To determine the subcellular localization, LMP-1::CemOrange2 (A; red) and CemOrange2::CUP-5 (D; red) expressing transgenic C. elegans were incubated with LTDR (B and E, blue) to label acidic compartments such as the lysosome. LMP-1 and CUP-5 colocalized with LTDR staining (C and F; magenta, arrowheads). Note, GLO-1 did not colocalize with LTDR (S1 Fig). CemOrange2::RAB-5 (G, red) and CemOrange2::RAB-7 (J, red) expressing animals were incubated with BSA::AlexaFluor647 (H and K, blue) to label the endo-lysosomal compartments. Both RAB-5 and RAB-7 colocalized with BSA::AlexaFluor647 (I and L, arrowheads). Transgenic animals expressing both CemOrange2::SKL (M, red) and DAF-22::GFP (N, blue) colocalized (O; magenta, arrowheads). mtCemOrange2 (P, red) expressing C. elegans were incubated with MitoTracker Deep Red (Q, blue) to label mitochondria. mtCemOrange2 colocalized with the MitoTracker stain (R; magenta, arrowheads). Transgenic animals expressing both CemOrange-1::TRAM-1 (S, red) and GFP::KDEL ER marker (T, blue) colocalized (U, magenta). AMAN-2::CemOrange2 (V, red) expressing animals were incubated with BODIPY C5 Ceramide-FL (C5 FL) (W, blue) to label lipid dense compartments such as the Golgi. AMAN-2 colocalized with C5 FL (X; magenta, arrowheads). AQP-1::CemOrange2 (Y) expressing animals were incubated in CellMask (Z; blue) to label phospholipid bilayers. AQP-1 colocalized with CellMask on the apical side of the plasma membrane (AA, magenta). Scale bar = 5 μm. (BB) Single XY plane confocal images of multiple transgenic C. elegans and their respective colocalization marker were analyzed for colocalization events (n ≥ 5). Colocalization was determined using the Manders correlation coefficient (MCC) algorithm in Volocity image analysis software (v6.3) analyzing the ratio of pixels in the CemOrange2 channel to pixels in the colocalization marker channel.

Fig 4. A live-animal sensor for lysosomal membrane integrity.

Pnhx-2lmp-1::CemOrange2 transgenic C. elegans were exposed to either PBS (A, C, E, G) or 5% tert-butyl hydroperoxide (t-BOOH; B and D) in PBS for 30 min or 500 mM LLOMe (F and H) in PBS for 30 min. Animals were visualized by confocal microscopy using a 40x PlanApo oil immersion objective (N.A.1.3) at Ex555 nm/Em565-590 nm over >20 z-planes. Representative confocal images of animals shown as a single XY plane. Transgenic C. elegans exposed to PBS alone (A, C, E, G) have punctate LMP-1::CemOrange2 expression (A and E, red). The same animals exposed to 5% t-BOOH (B) or 500 mM LLoMe (F) lose punctate LMP-1::CemOrange2 labeled structures. DIC images (C, D, G, H) displayed for reference. Scale bars = 25 μm.

Fig 5. SQST-1:: and ::LGG-1 CemOrange2 fusion proteins monitor autophagic flux.

Pnhx-2sqst-1::CemOrange2 (A, C, E; red) and Pnhx-2CemOrange2::lgg-1 (B, D, F; red) transgenic animals were treated with a diluent control (0.1% DMSO; A and B), 3-methyladenine (10 mM 3-MA, C and D), or 25 μM fluphenazine (E and F) for 16 hrs in liquid culture and analyzed using confocal microscopy over ≥ 20 z-planes Ex555 nm/Em565-590 nm. Representative confocal images of animals shown as a single XY plane. Scale bar = 25 μm. Multiple confocal images of transgenic animals (n ≥ 5 animals) expressing SQST-1::CemOrange2 (G) or CemOrange2::LGG-1 (H) after treatment with the above compounds, were quantified for number of puncta per μm2 imaged using the Quantification module in the Volocity image analysis software (v6.3). Statistical analysis of the drug-treated animals relative to diluent control was performed using an unpaired, 2-tailed t-test (*p<0.05).

Fig 6. CemOrange2 and three-color imaging.

Pnhx-2lmp-1::CemOrange2;Pnhx-2GFP::ATZ;Pnhx-2mKate2::lgg-1 transgenic C. elegans expressing sGFP::ATZ (A and B; green; Ex488 nm/Em500-540 nm), LMP-1::CemOrange2 (C and D; pseudocolored blue; Ex545 nm/ Em560-590 nm) and mKate2::LGG-1 (E and F; red; Ex594 nm/Em605-645 nm) were examined by confocal microscopy over >20 z-planes using a 40x PlanApo oil immersion objective (N.A. 1.3). Maximum intensity projections are shown (A, C, E, G). Scale bars = 25 μm. Magnified single XY regions (dashed box) are included to highlight colocalization events (B, D, F, H). Scale bars = 5 μm. mKate2::LGG-1 and LMP-1::CemOrange2 had multiple colocalization events (G and H; magenta merge, white arrowheads). Alpha-1 antitrypsin Z mutation (ATZ), a polymerizing protein which accumulates in the ER and is degraded, in part, by autophagy, show colocalization with mKate2::LGG-1 (G and H; orange merge, yellow arrowheads). Pnhx-2glo-1::CemOrange2;Pvha-6lmp-1::GFP transgenic C. elegans expressing LMP-1::GFP (I and J, green; Ex488 nm/Em500-540 nm) and GLO-1::CemOrange2 (M and N, red; Ex555 nm/Em560-590 nm) were stained with the acidic organelle fluorescent dye, LTDR (K and L, blue; Ex647 nm/Em660-700 nm). Animals were imaged by confocal microscopy over >20 z-planes and maximum intensity projections are shown (I, K, M, O). Scale bars = 25 μm. Magnified single XY regions (dashed box) are included to highlight colocalization events (J, L, N, P). Scale bars = 5 μm. Colocalization events are seen between LMP-1::GFP and LTDR (O and P, cyan merge, arrowheads) but not with GLO-1::CemOrange2.

Fig 7. A CemOrange2 marker co-localizes a mutant ABC transporter protein to the ER.

Alignment of the primary amino sequence from human (Hsa) ABCA3 (Accession: AAH20724.1) and C. elegans (Cel) ABT-4 (Accession: NP_503175.1) using the ClustalW algorithm (A). Only residues 100–136 of HsaABCA3 aligned with residues 161–200 from CelABT-4 are shown. The asterisk marks L101 (human numbering) or L162 (C. elegans numbering) in the alignment that is conserved between species. Pnhx-2abt-4::mKate2;Pnhx-2TRAM-1::CemOrange2 (B, D, F) or Pnhx-2abt-4L162P::mKate2;Pnhx-2TRAM-1::CemOrange2 (C, E, G) transgenic C. elegans strains were imaged by confocal microscopy using a 40x PlanApo oil immersion objective (N.A. 1.3) over >20 z-planes. Representative single XY regions are shown. ABT-4::mKate2 (D, pseudocolored blue) or ABT-4L162P::mKate2 (E, pseudocolored blue; Ex 594 nm/Em605-645 nm) and TRAM-1::CemOrange2 (B,C, orange; Ex549 nm/ Em560-580 nm), showed that ABT-4::mKate2 trafficked normally to the apical membrane in intestinal cells (F), whereas the single point mutation in ABT-4L162P triggered retention within the ER (G). Scale bar = 25 μm. (H) Colocalization between ABT-4 or ABT-4L162P and TRAM-1 (F and G, respectively) was determined using the Pearson correlation in Volocity image analysis software (v6.3) (***p<0.001; n≥10).