Fig. 5 | Nature Communications

Fig. 5

From: Bacterial encapsulins as orthogonal compartments for mammalian cell engineering

Fig. 5

Bioengineering of a melanosome by targeting melanin-generating tyrosinase to the encapsulin compartment. a Schematic of the detoxifying effects of compartmentalized melanin production by encapsulated tyrosinase from Bacillus megaterium targeted to the nanocompartment via fusion to the native cargo D. The substrate l-tyrosine enters the compartment via the pores in the nanoshell. b BN-PAGE showing on-gel production of melanin via tyrosinase expressed in HEK293T cells fused to Myc-tagged encapsulin-cargo D (MycD-BmTyr) to encapsulate it in the assembled nanoshell. Dark colorization of the band was observed after incubation with 2 mM l-tyrosine and 100 µM CuCl2 in PBS (pH 7.4) for 1 h at 37 °C. c Bright-field images of HEK293T cells expressing MycD-BmTyr with and without StrepTagII-modified shell (ASTII) after 48 h of expression. Twenty four hours post transfection, cells were supplemented with 1 mM l-tyrosine and 10 µM CuCl2. Cell protrusions (white arrows) were apparent indicating toxic effects of overexpression of non-encapsulated tyrosinase. Scale bar: 20 µm. d Corresponding luciferase-based viability assay of HEK293T cells treated as in c overexpressing MycD-BmTyr with or without ASTII after 48 h. (The bars represent the mean ± SEM, n = 6, p < 0.0001, unpaired t-test.) e Images of two tubular phantoms (transversal slice) obtained by multispectral optoacoustic tomography (MSOT). The phantoms were filled with ~107 cells in 1.5% low melting agar expressing MycD-BmTyr with ASTII (supplementation as in c and d) or containing highly concentrated ink (OD = 0.2) as control showing the intense contrast obtained between 690 nm and 900 nm from the melanin-producing encapsulins. The coefficients obtained from linear unmixing of the optoacoustic spectra with a melanin reference spectrum are displayed on the red colormap overlaid on the image obtained at 720 nm. The lower left inset shows a color photograph of the tubular phantom containing the cells. Scale bar: 3 mm

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