Genetic constructs

Mammalian codon-optimized MxEncA (UniProt:MXAN_3556) MxEncB, MxEncC, and MxEncD (UniProt: MXAN_3557, MXAN_4464, MXAN_2410) were custom synthesized by Integrated DNA Technologies and cloned into pcDNA 3.1 ( + ) Zeocin (Invitrogen) using restriction cloning or Gibson assembly. The MxEncA surface tags (FLAG or StrepTagII) were C-terminally appended using Q5® Site-Directed Mutagenesis (New England Biolabs). N-terminal Myc epitopes were added accordingly to the cargo proteins. Multigene expression of B, C, and D was achieved by generating a single reading frame containing all three genes separated by P2A peptides yielding BCD P2A . A “scarless” bicistronic construct encoding MycC and ASTII was custom synthesized by inserting a Ssp DnaE mini‐intein variant engineered for hyper‐N‐terminal autocleavage followed by a P2A peptide in between the genes as previously described41. For generating stable clones expressing MxEncABCD, MxEncAFLAG was cloned into the Cytomegalovirus promoter (CMV) driven expression cassette of pBudCE4.1 (Invitrogen) and BCD P2A was cloned into the elongation factor 1 alpha promoter (EF1a) driven expression cassette of the vector via restriction cloning. To generate AAV enabling multigene expression of MxEncAFLAG and MxB-Mms7ct, two strategies were employed: MxEncAFLAG was cloned upstream of an ECMV internal ribosome entry site (IRES) whereas MxB-Mms7ct was inserted downstream. The second approach employs MxB-Mms7ct followed by a P2A peptide and MxEncAFLAG. The two cassettes were subcloned into pAAV-CamKIIa (https://www.addgene.org/26969/) with BamHI and EcoRI. AAVs were custom prepared by the UNC Vector Core of the University of North Carolina at Chapel Hill. To test the bicistronic expression constructs used for the AAVs in HEK293T cells, the cassettes were also sub-cloned into the pcDNA 3.1 ( + ) Zeocin with EcoRI and NotI. To target PAmCherry1 and mEos4b as cargo to the encapsulin nanocompartments, the fluorescent proteins were C-terminally fused to 2 × GGGGS linkers followed by the minimal encapsulation signal LTVGSLRR (EncSig). To generate the destabilized version of mEos4b, the L106P mutant of FKBP12 (DD-N)43 was N-terminally appended to mEos4b-EncSig using Gibson-Assembly yielding DD-mEos4b-EncSig. For complementation of split PAmCherry1 inside the encapsulin nanoshell, amino acids 1–159 of PAmCherry1 were fused to MxEncC via a 2 × GGGGS linker and amino acids 160–236 of PAmCherry1 were directly fused to the C-terminus of MxEncB. For complementation of a split luciferase, the split part LgBit (NanoBiT system, Promega) was fused C-terminally to MxEncC via a 2 × GGGGS linker. SmBit was directly fused to the C-terminus of MxEncB. SmCSE55 (UniProt: Smal_0489) and APEX253 were fused to 2 × GGGGS linker followed by the minimal encapsulation signal. Mammalian codon-optimized Bacillus megaterium tyrosinase (BmTyr) was C-terminally appended to MycD separated by 2 × GGGGS linker in custom gene synthesis. C-terminally FLAG-tagged Mus musculus Zip14 was inserted into pcDNA 3.1 ( + ) or pIRES2-ZsGreen1 via restriction cloning. To yield secreted encapsulins, MxEncASTII was N-terminally fused to a human BM40 secretion peptide. In order to generate encapsulin derivatives featuring C-terminal acidic peptides of magnetotactic bacteria Mms proteins that are implicated in mediation of magnetite formation either the C-terminal peptide of Mms6 (YAYMKSRDIESAQSDEEVELRDALA) or Mms7 (YVWARRRHGTPDLSDDALLAAAGEE) of Magnetospirillum magneticum were fused either to the inward-facing N-terminus of MxEncAFLAG or to the C-terminus of either the MxEncB or C using Q5® Site-Directed Mutagenesis. For a complete list of the genetic constructs featuring their composition refer to Supplementary Table 1.

Cell culture

Low passage number HEK293T (ECACC: 12022001, obtained via Sigma-Aldrich) and CHO (ECACC: 85050302, obtained via Sigma-Aldrich) cells were cultured in advanced DMEM with 10 % FBS and penicillin–streptomycin at 100 µg/ml at 37 °C and 5% CO 2 . Cells were transfected with X-tremeGENE HP (Roche) according to the protocol of the manufacturer. DNA amounts (ratio shell to cargos) were kept constant in all transient experiments to yield reproducible DNA-Lipoplex formation. To generate a stable HEK293T cell line expressing MxEncABCD, cells were transfected with pBudCE4.1 MxEncABCD and stable transfectants were selected with 300 µg/ml Zeocin (InvivoGen).

Protein expression and lysis

Cells were harvested between 24 and 48 h post transfection. Cells were lysed with M-PER Mammalian Protein Extraction Reagent (Pierce Biotechnology) containing a mammalian protease inhibitor cocktail (SIGMA P8340, Sigma-Aldrich) according to the protocol of the manufacturer in all experiments using FLAG-tagged encapsulins. For lysis of cells expressing StrepTagII-modified encapsulins, cells were resuspended in Buffer W (150 mM NaCl, 100 mM Tris-Cl, pH 8.0) and exposed to four freeze–thaw cycles in LN 2 . After spinning down cell debris at 10,000 × g for 15 min, cell lysates were kept at 4 °C for downstream analyses. Protein concentrations of lysates were determined by measuring OD at 280 nm.

Co-immunoprecipitation of encapsulins

Cell lysates were incubated with Anti-FLAG® M2 Magnetic Beads or Anti-FLAG® M2 affinity gel (SIGMA M8823 and A2220, Sigma-Aldrich) according to the protocol of the manufacturer. After binding, the magnetic beads were washed four times on a magnetic separator rack (DYNAL separator, Invitrogen) with M-PER buffer. Bound FLAG-tagged encapsulins were eluted using M-PER buffer containing 100 µg/ml FLAG-peptide (SIGMA F3290, Sigma-Aldrich). In the case of encapsulins with an external StrepTagII, MagStrep “type3” XT beads or Strep-Tactin®XT resin (IBA Lifesciences) was used according to the protocol of the manufacturer. Proteins were eluted using Buffer BXT (150 mM NaCl, 100 mM Tris-Cl, pH 8.0, 50 mM Biotin). To analyze the eluted proteins, samples were mixed with SDS-PAGE sample buffer and incubated at 95 °C for 5 min. Samples were loaded onto pre-cast 12% Bio-Rad Mini-PROTEAN® TGX™ (Bio-Rad Laboratories) gels and run for 45 min at 200 V. Accordingly, gels were either directly silver-stained using SilverQuest™ Silver Staining Kit (Novex) according to the protocol of the manufacturer or immuno-blotted onto PVDF membranes. After blotting, membranes were blocked in 5% non-fat milk in TBS for 1 h at room temperature. Subsequently, membranes were incubated in TBS containing 5% non-fat milk and 1 µg/ml Monoclonal ANTI-FLAG® M2 antibody (SIGMA F1804, Sigma-Aldrich) or 1 µg/ml Anti-Myc Tag Antibody clone 9E10 (05–419, EMD Millipore) for 2 h at room temperature. After five washing cycles with TBS, membranes were incubated with anti-mouse IgG HRP-conjugate (SIGMA A5278, Sigma-Aldrich) for 1 h at room temperature in 5% non-fat milk in TBS. Protein bands were detected using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare Bio-Sciences AB) on a Fusion FX7/SL advance imaging system (Peqlab Biotechnologie GmbH). For dephosphorylation of protein material from the Co-IP, 10 units of calf intestinal phosphatase (New England Biolabs) were added to protein solutions in 1× CutSmart Buffer (New England Biolabs) and incubated for 1 h at 37 °C. For densitometric determination of SDS-PAGE bands, band intensity integrals were measured using ImageJ (NIH).

Blue Native gel electrophoresis and on-gel analyses

For detection of native encapsulin nanocompartments, the NativePAGE™ Novex® Bis-Tris Gel System (Life Technologies) was used. Either eluted material from the Co-IP/purification or whole cell lysates of cells expressing encapsulins in NativePAGE™ Novex® sample buffer were loaded onto pre-cast NativePAGE™ Novex® 3–12% Bis-Tris gels. NativeMark™ Unstained Protein Standard (Life Technologies) covering a size range between 20 and 1200 kDa was used as a marker. The total protein amount of whole cell lysates loaded per well was adjusted to ~1–3 µg. Blue native (BN) gels were run for 90–180 min at 150 V according to the protocol of the manufacturer. Gels loaded with samples from Co-IP/purification were silver-stained using SilverQuest™ Silver Staining Kit (Novex) or Coomassie-stained using Bio-Safe™ Coomassie Stain (Bio-Rad Laboratories). For protein detection, gels loaded with whole cell lysate samples were Coomassie-stained accordingly. For detection of iron-containing proteins, gels loaded with samples containing iron loaded encapsulins were Prussian Blue (PB) stained. Briefly, gels were incubated in 2% potassium hexacyanoferrate(II) in 10% HCl for 45 min. For 3,3′-diaminobenzidine-enhancement (DAB PB), gels were washed three times with ddH 2 O and incubated in 0.1 M phosphate buffer (pH 7.4) containing 0.025% DAB and 0.005% H 2 O 2 until dark-brown bands appeared. To stop DAB polymerization, gels were washed three times with ddH 2 O. For detection of fluorescent signals from native encapsulin bands (fluorescent cargos: mEos4b, PAmCherry1, split PAmCherry1 or mineralized CdS), unstained BN gels were imaged on a Fusion FX7/SL advance imaging system (Peqlab Biotechnologie GmbH) using the UV fluorescence mode. For on-gel detection of luminescence signal generated by encapsulated split NanoLuciferase, unstained BN gels were soaked in 1 ml of Nano-Glo® Luciferase substrate (Nano-Glo® Luciferase Assay, Promega) and imaged on a Fusion FX7/SL advance imaging system (Peqlab Biotechnologie GmbH) in chemiluminescence mode. For whole cell lysate luminescence detection, cell lysates were mixed with the substrate at a 1:1 ratio and luminescence readings were taken on a Centro LB 960 (Berthold Technologies) at 0.1 s acquisition time. For detection of APEX2 peroxidase activity inside encapsulins, unstained BN gels were incubated in 0.1 M phosphate buffer (pH 7.4) containing 0.025% DAB and 0.005% H 2 O 2 for 15 min until black bands appeared on the gel. For microscopic detection of DAB polymerization in cells expressing APEX2-loaded encapsulins, cells were fixed in 4% PFA in PBS for 15 min. Subsequently, cells were incubated in 0.1 M phosphate buffer (pH 7.4) containing 0.025% DAB and 0.005% H 2 O 2 for 5 min. The reaction was stopped by washing three times with PBS. For the on-gel detection of melanin generation associated with encapsulins, gels loaded with whole cell lysates of HEK293T cells expressing encapsulins loaded with tyrosinase were incubated in PBS containing 2 mM l-tyrosinase and 100 µM CuCl 2 for 1 h at 37 °C until a black encapsulin band became visible.

Size exclusion chromatography

Size exclusion chromatography (SEC) of purified AFLAG with or without DD-mEos4b-EncSig was performed on an Äkta Purifier (GE Healthcare) equipped with an analytical size exclusion column (Superose 6 10/300 GL, GE Healthcare) at 4 oC. For refractive index (RI) detection, a Viscotek TDA 305 triple array detector (Malvern Instruments) downstream of the column was used. In total, 100 µl samples were run at a flow rate of 0.4 ml/min in 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 7.4, at a concentration of 0.3 mg/ml.

Dynamic light scattering

Dynamic light scattering experiments were performed on a DynaPro NanoStar instrument and analyzed with DYNAMICS 7.1.9 software (Wyatt Technology). Measurements were performed at 22 °C using standard rectangular cuvettes containing 60 µl of protein sample in the concentration range between 0.15 and 0.5 mg/ml. For each measurement, 100 acquisitions with an acquisition time of 5 s were recorded.

Native mass spectrometry

Purified sample material from HEK293T cells expressing AFLAG, with and without co-expression of the photoactivatable fluorescent protein DD-mEos4b-EncSig, was buffer exchanged to 150 mM aqueous ammonium acetate, pH 7.5 using Micro Bio-Spin Columns with Bio-Gel P6 (Biorad, USA) following the manufacturer’s protocol for buffer exchange. Samples were analyzed at a concentration of 0.1–0.45 g/l, corresponding to an estimated monomer concentration ranging from 3 to 14 μM. Gold-coated nanoelectrospray needles were made in-house from borosilicate capillaries (Kwik-Fil, World Precision Instruments, Sarasota, FL) on a P97 puller (Sutter Instruments, Novato, CA) and being coated by using an Edwards Scancoat six pirani 501 sputter coater (Edwards Laboratories, Milpitas, USA). Measurements were carried out in positive ion mode on a modified Q-ToF 2 (Waters, UK) instrument71,72, operated at elevated pressure in the source region (~10 mbar), using Xenon as collision gas at 2*10−2 mbar in the collision cell. Capillary and sample cone voltage was set to 1400 V and 150 V, respectively. The voltage before the collision cell was either set to 100 V or 250–300 V, optimizing for desolvation of the intact complex or the subsequent ejection of subunits, respectively. Spectra were calibrated using an aqueous solution of cesium iodide (25 mg/ml) and exported from MassLynx. All further data analysis was performed with in-house developed python scripts (Python 3.6). When applicable, charges were assigned to charge state resolved peak series by extracting the top position for consecutive charge states and minimizing the standard deviation (SD) of the average mass by trying different charge states. Centroids for empty and cargo filled encapsulins (T = 3) were calculated using all data points above 40 % of the base peaks intensity in the appropriate region (m/z 30,000–40,000 for empty and m/z 35,000–45,000 for cargo filled encapsulins). The average was taken over three technical replicates and the error represents the standard deviation (SD). To estimate the mass from the m/z position, we fitted 77 empirical determined masses and their corresponding m/z positions to the equation Mass[kDa] = A*m/zB. These 77 proteins consist of encapsulin T = 1, which mass was determined in this study as well as 76 other assemblies, which were already measured and reported in previous publications73,74. The resulting formula Mass[kDa] = 1.63*10−6*m/z2.14 was used to calculate the mass from the average m/z positions of empty and cargo filled encapsulins (T = 3). Since mass and m/z positions do not follow a linear relation, we averaged the upper and lower error from the m/z dimension projected in the mass domain. Cargo load was estimated based on the difference between the predicted masses of empty and cargo loaded encapsulins (T = 3). The error of the mass difference was calculated using the equation σΔMass = (σMass12 + σMass22)1/2. The difference in mass was then divided by 41.4 kDa, the mass of the DD-mEos4b-EncSig monomer protein. The AFLAG monomer mass was calculated as the weighted average for the different proteoforms, using the summed intensities over the charge states for each species.

Complementation of split PAmCherry1 inside encapsulins

Cells transfected with C-PAs1 and B-PAs2 with or without AFLAG were seeded onto 8-well Poly-l-lysine-coated microscopy chips (Ibidi). Thirty-six hours post transfection, live cell confocal microscopy was conducted on a Leica SP5 system (Leica Microsystems). For photoactivation of split PAmCherry1, samples were illuminated with a 405 nm laser for 60 s at 40% laser power. The signal of complemented split PAmCherry1 was excited using the 561 nm laser. To quantify the complementation of split PAmCherry 1 with or without the encapsulin shell, the ratio of the total mean fluorescence after photoactivation divided by the signal before was calculated. ImageJ was used to quantify mean fluorescence values from randomly chosen areas on the well.

Multispectral optoacoustic tomography

Optoacoustic images of cells co-expressing ASTII and MycD-BmTyr were acquired on an inVision 256-TF system (iThera Medical GmbH). Briefly, ~107 HEK293T cells co-expressing the genes treated with 10 µM CuCl 2 and 1 mM l-tyrosine 24 h prior to the measurement were detached using trypsin, washed with PBS, embedded into 1 % low melting agar yielding a tubular phantom of ~ 300 µl volume. The cell phantom and an ink phantom (OD = 0.2) were placed in a custom-built sample holder and optoacoustic images were acquired for the range of wavelengths between 690 and 900 nm. Signals were reconstructed using ViewMSOT software suite (iThera Medical GmbH) and linearly unmixed using a reference spectrum for melanin.

Magnetic sorting

Cells were washed twice with PBS, detached with Accutase® (Sigma-Aldrich) and resuspended in DPBS supplemented with 10% fetal bovine serum (Gibco) prior to sorting. For magnetic sorting, columns filled with ferromagnetic spheres (MS columns, Miltenyi Biotec) were placed in an external magnetic field (OctoMACS separator, Miltenyi Biotec) and equilibrated with 1 ml DPBS containing 10% FBS. The column was loaded with cells and washed with 0.5 ml DPBS; the flow-through was collected as one fraction. After removing the column from the magnetic separator, cells were eluted with 1 ml DPBS. The total number of cells before sorting as well as the cell numbers in flow-through and eluate were determined with a Countess II FL Automated Cell Counter (Life Technologies).

Magnetic resonance imaging of cells

MR images were acquired at a Bruker BioSpec 94/20USR, 9.4T system equipped with a RF RES 400 1H 112/072 Quad TR AD resonator. For T 2 * measurements of cell pellets, 4*106 HEK293T cells were seeded 24 h prior to transfection on poly-l-lysine-coated 10 cm cell culture dishes. Twenty four hours post transfection, ferrous ammonium sulfate (FAS) was added to the medium yielding a concentration of 1 mM. Twenty four hours post iron addition, cells were washed three times with DPBS and detached with Accutase® and centrifuged at 500 × g for 4 min. The pellets were resuspended in 800 μl DPBS and transferred to cryobank vials (Thermo Scientific Nunc) containing 50 μl of solidified 1% agarose at the bottom. Cells were then spun down at 2000 × g for 2 min and immediately used for MRI. T 2 * measurements were conducted in a custom-made holder filled with DPBS to avoid susceptibility artifacts. T 2 * values were calculated based on a multiple gradient echo (MGE) sequence with a TR of 800 ms, 12 echoes with an echo spacing of 4.5 ms (3.5–58.5 ms), a flip angle of 50°, field of view of 65 × 65 mm and a matrix size of 256 × 256. Relaxation rates were calculated with the Image Sequence Analysis Tool from Bruker BioSpin MRI GmbH.

In vivo expression of encapsulins in murine brains

Mice were positioned in a stereotaxic frame, anesthetized with isoflurane, and implanted bilaterally with MRI compatible guide cannulae (Plastics One) that were stably fixated with dental cement. Injection cannulae (Plastics One) were connected via polyethylene tubing (PE-50), filled with silicone oil, connected to a PhD 2000 syringe pump (Harvard Apparatus) and backfilled with solutions containing AAV viral particles. Injection cannulae were inserted into the guide cannulae and lowered into the brain. A volume of 1 µl of viral particles was injected at 0.1 µl/min. Injection cannulae were subsequently retracted slowly and replaced with dummy cannulae (Plastics One) that screwed firmly into the guide cannula pedestals. All experiments on mice were conducted in accordance with the guidelines approved by the government of Upper Bavaria.

Immunohistochemistry

Three to six weeks after intracranial viral injection, mice were terminally anesthetized, perfused, and the brains were removed for cryosectioning. Brain slices were then blocked in SuperBlock (TBS) Blocking Buffer (Thermo Fisher Scientific) for 1 h at room temperature in a humidified chamber. Subsequently, brain slices were incubated in 5 µg/ml Monoclonal ANTI-FLAG® M2 antibody (SIGMA F1804, Sigma-Aldrich) in TBS for 2 h at room temperature. After 5 × 5 min washes with TBS, the brain slices were incubated in 1 µg/ml Goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody conjugated to Alexa Fluor 488 (A-11001, Invitrogen) in TBS for 1 h at room temperature in darkness. For nuclear counterstaining, DAPI was added at 300 nM for 5 min. Finally, the brains slices were washed five times with TBS. Brain slices were subsequently imaged on an EVOS FL Auto Cell Imaging System (Invitrogen) or a Leica SP 5 confocal microscope (Leica Microsystems).

In vivo MRI

HEK293T cells (~4*106) were seeded onto poly-l-lysine-coated 10 cm cell culture dishes. Twenty four hours after seeding, cells were transiently transfected at 70–80% confluency with DNA constructs encoding either AFLAG + BM7 or AFLAG + mEos4b-EncSig, as well as for both conditions Zip14 at 5% of the total DNA amount using X-tremeGENE™ (Roche). Twenty four hours post transfection, the cell culture medium was replenished with fresh medium containing 1 mM FAS. Twenty four hours after incubation with FAS, cells were washed gently three times with PBS, detached from the culture dishes after 5 min of treatment with a 1:1 solution of Accutase® (Sigma) and Trypsin, centrifuged for 5 min at 1200 × g and resuspended in growth media. Cell suspensions were backfilled into two injection cannulae (28 Gauge, Plastics One, Roanoke, VA, USA) connected via plastic tubing to 25 µl Hamilton glass syringes clamped in a remote dual syringe pump (PHD 22/2,000; Harvard Apparatus, Holliston, MA, USA). Injection cannulae (the side of injection for AFLAG + BM7 or control were switched between experiments) were then lowered into bilateral guide cannulae (22 Gauge, Plastics One, Roanoke, VA) that were previously implanted in Sprague–Dawley rats75. Rats were then centered in the bore of a 7T 20 cm inner diameter, horizontal bore magnet (Bruker BioSpin MRI GmbH, Ettlingen, Germany) and gradient echo scans (FOV = 2.5 cm × 2.5 cm, matrix size = 256 × 256; seven slices with 1 mm slice thickness) were taken at a TR = 800 ms and different echo times (5, 10, 15, 20, 25 ms) to compute relaxation rate maps and perform ROI analysis (circular ROIs of 1 mm diameter placed on injection sites) using custom routines in Matlab (Mathworks, Natick, MA, USA). All procedures on rats were conducted in accordance with National Institutes of Health guidelines and with the approval of the MIT Committee on Animal Care.

Cell viability assays

Iron-related cytotoxicity was monitored via the Roche Cytotoxicity Detection Kit (LDH) (Roche Diagnostics) according to the protocol of the manufacturer. Briefly, 7.5*105 HEK293T cells were seeded on poly-l-lysine-coated 24-well plates. Twenty four hours post seeding, cells were transfected with different combinations of genes using X-tremeGENE HP (Roche). The Zip14 DNA amount was kept constant in all samples expressing Zip14 (5% of total DNA). For expression of combinations of AFLAG with cargo proteins, 60% of the total DNA amount was encoding AFLAG and the remaining 35% were used for the respective cargo molecule. 24 h post transfection, FAS was added to the medium from a 100 mM stock solution yielding a final concentration of 2.5 mM. Twenty four hours post addition of FAS, cells were assayed for LDH release. In order to evaluate gene-related toxicity in the absence of iron, the assay was performed accordingly but without iron supplementation and cells were assayed 48 h post transfection. The Luciferase-based viability assay (RealTime-Glo™ MT Cell Viability Assay, Promega) was performed according to the protocol of the manufacturer in 96-well plate format as an endpoint measurement. Luminescence readings were taken on a Centro LB 960 (Berthold Technologies) at 0.5 s acquisition time.

Electron microscopy

Please refer to Supplementary Methods in the Supplementary Information for a detailed description of the electron microscopy techniques used.

Data availability

Data are available upon reasonable request to the corresponding author. The cryo-EM maps of non-iron loaded and iron loaded encapsulins in HEK293T cells have been deposited under EMDB-4392 and 4393 respectively.