Real-time investigation of dynamic protein crystallization in living cells

Abstract

X-ray crystallography requires sufficiently large crystals to obtain structural insights at atomic resolution, routinely obtained in vitro by time-consuming screening. Recently, successful data collection was reported from protein microcrystals grown within living cells using highly brilliant free-electron laser and third-generation synchrotron radiation. Here, we analyzed in vivo crystal growth of firefly luciferase and Green Fluorescent Protein-tagged reovirus μNS by live-cell imaging, showing that dimensions of living cells did not limit crystal size. The crystallization process is highly dynamic and occurs in different cellular compartments. In vivo protein crystallization offers exciting new possibilities for proteins that do not form crystals in vitro.

FIG. 1.

Time-resolved in vivo crystal growth of firefly luciferase in Sf9 insect cells recorded by life-cell imaging with DIC optics. Cells were plated to 40% confluence on glass coverslips and infected with recombinant P3 luciferase virus stocks. 3 days p.i. cells were put on the live cell microscope and imaged for 67 h. (a) Luciferase crystals grew slowly over the entire period of 67 h from 70 to 100 μm in length. (b) Luciferase in vivo crystallization can be dynamic. In the example show, the crystal shrinks within 33 h from 61 μm to about the cell diameter (15 μm) and starts growing again to a final length of 37 μm. (Multimedia view) [URL: http://dx.doi.org/10.1063/1.4921591.1] [URL: http://dx.doi.org/10.1063/1.4921591.2]

FIG. 2.

Morphology and intracellular localization of in vivo firefly luciferase crystals. (a) Luciferase crystals can be extraordinarily long. The dimensions of the luciferase crystal inside a living cell pictured are 206 μm × 3 μm. Note that the diameter of the Sf9 cell body is only 21 μm. (b) Dead cells in the culture were identified by strong red nuclear fluorescence by addition of the membrane impermeable DNA-binding dye propidium iodide (at 500 ng/ml) to the culture medium at 4 days p.i. with recombinant baculovirus. Cells harboring crystals show no nuclear fluorescence establishing that their plasma membranes were intact. (c) Bodipy 558/568 staining a membrane surrounding a luciferase crystal. A 3D reconstruction of a z-stack of confocal fluorescence images is shown. (d) Firefly luciferase crystals are surrounded by peroxisomal membranes. Cells were co-infected with recombinant firefly luciferase and baculovirus expressing the peroxisomal membrane marker protein Pex26 fused to mCherry. Confocal images recording the DIC and the mCherry fluorescence channels were taken 4 days p.i. Note that the internal membrane surrounding the crystal is clearly labeled with the peroxisomal marker protein. (Multimedia view) [URL: http://dx.doi.org/10.1063/1.4921591.3] [URL: http://dx.doi.org/10.1063/1.4921591.4]

FIG. 3.

Stability of firefly luciferase in vivo crystals. Cells were imaged 4 days p.i. with recombinant luciferase baculovirus on a live cell imaging system. (a) The cell is lysed during image acquisition by the baculovirus. Immediately after cell membrane rupture, the luciferase crystal starts to dissolve. It may take several hours for the crystal to dissolve, likely depending on how much of the, presumably protective, membranes are still left around the crystal. (b) During image acquisition, the medium was exchanged by culture medium supplemented with 0.1% Triton-X 100. In this case, cellular membranes were immediately disrupted and crystals dissolved within 30 min. (c) During image acquisition, the medium was exchanged for a crystal extraction buffer supplemented with 0.1% Triton-X 100. Cellular membranes were quickly disrupted like in (b), but the crystals dissolved much slower. Crystal length was reduced on average only by 30% during 1 h of imaging. (d) During ongoing image acquisition, medium was supplemented with 100 μM membrane-permeable D-Luciferin ethyl ester. This results in a very fast breakdown of the luciferase crystals within 70 s in the intact cells, probably caused by activation of the enzyme. (Multimedia view) [URL: http://dx.doi.org/10.1063/1.4921591.5] [URL: http://dx.doi.org/10.1063/1.4921591.6] [URL: http://dx.doi.org/10.1063/1.4921591.7] [URL: http://dx.doi.org/10.1063/1.4921591.8]

FIG. 4.

Time-resolved in vivo crystal growth of GFP-μNS in Sf9 insect cells recorded by life-cell imaging with DIC optics and GFP fluorescence. Crystal growth was imaged 3 days p.i. over night. (a) GFP-μNS is expressed in the cytoplasm and displays a ubiquitous diffuse fluorescence together with a variable number of dots with higher fluorophore concentration. (b) Some of those dots apparently act as crystallization points and start to grow by accumulating more protein. (c) Crystals are fully grown. Background fluorescence in the cytoplasm is still visible as well as several crystallization nuclei that did not grow out into a full crystal. (Multimedia view) [URL: http://dx.doi.org/10.1063/1.4921591.9]

FIG. 5.

Morphology of GFP-μNS in vivo crystals. Cells were infected with recombinant GFP-μNS baculovirus and incubated for 3 days. Subsequently, cells were imaged on a confocal fluorescence microscope system. (a) Mostly, rod-like crystals can be seen, routinely with several crystals per cell. Crystals inside cells can be separated (arrow 1) or lying parallel to each other (arrow 2), or they arranged in a star-like fashion (arrow 3). The length of the crystals does not exceed the diameter of the cell body. The diameter of the crystals varies between <1 and 5 μm. (b) The fluorescent GFP-μNS crystals show a hexagonal profile in confocal cross sections (arrows). (c) A 3D reconstruction of a z-stack of confocal fluorescence images is shown. Several packs of crystals in a star like arrangement can be seen. (d) Dead cells were identified by adding the membrane impermeable dye propidium iodide (500 ng/ml) at 4 days p.i. Note that crystals are visible in both cells with or without red nuclear fluorescence, demonstrating that GFP-μNS crystals are stable after cell lysis caused by baculovirus reproduction. (Multimedia view) [URL: http://dx.doi.org/10.1063/1.4921591.10]

FIG. 6.

Surface morphology and diffraction quality of GFP-μNS crystals. (a) Extracted GFP-μNS crystals were spotted onto a sample carrier and applied to SEM. The depicted crystal has a smooth and highly regular surface and a size of 15 μm × 2 μm. Moreover, it retains its rod-shaped morphology even under vacuum conditions, indicating its remarkable intrinsic stability. (b) GFP-μNS crystals were pelleted in a glass capillary and analyzed by synchrotron powder diffraction experiments. The pattern reveals weak diffraction of approximately 30 Å, confirming a crystalline state.