A Diffraction-Quality Protein Crystal Processed as an Autophagic Cargo Hidekazu Tsutsui, Yuka Jinno, Keiko Shoda, Akiko Tomita, Makoto Matsuda, Eiki Yamashita, Hiroyuki Katayama, Atsushi Nakagawa, Atsushi Miyawaki  Molecular Cell  Volume 58, Issue 1, Pages 186-193 (April 2015) DOI: 10.1016/j.molcel.2015.02.007 Copyright © 2015 Elsevier Inc. Terms and Conditions

Molecular Cell 2015 58, 186-193DOI: (10.1016/j.molcel.2015.02.007) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 1 h41pkve6, a Crystal-Forming Fluorescent Protein (A) Transmission (T), fluorescence (FL), and merged images of an HEK293 cell bearing a crystal of h41pkve6 protein. (B) A crystal within a cell was excited with linearly polarized light (left). Fluorescence intensity is plotted as a function of polarization angle (right). (C) An h41pkve6 crystal was photo-activated (photo-converted) from green to red with irradiation at 405 nm. Based on this, h41pkve6 was renamed Xpa. See also Movie S1. (D) Packing of crystal structures determined with diffraction from a single cell. The crystal contained two molecules in its asymmetrical unit, which are colored in green and red. A unit cell is boxed with a dotted line. See also Figure S2. (E) Tetrameric structure of h41pkve6 generated by crystallographic symmetry. See also Table 1. (F) A fluorescence image of a hippocampal neuron at 12 days in vitro (culture) bearing a rod-shaped crystal of Xpa. (G) Dependency of polarization angle of the h41pkve6 crystal grown in a neuron. (H) Intra-cytoplasmic crystallization process revealed by long-term imaging. Differential interference contrast (DIC) and fluorescence (FL) images of HEK cells at the indicated times. See also Figure S3B and Movie S2. (I) Cytoplasmic fluorescence intensity (green filled squares) and axial length of the crystal (black open circles) observed in the transfected HEK cell shown in (H). (J) Distribution of crystallization time points for 34 crystals time-lapse imaged. Time zero corresponds to day 2 post-transfection, at which crystallization became detectable. All scale bars = 5 μm. Molecular Cell 2015 58, 186-193DOI: (10.1016/j.molcel.2015.02.007) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 2 Autophagic Responses to a Protein Crystal Grown within the Cell (A–C) TEM micrographs of an HEK293 cell bearing a Xpa crystal. The membrane structure that surrounds the crystal is indicated by arrowheads. Scale bars = 5 μm (A), 200 nm (B), 100 nm (C). Pitch of the observed lattice pattern was 7–8 nm. We did not identify the origin of the observed lattice, but viewing from the b-axis is anticipated to provide an aligned stripe pattern (see Figure S2). (D) Cells bearing XpaH62Q crystals were subjected to immunocytochemical analysis using anti-AP1, anti-EEA1, and anti-LAMP-1 antibodies. (E) Two models of the formation of a Xpa(H62Q) crystal. Top: non-selective autophagy. Bottom: selective autophagy. Lysosomal membranes are indicated by thick black lines. (F) LAMP-1 immunocytochemistry of an XpaH62Q crystal grown in the presence of 50 μM LY294002, a PI3-kinase inhibitor. Scale bars (D and F) = 10 μm. The images (D and F) each are representative of more than four different cells. Molecular Cell 2015 58, 186-193DOI: (10.1016/j.molcel.2015.02.007) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 3 Response to Crystals via Selective Macroautophagy (A–K) A schematic model for cellular response to a protein crystal (drawn as a green diamond). (A and B) After transfection, protein concentration is increased. (C) Crystal nucleation. (D) Crystal growth. The thick arrow indicates that the growth process is extremely rapid. (E) Recruitment of ubiquitin (blue) to the crystal. (F) Recruitment of p62 (red) to the ubiquitinated crystal. (G) Association with autophagosomal membranes. LC3 is indicated by a small black dot. (H) Encapsulated by autophagosomes. (I) Lysosomal fusion to autophagosomes. Lysosomes are indicated by gray dots. (J) Formation of autolysosomes. Gray color indicates acidity. (K) A protein crystal inside the lysosomal lumen. Gray color indicates acidity. (L) A crystal (XpaH62Q) exhibiting anti-ubiquitin immunofluorescence, which was grown in the presence of LY294002. (M) A crystal (XpaH62Q) exhibiting anti-p62 immunofluorescence, which was grown in the presence of LY294002. (N) A crystal (XpaH62Q) decorated with anti-LC3 immunofluorescence, which was grown without LY294002. This type of anti-LC3 immunolabeling was observed occasionally (3/70 crystals). (O) A crystal (XpaH62Q) associated with LC3-mCherry, which was grown in the presence of Bafilomycin A1. Similar images were obtained frequently (10/13 crystals). Scale bars (L–O) = 5 μm. The images (L, M, and O) each are representative of more than five different cells. Molecular Cell 2015 58, 186-193DOI: (10.1016/j.molcel.2015.02.007) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 4 Intra-Nuclear Crystallization (A) Intra-nuclear crystallization process revealed by long-term imaging. Differential interference contrast (DIC) and fluorescence (FL) images of HEK cells at the indicated times. Time zero corresponds to day 2 post-transfection. See also Movie S3. (B) LAMP-1 immunocytochemistry of a cell bearing an intra-nuclear XpaH62Q crystal. Scale bars = 5 μm. Similar immunocytochemical data were obtained in two other experiments with different cells. Molecular Cell 2015 58, 186-193DOI: (10.1016/j.molcel.2015.02.007) Copyright © 2015 Elsevier Inc. Terms and Conditions