Journal: Journal of Cell Science

Article Title: CryoAPEX – an electron tomography tool for subcellular localization of membrane proteins

doi: 10.1242/jcs.222315

Figure Lengend Snippet: OSER as a system for evaluating membrane preservation and staining specificity. (A) Flowchart describing cryoAPEX. (B) Schematic of APEX-tagged ERM expressed on the cytosolic face of the ER membrane. (C) The reorganized ER morphology in chemically fixed, DAB reacted ERM–APEX2-expressing cells that were processed via traditional chemical fixation and alcohol dehydration (a–c) or by cryoAPEX (d–f) was compared to ERM–APEX2 expressing cells that were cryofixed live and without the DAB reaction (g–i). The live cryofixed cells (g–i) represent the best attainable ultrastructural preservation and serve here as the metric for evaluating membrane preservation obtained via the two APEX-based detection protocols (a–f). The high specificity of staining obtained by cryoAPEX is exemplified in the images in panels j–l. Here, thin-section images of cells expressing ERM–APEX2 processed by cryoAPEX show preferential staining of the reorganized ER (j, orange asterisks) and the outer mitochondrial membrane (red asterisks in j and orange arrowheads in k–l, respectively) but not of the mitochondrial cristae (k, red arrowheads). (D) Post-staining with heavy metals improves definition of preferentially stained membranes. Post-staining of thin sections with heavy metals using uranyl acetate and Sato's lead solution following cryoAPEX provides additional contrast, thereby improving resolution. Shown are thin sections of the same cell imaged in Fig. 2Cd before (a,c,e) and after (b,d,f) post-staining. Comparison of panels e and f clearly shows improved definition and the resolution of membranes at high magnifications in post-stained samples (f). UA, uranyl acetate.

Article Snippet: As additional controls for the specificity of the ERM–APEX2 staining for the ER, we assessed localization of three organellar markers – namely, mito-V5–APEX2 that targets to the mitochondrial matrix (Addgene plasmid # 72480 ; Lam et al., 2015 ); CAAX–APEX2 that targets to the plasma membrane ( Lam et al., 2015 ), and ManII–APEX2 that targets to the Golgi lumen (see Materials and Methods).

Techniques: Preserving, Staining, Expressing

Journal: Journal of Cell Science

Article Title: CryoAPEX – an electron tomography tool for subcellular localization of membrane proteins

doi: 10.1242/jcs.222315

Figure Lengend Snippet: HYPE localizes to the lumenal face of the ER membrane as periodic foci. (A) An image of a thin section of HEK-293T cells expressing HYPE–APEX2 and processed by cryoAPEX reveal staining of the ER tubules in a well-preserved (dense) cytoplasmic background. (B,C) Higher magnification images of a small section of the peripheral ER (demarcated by yellow box in A and shown in B, with further magnification of red box in B shown in C) exhibits periodic foci of APEX2-generated density (B, red box and C, white arrowheads showing periodicity between the HYPE foci). (D) Center-to-center density measurements showing the distance (in nm, blue lines) between the HYPE-specific foci (yellow circles) in C. (E) Untransfected control HEK-293T cells processed in an identical manner show the lack of APEX2-generated density within the ER lumen. (F,G) Higher magnification images of a small section of E (demarcated by yellow box and shown in F, with further magnification of red box in F shown in G) clearly shows the lack of density on the lumenal face. Additionally, density corresponding to ribosomes on the cytoplasmic face of the ER membrane is evident (G, white arrowheads). Thus, HYPE is detected only along the lumenal face of the ER membrane and never on the cytosolic face.

Article Snippet: As additional controls for the specificity of the ERM–APEX2 staining for the ER, we assessed localization of three organellar markers – namely, mito-V5–APEX2 that targets to the mitochondrial matrix (Addgene plasmid # 72480 ; Lam et al., 2015 ); CAAX–APEX2 that targets to the plasma membrane ( Lam et al., 2015 ), and ManII–APEX2 that targets to the Golgi lumen (see Materials and Methods).

Techniques: Expressing, Staining, Generated

Journal: Journal of Cell Science

Article Title: CryoAPEX – an electron tomography tool for subcellular localization of membrane proteins

doi: 10.1242/jcs.222315

Figure Lengend Snippet: Assessing localization of HYPE in subcellular compartments other than the ER. (A) Localization of HYPE to the nuclear envelope (NE). Images of thin sections from cells transfected with HYPE–APEX2 and processed by cryoAPEX show HYPE-specific density within the perinuclear space of the nuclear envelope (a–c). At higher magnification, this staining shows a pattern similar to that seen along the lumenal face of the ER membrane (c; compare red and yellow arrowheads within the NE and the ER, respectively). The same untransfected cells as used in Fig. 3E processed in this manner exhibit no membrane-associated staining within the perinuclear space of the nuclear envelope (d–f and white arrowheads in f). (B) HYPE does not localize to the mitochondria. Cells transfected with HYPE–APEX2 were processed by cryoAPEX in the presence of osmium tetroxide but without addition of uranyl acetate or tannic acid. Thin sections of these cells showed a distinct lack of mitochondrial membrane staining, making it difficult to visualize mitochondria at low magnifications (red asterisks in a). Magnified images of well-preserved ER–mitochondrial junctions (MAMS; demarcated by red box in a with further magnification of red box area shown in b) clearly show ER tubules in close contact with two adjacent mitochondria (b). A further magnified image of the MAMS shows the HYPE–APEX2 staining of the ER but no apparent staining within the inner or outer mitochondrial membranes (b, yellow box; and c, magnified image of the area within this yellow box). The periodic distribution of HYPE is retained even at the MAMS (c, white arrowheads). MITO, mitochondrion; IMM, inner mitochondrial membrane; OMM, outer mitochondrial membrane; MAMS, mitochondria-associated membranes. (C) HYPE does not localize to the plasma membrane (PM). Images of a cell expressing HYPE–APEX2 reveal absence of plasma membrane staining (a). Images of ER–PM junctions at high magnifications show well-preserved junctions where the ER makes extended contacts with the plasma membrane (red box in a; yellow box in b; white bar in c). b shows higher magnification of red box area in a, c shows higher magnification of yellow box area in b. Staining was contained within the cortical ER tubules with no apparent staining of the plasma membrane (c, black arrowheads). (D) HYPE does not enter the secretory pathway. To assess whether HYPE enters the secretory pathway, the Golgi apparatus was imaged (a–d). Images from thin sections of HYPE–APEX2-transfected cells show an area where ER tubules are interspersed within Golgi stacks (a, region within yellow box). Higher magnification of this region (b shows higher magnification of yellow box area in a, c shows higher magnification of red box area in b) shows the typical stacked morphology of the Golgi apparatus devoid of any osmicated DAB density within its lumen. Further magnification of a well-preserved ER–Golgi junction (d) shows a region of extensive contact between the two organelles where the DAB density was restricted to the ER lumen and shows no apparent staining of the Golgi apparatus (b, area within red box; red arrowheads in c and d indicate the ER–Golgi junction).

Article Snippet: As additional controls for the specificity of the ERM–APEX2 staining for the ER, we assessed localization of three organellar markers – namely, mito-V5–APEX2 that targets to the mitochondrial matrix (Addgene plasmid # 72480 ; Lam et al., 2015 ); CAAX–APEX2 that targets to the plasma membrane ( Lam et al., 2015 ), and ManII–APEX2 that targets to the Golgi lumen (see Materials and Methods).

Techniques: Transfection, Staining, Expressing

Journal: Journal of Cell Science

Article Title: CryoAPEX – an electron tomography tool for subcellular localization of membrane proteins

doi: 10.1242/jcs.222315

Figure Lengend Snippet: Superior ultrastructural preservation enables the tracking of HYPE's subcellular localization via serial sectioning. To demonstrate the consistency of the membrane ultrastructure preservation obtained by cryoAPEX, cells expressing HYPE–APEX2 were serially sectioned and a specific area (panel A, yellow box) was imaged. Multiple ribbons containing between 10 and 20 serial sections of 90 nm thickness were collected, screened and imaged. Representative images of eight serial sections showing ER localization of HYPE are presented (images serially numbered 1–8). Sections exhibit a dense well-preserved cytoplasm with undisrupted membrane ultrastructure of organelles such as mitochondria and Golgi complex in close proximity to the ER tubules containing HYPE–APEX2 density. Thus, we can follow HYPE localization through the volume of the cell without loss of contextual information. Scale bars: 5 μm (A); 8 μm (magnifcations 1–8).

Article Snippet: As additional controls for the specificity of the ERM–APEX2 staining for the ER, we assessed localization of three organellar markers – namely, mito-V5–APEX2 that targets to the mitochondrial matrix (Addgene plasmid # 72480 ; Lam et al., 2015 ); CAAX–APEX2 that targets to the plasma membrane ( Lam et al., 2015 ), and ManII–APEX2 that targets to the Golgi lumen (see Materials and Methods).

Techniques: Preserving, Expressing

Journal: Journal of Cell Science

Article Title: CryoAPEX – an electron tomography tool for subcellular localization of membrane proteins

doi: 10.1242/jcs.222315

Figure Lengend Snippet: EM tomographic reconstruction of the ER exhibiting HYPE–APEX2 density. Tilt-series from HEK-293T cells expressing HYPE–APEX2 and processed by cryoAPEX were collected for 3D reconstruction of HYPE-specific density. (A) An image of the whole HYPE–APEX2-expressing cell showing an area containing ER tubules from where the tilt-series was collected (panel a and magnified red box). Panel b shows a snapshot from the movie (see Movie 1) showing the reconstructed tomogram of HYPE density within the ER lumen. Panel c shows a snapshot from the movie (see Movie 2) showing the reconstructed tomogram of the thresholded HYPE density (in maroon). (B) An additional view of the 3D model of the ER membrane (blue) and the HYPE density within (gold) generated and visualized with the IMOD ‘Isosurface’ tool (panels a–c). (C) Snapshots of a segment of the ER showing the HYPE–APEX2 density (gold) modeled within the lumen of the ER membrane (blue) visualized from the top of the indicated clipping plane (panels a–c). Red arrowheads in b show magnified images of different regions within this segment show HYPE's periodic density pattern along the lumenal walls. Red arrowheads in c show another view exposing the full face of the density (gold) using visualization tools that make the membrane transparent. This pattern of HYPE-specific density is more apparent when the clipping plane is moved downward in the z-direction, progressively shaving through the depths of the different layers (panel d, slices i–xi), and is most apparent when visualized in the thinnest slice (slice xi). (D) A clipping plane that moves in a head-to-tail direction shows HYPE's density pattern on the ER membrane from a front-on perspective (slices 1–8).

Article Snippet: As additional controls for the specificity of the ERM–APEX2 staining for the ER, we assessed localization of three organellar markers – namely, mito-V5–APEX2 that targets to the mitochondrial matrix (Addgene plasmid # 72480 ; Lam et al., 2015 ); CAAX–APEX2 that targets to the plasma membrane ( Lam et al., 2015 ), and ManII–APEX2 that targets to the Golgi lumen (see Materials and Methods).

Techniques: Expressing, Generated