Ribosome-associated vesicles: A dynamic subcompartment of the endoplasmic reticulum in secretory cells

Abstract

The endoplasmic reticulum (ER) is a highly dynamic network of membranes. Here, we combine live-cell microscopy with in situ cryo-electron tomography to directly visualize ER dynamics in several secretory cell types including pancreatic β-cells and neurons under near-native conditions. Using these imaging approaches, we identify a novel, mobile form of ER, ribosome-associated vesicles (RAVs), found primarily in the cell periphery, which is conserved across different cell types and species. We show that RAVs exist as distinct, highly dynamic structures separate from the intact ER reticular architecture that interact with mitochondria via direct intermembrane contacts. These findings describe a new ER subcompartment within cells.

Fig. 1. Identification of ER-derived vesicles in secretory cells.

(A) Live-cell super-resolution STED imaging of insulin-secreting INS-1E cells expressing ER marker mNeon-KDEL. Representative individual optical slices at different planes within the cell including the cell top (left), center (middle), and bottom (right) demonstrate punctate structures primarily in the cell periphery (cell top and bottom), in addition to an extensive reticular distribution throughout the cells. Scale bars, 5 μm. Insets show enlarged images of individual mNeon-KDEL puncta (arrowheads). (B) HiLo imaging of INS-1E cells expressing mNeon-KDEL confirms numerous punctate structures (see movies S2 and S3). Scale bar, 2 μm. (C to E) mNeon-KDEL–labeled puncta demonstrate dynamic movement throughout the cell [including within the boxed region in (B)] using HiLo microscopy. Movement of a mNeon-KDEL punctum is indicated by the following: (C) the horizontal line (in red) to show distance traveled (scale bar, 2 μm), (D) a kymograph of motion across time, and (E) accompanying time-lapse images that show movement at specific time points in the kymograph, as indicated by the red arrows (scale bar, 2 μm). (F) Representative HiLo images of INS-1E cells expressing both mNeon-KDEL (in green) and ER membrane marker Halo-Sec61β (in red). Scale bar, 10 μm. Magnified region of interest showing dual-labeled punctate structures within a peripheral process. Scale bar, 5 μm. (G) Representative fluorescent line intensity profiles for mNeon-KDEL and Halo-Sec61β channels along the direction of the white line drawn across a puncta showing colocalization of the two ER markers. a.u., arbitrary units.

Fig. 2. Visualization of ER-derived RAVs by cryo-CLEM and cryo-ET.

(A) Cryo-CLEM show calreticulin-EYFP localizes to vesicles associated with ribosome-like particles termed RAVs in INS-1E cells. A cryo–tomographic slice was overlaid on an epifluorescence image with calreticulin-EYFP fluorescence in RAVs (in yellow) and MitoTracker Red–labeled mitochondria (in red). Scale bar, 2 μm. (B) Enlarged view of the green box in (A) with and without fluorescence overlays show calreticulin-EYFP fluorescence localizes to RAVs with a mitochondrion found alongside (labeled M). Pixels on the detector represent 2.6 Å at the specimen level; images are nonmontaged. Scale bar, 250 nm. (C) Cryo–tomographic slice demonstrating a RAV alongside a lamella of conventional ER and mitochondria (indicated by M). Light green arrows indicate cisternae of the ER network. Scale bar, 200 nm. (D) Isosurface of the ER-RAV association in (C) highlighting contact between the two structures (ER, light blue; RAV, dark blue; ribosomes, yellow). Scale bar, 200 nm. (E) Original cryo-tomographic slice featuring an enlarged view of the area highlighted in the orange box from (C). Light green arrows highlights an ER cisterna sandwiched between the RAV and mitochondrion (M) alongside; the orange arrow points to the site of contact between the ER network and RAV. Scale bar, 100 nm. (F) Isosurface showing RAV-like structures attached to ER in mouse embryonic fibroblasts (MEFs) (ER membranes, light blue; ribosomes, yellow). Scale bar, 200 nm. (G) Additional, enlarged isosurface view of segmented ER membranes from the yellow box in (F) highlighting attachment sites of RAV-like structures to ER cisternae (indicated by blue, green, and red arrows) including via three-way junctions. Scale bar, 100 nm. (H to J) Original cryo-tomographic slices featuring views of the thin ER tubules associated with the RAV-like structures highlighted by the respectively colored arrows in (F) and (G). Panels (A to E) in INS-1E cells; Panels (F to J) in MEFs. Scale bars, 100 nm.

Fig. 3. Subtomogram average of RAV-bound ribosomal complex.

(A) Average of 1362 manually selected subvolumes of RAV-bound electron-dense particles. Reference-free alignment reveals that these particles strongly resemble 80S mammalian ribosomes. Putative density assignments: yellow, 40S subunit; light blue, 60S subunit; purple, tRNA-eEF1a-GTP ternary complex; gray, averaged portion of RAV membrane associated with the bound ribosome; dark blue, translocon; green, TRAP; red, OST. (B) Subtomogram average fitted over a color-coded, high-resolution atomic model of the mammalian ribosome-Sec61 complex (EMD 2644) (31), revealing similarity between the structures. Yellow, 40S subunit; blue, 60S subunit; red, Sec61. Additional densities potentially representing OST and TRAP are also evident (in gray). (C) Illustration of subtomogram averaged RAV-bound ribosomes mapped onto their original positions on a RAV. Bound particles show an ordered spiral arrangement indicative of polysomes. This illustration was created by mapping a bound-ribosome subtomogram average into the original locations of the subtomograms containing putative ribosomes. (D) Enlarged view of the spiral polysome arrangement. Orientations of ribosomal exit tunnels (in red) are depicted relative to the sphere in (C) and (D). (E) GFP-Sec61β localizes to RAVs by cryo-CLEM. A cryo-tomographic slice correlated with an epifluorescence image demonstrating GFP-Sec61β fluorescence in RAVs (in green) and MitoTracker Red–labeled mitochondrion (in red); 500-nm fiducial blue fluorospheres (in blue) are also evident. Scale bar, 2 μm. (F) Enlarged cryo-tomogram views of the red boxed area in (E) with and without fluorescence overlays. GFP-Sec61β fluorescence localizes to the population of RAVs. Pixels on the detector represent 2.6 Å at the specimen level; images are nonmontaged. Scale bar, 300 nm.

Fig. 4. Distribution of RAVs in the cell periphery.

INS-1E cells were grown directly on EM grids. (A) Overview cryo-EM image of a thin protrusion extending from the periphery of the cell body and containing accumulations of secretory granules, ribosomes, and cytoskeleton. Scale bar, 2 μm. Inset shows a representative phase-contrast image of an INS-1E cell grown on an EM grid; this image is from a different cell than the one imaged in the remainder of the figure. Scale bar, 10 μm. (i) At the junction between the cell body and the protrusion, there are accumulations of mitochondria, small dense-core secretory granules, and RAVs. (ii) Extensive microtubule tracks run lengthwise along the protrusion and appear to transport secretory granules and mitochondria toward (iii) the expanded tip of the protrusion. These tips are packed with mature dense-core secretory granules and mitochondria and are absent of RAVs. Scale bars, 200 nm. (Note that white circles correspond to 2-μm holes of the QUANTIFOIL holey carbon film onto which the cells are attached). (B) An additional view of the same junctional region between the cell body and protrusion highlighted in (i) featuring an abundance of RAVs (indicated by arrows) and free, cytoplasmic ribosomes. Scale bar, 500 nm. Pixels on the detector represent 2.6 Å at the specimen level; images are montaged in the overview with nonmontaged images in the remaining panels. Images are representative of n > 3 independent experiments.

Fig. 5. Cryo-tomography of cryo-FIB milled INS-1E cells reveals the intact ER and Golgi apparatus secretory complex.

(A) Cryo–tomographic slice of a cryo-FIB milled region in the cell center exhibiting flattened ER cisternae (labeled ER) and coated membrane sites of vesicle budding (labeled B). Alongside these apparent exit sites are both coated and uncoated vesicles (labeled C and U, respectively) and microtubules crisscrossing the field (labeled MT). Scale bar, 100 nm. (B) Colorized 3D segmentation of the same field demonstrating interconnected ER (in blue) and Golgi apparatus (in red). Interspersed throughout are coated vesicles (in green), uncoated vesicles (in yellow), and microtubules (in magenta). (i to iii) Enlarged panels highlighting key structural features: (i) segmented ER cisterna within the larger ER network with an intact uncoated vesicle alongside; (ii) Golgi cisterna including a site of coated membrane budding with numerous uncoated vesicles nearby; (iii) additional view of the Golgi membrane network featuring a site of coated membrane budding and uncoated vesicles. Scale bars, 100 nm. (C) Cryo–tomographic slices comparing the appearance and dimensions of a RAV (~200-nm diameter) with a conventional coat protein complex (COP)–coated vesicle (~50-nm diameter). Scale bar, 100 nm (corresponds both to the main panel and the inset). (D) Scatter plot of the distribution of diameters of vesicular structures including RAVS across several cell types and species [rat INS-1E–coated vesicles, INS-1E uncoated vesicles, INS-1E RAVs, mouse embryonic fibroblast (MEF) RAVs, and human primary fibroblast RAVs]. Asterisks represent the median diameter, and triangles represent the mean diameter of the respective vesicles.

Fig. 6. RAV- and ER-mitochondrial interactions imaged by cryo-EM and cryo-ET.

(A) Cryo-EM image of interactions between RAVs and nearby mitochondria (labeled M) in INS-1E cells. Inset shows enlarged view of the interface between RAV and mitochondrial membranes. Note the absence of ribosomes at the tight interface (13-nm width). Scale bar, 500 nm. (B) Site of RAV-mitochondrion interaction in which RAV and mitochondrial membranes extend, acquiring an “hourglass-like” morphology at the single point of contact (within the boxed area). Scale bar, 500 nm. (C) Cryo–tomographic slice of a MEF, revealing an ER-mitochondrial contact site. Scale bar, 500 nm. (D) Segmentation of the ER-mitochondrial association in MEFs highlighting a similar hourglass-like point of membrane contact (ER, dark blue; mitochondrion, light blue) point of contact. (E) Enlarged view of the ER-mitochondrial contact in (C) showing the extension and deformation of both the OMm and ER membrane. Both the inner mitochondrial membrane (IMm) and mitochondrial cristae remain unchanged.