Extended Data Figure 2. Electron microscopy images of an extruded exopher.

a. A membrane GFP reporter reveals that exophers contain membrane. Strain is ZB4524 bzEx242 [Pmec-4PH(plcDelta)::GFP]³⁵. PVM neuron. S, soma; E, exopher. Scale bar, 2 μm.

b. General relationship of exopher to ALMR soma. Schematic view from a lateral aspect (anterior to the left). The ALMR soma remains connected to its primary dendrite as represented here, projecting leftward. Several smaller membrane-bound tubes extend away from the soma, containing small items expelled from the ALMR soma, such as the large vesicle shown along the path between the soma and the exopher. The ALMR nucleus (N) remains intact, but pushed to an eccentric position by other cytoplasmic inclusions. The soma contains intact rough endoplasmic reticulum (RER), mitochondria (M), small vesicles (not shown), and larger protein aggregates (A).

The exopher comprises heterogeneous contents. The exterior of the exopher is completely bounded by the hypodermal plasma membrane, so that none of the exopher contents are immediately in contact with the hypodermal cytoplasm. Often, a double membrane is observed in which the exopher is likely to supply the inner bilayer, and the hypodermis is likely to contribute the outer bilayer. Additional, separate membrane-bound items lie peripherally (not shown; but see panel h), whose provenance is uncertain, but may represent breakdown products from an earlier, larger stage of the exopher. Most internal contents of the exopher also have their own membrane boundaries, but some diffuse material (not shown; but see panels c, e) fills the spaces around those membrane-bound objects. Membrane-bound contents include portions of the neuronal cytoplasm holding intact RER, large protein aggregates (A), and complex whorls of membrane (W) that generally seem to enclose empty space. Two lysosomes (L) are shown here, one lying separately, nearby the exopher, and a second one in the process of fusing into the exopher’s outer membrane. Other small lysosomes are seen within the exopher (not shown). A tube is shown extending away (far right) towards the pseudocoelom, which might offer one potential route for elimination of contents that cannot be degraded during hypodermal transit. Cartoon designed by and published with permission of C. Crocker.

Panelsc,d,f,h are TEM views of a single exopher, emitted from the cell body in panel e.

c. The exopher is fully embedded within the hypodermis. Strain is Is[p_mec-4mCh1]; Is[p_mec-4GFP]. TEM of thin transverse section, seen from anterior aspect (thus left/right are reversed). Shown is an exopher (E) fully embedded within the hypodermis (H). Animal cuticle shown on the left (C). The exopher is ~1.5 μm across, similar in size to the excretory canal (EC). The exopher lies closer to the pseudocoelom (P), while the ALMR neuron soma lies closer to the cuticle (see panel e). Jagged white spaces running vertically through the hypodermis are an artifact where the tissue cracked during processing. Scale bar, 2 μm.

d. Exophers are complex and heterogeneous structures with multiple membranes. The exopher is characterized by many small round protrusions and involutions. The main exopher complex has a complete plasma membrane surrounding it, isolating its contents from the hypodermal cytoplasm. Involuted portions of the exopher can have multiple membrane layers. In this thin section, the exopher displays structures such as protein aggregates (A), rough endoplasmic reticulum (RER), and a possible small lysosome, in addition to loose material floating inside the exopher membrane within a less electron dense fluid matrix. Scale bar, 1 μm.

e. The originating touch neuron soma remains intact. The ALMR neuron still has intact cell and nuclear membranes. Small mitochondria (M) are visible inside the soma, as are protein aggregates (A). Note that the aggregate within the soma is not membrane bound, resembling a mammalian aggresome⁴². The electron density of the neuronal cytoplasm is darker than that of the surrounding hypodermis, and the mitochondria of the hypodermis are far larger than those of the neuron. Scale bar, 2 μm.

f. The exopher is surrounded by a continuous membrane and contains electron-lucent materials and electron dense membrane whorls (W). The exterior of the exopher is completely bounded by the hypodermal plasma membrane, so that none of the exopher contents are immediately in contact with the hypodermal cytoplasm. The exopher contains complex whorls of membrane (W) that enclose an electron-lucent lumen. Within the exopher membrane, the exopher lumen also is electron-lucent, with some diffuse free-floating material. Scale bar is 1μm.

g. Fluorescent images demonstrate exopher features similar to the TEM ultrastructure. Fluorescent microscopy of exophers have irregular shapes and round protrusions, and an irregular distribution of distinct fluorescent signals that resemble the heterogeneous domains we see by TEM. Shown is an exopher in a day 2 animal expressing a soluble YFP, a CFP PolyQ128 fusion, and an aggregation prone mCherry. The mCherry has a bright spot that excludes the other two signals (arrow). The Q128 CFP is localized in the middle portion of the exopher, and the YFP is most strongly localized to the bottom portion of the exopher. The YFP signal also forms a dim ring around the mCherry spot. View is from a lateral aspect, as in panel a. Scale bar, 2 μm.

h. Serial sections of the exopher reveal a complex and heterogeneous structure. Seven views (numbered 1–7) are shown from 50 serial thin sections that traverse the main region of the exopher. As one approaches the edge of the exopher, additional membrane-bound objects can be seen at the fringe of the main body (see panels 6, 7), which likely represent portions that have decayed from the original larger object, and are perhaps more easily phagocytosed by the hypodermis, or shuttled along a tube for release into the pseudocoelom. A small electron dense lysosome (L) lies beside the exopher in panel 5. Scale bar, 1 μm.