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. 2015 Oct 12;211(1):27–37. doi: 10.1083/jcb.201504136

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© 2015 Hyenne et al.

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Figure 3. — RAL-1 affects different steps of exosome secretion. (A) Quantitative electron microscopy analysis of MVBs in epidermal cells: (1 and 2) density (1) and diameter (2) of MVBs, (3) number of ILVs per MVB, (4) ILV diameter, and (5) distance between MVB and the APM. (B) MVB density is decreased in ral-1(tm5205) compared with the WT and is increased in ral-1(RNAi) compared with control(RNAi). (C) The density of VHA-5::RFP puncta is decreased in ral-1(tm5205) compared with the WT but is unaffected in ral-1(RNAi). (D–F) In ral-1(tm5205) mutants, MVBs have an abnormal size (E) and ILV content (F). (G) In ral-1(RNAi) animals, 57% of MVBs are within 50 nm of the apical plasma membrane, compared with 20% in control animals. (H and I) Two MVBs in proximity of the apical plasma membrane from control (H) and ral-1(RNAi) (I) animals. MVBs from ral-1(RNAi) animals can form a hemifusion diaphragm (I) with the apical plasma membrane. (J and J’) EVs purified from 4T1 mammalian cells and observed by electron microscopy. (K) Depletion of either RalA or RalB by shRNA leads to a decrease in the number of EVs observed by electron microscopy compared with control shRNA (P < 0.0001 between sh control and either sh RalA or sh RalB; pool of four independent purifications, Mann-Whitney test). (L and M) Western blot of cell lysates and secreted EVs. One representative experiment (L) and pooled quantification (M) of four independent purifications (P < 0.03 between sh control and either sh RalA or sh RalB for each marker, Mann-Whitney test). Numbers in or above the bars indicate the number of animals (C), MVBs (E, F, and M) or fields (K) analyzed. APM, apical plasma membrane; CL, cell lysate; Cu, cuticle; Cy, cytoplasm; MVBm, MVB outer membrane. Errors bars, SEM.