Hrs- and CD63-dependent competing mechanisms make different sized endosomal intraluminal vesicles

Abstract

Multivesicular endosomes/bodies (MVBs) contain intraluminal vesicles (ILVs) that bud away from the cytoplasm. Multiple mechanisms of ILV formation have been identified, but the relationship between different populations of ILVs and MVBs remains unclear. Here, we show in HeLa cells that different ILV subpopulations can be distinguished by size. EGF stimulation promotes the formation of large ESCRT-dependent ILVs, whereas depletion of the ESCRT-0 component, Hrs, promotes the formation of a uniformly sized population of small ILVs, the formation of which requires CD63. CD63 has previously been implicated in ESCRT-independent sorting of PMEL in MVBs and transfected PMEL is present on the small ILVs that form on Hrs depletion. Upregulation of CD63-dependent ILV formation by Hrs depletion indicates that Hrs and CD63 regulate competing machineries required for the generation of distinct ILV subpopulations. Taken together our results indicate that ILV size is influenced by their cargo and mechanism of formation and suggest a competitive relationship between ESCRT-dependent and -independent mechanisms of ILV formation within single MVBs.

Keywords: CD63; Hrs; cholesterol; intraluminal vesicle; multivesicular endosome.

Figure 1

Depletion of Hrs and Tsg101 but not CD63 or ceramide affects the morphology of MVBs in resting HeLa cells. HeLa cells were treated with non-targeting control, Hrs, Tsg101 or CD63 siRNA or were treated with the sphingomyelinase inhibitor, GW4869, to deplete ceramide. Cells were then incubated for 2 h in the presence of BSA-gold and prepared for conventional EM. Clathrin coats in Tsg101-depleted cells are shown by arrows (A). Knockdown efficiency was analysed by western blotting (B). Scale bar: 100 nm.

Figure 2

CD63 or ceramide depletion does not affect MVB diameter or ILV number. HeLa cells were treated with non-targeting control, Hrs, Tsg101 or CD63 siRNA or were treated with the sphingomyelinase inhibitor, GW4869, to deplete ceramide. Cells were then incubated for 2 h with BSA-gold and analysed by conventional EM. Diameters and ILV numbers of BSA-gold-containing MVBs were quantified in random sections. Hrs or Tsg101 depletion caused enlargement of MVBs (***p < 0.001), whilst CD63 or ceramide depletion had no effect on MVB size compared to control cells (A). CD63 or ceramide depletion had no significant effect on the number of ILVs per MVB (B). ***p < 0.001. Data shown are mean ± SD (50–100 MVBs/condition, 3 independent experiments).

Figure 3

EGF stimulation promotes the generation of large ILVs. HeLa cells were incubated with 10 nm BSA-gold for 2 h at 37°C and stimulated with or without EGF and 5 nm anti-EGFR gold for the final 25 min prior to fixation. Cells were analysed by conventional EM (A) and ILV sizes in BSA-gold-containing MVBs measured (B). Data shown are the mean ± SD (>200 ILVs/condition, 3 independent experiments). Scale bar: 100 nm.

Figure 4

Large ILVs are only found in MVBs that contain EGFR. HeLa cells were incubated with 10 nm BSA-gold for 2 h at 37°C and stimulated with or without EGF and 5 nm anti-EGFR gold for the final 25 min prior to fixation. Cells were analysed by conventional EM (A) and the sizes of 100 ILVs in EGFR-gold positive and -negative MVBs were measured (B). Scale Bar: 200nm.

Figure 5

Hrs-depleted HeLa cells do not display enlarged ILVs following EGF stimulation. Control, Hrs- or Tsg101-depleted cells were incubated with 10 nm BSA-gold for 2 h at 37°C and stimulated with EGF and 5 nm anti-EGFR-gold for 25 min prior to fixation. Cells were analysed by conventional EM (A). Arrowheads indicate EGFR-gold. Endosomal clathrin coats are indicated by arrows. The diameters of all ILVs within 20 MVBs were analysed (B). Binning the ILV diameters to either <40 or >40 nm (C) reveals a greatly increased number of smaller ILVs/MVB in Hrs-depleted cells. Data are mean ± SD (2 independent experiments). ***p < 0.001. Scale bar: 200 nm

Figure 6

High-pressure freezing reveals that ILVs may be connected by intramembranous material. Control or Hrs-depleted HeLa cells were incubated with 10 nm BSA-gold for 2 h at 37°C. Cells were stimulated with EGF and 5 nm anti-EGFR-gold for 25 min prior to fixation and then analysed by conventional EM. Endosomal clathrin coats are shown by arrows. Intramembranous fibrils are shown by arrowheads. Scale bar: 100 nm

Figure 7

Small ILVs still form in Hrs-depleted cells cultured in delipidated serum. Control or Hrs-depleted cells were cultured in the presence of LPDS prior to EGF stimulation for 25 min and prepared for conventional EM. Diameters of all ILVs within 20 MVBs were analysed/specimen. The ILVs of Hrs-depleted LPDS-treated cells had the same mean diameter as those cultured in full serum (A) but had a reduced number of ILVs per MVB (B). Data shown are the mean ± SD (approximately 20 MVBs/condition).

Figure 8

CD63 and PMEL are cargos for small ILVs generated following Hrs depletion. Control or Hrs-depleted cells were prepared for cryo-immuno EM and ultrathin frozen sections labelled with anti-CD63 antibody (A). CD63 was present on ILVs in control cells and on small ILVs in Hrs-depleted cells. Control or HeLa cells depleted of Hrs were transfected with PMEL (B). Cryo-immuno EM of these cells with anti-PMEL antibody revealed that PMEL was present on small ILVs in Hrs-depleted cells but the tight packing of ILVs and PMEL-induced fibrils in control and Hrs-depleted cells prevented the determination of whether it was exclusively on small ILVs. Scale bar: 200 nm

Figure 9

Simultaneous depletion of Hrs and CD63 prevents the formation of small ILVs. HeLa cells were depleted of Hrs or CD63 alone or Hrs and CD63 and were incubated in the presence of EGF and anti-EGFR gold for 25 min prior to fixation. Cells were analysed by conventional EM (A). Binning the ILV diameters to either <40 nm or >40 nm revealed that simultaneous CD63 depletion prevented the increase in number of smaller ILVs/MVB induced by Hrs depletion (B). Data shown are the mean ± SD (>40 MVBs/condition, 2 independent experiments). Scale bar: 200 nm.