CHMP5 is essential for late endosome function and down-regulation of receptor signaling during mouse embryogenesis

Abstract

Charged MVB protein 5 (CHMP5) is a coiled coil protein homologous to the yeast Vps60/Mos10 gene and other ESCRT-III complex members, although its precise function in either yeast or mammalian cells is unknown. We deleted the CHMP5 gene in mice, resulting in a phenotype of early embryonic lethality, reflecting defective late endosome function and dysregulation of signal transduction. Chmp5-/- cells exhibit enlarged late endosomal compartments that contain abundant internal vesicles expressing proteins that are characteristic of late endosomes and lysosomes. This is in contrast to ESCRT-III mutants in yeast, which are defective in multivesicular body (MVB) formation. The degradative capacity of Chmp5-/- cells was reduced, and undigested proteins from multiple pathways accumulated in enlarged MVBs that failed to traffic their cargo to lysosomes. Therefore, CHMP5 regulates late endosome function downstream of MVB formation, and the loss of CHMP5 enhances signal transduction by inhibiting lysosomal degradation of activated receptors.

Figure 1.

Characterization of CHMP5. (a) Protein sequence alignment of CHMP5 homologues from mouse, human, D. melanogaster, C. elegans, A. thaliana, and yeast. (b) Northern blot analysis of adult mouse tissues.

Figure 2.

Generation of Chmp5^−/− embryos. (a and b) Targeting of the Chmp5 gene and analysis of ES clones. The targeting vector is described in Materials and methods. Note that the first two exons of the Bag1 gene are represented by two horizontally lined boxes. Genomic DNA was digested with XbaI or BamHI and hybridized with probe B or A, respectively. The hybridized DNA fragments were 19 kb (XbaI) and 6.1 kb (BamHI) for the wild-type allele and 14 kb (XbaI) and 4.5 kb (BamHI) for the targeted allele. (c) Wild-type (+/+) and mutant (−/−) ES cell lysates were immunoblotted with antibodies specific for CHMP5 and GAPDH4.

Figure 3.

Abnormal development of Chmp5 mutant embryos. (a) Morphological analysis of Chmp5 mutant embryos. Lateral view of wild-type (+/+) embryos and mutant (−/−) littermates at E7.5, E7.75, E8.5, and E9.25. (b) Lateral, front, and ventral view of E8.75 Chmp5 mutant embryos. The yolk sac was removed from the embryos. Al, allantois; Am, amnion; Hf, head fold; M, midline. (c) Histological analysis of E8.5 Chmp5 mutant embryos. Ht, heart; S, somite. (d) Abnormal heart formation in Chmp5 mutant embryos revealed by whole-mount in situ hybridization with the cardiac marker Nkx2.5. (top) Arrows and arrowhead indicate heart tissue. Massive cell death in Chmp5 mutant embryos revealed by TUNEL staining of E8.5 embryos. (bottom) Arrow indicates apoptotic cells. Bars: (c) 50 μm; (d) 100 μm.

Figure 4.

CHMP5 is essential for biogenesis of lysosomes from mature MVBs. (a–c) Immunofluorescence analysis of Chmp5 ^−/− primary embryonic cells. Transferrin receptor (TfR), CA-MPR, LBPA, and LAMP1 are markers for early endosome, late endosome/MVB, and MVB/lysosome, respectively. Arrowheads indicate phase-lucent vacuolar structures present in the Chmp5 ^−/− cells. (d) Induction of MHC II expression by IFN-γ. Primary cells derived from E9.0 embryos were incubated with the indicated amount of IFN-γ for 48 h (top) or incubated with 1 μg IFN-γ for the indicated time points (bottom). Expression of MHC II molecules was analyzed by immunoblotting with anti-MHC II (I-Aβ) antibody. (e) Wild-type or Chmp5 ^−/− primary embryonic cells were stained with anti-MHC II (I-Aβ) antibody. (f) Transmission electron microscopy analysis of MVBs in the endodermal cells of E8.25 embryos. The inset shows an internal vesicle. (g) The fraction of MVBs per total cell volume was measured in endodermal layer of wild-type embryos (+/+) and mutant (−/−) littermates. Approximately 120 MVBs from the sections of six wild-type or Chmp5 ^−/− embryos were counted. Error bars represent the SD. Bars: (a–c and e) 10 μm; (f) 500 nm.

Figure 5.

CHMP5 plays a role in endosomal transport to lysosomes. (a and b) HRP degradation is blocked in Chmp5 ^−/− cells. ES cells cultured on 0.2% gelatin-coated plates were incubated in medium containing 100 μg/ml HRP for 1 h and then placed in normal medium (time 0). (a) Cells were lysed at the indicated time points and immunoblotted with anti-HRP antibody. (b) Alternatively, the amount of HRP remaining inside the cells was quantitatively measured by flow cytometry, and the percentage of cellular HRP was calculated based on mean fluorescence intensity. (c) Transmission electron microscopy analysis of internalized HRP in MVBs of Chmp5 ^−/− cells. After incubation with HRP for 2 h, ES cells were fixed, sectioned, and stained with gold-conjugated anti-HRP antibody. Arrowheads indicate HRP-gold particles. (d) Lysosomal hydrolase activity in Chmp5 ^−/− cells. Wild-type (+/+; 1) and mutant (−/−; 2 and 3) ES cells were lysed and immunoblotted with antibodies specific to cathepsin D and L. Bar, 100 nm.

Figure 6.

CHMP5 plays a role in down-regulation of TGFβ receptors. (a) Subcellular distribution of TβRII in CHMP5 knockdown cells. NIH3T3 cells were transfected with extracellularly HA-TβRII in the absence or presence of murine CHMP5 shRNA (Sh2). 48 h later, cells were incubated with HA antibody, stimulated with 5 ng/ml TGFβ for 1 h, and immunostained with anti-LAMP1 antibody. (b) HEK293 cells were transfected with COOH-terminal HA-TβRII in the absence or presence of human CHMP5 shRNA (Sh3). 48 h after transfection, cells were treated with 10 ng/ml TGFβ for 5 h or left untreated, and fractionated on Percoll gradients. Transferrin receptor (TfR), Rab7, or LAMP1 was used as a marker for the plasma membrane, early or recycling endosomes, late endosomes/MVBs, or lysosomes, respectively. Each fraction was treated with proteinase K or left untreated, and HA-TβRII was analyzed by immunoblotting with anti-HA antibody. (c) NIH3T3 cells were transfected with TβRII-HA, together with control vector or murine CHMP5 shRNA (Sh2). 48 h later, cells were labeled with S³⁵ methionine in the absence or presence of 0.4 mM chloroquine, and then chased for the indicated times in medium containing unlabeled methionine and 5 ng/ml TGFβ. S³⁵-labeled TβRII was immunoprecipitated with anti-HA antibody, and then analyzed by phosphorimaging. Alternatively, the HA-TβRII expression was quantified and graphed as receptor quantity (percentage of time 0) versus time. (d) HEK293 cells were transfected with control vector (Vector), CHMP5 expression vector (CHMP5), or human CHMP5 shRNAs (Sh3) and immunoblotted with antibodies specific to CHMP5 and GAPDH4 (top). HEK293 cells were transfected with TβRI-HA and TβRII-HA, together with control vector, CHMP5, or CHMP5-Sh3. 48 h after transfection, cells were treated with 10 ng/ml TGFβ for 5 h, or left untreated in the presence of 20 μM cyclohexamide before lysis, and then immunoblotted with antibodies specific to HA and GAPDH4. (e) HEK293 cells were transfected with extracellularly HA-TβRII, together with control vector or human CHMP5 shRNA (Sh3). 48 h after transfection, HA-TβRII expression was analyzed with anti-HA antibody by using flow cytometry. (f) NIH3T3 cells were incubated with biotin-conjugated HRP in medium containing protease inhibitors for 3 h to label preexisting lysosomes. 48 h after transfection with murine CHMP5 shRNAs (Sh1 and Sh2), cells were incubated with streptavidin for 10 min, washed, and chased for the indicated time. Cells were lysed with biotin-containing lysis buffer and HRP enzymatic activity was measured using ELISA on anti–streptavidin-coated plates. Endogenous murine CHMP5 protein level was analyzed by immunoblotting with anti-CHMP5 antibody (top). Error bars represent the SD. n = 3.

Figure 7.

CHMP5 regulates multiple signaling pathways. (a) Hyperactivation of multiple signaling pathways in Chmp5 ^−/− embryos. Extracts from E8.5 wild-type (+/+) and mutant (−/−) embryos were immunoblotted with antibodies specific to phosphotyrosine (pY), phospho-Erk1/2 (P-Erk1/2), phospho-Smad2 (P-Smad2), Smad2, Smad4, and GAPDH4. (b) Nuclear localization of phosphorylated Smad2 in Chmp5 ^−/− primary embryonic cells. Wild-type (+/+) and Chmp5 ^−/− (−/−) cells were stained with anti–phospho-Smad2 antibody. Images were obtained by a fluorescence microscope using a 40× objective. (c) NMuMg cells were transfected with the 3TP-lux reporter together with the indicated amounts of CHMP5 expression vector. Cells were treated with 1 ng/ml TGFβ for 24 h before lysis and then analyzed for luciferase activity. (d) NMuMG cells were transfected with murine CHMP5 shRNAs (Sh2 and Sh1) or control vector (Vector) and assayed as in c. Endogenous CHMP5 protein level was analyzed by immunoblotting with anti-CHMP5 antibody. (e and f) Distributions of TβRII and SARA in Chmp5 ^−/− primary embryonic cells. Wild-type (+/+) and Chmp5 ^−/− (−/−) cells were stained with antibodies specific for TβRII (e) and SARA (f). (g) HEK293 cells were transfected with pBIIX-luc reporter together with the indicated amounts of CHMP5 expression vector. Cells were treated with 10 ng/ml TNFα or10 ng/ml IL-1β for 4 h before lysis and then analyzed for luciferase activity. Alternatively, cells were transfected with pBIIX-luc reporter, CD4-TLR4 expression vector, and the indicated amounts of CHMP5 expression vector. Luciferase activity was assayed 24 h after transfection. (c, d, and g) Error bars represent the SD. n = 3. Bars, 20 μm.