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. 2015 Nov 9;211(3):605-17.
doi: 10.1083/jcb.201505108.

Endosomal sorting of Notch receptors through COMMD9-dependent pathways modulates Notch signaling

Haiying Li  1 , Yeon Koo  2 , Xicheng Mao  1 , Luis Sifuentes-Dominguez  3 , Lindsey L Morris  1 , Da Jia  1 , Naoteru Miyata  1 , Rebecca A Faulkner  1 , Jan M van Deursen  4 , Marc Vooijs  5 , Daniel D Billadeau  6 , Bart van de Sluis  7 , Ondine Cleaver  2 , Ezra Burstein  8
Affiliations

Endosomal sorting of Notch receptors through COMMD9-dependent pathways modulates Notch signaling

Haiying Li et al. J Cell Biol. .

Abstract

Notch family members are transmembrane receptors that mediate essential developmental programs. Upon ligand binding, a proteolytic event releases the intracellular domain of Notch, which translocates to the nucleus to regulate gene transcription. In addition, Notch trafficking across the endolysosomal system is critical in its regulation. In this study we report that Notch recycling to the cell surface is dependent on the COMMD-CCDC22-CCDC93 (CCC) complex, a recently identified regulator of endosomal trafficking. Disruption in this system leads to intracellular accumulation of Notch2 and concomitant reduction in Notch signaling. Interestingly, among the 10 copper metabolism MURR1 domain containing (COMMD) family members that can associate with the CCC complex, only COMMD9 and its binding partner, COMMD5, have substantial effects on Notch. Furthermore, Commd9 deletion in mice leads to embryonic lethality and complex cardiovascular alterations that bear hallmarks of Notch deficiency. Altogether, these studies highlight that the CCC complex controls Notch activation by modulating its intracellular trafficking and demonstrate cargo-specific effects for members of the COMMD protein family.

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Figures

Figure 1.
Figure 1.
Identification of COMMD9-interacting proteins. (A) Tandem affinity purification of COMMD9 (HA and TB tagged) in HEK293 cells was followed by SDS-PAGE and silver staining. The location of the bait, or the control polypeptide expressed by the empty vector, is marked by an asterisk. Other coprecipitated bands are evident in the COMMD9 lane. Molecular mass markers (in kD) are noted. (B) Top hits identified by LC/MS-MS. (C) Confirmation of the endogenous interaction between COMMD9 and Notch2. COMMD9 was immunoprecipitated with specific antibodies, and the precipitates were immunoblotted for Notch2 (using an antibody that is specific for its C-terminal region) and other proteins as indicated. Molecular mass markers (in kD) are noted. (D and E) COMMD9 interacts with Notch1 but not Notch3. Endogenous COMMD9 was immunoprecipitated and associated proteins were immunoblotted for Notch1 (D, two individual antibodies that are specific for Notch1 C terminus) and Notch3 (E). Molecular mass markers (in kD) are noted.
Figure 2.
Figure 2.
COMMD9 regulates the surface localization of Notch2. (A) Generation of COMMD9-deleted HeLa cells with CRISPR/Cas9 technology (Cr-COMMD9). Western blots for COMMD9 are shown in three clones derived using two different guide RNA sequences (gRNA). Molecular mass markers (kD) are noted. (B) Localization of Notch2 in parental HeLa cells and in Cr-COMMD9 clones was assessed by immunofluorescence staining. Bars: (top) 20 μm; (bottom) 5 µm. (C and D) Surface Notch2 was also assessed by biotinylation, precipitation, and immunoblotting as described in the Materials and methods section (C). The immunoblots for N-cadherin serve as loading controls. Molecular mass markers (in kD) are noted. Semiquantitation was performed by densitometry analysis, and the averages of three independent iterations are presented (D). Error bars represent the SEM. *, P < 0.05. (E) HA-COMMD9 was reintroduced using a lentiviral vector resulting in rescued expression of COMMD9 in clones 1 and 3, as shown here by immunoblotting. Molecular mass markers (in kD) are noted. (F and G) Notch2 surface expression was assessed by biotinylation and immunoblotting (E). Molecular mass markers (in kD) are noted. Semiquantitation by densitometry analysis and the averages of three independent iterations are presented (G). Error bars represent the SEM. *, P < 0.05.
Figure 3.
Figure 3.
COMMD9 deficiency impairs Notch-dependent gene expression. (A) U2OS cells stably deficient in COMMD9 or expressing HA-COMMD9 were generated using lentiviral vectors. COMMD9 Western blots with molecular mass markers (in kD) are shown. (B) U2OS cells shown in Fig. 3 A were stimulated with Jagged1 (plate bound), and the induction of HEY1 mRNA was determined by qRT-PCR. Triplicate samples were averaged, and error bars represent the SEM. *, P < 0.05. (C) The same cells shown in Fig. 3 A were transfected with a Notch-responsive luciferase reporter. Luciferase activity was determined and normalized against the control conditions. Triplicate samples were averaged. Error bars represent the SEM. *, P < 0.05. (D) Immortalized MEFs obtained from wild-type (WT) or COMMD9-deficient (KO) littermate embryos were used. Cells were stimulated with Jagged1 (plate bound) or TGF-β (soluble), and the induction of Notch-responsive genes (Hey1, Hey2, and Hes1) or TGF-β responsive genes (Junb) was assessed by qRT-PCR. Triplicate samples were averaged. Error bars represent the SEM. *, P < 0.05. (E) Furthermore, COMMD9 expression was rescued in the KO line using a lentivirus, and the responsiveness of this control line was examined and compared against an empty vector control. Triplicate samples were averaged. Error bars represent the SEM. *, P < 0.05. (F) MEFs derived from an embryo with a conditional Commd9 allele (F/F) were used to derive Commd9-deficient cells (−/−). These cells were stimulated with plate-bound Jagged1 (left) or cell-bound Jagged1 (right) and the induction of Notch responsive genes (Hey1, Hey2, and Hes1) was monitored by qRT-PCR. Triplicate samples were averaged. Error bars represent the SEM. *, P < 0.05.
Figure 4.
Figure 4.
COMMD9 deficiency leads to lysosomal degradation of Notch. (A) HEK293 cells were transfected with a Notch2 expression vector; siRNA was used to concurrently silence COMMD9 expression. Notch2 expression was monitored using antibodies that recognize the extracellular domain (Notch2-N) or an intracellular epitope (Notch2-C). Molecular mass markers (in kD) are shown. (B) HEK293 cells were transfected with a Jagged1 expression vector; siRNA was used to concurrently silence COMMD9 expression. Jagged1 expression was evaluated using Jagged1 or FLAG antibodies. Molecular mass markers (in kD) are shown. (C) Levels of Notch1 and Notch2 expression in two Commd9-deleted cell lines (−/−) and their isogenic controls (F/F) were determined by immunoblotting. Molecular mass markers (in kD) are shown. (D) Using Commd9 F/F and −/− MEFs, total Notch1 and Notch2 levels (whole-cell lysates [WCLs]) were determined in by immunoblotting (left). In addition, surface levels of both proteins (plasma membrane [PM]) were also determined by biotinylation and immunoblotting (right). Molecular mass markers (in kD) are shown. (E) The reduced expression of Notch2 shown in Fig. 4 C is rescued by Bafilomycin A1 (BafA) treatment, an inhibitor of endolysosomal acidification that impairs lysosomal proteases. Molecular mass markers (in kD) are shown.
Figure 5.
Figure 5.
COMMD9 deficiency leads to embryonic lethality and complex cardiovascular abnormalities. (A) Genotyping results at different developmental stages demonstrate that Commd9-deficient mice (gene trap knockout [KO] model) are not viable at birth. Expected Mendelian rates are seen at E10.5. (B) KO embryos display neural tube edema (white arrows), seen by E10.5 (left). This is followed by punctate hemorrhages (yellow arrows), seen at E11.5 (right). WT, wild type. Bar, 500 µm. (C) KO embryos eventually developed widespread hemorrhages that are apparent by E11.5 and at later stages. Bar, 500 µm. (D) Cross-sectional histologic analysis demonstrated small hypoplastic hearts in all KO embryos. Bar, 200 µm. (E) Areas of unilateral narrowing of the dorsal aorta were commonly seen in mutant embryos (arrow). Bar, 100 µm. (F) Hearts were isolated from somite-matched E10.5 embryos. RNA was extracted from these organs, and qRT-PCR was used to determine the relative level of expression of two Notch target genes (Hey1 and Hey2) and Commd9.
Figure 6.
Figure 6.
COMMD9 interacts and colocalizes with COMMD5. (A) Colocalization of COMMD9 and COMMD5 was evaluated in HeLa cells after CRISPR-mediated deletion and rescue of COMMD9 expression (Fig. 2 D; HA tagged, clone 3). Immunofluorescence staining for COMMD9 (HA) and COMMD5 was followed by confocal microscopy imaging. Bar, 10 µm. (B) In vitro binding between the indicated recombinant COMMD proteins. The indicated combinations of COMMD proteins were expressed in E. coli as described in the Materials and methods section. After lysis, one of the proteins (fused to the GST or MBP tag) was precipitated, and the presence of the partner COMMD protein was assessed by Coomassie blue staining. Molecular mass markers (in kD) are shown.
Figure 7.
Figure 7.
COMMD9 and its partner COMMD5 regulate Notch2. (A and B) The effects of COMMD9 deficiency on COMMD5 and COMMD10 interactions with the CCC complex were evaluated in two CRISPR clones. Endogenous input levels for several COMMD proteins were not substantially affected (A). In contrast, COMMD5 and COMMD10 interactions with the CCC complex subunit CCDC22 were lost in COMMD9-deficient cells. COMMD4, COMMD6, and COMMD8 were unaffected (B). Molecular mass markers (in kD) are shown. (C) In HEK 293T cells, each individual COMMD gene was silenced using siRNA transfection. Deficiency of either COMMD5 or COMMD9 led to reduced Notch2 expression. All other siRNA treatments did not affect this receptor. Molecular mass markers (in kD) are shown. (D) In vitro binding between an immunopurified Notch2 C-terminal polypeptide and recombinant COMMD complexes was evaluated by coprecipitation. Only COMMD5-containing complexes bound to Notch2. Molecular mass markers (in kD) are shown.
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