Figure 8.
Human NOTCH3 localizes to different endosomal subdomains and can be activated by VPS4EQ. (A and B) Localization of human Notch3 expressed in hTERT-RPE-1 cells compared with expressed mRFP-clathrin light chain in EEA1 positive endosome showing either separate localization of Notch and clathrin, green arrow (A) or overlapping localization, white arrow (B). (C and D) Localization of endogenous human NOTCH3 in MCF7 cells compared with endogenous clathrin heavy chain (CHC) in EEA1 positive endosome showing either separate localization of NOTCH3 and clathrin, green arrow (C), or overlapping localization, white arrow (D). (E) Scoring of % NOTCH3 localization with clathrin in EEA1 endosomes when expressed in htert-RPE-1 cells or endogenous NOTCH3 and clathrin in MCF7 cells. Scoring of overlapping localization was performed on 11 and 16 cells respectively in five optical Z sections per cell, total numbers of endosomes scored, are indicated. (F) In hTert-RPE1 cells, EGFP-tagged VPS4EQ expression causes enlarged EEA1 positive endosomes with ring-like structures surrounding an enlarged central lumen. Expressed NOTCH3 localized on the perimeter of the organelle (red arrows). VPS4 is localized to sub-regions of the organelle either adjacent to or separate from NOTCH3 and is also localized to EEA1 negative structures. (G) NOTCH3 is localized in EEA1-positive endosome, adjacent to the clathrin-positive domain in cells without VPS4EQ expression. (H) An enlarged clathrin-positive domain in cell coexpressing NOTCH3 and VPS4EQ. NOTCH3 puncta (red arrows) are localized within clathrin marked region, either adjacent or separate from the VPS4EQ occupied region. (I) VPS4EQ expression increases % of clathrin stained puncta that also have NOTCH3 localization (n = 10 cells scored in Z sections through the cytoplasmic region, a total of 1,505 and 703 clathrin puncta scored, for the presence of NOTCH3, in NOTCH3 and NOTCH3 + VPS4EQ expressing cells, respectively). (J and K) VPS4EQ induced the NOTCH3 signal (J) and decreased relative sensitivity to BB-94 (K). * indicates P < 0.05 by two tailed t test, error bars are SEM, sample sizes are indicated on figure.

Human NOTCH3 localizes to different endosomal subdomains and can be activated by VPS4 EQ . (A and B) Localization of human Notch3 expressed in hTERT-RPE-1 cells compared with expressed mRFP-clathrin light chain in EEA1 positive endosome showing either separate localization of Notch and clathrin, green arrow (A) or overlapping localization, white arrow (B). (C and D) Localization of endogenous human NOTCH3 in MCF7 cells compared with endogenous clathrin heavy chain (CHC) in EEA1 positive endosome showing either separate localization of NOTCH3 and clathrin, green arrow (C), or overlapping localization, white arrow (D). (E) Scoring of % NOTCH3 localization with clathrin in EEA1 endosomes when expressed in htert-RPE-1 cells or endogenous NOTCH3 and clathrin in MCF7 cells. Scoring of overlapping localization was performed on 11 and 16 cells respectively in five optical Z sections per cell, total numbers of endosomes scored, are indicated. (F) In hTert-RPE1 cells, EGFP-tagged VPS4EQ expression causes enlarged EEA1 positive endosomes with ring-like structures surrounding an enlarged central lumen. Expressed NOTCH3 localized on the perimeter of the organelle (red arrows). VPS4 is localized to sub-regions of the organelle either adjacent to or separate from NOTCH3 and is also localized to EEA1 negative structures. (G) NOTCH3 is localized in EEA1-positive endosome, adjacent to the clathrin-positive domain in cells without VPS4EQ expression. (H) An enlarged clathrin-positive domain in cell coexpressing NOTCH3 and VPS4EQ. NOTCH3 puncta (red arrows) are localized within clathrin marked region, either adjacent or separate from the VPS4EQ occupied region. (I) VPS4EQ expression increases % of clathrin stained puncta that also have NOTCH3 localization (n = 10 cells scored in Z sections through the cytoplasmic region, a total of 1,505 and 703 clathrin puncta scored, for the presence of NOTCH3, in NOTCH3 and NOTCH3 + VPS4EQ expressing cells, respectively). (J and K) VPS4EQ induced the NOTCH3 signal (J) and decreased relative sensitivity to BB-94 (K). * indicates P < 0.05 by two tailed t test, error bars are SEM, sample sizes are indicated on figure.

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