Figure 4. rumi is required in the signal-receiving cell upstream of the S3 cleavage.

(A and A’) Ectopic expression of the full length Notch (N^FL) induces aberrant Cut (red) expression in MARCM clones of a wild-type chromosome that are close to the dorso-ventral boundary (arrows in A’). GFP (green) marks the clones that ectopically express N^FL in (A) and (B).

(B and B’) Ectopic expression of N^FL in MARCM rumi clones does not induce Notch signaling. Note that in rumi mutant cells Cut (red) expression is lost in the prospective wing margin despite N^FL overexpression (arrows in B’).

(C and C’) Ectopic expression of N^ECN induces Cut (red) expression in rumi cells. GFP marks MARCM rumi clones that ectopically express N^ECN, suggesting that the Rumi function is required upstream of the S3 cleavage of Notch.

(D) Western blots showing that Notch processing is altered in the absence of Rumi function at the restrictive temperature. Anti-N^ICD antibody was used in all blots. The top bands in each blot correspond to full length Notch, which is around 300 kDa. In wing disc extracts one predominant band that corresponds to Notch cleavage product is visible in both wild-type (wt) and in rum^Δ26/Δ26 (rumi) larvae kept at 18°C. This band is not detected in extracts of rumi larvae kept at 28°C for 10 hours prior to dissection (left arrow). In brain extracts four ~120 kDa fragments are visible in wt larvae and in rumi larvae that were kept at 18°C. The upper two bands however are lost in rumi larvae kept at 28°C (right arrow).

(E) RNAi-mediated knockdown of Rumi and Kuz results in similar Notch processing defects in S2 cells. Western blotting with anti-N^ICD on protein extracts from S2 cells raised at 28°C is used to determine the pattern of Notch cleavage. Control cells (EGFP dsRNA) show two cleavage products (arrow). Addition of a Furin inhibitor (FI) does not alter this pattern. However, treatment of S2 cells with dsRNA against Rumi or Kuz results in the loss of the upper cleavage product, strongly suggesting that the Kuz-mediated S2 cleavage of Notch is affected by the loss of Rumi at the restrictive temperature. Note that three different kuz dsRNAs produce the same results.