Salivary glands and eye imaginal discs using ey-GAL4 driver. Immunocytochemical analysis revealed that Importin-a3 and Notch-ICD indeed co-localized in cell nuclei (Figure 1D1?F4).mutation (NAx-16172) which displays a shortened longitudinal vein V (L5), we noticed an extension of L5 vein upto the wing margin in Autophagy inhibitor transheterozygous combination with imp-a3 mutations (Figure 2C1?C4). The wing vein thickening phenotype of Delta (Dl) and the wing notching phenotype of the dominant negative mutation of Serrate (SerBd-G) were also enhanced by reducing the dose of imp-a3 (Figure 2D1?F4). A transheterozygous combination of dx, which is a cytoplasmic modulator of Notch activity, and imp-a3 alleles resulted in normal wings (data not shown) whereas dx in hemizygous combination with imp-a3 alleles showed wing phenotype that consist of extra vein material at the distal ends of wing veins (Figure 2G1?G4). These observations confirm a functional relationship between imp-a3 and Notch consistent with their molecular interactions.imp-a3 Mutant Cells have Elevated Notch Protein LevelsTo further analyze the molecular implications of Importin-a3 and Notch interaction in vivo, we examined the effects of imp-a3 loss-of-function on the endogenous Notch protein. We generated imp-a3 loss-of-function clones in two different larval tissues, eyeantennal imaginal discs and salivary glands, using imp a3D93 null mutant [26] and the FLP/FRT system [27]. The FLP activity that is under the control of eyeless promoter (ey-FLP) was used to induce somatic recombination events in eye discs, while hsFLP was used to generate somatic clones in salivary glands. Salivary glands begin to develop at 4.5 hours of development and complete 1315463 by 10 hours of embryonic development. Thus, to generate somatic clones in salivary glands, 4? hours old embryos were subjected to a single heat shock (37uC for 45 min). The imp-a3 mutant clones in either tissues were identified by the absence of GFP expression. Notch protein levels in the cytoplasm were strongly elevated in imp-a3 mutant cells compared with the surrounding wild-type cells in both salivary glands and eye-antennal discs (Figure 3A1?C4). These elevated Notch protein levels in imp-a3 mutant cells may be due to the lack of nuclear transport of Notch-ICD in the absence of Importin-a3.Gain-of-function Effect of imp-a3 on Notch ProteinIn parallel with the imp-a3 loss-of-function analysis, we also determined the gain-of-function effect of imp-a3 by monitoring the effects of its ectopic expression on Notch localization. We examined the distribution of endogenous Notch protein in wing imaginal discs in which imp-a3 expression was driven by en-GAL4 driver. Expression of UAS-HA-imp-a3 with en-GAL4 driver resulted in cytoplasmic aggregates of Notch protein in posterior compartment cells of wing discs (Figure 3D1?D3, also see Figure S1F1?S1F3). Since en-GAL4 is a posterior compartment specific driver, cells from anterior compartment serve as an internal control (Figure 3D1?D3, and S1E1 1E3). This effect is specific for impa3 since when the other two importins, imp-a1 and imp-a2, were overexpressed in wing discs using en-GAL4 driver, cytoplasmic aggregates of Notch were never observed (see Figure S1A1 1D3). Thus, this effect on Notch accumulation is a specific consequence of imp-a3 expression. Our analysis does not exclude the possibility that these aggregates may be some kind of vesicular structures which remains to be determined.Genetic Interactions.Salivary glands and eye imaginal discs using ey-GAL4 driver. Immunocytochemical analysis revealed that Importin-a3 and Notch-ICD indeed co-localized in cell nuclei (Figure 1D1?F4).mutation (NAx-16172) which displays a shortened longitudinal vein V (L5), we noticed an extension of L5 vein upto the wing margin in transheterozygous combination with imp-a3 mutations (Figure 2C1?C4). The wing vein thickening phenotype of Delta (Dl) and the wing notching phenotype of the dominant negative mutation of Serrate (SerBd-G) were also enhanced by reducing the dose of imp-a3 (Figure 2D1?F4). A transheterozygous combination of dx, which is a cytoplasmic modulator of Notch activity, and imp-a3 alleles resulted in normal wings (data not shown) whereas dx in hemizygous combination with imp-a3 alleles showed wing phenotype that consist of extra vein material at the distal ends of wing veins (Figure 2G1?G4). These observations confirm a functional relationship between imp-a3 and Notch consistent with their molecular interactions.imp-a3 Mutant Cells have Elevated Notch Protein LevelsTo further analyze the molecular implications of Importin-a3 and Notch interaction in vivo, we examined the effects of imp-a3 loss-of-function on the endogenous Notch protein. We generated imp-a3 loss-of-function clones in two different larval tissues, eyeantennal imaginal discs and salivary glands, using imp a3D93 null mutant [26] and the FLP/FRT system [27]. The FLP activity that is under the control of eyeless promoter (ey-FLP) was used to induce somatic recombination events in eye discs, while hsFLP was used to generate somatic clones in salivary glands. Salivary glands begin to develop at 4.5 hours of development and complete 1315463 by 10 hours of embryonic development. Thus, to generate somatic clones in salivary glands, 4? hours old embryos were subjected to a single heat shock (37uC for 45 min). The imp-a3 mutant clones in either tissues were identified by the absence of GFP expression. Notch protein levels in the cytoplasm were strongly elevated in imp-a3 mutant cells compared with the surrounding wild-type cells in both salivary glands and eye-antennal discs (Figure 3A1?C4). These elevated Notch protein levels in imp-a3 mutant cells may be due to the lack of nuclear transport of Notch-ICD in the absence of Importin-a3.Gain-of-function Effect of imp-a3 on Notch ProteinIn parallel with the imp-a3 loss-of-function analysis, we also determined the gain-of-function effect of imp-a3 by monitoring the effects of its ectopic expression on Notch localization. We examined the distribution of endogenous Notch protein in wing imaginal discs in which imp-a3 expression was driven by en-GAL4 driver. Expression of UAS-HA-imp-a3 with en-GAL4 driver resulted in cytoplasmic aggregates of Notch protein in posterior compartment cells of wing discs (Figure 3D1?D3, also see Figure S1F1?S1F3). Since en-GAL4 is a posterior compartment specific driver, cells from anterior compartment serve as an internal control (Figure 3D1?D3, and S1E1 1E3). This effect is specific for impa3 since when the other two importins, imp-a1 and imp-a2, were overexpressed in wing discs using en-GAL4 driver, cytoplasmic aggregates of Notch were never observed (see Figure S1A1 1D3). Thus, this effect on Notch accumulation is a specific consequence of imp-a3 expression. Our analysis does not exclude the possibility that these aggregates may be some kind of vesicular structures which remains to be determined.Genetic Interactions.
