Regulation of Notch signaling by multiple Ankyrin repeat containing protein Mask

Abstract

Background: Notch pathway is an evolutionarily conserved, highly pleiotropic signaling system that governs diverse developmental processes. Its diverse functions are attributed to the intricate regulatory mechanisms that finely tune the pathway. While several known elements contribute to maintaining cellular homeostasis by modulating Notch signaling, many unidentified components likely play significant roles in its regulation, necessitating further exploration.

Methods: To identify novel regulators of Notch-intracellular domain (Notch-ICD), we carried out a yeast two-hybrid screen and identified Multiple Ankyrin repeat single KH domain containing protein (Mask) as an interacting partner of Notch-ICD. Physical interaction between these two proteins was further validated by co-immunoprecipitation experiments. Moreover, cellular studies using immunocytochemistry reveals that Mask plays important role in Notch turnover and protect from degradation. To inhibit lysosomal degradation, chloroquine was introduced in the food at a concentration of 1 mg/ml.

Results: Immunocytochemical analyses revealed that Notch and Mask co-localised within the same subcellular compartments. Different alleles of mask showed strong genetic interactions with Notch pathway components in transheterozygous combinations. Loss- and gain-of-function studies of Mask demonstrated that it plays a regulatory role in Notch signaling. Specifically, the absence of Mask results in downregulation of Notch target genes, although it does not significantly affect endogenous Notch protein levels. Our data suggest that Mask positively regulates Notch signaling by stabilizing Notch-ICD and protecting it from lysosomal degradation. Treatment with 1 mg/ml chloroquine can mitigate the Mask loss-mediated Notch intracellular domain degradation. This study presents a novel mode of Notch signaling regulation mediated by the Ankyrin repeat-containing protein Mask.

Conclusion: Here we provide evidence that Mask physically binds with Notch-ICD and positively regulates Notch signaling pathway by protecting it from degradation. Mask genetically interacts with Notch and Notch pathway components and absence of Mask affects the Notch signaling pathway thus it may control the hyper activation of the signaling.

Keywords: Drosophila; Ankyrin repeat; Cell signaling; Endocytosis; Mask; Notch.

Fig. 1

Drosophila Notch binds with Mask. A Schematic representation of the domain organization of Mask and ANKHD1, the human ortholog of Drosophila Mask. Different domains and boundary residues are marked at the bottom. Ankyrin repeat 1 (ANK1), Ankyrin repeat 2 (ANK2), nuclear export signal (NES); nuclear localization signal (NLS), K homology domain (KH). ANKHD1 has 77% and 81% sequence similarity to ANK1 and ANK2, and 51% similarity to the KH domain of Drosophila Mask. The second ankyrin repeat domain of Mask is important and sufficient to bind with Notch ICD. B-C Co-immunoprecipitation of Notch-ICD and Mask was carried out with wing disc lysate overexpressing both the protein using vg-GAL4. (+) symbol indicates the presence and (-) shows the absence of the respective reagent. B Notch protein was immunoprecipitated by overexpressed Mask and detected by the anti-Notch antibody (C17.9 C6). There was absence of the Notch band in the negative control lane. C In the other direction, Mask was immunoprecipitated by Notch and detected by the anti-Mask antibody generated in our lab. Negative control lane shows the absence of the Mask protein band. Absence of IP antibody performed as a negative control. D Domain specific interaction was studied using GST pull-down assay. GST pull-down assay was performed with lysate of salivary glands in which Notch-ICD was overexpressed using salivary gland specific sgs-GAL4 and purified recombinant GST-ANK1 (amino acids 546–1043), GST-ANK2 (amino acids 2312–2644) and GST-KH (amino acids 3036–3100) and other control as indicated. GST pulled down proteins were analysed by western blotting with anti-Notch (C17.9 C6) antibodies. GST-ANK2 protein pulled down the Notch-ICD, M indicates the marker lane. E-O Co-localization of Mask and Notch-ICD in wing disc. Expression of Mask is homogeneous throughout the wing disc similar to the expression of Notch. Endogenous Mask co-localises with endogenous Notch-ICD in wing disc, I-L are high magnification images of the white marked region of G. Images in G and K are merge of those in E, F and I, J respectively. Images in H and L are merged with DAPI and G and K respectively. Images in M–O are some colocalization point from image K. Arrowhead shows the colocalization point in image K. P shows the Pierson’s correlation coefficient value of Mask and Notch colocalization, that is approximately 0.5, calculated using FIJI. Scale bars, 100 µm (E–H), 20 µm (I-L)

Fig. 2

Genetic interaction of mask and Notch pathway components. (A1-G4) Representative wings from individuals with indicated genotype. Wings from wild type individuals (A1) and mask heterozygous (A2-A4) showed normal wing phenotype. Wings from N^{54 l9} heterozygotes (B1) showed wing nicking phenotype, which was enhanced in trans-heterozygous combination with mask allele (B2-B4). Wings from male N^nd−3 hemizygotes (C1) showed nicking phenotype which was enhanced in combination with mask allele (C2-C4). Gain-of-function allele of Notch N^AX−16172 shows shortened the longitudinal vein L5 and L4 (D1), which in combination with different allele of mask rescued the length of L4 completely (D2-D4). Hemi-zygotes of dx allele dx¹⁵² showed vein thickening in the distal region of the wing blade and sporadic wing nicking phenotype (E1), which was enhanced by combination with different mask loss-of-function allele (E2-E4). Dominant negative Notch showed wing nicking phenotype in heterozygous condition (F1), which was enhanced in trans-heterozygous condition with different mask allele (F2-F4). Dominant negative Mastermind, MamH in heterozygous condition shows wing notching phenotype (G1), which was enhanced in trans-heterozygous combination with different mask allele (G2-G4). Scale bar 500 µm (A1-G4). (H) Represents the percentage of flies showing wing nicking phenotype of different mask allele with combination of N^{N54 l9}, N^Nd−3 and dx¹⁵² respectively. (I) Represents the length of L4 in N^AX−16172 and combination with different mask allele in male and female. (J) Represents the wing area in C96-GAL4 driven Dominant-negative-Notch with combination of different mask allele. For every set of experiment at least 200 flies were observed and measurement of wing area and vein length was measured for at least 25 flies and the data was employed for statistical analysis. Significance was determined using one-way analysis of variance (ANOVA) with Tukey’s multiple comparison post-test, with a p-value < 0.05 considered statistically significant (**p < 0.01, ***p < 0.001). Scale bar 500 µm

Fig. 3

Reducing Mask level down regulates Notch signaling activity. A-H Representative images from third instar wing imaginal disc show the expression of Cut and NRE-GFP upon reducing the level of Mask using posterior domain specific engrailed-GAL4. Abrogating Mask in the posterior domain using Mask-RNAi results into abolished Cut (A-D) and NRE-GFP (E–H) expression. A and E shows the RFP marked posterior domain where Mask was downregulated. B and F shows the abrogated Cut and NRE-GFP expression upon Mask downregulation, respectively. C and G is the merged images of A,B and E,F respectively. D and H are the merged images of C and G with DAPI respectively. B’ and F’ are the intensity of Cut and NRE-GFP along with DV boundary, respectively. Q represents the relative intensity of Cut and NRE-GFP at the anterior and posterior domain. A total number of disc examined = 20, for each case. A total number of 5 discs were used for quantification in each case followed by unpaired t-test to determine the significance of our findings. (I-P) Downregulation of Mask using patched-GAL4 along with AP boundary shows the abrogated Cut (I-L) expression at the AP-DV junction but no change at the level of Notch protein (M-P). I and M shows the GFP marked AP boundary where Mask was downregulated. J and N show the level of Cut and Notch protein respectively, upon downregulation of Mask at AP boundary. K, O is the merged images of I,J and M, N respectively. L and P are the merged images with DAPI. J’ and N’ show the intensity of Cut and Notch at DV boundary. (R-W) Loss-of-function clones of mask using mask^10.22 allele were generated with FLP/FRT system; mask.^10.22 clones were marked by the absence of Green Fluorescent Protein (GFP) expression. Cut staining in wing disc of such clones display significant reduction in the expression (S); whereas Notch staining shows no significant change in expression and localization of Notch protein (V). Scale bars, 20 µm (A-P) and (R-W). Unpaired t-test was performed to determine the p-value (**p < 0.01, ***p < 0.001)

Fig. 4

mask shows epistatic interaction with Notch and is required for Notch mediated downstream target gene expression. (A-N) Downregulation of Mask at AP and DV boundary using dpp-GAL4 and C96-GAL4 respectively results in significant reduction of Cut expression. The overexpression of Notch-ICD results into excessive Cut expression at AP (A) and DV (C) boundary. This overexpressed Cut is significantly rescued upon downregulation of Mask using Mask RNAi in the same background in AP (K) and DV (M) boundary respectively. F and H shows the effect of downregulation of Mask upon Cut expression pattern at AP (F) and DV (H) boundary. B and D show that NICD overexpression does not affect the expression of Mask in AP and DV boundary. G and L are the expression pattern of Mask upon Mask downregulation and Notch overexpression plus Mask down regulation at AP boundary. I and N are the expression pattern of Mask upon Mask downregulation and Notch overexpression plus Mask down regulation at DV boundary. E represents the effect of NICD overexpression at the DV boundary using C96-GAL4, J represents the Mask downregulation with C96-GAL4. O represents that Mask downregulation results in the reduction of Notch overexpression phenotypes at the wing margin.. A’, F’, K’ and C’, H’, M’ show the intensity profiling of Cut at the AP and DV boundary, respectively. P represents the average intensity of Cut in Notch overexpressed, Mask downregulation and Mask downregulation in Notch overexpressed background at AP and DV boundary. A total 15 discs for each case were observed and the result was consistent. ImageJ software was utilized for intensity profiling by calculating the mean intensity (integrated density divided by the area of the domain). Quantification was done on a total of five discs for each condition. Significance was determined using one-way analysis of variance (ANOVA) with Tukey’s multiple comparison post-test, with a p-value < 0.05 considered statistically significant (**p < 0.01, ***p < 0.001). Q shows the percentage of wing nicking phenotype in Notch overexpressed, Mask downregulation and Mask downregulation in Notch overexpressed background. Profile plot analysis was done by FIJI software. Scale bar 20 µm (A-N)

Fig. 5

Effect of Mask overexpression on Notch signalling activity. (A-L) engrailed-GAL4 driven Mask overexpression at the posterior domain (marked with GFP A, E, I) of third inster wing imaginal disc resulted in the reduction at the expression of Notch target Cut (B), Dpn (F) and Vg (J). C, G and K represents the merged images of AB, EF and IJ respectively. D, H and L represents the merged images with DAPI. B’, F’ and J’ shows the intensity profiling of Cut, Dpn and Vg respectively. (M) compares the average intensity of Cut, Dpn, and Vg per unit area between the anterior and posterior domains when Mask is overexpressed in the posterior domain. A total of 15 discs was examine for each case, and five out of them are used to quantify the intensity. Intensity data shows that increased dosage of Mask significantly reduces the level of Notch target gene expression at the posterior domain. Unpaired t-test was performed to determine the p-value (*p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 6

Blocking caspase activity to inhibit cell death does not alleviate the loss of Notch target gene expression induced by Mask overexpression. (A-L) Represents the third inster wing imaginal disc overexpressing both Mask and p35 the caspase blocker using posterior domain specific en-GAL4 tagged with GFP (A,E,I). (B, F, J) Co expression of Mask and p35 minorly rescued the abolished Notch target genes but not completely. (C,G,K) represents the merged images from AB, EF and IJ respectively. (D, H, L) Shows the merged image with DAPI. B’, F’, J’ represents the intensity profiling of Cut, Dpn and Vg respectively. (M) compares the average intensity of Cut, Dpn, and Vg per unit area between the anterior and posterior domains when Mask and p35 is overexpressed in the posterior domain. Total 20 (n = 20) imaginal discs were examine out of them five were used to quantify the intensity. An unpaired t-test was subsequently conducted to assess the significance of the results (**p < 0.01, ***p < 0.001). (N) adult wing image shows improvement in loss of wing blade area but not completely rescued, where both Mask and p35 was overexpressed using en-GAL4 driver. (O) Blocking JNK signalling by dominant negative form of basket (UAS-Basket-DN) rescued the wing phenotype completely. (P)Graph showing the percentage of wing area among different genotype mentioned. (Q-S) Overexpression of Mask at posterior domain (marked with GFP) (Q), trigger JNK activation, reported by TRE-JNK reporter line(R), (S) is the merged image of Q and R. (T-V) Overexpression of Basket-DN rescues the loss of Cut (U) due to Mask overexpression. (T) GFP marked the domain and (V) shows the merged image of T and U. (R’, U’) Graph shows the intensity profiling of TRE JNK in Mask overexpression condition and profiling of Cut upon Basket-DN expression in Mask overexpression background. (R’’, U’’) Graph shows the Comparison between anterior and posterior domain specific expression of TRE-JNK and Cut, respectively. A total 15 imaginal discs were examined and out of them total five discs were used to quantify the intensity for R’’, and for U’’ total 10 imaginal discs were examined and 5 out of them were used to quantify the intensity. Statistical analyses were carried out to determine the significance of the data by performing unpaired t-test and determine the p-value (*p < 0.05, **p < 0.01, ***p < 0.001). Scale bar 20 µm (A-L) and (Q–V)

Fig. 7

Excess Mask accumulates endogenous Notch proteins and affects negatively. (A-F) Mask overexpression at the posterior domain using en-GAL4, domain marked with anti-Mask stain (A) or GFP expression (D). (B, E) Accumulation of Notch was observed at the posterior domain. (C, F) represents the merged images with AB and DE respectively. (B’) Graph shows the relative intensity difference of Notch between anterior and posterior domain in Mask overexpression condition. (B’’) Intensity profiling shows the increased intensity of Notch at the posterior domain where Mask was overexpressed. (G-I) en-GAL4 mediated Mask overexpression results into elevated Notch level. G represents the posterior domain marked with RFP. H shows the increased Notch level at the posterior domain. I represents the merged images of G and H. (J) Graph shows the relative fold change of Notch RNA level upon Mask overexpression compared to wild type. (K) Shows that elevated Mask level results into reduced transcript level of kuzbanian transcript compared to the control one. Statistical analyses were carried out to determine the significance of the data by performing unpaired t-test and determine the p-value (*p < 0.05, **p < 0.01, ***p < 0.001). Scale bars 20 µm (A-F) and (G-I)

Fig. 8

Abrogated Mask expression affects Notch-ICD stability. (A-C) Overexpression of Notch at AP boundary by dpp-GAL4. Notch express along with the AP boundary and visible with bright nuclear dot like expression (A). (D-F) Treatment with 1 mg/ml concentration of chloroquine in Notch-ICD overexpression results into subtle increase of Notch signal. G represents the down regulation of Mask in the same Notch overexpression background results compromised expression of Notch-ICD. Majority of the cells of the AP boundary cannot sustain Notch-ICD expression (G). B,E and H represents the expression of Mask. (J-L) Treatment with 1 mg/ml concentration of chloroquine in coexpression of Notch-ICD and Mask RNAi larvae shows a significant restoration of the expression of Notch-ICD. Improved expression of Notch-ICD (J). (M) shows the average intensity of Notch protein control and experimental condition. Statistical analysis shows that treatment with chloroquine significantly restores the Notch-ICD level. Total 15 imaginal discs were examined and out of them total five discs were used to quantify the intensity. Statistical analyses were carried out to determine the significance of the data by performing unpaired t-test and determine the p-value (*p < 0.05, **p < 0.01, ***p < 0.001). Scale bar 100 µm (A-L). (N)Western blot analysis shows 1 mg/ml chloroquine treatment results into increased amount of Notch-ICD and full length receptor (lane 3) compared to control untreated group (lane 2). vg-GAL4 mediated downregulation of Mask results into decreased intensity of Notch-ICD (120 kDa) (lane 4) but full length Notch was broadly unaffected. Treatment with 1 mg/ml chloroquine restores the diminished Notch-ICD level (lane 5)

Fig. 9

Mask protects Notch-ICD from lysosomal degradation. (A-P) In MARCM clone analysis, the GFP positive cells are mask null and Notch-ICD overexpressed in the same cell. Loss of mask cells are with compromised Notch-ICD expression (B). Total GFP positive clonal cell versus Notch-ICD expressed cells are lower. (F) is the lower zoom image of image (B). C and G represents the merged images of AB and EF respectively. D and H are the merged images with DAPI. (I-P) 1 mg/ml concentration of chloroquine treatment in the same genetic background improves the Notch-ICD signal in the mask mutant GFP positive cells. Total GFP positive clonal cell versus number of notch-ICD expressing cells are increased compared to the control clone. (N) is the lower magnified image of (J). K and O are the merged images of IJ and MN. L and P are the merged images with DAPI. Q represents the percentage of cells showing overexpressed Notch in MARCM positive clones in controlled and chloroquine treated condition. Data shows a significant increase in the overexpressed Notch positive cells in chloroquine treated group compared to the control one. Statistical analyses were carried out to determine the significance of the data by performing unpaired t-test and determine the p-value (*p < 0.05, **p < 0.01, ***p < 0.001). Scale bars 20 µm (A-P)

Fig. 10

A model illustrating the potential mechanism by which Mask regulates the Notch signaling pathway. Mask interacts with Notch-ICD, stabilizing it and preventing its degradation. In the absence of Mask, Notch-ICD undergoes degradation, highlighting Mask’s role in positively regulating Notch signaling