Characterization of a novel gene involved in border cell migration

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1 Characterization of a novel gene involved in border cell migration Wei-Hao Li, He-Yen Chou and Li-Mei Pai Graduate Institute of Basic Medical Science Chang-Gung University, Tao-Yuan, Taiwan, R.O.C. Abstract The protein no.75 was identified through interaction with D-ADI1 in yeast-two-hybrid. No. 75 is a novel PDZ protein. Protein 75 C-terminal was similar to Syntenin in mammalian. Expression of no.75 RNAi in border cell led to the delay of border cell migration in Drosophila. Furthermore, knockdown of no. 75 resulted in border cell cluster tightly connected, forming an orb shape. We further analyzed the junction protein distribution in no.75 knockdown border cells. Interestingly, the normal localization of integrin at the basolateral junction between border cells was altered. Integrin did not evenly localize to each junction between border cells and border cells in no.75 knockdown border cells. Our data suggest that protein no.75 may be involved in integrin trafficking to cell junction. Moreover, reduction of p-fak signal was observed in this delayed border cells border. In sum, protein no. 75 may affect border cell migration through regulation of integrin mediated signaling transduction. Results Fig.1 Cloning of protein 75 and the interaction between P.75 and D-ADI1. Protein75 gene was cloned from Drosophila ovary cdna. The central region of PBC was a antigen for protein 75 antibody (A). Different ovary extract was analyzed by immunoblotting. Protein 75 signal was detected between 170kd and 130kd markers, and Tubulin Gal4:: UAS HA-75 was a positive control (asterisk). WT (OreR) signal was stronger than that of depletion of one copy (df/+)(b). Furthermore, full length protein 75 were constructed into yeast-two-hybrid expressing plasmid (pgadt7 or pgbkt7). Yeast co-transformed with an expression vector encoding GAL4 AD fused to the protein 75 full length and a expression vector encoding GAL4-BD fused to ADI1. Co-transformed with empty vector pgadt7 and pgbkt7 as a negative control. And co-transformed of Gal4 -pcl1 and pgbkt7 is a positive control. Yeast can grow on G3 plate while co-transformed with pgadt7-75 and pgbkt7-d-adi1. So we suggest protein 75 interacts with D-ADI1. Fig. 2 Border cell migration delay in UAS-protein 75 RNAi transgenic fly Analysis border cell migration in egg chambers expression protein 75RNAi driven by C306 GAL4 at % and 57% of stage 10 egg chambers showed delay of border cell migration in RNAi-1 and RNAi-2 expressing egg chambers, respectively.

2 Fig. 3 Expression of UAS-HA-75 transgene rescued the delay of border cell migration. 70% of S10 egg chamber showed delay of border cell migration while UAS-protein 75 RNAi and UAS-GFP were co-expressed in protein 75 heterozygous background. This defect could be rescued while replacing UAS-GFP with UAS HA-P.75. Fig.4 The protein 75 contains a PH and a PDZ domain There are two protein 75 isoforms in Drosophila. PB isoform contains 994 a.a. According to protein motif analysis, there are a PH domain in the N-terminus and a PDZ domain in the C-terminus. However, the PC isoform lacks the C-terminal PDZ domain. The C-terminus of Protein 75 is similar to the C-terminus of Syntenin in blast alignment. This region includes partial sequence of first PDZ domain and entire second PDZ domain of syntenin. This region is 26% identities and 50 positive between protein 75 and Syntenin. Fig. 5 Delayed-border cells displayed a compact cluster phenotype and an alteration of its integrin distribution. In WT border cell cluster, integrin was evenly distributed in each junction between border cell and border cell (A). And the border cell cluster showed a spreading morphology. However, expression protein 75 RNAi in border cells led to migration delay, and border cell cluster showed a compact morphology (B). Furthermore, the integrin signal was only localized in some junction between border cell and border cell. Fig. 6 Reduction of p-fak signal in protein 75 depleted border cells. Significant and continuous p-fak signal was distributed in cell junction between nurse cells and follicle cells in wild-type egg chamber (A). When Protein 75 RNAi expressed in border cells, p-fak signal became weak, and discontinuous (B). Conclusion A. Protein 75 was a PDZ containing protein which interacted with D-ADI1. B. Expression of protein 75 RNAi led to stage 10 border cell migration delay, and morphology change of the cluster. C. The cluster of delayed-border cells displayed abnormal integrin distribution and reduction of p-fak signaling. Fig.1 Cloning of protein 75 and the interaction between P.75 and D-ADI1.

3 Fig. 2 Border cell migration delay in UAS-protein 75 RNAi transgenic fly Fig. 3 Expression of UAS-HA-75 transgene rescued the delay of border cell

4 migration. Fig.4 The protein 75 contains a PH and a PDZ domain

5 Fig. 5 Delayed-border cells displayed a compact cluster phenotype and an alteration of its integrin distribution.

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7 Fig. 6 Reduction of p-fak signal in protein 75 depleted border cells.

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