Detection of RTK Pathway Activation in Drosophila Using Anti-dpERK Immunofluorescence Staining
In Drosophila, like in other metazoans, receptor tyrosine kinase (RTK) signaling pathways control diverse cellular processes such as migration, growth, fate determination, and differentiation (Shilo, Development 132:4017–4027, 2005). Activation of RTKs by
- PDF / 1,621,680 Bytes
- 8 Pages / 504 x 720 pts Page_size
- 91 Downloads / 164 Views
The monoclonal antibody recognizing dpERK was originally raised using a synthetic 11-amino acid long phospho-peptide conforming to the vertebrate MAPK/Erk activation loop (3). Due to the high evolutionary conservation of the activation loop, this antibody crossreacts with active (but not inactive) MAPK/Erk in different species, including Drosophila (2, 4). In flies, a single gene called rolled encodes the only MAPK/Erk family member, which serves as a downstream effector for all known RTK pathways (5).
Rony Seger (ed.), MAP Kinase Signaling Protocols: Second Edition, Methods in Molecular Biology, vol. 661, DOI 10.1007/978-1-60761-795-2_24, © Springer Science+Business Media, LLC 2010
401
402
Helman and Paroush
Accordingly, anti-dpERK staining is currently the method of choice for tracking and investigating the kinetics and dynamics of RTK signaling during Drosophila development (6) (Fig. 1). One feature renders the anti-dpERK antibody superior to related antibodies available for activated MAPK/JNK and MAPK/ p38 (7, 8): it stains intact tissues at various stages of development (oogenesis, embryogenesis, larval, and adult) (9–13). Hence, this antibody has served as an effective tool for exploring RTK signal transduction pathways in vivo (1). For example, quantification of the dynamic dpERK gradients formed in response to the Torso RTK pathway at the termini of syncytial Drosophila embryos has pointed to nuclear trapping as a novel mechanism for restricting diffusion of activated MAPK (14). In this context, the nuclei function to limit the spatial extent of receptor activation and to sharpen dpERK gradients. Anti-dpERK staining has also proved useful in genetic epistasis experiments, e.g., to establish whether gene functions that impinge on RTK pathways do so upstream, in parallel or downstream of MAPK/Erk (15). In general, combining fluorescent antibody staining together with confocal microscopy enables the simultaneous analysis, at high resolution, of two or more distinct signals in the same sample. Given that RTK signaling pathways regulate multiple
Fig. 1. Anti-dpERK staining is an effective readout for multiple RTK signaling pathways in Drosophila embryogenesis. (a–d) Confocal images of Drosophila embryos at different stages of development, stained for dpERK. In all panels, anterior is to the left. (a) Staining is evident at both termini of stage 4 syncytial blastoderm embryo, in regions where the Torso RTK pathway is active. (b, c) MAPK/Erk is phosphorylated and activated in response to two waves of EGFR-mediated signaling. Lateral view of stage 5 blastoderm (b) and ventral view of stage 10 (c) embryos; note the staining in the ventral ectoderm, in domains straddling the ventral midline. (d) At stage 12, staining is detected in tracheal branches (demarcated by dashed lines), specifically in posterior lateral migrating tip cells in which the FGFR pathway is active (arrowheads ).
Detection of RTK Pathway Activation in Drosophila
403
processes at different developmental stages in Drosophila, it is often u
Data Loading...
