Supplemental Experimental Procedures s2

Supplemental Experimental Procedures

Expression clones were made in the pSM vector, a derivative of pPD49.26 (A. Fire) with extra cloning sites (S. McCarroll and C.I. Bargmann, unpublished data). The plasmids and transgenic strains (0.5-30ng µl-1) were generated using standard techniques and coinjected with markers Punc122::gfp, Punc122::dsRed or Podr1::gfp (15- 30ng µl-1): mgIs18 [Pttx-3::gfp],wyIs45 [Pttx3::gfp::rab3], olaEx54 [Pttx3::mig10A::gfp, Pttx3::rab3::mch], olaEx324 [Pttx3::mig10B::gfp], wyEx1938 [Pttx3::mig10C::gfp], wyIs97 [Punc86::myr-gfp, Punc86::mch::rab3], olaEx83 [Fosmid mig10A mig10B], olaEx110 [Fosmid mig10B], olaEx105 [Fosmid mig10A(-) mig10B(+)], olaEx142 [Fosmid mig10A(+) mig10B(-)], olaEx435 [Fosmid mig10B Swap A], olaEx53 [Pttx3::mig10B::gfp, Pttx3::rab3::mch], olaEx317 [Pttx3::mig10B n-terminus], olaEx734 [Pttx3::mig10B n-terminus, Pttx3::mig10B::gfp], olaEx878 [Pttx3::mig10A n-terminus], olaEx207 [Pttx3::mig10B n-terminus::gfp], olaEx602 [Pmig10b::gfp, Pttx3::rfp], olaEx885 [Pmig10a::gfp, Pttx3::rfp, Ptph1::mch], olaEx889 [Pmig10c::gfp, Pttx3::rfp, Ptph1::mch], vsIs45 [Ptph-1::gfp], olaEx253 [Ptph-1::mig-10A::gfp], olaEx445 [Ptph-1::mig-10B::gfp], olaEx190 [Ptph-1::mig-10C::gfp], wyEx2110 [Punc-86::mig-10A::gfp], wyEx2404 [Punc-86::mig-10B::gfp], wyEx2122 [Punc-86::mig-10C::gfp], olaEx309 [Punc-86::mig-10A], olaEx310 [Punc-86::mig-10A], olaEx214 [Punc-86::mig-10B], olaEx312 [Punc-86::mig-10B], olaEx313 [Punc-86::mig-10B], olaEx294 [Pttx-3::UtrCH::gfp], olaEx1059 [Pttx-3::gfp::snn-1A; Pttx-3::gfp::rab-3], olaEx581 [Pttx-3::ced-5::gfp], olaEx207 [Pttx-3::mig-10B n-terminal helix::gfp].

Detailed subcloning information will be provided upon request. For the MIG-10 isoforms, individual isoforms (A, B and C) were isolated by PCR from cDNA collected from a mixed stage population. To determine MIG-10B expression, the region 6kb upstream of the mig-10b start site was amplified by PCR and cloned upstream of GFP. To determine MIG-10A expression, the region 12kb upstream of the mig-10a start site was amplified by PCR and was transcriptionally fused to GFP. To determine MIG-10C expression, the region 4kb upstream of the mig-10c start site was used for a transcriptional fusion to GFP. For the MIG-10B N-terminal helix used in the MIG-10B dominant negative experiments, we cloned amino acids 1-41 downstream of the ttx-3 promoter.

For MIG-10B:GFP in AIY, we injected the plasmid at varying concentrations ranging from 15 ng/uL to 30 ng/uL. Four lines were examined for each injection condition, (for a total of 14 lines) and we observed the same localization pattern in all transgenic lines and conditions. To examine mutant phenotypes, we used a single transgenic line for crosses (instead of performing new injections directly into mutants and generating new transgenic lines). For MIG-10A:GFP, MIG-10B:GFP and MIG-10C:GFP in NSM, we injected the plasmids at 30 ng/uL and examined three or more lines for each injection. For MIG-10B:GFP in HSN, we injected the plasmid at 0.5 ng/uL and observed the same localization pattern in all transgenic lines. For MIG-10A in AIY, we injected the plasmid at varying concentrations between 15 ng/uL and 30 ng/uL. A total of seven lines were examined, and we observed the same localization pattern in all transgenic lines. For MIG-10A:GFP in HSN, we injected the plasmid at 0.5 ng/uL, examined three lines and observed the same localization pattern in all transgenic lines. For MIG-10C:GFP in AIY, we injected the plasmid at 30 ng/uL, examined three lines and observed the same localization pattern in all transgenic lines. For MIG-10C:GFP in HSN, we injected the plasmid at 0.5 ng/uL, examined five lines and observed the same localization pattern in all transgenic lines.

Supplemental References

Deleage, G., Blanchet, C., and Geourjon, C. (1997). Protein structure prediction. Implications for the biologist. Biochimie 79, 681-686.

Geourjon, C., and Deleage, G. (1995). SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. Comput Appl Biosci 11, 681-684.

Lee, H.S., Lim, C.J., Puzon-McLaughlin, W., Shattil, S.J., and Ginsberg, M.H. (2009). RIAM activates integrins by linking talin to ras GTPase membrane-targeting sequences. J Biol Chem 284, 5119-5127.

Rost, B., Sander, C., and Schneider, R. (1994). PHD--an automatic mail server for protein secondary structure prediction. Comput Appl Biosci 10, 53-60.