_:b432058335 . _:b432058459 . _:b432058499 . _:b432058403 . _:b432058492 . . _:b432058300 . . _:b432058423 . . . _:b7626541 . _:b432058541 . . . _:b432058341 . . . _:b432058363 . . . _:b7626516 . . . _:b7626530 . _:b432058342 . _:b432058447 . . _:b432058419 . _:b432058348 . . . . _:b432058477 . _:b7626532 . . . . _:b432058416 . . _:b432058544 . _:b432058535 . _:b432058500 . _:b432058334 . _:b432058412 . _:b432058413 . _:b432058414 . _:b432058415 . _:b432058408 . _:b432058409 . _:b432058410 . _:b432058411 . . _:b432058404 . _:b432058443 . _:b432058405 . _:b432058406 . _:b432058407 . _:b432058344 . _:b432058400 . _:b432058401 . _:b432058402 . _:b432058403 . _:b432058428 . _:b432058429 . _:b432058430 . _:b432058431 . _:b432058424 . _:b432058519 . _:b432058425 . _:b432058426 . _:b432058427 . _:b432058420 . _:b432058421 . _:b7626525 "Both the in\u00A0vivo genetic evidence described above, along with the observation that Ft cadherin domains can be phosphorylated by Fj (Figure\u00A01K; [>>13<<]), suggest that Fj might also act on Ft." . _:b432058422 . _:b432058423 . _:b432058416 . . _:b432058417 . _:b432058418 . _:b432058419 . _:b432058380 . _:b432058381 . _:b432058382 . _:b432058383 . _:b432058376 . _:b432058377 . _:b432058378 . _:b432058362 . _:b432058379 . _:b432058436 . _:b432058372 . _:b432058373 . _:b432058374 . _:b432058490 . _:b432058375 . _:b432058368 . _:b432058369 . _:b432058391 . _:b432058407 . _:b432058370 . _:b432058371 . _:b432058396 . _:b432058397 . _:b432058398 . _:b432058399 . _:b432058392 . _:b432058393 . _:b7626522 . _:b432058394 . _:b432058395 . . _:b432058388 . _:b432058389 . _:b432058390 . _:b432058391 . _:b432058384 . _:b432058385 . _:b432058386 . _:b432058387 . . _:b432058476 . _:b432058477 . _:b7626521 . _:b432058478 . _:b432058479 . _:b432058472 . _:b432058473 . _:b432058474 . _:b432058475 . _:b432058468 . _:b432058469 . _:b432058470 . _:b432058543 . _:b432058471 . _:b432058464 . _:b432058465 . _:b432058466 . . _:b432058467 . _:b432058492 . _:b432058493 . _:b432058494 . _:b432058495 . _:b432058488 . _:b432058489 . _:b432058490 . _:b432058491 . _:b432058484 . _:b432058485 . . _:b432058486 . _:b432058487 . _:b432058480 . _:b432058481 . _:b432058482 . . _:b432058483 . _:b432058444 . _:b432058445 . _:b432058446 . _:b432058447 . _:b432058440 . _:b432058441 . _:b432058442 . _:b7626532 "And, consistent with this, overexpression of evenly expressed fj, which should tend to flatten a Ft-Ds gradient of activity, decreases wing size [5, 11, 18\u2013>>20<<]. Further, the reduction in wing size we now see in ds\u2212 fj\u2212, Act-ds-EGFP wings is again consistent because Ds-EGFP is, presumably, evenly distributed. Overall, our results are consistent with the hypothesis that graded Fj activity (and" . _:b432058393 . _:b432058444 . _:b432058443 . _:b432058436 . . _:b432058437 . . _:b7626544 "Primary antibodies used for histology were rat anti-Fj [>>12<<], rabbit anti-Ds [6], chicken anti-GFP (Chemicon), mouse monoclonal anti-Fmi (Drosophila Studies Hybridoma Bank) (Usui et\u00A0al." . _:b432058438 . _:b432058439 . _:b432058351 . . _:b432058432 . _:b432058433 . _:b432058508 . . _:b432058513 . _:b432058434 . _:b432058435 . _:b432058460 . _:b432058461 . _:b432058462 . _:b432058463 . _:b432058468 . _:b432058456 . _:b432058457 . . _:b432058458 . _:b432058459 . _:b7626545 . _:b432058452 . _:b432058453 . _:b432058524 . _:b432058454 . _:b432058455 . _:b432058448 . _:b432058336 . _:b432058449 . _:b432058388 . _:b432058450 . . _:b432058451 . . _:b432058540 . _:b432058541 . _:b432058542 . _:b432058543 . . _:b432058536 . _:b432058537 . . _:b432058538 . _:b432058539 . _:b7626549 . _:b432058532 . _:b432058533 . _:b432058534 . _:b432058535 . _:b432058528 . _:b432058529 . _:b432058530 . _:b432058531 . _:b432058401 . . _:b432058548 . _:b432058549 . _:b432058475 . _:b432058550 . . . _:b432058544 . _:b432058545 . _:b432058546 . _:b432058547 . _:b432058508 . _:b432058509 . _:b432058510 . . _:b432058511 . _:b432058354 . _:b432058504 . _:b432058505 . _:b432058506 . _:b432058507 . _:b432058500 . _:b432058501 . _:b432058502 . _:b432058503 . . _:b432058496 . _:b432058497 . _:b432058498 . _:b432058545 . _:b432058499 . . _:b432058524 . _:b432058525 . _:b432058526 . _:b432058527 . _:b432058520 . _:b432058521 . _:b432058522 . _:b432058523 . _:b432058516 . _:b432058517 . _:b432058518 . _:b432058519 . _:b7626534 . _:b432058337 . _:b432058512 . _:b432058297 . _:b432058513 . _:b432058514 . _:b432058515 . . _:b7626529 . . _:b432058480 . . . _:b432058465 . _:b432058369 . . _:b7626540 "The Golgi-tethered forms were made by swapping the appropriate region of the coding sequence into GNT-Fj [>>12<<]." . . _:b432058322 . . _:b432058498 . . _:b7626535 "Fj has recently been found to act in the Golgi [>>12<<] and mediate the phosphorylation of a subset of cadherin repeats of both Ft and Ds [13], but the function in\u00A0vivo of this phosphorylation was not known." . _:b432058548 . _:b432058298 . _:b432058438 . _:b432058381 . _:b7626516 . _:b7626522 . _:b7626521 . . . _:b432058516 . _:b432058529 . . _:b432058507 . . _:b7626521 "Ft and Ds can form heterodimers, intercellular bridges conveying polarity information from cell to cell [4\u2013>>7<<], and we now tested whether phosphorylation of Ds and/or Ft by Fj might regulate this heterodimerization." . _:b432058534 . . . _:b432058301 . _:b432058296 . _:b432058504 . . . _:b432058353 . . _:b7626550 "For western blots, rat or rabbit anti-Fj [>>12<<], mouse anti-GFP (Abcam), mouse anti \u03B1-tubulin DM1A (Sigma), mouse anti-Myc (Santa Cruz), mouse anti-HA (Roche), and secondaries conjugated to horseradish peroxidase (Dako) were used." . . . . _:b7626537 _:b7626538 . _:b7626537 _:b7626539 . _:b7626537 _:b7626540 . _:b7626537 _:b7626541 . _:b7626539 "fj constructs were expressed under upstream activating sequence (UAS) control [>>23<<] in transgenic flies, under the copper-inducible metallothionein promoter in the vector pMK33B for tissue culture experiments, and in the pENTR vector for the coimmunoprecipitation experiments in which they were additionally Myc tagged." . _:b7626537 _:b7626542 . _:b7626537 _:b7626543 . _:b7626537 _:b7626544 . _:b7626537 _:b7626545 . _:b432058458 . _:b7626537 _:b7626546 . _:b7626537 _:b7626547 . _:b7626537 _:b7626548 . _:b7626537 _:b7626549 . _:b7626537 _:b7626550 . _:b432058380 . _:b432058523 . _:b432058527 . _:b432058404 . . _:b7626519 . _:b7626515 _:b7626536 . . "10.1016%2Fj.cub.2010.03.056" . _:b432058503 . _:b7626515 _:b7626528 . _:b7626515 _:b7626529 . _:b7626515 _:b7626530 . . _:b7626515 _:b7626531 . _:b7626515 _:b7626532 . _:b7626515 _:b7626533 . _:b7626515 _:b7626534 . _:b7626515 _:b7626535 . . . . . _:b432058532 . . . _:b432058505 . _:b7626543 . _:b7626515 _:b7626520 . _:b432058355 . _:b7626515 _:b7626521 . _:b432058449 . _:b7626515 _:b7626522 . _:b7626515 _:b7626523 . _:b7626515 _:b7626524 . _:b7626515 _:b7626525 . _:b7626515 _:b7626526 . _:b7626515 _:b7626527 . . _:b7626515 _:b7626516 . _:b7626515 _:b7626517 . _:b7626515 _:b7626518 . _:b7626515 _:b7626519 . . . . _:b432058474 . _:b432058455 . . . _:b432058533 . _:b432058432 . . _:b432058521 . . _:b432058396 . _:b432058502 . _:b432058424 . . _:b7626533 . . _:b7626532 . . _:b432058299 . . _:b7626531 . . _:b7626530 . _:b432058375 . . _:b432058473 . . . _:b432058441 . . . . _:b7626542 "ds-EGFP and point mutants were subcloned into pAttB-Act-FRT-polyA-FRT (derived from pAct-FRT-polyA-FRT [>>25<<]), and transgenes were integrated into the same landing site (VK26 at 96F3 [26]) by BestGene." . _:b432058461 . . . . _:b432058293 . . . _:b432058517 . . _:b7626516 . _:b432058418 . . . . _:b432058303 . _:b432058463 . _:b432058373 . _:b432058359 . _:b432058440 . . . . . _:b432058309 . . _:b432058400 . _:b432058397 . . _:b432058497 . . _:b432058302 . _:b432058338 . _:b432058292 . _:b432058547 . . _:b7626516 . . . . . _:b432058408 . . _:b432058389 . _:b432058484 . . _:b432058454 . _:b432058326 . . . _:b7626538 . _:b7626528 . . . _:b432058290 "22"^^ . _:b7626516 . _:b7626534 . . . _:b7626534 . _:b7626536 . _:b432058291 "21"^^ . _:b432058373 . . _:b7626519 "The region shows homology to a kinase-active site [>>17<<] containing the essential aspartate residues required for kinase function, as well as other conserved residues nearby." . _:b432058293 "19"^^ . . _:b432058372 . _:b432058375 . _:b432058292 "20"^^ . _:b432058374 . _:b432058294 "17"^^ . _:b432058369 . _:b432058295 "16"^^ . _:b432058368 . _:b432058296 "16"^^ . _:b432058303 "13"^^ . _:b432058540 . _:b432058371 . . _:b432058297 "16"^^ . _:b432058302 "13"^^ . _:b7626532 . _:b432058370 . _:b432058298 "16"^^ . _:b432058299 "14"^^ . _:b7626521 . _:b432058431 . _:b432058381 . _:b432058300 "14"^^ . _:b432058380 . . _:b432058301 "14"^^ . _:b432058420 . . _:b432058383 . _:b432058331 . . _:b432058319 . _:b432058382 . _:b7626517 . _:b7626516 . _:b432058377 . _:b7626516 . _:b432058317 . _:b432058376 . . _:b432058460 . _:b432058310 "10"^^ . . _:b432058379 . _:b432058311 "9"^^ . _:b432058309 "10"^^ . _:b7626518 . _:b432058378 . _:b432058308 "11"^^ . _:b432058389 . _:b432058307 "11"^^ . _:b432058388 . _:b432058476 . _:b432058538 . _:b432058391 . _:b432058306 "12"^^ . _:b432058390 . _:b432058305 "12"^^ . . _:b432058385 . _:b432058304 "13"^^ . _:b432058384 . _:b432058318 "9"^^ . _:b432058387 . _:b432058319 "8"^^ . _:b7626515 "results and discussion" . _:b432058386 . _:b432058316 "9"^^ . . _:b7626547 . _:b432058397 . _:b432058317 "9"^^ . _:b432058396 . _:b432058314 "9"^^ . . _:b432058399 . _:b432058330 . _:b432058315 "9"^^ . _:b432058398 . _:b432058312 "9"^^ . _:b432058494 . _:b432058482 . _:b432058393 . _:b432058313 "9"^^ . _:b432058392 . _:b432058326 "8"^^ . _:b432058395 . _:b432058327 "8"^^ . . _:b432058394 . _:b432058324 "8"^^ . _:b432058546 . _:b432058325 "8"^^ . _:b432058405 . _:b432058404 . _:b432058322 "8"^^ . _:b432058407 . _:b432058323 "8"^^ . _:b432058368 . _:b432058429 . _:b432058320 "8"^^ . _:b432058406 . _:b432058401 . _:b432058321 "8"^^ . _:b432058522 . _:b432058549 . _:b432058334 "7"^^ . _:b432058400 . _:b432058335 "7"^^ . _:b432058403 . _:b432058312 . _:b432058332 "7"^^ . _:b432058402 . _:b432058333 "7"^^ . _:b432058413 . . . _:b432058330 "8"^^ . _:b432058412 . _:b7626525 . _:b432058331 "8"^^ . _:b432058415 . _:b432058371 . _:b432058328 "8"^^ . _:b432058414 . _:b7626524 "The GNT-Fj form, which is not secreted from cells, was at least as potent as wild-type Fj in inhibiting Ds binding to Ft (Figure\u00A02D), consistent with Fj mediating its effect in the Golgi [>>12<<], and not at the cell surface." . _:b432058412 . _:b7626527 . _:b432058329 "8"^^ . . _:b432058409 . _:b432058342 "7"^^ . _:b7626526 . _:b432058408 . _:b432058343 "6"^^ . . _:b432058411 . _:b432058340 "7"^^ . _:b7626520 . _:b432058410 . _:b432058341 "7"^^ . _:b432058421 . _:b432058338 "7"^^ . _:b432058420 . _:b432058356 . _:b432058339 "7"^^ . _:b432058423 . _:b432058336 "7"^^ . _:b432058422 . _:b432058337 "7"^^ . . _:b432058417 . _:b432058350 "6"^^ . . _:b432058416 . . _:b432058351 "6"^^ . . _:b432058419 . _:b432058405 . _:b432058348 "6"^^ . _:b432058418 . _:b432058349 "6"^^ . _:b432058429 . . _:b432058512 . _:b432058346 "6"^^ . _:b432058428 . . . _:b432058347 "6"^^ . _:b432058471 . _:b432058431 . _:b432058344 "6"^^ . _:b432058430 . _:b432058345 "6"^^ . _:b432058425 . . . . _:b432058424 . . _:b432058357 "5"^^ . _:b432058442 . _:b432058427 . . _:b432058356 "6"^^ . . _:b432058426 . _:b432058359 "5"^^ . _:b432058515 . _:b432058437 . _:b432058358 "5"^^ . _:b432058314 . _:b432058354 "6"^^ . _:b432058528 . _:b432058436 . . _:b432058355 "6"^^ . . _:b432058439 . _:b432058352 "6"^^ . _:b432058438 . _:b7626545 "Primary antibodies used for histology were rat anti-Fj [12], rabbit anti-Ds [>>6<<], chicken anti-GFP (Chemicon), mouse monoclonal anti-Fmi (Drosophila Studies Hybridoma Bank) (Usui et\u00A0al." . _:b432058353 "6"^^ . _:b432058325 . _:b432058433 . _:b432058432 . _:b432058365 "5"^^ . . _:b432058435 . _:b432058364 "5"^^ . . _:b432058434 . _:b432058367 "5"^^ . . . _:b432058445 . _:b432058366 "5"^^ . _:b432058444 . _:b432058361 "5"^^ . . _:b432058447 . _:b432058360 "5"^^ . . _:b432058446 . _:b432058363 "5"^^ . _:b432058373 "4"^^ . . _:b432058441 . . _:b432058362 "5"^^ . _:b432058372 "4"^^ . _:b432058440 . _:b432058483 . _:b432058375 "4"^^ . _:b432058443 . _:b432058374 "4"^^ . . _:b432058442 . . _:b432058369 "5"^^ . _:b432058430 . _:b432058453 . _:b432058368 "5"^^ . _:b432058452 . _:b432058371 "5"^^ . _:b432058455 . _:b432058370 "5"^^ . _:b432058439 . _:b432058454 . _:b432058381 "4"^^ . _:b432058345 . _:b432058449 . . _:b432058380 "4"^^ . _:b432058448 . _:b432058434 . . _:b432058383 "4"^^ . _:b7626520 "this domain comprises amino acids 432 to 508. The region shows homology to a kinase-active site [17] containing the essential aspartate residues required for kinase function, as well as other conserved residues nearby. Ishikawa et\u00A0al. [>>13<<] mutated amino acids 490\u2013492 of Fj, which includes the putative Mn2+-binding site (D490), and found that this protein could no longer phosphorylate Ft or Ds cadherin domains and was nonfunctional in\u00A0vivo." . _:b432058451 . _:b432058382 "4"^^ . _:b432058383 . _:b432058450 . _:b432058377 "4"^^ . . _:b432058402 . . _:b432058461 . _:b432058376 "4"^^ . . . _:b432058460 . _:b432058379 "4"^^ . _:b432058463 . _:b432058479 . _:b432058378 "4"^^ . _:b432058462 . _:b432058501 . _:b432058389 "4"^^ . _:b432058394 . . _:b432058457 . _:b432058388 "4"^^ . . _:b432058456 . _:b432058391 "4"^^ . _:b432058472 . _:b7626527 "A secreted form of HA-tagged Ds that contains the first five cadherin domains (Ds1-5sec), including amino acid S236, which is phosphorylated by Fj [>>13<<], was expressed in cells in the presence of either Golgi-tethered GNT-fj or (as controls) forms of GNT-fj mutated in the kinase domain." . _:b432058411 . _:b432058459 . _:b432058304 . _:b432058390 "4"^^ . _:b432058414 . _:b432058458 . . _:b432058462 . _:b432058385 "4"^^ . _:b432058451 . . _:b432058469 . _:b432058384 "4"^^ . _:b432058468 . _:b432058387 "4"^^ . _:b7626543 "ds-EGFP and point mutants were subcloned into pAttB-Act-FRT-polyA-FRT (derived from pAct-FRT-polyA-FRT [25]), and transgenes were integrated into the same landing site (VK26 at 96F3 [>>26<<]) by BestGene. The following genotypes were used:" . _:b432058471 . _:b432058386 "4"^^ . . _:b7626533 . _:b432058470 . _:b432058397 "4"^^ . _:b432058465 . _:b7626536 "Fj has recently been found to act in the Golgi [12] and mediate the phosphorylation of a subset of cadherin repeats of both Ft and Ds [>>13<<], but the function in\u00A0vivo of this phosphorylation was not known." . _:b7626532 . _:b432058537 . _:b432058396 "4"^^ . . _:b432058464 . _:b432058399 "4"^^ . _:b432058467 . . _:b432058398 "4"^^ . _:b432058466 . _:b7626523 "We cotransfected Drosophila S2 cells with fj or GNT-fj, a Golgi-tethered form of the protein that is more active in\u00A0vivo [>>12<<], plus either ft or ds." . _:b432058393 "4"^^ . _:b7626531 "Recent data suggest that steeply graded Fj distribution and, consequently, steeply graded Ft-Ds activity promote growth [3, 18, >>20<<]. And, consistent with this, overexpression of evenly expressed fj, which should tend to flatten a Ft-Ds gradient of activity, decreases wing size [5, 11, 18\u201320]." . _:b432058477 . _:b432058392 "4"^^ . . _:b432058476 . _:b7626531 . _:b432058395 "4"^^ . _:b432058488 . . _:b7626530 . _:b432058479 . _:b432058394 "4"^^ . _:b432058478 . . _:b432058405 "4"^^ . _:b432058473 . . _:b432058404 "4"^^ . _:b432058427 . . . _:b432058407 "3"^^ . _:b432058472 . _:b432058475 . _:b432058406 "4"^^ . . _:b432058474 . _:b432058401 "4"^^ . _:b432058485 . _:b432058400 "4"^^ . . _:b432058484 . _:b432058399 . _:b432058403 "4"^^ . . _:b432058487 . . _:b432058402 "4"^^ . _:b432058413 "3"^^ . _:b432058486 . _:b432058412 "3"^^ . _:b432058481 . _:b7626546 "(PBS) 0.1% Triton X-100 prior to immunolabeling. Primary antibodies used for histology were rat anti-Fj [12], rabbit anti-Ds [6], chicken anti-GFP (Chemicon), mouse monoclonal anti-Fmi (Drosophila Studies Hybridoma Bank) (Usui et\u00A0al. [>>28<<]), and a rat serum against the intracellular domain of Ft, which was generated with a His-tagged fusion protein corresponding to amino acids 4665\u20134859." . _:b432058415 "3"^^ . _:b432058480 . . _:b432058414 "3"^^ . _:b432058483 . _:b432058409 "3"^^ . _:b432058482 . . _:b432058408 "3"^^ . _:b432058493 . _:b432058411 "3"^^ . _:b432058492 . _:b432058410 "3"^^ . _:b432058495 . _:b432058421 "3"^^ . _:b432058494 . . _:b432058420 "3"^^ . _:b432058489 . _:b432058423 "3"^^ . _:b432058488 . . . . _:b432058422 "3"^^ . _:b432058491 . _:b432058417 "3"^^ . _:b432058490 . . _:b432058501 . _:b432058416 "3"^^ . _:b432058360 . _:b432058419 "3"^^ . _:b432058500 . _:b432058418 "3"^^ . _:b432058503 . . _:b432058429 "3"^^ . _:b432058502 . . _:b432058428 "3"^^ . _:b7626538 "regions were verified by sequencing. ds and ft constructs were expressed under control of the Act5C promoter in both tissue culture experiments and transgenic flies. Full-length ds was created from ds cDNA fragments (gift from M. Noll) [>>22<<]. The untagged form lacks the final 303 aa because of a premature stop codon in the original cDNA, which was corrected in the Ds-EGFP form." . _:b432058497 . _:b432058431 "3"^^ . _:b432058496 . _:b432058430 "3"^^ . _:b432058499 . . _:b432058425 "3"^^ . _:b432058498 . _:b432058518 . _:b432058424 "3"^^ . _:b432058509 . . _:b432058398 . _:b432058508 . _:b432058427 "3"^^ . _:b432058457 . . _:b432058511 . _:b432058426 "3"^^ . _:b432058437 "3"^^ . _:b432058510 . . _:b432058406 . . _:b432058505 . _:b432058436 "3"^^ . _:b432058439 "3"^^ . _:b432058504 . _:b432058327 . _:b432058507 . _:b432058340 . _:b432058438 "3"^^ . . . _:b432058433 "3"^^ . _:b432058506 . _:b432058432 "3"^^ . _:b432058517 . _:b432058435 "3"^^ . _:b432058516 . . . _:b432058519 . _:b432058434 "3"^^ . _:b432058445 "3"^^ . _:b432058518 . . _:b432058513 . _:b432058444 "3"^^ . . _:b432058415 . . _:b432058512 . _:b432058447 "3"^^ . . _:b432058386 . _:b432058446 "3"^^ . _:b432058515 . . _:b432058332 . _:b432058370 . _:b432058441 "3"^^ . _:b432058514 . _:b7626518 "fj clones do not repolarize the surrounding wild-type hairs [>>7<<], arguing that Fj acts by changing the activity of Ds and Ft inside the clones\u2014and not by acting (for example, as a secreted protein) directly on Ds and/or Ft in neighboring wild-type cells." . _:b432058313 . _:b432058525 . _:b432058440 "3"^^ . _:b432058443 "3"^^ . _:b432058524 . _:b432058324 . _:b432058527 . _:b432058442 "3"^^ . _:b432058526 . _:b432058453 "3"^^ . _:b432058521 . _:b432058452 "3"^^ . _:b432058520 . _:b432058455 "3"^^ . _:b432058523 . _:b432058454 "3"^^ . _:b432058522 . _:b432058449 "3"^^ . _:b432058533 . _:b432058448 "3"^^ . _:b432058532 . _:b432058451 "3"^^ . _:b432058535 . _:b432058450 "3"^^ . _:b432058291 . . _:b432058478 . _:b432058461 "2"^^ . _:b432058534 . _:b432058460 "2"^^ . _:b432058529 . . _:b432058435 . _:b432058463 "2"^^ . _:b432058528 . . _:b432058462 "2"^^ . _:b432058531 . _:b432058457 "2"^^ . _:b432058530 . _:b432058469 . _:b432058541 . . _:b432058456 "3"^^ . _:b432058525 . _:b432058459 "2"^^ . _:b432058540 . _:b432058486 . _:b432058536 . _:b432058458 "2"^^ . _:b432058543 . _:b432058469 "2"^^ . _:b432058542 . _:b432058468 "2"^^ . _:b432058537 . _:b432058471 "2"^^ . _:b432058536 . . _:b432058470 "2"^^ . _:b432058539 . . _:b432058465 "2"^^ . _:b432058538 . _:b7626529 "To assay the activity of the transgenes, we looked at their effects on wing size, a parameter modulated by Fj, Ds, and Ft through their regulation of the Warts/Hippo pathway [1, >>3<<]. Loss of ds activity did not much alter wing area (Figure\u00A04F) but did affect wing shape (Figure\u00A04B)." . _:b432058433 . _:b432058378 . _:b432058464 "2"^^ . _:b432058549 . . _:b432058467 "2"^^ . _:b432058548 . _:b432058382 . _:b432058466 "2"^^ . _:b432058477 "2"^^ . _:b432058550 . _:b7626537 "experimental procedures" . _:b432058476 "2"^^ . _:b432058545 . . . _:b432058479 "2"^^ . _:b432058544 . _:b432058478 "2"^^ . _:b432058547 . _:b432058473 "2"^^ . . _:b432058546 . _:b432058472 "2"^^ . _:b432058475 "2"^^ . _:b432058366 . _:b432058474 "2"^^ . . _:b432058485 "2"^^ . . _:b432058484 "2"^^ . _:b432058487 "2"^^ . _:b7626530 "However, uniform Ds expression reduces the area of the wing [5, 18\u2013>>20<<], which we also see if we express Act-ds-EGFP uniformly in a ds background (Figures 4C and 4F)." . _:b432058445 . _:b432058486 "2"^^ . . _:b432058510 . _:b432058481 "2"^^ . _:b432058487 . _:b432058448 . _:b432058480 "2"^^ . _:b432058483 "2"^^ . . _:b432058377 . _:b432058539 . _:b432058482 "2"^^ . _:b432058493 "2"^^ . _:b432058421 . _:b432058492 "2"^^ . _:b432058495 "2"^^ . . _:b432058494 "2"^^ . . _:b432058372 . _:b432058489 "2"^^ . _:b432058488 "2"^^ . . . _:b432058395 . _:b432058491 "2"^^ . _:b432058300 . _:b432058490 "2"^^ . _:b432058301 . _:b432058321 . _:b432058302 . _:b432058303 . _:b432058339 . _:b432058501 "2"^^ . _:b432058296 . _:b432058297 . _:b432058298 . _:b432058299 . _:b432058292 . _:b432058500 "2"^^ . _:b432058293 . _:b432058294 . _:b432058295 . _:b432058503 "2"^^ . _:b432058290 . _:b432058514 . _:b432058291 . _:b432058502 "2"^^ . _:b7626533 "We note that DsS>Dx3 might be expected to mimic the effect of uniform Fj on the wing, which reduces wing size [18, >>20<<], and\u00A0indeed, ds\u2212, dsS>Dx3-EGFP wings are smaller than ds\u2212, ds-EGFP wings (Figures 4C, 4E, and 4F)." . _:b432058497 "2"^^ . _:b432058496 "2"^^ . _:b432058307 . _:b432058499 "2"^^ . _:b432058498 "2"^^ . _:b432058323 . _:b432058452 . _:b432058509 "2"^^ . _:b432058508 "2"^^ . _:b432058470 . _:b432058511 "2"^^ . . _:b432058510 "2"^^ . _:b432058348 . _:b432058349 . _:b432058350 . _:b432058351 . _:b432058505 "2"^^ . _:b432058344 . _:b432058345 . _:b432058346 . _:b432058347 . _:b432058504 "2"^^ . _:b432058340 . _:b432058341 . _:b432058342 . _:b432058343 . _:b432058507 "2"^^ . _:b432058336 . _:b432058337 . _:b432058338 . _:b432058339 . _:b432058506 "2"^^ . _:b432058364 . _:b432058365 . _:b432058366 . _:b432058367 . _:b432058517 "2"^^ . _:b432058360 . _:b432058361 . _:b432058362 . _:b432058363 . _:b432058516 "2"^^ . _:b432058356 . _:b432058357 . _:b432058358 . _:b432058387 . _:b432058359 . _:b432058519 "2"^^ . _:b432058352 . _:b432058353 . _:b432058354 . _:b432058355 . _:b432058518 "2"^^ . _:b432058316 . . _:b432058317 . _:b432058318 . _:b432058550 . _:b432058319 . _:b432058513 "2"^^ . _:b432058312 . _:b432058313 . _:b432058314 . _:b432058315 . _:b432058512 "2"^^ . _:b432058308 . _:b432058410 . _:b432058530 . _:b432058309 . _:b432058310 . _:b432058361 . _:b432058311 . _:b432058515 "2"^^ . _:b432058293 . _:b432058304 . _:b432058305 . _:b432058428 . _:b432058306 . _:b432058307 . . _:b432058292 . _:b432058332 . _:b432058514 "2"^^ . _:b432058333 . _:b432058334 . _:b432058335 . _:b432058525 "2"^^ . _:b432058295 . _:b432058328 . . _:b432058329 . . _:b432058330 . _:b432058331 . _:b432058524 "2"^^ . _:b432058294 . _:b432058324 . _:b432058325 . . _:b432058326 . _:b432058327 . _:b432058527 "2"^^ . _:b432058320 . _:b432058321 . _:b432058322 . _:b432058323 . _:b432058526 "2"^^ . . . _:b432058521 "2"^^ . _:b432058291 . _:b432058520 "2"^^ . _:b432058290 . _:b432058437 . _:b432058523 "2"^^ . _:b432058301 . _:b432058467 . . _:b432058531 . _:b432058522 "2"^^ . _:b432058300 . . . _:b432058533 "2"^^ . _:b432058303 . . _:b432058532 "2"^^ . _:b432058302 . _:b7626537 . _:b7626542 . _:b432058535 "2"^^ . _:b432058297 . . . _:b432058534 "2"^^ . _:b432058296 . _:b432058493 . _:b432058529 "2"^^ . _:b432058299 . _:b432058528 "2"^^ . _:b432058298 . _:b432058531 "2"^^ . _:b432058309 . _:b7626549 "To stain for Ds specifically at the cell surface, we immunolabeled cells expressing Ds-EGFP in the absence of detergent with an anti-Ds primary antibody raised against the extracellular domain of the protein [>>6<<] followed by an RRX-conjugated secondary antibody. Cells were then labeled with anti-GFP and secondary antibodies in the presence of detergent to label total Ds." . _:b432058530 "2"^^ . _:b432058308 . _:b7626515 . _:b432058541 "2"^^ . _:b432058311 . _:b432058367 . _:b7626539 . _:b7626546 . _:b432058540 "2"^^ . _:b432058310 . . _:b432058543 "2"^^ . _:b432058305 . . _:b432058506 . _:b432058328 . _:b432058542 "2"^^ . _:b432058304 . _:b432058305 . _:b432058537 "2"^^ . _:b432058307 . _:b432058536 "2"^^ . _:b432058306 . _:b432058539 "2"^^ . _:b432058317 . _:b7626548 . _:b432058538 "2"^^ . _:b432058316 . . _:b432058549 "2"^^ . _:b432058319 . . _:b432058548 "2"^^ . _:b432058318 . . . _:b432058313 . _:b432058550 "2"^^ . _:b432058312 . _:b432058290 . _:b432058545 "2"^^ . _:b432058315 . . _:b7626534 . _:b432058544 "2"^^ . _:b432058314 . _:b432058320 . _:b432058325 . _:b432058374 . _:b432058547 "2"^^ . _:b432058546 "2"^^ . _:b432058324 . . _:b432058456 . _:b432058327 . _:b432058326 . . _:b432058321 . . _:b432058320 . _:b432058323 . _:b7626541 "Alleles used are described in FlyBase [>>24<<]. Loss-of-function clones that simultaneously express a protein under UAS control were generated by heat shocking third instar larvae for 1 hr at 37\u00B0C." . _:b7626524 . _:b432058322 . _:b432058426 . _:b432058333 . _:b7626526 "Because Fj is known to phosphorylate Ds cadherin domains (Figure\u00A01J; [>>13<<]), its effects are most likely implemented via changes in the mutual binding affinity of Ft and Ds." . _:b432058332 . _:b432058346 . _:b7626523 . . _:b432058335 . _:b432058379 . _:b432058334 . _:b432058376 . . _:b432058329 . _:b7626528 "Ishikawa et\u00A0al. [>>13<<] identified three conserved serines (S236, S561, and S881) in Ds cadherin domains that can be phosphorylated by Fj in\u00A0vitro or in cell-based assays (Figure\u00A03A)." . _:b432058328 . _:b432058466 . _:b432058331 . _:b432058330 . . _:b432058341 . _:b432058340 . . _:b432058520 . _:b432058343 . _:b7626522 . _:b432058342 . _:b7626534 "fj was first shown to interact with ds by Waddington [21], and subsequent studies showed that it regulates the functions of ds and ft [4, 6\u20139, >>12<<]. Fj has recently been found to act in the Golgi [12] and mediate the phosphorylation of a subset of cadherin repeats of both Ft and Ds [13], but the function in\u00A0vivo of this phosphorylation was not known." . _:b7626521 . _:b432058337 . _:b432058511 . . . _:b432058336 . _:b432058409 . . _:b432058339 . _:b432058357 . _:b432058338 . _:b432058349 . _:b432058464 . _:b432058348 . _:b7626517 "ectoDs), an active form of Ds lacking the intracellular domain [>>7<<]. These clones reversed the polarity of hairs behind the clone (Figure\u00A01A), but coexpression of UAS." . _:b432058351 . _:b432058350 . _:b432058345 . _:b7626550 . _:b432058344 . _:b432058347 . _:b432058346 . _:b432058357 . _:b432058496 . _:b432058392 . . _:b432058356 . "PMC0" . _:b432058359 . . _:b7626544 . _:b432058358 . _:b432058481 . _:b432058294 . . _:b432058353 . . _:b7626516 "Extensive use of this experimental assay has given insight into how these three molecules act to build planar polarity [6\u20139, 14\u2013>>16<<], but it has not been clear whether Fj acts on Ds, on Ft, or on both molecules." . _:b432058352 . _:b432058453 . _:b432058355 . _:b432058354 . . _:b432058364 . _:b432058365 . _:b432058491 . _:b432058318 . _:b432058364 . _:b432058367 . _:b432058366 . . _:b432058361 . _:b7626534 . _:b432058360 . _:b432058295 . _:b7626535 . _:b432058363 . _:b432058362 . _:b432058413 . _:b432058316 . _:b432058343 . _:b7626531 . _:b7626529 . _:b432058422 . _:b7626540 . . . _:b432058489 . . . . _:b432058333 . . _:b432058495 . . _:b432058315 . _:b7626516 . . _:b7626517 . _:b432058542 . _:b7626530 . _:b7626518 . _:b7626519 . _:b7626532 . _:b432058329 . . _:b432058446 . _:b7626548 "FLAG-tagged Flamingo (Fmi) [27], a cadherin-containing protein that will bind homophilically [>>28<<], was used as a control in cell aggregation assays." . _:b7626515 . _:b7626524 . _:b432058526 . _:b7626525 . . _:b7626526 . _:b432058308 . _:b7626527 . _:b432058306 . _:b7626520 . _:b432058311 . _:b7626521 . . . . _:b7626522 . _:b432058384 . _:b432058310 . _:b7626523 . . _:b7626532 . _:b7626533 . _:b7626522 "When Drosophila S2 cells were separately transfected with Ft and Ds and then mixed together, they formed cell aggregates; Ds and Ft appeared to stabilize each other's localization at the cell membrane (Figure\u00A02A) [4\u2013>>6<<]. We cotransfected Drosophila S2 cells with fj or GNT-fj, a Golgi-tethered form of the protein that is more active in\u00A0vivo [12], plus either ft or ds. fj was expressed under the control of a Cu2+-inducible promoter, whereas ft and ds were" . _:b7626534 . . _:b432058349 . _:b7626535 . _:b7626528 . _:b432058450 . _:b7626534 . _:b7626529 . _:b7626530 . _:b432058485 . _:b7626531 . _:b7626540 . . _:b7626541 . . . _:b7626542 . . _:b432058417 . _:b432058385 . _:b7626543 . _:b7626536 . . _:b432058365 . _:b7626537 . _:b7626538 . _:b7626539 . _:b7626548 . _:b432058390 . _:b7626549 . _:b432058509 . _:b7626550 . _:b7626547 "FLAG-tagged Flamingo (Fmi) [>>27<<], a cadherin-containing protein that will bind homophilically [28], was used as a control in cell aggregation assays." . _:b7626544 . _:b432058358 . . _:b7626545 . _:b432058350 . . _:b7626546 . _:b7626547 . . . _:b432058352 . _:b432058347 . . . . _:b432058425 . . . . . .