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First published online 6 February 2007
doi: 10.1242/jcs.03382

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A large complex containing Patched and Smoothened initiates Hedgehog signaling in Drosophila

¹ Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, USA
² Department of Biomedical Sciences, Florida State University, Tallahassee, FL 32306, USA

View larger version (58K): [in this window] [in a new window]   Fig. 1. A large complex involving Ptc, Smo and Sxl in Drosophila embryos. (A) Ci, Cos2, Ptc, Smo and Sxl immunoprecipitates from wild-type 0- to 12-hour embryonic extracts probed for Sxl, Ptc and Smo. Input lane (I) is 5% of extract used. The table gives the percentage of protein immunoprecipitated from an average of two or more experiments. (B) Fractionation of cytoplasmic extract from wild-type embryos analyzed by western blot using the antibodies in A as well as anti-Fu. Arrows at top show the elution position of the given size marker. Several of the Hh components are phosphorylated (asterisk) and migrate as doublets (Cos2 and Fu); Smo is also phosphorylated and does not migrate as a discrete band. `F' represents total extract from adult females used as a marker for each protein. Three arbitrary complex types of changing Hh components (Complex A-C) can be described. (C) Immunoprecipitation of fractions from each complex type with antibodies to known Hh components show that Ptc and other Hh components, as well as Sxl are associated. (D) Controls for immunoprecipitates of Hh complex components using antibodies to BicD, Dlg and Fz.

View larger version (65K): [in this window] [in a new window]   Fig. 2. Ptc association within the Hh complex responds to activation state of the pathway. Ci, Cos2, Smo and Sxl immunoprecipitated from embryos expressing a different Ptc variant followed by western blot analysis for Sxl, Ptc and Smo. Efficiency of IP is relative to the 5% extract in the input lane (I). (A) Embryos expressing Ptc1130X. (B) Embryos expressing PtcD584N. (C) Embryos overexpressing wild-type Ptc. (D) Embryos expressing PtcLoop2.

View larger version (90K): [in this window] [in a new window]   Fig. 3. Association of Ptc with the Hh complex is independent of Sxl. (A-F) Male salivary gland cells with the SmoD16 deletion allele (and PKAH2). Ci and Ptc colocalize at the plasma membrane and vesicular network (C and F colocalized pixels only). (G) Adult male and female extracts treated with anti-Ci and Cos2 show that Cos2 immunoprecipitates 16% of the Ptc in males, 22% in females; Ci immunoprecipitates 1% of the Ptc in males and 16% in females.

View larger version (101K): [in this window] [in a new window]   Fig. 4. Ptc can alter Sxl subcellular location. (A-F) Embryos expressing different Ptc variants stained for Sxl (green, A-F), and propidium iodide (PI; red) merged with Sxl (A'-F'). (A') Sxl is primarily nuclear in wild-type (wt) embryos. Ptc+ (B') and PtcLoop2 (F') do not completely titrate Sxl out of the nucleus giving a diffuse image; the PI signal is yellow to orange. (C') PtcD584N resembles the wild type and Sxl is more distinctly nuclear. (D',E') Ptc1130X and the carboxyl half of Ptc, PtcC, strongly titrate Sxl out of the nucleus; the PI signal is more red. (G-L) Effects of Ptc variants on Hh signaling in embryos reported by full-length Ci (red) and Wg (blue) expression. Relative to wt (G), Ci levels are increased by the expression of PtcD584N (I) and PtcC (K), decreased by Ptc+ (H) and PtcLoop2 (L). The increase caused by Ptc1130X (J) is very modest. Wg expression is depressed by Ptc+ and PtcLoop2 (H' and L'; arrowheads mark disrupted Wg stripe), elevated by PtcC (K') and modestly elevated by PtcD584N (I'). The feedback between Ci and Wg (Lessing and Nusse, 1998) appears most affected by Ptc1130X and Wg levels are not strongly elevated (J'). This is more evident as the embryos get older and the Wg levels drop. Embryos scanned at similar settings with a 40x objective. Bars, 20 µm.

View larger version (125K): [in this window] [in a new window]   Fig. 5. Colocalization of Ptc and Smo, with Ci and Sxl. Wild-type female and male salivary gland cells stained for Ci and Ptc or Ci and Smo (and Sxl in females) in the presence and absence of Hh. Female glands in the absence of Hh stained for Sxl, Smo and Ci (A-C) or Sxl, Ptc and Ci (G-I). Male glands in the absence of Hh signal stained for Smo and Ci (D,E) or Ptc and Ci (J,K). Female glands expressing Hh stained for Sxl, Smo and Ci (M-O) or Sxl, Ptc and Ci (S-U). Male glands expressing Hh stained for Smo and Ci (P,Q) or Ptc and Ci (V,W). `Col.' panels (A'-C',F,G'-I',L,M'-O',R,S'-U',X) show only the pixels that are common between two proteins. Note the extensive colocalization close to the plasma membrane in most panels.

View larger version (124K): [in this window] [in a new window]   Fig. 6. Colocalization analyses of Hh components when endocytosis is blocked. Salivary gland cells expressing the dominant negative form of Shi, ShiK44A, or Rab5, Rab5SN stained for Ci and Ptc or Ci and Smo (and Sxl in females). Female glands expressing ShiK44A stained for Sxl, Smo and Ci (A-C) or Sxl, Ptc and Ci (G-I). Male glands expressing ShiK44A stained for Smo and Ci (D,E) or Ptc and Ci (J,K). `Col.' panels (A'-C',F,G'-I',L) show only pixels common to the indicated proteins. (M-O,S-U) Female glands expressing Rab5SN stained for Sxl, Smo and Ci (M-O) or Sxl, Ptc and Ci (S-U). `Col.' panels (M'-O',P'-R') show only pixels common to both proteins. All sections are from near the plasma membrane. Percentages of all colocalizations are given in Fig. 7.

View larger version (38K): [in this window] [in a new window]   Fig. 7. Colocalization percentages of Ci and Sxl with Ptc and Smo in both sexes. (A) Colocalization of full-length Ci with the two Hh membrane components, Ptc and Smo (1), as well as Sxl (2), in wild-type salivary glands in the absence (–Hh) and presence of Hh (+Hh) at the plasma membrane (PM) and within the cell. Colocalization of the proteins at the plasma membrane (B) and within the cell (C), in salivary glands of the wild type or animals expressing Shi, Rab5 or Rab7 mutant proteins. Males and females show differences in amounts. Plasma membrane optical sections include region near the membrane. Sections taken within the cell include a cross section of the nucleus. Percentages reflect the average of at least two, usually three separate optical sections.

View larger version (35K): [in this window] [in a new window]   Fig. 8. Ptc and Sxl are endocytosed with Hh signaling components. Ci, Cos2, Smo or Sxl IP of embryo (0- to 12-hour) extracts expressing the dominant negative Shi (ShiK44A), Rab5 (Rab5SN) or gain-of-function Rab7 (Rab7QL) variant probed for Ptc and Sxl. Percentage of Sxl (A) and Ptc (B) immunoprecipitated for each variant compared with wild-type (wt) data of Fig. 1. The average and standard error are the mean of two or more data sets. Error bars represent ±1 s.e. White bars, wild type; light gray bars, Shi; white/black dot bars, Rab5; dark gray bars, Rab7.

View larger version (50K): [in this window] [in a new window]   Fig. 9. Model of endocytosis and Hh signaling. In the absence of Hh Ptc, Smo and the cytoplasmic components are endocytosed. All the components including some of the Ptc, are degraded; Ci is proteolyzed to its repressor form (Ci75). Most of the Ptc recycles to the plasma membrane (broken arrow) permitting Ptc to repeat the cycle and regulate Smo levels in a `catalytic' manner. In the presence of Hh the initial events are similar. Ptc bound to Hh is now sorted for degradation while Smo splits apart, is activated and full-length Ci (Ci155) is generated. Membrane and microtubule association of Cos2 and Fu decreases upon Hh signaling (Stegman et al., 2004) favoring their early release from Smo and vesicles. Activated Smo recycles to the plasma membrane where it activates more Ci; Cos2 and Fu are destabilized (Lum et al., 2003; Ruel et al., 2003), suggesting that once Ci is activated, Cos2 and Fu are degraded. Shi, Rab5 and Rab7 denote where these components function within the endocytic cycle.