QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online January 12, 2006
doi: 10.1242/10.1242/jcs.02743

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Müller, D. Articles by Preiss, A. Search for Related Content PubMed PubMed Citation Articles by Müller, D. Articles by Preiss, A. Social Bookmarking                         What's this?

A molecular link A molecular link between Hairless and Pros26.4, a member of the AAA-ATPase subunits of the proteasome 19S regulatory particle in Drosophila

Universität Hohenheim, Institut für Genetik (240), Garbenstr. 30, 70599 Stuttgart, Germany

View larger version (29K): [in a new window]   Fig. 1. Molecular interactions between Hairless and Pros26.4. (A) Schematic drawing of Hairless deletion constructs. HFL, full-length Hairless (1076 aa), with SBD, Suppressor-of-Hairless-binding domain; GBD, Groucho-binding domain; CBD, binding site of the C-terminal binding protein CtBP. C1, N-terminal truncation (929 aa); C2, deletion of the Su(H)-binding domain (HS; 885 aa); C3, deletion of acidic domain (867 aa); CX, internal deletion within the C-terminal third of the Hairless protein (751 aa); C6, C-terminal truncation of 15 amino acids including the CtBP-binding domain (HC; 1061 aa) [compare with Maier et al. (Maier et al., 1997)]. (B) Quantification of the interaction of Pros26.4 (pJG-S4) with Hairless and its deletion constructs. The quantitative yeast two-hybrid assay shows that the Hairless full-length construct (HFL) as well as all Hairless deletion constructs, with the exception of CX, retain interaction capacity. Control was empty vector (pEG). Values represent Miller units. (C) Drawing of Pros26.4 protein; the conserved AAA-ATPase domain (AAA) is located in the C-terminal half; ATP-bindings sites (`Walker'-motifs A and B, and motif C) are highlighted (Confalonieri and Duguet, 1995). Below, the deletion constructs S4-I to S4-V in pJG are shown. Numbers refer to included codons. (D) Interactions of Pros26.4 deletion constructs and Hairless HFL in pEG were quantified: only S4-I, which contains the N-terminal half but lacks the ATPase domain, retains its binding activity, which is in contrast with any of the other constructs. Control was empty vector (pEG). (E) Co-immunoprecipitation of Pros26.4 and Hairless from embryonic extracts. Protein extracts from Drosophila embryos were taken for immunoprecipitation using anti-Hairless antiserum (IP H). Precipitates were probed with either Hairless antiserum (left) or Pros26.4 antiserum (anti-S4; right). For comparison, total embryonic extracts were loaded in the next lane (in, input about 12% of IP). As control (co), the precipitation was performed with unrelated antiserum. Molecular mass is given in kDa.

View larger version (78K): [in a new window]   Fig. 2. Pros26.4 transcription during development. (A) Maternal mRNA is uniformly distributed in a stage-4 embryo (yo, yolk; pc, pole cells). (B) With the onset of neurogenesis, transcripts start to accumulate in presumptive neuronal cells (arrow; stage 11). (C) Nervous-system-specific accumulation of Pros26.4 mRNA is apparent with germ-band retraction (br, brain; vc, ventral chord). (D) At the time of dorsal closure (stage 14), expression is mainly detected in the ventral chord (vc) and the brain (br). (E) A close-up of the developing head of a stage-12 embryo shows a modulated expression in the brain (br). (F) A dorsal view on an extended germ band embryo shows the modulated expression (arrow) in the nervous system anlage (stage 10). (G) A ventral view on a stage-11 embryo shows accumulation of Pros26.4 mRNA in presumptive neuroblasts (arrow). (H) A dorsal view on a stage-14 embryo highlights the strong mRNA expression in the two brain lobes (br) and also enrichment in the posterior spiracles (ps). Embryos are oriented with anterior left and dorsal up unless otherwise noted. Stages are according to Campos-Ortega and Hartenstein (Campos-Ortega and Hartenstein, 1997). (I) Pros26.4 mRNA transcription levels remain high in proliferating neuroblasts of the ventral chord (arrow) and also in the ring gland (rg) of third instar larvae. (J) In the eye disc (ed) expression is mainly observed in cells anterior to the morphogenetic furrow, whereas in the antennal disc (ad) mRNA distribution is more uniform. (K) In the wing disc, expression is observed all over in a modulated pattern; it is weaker in presumptive vein areas (arrow) and the zone of non-proliferation (asterisk). (L) A likewise modulated expression is observed in the leg disc. (M-P) Expression of Pros28.1, which encodes the -subunit of the catalytic 20S core of the proteasome, is very similar when compared with that of Pros26.4.

View larger version (67K): [in a new window]   Fig. 3. Pros26.4 protein expression. (A-B") Pros26.4 protein expression during oogenesis is shown in red. The nuclei are shown in green by use of a histone H2A-GFP reporter line (A,A" and B, B"). (A,A') In early follicles, Pros26.4 protein is detected uniformly in the cytoplasm of all cell types, the somatic follicle cells (small arrow) as well as the oocyte and nurse cells which both belong to the germ line. In stage 9 (right follicle) and following, conspicuous accumulation of Pros26.4 protein is observed in the germinal vesicle (white arrow). Earlier follicles, for example the one to the left (stage 8), do not reveal this accumulation. Moreover, nurse-cell nuclei are largely devoid of the protein (open arrowheads). (B) A close-up of the germinal vesicle (white arrow) in a stage-10 follicle shows lower levels of Pros26.4 protein. Nurse-cell nuclei (open arrowhead) and follicle-cell nuclei (small open arrow) are largely devoid of Pros26.4 protein. (C-E) Protein expression in embryos is uniform and can be detected in the cytoplasm and also in nuclei starting from blastoderm stage (C, stage 5, arrow indicates pole cells), throughout germ band extension (D, stage 11) and retraction, and also during dorsal closure (E, stage 14). At this later stage, the protein is enriched in the neuromeres of the ventral chord (open arrowheads) and the midgut (white arrow). Nuclear accumulation is mostly apparent in the nuclei of the amnio-serosa (small arrow).

View larger version (133K): [in a new window]   Fig. 4. Disruption of the retina upon depletion of Pros26.4 (A) Scanning-electron micrograph of a wild-type eye highlights the regular array of the ommatidia. (B) A tangential section through a wild-type eye reveals the crystalline-like structure of the retina. In each ommatidium, seven photoreceptor cells are discernible by the centrally located, darkly stained rhabdomeres. (C) Eye imaginal discs from wild-type third instar larvae contain few apoptotic cells as visualized by Acridine Orange staining (bright dots, examples are marked by arrows). (D) Depletion of Pros26.4 within the differentiating eye field by respective overexpression of the dsS4 construct (gmr-Gal4>UAS-dsS4) results in a graded fusion of the ommatidia from posterior (arrowhead) to anterior (left). (E) Underlying is a degeneration of retinula cells as seen in tangential sections of the eye. Whereas the ommatidial array is still visible at the anterior (left half, compare with B), the tissue is completely deranged at the posterior side and ommatidial structure is no longer discernible (arrowhead). (F) Larvae of the same genotype show a dramatic increase in cell death in their eye imaginal discs as visualized with Acridine Orange (bright dots, examples marked by arrows). (G) Only remnants of the head remain after knock-down of Pros26.4 within the developing eye (ey-Gal4>UAS-dsS4): parts of the antenna (an, 3rd antennal segment; ar, arista) are present and a fully developed labrum (lb) with pedipalpi (pd) that arise from different imaginal discs. (H) Cell death can be rescued to almost wild-type pattern by overexpressing baculoviral p35 protein in larval eye discs with reduced Pros26.4 activity (compare with C and F). Posterior is on the right side in all pictures except for G) which shows a frontal view.

View larger version (172K): [in a new window]   Fig. 5. Genetic interactions with Notch, Su(H) and Hairless. (A) Overexpression of GFP in the gmr-pattern results in normal eyes and was used as control (GMR>GFP). Upper panel, side view with posterior at the right; lower panel, top view. (B) RNAi to Pros26.4 causes a posterior to anterior degeneration of the retina visible by glossy appearance and adhering necrotic tissue in the posterior half of the eye (arrow) (GMR>dsS4). (C) Overexpression of Hairless results in smaller eyes with irregular ommatidia (GMR>H). (D) This phenotype is strongly enhanced by reduction of Pros26.4 activity: eyes are much smaller and have an overall glossy appearance due to ommatidial fusion (arrow) (GMR>H, dsS4). (E) Overexpression of Su(H) causes hypertrophy of the eye, which is apparent in the top view [white arrow; GMR>Su(H)]. (F) The overgrowth phenotype remains unchanged by knock-down of Pros26.4, whereas retina degeneration and necrosis, which happens only later in development, is clearly visible [arrow; GMR>Su(H), dsS4]. (G) Overgrowth is even more pronounced when NICD is overexpressed (white arrow; GMR>N). (H) Again, this phenotype remains unchanged by reduction of Pros26.4 activity. Genotypes are (A) gmr-Gal4 / UAS-GFP; (B) gmr-Gal4 / +, UAS-dsS4 / +; (C) gmr-Gal4 UAS-H / UAS-GFP; (D) gmr-Gal4 UAS-H / +, UAS-dsS4 / +; (E) gmr-Gal4 / +, UAS-Su(H) / +; (F) gmr-Gal4 / +, UAS-dsS4 UAS-Su(H) / +; (G) gmr-Gal4 / +, UAS-NICD / TM3 Sb; (H) gmr-Gal4 / +, UAS-dsS4 UAS-NICD / +. Crosses were maintained at 25°C (A-D) and 18°C (E-H).

View larger version (115K): [in a new window]   Fig. 6. Protein stabilization after depletion of Pros26.4. Uniform overexpression of Hairless (red) was induced by a heat shock pulse and detected one hour and 12 hours post-induction, respectively. (A,A') DIAP1 was overexpressed along the antero-posterior boundary of the imaginal disc and detected with anti-DIAP1 antibodies (green). Hairless remains at slightly elevated levels in the DIAP1 expressing cells after 12 hours (arrow). Genotype: UAS-DIAP1 / +, ptc-Gal4/+, hsH / +. (B) The same experiment was performed while depleting Pros26.4 along the antero-posterior boundary by RNAi. Accumulation of Hairless protein along the border is visible already one hour post-induction (arrow). (B') At 12 hours post-induction, stabilization of Hairless is very obvious along the antero-posterior boundary (arrow). Genotype: UAS-DIAP1 / +, ptc-Gal4 / +, UAS-dsS4 hsH / +. (C-D') Su(H) protein expression (red) was induced uniformly by heat shock. (C) A very subtle stabilization along the antero-posterior boundary was observed by DIAP1 overexpression (green) at 1 hour post induction (arrow) which was no longer detectable 12 hours later (C'). (D,D') Simultaneous knock-down of Pros26.4 did not cause any specific Su(H) accumulation. Genotypes: UAS-DIAP1 / +, ptc-Gal4 / +, hsSu(H) / + in C) and UAS-DIAP1 / +, ptc-Gal4 / +, UAS-dsS4 hsSu(H) / + in D. (E–F') Intracellular domain of Notch was induced all over by heat shock. Little effect on the stability of Notch (red) was observed by overexpression of either DIAP1 alone (E,E') or by accompanying knock-down of Pros26.4 (F,F'). Genotypes are UAS-DIAP1 / +, ptc-Gal4 / +, hsNICD / + in E and UAS-DIAP1, ptc-Gal4 / +, UAS-dsS4 hsNICD / + in F. Expression of the respective proteins was detected by immunostaining with DIAP1 antibodies (green) and Hairless (red, A-B'), Su(H) (red, C-D') and Notch (red, E-F'). Arrows point to antero-posterior boundary where dsS4 and/or DIAP1 were overexpressed.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter