Proteasome, but not autophagy, disruption results in severe eye and wing dysmorphia: a subunit- and regulator-dependent process in Drosophila.

Proteasome-dependent and autophagy-mediated degradation of eukaryotic cellular proteins represent the two major proteostatic mechanisms that are critically implicated in a number of signaling pathways and cellular processes. Deregulation of functions engaged in protein elimination frequently leads to development of morbid states and diseases. In this context, and through the utilization of GAL4/UAS genetic tool, we herein examined the in vivo contribution of proteasome and autophagy systems in Drosophila eye and wing morphogenesis. By exploiting the ability of GAL4-ninaE. GMR and P{GawB}Bx(MS1096) genetic drivers to be strongly and preferentially expressed in the eye and wing discs, respectively, we proved that proteasomal integrity and ubiquitination proficiency essentially control fly's eye and wing development. Indeed, subunit- and regulator-specific patterns of severe organ dysmorphia were obtained after the RNAi-induced downregulation of critical proteasome components (Rpn1, Rpn2, α5, β5 and β6) or distinct protein-ubiquitin conjugators (UbcD6, but not UbcD1 and UbcD4). Proteasome deficient eyes presented with either rough phenotypes or strongly dysmorphic shapes, while transgenic mutant wings were severely folded and carried blistered structures together with loss of vein differentiation. Moreover, transgenic fly eyes overexpressing the UBP2-yeast deubiquitinase enzyme were characterized by an eyeless-like phenotype. Therefore, the proteasome/ubiquitin proteolytic activities are undoubtedly required for the normal course of eye and wing development. In contrast, the RNAi-mediated downregulation of critical Atg (1, 4, 7, 9 and 18) autophagic proteins revealed their non-essential, or redundant, functional roles in Drosophila eye and wing formation under physiological growth conditions, since their reduced expression levels could only marginally disturb wing's, but not eye's, morphogenetic organization and architecture. However, Atg9 proved indispensable for the maintenance of structural integrity of adult wings in aged flies. In toto, our findings clearly demonstrate the gene-specific fundamental contribution of proteasome, but not autophagy, in invertebrate eye and wing organ development

RNAi-mediated disruption of the 19S proteasome regulatory cap results in highly dysmorphic fly wings.

<p>Stereo-microscopical (a, c, e and g) and Optical (b, d, f and h) images, demonstrating severely dysmorphic wing phenotypes obtained under Rpn1, 2 and 6 deficient cellular conditions. (a and b) P{GawB}Bx^MS1096 single transgenic adult fly wings (utilized as control organ-tissues), structured by a marginal vein encompassing the length of the anterior wing margin, four longitudinal veins (L2–L5), two incomplete longitudinal veins (L1 and L6) and two transverse veins (cv-a {anterior cross-vein} and cv-p {posterior cross-vein}). (c and d) P{GawB}Bx^MS1096/UAS-Rpn1_RNAi double transgenic fly wings, characterized by reduced expression of Rpn1 19S proteasome subunit. (e and f) P{GawB}Bx^MS1096/UAS-Rpn2_RNAi double transgenic fly wings, carrying downregulated Rpn2 protein levels. (g and h) P{GawB}Bx^MS1096/UAS-Rpn6_RNAi double transgenic flies, producing wings with decreased Rpn6 cellular contents. Scale Bars: 200 µm.</p

The structural integrity of 20S proteasome core particle is critically implicated in Drosophila eye morphogenesis.

<p>Stereo-microscopical (a, e and i), Optical (d, h and l {semi-thin sections}) and Scanning Electron Microscopy (SEM) (b, c, f, g, j and k) images, revealing the damaged architectural pattern of fly eye in 20S proteasome deficient environments (arrow). (a–d) GAL4-ninaE.GMR/UAS-alpha5_RNAi double transgenic fly eyes, carrying reduced α5 protein levels. (e–h) GAL4-ninaE.GMR/UAS-dbeta5_RNAi double transgenic fly eyes, characterized by downregulated β5 proteasome core protein expression levels. (i–l) GAL4-ninaE.GMR/UAS-beta6_RNAi double transgenic flies, producing eyes with decreased β6 cellular contents. Dashed circle: ommatidium. Scale Bars: 50 µm.</p

Downregulation of autophagy is associated with mild Drosophila wing lesions.

<p>Stereo-microscopical (a, c, e, g and i) and Optical (b, d, f, h and j) images, demonstrating that, attenuation of autophagy can induce only marginally pathological phenotypes of fly wing formation (a–j) (arrows). (a and b) P{GawB}Bx^MS1096/UAS-Atg1_RNAi double transgenic fly wings, characterized by reduced Atg1 protein contents. (c and d) P{GawB}Bx^MS1096/UAS-Atg4_RNAi double transgenic fly wings, produced in Atg4 deficient cellular environments. (e and f) P{GawB}Bx^MS1096/UAS-Atg7_RNAi double transgenic fly wings, carrying downregulated Atg7 expression levels. (g and h) P{GawB}Bx^MS1096/UAS-Atg9_RNAi double transgenic fly wings, developed under cellular conditions of attenuated Atg9 autophagic protein synthesis. (i and j) P{GawB}Bx^MS1096/UAS-Atg18_RNAi double transgenic fly wings, bearing decreased amount of Atg18 protein. Asterisks (b {Atg1}, d {Atg4}, f {Atg7} and j {Atg18}): double (heterozygote) transgenic fly populations, containing both wild-type and mutant (at a low frequency) wing phenotypes. Scale Bars: 200 µm.</p

Disintegration of 20S proteasome core particle leads to fly wing dysmoprhia.

<p>Stereo-microscopical (a, c, e, g, i and k) and Optical (b, d, f, h, j and l) images, illustrating the critical roles of α- and β-based rings of the 20S proteasome catalytic core in wing morphogenesis. (a and b) P{GawB}Bx^MS1096/UAS-alpha5_RNAi double transgenic fly wings, carrying reduced cellular contents of α5 proteasome subunit. (c and d) P{GawB}Bx^MS1096/UAS-dbeta5_RNAi double transgenic fly wings, characterized by downregulated β5 expression levels. (e and f) P{GawB}Bx^MS1096/UAS-beta6_RNAi double transgenic flies, producing wings with diminished amount of β6 20S proteasome protein. (g and h) P{GawB}Bx^MS1096/UAS-Prosbeta21 double transgenic fly wings, overexpressing the conditionally (temperature sensitive {ts}) mutant β2^ts proteasome subunit. (i and j) P{GawB}Bx^MS1096/UAS-Pros261 double transgenic fly wings, carrying increased levels of conditionally mutant β6^ts protein specifically in the wing. (k and l) P{GawB}Bx^MS1096/UAS-Prosbeta21 - UAS-Pros261 triple transgenic flies, bearing dysmorphic wings due to organ-specific overexpression of defective (conditionally mutant) β2^ts and β6^ts 20S proteasome components. Scale Bars: 200 µm.</p

The β2 and β6 20S proteasome core subunits play essential roles in fly eye development.

<p>Stereo-microscopical (a, e and i), Optical (d, h and l {semi-thin sections}) and Scanning Electron Microscopy (SEM) (b, c, f, g, j and k) images, demonstrating the critical contribution of β2 and β6 20S proteasome core subunits in fly eye morphogenesis. (a–d) GAL4-ninaE.GMR/UAS-Prosbeta21 double transgenic fly eyes, overexpressing the conditionally (temperature sensitive {ts}) mutant β2^ts proteasome core protein form. (e–h) GAL4-ninaE.GMR/UAS-Pros261 double transgenic fly eyes, characterized by upregulated mutant β6^ts expression levels. (i–l) GAL4-ninaE.GMR/UAS-Prosbeta21 - UAS-Pros261 triple transgenic flies, bearing increased eye-specific contents of mutant (defective) β2^ts and β6^ts proteasome subunit forms. Dashed circle: ommatidium. Arrow: lesion area(s). Scale Bars: 50 µm.</p

RNAi-mediated elimination of critical autophagic proteins does not harm fly eye architectural structure.

<p>Stereo-microscopical (a, c, e, g and i) and Scanning Electron Microscopy (SEM) (b, d, f, h and j) images, illustrating the surface morphology and structural organization of Drosophila compound eye in Atg1, 4, 7, 9 and 18 deficient cellular environments. (a and b) GAL4-ninaE.GMR/UAS-Atg1_RNAi double transgenic fly eyes, characterized by likely reduced Atg1 protein contents. (c and d) GAL4-ninaE.GMR/UAS-Atg4_RNAi double transgenic fly eyes, carrying downregulated Atg4 expression levels. (e and f) GAL4-ninaE.GMR/UAS-Atg7_RNAi double transgenic fly eyes, likely producing diminished amount of Atg7 autophagic protein. (g and h) GAL4-ninaE.GMR/UAS-Atg9_RNAi double transgenic fly eyes, probably containing decreased Atg9 protein levels. (i and j) GAL4-ninaE.GMR/UAS-Atg18_RNAi double transgenic fly eyes, likely developed in an Atg18 deficient cellular background. Scale Bars: 50 µm.</p

RNAi-mediated reduction of Atg9 protein levels is tightly associated with loss of wing’s structural integrity in aged Drosophila flies.

<p>(A) Wings from 70 days-old single (homozygote) transgenic (both male and female) flies carrying only the P{GawB}Bx^MS1096 genetic driver present (in their vast majority) with a generally physiological morphology and structure. (B) Optical images (a–g) of double (heterozygote) transgenic wings, carefully isolated from 10 to 70 days-old female flies that were produced by Atg9_RNAi homozygote females crossed with P{GawB}Bx^MS1096 homozygote males, clearly demonstrate the age-dependent structural fragility of the organ (broken wings). Arrows: wing dysmorphic features. Scale Bars: 200 µm.</p

Downregulation of Rpn1 and Rpn2 19S cap proteasome subunits severely affects eye morphogenesis.

<p>(A) Confocal Laser Scanning Microscopy (CLSM) images, illustrating the organ-specific and targeted overexpression of EGFP (enhanced green fluorescent protein) reporter protein exclusively in the (a) eye disc of double transgenic flies carrying the GAL4-ninaE.GMR/UAS-2xEGFP strain genotype and (b) dorsal wing disc of double transgenic flies characterized by the P{GawB}Bx^MS1096/UAS-2xEGFP strain genotype, and therefore reflecting the functional reliability and efficiency of the cell type-dependent genetic scheme employed in the present study. (B) Stereo-microscopical (a, e and i), Optical (d, h and l {semi-thin sections}) and Scanning Electron Microscopy (SEM) (b, c, f, g, g′, j and k) images, demonstrating Drosophila compound eye morphology and structural organization in Rpn1 and Rpn2 functionally deficient cellular environments. (a–d) GAL4-ninaE.GMR single transgenic adult fly compound eyes (dashed circle: ommatidium; 1–7: photoreceptor cells) (utilized as control condition; identical phenotypes are also observed for single transgenic flies grown at 29°C, and for triple transgenic flies overexpressing the β2^ts and β6^ts -conditional- mutant proteasome components raised at 25°C {data not shown}). (e–h) GAL4-ninaE.GMR/UAS-Rpn1_RNAi double transgenic fly eyes, carrying reduced Rpn1 protein contents. (i–l) GAL4-ninaE.GMR/UAS-Rpn2_RNAi double transgenic fly eyes, characterized by downregulated Rpn2 proteasome subunit expression levels. Arrows: lesion area(s). Scale Bars: 50 µm.</p