Drosophila signal peptidase complex member Spase12 is required for development and cell differentiation

Abstract

It is estimated that half of all proteins expressed in eukaryotic cells are transferred across or into at least one cellular membrane to reach their functional location. Protein translocation into the endoplasmic reticulum (ER) is critical to the subsequent localization of secretory and transmembrane proteins. A vital component of the translocation machinery is the signal peptidase complex (SPC)--which is conserved from yeast to mammals--and functions to cleave the signal peptide sequence (SP) of secretory and membrane proteins entering the ER. Failure to cleave the SP, due to mutations that abolish the cleavage site or reduce SPC function, leads to the accumulation of uncleaved proteins in the ER that cannot be properly localized resulting in a wide range of defects depending on the protein(s) affected. Despite the obvious importance of the SPC, in vivo studies investigating its function in a multicellular organism have not been reported. The Drosophila SPC comprises four proteins: Spase18/21, Spase22/23, Spase25 and Spase12. Spc1p, the S. cerevisiae homolog of Spase12, is not required for SPC function or viability; Drosophila spase12 null alleles, however, are embryonic lethal. The data presented herein show that spase12 LOF clones disrupt development of all tissues tested including the eye, wing, leg, and antenna. In the eye, spase12 LOF clones result in a disorganized eye, defective cell differentiation, ectopic interommatidial bristles, and variations in support cell size, shape, number, and distribution. In addition, spase12 mosaic tissue is susceptible to melanotic mass formation suggesting that spase12 LOF activates immune response pathways. Together these data demonstrate that spase12 is an essential gene in Drosophila where it functions to mediate cell differentiation and development. This work represents the first reported in vivo analysis of a SPC component in a multicellular organism.

Figure 1. spase12 mutant alleles.

(A) spase12^d4 is a 4 kb deletion (purple shaded region). (B) spase12^EY10774 contains a transposon inserted in the second exon. (B) spase12^C24 is a 303 bp deletion (purple shaded region). spase12 GR (A) is a 29 kb genomic construct.

Figure 2. spase12 LOF results in a disorganized eye, loss of pigmentation, and melanotic mass formation.

(A) yw ey-flp/+; FRT 82B P[w+] cl/FRT 82B where w+ marks control tissue and w- marks the clone. yw ey-flp/+; FRT 82B P[w+] cl/FRT 82B spase12^d4 P[w+] (ey-flp; spase12^d4) (B), yw ey-flp/+; FRT 82B P[w+] cl/FRT 82B spase12^EY10774 P[w+] (ey-flp; spase12^EY10774) (C) and yw ey-flp/+; FRT 82B P[w+] cl/FRT 82B spase12^C24 (ey-flp; spase12^C24) (D) eyes are disrupted compared to the control (A). Clonal tissue in ey-flp; spase12^d4 eyes (B) appears lighter in color than spase12^d4/+ which exhibits a strong P[w+] eye color (B''). spase12^EY10774/+ (C''). spase12^C24/+ (D''). Examples of melanotic masses in ey-flp; spase12^d4 (B'), spase12^EY10774 (C'), and spase12^C24 (D') mosaic eyes. A single copy of spase12 GR rescues spase12^d4 in yw ey-flp/+; FRT 82B P[w+] cl/FRT 82B spase12^d4 P[w+], spase12 GR P[w+] (E). spase12^rev complements spase12^d4 (F). Ubiquitous expression of UAS-spase12 rescues spase12 phenotypes in w; ubi-gal4/UAS-spase12; spase12^d4 P[w+]/FRT 82B spase12^C24 flies (G).

Figure 3. spase12 LOF disrupts development in multiple tissues.

yw ubx-flp/+; FRT 82B (M) P[w+ ubi-GFP]/FRT control (A), yw ubx-flp/+; FRT 82B (M) P[w+ ubi-GFP]/FRT 82B spase12^C24 wings are crumpled and melanized (A'). Clones in the distal portion of yw hs-flp/+; FRT 82B (M) P[w+ ubi-GFP]/FRT 82B spase12^C24 legs (B') are twisted and stunted compared to yw hs-flp/+; FRT 82B (M) P[w+ ubi-GFP]/FRT 82B control (B). yw hs-flp/+; FRT 82B (M) P[w+ ubi-GFP]/FRT 82B control (C). Clones in the head region of yw hs-flp/+; FRT 82B (M) P[w+ ubi-GFP]/FRT 82B spase12^C24 result in eye, bristle, and antennal defects as well as melanotic mass formation (C').

Figure 4. spase12 adult eyes have ectopic and missing rhabdomeres and polarity defects.

Thin plastic sections of yw ey-flp/+; FRT 82B P[w+] cl/FRT 82B control (A), yw ey-flp/+; FRT 82B P[w+] cl/FRT 82B spase12^d4 P[w+] (ey-flp; spase12^d4) (B), yw ey-flp/+; FRT 82B P[w+] cl/FRT 82B spase12^EY10774 P[w+] (ey-flp; spase12^EY10774) (C) and yw ey-flp/+; FRT 82B P[w+] cl/FRT 82B spase12^C24 (ey-flp; spase12^C24) (D) mosaic eyes are disorganized and exhibit spacing defects between ommatidia (red asterisks) and varying rhabdomere numbers. Red circles mark ommatidia with an ectopic inner rhabdomere while yellow circles mark ommatidia with a missing inner rhabdomeres. Polarity of individual ommatidia within ey-flp; 82B (A', A''), ey-flp; spase12^d4(B', B''), ey-flp; spase12^EY10774(C', C''), and ey-flp; spase12^C24 (D', D'') is represented by red arrows. Section through ey-flp; spase12^C24 melanotic mass (D''') reveals degenerating tissue with large black dots consistent with dying cells (red arrows).

Figure 5. spase12 LOF leads to increased apoptosis.

yw ey-flp/+; FRT 82B P[w+] cl/FRT 82B (ey-flp; 82B) control (A–A'') has limited Caspase expression and normal Elav expression compared to yw ey-flp/+; FRT 82B P[w+] cl/FRT 82B spase12^C24 (ey-flp; spase12^C24) (B–B'') mosaic discs in which Caspase is upregulated and Elav expression strongly disrupted.

Figure 6. Loss of spase12 leads to defects in cell differentiation.

yw hs-flp/+; FRT 82B (M) P[w+ ubi-GFP]/FRT 82B spase12^C2448 APF eye discs stained with Armadillo (Arm) (A, B, C). GFP negatively marks clones (A', B', C'). Red dashed boxes outline representative ommatidia which are highlighted in A'', B'', C''. Support cells are color-coded according to their identity: interommatidial bristles (magenta), secondary pigment cells (cyan) and tertiary pigment cells (green) (A''', B''', C'''). The center of each ommatidium contains four cone cells, which strongly express Arm, surrounded by two primary pigment cells. spase12^C24 mutant tissue (B, B' and C, C') exhibit multiple defects: ommatidia missing one or both primary pigment cells (stars), ectopic IOBs (arrowheads), ectopic primary pigment cell (asterisk), and gaps in the support cell structure that allow contact between primary pigment cells of neighboring ommatidia (red arrows). (B''') A spase12^C24 mutant ommatidium is misshapen and has a cluster of three IOBs. Additionally, support cells are not properly placed and the identity of three support cells (purple) cannot be determined by their shape or placement. (C''') A spase12^C24 mutant ommatidium fails to maintain the appropriate pattern of cell types at the vertices and possesses an ectopic primary pigment cell (asterisk). A secondary pigment cell (red arrow) does not fully extend to separate one ommatidium from its neighbor and one side of the ommatidium has two secondary pigment cells rather than one (white arrow).

Figure 7. spase12 LOF results in ectopic IOBs and ommatidial fusions.

Scanning electron microscopy (SEM) of (A) yw ey-flp/+; FRT 82B P[w+] cl/FRT 82B control and (B) yw ey-flp/+; FRT 82B P[w+] cl/FRT 82B spase12^C24at 200X, (A', B') 1000X, and (A'', B'') 2000X. Adult ey-flp; spase12^C24 eyes (B–B'') are disorganized with ectopic interommatidial bristles (red arrows) and ommatidial fusions (white arrows).