Minor proteins and enzymes of the Drosophila eggshell matrix

Abstract

The Drosophila eggshell provides an in vivo model system for extracellular matrix assembly, in which programmed gene expression, cell migrations, extracellular protein trafficking, proteolytic processing, and cross-linking are all required to generate a multi-layered and regionally complex architecture. While abundant structural components of the eggshell are known and are being characterized, less is known about non-abundant structural, regulatory, and enzymatic components that are likely to play critical roles in eggshell assembly. We have used sensitive mass spectrometry-based analyses of fractionated eggshell matrices to validate six previously predicted eggshell proteins and to identify eleven novel components, and have characterized the expression patterns of many of their mRNAs. Among these are several putative structural or regulatory (non-enzymatic) proteins, most larger in mass than the major eggshell proteins and often showing preferential expression in follicle cells overlying specific structural features of the eggshell. Of particular note are the putative enzymes, some likely to be involved in matrix cross-linking (two yellow family members previously implicated in eggshell integrity, a heme peroxidase, and a small-molecule oxidoreductase) and others possibly involved in matrix proteolysis or adhesion (proteins related to cathepsins B and D). This work provides a framework for future molecular studies of eggshell assembly.

Building up the Drosophila eggshell: First of all the eggshell genes must be transcribed

“…The transcript of the VM26A.2 gene, encoding the most abundant component of the vitelline membrane, is detected in all follicle cells surrounding the oocyte from stage 8. The same expression pattern was observed for the VM26A.1 gene and for the CG9050 gene (Burke et al, 1987;Popodi et al, 1988, Fakhouri et al, 2006. Conversely, the VM32E gene shows a peculiar spatial transcriptional profile and its expression is restricted at stage 10 of oogenesis (Gargiulo et al, 1991).…”

“…Among these, the Femcoat protein is specifically expressed in the follicle cells at late stages and it is required for endochorion formation (Kim et al, 2002). In a recent effort to find unknown proteins playing relevant structural or regulatory roles in eggshell biogenesis, 11 new distinct proteins have been identified as structural components of the eggshell (Fakhouri et al, 2006). Among these three are putative vitelline membrane proteins, seven are putative chorion components and one is a novel protein.…”

“…In addition, some minor chorion genes are expressed in a more localized manner (Parks and Spradling, 1987). Interestingly, among the putative chorion genes recently identified, several show preferential expression in follicle cells covering specific regions of the eggshell suggesting they encode components that could contribute to specific structural features of the eggshell (Fakhouri et al, 2006).…”

Mutation of TweedleD, a member of an unconventional cuticle protein family, alters body shape in Drosophila

“…6) and is no longer detectable by the end of the last larval stage. The lack of any shape alteration in TwdlD 1 embryos (data not shown) presumably reflects the fact that the surrounding eggshell is a protein-based extracellular matrix distinct from cuticle (30). Within the eggshell, however, the embryonic cuticle structure is clearly affected, as is evident upon examination of newly hatched first-instar larvae (see Fig.…”

Accumulation of the Drosophila Torso-like protein at the blastoderm plasma membrane suggests that it translocates from the eggshell

“…Of note, our experiments also showed variable patterns of vitelline membrane proteins, as determined by confocal microscopy. Thus, for example, the locations of Nasrat and VM32E, which have been assigned to the vitelline membrane by electron microscopy (Andrenacci et al, 2001) and identified as eggshell proteins by proteomics (Fakhouri et al, 2006), often do not overlap when viewed by confocal microscopy (Jiménez et al, 2002). This observation might indicate heterogeneity within the vitelline membrane or differences in antibody accessibility or recognition caused by sample processing and/or distinct fixation procedures used for confocal microscopy or for electron microscopy.…”