Characterization of dec-1 Protein Interactions during Eggshell Assembly in Drosophila

Steven Koutnik
Marquette University
Mentor:  Dr. Gail Waring

The assembly of ordered complex structures is paramount to the development of all species.  Using Drosophila eggshell assembly as an experimental system the goal of this study is to add to the understanding of how complex extracellular structures are made through discovering which eggshell proteins interact and how those interactions are achieved. Drosophila eggshell proteins are secreted into the extracellular space between the oocyte and overlaying follicle cells during late oogenesis and form a multilayered structure.  The two major eggshell layers are the vitelline membrane (VM) and the endochorion.  Despite knowing several proteins that are present in the eggshell during its assembly, very little is known about how they interact.  The focus of this study was s60, a protein which is found in both the VM and the endochorion layers in late stage 14 egg chambers.

s60 is cleaved from its precursor, s80, in the VM during late stage 13 and early stage 14 of oogenesis.  By being in both the VM and the endochorion layers s60 may be engaged in different protein interactions.  Eggshell fragments can be recovered from homogenized egg chambers by low speed centrifugation. In order to determine what is necessary to release s60 from the eggshell, stage 14 egg chambers were exposed to solubilizing agents and pelleted at 12,000 rpm for 10 minutes.  The proteins in the supernatant and pellet fractions were then solubilized in 2% SDS at 95ºC in the presence of a reducing agent and separated by SDS-PAGE.  Using Western Blot analysis the relative amounts of s60 in the pellet and supernatant fractions were evaluated. The release of endochorion proteins was monitored by s36, a major endochorion protein; the release of VM proteins was monitored by sV23, a major VM protein. 

Stage 14 egg chambers were disrupted by homogenization in Tris buffered saline (TBS), 1% SDS in TBS at room temperature, or 5% beta-mercaptoethanol (BME) in TBS.  With the egg chambers disrupted in TBS it was found that heat (95ºC for three minutes) was insufficient to displace any of the eggshell proteins.  1% SDS at room temperature released most s80, s60, and s36; sV23 was in the pellet under these conditions.  Inconsistent results were observed with the egg chambers disrupted in 5% BME. The endochorion protein, s36, was found in the pellet; sV23 was found in the supernatant; however, depending upon the experiment, s60 was found either in the pellet or the supernatant. This discrepancy needs to be resolved. Since the movement of s60 from the VM to the endochorion is a dynamic process, more precise staging of the stage 14 egg chambers may be critical. If s60 is differentially released, to aid in determining whether the released fraction comes from the VM or endochorion layer, an endochorion deficient mutant (DC238) will be used.  Consistent with its morphological phenotype, the accumulation of s36 was found to be severely reduced in DC238 mutant egg chambers. 
 
 

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