Developmental pattern formation is orchestrated by diffusible signaling molecules, termed morphogens, that form gradients from which cells can determine positionally appropriate fates. Stochasticity in morphogen binding, signal transduction, and gene expression create local cell-to-cell variability in the readout of morphogen gradients. However, little is known about the actual levels of noise in morphogen gradient responses, or the mechanisms that might control it. To investigate this, I quantified the transcriptional activity noise, protein expression noise, and protein half-life of optomotor blind (omb), one of the downstream targets of the morphogen Dpp in the Drosophila larval wing imaginal disc. Using combined fluorescence in situ hybridization (FISH) with intronic probes, immunofluorescence, image segmentation and image analysis, I observed a very high level of transcriptional variability characterized by coefficients of variation (CV) as high as ~110%, in the cells in which omb plays a central role in specifying the location of wing vein primordium L5. However, the half-life of the Omb protein was found to be very long, ~ 6 hours, which would be expected to provide significant temporal filtering of the transcriptional noise. I showed that the reduction in noise from transcript to protein is sufficient to account for the precision of the positional information that patterns vein L5.

I also investigated why the positioning of vein L5 is remarkably robust to genetic manipulation that change the shape of the Dpp morphogen gradient. I observed that patters of Dpp signaling and Omb expression are not constant during larval development, but change continuously, and not always in concert with each other. By taking into account the long half-life of Omb it was possible to build a model that explains both these movements and the remarkable robustness of L5 patterning to changes in Dpp gradient shape.

The endomembrane system is the defining feature of eukaryotic cells and ensures that the appropriate proteins are sorted and transported to their correct subcellular location where they perform their biological functions. The coat protein complex II (COPII) mediates the formation of COPII vesicles, which are responsible for exporting proteins and lipids from the endoplasmic reticulum (ER). The core COPII coat consists of five proteins: the small GTPase Sar1, the Sec23/24 heterodimer, and the Sec13/31 heterotetramer. The recognition and subsequent packaging of cargo into COPII vesicles is mediated by interaction with the Sec24 subunit.

The development of a cell-free vesicle budding reaction has facilitated a biochemical analysis of COPII trafficking. Initial experiments examining COPII packaging of mammalian cargo proteins suggested that while some cargo proteins could be packaged directly by the core COPII components, other cargo proteins seemed to require additional cytosolic factors for efficient packaging. Whether auxiliary cytosolic factors exist to promote the COPII packaging of a specific subset of cargo proteins is the central question that motivated the work described in this dissertation.

This question was first approached using Vangl2, a cell surface localized transmembrane protein involved in planar cell polarity signaling, as a model cargo molecule. However, during the course of characterizing COPII packaging of Vangl2 in vitro, it was determined that this was not the appropriate system to address this question. As a result, proTGFα, the precursor form of transforming growth factor alpha, was chosen as a model cargo protein to continue to address the central question.

Here, I describe the key observations that show the existence of auxiliary cytosolic factor(s) that are required for efficient COPII packaging of specific cargo. In the particular case of proTGFα, both its recruitment by the COPII machinery and its final packaging into COPII vesicles require the cooperation of one or more auxiliary cytosolic factors and a transmembrane cargo receptor, Cornichon-1. Moreover, I present evidence showing that interaction of cargo proteins with the COPII coat is insufficient for packaging into COPII vesicles. This suggests that cargo recruitment and vesicle packaging are two distinct steps of COPII vesicle formation. Taken together, these observations provide novel insights into the molecular mechanism of COPII vesicle formation in mammals.