No abstract

The dorsoventral (DV) patterning of the Drosophila embryo depends on the nuclear localization gradient of Dorsal (Dl), a protein related to the mammalian NF-B transcription factors. Current understanding of how the Dl gradient works has been derived from studies of its transcriptional interpretation, but the gradient itself has not been quantified. In particular, it is not known whether the Dl gradient is stable or dynamic during the DV patterning of the embryo. To address this question, we developed a mathematical model of the Dl gradient and constrained its parameters by experimental data. Based on our computational analysis, we predict that the Dl gradient is dynamic and, to a first approximation, can be described as a concentration profile with increasing amplitude and constant shape. These timedependent properties of the Dl gradient are different from those of the Bicoid and MAPK phosphorylation gradients, which pattern the anterior and terminal regions of the embryo. Specifically, the gradient of the nuclear levels of Bicoid is stable, whereas the pattern of MAPK phosphorylation changes in both shape and amplitude. We attribute these striking differences in the dynamics of maternal morphogen gradients to the differences in the initial conditions and chemistries of the anterior, DV, and terminal systems.computational modeling ͉ Drosophila ͉ systems biology ͉ parameter estimation A tissue patterned by morphogen gradients can change its transcriptional state, grow, or deform either in response to the gradients or independently of them (1-3). When these changes are much slower than the dynamics of the gradient, a tissue responds to a stable signal. Transcriptional interpretation of such signals can rely on differences in the expression thresholds of target genes with respect to the spatially distributed repressors or activators (2, 4). A different strategy for signal interpretation is required when the formation of positional information becomes intertwined with the dynamics of the patterned system (2, 5). Here, we suggest that the dorsoventral (DV) patterning of the Drosophila embryo operates in this regime.The DV patterning of the Drosophila embryo depends on the nuclear localization gradient of Dorsal (Dl), a protein related to the NF-B family of transcription factors (6-10). Transcriptional interpretation of the Dl gradient depends on the differences in the affinities of the Dl binding sites in the Dl-target genes and several gene expression and signaling cascades initiated by Dl (6,11,12). A ventral-to-dorsal occupancy gradient of the Toll cell surface receptor provides the activating signal for the DV patterning (13). In the absence of this signal, Dl is sequestered in the cytoplasm, in complex with an inhibitory protein I-B, called Cactus (Cact) in Drosophila. In response to Toll signaling, the Dl-Cact complex dissociates, Cact is degraded, and Dl enters the nucleus to control gene expression. In the current model of DV patterning, positional information is established by the spatial pattern of Toll occ...

Two lipid membrane sculpting BAR domain proteins, PICK1 and ICA69, play a key role early in the biogenesis of peptide hormone secretory vesicles and are critical for normal growth and metabolic homeostasis.

BAR domains are dimeric protein modules that sense, induce, and stabilize lipid membrane curvature. Here, we show that membrane curvature sensing (MCS) directs cellular localization and function of the BAR domain protein PICK1. In PICK1, and the homologous proteins ICA69 and arfaptin2, we identify an amphipathic helix N-terminal to the BAR domain that mediates MCS. Mutational disruption of the helix in PICK1 impaired MCS without affecting membrane binding per se. In insulin-producing INS-1E cells, super-resolution microscopy revealed that disruption of the helix selectively compromised PICK1 density on insulin granules of high curvature during their maturation. This was accompanied by reduced hormone storage in the INS-1E cells. In Drosophila, disruption of the helix compromised growth regulation. By demonstrating size-dependent binding on insulin granules, our finding highlights the function of MCS for BAR domain proteins in a biological context distinct from their function, e.g., at the plasma membrane during endocytosis.

Rab2 is a conserved Rab GTPase with a well-established role in secretory pathway function and phagocytosis. Here we demonstrate that Drosophila Rab2 is recruited to late endosomal membranes, where it controls the fusion of LAMP-containing biosynthetic carriers and lysosomes to late endosomes. In contrast, the lysosomal GTPase Gie/Arl8 is only required for late endosome-lysosome fusion, but not for the delivery of LAMP to the endocytic pathway. We also find that Rab2 is required for the fusion of autophagosomes to the endolysosomal pathway, but not for the biogenesis of lysosome-related organelles. Surprisingly, Rab2 does not rely on HOPS-mediated vesicular fusion for recruitment to late endosomal membranes. Our work suggests that Drosophila Rab2 is a central regulator of the endolysosomal and macroautophagic/autophagic pathways by controlling the major heterotypic fusion processes at the late endosome.