A novel broad spectrum venom metalloproteinase autoinhibitor in the rattlesnake Crotalus atrox evolved via a shift in paralog function.

The complexity of snake venom composition reflects adaptation to the diversity of prey and may be driven at times by a coevolutionary arms race between snakes and venom-resistant prey. However, many snakes are also resistant to their own venom due to serum-borne inhibitors of venom toxins, which raises the question of how snake autoinhibitors maintain their efficacy as venom proteins evolve. To investigate this potential three-way arms race among venom, prey, and autoinhibitors, we have identified and traced the evolutionary origin of serum inhibitors of snake venom metalloproteinases (SVMPs) in the Western Diamondback rattlesnake Crotalus atrox which possesses the largest known battery of SVMP genes among crotalids examined. We found that C. atrox expresses five members of a Fetuin A-related metalloproteinase inhibitor family but that one family member, FETUA-3, is the major SVMP inhibitor that binds to approximately 20 different C. atrox SVMPs and inhibits activities of all three SVMP classes. We show that the fetua-3 gene arose deep within crotalid evolution before the origin of New World species but, surprisingly, fetua-3 belongs to a different paralog group than previously identified SVMP inhibitors in Asian and South American crotalids. Conversely, the C. atrox FETUA-2 ortholog of previously characterized crotalid SVMP inhibitors shows limited activity against C. atrox SVMPs. These results reveal that there has been a functional evolutionary shift in the major SVMP inhibitor in the C. atrox lineage as the SVMP family expanded and diversified in the Crotalus lineage. This broad-spectrum inhibitor may be of potential therapeutic interest.

BAR-SH3 sorting nexins are conserved interacting proteins of Nervous wreck that organize synapses and promote neurotransmission.

Nervous wreck (Nwk) is a conserved F-BAR protein that attenuates synaptic growth and promotes synaptic function in Drosophila. In an effort to understand how Nwk carries out its dual roles, we isolated interacting proteins using mass spectrometry. We report a conserved interaction between Nwk proteins and BAR-SH3 sorting nexins, a family of membrane-binding proteins implicated in diverse intracellular trafficking processes. In mammalian cells, BAR-SH3 sorting nexins induce plasma membrane tubules that localize NWK2, consistent with a possible functional interaction during the early stages of endocytic trafficking. To study the role of BAR-SH3 sorting nexins in vivo, we took advantage of the lack of genetic redundancy in Drosophila and employed CRISPR-based genome engineering to generate null and endogenously tagged alleles of SH3PX1. SH3PX1 localizes to neuromuscular junctions where it regulates synaptic ultrastructure, but not synapse number. Consistently, neurotransmitter release was significantly diminished in SH3PX1 mutants. Double-mutant and tissue-specific-rescue experiments indicate that SH3PX1 promotes neurotransmitter release presynaptically, at least in part through functional interactions with Nwk, and might act to distinguish the roles of Nwk in regulating synaptic growth and function.

Depletion or over-expression of Sh3px1 results in dramatic changes in cell morphology.

The mammalian Sorting Nexin 9 (Snx9) family consists of three paralogs: Snx9, Snx18 and Snx33. Most of the published literature to date has centered on the role of Snx9 in clathrin-mediated endocytosis (CME). Snx9 contains an Sh3 domain at its N-terminus and has been shown to interact with Dynamin and actin nucleation factors via this domain. In addition to the Sh3 domain, Snx9 also contains a C-terminal BAR domain. BAR domains are known to sense and/or induce membrane curvature. In addition to endocytosis, recent studies have implicated the Snx9 family in diverse processes such as autophagy, macropinocytosis, phagocytosis and mitosis. The Snx9 family is encoded by a single gene in Drosophila called sh3px1. In this report, we present our initial characterization of sh3px1. We found that depletion of Sh3px1 from Drosophila Schneider 2 (S2) cells resulted in defective lamellipodia formation. A similar phenotype has been reported upon depletion of Scar, the actin nucleation factor implicated in forming lamellipodia. In addition, we demonstrate that over-expression of Sh3px1 in S2 cells results in the formation of tubules as well as long protrusions. Formation of these structures required the C-terminal BAR domain as well as the adjacent Phox homology (PX) domain of Sh3px1. Furthermore, efficient protrusion formation by Sh3px1 required the actin nucleation factor Wasp. Tubules and protrusions were also generated upon over-expressing the mammalian orthologs Snx18 and Snx33 in S2 cells. By contrast, over-expressing Snx9 mostly induced long tubules.

Highly specific and efficient CRISPR/Cas9-catalyzed homology-directed repair in Drosophila.

We and others recently demonstrated that the readily programmable CRISPR/Cas9 system can be used to edit the Drosophila genome. However, most applications to date have relied on aberrant DNA repair to stochastically generate frameshifting indels and adoption has been limited by a lack of tools for efficient identification of targeted events. Here we report optimized tools and techniques for expanded application of the CRISPR/Cas9 system in Drosophila through homology-directed repair (HDR) with double-stranded DNA (dsDNA) donor templates that facilitate complex genome engineering through the precise incorporation of large DNA sequences, including screenable markers. Using these donors, we demonstrate the replacement of a gene with exogenous sequences and the generation of a conditional allele. To optimize efficiency and specificity, we generated transgenic flies that express Cas9 in the germline and directly compared HDR and off-target cleavage rates of different approaches for delivering CRISPR components. We also investigated HDR efficiency in a mutant background previously demonstrated to bias DNA repair toward HDR. Finally, we developed a web-based tool that identifies CRISPR target sites and evaluates their potential for off-target cleavage using empirically rooted rules. Overall, we have found that injection of a dsDNA donor and guide RNA-encoding plasmids into vasa-Cas9 flies yields the highest efficiency HDR and that target sites can be selected to avoid off-target mutations. Efficient and specific CRISPR/Cas9-mediated HDR opens the door to a broad array of complex genome modifications and greatly expands the utility of CRISPR technology for Drosophila research.