Actin networks undergo rearrangements that influence cell shape. Actin network organization is regulated by a host of actin-binding proteins. The Drosophila synaptotagmin-like protein, bitesize (Btsz), organizes actin at epithelial cell apical junctions in a manner that depends on its interaction with the actin-binding protein, moesin. Using RNAi, we showed that Btsz functions at earlier, syncytial stages of Drosophila embryo development. Btsz is required to stabilize pseudo-cleavage furrows, preventing metaphase spindle collisions and nuclear fallout prior to cellularization. While previous studies have focused on Btsz function through moesin, we find that phosphorylated moesin localized to the nuclear envelope and was not enriched at pseudo-cleavage furrows, suggesting a moesin-independent function for Btsz in syncytial embryos. Consistent with this, mutants that affected all moesin-binding domain isoforms did not recapitulate pan-isoform Btsz depletion and we find that the C-terminal half of Btsz cooperatively binds to and bundles F-actin. We propose that synaptotagmin-like proteins directly regulate actin organization during syncytial Drosophila development.

Neuronal extracellular vesicles (EVs) are locally released from presynaptic terminals, carrying cargoes critical for intercellular signaling and disease. EVs are derived from endosomes, but it is unknown how these cargoes are directed to the EV pathway rather than for conventional endolysosomal degradation. Here, we find that endocytic machinery plays an unexpected role in maintaining a release-competent pool of EV cargoes at synapses. Endocytic mutants, including nervous wreck (nwk), shibire/dynamin, and AP-2, unexpectedly exhibit local presynaptic depletion specifically of EV cargoes. Accordingly, nwk mutants phenocopy synaptic plasticity defects associated with loss of the EV cargo synaptotagmin-4 (Syt4) and suppress lethality upon overexpression of the EV cargo amyloid precursor protein (APP). These EV defects are genetically separable from canonical endocytic functions in synaptic vesicle recycling and synaptic growth. Endocytic machinery opposes the endosomal retromer complex to regulate EV cargo levels and acts upstream of synaptic cargo removal by retrograde axonal transport. Our data suggest a novel molecular mechanism that locally promotes cargo loading into synaptic EVs.

Neuronal extracellular vesicles (EVs) play important roles in intercellular communication and pathogenic protein propagation in neurological disease. However, it remains unclear how cargoes are selectively packaged into neuronal EVs. Here, we show that loss of the endosomal retromer complex leads to accumulation of EV cargoes including amyloid precursor protein (APP), synaptotagmin-4 (Syt4), and neuroglian (Nrg) at Drosophila motor neuron presynaptic terminals, resulting in increased release of these cargoes in EVs. By systematically exploring known retromer-dependent trafficking mechanisms, we show that EV regulation is separable from several previously identified roles of neuronal retromer. Conversely, mutations in rab11 and rab4, regulators of endosome-plasma membrane recycling, cause reduced EV cargo levels, and rab11 suppresses cargo accumulation in retromer mutants. Thus, EV traffic reflects a balance between Rab4/Rab11 recycling and retromer-dependent removal from EV precursor compartments. Our data shed light on previous studies implicating Rab11 and retromer in competing pathways in Alzheimer’s disease, and suggest that misregulated EV traffic may be an underlying defect.

Fluorescent protein tags have revolutionized cell and developmental biology, and in combinationwith binaryexpressionsystems theyenable diverse tissue-specific studies of protein function. However these binary expression systems often do not recapitulate endogenous protein expression levels, localization, binding partners and/or developmental windows of gene expression. To address these limitations, we have developed a method called T-STEP (tissuespecific tagging of endogenous proteins) that allows endogenous loci to be tagged in a tissue specificmanner. T-STEPusesacombination of efficient CRISPR/Cas9-enhanced gene targeting and tissue-specific recombinase-mediated tag swapping to temporally and spatially label endogenous proteins. We have employed this method to GFP tag OCRL (a phosphoinositide-5-phosphatase in the endocytic pathway) and Vps35 (a Parkinson’s disease-implicated component of the endosomal retromer complex) in diverse Drosophila tissues including neurons, glia, muscles and hemocytes. Selective tagging of endogenous proteins allows, for the first time, cell type-specific live imaging and proteomics in complex tissues.

Exosomes are small secretory vesicles (50-150 nm in diameter) of endocytic origin that have been recently identified as a novel intercellular communication pathway. They carry select cargo that impact the recipient cell’s metabolism and signaling pathways. The tissue-specificity of targeting and intracellular trafficking after uptake to the target cell are not fully understood and likely involve novel molecular cross-talk. Given that cultured tissue cells are used for the majority of exosome research (out of necessity), the Drosophila melanogaster larval neuromuscular junction (NMJ) offers a truly unique in vivo experimental system to address the biology of exosomes including their formation, release, uptake and trafficking. Here, I identify the membrane remodeling protein Amphiphysin as required for exosome trafficking in the recipient muscle cell. Furthermore, I show that the v-SNARE Ykt6 plays a role in neuronal exosome release. Additionally, I provide evidence that suggests a cargo-dependent exosome trafficking mechanism in the recipient muscle cells. Finally, I also developed a split-GFP reporter system to visualize post-synaptic exosomal trafficking and describe its in vivo application.

Fluorescent protein tags have revolutionized cell and developmental biology, and in combination with binary expression systems they enable diverse tissue-specific studies of protein function. However these binary expression systems often do not recapitulate endogenous protein expression levels, localization, binding partners and/or developmental windows of gene expression. To address these limitations, we have developed a method called T-STEP (tissue-specific tagging of endogenous proteins) that allows endogenous loci to be tagged in a tissue specific manner. T-STEP uses a combination of efficient CRISPR/Cas9-enhanced gene targeting and tissue-specific recombinase-mediated tag swapping to temporally and spatially label endogenous proteins. We have employed this method to GFP tag OCRL (a phosphoinositide-5-phosphatase in the endocytic pathway) and Vps35 (a Parkinson's disease-implicated component of the endosomal retromer complex) in diverse Drosophila tissues including neurons, glia, muscles and hemocytes. Selective tagging of endogenous proteins allows, for the first time, cell type-specific live imaging and proteomics in complex tissues.