Wnt signaling plays critical roles during synaptic development and plasticity. However, the mechanisms by which Wnts are released travel to target cells unresolved. During development, secretion of Drosophila Wnt1, Wingless, requires function Evenness Interrupted (Evi)/Wls, a Wingless-binding protein that is secreted along with Wingless at neuromuscular junction. Given Evi transmembrane protein, these studies suggested presence novel vesicular mechanism trans-synaptic communication,...

O-Fucose is an unusual form of glycosylation found on epidermal growth factor-like (EGF) repeats and thrombospondin type 1 (TSRs) in many secreted transmembrane proteins. Recently O-fucose EGF was shown to play important roles Notch signaling. In contrast, physiological for TSRs are unknown. the accompanying paper (Luo, Y., Nita-Lazar, A., Haltiwanger, R. S. (2006) J. Biol. Chem. 281, 9385–9392), we demonstrated that enzyme distinct from protein O-fucosyltransferase adds TSRs. A known...

Sialylation is an important carbohydrate modification of glycoconjugates in the deuterostome lineage animals. By contrast, evidence for sialylation protostomes has been scarce and somewhat controversial. In present study, we characterize a Drosophila sialyltransferase gene, thus providing experimental presence protostomes. This gene encodes functional alpha2-6-sialyltransferase (SiaT) that closely related to vertebrate ST6Gal family, indicating ancient evolutionary origin this family....

Human C1 inhibitor (hC1INH) is a therapeutic N, O-glycoprotein with growing number of clinical applications, but the current natural supplies are not likely to meet demands. Therefore, recombinant approaches interest, whereby specific attention has be paid generated glycosylation patterns. Here, N,O-glycoprotein was expressed in mammary gland transgenic rabbits and subjected glycan analysis. After release N-glycans recombinant-rabbit human (rhC1INH) by...

Although the function of many glycoproteins in nervous system fruit flies is well understood, information about glycosylation profile and glycan attachment sites for such proteins scarce. In order to fill this gap facilitate analysis N-linked system, we have performed an extensive survey membrane-associated their N-glycosylation isolated from adult Drosophila brain. Following subcellular fractionation trypsin digestion, used different lectin affinity chromatography steps isolate...

In vertebrates, sialylated glycans participate in a wide range of biological processes and affect the development function nervous system. While complexity glycosylation functional redundancy among sialyltransferases provide obstacles for revealing roles sialylation mammals, Drosophila possesses sole vertebrate-type sialyltransferase, sialyltransferase (DSiaT), with significant homology to its mammalian counterparts, suggesting that could be suitable model investigate sialylation. To explore...

Neuronal extracellular vesicles (EVs) play important roles in intercellular communication and pathogenic protein propagation 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 EV including amyloid precursor (APP), synaptotagmin-4 (Syt4), neuroglian (Nrg) at Drosophila motor neuron presynaptic terminals, resulting increased release these By systematically...

Neuronal extracellular vesicles (EVs) are locally released from presynaptic terminals, carrying cargoes critical for intercellular signaling and disease. EVs derived endosomes, but it is unknown how these directed to the EV pathway rather than conventional endolysosomal degradation. Here, we find that endocytic machinery plays an unexpected role in maintaining a release-competent pool of at synapses. Endocytic mutants, including nervous wreck (nwk), shibire/dynamin, AP-2, unexpectedly...

Extracellular vesicles (EVs) are released by many cell types, including neurons, carrying cargoes involved in signaling and disease. It is unclear whether EVs promote intercellular or serve primarily to dispose of unwanted materials. We show that loss multivesicular endosome-generating endosomal sorting complex required for transport (ESCRT) machinery disrupts release EV from Drosophila motor neurons. Surprisingly, ESCRT depletion does not affect the activities cargo Synaptotagmin-4 (Syt4)...

Exosomes, small secreted microvesicles, are implicated in intercellular communication diverse cell types, transporting protein, lipid and nucleic acid cargo that impact the physiology of recipient cells. Besides signaling function exosomes they also serve as a mechanism to dispose obsolete cellular material.1 Particularly exciting is involvement exosomal nervous system, this has important implications for brain development function. The properties beginning entice biomedical community since...

ABSTRACT Fluorescent protein tags have revolutionized cell and developmental biology, in combination with binary expression systems they enable diverse tissue-specific studies of function. However these often do not recapitulate endogenous levels, localization, binding partners and/or windows gene expression. To address limitations, we developed a method called T-STEP (tissue-specific tagging proteins) that allows loci to be tagged tissue specific manner. uses efficient CRISPR/Cas9-enhanced...

Abstract The family of mammalian O-mannosyltransferases includes two enzymes, POMT1 and POMT2, which are thought to be essential for muscle neural development. Similar organisms, Drosophila has O-mannosyltransferase genes, rotated abdomen (rt) DmPOMT2, encoding proteins with high homology their counterparts. previously reported mutant phenotype the rt gene a clockwise rotation defects in embryonic No mutants have been described so far DmPOMT2 locus. In this study, we determined that mutation...

Membranes form elaborate structures that are highly tailored to their specialized cellular functions, yet the mechanisms by which these shaped remain poorly understood. Here, we show conserved membrane-remodeling C-terminal Eps15 Homology Domain (EHD) protein Past1 is required for normal assembly of subsynaptic muscle membrane reticulum (SSR) at Drosophila melanogaster larval neuromuscular junction (NMJ). past1 mutants exhibit altered NMJ morphology, decreased synaptic transmission, reduced...

The large-scale production of recombinant biopharmaceutical glycoproteins in the milk transgenic animals is becoming more widespread. However, comparison with bacterial, plant cell, or cell culture systems, little known about glycosylation machinery mammary gland, and hence on produced animals. Here influence presented several lactation parameters N-glycosylation C1 inhibitor (rhC1INH), a human serum glycoprotein, expressed rabbits. Enzymatically released N-glycans series rhC1INH samples...

Pompe disease is a lysosomal glycogen storage disorder characterized by acid alpha-glucosidase (GAA) deficiency. More than 110 different pathogenic mutations in the gene encoding GAA have been observed. Patients with this are being treated intravenous injection of recombinant forms enzyme. Focusing on approaches to produce enzyme means that specific attention has be paid generated glycosylation patterns. Here, human was expressed mammary gland transgenic rabbits. The N-linked glycans...

ABSTRACT Neuronal extracellular vesicles (EVs) play important roles in intercellular communication and pathogenic protein propagation 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 EV Amyloid Precursor Protein (APP) Synaptotagmin-4 (Syt4) at Drosophila motor neuron presynaptic terminals, resulting increased release these By systematically exploring known...

The majority of synaptic plasma membrane components are glycosylated. It is now widely accepted that this post-translational modification crucial during the establishment, maintenance and function nervous system. Despite its significance, structural information about glycosylation system specific glycoproteins very limited. In present study major glycan structures chicken (SPM) associated glycoprotein glycans were determined. N-glycans released by hydrazinolysis, labelled with...

Abstract Neuronal extracellular vesicles (EVs) can be locally released from presynaptic terminals, carrying cargoes that are important in intercellular signaling and disease. EVs derived endosomes, but it remains unclear how synaptic directed to the EV pathway, rather than undergoing conventional retrograde endosomal transport degradation. Here, we find clathrin-mediated endocytic machinery plays an unexpected role maintaining a release-competent pool of cargoes. Endocytic mutants, including...

Abstract Fluorescent protein tags have revolutionized cell and developmental biology, in combination with binary expression systems they enable diverse tissue-specific studies of function. However these often do not recapitulate endogenous levels, localization, binding partners, windows gene expression. To address limitations, we developed a method called T-STEP ( T issue- S pecific agging E ndogenous P roteins) that allows loci to be tagged tissue specific manner. uses efficient targeting...

Extracellular vesicles (EVs) are released by many cell types including neurons, carrying cargoes involved in signaling and disease. It is unclear whether EVs promote intercellular or serve primarily to dispose of unwanted materials. We show that loss multivesicular endosome-generating ESCRT (endosomal sorting complex required for transport) machinery disrupts release EV from Drosophila motor neurons. Surprisingly, depletion does not affect the activities cargo Synaptotagmin-4 (Syt4) only...