Inactivation of clathrin heavy chain inhibits synaptic recycling but allows bulk membrane uptake

Synaptic vesicle reformation depends on clathrin, an abundant protein that polymerizes around newly forming vesicles. However, how clathrin is involved in synaptic recycling in vivo remains unresolved. We test clathrin function during synaptic endocytosis using clathrin heavy chain (chc) mutants combined with chc photoinactivation to circumvent early embryonic lethality associated with chc mutations in multicellular organisms. Acute inactivation of chc at stimulated synapses leads to substantial membrane internalization visualized by live dye uptake and electron microscopy. However, chc-inactivated membrane cannot recycle and participate in vesicle release, resulting in a dramatic defect in neurotransmission maintenance during intense synaptic activity. Furthermore, inactivation of chc in the context of other endocytic mutations results in membrane uptake. Our data not only indicate that chc is critical for synaptic vesicle recycling but they also show that in the absence of the protein, bulk retrieval mediates massive synaptic membrane internalization

Recombineering-mediated tagging of Drosophila genomic constructs for in vivo localization and acute protein inactivation

Studying gene function in the post-genome era requires methods to localize and inactivate proteins in a standardized fashion in model organisms. While genome-wide gene disruption and over-expression efforts are well on their way to vastly expand the repertoire of Drosophila tools, a complementary method to efficiently and quickly tag proteins expressed under endogenous control does not exist for fruit flies. Here, we describe the development of an efficient procedure to generate protein fusions at either terminus in an endogenous genomic context using recombineering. We demonstrate that the fluorescent protein tagged constructs, expressed under the proper control of regulatory elements, can rescue the respective mutations and enable the detection of proteins in vivo. Furthermore, we also adapted our method for use of the tetracysteine tag that tightly binds the fluorescent membrane-permeable FlAsH ligand. This technology allows us to acutely inactivate any tagged protein expressed under native control using fluorescein-assisted light inactivation and we provide proof of concept by demonstrating that acute loss of clathrin heavy chain function in the fly eye leads to synaptic transmission defects in photoreceptors. Our tagging technology is efficient and versatile, adaptable to any tag desired and paves the way to genome-wide gene tagging in Drosophila

The role of Clathrin and Dynamin in synaptic vesicle endocytosis

Maintenance of proper neuronal communication relies on the constant supply and availability of synaptic vesicles at the nerve terminal. In neurons different modes of synaptic vesicle recycling coexist. The most common and best studied of these mechanisms is clathrin mediated endocytosis. During this process several proteins and lipids cooperate in a very structured manner to form new vesicles by budding from the plasma membrane. Our studies aimed to determine the role of clathrin anddynamin in synaptic vesicle endocytosis in vivo. Interestingly, althoughclathrin and dynamin binding partners, localization in the cell and 3D structure are well known the effect of their complete loss of function during synaptic vesicle recycling as well as their possible interaction have not been studied in vivo in any organism.To investigate the role of endocytic proteins like clathrin and dynamin in synaptic vesicle recycling in vivo it is recommended to use null or severe hypomorphic alleles of these genes. However, both clathrin and dynamin null mutants die early in embryonic development, precluding a detailed analysis of mutant synaptic terminals. To overcome this issue and thus to understand the functions of clathrin and dynamin in vivo, wehave adapted an acute inactivation strategy termed FlAsH-FALI (fluorescein assisted light inactivation using the membrane permeable reagent FlAsH) for use at synapses in fruit flies. We developed a recombineering strategy allowing us to very efficiently modify genomic DNA fragments so that they encode a protein of interest fused to a tetracysteine tag, which is required for binding of the FlAsH reagent, under native promoter control. We used this technology to acutely inactivate several endocytic proteins at Drosophila synapses, including Clathrin and Dynamin.To investigate the function of Clathrin in synaptic vesicle recycling inwe have combined Drosophila clathrin heavy chain (chc) null mutant with acute FlAsH-FALI photoinactivation of CHC. Our results indicate that theabsence of Clathrin leads to formation of huge submembrane structures upon nerve stimulation. Vesicle cycling from these structures is abolished, resulting in dramatic defects in neurotransmitter release. Additionally, photoinactivation of Clathrin in other endocytic mutants, each characterized by highly reduced or abolished levels of CME like synaptojanin, dap160 or shibirets1, also leads to the appearance of large membrane invaginations. Taken together these data indicate that Clathrin is essential for the formation of synaptic vesicles in vivo andthat in the absence of this protein bulk endocytosis prevails.Due to qualitative similarities between the phenotypes of photoinactivated Clathrin and dynamin 1 knockout mice neurons we proposethat dynamin may coordinate together with clathrin the formation of synaptic vesicles of fixed size and shape. To test this hypothesis we also employed the FlAsH-FALI technique and photoinactivated Dynamin in the shi12-12B null mutant background (shibire being the fly gene encoding Dynamin). Inactivation of Dynamin, similarly to Clathrin inactivation, results in the formation of large submembrane structures upon stimulation. Interestingly, this is at odds with the phenotype of shits1 temperature sensitive mutant which shows a complete block in endocytosis at restrictive temperature and where Dynamin is locked in metastable aggregated state and wraps around nascent vesicle necks at the plasma membrane. Thus, the protein is still present and may thus still be in state to stabilize its downstream targets such as Clathrin. Moreover photoinactivation of Dynamin in shits1 mutant at the restrictive temperature also leads to the formation of large submembranestructures upon stimulation, reinforcing the idea that shits1 does not confer a dynamin null phenotype. Based on our data we propose a model where Dynamin may coordinate together with Clathrin the formation of synaptic vesicles of fixed size and shape in vivo.status: publishe

Dynamin photoinactivation blocks Clathrin and α-adaptin recruitment and induces bulk membrane retrieval

Dynamin is a well-known regulator of synaptic endocytosis. Temperature-sensitive dynamin (shi(ts1)) mutations in Drosophila melanogaster or deletion of some of the mammalian Dynamins causes the accumulation of invaginated endocytic pits at synapses, sometimes also on bulk endosomes, indicating impaired membrane scission. However, complete loss of dynamin function has not been studied in neurons in vivo, and whether Dynamin acts in different aspects of synaptic vesicle formation remains enigmatic. We used acute photoinactivation and found that loss of Dynamin function blocked membrane recycling and caused the buildup of huge membrane-connected cisternae, in contrast to the invaginated pits that accumulate in shi(ts1) mutants. Moreover, photoinactivation of Dynamin in shi(ts1) animals converted these pits into bulk cisternae. Bulk membrane retrieval has also been seen upon Clathrin photoinactivation, and superresolution imaging indicated that acute Dynamin photoinactivation blocked Clathrin and α-adaptin relocalization to synaptic membranes upon nerve stimulation. Hence, our data indicate that Dynamin is critically involved in the stabilization of Clathrin- and AP2-dependent endocytic pits.status: publishe

A novel X-chromosome EMS screen identifies genes implicated in neuronal communication

status: publishe

Loss of Skywalker Reveals Synaptic Endosomes as Sorting Stations for Synaptic Vesicle Proteins

Exchange of proteins at sorting endosomes is not only critical to numerous signaling pathways but also to receptor-mediated signaling and to pathogen entry into cells; however, how this process is regulated in synaptic vesicle cycling remains unexplored. In this work, we present evidence that loss of function of a single neuronally expressed GTPase activating protein (GAP), Skywalker (Sky) facilitates endosomal trafficking of synaptic vesicles at Drosophila neuromuscular junction boutons, chiefly by controlling Rab35 GTPase activity. Analyses of genetic interactions with the ESCRT machinery as well as chimeric ubiquitinated synaptic vesicle proteins indicate that endosomal trafficking facilitates the replacement of dysfunctional synaptic vesicle components. Consequently, sky mutants harbor a larger readily releasable pool of synaptic vesicles and show a dramatic increase in basal neurotransmitter release. Thus, the trafficking of vesicles via endosomes uncovered using sky mutants provides an elegant mechanism by which neurons may regulate synaptic vesicle rejuvenation and neurotransmitter release.status: publishe

Brain-Specific Biomarkers as Mortality Predictors after Aneurysmal Subarachnoid Haemorrhage

Aneurysmal subarachnoid haemorrhage (aSAH) is a serious condition with a high mortality and high permanent disability rate for those who survive the initial haemorrhage. The purpose of this study was to investigate markers specific to the central nervous system as potential in-hospital mortality predictors after aSAH. In patients with an external ventricular drain, enolase, S100B, and GFAP levels were measured in the blood and cerebrospinal fluid (CSF) on days 1, 2, and 3 after aSAH. Compared to survivors, non-survivors showed a significantly higher peak of S100B and enolase levels in the blood (S100B: 5.7 vs. 1.5 ng/mL, p = 0.031; enolase: 6.1 vs. 1.4 ng/mL, p = 0.011) and the CSF (S100B: 18.3 vs. 0.9 ng/mL, p = 0.042; enolase: 109.2 vs. 6.1 ng/mL, p = 0.015). Enolase showed the highest level of predictability at 1.8 ng/mL in the blood (AUC of 0.873) and 80.0 ng/mL in the CSF (AUC of 0.889). The predictive ability of S100B was also very good with a threshold of 5.7 ng/mL in the blood (AUC 0.825) and 4.5 ng/mL in the CSF (AUC 0.810). In conclusion, enolase and S100B, but not GFAP, might be suitable as biomarkers for the early prediction of in-hospital mortality after aSAH