Comprehensive study of SNAREs involved in the post-Golgi transport in photoreceptors.

Polarized transport is essential for the construction of multiple plasma membrane domains within cells. photoreceptors serve as excellent model systems for studying the mechanisms of polarized transport. We conducted a comprehensive soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) screening of the fly genome using RNAi knockdown and CRISPR/Cas9 somatic knockout combined with the CoinFLP system to identify SNAREs involved in post-Golgi trafficking.

RudLOV is an optically synchronized cargo transport method revealing unexpected effects of dynasore.

Live imaging of secretory cargoes is a powerful method for understanding the mechanisms of membrane trafficking. Inducing the synchronous release of cargoes from an organelle is key for enhancing microscopic observation. We developed an optical cargo-releasing method, 'retention using dark state of LOV2' (RudLOV), which enables precise spatial, temporal, and quantity control during cargo release.

Golgi clustering by the deficiency of COPI-SNARE in photoreceptors.

A comprehensive study of soluble -ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in the fly genome by RNAi in photoreceptors indicated that knockdown of any of the COPI-SNAREs, , , and , resulted in the same characteristic phenotypes: Golgi stacks gathering on their -side, laterally expanded Golgi cisternae, and a reduced number of discrete Golgi stacks. These Golgi stacks are reminiscent of mammalian Golgi ribbons and Brefeldin A (BFA)-bodies in S2 cells. As previously reported, BFA suppresses -Golgi network (TGN) fission and Golgi stack separation to form a BFA-body, which is a cluster of Golgi stacks cored by recycling endosomes.

Functions of neuronal Synaptobrevin in the post-Golgi transport of Rhodopsin in Drosophila photoreceptors.

Polarized transport is essential for constructing multiple plasma membrane domains in the cell. Drosophila photoreceptors are an excellent model system to study the mechanisms of polarized transport. Rab11 is the key factor regulating the post-Golgi transport of rhodopsin 1 (Rh1; also known as NinaE), a photoreceptive protein, to the rhabdomere, a photoreceptive plasma membrane.

Stratum is required for both apical and basolateral transport through stable expression of Rab10 and Rab35 in photoreceptors.

Post-Golgi transport for specific membrane domains, also termed polarized transport, is essential for the construction and maintenance of polarized cells. Highly polarized photoreceptors serve as a good model system for studying the mechanisms underlying polarized transport. The Mss4 ortholog, Stratum (Strat), controls basal restriction of basement membrane proteins in follicle cells, and Rab8 acts downstream of Strat.

Sec71 separates Golgi stacks in S2 cells.

Golgi stacks are the basic structural units of the Golgi. Golgi stacks are separated from each other and scattered in the cytoplasm of cells. Here, we report that the ARF-GEF inhibitor Brefeldin A (BFA) induces the formation of BFA bodies, which are aggregates of Golgi stacks, -Golgi networks and recycling endosomes.

Recycling endosomes associate with Golgi stacks in sea urchin embryos.

The trans-Golgi network (TGN) and recycling endosome (RE) have been recognized as sorting centers, the former for newly synthesized and the latter for endocytosed proteins. However, recent findings have revealed that TGN also receives endocytosed materials and RE accepts newly synthesized proteins destined to the plasma membrane. Recently, we reported that in both and microtubule-disrupted HeLa cells, REs are associated with the trans-side of Golgi stacks.

Rab10, Crag and Ehbp1 regulate the basolateral transport of NaKATPase in photoreceptors.

Cells are often polarized and have multiple plasma membrane domains. To establish and maintain these domains, polarized transport is essential, and its impairment results in genetic disorders. Nevertheless, the underlying mechanisms of polarized transport have not been elucidated.

Recycling endosomes attach to the trans-side of Golgi stacks in and mammalian cells.

Historically, the trans-Golgi network (TGN) has been recognized as a sorting center of newly synthesized proteins, whereas the recycling endosome (RE) is a compartment where endocytosed materials transit before being recycled to the plasma membrane. However, recent findings revealed that both the TGN and RE connect endocytosis and exocytosis and, thus, are functionally overlapping. Here we report, in both and microtubule-disrupted HeLa cells, that REs are interconvertible between two distinct states, namely Golgi-associated REs and free REs.

ER membrane protein complex is required for the insertions of late-synthesized transmembrane helices of Rh1 in photoreceptors.

Most membrane proteins are synthesized on and inserted into the membrane of the endoplasmic reticulum (ER), in eukaryote. The widely conserved ER membrane protein complex (EMC) facilitates the biogenesis of a wide range of membrane proteins. In this study, we investigated the EMC function using photoreceptor as a model system.

Syndapin constricts microvillar necks to form a united rhabdomere in photoreceptors.

photoreceptors develop from polarized epithelial cells that have apical and basolateral membranes. During morphogenesis, the apical membranes subdivide into a united bundle of photosensory microvilli (rhabdomeres) and a surrounding supporting membrane (stalk). By EMS-induced mutagenesis screening, we found that the F-Bin/Amphiphysin/Rvs (F-BAR) protein syndapin is essential for apical membrane segregation.

Parcas is the predominant Rab11-GEF for rhodopsin transport in photoreceptors.

Rab11 is essential for polarized post-Golgi vesicle trafficking to photosensitive membrane rhabdomeres in photoreceptors. Here, we found that Parcas (Pcs), recently shown to have guanine nucleotide exchange (GEF) activity toward Rab11, co-localizes with Rab11 on the trans-side of Golgi units and post-Golgi vesicles at the base of the rhabdomeres in pupal photoreceptors. Pcs fused with the electron micrography tag APEX2 localizes on 150-300 nm vesicles at the trans-side of Golgi units, which are presumably fly recycling endosomes.

The roles of Syx5 in Golgi morphology and Rhodopsin transport in Drosophila photoreceptors.

SNAREs (SNAP receptors) are the key components of protein complexes that drive membrane fusion. Here, we report the function of a SNARE, Syntaxin 5 (Syx5), in the development of photoreceptors in Drosophila In wild-type photoreceptors, Syx5 localizes to cis-Golgi, along with cis-Golgi markers: Rab1 and GM130. We observed that Syx5-deficient photoreceptors show notable accumulation of these cis-Golgi markers accompanying drastic accumulation of vesicles between endoplasmic reticulum (ER) and Golgi cisternae.

Rab6 functions in polarized transport in Drosophila photoreceptors.

Selective membrane transport pathways are essential for cells in situ to construct and maintain a polarized structure comprising multiple plasma membrane domains, which is essential for their specific cellular functions. Genetic screening in Drosophila photoreceptors harboring multiple plasma membrane domains enables the identification of genes involved in polarized transport pathways. Our genome-wide high-throughput screening identified a Rab6-null mutant with a rare phenotype characterized by a loss of 2 apical transport pathways with an intact basolateral transport.

Rab6 Is Required for Multiple Apical Transport Pathways but Not the Basolateral Transport Pathway in Drosophila Photoreceptors.

Polarized membrane trafficking is essential for the construction and maintenance of multiple plasma membrane domains of cells. Highly polarized Drosophila photoreceptors are an excellent model for studying polarized transport. A single cross-section of Drosophila retina contains many photoreceptors with 3 clearly differentiated plasma membrane domains: a rhabdomere, stalk, and basolateral membrane.

dPob/EMC is essential for biosynthesis of rhodopsin and other multi-pass membrane proteins in Drosophila photoreceptors.

In eukaryotes, most integral membrane proteins are synthesized, integrated into the membrane, and folded properly in the endoplasmic reticulum (ER). We screened the mutants affecting rhabdomeric expression of rhodopsin 1 (Rh1) in the Drosophila photoreceptors and found that dPob/EMC3, EMC1, and EMC8/9, Drosophila homologs of subunits of ER membrane protein complex (EMC), are essential for stabilization of immature Rh1 in an earlier step than that at which another Rh1-specific chaperone (NinaA) acts. dPob/EMC3 localizes to the ER and associates with EMC1 and calnexin.

GPI biosynthesis is essential for rhodopsin sorting at the trans-Golgi network in Drosophila photoreceptors.

Sorting of integral membrane proteins plays crucial roles in establishing and maintaining the polarized structures of epithelial cells and neurons. However, little is known about the sorting mechanisms of newly synthesized membrane proteins at the trans-Golgi network (TGN). To identify which genes are essential for these sorting mechanisms, we screened mutants in which the transport of Rhodopsin 1 (Rh1), an apical integral membrane protein in Drosophila photoreceptors, was affected.

Regulation of arrestin translocation by Ca2+ and myosin III in Drosophila photoreceptors.

Upon illumination several phototransduction proteins translocate between cell body and photosensory compartments. In Drosophila photoreceptors arrestin (Arr2) translocates from cell body to the microvillar rhabdomere down a diffusion gradient created by binding of Arr2 to photo-isomerized metarhodopsin. Translocation is profoundly slowed in mutants of key phototransduction proteins including phospholipase C (PLC) and the Ca(2+)-permeable transient receptor potential channel (TRP), but how the phototransduction cascade accelerates Arr2 translocation is unknown.

Arrestin translocation is stoichiometric to rhodopsin isomerization and accelerated by phototransduction in Drosophila photoreceptors.

Upon illumination, visual arrestin translocates from photoreceptor cell bodies to rhodopsin and membrane-rich photosensory compartments, vertebrate outer segments or invertebrate rhabdomeres, where it quenches activated rhodopsin. Both the mechanism and function of arrestin translocation are unresolved and controversial. In dark-adapted photoreceptors of the fruitfly Drosophila, confocal immunocytochemistry shows arrestin (Arr2) associated with distributed photoreceptor endomembranes.

Ca2+-dependent metarhodopsin inactivation mediated by calmodulin and NINAC myosin III.

Phototransduction in flies is the fastest known G protein-coupled signaling cascade, but how this performance is achieved remains unclear. Here, we investigate the mechanism and role of rhodopsin inactivation. We determined the lifetime of activated rhodopsin (metarhodopsin = M( *)) in whole-cell recordings from Drosophila photoreceptors by measuring the time window within which inactivating M( *) by photoreisomerization to rhodopsin could suppress responses to prior illumination.

Calcium-activated Myosin V closes the Drosophila pupil.

Approximately 40 years ago, an elegant automatic-gain control was revealed in compound eye photoreceptors: In bright light, an assembly of small pigment granules migrates to the cytoplasmic face of the photosensitive membrane organelle, the rhabdomere, where they attenuate waveguide propagation along the rhabdomere. This migration results in a "longitudinal pupil" that reduces rhodopsin exposure by a factor of 0.8 log units.

Myosin V, Rab11, and dRip11 direct apical secretion and cellular morphogenesis in developing Drosophila photoreceptors.

Sensory neuron terminal differentiation tasks apical secretory transport with delivery of abundant biosynthetic traffic to the growing sensory membrane. We recently showed Drosophila Rab11 is essential for rhodopsin transport in developing photoreceptors and asked here if myosin V and the Drosophila Rab11 interacting protein, dRip11, also participate in secretory transport. Reduction of either protein impaired rhodopsin transport, stunting rhabdomere growth and promoting accumulation of cytoplasmic rhodopsin.