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Endogenously tagged rab proteins: a resource to study membrane trafficking in Drosophila.

ABSTRACT: Membrane trafficking is key to the cell biological mechanisms underlying development. Rab GTPases control specific membrane compartments, from core secretory and endocytic machinery to less-well-understood compartments. We tagged all 27 Drosophila Rabs with YFP(MYC) at their endogenous chromosomal loci, determined their expression and subcellular localization in six tissues comprising 23 cell types, and provide this data in an annotated, searchable image database. We demonstrate the utility of these lines for controlled knockdown and show that similar subcellular localization can predict redundant functions. We exploit this comprehensive resource to ask whether a common Rab compartment architecture underlies epithelial polarity. Strikingly, no single arrangement of Rabs characterizes the five epithelia we examine. Rather, epithelia flexibly polarize Rab distribution, producing membrane trafficking architectures that are tissue- and stage-specific. Thus, the core machinery responsible for epithelial polarization is unlikely to rely on polarized positioning of specific Rab compartments.

SUBMITTER: Dunst S

PROVIDER: S-EPMC4431667 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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Endogenously tagged rab proteins: a resource to study membrane trafficking in Drosophila.

Dunst Sebastian S Kazimiers Tom T von Zadow Felix F Jambor Helena H Sagner Andreas A Brankatschk Beate B Mahmoud Ali A Spannl Stephanie S Tomancak Pavel P Eaton Suzanne S Brankatschk Marko M

Developmental cell 20150501 3

Membrane trafficking is key to the cell biological mechanisms underlying development. Rab GTPases control specific membrane compartments, from core secretory and endocytic machinery to less-well-understood compartments. We tagged all 27 Drosophila Rabs with YFP(MYC) at their endogenous chromosomal loci, determined their expression and subcellular localization in six tissues comprising 23 cell types, and provide this data in an annotated, searchable image database. We demonstrate the utility of t ...[more]

PMID: 25942626

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Project description:UnlabelledRas-associated binding (Rab) proteins and Rab-associated proteins are key regulators of vesicle transport, which is essential for the delivery of proteins to specific intracellular locations. More than 60 human Rab proteins have been identified, and their function has been shown to depend on their interaction with different Rab-associated proteins regulating Rab activation, post-translational modification and intracellular localization. The number of known inherited disorders of vesicle trafficking due to Rab cycle defects has increased substantially during the past decade. This review describes the important role played by Rab proteins in a number of rare monogenic diseases as well as common multifactorial human ones. Although the clinical phenotype in these monogenic inherited diseases is highly variable and dependent on the type of tissue in which the defective Rab or its associated protein is expressed, frequent features are hypopigmentation (Griscelli syndrome), eye defects (Choroideremia, Warburg Micro syndrome and Martsolf syndrome), disturbed immune function (Griscelli syndrome and Charcot-Marie-Tooth disease) and neurological dysfunction (X-linked non-specific mental retardation, Charcot-Marie-Tooth disease, Warburg Micro syndrome and Martsolf syndrome). There is also evidence that alterations in Rab function play an important role in the progression of multifactorial human diseases, such as infectious diseases and type 2 diabetes. Rab proteins must not only be bound to GTP, but they need also to be 'prenylated'-i.e. bound to the cell membranes by isoprenes, which are intermediaries in the synthesis of cholesterol (e.g. geranyl geranyl or farnesyl compounds). This means that isoprenylation can be influenced by drugs such as statins, which inhibit isoprenylation, or biphosphonates, which inhibit that farnesyl pyrophosphate synthase necessary for Rab GTPase activity.ConclusionAlthough protein-trafficking disorders are clinically heterogeneous and represented in almost every subspeciality of pediatrics, the identification of common pathogenic mechanisms may provide a better diagnosis and management of patients with still unknown Rab cycle defects and stimulate the development of therapeutic agents.

Project description:The outer segment is a specialized compartment of vertebrate rod and cone photoreceptor cells where phototransduction takes place. In rod cells it consists of an organized stack of disks enclosed by a separate plasma membrane. Although most proteins involved in phototransduction have been identified and characterized, little is known about the proteins that are responsible for outer segment structure and renewal. In this study we used a tandem mass spectrometry-based proteomics approach to identify proteins in rod outer segment preparations as an initial step in defining their roles in photoreceptor structure, function, renewal, and degeneration. Five hundred and sixteen proteins were identified including 41 proteins that function in rod and cone phototransduction and the visual cycle and most proteins previously shown to be involved in outer segment structure and metabolic pathways. In addition, numerous proteins were detected that have not been previously reported to be present in outer segments including a subset of Rab and SNARE proteins implicated in vesicle trafficking and membrane fusion. Western blotting and immunofluorescence microscopy confirmed the presence of Rab 11b, Rab 18, Rab 1b, and Rab GDP dissociation inhibitor in outer segments. The SNARE proteins, VAMP2/3, syntaxin 3, N-ethylmaleimide-sensitive factor, and Munc 18 detected in outer segment preparations by mass spectrometry and Western blotting were also observed in outer segments by immunofluorescence microscopy. Syntaxin 3 and N-ethylmaleimide- sensitive factor had a restricted localization at the base of the outer segments, whereas VAMP2/3 and Munc 18 were distributed throughout the outer segments. These results suggest that Rab and SNARE proteins play a role in vesicle trafficking and membrane fusion as part of the outer segment renewal process. The data set generated in this study is a valuable resource for further analysis of photoreceptor outer segment structure and function.

Dataset Information

Endogenously tagged rab proteins: a resource to study membrane trafficking in Drosophila.

Publications

Endogenously tagged rab proteins: a resource to study membrane trafficking in Drosophila.

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