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1.
J Cell Sci ; 129(7): 1416-28, 2016 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-26906417

RESUMO

During bidirectional transport, individual cargoes move continuously back and forth along microtubule tracks, yet the cargo population overall displays directed net transport. How such transport is controlled temporally is not well understood. We analyzed this issue for bidirectionally moving lipid droplets in Drosophila embryos, a system in which net transport direction is developmentally controlled. By quantifying how the droplet distribution changes as embryos develop, we characterize temporal transitions in net droplet transport and identify the crucial contribution of the previously identified, but poorly characterized, transacting regulator Halo. In particular, we find that Halo is transiently expressed; rising and falling Halo levels control the switches in global distribution. Rising Halo levels have to pass a threshold before net plus-end transport is initiated. This threshold level depends on the amount of the motor kinesin-1: the more kinesin-1 is present, the more Halo is needed before net plus-end transport commences. Because Halo and kinesin-1 are present in common protein complexes, we propose that Halo acts as a rate-limiting co-factor of kinesin-1.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Cinesinas/metabolismo , Gotículas Lipídicas/metabolismo , Animais , Animais Geneticamente Modificados , Transporte Biológico , Drosophila melanogaster/metabolismo
2.
Cell ; 156(1-2): 109-22, 2014 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-24439372

RESUMO

Interactions between commensals and the host impact the metabolic and immune status of metazoans. Their deregulation is associated with age-related pathologies like chronic inflammation and cancer, especially in barrier epithelia. Maintaining a healthy commensal population by preserving innate immune homeostasis in such epithelia thus promises to promote health and longevity. Here, we show that, in the aging intestine of Drosophila, chronic activation of the transcription factor Foxo reduces expression of peptidoglycan recognition protein SC2 (PGRP-SC2), a negative regulator of IMD/Relish innate immune signaling, and homolog of the anti-inflammatory molecules PGLYRP1-4. This repression causes deregulation of Rel/NFkB activity, resulting in commensal dysbiosis, stem cell hyperproliferation, and epithelial dysplasia. Restoring PGRP-SC2 expression in enterocytes of the intestinal epithelium, in turn, prevents dysbiosis, promotes tissue homeostasis, and extends lifespan. Our results highlight the importance of commensal control for lifespan of metazoans and identify SC-class PGRPs as longevity-promoting factors.


Assuntos
Proteínas de Transporte/metabolismo , Drosophila melanogaster/microbiologia , Drosophila melanogaster/fisiologia , Imunidade Inata , Longevidade/imunologia , Modelos Animais , Animais , Citocinas/imunologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/imunologia , Disbiose/imunologia , Disbiose/microbiologia , Fatores de Transcrição Forkhead/metabolismo , Homeostase , Intestinos/imunologia , Intestinos/microbiologia , Transcriptoma
3.
J Vis Exp ; (40)2010 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-20613707

RESUMO

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000 g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.


Assuntos
Centrifugação/métodos , Drosophila/química , Drosophila/embriologia , Ágar/química , Animais , Embrião não Mamífero/química , Organelas/química
4.
Cell ; 135(6): 1098-107, 2008 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-19070579

RESUMO

The microtubule motor kinesin-1 plays central roles in intracellular transport. It has been widely assumed that many cellular cargos are moved by multiple kinesins and that cargos with more motors move faster and for longer distances; concrete evidence, however, is sparse. Here we rigorously test these notions using lipid droplets in Drosophila embryos. We first employ antibody inhibition, genetics, biochemistry, and particle tracking to demonstrate that kinesin-1 mediates plus-end droplet motion. We then measure how variation in kinesin-1 expression affects the forces driving individual droplets and estimate the number of kinesins actively engaged per droplet. Unlike in vitro, increased motor number results in neither longer travel distances nor higher velocities. Our data suggest that cargos in vivo can simultaneously engage multiple kinesins and that transport properties are largely unaffected by variation in motor number. Apparently, higher-order regulatory mechanisms rather than motor number per se dominate cargo transport in vivo.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/citologia , Drosophila/metabolismo , Cinesinas/metabolismo , Metabolismo dos Lipídeos , Animais , Transporte Biológico , Dineínas/metabolismo , Embrião não Mamífero/metabolismo , Lipídeos/química , Óptica e Fotônica/instrumentação , Óptica e Fotônica/métodos
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