Live imaging of synapse development and measuring protein dynamics using two-color fluorescence recovery after photo-bleaching at Drosophila synapses

Füger, Petra; Behrends, Laila B; Mertel, Sara; Sigrist, Stephan J.; Rasse, Tobias M.

doi:10.1038/nprot.2007.472

Cited by 59 publications

(55 citation statements)

References 35 publications

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“…Animals were anesthetized with Suprane in a custom chamber that allows imaging through the cuticle of live third instar larvae (Fuger et al 2007) with a 63· oil objective. Camera settings were adjusted so that the aggregate to be bleached was just below saturation.…”

Section: Frap and Live Imagingmentioning

confidence: 99%

Huntingtin Aggregation Kinetics and Their Pathological Role in aDrosophilaHuntington’s Disease Model

et al. 2012

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Huntington's disease is a neurodegenerative disorder resulting from expansion of a polyglutamine tract in the Huntingtin protein. Mutant Huntingtin forms intracellular aggregates within neurons, although it is unclear whether aggregates or more soluble forms of the protein represent the pathogenic species. To examine the link between aggregation and neurodegeneration, we generated Drosophila melanogaster transgenic strains expressing fluorescently tagged human huntingtin encoding pathogenic (Q138) or nonpathogenic (Q15) proteins, allowing in vivo imaging of Huntingtin expression and aggregation in live animals. Neuronal expression of pathogenic Huntingtin leads to pharate adult lethality, accompanied by formation of large aggregates within the cytoplasm of neuronal cell bodies and neurites. Live imaging and Fluorescence Recovery After Photobleaching (FRAP) analysis of pathogenic Huntingtin demonstrated that new aggregates can form in neurons within 12 hr, while preexisting aggregates rapidly accumulate new Huntingtin protein within minutes. To examine the role of aggregates in pathology, we conducted haplo-insufficiency suppressor screens for Huntingtin-Q138 aggregation or Huntingtin-Q138-induced lethality, using deficiencies covering 80% of the Drosophila genome. We identified two classes of interacting suppressors in our screen: those that rescue viability while decreasing Huntingtin expression and aggregation and those that rescue viability without disrupting Huntingtin aggregation. The most robust suppressors reduced both soluble and aggregated Huntingtin levels, suggesting toxicity is likely to be associated with both forms of the mutant protein in Huntington's disease.

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Section: Frap and Live Imagingmentioning

confidence: 99%

Huntingtin Aggregation Kinetics and Their Pathological Role in aDrosophilaHuntington’s Disease Model

et al. 2012

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“…In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution [1][2][3] . While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described 4,5,6,7,8 , the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm 1 , (2) a high survival rate of > 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days 2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels.…”

Section: Introductionmentioning

confidence: 99%

“…non-dissected) allowing observation to occur over a number of days 1 . The accompanying video details the function of individual parts of the in vivo imaging chamber 2,3 , the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.…”

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confidence: 99%

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Zhang

Füger

Hannan

et al. 2010

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Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution [1][2][3] . While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described 4,5,6,7,8 , the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm 1 , (2) a high survival rate of > 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days 2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter 1 in stable transgenic lines and the exon trap line FasII-GFP , the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

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“…The fly embryo is an excellent system for such studies as it is small and optically clear allowing deep, high-resolution imaging in vivo [1][2][3] . Other stages of fly development have proven to be less tractable, requiring anaesthetization 4 , dissection and short term culture 5,6 , or the creation of windows in the cuticle for imaging 7,8 . These manipulations usually compromise animal development in the long term or affect the animal in ways that limit imaging to short periods.…”

Section: Introductionmentioning

confidence: 99%

Imaging Through the Pupal Case of Drosophila melanogaster

Keroles

Dusseault

Liu

et al. 2014

JoVE

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The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 µm of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues. Video LinkThe video component of this article can be found at

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Live imaging of synapse development and measuring protein dynamics using two-color fluorescence recovery after photo-bleaching at Drosophila synapses

Cited by 59 publications

References 35 publications

Huntingtin Aggregation Kinetics and Their Pathological Role in aDrosophilaHuntington’s Disease Model

Huntingtin Aggregation Kinetics and Their Pathological Role in aDrosophilaHuntington’s Disease Model

<em>In vivo</em> Imaging of Intact <em>Drosophila</em> Larvae at Sub-cellular Resolution

Imaging Through the Pupal Case of <em>Drosophila melanogaster</em>

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