Published: March 29th, 2013
A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.
The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.
1. Preparing Flies for Dissection
2. Preparation of Oocytes for Imaging Using an Upright Compound Microscope
3. Preparation of Oocytes for Imaging Using an Inverted Compound Microscope
4. Live Imaging
5. Troubleshooting and Common Problems
Note: Some confocal software packages allow for cell tracking, but in general it is better to abandon imaging of drifting oocytes and instead use another specimen.
Imaging of GFP-labeled proteins will vary depending on how strongly the tagged protein is expressed. A mid-stage oocyte expressing reticulon-like 1 (Rtnl-1), exon tagged with GFP8,9 produces strong signal. Rtnl-1 appears to co-localize with the long microtubules present during ooplasmic streaming10 (Figure 1A). Rtnl-1 is a good marker for observing streaming, and is also an indicator of microtubule organization in late stage oocytes.
Ooplasmic streaming in a wildtype egg chamber is evident as noticeable movement of autofluorescent yolk vesicles when using a capture rate of one frame every 10 sec (Figure 1B). A maximum projection of five frames can be generated to mark the path of moving vesicles.
Figure 1. Live imaging of both autofluorescent and GFP-tagged structures. A) A single time-lapse image of a stage 11 oocyte expressing Rtnl1-GFP at 400X. Rtnl1-GFP fibers move with a wave-like motion in streaming oocytes. B) A five frame maximum projection of a stage 10B egg chamber in which the autofluorescent yolk vesicles were imaged every 10 sec for 50 sec total, at 400X magnification. Scale bar = 30 μm.
Drosophila oocytes are a versatile model system for addressing a variety of questions related to the function and organization of cells and subcellular components. A complete understanding of protein function requires the monitoring of both spatial and temporal aspects of protein behavior. Advances in both imaging systems and the genetic tools available to Drosophilists make it possible for even small labs to perform high quality imaging experiments in living oocytes. Here I outline a protocol to analyze the behavior of GFP-tagged or autofluorescent proteins and structures during mid- to late-oogenesis that can be used in conjunction with a variety of genetic backgrounds to probe protein function.
In this protocol I describe the process by which oocytes are prepared for imaging and the basic confocal settings that will enable acquisition of time lapse video of fluorescent proteins and structures. Additional details on oocyte preparation are described by Weil et al.6 A wide variety of fly strains expressing proteins that have been endogenously tagged via exon-trapping are available8, and infinitely more protein variants can be engineered and reintroduced to flies.
In working with living tissue, the health of the specimens is of paramount importance. Egg chambers from stage 10B can complete oogenesis in an essentially autonomous fashion11, making them ideal for short term imaging studies. Care must be taken at several key steps to get high quality data. During dissection it is important to manipulate ovaries and oocytes as little as possible, and to minimize the amount of time between dissection and the completion of imaging. Ideally, a small dissection station should be located in the same room that houses the confocal scope, and only a few egg chambers at a time should be imaged. The number recommended here (5-8) ensures that dissection time is minimized while maximizing the likelihood of obtaining good quality images. I recommend capturing a short series of images to assess image quality and confirm that capture settings are optimized, before capturing a longer time lapse sequence. Most software allows individual frames to be saved, and these images can then be analyzed in a variety of ways using ImageJ12 or other software.
I have nothing to disclose.
This work was supported by grant GM096076 from the NIH AREA program.
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