細胞間腔の形態形成と単一細胞レベルでのIn Vivo偏光膜生合成のためのモデルとして線虫腸: 抗体の汚損、RNAi 損失の機能解析、イメージングによるラベリング

The overall goal of this combination of standard methods is to demonstrate how the C.elegans intestine can be used for the in vivo analysis of polarized membrane biogenesis and lumen morphogenesis at the single-cell and sub-cellular level. This method can help answer key questions in the polarity and biogenesis fields such as how apical versus basolateral membrane domains and other sub-cellular asymmetries are established and maintained during and after morphogenesis. The main advantage of this model system is that this transparent single layer of only 20 cells can survive in vivo tissue chamber to dissect single cell polarized membrane and lumen biogenesis.

The techniques can be extended to the analysis of other C.Elegans morphogenesis phenotypes the focus here is on the analysis of membrane biogenesis, specifically on apical membrane and lumen biogenesis. The procedures include refined imaging techniques in fixed and alive animals to distinguish membrane domains and sub-cellular components and to identify lumen morphogenesis phenotypes in a C.elegans intestine. This video on intestinal tubulogenesis is accompanied by a video on excretory canal tubulogenesis mutually informative models where the in vivo analysis of polarized membrane biogenesis.

With regard to labeling the generation of transgenic animals with fluorescently labeled fusion proteins for live imaging is described in the accompanying video on the analysis of excretory canal tubulogenesis. Such animals can be directly used in RNAi experiments. Third generation is typically the first step in this combination of procedures.

This video starts with the alternative labeling method antibody staining for which animals must be fixed. The different labeling procedures can be combined with immunohistochemistry typically being used for the final imaging analysis. This protocol also has examples of intestine specific membrane markers promoters and other resources useful for both labeling procedures.

After demonstrating immunohistochemistry this video uses a transgenic strain that labels the intestinal apical membrane with ERM-1 GFP to search for polarity and lumen morphogenesis phenotypes using RNAi. First prepare a slide to fix the worms. Pipette 30 microliters of poly-L-lysine onto a slide and then set a second slide onto the other and drop them together to spread the solution evenly onto both.

Now peel apart the two slides and let them air dry for 30 minutes. Next place a flat metal block into a container filled with liquid nitrogen. Wash the plates with M9 solution and transfer about 10 microliters of M9 containing the worms.

Alternatively pick the worms and add M9 to the slide. Once the worms are transferred gently drop a cover slip cross ways onto them such that the cover slip over hangs one edge. Then gently press the cover slip without making any lateral movements.

Now immediately transfer the slide to the metal block in liquid nitrogen and let it freeze. After five minutes flick off the cover slip using the overhanging edge. Do this swiftly to get the proper crack of the cuticle.

Next immerse the slide in a methanol filled glass Coplin jar at minus 20 degrees Celsius. After five minutes transfer the slide to an acetone filled glass Coplin jar for another five minutes at the same tempature. The slides can be stained directly or stored at minus 20 degrees Celsius.

Remove the slide from acetone and let it air dry at room temperature. Next mark the location for a circular wall of petroleum jelly on the underside of the slide then build up the jelly wall. Do not rub off the jelly during this procedure.

Next prepare a wet chamber in a bin with wet paper towels and place the slide onto a rack within. There should be no contact between the towels and slide or between adjacent slides. Now pipette enough PBS into the jelly circle to cover the area.

Gently place a pipette tip at the edge of the circle and allow fluid to smoothly disperse over the worms. Then close the lid and wait five minutes. After five minutes tilt the slide and slowly aspirate the PBS taking care not to lose worms from the slide.

Next carefully fill the jelly circle with freshly prepared blocking solution. And let the slide incubate for 15 minutes at room tempature. After 15 minutes replace the blocking solution with blocking solution containing the primary antibody.

And incubate the slide at four degrees Celsius overnight. The next day remove the primary antibody and wash the slide by applying and removing blocking solution three times. Allow each block application to incubate for 10 minutes.

Next add the secondary antibody diluted in blocking solution and incubate the slide for an hour at room temperature. Thereafter wash the slide two times with blocking solution and then once with PBS. Then remove as much of the PBS as possible without permitting the specimen to dry out and carefully remove the jelly around the specimen.

Apply a drop of mounting medium onto the specimen followed by a cover slip and a nail polish seal. Then store the slides in the dark at four degrees Celsius. Transfer the RNAi library plate from minus 80 degrees Celsius to dry ice.

Remove the sealing tape. And transfer the clone of interest onto selective auger. Then streak out the clone and grow the bacteria over night.

Before returning the library plate to the minus 80 degrees Celsius freezer apply new sealing tape. The next day inoculate colonies from the plate into one milliliter aliquots of liquid medium containing ampicillin. Then shake them for about 14 hours at 37 degrees Celsius.

The following day seed 200 microliters per clone onto separate RNAi plates. Let the plates dry and induce the RNAi production with an overnight incubation at room tempature. A day later transfer five L4 stage worms to each RNAi plate labeled with the specific gene that is targeted.

Then transfer the plates to an incubator typically at 20 degrees Celsius. First examine control versus RNAi animals under bright light at low and high magnification. Next examine control versus RNAi animals under fluorescent light under the appropriate filters.

Systematically scan a plate with RNAi animals from the upper left to the lower right of the entire plate searching for phenotypes. Look for animals of interest. For a closer examination of the selected RNAi phenotypes focus on the intestine and use the the zoom to assess tubulogenesis and lumen morphogenesis phenotypes.

To examine the worms of interest under confocal microscopy first mount and immobilize them by hand make a thin circle of vacuum grease or petroleum jelly on a glass slide. The grease layer should be thin not thicker than 0.1 millimeter. Next add about 3.5 microliters of 10 millimolar sodium azide into the middle of the circle.

Then swiftly pick worms of interest into the solution under a dissecting microscope before the solution dries. Then gently apply a cover slip and image the animals within 30 minutes. Use mild pressure and under the microscope verify that the worms are not floating in solution.

Under the confocal microscope find the worms under low power and focus. Then use a 60 or 100X oil immersion objective to view and image the intestine. Next examine the animal under fluorescent light with the appropriate channel to select areas that display the cellular defect of interest.

Move swiftly to avoid photo bleaching. Then switch to laser scanning and restrict the image to the intestine by setting scanning boundaries at its dorsal and ventral side. Capture an image series and later merge the images into a single projection image.

This protocol describes how to molecularly analyze and visually dissect polarized membrane biogenesis in the developing C.elegans intestine where apical membrane and lumen morphogenesis coincide. Apical and basolateral membrane domains, membrane associated junctions and the intestinal lumen could be resolved and their relation to each other could be examined throughout development. These sub-cellular components could be labeled by fluorescently tagged antibodies or fluorescent fusion proteins for double or triple labeling as described in the accompanying paper.

Gross lumen defects could be quickly scanned by using a dissecting fluorescence microscope. The apical membranes cytoskeleton linker ERM-1 coupled to GFP is a robustly expressed marker useful for a RNAi screen. An array of novel intestinal lumen morphogenesis and polarity phenotypes along with their underlying gene defects were uncovered when using this marker.

Then higher magnification images acquired by a confocal laser scanning were used to more carefully examine the phenotypes to better understand the underlying defects. Some phenotypes could be quantified. Quantification was used to compare such defects and track their progression through development.

After watching this video you should have a good understanding of how to label intestinal membrane and endo membrane compartments by immuno staining and how to analyze function RNAi phenotypes of polarized membrane and lumen biogenesis.