Freeze Crack ~UPD~ C Elegans Labs
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The C. elegans germline makes an excellent model for studying meiosis, in part due to the ease of conducting cytological analyses on dissected animals. Whole mount preparations preserve the structure of meiotic nuclei, and importantly, each gonad arm contains all stages of meiosis, organized in a temporal-spatial progression that makes it easy to identify nuclei at different stages. Adult hermaphrodites have two gonad arms, each organized as a closed tube with proliferating germline stem cells at the distal closed end and cellularized oocytes at the proximal open end, which join in the center at the uterus. Dissection releases one or both gonad arms from the body cavity, allowing the entirety of meiosis to be visualized. Here, a common protocol for immunofluorescence against a protein of interest is presented, followed by DAPI staining to mark all chromosomes. Young adults are immobilized in levamisole and quickly dissected using two syringe needles. After germline extrusion, the sample is fixed before undergoing a freeze crack in liquid nitrogen, which helps permeabilize the cuticle and other tissues. The sample can then be dehydrated in ethanol, rehydrated, and incubated with primary and secondary antibodies. DAPI is added to the sample in the mounting medium, which allows reliable visualization of DNA and makes it easy to find animals to image under a fluorescent microscope. This technique is readily adopted by those familiar with handling C. elegans after a few hours spent practicing the dissection method itself. This protocol has been taught to high-schoolers and undergraduates working in a research lab and incorporated into a course-based undergraduate research experience at a liberal arts college.
There are several different methods used for antibody staining in C. elegans (reviewed in our previous work4). To stain intact 'worms,' methods were developed to freeze and thaw in a relatively hard fixative (formaldehyde or glutaraldehyde); the freeze-thaw cycles help to crack the cuticle to allow rapid penetration of the fixative5. After fixation, the cuticle was permeabilized to allow penetration of the antibody; methods included treatment with reducing agents, collagenase, or both5-7. These treatments preserved morphology, but often reduced or destroyed antibody recognition. Alternative methods include dissection8 to allow antibody penetration.
Common problems encountered with freeze-cracking using any fixation condition are illustrated in the final figure (Figure 4). The most common problem is twisting of the sample due to relative motion of the two slides during compression. The fact that the body of the worm is twisted just posterior of the pharynx can be seen by the morphology of the stained tissues. This image also shows the problem with background staining, due to antibody sticking to the poly-lysine coating the slide. Note, however, that all of these images were collected using slides made during the first antibody staining attempts of undergraduates in a cell biology laboratory course. Even with practice, many individual worms will show the problems seen in Figure 4. However, on any one slide, worms like those seen in Figures 2 and 3 can be found and examined.
Figure 1. Outline of procedures. Flow chart indicating steps to perform the complete freeze-cracking procedure. Alternatives for varied fixation conditions and numbers of worms are indicated.
For most immunostaining experiments, embryos were fixed on slides using the freeze-crack methanol procedure and incubated with primary antibodies and fluorochrome-conjugated secondary antibodies(Leung et al., 1999); embryos were fixed for PIE-1 immunostaining as described(Mello et al., 1996). The following primary antibodies/antisera and dilutions were used: chicken anti-GFP, 1:200 (Chemicon); mouse anti-HMP-1, 1:10(Costa et al., 1998); rabbit anti-HMR-1, 1:10 (Costa et al.,1998); rabbit anti-LAD-1, 1:300(Chen et al., 2001); rabbit anti-PAR-1, 1:30 (Guo and Kemphues,1995); rabbit anti-PAR-2, 1:3(Boyd et al., 1996); mouse anti-PAR-3, 1:20 (this study); rabbit anti-PAR-6, 1:20(Hung and Kemphues, 1999);mouse anti-PIE-1, 1:10 (Mello et al.,1996); rat anti-PKC-3, 1:10(Tabuse et al., 1998); rabbit anti-PGL-1, 1:1000 (Kawasaki et al.,1998). Images of immunostained embryos were captured on a Deltavision microscope (Applied Precision) and deconvolved. Where not indicated, immunostaining observations were based on the analysis of >15 embryos at the appropriate stage.
When we set out on this project TurboID had not yet been successfully applied to identify the proximity interactors of specific proteins in C. elegans (two such studies were recently published by the Feldman and de Bono labs, [13,20]). To explore the potential for TurboID to map centrosomal protein-protein interactions we chose two target proteins for our proof of principle experiments, the PCM scaffolding protein SPD-5 and the Polo-like kinase PLK-1. As the major structural component of centrosomes in C. elegans [6,21], SPD-5 displays no detectable exchange with the cytoplasmic pool once incorporated into the centrosome . Moreover, fluorescence correlation spectroscopy has shown that SPD-5 is largely monomeric in the cytoplasm, with only a small fraction associated with the PP2A regulatory proteins RSA-1 and RSA-2 . Consistent with this, our efforts to identify SPD-5 interactors by tandem affinity purification using the localization and affinity purification (LAP) tag  were unsuccessful. In contrast, Plk1 dynamically localizes to multiple mitotic structures, with almost complete exchange at centrosomes within seconds after photobleaching. A key regulator of mitotic events, its behavior is thought to be the result of transient interactions with its numerous substrates throughout the cell [24,25]. SPD-5 and PLK-1 both therefore in different ways represent challenging subjects for interaction biochemistry. We began by generating transgenic strains for each protein expressing a GFP-TurboID fusion under endogenous regulatory sequences by Mos1 transposon mediated insertion at a defined chromosomal locus , along with corresponding control strains lacking the SPD-5/PLK-1 coding sequence (Fig 1B). Both fusions localized similarly to the endogenous protein in early embryos, with GFP:TurboID:SPD-5 distributed throughout the PCM while GFP:TurboID:PLK-1 was heavily concentrated at centrioles as well as at other cellular locations, including kinetochores and the spindle midzone (Fig 1C and 1E). Fluorescent streptavidin probes showed biotinylation signal coincident with GFP fluorescence in the same locations, indicating functionality of the biotin ligase. In contrast, there was little biotinylation signal in control strains beyond weak mitochondrial staining also seen in N2 wild-type embryos. Interestingly, biotinylation staining, both specific and non-specific, was not noticeably increased upon addition of supplemental biotin, nor were there any deleterious consequences to growing TurboID strains on biotin-producing OP50 bacteria (Embryonic viability 99.4%, n = 1890, GFP:TurboID:SPD-5; 99.9%, n = 2622 GFP:TurboID:PLK-1). We therefore performed C. elegans liquid cultures and embryo isolations under standard conditions, with three independent replicates for both experimental (GFP:TurboID:SPD-5/PLK-1) and control (GFP:TurboID) conditions.
ELP-1 localizes to body wall muscle in hermaphrodites. (A) Near the muscle cell membrane, ELP-1::GFP is prominent in lines (small arrows) oriented with the longitudinal axis of the cell. ELP-1::GFP is also associated with an array of fluorescent filaments (jagged arrow). (B) Affinity-purified antibodies against ELP-1 also stain a linear array near the cell surface. Bar represents 10 μm. Within these "lines" there often is a repeating unit of fluorescence that is apparent in both transgenic animals (C) and in antibody-stained animals (D). Bar represents 2.5 μm. (E) Dense bodies (arrowheads) are shown at the muscle cell surface by phase contrast microscopy. ELP-1::GFP expression was examined via fluorescence light microscopy (F) and the two images taken at the same focal plane were overlaid in Panel G. Bar represents 1 μm. Muscle cells that were double-stained with antibodies against ELP-1 (H, J) and PAT-3/β-integrin (MH25) (I, and J) show overlapping staining patterns. The integrin puncta are superimposed upon the more linear staining pattern of ELP-1. When the membrane is removed by the freeze-cracking procedure (outlined in white in panel J), integrin is lost and the ELP-1 antigen remains. Bar represents 10 μm.
Immunofluorescent staining of worms was carried out following the methods of Miller and Shakes (1995) . A mixed population of worms was freeze-cracked by immersion in liquid nitrogen, fixed in ice-cold methanol (15 min) and in ice-cold acetone (10 min). Animals were washed twice in PBT [PBS containing 0.1% Triton-X (v/v) and 0.1% BSA (w/v)] and stained with primary antibodies overnight at 4°C. The primary antibodies used were the affinity-purified anti-ELP-1 antibodies (diluted 1:50 in PBST with BSA) and a mouse monoclonal anti-PAT-3 antibody MH25 (a gift from Michelle Hresko, Washington University School of Medicine, St. Louis, ) (diluted 1:250 in PBST). The worms were washed three times in PBST and then incubated with the secondary antibodies: Cy2-conjugated donkey anti-rabbit [1:250] or Cy3-conjugated donkey anti-mouse [1:250] in PBT buffer overnight at 4°C. Lastly, the slides were washed three times in 1× PBT and mounted in PBT buffer. 2b1af7f3a8