Conditional immobilization for live imaging C. elegans using auxin-dependent protein depletion

The visualization of biological processes using fluorescent proteins and dyes in living organisms has enabled numerous scientific discoveries. The nematode Caenorhabditis elegans is a widely used model organism for live imaging studies since the transparent nature of the worm enables imaging of nearly all tissues within a whole, intact animal. While current techniques are optimized to enable the immobilization of hermaphrodite worms for live imaging, many of these approaches fail to successfully restrain the smaller male worms. To enable live imaging of worms of both sexes, we developed a new genetic, conditional immobilization tool that uses the auxin inducible degron (AID) system to immobilize both hermaphrodites and male worms for live imaging. Based on chromosome location, mutant phenotype, and predicted germline consequence, we identified and AID-tagged three candidate genes (unc-18, unc-104, and unc-52). Strains with these AID-tagged genes were placed on auxin and tested for mobility and germline defects. Among the candidate genes, auxin-mediated depletion of UNC-18 caused significant immobilization of both hermaphrodite and male worms that was also partially reversible upon removal from auxin. Notably, we found that male worms require a higher concentration of auxin for a similar amount of immobilization as hermaphrodites, thereby suggesting a potential sex-specific difference in auxin absorption and/or processing. In both males and hermaphrodites, depletion of UNC-18 did not largely alter fertility, germline progression, nor meiotic recombination. Finally, we demonstrate that this new genetic tool can successfully immobilize both sexes enabling live imaging studies of sexually dimorphic features in C. elegans. ARTICLE SUMMARY C. elegans is a powerful model system for visualizing biological processes in live cells. In addition to the challenge of suppressing the worm movement for live imaging, most immobilization techniques only work with hermaphrodites. Here, we describe a new genetic immobilization tool that conditionally immobilizes both worm sexes for live imaging studies. Additionally, we demonstrate that this tool can be used for live imaging the C. elegans germline without causing large defects to germline progression or fertility in either sex.

immobilization tool that conditionally immobilizes both worm sexes for live imaging studies. 1 Additionally, we demonstrate that this tool can be used for live imaging the C. elegans germline 2 without causing large defects to germline progression or fertility in either sex. 3

INTRODUCTION 1
The discovery of green fluorescent protein (GFP) and subsequent proliferation of both 2 engineered and additional fluorescent proteins has revolutionized biological research by 3 enabling direct visualization of the biological processes occurring within living organisms. This 4 breakthrough changed how scientists viewed cellular processes from snapshots in time 5 generated by fixed images to the complete dynamic picture occurring in real-time within the 6 organism (CHALFIE 2009). Live imaging experiments are now performed in nearly every model 7 system spanning all kingdoms of life and can be found in a diverse range of biological fields 8 from molecular biology to systems and synthetic biology (REMINGTON 2011). 9 For decades, researchers have been curating protocols and technologies to facilitate 10 imaging live Caenorhabditis elegans worms. This small, soil-dwelling nematode is completely 11 transparent, thereby making it ideal for live imaging since the intact worm can be placed on a 12 slide and imaged without requiring any tissue dissection or tissue clarification. However, 13 immobilizing the worms on slides can be challenging since wild type worms are highly mobile 14 and spend their life traveling in a sinusoidal pattern across plates eating lawns of bacteria. 15 Further, worms display phototaxis when exposed to light stimulus, avoiding ultraviolet (340nm), 16 blue (470nm), and green (500nm) wavelengths of light (WARD et al. 2008). This phototaxis 17 behavior is problematic for live imaging since most of these experiments visualize proteins with 18 GFP, which is typically excited by 488nm light. Thus, this stimulus used to excite GFP also 19 triggers avoidance behavior from the worms. 20 To enable immobilization of C. elegans, custom microfluidic devices have been 21 successfully used to keep worms immobile and alive for imaging experiments anywhere from 22 hours to days (SAN-MIGUEL AND LU 2013). While these devices work very well at gently 23 immobilizing the worms, the generation of these devices requires a fabrication facility with the 24 appropriate equipment and expertise in photolithography, which is used to generate the 25 negative molds that create the plastic microfluidic chips. Thus, for many labs without access to 26 worms. Then, worms were carefully pipetted with low bind tips (Genesee Scientific, cat no. 23-1 121RS) to NGM plates. Worms were monitored for recovery at 24 hours post imaging. Any 2 worms that did not survive after the recovery were excluded from any analysis. For experiments 3 with worms imaged at 60x, 0.08% tricaine (Ethyl 3-aminobenzoate methanesulfonate; Sigma-4 Aldrich, cat. no. E10521-50G) and 0.008% tetramisole hydrochloride (Sigma-Aldrich, cat. no. 5 T1512-10G) were added to the M9 imaging media and agarose pads. The addition of the 6 anesthetics did not interfere with the recovery of the worms post imaging. 7 8 Immunohistochemisty. Immunofluorescence was performed as described in (LIBUDA et al. 9 2013). Briefly, gonads were dissected in egg buffer with 0.1% Tween20 on to VWR superfrost 10 plus slides from 18-24hr post L4 progeny worms from parental worms on NGM only plates or 11 NGM plates with 1mM or 10mM auxin. Dissected gonads were fixed in 5% paraformaldehyde 12 for 5 minutes, flash frozen in liquid nitrogen, and fixed for 1 minute in 100% methanol at -20ºC. 13 Slides were washed three times in PBS+0.1% Tween20 (PBST) for 5 minutes each and incubated overnight in a humid chamber with a parafilm cover. Slides were then washed three 17 times in PBST for 10 minutes each and incubated with secondary antibodies (goat anti-rabbit 18 AlexaFluor488, ThermoFisher, cat. no. A11034) at 1:200 dilution for 2 hours in a humid 19 chamber with a parafilm cover. Slides were washed two times in PBST then incubated with 20 2µg/mL DAPI for 15-20 minutes in a humid chamber. Prior to mounting slides were washed 21 once more in PBST for 10 minutes and mounted using Vectashield with a 22x22mm coverslip 22 (no. 1.5). Slides were sealed with nail polish and stored at 4ºC until imaged. 23 EdU staining was performed as described in (ALMANZAR et al. 2021) with minor changes. 24 Briefly, worms were washed three times in PBS + 0.1% TritonX. Then, worms were incubated 25 for 1.5 hours nutating in PBS + 0.1% TritonX with 4mM 5-Ethynyl-2'-deoxyuridine (EdU), which 26 was diluted from a stock 10mM EdU in distilled water from the Invitrogen Click-iT Edu Alexa 1 Fluor 488 imaging kit (Invitrogen, cat. no. C10338). Worms were washed two times in PBS + 2 0.1% TritonX for 1-2 minutes each then plated onto either NGM or NGM with 1mM or 10mM 3 auxin plates. Time was noted when worms were removed from EdU to start chase time course 4 of the EdU staining. Both male and hermaphrodite worms were dissected and fixed as 5 described above at 0, 10 and 24 hours post removal from EdU and only hermaphrodites were 6 dissected at 48 hours post EdU removal. At each time point 15-20 worms were dissected and 7 washed three times in PBST. Then, slides were either immediately processed with the Click-iT 8 reaction or held in PBST overnight at 4ºC and the Click-iT reaction was performed the next day. 9 The Click-iT reaction was performed as described in the kit manual except the volumes in the 10 Click-iT reaction mix were reduced. All slides were incubated with 50µL of the Click-iT reaction 11 mix containing 43µL 1x Click-iT reaction buffer, 2 µL CuSO4, 0.2µL AlexaFluor488, and 5µL 12 reaction buffer additive. Slides were incubated for 30 minutes in a humid chamber with a 13 parafilm cover. Then washed three times in PBST for 10 minutes each and incubated with 14 2µg/mL DAPI in water for 20 minutes with a parafilm cover. Slides were washed once in PBST 15 for 10 minutes then mounted in Vectashield using 22x22mm coverslip (no. 1.5) and sealed 16 using nail polish. All slides were stored at 4ºC and imaged within gonad was analyzed for RAD-51 foci per nucleus, which was determined by DAPI morphology. 7 The pachytene region was defined by the first row that did not contain more than 1-2 transition 8 zone half-moon like nuclei and the last row that contained all pachytene nuclei with the 9 occasional single diplotene nucleus. These criteria were used for establishing the pachytene 10 region in both hermaphrodites and males. To determine the position of the EdU staining within 11 the germline, the EdU gonad images were max intensity z-projected in FIJI. Then, the position 12 of the EdU straining front was determined by the last nucleus within the germline labeled with 13 EdU. Max intensity z-projection montages and movies were made in FIJI, and only GFP::SYP-2 14 movies were stabilized using the FIJI plugin "StackRegJ_" 15 (https://research.stowers.org/imagejplugins/). This stabilization was necessary to reduce the 16 motion of the germline inside in the worm and generate a stable movie for viewing. Additionally, 17 photobleach correction was applied to the GFP::SYP-2 male movie using the photobleach 18 correction application in FIJI. All images and movies have been slightly adjusted for brightness 19 and contrast using FIJI. 20 21 Statistics. All statistical tests were performed using Prism. For the worm tracking assay, the 22 average speed of each worm was calculated in the FIJI plugin "wrMTrck" and the multiple 23 comparisons Kruskal-Wallis test was performed to determine statistical differences between 24 each genotype assayed. For the fertility assay, brood size was determined by summing living 25 progeny and dead eggs and statistical differences were determined using 2way ANOVA with 26 Dunnett's multiple comparisons test. For the RAD-51 and DAPI body quantification, statistical 1 differences were determined using the nonparametric Mann-Whitney test. Each test used is 2 indicated in the Results section next to the reported p-value and all n values are reported in the 3 figure legends. 4 5 Data Availability. All strains are available for request. All supplemental materials are available 6 at Figshare. Figure S1 shows a diagram of the steps used to immobilize worms for live imaging. 7 Table S1 is contains all the primers and sgRNAs used for CRIPSR generations of unc-18::AID, 8 unc-104::AID and AID::unc-52. Movie S1 is a brightfield timelapse of an immobilized 9 hermaphrodite at 10x magnification. Movie S2 is a timelapse of SYP-2::GFP in an immobilized 10 hermaphrodite at 60x magnification. Movie S3 is an entire germline view of SYP-2::GFP at 60x 11 in an immobilized hermaphrodite. Movie S4 is a brightfield timelapse of an immobilized male at 12 20x magnification. Movie S5 is a timelapse of SYP-2::GFP in an immobilized male at 60x 13 magnification. Movie S6 is an entire germline view of SYP-2::GFP at 60x in an immobilized 14 male. 15

Reversible paralysis from auxin-dependent depletion of UNC-18 and UNC-104 18
To conditionally immobilize worms, we used the auxin-inducible degron (AID) system, which has 19 been used in multiple different worm tissues to selectively deplete proteins of interests at 20 protein Transport Inhibitor Response 1 (TIR1), which can be regulated using tissue-specific 24 promotors; and (3) the plant hormone auxin, which can be absorbed externally by the worms 25 ( Figure 1A). Auxin exposure promotes the binding of TIR1 to the degron sequence. TIR1 is able 26 to interact with components of the endogenous SCF E3 ubiquitin ligase complex generating a 1 functional complex that can ubiquitinate the degron tagged protein and target it for proteasome-2 mediated degradation (NATSUME AND KANEMAKI 2017). Additionally, this protein degradation is 3 completely reversible once the worms are removed from auxin, such that after a period of time 4 the degron tagged protein can return to wild type levels (ZHANG et al. 2015). 5 To conditionally immobilize worms, we combined the AID system with genes that cause 6 severe worm paralysis when mutated and applied this immobilization to visualize the germline in 7 live animals. To narrow down the candidate list of genes, we focused on Chromosomes X and II 8 since we wanted to use this system to study the C. elegans germline and these chromosomes 9 are mainly devoid of germline expressed genes (REINKE et al. 2000). We then obtained mutants 10 from all the identified genes on these chromosomes that were indicated on the CGC as being 11 homozygous viable and severely paralyzed. Additionally, we avoided any genes that had the 12 potential to alter the germline, vulval development, or vulval function. From these candidates, 13 we selected three genes to tag with the AID sequence: unc-104, unc-18, and unc-52 (  Using CRISPR/Cas9, each of these genes were tagged with the AID sequence and 20 genetic crosses were performed to incorporate TIR1. Two different TIR1 constructs were used 21 in this study: 1) rgef-1p::TIR1, which expresses only in neurons; and, 2) eft-3p::TIR1, which has 22 pan-somatic expression. For all experiments, we found that animals needed to be grown for a 23 single generation on plates containing nematode growth media (NGM) with auxin to exhibit the 24 strongest paralysis phenotype (see Methods). 25 We first assayed unc-104::AID and unc-18:AID using the neuron-specific expression of 1 TIR1 (rgef-1p::TIR1) and AID::unc-52 with pan-somatic expression of TIR1 (eft-3p::TIR1) 2 ( Figure 1B). AID::unc-52 displayed no changes in mobility when grown on auxin plates, thus this 3 gene was excluded from any further studies. Both unc-104::AID and unc-18::AID display 4 significant decreases in mobility on auxin plates, which we assayed by tracking the motion of 5 the worms on normal NGM plates and NGM with 1mM auxin ( Figure 1C, P<0.0001, Kruskal-6 Wallis). We noticed that unc-104::AID did not display as strong of a mobility defect as unc-7 18::AID when depleted using the neuron specific TIR1 (median average speed: 3.604 and 2.927 8 pixels/second, respectively). Moreover, depleting UNC-104::AID with the pan-somatic eft- 3 9 driven TIR1 exhibited a similar degree of mobility defects to neuron-specific depletion of unc-10 104::AID with the rgef-1 driven TIR1 (P>0.999, Kruskal-Wallis multiple comparisons test). 11 Although the effects of each driver on UNC-104::AID depletion were statistically 12 indistinguishable, the median average speed was slightly lower with the pan-somatic eft-3 driver 13 (neuron-specific rgef-1 driven TIR1: 3.604 pixels/second; pan-somatic eft-3 driven TIR1: 2.625 14 pixels/second). 15 With this worm immobilization technique, worms can be recovered post-imaging and 16 assayed for viability off auxin. We found that upon removal from auxin for 18-24 hours both unc-17 104::AID and unc-18::AID worms recovered some degree of normal motion ( Figure 1C). unc-18 104::AID with TIR1 driven by both the neuron-specific rgef-1 promoter or the pan-somatic eft-3 19 promoter recovered motion to levels statistically indistinguishable from wild type, but the overall 20 median average speed in these animals was lower than wild type worms (wild type: 20.34 21 pixel/second; rgef-1p:TIR1; unc-104::AID: 7.669 pixel/second; eft-3p::TIR1; unc-104::AID: 10. 16 22 pixel/second). unc-18::AID worms were able to recover some motion off auxin compared to the 23 unc-18::AID in the presence of auxin; however, this recovered motion was significantly slower 24 than wild type worms (median average speed for unc-18::AID on auxin: 2.927 pixel/second; 25 unc-18::AID recovery: 5.634 pixel/second; wild type: 20.34 pixel/second; P<0.0001, Kruskal-26 Wallis). Taken together, these motion recovery experiments suggest that unc-18::AID may 1 require more time to completely recover wild type motion compared to the unc-104::AID worms. 2 Overall, both unc-104::AID and unc-18::AID are able to both partially recover movement after 3 removal from the auxin treatment. 4

UNC-104::AID depletion has slight behavioral and fertility defects 6
To examine the effectiveness of this conditional immobilization system for live imaging, we 7 focused on implementing this system for live imaging the C. elegans germline. Since the effects 8 of UNC-104 or UNC-18 loss on germline function are unknown, we examined multiple aspects 9 of germline biology to determine if a loss of UNC-104 or UNC-18 causes germline-specific 10 defects. We began by assaying the fertility of hermaphrodite worms containing unc-104::AID 11 and unc-18::AID under both depletion (in the presence of auxin) and wildtype conditions ( Figure  12 2). For these fertility assays, we counted the number of living progeny, dead eggs and 13 unfertilized eggs from hermaphrodite worms that were moved each day for five days to new 14 plates (see Methods). Scoring fertility over multiple days allowed for observation of most of the 15 hermaphrodite reproductive lifespan, which begins with a large abundance of living progeny and 16 subsequently ends with unfertilized eggs once the hermaphrodite sperm is depleted (WARD AND 17 CARREL 1979). To compare the fertility of each genotype, we calculated brood size of each 18 worm, which is the sum of living progeny and dead eggs. 19 In the absence of auxin, all genotypes displayed brood sizes that were indistinguishable 20 from wild type worms through all five days of scoring ( Figure 2A). In particular, wild type worms 21 display similar brood sizes both on and off auxin throughout all five days. Additionally, the 22 number of dead eggs was also similar between the on and off auxin wild type worms suggesting 23 that oocyte viability is not being altered ( Figure 2B). Further, the cumulative sum of the brood 24 size for wild type both on and off auxin (no auxin average cumulative brood size for 12 worms:  ensures a continued depletion of UNC-18::AID throughout the imaging. Using M9 media, we 24 made 7-9% agarose pads and this pad was then transferred and carefully laid over the top of 25 the worm. Whatman paper was used to wick away any excess liquid between the coverslip and 26 agarose pad. Then, the coverslip-worm-agar pad sandwich was sealed to a slide using Vaseline 1 to prevent drying out of the agarose pad during live imaging ( Figure S1). 2 For brightfield imaging, worms were imaged every 90 seconds for a total of 60 minutes. 3 During that time, mounted hermaphrodite worms are able to subtly move their heads and some 4 of the worms continued to ovulate oocytes that would stack up in a pile next to the worm (2-3 5 oocytes/60 min, 7 worms, Figure 4A, Movie S1). This continued ovulation of the hermaphrodite 6 is an excellent indicator that germline progression is not being impeded by having the worms 7 mounted underneath an agar pad. Additionally, the movement of the germline can be seen by 8 directly looking at the germline nuclei using a fluorescently tagged component of the 9 synaptonemal complex (SC), SYP-2::GFP, which is a meiotic chromosome structure that 10 assembles between homologous chromosomes in the germline from late transition zone to 11 diakinesis ( Figure 4B, Movie S2). To minimize motion of the worm at 60x, we included 0.08% 12 tricaine and 0.008% tetramisole anesthetics, which is at a concentration nearly 10-fold lower One of the unique features of our conditional immobilization system using unc-18::AID; gref-2 1p::TIR1 is that it works well with whole, intact male worms. From our immobilization 3 experiments, we found that male worms require a higher concentration of auxin to induce a 4 more robust immobilization than hermaphrodite worms ( Figure 5A). Male worms grown on 5 plates containing 10mM auxin exhibited a more severe immobilization phenotype and a tighter 6 distribution of average speeds than male worms on 1mM auxin (median average speed 1.251 7 and 4.809 pixels/second, respectively). Further, male worms removed from auxin for 18-24 8 hours recovered sinusoidal movements close to wild type levels regardless of the initial auxin 9 concentration. These results suggest a sexual dimorphic difference in auxin sensitivity in C. 10 elegans, where male worms may absorb and process auxin differently than hermaphrodite 11 worms. Future studies focused on auxin processing in both C. elegans sexes may reveal the 12 mechanisms behind this intriguing sexual dimorphism. Based on these analyses which revealed 13 a more robust immobilization at higher auxin concentrations, we proceeded with using 10mM 14 auxin to immobilize male worms for live imaging. To enable live imaging of male worms, we mounted the worms using the same steps as 12 described above for the hermaphrodite worms except we used 10mM auxin in the M9 media 13 instead of 1mM auxin to maintain depletion of UNC-18::AID during imaging ( Figure S1). All live 14 imaging experiments were performed using the same imaging settings as the hermaphrodites 15 for except for the brightfield timelapses, which due to the smaller size of the male worms were 16 captured at 20x magnification instead of 10x ( Figure 6). Notably, the same mounting and 17 immobilization method worked as efficiently to immobilize the male worms as it did with the 18 hermaphrodite worms ( Figure 6A, Movie S4). Further, using SYP-2::GFP with the unc-18::AID; 19 rgef-1::TIR1 conditional immobilization technique, we were able to observe both the directional 20 motion of germline progression and the chromosome motion within each nucleus ( Figure 6B, 21 Movie S5, S6). 22

DISCUSSION 24
The transparent nature of the C. elegans worm makes this model organism ideal for live 25 imaging studies, however, effectively and reliably immobilizing the worm without injury has been 26 a challenge for many C. elegans labs seeking to do live imaging experiments. We developed 1 and validated a new tool that enables conditional immobilization of C. elegans for live imaging 2 the germline. This conditional immobilization tool uses the auxin inducible degron system, which 3 we show works for immobilizing both hermaphrodite and male worms. Notably, we found that 4 depletion of the gene product responsible for this immobilization phenotype does not cause any 5 significant changes within the germline of either sex. Finally, with this tool we were able to 6 demonstrate that both male and hermaphrodite worms can be minimally restrained as whole 7 animals with an agar pad and imaged live for at least two hours (Movies S4 and S6). 8 The conditional immobilization technique described here to immobilize worms enhances 9 the existing toolkit for live imaging worms. While many modalities exist from microfluidic chips to 10 pharmaceuticals for immobilization of the worm, here we present an accessible genetic tool that 11 can be used to easily immobilize worms and can be implemented in any lab without needing to 12 purchase specialized equipment or use hazardous chemicals. Notably, the use of anesthetics 13 has been widespread for live imaging studies; however, male C. elegans are known to respond 14 differently than hermaphrodites to different chemicals and toxins including the widely used  Table S1 for CRISPR details). Moreover, there are likely more genes within the C. elegans 22 genome that could be tagged with AID and used to induce analogous immobilization to UNC- 18 23 depletion. Future expansion of the tool kit for the conditional immobilization of worms will 24 provide even greater genetic flexibility for use of this system in a multitude of live imaging 25 studies. 26

ACKNOWLEDGEMENTS 1
We thank the CGC for strains, which is funded by NIH Office of Research Infrastructure 2 Programs (P40 OD010440). We thank members of the Libuda Lab, especially N. Kurhanewicz,3 A. Naftaly and E. Toraason, for discussion and comments on the manuscript. This comprehensive description see the Methods section "Conditional immobilization for live 5 imaging". 6 7 Movie S1. Brightfield 10x timelapse movie of an immobilized hermaphrodite worm. A 1 hermaphrodite worm containing unc-18::AID; rgef-1p::TIR1 that was immobilized on 1mM auxin 2 and mounted under an agar pad. Images were captured every 90 seconds for 60 minutes. 3 4 Movie S2. SYP-2::GFP in an immobilized hermaphrodite worm. Timelapse images were 5 taken at 60x and images were captured every 5 seconds for 65 minutes. Movie was stabilized 6 for viewing (see Methods for details). 7 8 Movie S3. Whole germline view of SYP-2::GFP in an immobilized hermaphrodite worm. 9 Timelapse images were taken at 60x and images were captured every 5 seconds for 2 hours. 10 The start of the germline occurs out of view of the camera on the left and germline progresses 11 from left (transition zone/early pachytene) to right (late pachytene/diplotene) within the movie. 12 Then, the germline makes a U-turn bend, which is out of view of the camera. Movie S6. Whole germline view of SYP-2::GFP in an immobilized male worm. Timelapse 26 images were taken at 60x and images were captured every 5 seconds for 2 hours. Arrowhead 27 indicates the approximate start of the germline within the mid-bottom region of the movie. 28 Germline progression initially starts off moving to the left, then the germline makes a U-turn 29 bend at the transition zone on the left side of the movie. After this bend, the germline 30 progression moves from the left (early pachytene) to right (late pachytene/diplotene) within the 31 movie. The end of the germline (condensation and mature sperm) occurs farther off to the right 32 outside of the camera view at this magnification. Movie was stabilized and corrected for 33 photobleaching for viewing (see Methods for details). 34 35