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

Abstract

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.

Male gonad-enriched microRNAs function to control sperm production inC. elegans

“…To measure meiotic progression, we exposed control and mutant males to an EdU pulse for 2.5 hours followed by a 10-hour chase. In this way, the progression of a set of cells through meiosis can be monitored (Jaramillo-Lambert et al 2007;Kocsisova et al 2018;Almanzar et al 2021;Cahoon and Libuda 2021). We investigated the rate of meiotic progression in the mir-235; mir-58.1 mir-83 him-8; mir-4807-4810.1 multiply mutant males, which displayed the strongest reduction in sperm number and in transition zone length (Figure 4A and 5B).…”