CRISPR/Cas9 Mediated Fluorescent Tagging of SPE-38 Reveals a Complete Localization Pattern in Live Spermatozoa

Overview

Journal Biomolecules

Publisher MDPI

Specialties Biochemistry
Molecular Biology

Date 2023 May 16

PMID 37189371

Authors

Yamei Zuo

Xue Mei

Andrew Singson

Affiliations

Soon will be listed here.

Abstract

The gene encodes a four-pass transmembrane molecule that is required in sperm for fertilization. In previous work, the localization of the SPE-38 protein was examined using polyclonal antibodies on spermatids and mature amoeboid spermatozoa. SPE-38 is localized to unfused membranous organelles (MOs) in nonmotile spermatids. Different fixation conditions revealed that SPE-38 either localized to fused MOs and the cell body plasma membrane or the pseudopod plasma membrane of mature sperm. To address this localization paradox in mature sperm, CRISPR/Cas9 genome editing was used to tag endogenous SPE-38 with fluorescent wrmScarlet-I. Homozygous male and hermaphrodite worms encoding SPE-38::wrmScarlet-I were fertile indicating the fluorescent tag does not interfere with SPE-38 function during sperm activation or fertilization. We found that SPE-38::wrmScarlet-I localized to MOs in spermatids consistent with previous antibody localization. In mature and motile spermatozoa we found SPE-38::wrmScarlet-I in fused MOs, the cell body plasma membrane, and the pseudopod plasma membrane. We conclude that the localization pattern observed with SPE-38::wrmScarlet-I represents the complete distribution of SPE-38 in mature spermatozoa and this localization pattern is consistent with a hypothesized role of SPE-38 directly in sperm-egg binding and/or fusion.

Citing Articles

SPE-51, a sperm-secreted protein with an immunoglobulin-like domain, is required for fertilization in C. elegans.

Mei X, Maniates K, Looper A, Krauchunas A, Druzhinina M, Dharia S Curr Biol. 2023; 33(14):3048-3055.e6.

PMID: 37453427 PMC: 10528068. DOI: 10.1016/j.cub.2023.06.029.

References

Krauchunas A, Marcello M, Singson A . The molecular complexity of fertilization: Introducing the concept of a fertilization synapse. Mol Reprod Dev. 2016; 83(5):376-86. PMC: 4993202. DOI: 10.1002/mrd.22634. View

Singson A . Every sperm is sacred: fertilization in Caenorhabditis elegans. Dev Biol. 2001; 230(2):101-9. DOI: 10.1006/dbio.2000.0118. View

Frokjaer-Jensen C, Davis M, Ailion M, Jorgensen E . Improved Mos1-mediated transgenesis in C. elegans. Nat Methods. 2012; 9(2):117-8. PMC: 3725292. DOI: 10.1038/nmeth.1865. View

Caroti F, Thiels W, Vanslambrouck M, Jelier R . Wnt Signaling Induces Asymmetric Dynamics in the Actomyosin Cortex of the Endomesodermal Precursor Cell. Front Cell Dev Biol. 2021; 9:702741. PMC: 8484649. DOI: 10.3389/fcell.2021.702741. View

Takayama J, Onami S . The Sperm TRP-3 Channel Mediates the Onset of a Ca(2+) Wave in the Fertilized C. elegans Oocyte. Cell Rep. 2016; 15(3):625-637. DOI: 10.1016/j.celrep.2016.03.040. View

Umeda R, Satouh Y, Takemoto M, Nakada-Nakura Y, Liu K, Yokoyama T . Structural insights into tetraspanin CD9 function. Nat Commun. 2020; 11(1):1606. PMC: 7105497. DOI: 10.1038/s41467-020-15459-7. View

Geldziler B, Marcello M, Shakes D, Singson A . The genetics and cell biology of fertilization. Methods Cell Biol. 2011; 106:343-75. PMC: 3275088. DOI: 10.1016/B978-0-12-544172-8.00013-X. View

Bindels D, Haarbosch L, van Weeren L, Postma M, Wiese K, Mastop M . mScarlet: a bright monomeric red fluorescent protein for cellular imaging. Nat Methods. 2016; 14(1):53-56. DOI: 10.1038/nmeth.4074. View

Gleason E, Lindsey W, Kroft T, Singson A, LHernault S . spe-10 encodes a DHHC-CRD zinc-finger membrane protein required for endoplasmic reticulum/Golgi membrane morphogenesis during Caenorhabditis elegans spermatogenesis. Genetics. 2005; 172(1):145-58. PMC: 1456142. DOI: 10.1534/genetics.105.047340. View

10.

KELLY W, Xu S, Montgomery M, Fire A . Distinct requirements for somatic and germline expression of a generally expressed Caernorhabditis elegans gene. Genetics. 1997; 146(1):227-38. PMC: 1207937. DOI: 10.1093/genetics/146.1.227. View

11.

Mine-Hattab J . When fixation creates fiction. Elife. 2023; 12. PMC: 9934853. DOI: 10.7554/eLife.85671. View

12.

Lee C, Vitriol E, Shim S, Wise A, Velayutham R, Zheng J . Dynamic localization of G-actin during membrane protrusion in neuronal motility. Curr Biol. 2013; 23(12):1046-56. PMC: 3712510. DOI: 10.1016/j.cub.2013.04.057. View

13.

Nelson G, Ward S . Vesicle fusion, pseudopod extension and amoeboid motility are induced in nematode spermatids by the ionophore monensin. Cell. 1980; 19(2):457-64. DOI: 10.1016/0092-8674(80)90520-6. View

14.

Singaravelu G, Chatterjee I, Rahimi S, Druzhinina M, Kang L, Xu X . The sperm surface localization of the TRP-3/SPE-41 Ca2+ -permeable channel depends on SPE-38 function in Caenorhabditis elegans. Dev Biol. 2012; 365(2):376-83. PMC: 3337337. DOI: 10.1016/j.ydbio.2012.02.037. View

15.

Kadandale P, Chatterjee I, Singson A . Germline transformation of Caenorhabditis elegans by injection. Methods Mol Biol. 2008; 518:123-33. PMC: 2796118. DOI: 10.1007/978-1-59745-202-1_10. View

16.

Nelson G, Roberts T, Ward S . Caenorhabditis elegans spermatozoan locomotion: amoeboid movement with almost no actin. J Cell Biol. 1982; 92(1):121-31. PMC: 2111997. DOI: 10.1083/jcb.92.1.121. View

17.

Xu X, Sternberg P . A C. elegans sperm TRP protein required for sperm-egg interactions during fertilization. Cell. 2003; 114(3):285-97. DOI: 10.1016/s0092-8674(03)00565-8. View

18.

Krauchunas A, Werner M, Britt N, Chen D, Maddox A, Singson A . CYLC-2 localizes to sperm. MicroPubl Biol. 2020; 2020. PMC: 7533102. DOI: 10.17912/micropub.biology.000314. View

19.

Praitis V, Casey E, Collar D, Austin J . Creation of low-copy integrated transgenic lines in Caenorhabditis elegans. Genetics. 2001; 157(3):1217-26. PMC: 1461581. DOI: 10.1093/genetics/157.3.1217. View

20.

Heppert J, Dickinson D, Pani A, Higgins C, Steward A, Ahringer J . Comparative assessment of fluorescent proteins for in vivo imaging in an animal model system. Mol Biol Cell. 2016; 27(22):3385-3394. PMC: 5221575. DOI: 10.1091/mbc.E16-01-0063. View