bioRxiv preprint doi: https://doi.org/10.1101/2021.08.16.456347; this version posted August 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 C2cd6-encoded CatSper Targets Sperm Calcium Channel to 2 Ca2+ Signaling Domains in the Flagellar Membrane 3 4 Jae Yeon Hwang1, Huafeng Wang1, Yonggang Lu2, Masahito Ikawa2, and Jean-Ju Chung1,3,* 5 6 1Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT 7 06510, USA 8 2Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 5650871, Japan 9 3Department of Gynecology and Obstetrics, Yale School of Medicine, New Haven, CT 06510, 10 USA 11 *Correspondence: [email protected] 12 13 Highlights 14 CatSper encoded by C2cd6 is a C2 membrane-associating domain containing protein 15 CatSper loss-of-function impairs sperm hyperactivation and male fertility 16 CatSper adopts ciliary trafficking machineries for flagellar targeting via C2 domain 17 CatSper targets the CatSper channel into nanodomains of developing sperm flagella 18 19 20 SUMMARY 21 In mammalian sperm cells, regulation of spatiotemporal Ca2+ signaling relies on the quadrilinear 22 Ca2+ signaling nanodomains in the flagellar membrane. The sperm-specific, multi-subunit 23 CatSper Ca2+ channel, which is crucial for sperm hyperactivated motility and male fertility, 24 organizes the nanodomains. Here, we report CatSper, the C2cd6-encoded membrane- 25 associating C2 domain protein, can independently migrate to the flagella and serve as a major 26 targeting component of the CatSper channel complex. CatSper loss-of-function in mice 27 demonstrates that it is essential for sperm hyperactivated motility and male fertility. CatSper 28 targets the CatSper channel into the quadrilinear nanodomains in the flagella of developing 29 spermatids, whereas it is dispensable for functional channel assembly. CatSper interacts with 30 ciliary trafficking machinery in a C2-dependent manner. These findings provide insights into the 31 CatSper channel trafficking to the Ca2+ signaling nanodomains and the shared molecular 32 mechanisms of ciliary and flagellar membrane targeting. 33 34 Keywords: Domain organization, Flagellar membrane targeting, Sperm motility, Ca2+ channel, 35 CatSper, Ca2+ signaling, Male fertility bioRxiv preprint doi: https://doi.org/10.1101/2021.08.16.456347; this version posted August 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 36 Introduction 37 Compartmentalization of the plasma membrane into distinct domains is an important mechanism 38 to control signaling. Cilia and flagella are microtubule-based projections generated from basal 39 bodies and ensheathed by the specialized membrane of distinctive lipid compositions (Garcia III 40 et al., 2018; Walters et al., 2020). A conserved intraflagellar transporter (IFT) system assembles 41 the axoneme - the core structure common to both cilia and flagella - whereas ciliary and flagellar 42 membranes contain concentrated ion channels and membrane receptors for signal transduction 43 (Pablo et al., 2017; Trötschel et al., 2020; Wang et al., 2021). Over the past decades, our 44 increased knowledge of ciliary targeting of membrane proteins has established that vesicular 45 transportation and IFTs enable the cargoes to pass transition zone and membrane diffusion 46 barrier (Nachury and Mick, 2019; Nachury et al., 2010). Cilia function as sensory antenna are 47 crucial for diverse biological processes including phototransduction, olfaction, and Hedgehog 48 signaling. 49 As specialized cilia, flagella of mammalian sperm have one particular task. The flagellum provides 50 propulsion for the sperm cells to reach and fertilize the eggs in the female reproductive tract. For 51 this arduous journey, flagellar membrane proteins also play essential roles in signal transduction 52 to regulate sperm motility, highlighting their importance in male fertility (Wang et al., 2021). 53 However, how flagellar membrane assembly is coordinated with other early spermiogenesis 54 process remains largely unknown. Just like cilium biogenesis, IFT systems are predicted to deliver 55 flagellar membrane proteins. Yet, the absence of IFT components compromises axonemal growth 56 and results in very short or no flagella (Avidor-Reiss and Leroux, 2015), making it difficult to 57 directly test this idea. It remains unclear whether flagellar membrane targeting uses the same 58 ciliary membrane targeting machinery or distinct motors, and how it is achieved. 59 In mammalian sperm cells, spatiotemporal regulation of Ca2+ signaling relies on the quadrilinear 60 organization of the Ca2+ signaling nanodomains in the flagellar membrane (Chung et al., 2014; 61 Hwang et al., 2019), where the sperm-specific, multi-subunit CatSper Ca2+ channel complexes 62 assemble into zigzag rows in each quadrant (Zhao et al., 2021). Previous studies have found that 63 only spermatozoa with four intact CatSper nanodomains can develop hyperactivated motility, 64 undergo acrosome reaction, and successfully arrive at the fertilizing site (Chung et al., 2017; 65 Chung et al., 2014; Ded et al., 2020). Thus, flagellar targeting of the CatSper channel and its 66 localization into the quadrilinear nanodomains is crucial for sperm motility and male fertility. 67 However, its complexity in composition (Hwang et al., 2019; Lin et al., 2021; Zhao et al., 2021), 68 the inseparable loss-of-function phenotypes of each transmembrane (TM) subunit in mice (Chung 69 et al., 2011; Hwang et al., 2019; Qi et al., 2007), and the lack of heterologous systems for 70 functional reconstitution have limited our understanding of the mechanism of the CatSper 71 assembly and delivery to flagella. 72 Here, we report CatSper (tau, CatSper Targeting subunit) containing C2 membrane-associating 73 domain encoded by C2CD6 is critical for CatSper flagellar targeting and trafficking into the 74 quadrilinear nanodomains. CatSper loss-of-function studies in mice reveal that CatSper targets 75 the preassembled CatSper complexes to elongating flagella, where CatSper links the channel- 76 carrying vesicles and motor proteins. C2 domain truncation of CatSper is sufficient to 77 compromise the spatiotemporal trafficking of the CatSper channel into the nanodomains in 78 developing spermatids and during sperm maturation. We demonstrate that mutant sperm still form 79 the CatSper channelalbeit significantly decreased, and conduct calcium, but they fail to bioRxiv preprint doi: https://doi.org/10.1101/2021.08.16.456347; this version posted August 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 80 hyperactivate, rendering the male infertile. These findings provide new insight into the molecular 81 mechanisms of flagellar targeting of membrane proteins in general and organizing the CatSper 82 Ca2+ signaling nanodomains in mammalian spermatozoa. 83 84 Results 85 CatSper is a New CatSper Component with the Membrane-Associating C2 Domain 86 The unique quadrilinear CatSper nanodomains within flagellar membrane suggest that the 87 CatSper channel complex might require a membrane-targeting molecule specialized for this task. 88 C2CD6 (previously known as ALS2CR11) was identified as one of the proteins significantly 89 reduced in CatSper1-null spermatozoa that lack the entire CatSper channel complex (Hwang et 90 al., 2019; Zhao et al., 2021). The testis-specific C2CD6 gene encodes two isoforms: both long 91 (>200 kDa) and short (~70 kDa) forms contain the conserved membrane-associating C2 domain 92 in both mouse and humans (Figures 1A and 1B). Thus, we hypothesize that C2CD6 is a new 93 CatSper component that mediates CatSper trafficking to the flagellar membrane and/or spatial 94 partitioning to the nanodomains. 95 We first examined whether C2CD6expression is CatSper-dependent (Figures 1C-1F). 96 Immunoblot analysis of sperm from a mouse cauda epididymis showed that protein levels of both 97 long and short forms of C2CD6 are severely reduced in CatSper1- and CatSperd-null sperm that 98 lack the entire CatSper channel, compared with those in WT sperm (Figures 1C and 1D, right). 99 C2CD6 is localized in the principal piece in both mouse and human sperm (Figures 1D and 1E). 100 3D structured illumination microscopy (SIM) further revealed a quadrilateral arrangement of 101 C2CD6 (Figure 1F), a hallmark of the CatSper Ca2+ signaling domain (Chung et al., 2017). In 102 addition, the decreased C2CD6 proteins distribute discontinuously (Figures 1C and 1D, middle) 103 in Efcab9-null sperm that contain overall only ~ 20% of the protein levels of CatSper subunits and 104 exhibit a fragmented pattern of the linear CatSper nanodomains (Hwang et al., 2019). All these 105 results suggest that C2CD6 is associated with the CatSper channel complex in cauda sperm. 106 C2CD6 is a newly identified bona fide CatSper component – we herein name it CatSper. 107 Intriguingly, in CatSper1- or CatSperd-null sperm, CatSper is not completely absent, but still 108 detected despite having even lower the protein levels compared to those of Efcab9-null sperm 109 (Figures 1C and 1D). This minor yet obvious presence of CatSperin the absence of the channel 110 complex distinguishes it from all the other previously reported