*2.9. Infertility*

The sperm tail has microtubule arrangemen<sup>t</sup> (9 × 2 + 2), which is similar to that of motile cilia [55]. Therefore, infertility is frequently observed in males with PCD [54]. There are some differences, however, between the sperm tail and motile cilia, including cell type-specific axonemal proteins

and accessory structures specific to the sperm tail, such as the mitochondrial sheath, fibrous sheath, and outer dense fibers [55]. Mutations in genes causative of PCD are not always associated with male infertility and vice versa [55]. The impairment of the sperm tail affects sperm motility. The impairment of motile and primary cilia also affects the function of the reproductive tract in both males and females [54,56]. Structural and functional studies of cilia associated with infertility constitute an important area in reproductive research [55,56].

### *2.10. Roles of Primary Cilia in Cancer*

Primary cilia in cultured mouse 3T3 fibroblasts and human retinal pigment epithelial (RPE1) cells can be disassembled and assembled by serum stimulation and deprivation, respectively [18,19,94]. Aurora A kinase (AURKA), one of the most important mitotic kinases for cell-cycle control [95], plays important roles in deciliation by serum stimulation [96,97]. AURKA is activated by serum stimulation through Ca<sup>2</sup>+/calmodulin signaling, the non-canonical WNT pathway, and phosphatidylinositol signaling [97–100]. Serum stimulation also activates AURKA through the pathway involving epidermal growth factor receptor (EGFR), ubiquitin-specific peptidase 8 (USP8), and trichoplein (TCHP) (described in the next section) [33,34,101]. Activated AURKA phosphorylates itself and target proteins during G1 phase, which stimulates the disassembly of primary cilia [97]. Several proteins associated with AURKA and ciliogenesis have been identified, including histone deacetylase 6 [94] and nudE neurodevelopment protein 1 (NDE1) [102]. In response to serum stimulation, NDE1 localizes at the basal body and suppresses ciliogenesis by tethering dynein light chain 1 [103]. Under serum deprivation conditions, cyclin-dependent kinase 5 is activated and phosphorylates NDE1. Phosphorylated NDE1 is then recognized and ubiquitylated by the E3 ligase complex SCFFBXW7, resulting in ciliogenesis [104,105]. Importantly, forced ciliation in cells growing under serum stimulation conditions can cause cell-cycle arrest [33,34,101,102,106]. These findings sugges<sup>t</sup> that the primary cilium can act as a negative regulator of the cell cycle and may be a tumor suppressor organelle [3–6,9,107–109]. In fact, the suppression of primary cilia function is associated with tumorigenesis, cell proliferation, and metastasis in many cancers, including glioblastoma [110], esophageal cancer [111], colon cancer [112], cholangiocarcinoma [113,114], pancreatic ductal adenocarcinoma [115], clear cell renal carcinoma [116], prostate cancer [117], ovarian cancer [118,119], melanoma [120], and chondrosarcoma [121] (Table 2). However, primary cilia can promote tumor progression under certain conditions. In medulloblastoma and basal cell skin carcinoma caused by gain-of-function mutation of SMO, primary cilia convert the GLI transcription factors GLI2 and GLI3 to their activated forms, inducing their translocation to the nucleus, increased transcription of Hedgehog target genes, and promotion of cell proliferation [122,123]. In contrast, primary cilia of medulloblastoma and basal cell skin carcinoma caused by gain-of-function mutation of GLI2 increases the activity of GLI3 as a transcriptional repressor, resulting in suppression of the proliferation of these cancer cells [122,123]. Further work will thus be necessary to fully understand the context-dependent roles of primary cilia in cell proliferation.


### **Table 2.** The roles of primary cilia in cancer.

PC—primary cilia; KD—knockdown; GOF—gain-of-function.

### **3. Roles of E3 Ubiquitin Ligases and DUBs in Ciliogenesis, Ciliopathy, and Cancer**

The structure and function of cilia are dynamically regulated by many ciliary proteins through posttranslational modification [2,4–6,25]. Ubiquitination and deubiquitination of ciliary proteins by E3 ubiquitin ligase and DUBs, respectively, are crucial for the dynamic regulation of cilia [4–6,31–34]. In this section, we briefly describe the role of E3 ubiquitin ligases and DUBs in ciliogenesis, ciliopathy, and cancer (Table 3).

**Figure 1.** Suppression of Kctd17 in zebrafish impairs ciliogenesis in Kupffer's vesicle and induces situs inversus (**A**) Staining of acetylated alpha-tubulin in the cilia of Kupffer's vesicle at 12 h post-fertilization (hpf) of wild-type (control) or Kctd17 knockout (KO) zebrafish. (**B**) Distribution of the cilia length in Kupffer's vesicle at 12 hpf in control or Kctd17 KO or knockdown (KD) zebrafish. (**C**) Representative in vivo images of control and Kctd17 KO zebrafish at 3 days post-fertilization (dpf). Control and Kctd17 KO zebrafish show rightward and leftward looping of the heart, respectively. Please see Video S1. (**D**) At 3 dpf, rightward and leftward looping of the heart is observed in about 95% of the control zebrafish and about 20% of the Kctd17 KO zebrafish, respectively. Scale bar: 20 μm (**A**), 200 μm (**C**).


**Table 3.** The roles of E3 ubiquitin ligases and deubiquitinases in ciliogenesis, ciliopathy, and cancer.

DUBs—deubiquitinases; KD—knockdown; KO—knockout; OE—overexpression; LOF—loss-of-function.
