CFAP65

CFAP65 is a conserved cilia- and flagella-associated protein that functions as a structural component of the C2a projection of the ciliary/flagellar central apparatus and is essential for sperm flagellar assembly (PMID:33988244, PMID:39092789). In the Chlamydomonas axoneme, the CFAP65 ortholog FAP65 localizes to the C2a projection together with FAP70 (CFAP70) and FAP147 (MYCBPAP), with FAP70 required for its retention in the axoneme (PMID:33988244); the CFAP65–MYCBPAP–CFAP70 association reconstitutes this C2a complex in the mammalian sperm central apparatus (PMID:39092789, PMID:39704931). During spermiogenesis CFAP65 organizes a cytoplasmic protein network with MNS1, RSPH1, TPPP2, ZPBP1, and SPACA1 and is required for acrosome biogenesis, manchette development, and mitochondrial sheath assembly, such that its loss produces hyper-constricted spermatid heads, abnormal manchette, and impaired flagellar elongation (PMID:34231842). In multiciliated cells, the ortholog Ccdc108 interacts with the IFT-B complex to control reciprocal centriolar recruitment and ciliary targeting, governing basal body migration and docking and establishment of the apical actin network via Drg1 and activated RhoA (PMID:35201641). Biallelic loss-of-function mutations in CFAP65 cause male infertility with multiple morphological abnormalities of the sperm flagella (MMAF), with absent central pair, acrosome hypoplasia, and disrupted mitochondrial sheath, recapitulated in knockout mice (PMID:31413122, PMID:31501240).

1. 2012 Low

   An early genetic clue placed CCDC108/CFAP65 in male reproductive function, raising the question of whether it acts in sperm motility.

   Evidence Mapping of a chromosomal inversion breakpoint disrupting CCDC108 in Rose-comb chickens, with testis expression and an MSP domain

   PMID:22761584

   Open questions at the time

   • Indirect breakpoint association only, no direct functional test of the protein
   • Postulated motility role not demonstrated mechanistically
2. 2017 Medium

   A non-ciliary role was probed by asking whether CFAP65 participates in mitochondrial retrograde signaling in somatic cells.

   Evidence siRNA knockdown and epistasis analysis in MKN45 gastric cancer cells placing CFAP65 in a TFAM-mtDNA-calcium-CFAP65-PCK1 axis

   PMID:29259235

   Open questions at the time

   • Mechanism of how CFAP65 transduces calcium signaling to PCK1 unresolved
   • Relationship to the ciliary/flagellar function unclear
   • Single cell line, single lab
3. 2019 High

   Whether CFAP65 loss causes human disease was answered by establishing it as a cause of MMAF-associated male infertility with reciprocal mouse evidence.

   Evidence Whole-exome sequencing of an MMAF cohort plus a CRISPR-Cas9 knockout mouse, with TEM and immunostaining of patient spermatozoa

   PMID:31413122 PMID:31501240

   Open questions at the time

   • Molecular function underlying flagellar defects not yet defined at this stage
   • Genotype-phenotype variability across mutations not characterized
4. 2021 High

   The cell-biological basis of the sperm phenotype was defined by showing CFAP65 is required for acrosome biogenesis, manchette development, and mitochondrial sheath assembly, acting within a cytoplasmic partner network.

   Evidence Cfap65 knockout mouse with histology, IF and EM of spermatids, plus endogenous co-IP and immunostaining identifying MNS1, RSPH1, TPPP2, ZPBP1, SPACA1

   PMID:34231842

   Open questions at the time

   • Whether the partner network is a single complex or transient associations unknown
   • Direct biochemical contacts within the network not mapped
5. 2021 High

   The structural identity of CFAP65 was established by placing its Chlamydomonas ortholog in the C2a projection of the central apparatus.

   Evidence Cryo-EM, mass spectrometry of fap70 mutant axonemes, and co-IP with HA-FAP70 in Chlamydomonas

   PMID:33988244

   Open questions at the time

   • High-resolution position of CFAP65 within C2a not resolved at the residue level
   • Whether the mammalian assembly mirrors Chlamydomonas not directly shown here
6. 2022 High

   A distinct multiciliated-cell function was revealed by showing the ortholog Ccdc108 mediates basal body migration and docking through reciprocal regulation with the IFT-B complex.

   Evidence Xenopus morpholino knockdown, co-IP with IFT-B, live imaging and immunofluorescence localization

   PMID:35201641

   Open questions at the time

   • Direct IFT-B subunit contacted by CFAP65 not pinpointed
   • How a central-apparatus protein also acts at basal bodies mechanistically unclear
   • Morpholino-based, not validated by genetic knockout
7. 2024 Medium

   The mammalian C2a complex was confirmed by demonstrating CFAP65 physically binds MYCBPAP alongside CFAP70.

   Evidence Endogenous IP-MS in MYCBPAP transgenic mice and in Mycbpap-knockout/control mouse testes

   PMID:39092789 PMID:39704931

   Open questions at the time

   • Stoichiometry and assembly order of the CFAP65-CFAP70-MYCBPAP complex unknown
   • Direct binary interfaces not mapped

• How CFAP65's roles as a structural central-apparatus subunit, an IFT-B-interacting basal body regulator, and a putative mitochondrial retrograde signaling factor are mechanistically reconciled remains unresolved.
• No structural model defining CFAP65's binding interfaces
• No biochemical reconstitution of the C2a or cytoplasmic network
• Connection between somatic signaling and ciliary roles unestablished