id: Q8NHU2
gene_symbol: CFAP61
product_type: PROTEIN
status: IN_PROGRESS
taxon:
  id: NCBITaxon:9606
  label: Homo sapiens
description: >-
  CFAP61 (Cilia- and flagella-associated protein 61; formerly C20orf26) is a structural
  scaffold
  protein and conserved component of the calmodulin- and radial spoke-associated complex
  (CSC)
  in motile cilia and flagella. The CSC, composed of CFAP61, CFAP91/MAATS1, and CFAP251/WDR66,
  connects the radial spokes to the nexin-dynein regulatory complex (N-DRC), mediating
  mechanochemical signal transduction that coordinates dynein motor activity and ciliary/flagellar
  beating. CFAP61 is specifically required for the stable assembly of radial spoke
  3 (RS3) and
  contributes to RS2 base formation. In sperm, loss of CFAP61 causes collapse of axonemal
  architecture with loss of central pair, radial spokes, and inner dynein arms, resulting
  in
  multiple morphological abnormalities of the flagella (MMAF) and male infertility
  (SPGF84).
  Despite containing a Rossmann-like FAD/NAD(P)-binding superfamily fold (residues
  665-998),
  CFAP61 has no enzymatic activity; this domain serves as a structural scaffold for
  protein-protein interactions within the axoneme. The protein also has a CFAP61-specific
  N-terminal domain and a C-terminal dimerisation domain. CFAP61 is expressed in all
  tissues
  with motile cilia, including respiratory epithelium, fallopian tubes, and testis,
  though
  disease phenotypes from human mutations are restricted to male infertility without
  classical
  primary ciliary dyskinesia symptoms.

alternative_products:
- name: '1'
  id: Q8NHU2-1
- name: '3'
  id: Q8NHU2-3
  sequence_note: VSP_003799, VSP_003800
- name: '4'
  id: Q8NHU2-4
  sequence_note: VSP_003798
- name: '5'
  id: Q8NHU2-5
  sequence_note: VSP_003802, VSP_003803, VSP_003804
- name: '6'
  id: Q8NHU2-6
  sequence_note: VSP_003802, VSP_013682, VSP_013683

existing_annotations:

# === IDA/IMP annotations (experimental, human/mouse) ===

- term:
    id: GO:0005930
    label: axoneme
  evidence_type: IDA
  original_reference_id: PMID:28282151
  review:
    summary: >-
      CFAP61 was identified in the axoneme by quantitative proteomic analysis of human
      airway cilia.
      This is direct experimental evidence for axonemal localization in human respiratory
      epithelial cilia.
    action: ACCEPT
    reason: >-
      Direct proteomic detection in purified human airway ciliary axonemes provides
      strong evidence
      for axonemal localization. This is consistent with CFAP61's known role as a
      structural component
      of the calmodulin- and spoke-associated complex (CSC), which is an integral
      axonemal complex.
    supported_by:
    - reference_id: PMID:28282151
      supporting_text: >-
        ...Quantitative Proteomic Analysis of Human Airway Cilia Identifies Previously
        Uncharacterized
        Proteins of High Abundance...
    - reference_id: PMID:37258679
      supporting_text: >-
        ...a full-length RS3, which we show is built on a base of CFAP61, CFAP91 and
        CFAP251...

- term:
    id: GO:0005930
    label: axoneme
  evidence_type: IDA
  original_reference_id: PMID:36659204
  review:
    summary: >-
      Cfap61 was detected in the axoneme of mouse sperm by immunofluorescence/immunohistochemistry,
      initially at the neck of elongating spermatids, later at the midpiece of mature
      sperm.
    action: ACCEPT
    reason: >-
      Direct immunolocalization in mouse sperm flagella axoneme. Transferred to human
      by MGI
      annotation. Consistent with human proteomic data (PMID:28282151) and the structural
      role
      of CFAP61 in the CSC complex.
    supported_by:
    - reference_id: PMID:36659204
      supporting_text: >-
        ...Cfap61 is initially localized at the neck of sperm, where it potentially
        functions
        in flagellum formation, and is later localized to the midpiece of the sperm...

- term:
    id: GO:0036126
    label: sperm flagellum
  evidence_type: IDA
  original_reference_id: PMID:36659204
  review:
    summary: >-
      Cfap61 protein was detected in sperm flagella by immunofluorescence in mouse,
      and separately
      confirmed in human sperm flagella by immunofluorescence with anti-CFAP61 antibodies.
    action: ACCEPT
    reason: >-
      Direct immunolocalization data in both mouse (PMID:36659204) and human (PMID:35174165)
      sperm.
      CFAP61 localizes along the full length of human sperm flagella (in contrast
      to mouse where it
      is more restricted to midpiece in mature sperm). Core localization for this
      protein.
    additional_reference_ids:
    - PMID:35174165
    supported_by:
    - reference_id: PMID:36659204
      supporting_text: >-
        ...Cfap61 is initially localized at the neck of sperm, where it potentially
        functions
        in flagellum formation, and is later localized to the midpiece of the sperm...
    - reference_id: PMID:35174165
      supporting_text: >-
        ...The specific signals of CFAP61, co-localizing with alpha-Tubulin, were
        detected on the sperm tails from a
        fertile control...

      full_text_unavailable: true
- term:
    id: GO:0120316
    label: sperm flagellum assembly
  evidence_type: IMP
  original_reference_id: PMID:35174165
  review:
    summary: >-
      Biallelic loss-of-function variants in CFAP61 (frameshift and nonsense) cause
      MMAF with
      severely disorganized axonemal ultrastructures in human sperm, including missing
      central pair,
      radial spokes, and inner dynein arms.
    action: ACCEPT
    reason: >-
      Multiple independent human families with biallelic CFAP61 variants show MMAF
      phenotype.
      TEM revealed severely disorganized axonemes. Both CFAP61 and CFAP251 signals
      were absent
      from patient sperm tails. This is strong IMP evidence that CFAP61 is required
      for normal
      sperm flagellum assembly.
    supported_by:
    - reference_id: PMID:35174165
      supporting_text: >-
        ...variants, c.451_452del (p.I151Nfs*4) in family 1 and c.847C > T (p.R283*)
        in family 2
        and 3, were identified recessively co-segregating with the MMAF phenotype.
        Transmission
        electron microscopy analyses revealed severe disorganized axonemal ultrastructures...

- term:
    id: GO:0030317
    label: flagellated sperm motility
  evidence_type: IMP
  original_reference_id: PMID:34792097
  review:
    summary: >-
      Cfap61 knockout mice show severely impaired sperm motility. Human patients with
      CFAP61
      splice variant also show asthenozoospermia. The protein is required for axoneme
      stability
      during spermiogenesis, and loss leads to axoneme scattering and disrupted IFT
      protein distribution.
    action: ACCEPT
    reason: >-
      Strong genetic evidence from both mouse knockout and human mutation that CFAP61
      is required
      for flagellated sperm motility. The mechanism is indirect -- CFAP61 is needed
      for radial spoke
      assembly, and without intact radial spokes the axoneme becomes unstable and
      motility is lost.
      The qualifier acts_upstream_of_or_within (in GOA) is appropriate since CFAP61's
      role is
      structural rather than directly in the motility machinery.
    supported_by:
    - reference_id: PMID:34792097
      supporting_text: >-
        ...CFAP61 is required for sperm flagellum formation and male fertility in
        human and mouse...
        the assembly of radial spoke components is impaired. As spermiogenesis progresses,
        the
        axoneme in Cfap61-/- cells becomes unstable and scatters...

- term:
    id: GO:0120316
    label: sperm flagellum assembly
  evidence_type: IMP
  original_reference_id: PMID:34792097
  review:
    summary: >-
      Cfap61 knockout mice show MMAF phenotype with impaired radial spoke assembly
      during
      early spermatid development, progressing to axoneme instability and scattering.
    action: ACCEPT
    reason: >-
      Complementary to the human IMP evidence from PMID:35174165. The mouse knockout
      provides detailed mechanistic insight: CFAP61 interacts with the CSC, radial
      spoke
      stalk and head, and its absence impairs radial spoke assembly early in spermiogenesis.
    supported_by:
    - reference_id: PMID:34792097
      supporting_text: >-
        ...We find that CFAP61 interacts with the CSC, radial spoke stalk and head.
        During early
        stages of Cfap61-/- spermatid development, the assembly of radial spoke components
        is impaired...

# === IBA annotations (phylogenetic) ===

- term:
    id: GO:0036126
    label: sperm flagellum
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  review:
    summary: >-
      Phylogenetically inferred sperm flagellum localization based on PANTHER tree
      analysis.
      This is now independently confirmed by direct experimental evidence (IDA from
      PMID:36659204
      and IF data from PMID:35174165).
    action: ACCEPT
    reason: >-
      The IBA annotation is correct and consistent with the IDA evidence. The phylogenetic
      inference is sound given the highly conserved CSC function across species, and
      is
      independently confirmed by direct experimental evidence.

- term:
    id: GO:0120316
    label: sperm flagellum assembly
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  review:
    summary: >-
      Phylogenetically inferred involvement in sperm flagellum assembly. Confirmed
      by
      direct IMP evidence from human and mouse studies.
    action: ACCEPT
    reason: >-
      Correct inference, independently confirmed by direct experimental evidence (IMP
      from
      PMID:34792097 and PMID:35174165). Sperm flagellum assembly is a core function
      of CFAP61.

# === IEA annotations (electronic) ===

- term:
    id: GO:0005856
    label: cytoskeleton
  evidence_type: IEA
  original_reference_id: GO_REF:0000044
  review:
    summary: >-
      Electronic annotation mapping from UniProt subcellular location. CFAP61 is indeed
      a
      cytoskeletal protein as a component of the axoneme.
    action: MODIFY
    reason: >-
      While technically correct, 'cytoskeleton' is too general. CFAP61 specifically
      localizes
      to the axoneme, which is already annotated with stronger evidence. The more
      specific
      term 'axoneme' (GO:0005930) is preferred and has IDA evidence.
    proposed_replacement_terms:
    - id: GO:0005930
      label: axoneme

- term:
    id: GO:0005929
    label: cilium
  evidence_type: IEA
  original_reference_id: GO_REF:0000044
  review:
    summary: >-
      Electronic annotation from UniProt subcellular location. CFAP61 is present in
      cilia.
    action: MODIFY
    reason: >-
      CFAP61 is specifically a component of motile cilia (not primary/immotile cilia).
      The term 'motile cilium' (GO:0031514) would be more accurate and is already
      present
      as an ISS annotation.
    proposed_replacement_terms:
    - id: GO:0031514
      label: motile cilium

- term:
    id: GO:0005930
    label: axoneme
  evidence_type: IEA
  original_reference_id: GO_REF:0000044
  review:
    summary: >-
      Electronic annotation of axoneme localization from UniProt. Correct and supported
      by
      direct experimental evidence.
    action: ACCEPT
    reason: >-
      Correct annotation, consistent with IDA evidence from PMID:28282151 and PMID:36659204.
      Axoneme localization is a core aspect of CFAP61 function as a CSC component.

# === ISS annotations (sequence similarity) ===

- term:
    id: GO:0003341
    label: cilium movement
  evidence_type: ISS
  original_reference_id: GO_REF:0000024
  review:
    summary: >-
      Transferred from Tetrahymena FAP61 ortholog (UniProtKB:A8IF44). In Tetrahymena,
      loss of FAP61 reduces cell swimming and affects ciliary waveform. In Chlamydomonas,
      CSC disruption impairs flagellar motility.
    action: ACCEPT
    reason: >-
      Well-supported ISS annotation. The role of CFAP61 in cilium movement is clearly
      established in multiple organisms through the CSC complex function. While direct
      evidence in human respiratory cilia is not yet available, the conserved mechanism
      of radial spoke-mediated motility regulation makes this a strong inference.
      Patients with CFAP61 mutations notably do not present PCD symptoms, suggesting
      possible redundancy in respiratory cilia, but the protein is present in airway
      cilia (PMID:28282151).
    supported_by:
    - reference_id: PMID:25694453
      supporting_text: >-
        ...loss of either FAP61 or FAP251 reduces cell swimming and affects the ciliary
        waveform and that RS3 is either missing or incomplete, whereas RS1 and RS2
        are unaffected...
    - reference_id: PMID:21613541
      supporting_text: >-
        ...analysis of both flagellar beating and microtubule sliding in vitro demonstrates
        that the CSC plays a critical role in modulating dynein activity...

- term:
    id: GO:0031514
    label: motile cilium
  evidence_type: ISS
  original_reference_id: GO_REF:0000024
  review:
    summary: >-
      Transferred from Tetrahymena ortholog. CFAP61 is a component of motile cilia,
      confirmed
      by its proteomic identification in human airway cilia.
    action: ACCEPT
    reason: >-
      Correct and well-supported. CFAP61 was directly identified in human airway motile
      cilia by proteomics (PMID:28282151), confirming the ISS transfer. Motile cilium
      is
      the appropriate specific CC term for this protein.
    supported_by:
    - reference_id: PMID:28282151
      supporting_text: >-
        ...Quantitative Proteomic Analysis of Human Airway Cilia Identifies Previously
        Uncharacterized Proteins of High Abundance...

- term:
    id: GO:0044782
    label: cilium organization
  evidence_type: ISS
  original_reference_id: GO_REF:0000024
  review:
    summary: >-
      Transferred from Chlamydomonas (Q23F13) and Tetrahymena (Q24DE2) orthologs.
      CFAP61 is
      involved in the structural organization of cilia through its role in radial
      spoke assembly.
    action: MODIFY
    reason: >-
      While CFAP61 does contribute to cilium organization, the term is very broad.
      The more specific term 'radial spoke assembly' (GO:0062177) better captures
      the
      actual function of CFAP61 within the CSC. CFAP61 is specifically required for
      RS3
      assembly and contributes to RS2 base formation.
    proposed_replacement_terms:
    - id: GO:0062177
      label: radial spoke assembly

- term:
    id: GO:0001536
    label: radial spoke stalk
  qualifier: colocalizes_with
  evidence_type: ISS
  original_reference_id: GO_REF:0000024
  review:
    summary: >-
      Transferred from Tetrahymena FAP61 ortholog. The CSC localizes at the base of
      radial
      spokes and CFAP61 specifically contributes to RS3 stem structure. In Liu et
      al. 2021,
      CFAP61 was shown to interact with radial spoke stalk and head.
    action: ACCEPT
    reason: >-
      The qualifier 'colocalizes_with' is appropriate for this ISS annotation. CFAP61
      as
      a CSC component is located at the base of radial spokes and interacts with stalk
      proteins. The colocalizes_with qualifier correctly indicates proximity/association
      rather than being part_of the radial spoke stalk per se.
    supported_by:
    - reference_id: PMID:25694453
      supporting_text: >-
        ...FAP61-null cilia lack an adjacent portion of the RS3 stem region...
    - reference_id: PMID:34792097
      supporting_text: >-
        ...CFAP61 interacts with the CSC, radial spoke stalk and head...
    - reference_id: PMID:37258679
      supporting_text: >-
        ...a full-length RS3, which we show is built on a base of CFAP61, CFAP91 and
        CFAP251... then links the RS3 subunits CFAP251 and CFAP61...

- term:
    id: GO:0005930
    label: axoneme
  evidence_type: ISS
  original_reference_id: GO_REF:0000024
  review:
    summary: >-
      Transferred from Tetrahymena ortholog. Axonemal localization is confirmed by
      multiple lines of evidence in human and mouse, including direct cryo-EM resolution
      of CFAP61 within the human respiratory-cilia axoneme (PMID:37258679, PDB 8J07).
    action: ACCEPT
    reason: >-
      Correct annotation, consistent with IDA evidence from PMID:28282151 and PMID:36659204.
      Axoneme localization is a core aspect of CFAP61 function. The cryo-EM structure of the
      human axoneme (PMID:37258679) directly resolves CFAP61 as part of the RS3 base,
      providing structural confirmation of axonemal localization and architecture.
    supported_by:
    - reference_id: PMID:37258679
      supporting_text: >-
        ...a full-length RS3, which we show is built on a base of CFAP61, CFAP91 and
        CFAP251...

# === NEW proposed annotations ===

- term:
    id: GO:0005200
    label: structural constituent of cytoskeleton
  evidence_type: ISS
  original_reference_id: PMID:25694453
  review:
    summary: >-
      CFAP61 functions as a structural scaffold within the axonemal CSC complex. It
      has no
      known enzymatic activity despite containing a Rossmann-like fold domain. Its
      loss
      specifically disrupts the structural integrity of radial spoke 3 in Tetrahymena
      and
      the entire axoneme in mammalian sperm. This is a molecular function annotation
      that
      is currently missing from GOA.
    action: NEW
    reason: >-
      CFAP61 has no molecular function annotation in GOA. Its role as a structural
      scaffold
      in the axoneme CSC is well established. The protein contains a Rossmann-like
      FAD/NAD(P)-binding fold that functions as a scaffolding domain rather than an
      enzyme.
      GO:0005200 (structural constituent of cytoskeleton) is the most appropriate
      MF term
      for a non-enzymatic structural component of the axoneme.
    supported_by:
    - reference_id: PMID:25694453
      supporting_text: >-
        ...FAP61-null cilia lack an adjacent portion of the RS3 stem region...
    - reference_id: PMID:34792097
      supporting_text: >-
        ...CFAP61 is a conserved component of the calmodulin- and radial spoke-associated
        complex (CSC) of cilia... CFAP61 interacts with the CSC, radial spoke stalk
        and head...
    - reference_id: file:human/CFAP61/CFAP61-deep-research-falcon.md
      supporting_text: CFAP61 is best supported as a structural/assembly and stabilization factor for the sperm flagellar axoneme, acting through the CSC-radial spoke system.

- term:
    id: GO:0062177
    label: radial spoke assembly
  evidence_type: ISS
  original_reference_id: PMID:25694453
  review:
    summary: >-
      CFAP61 is required for radial spoke 3 assembly in Tetrahymena and for radial
      spoke
      component assembly during spermiogenesis in mouse. This process annotation is
      not
      currently in GOA but is a core function of the protein.
    action: NEW
    reason: >-
      Radial spoke assembly is the primary biological process function of CFAP61 within
      the CSC. In Tetrahymena, FAP61 loss specifically ablates RS3 stem structure.
      In
      mouse spermatids, CFAP61 loss impairs radial spoke component assembly during
      early
      spermiogenesis. This is more specific and informative than the existing 'cilium
      organization' annotation.
    supported_by:
    - reference_id: PMID:25694453
      supporting_text: >-
        ...loss of either FAP61 or FAP251 reduces cell swimming and affects the ciliary
        waveform and that RS3 is either missing or incomplete, whereas RS1 and RS2
        are unaffected...
    - reference_id: PMID:34792097
      supporting_text: >-
        ...During early stages of Cfap61-/- spermatid development, the assembly of
        radial
        spoke components is impaired...
    - reference_id: PMID:37258679
      supporting_text: >-
        ...a full-length RS3, which we show is built on a base of CFAP61, CFAP91 and
        CFAP251...
    - reference_id: file:human/CFAP61/CFAP61-deep-research-falcon.md
      supporting_text: Its pathogenic loss disrupts assembly and/or maintenance of radial spokes and associated axonemal complexes, producing axoneme instability and disorganization.

core_functions:
- description: >-
    CFAP61 functions as a structural scaffold within the calmodulin- and spoke-associated
    complex (CSC), providing the structural basis for radial spoke 3 (RS3) assembly
    and
    contributing to RS2 base stability. Through the CSC, CFAP61 mediates the connection
    between radial spokes and the nexin-dynein regulatory complex (N-DRC), enabling
    mechanochemical signal transduction that coordinates dynein motor activity and
    ciliary
    beating. The protein contains a Rossmann-like FAD/NAD(P)-binding fold that serves
    as a structural scaffold rather than an enzyme active site, an N-terminal
    axoneme-targeting domain, and a C-terminal dimerisation domain.
  molecular_function:
    id: GO:0005200
    label: structural constituent of cytoskeleton
  directly_involved_in:
  - id: GO:0062177
    label: radial spoke assembly
  - id: GO:0120316
    label: sperm flagellum assembly
  locations:
  - id: GO:0005930
    label: axoneme
  - id: GO:0036126
    label: sperm flagellum
  - id: GO:0031514
    label: motile cilium
  supported_by:
  - reference_id: PMID:25694453
    supporting_text: >-
      ...FAP61-null cilia lack an adjacent portion of the RS3 stem region...
      loss of either FAP61 or FAP251 reduces cell swimming and affects the
      ciliary waveform and that RS3 is either missing or incomplete...
  - reference_id: PMID:34792097
    supporting_text: >-
      ...CFAP61 is a conserved component of the calmodulin- and radial
      spoke-associated complex (CSC) of cilia... CFAP61 interacts with the CSC,
      radial spoke stalk and head...
  - reference_id: PMID:22740634
    supporting_text: >-
      ...the CSC connects three major axonemal complexes involved in dynein
      regulation: RS2, the nexin-dynein regulatory complex (N-DRC), and RS3S...
  - reference_id: file:human/CFAP61/CFAP61-deep-research-falcon.md
    supporting_text: CFAP61 is currently understood as a mammalian component of the calmodulin- and spoke-associated complex that is physically and functionally associated with the radial spoke system.

- description: >-
    In sperm, CFAP61 is essential for flagellum morphogenesis and motility.
    Loss of CFAP61 function causes multiple morphological abnormalities of the
    flagella (MMAF) and male infertility (SPGF84). CFAP61 interacts with
    intraflagellar transport proteins (IFT22, IFT81, WDR35) and its loss
    disrupts IFT protein distribution during spermiogenesis.
  molecular_function:
    id: GO:0005200
    label: structural constituent of cytoskeleton
  directly_involved_in:
  - id: GO:0120316
    label: sperm flagellum assembly
  - id: GO:0030317
    label: flagellated sperm motility
  locations:
  - id: GO:0036126
    label: sperm flagellum
  - id: GO:0005930
    label: axoneme
  supported_by:
  - reference_id: PMID:34792097
    supporting_text: >-
      ...CFAP61 is required for sperm flagellum formation and male fertility
      in human and mouse... the assembly of radial spoke components is impaired.
      As spermiogenesis progresses, the axoneme in Cfap61-/- cells becomes unstable
      and scatters, and the distribution of intraflagellar transport proteins is disrupted...
  - reference_id: PMID:35174165
    supporting_text: >-
      ...CFAP61 and CFAP251 signals were absent from sperm tails of the patients,
      which suggested the loss of functional CSC in sperm flagella...
  - reference_id: file:human/CFAP61/CFAP61-deep-research-falcon.md
    supporting_text: Biallelic CFAP61 loss-of-function variants are an established cause of multiple morphological abnormalities of the flagella.

proposed_new_terms:
- proposed_name: calmodulin- and spoke-associated complex
  proposed_definition: >-
    A protein complex associated with the radial spokes of motile cilia and flagella,
    composed of CFAP61, CFAP91/MAATS1, and CFAP251/WDR66 (mammalian nomenclature;
    FAP61/CaM-IP3, FAP91/CaM-IP2, FAP251/CaM-IP4 in Chlamydomonas). The CSC connects
    radial spoke 2 (RS2), radial spoke 3 (RS3), and the nexin-dynein regulatory complex
    (N-DRC), mediating mechanochemical signal transduction for coordinated ciliary
    beating.
  justification: >-
    The CSC is a well-characterized multi-protein complex with a defined composition
    and
    function in motile cilia across eukaryotes. It has been the subject of numerous
    studies since its identification in Chlamydomonas (PMID:21613541). No GO CC term
    currently exists for this complex. Having a term would allow annotation of CFAP61,
    CFAP91, and CFAP251 as part_of the CSC.
  proposed_parent:
    id: GO:0005930
    label: axoneme
  supported_by:
  - reference_id: PMID:21613541
    supporting_text: >-
      ...We previously identified a CaM and Spoke associated Complex (CSC) and
      provided evidence that this complex mediates regulatory signals between
      the radial spokes and dynein arms...
  - reference_id: PMID:22740634
    supporting_text: >-
      ...the CSC connects three major axonemal complexes involved in dynein
      regulation: RS2, the nexin-dynein regulatory complex (N-DRC), and RS3S...

suggested_questions:
- question: >-
    Why do human patients with biallelic CFAP61 loss-of-function mutations not present
    with primary ciliary dyskinesia (PCD) symptoms affecting respiratory cilia, despite
    CFAP61 being present in airway cilia? Is there functional redundancy in respiratory
    cilia that compensates for CFAP61 loss?
- question: >-
    What is the functional significance of the Rossmann-like FAD/NAD(P)-binding fold
    in
    CFAP61? Does it bind any cofactor or ligand, or is it purely a structural scaffold?
    Has anyone tested for oxidoreductase or other enzymatic activity?
- question: >-
    How does CFAP61 interact with intraflagellar transport (IFT) proteins WDR35, IFT22,
    and IFT81? Is CFAP61 itself an IFT cargo, or does it play a role in organizing
    IFT
    within the axoneme?

suggested_experiments:
- hypothesis: >-
    The Rossmann-like domain of CFAP61 has no catalytic activity and functions purely
    as a protein-protein interaction scaffold.
  description: >-
    Express and purify the isolated Rossmann-like domain (residues 665-998) of CFAP61
    and test for FAD/NAD(P) binding by isothermal titration calorimetry or thermal
    shift
    assay. Test for oxidoreductase activity using standard spectrophotometric assays.
    If no cofactor binding or activity is detected, use the domain for pull-down
    experiments to identify its binding partners within the CSC/radial spoke machinery.
  experiment_type: biochemical assay

- hypothesis: >-
    CFAP61 function in respiratory cilia is compensated by redundant mechanisms, explaining
    the absence of PCD in patients with CFAP61 mutations.
  description: >-
    Generate conditional Cfap61 knockout in mouse airway epithelial cells. Analyze
    ciliary beating frequency and waveform by high-speed video microscopy. Perform
    cryo-ET of respiratory cilia to determine whether RS3 is affected. Compare with
    Cfap91 and Cfap251 knockouts to test whether CSC component redundancy differs
    between tissues.
  experiment_type: mouse genetics / ciliary physiology

references:
- id: GO_REF:0000024
  title: Manual transfer of experimentally-verified manual GO annotation data to
    orthologs by curator judgment of sequence similarity
  findings: []
- id: GO_REF:0000033
  title: Annotation inferences using phylogenetic trees
  findings: []
- id: GO_REF:0000044
  title: Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular 
    Location vocabulary mapping, accompanied by conservative changes to GO terms
    applied by UniProt
  findings: []
- id: PMID:28282151
  title: Quantitative Proteomic Analysis of Human Airway Cilia Identifies 
    Previously Uncharacterized Proteins of High Abundance.
  findings:
  - statement: CFAP61 was identified in human airway ciliary axonemes by 
      quantitative LC/MS proteomics.
    supporting_text: >-
      ...Quantitative Proteomic Analysis of Human Airway Cilia Identifies Previously
      Uncharacterized Proteins of High Abundance...
- id: PMID:34792097
  title: CFAP61 is required for sperm flagellum formation and male fertility in 
    human and mouse.
  findings:
  - statement: >-
      CFAP61 is a conserved CSC component. A splice variant causes MMAF in human.
      Cfap61 knockout mice are infertile with impaired radial spoke assembly during
      spermiogenesis and subsequent axoneme instability.
    supporting_text: >-
      ...CFAP61 is a conserved component of the calmodulin- and radial spoke-associated
      complex (CSC) of cilia... the assembly of radial spoke components is impaired.
      As
      spermiogenesis progresses, the axoneme in Cfap61-/- cells becomes unstable and
      scatters,
      and the distribution of intraflagellar transport proteins is disrupted...
  - statement: >-
      CFAP61 interacts with the CSC, radial spoke stalk, and head proteins including
      CFAP91, RSPH3A, ROPN1, ROPN1L, RSPH9, DYNLT1, DYNC1I2, TUBB3, and IFT proteins.
    supporting_text: >-
      ...CFAP61 interacts with the CSC, radial spoke stalk and head...
  - statement: >-
      This study reveals an organ-specific mechanism of axoneme stabilization. Cfap61
      knockout mice show infertility but not other PCD symptoms.
    supporting_text: >-
      ...We generated Cfap61 knockout mice that recapitulate the infertility phenotype
      of
      the human CFAP61 mutation, but without other symptoms usually observed in PCD...
- id: PMID:35174165
  title: Biallelic Variants in CFAP61 Cause Multiple Morphological Abnormalities
    of the Flagella and Male Infertility.
  findings:
  - statement: >-
      Biallelic CFAP61 variants (frameshift p.I151Nfs*4 and nonsense p.R283*) cause
      MMAF
      with severely disorganized axonemes in three Pakistani families with 11 infertile
      males.
    supporting_text: >-
      ...variants, c.451_452del (p.I151Nfs*4) in family 1 and c.847C > T (p.R283*)
      in family 2
      and 3, were identified recessively co-segregating with the MMAF phenotype. Transmission
      electron microscopy analyses revealed severe disorganized axonemal ultrastructures,
      and
      missings of central pair, RSs, and inner dynein arms were also observed...
  - statement: >-
      Both CFAP61 and CFAP251 proteins are absent from patient sperm tails, indicating
      loss of the entire CSC complex.
    supporting_text: >-
      ...CFAP61 and CFAP251 signals were absent from sperm tails of the patients,
      which
      suggested the loss of functional CSC in sperm flagella...
- id: PMID:36659204
  title: Absence of murine CFAP61 causes male infertility due to multiple 
    morphological abnormalities of the flagella.
  findings:
  - statement: >-
      Cfap61 knockout mice show MMAF with short, coiled, and irregular flagella. Cfap61
      localizes first to the neck, then to the midpiece of mature sperm.
    supporting_text: >-
      ...Cfap61 is initially localized at the neck of sperm, where it potentially
      functions
      in flagellum formation, and is later localized to the midpiece of the sperm...
- id: PMID:37258679
  title: Axonemal structures reveal mechanoregulatory and disease mechanisms.
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: >-
      Cryo-EM atomic model of the human respiratory-cilia axoneme (PDB 8J07) directly
      resolves CFAP61 as a structural subunit at the base of radial spoke 3 (RS3),
      confirming its axonemal localization and radial-spoke association; the GTP/GDP/Mg
      ligands in the structure belong to axonemal tubulin, not CFAP61, so no GTPase/
      nucleotide-binding function is attributable to CFAP61.
  findings:
  - statement: >-
      In the human respiratory-cilia axoneme, the full-length RS3 is built on a base of
      CFAP61, CFAP91 and CFAP251; CFAP91 links the RS3 subunits CFAP251 and CFAP61,
      defining the architecture of the calmodulin- and spoke-associated complex within
      the axoneme.
    supporting_text: >-
      ...a full-length RS3, which we show is built on a base of CFAP61, CFAP91 and
      CFAP251... then links the RS3 subunits CFAP251 and CFAP61...
- id: PMID:35387802
  title: Biallelic CFAP61 variants cause male infertility in humans and mice
    with severe oligoasthenoteratozoospermia.
  findings:
  - statement: >-
      Additional CFAP61 pathogenic variants (R552C, D971N, splice site, G556R) identified
      in OAT patients with MMAF, absence of central pair, and mitochondrial sheath
      malformation.
    supporting_text: >-
      ...CONCLUSIONS: Our findings indicate that CFAP61 is essential for spermatogenesis
      and that biallelic
      CFAP61 variants lead to male infertility in humans and mice with OAT...
- id: PMID:25694453
  title: The CSC proteins FAP61 and FAP251 build the basal substructures of 
    radial spoke 3 in cilia.
  findings:
  - statement: >-
      In Tetrahymena, FAP61 is required for RS3 stem assembly. FAP61-null cilia have
      missing or incomplete RS3 while RS1 and RS2 are unaffected. Loss reduces cell
      swimming and alters ciliary waveform.
    supporting_text: >-
      ...loss of either FAP61 or FAP251 reduces cell swimming and affects the ciliary
      waveform and that RS3 is either missing or incomplete, whereas RS1 and RS2 are
      unaffected. Specifically, FAP251-null cilia lack an arch-like density at the
      RS3
      base, whereas FAP61-null cilia lack an adjacent portion of the RS3 stem region...
- id: PMID:22740634
  title: The CSC connects three major axonemal complexes involved in dynein 
    regulation.
  findings:
  - statement: >-
      The CSC connects RS2, the N-DRC, and RS3 stand-in (RS3S) in Chlamydomonas,
      providing a structural link for signal transduction from radial spokes to dynein
      motors.
    supporting_text: >-
      ...the CSC connects three major axonemal complexes involved in dynein regulation:
      RS2, the nexin-dynein regulatory complex (N-DRC), and RS3S...
- id: PMID:21613541
  title: The CSC is required for complete radial spoke assembly and wild-type 
    ciliary motility.
  findings:
  - statement: >-
      The CSC was first characterized as a calmodulin- and spoke-associated complex
      in
      Chlamydomonas. It is required for RS2 assembly and modulates dynein activity.
    supporting_text: >-
      ...We previously identified a CaM and Spoke associated Complex (CSC) and provided
      evidence that this complex mediates regulatory signals between the radial spokes
      and dynein arms... the CSC is required for spoke assembly and wild-type motility...
- id: PMID:36552811
  title: Cfap91-Dependent Stability of the RS2 and RS3 Base Proteins and 
    Adjacent Inner Dynein Arms in Tetrahymena Cilia.
  findings:
  - statement: >-
      Cfap91 is required for stable docking of RS3 and RS2 base proteins. Loss of
      Cfap91
      causes significant reduction of Cfap61 and Cfap251 levels at the axoneme.
    supporting_text: >-
      ...The localization studies confirmed that the level of RS3-specific proteins,
      Cfap61
      and Cfap251, as well as RS2-associated Cfap206, are significantly diminished
      in
      Tetrahymena CFAP91-KO cells...
- id: PMID:37982895
  title: CFAP61 knockdown aggravates male infertility by inhibiting testosterone
    secretion by Leydig cells via the MAPK/COX-2 pathway.
  findings:
  - statement: >-
      In vitro knockdown of CFAP61 in Leydig cells reduced testosterone secretion
      via
      MAPK/COX-2 pathway activation. Physiological relevance is uncertain.
    supporting_text: >-
      ...CFAP61 knockdown reduced the Leydig cell viability and testosterone secretion
      and enhanced apoptosis...
- id: file:human/CFAP61/CFAP61-notes.md
  title: CFAP61 review notes
  findings:
  - statement: >-
      CFAP61 contains a Rossmann-like FAD/NAD(P)-binding superfamily fold (residues
      665-998)
      that functions as a structural scaffold, not an enzyme. The BioReason-Pro paper
      highlights this as an example of a domain that could mislead automated annotation
      pipelines into predicting enzymatic function.
- id: file:human/CFAP61/CFAP61-deep-research-falcon.md
  title: Falcon deep research report for CFAP61
  findings:
  - statement: Falcon research supports CFAP61 as a non-enzymatic axonemal CSC/radial-spoke structural factor needed for sperm flagellum assembly, radial spoke assembly, and sperm motility.
    supporting_text: CFAP61 is best supported as a structural/assembly and stabilization factor for the sperm flagellar axoneme, acting through the CSC-radial spoke system.