SPC24

SPC24 (PF3D7_1227600) is a highly divergent but genuine subunit of the outer kinetochore NDC80 complex in Plasmodium, functioning in chromosome segregation during the parasite's proliferative life-cycle stages (PMID:32501284). Identified by deep-homology searching together with live-cell imaging in P. berghei, it completes the canonical tetrameric NDC80 assembly, in which SPC24 pairs with SPC25 as a heterodimer that joins the NDC80–NUF2 heterodimer (PMID:32501284). Affinity purification of SPC24 from synchronised mitotic gametocytes recovers NDC80, NUF2, and SPC25 as the dominant partners, confirming the SPC24–SPC25 heterodimer and its incorporation into the tetramer in the malaria parasite (PMID:36006241). During male gametogenesis, SPC24 localises laterally along EB1-decorated spindle microtubules throughout endomitosis rather than forming end-on attachments, a pattern shared with NDC80, SPC25, and AKiT1 and lost in an EB1-null background (PMID:37208365). Cross-linking immunoprecipitation places SPC24 at the interface between the outer NDC80 complex and apicomplexan-specific inner kinetochore AKiT proteins (PMID:36006241) and in proximity to the chromosomal passenger complex kinase ARK1 (PMID:42323312). Beyond these findings, no in vitro reconstitution or targeted mutagenesis of SPC24 itself has been characterized in the available corpus.

1. 2020 Medium

   Whether Plasmodium possesses a complete canonical NDC80 kinetochore complex was unresolved because its subunits are highly divergent; identifying SPC24 established that the parasite encodes a genuine, if divergent, tetrameric NDC80 assembly.

   Evidence Bioinformatic deep-homology search combined with NDC80-GFP live-cell imaging in P. berghei

   PMID:32501284

   Open questions at the time

   • Identification rests on homology plus imaging of NDC80, not on direct functional or structural analysis of SPC24 itself
   • No mutagenesis or knockout of SPC24 to test its requirement
   • No in vitro reconstitution of the SPC24–SPC25 heterodimer
2. 2022 Medium

   Whether the homology-inferred SPC24 physically assembles into the NDC80 tetramer in the parasite was untested; affinity purification confirmed SPC24 co-purifies with NDC80, NUF2, and SPC25 as its principal partners.

   Evidence SPC24-3xHA affinity purification with label-free quantitative mass spectrometry from synchronised mitotic gametocytes

   PMID:36006241

   Open questions at the time

   • Single-lab Co-IP without reciprocal SPC25-side validation of the heterodimer
   • Stoichiometry and direct binary interfaces not resolved
   • No structural model of the parasite tetramer
3. 2022 Low

   Whether the outer NDC80 complex connects to the apicomplexan-specific inner kinetochore was open; cross-linking placed SPC24 at the outer–inner interface with AKiT2–6.

   Evidence Cross-linking immunoprecipitation (SPC24-3xHA) with quantitative MS comparing low vs. high cross-linking conditions

   PMID:36006241

   Open questions at the time

   • Cross-linking MS enrichment only, with no orthogonal validation of a direct SPC24–AKiT interaction
   • Cannot distinguish direct from indirect association
   • Interaction interfaces and functional consequence unknown
4. 2023 Medium

   The mode of kinetochore–spindle attachment in Plasmodium was undefined; imaging showed SPC24-containing kinetochores attach laterally, not end-on, along EB1-decorated microtubules throughout endomitosis.

   Evidence IFA and ultrastructure expansion microscopy of SPC24-4Myc parasites, including an EB1-null comparison, in P. yoelii male gametogenesis

   PMID:37208365

   Open questions at the time

   • Shown for male gametogenesis; attachment mode in other proliferative stages not established
   • Mechanistic basis of EB1-dependence for SPC24 localisation not resolved
   • Whether SPC24 directly contacts microtubules or EB1 not determined
5. 2026 Low

   Whether SPC24 lies near the chromosomal passenger complex regulatory hub was unknown; cross-linking placed SPC24 in the ARK1 interaction neighbourhood at the kinetochore during male gametogenesis.

   Evidence ARK1 cross-linking immunoprecipitation with quantitative MS under incremental cross-linking conditions in P. berghei

   PMID:42323312

   Open questions at the time

   • Detected only under high cross-linking, indicating a labile or indirect proximity with no orthogonal validation
   • No evidence SPC24 is an ARK1 substrate or direct binding partner
   • Functional role of the SPC24–ARK1 proximity unknown

• The intrinsic contribution of SPC24 to kinetochore assembly and chromosome segregation remains untested by direct loss-of-function or structural analysis.
• No SPC24 conditional knockout phenotype
• No structure of the parasite SPC24–SPC25 heterodimer or tetramer
• Direct binding partners and microtubule/EB1 contacts unresolved