CEN3

CEN3 (PF3D7_1027700) is a calcium-binding centrin that associates with the centrosome-equivalent centriolar plaque of Plasmodium during cell division (PMID:18693242, PMID:34535568). It is expressed across sporozoite, asexual blood, gametocyte, and oocyst stages and localizes in close proximity to the nucleus, specifically to the extranuclear compartment of the centriolar plaque — a position distinct from the intranuclear region where microtubules are nucleated (PMID:18693242, PMID:34535568). At this site CEN3 physically interacts with CEN1 (by reciprocal co-immunoprecipitation) and co-localizes with the Sfi1-like plaque protein PfSlp, and in the P. berghei ortholog it forms part of a multi-protein centrin complex including CEN-1, CEN-2, and CEN-4 (PMID:37130129, PMID:30541825). In P. berghei, cen-3 could not be deleted, indicating the gene is essential for asexual blood-stage development (PMID:30541825). Biochemically, recombinant CEN3 undergoes calcium-inducible liquid-liquid phase separation in vitro, forming droplets that fuse and wet surfaces; unlike CEN1, CEN3 condensates mature partially irreversibly toward a gel-like state, a temperature-dependent process, and this behavior depends on its disordered N-terminus and EF-hand calcium-binding motifs, establishing Ca2+-specific condensation as a defining property (PMID:38150475). Beyond its centriolar-plaque localization, essentiality, centrin-complex membership, and calcium-driven condensation, the precise cellular function of CEN3 in centriolar plaque biology has not been mechanistically resolved in the available corpus.

1. 2008 Medium

   Established that CEN3 is a broadly expressed centrin positioned at the parasite centrosome, placing it at the machinery of cell division.

   Evidence Immunoelectron and confocal/widefield immunofluorescence microscopy with anti-centrin and anti-alpha-tubulin antibodies in P. falciparum

   PMID:18693242

   Open questions at the time

   • Antibody recognizes multiple centrins and does not exclusively report CEN3
   • No functional role assigned beyond spatial association with the centrosome
   • Sub-compartment within the centriolar plaque not resolved
2. 2019 Medium

   Tested whether CEN3 is dispensable and whether it acts alone; the ortholog proved essential and was shown to sit within a multi-centrin complex.

   Evidence Failed cen-3 gene deletion (essentiality inference) and multi-protein complex formation analysis with PbCEN-4 in P. berghei

   PMID:30541825

   Open questions at the time

   • Essentiality inferred from failed knockout rather than conditional depletion
   • Stoichiometry and architecture of the centrin complex not defined
   • Direct versus indirect nature of CEN3 contacts within the complex unresolved
3. 2021 High

   Resolved where within the centriolar plaque CEN3 acts, distinguishing its extranuclear position from the intranuclear microtubule-nucleating compartment.

   Evidence Super-resolution STED microscopy and correlative light/electron tomography with affinity-purified anti-PfCentrin3 antibody in P. falciparum NF54

   PMID:34535568

   Open questions at the time

   • Functional consequence of extranuclear localization not established
   • Dynamics of CEN3 recruitment during the cell cycle not tracked
   • Molecular tethers holding CEN3 in the extranuclear compartment unknown
4. 2023 Medium

   Defined CEN3's physical partners at the plaque, connecting it to CEN1 and the Sfi1-like protein PfSlp.

   Evidence Reciprocal co-immunoprecipitation (PfCen1-GFP pulldown) and dual-color STED super-resolution microscopy in P. falciparum

   PMID:37130129

   Open questions at the time

   • No cross-linking or quantitative enrichment analysis performed
   • Whether the CEN3-CEN1 interaction is direct not shown
   • Functional role of the CEN3-PfSlp co-localization not tested
5. 2023 High

   Provided a biophysical mechanism for CEN3 accumulation by showing calcium-specific, EF-hand- and N-terminus-dependent phase separation, and distinguished CEN3 from CEN1 by its partial condensate irreversibility.

   Evidence In vitro reconstitution with recombinant tagged and untagged PfCen3, turbidity/light-scattering, live droplet fusion imaging at varying temperatures, and EDTA dissolution assays

   PMID:38150475

   Open questions at the time

   • Phase separation demonstrated in vitro only, not confirmed at the centriolar plaque in vivo
   • Physiological consequence of irreversible condensate maturation unknown
   • Whether CEN3 condensation recruits or scaffolds its complex partners not tested

• How Ca2+-driven CEN3 condensation is coupled to its essential role in centriolar plaque function and parasite division remains unresolved.
• No conditional depletion linking loss of CEN3 to a specific division defect
• In vitro phase separation not yet connected to in vivo condensate formation
• Direct molecular activity of CEN3 within the plaque undefined