CEN4

CEN4 (PF3D7_1105500) is an alveolate-specific, calcium-binding centrin that acts at the parasite centriolar plaque/microtubule-organizing center (MTOC) during the repeated rounds of nuclear division that drive Plasmodium proliferation (PMID:18693242, PMID:30541825). In P. falciparum it is expressed across sporozoite, asexual blood, gametocyte, and oocyst stages and localizes in close proximity to the nucleus in association with the centrosome, co-staining with alpha-tubulin (PMID:18693242). Live super-resolution and GFP imaging in P. berghei show CEN4 is cytoplasmic outside mitosis but is dynamically recruited to the centriolar plaque during nuclear division, where its foci duplicate ahead of each division and appear as one or two foci per nucleus consistent with asynchronous mitosis (PMID:30541825, PMID:32501284). CEN4 assembles into a multi-protein complex with the other Plasmodium centrins (PbCen1, 2, and 3), and genetic knockout of the P. berghei ortholog establishes that CEN4 is dispensable for asexual proliferation (PMID:30541825). Recombinant PfCEN4 undergoes calcium-inducible, EDTA-reversible liquid-liquid phase separation into fusogenic droplets and can form co-condensates with other centrins, and it does so despite lacking a predicted N-terminal intrinsically disordered region, indicating IDR-independent condensation for this paralog (PMID:38150475).

1. 2008 Medium

   Establishing where CEN4 acts and whether it is functional was the first question; localization to the centrosome near the nucleus across multiple life-cycle stages placed CEN4 at the parasite MTOC and demonstrated its expression throughout the life cycle.

   Evidence Immunofluorescence and immunoelectron microscopy with anti-centrin/anti-alpha-tubulin co-staining, plus growth complementation in a Leishmania centrin knockout line, in P. falciparum

   PMID:18693242

   Open questions at the time

   • The complementation assay tested all four PfCENs and did not resolve whether CEN4 specifically rescued the Leishmania defect
   • Static imaging did not reveal how localization changes through the cell cycle
   • No interaction partners or complex membership defined
2. 2019 High

   Whether CEN4 localization is dynamic, whether it partners with other centrins, and whether it is required for growth were resolved: CEN4 relocates from cytoplasm to the centriolar plaque during nuclear division, joins a centrin complex, and is dispensable for proliferation.

   Evidence Super-resolution SIM live-cell imaging, co-immunoprecipitation/complex analysis, and genetic knockout in P. berghei

   PMID:30541825

   Open questions at the time

   • The molecular trigger for cytoplasm-to-MTOC recruitment is not defined
   • Dispensability for asexual proliferation leaves open roles in sexual or mosquito stages
   • Stoichiometry and architecture of the centrin complex not resolved
3. 2020 Medium

   The timing of CEN4 behavior relative to mitosis was clarified: CEN4 foci duplicate before each nuclear division, marking the parasite's preparation for successive rounds of division.

   Evidence Live-cell GFP imaging of CEN4-GFP as an MTOC/centriolar plaque marker in P. berghei

   PMID:32501284

   Open questions at the time

   • Whether CEN4 duplication is a cause or a consequence of centriolar plaque duplication is not established
   • Mechanism controlling asynchronous foci numbers per nucleus unknown
4. 2023 Medium

   How CEN4 might organize material at the MTOC was addressed biochemically: recombinant CEN4 undergoes calcium-inducible, reversible phase separation and co-condenses with other centrins, providing a physical basis for centrin assembly.

   Evidence In vitro turbidity assays, widefield microscopy of droplet formation/fusion, EDTA reversibility, and IUPred3 IDR prediction with recombinant PfCEN4

   PMID:38150475

   Open questions at the time

   • LLPS was shown in vitro only; its occurrence and function at the parasite centriolar plaque in vivo is not demonstrated
   • The sequence determinant driving CEN4 condensation without an N-terminal IDR is not identified
   • Functional consequence of centrin co-condensation for MTOC activity untested

• The specific mechanistic function of CEN4 at the centriolar plaque and its dedicated (non-redundant) role, given its dispensability for asexual growth, remain unresolved.
• No defined biochemical activity linking CEN4 to centriolar plaque duplication or spindle organization
• Role in sexual and mosquito-stage development not directly tested despite expression there
• Whether in vitro LLPS reflects a physiological condensation mechanism in vivo is unknown