EB1

TgEB1 is a microtubule plus end-binding protein that governs mitotic spindle integrity and accurate chromosome segregation in Toxoplasma gondii (PMID:26466679). A C-terminal nuclear localization signal confines it to the nucleoplasm during interphase, and upon mitosis it redistributes onto the intranuclear spindle microtubules and later associates with subpellicular microtubules at the growing end of daughter buds during karyokinesis (PMID:26466679). Its essential mitotic role is to bundle spindle microtubules so that kinetochore clustering is maintained: depletion causes disintegration of kinetochore clustering (PMID:26466679), while overexpression of a high-MT-affinity mutant overstabilizes microtubule bundles and avulses clustered kinetochores, showing that MT-bundling activity must be tightly tuned (PMID:26466679). Recruitment of TgEB1 to the spindle is downstream of the centrosome inner core, as loss of TgCep250L1 abolishes spindle localization and yields unsegregated nuclei (PMID:36069445). The MT-binding mechanism is conserved, operating on the same principles when expressed heterologously in Xenopus (PMID:26466679).

1. 2015 High

   Establishing where and when a microtubule plus-end binder acts across the cell cycle showed that TgEB1 is spatially partitioned by an NLS, tying its activity to specific mitotic structures.

   Evidence Live imaging and endogenous tagging with identification of a C-terminal NLS in Toxoplasma

   PMID:26466679

   Open questions at the time

   • Does not identify the plus-end recognition motif or MT-binding partners
   • Mechanism of NLS-driven nucleoplasmic-to-spindle switch during mitosis not resolved
2. 2015 High

   Whether TgEB1 is functionally required for mitosis was answered by depletion, which disrupted kinetochore clustering and defined its role in accurate chromosome segregation.

   Evidence Conditional depletion of TgEB1 with kinetochore-clustering readout in Toxoplasma

   PMID:26466679

   Open questions at the time

   • Does not establish whether the defect is direct MT-bundling loss or an indirect spindle collapse
   • No quantification of segregation error rate or downstream viability
3. 2015 High

   Whether MT-affinity is a regulated parameter was tested with a gain-of-function mutant, showing that excessive bundling activity is itself pathological by avulsing kinetochores.

   Evidence Overexpression of a high-MT-affinity TgEB1 mutant with microtubule and kinetochore phenotype analysis in Toxoplasma

   PMID:26466679

   Open questions at the time

   • Does not identify the physiological regulator that tunes MT-affinity in wild-type cells
   • Structural basis of the affinity-increasing mutation not defined
4. 2015 Medium

   Whether the MT-binding mechanism is species-specific or conserved was addressed by heterologous expression, indicating a conserved plus-end binding principle.

   Evidence Heterologous expression in a Xenopus system with MT-binding assay

   PMID:26466679

   Open questions at the time

   • Single heterologous experiment, not independently replicated
   • Does not test whether the mitotic bundling function is conserved, only MT association
5. 2022 Medium

   What positions TgEB1 on the spindle was answered by centrosome inner-core epistasis, placing TgEB1 spindle recruitment downstream of TgCep250L1.

   Evidence Conditional degradation of TgCep250L1 with TgEB1 localization by fluorescence and ultrastructure expansion microscopy in Toxoplasma

   PMID:36069445

   Open questions at the time

   • Does not establish whether recruitment is direct or via intermediate spindle-nucleating factors
   • Single lab, single study without reciprocal validation

• How TgEB1's MT-bundling affinity is physiologically tuned during the cell cycle, and the molecular link between the centrosome inner core and TgEB1 spindle loading, remain unresolved.
• No identified regulator of TgEB1 MT-affinity
• No direct biochemical partner connecting TgCep250L1 to TgEB1
• No structural model of the MT-binding interface