Mst27D (CG31907) is a spermatid-specific member of the EB1/RP-EB microtubule-associated protein family. It belongs to a fly-lineage EB subfamily that also contains Drosophila Eb1. Its N-terminal calponin-homology (CH) domain binds microtubules. Its divergent C-terminal region (residues 151-424) lacks the EB1 C-terminal (EBH) domain and the acidic EEY/F tail of canonical EB proteins; it mediates homodimerization and binds the nuclear pore protein Nup358. Endogenous Mst27D accumulates after meiosis. It localizes to the nuclear envelope region that still holds nuclear pore complexes, and to the microtubule bundle of the adjacent dense complex. Unlike Eb1, which tracks growing microtubule plus ends, Mst27D binds along the whole microtubule lattice. At high concentration it bundles microtubules. In spermatids it links nuclear pore complexes to the dense-complex microtubules and is needed to bundle these progressively into a single bundle. Null mutants fail to form this bundle and their spermatid nuclei elongate abnormally.
Summary: Native Mst27D is on the nuclear envelope next to the nuclear pores and on the dense-complex microtubule bundle. It is largely absent from the basal body, which is the spermatid MTOC.
Reason: The MTOC IBD sits at the eukaryote-wide node PTN000065701 and records canonical EB localization at centrosomes and spindle pole bodies. Mst27D is in the fly-lineage subfamily PTHR10623:SF6 with Eb1. Unlike Eb1, it has lost the EBH domain, which recruits partners in canonical EBs, and it does not track plus ends. The target data place it away from the MTOC. Live imaging found the basal-body region "largely devoid of Mst27D-EGFP". The dense-complex bundle that Mst27D organizes "did not seem to be nucleated by the centrosome/basal body". Mst27D organizes microtubules nucleated elsewhere but does not itself reside at an MTOC. The annotation is therefore an over-propagation, though Mst27D remains a microtubule-associated protein.
Propagation Review
Root cause:PROPAGATION BAD
Failure modes:CONTEXT OR TISSUE MISMATCH
Sources checked:
PANTHER:PTN000065701 · PTN000065701 SUPPORTS SOURCE BUT NOT TARGET
Canonical EB MTOC localization is well supported in donors. For Mst27D, live imaging shows it is largely absent from the basal body and lacks EBH-mediated recruitment.
Summary: Mst27D is enriched on the cytoplasmic microtubule bundle of the spermatid dense complex and decorates microtubules along their length in S2R+ cells.
Reason: Microtubule association by the CH domain is the feature conserved across the EB family. The PTN000065701 assertion is supported in the target. In spermatids, Mst27D-mCherry is enriched on the dense-complex microtubule bundles. In cultured cells, full-length Mst27D binds microtubules along their length. These are cytoplasmic, non-spindle microtubules. They are the place where the core Mst27D activity occurs.
Summary: The Mst27D CH domain binds microtubules along the lattice rather than at plus ends. The correct term is the more specific child microtubule lateral binding.
Reason: The InterPro2GO mapping is correct; Mst27D binds microtubules. The target data show the mode of binding. Full-length Mst27D, and a chimera carrying the Mst27D CH domain on the Eb1 dimerization region, both decorate microtubules along their length, whereas Eb1 forms plus-end comets. GO:0099609 (microtubule lateral binding, "binding to the side of a microtubule") is an is_a child of GO:0008017 and describes this activity more precisely. It is used as the core molecular function.
Mst27D binds all along MTs, contrasting with Eb1’s preference for growing MT plus ends
GO:0031110 regulation of microtubule polymerization or depolymerization
IBA GO_REF:0000033
UNDECIDED
Summary: The proposed effect of Mst27D bundling on microtubule stability has not been tested. No target data address polymerization or depolymerization directly.
Reason: Canonical EBs regulate microtubule dynamics at growing plus ends. Mst27D has lost plus-end tracking and the EBH domain, which argues against inheriting that mechanism. However, the authors propose that Mst27D-driven bundling stabilizes dense-complex microtubules, and demecolcine shows that dense-complex formation needs dynamic polymerization. This could be a different route to regulating dynamics, but no assay of microtubule growth or shrinkage with and without Mst27D has been done. The ancestral mechanism is unlikely to apply. Whether a bundling-based effect on stability exists is unknown. This review neither accepts nor removes the term.
Propagation Review
Root cause:UNRESOLVED
Sources checked:
PANTHER:PTN000065701 · PTN000065701 UNRESOLVED
The plus-end mechanism behind the donors' term has diverged in Mst27D. A bundling-based stabilization effect has been proposed but not tested.
Summary: Full-length Mst27D binds the microtubule lattice and is not enriched at growing plus ends. Comets formed only by an isolated C-terminal fragment.
Reason: The plus-end IBD at PTN000065701 records canonical EB plus-end tracking. Mst27D shares PTHR10623:SF6 with Eb1, but its CH domain has diverged. Grafting the Mst27D CH domain onto the Eb1 C-terminal dimerization region gives lattice-wide binding, not comets. The microtubule-binding domain therefore no longer prefers plus ends. Comets were seen only with Mst27D_CT-EGFP, an isolated C-terminal fragment that has no microtubule-binding domain. The authors attribute these comets to possible heterodimerization with endogenous Eb1. Mst27D also lacks the EBH domain and EEY/F tail. Plus-end localization is therefore an inherited EB trait that Mst27D has lost.
Propagation Review
Root cause:PROPAGATION BAD
Failure modes:CONTEXT OR TISSUE MISMATCH
Sources checked:
PANTHER:PTN000065701 · PTN000065701 SUPPORTS SOURCE BUT NOT TARGET
Domain-swap experiments place the loss of plus-end preference in the Mst27D CH domain. The EBH domain is also absent.
Summary: Mst27D recruits Nup358, and with it nuclear pore complexes, to microtubules. Its C-terminal region binds Nup358, and co-expressed Nup358 is drawn into Mst27D-induced microtubule cables.
Reason: In canonical EBs this process is +TIP recruitment through the EBH domain, which Mst27D lacks. The general process is nevertheless supported in the target through a divergent C-terminal region. Mst27D's C-terminal region binds Nup358. When overexpressed, Mst27D draws co-expressed mCherry-Nup358 into microtubule cables. In spermatids, Mst27D links NPCs to the dense-complex microtubule bundle. Mst27D is the protein that brings a partner to the microtubule, so this is participation, not just a requirement.
Summary: The Mst27D CH domain binds along the microtubule lattice. Mst27D lacks plus-end preference, so lateral binding is the accurate activity.
Reason: The donors include both fly SF6 paralogs, Eb1 (FBgn0027066) and CG18190 (FBgn0034403). Both keep the EBH domain and plus-end tracking. Mst27D lacks the EBH domain and the EEY/F tail. In a domain swap, the Mst27D CH domain on the Eb1 dimerization region binds the lattice, not plus ends. The activity Mst27D inherited is binding to microtubules, and in Mst27D it has become lateral binding, not plus-end binding. Lateral binding is captured by the GO:0099609 replacement on the microtubule binding row. This plus-end term over-annotates Mst27D, as does the matching microtubule plus-end location row.
Propagation Review
Root cause:PROPAGATION BAD
Failure modes:CONTEXT OR TISSUE MISMATCH
Sources checked:
PANTHER:PTN000065701 · PTN000065701 SUPPORTS SOURCE BUT NOT TARGET
Donor plus-end binding is well supported. In Mst27D, plus-end preference is lost at the CH domain and the EBH domain is absent.
Summary: Endogenous Mst27D is made only after meiosis, in spermatids that do not divide. Null mutants have no defects before meiosis ends.
Reason: The spindle-assembly IBD at PTN000065701 is ancestral EB biology, seeded in flies by Eb1. The target evidence directly argues against it. Endogenous Mst27D accumulation starts after meiosis, and null mutants show no abnormality until after meiosis. Postmeiotic spermatids build no spindles. Spindle enrichment was seen only with transgenes that expressed Mst27D prematurely. Spindle assembly was therefore not inherited as a native Mst27D role.
Propagation Review
Root cause:PROPAGATION BAD
Failure modes:CONTEXT OR TISSUE MISMATCH
Sources checked:
PANTHER:PTN000065701 · PTN000065701 SUPPORTS SOURCE BUT NOT TARGET
Native postmeiotic expression and a stage-resolved null phenotype show that Mst27D diverged from the ancestral spindle role.
Summary: Native Mst27D is present only in postmeiotic spermatids, which have no spindle. It localizes to the nuclear envelope next to the nuclear pores and to the dense complex.
Reason: The spindle-midzone IBD comes from yeast EB donors (Mal3, Bim1). Endogenous Mst27D first accumulates after both meiotic divisions, so the protein is absent when a spindle midzone exists. The spindle signal seen in meiotic cells came from prematurely expressed transgenes, not native protein. This is the same basis as the spindle-assembly removal, and the two rows are treated together.
Propagation Review
Root cause:PROPAGATION BAD
Failure modes:CONTEXT OR TISSUE MISMATCH
Sources checked:
PANTHER:PTN000065701 · PTN000065701 SUPPORTS SOURCE BUT NOT TARGET
Native Mst27D is postmeiotic, so it does not coexist with a spindle midzone.
IMP PMID:37428798 Nuclear elongation during spermiogenesis depends on physical...
NEW
Summary: Mst27D is required to bundle the dense-complex microtubules progressively, and it bundles microtubules itself when expressed in cultured cells.
Reason: Mst27D does part of the work of this process. It is not only necessary for it. Null spermatids fail to form the dense-complex bundle. In S2R+ cells, high Mst27D levels bundle microtubules into cables, with either EGFP or monomeric mCherry tags. A chimera that cannot reach NPCs still bundles. The authors propose that an Mst27D dimer cross-links two microtubules through its two CH domains. The perturbation and gain-of-function data together support direct participation.
MT bundling was also obtained with the chimeric protein Mst27D_CH-Eb1_CT-EGFP, indicating that the MT bundling activity does not require the concentration of Mst27D by recruitment to NPCs
IMP PMID:37428798 Nuclear elongation during spermiogenesis depends on physical...
NEW
Summary: Mst27D is the physical linker between nuclear pores and the dense-complex microtubule bundle, and this linkage shapes the elongating spermatid nucleus.
Reason: This is not a downstream phenotype. Mst27D is a structural link in the force-bearing connection between the nuclear envelope and the microtubule bundle, and the nucleus elongates along that bundle. It does part of the work of nuclear shaping. Nup358 is needed to bring Mst27D to the nuclear envelope. Without Mst27D, nuclear elongation is abnormal. Spag4 forms a separate nuclear-envelope-to-microtubule connection at the basal body, and Mst27D acts independently of it.
IDA PMID:37428798 Nuclear elongation during spermiogenesis depends on physical...
NEW
Summary: In spermatids, Mst27D is restricted to the nuclear envelope region that still holds nuclear pores, and Nup358 is needed for this enrichment.
Reason: Live imaging of tagged Mst27D in spermatids shows that it is restricted to the nuclear envelope region containing NPCs. Depleting Nup358 reduces this enrichment. Mst27D's linker function is carried out at this nuclear envelope face, where it connects pores to cytoplasmic microtubules.
Nup358 is clearly required for normal enrichment of Mst27D on the NE
Core Functions
Binds the microtubule lattice through its N-terminal CH domain and, as a homodimer, bundles the perinuclear microtubules of the spermatid dense complex into a single bundle.
In Mst27D null mutants, this bundling process does not occur and nuclear elongation is abnormal.
Links Nup358-containing nuclear pore complexes on the spermatid nuclear envelope to the dense-complex microtubule bundle. Its C-terminal region binds Nup358. This linkage is needed for normal elongation of the spermatid nucleus.
Q: Does the Mst27D CH domain bind microtubules directly in vitro? Is its lattice-wide binding caused by specific substitutions in the CH domain, compared with the plus-end-tracking CH domain of Eb1?
Q: Is the concentration of endogenous Mst27D in spermatids high enough to cross-link microtubules directly? Or does Mst27D bundle mainly by concentrating other bundling factors at the nuclear envelope?
Suggested Experiments
Experiment: Reconstitute purified full-length Mst27D with dynamic microtubules (TIRF). Test for lattice binding versus plus-end tracking, for microtubule bundling, and for effects on growth and catastrophe rates. This would test the bundling activity directly and decide the IBA term for regulation of microtubule polymerization or depolymerization.
Type: in vitro reconstitution (TIRF microscopy)
External Prediction Reviews
These computational predictions are reviewed separately from the GOA annotation set used for this review. The assessments below are from this project and do not constitute official GO annotations or endorsement by GO/UniProt. They are not included in the existing annotation review above.
Microtubule binding is directly supported by CH-domain experiments, full-length localization and the bundling phenotype; plus-end-specific and polymerization-dynamics claims are unsupported EB1-family carry-over.
Review rationale: Microtubule binding is directly supported by CH-domain experiments, full-length localization and the bundling phenotype. The same broad term already appears in GOA. Although GOA also contains plus-end binding, primary evidence shows that Mst27D binds along the lattice rather than sharing EB1’s preference, so the unsupported narrower annotation is not grounds for LSP. ProtNLM correctly captures the established broad activity; the gene review refines the IEA row to the more specific microtubule lateral binding (GO:0099609), which is not yet in GOA. The OpenScientist audit also retained GO:0008017 while identifying plus-end, spindle and dynamics terms as EB1/MAPRE-family IBA carry-over.
Supporting Evidence:
PMID:37428798: "Moreover, Mst27D binds all along MTs, contrasting with Eb1’s preference for growing MT plus ends."
PMID:37428798: "full length Mst27D-EGFP associated with MTs all along their length"
PMID:37428798: "In Mst27D null mutants, this bundling process does not occur and nuclear elongation is abnormal."
FlyBase gene description for Mst27D (DROME)FlyBase
FlyBase gene description for Mst27D (DROME)
Male specific transcript 27D (Mst27D) encodes a protein required for normal elongation of the spermatid nucleus from spherical to needle shape. Mst27D is transcribed exclusively in mid to late spermatocytes.