Entry created on 1 July 2019 (Revision 1.0) Annotator: Rita Pancsa
This entry is part of a multi-component system encompassing the following entries: Q9W4I7 P16554
Basic protein information
Accession P16554
Common name NUMB
Gene NUMB
Organism Drosophila melanogaster
Uniprot name Protein numb
Basic LLPS information
Organelle cytoplasmic protein granule; basal Numb-Pon crescent in dividing neuroblasts
Type of experimental evidence
Joined entry Q9W4I7 P16554
Protein region(s) mediating LLPS
65
-
203
PTB domain
Based on the experimental results of the following publication: 29467404
Molecular features viewer
PDB structures
View on PDBe
Extended LLPS information
Functional description
Uneven distribution and local concentration of protein complexes on distinct membrane cortices is a fundamental property in numerous biological processes, including Drosophila neuroblast (NB) asymmetric cell divisions (ACD) and cell polarity in general. In NBs, the cell fate determinant Numb forms a basal crescent together with Pon and is segregated into the basal daughter cell to initiate its differentation. Numb PTB domain, using two distinct binding surfaces, recognizes repeating motifs within Pon in a previously unrecognized mode. Several repeating motifs have been found in Pon: type A „FxNxx[F/L]” motif and type B „NP[F/Y]E[V/I]xR” motif; the isolated motifs barely interact with Numb, however, the proper combination of both motifs dramatically increases the interaction with Numb PTB. The multivalent Numb-Pon interaction leads to high binding specificity and LLPS of the complex both in vitro and in living cells. The direct interaction between Pon and Numb PTB is responsible for the correct localization of Numb during ACD. The proper targeting and local concentration of Numb by Pon on the basal cortex is essential for its subsequent inhibition of Notch signaling. Such phase-transition-mediated protein condensations on distinct membrane cortices may be a general mechanism for various cell polarity regulatory complexes (PMID:29467404).
Literature supporting the LLPS: 29467404
Functional class of membraneless organelle: regulator of spatial patterns
Binding partners (at biological protein concentrations)
1) Pon N-terminal fragment (strictly required for LLPS)
Type of RNA(s) required/used for the LLPS at biological protein concentrations
RNA not required.
Molecular interaction types contributing to LLPS
multivalent domain-motif interactions (PMID:29467404)
Determinants of phase separation and droplet properties
1) protein concentration 2) valency of Pon 3) stoichiometry of the components
Membrane cluster No
Partner-dependent Yes
RNA-dependent No
PTM required No
Domain-motif interactions Yes
Discrete oligomerization No
Regulation and disease
Post-translational modifications affecting LLPS
Position Residue PTM Effect Reference Modifying enzyme Notes
Isoforms known to affect LLPS
Isoform Effect Reference
All known isoforms containing sequence changes in the LLPS region(s)
Position type Isoform names from UniProt
Disease mutations affecting LLPS
Mutation dbSNP Disease OMIM Effect Reference Notes
Experimental information
Experimental techniques applied to prove/investigate LLPS
Specific physical interaction was confirmed between Numb PTB and an N-terminal fragment (amino acids (aa) 1–228) of Pon (mutation, truncation) in vitro by pull-down assays (protein-protein interaction detection assay). Proper combination and valency of type A “FxNxx[F/L]” and type B “NP[F/Y]E[V/I]xR” motifs (mutation) dramatically increased the interaction with Numb PTB in vitro (physical interaction confirmed by ITC and pull-down protein-protein interaction detection assays). The complex structures of Numb PTB with either Pon A2B2 or Pon B1A2 was solved by X-ray crystallography. Numb PTB and Pon A1B3 formed liquid droplets (morphology, particle size and count by microscopy) in vitro and droplet formation was protein concentration-dependent. High-affinity monovalent Pon peptide (change in the concentration of a small molecule) dispersed the droplets in vitro (microscopy). Fluorescently tagged Pon and Numb showed co-localization in vitro by epifluorescence microscopy. In vivo overexpression of GFP-fused Pon and Cherry-fused Numb PTB in HeLa cells led to bright puncta in the nucleus (protein localization) showing the co-localization of the two proteins with time-lapse microscopy. Overexpression of only one of the fusion proteins, or both fusion proteins but with mutations perturbing the interaction in at least one of them did not result in in vivo puncta formation. In vivo, in transgenic flies, overexpression of the Flag-tagged wild type (creation of a fusion protein) or mutant forms showed that during neuroblast division Pon together with Numb is basally localized (protein localization) and is segregated into the basal daughter cell after assymmetric cell division. The interaction between Pon and Numb PTB is responsible for the correct, basal localization of Numb (protein localization, microscopy) as it is disturbed for Pon-binding deficient Numb mutants. PMID:29467404.
Experimental observations supporting the liquid material state of the condensate
morphological traits (PMID:29467404) sensitivity to 1,6-hexanediol (PMID:29467404) dynamic exchange of molecules with surrounding solvent (PMID:29467404)