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Related publication: Culurgioni S., Mari S., Bonetti P., Gallini S., Bonetto G., Brennich M., Round A., Nicassio F. & Mapelli M. Insc:LGN tetramers promote asymmetric divisions of mammary stem cells. Nat. Commun. 9, 1025 (2018). DOI: 10.1038/s41467-018-03343-4
Publication keywords: Mammary stem cells; Cell divisions; Asymmetric fate
In stem cells, asymmetric cell division (ACD) generates two distinct cells: a stem cell, and a cell committed to differentiate. Defects in this delicate and extremely coordinated process can cause cell over-proliferation and cancer. How stem cell ACDs are executed remains largely unclear. The knowledge of the architecture of key ACD players constitutes a remarkable advance in the understanding of the operational principles of asymmetric divisions. An international research group determined the three-dimensional structure of the Insc:LGN complex, which revealed that oligomerisation and clustering of LGN (spindle orientation protein) and Insc are essential for ACDs.
To obtain an optimal structural characterisation of this complex, several tens of crystals obtained in different conditions were tested on the Macromolecular Crystallography (MX) beamlines I04 and I04-1, but only two diffracted to 3.4 Å and 4.0 Å resolution. The final combination of the two datasets allowed the structural determination. The group showed that LGN forms intertwined tetramers with Insc, which are markedly stable. Most importantly, in mammary stem cells, the tetrameric molecular assembly suffices to promote asymmetric cell divisions, and reverts the over-proliferation caused by loss of the tumour suppressor gene p53.
Stem cells are characterised by two unique properties: (I) the ability of engendering all differentiated cell types, and (II) the capacity of self-renewal through several rounds of cell divisions keeping an undifferentiated state. Specifically, stem cells divide asymmetrically, generating a daughter stem cell (identical to the mother) attached to the maternal niche, and a daughter cell inclined to relocate and differentiate. This asymmetric cell division (ACD) requires the spatial coordination between the division plane and cell polarity cues. The current view is that cell polarity is established by Par proteins (Par3:Par6:aPKC) at the apical site, while the division plane is defined by the position of the mitotic spindle regulated by the Ga:LGN:NuMA spindle orientation complex. The protein Inscuteable (Insc) links these two cellular processes as a result of its concomitant binding to Par3 and LGN.
The Drosophila melanogaster LGNTPR and InscASYM polypeptides were coexpressed in bacteria and purified as a 2:2 stoichiometry complex with an experimentally measured mass of 180 kDa (Fig. 1a). After the identification of the first crystal hits, extensive efforts dedicated to the optimisation of the crystallisation condition yielded plate-shaped crystals diffracting to 3.3-4.0 Å. Despite the low resolution, the good data quality and the consistency across the whole data collection resulted in high redundancy datasets, and permitted the generation of electron density maps that were suitable for structure determination. The overall architecture of the LGNTPR:InscASYM complex consists of an intertwined hetero-tetramer, where each Insc chain contacts one TPR domain (LGN-A) through its InscPEPT region, and the other (LGN-B) with its four-helix bundle. The final portion of each InscASYM is organised in Armadillo repeats that contact one another, further stabilising the assembly (Fig. 1b and 1d). The two TPR domains are complementarily juxtaposed in a head-to-head interaction, forming a cylinder open on one side; the cylinder is closed by the Insc helix bundle, increasing the steadiness of the complex.
To determine the functional relevance of the InscASYM-dependent LGN oligomerisation for stem cell asymmetric divisions, we analysed the ectopic expression of different constructs of Insc in murine mammary stem cells (MaSCs) depleted of the tumour suppressor gene p53 (p53-KO). Notably, p53- KO MaSCs show low Insc expression, and replicate abnormally through cycles of symmetric divisions, causing over-proliferation2. Restoring Insc expression in p53-KO MaSCs increases the number of ACDs (Fig. 3a), and consistently decreases the number of mammospheres formed in sphere-forming assays (Fig. 3b). Notably, the Insc region encompassing the ASYM domain is sufficient to reduce the sphere-forming efficiency, while the the InscASYM domain lacking the four-helix-bundle, and forming a 1:1 complex with LGN, is not (Fig. 3b). These results clearly demonstrate that the oligomerisation of InscASYM with LGN is essential for the asymmetric functions of Insc in stem cell divisions.
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