Molecular basis of the STIL coiled coil oligomerization explains its requirement for de-novo formation of centrosomes in mammalian cells

Ahuvit David, Hadar Amartely, Noa Rabinowicz, Mai Shamir, Assaf Friedler*, Shai Izraeli

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

16 Scopus citations

Abstract

The STIL protein is essential for centriole replication and for the nonlated, de novo centriole biogenesis that is required for mammalian embryogenesis. Here we performed quantitative biophysical and structural analysis of the central short coiled coil domain (CCD) of STIL that is critical for its function. Using biophysical, biochemical and cell biology approaches, we identified the specific residues in the CCD that mediate the oligomerization, centrosomal localization and protein interactions of STIL. We characterized the structural properties of the coiled coil peptide using circular dichroism spectroscopy and size exclusion chromatography. We identified two regions in this domain, containing eight hydrophobic residues, which mediate the coiled coil oligomerization. Mutations in these residues destabilized the coiled coil thermodynamically but in most cases did not affect its secondary structure. Reconstituting mouse embryonic fibroblasts lacking endogenous Stil, we show that STIL oligomerization mediated by these residues is not only important for the centrosomal functions of STIL during the canonical duplication process but also for de-novo formation of centrosomes.

Original languageAmerican English
Article number24296
JournalScientific Reports
Volume6
DOIs
StatePublished - 14 Apr 2016

Bibliographical note

Funding Information:
AF is supported by grants from the Israel Science Foundation (ISF), the Israel Cancer Research Foundation (ICRF) and by the Minerva Centre for Bio-Hybrid Complex Systems. HA is supported by the Dalia and Dan Meydan fellowship for Ph.D students. AD is supported by an Eshkol grant from the Israel Science Ministry. SI is supported by grants from the Israel Science Foundation and Israel Science Ministry. NU and SI are supported by grants from the the Israel Cancer Research Foundation (ICRF). We thank Rolf Marshelek for providing the Streptagged pEXPR-IBA105 plasmid and Philip Beachy for a Shh-N encoding plasmid. These studies were performed as partial requirements of AD and HA for a PhD degree of Tel Aviv and Hebrew universities, respectively.

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