A micrometer-scale self-assembled platform around a human centriole: How does it form, and what to make of it after all?

A micrometer-scale self-assembled platform around a human centriole: How does it form, and what to make of it after all?

As an electron-dense, membrane-less organelle (approximately 100–300 aL in volume before undergoing mitotic expansion), the centrosome plays a critical role in promoting various cellular events, including cell division, cell motility, and intracellular signaling1,2. Multiple super-resolution imaging studies have revealed that pericentriolar material (PCM) proteins are highly organized, exhibiting layers of concentrically localized patterns around a centriole3-6. Yet how PCM scaffold proteins organize the PCM space remains largely unknown. Given that a micrometer-scale PCM is densely packed with a few hundred copies of several hundred different proteins7, properly organizing the PCM space is essential to achieve its functional proficiency8,9. Aberrant centrosome function predisposes humans to various diseases, including cancer, microcephaly, and neurological disorders10-13. Thus, unraveling the mechanism underlying PCM organization may reveal the fundamental principles governing centrosome functionality and could help determine the etiology of centrosome-associated human disorders. 

A self-assembled cylindrical Cep63•Cep152 platform formed around a human centriole.

My group stumbled into this direction of research when we unexpectedly observed that the coexpression of a polo-like kinase 4 (Plk4)-binding protein, Cep15214,15, and its binding target, Cep6316, form a sphere-like assembly in the cytosol17. Plk4 plays a key role in centriole biogenesis by triggering downstream events, including the cartwheel-like Sas6 self-assembly found in the lumen of a centriole18,19. Notably, the expression of each Cep63 or Cep152 resulted in only cytosolic aggregates, suggesting that these two proteins work together to form a higher-order assembly. Subsequent studies with Cep63 and Cep152 revealed that these two long coiled-coil-encoding proteins interact with each other only through a short region in their C-terminal and middle region (i.e., Cep63-C and Cep152-M), respectively, by forming a heterotetrameric four-helical bundle arranged in an anti-parallel manner20. Further studies with purified recombinant proteins showed that these proteins can cooperatively generate a micrometer-scale “cylindrical” self-assembly in a Cep63 N-terminus-in and Cep152 N-terminus-out fashion on a slide glass. The architectural feature of the self-assembly resembles the cylindrical localization patterns of Cep63 and Cep152 around a centriole5,6. (Note several studies call this localization pattern “toroidal.”) Thus, unlike intermediate filament-forming long coiled-coil proteins, such as keratin, lamin, and vimentin, which form a long cable-like structure in an open cytosolic environment, Cep63 and Cep152 cooperatively generate a PCM architecture by self-assembling them around a centriole.

The assembly occurs through a stepwise and reversible process.

How do Cep63 and Cep152 self-assemble into a higher-order cylindrical architecture? In the Ahn, JI, et al. paper in Communications Biology (DOI: 10.1038/s42003-023-05067-8), our group combined MINFLUX nanoscopy with biophysical and structural analyses and theoretical modeling to demonstrate that the Cep63•Cep152 complex undergoes a concentration-dependent and reversible tetramer–octamer–hexadecamer transition to generate a cylindrical self-assembly21 (Fig. 1A). The formation of the hexadecameric Cep63•Cep152 complex was cooperative. With our experimental systems, we failed to detect a Cep63•Cep152 complex greater than the hexadecamer. Notably, although the assembly process appears highly dynamic, the self-assembled architecture remained stable for up to five days under physiological buffer conditions20. These two contrasting properties—intrinsic dynamics and steady-state stability—are the distinguishing characteristics of a self-organizing system22.

The octameric Cep152 (2D) mutant misorganizes the Cep63•Cep152 platform.

The significance of the stepwise self-assembly process becomes apparent from the experiment carried out with the I1279D and L1286D (2D) mutant, which forms a stable octameric form (i.e., having no reversible tetramer–octamer–hexadecamer transition) without any apparent propensity for self-association. Further characterization of the 2D mutant revealed that it forms severely defective cylindrical self-assemblies in vitro and misorganizes the pericentriolar Cep63•Cep152 platform in vivo, exhibiting a significantly reduced number of Cep152 molecules scattered in a wider radial space21 (Fig. 1B). These findings suggest that the ability of Cep152 to undergo stepwise formation into higher-molecular-weight complexes is essential for the dynamic recruitment and assembly of the Cep63•Cep152 building block around a centriole.

Effect of misorganizing the Cep63•Cep152 platform on Plk4 function and beyond.

Misorganizing the Cep63•Cep152 platform had far-reaching consequences, directly altering Plk4-dependent centriole biogenesis. This is because the functionality of Plk4, which binds to the N-terminus of Cep15214,15,23, is determined by the amount of Plk4 undergoing trans-autophosphorylation-induced liquid–liquid phase separation (LLPS) and ensuing ring-to-dot relocalization to the procentriole assembly site24-26. Moreover, since the LLPS is concentration-dependent, the dimension of the PCM space where Plk4 is distributed and the organizational pattern of Plk4 on the Cep63•Cep152 platform directly impact the efficiency of Plk4’s LLPS. Therefore, we propose that the organizational properties of the Cep63•Cep152 platform determine the timing of Plk4 activation and Plk4-dependent centriole biogenesis. 

Intriguingly, multiple cancer-associated Cep63 and Cep152 mutations are found in human cancer tissues (Catalogue of Somatic Mutations in Cancer; https://cancer.sanger.ac.uk/cosmic) but not in the gnomAD (https://gnomad.broadinstitute.org), which generally represents wildtype variants. Several of these mutations are present within the regions forming the heterotetrameric Cep63•Cep152 complex. Thus, investigating the mutations’ significance could offer a deeper understanding about the architecture–function relationship of the Cep63•Cep152 complex. It may also help uncover new principles of building the Cep63•Cep152 self-assembly and provide valuable insights into the causes of PCM-associated human disorders.


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