Centriolar satellites comprise cytoplasmic granules that are located around the centrosome. Their molecular identification was first reported more than a quarter of a century ago. These particles are not static in the cell but instead constantly move around the centrosome. Over the last decade, significant advances in their molecular compositions and biological functions have been achieved due to comprehensive proteomics and genomics, super-resolution microscopy analyses and elegant genetic manipulations. Centriolar satellites play pivotal roles in centrosome assembly and primary cilium formation through the delivery of centriolar/centrosomal components from the cytoplasm to the centrosome. Their importance is further underscored by the fact that mutations in genes encoding satellite components and regulators lead to various human disorders such as ciliopathies. Moreover, the most recent findings highlight dynamic structural remodelling in response to internal and external cues and unexpected positive feedback control that is exerted from the centrosome for centriolar satellite integrity.

Anchoring microtubules to the centrosome is critical for cell geometry and polarity, yet the molecular mechanism remains unknown. Here we show that the conserved human Msd1/SSX2IP is required for microtubule anchoring. hMsd1/SSX2IP is delivered to the centrosome in a centriolar satellite-dependent manner and binds the microtubule-nucleator γ-tubulin complex. hMsd1/SSX2IP depletion leads to disorganised interphase microtubules and misoriented mitotic spindles with reduced length and intensity. Furthermore, hMsd1/SSX2IP is essential for ciliogenesis, and during zebrafish embryogenesis, knockdown of its orthologue results in ciliary defects and disturbs left-right asymmetry. We propose that the Msd1 family comprises conserved microtubule-anchoring proteins.

The reconstitution of extracellular matrix (ECM) components in three-dimensional (3D) cell culture environments with microscale precision is a challenging issue. ECM microparticles would potentially be useful as solid particulate scaffolds that can be incorporated into 3D cellular constructs, but technologies for transforming ECM proteins into cell-sized stable particles are currently lacking. Here, we describe new processes to produce highly condensed collagen microparticles by means of droplet microfluidics or membrane emulsification. Droplets of an aqueous solution of type I collagen were formed in a continuous phase of polar organic solvent followed by rapid dissolution of water molecules into the continuous phase because the droplets were in a non-equilibrium state. We obtained highly unique, disc-shaped condensed collagen microparticles with a final collagen concentration above 10% and examined factors affecting particle size and morphology. After testing the cell-adhesion properties on the collagen microparticles, composite multicellular spheroids comprising the particles and primary rat hepatocytes were formed using microfabricated hydrogel chambers. We found that the ratio of the cells and particles is critical in terms of improvement of hepatic functions in the composite spheroids. The presented methodology for incorporating particulate-form ECM components in multicellular spheroids would be advantageous because of the biochemical similarity with the microenvironments in vivo.

Centrioles are the major constituents of the animal centrosome, in which Plk4 kinase serves as a master regulator of the duplication cycle. Many eukaryotes also contain numerous peripheral particles known as centriolar satellites. While centriolar satellites aid centriole assembly and primary cilium formation, it is unknown whether Plk4 plays any regulatory roles in centriolar satellite integrity. Here we show that Plk4 is a critical determinant of centriolar satellite organisation. Plk4 depletion leads to the dispersion of centriolar satellites and perturbed ciliogenesis. Plk4 interacts with the satellite component PCM1, and its kinase activity is required for phosphorylation of the conserved S372. The nonphosphorylatable PCM1 mutant recapitulates phenotypes of Plk4 depletion, while the phosphomimetic mutant partially rescues the dispersed centriolar satellite patterns and ciliogenesis in cells depleted of PCM1. We show that S372 phosphorylation occurs during the G1 phase of the cell cycle and is important for PCM1 dimerisation and interaction with other satellite components. Our findings reveal that Plk4 is required for centriolar satellite function, which may underlie the ciliogenesis defects caused by Plk4 dysfunction.

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Human Msd1/SSX2IP is a component of centriolar satellites and essential for microtubule anchoring to the centrosome. Anchoring defects lead to abnormal accumulation of centriolar components at centriolar satellites, which interferes with centriole assembly. Loss of Msd1/SSX2IP produces supernumerary centriole precursors specifically in tumor cells.

Transition metal-mediated templating and self-assembly have shown powerful potentials for the synthesis of interlocked molecules. These two strategies were combined in designing and preparing a new type of coordination catenanes incorporating Cu(I) and Pd(II) metal centers. The ligand designed here contains a phenanthroline core and pyridine sidearms (compound 1). Using this phenanthroline-pyridine conjugated ligand, two approaches were examined, which were shown to be surprisingly efficient for the catenane synthesis: the entwining route (entwining of two ligands around Cu(I) followed by Pd(II) clipping) and the threading approach (Cu(I)-templated threading of a cyclic ligand on an acyclic ligand followed by the Pd(II) clipping of the second ring). In the former method, stepwise treatment of 1 with Cu(CH(3)CN)(4)PF(6) (templating center) and enPd(NO(3))(2) (assembling center) gives rise to the quantitative formation of CuPd(2) catenane 18. In the latter method, Cu(I) templates the threading of phenanthroline-containing macrocycle 2 on ligand 1, which is followed by Pd(II) clipping to give hetero catenane 20. In both approaches, the formation of catenanes is convincing thanks to the strong templating effect of Cu(I), while the ring closure steps are efficiently furnished by Pd(II)-directed self-assembly.

Synthesis of [2]catenane 6 has been successfully achieved by the combination of Pd complex 1 and pyridines 2 and 3 at a molar ratio of 2:1:1 in D20. A mixture of square molecule 4 (prepared from 1 and 2) and macrocycle 5 (obtained from 1 and 3), in which the final ratio of 1, 2, and 3 was kept 2:1:1 reorganizes in D2O/CD3OD (1:1) to form 6 within one day. However, the same mixture in D2O shows the formation of novel [3]catenane 7 along with the [2]catenane. In order to make 7, the theoretical ratio of components 1, 2, and 3 should be 3:1:2. Thus, deliberately maintaining such ratio of the above-mentioned molecules, a higher proportion of the [3]catenane is observed in D2O as found from 1H NMR spectra of the system. Reorganization of the twelve components to form [3]catenane is supported by studies with the DOSY method. This method is a first attempt to separate, from a mixture, either catenanes or any other supramolecular self-assembly structures. CSI-MS studies further support the assigned catenane super structures 6 and 7. All the results indicate that the [2]catenane is thermodynamically the most stable structure, while the [3]catenane is a meta-stable self-assembly.