Schmidt Cernohorska, M. | Search bioRxiv

bioRxiv2020

The BBSome assembly is spatially controlled by BBS1 and BBS4 in human cells

Bardet-Biedl Syndrome (BBS) is a pleiotropic ciliopathy caused by dysfunction of primary cilia. Most BBS patients carry mutations in one of eight genes encoding for subunits of a protein complex, BBSome, which mediates the trafficking of ciliary cargoes. Although, the structure of the BBSome has been resolved recently, the mechanism of assembly of this complicated complex in living cells is poorly understood. We generated a large library of human retinal epithelial cell lines deficient in particular BBSome subunit and expressing another subunit tagged with a fluorescent protein. We performed a comprehensive analysis of these cell lines using biochemical and microscopy approaches. Our data revealed that the BBSome formation is a sequential process including a step of the pre-BBSome assembly at pericentriolar satellites nucleated by BBS4, followed by the translocation of the BBSome into the ciliary base mediated by BBS1.

cell biology↗

bioRxiv2020

Prospects and limitations of expansion microscopy in chromatin ultrastructure determination

Expansion Microscopy (ExM) is a method to magnify physically a specimen with preserved ultrastructure. It has the potential to explore structural features beyond the diffraction limit of light. The procedure has been successfully used for different animal species, from isolated macromolecular complexes through cells to tissue slices. Expansion of plant-derived samples is still at the beginning, and little is known whether the chromatin ultrastructure becomes altered by physical expansion. In this study, we expanded isolated barley nuclei and compared whether ExM can provide a structural view of chromatin comparable with super-resolution microscopy. Different fixation and denaturation/digestion conditions were tested to maintain the chromatin ultrastructure. We achieved up to [~]4.2-times physically expanded nuclei corresponding to a maximal resolution of [~]50-60 nm when imaged by wild-field (WF) microscopy. By applying structured illumination microscopy (SIM, super-resolution) doubling the WF resolution the chromatin structures were observed at a resolution of [~]25-35 nm. WF microscopy showed a preserved nucleus shape and nucleoli. Moreover, we were able to detect chromatin domains, invisible in unexpanded nuclei. However, by applying SIM we observed that the preservation of the chromatin ultrastructure after expansion was not complete and that the majority of the tested conditions failed to keep the ultrastructure. Nevertheless, using expanded nuclei we detected successfully centromere repeats by fluorescence in situ hybridization (FISH) and the centromere-specific histone H3 variant CENH3 by indirect immunostaining. However, although these repeats and proteins were localized at the correct position within the nuclei (indicating a Rabl orientation) their ultrastructural arrangement was impaired.

cell biology↗