Biology subjects

Chatzifrangkeskou, M.

Publications and source records attributed to Chatzifrangkeskou, M..

4 recordsLinked to original sources

bioRxiv2022

Development of a Xenopus-based assay for high-throughput evaluation of mucociliary flow

Motile cilia are organelles lining the surfaces of major organs of the human body and generate directional fluid flow. Ciliary dysfunction has been linked to an emerging class of multisystem disorders, collectively known as motile ciliopathies. Drug screening for ciliopathies is challenging due to the unavailability of high-throughput assays that can evaluate ciliary flow generation. Here, we describe the development of a unique assay that enables the direct and rapid evaluation of mucociliary flow, which simultaneously facilitates high-throughput screening of potential therapeutic agents for motile ciliopathies. The assay relies on the ability of Xenopus tadpoles to promote mixing of a two-phase differential density aqueous mixture, through the robust flow generated by the mucociliary epithelium on their epidermis. We show that the rate of phase mixing is proportional to the rate of cilia-driven flow, therefore it directly represents the effectiveness of flow generation. We also demonstrate that the assay can detect changes in ciliary flow elicited by defects in cilia, CBF modulation and rotational polarity, providing an ideal assay for the identification of CBF-modulating compounds, as potential drugs for motile ciliopathies. Importantly we use the assay to show that CBF modulating drugs can improve flow generation and could thus be used as a potential therapeutic approach in PCD patients. The assay we have developed thus represents a powerful new tool for research, as well as drug development.

cell biology↗

bioRxiv2022

The apical Ciliary Adhesion complex is established at the basal foot of motile ciliaand depends on the microtubule network

The Ciliary Adhesion (CA) complex forms in close association with the basal bodies of cilia during the early stages of ciliogenesis and is responsible for mediating complex interactions with the actin networks of multiciliated cells (MCCs). However, its precise localization with respect to basal body accessory structures and the interactions that lead to its establishment in MCCs are not well understood. Here, we studied the distribution of the CA proteins using super-resolution imaging and possible interactions with the microtubule network. The results of this study reveal that the apical CA complex forms at the distal end of the basal foot and depends on microtubules. Our data also raise the possibility that CAs may have additional roles in the regulation of the organization of the microtubule network of MCCs.

cell biology↗

bioRxiv2021

53BP1 mediated recruitment of RASSF1A at ribosomal DNA breaks facilitates local ATM signal amplification

DNA lesions occur across the genome and constitute a threat to cell viability; however, damage at specific genomic loci has a disproportionally greater impact on the overall genome stability. The ribosomal RNA gene repeats (rDNA) are emerging fragile sites due to repetitive nature, clustering and high transcriptional activity. Notably, recent progress in understanding how the rDNA damage response is organized has highlighted the key role of adaptor proteins in the response. Here we identify that the scaffold and tumor suppressor, RASSF1A is recruited at sites of damage and particularly enriched at rDNA breaks. Employing targeted nucleolar DNA damage, we find that RASSF1A recruitment requires ATM activity and depends on the 53BP1. At sites of damage RASSF1A facilitates local ATM signal establishment and rDNA break repair. RASSF1A silencing, a common epigenetic event during malignant transformation, results in persistent breaks, rDNA copy number alterations and decreased cell viability. Moreover, meta-analysis of a lung adenocarcinoma cohort showed that epigenetic silencing of the scaffold leads in rDNA copy number discrepancies. Overall, we present evidence that RASSF1A acts as a DNA repair factor and offer mechanistic insight in how the nucleolar DNA damage response is organized.

cell biology↗

bioRxiv2019

RASSF1A Is Required For The Maintenance Of Nuclear Actin Levels

Nuclear actin participates in many essential cellular processes including gene transcription, chromatic remodelling and mRNA processing. Actin shuttles into and out the nucleus through the action of dedicated transport receptors importin-9 and exportin-6, but how this transport is regulated remains unclear. Here we show that RASSF1A is a novel regulator of actin nucleocytoplasmic trafficking and is required for the active maintenance of nuclear actin levels through supporting binding of exportin-6 (XPO6) to RAN GTPase. RASSF1A (Ras association domain family 1 isoform A) is a tumour suppressor gene frequently silenced by promoter hypermethylation in all major solid cancers. Specifically, we demonstrate that endogenous RASSF1A localises to the nuclear envelope (NE) and is required for nucleo-cytoplasmic actin transport and the concomitant regulation of Myocardin-related transcription factor A (MRTF-A), a coactivator of the transcription factor serum response factor (SRF). The RASSF1A/RAN/XPO6/nuclear actin pathway is aberrant in cancer cells where RASSF1A expression is lost and correlates with reduced MTRF/SRF activity leading to cell adhesion defects. Taken together, we have identified a previously unknown mechanism by which the nuclear actin pool is regulated and uncovered a previously unknown link of RASSF1A and MTRF/SRF in tumour suppression.

cell biology↗