Role of CSA and CSB proteins in cytokinesis

Abstract

Cockayne syndrome (CS) is a genetic disease inherited in an autosomal recessive pattern. CS patients are characterized by photosensitivity, severe growth retardation, cachectic dwarfism, feature of premature aging and progressive neurological abnormalities of the central nervous system including microcephaly, cerebellar atrophy and demyelinating peripheral neuropathy. The average life span of children with this syndrome is about 12 years of age. CS patients have been assigned to two genetic complementation groups (CS-A and CS-B), whose corresponding genes (csa and csb) have been cloned and characterized (Henning et al 1995; Troelstra et al., 1992; Lehmann, 1982). CSA and CSB proteins have critical roles in a sub pathway of nucleotide excision repair known as transcription-coupled repair (TCR). Although a defect in TCR pathway could potentially account for the enhanced photosensitivity of CS patients, other pathological features including neurological dysfunctions may not solely explained by a DNA repair defect and requires additional explanations. Accordingly, in the last years it has been suggested that CSA and CSB proteins play multiple pleiotropic functions. More recently, we and others have demonstrated that CSB mediates the transcriptional programs following exposure to cellular stressors such as UV, oxidative damage, inflammation and hypoxia. Therefore, abnormalities in the regulation of RNA pol I and II mediated transcription might provide plausible explanations for many of the somatic features, including aspects of neurological symptoms associated with CS. Observation of neurological symptoms detected either at birth or during early childhood raises the possibility that CSB may have a crucial role in the transcriptional programs that govern the plasticity and the maintenance of the central nervous system during (perinatal and postnatal) pediatric life. Our recent studies showed that CSB suppression affects the neuronal differentiation capability of human neural progenitor cells. CSB also plays a critical role in cell robustness, negatively modulating p53 activity after cellular stress, including DNA damage and hypoxia and counteracting p53-independent apoptosis. Finally, CSB, as anti-apoptotic factor is over-expressed in a variety of cancer cells and tissues, so it represents a strategic target for anticancer therapy: the inhibition or down regulation of CSB in cancer cells makes these cells hypersensitive to a variety of commonly used cancer chemotherapeutic agents. Interesting, an unknown extranuclear role of CSA and CSB proteins is emerging. This study reveals an unexpected subcellular localization and biological function of CSA and CSB proteins in cytokinesis. We discovered, in several tumoral cells line and in Cockayne fibroblast, that CSA and CSB proteins have a different cytoplasmic localization during mitosis in cytoskeletal structures such as the centrosomes and the midbody (the intercellular microtubule bridge connecting two daughter cells at the end of cytokinesis). Immunofluorescence (IF) staining for CSA and CSB during mitosis shows that in the early stages the cytoplasmic localization seems to be consequent of chromosomes condensation. However, from the onset of cleavage furrow the proteins take progressively a specific position. In particular, while during metaphase CSA and CSB exit from the nucleus yet condensed, in anaphase, during cleavage furrow contraction, they are associated with the spindle midzone; then, in telophase, they accumulate at the midbody, where they have a characteristic ring-like arrangement embraced by Aurora B in the so called bulge-zone. These observations suggest a potential role of CSA and CSB protein in the organization and functioning of cytoskeleton-mediated cellular activities encompassing polarity, motility and cellular division. Depletion of CSB proteins in tumoral cells line and CSA and CSB mutations in Cockayne fibroblasts, results in cytokinesis failure. In particular, we show the presence of syncytium-like cells, the increase of cells number at the midbody stage and abscission defects associated with the increase of aberrant mitosis and with the accumulation of multi- binucleated cells. These signs are usually observed in cells with impaired abscission, that support the role of this proteins in last stage of cell division. To investigate the mechanism of CSA and CSB protein in the abscission, we analyzed the spatiotemporal localization of a series of structural and functional proteins sequentially recruited during cytokinesis to assure proper cell division. In all cells analyzed at the early stages of cytokinesis, we observed that the localization patterns for all proteins examined are not affected during midzone formation and cleavage furrow ingression in the CSA and CSB mutated cells, indicating that the proteins are not mainly involved in the early events of cytokinesis. In contrast, in the subsequent stage of midbody formation, PLK1 and PRC1 proteins can be detected at the midbody but their distribution pattern is altered, becoming dispersed along the midbody microtubules and the midbodies appear elongated. Furthermore, by analyzing the proteins involved in abscission, in CSA and CSB mutated cells the critical abscission factors as ALIX and Spastin present several defects of organization localizing itself along the midbody. PLK1 and PRC1 proteins are closely associated in the regulation of midzone and midbody formation; the ubiquitination of both proteins it is required for the cytokinesis completion. CSA protein is a component of a CRL4 E3 ubiquitin ligase complex while CSB protein, whit a Cterminal UBD, is a substrate of CSA. We speculate that in CSA and CSB mutated cells, PRC1 and PLK1 proteins are delocalized because of the lack of CSA protein that don’t permit the protein degradation. Overall, these results indicate that CSA and CSB protein play a role at the terminal stage of cytokinesis by controlling the morphology of the midbody and organizing the localization of a few critical regulators of the abscission.

La sindrome di Cockayne (CS) è una malattia genetica autosomica recessiva. I pazienti CS sono caratterizzati da fotosensibilità cutanea, grave ritardo nella crescita, nanismo e cachessia, caratteristiche di invecchiamento precoce e da anomalie neurologiche del sistema nervoso centrale, tra cui microcefalia e atrofia cerebellare. La durata media della vita dei bambini affetti da questa sindrome è di circa 12 anni di età. I pazienti CS sono stati assegnati a due gruppi genetici di complementazione (CS-A e CS-B), i cui corrispondenti geni (csa e csb) sono stati clonati e caratterizzati. Le proteine CSA e CSB hanno un ruolo critico nel sub-pathway di riparazione per escissione nucleotidica (TCR). Anche se un difetto nel TCR potenzialmente spiega la maggiore fotosensibilità dei pazienti CS, altre caratteristiche patologiche, tra cui le disfunzioni neurologiche potrebbero non possono essere spiegate solo da un difetto di riparazione del DNA. Di conseguenza, negli ultimi anni è emerso che CSA e CSB svolgono funzioni pleiotropiche. Recentemente, abbiamo dimostrato che CSB media i programmi trascrizionali in seguito ad esposizione a fattori di stress cellulari come UV, danno ossidativo, infiammazione e ipossia. Inoltre abbiamo dimostrato che la soppressione CSB colpisce la capacità di differenziamento neuronale di cellule progenitrici neurali umane. CSB modula negativamente l'attività di p53 dopo stress cellulare e contrasta l'apoptosi in maniera p53-indipendente. Infine, CSB, come fattore anti-apoptotico è sovraespresso in una varietà di cellule tumorali e tessuti, quindi rappresenta un obiettivo strategico per la terapia antitumorale: l'inibizione o down regulazione di CSB nelle cellule tumorali rende queste cellule ipersensibili ad una varietà di agenti chemioterapici comunemente utilizzati. Infine, questo studio rivela una nuova localizzazione subcellulare delle proteine CSA eCSB e il loro coinvolgimento nella citodieresi. Infatti, tramite studi di IF abbiamo dimostrato che la soppressione di CSA e CSB in diverse linee tumorali e la mutazione delle stesse in fibroblasti Cockayne determina il fallimento della citodieresi e un aumento di cellule bi-multinucleate.