Please use this identifier to cite or link to this item: http://hdl.handle.net/2067/3031
Title: Role of CSA and CSB proteins in cytokinesis
Other Titles: Ruolo delle proteine CSA e CSB nella citodieresi
Authors: Costantino, Michele
Keywords: Cytokinesis;Cokayne sindrome;Mitosis;Multinucleated cells;CSA;CSB;BIO/11;Citodieresi;Sindrome di Cokayne;Mitosi;Cellule multinucleate
Issue Date: 6-May-2016
Publisher: Università degli studi della Tuscia - Viterbo
Series/Report no.: Tesi di dottorato di ricerca. 28. ciclo
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.
Description: 
Dottorato di ricerca in Genetica e biologia cellulare
URI: http://hdl.handle.net/2067/3031
Appears in Collections:Archivio delle tesi di dottorato di ricerca

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