CP ASSEMBLY IN CILIA

"Role of the tubulin posttranslational modifications and microtubule severing protein, katanin in the cilia central pair assembly."

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 Obiettivo del progetto (Objective)

'Cilia are microtubule (MT)-based external cell extensions that perform sensory and locomotory functions. Disruption of ciliary beating leads to human disorders such as infertility, airways diseases and hydrocephalus. The central pair of MTs (CP) apparatus is required for proper cilia motility yet the molecular mechanism that regulates its assembly is unknown. Loss of function mutations of MTs severing protein katanin or changes in the levels of MTs posttranslational modifications (PTMs) result in assembly of short immotile cilia that lack CP. Tubulin PTMs affect activity of katanin toward cytoplasmic MTs but the role of katanin and tubulin PTMs in ciliogenesis is unknown. Thus our main goal is to determine the role of katanin and tubulin PTMs and their reciprocal relationships during ciliogenesis, and their effect on the assembly and/or stability of CP MTs. We intend to identify katanin ciliary localization domain and ciliary interacting proteins. We will use a well established model to analyze motile cilia, a ciliate Tetrahymena thermophila. We will take advantage of high resolution microscopy for localization studies. We will perform mutagenesis analysis of katanin and b-tubulin in order to identified the key amino acid(s) involved in ciliary localization and interactions between these two proteins in cilia. To determine if defects in cilia caused by b-tubulin or katanin mutations can be rescued by parallel mutations in katanin and b-tubulin, respectively and restore disrupted protein interactions we will mutate katanin (or b-tubulin) and express under inducible promoter in wild-type and loss of function mutation background of partner protein. The outcome of the project will have likely medical implications. The project links different aspects of my postdoctoral research and will enhance the research potential of the Host Organization, provide foundation for competitive research program and will become a stepping stone into building an independent research group.'

Introduzione (Teaser)

Cilia are microtubules-based cell protrusions that perform sensory and locomotory functions in diverse cell types in organisms. Recent findings suggest that in a number of genetic disorders, the underlying cause may be a dysfunctional molecular mechanism in the cilia structures.

Descrizione progetto (Article)

A cilium (the plural is cilia) is an organelle found in eukaryotic cells. There are two types of cilia: motile cilia and non-motile or primary cilia, which typically serve as sensory organelles. In humans, primary cilia are found on nearly every cell in the body. The microtubule skeleton of cilia is composed of 9 doublets of peripheral microtubules. Motile cilia consist of two additional central microtubules, so-called central pair (CP).

Earlier observations indicated that lack or mutation of microtubule severing protein p60 katanin or its regulatory subunit, p80, leads to formation of cilia that lack CP microtubules. The EU-funded CP ASSEMBLY IN CILIA project was initiated to investigate the role of the microtubule severing protein, katanin and tubulin modifications in the assembly of CP microtubules.

During the initial two years, researchers obtained interesting data concerning katanins and gamma-tubulin localisation. Analysis was carried out with wild type and mutant cells at the cellular and ultrastructural level. They determined that the changes in the level of tubulin post-translational modifications do not affect the localisation of gamma-tubulin. However, the modifications caused the accumulation of katanin p60 leading to protein mislocalisation. Interestingly, changes in the levels of the modifications did not influence the localisation of katanin regulatory subunit, p80.

Project also aims to investigate the localisation of other katanin-like proteins. Moreover, researchers currently perform mutagenesis analysis of katanin 1 in order to identify motifs or domains required for function of this protein in cilia.

Results of CP ASSEMBLY IN CILIA project are important as cilia defects adversely affect numerous critical developmental signalling pathways essential to cellular development. These fundamental studies help to understand the multi-symptom nature of a large set of syndromes and diseases.