Focus of Research (03/2013-)
The packaging of chromatin into the nucleus of a eukaryotic cell requires an extraordinary degree of compaction and physical organization. In recent years, it has been shown that this organization is dynamically orchestrated to regulate responses to exogenous stimuli as well as to guide complex cell-type-specific developmental programmes. Although the nucleosomes represent the basic unit of chromatin compaction, they constitute only the first level of chromatin organization. The amount of condensation needed for a typical genome to be fitted into an interphase nucleus or set of metaphase chromosomes, indicates that there are additional “higher order” levels of chromatin organization. In the context of chromatin, higher-order structure may be defined as any reproducible conformation of nucleosomes in 3D space. The most evident form of higher-order chromatin structure is the mitotic/meiotic chromosomes in which the DNA is highly compacted. Studies on 3D spatial locations and transcriptional competence of genes in respect to their chromosome territories have provided some important insights on the importance of this level of chromatin organization on regulation of gene expression.
I’m currently exploiting the LacO/LacI system in Arabidopsis to investigate in vivo the repositioning of alleles of a Polycomb target gene, FLC, during an environmentally-triggered silencing process involved in the control of flowering time – vernalization how FLC and its components are organized within the three-dimensional space of the nucleus.
Focus of Research (02/2012-02/2013)
Protein scaffolds in the coupling of synaptic exocytosis and endocytosis
Postdoctoral Research Project:
Synaptic transmission involves the stimulus-evoked release of neurotransmitter from synaptic vesicles (SVs) fusing at presynaptic active zone (AZ) membranes. The molecular machineries for SV exocytosis and endocytosis have been studied independently in some detail. For long-term performance of synapses, however, the demand of new release-ready SVs at active zones must be coordinated with endocytic retrieval to maintain exo-endocytic balance as well as the compositional identity of SVs and of the plasma membrane. Recent data indicate that at many synapses SV exocytosis is tightly coupled to endocytosis, and that synapses have evolved a specialized apparatus involving multidomain protein scaffolds to comply with these demands.
NeuroCure laboratories have identified and characterized scaffold proteins (Intersectin/Dap160; ELKS/Bruchpilot; Git), which serve as prime candidates to operate in exo-endocytic coupling at vertebrate and invertebrate synapses. Bruchpilot, a fly relative of the mammalian AZ proteins ELKS, constitutes a central component of the AZ scaffold and loss of function of brp causes defects in short-term plasticity due to impaired SV exocytosis. Preliminary data indicate that Brp associates with the endocytic fissioning enzyme dynamin as well with other endocytic proteins, and with NSF, a crucial factor for disassembly of post-fusion SNARE complexes. Conversely, super-resolution microscopy and functional data indicate that the endocytic scaffold protein intersectin not only regulates early stages of clathrin-dependent SV endocytosis, but also acts as a crucial factor for the recruitment of release-ready vesicles at AZs.
Using a multidisciplinary approach, my project aims to combine cell biological, biochemical, physiological, genetic, and acute perturbation approaches with super-resolution light microscopy (SRLM) and optical imaging techniques to investigate the structural and functional organization of multidomain scaffolds at synapses and their role in exo-endocytic coupling. Results from this project are expec ted to unravel the molecular organization of presynaptic scaffolds in SV cycling with important implications for the understanding of neurological disorders including autism and schizophrenia.
Rosa S., Ntoukakis V., Ohmido N., Pendel A. , Abranches R. and Shaw P. Auxin regulates chromatin dynamics and histone acetylation in root development (submitted to Developmental Cell)
Rosa S., Shaw P. “Opening chromatin to transcription: the different levels of regulation” Review (in prep)
De Lucia F.+, Rosa S.+, Mylne J., Ohmido N., Kato N., Shaw P. and Dean C. Cold-induced physical clustering of FLC alleles is an early event in vernalization (submitted to Nature Cell Biology). (+Co-first authorship)
Pires A., Rosa S., Castanheira S., Fevereiro P. and Abranches R. (2012) Expression of a recombinant human erythropoietin in suspension cell cultures of Arabidopsis, tobacco and Medicago. Plant Cell, Tissue and Organ Culture.
Ntoukakis, V., Rosa, S.,Pendle, A., Bell, E.M., Ohmido, N., Dolan, L., and Shaw P. J. H2A.Z controls a position-dependent cell fate switch in Arabidopsis (in prep)