Fusione cellula-cellula in colture a breve termine di cellule di vertebrati e invertebrati: analisi ultrastrutturale, cellulare e molecolare e ruolo della ECM, di fattori di crescita e di miRNA

Responsabili di progetto

Francesca Zito, Igor Kireev

Accordo

RUSSIA - RFBR-suspended - Russian Foundation for Basic Research

Bando

CNR/RFBR 2015-2017

Dipartimento

Scienze biomediche

Area tematica

Scienze biomediche

Stato del progetto

Nuovo

Proposta di ricerca

In multicellular organisms, cell fusion is a fundamental mechanism devoted to shape organs, such as muscle and bone, and it has been implicated in some pathological conditions (Aguilar et al 2013 Trends Genetics 29, 427-437). Indeed, cell fusion failure in humans might be associated with diseases such as myo- and osteopathies.
Cell fusion is a genetically programmed process, in which different cells share some common steps, as they follow a well-ordered ritual in order to fuse: migration, adhesion and fusion. Each step requires the activation of specific signalling transduction pathways leading up to the eventual fusion event. Fused cells undergo dramatic changes in ultrastructure and behaviour, and acquire new developmental fates. The molecular mechanisms that mediate cell fusion are currently under investigation in a number of cells, although the fusion machinery in its complexity remains to be characterized. Dynamic reorganization of the cytoskeleton is expected to be involved in cell fusion events, controlling the direction of cell migration through the formation of filopodia and regulating fusion-pore formation during syncytium organization.
Recently, miRNAs have been identified as critical regulators of cell fusion in osteoclastogenesis (Xie et al 2014 BBRC 446:98-104). They negatively regulate protein expression by binding to target sites in mRNAs, inducing repression of mRNA translation or transcript destabilization and decay.
It is also well acknowledged that complex cell movements require appropriate interactions of cells with the extracellular environment, including soluble factors, such as growth factors (GF), and extracellular matrix (ECM) molecules. Each of these components influences many aspects of cellular behaviour, including cell shape and motility, cytoskeleton organization, cell proliferation and differentiation (Goody et al 2010 Mol Reprod Dev 77:475-488).
The present collaboration aims to study the cell fusion process of in vitro cultured cells from vertebrates and invertebrates. The choice of an in vitro cell culture approach is supported by several strengths: the possibility to carefully control both media and matrix of the cultures, to easily manipulate cellular components with drugs, siRNA or DNA constructs, to analyze cell fusion in the absence of other cell types. As a general rule, stabilized cell lines provide a valuable experimental system for in vitro investigations of cellular processes, although some concerns may arise regarding their phenotypic state and differences in relation to their cognate primary cells. Indeed, primary cells have an obvious attractiveness in terms of a better mirroring of the in vivo behaviour, thus providing data that can potentially be extrapolated to current in vivo models. For these reasons, we will use a stabilized vertebrate cell line, the mouse macrophage cells RAW264.7, and a primary invertebrate cell line, the Paracentrotus lividus sea urchin primary mesenchyme cells (PMCs).
Macrophages provide an example of cells undergoing fusion, which leads to the formation of multinucleated osteoclasts in bone. The cell fusion leading to osteoclasts is critically involved in bone homeostasis, and mutations in key components of signalling pathways underlying this process may lead to severe osteoclast phenotypes and hence to serious bone diseases. Thus, increasing knowledge of cell fusion mechanisms might contribute to a better understanding of bone biology and pathology. In particular, the macrophage cell line RAW 264.7 is widely used for studies of osteoclast differentiation, since become multinucleated cells when exposed to M-CSF and RANKL (Xing et al 2012 World J Orthop 3:212-222).
The sea urchin embryo has been used as a valuable model for developmental biology studies, owing to its rapid embryogenesis, its transparency and simplicity in shape and organization. Furthermore, the recent sequencing of the entire sea urchin genome has greatly advanced our knowledge on the molecular basis of developmental processes. The first event of cell fusion occurs during gastrulation by the PMCs and leads eventually to the formation of the embryonic skeleton. The gene regulatory network (GRN) underlying the differentiation of PMCs as well as the skeleton synthesis is currently understood in great detail (Ettensohn 2009 Development 136:11-21). Nevertheless, although the dynamics of PMC fusion have been extensively analysed in vivo, the GRN activating this process and the ECM/GF components involved are relatively unknown. Recent research has uncovered few fusion-associated candidate genes in PMCs, homologous to myoblast and/or macrophages cell fusion genes, and some apparently conserved miRNAs. A great advantage is the chance to isolate PMCs from the embryo, culture them in vitro for short times and induce them to fuse and synthesize the skeleton, reproducing the in vivo PMCs behaviour. Furthermore, the comparatively small number of total miRNAs and the lack of redundancy of multiple miRNA families in the sea urchin will simplify the study of their role in cell fusion.
Thus, cell fusion process is critical for both osteoclasts and PMCs function and a deep knowledge of ultrastructural cellular events coupled with the functional properties of specific molecules can lead to a better understanding of molecular mechanisms underlying this fundamental cellular event.
The focus of this collaboration is to define the role of ECM and GF molecules as well as miRNAs in the cell fusion process in terms of 1) ultrastructural analyses of the cells with a wide array of imaging methods, including traditional approaches to live-cell imaging and more advanced techniques, such as super-resolution fluorescence microscopy and CLEM, which is the expertise of the Russian group, and 2) molecular analyses measuring variations in the expression levels of cell fusion specific genes and proteins in sea urchin and cell lines, which is the expertise of the Italian group.

Obiettivi della ricerca

The main objective of this project is to outline a scenario, as complete as possible, of the cell fusion process. At the same time, the use of in vitro cultures of mouse RAW 264.7 and sea urchin PMCs will allow the identification of common features between stabilized and primary cell lines, as well as the uncovering of conserved mechanisms governing this process in vertebrates and invertebrates. Two strategies will be adopted to unravel different steps of the cell fusion and to get information on factors regulating this process. One consists of experiments in which samples from different steps of cell fusion will be analysed for ultrastructural modifications of cytoskeleton and organelles organization (Russian group) and for the expression of molecules known to regulate cell fusion (Italian group), in normal conditions and in the presence of different ECM and/or GF molecules. The second strategy concerns the execution of loss or gain of function experiments (employing neutralizing antibodies, protease inhibitors, pre-miRNAs, anti-miRNAs, siRNAs) to determine the effects of manipulating specific factors on cell fusion, at ultrastructural, cellular and molecular levels. A valuable outcome of this project will be a wider knowledge on the subject of interest using the resources and the expertise available in two countries and the dissemination of the results will be a crucial step for the success of the project and for the sustainability of outputs in the long term.

Ultimo aggiornamento: 24/04/2024