IFOM Centre of Cell Oncology and Ultrastructure

Research project

The IFOM Cell Oncology and Ultrastructure unit is based in Genova and operates since January 2003 thanks to the generous contributions of the Italian Foundation for Cancer Research and of the University of Genova. The unit rests on state-of-the-art equipment for ultrastructural analysis on plastic embedded samples, immuno-gold localizations on ultrathin cryo-sections, pre-embedding immuno-peroxidase, 3D EM tomography. However, the most common cell and molecular biology techniques are implemented in the lab and routinely used. Beside performing own research the unit provides expertise to other groups at IFOM to solve scientific problems by the use of ultrastructural analysis.

fig.1 Upon ligand binding EGFR co-localizes with membrane rafts. HeLa cell stimulated with EGF at 0°C. Membrane raft identified by cholera toxin B-subunit labeling with HRP (dense plaque). EGFR identified by immunogold [+zoom]

Major ongoing projects: RTK receptor internalization
The engagement, at the plasma membrane, of receptor tyrosine kinases (RTKs) by their cognate ligands has two major consequences: 1. The kinase activity of RTKs is stimulated with ensuing receptor auto-phosphorylation and recruitment/activation of the signal transduction machinery responsible for the modulation of cellular functions as diverse as proliferation, differentiation and apoptosis. Membrane microdomains, the so-called lipid rafts, function as platforms to concentrate receptors and assemble the signal transduction machinery. 2. Activated receptors trigger their own endocytosis, a complex process which participates to the propagation of intracellular signals, but whose ultimate goal is to extinguish signaling through removal of receptors from the cell surface. Impairment of endocytic control is likely going to play a role in hyperproliferative conditions, first and foremost cancer. How the site of signaling by receptor tyrosine kinases (RTK) is connected to the site of their internalization is largely unknown. The RTK endocytosis is carried out by different specialized structures, e.g. clathrin coated pits/vesicles and/or caveolae. Whereas the latter are known to originate from raft regions of the plasma membrane, the former have been so far considered to be raft independent. We have recently shown that the epidermal growth factor receptor may be internalized through clathrin-coated pits that form within rafts. Thus, specialized membrane microdomains have the ability to assemble both the molecular machineries necessary for intracellular propagation of effectors signal and for receptor internalization.

Download a 3D tomography movie of an early endosomes immuno-gold labeled for an internalized receptor. [.mov, 6.7 Mb]

Melanosome biogenesis
For many years the melanosome biogenesis has been considered as a bipartite pathway, in which tyrosinase-charged vesicles, budding off the TGN, would fuse with smooth endoplasmic reticulum-derived pre-melanosomes. However, recently published reports and our unpublished data do not support this hypothesis, and suggest that endosomes may play a role as intermediate steps in the biogenesis of the melanosomes. Our results stem from the study of a melanosome protein, i.e. OA1 (Ocular Albinism 1). OA1 is a melanoma/melanocyte differentiation antigen. Elevated levels of OA1-specific T cells have been found in patients with metastatic malignant melanoma. OA1 has been found: i. To bear homology with members of the GPCR superfamily and to bind to heterotrimeric G proteins (GΒ and Gα_i); ii. To associate exclusively to intracellular membranes, unlike all other known GPCRs, including those present in melanosomes. Furthermore, based on electron microscopy analysis of RPE from ^OA1-/y transgenic mice, and the rate of melanosome maturation in patient skin melanocytes transduced in vitro with OA1, we have proposed that OA1 participates to the melanosome biogenesis and may function as possible negative controller of its maturation. In spite of this knowledge, the lack of information on the site and the mechanism of action of OA1, in particular the downstream signaling pathway and the ligands regulating its activity, hampers the full interpretation of these data. We are presently addressing the question of the putative site of action by defining the OA1 sorting pathway.

Development of innovative correlative imaging approaches
In collaboration with unit Diaspro. Correlative light/electron microscopy allows the simultaneous observation of a given subcellular structure by fluorescence light microscopy and electron microscopy. The use of this approach is becoming increasingly frequent in cell biology. Here we report on a new high data output correlative light/electron microscopy method, based on the use of cryosections. We successfully applied the method to analyse the structure of rough and smooth Russell’s bodies, used as model systems. The major advantages of our method are: (1) the possibility to correlate several hundreds of events at the same time; (2) the possibility to perform 3D correlation; (3) the possibility to immuno-label both endogenous and recombinantly expressed proteins, at the same time; (4) the possibility to combine the high-data analysis capability of FLM, with the high precision-accuracy of TEM in a CLEM hybrid morphometry analysis. We have identified and optimized critical steps in sample preparation, defined routines for sample analysis and re-tracing of regions of interest, developed software for semi/fully-automatic 3D reconstruction, and defined preliminary conditions for an hybrid light/electron microscopy morphometry approach. We are currently working to further improve this approach by automation of specific steps.