Antonella De Matteis
Cell Biology and Disease Mechanisms Program Coordinator, TIGEM
Signalling in Membrane Trafficking
Membrane trafficking maintains cell organization, organelle homeostasis, and intercellular communication. Its important role is confirmed by the serious consequences that result from its disruption, such as those caused by inherited defects of the trafficking machineries. The control mechanisms ensuring correct function of trafficking machineries are now known.
Over the years we have investigated the interplay between signalling and trafficking, in particular at the Golgi complex, a central sorting station in membrane trafficking pathways. We have demonstrated that what was considered to be a “constitutive” process, i.e. membrane trafficking to, through, and out of the Golgi complex, is actually a highly regulated process controlled by secondary messengers and plasma membrane receptors. We then focused on the role of phosphoinositides in Golgi complex, studying both its regulation of local phosphoinositide metabolism and phosphonositide effectors. We have established a link between the GTPase ARF and the regulation of phosphoinositide metabolism in Golgi by identifying a specific PI4 kinase isoform as a novel ARF effector. FAPP proteins are among the phosphoinositide effectors that we have identified at the Golgi; we have shown that they are involved in Golgi-to-plasma membrane trafficking and glycosphingolipid synthesis at the Golgi complex, thanks to FAPP2-operated non-vesicular transport of glucosylceramide.
Mendelian Disorders of Membrane Trafficking: Molecular Mechanisms and Drug Target Identification
One of our group’s missions is to “apply” our basic knowledge of membrane trafficking to the study of Mendelian disorders, diseases that arise from defects in membrane trafficking machinery. Our aim is to clarify the cellular and molecular pathogeneses of these disorders and to identify candidate drug targets. We are presently focusing on two disorders: Lowe syndrome and spondylo-epiphyseal dysplasia (SED) tarda.
Oculocerebrorenal syndrome of Lowe (OCRL), or Lowe syndrome, is a rare X-linked genetic disease caused by mutations in the ocrl1 gene that is characterized by congenital cataracts, renal Fanconi’s syndrome (low molecular weight proteinuria, tubular acidosis) and mental retardation. OCRL-1 is a PtdIns(4,5)P2 5-phosphatase localized at the plasma membrane, in endosomal compartments and at the Golgi complex. We have shown that its activity is required for different trafficking pathways that intersect early endosomes, including that of megalin, which controls protein reabsorption in proximal tubular cells at the kidney. Non-functional OCRL causes a “traffic jam” at the early endosomes as a consequence of the accumulation of PtdIns(4,5)P2 and dysregulation of actin assembly in the organelle compartment.
X-linked SED tarda is caused by mutations in sedlin, a component of the multi-molecular TRAPP complex, which has been conserved from yeast to mammals. SED tarda is characterized by a disproportionately short stature, short trunk and osteoarthritis. The main defect of chondrogenesis is the chondrocytes’ inability to correctly secrete and assemble extracellular matrix components. Indeed, our studies have uncovered a role for sedlin in the trafficking of selective cargoes from the endoplasmic reticulum to the Golgi complex. In particular, we have shown that sedlin is required for the efficient and selective export of procollagen from the endoplasmic reticulum. Derangement of this process due to sedlin mutation may explain the defective chondrogenesis underlying SED tarda.
High Content Screening Facility
Approximately 6,000 human genetic disorders are known to exist. According to the OMIM database, the responsible gene for over 30% of the currently known genetic diseases has been identified. While molecular mechanisms are becoming increasingly recognizable thanks to persistent research efforts, therapeutic treatments have yet to see such progress. Since each genetic disease is particular in its rarity, genetic disorders as a whole have been, until very recently, neglected by the profit-driven structure of the system for drug development. As a result, the number of genetic diseases left untreated is still overwhelming. Thus, it is necessary to develop and implement a non-profit-driven process in order to discover effective treatments for genetic diseases.
Cell-based High Content Screening (HCS) has rapidly gained relevance, as it is being used in almost all of the pre-clinical steps of the drug discovery process. HCS is an analysis tool used to acquire, manage, and search multi-parametric information regarding the composite phenotype of cells. Thus HCS is important becaus eit allows for the simultaneous analysis of the impact of a given perturbation (gene modification or exposure to a drug) on such composite phenotypes. For these reasons, HCS is used during secondary and tertiary screening procedures to support hit-to-lead processes and mode-of-action studies, but also as a tool for primary screening in which thousands of compounds may be identified.
The HCS Facility at TIGEM-Pozzuoli has been tremendously upgraded with the integration of two high content microscopes (Opera and Operetta) with robotic plate-handlers, a liquid handling station (Zephyr) and a plate washer. This configuration significantly increases the throughput procedures executed in the facility. In addition we have a dedicated a cell culture room and a liquid handling station (StartLet Hamilton) to manipulation of compounds, siRNAs and cell lines, making our HCS facility suitable for DNA/RNAi-based high content screening. The volume of images and datapoints generated by the HCS Facility requires the HCS staff to collaborate with the Informatics and Bioinformatics TIGEM cores.
The main activities of the HCS Facility are
- Generation, development, validation and screening of disease cellular models of genetic diseases
- Identification of correctors for genetic diseases (small molecule libraries).
- Identification of drug targets for genetic diseases (druggable genome siRNA library).
- Identification of molecular pathways controlling specific cell functions (siRNA libraries).
- Supporting TIGEM researchers in the developing and realization of screening projects
Specific (HC)-cell based assays:
- Nuclear translocation assay
- Lysosomal morphometrics
- Lysosomal clearance
- Cell cycle (DNA content)
- Lysosomal membrane permeabilization (LMP)
- mTOR activity assay
- Focus small molecules libraries (around 1500 compounds; FDA compounds, kinase and phosphatase inhibitors, nuclear receptor ligands)
- siRNA libraries (11.000 genes, including druggable genome)
- microRNAs (1500)
StartLet liquid handler
Zephyr liquid handler
Multidrops Plate washers
Specialized cell culture room
The HCS Facility also conducts research focused on the discovery of new 'druggable' targets and the development of pharmacological strategies to treat rare genetic diseases by using cell biology approaches and high-content imaging technology. To identify new 'druggable' targets we rely on RNAi-based screenings. We have developed automated protocols to transfect cells with siRNA oligonucleotides using the Hamilton Startlet liquid handling station. We house various molecule libraries, but our expanded version of an RNAi-collection is especially important for targeting the druggable genome. Indeed, this approach is particularly useful in helping us understand the signal transduction pathways involved in the pathogenesis of rare diseases.
Using such strategies demands relevant cellular models of genetic diseases. Thus we are using CRISPR technology, siRNA-mediated silencing and overexpression of fluorescent reporter genes to generate suitable cellular models and multi-parametric read-outs to be used in several screening projects.
Staff Scientist: Cathal Wilson
PostDoc:, Raffaele La Montagna, Leopoldo Staiano, Rossella Venditti, Ivan Castello
Ph.D. Student: Francesca Zappa, Maria Chiara Masone, Mario Failli
Pre-doc: Valentina Morra
Technician: Michele Santoro, Giuseppe di Tullio
Venditti R, Scanu T, Santoro M, Di Tullio G, Spaar A, Gaibisso R, Beznoussenko GV, Mironov AA, Mironov A, Jr., Zelante L, Piemontese MR, Notarangelo A, Malhotra V, Vertel BM, Wilson C, De Matteis MA (2012). Sedlin controls the ER export of procollagen by regulating the Sar1 cycle. Science. 337(6102):1668-1672.
Vicinanza M, Di Campli A, Polishchuk E, Santoro M, Di Tullio G, Godi A, Levtchenko E, De Leo MG, Polishchuk R, SandovalL, Marzolo MP, De Matteis MA (2011). OCRL controls trafficking through early endosomes via PtdIns4,5P2-dependent regulation of endosomal actin. EMBO J. 30(24): 4970–4985. doi:10.1038/emboj.2011.354.
De Matteis MA, Luini A (2011). Mendelian Disorders of membrane trafficking. N Eng J Med. 365: 927-938. doi: 10.1056/NEJMra0910494.
D'Angelo G, Polishchuk E, Di Tullio G, Santoro M, Di Campli A, Godi A, West G, Bielawski J, Chuang CC, van der Spoel AC, Platt FM, Hannun YA, Polishchuk R, Mattjus P, De Matteis MA (2007). Glycosphingolipid synthesis requires FAPP2 transfer of glucosylceramide. Nature. 449: 62-67. doi:10.1038/nature06097.
D'Angelo G, Uemura T, Chuang CC, Polishchuk E, Santoro M, Ohvo-Rekilä H, Sato T, Di Tullio G, Varriale A, D'Auria S, Daniele T, Capuani F, Johannes L, Mattjus P, Monti M, Pucci P, Williams RL, Burke JE, Platt FM, Harada A, De Matteis MA (2013). Vesicular and non-vesicular transport feed distinct glycosylation pathways in the Golgi. Nature. 501:116-20.393-404. doi:10.1038/nature12423.