Our long term goal is to characterize regulatory mechanisms of membrane trafficking and their role in infection and human disease. For this, we use state-of-the-art techniques, such as live-cell imaging and confocal microscopy and a broad range of molecular biology, biochemistry and cell biology methods to advance the knowledge in this area. We focus on the study of small GTPases of the Rab and Arf families of the Ras superfamily, as these have been shown to be master regulators of all steps of membrane trafficking. Mutations in several proteins of these families lead to diseases affecting mainly neurons, cilia and lysosome-related organelles. Moreover, several of these proteins have been implicated in tumorigenesis. Furthermore, Rab and Arf proteins have been shown to play essential roles in infection, since several microbes can subvert these proteins in order to escape degradation and evade the immune system. Thus, the study of the mechanisms of diseases caused by mutations in Rab and Arf proteins, as well as the subversion of these proteins by microbes can shed light on the etiology of these diseases and allow a better understanding of the protein functions.
ciliopathies, cell migration and adhesion, cancer, Arl, Rab, endocytic recycling traffic, lysosomes, lysosome-related organelles, regulated secretion, pigmentation, melanin, melanocyte, keratinocyte.
i. Regulation of endocytic recycling traffic: implications for ciliopathies and cancer
Primary cilia are mechanosensory organelles that detect and transmit chemical and mechanical signals from the extracellular environment to the cell’s interior and are present in almost all cell types in mammals. Defects in primary cilia lead to diseases collectively known as ciliopathies, which are characterized by obesity, polydactyly, renal cysts, retinal degeneration and neurological defects. In particular, we are interested in studying the role of Arl13b and Rab35, which regulate endocytic recycling traffic, in ciliary cargo trafficking. With these studies, we expect to shed light on the importance of the endocytic recycling pathway in the assembly and maintenance of primary cilia and the etiology of ciliopathies.
Several processes depend on the endocytic recycling pathway, including cell adhesion and migration. These processes are subverted by cancer cells to disseminate and form metastases distantly from the primary tumor. We found that Arl13b regulates cell migration through the interaction with the actin cytoskeleton in non-pathological conditions. We now aim to characterize the role of Arl13b in cancer cell adhesion and migration and establish whether Arl13b can serve as a new biomarker for cancer invasiveness and/or a new target for anti-cancer therapies.
ii. Regulated secretion and pigmentary disorders
Among the diseases caused by defects in membrane trafficking, there are several affecting the secretion of lysosome-related organelles (LROs). We use melanosomes as a model of LROs in order to define the molecular mechanism involved in melanin exocytosis and transfer to adjacent keratinocytes. These processes are at the core of skin pigmentation, since melanin has to be transferred from melanocytes, where it is synthesized, to keratinocytes, where it forms a cap over the cells’ nuclei to shield it from UV radiation. In particular, we found that Rab11b is essential for melanin exocytosis by melanocytes and now aim to identify other molecular players involved in this function. Moreover, we aim to define the route followed by melanin upon uptake by keratinocytes. Therefore, our work has implications for human diseases that cause pigmentation defects and can also have cosmetic applications.
Furthermore, we are investigating the molecular machinery that regulates lysosome exocytosis, a process that is required for plasma membrane repair and cell survival after injury.
Impairing tumor progression through the inactivation of the small G protein Arl13b
GTP-binding proteins act as molecular switches, cycling between active guanosine 5′-triphosphate (GTP)-bound and inactive guanosine 5’-diphosphate (GDP)-bound conformations. The cycling between these two states is regulated by guanine nucleotide-exchange factors (GEFs), and by GTPase-activating proteins (GAPs). GEFs activate small G proteins by catalyzing the exchange of GDP for GTP, whereas GAPs inactivate small G proteins by catalyzing GTP hydrolysis.
We and others found that the GTP-binding protein Arl13b plays an important role in tumor progression, by positively regulating cancer cell growth, migration and invasion and that it could be targeted as an anti-cancer therapy. However, there are no known molecules that modulate the activity of Arl13b. Therefore, this project aims to identify GAPs that promote the inactive conformation of Arl13b. This will allow the identification of molecules with therapeutic potential to inhibit Arl13b activity and impair cancer progression.
“Molecular mechanisms of cell migration and invasion: developing a new strategy to impair tumor progression"
01/04/2016 – 31/03/2019
Fundação para a Ciência e a Tecnologia (PTDC/BIM-MEC/4905/2014)
Most cancer-related deaths are caused by metastases originating from a primary tumor. Metastasis formation involves cancer cell migration and invasion of surrounding issues. For tumor cells to acquire an invasive phenotype, major changes in cell shape and migration properties must occur, involving actin cytoskeleton and actin-binding proteins that underpin protrusions. Thus, the actin cytoskeleton and actin-binding proteins represent major regulators of cell migration, invasion, polarity and growth that are often subverted by metastatic tumor cells. Additionally, changes in membrane trafficking, including the directed recycling of integrin adhesion molecules to the leading edge of migrating cells has emerged in recent years as being involved in cancer invasion and metastasis.
In this project, we aim to uncover the role of the GTP-binding protein Arl13b and the non-muscular myosin IIA (NMIIA) in tumor dissemination in vivo and correlate Arl13b and NMIIA expression with cancer progression in patient samples. Furthermore, we aim to define the molecular mechanism by which Arl13b and NMIIA regulate cell migration and invasion. This will lead to an increased understanding of the molecular and cellular basis of tumor progression and might lead to the development of more effective strategies to target invasion and metastasis of cancer cells for therapeutic purposes.
“Regulation of endocytic recycling traffic: implications for ciliogenesis, cell migration and cytokinesis”
01/06/2015 – 31/05/2020
Fundação para a Ciência e a Tecnologia (IF/00501/2014/CP1252/CT0001)
The endocytic recycling pathway has gained attention in recent years and is now known to be used by the cell not only to recycle internalized receptors but also in essential functions such as cytokinesis, cell migration and the formation and maintenance of primary cilia. However, how endocytic recycling is regulated to achieve these diverse functions is poorly understood. Importantly, defects in the referred processes can lead to diseases, such as ciliopathies, in the case of cilia formation, and cancer, in the case of cell migration and cytokinesis.
The main goal of this project is to understand how endocytic recycling traffic is regulated to control diverse essential cellular processes like ciliogenesis, cell migration and cytokinesis. This knowledge is crucial to understand the etiology of diseases caused by defects in these processes, namely cancer and ciliopathies, as well as to find novel therapies for these diseases.
“Molecular mechanisms of melanin transfer in skin pigmentation”
01/04/2014 – 30/09/2015
Fundação para a Ciência e a Tecnologia (EXPL/BEX-BCM/0379/2013)
The epidermal-melanin unit is composed by one melanocyte, stably located at the basal layer of the epidermis, and 30-40 surrounding keratinocytes. Melanocytes synthesize and store melanin in specialized organelles termed melanosomes. After melanosome maturation, the melanin is transferred to keratinocytes, where it migrates to the apical perinuclear region of the cell to form a protective cap, shielding the DNA from ultraviolet radiation-induced damage. However, the molecular mechanisms underlying intercellular transfer of melanin remain poorly understood. Present hypotheses include transfer via shedding of membrane-bound vesicles, cytophagocytosis, coupled exocytosis and endocytosis, and direct delivery through filopodia. We have investigated the molecular mechanisms directing melanin transfer from skin melanocytes and found evidence to support melanin secretion by melanocytes with subsequent endocytosis by keratinocytes as the main form of transfer. Additionally, we have identified Rab11b as a key regulator of melanin exocytosis and transfer to keratinocytes. With this project we aim to expand the current knowledge on the mechanism of melanin transfer by finding additional molecular factors involved in this process, using our existing model systems in conjunction with novel in vitro models. In particular, we aim to implicate Rab11b effectors and validate our findings using 3-dimensional (3D) organotypic epidermal cell cultures.
“A new approach to fight tuberculosis”
01/01/2012 – 30/06/2015
Fundação para a Ciência e a Tecnologia (HMSP-ICT/0024/2010)
Tuberculosis kills 1.5 million people every year and it is estimated that one third of the world population is infected by Mycobacterium tuberculosis (Mtb). Recently, macrophage apoptosis was described as a novel defense mechanism against tuberculosis. Interestingly, it was also observed that ongoing membrane repair abrogates damage to the macrophages and prevents release of intracellular mycobacteria by resealing large pores in cellular membranes. However, virulent Mtb can subvert membrane repair, constituting an immune-evasion strategy. Therefore, membrane repair could be the critical mechanism that results in impermeability of the apoptotic macrophage leading to containment of Mtb and its products within the phagosome. The aims of this project are the characterization of the mechanisms involved in plasma membrane repair and the identification of mycobacterial factors that interfere with this process.
“Molecular mechanisms of melanosome transfer and processing by keratinocytes”
01/12/2011 – 31/08/2014
Fundação para a Ciência e a Tecnologia (PTDC/BIA-BCM/111735/2009) The “Epidermal-Melanin Unit” comprises the functional complex in which melanocytes and keratinocytes co-operate in a synergistic fashion giving rise to human skin pigmentation. Melanin, a dark pigment, is synthesized in melanocytes and packaged into organelles termed melanosomes before subsequently being transferred to neighboring keratinocytes. These processes form the basis of skin pigmentation which is critical for photo-protection against ultra-violet damage. Whereas the biosynthesis of melanin, and the biogenesis of melanosomes have been extensively characterized, the mechanism of melanin transfer from melanocytes to keratinocytes remains controversial. Recent transmission electron microscopy analysis of human skin samples suggests that the prevalent mechanism of melanin transfer is melanin exocytosis by melanocytes followed by phagocytosis by keratinocyes. Three lines of evidence support this mechanism:
1) the presence of naked melanin in the extra-cellular space between melanocytes and keratinocytes, 2) within keratinocytes, melanin is surrounded by a single membrane bilayer and 3) cryo-immuno EM revealed that this membrane lacks the melanosomal membrane protein TYRP1. The aim of this project is to validate this hypothesis and identify the molecular players involved in this process utilizing both 2D and 3D in vitro models of the epidermal-melanin unit. Using these systems we aim to determine the molecular basis of melanin transfer by depleting proteins involved in membrane trafficking (Rab GTPases and SNAREs) using siRNA technology and determining the effect of depletion on both melanin exocytosis by melanocytes and melanin uptake by keratinocytes. Thus, this project will provide a better understanding of the mechanisms involved in skin pigmentation which could have implications for the pharmaceutical and cosmetic industries as well as skin cancer prevention.
“Molecular dissection of the intracellular role of Plasmodium in macrophages and dendritic cells”
01/04/2010 – 30/09/2013
Fundação para a Ciência e a Tecnologia (PTDC/SAU-MII/104622/2008)
Malaria infection in tropical regions continues to be a very significant cause of morbidity and mortality with social e economical consequences. The blood stage of the parasite life cycle is the one responsible for the symptoms and the pathology of the disease. Although important scientific progress has been achieved, a vaccine is still not available and there is an increase in drug resistance. Phagocytic cells like macrophages and dendritic cells (DCs) internalize Plasmodium-infected erythrocytes. After internalization, the parasite resides within a membrane-bound vacuole or phagosome. Phagosomes then mature by interchanging membrane and soluble material with organelles from the endocytic pathway. Rab GTPases are involved in the regulation of membrane traffic along the endocytic pathway. However, very little is known about the involvement of Rab proteins in the phagocytic pathway of macrophages and DCs after internalization of Plasmodium-infected erythrocytes. In this project we propose to study the intracellular route of Plasmodium-infected erythrocytes in macrophages and DCs and the role of Rab proteins in this process. Furthermore, we aim to define the role of Rab proteins in MHC class II-dependent antigen presentation by Plasmodium-infected DCs.
“Subversion of the host endocytic pathway by Plasmodium sporozoites”
01/02/2010 – 31/07/2013
Fundação para a Ciência e a Tecnologia (PTDC/SAU-MII/108206/2008)
Malaria starts with the infection of the host liver by Plasmodium sporozoites, the parasite form transmitted by infected mosquitoes. Sporozoites migrate through several hepatocytes before finally infecting one where they develop inside a parasitophorous vacuole (PV) and replicates into thousand of merozoites, the blood infective forms. As the development of Plasmodium sporozoites inside hepatocytes is an obligatory step before the onset of disease, understanding the parasite’s requirements during this period is crucial for the development of any form of early intervention.
In the case of malaria, the PV corresponds to a new and induced host cell compartment. The fusion of host cell organelles with the PV depends on the nature of its membrane, the characteristics of which are initially determined at the moment of the parasite’s internalization into the host cell. Phosphoinositides (PIs) are key components in the regulation of membrane trafficking and are fundamental to the function and identity of the organelles of eukaryotic cells. Host cell organelles can be systematically taken over by many pathogens by subversion of pathways necessary for their penetration into the host cells and/or for their survival inside the cell. Our main goals are to characterize the (re)distribution of host organelles and PIs in the context of Plasmodium liver invasion and the interplay between Plasmodium parasites and host cell autophagy and characterize the involvement of different PIs during Plasmodium development.
“Role of Arl13b in endocytic trafficking”
01/10/2009 – 30/09/2013
Research Executive Agency (PIRG05-GA-2009-247726)
Intraflagellar transport (IFT), which ensures the transport of ciliary cargo towards the tip or the base of the cilium has been characterized in detail. However, we know much less about sorting and trafficking of cargo destined for cilia and also how ciliary cargo is turned over, back to the cytoplasm. Since cilia do not synthesize proteins, these have to be transported from intracellular compartments to that destination. Ciliary components also need to be recycled or degraded by compartments of the cell. Thus, intracellular trafficking is essential for the function and assembly of primary cilia. This project aims to determine the role of Arl13b in ciliary cargo trafficking and also the mechanism by which Arl13b could regulate endocytic trafficking.
- Proença JT, Barral DC, Gordo I. (2017) Commensal-to-pathogen transition: One-single transposon insertion results in two pathoadaptive traits in Escherichia coli - macrophage interaction. Sci Rep. Jul 3;7(1):4504. doi: 10.1038/s41598-017-04081-1
- Casalou C., Faustino A., Barral D.C. (2016) Arf proteins in cancer cell migration. Small GTPases. Oct; 7(4):270-82. doi: 10.1080/21541248.2016.1228792.
- Encarnação M., Espada L., Escrevente C., Mateus D., Ramalho J., Michelet X., Santarino I., Hsu V.W., Brenner M.B., Barral D.C., Vieira O.V. (2016) A Rab3a-dependent complex essential for lysosome positioning and plasma membrane repair. J Cell Biol. Jun 20;213(6):631-40. doi: 10.1083/jcb.201511093.
- Gonçalves S.A., Macedo D., Raquel H., Simões P.D., Giorgini F., Ramalho J.S., Barral D.C., Ferreira Moita L., Outeiro T.F. 2016. shRNA-Based Screen Identifies Endocytic Recycling Pathway Components That Act as Genetic Modifiers of Alpha-Synuclein Aggregation, Secretion and Toxicity. PLoS Genet. Apr 28;12(4):e1005995. doi: 10.1371/journal.pgen.1005995. eCollection 2016 Apr..
- Seixas C., Choi S.Y., Polgar N., Umberger N.L., East M.P., Zuo X., Moreiras H., Ghossoub R., Benmerah A., Kahn R.A., Fogelgren B., Caspary T., Lipschutz J.H., Barral D.C.(2015) Arl13b and the exocyst interact synergistically in ciliogenesis. Mol Biol Cell. Jan; 27(2): 308-20. doi:10.191/pii: mbc.E15-02-0061. Epub 2015 Nov 18.
- Thieleke-Matos C., Lopes da Silva M., Cabrita-Santos L., Portal M.D., Rodrigues I.P., Zuzarte-Luis V., Ramalho J.S., Futter C.E., Mota M.M., Barral D.C., Seabra M.C. (2015) Host cell autophagy contributes to Plasmodium liver development. Cell Microbiol. Mar; 18(3):437-50. doi: 10.1111/cmi.12524. Epub 2015 Nov 4
- Cláudio N., Pereira F.J.C., Barral D.C. (2014) Membrane traffic and disease. In: eLS. John Wiley and Sons, Ltd: Chichester.
- Chutna O., Gonçalves S., Villar-Piqué A., Guerreiro P., Marijanovic Z., Mendes T., Ramalho J., Emmanouilidou E., Ventura S., Klucken J., Barral D.C., Giorgini F., Vekrellis K., Outeiro T.F. (2014) The small GTPase Rab11 co-localizes with α-synuclein in intracellular inclusions and modulates its aggregation, secretion and toxicity. Hum Mol Genet. Dec 20; 23(25):6732-35. doi: 10.1093/hmg/ddu391. Epub 2014 Aug 4.
- Thieleke-Matos C., Lopes da Silva M., Cabrita-Santos L., Pires C.F., Ramalho J.S., Ikonomov O., Seixas E., Shisheva A., Seabra M.C., Barral D.C. (2014) Host PI(3,5)P2 activity is required for Plasmodium berghei growth during liver stage infection. Traffic. 15(10):1066-82. doi: 10.1111/tra.12190. Epub 2014 Jul 3.
- Casalou C., Seixas C. Portelinha A., Barros M., Pintado P., Ramalho J.S., Lopes S.S., Barral D.C. (2014) Arl13b and the non-muscle myosin heavy chain IIA are required for circular dorsal ruffle formation and cell migration. J Cell Sci. Jun 15;127(Pt 12):2709-22. doi: 10.1242/jcs.143446. Epub 2014 Apr 28.
- Tarafder A.K., Bolasco G., Correia M.S., Pereira F.J.C., Iannone L., Hume A.N., Kirkpatrick N., Picardo M., Torrisi M.R., Rodrigues I.P., Ramalho J.S., Futter C.E., Barral D.C., Seabra M.C. (2014) Rab11b mediates melanin transfer between donor melanocytes and acceptor keratinocytes via coupled exo/endocytosis. J Invest Dermatol. Apr;134(4):1056-66. doi: 10.1038/jid.2013.432. Epub 2013 Oct 18.
- Seixas E., Barros M., Seabra M.C., Barral D.C. (2013) Rab and Arf proteins in genetic diseases. Traffic. Aug;14(8):871-85. doi: 10.1111/tra.12072. Epub 2013 Apr 29.
- Barral D.C., Garg S., Casalou C., Watts G.F., Sandoval J.L., Ramalho J.S., Hsu V.W., Brenner M.B. (2012) Arl13b regulates endocytic recycling traffic. Proc Natl Acad Sci U S A. Dec 26;109(52):21354-9.doi: 10.1073/pnas.1218272110. Epub 2012 Dec 7.
- Lopes da Silva M., Thieleke-Matos C., Cabrita-Santos L., Ramalho J.S., Wavre-Shapton S.T., Futter C.E., Barral D.C., Seabra M.C. (2012) The Host Endocytic Pathway is Essential for Plasmodium berghei Late Liver Stage Development. Traffic. Oct;13(10):1351-63. doi: 10.1111/j.1600-0854.2012.01398.x. Epub 2012 Aug 3.
- Seixas E., Ramalho J.S., Mota L.J., Barral D.C., Seabra M.C. (2012) Bacteria and protozoa differentially modulate the expression of Rab proteins. PLoS ONE. 7(7):e39858. Epub 2012 Jul 20.
- Garg S., Sharma M., Ung C., Tuli A., Barral D.C., Hava D.L., Veerapen N., Besra G.S., Hacohen N., Brenner M.B. (2011) Lysosomal trafficking, antigen presentation, and microbial killing are controlled by the Arf-like GTPase Arl8b. Immunity. Aug 26;35(2):182-93. doi: 10.1016/j.immuni.2011.06.009. Epub 2011 Jul 28.
• Miguel Seabra, Centro de Estudos de Doenças Crónicas, PT
• Susana Lopes, Centro de Estudos de Doenças Crónicas, PT
• Paula Macedo, Centro de Estudos de Doenças Crónicas, PT
• Ana Félix, Centro de Estudos de Doenças Crónicas and Instituto Português de Oncologia de Lisboa Francisco Gentil, PT
• Catarina Oliveira, Centro de Neurociências e Biologia Celular, PT
• Henrique Girão, Instituto Biomédico de Investigação da Luz e Imagem, PT
• Arnaud Echard, Institut Pasteur, FR
• Graça Raposo, Institut Curie, FR
• Helena Florindo, Faculdade de Farmácia da Universidade de Lisboa, PT
• Ricardo Silvestre, Instituto de Investigação em Ciências da Vida e da Saúde, PT
• Oliver Blacque, University College Dublin, IRL
If you would like to apply for a position please contact Duarte Barral (duarte.barral(at)nms.unl.pt).