Membrane Traffic in Disease

Duarte Barral 12-6-2013 - 7557

Duarte C. Barral

Principal Investigator
Graduated in Biology from the University of Lisbon, Portugal.
PhD in Biology from Imperial College London, UK, under the supervision of Miguel Seabra.
Post-doctoral fellow at the lab of Michael B. Brenner at Brigham and Women´s Hospital, Harvard Medical School, Boston, USA.

CV

 

Location:

CEDOC
Campus Sant'Ana
Pólo de Investigação, NMS, UNL
Rua Câmara Pestana, nº 6
Lab 2.7
1150-082 Lisboa, Portugal

Phone: (+351) 218 803 101 (Ext. 26033)
Lab ext: 26053
Fax: (+351) 218 851 920
E-mail: duarte.barral(at)nms.unl.pt

Main Interests:

Cell 2 Our long term goal is to uncover 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. Thus, the study of the mechanisms of diseases caused by mutations in Rab and Arf proteins, can shed light on the etiology of these diseases and allow a better understanding of the protein functions.

Keywords:

cell migration and adhesion, cancer, Arl, Rab, endocytic recycling traffic, lysosomes, lysosome-related organelles, regulated secretion, pigmentation, melanin, melanocyte, keratinocyte.

Research Areas:
i. Cancer and ciliopathies

Several cellular functions, such as cell adhesion and migration are subverted by cancer cells to disseminate and form metastases, distant from the primary tumor. Moreover, Rab and Arf family small GTPases have been implicated in different types of cancer. We are currently investigating the role of these proteins in breast cancer and melanoma progression and metastasis, with the aim of characterizing the mechanisms involved and finding new biomarkers of cancer invasiveness and new therapeutic targets. This is a pressing need, as metastasis remains the main cause of cancer-related deaths.
New Image 3
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. Interestingly, primary cilia have been implicated in several types of cancer, either as negative or positive regulators. Therefore, we aim to understand the mechanisms behind the link between primary cilia function and cancer progression.

ii. Pigmentary disorders

db imagem pigmentacao 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 LRO in order to define the molecular mechanism involved in melanin exocytosis and transfer to adjacent keratinocytes. These processes ensure skin pigmentation, since melanin has to be transferred from melanocytes, where it is synthesized, to keratinocytes, within which it forms a cap over the cells’ nuclei to shield it from UV radiation. In particular, we aim to identify the other molecular players and molecular mechanisms involved in melanin exocytosis from melanocytes. Moreover, we aim to define the route followed by melanin upon uptake by keratinocytes and the process by which melanin forms a supra-nuclear cap or "parasol", shielding the nuclei of keratinocytes from UV radiation. Therefore, our work has implications for human diseases that cause pigmentation defects and can also have cosmetic applications.

Discover more about the Membrane Traffic in Disease Lab

. Casos Singulares - Canal S+ (Part 1) & Casos Singulares - Canal S+ (Part 2)
. Microorganismos e hospedeiros. Conhecer os fundamentos para encontrar novas maneiras de combater infeções.
. Parasita da malária e bactéria E. coli manipulam proteínas da célula hospedeira para evitarem a destruição
. Shining a Light on Black Holes in Keratinocytes
. Meet the Investigator: Maria Serrano Correia, PhD
. Meet the Investigator: Hugo Moreiras, MSc

Ongoing Projects:

LYSOCILLLYSOCIL: Excel in Rare Diseases’ Research: Focus on LYSOsomal Disorders and CILiopathies
01/11/2018 – 30/04/2022
European Union’s Horizon Twinning project 2020 (No 811087)
LYSOCIL is a Twinning Project led by NOVA Medical School (NMS) that aims to strengthen the research and innovation capacity of the Chronic Diseases Research Center (CEDOC-NMS|FCM) in research in rare diseases, helping it to become a national and internationally-recognized centre of excellence in chronic rare diseases research and innovation. For this project, CEDOC-NMS|FCM partnered up with two internationally-leading institutions, Münster University (WWU), a German institution with a strong research profile in basic, clinical and translational medicine connected to rare diseases, and the Telethon Institute of Genetics and Medicine (TIGEM), the leading Italian research center dedicated to understanding the molecular mechanisms behind rare genetic diseases. This partnership will boost the multidisciplinary knowledge and experience, as well as research and training practices and widen the collaborative networks to step up the excellence of the research of the involved partners. Our research group is a partner in this project.

imagem projecto Molecular Mechanisms of melanin internalization and processing by keratinocytes
01/10/2018 – 30/09/2021
Fundação para a Ciência e a Tecnologia
(PTDC/BIA-CEL/29765/2017)
The skin is the largest organ of the human body and provides protection against external aggressions. The outmost layer, the epidermis is composed mainly by two cell types: melanocytes and keratinocytes. Melanocytes synthesize the pigment melanin and localize to the basal layer. Keratinocytes are present in all the layers and are the final recipients of melanin. Melanin protects skin cells against ultraviolet (UV) radiation-induced damage, which can lead to the onset of skin cancer. Melanin synthesis occurs in specialized organelles called melanosomes, which share several features with lysosomes like low pH, the presence of lysosomal membrane proteins and catalytic enzymes and are thus considered lysosome-related organelles. Once fully mature and located at the tips of melanocyte dendrites, melanosomes are transferred to keratinocytes. We found evidence that the predominant model of melanin transfer is coupled exo/endocytosis, where the melanin core is exocytosed by melanocytes in a process dependent on the small GTPase Rab11b and the exocyst tethering complex, and then internalized by keratinocytes. Despite the crucial role of melanin uptake and processing by keratinocytes for skin pigmentation, the pathways involved have not been characterized. Moreover, after melanin is internalized by keratinocytes, little is known about how it accumulates at the supra-nuclear region of keratinocytes to shield the DNA from UV radiation. Therefore, we propose to study how melanin is internalized and processed by keratinocytes. Specifically, we aim to characterize the internalization route used by keratinocytes to uptake melanin, the processing of melanin within keratinocytes and the formation of a "parasol" of melanin granules over the nuclei of keratinocytes. Our initial characterization suggests that melanin is stored in specialized endocytic compartments within keratinocytes that are not highly acidic or degradative, allowing it to resist degradation for long periods and remain during the process of differentiation towards the superficial layers of the stratified epithelium. This study has the potential to provide entirely new concepts in the field of skin pigmentation. Pigmentation disorders cause a reduction in the quality of life of those affected due to their social impact. Also, with the modern lifestyle, photoprotection has become an important public health issue. Thus, this work can also lead to the identification of novel key regulators that will become drug targets for the pharma, biotech and cosmetic industries.

Completed Projects:

Imagem 2“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.
imagem projecto siteImpairing tumor progression through the inactivation of the small G protein Arl13b
NOVA Health
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.

Imagem 1 “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.

“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.

Selected Publications:
  • 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.
  • 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.

Collaborations

• Miguel Seabra, Centro de Estudos de Doenças Crónicas, PT
• Susana Lopes, Centro de Estudos de Doenças Crónicas, PT
• Cláudia Almeida, Centro de Estudos de Doenças Crónicas, PT
• Henrique Girão, Instituto Biomédico de Investigação da Luz e Imagem, PT
• Graça Raposo, Institut Curie, FR
• Marta Pojo, Instituto Português de Oncologia de Lisboa Francisco Gentil, PT
• Jacinta Serpa, Centro de Estudos de Doenças Crónicas e Instituto Português de Oncologia Francisco Gentil, PT
• Abel Oliva, Instituto de Tecnologia Química e Biológica António Xavier, PT
• Rune Matthiesen, Centro de Estudos de Doenças Crónicas, PT

Team photos

Retiro 2019 A

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Retiro DB 2017

foto descida do rio

Foto grupo1

Retiro DB 2016-1

Lab retreat 2015

IMG_0200

Lab retreat 2014

CEDOC - IGC 12-6-2013 - 7457

Laborat-IGC-31-01-2013----5057

Laborat-IGC-31-01-2013----4952

Postdoctoral Position at the Membrane Traffic in Infection and Disease Group - Duarte Barral Lab

We are always interested in highly motivated candidates! Therefore, if you would like to join the group send a motivation letter and your CV to duarte.barral(at)nms.unl.pt.