Stress & Cytoskeleton

Research Interests

Microtubules (Mts) are polarized polymers of α/β-tubulin heterodimers participating in a wide range of cell functions, e.g. cell division, cell signalling, intracellular transport and locomotion. How Mts accomplish this variety of functions is far from being completely understood. Although there is a increasing data concerning this subject, one of the most attractive questions in the field of tubulin/Mt biogenesis is still "how is the functional tubulin 3D-structure achieved in the cell?" and "how is this related with Mt biogenesis, function dynamics and recycling of tubulin heterodimers in vivo?" The maturation of tubulin heterodimers is a complex process involving the interaction of tubulins with several proteins: molecular chaperones (e.g. the cytosolic chaperonin containing TCP1- CCT) and the tubulin cofactors (TBCA-TBCE).
The main goal of the Stress and Cytoskeleton group is to understand how the components of the tubulin folding pathway control tubulin synthesis, flux, transport and heterodimers recycling and therefore are putative targets for regulation of the assembly of specialized Mt structures (i.e. cilia) and Mt dynamics.
Regarding the biological role of CCT, tubulin cofactors and related proteins research has been focused on studies of:
a) how tubulin cofactor E and CCT-subunits are involved in biogenesis and maintenance of cilia in the protozoa ciliate Tetrahymena.
b) how the knockdown of the TBCA gene perturbs the mammalian Mt cytoskeleton and its dynamics as well as in the investigation of how TBCA is able to respond to alterations of the native tubulin heterodimers pool.
c) the biological role of the recently described human Cofactor C related protein, TBCC-domain containing 1 (TBCCD1), which we propose to be a key regulator of centrosome positioning and consequently of internal cell organization.

We are also studing how Mt dynamics in mammalian cells is related to biophysical properties of these polymers, with the goal of establishing nano-devices where Mts and associated motor proteins are used as deliver target systems.
Finally, the group is investigating how rearrangements in the Mt cytoskeleton of the Apicomplexa protozoan parasite Besnoitia besnoiti that causes disease in bovine, are involved in the first steps of host cell invasion

Group Members

Sofia Nolasco	Postdoc
	Tel: 21 440 7947
Rita Cardoso	External Ph.D. Student
	Tel: 21 440 7947
Alexandra Tavares	Trainee
	Tel: 21 440 7947
Ruben Ramalho	2006 PDIGC PhD Student
	Tel: 21 440 7947

Research Project

Rate and effects of mutations in the protozoa Tetrahymena thermophila

This project was recently begun in close collaboration with the Evolutionary Biology group, lead by Isabel Gordo. Our aim is to measure several mutational properties of this ciliate. Towards this end, we will:
1) estimate the genomic mutation rate;
2) quantify fitness decline due to strong population bottleneck;
3) estimate the deleterious mutation rate and average effect of mutations that decrease fitness.
4) quantify the rate of fitness recovery and the rate at which compensatory mutations arise.

Funding

Fundação para a Ciencia e Tecnologia (FCT) Project Grant, Portugal

Collaborators

IGC Evolutionary Biology Group – Isabel Gordo

Research Project

Microtubule cytoskeleton behavior in the initial steps of host cell invasion by Besnoitia besnoiti and Toxoplasma gondii

Besnoitia besnoiti is a coccidian tissue cyst-forming protozoan of the phylum Apicomplexa responsible for bovine besnoitiosis, a pathology causing important losses in the cattle industry. Proliferation of these organisms occurs by invasion of a host cell followed by parasite growth and division until the host cell is lysed by the replicating parasites. Parasites that practice this life-style are not able to grow extracellularly, which makes them vulnerable if entry is prevented. Studying how parasites gain entry into their host cells is thus important for the design of improved therapies. We have been focused in the study of the Mt cytoskeleton of isolated tachyzoites of B. besnoiti and its characterization during the first steps of host cell invasion in vitro using indirect immunofluorescence and atomic force microscopy. We showed that although the host cell Mt cytoskeleton is not essential for parasite entrance, both Mt cytoskeletons might have an active role during the invasion. Experiments are in progress to identify the molecular mechanisms underlying the cross-talk between the two cytoskeletons. In these studies, we also intend to use T. gondii as a biological model, essentially because the genome is sequenced and multiple genetic tools are already available. The combination of these data with those coming from B. besnoiti, will improve our understanding of the basic biology of the Apicomplexan parasites. Furthermore, T. gondii is the most common cause of congenital neurological defects in humans, and a devastating opportunistic infection in immuno-compromised patients.

Funding

Fundação para a Ciencia e Tecnologia (FCT) Project Grant, Portugal

Collaborators

Instituto de Investigação Científica Tropical (CIISA) - Alexandre Leitão
Instituto Superior Técnico - Luís Melo
Laboratório de Parasitologia, Núcleo da Mitra, Universidade de Évora, ICAM, Portugal - Helder Cortes

Research Project

Study of physical properties of microtubules by AFM techniques. Use of microstructured actuators for directional control of microtubules

Microtubules are powerful spatial organizers in the cell which interact with multiple proteins and organelles and are involved in a variety of functions from cell division, compartmentalization, signal transducing, polarity to morphogenesis. The possibility of using them to direct the position and movement of other molecules makes these polymers attractive candidates for bionanotechnological applications, such as biomimetic actuators, sensors, etc. Thus, the control and manipulation of microtubules is an important part of the ongoing effort to produce new MEMS (microelectromechanical systems) that integrate biological components for use in medical and biological applications. We propose to use static electric fields to control the direction of microtubules adsorbed to a substrate and have so far demonstrated this method applied to a bulk sample of microtubules adsorbed to functionalized mica. To achieve micron-level control of microtubule structures, we have, during this year, designed and constructed a microstructured substrate containing multiple capacitor arrays to achieve locally controlled adsorption of microtubules. The device is currently undergoing its first testing and optimization cycle.

Funding

Fundação para a Ciencia e Tecnologia (FCT) Project Grant, Portugal

Collaborators

Instituto Superior Técnico - Luís Melo
INESC-NM - Susana Freitas

Research Project

The puzzling players (tubulin cofactors) of the tubulin folding pathway

It is well established that Mts and their building blocks- tubulin heterodimers- are crucial targets in cancer development and cancer therapy by using antimitotic agents. Expression of some tubulin isotypes is restricted to specific tissues, whereas other isotypes are constitutively expressed, resulting in a unique pattern of expression for each tissue. Tumor cells often express a different complement of beta-tubulin isotypes than their normal counterparts. The functional significance of variations in tubulin isotype expression in both normal and tumor cells is not clearly understood. Furthermore, when cells are submitted to a long exposure to antimitotic agents (e.g. taxol) they tend to express isotypes that contribute to a more dynamic behaviour of microtubules. However the proteins/factors that control this response are not known. In this context we propose that the components of the tubulin folding pathway are privileged targets to participate in this regulation and experiments will be carried on to test this hypothesis.
There are growing evidences that some of tubulin cofactor may play roles outside of the tubulin folding pathway. In this context we are also focused in developing experiments to identify/clarify these new functions.

Collaborators

Facultad de Medicina, Departamento de Biologia Molecular, Universidad de Cantabria, Spain - Juan Zabala

Publications

(Selected) Updated June (2010).

Seixas C, Cruto T, Tavares A, Gaertig J, Soares H. (2010). CCTalpha and CCTdelta chaperonin subunits are essential and required for cilia assembly and maintenance in Tetrahymena PLoS One 5(5) :e10704

Gonçalves, J, Nolasco,S, Nascimento, R, Lopez Fanarraga, M, Zabala, JC and Soares, H. (2010). TBCCD1, a new centrosomal protein is required for centrosome and Golgi apparatus positioning. EMBO Reports 11 :194-200

Ramalho, RR, Soares, H and Melo, LV (2007). Microtubule behavior under strong electromagnetic fields. Mat. Sci. Eng. C. 27 :1207–1210

Reis, Y, Cortes, H, Viseu Melo, L, Fazendeiro, I, Leitao, A and Soares, H. (2006). Microtubule cytoskeleton behavior in the initial steps of host cell invasion by Besnoitia besnoiti FEBS Lett 580 :4673-82

Nolasco S, Bellido J, Gonçalves J, Zabala JC and Soares, H. (2005). Tubulin cofactor A gene silencing in mammalian cells induces changes in microtubule cytoskeleton, cell cycle arrest and cell death FEBS Lett 579 :3515-3524

Seixas, C., Casalou, C., Melo, L.V., Nolasco, S., Brogueira, P. and Soares, H. (2003). Subunits of the chaperonin CCT are associated with Tetrahymena microtubule structures and are involved in cilia biogenesis. Exp. Cell Res. 290 :303-321

Domingues, C., Soares, H., Rodrigues-Pousada, C. and Cyrne, L. (1999). Structure of Tetrahymena CCTq gene and its expression under colchicine treatment. Bioch. Bioph. Acta. 1446 :443-449

Soares, H., Cyrne, L., Casalou, C., Ehmann, B. and Rodrigues-Pousada, C. (1997). The third member of Tetrahymena CCT-subunit gene family, TpCCTa, encodes a component of the hetero-oligomeric chaperonin complex. Biochem J., 326 :21-29

Soares, H., Penque, D., Mouta, C, and Rodrigues-Pousada, C. (1994). A Tetrahymena orthologue of the mouse chaperonin subunit CCTg and its co-expression with tubulin during cilia recovery. J. Biol. Chem. 269 :29299-29307

Dujon B, (...) Soares, H. (...) and Mewes HW (107 authors) (1994). Complete DNA sequence of yeast chromosome XI Nature 369 :371-378