Please cite this article in press as: Midlej, V., et al., Mitosomal chaperone modulation during the life cycle of the pathogenic protist Giardia intestinalis. Eur. J. Cell Biol. (2016), http://dx.doi.org/10.1016/j.ejcb.2016.08.005 ARTICLE IN PRESS G Model EJCB-50900; No. of Pages 12 European Journal of Cell Biology xxx (2016) xxx–xxx Contents lists available at ScienceDirect European Journal of Cell Biology journal homepage: www.elsevier.com/locate/ejcb Research paper Mitosomal chaperone modulation during the life cycle of the pathogenic protist Giardia intestinalis Victor Midlej a,b,∗ , Luciana Penha b , Rosane Silva b , Wanderley de Souza b,d , Marlene Benchimol a,b,c,d,∗∗ a Instituto de Ciências Biológicas, Programa de Ciências Morfológicas, Universidade Federal do Rio de Janeiro, Av. Brg. Trompowski, Rio de Janeiro – RJ, 21044-020, Brazil b Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Av. Brg. Trompowski, Rio de Janeiro – RJ, 21044-020, Brazil c Universidade UNIGRANRIO, R. Prof. José de Souza Herdy, 1160 – Jardim Vinte e Cinco de Agosto, Duque de Caxias – RJ, 25071-202, Brazil d Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens-INBEB e Centro Nacional de Bioimagens-CENABIO, Av. Brg. Trompowski, Rio de Janeiro – RJ, 21044-020, Brazil a r t i c l e i n f o Article history: Received 5 April 2016 Received in revised form 10 August 2016 Accepted 29 August 2016 Keywords: Giardia intestinalis Mitosomes Chaperones Encystation Cysts Differentiation a b s t r a c t The mitosome is a double-membrane enveloped organelle that is found in few unicellular eukaryotes, one of which is the human intestinal parasitic protist Giardia intestinalis, which also lacks mitochondria and peroxisomes. This flagellated protist grows in vitro as trophozoites and under some conditions, differen- tiates into cysts, which are characterized by the absence of externalized flagella, a round shape, and the presence of a cyst wall. The presence and distribution of mitosomal proteins, such as giardial iron-sulfur cluster protein (GiIscU), heat-shock protein 70 (mit-HSP70) and giardial chaperonin 60 (GiCpn60), during the process of trophozoite-to-cyst transformation was tracked using confocal laser scanning microscopy and western blotting. During the early stages of the differentiation process (∼12 h), there was a signif- icant decrease in the extent of chaperone labeling in the cells, which disappeared after 21 h but was recovered during the cyst stage; IscU labeling remained present throughout the differentiation process. This finding was confirmed by mRNA expression analysis, thus indicating that a process modulates the expression of mitosomal chaperones during the G. intestinalis life cycle. Microscopy techniques, such as structured illumination and electron tomography, revealed a novel profile for central mitosomes, as well as the presence of both rounded and elongated mitosomes. © 2016 Elsevier GmbH. All rights reserved. 1. Introduction Mitochondria are organelles that are thought to have evolved from an alpha-proteobacterial endosymbiont approximately two billion years ago (Gray et al., 1998). The mitochondrion was long thought to be absent in some unicellular eukaryotes. These species were grouped as paraphyletic organisms without mitochondria and were classified into the Kingdom Archezoa (Cavalier-Smith, Abbreviations: Cpn60, Chaperone 60; CW, cyst wall; CWP1, cyst wall protein 1; ECVs, encystation carbohydrate-containing vesicles; ESVs, encystation specific vesicles; HSP70, heat-shock protein 70; IscU, iron-sulfur cluster enzyme assembly; PVDF, polyvinylidene fluoride; SR-SIM, super resolution- structured illumination microscopy. ∗ Corresponding author at: Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil. ∗∗ Corresponding author at: UNIGRANRIO, Rio de Janeiro, Brazil. E-mail addresses: vmidlej@hotmail.com (V. Midlej), marlenebenchimol@gmail.com (M. Benchimol). 1987). The diplomonads, such as Giardia intestinalis, are among the most interesting Archezoa, as their phylogenetic position in eukary- otic trees has not been defined unequivocally (Hampl et al., 2009; Keeling et al., 2005). Current molecular data have disproven the Archezoa concept, in that all of the known Archezoa, including G. intestinalis and other enigmatic protists, have been shown to con- tain mitochondrion-related organelles (Hampl et al., 2006; Tovar et al., 2003). Mitosomes are double-membrane enveloped organelles that are related to mitochondria and have been identified in the human intestinal parasite G. intestinalis (Tovar et al., 2003). The name “mitosome” (synonym: crypton) was proposed to indicate that the organelles are highly reduced (cryptic) mitochondria (Tovar et al., 2003). The discovery of mitosomes in Giardia was strongly sup- ported by the identification of the protein machinery components responsible for iron-sulfur (Fe-S) cluster assembly (the IscS and IscU proteins) in this organelle (Tachezy et al., 2001). In addition to IscS and IscU, other proteins, such as the chaperone Cpn60 and http://dx.doi.org/10.1016/j.ejcb.2016.08.005 0171-9335/© 2016 Elsevier GmbH. 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