RESEARCH ARTICLE EhRab21 mobilization during erythrophagocytosis in Entamoeba histolytica Luis A. Constantino-Jonapa | Verónica Ivonne Hernández-Ramírez | Carlos Osorio-Trujillo | Patricia Talamás-Rohana Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del I.P.N., Avenida Instituto Politécnico Nacional No. 2508, Col. San Pedro Zacatenco, Delegación Gustavo A. Madero, CDMX, CP 07360, Mexico Correspondence Patricia Talamás-Rohana, Ave. IPN No. 2508, Col. San Pedro Zacatenco, CDMX 07360. Email: ptr@cinvestav.mx Review Editor: Prof. Alberto Diaspro Funding information Basic Science Research Project, CONACYT, MEXICO, Grant/Award Number: 221136-M- 1370 Abstract Rab proteins are present in all eukaryotic lineages and regulate vesicular trafficking. Entamoeba histolytica has approximately 100 genes encoding Rab proteins, among which 16 have homology with human Rab proteins. Human Rab21 participates in integrin recycling, and thus amoebic Rab21 was believed to regulate the mobilization of Ehβ1FNR (integrin-like fibronectin receptor related with human integrin β1). We analyzed the distribution of EhRab21 using a polyclonal antibody produced with a specific peptide against the amoebic Rab protein, using confocal microscopy and specific probes for different organelles. EhRab21 was not associated with Ehβ1FNR in fibronectin-stimulated trophozoites. However, EhRab21 was relocalized to lyso- somes in erythrophagocytosis assays and was also found in Golgi-positive structures and the nuclear periphery. These results suggest that EhRab21, unlike its human homologue, is not pre- sent in the recycling pathway. However, according to the results, EhRab21 may regulate the trafficking between lysosomes and the Golgi apparatus. KEYWORDS Golgi apparatus, lysosomes, nucleus, vesicular trafficking 1 | INTRODUCTION One of the major characteristics of eukaryotic cells is cellular compart- mentalization, which separates the major biochemical processes to improve regulation (Martin & Koonin, 2006). This system is called the endomembrane system, which can be divided into exocytic and endo- cytic pathways. Exocytosis is the export of products to the surface while endocytosis is the entrance route to the cell, important for the uptake of nutrients and recycling of the plasma membrane (Repnik, Cesen, & Turk, 2013). These processes require regulating mechanisms for the correct function and delivery of specific molecules to individual organelles, achieved by co-operative proteins, including soluble NSF attachment protein receptor (SNARE)s, vesicle coat complexes and importantly, Rab GTPases (Cai, Reinisch, & Ferro-Novick, 2007; Stenmark, 2009; Sudhof & Rothman, 2009).Rab proteins are the largest subfamily of GTPases of the Ras superfamily, with 11 identified in yeast and >60 members in humans that can be classified into several phylogenetic and functional groups (Pereira-Leal & Seabra, 2001). The genomes of all eukaryotes encode multiple members of the Rab family, and even the simplest eukaryotes have more than 10 Rab proteins (Diekmann et al., 2011; Pereira-Leal, 2008; Zhang et al., 2007). Therefore, it has been established that Rab proteins have been present since the appearance of the endomembrane system, and it has been hypothe- sized that they were present in the last eukaryotic cell ancestor (LECA) (Klopper, Kienle, Fasshauer, & Munro, 2012). Rab5, Rab20, Rab21, Rab22, Rab24, and Rab50 likely form an ancient endocytic clade that is predicted to be present in LECA (Elias, Brighouse, Gabernet-Castello, Field, & Dacks, 2012). In the enteric protozoan parasite Entamoeba histolytica, approximately 100 Rab pro- teins are present, and from those, only Rab21 and Rab5 correspond to the ancient endocytic clade (Saito-Nakano, Loftus, Hall, & Nozaki, 2005). EhRab5 participates in phagocytosis, unlike its human homo- logue, which is involved in endocytosis (Saito-Nakano, Yasuda, Nakada-Tsukui, Leippe, & Nozaki, 2004). Alternatively, EhRab21 has been reported to be an actin modulator in fibronectin-stimulated tro- phozoites (Emmanuel, Nakano, Nozaki, & Datta, 2015), suggesting that EhRab21 could participate in Ehβ1FNR mobilization; however this Received: 16 June 2017 Revised: 3 May 2018 Accepted: 25 May 2018 DOI: 10.1002/jemt.23069 Microsc Res Tech. 2018;1–12. wileyonlinelibrary.com/journal/jemt © 2018 Wiley Periodicals, Inc. 1