Molecular & Biochemical Parasitology 115 (2001) 1–17 www.parasitology-online.com. Reviews: Parasite cell Biology: 1 The flagellum and flagellar pocket of trypanosomatids Scott M. Landfear *, Marina Ignatushchenko Department of Molecular Microbiology and Immunology, Oregon Health Sciences Uni6ersity, Portland, OR 97201, USA Received 9 November 2000; received in revised form 26 January 2001; accepted 5 March 2001 Abstract The flagellum and flagellar pocket are distinctive organelles present among all of the trypanosomatid protozoa. Currently, recognized functions for these organelles include generation of motility for the flagellum and dedicated secretory and endocytic activities for the flagellar pocket. The flagellar and flagellar pocket membranes have long been recognized as morphologically separate domains that are component parts of the plasma membrane that surrounds the entire cell. The structural and functional specialization of these two membranes has now been underscored by the identification of multiple proteins that are targeted selectively to each of these domains, and non-membrane proteins have also been identified that are targeted to the internal lumina of these organelles. Investigations on the functions of these organelle-specific proteins should continue to shed light on the unique biological activities of the flagellum and flagellar pocket. In addition, work has begun on identifying signals or modifications of these proteins that direct their targeting to the correct subcellular location. Future endeavors should further refine our knowledge of targeting signals and begin to dissect the molecular machinery involved in transporting and retaining each polypeptide at its designated cellular address. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Trypanosomatid protozoa; Flagellum; Flagellar Pocket; Organelle-specific proteins; Review 1. Introduction or partially with the more general cell biology of the flagellar pocket and flagellum. In contrast, recently Two distinctive features in the cell biology of try- published material has increased the number of proteins panosomatid protozoa are the presence of flagella in at known to reside within the membranes or lumina of the least some life cycle stages and the existence of a flagellum or flagellar pocket and has given us novel prominent invagination of the plasma membrane called molecular markers with which to probe the biological the flagellar pocket [1]. The purpose of this review is to functions of these organelles. provide an overview of recent as well as some longer The surface membrane of kinetoplastid protozoa, standing discoveries concerning the nature of these two including Trypanosoma brucei, Trypanosoma cruzi, closely apposed organelles. This article is organized Leishmania species, Crithidia fasciculata and others has largely around specific proteins that have been shown been divided into three morphologically distinct subdo- to reside in either the flagellum or the flagellar pocket. mains [2]: the flagellar membrane, the flagellar pocket, The reason for this approach is that a number of and the pellicular plasma membrane (Fig. 1). It is now excellent reviews already exist [2–6] that deal wholely recognized that each of these domains represents a Abbre6iations: BSA, Bovine serum albumen; CRAM, cysteine-rich acidic integral membrane protein; ER, endoplasmic reticulum; ESAG, expression site associated gene; FCaBP, flagellar calcium binding protein; GFP, green fluorescent protein; GPI, glycosylphosphatidylinositol; HDL, high density lipoprotein; HRP, horse radish peroxidase; IFT, intraflagellar transport; LDL, low density lipoprotein; LPG, lipophosphogly- can; MVT, multivesicular tubule; PFR, paraflagellar rod; PPG, proteophosphoglycan; sAP, soluble acid phosphatase; SDS, sodium dodecylsul- fate; Tf, transferrin; TFBP, transferrin binding protein; TLTF, T lymphocyte triggering factor; VSG, variant surface glycoprotein. * Corresponding author. Tel.: +1-503-4942426; fax: +1-503-4946862. E-mail address: [email protected] (S.M. Landfear). 0166-6851/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S0166-6851(01)00262-6 2 S.M. Landfear, M. Ignatushchenko / Molecular & Biochemical Parasitology 115 (2001) 1–17 Fig. 1. Subcellular structure of a bloodstream form African trypanosome (reproduced from [5] by copyright permission of Elsevier Science Ltd.). The abbreviations are: az, adhesion zone at the entrance of the flagellar pocket; cv, coated vesicles; er, endoplasmic reticulum; fl, flagellum; fp, flagellar pocket; gl, glycosome, a membrane bound organelle involved in glycolysis and other metabolic pathways; go, Golgi apparatus; k, kinetoplast containing highly catenated kinetoplast DNA; l, lysosome; m, mitochondrion; mt, subpellicular microtubules; n, nucleus, sc, surface coat containing variant surface glycoprotein; tv, tubulovesicular structure. The length of the cell is approximately 20 mm. The cell surface membrane can be divided into the pellicular plasma membrane surrounding the cell body, the flagellar pocket membrane, and the flagellar membrane. highly specialized membrane with distinctive functions activities such as the attachment of parasites to the and unique protein and possibly lipid compositions. endothelium of their insect hosts [1], and it may also Thus, the pellicular plasma membrane surrounds the be a specialized sensory organelle. The flagellar body of the cell and is attached to a dense corset of pocket, a deep invagination at the base of the flagel- highly stable, cross-linked microtubules. This mem- lum (Fig. 3) is responsible for uptake of larger nutri- brane contains many of the permeases that mediate ents via receptor-mediated endocytosis, for secretion uptake of nutrients via classical transporter cycles, it of proteins into the extracellular medium, and for in- provides the cell body with its shape, and in some tegration of membrane proteins into the cell surface. cases, it is densely covered with a protein or glycol- It is noteworthy that these three membranes are phys- ipid coat that protects the parasite against host im- ically contiguous, and all constitute part of the mune responses, as in the case of the variant surface plasma membrane despite their highly differentiated glycoproteins (VSGs) of T. brucei [7] and the abun- biological functions. The identification of proteins dant glycolipid lipophosphoglycan (LPG) of Leishma- that are localized discretely to one of these plasma nia species [8]. The flagellum (Fig. 2) is the classical membrane components has helped to delineate the motility organelle that moves the parasite forward by distinct functions of each of these membrane surfaces. wave-like beats of the microtubule-based flagellar ax- We are now beginning to understand how each com- oneme, but it is also involved in additional biological partment is maintained as a unique entity by identify- Fig. 2. The cytoskeleton of the flagellum and paraflagellar rod of L. mexicana (reproduced from [44], by copyright permission of Elsevier Science Ltd.). (A) A whole-mount negatively stained cytoskeleton is shown, including the basal body ‘b’ where the flagellum initiates, the flagellar axoneme ‘a’ which is the microtubule-based structure that generates flagellar motility, and the fibrous paraflagellar rod ‘p’ that runs adjacent to the axoneme. The subpellicular microtubules that lie underneath the pellicular plasma membrane can be seen in the cell body region at the left of the figure. (B) A transverse view of the flagellar cytoskeleton showing the ‘9+2’ arrangement of singlet and doublet axonemal microtubules. The fibrous paraflagellar rod is underneath the axoneme and consists of the proximal, intermediate, and distal domains (labeled ‘pd’, ‘id’, and ‘dd’, respectively). S.M. Landfear, M. Ignatushchenko / Molecular & Biochemical Parasitology 115 (2001) 1–17 3 rest of the surface bilayer is the observation that several well-characterized membrane proteins in various trypanosomatids are localized discretely in the mem- brane of this organelle. A number of Ca+2-binding proteins of both T. cruzi and T. brucei, receptor- -adenylate cyclases of T. brucei, and a glucose transporter isoform in L. enriettii are all present in the flagellar membrane, while they occur at low or undetectable levels on the pellicular plasma membrane. In addition, earlier biochemical studies [9] detected specific proteins on SDS-polyacrylamide gels using a membrane fraction from purified flagella, although this fraction was not directly compared with the pellicular plasma membrane to determine which of these proteins was truly flagellar-specific. Furthermore, studies on the lipid composition of flagellar membranes of several micro-organisms [10] have revealed high sterol/ phospholipid ratios compared with other membranes, and this pattern of differential lipid content appears to apply in trypanosomatids, as well, where the sterol- binding antibiotic filipin intercalates to a high degree in the flagellar membrane [9]. A question of central importance is to determine how proteins are selectively targeted to or excluded from the flagellar membrane, and studies on several of the flagellar membrane proteins are beginning to elucidate this process. Another intriguing question has to do with the biological significance of flagellar localization; that is, what are the specific functions of proteins that reside exclusively or preferentially in this membrane? 2.1. Flagellar Ca+2-binding proteins Fig. 3. A thin section electron micrograph through the flagellar pocket of a bloodstream form T.