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Naegleria: a Classic Model for De Novo Basal Body Assembly Lillian K

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Naegleria: a Classic Model for De Novo Basal Body Assembly Lillian K Fritz‑Laylin and Fulton Cilia (2016) 5:10 DOI 10.1186/s13630-016-0032-6 Cilia REVIEW Open Access Naegleria: a classic model for de novo basal body assembly Lillian K. Fritz‑Laylin1* and Chandler Fulton2 Abstract The amoeboflagellate Naegleria was one of the first organisms in which de novo basal body/centriole assembly was documented. When in its flagellate form, this single-celled protist has two flagella that are templated by two basal bodies. Each of these basal bodies is structurally well conserved, with triplet microtubules and well-defined proximal cartwheel structures, similar to most other eukaryotic centrioles. The basal bodies are anchored to the nucleus by a single, long striated rootlet. The Naegleria genome encodes many conserved basal body genes whose expression is induced prior to basal body assembly. Because of the rapid and synchronous differentiation from centriole-less amoe‑ bae to temporary flagellates with basal bodies, Naegleria offers one of the most promising systems to study de novo basal body assembly, as well as the mechanisms regulating the number of centrioles assembled per cell. The organism features of Naegleria centrioles is the speed at which dif- Naegleria gruberi is a free-living protist easily isolated ferentiating cells turn on the genes, synthesize the pro- from freshwater sources around the world [1–3]. Naeg- teins, and assemble two canonical basal bodies without leria’s reproductive form is a 15-µm predatory amoeba any pre-existing “template” precursors. Naegleria syn- that feeds on bacteria (Fig. 1). However, when faced thesizes and assembles centriole components only dur- with environmental signals such as nutritional, temper- ing the transition to its temporary flagellate form; in the ature, osmotic, and/or pH shifts, Naegleria undergoes laboratory, at least, it can live for years as reproducing an astounding metamorphosis from a crawling amoeba amoebae or resting cysts without ever using centrioles. to a streamlined flagellate capable of swimming for sev- Naegleria has been developed as a model to study its eral hours before reverting to an amoeba [2, 3]. Only the incredibly rapid, synchronous, and reproducible dif- amoebae reproduce, and their mitosis involves no centri- ferentiation from one cell phenotype to a very different oles [4]. The amoeba-to-flagellate differentiation requires one. Protocols have been developed for straightforward de novo assembly of basal bodies and flagella, including control of this process [2, 3], a methodology that opened transcription and translation of their molecular compo- the door to understanding the roles transcription and nents, even including tubulin (Fig. 1) [5–9]. Despite the translation play in de novo centriole assembly [10], and complexity of this task, Naegleria cells accomplish the tracing the expression, translation, and localization of amoeba-to-flagellate conversion in about an hour [2, 3]. individual proteins during differentiation [5–8]. More This developmental feat led to one of the first discover- recently, genome sequencing has revealed that Naegle- ies of de novo basal body assembly [4], at a time when ria has many canonical centriole/basal body genes, and even the concept of de novo centriole assembly was met microarray analysis of differentiation has also led to the with scepticism. To this day, one of the most interesting prediction of novel centriole genes [9, 11]. Naegleria is a member of the heteroloboseans, a clade composed of a wide variety of amoebae, flagellates, and *Correspondence: [email protected] amoeboflagellates, of whichNaegleria is the best-studied 1 Department of Cellular and Molecular Pharmacology, University example [11]. The heteroloboseans are distantly related of California, San Francisco, CA 94158, USA Full list of author information is available at the end of the article to two other groups, the jacobids, and the euglenozoans © 2016 Fritz-Laylin and Fulton. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons. org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Fritz‑Laylin and Fulton Cilia (2016) 5:10 Page 2 of 6 Fig. 1 Naegleria differentiation. Amoebae can differentiate into flagellates, during which time they assemble basal bodies, flagella, flagellar rootlets, and a cortical microtubule cytoskeleton de novo. This process takes about an hour, and includes transcription and translation of basal body and flagella genes, including flagellar tubu‑ lin [5–9]. This process has been experimentally optimized to be highly synchronous and temporally reproducible [2, 3, 20, 25] Fig. 2 Naegleria basal body structure. Schematic of both Naegleria that include the parasitic trypanosomes [12]. The ances- basal bodies drawn in longitudinal section, including the single rhizo‑ tor of these three clades diverged from other eukaryotic plast (striated rootlet) that connects both basal bodies to the nucleus. lineages somewhere during the past 1–3 billion years [11, Electron micrographs of cross sections of the flagellar‑basal body apparatus highlighting Y‑shaped links (top), transition fibers (middle) 13]. and cartwheel are adapted from figure 5 of [18] Despite the eons that separate Naegleria from ani- mal and fungal lineages, analysis of its fully sequenced genome indicates that Naegleria represents a sophisti- cated and surprisingly complex modern eukaryote, with Consistent with this canonical centriole ultrastructure, about 16,000 genes including complete actin and micro- the Naegleria genome encodes many conserved centriole tubule cytoskeletons, mitotic and meiotic machinery, components, including γ-, δ-, and ε-tubulins, and SAS-6 transcription factors [14], membrane trafficking, exten- [11]. These and other core components are readily rec- sive networks of signaling machinery (including hun- ognized, although some Naegleria orthologs have exten- dreds of protein kinases and small GTPases), and both sively diverged from those of commonly studied species. aerobic and anaerobic metabolic pathways [11]. Based on a seminal electron microscopy study of Nae- The genus Naegleria has about 40 species that are gleria basal bodies and flagella [18], transition zones defined mainly by differences in extrachromosomal DNA also appear well conserved. Although electron micro- sequences [15]. Many of these have very similar life his- graphs revealing details of the lumen of the transition tories, although there are some less-studied species that zone are not available, the published data clearly show appear to have other options in their life cycles (such electron densities representing both basal and terminal as division in flagellates [1]). Clonal strains of two mor- plates [18]. Fibrous links between microtubule doublets phologically very similar free-living species have been and the membrane can be seen at the level of the basal used for almost all studies of basal body development plate, likely corresponding to the Y-shaped links seen at and form. One is N. gruberi strain NEG (the strain for this location in other organisms, connecting microtu- which we have a draft genome [11]); the other was also bule doublets to the ciliary neck. Proximal to the termi- known as N. gruberi strain NB-1 until a difference in nal plate, fibers radiate from microtubule triplets into the ITS sequence caused it to be redefined as N. pringsheimi cytoplasm, which are likely transition fibers [18]. [15]. Herein when we refer to Naegleria we are referring to studies in strains NEG and NB-1. (The opportunistic Additional basal body structures or accessory human pathogen N. fowleri has a similar life cycle, and structures when it forms flagellates the basal bodies appear to be Naegleria’s dual basal bodies are connected to its nucleus formed de novo [16, 17]). by a slender, long (up to 15 microns) striated rootlet called a rhizoplast (Fig. 2) [18–20]. One end of the rhizo- Basic basal body structure plast is tightly adhered to the proximal end of the basal Mature Naegleria flagellates typically have two basal bod- bodies via a striated wedge-shaped structure, while the ies that are anchored at the plasma membrane and tem- other end runs along the nucleus, terminating in a pocket plate motile flagella [18]. The two basal bodies appear within the nuclear envelope [18]. structurally equivalent, with triplet microtubules and a The strength of the attachment of the rhizoplast to clear luminal cartwheel at the proximal end (Fig. 2) [18]. the basal bodies is evident by the ability of the two to be Fritz‑Laylin and Fulton Cilia (2016) 5:10 Page 3 of 6 purified intact [19, 21]. Even the complex of nucleus and basal body [4, 25], this organism clearly does not require flagellar apparatus (basal bodies, rootlets, flagella) are a basal body or centriole for its normal growth. Mitosis sufficiently attached to be co-isolated [18]. Purified rhiz- in Naegleria is intranuclear, and the microtubules do not oplasts appear to be at least 50 % composed of a single focus to the poles [4, 24, 26]. It is clear that the basal body 170KD protein, and have been suggested to be related to does not assume
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