Identification of α-11 giardin as a flagellar and surface component of Giardia lamblia

Giardia lamblia is a protozoan pathogen with distinct cytoskeletal structures, including median bodies and eight flagella. In this study, we examined components comprising G. lamblia flagella. Crude flagellar extracts were prepared from G. lamblia trophozoites, and analyzed by two-dimensional (2-D) gel electrophoresis. The 19 protein spots were analyzed by MALDI–TOF mass spectrometry, identifying ten metabolic enzymes, six distinct giardins, Giardia trophozoite antigen 1, translational initiation factor eIF-4A, and an extracellular signal-regulated kinase 2. Among the identified proteins, we studied α-11 giardin which belongs to a group of cytoskeletal proteins specific to Giardia. Western blot analysis and real-time PCR indicated that expression of α-11 giardin is not significantly increased during encystation of G. lamblia. Immunofluorescence assays using anti-α-11 giardin antibodies revealed that α-11 giardin protein mainly localized to the plasma membranes and basal bodies of the anterior flagella of G. lamblia trophozoites, suggesting that α-11 giardin is a genuine component of the G. lamblia cytoskeleton.     

Giardia spp.: Distribution of contractile proteins in the attachment organelle

Giardia spp. trophozoites isolated from rat small intestine were examined by light microscopy, electron microscopy, SDS-gel electrophoresis, and immunocytochemistry. In SDS-gels of protein extracts of isolated Giardia spp. trophozoites protein bands corresponding to myosin, α-actinin, and actin were identified by comigration with avian myofibril proteins and molecular weight standards. Actin was specifically identified in SDS-gels by immunoautoradiography. Immunostaining for actin, α-actinin, myosin, and tropomyosin in trophozoites was demonstrated in the periphery of the ventral disc in an area corresponding to the lateral crest. Electron-dense fibrillar was observed in the lateral crest of the ventral disc by electron microscopy. Immunostaining for actin and α-actinin was also observed in the area of the median body, a microtubular organelle, and in electron-dense fibrillar material associated with the intracellular axonemes of the posterior-lateral flagella. The localization of these contractile proteins in the ventral disc suggests that they may play an important role in the mechanism of trophozoite attachment.     

Identification of Obscure yet Conserved Actin-Associated Proteins in Giardia lamblia

Consistent with its proposed status as an early branching eukaryote, Giardia has the most divergent actin of any eukaryote and lacks core actin regulators. Although conserved actin-binding proteins are missing from Giardia, its actin is utilized similarly to that of other eukaryotes and functions in core cellular processes such as cellular organization, endocytosis, and cytokinesis. We set out to identify actin-binding proteins in Giardia using affinity purification coupled with mass spectroscopy (multidimensional protein identification technology [MudPIT]) and have identified >80 putative actin-binding proteins. Several of these have homology to conserved proteins known to complex with actin for functions in the nucleus and flagella. We validated localization and interaction for seven of these proteins, including 14-3-3, a known cytoskeletal regulator with a controversial relationship to actin. Our results indicate that although Giardia lacks canonical actin-binding proteins, there is a conserved set of actin-interacting proteins that are evolutionarily indispensable and perhaps represent some of the earliest functions of the actin cytoskeleton.     

Naegleria gruberi De Novo Basal Body Assembly Occurs via Stepwise Incorporation of Conserved Proteins

Centrioles and basal bodies are discrete structures composed of a cylinder of nine microtubule triplets and associated proteins. Metazoan centrioles can be found at mitotic spindle poles and are called basal bodies when used to organize microtubules to form the core structure of flagella. Naegleria gruberi, a unicellular eukaryote, grows as an amoeba that lacks a cytoplasmic microtubule cytoskeleton. When stressed, Naegleria rapidly (and synchronously) differentiates into a flagellate, forming a complete cytoplasmic cytoskeleton de novo, including two basal bodies and flagella. Here, we show that Naegleria has genes encoding conserved centriole proteins. Using novel antibodies, we describe the localization of three centrosomal protein homologs (SAS-6, γ-tubulin, and centrin-1) during the assembly of the flagellate microtubule cytoskeleton. We also used these antibodies to show that Naegleria expresses the proteins in the same order as their incorporation into basal bodies, with SAS-6 localizing first, followed by centrin and finally γ-tubulin. The similarities between basal body assembly in Naegleria and centriole assembly in animals indicate that mechanisms of assembly, as well as structure, have been conserved throughout eukaryotic evolution.The beautiful and enigmatic pinwheel structures of centrioles and basal bodies have captured the imaginations of cell biologists for over a century. These small (∼1-μm) organelles are composed largely of a cylinder of nine microtubule triplets (11). The surrounding amorphous material harbors the microtubule-organizing activities of the centrosome, placing centrioles at the hub of the microtubule cytoskeleton. Metazoan centrosomes define mitotic spindle poles, and their centrioles are called basal bodies when used to form cilia (29). Moreover, in 1900 Meeves showed in a series of classical experiments that centrioles and basal bodies are interconvertible structures (34). Centrioles must replicate exactly once per cell cycle, as duplication errors can lead to problems with chromosome segregation and cell morphology (17).Virtually all animal cells have a pair of centrosomal centrioles that duplicate via “templated” assembly, with the new centriole developing perpendicular and attached to a preexisting centriole (4). Centrioles can also be formed “de novo” in cytosol devoid of preexisting centrioles and basal bodies (20). In addition to many in vivo examples (20), terminally differentiated fibroblasts held in S phase can assemble centrioles de novo after removal of preexisting centrioles by laser microsurgery (15).The amoeboflagellate Naegleria gruberi grows as an amoeba that completely lacks a cytoplasmic microtubule cytoskeleton. However, when exposed to stressors such as temperature, osmotic, or pH changes, Naegleria rapidly differentiates into a flagellate, forming a complete cytoplasmic cytoskeleton from scratch, including two basal bodies and flagella (8). This differentiation occurs synchronously, with approximately 90% of cells growing visible flagella in a 15-min window (T₅₀ = 65 min after initiation of differentiation). As part of this differentiation, Naegleria has been shown to assemble the pinwheel structure of the basal bodies de novo, about 10 min before flagella are seen (11).Two centrosomal proteins that have been studied during Naegleria differentiation are centrin and γ-tubulin. Centrin is a calcium-binding phosphoprotein that is an integral component of the wall and lumen of basal bodies and of the pericentriolar lattice in many organisms (4, 19). During differentiation, Naegleria induces synthesis of centrin protein, which then localizes specifically to basal body structures throughout differentiation (18). γ-Tubulin is a general microtubule nucleation factor that localizes to microtubule-organizing centers (MTOCs) of many types. Surprisingly, Naegleria''s γ-tubulin homolog has been reported to localize to basal body precursor complexes and then move to the other end of the cell before disappearing completely (32).A third protein that has come under recent scrutiny for its role in centriole duplication is SAS-6, a functionally conserved coiled-coil protein required for the formation of diverse basal body precursor structures (7, 21,–23, 31). In Caenorhabditis elegans and Drosophila melanogaster, SAS-6 is recruited at S phase to form the “central tube,” a cylindrical basal body precursor that lacks microtubules (22, 23). SAS-6 is also required for the formation of the flat ring or cartwheel with nine radiating spokes, which is the first structure to be formed in the Chlamydomonas basal body (21).To determine if Naegleria is likely to have typical basal body components, we identified conserved basal body genes in the Naegleria genome. We also made antibodies to and localized Naegleria''s homologs of SAS-6 and γ-tubulin. Finally, we have determined the order of expression and incorporation of these proteins, as well as centrin, during Naegleria de novo basal body assembly.     

The nuclei of <Emphasis Type="Italic">Giardia lamblia</Emphasis>—new ultrastructural observations

Giardia lamblia is a parasite possessing a complex cytoskeleton and an unusual morphology of bearing two nuclei. Here, the interphasic nuclei of trophozoites, using field emission scanning electron microscopy, routine scanning and transmission electron microscopy, immunocytochemistry, and 3D reconstruction, are presented. An approach using plasma-membrane extraction allowed the observation of the two nuclei still attached in their original positions. The observations are as follows: (1) Giardia nuclei and cytoskeleton were studied in demembranated cells by routine scanning electron microscopy and field emission; (2) both nuclei are anchored to basal bodies of the anterior flagella and to the descending posterior-lateral and ventral flagella, at the right and left nuclei, respectively, in cells attached by its ventral disc; (3) this attachment occurs by proteinaceous links, which were labeled by anti-actin and anti-centrin but not by anti-dynein or anti-tubulin antibodies; (4) fibrilar connections between the nuclei and the disc were also observed; and (5) nuclei exhibited a pendular movement when living cells were treated with cytochalasin, although the nuclei were still connected by their anterior region. Our analysis indicated that the nuclei have a defined position, and fibrils perform an anchoring system. This raises the possibility of a mechanism for nuclei-fidelity migration during mitosis.     

The cell biology of peritrichous flagella in Bacillus subtilis

Bacterial flagella are highly conserved molecular machines that have been extensively studied for assembly, function and gene regulation. Less studied is how and why bacteria differ based on the number and arrangement of the flagella they synthesize. Here we explore the cell biology of peritrichous flagella in the model bacterium Bacillus subtilis by fluorescently labelling flagellar basal bodies, hooks and filaments. We find that the average B. subtilis cell assembles approximately 26 flagellar basal bodies and we show that basal body number is controlled by SwrA. Basal bodies are assembled rapidly (< 5 min) but the assembly of flagella capable of supporting motility is rate limited by filament polymerization (> 40 min). We find that basal bodies are not positioned randomly on the cell surface. Rather, basal bodies occupy a grid‐like pattern organized symmetrically around the midcell and that flagella are discouraged at the poles. Basal body position is genetically determined by FlhF and FlhG homologues to control spatial patterning differently from what is seen in bacteria with polar flagella. Finally, spatial control of flagella in B. subtilis seems more relevant to the inheritance of flagella and motility of individual cells than the motile behaviour of populations.     

Identification of Giardia lamblia DHHC Proteins and the Role of Protein S-palmitoylation in the Encystation Process

Protein S-palmitoylation, a hydrophobic post-translational modification, is performed by protein acyltransferases that have a common DHHC Cys-rich domain (DHHC proteins), and provides a regulatory switch for protein membrane association. In this work, we analyzed the presence of DHHC proteins in the protozoa parasite Giardia lamblia and the function of the reversible S-palmitoylation of proteins during parasite differentiation into cyst. Two specific events were observed: encysting cells displayed a larger amount of palmitoylated proteins, and parasites treated with palmitoylation inhibitors produced a reduced number of mature cysts. With bioinformatics tools, we found nine DHHC proteins, potential protein acyltransferases, in the Giardia proteome. These proteins displayed a conserved structure when compared to different organisms and are distributed in different monophyletic clades. Although all Giardia DHHC proteins were found to be present in trophozoites and encysting cells, these proteins showed a different intracellular localization in trophozoites and seemed to be differently involved in the encystation process when they were overexpressed. dhhc transgenic parasites showed a different pattern of cyst wall protein expression and yielded different amounts of mature cysts when they were induced to encyst. Our findings disclosed some important issues regarding the role of DHHC proteins and palmitoylation during Giardia encystation.     

Changes in the N-glycome, glycoproteins with Asn-linked glycans, of Giardia lamblia with differentiation from trophozoites to cysts

Giardia lamblia is present in the intestinal lumen as a binucleate, flagellated trophozoite or a quadranucleate, immotile cyst. Here we used the plant lectin wheat germ agglutinin (WGA), which binds to the disaccharide di-N-acetyl-chitobiose (GlcNAc₂), which is the truncated Asn-linked glycan (N-glycan) of Giardia, to affinity purify the N-glycomes (glycoproteins with N-glycans) of trophozoites and cysts. Fluorescent WGA bound to the perinuclear membranes, peripheral acidified vesicles, and plasma membranes of trophozoites. In contrast, WGA bound strongly to membranes adjacent to the wall of Giardia cysts and less strongly to the endoplasmic reticulum and acidified vesicles. WGA lectin-affinity chromatography dramatically enriched secreted and membrane proteins of Giardia, including proteases and acid phosphatases that retain their activities. With mass spectroscopy, we identified 91 glycopeptides with N-glycans and 194 trophozoite-secreted and membrane proteins, including 42 unique proteins. The Giardia oligosaccharyltransferase, which contains a single catalytic subunit, preferred N glycosylation sites with Thr to those with Ser in vivo but had no preference for flanking amino acids. The most-abundant glycoproteins in the N-glycome of trophozoites were lysosomal enzymes, folding-associated proteins, and unique transmembrane proteins with Cys-, Leu-, or Gly-rich repeats. We identified 157 secreted and membrane proteins in the Giardia cysts, including 20 unique proteins. Compared to trophozoites, cysts were enriched in Gly-rich repeat transmembrane proteins, cyst wall proteins, and unique membrane proteins but had relatively fewer Leu-rich repeat proteins, folding-associated proteins, and unique secreted proteins. In summary, there are major changes in the Giardia N-glycome with the differentiation from trophozoites to cysts.     

Giardia intestinalis: Aphidicolin influence on the trophozoite cell cycle

This study is a thorough examination of the effects of the DNA polymerase inhibitor aphidicolin on the nuclear cycle and cell cycle progression characteristics, as well as their reversibility, in Giardia intestinalis. Giardia trophozoites are arrested in the G1/S-junction after aphidicolin treatment according to their DNA content. However, cell growth continues and trophozoites arrested with aphidicolin resemble cells in the G2 phase and trophozoites in ageing cultures. Extensive treatment with aphidicolin causes side effects and we detected positive signals for phosphorylated histone H2A, which, in mammalian cells, is involved in a signalling pathway triggered as a reaction to double stranded DNA breaks. These results suggest that aphidicolin causes dissociation of the nuclear and cytoplasmic cycles, a phenomenon that has also been described for other inhibitors in mammalian cell lines. Thus, if aphidicolin is used for synchronization of Giardia trophozoites, this fact must be accounted for, and treatment with aphidicolin must be minimal.     

Peroxiredoxin 1 is involved in disassembly of flagella and cilia

Cilia/flagella are evolutionarily conserved cellular organelles. In this study, we demonstrated that Dunaliella salina Peroxiredoxin 1 (DsPrdx1) localized to the flagella and basal bodies, and was involved in flagellar disassembly. The link between DsPrdx1 and flagella of Dunaliella salina (D. salina) encouraged us to explore the function of its human homologue, Homo sapiens Peroxiredoxin 1 (HsPrdx1) in development and physiology. Our results showed that HsPrdx1 was overexpressed, and cilia were lost in esophageal squamous cell carcinoma (ESCC) cells compared with the non-cancerous esophageal epithelial cells Het-1A. Furthermore, when HsPrdx1 was knocked down by short hairpin RNA (shRNA) lentivirus in ESCC cells, the phenotype of cilia lost can be reversed, and the expression levels of tumor suppressor genes LKB1 and p-AMPK were increased, and the activity of the oncogene Aurora A was inhibited compared with those in cells transfected with scrambe-shRNA lentivirus. These findings firstly showed that Prdx1 is involved in disassembly of flagella and cilia, and suggested that the abnormal expression of the cilia-related gene including Prdx1 may affect both ciliogenesis and cancernogenesis.     

The Giardia ventrolateral flange is a lamellar membrane protrusion that supports attachment

Attachment to the intestinal epithelium is critical to the lifestyle of the ubiquitous parasite Giardia lamblia. The ventrolateral flange is a sheet-like membrane protrusion at the interface between parasites and attached surfaces. This structure has been implicated in attachment, but its role has been poorly defined. Here, we identified a novel actin associated protein with putative WH2-like actin binding domains we named Flangin. Flangin complexes with Giardia actin (GlActin) and is enriched in the ventrolateral flange making it a valuable marker for studying the flanges’ role in Giardia biology. Live imaging revealed that the flange grows to around 1 μm in width after cytokinesis, then remains uniform in size during interphase, grows in mitosis, and is resorbed during cytokinesis. A flangin truncation mutant stabilizes the flange and blocks cytokinesis, indicating that flange disassembly is necessary for rapid myosin-independent cytokinesis in Giardia. Rho family GTPases are important regulators of membrane protrusions and GlRac, the sole Rho family GTPase in Giardia, was localized to the flange. Knockdown of Flangin, GlActin, and GlRac result in flange formation defects. This indicates a conserved role for GlRac and GlActin in forming membrane protrusions, despite the absence of canonical actin binding proteins that link Rho GTPase signaling to lamellipodia formation. Flangin-depleted parasites had reduced surface contact and when challenged with fluid shear force in flow chambers they had a reduced ability to remain attached, confirming a role for the flange in attachment. This secondary attachment mechanism complements the microtubule based adhesive ventral disc, a feature that may be particularly important during mitosis when the parental ventral disc disassembles in preparation for cytokinesis. This work supports the emerging view that Giardia’s unconventional actin cytoskeleton has an important role in supporting parasite attachment.     

Drosophila Bld10 Is a Centriolar Protein That Regulates Centriole, Basal Body, and Motile Cilium Assembly

Cilia and flagella play multiple essential roles in animal development and cell physiology. Defective cilium assembly or motility represents the etiological basis for a growing number of human diseases. Therefore, how cilia and flagella assemble and the processes that drive motility are essential for understanding these diseases. Here we show that Drosophila Bld10, the ortholog of Chlamydomonas reinhardtii Bld10p and human Cep135, is a ubiquitous centriolar protein that also localizes to the spermatid basal body. Mutants that lack Bld10 assemble centrioles and form functional centrosomes, but centrioles and spermatid basal bodies are short in length. bld10 mutant flies are viable but male sterile, producing immotile sperm whose axonemes are deficient in the central pair of microtubules. These results show that Drosophila Bld10 is required for centriole and axoneme assembly to confer cilium motility.     

Dealing with several flagella in the same cell

Flagella are sophisticated organelles found in many eukaryotic microbes where they perform functions related to motility, signal detection, or cell morphogenesis. In many cases, several flagella are present per cell, and these can have a different composition, length, age, or function, raising the question of how this is managed. When the flagella are equivalent and constructed simultaneously such as in Chlamydomonas or Naegleria, we propose an equal access model where molecular components have free access to each organelle. By contrast, Trypanosoma and Leishmania contain temporally distinct organelles and elongate a new flagellum whilst maintaining the existing one. The equal access model could function providing that the mature flagellum is “locked” so that it can no longer be elongated or shortened. Alternatively, access of flagellar components could be restricted at the level of the basal body, the transition zone, or the loading on intraflagellar transport trains. In organisms that contains flagella of different age and composition such as Giardia, a temporal dimension is necessary, with the production of protein components of flagella spreading over one or more cell cycles. In the future, deciphering the molecular mechanisms involved in these processes should reveal new insights in flagellum assembly and function.     

A Detailed, Hierarchical Study of Giardia lamblia's Ventral Disc Reveals Novel Microtubule-Associated Protein Complexes

Giardia lamblia is a flagellated, unicellular parasite of mammals infecting over one billion people worldwide. Giardia''s two-stage life cycle includes a motile trophozoite stage that colonizes the host small intestine and an infectious cyst form that can persist in the environment. Similar to many eukaryotic cells, Giardia contains several complex microtubule arrays that are involved in motility, chromosome segregation, organelle transport, maintenance of cell shape and transformation between the two life cycle stages. Giardia trophozoites also possess a unique spiral microtubule array, the ventral disc, made of approximately 50 parallel microtubules and associated microribbons, as well as a variety of associated proteins. The ventral disc maintains trophozoite attachment to the host intestinal epithelium. With the help of a combined SEM/microtome based slice and view method called 3View® (Gatan Inc., Pleasanton, CA), we present an entire trophozoite cell reconstruction and describe the arrangement of the major cytoskeletal elements. To aid in future analyses of disc-mediated attachment, we used electron-tomography of freeze-substituted, plastic-embedded trophozoites to explore the detailed architecture of ventral disc microtubules and their associated components. Lastly, we examined the disc microtubule array in three dimensions in unprecedented detail using cryo-electron tomography combined with internal sub-tomogram volume averaging of repetitive domains. We discovered details of protein complexes stabilizing microtubules by attachment to their inner and outer wall. A unique tri-laminar microribbon structure is attached vertically to the disc microtubules and is connected to neighboring microribbons via crossbridges. This work provides novel insight into the structure of the ventral disc microtubules, microribbons and associated proteins. Knowledge of the components comprising these structures and their three-dimensional organization is crucial toward understanding how attachment via the ventral disc occurs in vivo.     

Mechanosignaling between central apparatus and radial spokes controls axonemal dynein activity

Cilia/flagella are conserved organelles that generate fluid flow in eukaryotes. The bending motion of flagella requires concerted activity of dynein motors. Although it has been reported that the central pair apparatus (CP) and radial spokes (RSs) are important for flagellar motility, the molecular mechanism underlying CP- and RS-mediated dynein regulation has not been identified. In this paper, we identified nonspecific intermolecular collision between CP and RS as one of the regulatory mechanisms for flagellar motility. By combining cryoelectron tomography and motility analyses of Chlamydomonas reinhardtii flagella, we show that binding of streptavidin to RS heads paralyzed flagella. Moreover, the motility defect in a CP projection mutant could be rescued by the addition of exogenous protein tags on RS heads. Genetic experiments demonstrated that outer dynein arms are the major downstream effectors of CP- and RS-mediated regulation of flagellar motility. These results suggest that mechanosignaling between CP and RS regulates dynein activity in eukaryotic flagella.     

De novo formation of basal bodies during cellular differentiation of Naegleria gruberi: Progress and hypotheses

Under defined laboratory conditions, Naegleria gruberi undergo an amoeba-to-flagellate differentiation. During this differentiation, N. gruberi changes its shape from an amorphous amoeba to a regular shaped flagellate and forms de novo a flagellar apparatus, which is composed of two basal bodies, two flagella, a flagellar rootlet, and cytoplasmic microtubules. The entire process is accomplished within 2 h after initiation of differentiation and more than 95% of cells in the population undergo this differentiation. This rapid and synchronous differentiation of N. gruberi provides us with a unique system in which we can study the process of de novo basal body assembly. In this review, I summarize recent findings associated with de novo basal body assembly and propose a hypothesis to explain how N. gruberi assemble two basal bodies per cell, which is what happens in the majority of cells.     

Giardia lamblia aurora kinase: a regulator of mitosis in a binucleate parasite

Giardia lamblia is a major cause of diarrhoeal disease worldwide. Since it has no known toxin, the ability of trophozoites to colonise the human small intestine is required for its pathogenesis. Mitosis in this protozoan parasite is a unique challenge because its two equivalent nuclei and complex cytoskeleton must be duplicated and segregated accurately. Giardial mitosis is a complex and rapid event that is poorly understood at the cellular and molecular levels. Higher eukaryotes have one to three members of the highly conserved Ser/Thr aurora kinase (AK) family that regulate key aspects of mitosis and cytokinesis. Giardia has a single AK orthologue (gAK) with 61% similarity to human AK A. In addition to the conserved active site residues, activation loop and destruction-box motifs characteristic of AKs, gAK contains a unique insert near the active site region. We epitope-tagged gAK at its C-terminus and expressed it under its own promoter. During interphase, gAK localises exclusively to the nuclei, but is not phosphorylated as shown by lack of staining with an antibody specific to phosphorylated AK A (pAK). In contrast, during mitosis pAK localises to the basal bodies/centrosomes and co-localises with tubulin to the spindle. During specific stages of mitosis, giardial pAK also localised dynamically to cytoskeletal structures unique to Giardia: the paraflagellar dense rods of the anterior flagella and the median body, whose functions are unknown, as well as to the parent attachment disc. Two AK inhibitors significantly decreased giardial growth and increased the numbers of cells arrested in cytokinesis. These inhibitors appeared to increase microtubule nucleation and cell-ploidy. Our data show that gAK is phosphorylated in mitosis and suggest that it plays an important role in the Giardia cell cycle. The pleiotropic localisation of AK suggests that it may co-ordinate the reorganisation and segregation of tubulin-containing structures in mitosis. We believe this is the first report of a signalling protein regulating cell division in Giardia.     

Export of cyst wall material and Golgi organelle neogenesis in Giardia lamblia depend on endoplasmic reticulum exit sites

Giardia lamblia parasitism accounts for the majority of cases of parasitic diarrheal disease, making this flagellated eukaryote the most successful intestinal parasite worldwide. This organism has undergone secondary reduction/elimination of entire organelle systems such as mitochondria and Golgi. However, trophozoite to cyst differentiation (encystation) requires neogenesis of Golgi‐like secretory organelles named encystation‐specific vesicles (ESVs), which traffic, modify and partition cyst wall proteins produced exclusively during encystation. In this work we ask whether neogenesis of Golgi‐related ESVs during G. lamblia differentiation, similarly to Golgi biogenesis in more complex eukaryotes, requires the maintenance of distinct COPII‐associated endoplasmic reticulum (ER) subdomains in the form of ER exit sites (ERES) and whether ERES are also present in non‐differentiating trophozoites. To address this question, we identified conserved COPII components in G. lamblia cells and determined their localization, quantity and dynamics at distinct ERES domains in vegetative and differentiating trophozoites. Analogous to ERES and Golgi biogenesis, these domains were closely associated to early stages ofnewly generated ESV. Ectopic expression of non‐functional Sar1 GTPase variants caused ERES collapse and, consequently, ESV ablation, leading to impaired parasite differentiation. Thus, our data show how ERES domains remain conserved in G. lamblia despite elimination of steady‐state Golgi. Furthermore, the fundamental eukaryotic principle of ERES to Golgi/Golgi‐like compartment correspondence holds true in differentiating Giardia presenting streamlined machinery for secretory organelle biogenesis and protein trafficking. However, in the Golgi‐less trophozoites ERES exist as stable ER subdomains, likely as the sole sorting centres for secretory traffic.     

THE CYTOSKELETON OF THE NAKED GREEN FLAGELLATE SPERMATOZOPSIS SIMILIS (CHLOROPHYTA):FLAGELLAR AND BASAL BODY DEVELOPMENTAL CYCLE1

Flagellar and basal body development during cell division was studied in the biflagellate green alga Spermatozopsis similis Preisig et Melkonian by light microscopy of immobilized living cells, statistical analysis of flagellar lengths during the cell cycle, and electron microscopy of cells and isolated cytoskeletons. Interphase cells display two flagella of unequal/subequal length. An eyespot located in an anterior lobe of the chloroplast is connected to the basal body bearing the shorter flagellum by means of a five-stranded microtubular root. Until cell division, the two parental flagella attain the same length. During cell division, each cell forms two new flagella that grow to a length of 1.5 μm before they are distributed in a semiconservative fashion together with the parental flagella to the two progeny cells at cytokinesis. During the following interphase, the flagella newly formed during the preceding cell division grow to attain the same length as the parental flagella until the subsequent cell division. The shorter of the two flagella of a cell thus represents the developmentally younger flagellum, which transforms to the mature state during two consecutive cell cycles. Interphase cells display only two flagella-bearing basal bodies; two nascent basal bodies are formed during cell division and are connected to the microtubular d-roots of respective parental basal bodies with which the newly formed basal bodies are later distributed to the progeny cells. During segregation, basal body pairs shaft into the 11/5 o'clock direction, thus conserving the 1/7 o'clock configuration of basal body pairs of interphase cells. Prior to chloroplast and cell division, an eyespot is newly formed near the cell posterior in close association with a 1s microtubular root, while the parental eyespot is retained. During basal body segregation, eyespot-root connections for both the old and newly formed eyespots are presumably lost, and new associations of the eyespots with the 2s roots of the newly formed basal bodies are established during cytokinesis. The significance of this “eyespot-flagellar root developmental cycle” for the absolute orientation of the progeny cells is discussed.     

THE FINE STRUCTURE OF GIARDIA MURIS

Giardia is a noninvasive intestinal zooflagellate. This electron microscope study demonstrates the fine structure of the trophozoite of Giardia muris in the lumen of the duodenum of the mouse as it appears after combined glutaraldehyde and acrolein fixation and osmium tetroxide postfixation. Giardia muris is of teardrop shape, rounded anteriorly, with a convex dorsal surface and a concave ventral one. The anterior two-thirds of the ventral surface is modified to form an adhesive disc. The adhesive disc is divided into 2 lobes whose medial surfaces form the median groove. The marginal grooves are the spaces between the lateral crests of the adhesive disc and a protruding portion of the peripheral cytoplasm. The organism has 2 nuclei, 1 dorsal to each lobe of the adhesive disc. Between the anterior poles of the nuclei, basal bodies give rise to 8 paired flagella. The median body, unique to Giardia, is situated between the posterior poles of the nuclei. The cytoplasm contains 300-A granules that resemble particulate glycogen, 150- to 200-A granules that resemble ribosomes, and fusiform clefts. The dorsal portion of the cell periphery is occupied by a linear array of flattened vacuoles, some of which contain clusters of dense particles. The ventrolateral cytoplasm is composed of regularly packed coarse and fine filaments which extend as a striated flange around the adhesive disc. The adhesive disc is composed of a layer of microtubules which are joined to the cytoplasm by regularly spaced fibrous ribbons. The plasma membrane covers the ventral and lateral surfaces of the disc. The median body consists of an oval aggregate of curved microtubules. Microtubules extend ventrally from the median body to lie alongside the caudal flagella. The intracytoplasmic portions of the caudal, lateral, and anterior flagella course considerable distances, accompanied by hollow filaments adjacent to their outer doublets. The intracytoplasmic portions of the anterior flagella are accompanied also by finely granular rodlike bodies. No structures identifiable as mitochondria, smooth endoplasmic reticulum, the Golgi complex, lysosomes, or axostyles are recognized.