CEP164C regulates flagellum length in stable flagella

Cilia and flagella are required for cell motility and sensing the external environment and can vary in both length and stability. Stable flagella maintain their length without shortening and lengthening and are proposed to “lock” at the end of growth, but molecular mechanisms for this lock are unknown. We show that CEP164C contributes to the locking mechanism at the base of the flagellum in Trypanosoma brucei. CEP164C localizes to mature basal bodies of fully assembled old flagella, but not to growing new flagella, and basal bodies only acquire CEP164C in the third cell cycle after initial assembly. Depletion of CEP164C leads to dysregulation of flagellum growth, with continued growth of the old flagellum, consistent with defects in a flagellum locking mechanism. Inhibiting cytokinesis results in CEP164C acquisition on the new flagellum once it reaches the old flagellum length. These results provide the first insight into the molecular mechanisms regulating flagella growth in cells that must maintain existing flagella while growing new flagella.     

Flagellar transformation in the heterokontEpipyxis pulchra (Chrysophyceae): Direct observations using image enhanced light microscopy

Summary Cells ofEpipyxis pulchra possess two heteromorphic flagella that differ markedly in function, particularly during motility and prey capture. Flagellar heterogeneity is achieved during the course of at least three cell cycles. Prior to cell division, cells produce two new long, hairy flagella while the parental long flagellum is transformed into a new short, smooth flagellum. The parental short flagellum remains a short flagellum for this and subsequent cell division cycles. Although flagellar transformation requires only two cell cycles, developmental differences exist between daughter cells and the maturation of a flagellum/basal body requires at least three cycles.     

Development of the flagellar apparatus during the cell cycle in unicellular algae

Summary Recent evidence has shown that algal cells acquire different flagella and a heterogeneous basal apparatus through the prolonged development of these structures over more than one cell cycle. A system for numbering algal flagella and basal bodies, which is based on developmental studies, is discussed along with the various means by which the flagellar/basal body developmental cycle can be determined. We review the information now available on development of the separate components of the flagellar apparatus-this comes particulary from the Chlorophyta and the Chromophyta-and attempt to elucidate any information which may help in phylogenetic comparisons. New data is provided on developmental changes in the cartwheel part of the basal body and basal body-associated connecting fibrils in green algae.Abbreviations Bb basal body - d right (dexter) root - df right fibrils connecting Bb triplets to microtubular and/or fibrous roots - EM electron microscopy - F flagellum - IMF immunofluorescence microscopy - LM light microscopy - NBBC nucleus-basal body connector - s left (sinister) root - sf 3left fibrils connecting Bb triplets to microtubular and/or fibrous roots. See Nomenclature section of Introduction for the numbering of basal bodies and their flagella; the same numbers apply to Bb-associated d and s roots, and df and sf fibrils     

The cytology and ultrastructure of zoospores of Hydrurus foetidus (Chrysophyceae)

The unusual tetrahedral shape of Hydrurus foetidus (Vill.) Trev. zoospores is associated with a complex skeletal system of microtubules extending from a broad flagellar root (up to 19 microtubules) into each of three, pointed anterior processes. The posterior end, also pointed and supported by a separate set of microtubules, contains a single large chloroplast with a prominent posterior furrow containing mitochondrial elements. A large immersed pyrenoid is penetrated by paired thylakoids. There is no eyespot. Numerous large Golgi bodies occur immediately anterior to the nucleus and up to 5–6 contractile vacuoles lie near the cell surface at the anterior end. Two terminally inserted flagella extend from the cell surface, a long one serving for cell locomotion, and the other vestigial with an axonemal pattern of 9+0. The flagellar root system consists of: (1) a thin, striated rhizoplast extending from the basal body of the long flagellum and ramifying over the surface of a conspicuous, anteriorly directed, conical projection of the nucleus; (2) a broad microtubular root which emanates from near the basal body of the long flagellum and appears to function as a MTOC; (3) a compound root, consisting of a striated fiber and two associated microtubules, which runs alongside the basal body of the stubby flagellum before terminating at the cell surface; and (4) a short two-membered microtubular root, also associated with the basal body of the stubby flagellum. Other components of the flagellar apparatus include a large dense body near the proximal end of the basal body of the short flagellum, and a small, dense, core-like structure closely associated with one of its triplet fibers. The flagellar apparatus of H. foetidus is remarkably similar in ultrastructure to that of Chrysonebula holmesii Lund.     

THE FLAGELLAR DEVELOPMENT CYCLE OF THE UNIFLAGELLATE PELAGOMONAS CALCEOLATA (PELAGOPHYCEAE)1

Vegetative cells of Pelagomonas calceolata Andersen & Saunders were confirmed to possess a reduced flagellar apparatus, consisting of a single basal body/flagellum that is not accompanied by either flagellar roots or a barren basal body. Just prior to division, the parental flagellum retracts (or is abscised) as two new basal bodies/flagella arise de novo. During cytokinesis the parental basal body segregates with a new basal body/flagellum, briefly producing a progeny cell typical of other known uniflagellates (i.e. containing a basal body/flagellum and accompanying barren basal body). The parental basal body then disintegrates or "transforms" out of existence, leaving both progeny cells with a single basal body/flagellum (i.e. neither progeny cell possesses any vestige of a mature flagellum/basal body ). Pelagomonas calceolata belongs to a lineage of chromophyte algae characterized by having a reduced flagellar apparatus, but it is the only known species, not only in this lineage but among all eukaryotes, to have undergone the complete elimination of the mature flagellum /basal body .     

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.     

Structural study of the basal bodies of the flagella of Bacillus brevis var. G.-B. P+

The structural organisation of the flagellum basal body was studied in Bacillus brevis var. G.-B. P+ by electron microscopy. It was compared with that of Escherichia coli MS 1350. The basal body of a B. brevis flagellum contains, in addition to two pairs of rings on a rod, another ring-like structure (d = 13.6 nm, h = 4.3 nm) which we referred to as a "collar". The collar makes the basal body of B. brevis different from that of B. subtilis, another Gram-positive bacterium. The collar seems to fasten the flagellum of B. brevis to the cell wall. We have concluded that the basal body can differ not merely among bacterial systematic groups, but also among bacteria belonging to one and the same genus. The role of individual elements in the structure of the basal body of bacterial flagella is discussed.     

Non‐equivalence in old‐ and new‐flagellum daughter cells of a proliferative division in Trypanosoma brucei

Differentiation of Trypanosoma brucei, a flagellated protozoan parasite, between life cycle stages typically occurs through an asymmetric cell division process, producing two morphologically distinct daughter cells. Conversely, proliferative cell divisions produce two daughter cells, which look similar but are not identical. To examine in detail differences between the daughter cells of a proliferative division of procyclic T. brucei we used the recently identified constituents of the flagella connector. These segregate asymmetrically during cytokinesis allowing the new‐flagellum and the old‐flagellum daughters to be distinguished. We discovered that there are distinct morphological differences between the two daughters, with the new‐flagellum daughter in particular re‐modelling rapidly and extensively in early G1. This re‐modelling process involves an increase in cell body, flagellum and flagellum attachment zone length and is accompanied by architectural changes to the anterior cell end. The old‐flagellum daughter undergoes a different G1 re‐modelling, however, despite this there was no difference in G1 duration of their respective cell cycles. This work demonstrates that the two daughters of a proliferative division of T. brucei are non‐equivalent and enables more refined morphological analysis of mutant phenotypes. We suggest all proliferative divisions in T. brucei and related organisms will involve non‐equivalence.     

DEVELOPMENT OF THE FLAGELLAR APPARATUS OF NAEGLERIA

Flagellates of Naegleria gruberi have an interconnected flagellar apparatus consisting of nucleus, rhizoplast and accessory filaments, basal bodies, and flagella. The structures of these components have been found to be similar to those in other flagellates. The development of methods for obtaining the relatively synchronous transformation of populations of Naegleria amebae into flagellates has permitted a study of the development of the flagellar apparatus. No indications of rhizoplast, basal body, or flagellum structures could be detected in amebae. A basal body appears and assumes a position at the cell surface with its filaments perpendicular to the cell membrane. Axoneme filaments extend from the basal body filaments into a progressive evagination of the cell membrane which becomes the flagellum sheath. Continued elongation of the axoneme filaments leads to differentiation of a fully formed flagellum with a typical "9 + 2" organization, within 10 min after the appearance of basal bodies.     

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.     

The flagellar developmental cycle in algae: flagellar transformation inCyanophora paradoxa (Glaucocystophyceae)

Summary Flagellar development during cell division was studied inCyanophora paradoxa using agarose-embedded cells, Nomarski optics and electronic flash photography. The cells bear two heterodynamic and differently oriented (anterior and posterior) flagella. Prior to cell division, cells produce two new anterior flagella while the parental anterior flagellum transforms into a posterior flagellum. The parental posterior flagellum remains a posterior flagellum throughout this and subsequent cell divisions. The development of a single flagellum thus extends through at least two cell cycles and flagellar heterogeneity is achieved by semiconservative distribution of the flagella during cell division. Based on these principles a universal numbering system for basal bodies and flagella of eukaryotic cells is proposed.     

Cryo-electron tomography and 3-D analysis of the intact flagellum in Trypanosoma brucei

Trypanosoma brucei is a uni-cellular protist that causes African sleeping sickness. These parasites have a flagellum that is attached to the cell body and is indispensible for its motility. The flagellum consists of a canonical 9+2 axoneme and a paraflagellar rod (PFR), an intricate tripartite, fibrous structure that is connected to the axoneme. In this paper we describe results from cryo-electron tomography of unperturbed flagella. This method revealed novel structures that are likely involved in attaching the flagellum to the cell. We also show the first cryo-electron tomographic images of a basal body in situ, revealing electron dense structures inside its triplet microtubules. Sub-tomogram averaging of the PFR revealed that its distal region is organized as an orthorhombic crystal.     

Cell division of Giardia intestinalis: flagellar developmental cycle involves transformation and exchange of flagella between mastigonts of a diplomonad cell

Giardia intestinalis is a binucleated diplomonad possessing four pairs of flagella of distinct location and function. Its pathogenic potential depends on the integrity of a complex microtubular cytoskeleton that undergoes a profound but poorly understood reorganization during cell division. We examined the cell division of G. intestinalis with the aid of light and electron microscopy and immunofluorescence methods and present here new observations on the reorganization of the flagellar apparatus in the dividing Giardia. Our results demonstrated the presence of a flagellar maturation process during which the flagella migrate, assume different position, and transform to different flagellar types in progeny until their maturation is completed. For each newly assembled flagellum it takes three cell cycles to become mature. The mature flagellum of Giardia is the caudal one that possesses a privileged basal body at which the microtubules of the adhesive disk nucleate. In contrast to generally accepted assumption that each of the two diplomonad mastigonts develops separately, we found that they are developmentally linked, exchanging their cytoskeletal components at the early phase of mitosis. The presence of the flagellar maturation process in a metamonad protist Giardia suggests that the basal body or centriole maturation is a universal phenomenon that may represent one of the core processes in a eukaryotic cell.     

On Flagellar Structure in Certain Flagellates

This paper describes the structure of the flagella, basal bodies, and some of the associated fibre systems in three genera of complex flagellates, Trichonympha, Pseudotrichonympha, and Holomastigotoides. Three groups of longitudinal fibres occur in a flagellum: two central and nine outer fibres such as have been repeatedly described in other material, and an additional set of nine smaller secondary fibres not previously identified as such. Each central fibre shows a helical substructure; the pair of them are enveloped in a common sheath. Each outer fibre is a doublet with one subfibre bearing projections—called arms—that extend toward the adjacent outer fibre. The basal body is formed by a cylinder of nine triplet outer fibres. Two subfibres of each triplet continue into the flagellum and constitute the doublets. The third subfibre terminates at the transition of basal body to flagellum, possibly giving rise to the nine radial transitional fibres that seem to attach the end of the basal body to the surface of the organism. The central and secondary flagellar fibres are not present in the lumen of the basal body, but other complex structures occur there. The form of these intraluminal structures differs from genus to genus. The flagellar unit is highly asymmetrical. All the flagella examined have possessed the same one of the two possible enantiomorphic forms. At least two systems of fibres are associated with the basal bodies of all three genera.     

Computer simulation of flagellar movement. VI. Simple curvature-controlled models are incompletely specified.

Computer simulation is used to examine a simple flagellar model that will initiate and propagate bending waves in the absence of viscous resistances. The model contains only an elastic bending resistance and an active sliding mechanism that generates reduced active shear moment with increasing sliding velocity. Oscillation results from a distributed control mechanism that reverses the direction of operation of the active sliding mechanism when the curvature reaches critical magnitudes in either direction. Bend propagation by curvature-controlled flagellar models therefore does not require interaction with the viscous resistance of an external fluid. An analytical examination of moment balance during bend propagation by this model yields a solution curve giving values of frequency and wavelength that satisfy the moment balance equation and give uniform bend propagation, suggesting that the model is underdetermined. At 0 viscosity, the boundary condition of 0 shear rate at the basal end of the flagellum during the development of new bends selects the particular solution that is obtained by computer simulations. Therefore, the details of the pattern of bend initiation at the basal end of a flagellum can be of major significance in determining the properties of propagated bending waves in the distal portion of a flagellum. At high values of external viscosity, the model oscillates at frequencies and wavelengths that give approximately integral numbers of waves on the flagellum. These operating points are selected because they facilitate the balance of bending moments at the ends of the model, where the external viscous moment approaches 0. These mode preferences can be overridden by forcing the model to operate at a predetermined frequency. The strong mode preferences shown by curvature-controlled flagellar models, in contrast to the weak or absent mode preferences shown by real flagella, therefore do not demonstrate the inapplicability of the moment-balance approach to real flagella. Instead, they indicate a need to specify additional properties of real flagella that are responsible for selecting particular operating points.     

A nucleus-basal body connector in Chlamydomonas reinhardtii that may function in basal body localization or segregation

We have isolated a nucleus-basal body complex from Chlamydomonas reinhardtii. The complex is strongly immunoreactive to an antibody generated against a major protein constituent of isolated Tetraselmis striata flagellar roots (Salisbury, J. L., A. Baron, B. Surek, and M. Melkonian, J. Cell Biol., 99:962-970). Electrophoretic and immunoelectrophoretic analysis indicates that, like the Tetraselmis protein, the Chlamydomonas antigen consists of two acidic isoforms of approximately 20 kD. Indirect immunofluorescent staining of nucleus- basal body complexes reveals two major fibers in the connector region, one between each basal body and the nucleus. The nucleus is also strongly immunoreactive, with staining radiating around much of the nucleus from a region of greatest concentration at the connector pole. Calcium treatment causes shortening of the connector fibers and also movement of nuclear DNA towards the connector pole. Electron microscopic observation of negatively stained nucleus-basal body complexes reveals a cluster of approximately 6-nm filaments, suspected to represent the connector, between the basal bodies and nuclei. A mutant with a variable number of flagella, vfl-2-220, is defective with respect to the nucleus-basal body association. This observation encourages us to speculate that the nucleus-basal body union is important for accurate basal body localization within the cell and/or for accurate segregation of parental and daughter basal bodies at cell division. A physical association between nuclei and basal bodies or centrioles has been observed in a variety of algal, protozoan, and metazoan cells, although the nature of the association, in terms of both structure and function, has been obscure. We believe it likely that fibrous connectors homologous to those described here for Chlamydomonas are general features of centriole-bearing eucaryotic cells.     

Reconstruction of the flagellar apparatus in Ploeotia costata (Euglenozoa) and its relationship to other euglenoid flagellar apparatuses

The flagellar apparatus of Ploeotia costata Farmer and Triemer was reconstructed using serial sectioning and TEM. The flagellar apparatus is similar to other euglenoids having two flagella arising from basal bodies connected by a striated fiber, and three asymmetrically arranged roots. The flagella emerge subapically from between the two ventral pellicle strips. The dorsal flagellum is 1/2 the body length and actively pulls the cell, while the ventral flagellum is twice the body length and drags along the substrate surface. The ventral and dorsal roots are on the opposite sides of their respective basal bodies, while the intermediate root is associated with the ventral flagellum on the side closest to the dorsal basal body. The dorsal root lines the dorsal side of the reservoir and after giving rise to the dorsal band lines the right side of the reservoir/canal. The ventral and intermediate roots join at the reservoir forming the intermediate-ventral root, which lines the left and ventral sides of the reservoir/canal. There was no evidence of a microtubule-reinforced pocket in P. costata. Comparisons with Ploeotia vilrea, Lentomonas applanatum, and related flagellar apparatuses led to the conclusion that the basic euglenoid flagellar structure is symplesiomorphic but with enough variation to be taxonomically diagnostic.     

New structural features of the flagellar base in Salmonella typhimurium revealed by rapid-freeze electron microscopy.

The structure of the flagellar base in Salmonella typhimurium has been studied by rapid-freeze techniques. Freeze-substituted thin sections and freeze-etched replicas of cell envelope preparations have provided complementary information about the flagellar base. The flagellar base has a bell-shaped extension reaching as far as 50 nm into the bacterial cytoplasm. This structure can be recognized in intact bacteria but was studied in detail in cell envelopes, where some flagella lacking parts of the bell were helpful in understanding its substructure. Structural relationships may be inferred between this cytoplasmic component of the flagellum and the recently described flagellar intramembrane particle rings as well as the structures associated with the basal body in isolated, chemically fixed flagella.