Directed movements of ciliary and flagellar membrane components: a review.

The ability to rapidly translocate polystyrene microspheres attached to the surface of a plasma membrane domain reflects a unique form of cellular force transduction occurring in association with the plasma membrane of microtubule based cell extensions. This unusual form of cell motility can be utilized by protistan organisms for whole cell locomotion, the early events in mating, and transport of food organisms along the cell surface, and possibly intracellular transport of certain organelles. Since surface motility is observed in association with cilia and flagella of algae, sea urchin embryos and cultured mammalian cells, it is likely that it serves an additional role beyond those already cited; this is likely to be the transport of precursors for the assembly and turnover of ciliary and flagellar membranes and axonemes. In the case of the Chlamydomonas flagellum, where surface motility has been most extensively studied, it appears that cross-linking of flagellar surface exposed proteins induces a transmembrane signaling pathway that activates machinery for moving flagellar membrane proteins in the plane of the flagellar membrane. This signaling pathway in vegetative Chlamydomonas reinhardtii appears to involve an influx of calcium, a rise in intraflagellar free calcium concentration and a change in the level of phosphorylation of specific membrane-matrix proteins. It is hypothesized that flagellar surface contact with a solid substrate (during gliding), a polystyrene microsphere or another flagellum (during mating) will all activate a signaling pathway similar to the one artificially activated by the use of monoclonal antibodies to flagellar membrane glycoproteins. A somewhat different signaling pathway, involving a transient rise in intracellular cAMP level, may be associated with the mating of Chlamydomonas gametes, which is initiated by flagellum-flagellum contact. The hypothesis that the widespread observation of microsphere movements on various ciliary and flagellar surfaces may reflect a mechanism normally utilized to transport axonemal and membrane subunits along the internal surface of the organelle membrane presents a paradox in that one would expect this to be a constitutive mechanism, not one necessarily activated by a signaling pathway.     

Molecular determinants of ciliary membrane localization of Trypanosoma cruzi flagellar calcium-binding protein

The flagellar calcium-binding protein (FCaBP) of Trypanosoma cruzi is localized to the flagellar membrane in all life cycle stages of the parasite. Myristoylation and palmitoylation of the N terminus of FCaBP are necessary for flagellar membrane targeting. Not all dually acylated proteins in T. cruzi are flagellar, however. Other determinants of FCaBP therefore likely contribute to flagellar specificity. We generated T. cruzi transfectants expressing the N-terminal 24 or 12 amino acids of FCaBP fused to GFP. Analysis of these mutants revealed that although amino acids 1-12 are sufficient for dual acylation and membrane binding, amino acids 13-24 are required for flagellar specificity and lipid raft association. Mutagenesis of several conserved lysine residues in the latter peptide demonstrated that these residues are essential for flagellar targeting and lipid raft association. Finally, FCaBP was expressed in the protozoan Leishmania amazonensis, which lacks FCaBP. The flagellar localization and membrane association of FCaBP in L. amazonensis suggest that the mechanisms for flagellar targeting, including a specific palmitoyl acyltransferase, are conserved in this organism.     

On self synchronization--aspects of flagellar and ciliary beating

Mathematical models involving hydrodynamically coupled oscillators are proposed to explain some self-synchronization phenomena observed in flagella and cilia. It is shown using elementary mathematical analysis and computer simulations that the behaviors of the models proposed are similar to those observed in experiment.     

Ultrastructures and evolutionary modalities of flagellar and ciliary systems in protists

Summary— As both the ultrastructure and function of flagella and cilia have been for the main part remarkably conserved during Eukaryote, evolution, the question arises as to whether the variations observed at the organite ultrastural level, or at the level of the development of a flagellar or ciliary cellular system could be considered as systematic or phylogenetic criteria. With regard to the fundamental structure, the known variations concern: 1) the kinetosome (length, position and number of cartwheels, number of triplets, and respective lengths of the microtubules); 2) the transition zone (various structural types); 3) the axoneme (number of doublets, central tubules, arms); 4) the paraxonemal formations (presence, position, structure); 5) the membrane (intramembranous particles; intramembranous particles; addition of components, mastigonemes, scales); 6) the fibres associated with the kinetosomes. Some of these variations are characteristic of taxa, and are considered as phylogenetic markers. Regarding the variations in the number of ciliary or flagellar units per cell, the following can be distinguished: cells with only one kinetosome (Ks), carrying one flagellum; cells with 2 neighbouring Ks (primary Ks = 2A), only one Ks, or the 2 Ks, bearing a flagellum; cells with numerous flagella or cilia. We consider that this configuration can result from: a) addition of new Ks around the primary Ks, forming a primary group (PG): either by replication of the primary couple (2 × 2A in Karotomorpha; 2 × 2A in Phaeopolykrikos; 4(2 × 2A) in Polkrikos), or by addition of new Ks (N) without copy or replication of the primary Ks (2A + 2N in Polytomella; 2A +3N in Tetratrichomonas; 2A + 3N in Tetratrichomonas; 2A + 4N in Hexamastix; 2A + 6N in Pyramimonas octopus). b) amplification of each constituent of the primordial couple in opalinids. c) amplification of the primary group [yx(AA + 2N) with y = 2 in diplomonadida and y > 2 in calonymphidae. d) appearance in morphogenetic fields situated outside the primary group (but possibly related to it) of new Ks which then form secondary groups (SG) where they are arranged in polarized rows composed of juxtaposed cortical units (monokinetids). In this case, PG can be of type 2A+2N (1PG+1SG in lophomonadida; 1PG+2SG in some trichonymphina), or can lose some Ks, resulting in reduced PG (4 reduced PG+4GS in Staurojoenina, n reduced PG + nGS in spirotrichonymphina). The PG may even disappear; either partially leaving remnants in the form of MTOCs in Stephanopogon, or completely, as in cilliates where the general ciliature would represent on the SG. From the above, it is seen that: a) the pluriflagellar state in protists depends on only a small number of factors; b) the same factors are used in different groups; c) different states have been obtained within different groups. An analysis of the respective evolution of kinetosomes and centrioles is proposed.     

Functional protofilament numbering of ciliary, flagellar, and centriolar microtubules

This article discusses the current state of knowledge about the evolutionarily conserved structure of ciliary, flagellar and centriolar microtubules, and formally proposes a functional numbering convention for their protofilaments.     

In vitro polymerization of flagellar and ciliary outer fiber tubulin into microtubules.

About 10--20% of the total protein in the outer fiber fraction was solubilized by sonication in a solution containing 5 mM MES, 0.5 mM MgSO4, 1.0 mM EGTA, 1.0 mM GTP, and 0 or 50 mM KC1 at pH 6.7. The sonicated extract was shown by analytical centrifugation to consist largely of a 6 S component (tubulin dimer), having a molecular weight of 103,000, as determined by gel filtration, and possessing a colchicine-binding activity of 0.8 mole per tubulin dimer. The tubulin fraction failed to polymerize into microtubules by itself. Addition of a small amount of the ciliary outer fiber fragments or reconstituted short brain microtubules, however, induced polymerization, as demonstrated by viscosity of flow birefringence changes as well as light or electron microscopic observations. The growth of heterogeneous microtubules upon mixing outer fiber tubulin with DEAE-dextran-decorated brain microtubules was observed by electron microscopy. Microtubules were reconstituted from outer fiber tubulin without addition of any nuclei fraction when a concentrated tubulin fraction was warmed at 35degree. A few doublet-like microtubules or pairs of parallel singlet microtubules that were closely aligned longitudinally could be observed among many singlet microtubules. Unlike other fiber microtubules, the reconstituted polymers were depolymerized by exposure to Ca2+ ions, high or low ionic strength, colchicine, low temperature or SH reagents. No microtubules were assembled under these conditions.     

Phosphorothioate analogs of ATP as the substrates of dynein and ciliary or flagellar movement

The phosphorothioate analog of ATP has a sulfur atom replacing a non-bridging oxygen atom of the triphosphate moiety of ATP. Due to the tetrahedral nature of the phosphorus atom, stereoisomers are known to exist, designated as the Sp and Rp isomers. We have reported [Shimizu & Furusawa (1986) Biochemistry 25, 5787] on the hydrolytic activity of the 22S dynein from Tetrahymena cilia towards the phosphorothioate analogs of ATP. In this paper, we extend our study and report on the microtubule-dynein dissociation by these analogs and on their ability to support sea urchin flagellar dynein enzymatic activity as well as ciliary or flagellar motility. It has been shown that the microtubule--22S-dynein complex is dissociated by the binding of ATP to the dynein enzymatic sites [Porter & Johnson (1983) J. Biol. Chem. 258, 6575]. We studied the dissociation by adenosine 5'-[alpha-thio]triphosphate (ATP[alpha S]), Sp or Rp, by light-scattering stopped-flow methods. The dissociation by (Sp)ATP[alpha S] was rapid and the rate of the light-scattering change by (Sp)ATP[alpha S] was a hyperbolic function of the nucleotide concentration, indicating that dissociation was a two-step process. On the other hand, (Rp)ATP[alpha S] up to 2 mM induced only slow and partial dissociation of the complex, while, in the presence of vanadate, it induced complete dissociation with a slightly higher rate (0.5 s-1). The adenosine 5'-[beta-thio]triphosphate (ATP[beta S]) isomers did not induce dissociation. The hydrolytic activity of the outer arm dynein from sea urchin sperm flagella towards these analogs was similar to that of 22S dynein. The ratios of Vmax (nmol.mg protein-1.min-1)/apparent Km (microM) of this dynein were 400-720, 53, 9.7, 0.62 and 0.028 for ATP, ATP[alpha S] (Sp or Rp), ATP[beta S] (Sp or Rp), respectively, in the presence of Mg2+ as the supporting cation. This dynein exhibited the same stereospecificity at beta phosphate as the 22S dynein or myosin. The detergent models of Tetrahymena or sea urchin spermatozoa were reactivated only by ATP or (Sp)ATP[alpha S] while other analogs were ineffective. The maximal beat frequency of the cilia or flagella reactivated by (Sp)ATP[alpha S] was one-quarter to one-half of that produced by ATP reactivation.     

A new automated method of image analysis: comparison of ciliary and flagellar beats

A new automated method of image analysis of sperm flagellar (human) and cilia (Dunaliella) bends is developed. This method permits an automatic determination of the line characterizing the flagellum. Two dynamic parameters are measured: the wave propagation velocity and the wave curvature radius. The data reveal similar patterns in the propagation of the principal and reverse waves between flagelated and ciliated cells. Conversely, differences are seen in principal wave curvature due perhaps to the presence of periaxonemal structures in the flagellum, absent in cilium. The identical patterns of reverse wave curvaturei in both systems may be linked to axonemal limitations.     

CEP290 tethers flagellar transition zone microtubules to the membrane and regulates flagellar protein content

Mutations in human CEP290 cause cilia-related disorders that range in severity from isolated blindness to perinatal lethality. Here, we describe a Chlamydomonas reinhardtii mutant in which most of the CEP290 gene is deleted. Immunoelectron microscopy indicated that CEP290 is located in the flagellar transition zone in close association with the prominent microtubule–membrane links there. Ultrastructural analysis revealed defects in these microtubule–membrane connectors, resulting in loss of attachment of the flagellar membrane to the transition zone microtubules. Biochemical analysis of isolated flagella revealed that the mutant flagella have abnormal protein content, including abnormal levels of intraflagellar transport proteins and proteins associated with ciliopathies. Experiments with dikaryons showed that CEP290 at the transition zone is dynamic and undergoes rapid turnover. The results indicate that CEP290 is required to form microtubule–membrane linkers that tether the flagellar membrane to the transition zone microtubules, and is essential for controlling flagellar protein composition.     

The future of Golgi research

This essay looks backward on the past three decades of research toward understanding the mechanism of macromolecular traffic through and within the Golgi apparatus with an eye to the future. I also explain why I feel the Golgi should continue to hold the attention of molecular cell biologists.