A new fla gene in Salmonella typhimurium—flaR—and its mutant phenotype-superhooks

Summary An electron microscopic examination of eight non-flagellate strains of S. typhimurium revealed that an H1 (amber) mutant produced flagellar hooks and basal structures indistinguishable from those associated with the proximal end of normal flagella. No flagella-related structures were seen in strains with mutations in fla genes A, B, C, D, F, K or M. A mutant of a new fla complementation group, flaR, produced abnormal structures, termed superhooks, which resemble the hooks of normal flagella assembled end-to-end. The mutant locus, flaR, maps between flaM and flaAIII.     

Basal structure and attachment of flagella in cells of Proteus vulgaris

Abram, Dinah (Purdue University, Lafayette, Ind.), Henry Koffler, and A. E. Vatter. Basal structure and attachment of flagella in cells of Proteus vulgaris. J. Bacteriol. 90:1337-1354. 1965.-The attachment of flagella to cells of Proteus vulgaris was studied electron microscopically with negatively stained and shadow-cast preparations of ghosts from standard cultures and from special cultures that produced "long forms." The flagellum, the basal portion of which is hooked, arises within the cell from a nearly spherical structure, 110 to 140 A in diameter. This structure appears to be associated with the cytoplasmic membrane; it may be a part of the membrane or a separate entity that lies just beneath the membrane. Flagella associated with cell walls free from cytoplasmic membrane frequently have larger bodies, 200 to 700 A in diameter, associated with their base. These structures probably consist at least partly of fragments of the cytoplasmic membrane, a portion of which folds around a smaller structure. Flagella in various stages of development were observed in long forms of P. vulgaris cells grown at low temperature. The basal structure of these flagella was similar to that of the long or "mature" flagella. Strands connecting the basal structures were observed in ghosts of long forms; these strands appear to be derived from the cytoplasmic membrane. Flagella were found to be attached to fragments of cell wall and to cytoplasmic membrane in a similar manner as they are attached to ghosts. In isolates of flagella that have been separated from the cells mechanically, the organelles often terminate in hooks which almost always appear naked, but have a different fine structure than the flagellum proper.     

Dissecting the sequential assembly and localization of intraflagellar transport particle complex B in chlamydomonas

Intraflagellar transport (IFT), the key mechanism for ciliogenesis, involves large protein particles moving bi-directionally along the entire ciliary length. IFT particles contain two large protein complexes, A and B, which are constructed with proteins in a core and several peripheral proteins. Prior studies have shown that in Chlamydomonas reinhardtii, IFT46, IFT52, and IFT88 directly interact with each other and are in a subcomplex of the IFT B core. However, ift46, bld1, and ift88 mutants differ in phenotype as ift46 mutants are able to form short flagella, while the other two lack flagella completely. In this study, we investigated the functional differences of these individual IFT proteins contributing to complex B assembly, stability, and basal body localization. We found that complex B is completely disrupted in bld1 mutant, indicating an essential role of IFT52 for complex B core assembly. Ift46 mutant cells are capable of assembling a relatively intact complex B, but such complex is highly unstable and prone to degradation. In contrast, in ift88 mutant cells the complex B core still assembles and remains stable, but the peripheral proteins no longer attach to the B core. Moreover, in ift88 mutant cells, while complex A and the anterograde IFT motor FLA10 are localized normally to the transition fibers, complex B proteins instead are accumulated at the proximal ends of the basal bodies. In addition, in bld2 mutant, the IFT complex B proteins still localize to the proximal ends of defective centrioles which completely lack transition fibers. Taken together, these results revealed a step-wise assembly process for complex B, and showed that the complex first localizes to the proximal end of the centrioles and then translocates onto the transition fibers via an IFT88-dependent mechanism.     

BASAL BODIES OF BACTERIAL FLAGELLA IN PROTEUS MIRABILIS : II. Electron Microscopy of Negatively Stained Material

This paper investigates further the question of whether the flagella of Proteus mirabilis emerge from basal bodies. The bacteria were grown to the stage of swarmer differentiation, treated lightly with penicillin, and then shocked osmotically. As a result of this treatment, much of the cytoplasmic content and also part of the plasma membrane were removed from the cells. When such fragmented organisms were stained negatively with potassium phosphotungstate, the flagella were found to be anchored—often by means of a hook—in rounded structures approximately 50 mµ wide, thus confirming Part I of our study. In these rounded structures a more brilliant dot was occasionally observed, which we interpret as being part of the basal granule. A prerequisite for the demonstration of the basal granules within the cells was, however, the removal of both the cytoplasm and the plasma membrane from their vicinity. In some experiments, the chondrioids were "stained" positively by the incorporation into them of the reduced product of potassium tellurite. The chondrioids were here observed to be more or less circular areas from which rodlike structures extended. The chondrioids adhered so firmly to the plasma membrane that they were carried away with it during its displacement by osmotic shocking, while the basal bodies were left behind. This observation disproves our previous suggestion that the flagella might terminate in the chondrioids. The basal bodies often occur in pairs, which suggest that they could be self-reproducing particles.     

Synthesis and assembly of the cytoskeleton of Naegleria gruberi flagellates

When Naegleria gruberi flagellates were extracted with nonionic detergent and stained by the indirect immunofluorescence method with AA-4.3 (a monoclonal antibody against Naegleria beta-tubulin), flagella and a network of cytoskeletal microtubules (CSMT) were seen. When Naegleria amebae were examined in the same way, no cytoplasmic tubulin-containing structures were seen. Formation of the flagellate cytoskeleton was followed during the differentiation of amebae into flagellates by staining cells with AA-4.3. The first tubulin containing structures were a few cytoplasmic microtubules that formed at the time amebae rounded up into spherical cells. The formation of these microtubules was followed by the appearance of basal bodies and flagella and then by the formation of the CSMT. The CSMT formed before the cells assumed the flagellate shape. In flagellate shaped cells the CSMT radiate from the base of the flagella and follow a curving path the full length of the cell. Protein synthetic requirements for the formation of CSMT were examined by transferring cells to cycloheximide at various times after initiation. One-half the population completed the protein synthesis essential for formation of CSMT 61 min after initiation of the differentiation. This is 10 min after the time when protein synthesis for formation of flagella is completed and 10-15 min before the time when the protein synthesis necessary for formation of the flagellate shape is completed.     

Three-dimensional organization of basal bodies from wild-type and delta-tubulin deletion strains of Chlamydomonas reinhardtii

Improved methods of specimen preparation and dual-axis electron tomography have been used to study the structure and organization of basal bodies in the unicellular alga Chlamydomonas reinhardtii. Novel structures have been found in both wild type and strains with mutations that affect specific tubulin isoforms. Previous studies have shown that strains lacking delta-tubulin fail to assemble the C-tubule of the basal body. Tomographic reconstructions of basal bodies from the delta-tubulin deletion mutant uni3-1 have confirmed that basal bodies contain mostly doublet microtubules. Our methods now show that the stellate fibers, which are present only in the transition zone of wild-type cells, repeat within the core of uni3-1 basal bodies. The distal striated fiber is incomplete in this mutant, rootlet microtubules can be misplaced, and multiflagellate cells have been observed. A suppressor of uni3-1, designated tua2-6, contains a mutation in alpha-tubulin. tua2-6; uni3-1 cells build both flagella, yet they retain defects in basal body structure and in rootlet microtubule positioning. These data suggest that the presence of specific tubulin isoforms in Chlamydomonas directly affects the assembly and function of both basal bodies and basal body-associated structures.     

Isolation of flagella from the archaebacterium Methanococcus voltae by phase separation with Triton X-114.

The flagella of Methanococcus voltae were isolated by using three procedures. Initially, cells were sheared to release the filaments, which were purified by differential centrifugation and banding in KBr gradients. Flagella were also prepared by solubilization of cells with 1% (vol/vol) Triton X-100 and purified as described above. Both of these techniques resulted in variable recovery and poor yield of flagellar filaments. Purification of intact flagella (filament, hook, and basal body) was achieved by using phase transition separation with Triton X-114. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified flagella revealed two major proteins, with molecular weights of 33,000 and 31,000. This result indicates the likely presence of two flagellins. The filament had a diameter of 13 nm. The basal structure consisted of a small knob, while a slight thickening of the filament immediately adjacent to this area was the only evidence of a hook region. Flagella from three other Methanococcus species were isolated by this technique and found to have the same ultrastructure as flagella from M. voltae. Isolation of flagella from three eubacteria and another methanogen (Methanospirillum hungatei [M. hungatii]) by the phase separation technique indicated that the detergent treatment did not affect the structure of basal bodies. Intact ring structures and well-differentiated hook regions were apparent in each of these flagellar preparations.     

Effect of Macromolecular Synthesis on the Coordinate Morphogenesis of Polar Surface Structures in Caulobacter crescentus

The Caulobacter polar surface structures (flagella, pili, and the deoxyribonucleic acid phage phiCbK receptors), which are expressed at proximal sites of swarmer cells in a coordinate manner (Shapiro, Annu. Rev. Microbiol., 30:377-407, 1976) could be blocked by a single mutation. The mutant C. crescentus CB13 ple-801 did not form these surface structures when grown at 35 degrees C. Upon shift down to 25 degrees C, the mutant cells initiated the formation of the surface structures. When mitomycin C was added to the mutant culture upon shift down from 35 to 25 degrees C, phiCbK receptor formation was inhibited to a minimal level. Rifampin and chloramphenicol completely inhibited phiCbK receptor formation when added to the mutant culture upon shift down. Deoxyribonucleic acid as well as ribonucleic acid and protein synthesis seem to be required for the formation of phiCbK receptors. Penicillin V also inhibited phiCbK receptor formation, indicating the involvement of cell wall synthesis. When the mutant CB13 ple-801 cells were shifted down briefly from 35 to 25 degrees C and then shifted up to 35 degrees C, flagella and phiCbK receptors were formed even at 35 degrees C to different extents depending on how long the cells were incubated at 25 degrees C. This formation of the surface structures at 35 degrees C was inhibited by rifampin. From these results, it appears that translation, assembly, or localization processes for the formation of the surface structures are not temperature sensitive at 35 degrees C in the pleiotropic mutant CB13 ple-801. The syntheses of deoxyribonucleic acid and the cell wall do not appear to be temperature sensitive either, since the mutant grows normally at 35 degrees C. It is suggested that there exists a regulatory step that commits the cells to initiate the synthesis of requisite ribonucleic acid for the formation of the polar surface structures.     

ULTRASTRUCTURE OF CEPHALEUROS VIRESCENS (CHROOLEPIDACEAE; CHLOROPHYTA). III. ZOOSPORES

Transmission electron microscopic examination of Cephaleuros virescens Kunze growing on leaves of Camellia spp. and Magnolia grandiflora L. indicates that unreleased zoospores in mature zoosporangia are similar to those produced by the related genus Phycopeltis epiphyton Millardet and unlike the quadriflagellate motile cells produced by taxa in other families of Chlorophyta. The zoospores bear four smooth isokont bilaterally “keeled” flagella containing typical “9 + 2” axonemes and lacking scales. Flagellar insertion is apical and the parallel basal bodies overlap laterally at two levels. A cross section through the four basal bodies shows a trapezoidal arrangement wherein the two upper (anterior) basal bodies are closer together than are the lower (posterior) two. Serial sections indicate that diagonally opposing upper and lower basal bodies anchor flagella which emerge from the same side of the apical papilla. Each of the four basal bodies is associated with a microtubular spline which extends beneath the plasmalemma to the posterior end of the zoospore. A distinct multilayered structure is associated with each of the lower basal bodies. A nucleus, mitochondria (two of which are closely associated with the nucleus and spline microtubules), a chloroplast, and cytoplasmic haematochrome droplets are present in each zoospore. Pyrenoids and eyespots are absent. Flagellar insertion is characterized by “reversed bilateral symmetry”; and zoospores with both right-handed and left-handed arrangements are produced. The ultrastructure of the zoospores clearly indicates that: 1) the mode of flagellar insertion: 2) morphology, number, and arrangement of multilayered structures, and 3) bilaterally keeled flagella are characteristic of the Chroolepidaceae.     

Length Control of the Flagellar Hook in a Temperature-Sensitive flgE Mutant of Salmonella enterica Serovar Typhimurium

The flagellar hook is a short, curved, extracellular structure located between the basal body and the filament. The hook is composed of the FlgE protein. In this study, we analyzed flagellum assembly in a temperature-sensitive flgE mutant of Salmonella enterica serovar Typhimurium. When the mutant cells were grown at 30°C, they produced flagella of a normal length (71% of the population) and short hooks without filaments (26%). At 37°C, 70% of the basal bodies lacked hooks, and intact flagella made up only 6% of the population. Mutant cells secreted monomeric FlgE in abundance at 37°C, suggesting that the mutant FlgE protein might be defective in polymerization at higher temperatures. The average length of the hooks in intact filaments was 55 nm, whereas after acid treatment, it was 45 nm. SDS-PAGE analysis of the hook-basal body showed that HAP1 was missing in the mutant but not in the wild type. We concluded that hook length in the mutant is controlled in the same way as in the wild type, but the hook appeared short after acid treatment due to the lack of HAP1. We also learned that the true length of the hook is possibly 45 nm, not 55 nm, as has been believed.     

Waveform analysis and structure of flagella and basal complexes from Bdellovibrio bacteriovorus 109J.

The structure of sheathed flagella from Bdellovibrio bacteriovorus was investigated. The first three periods of these flagella were characterized by progressively smaller wavelengths and amplitudes in periods more distal to the cell. The damped appearance was due to a single nonrandom transition between two helical structures within each filament. The intersection of the two helices, one of which was a threefold-reduced miniature of the other, occurred at a fixed distance along the filament and resulted in a shift in the flagellar axis. Flagella increased in length as the cells aged and assumed a constant miniature waveform at their distal ends. The core filament was the principal determinant of flagellar morphology. It was composed of 28,000- and 29,500-dalton polypeptides. The 28,000-dalton subunits were located in the cell-proximal segment of the filament, and the 29,500-dalton subunits were located in the more distal region. The heteromorphous appearance of bdellovibrio flagella arose from the sequential assembly of these subunits. The basal complex associated with core filaments was examined because of its potential involvement in sheath formation. Bdellovibrio basal organelles were generally similar to those of other gram-negative species, but appeared to lack a disk analogous to the outer membrane-associated L ring which is a normal component of gram-negative basal complexes.     

Flagellar waveform and rotational orientation in a Chlamydomonas mutant lacking normal striated fibers

The Chlamydomonas mutant vfl-3 lacks normal striated fibers and microtubular rootlets. Although the flagella beat vigorously, the cells rarely display effective forward swimming. High speed cinephotomicrography reveals that flagellar waveform, frequency, and beat synchrony are similar to those of wild-type cells, indicating that neither striated fibers nor microtubular rootlets are required for initiation or synchronization of flagellar motion. However, in contrast to wild type, the effective strokes of the flagella of vfl-3 may occur in virtually any direction. Although the direction of beat varies between cells, it was not observed to vary for a given flagellum during periods of filming lasting up to several thousand beat cycles, indicating that the flagella are not free to rotate in the mature cell. Structural polarity markers in the proximal portion of each flagellum show that the flagella of the mutant have an altered rotational orientation consistent with their altered direction of beat. This implies that the variable direction of beat is not due to a defect in the intrinsic polarity of the axoneme, and that in wild-type cells the striated fibers and/or associated structures are important in establishing or maintaining the correct rotational orientation of the basal bodies to ensure that the inherent functional polarity of the flagellum results in effective cellular movement. As in wild type, the flagella of vfl-3 coordinately switch to a symmetrical, flagellar-type waveform during the shock response (induced by a sudden increase in illumination), indicating that the striated fibers are not directly involved in this process.     

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.     

Nm23/NDP kinases in human male germ cells: role in spermiogenesis and sperm motility?

Nucleoside diphosphate (NDP) kinases, responsible for the synthesis of nucleoside triphosphates and produced by the nm23 genes, are involved in numerous regulatory processes associated with proliferation, development, and differentiation. Their possible role in providing the GTP/ATP required for sperm function is unknown. Testis biopsies and ejaculated sperm were examined by immunohistochemical and immunofluorescence microscopy using specific antibodies raised against Nm23-H5, specifically expressed in testis germinal cells and the ubiquitous NDP kinases A to D. Nm23-H5 was present in sperm extract, together with the ubiquitous A and B NDP kinases (but not the C and D isoforms) as shown by Western blotting. Nm23-H5 was located in the flagella of spermatids and spermatozoa, adjacent to the central pair and outer doublets of axonemal microtubules. High levels of NDP kinases A and B were observed at specific locations in postmeiotic germinal cells. NDP kinase A was transiently located in round spermatid nuclei and became asymmetrically distributed in the cytoplasm at the nuclear basal pole of elongating spermatids. The distribution of NDP kinase B was reminiscent of the microtubular structure of the manchette. In ejaculated spermatozoa, the proteins presented specific locations in the head and flagella. Nm23/NDP kinase isoforms may have specific functions in the phosphotransfer network involved in spermiogenesis and flagellar movement.     

Ultrastructural analysis of flagellar development in plurilocular sporangia of Ectocarpus siliculosus (Phaeophyceae)

Flagellar development in the plurilocular zoidangia of sporophytes of the brown alga Ectocarpus siliculosus was analyzed in detail using transmission electron microscopy and electron tomography. A series of cell divisions in the plurilocular zoidangia produced the spore-mother cells. In these cells, the centrioles differentiated into flagellar basal bodies with basal plates at their distal ends and attached to the plasma membrane. The plasma membrane formed a depression (flagellar pocket) into where the flagella elongated and in which variously sized vesicles and cytoplasmic fragments accumulated. The anterior and posterior flagella started elongating simultaneously, and the vesicles and cytoplasmic fragments in the flagellar pocket fused to the flagellar membranes. The two flagella (anterior and posterior) could be clearly distinguished from each other at the initial stage of their development by differences in length, diameter and the appendage flagellar rootlets. Flagella continued to elongate in the flagellar pocket and maintained their mutually parallel arrangement as the flagellar pocket gradually changed position. In mature zoids, the basal part of the posterior flagellum (paraflagellar body) characteristically became swollen and faced the eyespot region. Electron dense materials accumulated between the axoneme and the flagellar membrane, and crystallized materials could also be observed in the swollen region. Before liberation of the zoospores from the plurilocular zoidangia, mastigoneme attachment was restricted to the distal region of the anterior flagellum. Structures just below the flagellar membrane that connected to the mastigonemes were clearly visible by electron tomography.     

The Vfl1 Protein in Chlamydomonas localizes in a rotationally asymmetric pattern at the distal ends of the basal bodies

In the unicellular alga Chlamydomonas, two anterior flagella are positioned with 180 degrees rotational symmetry, such that the flagella beat with the effective strokes in opposite directions (Hoops, H.J., and G.B. Witman. 1983. J. Cell Biol. 97:902-908). The vfl1 mutation results in variable numbers and positioning of flagella and basal bodies (Adams, G.M.W., R.L. Wright, and J.W. Jarvik. 1985. J. Cell Biol. 100:955-964). Using a tagged allele, we cloned the VFL1 gene that encodes a protein of 128 kD with five leucine-rich repeat sequences near the NH(2) terminus and a large alpha-helical-coiled coil domain at the COOH terminus. An epitope-tagged gene construct rescued the mutant phenotype and expressed a tagged protein (Vfl1p) that copurified with basal body flagellar apparatuses. Immunofluorescence experiments showed that Vfl1p localized with basal bodies and probasal bodies. Immunogold labeling localized Vfl1p inside the lumen of the basal body at the distal end. Distribution of gold particles was rotationally asymmetric, with most particles located near the doublet microtubules that face the opposite basal body. The mutant phenotype, together with the localization results, suggest that Vfl1p plays a role in establishing the correct rotational orientation of basal bodies. Vfl1p is the first reported molecular marker of the rotational asymmetry inherent to basal bodies.     

Isolation and characterization of FliK-independent flagellation mutants from Salmonella typhimurium.

A flagellum of Salmonella typhimurium and Escherichia coli consists of three structural parts, a basal body, a hook, and a filament. Because the fliK mutants produce elongated hooks, called polyhooks, lacking filament portions, the fliK gene product has been believed to be involved in both the determination of hook length and the initiation of the filament assembly. In the present study, we isolated two mutants from S. typhimurium which can form flagella even in the absence of the fliK gene product. Flagellar structures were fractionated from these suppressor mutants and inspected by electron microscopy. The suppressor mutants produced polyhook-filament complexes in the fliK mutant background, while they formed flagellar structures apparently indistinguishable from those of the wild-type strain in the fliK+ background. Genetic and sequence analyses of the suppressor mutations revealed that they are located near the 3'-end of the flhB gene, which has been believed to be involved in the early process of the basal body assembly. On the basis of these results, we discuss the mechanism of suppression of the fliK defects by the flhB mutations and propose a hypothesis on the export switching machinery of the flagellar proteins.     

Functional and trafficking defects in ATP binding cassette A3 mutants associated with respiratory distress syndrome

Members of the ATP binding cassette (ABC) protein superfamily actively transport a wide range of substrates across cell and intracellular membranes. Mutations in ABCA3, a member of the ABCA subfamily with unknown function, lead to fatal respiratory distress syndrome (RDS) in the newborn. Using cultured human lung cells, we found that recombinant wild-type hABCA3 localized to membranes of both lysosomes and lamellar bodies, which are the intracellular storage organelles for surfactant. In contrast, hABCA3 with mutations linked to RDS failed to target to lysosomes and remained in the endoplasmic reticulum as unprocessed forms. Treatment of those cells with the chemical chaperone sodium 4-phenylbutyrate could partially restore trafficking of mutant ABCA3 to lamellar body-like structures. Expression of recombinant ABCA3 in non-lung human embryonic kidney 293 cells induced formation of lamellar body-like vesicles that contained lipids. Small interfering RNA knockdown of endogenous hABCA3 in differentiating human fetal lung alveolar type II cells resulted in abnormal, lamellar bodies comparable with those observed in vivo with mutant ABCA3. Silencing of ABCA3 expression also reduced vesicular uptake of surfactant lipids phosphatidylcholine, sphingomyelin, and cholesterol but not phosphatidylethanolamine. We conclude that ABCA3 is required for lysosomal loading of phosphatidylcholine and conversion of lysosomes to lamellar body-like structures.     

A phylogenetic analysis of the purple photosynthetic bacteria

It is proposed that gliding motility in bacteria is based on rotary assemblies located in the cell envelope and that these assemblies may be analogous to basal regions of bacterial flagella. This proposal rests on the following evidence: (i) Structures resembling flagellar basal regions have been demonstrated in cells ofCytophaga johnsonae andFlexibacter columnaris, and such structures are absent from one nonmotile mutant ofF. columnaris. (ii) The effects of inhibitors of energy metabolism on gliding motility are identical with their effects on prokaryotic fiagellar motility. (iii) The active movement of latex spheres along surfaces of gliding bacteria appears to depend on mechanisms responsible for motility and can be explained by the presence of rotary surface assemblies.     

The Microtubule plus end-tracking protein EB1 is localized to the flagellar tip and basal bodies in Chlamydomonas reinhardtii

Flagellar axonemes assemble and continuously turn over at the flagellar tip. The supply and removal of axonemal subunits at the tip are mediated by intraflagellar transport (IFT), a motility process essential for the assembly and maintenance of all eukaryotic flagella and cilia. IFT is characterized by the movement of large protein complexes (IFT particles) from the basal bodies to the flagellar tip by kinesin-II and from the tip back to the basal bodies by cytoplasmic dynein 1b. The IFT particles consist of approximately 16 polypeptides partitioned into two complexes, A and B, and associate with axonemal precursors/turn over products. The mechanisms by which IFT motor regulation and cargo loading/unloading occur at the flagellar tip are unknown. We identified a Chlamydomonas reinhardtii ortholog of the microtubule (MT) plus end-tracking protein EB1 [4] (CrEB1) and show here that CrEB1 localizes to the tip of flagella and to the proximal part of the basal bodies. Furthermore, we found that CrEB1 is depleted from flagella of the temperature-sensitive (ts) flagellar assembly-defective (fla) mutant fla11(ts) at the restrictive temperature. This depletion of CrEB1 is accompanied by a dramatic accumulation of IFT particle polypeptides near the flagellar tip.