Base Of Flagellum Research Articles

Isolation and Cs+ resistance mechanism of Escherichia coli strain ZX-1.

This research aims to elucidate the physiological mechanisms behind the accidental acquisition of high-concentration cesium ions (Cs+) tolerance of Escherichia coli and apply this understanding to develop bioremediation technologies. Bacterial Cs+ resistance has attracted attention, but its physiological mechanism remains largely unknown and poorly understood. In a prior study, we identified the Cs+/H+ antiporter TS_CshA in Microbacterium sp. TS-1, resistant to high Cs+ concentrations, exhibits a low Cs+ affinity with a Km value of 370 mM at pH 8.5. To enhance bioremediation efficacy, we conducted random mutagenesis of TS_cshA using Error-Prone PCR, aiming for higher-affinity mutants. The mutations were inserted downstream of the PBAD promoter in the pBAD24 vector, creating a mutant library. This was then transformed into E. coli-competent cells. As a result, we obtained a Cs+-resistant strain, ZX-1, capable of thriving in 400 mM CsCl-a concentration too high for ordinary E. coli. Unlike the parent strain Mach1™, which struggled in 300 mM CsCl, ZX-1 showed robust growth even in 700 mM CsCl. After 700 mM CsCl treatment, the 70S ribosome of Mach1™ collapsed, whereas ZX-1 and its derivative ΔZX-1/pBR322ΔAp remained stable. This means that the ribosomes of ZX-1 are more stable to high Cs+. The inverted membrane vesicles from strain ZX-1 showed an apparent Km value of 28.7 mM (pH 8.5) for Cs+/H+ antiport activity, indicating an approximately 12.9-fold increase in Cs+ affinity. Remarkably, the entire plasmid isolated from ZX-1, including the TS_cshA region, was mutation-free. Subsequent whole-genome analysis of ZX-1 identified multiple SNPs on the chromosome that differed from those in the parent strain. No mutations in transporter-related genes were identified in ZX-1. However, three mutations emerged as significant: genes encoding the ribosomal bS6 modification enzyme RimK, the phage lysis regulatory protein LysB, and the flagellar base component protein FlgG. These mutations are hypothesized to affect post-translational modifications, influencing the Km value of TS_CshA and accessory protein expression. This study unveils a novel Cs+ resistance mechanism in ZX-1, enhancing our understanding of Cs+ resistance and paving the way for developing technology to recover radioactive Cs+ from water using TS_CshA-expressing inverted membrane vesicles.

The Cby3/ciBAR1 complex positions the annulus along the sperm flagellum during spermiogenesis.

Proper compartmentalization of the sperm flagellum is essential for fertility. The annulus is a septin-based ring that demarcates the midpiece (MP) and the principal piece (PP). It is assembled at the flagellar base, migrates caudally, and halts upon arriving at the PP. However, the mechanisms governing annulus positioning remain unknown. We report that a Chibby3 (Cby3)/Cby1-interacting BAR domain-containing 1 (ciBAR1) complex is required for this process. Ablation of either gene in mice results in male fertility defects, caused by kinked sperm flagella with the annulus mispositioned in the PP. Cby3 and ciBAR1 interact and colocalize to the annulus near the curved membrane invagination at the flagellar pocket. In the absence of Cby3, periannular membranes appear to be deformed, allowing the annulus to migrate over the fibrous sheath into the PP. Collectively, our results suggest that the Cby3/ciBAR1 complex regulates local membrane properties to position the annulus at the MP/PP junction.

ULK4 and Fused/STK36 interact to mediate assembly of a motile flagellum.

Unc-51-like kinase (ULK) family serine-threonine protein kinase homologues have been linked to the function of motile cilia in diverse species. Mutations in Fused/STK36 and ULK4 in mice resulted in hydrocephalus and other phenotypes consistent with ciliary defects. How either protein contributes to the assembly and function of motile cilia is not well understood. Here we studied the phenotypes of ULK4 and Fused gene knockout (KO) mutants in the flagellated protist Leishmania mexicana. Both KO mutants exhibited a variety of structural defects of the flagellum cytoskeleton. Biochemical approaches indicate spatial proximity of these proteins and indicate a direct interaction between the N-terminus of LmxULK4 and LmxFused. Both proteins display a dispersed localization throughout the cell body and flagellum, with enrichment near the flagellar base and tip. The stable expression of LmxULK4 was dependent on the presence of LmxFused. Fused/STK36 was previously shown to localize to mammalian motile cilia, and we demonstrate here that ULK4 also localizes to the motile cilia in mouse ependymal cells. Taken together these data suggest a model where the pseudokinase ULK4 is a positive regulator of the kinase Fused/ STK36 in a pathway required for stable assembly of motile cilia.

The flagellar motor protein FliL forms a scaffold of circumferentially positioned rings required for stator activation

The flagellar motor stator is an ion channel nanomachine that assembles as a ring of the MotA5MotB2 units at the flagellar base. The role of accessory proteins required for stator assembly and activation remains largely enigmatic. Here, we show that one such assembly factor, the conserved protein FliL, forms an integral part of the Helicobacter pylori flagellar motor in a position that colocalizes with the stator. Cryogenic electron tomography reconstructions of the intact motor in whole wild-type cells and cells lacking FliL revealed that the periplasmic domain of FliL (FliL-C) forms 18 circumferentially positioned rings integrated with the 18 MotAB units. FliL-C formed partial rings in the crystal, and the crystal structure-based full ring model was consistent with the shape of the rings observed in situ. Our data suggest that each FliL ring is coaxially sandwiched between the MotA ring and the dimeric periplasmic MotB moiety of the stator unit and that the central hole of the FliL ring has density that is consistent with the plug/linker region of MotB in its extended, active conformation. Significant structural similarities were found between FliL-C and stomatin/prohibitin/flotillin/HflK/C domains of scaffolding proteins, suggesting that FliL acts as a scaffold. The binding energy released upon association of FliL with the stator units could be used to power the release of the plug helices. The finding that isolated FliL-C forms stable partial rings provides an insight into the putative mechanism by which the FliL rings assemble around the stator units.

Tetraselmis jejuensis sp. nov. (Chlorodendrophyceae), a Euryhaline Microalga Found in Supralittoral Tide Pools at Jeju Island, Korea.

We found the euryhaline microalga, Tetraselmis jejuensis sp. nov., which was adapted to supralittoral tide pools with salinities varying from 0.3–3.1%. Fifteen strains of T. jejuensis were isolated from Daejeong (DJ) and Yongduam (YO), and clonal cultures were established in the laboratory. Morphological characterization revealed that the cells have a compressed shape, four flagella emerging from a depression near the apex in two opposite pairs, a cup-shaped chloroplast containing one pyrenoid surrounded by starch, and eyespot regions not located near the flagellar base. T. jejuensis cells showed distinct characteristics compared to other Tetraselmis species. First, a regular subunit pattern with honeycomb-like structures was predominantly displayed on the surface in the middle of the cell body. Second, the pyrenoid was invaded by both cytoplasmic channels comprising electron-dense material separated from the cytoplasm, and two branches of small cytoplasmic channels (canaliculi) in various directions, which characterize the subgenus Tetrathele. Eyespot regions containing a large number of osmiophilic globules, packed closely together and arranged in subcircular close packing of diverse sizes, were dispersed throughout the chloroplast. In the phylogenetic analysis of small subunit (SSU) rDNA sequences, the 15 strains isolated from DJ and YO separated a newly branched clade in the Chlorodendrophyceae at the base of a clade comprising the T. carteriiformi/subcordiformis clade, T. chuii/suecica clade, and T. striata/convolutae clade. The strains in the diverging clade were considered to belong to the same species. The SSU rDNA sequences of the DJ and YO strains showed a maximum difference of 1.53% and 1.19% compared to Tetraselmis suecica (MK541745), the closest species of the family based on the phylogenetic analysis, respectively. Based on morphological, molecular, and physiological features, we suggest a new species in the genus Tetraselmis named Tetraselmis jejuensis, with the species name “jejuensis” referring to the collection site, Jeju Island, Korea.

The multi‐scale architecture of mammalian sperm flagella and implications for ciliary motility

Motile cilia are molecular machines used by a myriad of eukaryotic cells to swim through fluid environments. However, available molecular structures represent only a handful of cell types, limiting our understanding of how cilia are modified to support motility in diverse media. Here, we use cryo‐focused ion beam milling‐enabled cryo‐electron tomography to image sperm flagella from three mammalian species. We resolve in‐cell structures of centrioles, axonemal doublets, central pair apparatus, and endpiece singlets, revealing novel protofilament‐bridging microtubule inner proteins throughout the flagellum. We present native structures of the flagellar base, which is crucial for shaping the flagellar beat. We show that outer dense fibers are directly coupled to microtubule doublets in the principal piece but not in the midpiece. Thus, mammalian sperm flagella are ornamented across scales, from protofilament‐bracing structures reinforcing microtubules at the nano‐scale to accessory structures that impose micron‐scale asymmetries on the entire assembly. Our structures provide vital foundations for linking molecular structure to ciliary motility and evolution.

Fine-structural characterization and phylogeny of Sphaerodinium (Suessiales, Dinophyceae), with the description of an unusual type of freshwater dinoflagellate cyst

Two strains of Sphaerodinium were established from two mountain areas in Portugal and examined by light microscopy, scanning and transmission electron microscopy, and sequence analyses of nuclear-encoded SSU, ITS1–5.8S–ITS2 and LSU rDNA. Both strains were identified as S. polonicum var. tatricum on the basis of comparison with the original taxonomic descriptions within the genus. The two strains were nearly identical in morphology and ultrastructure, except for the presence of pseudograna-like thylakoid stacks within more rounded chloroplast lobes in one of them. Sexual reproduction occurred in culture batches and resting cysts with single or grouped processes with wide bases and distal platforms with slightly recurved margins were seen to develop by sudden retraction of planozygote cytoplasm. Morphological, fine-structural and molecular characters were compared with previously available information from S. cracoviense, allowing for a more robust characterization of the genus. Important characters include a type F eyespot, a pusule canal linking the transverse flagellar canal to a collecting chamber connected to regular pusular tubes, a ventral fibre extending from the proximal-right side of the longitudinal basal body, and a membranous, lamellar body with a honeycomb pattern near the flagellar base area. The latter two features are shared with Baldinia anauniensis.

Flagellar beating pattern of male gametes during the fertilization process of the marine green macroalga Bryopsis maxima (Ulvophyceae, Chlorophyta)

SUMMARYI studied the flagellar movements and mating behavior of biflagellate gametes produced by the green marine macroalga Bryopsis maxima using high‐speed video microscopy. This procedure allowed me to visualize the very rapid fertilization process in a marine ulvophycean. Discharged male and female gametes always swung their flagella backwards during forward swimming. They swam with a flagellar beat type pattern in which undulatory waves produced at the flagellar bases propagated towards the tips. Each beat cycle lasted 15–20 ms. Male gametes were immediately attracted to the vicinity of female gametes when the two mating types were mixed. As each male gamete swam past a female, it reversed direction towards the female by changing the beat pattern of one of its two flagella. The attraction of male gametes to the female ones was inhibited in calcium‐free seawater in both the presence and absence of 10 mM ethylene glycol‐bis(β‐aminoethyl ether)‐N,N,N′,N′‐tetraacetic acid, a calcium chelator, but attraction recovered in the presence of 10 mM CaCl2, suggesting that Ca2+ is necessary for the attraction response. The initial contact between the two gametes occurred when one of the male flagellar tips encountered a female flagellum. The male gamete kept in contact with the female gamete (i) at the flagellar tips, and then (ii) by anterior cell body contact. Flagella continued to beat and cell bodies oscillated as gametes maintained contact. In the final step, the gametes fused.

Mechanical induction of oscillatory movement in demembranated, immotile flagella of sea urchin sperm at very low ATP concentrations.

Oscillation is a characteristic feature of eukaryotic flagellar movement. The mechanism involves the control of dynein-driven microtubule sliding under self-regulatory mechanical feedback within the axoneme. To define the essential factors determining the induction of oscillation, we developed a novel experiment by applying mechanical deformation of demembranated, immotile sea urchin sperm flagella at very low ATP concentrations, below the threshold of ATP required for spontaneous beating. Upon application of mechanical deformation at above 1.5 µmoll-1 ATP, a pair of bends could be induced and was accompanied by bend growth and propagation, followed by switching the bending direction. For an oscillatory, cyclical bending response to occur, the velocity of bend propagation towards the flagellar tip must be kept above certain levels. Continuous formation of new bends at the flagellar base was coupled with synchronized decay of the preceding paired bends. Induction of cyclical bends was initiated in a constant direction relative to the axis of the flagellar 9+2 structure, and resulted in the so-called principal bend. In addition, stoppage of the bending response occasionally occurred during development of a new principal bend, and in this situation, formation of a new reverse bend did not occur. This observation indicates that the reverse bend is always active, opposing the principal bend. The results show that mechanical strain of bending is a central component regulating the bend oscillation, and switching of the bend direction appears to be controlled, in part, by the velocity of wave propagation.

Studies on Peridinium aciculiferum and Peridinium malmogiense (=Scrippsiella hangoei): comparison with Chimonodinium lomnickii and description of Apocalathium gen. nov. (Dinophyceae)

:The fine structure of the freshwater Peridinium aciculiferum and the closely related Baltic Sea species currently known as Scrippsiella hangoei were examined in serial sections. The species name Peridinium malmogiense is shown to be an earlier synonym of Scrippsiella hangoei and is restored as the name of the species. Although both species have been included in the genus Peridinium, their phylogenetic positions are within the Thoracosphaeraceae, close to the specialized predators known as the pfiesteriaceans and the photosynthetic freshwater Chimonodinium lomnickii. The fine-structural features of the two species proved to be very similar, including the details of flagellar bases and roots, and the type of pyrenoid, which consisted of dilated areas of the chloroplast crossed by two-thylakoid lamellae and not associated with starch sheaths. Comparison with Chimonodinium revealed significant differences, in particular the absence of an eyespot and any trace of microtubules associated with a peduncle, which contrast with the multilayered eyespot and the distinct microtubular basket (MB) of C. lomnickii. The absence of a MB in P. aciculiferum and P. malmogiense is regarded as a character loss within a group of species hypothesized to be derived from a MB-containing ancestor. A phylogenetic analysis based on concatenation of nuclear-encoded small subunit rDNA, internal transcribed spacers 1 and 2 including 5.8S sequences agreed with published phylogenies based on genes of the ribosomal operon in closely grouping P. aciculiferum, P. malmogiense and two other species of peridinioids with a similar amphiesmal plate arrangement: P. euryceps and P. baicalense. The four species are regarded as members of the same genus. While one of the closest known relatives of these four species is C. lomnickii, the variable association of this species to several other groups of species in published phylogenies and the differences in fine-structure revealed in the present work advise against transferring the studied species to Chimonodinium. The new genus Apocalathium is described with P. aciculiferum as type species.

Insight into the flagella type III export revealed by the complex structure of the type III ATPase and its regulator

FliI and FliJ form the FliI6FliJ ATPase complex of the bacterial flagellar export apparatus, a member of the type III secretion system. The FliI6FliJ complex is structurally similar to the α3β3γ complex of F1-ATPase. The FliH homodimer binds to FliI to connect the ATPase complex to the flagellar base, but the details are unknown. Here we report the structure of the homodimer of a C-terminal fragment of FliH (FliHC2) in complex with FliI. FliHC2 shows an unusually asymmetric homodimeric structure that markedly resembles the peripheral stalk of the A/V-type ATPases. The FliHC2-FliI hexamer model reveals that the C-terminal domains of the FliI ATPase face the cell membrane in a way similar to the F/A/V-type ATPases. We discuss the mechanism of flagellar ATPase complex formation and a common origin shared by the type III secretion system and the F/A/V-type ATPases.

The intraflagellar transport dynein complex of trypanosomes is made of a heterodimer of dynein heavy chains and of light and intermediate chains of distinct functions.

Cilia and flagella are assembled by intraflagellar transport (IFT) of protein complexes that bring tubulin and other precursors to the incorporation site at their distal tip. Anterograde transport is driven by kinesin, whereas retrograde transport is ensured by a specific dynein. In the protist Trypanosoma brucei, two distinct genes encode fairly different dynein heavy chains (DHCs; ∼40% identity) termed DHC2.1 and DHC2.2, which form a heterodimer and are both essential for retrograde IFT. The stability of each heavy chain relies on the presence of a dynein light intermediate chain (DLI1; also known as XBX-1/D1bLIC). The presence of both heavy chains and of DLI1 at the base of the flagellum depends on the intermediate dynein chain DIC5 (FAP133/WDR34). In the IFT140(RNAi) mutant, an IFT-A protein essential for retrograde transport, the IFT dynein components are found at high concentration at the flagellar base but fail to penetrate the flagellar compartment. We propose a model by which the IFT dynein particle is assembled in the cytoplasm, reaches the base of the flagellum, and associates with the IFT machinery in a manner dependent on the IFT-A complex.

Fine structure of sensory apparatus on the head of Cixiopsis punctatus.

The external morphology of the heads of adult male and female Cixiopsis punctatus (Matsumura) (Hemiptera: Fulgoromorpha: Tropiduchidae) was studied using scanning electron microscopy. Eleven types of sensilla or sensory organs were identified: trichoid sensilla on the pedicel, scape, maxillae, and labium; campaniform sensilla on the antennal pedicel, antennal scape, maxillae, and labium; plate organs on the antennal pedicel; coeloconic sensilla in Bourgoin's organ and styloconic sensilla on the expanded flagellar base; Evans' organ and button-like sensilla on the maxillary plates; basiconic sensilla, peg sensilla, and coin-shaped sensilla on the labium. Styloconic sensilla on the expanded flagellar base and peg sensilla located between the dorsal sensory field and the opening of the maxillae and mandibles were first reported in Tropiduchidae. The external morphology, distribution, and abundance of sensilla located on antennae, maxillae, and labium in C. punctatus were illustrated.

Ultrastructure and Molecular Phylogenetic Position of Neometanema parovale sp. nov. (Neometanema gen. nov.), a Marine Phagotrophic Euglenid with Skidding Motility

Heteronema is a commonly encountered genus of phagotrophic euglenids that contains very different morphotypes, including elongate gliding species and ovoid skidding forms. We report the first ultrastructural and sequence data from a culture of an ovoid skidding heteronemid, KM051. Cells were 8-23.5 μm long with 22 pellicular strips and a fibrous extracellular layer. The tubular extrusomes had dense centre sections. The feeding apparatus was barely visible by light microscopy, but included two microtubule-supported rods. The flagella had hollow, inflated transition zones, heteromorphic paraxonemal rods, and sheaths of flagellar hairs. The posterior flagellum bore a knob that, unusually, sat >2 μm distal to the flagellar base. No ultrastructural features were uniquely shared by KM051 and the elongate, gliding species Heteronema scaphurum. Conversely, the pellicular microtubule array resembles that in deep-branching primary osmotrophs (Aphagea). 18S ribosomal DNA (18S rDNA) phylogenies showed that KM051 is related to a recently obtained Heteronema c.f. exaratum sequence. These skidding heteronemids are not closely related to H. scaphurum, and instead are closely related to Dinema, Anisonema and specifically, Aphagea. The skidding species in Heteronema are transferred to Neometanema gen. nov. (along with most species of Metanema Klebs, 1893), with KM051 described as Neometanema parovale sp. nov.

Fine structure of sensory apparatus on the head ofCixiopsis punctatus

The external morphology of the heads of adult male and female Cixiopsis punctatus (Matsumura) (Hemiptera: Fulgoromorpha: Tropiduchidae) was studied using scanning electron microscopy. Eleven types of sensilla or sensory organs were identified: trichoid sensilla on the pedicel, scape, maxillae, and labium; campaniform sensilla on the antennal pedicel, antennal scape, maxillae, and labium; plate organs on the antennal pedicel; coeloconic sensilla in Bourgoin’s organ and styloconic sensilla on the expanded flagellar base; Evans’ organ and button-like sensilla on the maxillary plates; basiconic sensilla, peg sensilla, and coin-shaped sensilla on the labium. Styloconic sensilla on the expanded flagellar base and peg sensilla located between the dorsal sensory field and the opening of the maxillae and mandibles were first reported in Tropiduchidae. The external morphology, distribution, and abundance of sensilla located on antennae, maxillae, and labium in C. punctatus were illustrated.

Phase-Related Developmental Characteristics of Antennal Sensilla of Nymphal Laodelphax striatellus (Hemiptera: Delphacidae), a Serious Virus-Transmitting Insect Vector of Graminaceous Crops

Abstract The immature development of the antennal sensilla borne by the first—fifth instar nymphal Laodelphax striatellus (Fallén) (Hemiptera: Delphacidae) were studied using scanning electron microscopy. Sensillar types of nymphs did not vary, but the distribution pattern of sensilla gradually changed during the postembryonic development. Seven types of antennal sensilla were found on the antennae of each of the first—fifth instars. One Böhm bristle and one sensillum campaniformium I on the scape, one Böhm bristle and one sensillum campaniformium II on the pedicel, and one sensillum coeloconicum and three sensilla basiconica on the swollen flagellar base (main body) remain constant during development. The numbers of sensilla chaetica on the scape and pedicel and the numbers of sensilla trichodea, sensilla placodea, and sensilla basiconica on the pedicel gradually increased from the youngest to the oldest instars. In contrast, sensilla trichodea, which occurred on the swollen flagellar base of the first instar nymph, disappeared after the first molt, and one sensillum placodeum on the swollen flagellar base of the first instar nymph gradually degraded during development and finally disappeared after the third molt. The length and width of antennae increased during the development, except for the flagellar main body, which presented negative growth in its width after the first molt. The development of antennae seems according with the need for bearing sensilla.

Regulated Membrane Protein Entry into Flagella Is Facilitated by Cytoplasmic Microtubules and Does Not Require IFT

The membrane protein composition of the primary cilium, a key sensory organelle, is dynamically regulated during cilium-generated signaling [1, 2]. During ciliogenesis, ciliary membrane proteins, along with structural and signaling proteins, are carried through the multicomponent, intensely studied ciliary diffusion barrier at the base of the organelle [3-8] by intraflagellar transport (IFT) [9-18]. A favored model is that signaling-triggered accumulation of previously excluded membrane proteins in fully formed cilia [19-21] also requires IFT, but direct evidence is lacking. Here, in studies of regulated entry of a membrane protein into the flagellum of Chlamydomonas, we show that cells use an IFT-independent mechanism to breach the diffusion barrier at the flagellar base. In resting cells, a flagellar signaling component [22], the integral membrane polypeptide SAG1-C65, is uniformly distributed over the plasma membrane and excluded from the flagellar membrane. Flagellar adhesion-induced signaling triggers rapid, striking redistribution of the protein to the apical ends of the cells concomitantly with entry into the flagella. Protein polarization and flagellar enrichment are facilitated by cytoplasmic microtubules. Using a conditional anterograde IFT mutant, we demonstrate that the IFT machinery is not required for regulated SAG1-C65 entry into flagella. Thus, integral membrane proteins can negotiate passage through the ciliary diffusion barrier without the need for a motor.

Avalanche-like behavior in ciliary import

Cilia and flagella are microtubule-based organelles that protrude from the cell body. Ciliary assembly requires intraflagellar transport (IFT), a motile system that delivers cargo from the cell body to the flagellar tip for assembly. The process controlling injections of IFT proteins into the flagellar compartment is, therefore, crucial to ciliogenesis. Extensive biochemical and genetic analyses have determined the molecular machinery of IFT, but these studies do not explain what regulates IFT injection rate. Here, we provide evidence that IFT injections result from avalanche-like releases of accumulated IFT material at the flagellar base and that the key regulated feature of length control is the recruitment of IFT material to the flagellar base. We used total internal reflection fluorescence microscopy of IFT proteins in live cells to quantify the size and frequency of injections over time. The injection dynamics reveal a power-law tailed distribution of injection event sizes and a negative correlation between injection size and frequency, as well as rich behaviors such as quasiperiodicity, bursting, and long-memory effects tied to the size of the localized load of IFT material awaiting injection at the flagellar base, collectively indicating that IFT injection dynamics result from avalanche-like behavior. Computational models based on avalanching recapitulate observed IFT dynamics, and we further show that the flagellar Ras-related nuclear protein (Ran) guanosine 5'-triphosphate (GTP) gradient can in theory act as a flagellar length sensor to regulate this localized accumulation of IFT. These results demonstrate that a self-organizing, physical mechanism can control a biochemically complex intracellular transport pathway.

Ultrastructure of sensory equipments on the heads ofKallitaxila granulata(Stål) (Hemiptera: Fulgoromorpha: Tropiduchidae)

The polyphagous planthopper, Kallitaxila granulata (Stål) (Hemiptera: Fulgoromorpha: Tropiduchidae), has been recently introduced into southeastern China, the Philippine islands, and Hawaii, where it has done significant damage to agricultural and forest ecosystems. The external morphology of the heads of adult male and female K. granulata was examined using scanning electron microscopy. Seven types of sensilla were reported: trichoid sensilla and campaniform sensilla on the antennal pedicel, antennal scape and maxilla; plate organs on the antennal pedicel; coeloconic sensilla in Bourgoin's organ on the expanded flagellar base; ampullaceal sensilla on the antennal pedicel; wavy-pit sensilla on the antennal pedicel and antennal scape; and coin-shaped sensilla on each lateral side of the labium. Evans' organ was described as placoid sensilla sunk into shallow cuticular cavities below the antennae. The external morphology, distribution, and abundance of sensilla located on antennae, maxillae, and labium in K. granulata were illustrated, with a brief discussion of their taxonomic, phylogenetic, and putative functional significance.

Single-Molecule Investigation of the Flagellar Membrane Signaling Protein Entry Mechanism

The composition of the flagellar membrane differs from that of the plasma membranes--it is enriched with a unique set of membrane signaling proteins. How these flagellar membrane proteins, which are synthesized in the cytoplasm, enter the flagella across diffusion barriers at the flagellar base has become a significant point of inquiry. Using single-molecule fluorescence imaging methods, we have investigated the intraflagellar transport (IFT) processes of Pkd2-GFP, an important flagellar signaling protein responsible for polycystic kidney disease, and tested two proposed models. In Model I, vesicles carry the proteins to the flagellar base and fuse with the membrane before or after the diffusion barrier to allow the flagellar membrane proteins to enter by lateral diffusion; and, in Model II, the flagellar membrane proteins are bound to the BBSome-IFT-particle machinery (kinesin or dynein motors carry IFT particles, which, in turn, carry BBSomes) at the flagellar base and are carried through the basal body transition fibers into flagella. We have observed Brownian diffusion of Pkd2 at the Chlamydomonas flagellar entry region, suggesting that Model I more accurately describes the entry mechanism. We have further studied the turnaround mechanisms of the IFT machinery at the flagellar tip. Our preliminary results indicate that the IFT machinery does not dissociate, diffuse, and re-associate at the flagellar tip for the return trip to the cell body.