Basal body-associated nucleation center for the centrin-based cortical cytoskeletal network in Paramecium

The infraciliary lattice, a contractile cortical cytoskeletal network of Paramecium, is composed of a small number of polypeptides including centrins. Its overall pattern reflects a hierarchy of structural complexity, from assembly and bundling of microfilaments to formation of polygonal meshes arranged in a continuous network subtending the whole cell surface, with local differentiations in the shape and size of the meshes. To analyse how the geometry of this complex network is generated and maintained, we have taken two approaches. Firstly, using monoclonal antibodies raised against the purified network, we have shown that all the component polypeptides colocalize, in agreement with previous biochemical data indicating that the infraciliary lattice is formed of large complexes comprising all the component polypeptides. Secondly, by taking advantage of different experimental conditions leading to disassembly of the network, we have followed its reassembly. Cytological analysis of the process revealed 1) that the network regrows exclusively from specific infraciliary lattice organizing centers (ICLOC), precisely localized near each basal body and, 2) that the global organization is not precisely controlled by genetic information but by the basal body pattern. Finally, slight ultrastuctural differences between reassembled and control lattices suggest that the organization of the filament bundles is partly templated by that of the preexisting ones.     

Ca2+ -binding proteins and contractility of the infraciliary lattice in Paramecium

The infraciliary lattice (ICL) is the innermost cortical cytoskeletal network of Paramecium. Its meshes which run around the proximal end of basal bodies form a continuous contractile network beneath the cell surface. We had previously shown that the network, which could be recovered in a contracted form and selectively solubilized by EGTA from an ICL-enriched cell fraction, was principally composed of 23–24 kDa polypeptides cross-reacting with antibodies raised against the 22 kDa Ca²⁺ -binding proteins of the ecto-endoplasmic boundary (EEB), a contractile cytoskeletal network of another ciliate Isotricha prostoma. We show here 1) that the ICL also comprises a 220 kDa polypeptide; 2) that the 23–24 kDa polypeptides are resolved in 2D gels into 11 spots of acidic pI, 7 of which are both Ca²⁺ -binding and cross-reacting with the anti EEB polypeptides; 3) that the network displays a high Ca²⁺ -affinity as the treshold for solubilization/co-precipitation of both high and low MW polypeptides is around 10⁻⁸ M free Ca²⁺ ; 4) that in vivo contraction of the network occurs upon physiological increase of internal calcium concentration. The likely phylogenetic relationships of the 23–24 kDa ICL polypeptides with the calmodulin related family of Ca²⁺ -modulated polypeptides and the functions of the ICL in cell contractility and Ca²⁺ homeostasis are discussed.     

The Striated Bands of Paramecium Are Immunologically Distinct from the Centrin-Specific Infraciliary Lattice and Cytostomal Cord

The pellicle of Paramecium has three two-dimensionally arrayed systems that occupy separate but closely paralleling planes. All three systems are now distinguishable by their differing immunological properties. This study focused on the two deeper systems. The infraciliary lattice lies innermost and labels with centrin-specific antibodies. The middle system, the striated bands, is specifically labeled with a monoclonal antibody that we have raised to a 110 kDa conical antigen in P. multimicronucleatum. This antibody labels a similar geometric cortical pattern in at least two species, P. multimicronucleatum and P. tetraurelia. Centrin-specific structures appear to be net-like in the above two species but show a more interrupted pattern in P. caudatum. The cytostomal cord is an essentially unbranched extension of the net-like infraciliary lattice and, like it, is centrin-specific. The cord has a unique association with the alveolar sacs which suggest these calcium-storing compartments contribute to the calcium fluxes required for contraction of the cord. A structural rather than a contractile function is favored for the striated bands, based solely on their morphology.     

Immunolocalization of actin in Paramecium cells.

We have selected a conserved immunogenic region from several actin genes of Paramecium, recently cloned in our laboratory, to prepare antibodies for Western blots and immunolocalization. According to cell fractionation analysis, most actin is structurebound. Immunofluorescence shows signal enriched in the cell cortex, notably around ciliary basal bodies (identified by anti-centrin antibodies), as well as around the oral cavity, at the cytoproct and in association with vacuoles (phagosomes) up to several mum in size. Subtle strands run throughout the cell body. Postembedding immunogold labeling/EM analysis shows that actin in the cell cortex emanates, together with the infraciliary lattice, from basal bodies to around trichocyst tips. Label was also enriched around vacuoles and vesicles of different size including "discoidal" vesicles that serve the formation of new phagosomes. By all methods used, we show actin in cilia. Although none of the structurally well-defined filament systems in Paramecium are exclusively formed by actin, actin does display some ordered, though not very conspicuous, arrays throughout the cell. F-actin may somehow serve vesicle trafficking and as a cytoplasmic scaffold. This is particularly supported by the postembedding/EM labeling analysis we used, which would hardly allow for any large-scale redistribution during preparation.     

Structure and Protein Composition of a Basal‐Body Scaffold (“Cage”) in the Hypotrich Ciliate Euplotes

Cilia on the ventral surface of the hypotrich ciliate Euplotes are clustered into polykinetids or compound ciliary organelles, such as cirri or oral membranelles, used in locomotion and prey capture. A single polykinetid may contain more than 150 individual cilia; these emerge from basal bodies held in a closely spaced array within a scaffold or framework structure that has been referred to as a basal‐body “cage”. Cage structures were isolated free of cilia and basal bodies; the predominant component of such cages was found on polyacrylamide gels to be a 45‐kDa polypeptide. Antisera were raised against this protein band and used for immunolocalizations at the light and electron microscope levels. Indirect immunofluorescence revealed the 45‐kDa polypeptide to be localized exclusively to the bases of the ventral polykinetids. Immunogold staining of thin sections of intact cells further localized this reactivity to filaments of a double‐layered dense lattice that appears to link adjoining basal bodies into ordered arrays within each polykinetid. Scanning electron microscopy of isolated cages reveals the lower or “basal” cage layer to be a fine lacey meshwork supporting the basal bodies at their proximal ends; adjoining basal bodies are held at their characteristic spacing by filaments of an upper or “medial” cage layer. The isolated cage thus resembles a miniature test‐tube rack, able to accommodate varying arrangements of basal‐body rows, depending on the particular type of polykinetid. Because of its clear and specific localization to the basal‐body cages in Euplotes, we have termed this novel 45‐kDa protein “cagein”.     

Rapid downregulation of the Ca2+-signal after exocytosis stimulation in Paramecium cells: essential role of a centrin-rich filamentous cortical network, the infraciliary lattice

We analysed in Paramecium tetraurelia cells the role of the infraciliary lattice, a cytoskeletal network containing numerous centrin isoforms tightly bound to large binding proteins, in the re-establishment of Ca2+ homeostasis following exocytosis stimulation. The wild type strain d4-2 has been compared with the mutant cell line Delta-PtCenBP1 which is devoid of the infraciliary lattice ("Delta-PtCenBP1" cells). Exocytosis is known to involve the mobilization of cortical Ca2+-stores and a superimposed Ca2+-influx and was analysed using Fura Red ratio imaging. No difference in the initial signal generation was found between wild type and Delta-PtCenBP1 cells. In contrast, decay time was greatly increased in Delta-PtCenBP1 cells particularly when stimulated, e.g., in presence of 1mM extracellular Ca2+, [Ca2+]o. Apparent halftimes of f/f0 decrease were 8.5 s in wild type and approximately 125 s in Delta-PtCenBP1 cells, requiring approximately 30 s and approximately 180 s, respectively, to re-establish intracellular [Ca2+] homeostasis. Lowering [Ca2+]o to 0.1 and 0.01 mM caused an acceleration of intracellular [Ca2+] decay to t(1/2)=33 s and 28 s, respectively, in Delta-PtCenBP1 cells as compared to 8.1 and 5.6, respectively, for wild type cells. We conclude that, in Paramecium cells, the infraciliary lattice is the most efficient endogenous Ca2+ buffering system allowing the rapid downregulation of Ca2+ signals after exocytosis stimulation.     

An Sfi1p-like centrin-binding protein mediates centrin-based Ca2+ -dependent contractility in Paramecium tetraurelia

The previous characterization and structural analyses of Sfi1p, a Saccharomyces cerevisiae centrin-binding protein essential for spindle pole body duplication, have suggested molecular models to account for centrin-mediated, Ca2+-dependent contractility processes (S. Li, A. M. Sandercock, P. Conduit, C. V. Robinson, R. L. Williams, and J. V. Kilmartin, J. Cell Biol. 173:867-877, 2006). Such processes can be analyzed by using Paramecium tetraurelia, which harbors a large Ca2+ -dependent contractile cytoskeletal network, the infraciliary lattice (ICL). Previous biochemical and genetic studies have shown that the ICL is composed of diverse centrin isoforms and a high-molecular-mass centrin-associated protein, whose reduced size in the démaillé (dem1) mutant correlates with defective organization of the ICL. Using sequences derived from the high-molecular-mass protein to probe the Paramecium genome sequence, we characterized the PtCenBP1 gene, which encodes a 460-kDa protein. PtCenBP1p displays six almost perfect repeats of ca. 427 amino acids (aa) and harbors 89 potential centrin-binding sites with the consensus motif LLX11F/LX2WK/R, similar to the centrin-binding sites of ScSfi1p. The smaller (260-kDa) protein encoded by the dem1 mutant PtCenBP1 allele comprises only two repeats of 427 aa and 46 centrin-binding sites. By using RNA interference and green fluorescent protein fusion experiments, we showed that PtCenBP1p forms the backbone of the ICL and plays an essential role in its assembly and contractility. This study provides the first in vivo demonstration of the role of Sfi1p-like proteins in centrin-mediated Ca2+-dependent contractile processes.     

Drosophila Centrosomin Protein is Required for Male Meiosis and Assembly of the Flagellar Axoneme

Centrosomes and microtubules play crucial roles during cell division and differentiation. Spermatogenesis is a useful system for studying centrosomal function since it involves both mitosis and meiosis, and also transformation of the centriole into the sperm basal body. Centrosomin is a protein localized to the mitotic centrosomes in Drosophila melanogaster. We have found a novel isoform of centrosomin expressed during spermatogenesis. Additionally, an anticentrosomin antibody labels both the mitotic and meiotic centrosomes as well as the basal body. Mutational analysis shows that centrosomin is required for spindle organization during meiosis and for organization of the sperm axoneme. These results suggest that centrosomin is a necessary component of the meiotic centrosomes and the spermatid basal body.     

A human centrosomal protein is immunologically related to basal body- associated proteins from lower eucaryotes and is involved in the nucleation of microtubules

Isolation of centrosomes from human cells has revealed a proteic pattern which is both complex and specific. As the most prominent structural element of centrosomes in animal cells, the centriole which is present as two copies, is a highly conserved structure, we have attempted to identify centrosomal proteins on the basis of immunocross-reaction with proteins identified in basal bodies from lower eucaryotes. We report that two antibodies, one raised against the Ca(+)-binding protein centrin (Salisbury, J. L., A. T. Baron, B. Surek, and M. Melkonian. 1984. J. Cell Biol. 99:962-970) and the other directed against a 230-kD protein isolated from the infraciliary cytoskeletal lattice of the protozoan Polyplastron m., decorate the centrosome of human cultured cells, and identify one of the major centrosomal components revealed as a doublet of 62/64 kD. Moreover the nucleation reaction of microtubules, which can be efficiently produced on isolated centrosomes, is blocked by the antibodies, a result which strongly implicates the 62/64-kD protein in this centrosomal activity. We also show that the 62/64-kD protein remains insoluble in conditions (0.5 M KI or 8 M urea) which are capable of extracting most of the centrosomal proteins. Immunocytochemical localization by EM of isolated centrosomes revealed the association of this 62/64-kD doublet with the intercentriolar link and the pericentriolar lattice. Our results suggest that conservation of structure in the centrosome from divergent organisms could be matched by conservation of proteins and activity, evidence for the maintenance of a specific function, which could involve Ca2+, associated with the microtubule organizing centers.     

Polypeptide composition,assembly and phosphorylation patterns of the photosystem II antenna system of Chlamydomonas reinhardtii

In recent years major progress has been made in describing the gene families that encode the polypeptides of the light-harvesting antenna system of photosystem II (PSII). At the same time, advances in the biochemical characterization of these antennae have been hampered by the high degree of similarity between the apoproteins. To help interpret the molecular results, we have re-examined the composition, the assembly and the phosphorylation patterns of the light-harvesting antenna of PSII (LHCII) in the green alga Chlamydomonas reinhardtii Dang, using a non-Tris SDS-PAGE system capable of resolving polypeptides that differ by as little as 200 daltons. Research to date has suggested that in C. reinhardtii the LHCII comprises just four polypeptides (p11, p13, p16 and p17), and CP29 and CP26 just one polypeptide each (p9 and p10, respectively), i.e. a total of six polypeptides. We report here that these antenna systems contain at least 15 polypeptides, 10 associated with LHCII, 3 with CP29, and 2 with CP26. All of these polypeptides have been positively identified by means of appropriate antibodies. We also demonstrate substantial heterogeneity to the pattern of in-vitro phosphorylation, with major differences found among members of closely spaced and immunologically related polypeptides. Most intriguing is the fact that the polypeptides that cross-react with the anti-type 2 LHCII antibodies of higher plants (p16, and to a lesser extent p11) are not phosphorylated, whereas in higher plants these are the most highly phosphorylated polypeptides. Also, unlike in higher plants, CP29 is heavily phosphorylated. Phosphorylation does not appear to have any effect on the mobility of polypeptides on fully denaturing SDS-PAGE gels. To learn more about the accumulation and organization of the light-harvesting polypeptides, we have also investigated a chlorophyll b-less mutant, cbn1-48. The LHCII is almost completely lost in this mutant, along with at least some LHCI. But the accumulation of CP29 and CP26 and their binding to PSII core complexes, is relatively unaffected. As expected, the loss of antenna polypeptides is accompanied by a reduction of the size of large reaction-center complexes. Following in-vitro phosphorylation the number of phosphorylated proteins is greatly increased in the mutant thylakoids compared to wildtype thylakoids. We present a model of the PSII antenna system to account for the new polypeptide complexity we have demonstrated.This work was supported by National Institute of Health grant GM22912 to L.A.S. We would like to thank Anastasios Melis for helpful discussions.     

FLAGELLAR APPARATUS STRUCTURE IS SIMILAR BUT NOT IDENTICAL IN VOLVULINA STEINII,EUDORINA ELEGANS,AND PLEODORINA ILLINOISENSIS (CHLOROPHYTA): IMPLICATIONS FOR THE “VOLVOCINE EVOLUTIONARY LINEAGE”1

The colonial and multicellular members of the Volvocales can be arranged in order of increasing size and complexity as the “volvocine series.” This series is often assumed to reflect an evolutionary progression. The flagellar apparatuses of previously examined algae are not consistent with a simple lineage. The flagellar apparatuses of Astrephomene gubernaculifera Pocock, Gonium pectorale Müller, Platydorina caudata Kofoid, Volvox rousseletii G. S. West, and V. carteri f. weismannia (Powers) Iyengar differ from one another, and there is no apparent progression inflagellar apparatus features from the simple to complex colonial forms. We examined the flagellar apparatuses of Volvulina steinii Playfair, Eudorina elegans Ehr., and Pleodorina illinoisensis Kofoid and found them to be similar to one another. The basal bodies are connected by a distal fiber that is offset to the anti side of the cell. Two microtubular rootlets originate on the inside of the basal bodies and extend toward the syn side. The other two rootlets are oriented perpendicular to the first two and are anti-parallel to each other. A coarsely striated component underlies the four-membered rootlets and extends to the basal bodies. A proximal fiber complex connects the two basal bodies. This complex consists of a branched striated component on the cis side of each basal body. One part extends toward the anti side of the cell, while the other extends into a fibrous component that runs between basal bodies. An additional structure extends in the anti direction from the trans side of each basal body. A fibrous component extends past one basal body in all four species. This component goes past the trans basal body in Volvulina steinii and the cis basal body in E. elegans and P. illinoisensis. The flagellar apparatuses of these organisms are similar to those of G. pectorale and Volvox carteri but different from the other colonial volvocalean algae examined. The algae examined in this study plus G. pectorale and V. carteri probably share a common evolutionary history that postdates the transition from the unicellular to colonial habit. Such a shared evolutionary history is a requirement of the volvocine hypothesis. However, we have not observed progressive changes in the flagellar apparatus correlated with increasing cell number, differentiation, and sexual specialization. Thus, it is possible, but not certain, that G. pectorale, Volvulina steinii, E. elegans, P. illinoisensis, and Volvox carteri may form part of a volvocine lineage.     

CORTICAL ULTRASTRUCTURE OF PARAMECIUM AURELIA : Studies on Isolated Pellicles

Two methods have been devised for the isolation of large quantities of purified pellicles (cortical layers) of Paramecium aurelia. Pellicles isolated by both procedures, when examined by electron microscopy, were found to contain ciliary basal bodies, two types of cortical membranes, ribbons of microtubules, kinetodesmal fibers, and elements of the infraciliary lattice system. By electron microscopy, the extent of preservation of the various cortical structures when pellicles are isolated by each method has been characterized. Pellicles isolated in both ways have been utilized to investigate cortical morphology of Paramecium. Both phase-contrast and electron microscopic observations have been made. Many new ultrastructural features were observed and are reported herein. An interesting result of this study is the discovery in stock CD that the structure of cortical territories (the territory is the functional unit of cortical morphogenesis and physiology) may vary within a single organism. Features which show variation include number of parasomal sacs, microtubular ribbons, and basal bodies (and therefore cilia) per territory, number of microtubules per ribbon, and length of kinetodesmal fibers. The possible significance of these variations, with respect to territory replication, is discussed. In addition, preliminary observations on the solubility of various cortical organelles in the presence of a number of protein-denaturing agents are reported.     

Energetics of New Zealand's temperate parrots

Abstract

As in most parrots (Order: Psittaciformes) studied, New Zealand species have, independent of body mass, high basal rates of metabolism, low thermal conductances, and precisely regulated body temperatures. An analysis of covariance showed that basal rate in parrots correlates with body mass and thermal climate; temperate species have basal rates that are 21% higher than those of tropical species; and New Zealand's parrot have basal rates 32% higher than expected from body mass. Present information suggests that basal rate in parrots appears to be correlated neither with water availability in the environment nor with food habits. High basal rates in parrots are associated with large pectoral muscle masses. The very small pectoral muscle masses of the kakapo, Strigops habroptilus, predict that this flightless parrot has a low basal rate.     

FINE STRUCTURE OF MEMBRANOUS AND MICROFIBRILLAR SYSTEMS IN THE CORTEX OF PARAMECIUM CAUDATUM

An electron microscope study of the cortex of Paramecium caudatum has revealed new details pertinent to several unresolved problems. The lateral boundaries of the alveoli do not regularly follow the crests of the polygonal ridges and thus their staining with silver cannot account for the external lattice seen by light microscopists. A granulo-fibrillar material is present, however, within the peaks of the ridges, which would account for the external lattice if so stained. Perforations are present between adjacent alveoli which make the whole mosaic of alveolar sacs within the cell's cortex continuous—both the membranes and the lumen. A microfibrillar system exhibiting a cross-striated pattern lies in the superficial cortex. These bands are inserted at their ends in the epiplasm and have a fine structure and arrangement suggesting a muscular function. The infraciliary lattice is a branching system of fibers with electron-opaque posts at the center of each branching locus. This system is distinct from the striated bands in morphology and in space. The epiplasm is discontinuous along the crests of the ridges, which may account for the pellicles' disposition to tear along these lines. A three-dimensional drawing is presented to show the interrelationships between the above membranous and microfibrillar systems.     

A γ-tubulin that associates specifically with centrioles in HeLa cells and the basal body complex in Chlamydomonas

γ-Tubulin is a putative component of microtubule initiating material. To further explore its subcellular distribution in plant and animal cells, we have raised a polyclonal antibody, Rb27, directed towards a conserved region (EEFATEGTDRKDVFFY) of the γ-tubulin molecule. Immunoblotting of cell protein extracts with Rb27 reveals a polypeptide band of M_r 49 kD in HeLa and a 58 kD band in Chlamydomonas. Although these polypeptides are comparable in size to forms of γ-tubulin detected previously in mammalian and plant protein extracts by other antibodies to γ-tubulin, by immunofluorescence microscopy Rb27 gives localization patterns not previously seen. It localizes specifically with the centrioles in HeLa cells and with the basal body complex in Chlamydomonas. Other γ-tubulin antibodies label pericentriolar material. Because of the similarities in the size of the polypeptides recognized by our and other γ-tubulin antibodies, and a restricted co-localization with known microtubule-organizing centres in evolutionarily distant organisms, we propose that Rb27 recognizes a novel conserved γ-tubulin isotype.     

Rumen ciliates from Tanzanian short horn zebu cattle,Bos taurus indicus,and the infraciliature of Entodinium palmare n.sp. and Enoploplastron stokyi ()

Rumen ciliates of ten Tanzanian short horn zebu cattle were examined. A total of 15 genera and 46 species were identified, including a new Entodinium species. The ciliate density was 22.2×10⁴ ml^?1. The number of species per host and the diversity index showed high values, 33.8 and 2.80, respectively. Rumen ciliates had a low percentage composition of the genus Entodinium (7.0–25.0%) and a slightly higher percentage composition of the genera, Eudiplodinium (19.3%), Diplodinium (14.1%), and Ostracodinium (13.1%). Entodinium palmare n.sp., Eudiplodinium kenyensis, and Enoploplastron stokyi were found in all cattle examined. The former two species have been found only in African zebu cattle. Entodinium palmare n. sp. has a characteristic right surface of the body like the “palm of a hand” because of the concave part on the postero-dorsal part of the body, and has the same pattern of infraciliary bands as in other Entodinium species. Enoploplastron stokyi has a characteristic pattern of infraciliary bands analogous to those of Epidinium ecaudatum and Ostracodinium mammosum; with the right side of the adoral polybrachykinety gradually tapering and a slender short vestibular polybrachykinety.     

Structural inheritance in Paramecium: ultrastructural evidence for basal body and associated rootlets polarity transmission through binary fission

Abstract One main difference between basal bodies and centrioles resides in the expression of their polarity: centrioles display a structural nine‐fold radial symmetry, whereas basal bodies express a circumferential polarity, thanks to their asymmetric set of rootlets. The origin of this polarity during organelle duplication still remains under debate: is it intrinsic to the nine‐fold structure itself (i.e. the nine microtubular triplets are not equivalent) or imposed by its immediate environment at time of assembly? We have reinvestigated this problem using the Ciliate Paramecium, in which the pattern of basal body duplication is well known. In this cell, all basal bodies produced within ciliary rows appear immediately anterior to parental ones. Observations on cells fixed with the tannic acid protocol suggest that, to be competent for basal body assembly, parental basal bodies have to be individually associated with a complete set of rootlets (monokinetid structure). During pro‐basal body assembly, full microtubular triplets were detected according to a random circumferential sequence; during the whole process, the new basal body and its associated rootlets maintained structural relations with the parental monokinetid structure by way of specific links. These results strongly suggest that basal body and associated rootlets (kinetid) polarity is driven by its immediate environment and provide a basis for the structural heredity property observed by Sonneborn some decades ago.     

Comparison of the three-dimensional organization of unextracted and Triton-extracted human neutrophilic polymorphonuclear leukocytes

The three-dimensional organization of the cytoplasm of randomly migrating neutrophils was studied by stereo high-voltage electron microscopy. Examination of whole-mount preparations reveals with unusual clarity the structure of the cytoplasmic ground substance and cytoskeletal organization; similar clarity is not observed in conventional sections. An extensive three-dimensional network of fine filaments (microtrabeculae) approximately 7 to 17 nm in diameter extends throughout the cytoplasm and between the two cell cortices; it also comprises the membrane ruffles and filopodia. The granules are dispersed within the lattice and are surrounded by microtrabeculae. The lattice appears to include dense foci from which the microtrabeculae emerge. Triton X-100 dissolves the plasma membrane, most of the granules, and many of the microtrabecular strands and leaves as a more stable structure a cytoskeletal network composed of various filaments and microtubules. Heavy meromyosin-subfragment 1 (S1) decoration discloses actin filaments as the major filamentous component present in membrane ruffles and filopodia. Actin filaments, extending from the leading edge of the cells, are of uniform polarity, with arrowheads pointing towards the cell body. Likewise, the filaments forming the core of filopodia have the barbed end distal. End-to-side associations of actin filaments as well as fine filaments (2--3 nm) which are not decorated with S1 and link actin filaments are observed. The ventral cell cortex includes numerous substrate-associated dense foci with actin filaments radiating from the dense center. Virtually all the microtubules extend from the centrosome. An average of 35 +/- 7 microtubules originate near the pair of centrioles and radiate towards the cell periphery; microtubule fragments are rare. Intermediate filaments form an open network of single filaments in the perinuclear space. Comparison of Triton-extracted and unextracted cells suggest that many of the filamentous strands seen in unextracted cells have as a core a stable actin filament.     

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.     

TAC102 Is a Novel Component of the Mitochondrial Genome Segregation Machinery in Trypanosomes

Trypanosomes show an intriguing organization of their mitochondrial DNA into a catenated network, the kinetoplast DNA (kDNA). While more than 30 proteins involved in kDNA replication have been described, only few components of kDNA segregation machinery are currently known. Electron microscopy studies identified a high-order structure, the tripartite attachment complex (TAC), linking the basal body of the flagellum via the mitochondrial membranes to the kDNA. Here we describe TAC102, a novel core component of the TAC, which is essential for proper kDNA segregation during cell division. Loss of TAC102 leads to mitochondrial genome missegregation but has no impact on proper organelle biogenesis and segregation. The protein is present throughout the cell cycle and is assembled into the newly developing TAC only after the pro-basal body has matured indicating a hierarchy in the assembly process. Furthermore, we provide evidence that the TAC is replicated de novo rather than using a semi-conservative mechanism. Lastly, we demonstrate that TAC102 lacks an N-terminal mitochondrial targeting sequence and requires sequences in the C-terminal part of the protein for its proper localization.