Azadirachtin Disrupts Formation of Organised Microtubule Arrays During Microgametogenesis of Plasmodium Berghei

The Journal of Eukaryotic Microbiology. Nov-Dec, 2002  |  Pubmed ID: 12503686

Transmission of malaria parasites from vertebrate blood to the mosquito vector depends critically on the differentiation of the gametocytes into gametes. This occurs in response to environmental stimuli encountered by the parasite in the mosquito bloodmeal. Male gametogenesis involves three rounds of DNA replication and endomitosis, and the assembly de novo of 8 motile axonemes. Azadirachtin, a plant limnoid and insecticide with an unkown mode of action, specifically inhibits the release of motile gametes from activated microgametocytes but does not inhibit growth and replication of a sexual blood stages. We have combined confocal laser scanning microscopy and transmission electron microscopy to examine the effect of azadirachtin on the complex reorganisation of the microtubule cytoskeleton during gametogenesis in Plasmodium berghei. Neither the replication of the genome nor the ability of tubulin monomers to assemble into microtubules upon gametocyte activation were prevented by azadirachtin. However, the drug interfered with the formation of mitotic spindles and with the assembly of microtubules into typical axonemes. Our observations suggest that azadarachtin specifically disrupts the patterning of microtubules into more complex structures, such as mitotic spindles and axonemes.

Mining the Plasmodium Genome Database to Define Organellar Function: What Does the Apicoplast Do?

Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. Jan, 2002  |  Pubmed ID: 11839180

Apicomplexan species constitute a diverse group of parasitic protozoa, which are responsible for a wide range of diseases in many organisms. Despite differences in the diseases they cause, these parasites share an underlying biology, from the genetic controls used to differentiate through the complex parasite life cycle, to the basic biochemical pathways employed for intracellular survival, to the distinctive cell biology necessary for host cell attachment and invasion. Different parasites lend themselves to the study of different aspects of parasite biology: Eimeria for biochemical studies, Toxoplasma for molecular genetic and cell biological investigation, etc. The Plasmodium falciparum Genome Project contributes the first large-scale genomic sequence for an apicomplexan parasite. The Plasmodium Genome Database (http://PlasmoDB.org) has been designed to permit individual investigators to ask their own questions, even prior to formal release of the reference P. falciparum genome sequence. As a case in point, PlasmoDB has been exploited to identify metabolic pathways associated with the apicomplexan plastid, or 'apicoplast' - an essential organelle derived by secondary endosymbiosis of an alga, and retention of the algal plastid.

Actin Filament Turnover Removes Bundles from Drosophila Bristle Cells

Journal of Cell Science. Feb, 2002  |  Pubmed ID: 11861770

Drosophila bristle cells form enormous extensions that are supported by equally impressive scaffolds of modular, polarized and crosslinked actin filament bundles. As the cell matures and support is taken over by the secreted cuticle, the actin scaffold is completely removed. This removal begins during cell elongation and proceeds via an orderly series of steps that operate on each module. Using confocal and electron microscopy, we found that the approximately 500-filament modules are fractured longitudinally into 25-50-filament subbundles, indicating that module breakdown is the reverse of assembly. Time-lapse confocal analysis of GFP-decorated bundles in live cells showed that modules were shortened by subunit removal from filament barbed ends, again indicating that module breakdown is the reverse of assembly. Module shortening takes place at a fairly slow rate of approximately 1microm/hour, implying that maximally crosslinked modules are not rapidly depolymerized. Barbed-end depolymerization was prevented with jasplakinolide and accelerated with cycloheximide, indicating that barbed-end maintenance requires continuous protein synthesis. Subbundle adhesion was lost in the presence of cytochalasin, indicating that continuous actin polymerization is required. Thus, these polarized actin filament bundles are dynamic structures that require continuous maintenance owing to protein and actin filament turnover. We propose that after cell elongation, maintenance falls behind turnover, resulting in the removal of this modular cytoskeleton.

Cysteine and Serine Protease Inhibitors Block Intracellular Development and Disrupt the Secretory Pathway of Toxoplasma Gondii

Microbes and Infection / Institut Pasteur. Feb, 2002  |  Pubmed ID: 11880042

A number of cysteine and serine protease inhibitors blocked the intracellular growth and replication of Toxoplasma gondii tachyzoites. Most of these inhibitors caused only minor alterations to parasite morphology irrespective of the effects on the host cells. However, three, cathepsin inhibitor III, TPCK and subtilisin inhibitor III, caused extensive swelling of the secretory pathway of the parasite (i.e. the ER, nuclear envelope, and Golgi complex), caused the breakdown of the parasite surface membrane, and disrupted rhoptry formation. The disruption of the secretory pathway is consistent with the post-translational processing of secretory proteins in Toxoplasma, and with the role of proteases in the maturation/activation of secreted proteins in general. Interestingly, while all parasites in an individual vacuole (the clonal progeny of a single invading parasite) were similarly affected, parasites in different vacuoles in the same host cell showed different responses to these inhibitors. Such observations imply that there are major differences in the biochemistry/physiology between tachyzoites within different vacuoles and argue that adverse effects on the host cell are not always responsible for changes in the parasite. Treatment of established parasites also leads to an accumulation of abnormal materials in the parasitophorous vacuole implying that materials deposited into the vacuole normally undergo proteolytic modification or degradation. Despite the often extensive morphological changes, nothing resembling lysosomal bodies was seen in any treated parasites, consistent with previous observations showing that mother cell organelles are not recycled by any form of autophagic-lysosomal degradation, although the question of how the parasite recycles these organelles remains unanswered.

Cell Invasion by Theileria Sporozoites

Trends in Parasitology. Jan, 2003  |  Pubmed ID: 12488213

Coalignment of Plasma Membrane Channels and Protrusions (fibripositors) Specifies the Parallelism of Tendon

The Journal of Cell Biology. May, 2004  |  Pubmed ID: 15159420

The functional properties of tendon require an extracellular matrix (ECM) rich in elongated collagen fibrils in parallel register. We sought to understand how embryonic fibroblasts elaborate this exquisite arrangement of fibrils. We show that procollagen processing and collagen fibrillogenesis are initiated in Golgi to plasma membrane carriers (GPCs). These carriers and their cargo of 28-nm-diam fibrils are targeted to previously unidentified plasma membrane (PM) protrusions (here designated "fibripositors") that are parallel to the tendon axis and project into parallel channels between cells. The base of the fibripositor lumen (buried several microns within the cell) is a nucleation site of collagen fibrillogenesis. The tip of the fibripositor is the site of fibril deposition to the ECM. Fibripositors are absent at postnatal stages when fibrils increase in diameter by accretion of extracellular collagen, thereby maintaining parallelism of the tendon. Thus, we show that the parallelism of tendon is determined by the late secretory pathway and interaction of adjacent PMs to form extracellular channels.

Phosphorylated BRCA1 is Predominantly Located in the Nucleus and Mitochondria

Molecular Biology of the Cell. Feb, 2005  |  Pubmed ID: 15591126

Multiple copies of the mitochondrial genome in eukaryotic cells are organized into protein-DNA complexes called nucleoids. Mitochondrial genome repair mechanisms have been reported, but they are less well characterized than their nuclear counterparts. To expand our knowledge of mitochondrial genome maintenance, we have studied the localization of the BRCA1 protein, known to be involved in nuclear repair pathways. Our confocal and immunoelectron microscopy results show that BRCA1 is present in mitochondria of several human cancer cell lines and in primary breast and nasal epithelial cells. BRCA1 localization in mitochondria frequently overlapped that of nucleoids. Small interfering RNA-mediated knockdown of BRCA1 in human cancer cells (confirmed by Western blot) results in decreased nuclear, cytoplasmic, and mitochondrial staining after immunofluorescence microscopy, establishing the specificity of the BRCA1 immunolabeling. Furthermore, using cell fractionation, dephosphorylation, and enzyme protection experiments, we show that a 220-kDa phosphorylated isoform of BRCA1 is enriched in mitochondrial and nuclear fractions but reduced in cytoplasmic subcellular fractions. Submitochondrial fractionation confirmed the presence of BRCA1 protein in isolated mitoplasts. Because phosphorylation of BRCA1 and subsequent changes in subcellular localization are known to follow DNA damage, our data support a universal role for BRCA1 in the maintenance of genome integrity in both mitochondria and nucleus.

The Parkin Co-regulated Gene Product, PACRG, is an Evolutionarily Conserved Axonemal Protein That Functions in Outer-doublet Microtubule Morphogenesis

Journal of Cell Science. Dec, 2005  |  Pubmed ID: 16278296

Eukaryotic cilia and flagella are highly conserved structures composed of a canonical 9+2 microtubule axoneme. Comparative genomics of flagellated and non-flagellated eukaryotes provides one way to identify new putative flagellar proteins. We identified the Parkin co-regulated gene, or PACRG, from such a screen. Male mice deficient in PACRG are sterile, but its function has been little explored. The flagellated protozoan parasite Trypanosoma brucei possesses two homologues of PACRG. We performed RNA interference knockdown experiments of the two genes independently and both together. Simultaneous ablation of both proteins produced slow growth and paralysis of the flagellum with consequent effects on organelle segregation. Moreover, using transmission electron microscopy, structural defects were seen in the axoneme, with microtubule doublets missing from the canonical 9+2 formation. The occurrence of missing doublets increased toward the distal end of the flagellum and sequential loss of doublets was observed along individual axonemes. GFP fusion proteins of both PACRG homologues localised along the full length of the axoneme. Our results provide the first evidence for PACRG function within the axoneme, where we suggest that PACRG acts to maintain functional stability of the axonemal outer doublets of both motile and sensory cilia and flagella.

Adoptive Transfer of Myelin Basic Protein-induced Experimental Autoimmune Encephalomyelitis Between SJL and B10.S Mice: Correlation of Priming Milieus with Susceptibility and Resistance Phenotypes

Journal of Neuroimmunology. Apr, 2006  |  Pubmed ID: 16480778

To study the mechanisms of EAE resistance, we directly transfer MBP-primed EAE-susceptible SJL lymph node cells into EAE-resistant B10.S recipients and vice versa. These transfers were unsuccessful because of strong alloreactivity between the two strains. Neonatal tolerance to SJL antigens was induced in B10.S mice and in these hosts MBP-primed SJL lymph node cells readily induce development of adoptive EAE. Conversely, transfer of MBP-primed B10.S lymph node cells into EAE-susceptible (SJL x B10.S)F1 recipients failed to induce EAE. These results are consistent with the notion that the priming milieus in the donor mice affect the expression of susceptible and resistant phenotypes.

Flagellar Motility is Required for the Viability of the Bloodstream Trypanosome

Nature. Mar, 2006  |  Pubmed ID: 16525475

The 9 + 2 microtubule axoneme of flagella and cilia represents one of the most iconic structures built by eukaryotic cells and organisms. Both unity and diversity are present among cilia and flagella on the evolutionary as well as the developmental scale. Some cilia are motile, whereas others function as sensory organelles and can variously possess 9 + 2 and 9 + 0 axonemes and other associated structures. How such unity and diversity are reflected in molecular repertoires is unclear. The flagellated protozoan parasite Trypanosoma brucei is endemic in sub-Saharan Africa, causing devastating disease in humans and other animals. There is little hope of a vaccine for African sleeping sickness and a desperate need for modern drug therapies. Here we present a detailed proteomic analysis of the trypanosome flagellum. RNA interference (RNAi)-based interrogation of this proteome provides functional insights into human ciliary diseases and establishes that flagellar function is essential to the bloodstream-form trypanosome. We show that RNAi-mediated ablation of various proteins identified in the trypanosome flagellar proteome leads to a rapid and marked failure of cytokinesis in bloodstream-form (but not procyclic insect-form) trypanosomes, suggesting that impairment of flagellar function may provide a method of disease control. A postgenomic meta-analysis, comparing the evolutionarily ancient trypanosome with other eukaryotes including humans, identifies numerous trypanosome-specific flagellar proteins, suggesting new avenues for selective intervention.

Structural Asymmetry and Discrete Nucleic Acid Subdomains in the Trypanosoma Brucei Kinetoplast

Molecular Microbiology. Jun, 2007  |  Pubmed ID: 17511811

The mitochondrial genome of Trypanosoma brucei is contained in a specialized structure termed the kinetoplast. Kinetoplast DNA (kDNA) is organized into a concatenated network of mini and maxicircles, positioned at the base of the flagellum, to which it is physically attached. Here we have used electron microscope cytochemistry to determine structural and functional domains involved in replication and segregation of the kinetoplast. We identified two distinct subdomains within the kinetoflagellar zone (KFZ) and show that the unilateral filaments are composed of distinct inner and outer filaments. Ethanolic phosphotungstic acid (E-PTA) and EDTA regressive staining indicate that basic proteins and DNA are major constituents of the inner unilateral filaments adjoining the kDNA disc. This evidence for an intimate connection of the unilateral filaments in the KFZ with DNA provides support for models of minicircle replication involving vectorial export of free minicircles into the KFZ. Unexpectedly however, detection of DNA in the KFZ throughout the cell cycle suggests that other processes involving kDNA occur in this domain. We also describe a hitherto unrecognized, intramitochondrial, filamentous structure rich in basic proteins that links the kDNA discs during their segregation and is maintained between them for an extended period of the cell cycle.

An Essential Quality Control Mechanism at the Eukaryotic Basal Body Prior to Intraflagellar Transport

Traffic (Copenhagen, Denmark). Oct, 2007  |  Pubmed ID: 17645436

Constructing a eukaryotic cilium/flagellum is a demanding task requiring the transport of proteins from their cytoplasmic synthesis site into a spatially and environmentally distinct cellular compartment. The clear potential hazard is that import of aberrant proteins could seriously disable cilia/flagella assembly or turnover processes. Here, we reveal that tubulin protein destined for incorporation into axonemal microtubules interacts with a tubulin cofactor C (TBCC) domain-containing protein that is specifically located at the mature basal body transitional fibres. RNA interference-mediated ablation of this protein results in axonemal microtubule defects but no effect on other microtubule populations within the cell. Bioinformatics analysis indicates that this protein belongs to a clade of flagellum-specific TBCC-like proteins that includes the human protein, XRP2, mutations which lead to certain forms of the hereditary eye disease retinitis pigmentosa. Taken with other observations regarding the role of transitional fibres in cilium/flagellum assembly, we suggest that a localized protein processing capacity embedded at transitional fibres ensures the 'quality' of tubulin imported into the cilium/flagellum, and further, that loss of a ciliary/flagellar quality control capability may underpin a number of human genetic disorders.

The Hydrocephalus Inducing Gene Product, Hydin, Positions Axonemal Central Pair Microtubules

BMC Biology. 2007  |  Pubmed ID: 17683645

Impairment of cilia and flagella function underlies a growing number of human genetic diseases. Mutations in hydin in hy3 mice cause lethal communicating hydrocephalus with early onset. Hydin was recently identified as an axonemal protein; however, its function is as yet unknown.

High Cell Surface Expression of CD4 Allows Distinction of CD4(+)CD25(+) Antigen-specific Effector T Cells from CD4(+)CD25(+) Regulatory T Cells in Murine Experimental Autoimmune Encephalomyelitis

Journal of Neuroimmunology. Dec, 2007  |  Pubmed ID: 17920698

Analysis of T regulatory cells (Treg) and T effector cells (Teff) in experimental autoimmune encephalomyelitis is complicated by the fact that both cell types express CD4 and CD25. We demonstrate that encephalitogenic T cells, following antigen recognition, up-regulate cell surface expression of CD4. The CD4(high) sub-population contains all of the antigen response as shown by proliferation and cytokine secretion, and only these cells are capable of transferring EAE to naive animals. On the other hand, a FACS separable CD25(+) sub-population of cells displayed consistent levels of CD4 prior to and after antigen stimulation. These cells displayed characteristics of Treg, such as expressing high levels of the Foxp3 gene and the ability to suppress mitogenic T cell responses.

Anti-biotin Antibodies Offer Superior Organelle-specific Labelling of Mitochondria over Avidin or Streptavidin

Methods in Molecular Biology (Clifton, N.J.). 2008  |  Pubmed ID: 18287658

A number of endogenously biotinylated proteins are found in both cytosol and mitochondria of mammalian cells from many tissues, including liver, spleen, pancreas, kidney, and intestine. Therefore, caution should be taken when using the biotin detection system. Endogenous biotin can interfere with staining systems that employ the use of biotin-avidin- or biotin-streptavidin-based detection systems and may therefore result in high, non-specific background staining. Here, we show that this endogenous biotin reactivity can be deliberately exploited and used as a specific mitochondrial marker in both light and electron microscopy as well as for identifying mitochondrial fractions on Western blot.

Three-dimensional Cellular Architecture of the Flagellar Pocket and Associated Cytoskeleton in Trypanosomes Revealed by Electron Microscope Tomography

Journal of Cell Science. Apr, 2009  |  Pubmed ID: 19299460

This study uses electron tomography linked to a variety of other EM methods to provide an integrated view of the flagellar pocket and basal body area of the African trypanosome procyclic trypomastigote. We reveal the pocket as an asymmetric membranous 'balloon' with two boundary structures. One of these - the collar - defines the flagellum exit point. The other defines the entry point of the flagellum into the pocket and consists of both an internal transitional fibre array and an external membrane collarette. A novel set of nine radial fibres is described in the basal body proximal zone. The pocket asymmetry is invariably correlated with the position of the probasal body and Golgi. The neck region, just distal to the flagellum exit site, is a specialised area of membrane associated with the start of the flagellum attachment zone and signifies the point where a special set of four microtubules, nucleated close to the basal bodies, joins the subpellicular array. The neck region is also associated with the single Golgi apparatus of the cell. The flagellar exit point interrupts the subpellicular microtubule array with discrete endings of microtubules at the posterior side. Overall, our studies reveal a highly organised, yet dynamic, area of cytoplasm and will be informative in understanding its function.

Perturbation of Phosphatidylethanolamine Synthesis Affects Mitochondrial Morphology and Cell-cycle Progression in Procyclic-form Trypanosoma Brucei

Molecular Microbiology. May, 2009  |  Pubmed ID: 19400804

Phosphatidylethanolamine (PE) and phosphatidylcholine (PC) are the two major constituents of eukaryotic cell membranes. In the protist Trypanosoma brucei, PE and PC are synthesized exclusively via the Kennedy pathway. To determine which organelles or processes are most sensitive to a disruption of normal phospholipid levels, the cellular consequences of a decrease in the levels of PE or PC, respectively, were studied following RNAi knock-down of four enzymes of the Kennedy pathway. RNAi against ethanolamine-phosphate cytidylyltransferase (ET) disrupted mitochondrial morphology and ultrastructure. Electron microscopy revealed alterations of inner mitochondrial membrane morphology, defined by a loss of disk-like cristae. Despite the structural changes in the mitochondrion, the cells maintained oxidative phosphorylation. Our results indicate that the inner membrane morphology of T. brucei procyclic forms is highly sensitive to a decrease of PE levels, as a change in the ultrastructure of the mitochondrion is the earliest phenotype observed after RNAi knock-down of ET. Interference with phospholipid synthesis also impaired normal cell-cycle progression. ET RNAi led to an accumulation of multinucleate cells. In contrast, RNAi against choline-/ethanolamine phosphotransferase, which affected PC as well as PE levels, caused a cell division phenotype characterized by non-division of the nucleus and production of zoids.

Peptide-major Histocompatibility Complex Dimensions Control Proximal Kinase-phosphatase Balance During T Cell Activation

The Journal of Biological Chemistry. Sep, 2009  |  Pubmed ID: 19628870

T cell antigen recognition requires binding of the T cell receptor (TCR) to a complex between peptide antigen and major histocompatibility complex molecules (pMHC), and this recognition occurs at the interface between the T cell and the antigen-presenting cell. The TCR and pMHC molecules are small compared with other abundant cell surface molecules, and it has been suggested that small size is functionally important. We show here that elongation of both mouse and human MHC class I molecules abrogates T cell antigen recognition as measured by cytokine production and target cell killing. This elongation disrupted tyrosine phosphorylation and Zap70 recruitment at the contact region without affecting TCR or coreceptor binding. Contact areas with elongated forms of pMHC showed an increase in intermembrane distance and less efficient segregation of CD3 from the large tyrosine phosphatase CD45. These findings demonstrate that T cell antigen recognition is strongly dependent on pMHC size and are consistent with models of TCR triggering requiring segregation or mechanical pulling of the TCR.

Cell Morphogenesis of Trypanosoma Brucei Requires the Paralogous, Differentially Expressed Calpain-related Proteins CAP5.5 and CAP5.5V

Protist. Nov, 2009  |  Pubmed ID: 19656721

Proteins from the calpain super-family are involved in developmentally- and environmentally-regulated re-modelling of the eukaryotic cytoskeleton and the dynamic organisation of signal transduction cascades. In trypanosomatid parasites, calpain-related gene families are unusually large, but we have little insight into the functional roles played by these molecules during trypanosomatid lifecycles. Here we report that CAP5.5, a cytoskeletal calpain-related protein subject to strict stage-specific expression in the sleeping sickness parasite Trypanosoma brucei, is essential and required for correct cell morphogenesis of procyclic (tsetse mid-gut stage) T. brucei. Striking consequences of CAP5.5 RNA interference are the loss of protein from the posterior cell-end, organelle mis-positioning giving rise to aberrant cytokinesis, and disorganisation of the sub-pellicular microtubules that define trypanosome cell shape. We further report that the stage-specificity of CAP5.5 expression can be explained by the presence of a paralogue, CAP5.5V, which is required for cell morphogenesis in bloodstream T. brucei; RNAi against this paralogous protein results in a qualitatively similar phenotype to that described for procyclic CAP5.5 RNAi mutants. By comparison to recently described phenotypes for other procyclic trypanosome RNAi mutants, likely functions for CAP5.5 and CAP5.5V are discussed.

A Protocol for Patients with Cardiovascular Implantable Devices Undergoing Magnetic Resonance Imaging (MRI): Should Defibrillation Threshold Testing Be Performed Post-(MRI)

Journal of Interventional Cardiac Electrophysiology : an International Journal of Arrhythmias and Pacing. Jun, 2010  |  Pubmed ID: 20111895

Magnetic resonance imaging (MRI) in patients with Cardiovascular Implantable Electronic Devices (CIED) has not been approved by the Food and Drug Administration. Recent data suggests MRI as a relative rather than absolute contraindication in CIED patients. Recently, the American Heart Association has recommended defibrillation threshold testing (DFTT) in implantable cardioverter defibrillator (ICD) patients undergoing MRI. We evaluated the feasibility and safety of a protocol for MRI in CIED patients, incorporating the new recommendations on DFTT.

Beyond 9+0: Noncanonical Axoneme Structures Characterize Sensory Cilia from Protists to Humans

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. Sep, 2010  |  Pubmed ID: 20371625

The intracellular amastigote stages of parasites such as Leishmania are often referred to as aflagellate. They do, however, possess a short axoneme of cryptic function. Here, our examination of the structure of this axoneme leads to a testable hypothesis of its role in the cell biology of pathogenicity. We show a striking similarity between the microtubule axoneme structure of the Leishmania mexicana parasite infecting a macrophage and vertebrate primary cilia. In both, the 9-fold microtubule doublet symmetry is broken by the incursion of one or more microtubule doublets into the axoneme core, giving rise to an architecture that we term here the 9v (variable) axoneme. Three-dimensional reconstructions revealed that no particular doublet initiated the symmetry break, and moreover it often involved 2 doublets. The tip of the L. mexicana flagellum was frequently intimately associated with the macrophage vacuole membrane. We propose that the main function of the amastigote flagellum is to act as a sensory organelle with important functions in host-parasite interactions and signaling in the intracellular stage of the L. mexicana life cycle.

Basal Body Movements Orchestrate Membrane Organelle Division and Cell Morphogenesis in Trypanosoma Brucei

Journal of Cell Science. Sep, 2010  |  Pubmed ID: 20682637

The defined shape and single-copy organelles of Trypanosoma brucei mean that it provides an excellent model in which to study how duplication and segregation of organelles is interfaced with morphogenesis of overall cell shape and form. The centriole or basal body of eukaryotic cells is often seen to be at the centre of such processes. We have used a combination of electron microscopy and electron tomography techniques to provide a detailed three-dimensional view of duplication of the basal body in trypanosomes. We show that the basal body duplication and maturation cycle exerts an influence on the intimately associated flagellar pocket membrane system that is the portal for secretion and uptake from this cell. At the start of the cell cycle, a probasal body is positioned anterior to the basal body of the existing flagellum. At the G1-S transition, the probasal body matures, elongates and invades the pre-existing flagellar pocket to form the new flagellar axoneme. The new basal body undergoes a spectacular anti-clockwise rotation around the old flagellum, while its short new axoneme is associated with the pre-existing flagellar pocket. This rotation and subsequent posterior movements results in division of the flagellar pocket and ultimately sets parameters for subsequent daughter cell morphogenesis.

T Cells That Trigger Acute Experimental Autoimmune Encephalomyelitis Also Mediate Subsequent Disease Relapses and Predominantly Produce IL-17

Journal of Neuroimmunology. Jan, 2011  |  Pubmed ID: 20826011

Earlier studies showed that donor T cells that initiated a murine adoptive EAE persisted in the CNS of the recipients throughout the subsequent relapsing cycles. To clarify the functions of the persistent donor T cells in EAE relapsing disease, anti-Thy-1 antibodies were used to deplete these cells. Results showed that such treatment abrogated subsequent relapsing cycles in these animals. In addition, it was evident that a shift in cytokine profile occurred during acute and relapsing disease phases. These results unambiguously support the appropriateness of targeting T cells with specificity for the priming antigen in design of therapeutic approaches for MS.

Molecular and Structural Basis of ESCRT-III Recruitment to Membranes During Archaeal Cell Division

Molecular Cell. Jan, 2011  |  Pubmed ID: 21255729

Members of the crenarchaeal kingdom, such as Sulfolobus, divide by binary fission yet lack genes for the otherwise near-ubiquitous tubulin and actin superfamilies of cytoskeletal proteins. Recent work has established that Sulfolobus homologs of the eukaryotic ESCRT-III and Vps4 components of the ESCRT machinery play an important role in Sulfolobus cell division. In eukaryotes, several pathways recruit ESCRT-III proteins to their sites of action. However, the positioning determinants for archaeal ESCRT-III are not known. Here, we identify a protein, CdvA, that is responsible for recruiting Sulfolobus ESCRT-III to membranes. Overexpression of the isolated ESCRT-III domain that interacts with CdvA results in the generation of nucleoid-free cells. Furthermore, CdvA and ESCRT-III synergize to deform archaeal membranes in vitro. The structure of the CdvA/ESCRT-III interface gives insight into the evolution of the more complex and modular eukaryotic ESCRT complex.

Differential Levels of Resistance to Disease Induction and Development of Relapsing Experimental Autoimmune Encephalomyelitis in Two H-2b-restricted Mouse Strains

Journal of Neuroimmunology. May, 2011  |  Pubmed ID: 21482438

Besides the major histocompatibility complex (MHC) genes, background genes are believed to influence the encephalitogenicity of SJL(H-2(s)) and B10.S (H-2(s)) mice responding to myelin basic protein (MBP). A new mouse strain was constructed to study the effects of the SJL genetic background in mice responding to H-2(b)-restricted neuroantigens. Although the SJL.B (H-2(b)) mouse remained resistant to MBP in active EAE induction, the disease severity was uniformly higher in MOG-induced active EAE and in MBP-induced adoptive EAE when compared to those of B6 (H-2(b)) mice. Treatment of mice with anti-CD25 antibodies prior to immunization caused 60% of SJL.B mice to become susceptible to MBP-induced EAE while only 14% of B6 mice were converted. In addition, MOG-induced EAE in SJL.B mice followed a remitting-relapsing disease course while B6 mice only exhibited monophasic or chronic episodes. The new SJL.B mouse strain provides a valuable tool for studying EAE resistance and remitting-relapsing disease in H-2(b) mice.

The Formation and Positioning of Cilia in Ciona Intestinalis Embryos in Relation to the Generation and Evolution of Chordate Left-right Asymmetry

Developmental Biology. Apr, 2012  |  Pubmed ID: 22342242

In the early mouse embryo monocilia on the ventral node rotate to generate a leftward flow of fluid. This nodal flow is essential for the left-sided expression of nodal and pitx2, and for subsequent asymmetric organ patterning. Equivalent left fluid flow has been identified in other vertebrates, including Xenopus and zebrafish, indicating it is an ancient vertebrate mechanism. Asymmetric nodal and Pitx expression have also been identified in several invertebrates, including the vertebrates' nearest relatives, the urochordates. However whether cilia regulate this asymmetric gene expression remains unknown, and previous studies in urochordates have not identified any cilia prior to the larval stage, when asymmetry is already long established. Here we use Scanning and Transmission Electron Microscopy and immunofluorescence to investigate cilia in the urochordate Ciona intestinalis. We show that single cilia are transiently present on each ectoderm cell of the late neurula/early tailbud stage embryo, a time point just before onset of asymmetric nodal expression. Mapping the position of each cilium on these cells shows they are posteriorly positioned, something also described for mouse node cilia. The C. intestinalis cilia have a 9+0 ring ultrastructure, however we find no evidence of structures associated with motility such as dynein arms, radial spokes or nexin. Furthermore the 9+0 ring structure becomes disorganised immediately after the cilia have exited the cell, indicative of cilia which are not capable of motility. Our results indicate that although cilia are present prior to molecular asymmetries, they are not motile and hence cannot be operating in the same way as the flow-generating cilia of the vertebrate node. We conclude that the cilia may have a role in the development of C. intestinalis left-right asymmetry but that this would have to be in a sensory capacity, perhaps as mechanosensors as hypothesised in two-cilia physical models of vertebrate cilia-driven asymmetry.