Thierry Blisnick

Pfsbp1, a Maurer's cleft Plasmodium falciparum protein, is associated with the erythrocyte skeleton

Antibodies from hyperimmune monkey sera, selected by absorption to Plasmodium falciparum-infected erythrocytes, and elution at acidic pH, allowed us to characterize a novel parasite protein, Pfsbp1 (P. falciparum skeleton binding protein 1). Pfsbp1 is an integral membrane protein of parasite-induced membranous structures associated with the erythrocyte plasma membrane and referred to as Maurers clefts. The carboxy-terminal domain of Pfsbp1, exposed within the cytoplasm of the host cell, interacts with a 35 kDa erythrocyte skeletal protein and might participate in the binding of the Maurers clefts to the erythrocyte submembrane skeleton. Antibodies to the carboxy- and amino-terminal domains of Pfsbp1 labelled similar vesicular structures in the cytoplasm of Plasmodium chabaudi and Plasmodium berghei-infected murine erythrocytes, suggesting that the protein is conserved among malaria species, consistent with an important role of Maurers cleft-like structures in the intraerythrocytic development of malaria parasites.

The ciliary pocket: an endocytic membrane domain at the base of primary and motile cilia

Cilia and flagella are eukaryotic organelles involved in multiple cellular functions. The primary cilium is generally non motile and found in numerous vertebrate cell types where it controls key signalling pathways. Despite a common architecture, ultrastructural data suggest some differences in their organisation. Here, we report the first detailed characterisation of the ciliary pocket, a depression of the plasma membrane in which the primary cilium is rooted. This structure is found at low frequency in kidney epithelial cells (IMCD3) but is associated with virtually all primary cilia in retinal pigment epithelial cells (RPE1). Transmission and scanning electron microscopy, immunofluorescence analysis and videomicroscopy revealed that the ciliary pocket establishes closed links with the actin-based cytoskeleton and that it is enriched in active and dynamic clathrin-coated pits. The existence of the ciliary pocket was confirmed in mouse tissues bearing primary cilia (cumulus), as well as motile cilia and flagella (ependymal cells and spermatids). The ciliary pocket shares striking morphological and functional similarities with the flagellar pocket of Trypanosomatids, a trafficking-specialised membrane domain at the base of the flagellum. Our data therefore highlight the conserved role of membrane trafficking in the vicinity of cilia.

Molecular basis of tubulin transport within the cilium by IFT74 and IFT81.

Cilium Conundrum The cilium has emerged as the antenna of eukaryotic cells, having numerous functions in sensory reception and developmental signaling. Several disorders, such as polycystic kidney disease, are the result of compromised cilia structure. Bhogaraju et al. (p. 1009) elucidate how the intraflagellar transport machinery recognizes tubulin, a ciliary cargo that is integral to cilium maintenance and formation and is constantly turned over at the cilium tip. Two proteins cooperate to promote tubulin transport to and into cilia. Intraflagellar transport (IFT) of ciliary precursors such as tubulin from the cytoplasm to the ciliary tip is involved in the construction of the cilium, a hairlike organelle found on most eukaryotic cells. However, the molecular mechanisms of IFT are poorly understood. Here, we found that the two core IFT proteins IFT74 and IFT81 form a tubulin-binding module and mapped the interaction to a calponin homology domain of IFT81 and a highly basic domain in IFT74. Knockdown of IFT81 and rescue experiments with point mutants showed that tubulin binding by IFT81 was required for ciliogenesis in human cells.

Analysis of membrane proteins by two-dimensional electrophoresis: comparison of the proteins extracted from normal or Plasmodium falciparum-infected erythrocyte ghosts.

Parasite‐encoded membrane proteins translocated to the surface of infected erythrocytes or in specialized vesicles underneath (Maurers clefts) play a key role in the asexual life cycle of Plasmodium falciparum (a malaria‐causing protozoan), by mediating key steps such as red blood cell invasion, sequestration of infected cells in microcapillaries, and red blood cell rupture. A large‐scale analysis of these membrane proteins would therefore be of great help to gain knowledge of the different stages of the Plasmodium falciparum life cycle. In order to be able to detect and identify parasite‐encoded proteins directed to the red blood cell membrane, we first defined the conditions required for optimal extraction and separation of normal red blood cell ghost proteins by two‐dimensional gel electrophoresis. These conditions included the use of urea, thiourea and new zwitterionic detergents in the extraction and isoelectric focusing media. The optimized conditions were then applied to analyze normal and P. falciparum‐infected red blood cell ghosts. Several protein spots were found only in infected ghosts and are expected to represent parasite‐encoded proteins. These proteins are currently under investigation.

Proteomic Analysis Identifies Novel Proteins of the Maurer’s Clefts, a Secretory Compartment Delivering Plasmodium falciparum Proteins to the Surface of Its Host Cell

A novel method was validated for the efficient distinction between malaria parasite-derived and host cell proteins in mass spectrometry analyses. This method was applied to a ghost fraction from Plasmodium falciparum-infected erythrocytes containing the red blood cell plasma membrane, the erythrocyte submembrane skeleton, and the Maurer’s clefts, a Golgi-like apparatus linked to and addressing parasite proteins to the host cell surface. This method allowed the identification of 78 parasite proteins. Among these we identified seven novel proteins of the Maurer’s clefts based on immunofluorescence studies and proteinase K digestion assays. The products of six contiguous genes located on chromosome 5 were identified, and the location within the Maurer’s clefts was established for two of them. This suggests a clustering of genes encoding Maurer’s cleft proteins. Our study sheds new light on the biological function of the Maurer’s clefts, which are central to the pathogenesis and to the intraerythrocytic development of P. falciparum.

A role for erythrocyte band 3 degradation by the parasite gp76 serine protease in the formation of the parasitophorous vacuole during invasion of erythrocytes by Plasmodium falciparum

A purified Plasmodium falciparum serine protease (gp76) implicated in erythrocyte invasion, degrades human erythrocyte band 3 and glycophorin A. Inhibition studies using synthetic peptides derived from the presumed band 3 enzymatic cleavage sites and the observed uptake of fluorescent phospholipids following gp76 treatment, suggest that band 3 degradation by this serine protease participates in the formation of the parasitophorous vacuole by restructuring the red cell cytoskeleton. These results provide a rationale for the elaboration of specific inhibitors to block red cell invasion by malaria parasites.

1001 model organisms to study cilia and flagella

Most mammalian cell types have the potential to assemble at least one cilium. Immotile cilia participate in numerous sensing processes, while motile cilia are involved in cell motility and movement of extracellular fluid. The functional importance of cilia and flagella is highlighted by the growing list of diseases due to cilia defects. These ciliopathies are marked by an amazing diversity of clinical manifestations and an often complex genetic aetiology. To understand these pathologies, a precise comprehension of the biology of cilia and flagella is required. These organelles are remarkably well conserved throughout eukaryotic evolution. In this review, we describe the strengths of various model organisms to decipher diverse aspects of cilia and flagella biology: molecular composition, mode of assembly, sensing and motility mechanisms and functions. Pioneering studies carried out in the green alga Chlamydomonas established the link between cilia and several genetic diseases. Moreover, multicellular organisms such as mouse, zebrafish, Xenopus, Caenorhabditis elegans or Drosophila, and protists such as Paramecium, Tetrahymena and Trypanosoma or Leishmania each bring specific advantages to the study of cilium biology. For example, the function of genes involved in primary ciliary dyskinesia (due to defects in ciliary motility) can be efficiently assessed in trypanosomes.

Sialorphin, a natural inhibitor of rat membrane-bound neutral endopeptidase that displays analgesic activity

Sialorphin is an exocrine and endocrine signaling mediator, which has been identified by a genomic approach. It is synthesized predominantly in the submandibular gland and prostate of adult rats in response to androgen steroids and is released locally and systemically in response to stress. We now demonstrate that the cell surface molecule to which sialorphin binds in vivo in the rat kidney is the membrane-anchored neutral endopeptidase (neprilysin; NEP, EC 3.4.24.11). NEP plays an important role in nervous and peripheral tissues, as it turns off several peptide-signaling events at the cell surface. We show that sialorphin prevents spinal and renal NEP from breaking down its two physiologically relevant substrates, substance P and Met-enkephalin in vitro. Sialorphin inhibited the breakdown of substance P with an IC50 of 0.4–1 μM and behaved as a competitive inhibitor. In vivo, i.v. sialorphin elicited potent antinociceptive responses in two behavioral rat models of injury-induced acute and tonic pain, the pin-pain test and formalin test. The analgesia induced by 100–200 μg/kg doses of sialorphin required the activation of μ- and δ-opioid receptors, consistent with the involvement of endogenous opioid receptors in enkephalinergic transmission. We conclude that sialorphin protects endogenous enkephalins released after nociceptive stimuli by inhibiting NEP in vivo. Sialorphin is a natural systemically active regulator of NEP activity. Furthermore, our study provides evidence that it is a physiological modulator of pain perception after injury and might be the progenitor of a new class of therapeutic molecules.

Basal Body Positioning Is Controlled by Flagellum Formation in Trypanosoma brucei

To perform their multiple functions, cilia and flagella are precisely positioned at the cell surface by mechanisms that remain poorly understood. The protist Trypanosoma brucei possesses a single flagellum that adheres to the cell body where a specific cytoskeletal structure is localised, the flagellum attachment zone (FAZ). Trypanosomes build a new flagellum whose distal tip is connected to the side of the old flagellum by a discrete structure, the flagella connector. During this process, the basal body of the new flagellum migrates towards the posterior end of the cell. We show that separate inhibition of flagellum assembly, base-to-tip motility or flagella connection leads to reduced basal body migration, demonstrating that the flagellum contributes to its own positioning. We propose a model where pressure applied by movements of the growing new flagellum on the flagella connector leads to a reacting force that in turn contributes to migration of the basal body at the proximal end of the flagellum.

Plaque antibody selection: rapid immunological analysis of a large number of recombinant phage clones positive to sera raised against Plasmodium falciparum antigens

A library of Plasmodium falciparum genomic DNA on the lambda gt11 phage vector was screened for clones positive to a rabbit serum raised against a purified fraction of P. falciparum proteins and a pool of sera from malaria patients. The positive clones were characterized with antibodies purified by the plaque antibody selection technique. This technique consist of purifying specific antibodies on a nitrocellulose filter blotted directly on a lawn of plaques of an antigen-producing phage clone. The purified antibodies are then used as a probe in a Western blot of parasite protein extract, for preliminary characterization of the clones. Using this method, two different clones coding for P. falciparum antigens were identified with the rabbit serum and about 20 with the human sera. This method can be of general use, i.e. it is not limited to parasite systems, and facilitates the immunological analysis and identification of a large number of clones.