Analysis of swimmers' velocity during the underwater gliding motion following grab start.

The purpose of this study was to determine the swimmers' loss of speed during the underwater gliding motion of a grab start. This study also set out to determine the kinematical variables influencing this loss of speed. Eight French national-level swimmers participated in this study. The swimmers were filmed using 4 mini-DV cameras during the entire underwater phase. Using the DLT technique and the Dempster's anthropometric data, swimmer's movement have been identified. Two principal components analysis (PCA) have been used to study the relations between the kinematical variables influencing the loss of speed. The swimmers reached a velocity between 2.2 and 1.9 ms(-1) after their centre of mass covered a distance ranging between 5.63 and 6.01 m from the start wall. For this range of velocity, head position was included between 6.02 and 6.51 m. First PCA show that the kinematical parameters at the immersion (first image at which the swimmers' whole body was under water) are included in the first two components. Second PCA show that the knee, hip and shoulder angles can be included in the same component. The present study identified the optimal instant for initiating underwater leg movements after a grab start. This study also showed that the performance during the underwater gliding motion is determined as much by variables at the immersion as by the swimmer's loss of speed. It also seems that to hold the streamlined position the synergetic action of the knee, the hip and the shoulder is essential.

Apical membrane targeting of Nedd4 is mediated by an association of its C2 domain with annexin XIIIb.

Nedd4 is a ubiquitin protein ligase (E3) containing a C2 domain, three or four WW domains, and a ubiquitin ligase HECT domain. We have shown previously that the C2 domain of Nedd4 is responsible for its Ca(2+)-dependent targeting to the plasma membrane, particularly the apical region of epithelial MDCK cells. To investigate this apical preference, we searched for Nedd4-C2 domain-interacting proteins that might be involved in targeting Nedd4 to the apical surface. Using immobilized Nedd4-C2 domain to trap interacting proteins from MDCK cell lysate, we isolated, in the presence of Ca(2+), a approximately 35-40-kD protein that we identified as annexin XIII using mass spectrometry. Annexin XIII has two known isoforms, a and b, that are apically localized, although XIIIa is also found in the basolateral compartment. In vitro binding and coprecipitation experiments showed that the Nedd4-C2 domain interacts with both annexin XIIIa and b in the presence of Ca(2+), and the interaction is direct and optimal at 1 microM Ca(2+). Immunofluorescence and immunogold electron microscopy revealed colocalization of Nedd4 and annexin XIIIb in apical carriers and at the apical plasma membrane. Moreover, we show that Nedd4 associates with raft lipid microdomains in a Ca(2+)-dependent manner, as determined by detergent extraction and floatation assays. These results suggest that the apical membrane localization of Nedd4 is mediated by an association of its C2 domain with the apically targeted annexin XIIIb.

Different properties of two isoforms of annexin XIII in MDCK cells.

Annexins form a family of proteins that are widely expressed and known to bind membranes in the presence of calcium. Two isoforms of the annexin XIII subfamily are expressed in epithelia. We previously reported that annexin XIIIb is apically localized in MDCK cells and that it is involved in raft-mediated delivery of apical proteins. We have now analyzed the properties of annexin XIIIa, which differs from annexin XIIIb by a deletion of 41 amino acids in the amino-terminal domain, and is distributed both apically and basolaterally. Annexin XIIIa binding to membranes is independent of calcium but requires its myristoyl amino-terminal modification, as observed with annexin XIIIb. Our biochemical and functional data show that annexin XIIIa behaves differently in the apical and in the basolateral compartments. Whereas annexin XIIIa apically can associate with rafts independently of calcium, the basolateral pool requires calcium for this. Annexin XIIIa, like annexin XIIIb, stimulates apical transport of influenza virus hemagglutinin but, in contrast, only annexin XIIIa inhibits basolateral transport of vesicular stomatitis virus G protein. Our results suggest that annexin XIIIa and XIIIb have specific roles in epithelial cells, and because of their structural similarities, these isoforms offer interesting tools for unravelling the functions of annexins.

Protein expression in Drosophila Schneider cells.

We expressed recombinant secreted, membrane, and cytosolic proteins in stably transfected Drosophila Schneider (SL-3) cells. To allow easy cloning of N- and C-terminal fusion proteins containing epitope- and His-tags for the detection of recombinant proteins and purification by affinity chromatography we constructed new expression vectors. To exemplify the general applicability of protein expression in Schneider cells we characterized the expression system with respect to inducibility, localization of the recombinant proteins, yields of purified proteins, and presence of posttranslational and cotranslational modifications. Secreted proteins became quantitatively N-glycosylated in SL-3 cells and the N-glycan of a Golgi-resident membrane protein was found to be Endo-H-resistant. Myristoylation of AnxXIIIb, a member of the annexin family, could be demonstrated and glycosylphosphatidylinositol-anchored proteins containing their lipid anchor were expressed efficiently in SL-3 cells. Since generation of stable cell lines and mass culture of SL-3 cells is cheap and easy, they provide an attractive eukaryotic expression system.

Annexin XIIIb associates with lipid microdomains to function in apical delivery.

A member of the annexin XIII sub-family, annexin XIIIb, has been implicated in the apical exocytosis of epithelial kidney cells. Annexins are phospholipid-binding proteins that have been suggested to be involved in membrane trafficking events although their actual physiological function remains open. Unlike the other annexins, annexin XIIIs are myristoylated. Here, we show by immunoelectron microscopy that annexin XIIIb is localized to the trans-Golgi network (TGN), vesicular carriers and the apical cell surface. Polarized apical sorting involves clustering of apical proteins into dynamic sphingolipid-cholesterol rafts. We now provide evidence for the raft association of annexin XIIIb. Using in vitro assays and either myristoylated or unmyristoylated recombinant annexin XIIIb, we demonstrate that annexin XIIIb in its native myristoylated form stimulates specifically apical transport whereas the unmyristoylated form inhibits this route. Moreover, we show that formation of apical carriers from the TGN is inhibited by an anti-annexin XIIIb antibody whereas it is stimulated by myristoylated recombinant annexin XIIIb. These results suggest that annexin XIIIb directly participates in apical delivery.

Regulation of the heat-shock response depends on divalent metal ions in an hflB mutant of Escherichia coli.

HflB, also called FtsH, is an essential Escherichia coli protein involved in the proteolysis of the heat-shock regulator sigma 32 and of the phage regulator lambda cll. The hflB1(Ts) allele (formerly called ftsH1) conferring temperature-sensitive growth at 42 degrees C is suppressed by loss of the ferric-uptake repressor Fur and by anaerobic growth. We show here that suppression requires TonB-dependent Fe(III) transport in the hflB1(Ts) fur mutant during aerobic growth at 42 degrees C and Feo-dependent Fe(II) transport during anaerobic growth at 42 degrees C. Temperature-resistant growth of hflB1(Ts) strains is also observed at 42 degrees C in the presence of a high concentration of Fe(II), Ni(II), Mn(II) or Co(II) salts, but not in the presence of Zn(II), Cd(II), Cu(II), Mg(II), Ca(II) or Cr(III) salts. However, neither Ni(II) nor a fur mutation permits growth in the complete absence of HflB. The heat-shock response, evaluated by an htpG::lacZ fusion, is overinduced in hflB1(Ts) strains at 42 degrees C because of stabilization of sigma 32. Growth in the presence of Ni(II) or in the absence of the Fur repressor abolishes this overinduction in the hflB1(Ts) strain, and, in the hflB1(Ts) fur mutant, sigma 32 is no longer stabilized at 42 degrees C. These results reinforce the recent observation that HflB is a metalloprotease active against sigma 32 in vitro and suggest that it can associate functionally in vivo with Fe(II), Ni(II), Mn(II) and Co(II) ions.