Mannose binding lectin and lung collectins interact with Toll-like receptor 4 and MD-2 by different mechanisms

Background

We have previously shown that lung collectins, surfactant protein A (SP-A) and surfactant protein D, interact with Toll-like receptor (TLR) 2, TLR4, or MD-2. Bindings of lung collectins to TLR2 and TLR4/MD-2 result in the alterations of signaling through these receptors, suggesting the immunomodulatory functions of lung collectins. Mannose binding lectin (MBL) is another collectin molecule which has structural homology to SP-A. The interaction between MBL and TLRs has not yet been determined.

Methods

We prepared recombinant MBL, and analyzed its bindings to recombinant soluble forms of TLR4 (sTLR4) and MD-2.

Results

MBL bound to sTLR4 and MD-2. The interactions were Ca²⁺-dependent and inhibited by mannose or monoclonal antibody against the carbohydrate-recognition domain of MBL. Treatment of sTLR4 or MD-2 by peptide N-glycosidase F significantly decreased the binding of MBL. SP-A bound to deglycosylated sTLR4, and this property did not change in chimeric molecules of SP-A/MBL in which Glu¹⁹⁵–Phe²²⁸ or Thr¹⁷⁴–Gly¹⁹⁴ of SP-A were replaced with the corresponding MBL sequences.

General Significance

These results suggested that MBL binds to TLR4 and MD-2 through the carbohydrate-recognition domain, and that oligosaccharide moieties of TLR4 and MD-2 are important for recognition by MBL. Since our previous studies indicated that lung collectins bind to the peptide portions of TLRs, MBL and lung collectins interact with TLRs by different mechanisms. These direct interactions between MBL and TLR4 or MD-2 suggest that MBL may modulate cellular responses by altering signals through TLRs.     

Genetic structure of <Emphasis Type="Italic">Cerasus jamasakura</Emphasis>, a Japanese flowering cherry,revealed by nuclear SSRs: implications for conservation

The genetic resources of a particular species of flowering cherry, Cerasus jamasakura, have high conservation priority because of its cultural, ecological and economic value in Japan. Therefore, the genetic structures of 12 natural populations of C. jamasakura were assessed using ten nuclear SSR loci. The population differentiation was relatively low (F _ST, 0.043), reflecting long-distance dispersal of seeds by animals and historical human activities. However, a neighbor-joining tree derived from the acquired data, spatial analysis of molecular variance and STRUCTURE analysis revealed that the populations could be divided into two groups: one located on Kyusyu Island and one on Honshu Island. Genetic diversity parameters such as allelic richness and gene diversity were significantly lower in the Kyushu group than the Honshu group. Furthermore, STRUCTURE analysis revealed that the two lineages were admixed in the western part of Honshu Island. Thus, although the phylogeographical structure of the species and hybridization dynamics among related species need to be evaluated in detail using several marker systems, the Kyusyu Island and Honshu Island populations should be considered as different conservation units, and the islands should be regarded as distinct seed transfer zones for C. jamasakura, especially when rapid assessments are required.     

Mal3 Masks Catastrophe Events in Schizosaccharomyces pombe Microtubules by Inhibiting Shrinkage and Promoting Rescue

Schizosaccharomyces pombe Mal3 is a member of the EB family of proteins, which are proposed to be core elements in a tip-tracking network that regulates microtubule dynamics in cells. How Mal3 itself influences microtubule dynamics is unclear. We tested the effects of full-length recombinant Mal3 on dynamic microtubules assembled in vitro from purified S. pombe tubulin, using dark field video microscopy to avoid fluorescent tagging and data-averaging techniques to improve spatiotemporal resolution. We find that catastrophe occurs stochastically as a fast (<2.2 s) transition from constant speed growth to constant speed shrinkage with a constant probability that is independent of the Mal3 concentration. This implies that Mal3 neither stabilizes nor destabilizes microtubule tips. Mal3 does, however, stabilize the main part of the microtubule lattice, inhibiting shrinkage and increasing the frequency of rescues, consistent with recent models in which Mal3 on the lattice forms stabilizing lateral links between neighboring protofilaments. At high concentrations, Mal3 can entirely block shrinkage and induce very rapid rescue, making catastrophes impossible to detect, which may account for the apparent suppression of catastrophe by Mal3 and other EBs in vivo. Overall, we find that Mal3 stabilizes microtubules not by preventing catastrophe at the microtubule tip but by inhibiting lattice depolymerization and enhancing rescue. We argue that this implies that Mal3 binds microtubules in different modes at the tip and on the lattice.Microtubules are intrinsically dynamic self-assembling structures of tubulin subunits (1) whose polymerization is subject to extensive spatial and temporal control in cells partly through the activity of microtubule-associated proteins (2). In cells, the EB family of microtubule plus end-tracking proteins (+TIPs)² localizes at the plus end of growing but not shrinking microtubules. EB depletion increases catastrophe frequency and reduces microtubule length in many species (3–5), suggesting that EBs suppress microtubule catastrophes. It is, however, unclear from these cellular studies whether this activity is direct or indirect because the dynamic binding of EBs to other +TIPs proteins enhances the localization of all EB complex proteins, including EB1, to microtubule ends (6).To determine the direct effect of EB family proteins on microtubule dynamics, in vitro experiments are necessary. These have established that microtubule end tracking is an intrinsic property of the EB proteins and that other +TIP proteins such as CLIP170 are dependent upon EBs for their microtubule end localization (7–9). However, EB1 binding also directly alters the structure of growing microtubule tips (10). In vitro studies show that Mal3, the EB1 homologue in Schizosaccharomyces pombe, can also affect the structure of microtubules. Sandblad et al. (11) found localization of Mal3 along the (A-lattice) seam of B-lattice microtubules and proposed this as a potential mechanism for direct microtubule stabilization by the EBs. Des Georges et al. (12) showed that Mal3 binds to and specifically stabilizes the A-lattice protofilament overlap, promoting nucleation and assembly of A-lattice-containing microtubules.Several studies in vitro have all shown that EBs can affect microtubule dynamics (4, 7, 10, 13) but conflict over which parameter is affected. Thus although Bieling et al. (7) and Manna et al. (13) observed no effect on microtubule growth rates, Komarova et al. (4) and Vitre et al. (10) found an acceleration of growth. Manna et al. (13) found that EB1 inhibits catastrophe, yet the other studies observed that EBs trigger catastrophe events. There is clearly a need to resolve these apparent conflicts, especially as the same proteins in vivo appear to suppress catastrophe.To try to elucidate the mechanism by which EB proteins influence microtubule assembly, we developed a minimalist approach in which the potential for confounding factors to affect the data is reduced or eliminated. Our assay uses proteins from a single organism, S. pombe, and GMPCPP-stabilized microtubule seeds assembled from purified tubulin with only the seeds attached to the chamber surface. We used this system to measure the effects of unlabeled full-length Mal3 on the polymerization dynamics of unlabeled S. pombe microtubules. Microtubules were imaged using dark field microscopy to avoid fluorescent labeling (see Fig. 1A). We also developed a semiautomated analysis system that allows us to digitize a large number of events, which can then be processed by data averaging and filtering. This reduces noise, allowing us to examine the detailed kinetics of the catastrophic switch from growth to shrinkage. Using this system, we find that Mal3 has no direct effect upon the frequency or kinetics of catastrophe events but that it does reduce shrinkage rates and increase rescue frequency in a dose-dependent manner.Open in a separate windowFIGURE 1.In vitro S. pombe microtubule dynamics assay. A, schematic diagram of S. pombe microtubule dynamics assay. GMPCPP stabilized polarity-marked microtubule seed assembled from Alexa Fluor 488- and Alexa Fluor 680-labeled pig brain tubulin. Only the center of the seed is attached to the surface by anti-Alexa Fluor 488 antibody. Dynamic non-fluorescently labeled S. pombe microtubules grown from seeds were observed by dark field illumination. B, merged fluorescence images of GMPCPP stabilized, polarity-marked pig microtubule seed (pig Alexa-MT). Green, Alexa Fluor 488; red, Alexa Fluor 680. Polarity is indicated by − or +. The plus end of the seed has a longer Alexa Fluor 680-labeled region (upper panel), a dark field image showing pig microtubule seed plus elongated S. pombe microtubules (middle panel), and the merged images (lower panel). Red broken lines show the ends of the seed, and yellow broken lines show the ends of the elongated S. pombe microtubules. Arrows indicate the dynamic S. pombe microtubule elongated from the stabilized microtubule seed. Scale bar: 10 μm. C, kymographs of microtubule length change over time. The left panel shows a diagram of a typical example. Time is indicated by the vertical axis, and length is indicated by the horizontal axis. Rescue (r) and catastrophe (c) events are labeled. Regrowth of shrinking microtubules from the seed (yellow arrow) were not counted as rescues. Scale bars: vertical, 5 min; horizontal, 20 μm. + and − ends of microtubule are indicated. D, enlargement of catastrophe events from the yellow rectangle in C. Scale bars: vertical, 30 s; horizontal, 5 μm.     

Reconstitution in vitro of the catalytic portion (NtpA3-B3-D-G complex) of Enterococcus hirae V-type Na-ATPase

Enterococcus hirae vacuolar ATPase (V-ATPase) is composed of a soluble catalytic domain (V₁; NtpA₃-B₃-D-G) and an integral membrane domain (V₀; NtpI-K₁₀) connected by a central and peripheral stalk(s) (NtpC and NtpE-F). Here we examined the nucleotide binding of NtpA monomer, NtpB monomer or NtpD-G heterodimer purified by using Escherichia coli expression system in vivo or in vitro, and the reconstitution of the V₁ portion with these polypeptides. The affinity of nucleotide binding to NtpA was 6.6 μM for ADP or 3.1 μM for ATP, while NtpB or NtpD-G did not show any binding. The NtpA and NtpB monomers assembled into NtpA₃-B₃ heterohexamer in nucleotide binding-dependent manner. NtpD-G bound NtpA₃-B₃ forming V₁ (NtpA₃-B₃-D-G) complex independent of nucleotides. The V₁ formation from individual NtpA and NtpB monomers with NtpD-G heterodimer was absolutely dependent on nucleotides. The ATPase activity of reconstituted V₁ complex was as high as that of native V₁-ATPase purified from the V₀V₁ complex by EDTA treatment of cell membrane. This in vitro reconstitution system of E. hirae V₁ complex will be valuable for characterizing the subunit-subunit interactions and assembly mechanism of the V₁-ATPase complex.     

Vertical migration in streams: seasonal use of the hyporheic zone by the spinous loach <Emphasis Type="Italic">Cobitis shikokuensis</Emphasis>

Vertical hydrological connectivity between the surface stream and benthic and hyporheic zones plays a key ecological role in the biodiversity of lotic ecosystems because it allows surface and benthic organisms to use the hyporheic zone as a seasonal habitat and refuge. Use of the hyporheic zone by surface/benthic organisms has been well studied in invertebrates, but little is known about the importance of this connectivity for fishes. We investigated streambed surface and hyporheic densities (5–10, 15–20 and 20–25 cm below the streambed surface) of a stream fish, Cobitis shikokuensis, over a 20-month period in the Shigenobu River, southwestern Japan, to test the hypothesis that it uses the hyporheic zone for spawning and overwintering. In total, 1,804 individuals (13–58 mm total length) were captured from 33 streambed surface samplings and 102 individuals (10–46 mm total length) were present in 1,147 samples of 57 hyporheic samplings. Population densities in both zones peaked in late summer–early autumn due to the recruitment of age 0+ fish and a female with eggs was found in the hyporheic zone during the reproductive season. Both 0+ and older fish were absent from the streambed surface during winter, and fish densities were also lower in the hyporheic zone at this time. However, the vertical distribution of the fish tended to be skewed towards the deeper hyporheic layers from autumn to spring. These findings indicate that C. shikokuensis vertically migrates between the streambed surface and the hyporheic zone for spawning, rearing and overwintering, suggesting that the integrity of vertical hydrological connectivity in lotic systems is crucial for certain fish species.     

Activation of the JNK pathway by nanosecond pulsed electric fields

Nanosecond pulsed electric fields (nsPEFs) are increasingly recognized as a novel and unique tool in various life science fields, including electroporation and cancer therapy, although their mode of action in cells remains largely unclear. Here, we show that nsPEFs induce strong and transient activation of a signaling pathway involving c-Jun N-terminal kinase (JNK). Application of nsPEFs to HeLa S3 cells rapidly induced phosphorylation of JNK1 and MKK4, which is located immediately upstream of JNK in this signaling pathway. nsPEF application also elicited increased phosphorylation of c-Jun protein and dramatically elevated c-jun and c-fos mRNA levels. nsPEF-inducible events downstream of JNK were markedly suppressed by the JNK inhibitor SP600125, which confirmed JNK-dependency of these events in this pathway. Our results provide novel mechanistic insights into the mode of nsPEF action in human cells.     

Crystal Structure of the cis-Dimer of Nectin-1: implications for the architecture of cell-cell junctions

In multicellular organisms, cells are interconnected by cell adhesion molecules. Nectins are immunoglobulin (Ig)-like cell adhesion molecules that mediate homotypic and heterotypic cell-cell adhesion, playing key roles in tissue organization. To mediate cell-cell adhesion, nectin molecules dimerize in cis on the surface of the same cell, followed by trans-dimerization of the cis-dimers between the neighboring cells. Previous cell biological studies deduced that the first Ig-like domain of nectin and the second Ig-like domain are involved in trans-dimerization and cis-dimerization, respectively. However, to understand better the steps involved in nectin adhesion, the structural basis for the dimerization of nectin must be determined. In this study, we determined the first crystal structure of the entire extracellular region of nectin-1. In the crystal, nectin-1 formed a V-shaped homophilic dimer through the first Ig-like domain. Structure-based site-directed mutagenesis of the first Ig-like domain identified four essential residues that are involved in the homophilic dimerization. Upon mutating the four residues, nectin-1 significantly decreased cis-dimerization on the surface of cultured cells and abolished the homophilic and heterophilic adhesion activities. These results indicate that, in contrast with the previous notion, our structure represents a cis-dimer. Thus, our findings clearly reveal the structural basis for the cis-dimerization of nectins through the first Ig-like domains.