Direct Measurement of Helical Cell Motion of the Spirochete Leptospira

Leptospira are spirochete bacteria distinguished by a short-pitch coiled body and intracellular flagella. Leptospira cells swim in liquid with an asymmetric morphology of the cell body; the anterior end has a long-pitch spiral shape (S-end) and the posterior end is hook-shaped (H-end). Although the S-end and the coiled cell body called the protoplasmic cylinder are thought to be responsible for propulsion together, most observations on the motion mechanism have remained qualitative. In this study, we analyzed the swimming speed and rotation rate of the S-end, protoplasmic cylinder, and H-end of individual Leptospira cells by one-sided dark-field microscopy. At various viscosities of media containing different concentrations of Ficoll, the rotation rate of the S-end and protoplasmic cylinder showed a clear correlation with the swimming speed, suggesting that these two helical parts play a central role in the motion of Leptospira. In contrast, the H-end rotation rate was unstable and showed much less correlation with the swimming speed. Forces produced by the rotation of the S-end and protoplasmic cylinder showed that these two helical parts contribute to propulsion at nearly equal magnitude. Torque generated by each part, also obtained from experimental motion parameters, indicated that the flagellar motor can generate torque >4000 pN nm, twice as large as that of Escherichia coli. Furthermore, the S-end torque was found to show a markedly larger fluctuation than the protoplasmic cylinder torque, suggesting that the unstable H-end rotation might be mechanically related to changes in the S-end rotation rate for torque balance of the entire cell. Variations in torque at the anterior and posterior ends of the Leptospira cell body could be transmitted from one end to the other through the cell body to coordinate the morphological transformations of the two ends for a rapid change in the swimming direction.     

Protrusion Fluctuations Direct Cell Motion

Many physiological phenomena involve directional cell migration. It is usually attributed to chemical gradients in vivo. Recently, other cues have been shown to guide cells in vitro, including stiffness/adhesion gradients or micropatterned adhesive motifs. However, the cellular mechanism leading to these biased migrations remains unknown, and, often, even the direction of motion is unpredictable. In this study, we show the key role of fluctuating protrusions on ratchet-like structures in driving NIH3T3 cell migration. We identified the concept of efficient protrusion and an associated direction index. Our analysis of the protrusion statistics facilitated the quantitative prediction of cell trajectories in all investigated conditions. We varied the external cues by changing the adhesive patterns. We also modified the internal cues using drug treatments, which modified the protrusion activity. Stochasticity affects the short- and long-term steps. We developed a theoretical model showing that an asymmetry in the protrusion fluctuations is sufficient for predicting all measures associated with the long-term motion, which can be described as a biased persistent random walk.     

Protrusion Fluctuations Direct Cell Motion

Many physiological phenomena involve directional cell migration. It is usually attributed to chemical gradients in vivo. Recently, other cues have been shown to guide cells in vitro, including stiffness/adhesion gradients or micropatterned adhesive motifs. However, the cellular mechanism leading to these biased migrations remains unknown, and, often, even the direction of motion is unpredictable. In this study, we show the key role of fluctuating protrusions on ratchet-like structures in driving NIH3T3 cell migration. We identified the concept of efficient protrusion and an associated direction index. Our analysis of the protrusion statistics facilitated the quantitative prediction of cell trajectories in all investigated conditions. We varied the external cues by changing the adhesive patterns. We also modified the internal cues using drug treatments, which modified the protrusion activity. Stochasticity affects the short- and long-term steps. We developed a theoretical model showing that an asymmetry in the protrusion fluctuations is sufficient for predicting all measures associated with the long-term motion, which can be described as a biased persistent random walk.     

Crystal Structure of LipL32, the Most Abundant Surface Protein of Pathogenic Leptospira spp.

Spirochetes of the genus Leptospira cause leptospirosis in humans and animals worldwide. Proteins exposed on the bacterial cell surface are implicated in the pathogenesis of leptospirosis. However, the biological role of the majority of these proteins is unknown; this is principally due to the lack of genetic systems for investigating Leptospira and the absence of any structural information on leptospiral antigens. To address this, we have determined the 2.0-Å-resolution structure of the lipoprotein LipL32, the most abundant outer-membrane and surface protein present exclusively in pathogenic Leptospira species. The extracellular domain of LipL32 revealed a compact, globular, “jelly-roll” fold from which projected an unusual extended β-hairpin that served as a principal mediator of the observed crystallographic dimer. Two acid-rich patches were also identified as potential binding sites for positively charged ligands, such as laminin, to which LipL32 has a propensity to bind. Although LipL32 shared no significant sequence identity to any known protein, it possessed structural homology to the adhesins that bind components of the extracellular matrix, suggesting that LipL32 functions in an analogous manner. Moreover, the structure provides a framework for understanding the immunological role of this major surface lipoprotein.     

Direct Measurement of Potential Difference across the Human Red Blood Cell Membrane

The electrical potential difference across the human red cell membrane has been measured directly. A biological amplifier with neutralized input capacity was used. Human red cells in modified Ringer solution were impaled individually with 3 M KCl-filled glass microelectrodes. Movements of the microelectrodes were effected by Leitz micromanipulators. Results showed a potential difference of -8.0 ± 0.21 (SEM) mv, the inside being negative with respect to the outside. This value is approximately that calculated by using the Nernst equation considering the intracellular and extracellular chloride concentrations.

As a control, similar measurements were made on nylon microcapsules containing hemoglobin. The measured potential of -0.52 ± 0.02 (SEM) mv, which agreed very well with the value calculated on the basis of Donnan equilibrium, was much smaller in magnitude as compared to the results for the red cell, and there was evidence of fixed charges on the microcapsule membrane. There was no evidence of this in the case of the red cell.

     

Role of svp in Drosophila Pericardial Cell Growth

The Drosophila dorsal vessel is a segmentally repeated linear organ, in which seven-up (svp) is expressed in two pairs of cardioblasts and two pairs of pericardial cells in each segment. Under the control of hedgehog (hh) signaling from the dorsal ectoderm, svp participates in diversifying cardioblast identities within each segment. In this experiment, the homozygous embryos of svp mutants exhibited an increase in cell size of Eve positive pericardial cells (EPCs) and a disarranged expression pattern, while the cardioblasts pattern of svp-lacZ expression was normal. In the meantime, the DA1 muscle founders were absent in some segments in svp mutant embryos, and the dorsal somatic muscle patterning was also severely damaged in the late stage mutant embryos, suggesting that svp is required for the differentiation of Eve-positive pericardial cells and DA1 muscle founders and may have a role in EPC cell growth.     

A novel fibronectin type III module binding motif identified on C-terminus of Leptospira immunoglobulin-like protein, LigB

Infection by pathogenic strains of Leptospira hinges on the pathogen’s ability to adhere to host cells via extracellular matrix such as fibronectin (Fn). Previously, the immunoglobulin-like domains of Leptospira Lig proteins were recognized as adhesins binding to N-terminal domain (NTD) and gelatin binding domain (GBD) of Fn. In this study, we identified another Fn-binding motif on the C-terminus of the Leptospira adhesin LigB (LigBCtv), residues 1708-1712 containing sequence LIPAD with a β-strand and nascent helical structure. This motif binds to 15th type III modules (15F₃) (K_D = 10.70 μM), and association (k_on = 600 M⁻¹ s⁻¹) and dissociation (k_off = 0.0129 s⁻¹) rate constants represents a slow binding kinetics in this interaction. Moreover, pretreatment of MDCK cells with LigB_1706-1716 blocked the binding of Leptospira by 39%, demonstrating a significant role of LigB_1706-1716 in cellular adhesion. These data indicate that the LIPAD residues (LigB_1708-1712) of the Leptospira interrogans LigB protein bind 15F₃ of Fn at a novel binding site, and this interaction contributes to adhesion to host cells.     

Cell signaling during development of Dictyostelium

Continuous communication between cells is necessary for development of any multicellular organism and depends on the recognition of secreted signals. A wide range of molecules including proteins, peptides, amino acids, nucleic acids, steroids and polylketides are used as intercellular signals in plants and animals. They are also used for communication in the social ameba Dictyostelium discoideum when the solitary cells aggregate to form multicellular structures. Many of the signals are recognized by surface receptors that are seven-transmembrane proteins coupled to trimeric G proteins, which pass the signal on to components within the cytoplasm. Dictyostelium cells have to judge when sufficient cell density has been reached to warrant transition from growth to differentiation. They have to recognize when exogenous nutrients become limiting, and then synchronously initiate development. A few hours later they signal each other with pulses of cAMP that regulate gene expression as well as direct chemotactic aggregation. They then have to recognize kinship and only continue developing when they are surrounded by close kin. Thereafter, the cells diverge into two specialized cell types, prespore and prestalk cells, that continue to signal each other in complex ways to form well proportioned fruiting bodies. In this way they can proceed through the stages of a dependent sequence in an orderly manner without cells being left out or directed down the wrong path.     

A Microscopic Formulation for the Actin-Driven Motion of Listeria in Curved Paths

Using a generalized Brownian ratchet model that accounts for the interactions of actin filaments with the surface of Listeria mediated by proteins like ActA and Arp2/3, we have developed a microscopic model for the movement of Listeria. Specifically, we show that a net torque can be generated within the comet tail, causing the bacteria to spin about its long axis, which in conjunction with spatially varying polymerization at the surface leads to motions of bacteria in curved paths that include circles, sinusoidal-like curves, translating figure eights, and serpentine shapes, as observed in recent experiments. A key ingredient in our formulation is the coupling between the motion of Listeria and the force-dependent rate of filament growth. For this reason, a numerical scheme was developed to determine the kinematic parameters of motion and stress distribution among filaments in a self-consistent manner. We find that a 5-15% variation in polymerization rates can lead to radii of curvatures of the order of 4-20 μm, measured in experiments. In a similar way, our results also show that most of the observed trajectories can be produced by a very low degree of correlation, <10%, among filament orientations. Since small fluctuations in polymerization rate, as well as filament orientation, can easily be induced by various factors, our findings here provide a reasonable explanation for why Listeria can travel along totally different paths under seemingly identical experimental conditions. Besides trajectories, stress distributions corresponding to different polymerization profiles are also presented. We have found that although some actin filaments generate propelling forces that push the bacteria forward, others can exert forces opposing the movement of Listeria, consistent with recent experimental observations.     

盘基网柄菌突变型细胞allC的细胞周期异常

研究盘基网柄菌(Dictyostelium discoideum)细胞周期的相关问题可以为真核生物细胞周期调控研究提供理论基础。细胞计数和细胞倍增时间计算的结果表明,突变allC细胞的倍增时间为2.36 h,仅为KAx-3细胞倍增时间的1/3。进一步利用流式细胞术测定两种细胞的细胞周期,并结合实时荧光定量PCR技术测定cycB1和cdk1基因的相对表达量的比值,我们发现,培养16 h的allC细胞处于G2期的数目(1.51%)显著少于KAx-3细胞(16.61%)(P0.05)。allC细胞和KAx-3细胞的细胞周期素B1(cyclinB1,cycB1)基因相对表达量分别是2.5和0.24(P0.05),两者相差10倍。两种类型细胞中处于G2期的细胞数目差异十分明显,cycB1的相对表达量也存在显著差异,表明cycB1的过表达可能在一定程度上影响allC细胞的细胞周期正常的调控机制,与突变细胞的G2期异常有一定关系,但具体机制仍需进一步探究。     

Measurement of enzymatic and motile activities of Arabidopsis myosins by using Arabidopsis actins

There are two classes of myosin, XI and VIII, in higher plants. Myosin XI moves actin filaments at high speed and its enzyme activity is also very high. In contrast, myosin VIII moves actin filaments very slowly with very low enzyme activity. Because most of these enzymatic and motile activities were measured using animal skeletal muscle α-actin, but not plant actin, they would not accurately reflect the actual activities in plant cells. We thus measured enzymatic and motile activities of the motor domains of two Arabidopsis myosin XI isoforms (MYA2, XI-B), and one Arabidopsis myosin VIII isoform (ATM1), by using three Arabidopsis actin isoforms (ACT1, ACT2, and ACT7). The measured activities were different from those measured by using muscle actin. Moreover, Arabidopsis myosins showed different enzymatic and motile activities when using different Arabidopsis actin isoforms. Our results suggest that plant actin should be used for measuring enzymatic and motile activities of plant myosins and that different actin isoforms in plant cells might function as different tracks along which affinities and velocities of each myosin isoform are modulated.     

Direct Visualization of Protease Action on Collagen Triple Helical Structure

Enzymatic processing of extracellular matrix (ECM) macromolecules by matrix metalloproteases (MMPs) is crucial in mediating physiological and pathological cell processes. However, the molecular mechanisms leading to effective physiological enzyme-ECM interactions remain elusive. Only scant information is available on the mode by which matrix proteases degrade ECM substrates. An example is the enzymatic degradation of triple helical collagen II fragments, generated by the collagenase MMP-8 cleavage, during the course of acute inflammatory conditions by gelatinase B/MMP-9. As is the case for many other matrix proteases, it is not clear how MMP-9 recognizes, binds and digests collagen in this important physiological process. We used single molecule imaging to directly visualize this protease during its interaction with collagen fragments. We show that the initial binding is mediated by the diffusion of the protease along the ordered helix on the collagen ¾ fragment, with preferential binding of the collagen tail. As the reaction progressed and prior to collagen degradation, gelatin-like morphologies resulting from the denaturation of the triple helical collagen were observed. Remarkably, this activity was independent of enzyme proteolysis and was accompanied by significant conformational changes of the working protease. Here we provide the first direct visualization of highly complex mechanisms of macromolecular interactions governing the enzymatic processing of ECM substrates by physiological protease.     

Cell surface proteins in the early embryogenesis of Pleurodeles waltlii

Surface proteins in the first embryonic stages (8–32 cells, morula, blastula, early and late gastrula) of Pleurodeles waltlii were selectively labelled by ¹²⁵I using lactoperoxidase and glucose/glucose oxidase. Iodination was effected either on non-dissociated embryos or after their dissociation with EDTA. On the outer surface of non-dissociated embryos the two-dimensional electrophoresis revealed only three groups of ¹²⁵I-labelled proteins which did not change during all studied stages. Quite different results were obtained with the cells of dissociated embryos. In addition to the iodinated proteins of the embryonic outer surface seven major iodinated proteins were identified. These proteins originate from the regions of cell-cell contacts in intact embryo. Their two-dimensional pattern in dissociated cells changes between stages 8–32 cells and morula. The next important difference was observed during gastrulation, which corresponds in Pleurodeles waltlii to the first morphogenetic movements. Therefore the outside and inside cell surfaces of embryo are different already at stage 8–32 cells (and probably earlier), before the first step of morphogenesis. The changes of cell surface proteins at early embryonal development take place inside the embryo, in the regions of cell-cell interactions.     

Cell wall hydroxyproline-polysaccharide associations in Lupinus hypocotyls

Extraction of lupin hypocotyl cell walls with guanidine thiocynate, both before and after dilute acid treatment does not dissolve the hydroxyproline indicating that compounds containing this amino acid are probably covalently linked to insoluble wall constituents other than through acid labile arabinofuranose-hydroxyproline links. Dilute alkali does extract all of the wall hydroxyproline largely as non-dialysable material. Sequential extraction of cell walls with alkali at two temperatures (2° and 22–25°) removes most of the hemicellulose at the lower temperature but only dissolves the hydroxyproline at the higher temperature. Other studies show that the hydroxyproline containing polymer is co-precipitated with hemicellulose-B arabino-xylan. When cell walls from elongating and non-elongating hypocotyl sections are compared using this sequential extraction, the hemicellulose-B arabino-xylan containing hydroxyproline from the non-elongating wall has a much higher proportion of arabinoseand galactose than the same polymer from the elongating wall. Much more of the hydroxyproline from the elongating wall is dialysable. These results indicate more bonding of the hydroxyproline-containing glycoprotein within the wall of non-elongating tissue consistent with its suggested role in stopping cell elongation. It is suggested that the glycoprotein is linked to insoluble wall constituents such as cellulose through galactose or by direct protein to cellulose links.     

Bacillusanthracis Surface-Layer Proteins Assemble by Binding to the Secondary Cell Wall Polysaccharide in a Manner that Requires csaB and tagO

Bacillus anthracis, the causative agent of anthrax, requires surface (S)-layer proteins for the pathogenesis of infection. Previous work characterized S-layer protein binding via the surface layer homology domain to a pyruvylated carbohydrate in the envelope of vegetative forms. The molecular identity of this carbohydrate and the mechanism of its display in the bacterial envelope are still unknown. Analyzing acid-solubilized, purified carbohydrates by mass spectrometry and NMR spectroscopy, we identify secondary cell wall polysaccharide (SCWP) as the ligand of S-layer proteins. In agreement with the model that surface layer homology domains bind to pyruvylated carbohydrate, SCWP was observed to be linked to pyruvate in a manner requiring csaB, the only structural gene known to be required for S-layer assembly. B. anthracis does not elaborate wall teichoic acids; however, its genome harbors tagO and tagA, genes responsible for the synthesis of the linkage unit that tethers teichoic acids to the peptidoglycan layer. The tagO gene appears essential for B. anthracis growth and complements the tagO mutant phenotypes of staphylococci. Tunicamycin-mediated inhibition of TagO resulted in deformed, S-layer-deficient bacilli. Together, these results suggest that tagO-mediated assembly of linkage units tethers pyruvylated SCWP to the B. anthracis envelope, thereby enabling S-layer assembly and providing for the pathogenesis of anthrax infections.     

Cell proliferation in the seminiferous and epididymal epithelia of Sus domesticus

It is important to understand the proliferative activity of the different structures of the male reproductive apparatus in livestock species, such as Sus domesticus, to ensure reproductive efficiency. The main aims of this study were (a) to evaluate the proliferative activity of the spermatogonia in the different stages of the seminiferous cycle and (b) to study the cell proliferation in the epididymal epithelium in each region, identifying the different cells involved. For this, the testes and epididymis of three healthy, sexually mature Sus domesticus boars were used. The organs were processed for light microscopy, and immunohistochemical techniques were used to detect proliferating cell nuclear antigen. The cells immunostaining positively and negatively for proliferating cell nuclear antigen were counted and several parameters and indexes were calculated to evaluate the proliferation in both epithelia, taking into account the stage of the seminiferous epithelium cycle, and, in the case of the epididymal epithelium, the different regions and cells are the same. Finally, a contrast analysis of equality between pairs of means was carried out followed by a least significant differences test, in which differences were considered significant at P < 0.05. In the seminiferous epithelium, the greatest total number of spermatogonia and proliferating spermatogonia was observed in the postmeiotic stages (mainly VII and VIII). The proliferation index of the spermatogonia increased from the meiotic to postmeiotic stages. As regards the epididymal epithelium, the total proliferation index was higher in the caput. In each region, the clear and principal cells showed the highest proliferation index with respect to the total number of cells counted, whereas the proliferation index of each cell with respect to the same type was higher in the clear cells, followed by the narrow and principal cells. In conclusion, the proliferative activity of spermatogonia in the seminiferous epithelium of Sus domesticus is stage-dependent, and mainly occurs in the postmeiotic stages. In the epididymal epithelium, proliferative activity takes place in several cell types and is dependent on the anatomical region of the epididymis. We think that these results may be of importance for understanding the pathologic or reproductive processes in which cell proliferation is involved in the male reproductive system.     

Direct ESI-MS analysis of O-acyl glycosylated cardiolipins from the thermophilic bacterium Alicyclobacillus acidoterrestris

We used direct ESI-MS analysis to identify derivatives of cardiolipin molecular species (i.e. O-acyl glycosylated cardiolipins) from the thermophilic bacterium Alicyclobacillus acidoterrestris. We used triple-quadrupole type mass spectrometer for analysis of this complex lipid and enzymatic hydrolysis and ¹H and ¹³C NMR for the identification of these cardiolipin derivatives. These techniques enabled us to identify and quantify the specific molecular species profiles of derivatives of cardiolipin directly from lipid extracts of the bacterium including the identification of the sugar moiety as α-d-mannose and all five acyls including their positional isomers.