Gliding movement in Peranema trichophorum is powered by flagellar surface motility

A colorless euglenoid flagellate Peranema trichophorum shows unique unidirectional gliding cell locomotion on the substratum at velocities up to 30 micro m/s by an as yet unexplained mechanism. In this study, we found that (1) treatment with NiCl(2) inhibited flagellar beating without any effect on gliding movement; (2) water currents applied to a gliding cell from opposite sides caused detachment of the cell body from the substratum. With only the anterior flagellum adhering to the substratum, gliding movement continued along the direction of the anterior flagellum; (3) gentle pipetting induced flagellar severance into various lengths. In these cells, gliding velocity was proportional to the flagellar length; and (4) Polystyrene beads were translocated along the surface of the anterior flagellum. All of these results indicate that a cell surface motility system is present on the anterior flagellum, which is responsible for cell gliding in P. trichophorum.     

Forced expression of a dominant-negative chimeric tropomyosin causes abnormal motile behavior during cell division

Forced expression of the chimeric human fibroblast tropomyosin 5/3 (hTM5/3) in CHO cell was previously shown to affect cytokinesis [Warren et al., 1995: J. Cell Biol. 129:697-708]. To further investigate the phenotypic consequences of misexpression, we have compared mitotic spindle organization and dynamic 2D and 3D shape changes during mitosis in normal cells and in a hTM5/3 misexpressing (mutant) cell line. Immunofluorescence microscopy of wild type and mutant cells stained with monoclonal anti-tubulin antibody revealed that the overall structures of mitotic spindles were not significantly different. However, the axis of the mitotic spindle in mutant cells was more frequently misaligned with the long axis of the cell than that of wild type cells. To assess behavioral differences during mitosis, wild type and mutant cells were reconstructed in 2D and 3D and motion analyzed with the computer-assisted 2D and 3D Dynamic Image Analysis Systems (2D-DIAS, 3D-DIAS). Mutant cells abnormally formed large numbers of blebs during the later stages of mitosis and took longer to proceed from the start of anaphase to the start of cytokinesis. Furthermore, each mutant cell undergoing mitosis exhibited greater shape complexity than wild type cells, and in every case lifted one of the two evolving daughter cells off the substratum and abnormally twisted. These results demonstrate that misexpression of hTM5/3 in CHO cells leads to morphological instability during mitosis. Misexpression of hTM5/3 interferes with normal tropomyosin function, suggesting in turn that tropomyosin plays a role through its interaction with actin microfilaments in the regulation of the contractile ring, in the localized suppression of blebbing, in the maintenance of polarity and spatial symmetry during cytokinesis, and in cell spreading after cytokinesis is complete.     

PtdIns(3,4,5)P3 regulates spindle orientation in adherent cells

Cultured adherent cells divide on the substratum, leading to formation of the cell monolayer. However, how the orientation of this anchorage-dependent cell division is regulated remains unknown. We have previously shown that integrin-dependent adhesion orients the spindle parallel to the substratum, which ensures this anchorage-dependent cell division. Here, we show that phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P3) is essential for this spindle orientation control. In metaphase, PtdIns(3,4,5)P3 is accumulated in the midcortex in an integrin-dependent manner. Inhibition of phosphatidylinositol-3-OH kinase (PI(3)K) reduces the accumulation of PtdIns(3,4,5)P3 and induces spindle misorientation. Introduction of PtdIns(3,4,5)P3 to these cells restores the midcortical accumulation of PtdIns(3,4,5)P3 and proper spindle orientation. PI(3)K inhibition causes dynein-dependent spindle rotations along the z-axis, resulting in spindle misorientation. Moreover, dynactin, a dynein-binding partner, is accumulated in the midcortex in a PtdIns(3,4,5)P3-dependent manner. We propose that PtdIns(3,4,5)P3 directs dynein/dynactin-dependent pulling forces on spindles to the midcortex, and thereby orients the spindle parallel to the substratum.     

A new oligotrich (Ciliophora,Oligotrichia) from Argentina,with redefinition of Novistrombidium Song and Bradbury

A new oligotrich similar to Novistrombidium was discovered in plankton samples from an artificial tributary of the Salado River, Buenos Aires province, Argentina, in summer 2010. Propecingulum fistoleramalliei sp. n. has an obovate and anteriorly truncated body, with a conspicuous ventral furrow, is flattened ventrally, and has a prominent right apical protrusion. It temporarily attaches to the substratum by a posterior mucous thread. Rod-shaped extrusomes arranged equidistantly and insert directly above the girdle kinety. The macronucleus is globular to ellipsoidal. The contractile vacuole is located in the left, anterior quarter of the cell and the adoral zone is composed of 30–35 collar, 9–14 buccal, and two thigmotactic membranelles. The girdle kinety is dextrally spiraled and ventrally open; the ventral kinety is posterior to anterior end of the girdle kinety. The oral primordium develops posterior to the right thigmotactic membranelle and anterior the stripe of extrusomes above left, lateral portion of the girdle kinety. The SSUrDNA phylogeny confirms one more time that Novistrombidium is not monophyletic; consequently, we elevate the subgenus Propecingulum up to genus rank and redefine the genus Novistrombidium.     

Actin cytoskeleton dynamics and the cell division cycle

The network of actin filaments is one of the crucial cytoskeletal structures contributing to the morphological framework of a cell and which participates in the dynamic regulation of cellular functions. In adherent cell types, cells adhere to the substratum during interphase and spread to assume their characteristic shape supported by the actin cytoskeleton. This actin cytoskeleton is reorganized during mitosis to form rounded cells with increased cortical rigidity. The actin cytoskeleton is re-established after mitosis, allowing cells to regain their extended shape and attachment to the substratum. The modulation of such drastic changes in cell shape in coordination with cell cycle progression suggests a tight regulatory interaction between cytoskeleton signalling, cell–cell/cell–matrix adhesions and mitotic events. Here, we review the contribution of the actin cytoskeleton to cell cycle progression with an emphasis on the effectors responsible for the regulation of the actin cytoskeleton and integration of their activities with the cell cycle machinery.     

A Computer-based image analysis system for biocide screening

A computer-based image analysis system that measures the behavioral response of the marine diatom Amphora coffeaeformis to potentially toxic challenges is described. At any one time the system is capable of measuring in a population of about 80 cells, changes in the number motile, their speed, direction, and if they are turning, their angular velocity and acceleration. Chemical compounds that interfere with the generation of energy, protein or glycoprotein synthesis, calcium homeostasis and cytoskeletal activity, change the motile behavior of the cell. A pre-requisite of motility is that the cells be attached to a substratum. It is proposed that the system, which requires only a 1-2 minute data collection period per replicate, could be used to screen for potential antifouling activity in chemical agents, and it is speculated that it could be used also for measuring the physicochemical interaction between attached motile organisms and their substratum, as well as in environment toxicology measurements.     

Invertebrate myosin filament: Parallel subfilament arrangement in the wall of solid filaments from the honeybee, Apis mellifica

Transverse serial sections (100-140 nm thick) of solid myosin filaments of the honeybee, Apis mellifica, were photographed in a JEM-200 electron microscope at 200 kV. The images were digitized and computer processed by rotational filtering. 87% of the myosin filaments showed 6-fold symmetry in their power spectra, confirming the results of earlier works (Beinbrech et al., 1988, 1991). To determine if the subfilaments were arranged parallel to the filament backbone, two methods were used. First, the three images of each myosin filament in the three serial sections were superimposed. 85% of the resulting images showed a strong peak for 6-fold symmetry and the averaged images showed 6 pairs of subfilaments, which gives evidence for parallel arrangement of the subfilaments relative to the filament axis. This result was confirmed by the second method in which a 3-dimensional reconstruction was made. An average image was made from the images of the same 17 myosin filaments from each of the three sections. The data for the 3-dimensional reconstruction were collected by tracing the outlines of the structures in the three successive sections. The resulting stereo image shows a parallel arrangement of the subfilaments.     

Intracellular degradation of the complement C3b/C4b receptor in the absence of ligand

Human neutrophils (PMN) respond to various soluble stimuli by translocating intracellular complement C3b/C4b receptors (CR1) to the cell surface. Ligand-independent internalization of surface CR1 has been demonstrated previously, but the fate of total cellular CR1 during PMN stimulation has not been determined. In order to study the fate of CR1 during neutrophil activation, we have employed a unique approach for the quantitative analysis of intracellular antigens which allows simultaneous measurement of total cellular and surface membrane antigen pools. Stimulation of isolated PMN with N-formyl-Met-Leu-Phe or ionomycin resulted in a mean 7-fold increase in surface CR1 expression within 15 min. Total cellular CR1 decreased by as much as 45% within 15 min, with loss continuing for up to 1 h. Inclusion of NH4Cl during PMN stimulation inhibited the loss of total CR1 without affecting surface CR1 expression. Addition of phenylmethylsulfonyl fluoride inhibited loss of total CR1 and enhanced the stimulus-induced increases in surface CR1. These data suggest that intracellular degradation of CR1 occurs during stimulation of PMN and may involve proteolysis in an acidic intracellular compartment. Since our experiments were done with isolated PMN in the absence of serum and complement components, this degradation occurred in the absence of C3b, the ligand for CR1. To our knowledge, ligand-independent degradation of a cell surface receptor has not been previously detected.     

Cell adhesion to extracellular matrix regulates the life cycle of integrins.

The expression of alpha 5 beta 1 integrin on the surface of fibroblasts requires adhesion to substratum. We have examined the basis for this adhesion-dependent surface expression by comparing the life cycle of integrins in parallel cultures of adherent and nonadherent cells. Results of biosynthetic labeling experiments in NRK fibroblasts showed that the synthesis and biosynthetic processing of the beta 1 integrin subunit proceed in the absence of cell attachment; however, when examining the behavior of preexisting cell surface integrins, we observed that the alpha beta 1 integrins are internalized and degraded when adhesion to substratum is blocked. A kinetic analysis of integrin internalization in cycloheximide-treated NRK cells showed that each of the fibroblast integrins we examined (in both the beta 1 and beta 3 families) are lost from the cell surface after detachment from substratum. Thus, the default integrin life cycle in fibroblasts involves continuous synthesis, processing, transport to the cell surface, and internalization/degradation. Interestingly, studies with NIH-3T3 cells expressing alpha 1 beta 1 integrin showed that the loss of cell-surface alpha 5 beta 1 integrin is blocked by adhesion of cells to dishes coated with type IV collagen (a ligand for alpha 1 beta 1 integrin) as well as fibronectin. Similarly, adhesion of these cells to dishes coated with type IV collagen stabilizes the surface expression of alpha 5 beta 1 as well as alpha 1 beta 1 integrin. We propose that the adhesion of fibroblasts to extracellular matrix protein alters the integrin life cycle and permits retention of these proteins at the cell surface where they can play important roles in transmitting adhesion-dependent signals.     

Relationship of F-actin distribution to development of polar shape in human polymorphonuclear neutrophils

Polymerization of actin has been associated with development of polar shape in human neutrophils (PMN). To examine the relation of filamentous actin (F-actin) distribution to shape change in PMN, we developed a method using computerized video image analysis and fluorescence microscopy to quantify distribution of F-actin in single cells. PMN were labeled with fluorescent probe NBD-phallicidin to measure filamentous actin and Texas red to assess cell thickness. We show that Texas red fluorescence is a reasonable measure of cell thickness and that correction of the NBD-phallicidin image for cell thickness using the Texas red image permits assessment of focal F-actin content. Parameters were derived that quantify total F-actin content, movement of F-actin away from the center of the cell, asymmetry of F-actin distribution, and change from round to polar shape. The sequence of change in F-actin distribution and its relation to development of polar shape in PMN was determined using these parameters. After stimulation with chemotactic peptide at 25 degrees C, F-actin polymerized first at the rim of the PMN. This was followed by development of asymmetry of F-actin distribution and change to polar shape. The dominant pseudopod developed first in the region of lower F-actin concentration followed later by polymerization of actin in the end of the developed pseudopod. Asymmetric F-actin distribution was detected in round PMN before development of polar shape. Based upon these data, asymmetric distribution of F-actin is coincident with and probably precedes development of polar shape in PMN stimulated in suspension by chemotactic peptide.     

SYMMETRY,LOCOMOTION, AND THE EVOLUTION OF AN ANTERIOR END: A LESSON FROM SEA URCHINS

Bilaterally symmetrical, “regular” sea urchins in the Family Echinometridae (Class Echinoidea; Phylum Echinodermata) were found to lack a locomotor anterior. Heterocentrotus mammillatus and Echinometra mathaei were observed while locomoting. Members of both ellipsoidal species were found to proceed with their short or long axis foremost with statistically equivalent frequencies. This finding demonstrates that the evolution of bilateral symmetry is not always accompanied by the evolution of a locomotor “anterior” end. The elliptical echinometrid sea urchins provide a particularly appropriate study group for investigating the relationship between the evolution of body form and locomotor behavior. Although the radially symmetrical regular sea urchins, from which the echinometrids sprang, lack a locomotor anterior, all “irregular” echinoids, which are also derived from a regular ancestor but are bilaterally symmetrical, possess an “obligate” locomotor anterior. The symmetry and behavior exhibited by the elliptical echinometrid sea urchins therefore demonstrates that the first irregular echinoids (which exhibit bilateral symmetry by definition) need not have possessed a locomotor anterior as they do today.     

Hatching mechanism and accelerated hatching of the eggs of a sac-spawning euphausiid Nematoscelis difficilis

Observation of the sac-spawning euphausiid Nematoscelis difficilisHansen during shipboard laboratory incubations showed that itsembryos usually hatch as pseudometanauplius (PMN) or metanauplius(MN). The eggs, which have a minute perivitelline space, arespherical at spawning and become elliptical after the nauplius1 develops. When ready to hatch, the PMN or MN embryos extendand contract their first and second antennae in a swimming movement,breaking the chorion into almost equal halves joined by onesmall section in the anterior part of the chorion. The mandiblesplay a secondary role in cutting the chorion. Then the embryopushes itself backwards with the first and second antennae toescape from the chorion. This is known as ‘push-off’hatching. The embryos always hatch progressively from the distalend towards the proximal end of the ovigerous sac. The timebetween hatching of the first and last embryo may reflect thetime the females require to lay a clutch of eggs (<2.1 h).Development time to the PMN stage at 10°C was     

Mechanochemical aspects of axonemal dynein activity studied by in vitro microtubule translocation.

We have determined the relationship between microtubule length and translocation velocity from recordings of bovine brain microtubules translocating over a Paramecium 22S dynein substratum in an in vitro assay chamber. For comparison with untreated samples, the 22S dynein has been subjected to detergent and/or to pretreatments that induce phosphorylation of an associated 29 kDa light chain. Control and treated dyneins have been used at the same densities in the translocation assays. In any given condition, translocation velocity (v) shows an initial increase with microtubule length (L) and then reaches a plateau. This situation may be represented by a hyperbola of the general form v = aL/(L+b), which is formally analogous to the Briggs-Haldane relationship, which we have used to interpret our data. The results indicate that the maximum translocation velocity Vo(= a) is increased by pretreatment, whereas the length constant KL(= b), which corresponds to Km, does not change with pretreatment, implying that the mechanochemical properties of the pretreated dyneins differ from those of control dyneins. The conclusion that KL is constant for defined in vitro assays rules out the possibility that the velocity changes seen are caused by changes in geometry in the translocation assays or by the numbers of dyneins or dynein heads needed to produce maximal translocational velocity. From our analysis, we determine that f, the fraction of cycle time during which the dynein is in the force-generating state, is small--roughly 0.01, comparable to the f determined previously for heavy meromyosin. The practical limits of these mechanochemical changes imply that the maximum possible ciliary beat frequency is about 120 Hz, and that in the physiological range of 5-60 Hz, beat frequency could be controlled by varying the numbers of phosphorylated outer arm dyneins along an axonemal microtubule.     

Ciliary beating in three dimensions: Steps of a quantitative description

We document a novel approach for quantitative assessment of ciliary activity, exemplified in rapid three-dimensional cyclic motion of the frontal cirri of Stylonychia. Cells held under voltage-clamp control are stimulated by step pulses to elicit reproducible hyperpolarization- or depolarization-induced ciliary motor responses. High-speed video recording at 200 fields per second is used for imaging ciliary organelles of the same cell in two perspectives: the axial view and, following cell rotation by 90 degrees, the lateral view. From video sequences of typically 1 s, the contours of the cirral images are determined and digitized. Computer programs are established to (1) reduce an observed image to a "ciliary axis", (2) sort series of axes by template to generate an averaged ciliary cycle in 2D-projection, and (3) to associate the generalized axial and lateral 2D-images for generation of a sequence of three-dimensional images, which quantitatively represent the cycle in space and time. The method allows us to produce predetermined perspectives of images selected from the ciliary cycle, and to generate stereo views for graphical representation of ciliary motion. The approach includes a potential for extraction of the complete microtubular sliding program of a cilium under reproducible electric stimulation of the ciliary membrane.     

Image analysis of the burrowing mechanisms of Polyphysia crassa (Annelida: Polychaeta) and Priapulus cudatus (Priapulida)

The burrowing mechanisms of two soft-bodied marine invertebrates, Polyphysia crassa (Annelida: Polychaeta) and Priapulus caudatus (Priapulida) were re-examined and extended utilizing computer image analysis. Predetermined points on sequential photographs of animals burrowing in methylcellulose (an artificial medium of high transparency) were digitized and stored as x, y coordinates from which were calculated segment length, width, and volume in addition to producing real and straightened images of the animal on a plotter. Both species are adapted to soft substrata and both utilize a direct peristaltic wave to advance the body into a cavity or loosened area formed by anterior structures. Polyphysia displaces and loosens the substratum with lateral scraping movements of its anterior six segments (the "head region") while Priapulus makes a large cavity anteriorly by the forceful eversion and dilation of the praesoma. Unlike Priapulus, Polyphysia utilizes a different method of locomotion when moving on the mud surface as opposed to burrowing, the former involving two direct peristaltic waves at a time and negligible internal pressures resulting in nearly continuous advance. However when burrowing, Polyphysia , like Priapulus , utilizes a single direct peristaltic wave alternating with phasic pressure pulses which advance the animal in step-wise fashion.     

Cell cycle dependent TN-C promoter activity determined by live cell imaging

The extracellular matrix protein tenascin-C plays a critical role in development, wound healing, and cancer progression, but how it is controlled and how it exerts its physiological responses remain unclear. By quantifying the behavior of live cells with phase contrast and fluorescence microscopy, the dynamic regulation of TN-C promoter activity is examined. We employ an NIH 3T3 cell line stably transfected with the TN-C promoter ligated to the gene sequence for destabilized green fluorescent protein (GFP). Fully automated image analysis routines, validated by comparison with data derived from manual segmentation and tracking of single cells, are used to quantify changes in the cellular GFP in hundreds of individual cells throughout their cell cycle during live cell imaging experiments lasting 62 h. We find that individual cells vary substantially in their expression patterns over the cell cycle, but that on average TN-C promoter activity increases during the last 40% of the cell cycle. We also find that the increase in promoter activity is proportional to the activity earlier in the cell cycle. This work illustrates the application of live cell microscopy and automated image analysis of a promoter-driven GFP reporter cell line to identify subtle gene regulatory mechanisms that are difficult to uncover using population averaged measurements.     

ClC-3 and IClswell are required for normal neutrophil chemotaxis and shape change

Polymorphonuclear leukocytes undergo directed movement to sites of infection, a complex process known as chemotaxis. Extension of the polymorphonuclear leukocyte (PMN) leading edge toward a chemoattractant in association with uropod retraction must involve a coordinated increase/decrease in membrane, redistribution of cell volume, or both. Deficits in PMN phagocytosis and trans-endothelial migration, both highly motile PMN functions, suggested that the anion transporters, ClC-3 and ICl(swell), are involved in cell motility and shape change ( Moreland, J. G., Davis, A. P., Bailey, G., Nauseef, W. M., and Lamb, F. S. (2006) J. Biol. Chem. 281, 12277-12288 ). We hypothesized that ClC-3 and ICl(swell) are required for normal PMN chemotaxis through regulation of cell volume and shape change. Using complementary chemotaxis assays, EZ-TAXIScantrade mark and dynamic imaging analysis software, we analyzed the directed cell movement and morphology of PMNs lacking normal anion transporter function. Murine Clcn3(-/-) PMNs and human PMNs treated with anion transporter inhibitors demonstrated impaired chemotaxis in response to formyl peptide. This included decreased cell velocity and failure to undergo normal cycles of elongation and retraction. Impaired chemotaxis was not due to a diminished number of formyl peptide receptors in either murine or human PMNs, as measured by flow cytometry. Murine Clcn3(-/-) and Clcn3(+/+) PMNs demonstrated a similar regulatory volume decrease, indicating that the ICl(swell) response to hypotonic challenge was intact in these cells. We further demonstrated that ICl(swell) is essential for shape change during human PMN chemotaxis. We speculate that ClC-3 and ICl(swell) have unique roles in regulation of PMN chemotaxis; ICl(swell) through direct effects on PMN volume and ClC-3 through regulation of ICl(swell).     

Polymorphonuclear neutrophils enhance anti-CD3-induced T cell activation: the role of polymorphonuclear FC-receptors.

We investigated the effect of polymorphonuclear neutrophils (PMN) on anti-CD3 mAb (OKT3 and anti-Leu4)-mediated T cell activation. In the absence of monocytes, purified E-rosette-positive cells (further referred to as "T cells") require either solid-phase bound anti-CD3 or the combination of both a high concentration of soluble anti-CD3 and exogenous recombinant interleukin 2 (rIL-2) to proliferate. PMN cannot sustain T cell proliferation with soluble anti-CD3, but they markedly boost proliferation in the presence of soluble anti-CD3 and rIL-2. When PMN were added to T cell cultures stimulated with anti-CD3, this resulted in IL-2 receptor (IL-2R) expression and CD3 modulation. The mechanism of enhancement of anti-CD3-induced IL-2-responsiveness by PMN was further analyzed. A cellular T cell-PMN interaction was found to play a critical role and this was mediated through PMN Fc receptors (FcR). PMN bear two types of low-affinity FcR (FcRII and FcRIII). FcRII is known to bind mIgG1 (e.g., anti-Leu4) and FcRIII binds mIgG2a (e.g., OKT3). FcR involvement was demonstrated by two observations. Anti-FcRII mAb IV.3 inhibited the PMN signal for T cell activation with anti-Leu4. PMN bearing the second variant of FcRII which is unable to bind mIgG1 failed to promote anti-Leu4/IL-2-mediated T cell proliferation. Thus, PMN potentiate T cell responsiveness to IL-2 in the presence of anti-CD3 mAb and this potentiation by PMN requires interaction of anti-CD3 with PMN-FcR.     

A computational model of ameboid deformation and locomotion

Traditional continuum models of ameboid deformation and locomotion are limited by the computational difficulties intrinsic in free boundary conditions. A new model using the immersed boundary method overcomes these difficulties by representing the cell as a force field immersed in fluid domain. The forces can be derived from a direct mechanical interpretation of such cell components as the cell membrane, the actin cortex, and the transmembrane adhesions between the cytoskeleton and the substratum. The numerical cytoskeleton, modeled as a dynamic network of immersed springs, is able to qualitatively model the passive mechanical behavior of a shear-thinning viscoelastic fluid (Bottino 1997). The same network is used to generate active protrusive and contractile forces. When coordinated with the attachment-detachment cycle of the cell's adhesions to the substratum, these forces produce directed locomotion of the model ameba. With this model it is possible to study the effects of altering the numerical parameters upon the motility of the model cell in a manner suggestive of genetic deletion experiments. In the context of this ameboid cell model and its numerical implementation, simulations involving multicellular interaction, detailed internal signaling, and complex substrate geometries are tractable. Received: 5 January 1998 / Revised version: 23 March 1998 / Accepted: 26 March 1998