Tip Of Filament Research Articles

Reorganization of the Growth Pattern of Schizosaccharomyces pombe in Invasive Filament Formation

The organization and control of polarized growth through the cell cycle of Schizosaccharomyces pombe, a single-celled eukaryote, have been studied extensively. We have investigated the changes in these processes when S. pombe differentiates to form multicellular invasive mycelia and have found striking alterations to the behavior of some of the key regulatory proteins. Cells at the tips of invading filaments are considerably more elongated than cells growing singly and grow at one pole only. The filament tip follows a strict direction of growth through multiple cell cycles. A group of proteins involved in the growth process and actin regulation, comprising Spo20, Bgs4, activated Cdc42, and Crn1, are all concentrated at the growing tip, unlike their distribution at both ends of single cells. In contrast, several proteins implicated in microtubule-dependent organization of growth, including Tea1, Tea4, Mod5, and Pom1, all show the opposite effect and are relatively depleted at the growing end and enriched at the nongrowing end, although Tea1 appears to continue to be delivered to both ends. A third group acting at different stages of the cell cycle, including Bud6, Rga4, and Mid1, localize similarly in filaments and single cells, while Nif1 shows a reciprocal localization to Pom1.

Phylogeny, ultrastructure, histopathology and prevalence of Myxobolus oliveirai sp. nov., a parasite of Brycon hilarii (Characidae) in the Pantanal wetland, Brazil

This paper presents the morphological, histological and ultrastructural characteristics of Myxobolus oliveirai sp. nov., a parasite of the gill filaments in Brycon hilarii from the Brazilian Pantanal. Out of 216 B. hilariispecimens examined (126 wild and 90 cultivated), 38.1% of wild specimens (n = 48) were infected. The parasites form elongated plasmodia primarily in the tip of gill filaments, reaching about 3 mm in length. A thorough comparison with all the Myxobolus species described from South American hosts, as well as nearly all the Myxobolus species described so far is provided. Partial sequencing of the 18S rDNA gene revealed a total of 1,527 bp. The Myxobolus species parasite of B. hilarii did not match any of the Myxozoa available in GenBank. In the phylogenetic analysis, M. oliveirai sp. nov. composed a monophyletic group with eight other species: five species of Myxobolus parasites of mugilid fishes, two parasites of pangasiid and one of centrarchid. Infection prevalence values of the parasite revealed no significant differences between wet and dry seasons or between males and females. The importance of the infection to the farming of the host species is emphasized.

Virus takes the actin express

Viruses typically hitchhike around the cytoplasm on microtubules. [Ohkawa et al.][1] reveal that one type of virus can travel within the cell using actin filaments. ![Figure][2] A baculovirus (red) zooms along at the tip of a polymerizing actin filament (green). Once baculoviruses break

Modeling capping protein FRAP and CALI experiments reveals in vivo regulation of actin dynamics

To gain insights on cellular mechanisms regulating actin polymerization, we used the Virtual Cell to model fluorescence recovery after photobleaching (FRAP) and chromophore-assisted laser inactivation (CALI) experiments on EGFP-capping protein (EGFP-CP). Modeling the FRAP kinetics demonstrated that the in vivo rate for the dissociation of CP from actin filaments is much faster (approximately 0.1 s(-1)) than that measured in vitro (0.01-0.0004 s(-1)). The CALI simulation revealed that in order to induce sustainable changes in cell morphology after CP inactivation, the cells should exhibit anticapping ability. We included the VASP protein as the anticapping agent in the modeling scheme. The model predicts that VASP affinity for barbed ends has a cooperative dependence on the concentration of VASP-barbed end complexes. This dependence produces a positive feedback that stabilizes the complexes and allows sustained growth at clustered filament tips. We analyzed the range of laser intensities that are sufficient to induce changes in cell morphology. This analysis demonstrates that FRAP experiments with EGFP-CP can be performed safely without changes in cell morphology, because, the intensity of the photobleaching beam is not high enough to produce the critical concentration of free barbed ends that will induce filament growth before diffusional replacement of EGFP-CP occurs.

Atmospheric corrosion initiation on steel from predeposited NaCl salt particles in high humidity atmospheres

Corrosion initiation on steel from predeposited NaCl particles was observed in situ using an optical microscope. Corrosion did not initiate from small droplets (D<&sim45 μm) formed by the deliquescence of predeposited NaCl particles upon exposing to high humidity (RH>80%). Corrosion occurred under larger NaCl droplets and was accompanied by the formation of secondary droplets outside of the perimeter of the initial droplets. Sometimes, corrosion that initiated from the large NaCl droplets propagated in the form of filiform corrosion. Elemental mapping using energy dispersive X-ray analysis showed that Cl was concentrated at the filament tip, while Na migrated to the outside region of the secondary droplets. Under large NaCl droplets, there was evidence that corrosion initiated from slag based inclusions.

New insights into F-pilus structure, dynamics, and function

F-pili are thin, flexible filaments elaborated by F(+) cells of Escherichia coli. They belong to the class of Gram-negative pili that function in horizontal gene transfer. F-pili are initially required to establish contacts between DNA donor and recipient cells. Beyond that, F-pilus function, and that of other conjugative pili, has remained obscure and controversial. The idea that F-pili are dynamic structures was proposed 40 years ago. Initially, F-pili were thought to remain extended until another cell bound to the filament tip, whereupon the filament retracted to bring the contacted cell to the donor cell surface. Thereafter, secure surface-surface contacts would allow efficient DNA transfer. A later variant of this hypothesis was that F-pili are inherently dynamic, elongating and retracting even in the absence of exogenous signals. A very different hypothesis, also proposed first about 40 years ago, was that F-pili are conduits, presumably passive, for the transfer of DNA from donor to recipient. In this hypothesis, DNA transfer is not obligatorily coupled to F-pilus retraction. Here, we review recent data obtained by integrating long-established facts about the biology of F-pili with modern tools of fluorescence and electron microscopy. These data suggest that one function for F-pili is to search a large volume around donor cells in liquid culture for the presence of other cells. However, this may not be the only function. We show that F-pilin is also required at a second, largely undefined step occurring after cells have been brought into direct contact by F-pilus retraction.

Mechanical unfolding of bacterial flagellar filament protein by molecular dynamics simulation

Bacterial flagellum is a nano-scale motility device constructed by self-assembly. During construction of the cell-exterior filament (the ‘propeller’), subunit proteins (called flagellin) are thought to be exported through the hollow flagellum to the growing filament tip in an unfolded state. To gain insight into the unfolded state preceding any force-spectroscopy experiments on flagellin, we employed force-probe molecular dynamics simulations. Two schemes to attain an unfolded state suitable for efficient transport were examined: (i) stretching flagellin along its length; (ii) unzipping flagellin from its adjacently placed termini. Atomic-level unfolding pathways and the mechanical efforts involved under each scheme were obtained for the four-domain flagellin from S. typhimurium. Flagellin appeared stiffer and required larger unfolding forces when stretched as the relative sliding of β-strands require the breaking of multiple hydrogen bonds at once. In contrast, unzipping requires lower unfolding forces as it mainly involves unraveling β-sheets by breaking hydrogen bonds one by one.

Mechanisms Influencing the Timing and Success of Reproductive Migration in a Capital Breeding Semelparous Fish Species, the Sockeye Salmon

Two populations of homing sockeye salmon (Oncorhynchus nerka; Adams and Chilko) were intercepted in the marine approaches around the northern and southern ends of Vancouver Island (British Columbia, Canada) en route to a natal river. More than 500 salmon were nonlethally biopsied for blood plasma, gill filament tips, and gross somatic energy (GSE) and were released with either acoustic or radio transmitters. At the time of capture, GSE, body length, and circulating testosterone ([T]) differed between populations, differences that reflected known life-history variations. Within-population analyses showed that in Adams sockeye salmon, plasma glucose ([glu]), lactate ([lactate]), and ion concentrations were higher in the northern approach than in the southern approach, suggesting that the former was more stressful. GSE, [T], and gill Na(+),K(+)-ATPase activities also differed between the two locales, and each varied significantly with Julian date, suggesting seasonality. Despite these relative geographic differences, the timing of river entry and the ability to reach spawning areas were strongly correlated with energetic, reproductive, and osmoregulatory state. Salmon that delayed river entry and reached spawning areas had relatively high GSE and low [T] and gill ATPase. In contrast, salmon that entered the river directly but that ultimately failed to reach spawning areas had lower GSE and higher [T] and gill ATPase, and they also swam at significantly faster rates (failed fish approximately 20.0 km d(-1) vs. successful fish approximately 15.5 km d(-1)). Physiologically, salmon that did not enter the river at all but that presumably died in the marine environment exhibited high stress (plasma [glu] and [lactate]) and ionoregulatory measures (plasma [Na(+)], [Cl(-)], osmolality).

Self-organization of the MinE protein ring in subcellular Min oscillations

We model the self-organization of the MinE ring that is observed during subcellular oscillations of the proteins MinD and MinE within the rod-shaped bacterium Escherichia coli. With a steady-state approximation, we can study the MinE ring generically--apart from the other details of the Min oscillation. Rebinding of MinE to depolymerizing MinD-filament tips controls MinE-ring formation through a scaled cell shape parameter r. We find two types of E-ring profiles near the filament tip: either a strong plateaulike E ring controlled by one-dimensional diffusion of MinE along the bacterial length or a weak cusplike E ring controlled by three-dimensional diffusion near the filament tip. While the width of a strong E ring depends on r, the occupation fraction of MinE at the MinD-filament tip is saturated and hence the depolymerization speed does not depend strongly on r. Conversely, for weak E rings both r and the MinE to MinD stoichiometry strongly control the tip occupation and hence the depolymerization speed. MinE rings in vivo are close to the threshold between weak and strong, and so MinD-filament depolymerization speed should be sensitive to cell shape, stoichiometry, and MinE-rebinding rate. We also find that the transient to MinE-ring formation is quite long in the appropriate open geometry for assays of ATPase activity in vitro, explaining the long delays of ATPase activity observed for smaller MinE concentrations in those assays without the need to invoke cooperative MinE activity.

Morphology and evolution of umbral dots and their substructures

Substructures - dark lanes and tails - of umbral dots (UDs) were predicted by numerical simulations of magnetoconvection. We analyse a 6 h 23 min time series of broadband images of a large umbra in the active region NOAA 10634, acquired with the 1-m Swedish Solar Telescope, in the wavelength band around 602 nm. A 43 min part of this series was reconstructed with the MFBD method, reaching a spatial resolution of 0.14". We measure brightness, size, lifetime, and horizontal velocities of various umbral structures. Most (90 %) of UDs and bright point-like features in faint LBs split and merge, and their median lifetimes are 3.5 or 5.7 min, depending on whether the split or merge event is considered as the end of their life. Both UDs and features in faint LBs that do not split or merge are clearly smaller (0.15") than the average size (0.17") of all features. Horizontal motions of umbral bright small-scale features are directed either into the umbra or along faint LBs with mean horizontal velocities of 0.34 km/s. Features faster than 0.4 km/s appear mostly at the periphery of the umbra. The intensity of dark lanes, measured in four bright central UDs (CUDs), is by a factor 0.8 lower than the peak intensity of CUDs. The width of dark lanes is probably less than the resolution limit 0.14". The characteristic time of substructure changes of UDs is ~4 min. We observe narrow (0.14") bright and dark filaments connected with PUDs. Usually one dark and two bright filaments form a 0.4" wide tail attached to one PUD, resembling a short dark-cored penumbral filament. Our results indicate the similarity between PUDs and PGs located at the tips of bright penumbral filaments. The features seen in numerical MHD simulations are consistent with our observations of dark lanes in CUDs and tails attached to PUDs.

Mechanics model for actin-based motility

We present here a mechanics model for the force generation by actin polymerization. The possible adhesions between the actin filaments and the load surface, as well as the nucleation and capping of filament tips, are included in this model on top of the well-known elastic Brownian ratchet formulation. A closed form solution is provided from which the force-velocity relationship, summarizing the mechanics of polymerization, can be drawn. Model predictions on the velocity of moving beads driven by actin polymerization are consistent with experiment observations. This model also seems capable of explaining the enhanced actin-based motility of Listeria monocytogenes and beads by the presence of Vasodilator-stimulated phosphoprotein, as observed in recent experiments.

65Zn2+ transport by isolated gill epithelial cells of the American lobster, Homarus americanus

Gills are the first site of impact by metal ions in contaminated waters. Work on whole gill cells and metal uptake has not been reported before in crustaceans. In this study, gill filaments of the American lobster, Homarus americanus, were dissociated in physiological saline and separated into several cell types on a 30, 40, 50, and 80% sucrose gradient. Cells from each sucrose solution were separately resuspended in physiological saline and incubated in 65Zn2+ in order to assess the nature of metal uptake by each cell type. Characteristics of zinc accumulation by each kind of cell were investigated in the presence and absence of 10 mM calcium, variable NaCl concentrations and pH values, and 100 muM verapamil, nifedipine, and the calcium ionophore A23187. 65Zn2+ influxes were hyperbolic functions of zinc concentration (1-1,000 microM) and followed Michaelis-Menten kinetics. Calcium reduced both apparent zinc binding affinity (K (m)) and maximal transport velocity (J (max)) for 30% sucrose cells, but doubled the apparent maximal transport velocity for 80% sucrose cells. Results suggest that calcium, sodium, and protons enter gill epithelial cells by an endogenous broad-specificity cation channel and trans-stimulate metal uptake by a plasma membrane carrier system. Differences in zinc transport observed between gill epithelial cell types appear related to apparent affinity differences of the transporters in each kind of cell. Low affinity cells from 30% sucrose were inhibited by calcium, while high affinity cells from 80% sucrose were stimulated. 65Zn2+ transport was also studied by isolated, intact, gill filament tips. These intact gill fragments generally displayed the same transport properties as did cells from 80% sucrose and provided support for metal uptake processes being an apical phenomenon. A working model for zinc transport by lobster gill cells is presented.

A Simple Model for the Disintegration of Highly Charged Solvent Droplets during Electrospray Ionization

This work uses a minimalist model for deciphering the opposing effects of Coulomb repulsion and surface tension on the stability of electrosprayed droplets. Guided by previous observations, it is assumed that progeny droplets are ejected from the tip of liquid filaments that are formed as protrusions of an initially spherical parent. Nonspherical shapes are approximated as assemblies of multiple closely spaced beads. This strategy greatly facilitates the calculation of electrostatic and surface energies. For a droplet at the Rayleigh limit the model predicts that growth of a very thin filament is a spontaneous process with a negligible activation barrier. In contrast, significant barriers are encountered for the formation of larger diameter filaments. These different barrier heights favor highly asymmetric droplet fission because the dimensions of the filament determine those of the ejected droplet(s). Substantial charge accumulation occurs at the filament termini. This allows each progeny droplet to carry a significant fraction of charge, despite its very small volume. In the absence of a long connecting filament, relieving electrostatic stress through progeny droplet emission would be ineffective. The model predicts the prevalence of fission events leading to the formation of several progeny droplets, instead of just a single one. Ejection bursts are followed by collapse back to a spherical shape. The resulting charge depleted system is incapable of producing additional progeny droplets until solvent evaporation returns it to the Rayleigh limit. Despite the very simple nature of the model used here, all of these predictions agree with experimental data.

Effects of Structure of Rho GTPase-activating Protein DLC-1 on Cell Morphology and Migration

DLC-1 encodes a Rho GTPase-activating protein (RhoGAP) and negative regulator of specific Rho family proteins (RhoA-C and Cdc42). DLC-1 is a multi-domain protein, with the RhoGAP catalytic domain flanked by an amino-terminal sterile α motif (SAM) and a carboxyl-terminal START domain. The roles of these domains in the regulation of DLC-1 function remain to be determined. We undertook a structure-function analysis involving truncation and missense mutants of DLC-1. We determined that the amino-terminal SAM domain functions as an autoinhibitory domain of intrinsic RhoGAP activity. Additionally, we determined that the SAM and START domains are dispensable for DLC-1 association with focal adhesions. We then characterized several mutants for their ability to regulate cell migration and identified constitutively activated and dominant negative mutants of DLC-1. We report that DLC-1 activation profoundly alters cell morphology, enhances protrusive activity, and can increase the velocity but reduce directionality of cell migration. Conversely, the expression of the amino-terminal domain of DLC-1 acts as a dominant negative and profoundly inhibits cell migration by displacing endogenous DLC-1 from focal adhesions.

Arp2/3 branched actin network mediates filopodia-like bundles formation in vitro.

During cellular migration, regulated actin assembly takes place at the cell leading edge, with continuous disassembly deeper in the cell interior. Actin polymerization at the plasma membrane results in the extension of cellular protrusions in the form of lamellipodia and filopodia. To understand how cells regulate the transformation of lamellipodia into filopodia, and to determine the major factors that control their transition, we studied actin self-assembly in the presence of Arp2/3 complex, WASp-VCA and fascin, the major proteins participating in the assembly of lamellipodia and filopodia. We show that in the early stages of actin polymerization fascin is passive while Arp2/3 mediates the formation of dense and highly branched aster-like networks of actin. Once filaments in the periphery of an aster get long enough, fascin becomes active, linking the filaments into bundles which emanate radially from the aster's surface, resulting in the formation of star-like structures. We show that the number of bundles nucleated per star, as well as their thickness and length, is controlled by the initial concentration of Arp2/3 complex ([Arp2/3]). Specifically, we tested several values of [Arp2/3] and found that for given initial concentrations of actin and fascin, the number of bundles per star, as well as their length and thickness are larger when [Arp2/3] is lower. Our experimental findings can be interpreted and explained using a theoretical scheme which combines Kinetic Monte Carlo simulations for aster growth, with a simple mechanistic model for bundles' formation and growth. According to this model, bundles emerge from the aster's (sparsely branched) surface layer. Bundles begin to form when the bending energy associated with bringing two filaments into contact is compensated by the energetic gain resulting from their fascin linking energy. As time evolves the initially thin and short bundles elongate, thus reducing their bending energy and allowing them to further associate and create thicker bundles, until all actin monomers are consumed. This process is essentially irreversible on the time scale of actin polymerization. Two structural parameters, L, which is proportional to the length of filament tips at the aster periphery and b, the spacing between their origins, dictate the onset of bundling; both depending on [Arp2/3]. Cells may use a similar mechanism to regulate filopodia formation along the cell leading edge. Such a mechanism may allow cells to have control over the localization of filopodia by recruiting specific proteins that regulate filaments length (e.g., Dia2) to specific sites along lamellipodia.

Evidence for the Oligomeric State of ‘Elastic’ Titin in Muscle Sarcomeres

The giant protein titin has important roles in muscle sarcomere integrity, elasticity and contractile activity. The key role in elasticity was highlighted in recent years by single-molecule mechanical studies, which showed a direct relationship between the non-uniform structure of titin and the hierarchical mechanism of its force-extension behavior. Further advances in understanding mechanisms controlling sarcomere structure and elasticity require detailed knowledge of titin arrangement and interactions in situ. Here we present data on the structure and self-interactive properties of an ∼ 290 kDa ( ∼ 100 nm long) tryptic fragment from the I-band part of titin that is extensible in situ. The fragment includes the conserved ‘distal’ tandem Ig segment of the molecule and forms side-by-side oligomers with distinctive 4 nm cross-striations. Comparisons between these oligomers and the end filaments seen at the tips of native thick filaments indicate identical structure. This shows that end-filaments are formed by the elastic parts of six titin molecules connecting each end of the thick filament to the Z-line. Self-association of elastic titin into stiff end-filaments adds a further hierarchical level in the mechanism of titin extensibility in muscle cells. Self-association of this part of titin may be required to prevent interference of the individual flexible molecules with myosin cross-bridges interacting with actin.

Actin Bundling: Initiation Mechanisms and Kinetics

Bundling of rapidly polymerizing actin filaments underlies the dynamics of filopodial protrusions that play an important role in cell migration and cell-cell interaction. Recently, the formation of actin bundles has been reconstituted in vitro, and two scenarios of bundle initiation, involving binding of two filament tips and, alternatively, linking of the tip of one filament to the side of the other, have been discussed. A first theoretical analysis is presented indicating that the two mechanisms can be distinguished experimentally. While both of them result counterintuitively in comparable numbers of bundles, these numbers scale differently with the average bundle length. We propose an experiment for determining which of the two mechanisms is involved in the in vitro bundle formation.

Model of reduction of actin polymerization forces by ATP hydrolysis

The effects of hydrolysis of ATP-actin to ADP-actin on actin polymerization-based force generation are calculated using a multifilament two-state Brownian ratchet model. The model treats an ensemble of rigid parallel filaments growing against a hard, inert, diffusing obstacle held in an optical trap. The filaments stochastically grow, depolymerize and undergo transitions between polymerizing and depolymerizing tip states. The parameters in the model are obtained from literature values and a fit to the measured dependence of the polymerization rate on the free-actin concentration. For more than two filaments, the stall force per filament near the critical concentration is much less than the equilibrium ATP-actin stall force. By reducing the availability of free monomers, the obstacle causes filament tips to convert to the depolymerizing state, so that only a small fraction of the filaments contact the obstacle at a given time.

4Pi Microscopy of the Nuclear Pore Complex

To explore whether super-resolution fluorescence microscopy is able to resolve topographic features of single cellular protein complexes, a two-photon 4Pi microscope was used to study the nuclear pore complex (NPC). The microscope had an axial resolution of 110–130 nm and a two-color localization accuracy of 5–10 nm. In immune-labeled HeLa cells, NPCs could be resolved much better by 4Pi than by confocal microscopy. When two epitopes of the NPC, one localized at the tip of the cytoplasmic filaments and the other at the ring of the nuclear basket, were immune-labeled, they could be clearly resolved in single NPCs, with the distance between them determined to be 152 ± 30 nm. In cells expressing a green fluorescent protein construct localized at the NPC center, the distances between the ring of the nuclear filaments and the NPC center was 76 ± 12 ( Potorous tridactylus cells) or 91 ± 21 nm (normal rat kidney cells), whereas the distance between the NPC center and the tips of the cytoplasmic filaments was 84 ± 18 nm, all values in good agreement with previous electron or single-molecule fluorescence estimates. We conclude that super-resolution fluorescence microscopy is a powerful method for analyzing single protein complexes and the cellular nanomachinery in general.

Septin 7 Interacts with Centromere-associated Protein E and Is Required for Its Kinetochore Localization

Chromosome segregation in mitosis is orchestrated by dynamic interaction between spindle microtubules and the kinetochore. Septin (SEPT) belongs to a conserved family of polymerizing GTPases localized to the metaphase spindle during mitosis. Previous study showed that SEPT2 depletion results in chromosome mis-segregation correlated with a loss of centromere-associated protein E (CENP-E) from the kinetochores of congressing chromosomes (1). However, it has remained elusive as to whether CENP-E physically interacts with SEPT and how this interaction orchestrates chromosome segregation in mitosis. Here we show that SEPT7 is required for a stable kinetochore localization of CENP-E in HeLa and MDCK cells. SEPT7 stabilizes the kinetochore association of CENP-E by directly interacting with its C-terminal domain. The region of SEPT7 binding to CENP-E was mapped to its C-terminal domain by glutathione S-transferase pull-down and yeast two-hybrid assays. Immunofluorescence study shows that SEPT7 filaments distribute along the mitotic spindle and terminate at the kinetochore marked by CENP-E. Remarkably, suppression of synthesis of SEPT7 by small interfering RNA abrogated the localization of CENP-E to the kinetochore and caused aberrant chromosome segregation. These mitotic defects and kinetochore localization of CENP-E can be successfully rescued by introducing exogenous GFP-SEPT7 into the SEPT7-depleted cells. These SEPT7-suppressed cells display reduced tension at kinetochores of bi-orientated chromosomes and activated mitotic spindle checkpoint marked by Mad2 and BubR1 labelings on these misaligned chromosomes. These findings reveal a key role for the SEPT7-CENP-E interaction in the distribution of CENP-E to the kinetochore and achieving chromosome alignment. We propose that SEPT7 forms a link between kinetochore distribution of CENP-E and the mitotic spindle checkpoint.