Cycles Of Retraction Research Articles

Interaction of the cyclic-di-GMP binding protein FimX and the Type 4 pilus assembly ATPase promotes pilus assembly.

Type IVa pili (T4P) are bacterial surface structures that enable motility, adhesion, biofilm formation and virulence. T4P are assembled by nanomachines that span the bacterial cell envelope. Cycles of T4P assembly and retraction, powered by the ATPases PilB and PilT, allow bacteria to attach to and pull themselves along surfaces, so-called “twitching motility”. These opposing ATPase activities must be coordinated and T4P assembly limited to one pole for bacteria to show directional movement. How this occurs is still incompletely understood. Herein, we show that the c-di-GMP binding protein FimX, which is required for T4P assembly in Pseudomonas aeruginosa, localizes to the leading pole of twitching bacteria. Polar FimX localization requires both the presence of T4P assembly machine proteins and the assembly ATPase PilB. PilB itself loses its polar localization pattern when FimX is absent. We use two different approaches to confirm that FimX and PilB interact in vivo and in vitro, and further show that point mutant alleles of FimX that do not bind c-di-GMP also do not interact with PilB. Lastly, we demonstrate that FimX positively regulates T4P assembly and twitching motility by promoting the activity of the PilB ATPase, and not by stabilizing assembled pili or by preventing PilT-mediated retraction. Mutated alleles of FimX that no longer bind c-di-GMP do not allow rapid T4P assembly in these assays. We propose that by virtue of its high-affinity for c-di-GMP, FimX can promote T4P assembly when intracellular levels of this cyclic nucleotide are low. As P. aeruginosa PilB is not itself a high-affinity c-di-GMP receptor, unlike many other assembly ATPases, FimX may play a key role in coupling T4P mediated motility and adhesion to levels of this second messenger.

Asymmetric distribution of type IV pili triggered by directional light in unicellular cyanobacteria

The type IV pili (T4P) system is a supermolecular machine observed in prokaryotes. Cells repeat the cycle of T4P extension, surface attachment, and retraction to drive twitching motility. Although the properties of T4P as a motor have been scrutinized with biophysics techniques, the mechanism of regulation remains unclear. Here we provided the framework of the T4P dynamics at the single-cell level in Synechocystis sp. PCC6803, which can recognize light direction. We demonstrated that the dynamics was detected by fluorescent beads under an optical microscope and controlled by blue light that induces negative phototaxis; extension and retraction of T4P was activated at the forward side of lateral illumination to move away from the light source. Additionally, we directly visualized each pilus by fluorescent labeling, allowing us to quantify their asymmetric distribution. Finally, quantitative analyses of cell tracking indicated that T4P was generated uniformly within 0.2 min after blue-light exposure, and within the next 1 min the activation became asymmetric along the light axis to achieve directional cell motility; this process was mediated by the photo-sensing protein, PixD. This sequential process provides clues toward a general regulation mechanism of T4P system, which might be essentially common between archaella and other secretion apparatuses.

The tumor promoter-activated protein kinase Cs are a system for regulating filopodia.

Different protein kinase C (PKC) isoforms have distinct roles in regulating cell functions. The conventional (α, β, γ) and novel (δ, ɛ, η, θ) classes are targets of phorbol ester tumor promoters, which are surrogates of endogenous second messenger, diacylglycerol. The promoter‐stimulated disappearance of filopodia was investigated by use of blocking peptides (BPs) that inhibit PKC maturation and/or docking. Filopodia were partially rescued by a peptide representing PKC ɛ hydrophobic sequence, but also by a myristoylated PKC α/β pseudosubstrate sequence, and an inhibitor of T‐cell protein tyrosine phosphatase (TC‐PTP). The ability to turn over filopodia was widely distributed among PKC isoforms. PKC α and η hydrophobic sequences enhanced filopodia in cells in the absence of tumor promoter treatment. With transcriptional knockdown of PKC α, the content of PKC ɛ predominated over other isoforms. PKC ɛ could decrease filopodia significantly in promoter‐treated cells, and this was attributed to ruffling. The presence of PKC α counteracted the PKC ɛ‐mediated enhancement of ruffling. The results showed that there were two mechanisms of filopodia downregulation. One operated in the steady‐state and relied on PKC α and η. The other was stimulated by tumor promoters and relied on PKC ɛ. Cycles of protrusion and retraction are characteristic of filopodia and are essential for the cell to orient itself during chemotaxis and haptotaxis. By suppressing filopodia, PKC ɛ can create a long‐term “memory” of an environmental signal that may act in nature as a mnemonic device to mark the direction of a repulsive signal.

Bislang stärkster molekularer Motor zieht Bakterien über Oberflächen

Type IV pili (T4P) are filamentous cell surface structures found in many bacteria. They are involved in important processes including motility, adhesion, and biofilm formation. T4P are highly dynamic and undergo cycles of extension, adhesion, and retraction. The structure of the machine driving these cycles remained unknown. We recently elucidated the architecture of this machine in Myxococcus xanthus and showed that it spans the entire cell envelope. We propose a testable working model how the pilus filament is extended and retracted.

Automated analysis of filopodial length and spatially resolved protein concentration via adaptive shape tracking.

Filopodia are dynamic, actin-rich structures that transiently form on a variety of cell types. To understand the underlying control mechanisms requires precise monitoring of localization and concentration of individual regulatory and structural proteins as filopodia elongate and subsequently retract. Although several methods exist that analyze changes in filopodial shape, a software solution to reliably correlate growth dynamics with spatially resolved protein concentration along the filopodium independent of bending, lateral shift, or tilting is missing. Here we introduce a novel approach based on the convex-hull algorithm for parallel analysis of growth dynamics and relative spatiotemporal protein concentration along flexible filopodial protrusions. Detailed in silico tests using various geometries confirm that our technique accurately tracks growth dynamics and relative protein concentration along the filopodial length for a broad range of signal distributions. To validate our technique in living cells, we measure filopodial dynamics and quantify spatiotemporal localization of filopodia-associated proteins during the filopodial extension-retraction cycle in a variety of cell types in vitro and in vivo. Together these results show that the technique is suitable for simultaneous analysis of growth dynamics and spatiotemporal protein enrichment along filopodia. To allow readily application by other laboratories, we share source code and instructions for software handling.

α-Actinin links extracellular matrix rigidity-sensing contractile units with periodic cell-edge retractions.

During spreading and migration, the leading edges of cells undergo periodic protrusion-retraction cycles. The functional purpose of these cycles is unclear. Here, using submicrometer polydimethylsiloxane pillars as substrates for cell spreading, we show that periodic edge retractions coincide with peak forces produced by local contractile units (CUs) that assemble and disassemble along the cell edge to test matrix rigidity. We find that, whereas actin rearward flow produces a relatively constant force inward, the peak of local contractile forces by CUs scales with rigidity. The cytoskeletal protein α-actinin is shared between these two force-producing systems. It initially localizes to the CUs and subsequently moves inward with the actin flow. Knockdown of α-actinin causes aberrant rigidity sensing, loss of CUs, loss of protrusion-retraction cycles, and, surprisingly, enables the cells to proliferate on soft matrices. We present a model based on these results in which local CUs drive rigidity sensing and adhesion formation.

Structure and Dynamics of Filopodia Studied by Electron Cryo-Tomography and Single Molecule Fluorescence Imaging

Filopodia are cellular projections around 100nm in diameter and usually several micrometres in length. They are composed of fascin-bundled actin filaments and the molecular motor myosin‑10 localises at the extreme tip. Filopodia are important in cell migration and in making cell-cell contacts and have been linked with roles in diverse areas of biology including angiogenesis, and path-finding properties of neuronal growth cones. Filopodia can be induced in model cell lines by overexpresion of myosin‑10 and live-cell imaging shows that they undergo stochastic cycles of extension and retraction. Myosin‑10 transports cargo along the actin filament bundle towards the filopodial tip, forming a complex of proteins which are responsible for governing growth, retraction and adhesion. We have used a combination of optical and electron microscopy to study the dynamics and ultra-structure of myosin‑10 induced filopodia. Electron cryo-tomography of flash-frozen vitrified mammalian cells revealed the arrangement of actin filaments within the filopodia. Single molecule imaging by total internal reflection fluorescence microscopy allowed movement of myosin‑10 with the filopodia to be quantified and super-resolution localisation gave structural and mechanical information about the filopodium that could be correlated to our electron tomographic images.

Computer vision profiling of neurite outgrowth dynamics reveals spatiotemporal modularity of Rho GTPase signaling.

Rho guanosine triphosphatases (GTPases) control the cytoskeletal dynamics that power neurite outgrowth. This process consists of dynamic neurite initiation, elongation, retraction, and branching cycles that are likely to be regulated by specific spatiotemporal signaling networks, which cannot be resolved with static, steady-state assays. We present NeuriteTracker, a computer-vision approach to automatically segment and track neuronal morphodynamics in time-lapse datasets. Feature extraction then quantifies dynamic neurite outgrowth phenotypes. We identify a set of stereotypic neurite outgrowth morphodynamic behaviors in a cultured neuronal cell system. Systematic RNA interference perturbation of a Rho GTPase interactome consisting of 219 proteins reveals a limited set of morphodynamic phenotypes. As proof of concept, we show that loss of function of two distinct RhoA-specific GTPase-activating proteins (GAPs) leads to opposite neurite outgrowth phenotypes. Imaging of RhoA activation dynamics indicates that both GAPs regulate different spatiotemporal Rho GTPase pools, with distinct functions. Our results provide a starting point to dissect spatiotemporal Rho GTPase signaling networks that regulate neurite outgrowth.

Cdc42 and RhoA reveal different spatio-temporal dynamics upon local stimulation with Semaphorin-3A.

Small RhoGTPases, such as Cdc42 and RhoA, are key players in integrating external cues and intracellular signaling pathways that regulate growth cone (GC) motility. Indeed, Cdc42 is involved in actin polymerization and filopodia formation, whereas RhoA induces GC collapse and neurite retraction through actomyosin contraction. In this study we employed Förster Resonance Energy Transfer (FRET) microscopy to study the spatio-temporal dynamics of Cdc42 and RhoA in GCs in response to local Semaphorin-3A (Sema3A) stimulation obtained with lipid vesicles filled with Sema3A and positioned near the selected GC using optical tweezers. We found that Cdc42 and RhoA were activated at the leading edge of NG108-15 neuroblastoma cells during spontaneous cycles of protrusion and retraction, respectively. The release of Sema3A brought to a progressive activation of RhoA within 30 s from the stimulus in the central region of the GC that collapsed and retracted. In contrast, the same stimulation evoked waves of Cdc42 activation propagating away from the stimulated region. A more localized stimulation obtained with Sema3A coated beads placed on the GC, led to Cdc42 active waves that propagated in a retrograde manner with a mean period of 70 s, and followed by GC retraction. Therefore, Sema3A activates both Cdc42 and RhoA with a complex and different spatial-temporal dynamics.

Neurons Rho to Get in Shape for the Day

Linking structural changes in neurons to animal behavior has proven challenging. New findings by Pesakou etal. tie daily cycles of axon arbor extension and retraction, mediated by Rho activity, to circadian and seasonal patterns of behavior in the fruit fly.

How do epidermal matrix metalloproteinases support re-epithelialization during skin healing?

Epithelialization of normal wounds occurs by an orderly series of events whereby keratinocytes migrate, proliferate, and differentiate to restore the epidermal barrier function. Keratinocyte migration is one of the earliest and crucial events determining the efficiency of the overall wound repair process. In response to various stimuli including that of growth factors, cytokines and the extracellular matrix, activated keratinocytes at the edges of the wound undergo dramatic morphological changes according to their migratory behaviour through development of protrusive adhesion contacts and cytoskeleton rearrangements. These phenotypic changes are accompanied by the upregulated expression of a new set of genes, among which are adhesion receptors and specific matrix degrading enzymes named matrix metalloproteinases (MMPs). The tightly regulated spatial and temporal MMP expression is crucial for proper re-epithelialization. These multi-domain zinc-containing endopeptidases are necessary for the proper completion of multiple features of epidermal regeneration. They play a key role in the migration process by controlling the repeated cycles of keratinocyte attachment and retraction. In the meantime, they process, degrade or remodel the extracellular matrix often producing cleavages in a gain-of-function manner.

Blocking integrin inactivation as an anti-angiogenic therapy.

During angiogenesis, endothelial cell migration is coordinated by integrin‐mediated contact with the extra‐cellular matrix (ECM), coupled with receptor tyrosine kinase signalling to regulate dynamic cytoskeletal and plasma membrane reorganization. A recent paper by Vitorino et al (2015) defined a new MAP4K4–moesin–talin–β1‐integrin pathway that could be therapeutically exploited to suppress pathologic angiogenesis.

Probing of PNA-DNA Hybrid Duplex Stability with AFM Force Spectroscopy

Peptide nucleic acids (PNA) are synthetic polymers the neutral peptide backbone of which provides elevated stability to PNA-PNA and hybrid PNA-DNA duplex. It was demonstrated recently (J.Org.Chem.2011, 76, 5614-5627) that additional modification of the backbone such as incorporation of diethylene glycol (miniPEG) further increases the duplex thermal stability, so the hybrid duplex with 10 miniPEG units (γ-PNA) has the melting temperature 23°C higher compared to the regular PNA-DNA duplex. Here we applied AFM force spectroscopy to probe the strength of the γ-PNA-DNA duplex (5′GAGTAG GTAG-3’) containing ten miniPEG-modified units. Single-stranded PNA and DNA oligonucleotides containing terminal thiol groups were immobilized on amino-functionalized AFM tip and mica substrate, respectively via bifunctional PEG tethers and the interaction between the DNA and γ-PNA polymers was analyzed by multiple approach retraction cycles over various locations on the mica substrate. Such single molecule probing experiments produced force curves with well-defined rupture events corresponding to the dissociation of the duplex formed during the approach step. The experiments were performed at various pulling rates (300-3000 nm/sec) enabled us to characterize the hybrid duplex stability using dynamic force spectroscopy (DFS) approach. The DFS measurements yielded rupture forces varying in the range 60-70 pN. The data analyzed in the framework of the Bell-Evans approach yielded a dissociation constant, koff ∼ 10−9 sec−1 and rupture distance x-1∼ 1.7 nm. Similar published DFS measurements of DNA duplex are characterized by koff with ∼ 108 times less values, which is in line with elevated stabilities of the γ-PNA-DNA duplexes compared with the DNA duplexes.The work was supported by NIH grants 5P01 GM091743-03 and 5R01 GM096039-04 to YLL. γ-PNA was synthesized at PNA innovation.

Individual chain dynamics of a polyethylene melt undergoing steady shear flow

Individual molecule dynamics have been shown to influence significantly the bulk rheological and microstructural properties of short-chain, unentangled, linear polyethylene liquids undergoing high strain-rate flows. The objective of this work was to extend this analysis to a linear polyethylene composed of macromolecules of a much greater length and entanglement density; i.e., a liquid consisting of C400H802 molecules, with approximately ten kinks per chain at equilibrium, as calculated by the Z1 code of Kröger [Comput. Phys. Commun. 168, 209–232 (2005)]. To achieve this, we performed nonequilibrium molecular dynamics (NEMD) simulations of a model system using the well-established potential model of Siepmann et al. [Nature 365, 330–332 (1993)] for a wide range of Weissenberg numbers (Wi) under steady shear flow. A recent study by Baig et al. [Macromolecules 43, 6886–6902 (2010)] examined this same system using NEMD simulations, but focused on the bulk rheological and microstructural properties as calculated from ensemble averages of the chains comprising the macromolecular liquids. In so doing, some key features of the system dynamics were not fully elucidated, which this article aims to highlight. Specifically, it was found that this polyethylene liquid displays multiple timescales associated with not only the decorrelation of the end-to-end vector (commonly related to the Rouse time or disengagement time, depending on the entanglement density of the liquid), but also ones associated with the retraction and rotation cycles of the individual molecules. Furthermore, when accounting for these individual chain dynamics, the “longest” relaxation time of the system was higher by a factor of 1.7, independent of shear rate, when calculated self-consistently due to the coupling of relaxation modes. Brownian dynamics (BD) simulations were also performed on an analogous free-draining bead-rod chain model to compare the rotation and retraction dynamics of a single chain in dilute solution with individual molecular motions in the melt. These BD simulations revealed that the dynamics of the free-draining chain are qualitatively and quantitatively similar to those of the individual chains comprising the polyethylene melt at strain rates in excess of Wi ≈ 50, implying a possible breakdown of reptation theory in the high shear limit. An examination of the bulk-average properties revealed the effects of the chain rotation and retraction cycles upon commonly modeled microstructural properties, such as the distribution function of the chain end-to-end vector and the entanglement number density.

GMF Promotes Leading-Edge Dynamics and Collective Cell Migration In Vivo

Lamellipodia are dynamic actin-rich cellular extensions that drive advancement of the leading edge during cell migration. Lamellipodia undergo periodic extension and retraction cycles, but the molecular mechanisms underlying these dynamics and their role in cell migration have remained obscure. We show that glia-maturation factor (GMF), which is an Arp2/3 complex inhibitor and actin filament debranching factor, regulates lamellipodial protrusion dynamics in living cells. In cultured S2R(+) cells, GMF silencing resulted in an increase in the width of lamellipodial actin filament arrays. Importantly, live-cell imaging of mutant Drosophila egg chambers revealed that the dynamics of actin-rich protrusions in migrating border cells is diminished in the absence of GMF. Consequently, velocity of border cell clusters undergoing guided migration was reduced in GMF mutant flies. Furthermore, genetic studies demonstrated that GMF cooperates with the Drosophila homolog of Aip1 (flare) in promoting disassembly of Arp2/3-nucleated actin filament networks and driving border cell migration. These data suggest that GMF functions in vivo to promote the disassembly of Arp2/3-nucleated actin filament arrays, making an important contribution to cell migration within a 3D tissue environment.

Evolution of Land Use in the Brazilian Amazon: From Frontier Expansion to Market Chain Dynamics

Agricultural census data and fieldwork observations are used to analyze changes in land cover/use intensity across Rondônia and Mato Grosso states along the agricultural frontier in the Brazilian Amazon. Results show that the development of land use is strongly related to land distribution structure. While large farms have increased their share of annual and perennial crops, small and medium size farms have strongly contributed to the development of beef and milk market chains in both Rondônia and Mato Grosso. Land use intensification has occurred in the form of increased use of machinery, labor in agriculture and stocking rates of cattle herds. Regional and national demands have improved infrastructure and productivity. The data presented show that the distinct pathways of land use development are related to accessibility to markets and processing industry as well as to the agricultural colonization history of the region. The data analyzed do not provide any indication of frontier stagnation, i.e., the slowdown of agricultural expansion, in the Brazilian Amazon. Instead of frontier stagnation, the data analyzed indicate that intensification processes in consolidated areas as well as recent agricultural expansion into forest areas are able to explain the cycle of expansion and retraction of the agricultural frontier into the Amazon region. The evolution of land use is useful for scenario analysis of both land cover change and land use intensification and provides insights into the role of market development and policies on land use.

ER network dynamics are differentially controlled by myosins XI-K, XI-C, XI-E, XI-I, XI-1, and XI-2.

The endoplasmic reticulum (ER) of higher plants is a complex network of tubules and cisternae. Some of the tubules and cisternae are relatively persistent, while others are dynamically moving and remodeling through growth and shrinkage, cycles of tubule elongation and retraction, and cisternal expansion and diminution. Previous work showed that transient expression in tobacco leaves of the motor-less, truncated tail of myosin XI-K increases the relative area of both persistent cisternae and tubules in the ER. Likewise, transient expression of XI-K tail diminishes the movement of organelles such as Golgi and peroxisomes. To examine whether other class XI myosins are involved in the remodeling and movement of the ER, other myosin XIs implicated in organelle movement, XI-1 (MYA1),XI-2 (MYA2), XI-C, XI-E, XI-I, and one not, XI-A, were expressed as motor-less tail constructs and their effect on ER persistent structures determined. Here, we indicate a differential effect on ER dynamics whereby certain class XI myosins may have more influence over controlling cisternalization rather than tubulation.

Air‐breathing fishes

Air‐breathing fishes

The Role of Arp2/3 in DRG Growth Cones Motility

The Arp2/3 complex simultaneously controls nucleation of actin polymerization and filament branching. We used optical tweezers and video imaging to analyse the role of Arp2/3 in the motility of lamellipodia from developing growth cones (GCs) of isolated Dorsal Root Ganglia (DRG) neurons. Kymograph analysis shows that inhibition of Arp 2/3 complex with 100 µM CK548 completely abolished the lamellipodia motion. Surprisingly when the activity of Arp2/3 is inhibited by 25 µM CK548, lamellipodia shrank for 6-8 min but then reappears again and show an almost normal motility. The average period to complete protrusion and retraction cycles of lamellipodia 76 s in control, increased to 110 s after the recovery from the exposure to this drug. The drug CK548 decreased, initially, the ability of the lamellipodia to lift up in the axial direction, but after the recovery lamellipodia were able to lift 4 µm up above the coverslip as in control conditions. The force generated by lamellipodia and their maximal protrusion velocity decreased when the concentration of drug CK548 is increased from 25 to 100 µM. After the recovery of an almost normal motility, ruffles were present but had a different shape and were more fragmented and invaginated, suggesting an altered cytoskeleton. These results show that Arp 2/3 is important in maintaining the morphology and the motility of the neuronal growth cones but in neuronal lamellipodia several distinct pathways contribute to actin filament branching and to their cross-linkage.

Outside-In Assembly Pathway of the Type IV Pilus System in Myxococcus xanthus

Type IV pili (T4P) are ubiquitous bacterial cell surface structures that undergo cycles of extension, adhesion, and retraction. T4P function depends on a highly conserved envelope-spanning macromolecular machinery consisting of 10 proteins that localizes polarly in Myxococcus xanthus. Using this localization, we investigated the entire T4P machinery assembly pathway by systematically profiling the stability of all and the localization of eight of these proteins in the absence of other T4P machinery proteins as well as by mapping direct protein-protein interactions. Our experiments uncovered a sequential, outside-in pathway starting with the outer membrane (OM) PilQ secretin ring. PilQ recruits a subcomplex consisting of the inner membrane (IM) lipoprotein PilP and the integral IM proteins PilN and PilO by direct interaction with the periplasmic domain of PilP. The PilP/PilN/PilO subcomplex recruits the cytoplasmic PilM protein, by direct interaction between PilN and PilM, and the integral IM protein PilC. The PilB/PilT ATPases that power extension/retraction localize independently of other T4P machinery proteins. Thus, assembly of the T4P machinery initiates with formation of the OM secretin ring and continues inwards over the periplasm and IM to the cytoplasm.