Cycles Of Retraction Research Articles

Building permits-control of type IV pilus assembly by PilB and its cofactors.

Many bacteria produce type IV pili (T4P), surfaced-exposed protein filaments that enable cells to interact with their environment and transition from planktonic to surface-adapted states. T4P are dynamic, undergoing rapid cycles of filament extension and retraction facilitated by a complex protein nanomachine powered by cytoplasmic motor ATPases. Dedicated assembly motors drive the extension of the pilus fiber into the extracellular space, but like any machine, this process is tightly organized. These motors are coordinated by various ligands and binding partners, which control or optimize their functional associations with T4P machinery before cells commit to the crucial first step of building a pilus. This review focuses on the molecular mechanisms that regulate T4P extension motor function. We discuss secondary messenger-dependent transcriptional or post-translational regulation acting both directly on the motor and through protein effectors. We also discuss the recent discoveries of naturally occurring extension inhibitors as well as alternative mechanisms of pilus assembly and motor-dependent signaling pathways. Given that T4P are important virulence factors for many bacterial pathogens, studying these motor regulatory systems will provide new insights into T4P-dependent physiology and efficient strategies to disable them.

Multiple glacial refugia during Pleistocene climatic oscillations shape the genetic pattern of Machilus thunbergii across East Asia

Abstract Pleistocene climatic oscillations caused periodic decline and rise of sea levels, leading to dispersion and retraction cycles of island flora. Yet, the role of island refugia in the current Sino-Japanese Floristic Region remains poorly understood. In this study, we investigated the population genetic structure of the widespread Sino-Japanese Floristic Region tree Machilus thunbergii to infer the potential impact of island refugia. We collected 1378 samples from 64 locations across the distribution ranges. Using chloroplast DNA and microsatellite markers, we found a pronounced genetic differentiation between mainland and island populations, which can be divided further into two and three groups, respectively. Furthermore, comparable numbers of private alleles and haplotypes are present in both mainland and island populations. No essential current gene flow was detected between mainland and island populations after their separation 14 000 years ago. Such patterns are hypothesized to result from the influence of multiple glacial island refugia during Pleistocene climatic oscillations, with limited pollen and seed dispersal of the species. Our findings underscore that the islands and submerged land bridge can act as refugia for plants during glacial periods and have essentially shaped the genetic structure of M. thunbergii populations.

Bidirectional pilus processing in the Tad pilus system motor CpaF

The bacterial tight adherence pilus system (TadPS) assembles surface pili essential for adhesion and colonisation in many human pathogens. Pilus dynamics are powered by the ATPase CpaF (TadA), which drives extension and retraction cycles in Caulobacter crescentus through an unknown mechanism. Here we use cryogenic electron microscopy and cell-based light microscopy to characterise CpaF mechanism. We show that CpaF assembles into a hexamer with C2 symmetry in different nucleotide states. Nucleotide cycling occurs through an intra-subunit clamp-like mechanism that promotes sequential conformational changes between subunits. Moreover, a comparison of the active sites with different nucleotides bound suggests a mechanism for bidirectional motion. Conserved CpaF residues, predicted to interact with platform proteins CpaG (TadB) and CpaH (TadC), are mutated in vivo to establish their role in pilus processing. Our findings provide a model for how CpaF drives TadPS pilus dynamics and have broad implications for how other ancient type 4 filament family members power pilus assembly.

Enhancement of adhesion strength in viscoelastic unsteady contacts

We present a general energy approach to study the unsteady adhesive contact of viscoelastic materials. Under the assumption of infinitely short-range adhesive interactions, we exploit the principle of virtual work to generalize Griffith’s local energy balance at contact edges to the case of a non-conservative (viscoelastic) material, subjected to a generic contact time–history. We apply the proposed energy balance criterion to study the approach–retraction motion of a rigid sphere in contact with a viscoelastic half-space. A strong interplay between adhesion and viscoelastic hysteretic losses is reported which can lead to strongly increased adhesion strength, depending on the loading history. Specifically, two different mechanisms are found to govern the increase of pull-off force during either approach–retraction cycles and approach – full relaxation – retraction tests. In the former case, hysteretic losses occurring close to the circular perimeter of the contact play a major role, significantly enhancing the energy release rate. In the latter case, instead, the pull-off enhancement mostly depends on the glassy response of the whole (bulk) material which, triggered by the fast retraction after relaxation, leads to a sort of ‘frozen’ state and results in a flat-punch-like detachment mechanism (i.e., constant contact area). In this case, the JKR theory of adhesive contact cannot be invoked to relate the observed pull-off force to the effective adhesion energy, i.e. the energy release rate G, and strongly overestimates it. Therefore, a rigorous mathematical procedure is also proposed to correctly calculate the energy release rate in viscoelastic dissipative contacts.

Adhesive contact mechanics of bio-inspired pillars: Exploring hysteresis and detachment modes

Engineering technologies frequently draw inspiration from nature, as exemplified in bio-inspired adhesive surfaces. These surfaces present textures adorned by pillars, mimicking the topography found on the pads of certain animals renowned for their exceptional adhesive capabilities. The adhesive response is strongly influenced by the morphology of these pillars. In typical existing models, perfect bonding conditions are assumed between the pillar and the countersurface, and solely the detachment process of the pillar from the countersurface is investigated.The proposed model, based on the assumption that interactions at the interface are governed by van der Waals forces modeled by the Lennard-Jones potential law, enables the examination of the entire approach and retraction cycle, tracking the movement of the pillar towards and away from the countersurface.Our findings reveal that adhesive contact mechanics is primarily influenced by the geometry of the pillar and the potential presence of interfacial ’defects’, which in turn affect the distribution of contact pressure. Furthermore, we show that the detachment process may simultaneously involve various modes of separation, such as crack propagation from outer edge, crack propagation from inner defects, and uniform decohesion. This suggests that existing theoretical models alone cannot fully elucidate the complexity of detachment phenomena. Additionally, we anticipate the occurrence of hysteretic losses during the approach-retraction cycle, attributed to pull-in and pull-off contact jumps. Adhesive hysteresis is a phenomenon consistently observed in experiments but frequently overlooked in existing models.

Perforated red blood cells enable compressible and injectable hydrogels as therapeutic vehicles

Hydrogels engineered for medical use within the human body need to be delivered in a minimally invasive fashion without altering their biochemical and mechanical properties to maximize their therapeutic outcomes. In this regard, key strategies applied for creating such medical hydrogels include formulating precursor solutions that can be crosslinked in situ with physical or chemical cues following their delivery or forming macroporous hydrogels at sub-zero temperatures via cryogelation prior to their delivery. Here, we present a new class of injectable composite materials with shape recovery ability. The shape recovery is derived from the physical properties of red blood cells (RBCs) that are first modified via hypotonic swelling and then integrated into the hydrogel scaffolds before polymerization. The RBCs’ hypotonic swelling induces the formation of nanometer-sized pores on their cell membranes, which enable fast liquid release under compression. The resulting biocomposite hydrogel scaffolds display high deformability and shape-recovery ability. The scaffolds can repeatedly compress up to ∼87% of their original volumes during injection and subsequent retraction through syringe needles of different sizes; this cycle of injection and retraction can be repeated up to ten times without causing any substantial mechanical damage to the scaffolds. Our biocomposite material system and fabrication approach for injectable materials will be foundational for the minimally invasive delivery of drug-loaded scaffolds, tissue-engineered constructs, and personalized medical platforms that could be administered to the human body with conventional needle-syringe systems.

Characterization and classification of lakes based on geospatial analyses in the karst hydrogeological system of the Bambuí group, Lagoa Santa, Minas Gerais, Brazil

The karst of Lagoa Santa is a protected area located near the city of Belo Horizonte, in Minas Gerais state (Brazil), namely Lagoa Santa Karst Environmental Protection Area (EPA), one of the main research areas related to archeology, paleontology, speleology and hydrogeology in the Brazilian territory. The region is comprised by several lakes, because of its complex water system due to karst evolution. In addition, the karstic nature of the rocks in the region allows the existence of several other typical dissolution morphologies, such as: caves, speleothems, sinkholes, uvalas, among others. The morpho-structural characteristics, combined with the local geology, can provide important information about the behavior of lakes in view of the cycles of expansion and retraction of the water surface. Thus, this work aims to understand the water dynamics of the lakes present in the Karst of Lagoa Santa through analysis of spatial data between the years 1984 and 2020. Based on the surveys, 153 lakes were identified in the study region in the last 35 years: 40 perennial lakes, 89 intermittent lakes, 19 new lakes and 5 lakes that dried up completely. Also, this study compares the relationship of each lake with the type of relief depressions (Sinkhole or Uvala), rock masses and the main negative structural lineaments. The data obtained allowed the identification of which characteristics are diagnostic for perenniality or intermittence of existing lakes in the region, as well as the grouping of existing lakes into subgroups with similar geological and morpho-structural characteristics. The most important characteristics to define the water dynamics on lakes are the evolutionary state in karst depressions and the presence of structural lineaments, in which in more evolved depressions (uvalas) the lakes tend to be intermittent and in less evolved depressions (sinkholes) the lakes tend to be perennial. Finally, the water bodies associated with structural lineaments may be the condition for the permanence of water in some depressions, mainly in lakes over non-carbonate formations existing in the region.

The mechanism for polar localization of the type IVa pilus machine in Myxococcus xanthus.

Type IVa pili (T4aP) are widespread bacterial cell surface structures with important functions in motility, surface adhesion, biofilm formation, and virulence. Different bacteria have adapted different piliation patterns. To address how these patterns are established, we focused on the bipolar localization of the T4aP machine in the model organism Myxococcus xanthus by studying the localization of the PilQ secretin, the first component of this machine that assembles at the poles. Based on experiments using a combination of fluorescence microscopy, biochemistry, and computational structural analysis, we propose that PilQ, and specifically its AMIN domains, binds septal and polar peptidoglycan, thereby enabling polar Tgl localization, which then stimulates PilQ multimerization in the outer membrane. We also propose that the presence and absence of AMIN domains in T4aP secretins contribute to the different piliation patterns across bacteria.

Three-Dimensional Printing Enabled Droplet Microfluidic Device for Real-Time Monitoring of Single-Cell Viability and Blebbing Activity.

Droplet-based microfluidics with the characteristics of high throughput, low sample consumption, increasing reaction speed, and homogeneous volume control have been demonstrated as a useful platform for biomedical research and applications. The traditional fabrication methods of droplet microfluidics largely rely on expensive instruments, sophisticated operations, and even the requirement of an ultraclean room. In this manuscript, we present a 3D printing-based droplet microfluidic system with a specifically designed microstructure for droplet generation aimed at developing a more accessible and cost-effective method. The performance of droplet generation and the encapsulation capacity of the setup were examined. The device was further applied to measure the variation in cell viability over time and monitor the cell's blebbing activity to investigate its potential ability and feasibility for single-cell analysis. The result demonstrated that the produced droplets remained stable enough to enable the long-time detection of cell viability. Additionally, cell membrane protrusions featuring the life cycle of bleb initiation, expansion, and retraction can be well-observed. Three-dimensional printing-based droplet microfluidics benefit from the ease of manufacture, which is expected to simplify the fabrication of microfluidics and expand the application of the droplet approach in biomedical fields.

Chemo-mechanical diffusion waves explain collective dynamics of immune cell podosomes

Immune cells, such as macrophages and dendritic cells, can utilize podosomes, mechanosensitive actin-rich protrusions, to generate forces, migrate, and patrol for foreign antigens. Individual podosomes probe their microenvironment through periodic protrusion and retraction cycles (height oscillations), while oscillations of multiple podosomes in a cluster are coordinated in a wave-like fashion. However, the mechanisms governing both the individual oscillations and the collective wave-like dynamics remain unclear. Here, by integrating actin polymerization, myosin contractility, actin diffusion, and mechanosensitive signaling, we develop a chemo-mechanical model for podosome dynamics in clusters. Our model reveals that podosomes show oscillatory growth when actin polymerization-driven protrusion and signaling-associated myosin contraction occur at similar rates, while the diffusion of actin monomers drives wave-like coordination of podosome oscillations. Our theoretical predictions are validated by different pharmacological treatments and the impact of microenvironment stiffness on chemo-mechanical waves. Our proposed framework can shed light on the role of podosomes in immune cell mechanosensing within the context of wound healing and cancer immunotherapy.

Acoustic emission monitoring of naturally developed damage in large-scale low-speed roller bearings

This article presents an approach to identify naturally developed damage in low-speed bearings using waveform-similarity-based clustering of acoustic emissions (AEs) under fatigue loading. The approach is motivated by the notation that each recorded AE signal from a particular damage is defined by the convolution of the source signal, transfer function of the propagation path and transfer function of the utilised sensor, and may thusly be used to identify consistent AE sources, for example due to crack growth. A sequential clustering procedure is proposed, that is based on waveform cross-correlation. The supporting theoretical background of waveform similarity, rooted in an analytical formulation of waveform propagation and transmission in complex structures, is discussed. The presented methodology is evaluated through application to AE data obtained in a low-speed run-to-failure experiment utilising a densely instrumented purpose-built linear bearing segment. The implemented sensor system comprises arrays of three types of AE transducers, that is relatively low - (40–100 kHz), mid - (95–180 kHz) and high-frequency (180–580 kHz), that are situated on both the raceways and supporting substructures of either side of the bearing. Over the course of 225,000 cycles of extension and retraction, wear has been developed. A total of about ∼2,300,000 AE signals have been recorded. Analysis of the recorded data suggests the rate of degradation increases from around 70,000 cycles onwards. Highly consistent structures of clusters indicative of a localised defect in the raceway have been identified from around 170,000 cycles onwards. These clusters are characterised by hit-rates in the range of 1–2 hits per cycle and an average similarity of 93%, they comprise about half the AE activity for the periods they have been identified for. These results highlight that the proposed cross-correlation-based clustering of AE waveforms and identification of multi-channel formations in said clusters compose a suitable methodology for assessment of damage in low-speed roller bearings.

Sulfolobus acidocaldarius adhesion pili power twitching motility in the absence of a dedicated retraction ATPase.

Type IV pili are ancient and widespread filamentous organelles found in most bacterial and archaeal phyla. They support a wide range of fundamental functions including adhesion to substrates, DNA uptake, self-aggregation and motility. In particular, disassembly of type IV pili mediated by PilT-family ATPases allows pilus retraction, which is responsible for twitching motility that is important for surface colonization in bacteria. Archaea do not possess PilT retraction ATPases, and it was therefore hypothesized that archaeal cells are not capable of twitching motility. Yet, recent microscopic observations of live Sulfolobus acidocaldarius showed that cells exhibit short-range surface-motility that resembles bacterial twitching motility. S. acidocaldarius is a thermoacidophilic crenarcheon that encodes three different type IV pili systems with non-redundant functions. While archaella are rotating type IV pili that allow swimming motility, UV-inducible pili promote cell aggregation and DNA exchange upon exposure to UV light, and adhesion pili are responsible for surface attachment and biofilm formation. Here, using a combination of automated single cell tracking at high temperature, high-temperature fluorescence imaging, and genetic manipulations, we demonstrate that S. acidocaldarius exhibits bona fide twitching motility, and that this behavior depends specifically on adhesion pili. We also find that twitching motility is conserved in other Sulfolobales species. Our results show that adhesion pili are capable of retraction in the absence of a PilT retraction ATPase. While PilT-independent pilus retraction was previously observed in bacteria such as C. crescentus, our study suggests that in archaea, which do not encode a dedicated retraction ATPase, this might be a common mechanism for pilus retraction. Our results also suggest that the ancestral type IV pilus machinery (maybe in LUCA) was capable of dynamic retraction and extension cycles in the absence of a dedicated retraction ATPase.

Live Cell Imaging of the Twitching Motility of Cyanobacteria by High-Resolution Microscopy.

Many cyanobacteria show directional movement either toward or away from light sources. The cell movement, also known as twitching motility, is usually driven by type IV pili (T4P), a bacterial molecular machine. The machine generates a propulsion force through repeated cycles of extension and retraction of pilus filaments. Here, I describe a phototaxis assay for observing Synechocystis sp. PCC6803 and Thermosynechococcus vulcanus at the single-cell level with optical microscopy. By adding fluorescent beads, I also describe a method how to visualize the asymmetric activation of T4P during phototaxis.

Highly Deformable and Durable Hydrogels through Synergy of Covalent Crosslinks and Nanosheet‐Reinforced Dynamic Interactions toward Flexible Sensor

AbstractIntegration of both excellent mechanical performance and remarkable durability into composite hydrogel materials is highly demanded yet challenging. Herein, a synergistic strategy of combining covalent crosslinks with nanosheet‐reinforced dynamic interactions is developed for the design and fabrication of deformable and recoverable nanocomposite hydrogels. Layered double hydroxide nanosheets and a trace amount of covalent crosslinks are simultaneously incorporated into the network, providing profound synergy in mechanical enhancement and durability extension. The resultant materials displayed high deformability (stretchability >1100%, compressibility >90%), considerable springiness and resilience, rapid and excellent recovery over 2000 cycles of compression and retraction as well as a 100% hysteresis ratio after only 1 min during large stretching. Notably, these networks are also transparent and ionically conductive. To demonstrate the utility, a wearable strain sensor is constructed for sensing and detecting human motions (e.g., subtle movement of mouth‐opening and large motion of joint‐bending), exhibiting a linear response with high sensitivity (gauge factor as high as 7.66) over 100–300% strain. This line of research will inspire renewed interest and work into synergic exploitation of nanospecies‐reinforced interactions and covalent crosslinks into the design of high‐performance composite materials.

Actin Turnover Required for Adhesion-Independent Bleb Migration

Cell migration is critical for many vital processes, such as wound healing, as well as harmful processes, such as cancer metastasis. Experiments have highlighted the diversity in migration strategies employed by cells in physiologically relevant environments. In 3D fibrous matrices and confinement between two surfaces, some cells migrate using round membrane protrusions, called blebs. In bleb-based migration, the role of substrate adhesion is thought to be minimal, and it remains unclear if a cell can migrate without any adhesion complexes. We present a 2D computational fluid-structure model of a cell using cycles of bleb expansion and retraction in a channel with several geometries. The cell model consists of a plasma membrane, an underlying actin cortex, and viscous cytoplasm. Cellular structures are immersed in viscous fluid which permeates them, and the fluid equations are solved using the method of regularized Stokeslets. Simulations show that the cell cannot effectively migrate when the actin cortex is modeled as a purely elastic material. We find that cells do migrate in rigid channels if actin turnover is included with a viscoelastic description for the cortex. Our study highlights the non-trivial relationship between cell rheology and its external environment during migration with cytoplasmic streaming.

A computational modeling of invadopodia protrusion into an extracellular matrix fiber network

Invadopodia are dynamic actin-rich membrane protrusions that have been implicated in cancer cell invasion and metastasis. In addition, invasiveness of cancer cells is strongly correlated with invadopodia formation, which are observed during extravasation and colonization of metastatic cancer cells at secondary sites. However, quantitative understanding of the interaction of invadopodia with extracellular matrix (ECM) is lacking, and how invadopodia protrusion speed is associated with the frequency of protrusion-retraction cycles remains unknown. Here, we present a computational framework for the characterization of invadopodia protrusions which allows two way interactions between intracellular branched actin network and ECM fibers network. We have applied this approach to predicting the invasiveness of cancer cells by computationally knocking out actin-crosslinking molecules, such as α-actinin, filamin and fascin. The resulting simulations reveal distinct invadopodia dynamics with cycles of protrusion and retraction. Specifically, we found that (1) increasing accumulation of MT1-MMP at tips of invadopodia as the duration of protrusive phase is increased, and (2) the movement of nucleus toward the leading edge of the cell becomes unstable as duration of the retractile phase (or myosin turnover time) is longer than 1 min.

Impacting Droplet Can Mitigate Dust from PDMS Micro-Post Array Surfaces

In this paper, the impact mechanisms of a water droplet on hydrophobized micro-post array surfaces are examined and the influence of micro-post arrays spacing on the droplet behavior in terms of spreading, retraction, and rebounding is investigated. Impacting droplet behavior was recorded using a high-speed facility and flow generated in the droplet fluid was simulated in 3D geometry accommodating conditions of the experiments. Micro-post arrays were initially formed lithographically on silicon wafer surfaces and, later, replicated by polydimethylsiloxane (PDMS). The replicated micro-post arrays surfaces were hydrophobized through coating by functionalized nano-silica particles. Hydrophobized surfaces result in a contact angle of 153° ± 3° with a hysteresis of 3° ± 1°. The predictions of the temporal behavior of droplet wetting diameter during spreading agree with the experimental data. Increasing micro-post arrays spacing reduces the maximum spreading diameter on the surface; in this case, droplet fluid penetrated micro-posts spacing creates a pinning effect while lowering droplet kinetic energy during the spreading cycle. Flow circulation results inside the droplet fluid in the edge region of the droplet during the spreading period; however, opposing flow occurs from the outer region towards the droplet center during the retraction cycle. This creates a stagnation zone in the central region of the droplet, which extends towards the droplet surface onset of droplet rebounding. Impacting droplet mitigates dust from hydrophobized micro-post array surfaces, and increasing droplet Weber number increases the area of dust mitigated from micro-post arrays surfaces.

The PilB-PilZ-FimX regulatory complex of the Type IV pilus from Xanthomonas citri.

Type IV pili (T4P) are thin and flexible filaments found on the surface of a wide range of Gram-negative bacteria that undergo cycles of extension and retraction and participate in a variety of important functions related to lifestyle, defense and pathogenesis. During pilus extensions, the PilB ATPase energizes the polymerization of pilin monomers from the inner membrane. In Xanthomonas citri, two cytosolic proteins, PilZ and the c-di-GMP receptor FimX, are involved in the regulation of T4P biogenesis through interactions with PilB. In vivo fluorescence microscopy studies show that PilB, PilZ and FimX all colocalize to the leading poles of X. citri cells during twitching motility and that this colocalization is dependent on the presence of all three proteins. We demonstrate that full-length PilB, PilZ and FimX can interact to form a stable complex as can PilB N-terminal, PilZ and FimX C-terminal fragments. We present the crystal structures of two binary complexes: i) that of the PilB N-terminal domain, encompassing sub-domains ND0 and ND1, bound to PilZ and ii) PilZ bound to the FimX EAL domain within a larger fragment containing both GGDEF and EAL domains. Evaluation of PilZ interactions with PilB and the FimX EAL domain in these and previously published structures, in conjunction with mutagenesis studies and functional assays, allow us to propose an internally consistent model for the PilB-PilZ-FimX complex and its interactions with the PilM-PilN complex in the context of the inner membrane platform of the X. citri Type IV pilus.

On the role of vesicle transport in neurite growth: Modeling and experiments

The processes that determine the establishment of the complex morphology of neurons during development are still poorly understood. Here, we focus on the question how a difference in the length of neurites affects vesicle transport. We performed live imaging experiments and present a lattice-based model to gain a deeper theoretical understanding of intracellular transport in neurons. After a motivation and appropriate scaling of the model we present numerical simulations showing that initial differences in neurite length result in phenomena of biological relevance, i.e. a positive feedback that enhances transport into the longer neurite and oscillation of vesicles concentrations that can be interpreted as cycles of extension and retraction observed in experiments. Thus, our model is a first step towards a better understanding of the interplay between the transport of vesicles and the spatial organization of cells.

Competence pili in Streptococcus pneumoniae are highly dynamic structures that retract to promote DNA uptake.

The competence pili of transformable Gram-positive species are phylogenetically related to the diverse and widespread class of extracellular filamentous organelles known as type IV pili. In Gram-negative bacteria, type IV pili act through dynamic cycles of extension and retraction to carry out diverse activities including attachment, motility, protein secretion, and DNA uptake. It remains unclear whether competence pili in Gram-positive species exhibit similar dynamic activity, and their mechanism of action for DNA uptake remains unclear. They are hypothesized to either (1) leave transient cavities in the cell wall that facilitate DNA passage, (2) form static adhesins to enrich DNA near the cell surface for subsequent uptake by membrane-embedded transporters, or (3) play an active role in translocating bound DNA via dynamic activity. Here, we use a recently described pilus labeling approach to demonstrate that competence pili in Streptococcus pneumoniae are highly dynamic structures that rapidly extend and retract from the cell surface. By labeling the principal pilus monomer, ComGC, with bulky adducts, we further demonstrate that pilus retraction is essential for natural transformation. Together, our results suggest that Gram-positive competence pili in other species may also be dynamic and retractile structures that play an active role in DNA uptake.