Numerical modeling of F-.Actin bundles interacting with cell membranes

Actin is one of the most aboundant proteins in eukaryotic cells, where it forms a dendridic network (cytoskeleton) beneath the cell membrane providing mechanical stability and performing fundamental tasks in several functions, including cellular motility. The first step in cell locomotion is the protrusion of a leading edge, for which a significant deformation of the membrane is required: this step relies essentially on the forces generated by actin polymerization pushing the plasma membrane outward. Different types of structures can emerge from the plasma membrane, like lamellipodia (quasi-2d actin mesh) and filopodia (parallel actin bundles). The main topic of the research project is the dynamics of bundles of parallel actin filaments growing against barriers, either rigid (a wall) or flexible (a membrane). In the first part of the thesis, the dynamic behavior of bundles of actin filaments growing against a loaded wall is investigated through a generalized version of the standard multi filaments Brownian Ratchet model in which the (de)polymerizing filaments are treated not as rigid rods but as semi-flexible discrete wormlike chains with a realistic value of the persistence length. A Statistical Mechanics framework is built for bundles of actin filaments growing in optical trap apparatus (harmonic external load) and several equilibrium properties are derived from it, like the maximum force that the filaments can exert (stalling force) or the number of filaments in contact with the wall. Besides, Stochastic Dynamic simulations are employed to study the non-equilibrium relaxation of the bundle of filaments growing in the same optical trap apparatus, interpreting the system evolution by a suitable Markovian approach. Thanks to the observed time scale separation between the wall motion and the filament size relaxation, the optical trap set-up allows to extract the full velocity-load curve V(F) -- the velocity at which the obstacle moves when subject to the combined action of the polymerizing filaments and the external load F -- from a single experiment. The main finding is the observation of a systematic evolution of steady non-equilibrium states over three regimes of bundle lengths L. A first threshold length Λ marks the transition between the rigid dynamic regime (L Λ), where the velocity V(F,L) is an increasing function of the bundle length L at fixed load F, the enhancement being the result of an improved level of work sharing among the filaments induced by flexibility. A second critical length corresponds to the beginning of an unstable regime characterized by a high probability to develop escaping filaments which start growing laterally and thus do not participate anymore to the generation of the polymerization force. This phenomenon prevents the bundle from reaching at this critical length the limit behavior corresponding to Perfect Load Sharing. In the second part of the thesis, filaments growing against a flexible, deformable membrane are studied by means of Langevin dynamics simulations; the membrane is discretized into a dynamically triangulated network of tethered beads, while the filaments are described as chains of bonded monomers. Both the monomers in the filaments and the membrane beads, which interact with each other via a purely repulsive potential, are followed in space and time integrating its equations of motion with a second order accurate scheme. The elastic properties of the membrane are studied in detail via several methods, showing an unprecedentent level of agreement among them. The onset of filopodial protrusions is observed for N>1 filaments growing from beneath the membrane and pushing it upwards, with a velocity which is systematically larger for flexible filaments than for rigid ones. Since filaments are wrapped by the membrane in the protrusion, escaping filaments are not predicted nor observed in this case

On the force-velocity relationship of a bundle of rigid bio-filaments

In various cellular processes, bio-filaments like F-actin and F-tubulin are able to exploit chemical energy associated with polymerization to perform mechanical work against an obstacle loaded with an external force. The force-velocity relationship quantitatively summarizes the nature of this process. By a stochastic dynamical model, we give, together with the evolution of a staggered bundle of Nfrigid living filaments facing a loaded wall, the corresponding force-velocity relationship. We compute the evolution of the model in the infinite wall diffusion limit and in supercritical conditions (monomer density reduced by critical density ρ^1>1), and we show that this solution remains valid for moderate non-zero values of the ratio between the wall diffusion and the chemical time scales. We consider two classical protocols: the bundle is opposed either to a constant load or to an optical trap setup, characterized by a harmonic restoring force. The constant load case leads, for each F value, to a stationary velocity Vstat(F;Nf,ρ^1) after a relaxation with characteristic time τmicro(F). When the bundle (initially taken as an assembly of filament seeds) is subjected to a harmonic restoring force (optical trap load), the bundle elongates and the load increases up to stalling over a characteristic time τOT. Extracted from this single experiment, the force-velocity VOT(F;Nf,ρ^1) curve is found to coincide with Vstat(F;Nf,ρ^1), except at low loads. We show that this result follows from the adiabatic separation between τmicroand τOT, i.e., τmicro≈ τOT

On the Properties of a Bundle of Flexible Actin Filaments in an Optical Trap

We establish the Statistical Mechanics framework for a bundle of Nf living and uncrosslinked actin filaments in a supercritical solution of free monomers pressing against a mobile wall. The filaments are anchored normally to a fixed planar surface at one of their ends and, because of their limited flexibility, they grow almost parallel to each other. Their growing ends hit a moving obstacle, depicted as a second planar wall, parallel to the previous one and subjected to a harmonic compressive force. The force constant is denoted as trap strength while the distance between the two walls as trap length to make contact with the experimental optical trap apparatus. For an ideal solution of reactive filaments and free monomers at fixed free monomers chemical potential, we obtain the general expression for the grand potential from which we derive averages and distributions of relevant physical quantities, namely the obstacle position, the bundle polymerization force and the number of filaments in direct contact with the wall. The grafted living filaments are modeled as discrete Wormlike chains, with Factin persistence length, subject to discrete contour length variations to model single monomer (de)polymerization steps. Rigid filaments, either isolated or in bundles, all provide average values of the stalling force in agreement with Hill's predictions, independent of the average trap length. Flexible filaments instead, for values of the trap strength suitable to prevent their lateral escape, provide an average bundle force and an average trap length slightly larger than the corresponding rigid cases (few percents). Still the stalling force remains nearly independent on the average trap length, but results from the product of two strongly L dependent contributions: the fraction of touching filaments and the single filament buckling force.Comment: 21 pages, 8 figure

Kinetic Study of Laboratory Mutants of NDM-1 Metallo-beta-Lactamase and the Importance of an Isoleucine at Position 35.

peer reviewedTwo laboratory mutants of NDM-1 were generated by replacing the isoleucine at position 35 with threonine and serine residues: the NDM-1(I35T)and NDM-1(I35S)enzymes. These mutants were well characterized, and their kinetic parameters were compared with those of the NDM-1 wild type. Thekcat,Km, andkcat/Kmvalues calculated for the two mutants were slightly different from those of the wild-type enzyme. Interestingly, thekcat/Kmof NDM-1(I35S)for loracarbef was about 14-fold higher than that of NDM-1. Far-UV circular dichroism (CD) spectra of NDM-1 and NDM-1(I35T)and NDM-1(I35S)enzymes suggest local structural rearrangements in the secondary structure with a marked reduction of alpha-helix content in the mutants

Peripheral nervous system involvement in SARS-CoV-2 infection: a review of the current pediatric literature

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified as the pathogen responsible for the pandemic health emergency declared by the World Health Organization in March 2020. During the first part of the pandemic, adults showed mild to severe respiratory symptoms. Children seemed initially exempt, both from acute and subsequent complications. Hyposmia or anosmia were promptly identified as the main symptoms of acute infection, so neurotropism of SARS-CoV-2 was immediately suspected. (1, 2). As the emergency progressed, post infectious neurological complications were described also in pediatric population (3). Cases of cranial neuropathy in connection with acute SARS-CoV-2 infection have been reported in pediatric patients, as an isolate post infectious complication or in the context of the multisystem inflammatory syndrome in children (MIS-C) (4–6). Neuroinflammation is thought to be caused by several mechanisms, among which immune/autoimmune reactions (7), but so far, no specific autoantibody has been identified. SARS-CoV-2 can enter the central nervous system (CNS) directly and/or infect it retrogradely, through the peripheral nervous system (PNS), after replicating peripherally; several factors regulate invasion and subsequent neuroinflammation. Indeed, direct/secondary entry and replication can activate CNS-resident immune cells that, together with peripheral leukocytes, induce an immune response and promote neuroinflammation. In addition, as we will discuss in the following review, many cases of peripheral neuropathy (cranial and non-cranial) have been reported during or after SARS-CoV-2 infection. However, some authors have pointed out that the increase of cranial roots and ganglia in neurological imaging is not always observed in children with cranial neuropathy. (8). Even if a variety of case reports were published, opinions about an increased incidence of such neurologic diseases, linked to SARS-CoV-2 infection, are still controversial (9–11). Facial nerve palsy, ocular movements abnormalities and vestibular alterations are among the most reported issues in pediatric population (3–5). Moreover, an increased screen exposure imposed by social distancing led to acute oculomotion’s disturbance in children, not primarily caused by neuritis (12, 13). The aim of this review is to suggest food for thought on the role of SARS-CoV-2 in neurological conditions, affecting the peripheral nervous system to optimize the management and care of pediatric patients

Replicon Typing of Plasmids Encoding Resistance to Newer β-Lactams

Polymerase chain reaction–based replicon typing represents a novel method to describe the dissemination and follow the evolution of resistance plasmids. We used this approach to study 26 epidemiologically unrelated Enterobacteriaceae and demonstrate the dominance of incompatibility (Inc) A/C or Inc N-related plasmids carrying some emerging resistance determinants to extended-spectrum cephalosporins and carbapenems

Filament flexibility enhances power transduction of F-actin bundles

In various biological processes, semi-flexible filaments like F-actin exploit chemical energy associated to polymerization to perform mechanical work against a loaded external obstacle. The dynamics of fingerlike filopodial structures, characterized by the relationship between the obstacle velocity $V$ and the applied external load $F$, is generally interpreted by a brownian ratchet mechanism appropriate to fully rigid filaments. Based on the properties of the Wormlike Chain model, we propose an original and general multiscale approach to consider filament flexibility in the modelling. By stochastic dynamic simulations, we studied the dynamical relaxation of a bundle of $N_f$ supercritical semi-flexible filaments against an optical trap load ($F=-\kappa_TL$, where $L$ is the distance between the grafting wall and the obstacle mobile wall, and $\kappa_T$ the trap strength). For realistic values of the model parameters, the systematic motion of the trap is two/three orders of magnitude slower than the characteristic time between chemical events and this separation of time scales lets consider the optical trap relaxation as a sequence of non-equilibrium steady states for the bundle properties. The velocity-load relationship $V(F,\lambda)$ can then be generalized by introducing the degree of flexibility $\lambda$, defined as the ratio of the filaments contour length over the typical length beyond which flexibility effects are detectable. Our approach lets us map out the entire range of flexibility from the rigid to the escaping filament regime, in which the trap gets large enough for filaments to have a high probability to laterally escape, not participating anymore to the bundle polymerization force. We show that flexibility considerably enriches the theoretical scenario filling the gap between the multi-filaments brownian ratchet model and the mean field Perfect Load Sharing result

On the force–velocity relationship of a bundle of rigid bio-filaments

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