Arp2/3 complex- and formin-mediated actin cytoskeleton networks facilitate actin binding protein sorting in fission yeast.

While it is well-established that F-actin networks with specific organizations and dynamics are tightly regulated by distinct sets of associated actin-binding proteins (ABPs), how ABPs self-sort to particular F-actin networks remains largely unclear. We report that actin assembly factors Arp2/3 complex and formin Cdc12 tune the association of ABPs fimbrin Fim1 and tropomyosin Cdc8 to different F-actin networks in fission yeast. Genetic and pharmacological disruption of F-actin networks revealed that Fim1 is preferentially directed to Arp2/3-complex mediated actin patches, whereas Cdc8 is preferentially targeted to formin Cdc12-mediated filaments in the contractile ring. To investigate the role of Arp2/3 complex- and formin Cdc12-mediated actin assembly, we used four-color TIRF microscopy to observe the in vitro reconstitution of ABP sorting with purified proteins. Fim1 or Cdc8 alone bind similarly well to filaments assembled by either assembly factor. However, in 'competition' reactions containing both actin assembly factors and both ABPs, ∼2.0-fold more Fim1 and ∼3.5-fold more Cdc8 accumulates on Arp2/3 complex branch points and formin Cdc12-assembled actin filaments, respectively. These findings indicate that F-actin assembly factors Arp2/3 complex and formin Cdc12 help facilitate the recruitment of specific ABPs, thereby tuning ABP sorting and subsequently establishing the identity of F-actin networks in fission yeast.

Multi-monoubiquitylation controls VASP-mediated actin dynamics.

The actin cytoskeleton performs multiple cellular functions, and as such, actin polymerization must be tightly regulated. We previously demonstrated that reversible, non-degradative ubiquitylation regulates the function of the actin polymerase VASP in developing neurons. However, the underlying mechanism of how ubiquitylation impacts VASP activity was unknown. Here, we show that mimicking multi-monoubiquitylation of VASP at K240 and K286 negatively regulates VASP interactions with actin. Using in vitro biochemical assays, we demonstrate the reduced ability of multi-monoubiquitylated VASP to bind, bundle, and elongate actin filaments. However, multi-monoubiquitylated VASP maintained the ability to bind and protect barbed ends from capping protein. Finally, we demonstrate the electroporation of recombinant multi-monoubiquitylated VASP protein altered cell spreading morphology. Collectively, these results suggest a mechanism in which ubiquitylation controls VASP-mediated actin dynamics.

Highly flexible PEG-LifeAct constructs act as tunable biomimetic actin crosslinkers.

In vitro studies of actin filament networks crosslinked with dynamic actin binding proteins provide critical insights into cytoskeletal mechanics as well as inspiration for new adaptive materials design. However, discontinuous variance in the physiochemical properties of actin binding proteins impedes holistic relationships between crosslinker molecular parameters, network structure, and mechanics. Bio-synthetic constructs composed of synthetic polymer backbones and actin binding motifs would enable crosslinkers with engineered physiochemical properties to directly target the desired structure-property relationships. As a proof of concept, bio-synthetic crosslinkers composed of highly flexible polyethylene glycol (PEG) polymers functionalized with the actin binding peptide LifeAct, are explored as actin crosslinkers. Using bulk rheology and fluorescence microscopy, these constructs are shown to modulate actin filament network structure and mechanics in a contour length dependent manner, while maintaining the stress-stiffening behavior inherent to actin filament networks. These results encourage the design of more diverse and complex peptide-polymer crosslinkers to interrogate and control semi-flexible polymer networks.

Reconstitution of the transition from a lamellipodia- to filopodia-like actin network with purified proteins.

How cells utilize complex mixtures of actin binding proteins to assemble and maintain functionally diverse actin filament networks with distinct architectures and dynamics within a common cytoplasm is a longstanding question in cell biology. A compelling example of complex and specialized actin structures in cells are filopodia which sense extracellular chemical and mechanical signals to help steer motile cells. Filopodia have distinct actin architecture, composed of long, parallel actin filaments bundled by fascin, which form finger-like membrane protrusions. Elongation of the parallel actin filaments in filopodia can be mediated by two processive actin filament elongation factors, formin and Ena/VASP, which localize to the tips of filopodia. There remains debate as to how the architecture of filopodia are generated, with one hypothesis proposing that filopodia are generated from the lamellipodia, which consists of densely packed, branched actin filaments nucleated by Arp2/3 complex and kept short by capping protein. It remains unclear if different actin filament elongation factors are necessary and sufficient to facilitate the emergence of filopodia with diverse characteristics from a highly dense network of short-branched capped filaments. To address this question, we combined bead motility and micropatterning biomimetic assays with multi-color Total Internal Reflection Fluorescence microscopy imaging, to successfully reconstitute the formation of filopodia-like networks (FLN) from densely-branched lamellipodia-like networks (LLN) with eight purified proteins (actin, profilin, Arp2/3 complex, Wasp pWA, fascin, capping protein, VASP and formin mDia2). Saturating capping protein concentrations inhibit FLN assembly, but the addition of either formin or Ena/VASP differentially rescues the formation of FLN from LLN. Specifically, we found that formin/mDia2-generated FLNs are relatively long and lack capping protein, whereas VASP-generated FLNs are comparatively short and contain capping protein, indicating that the actin elongation factor can affect the architecture and composition of FLN emerging from LLN. Our biomimetic reconstitution systems reveal that formin or VASP are necessary and sufficient to induce the transition from a LLN to a FLN, and establish robust in vitro platforms to investigate FLN assembly mechanisms.

A photochemical route in the synthesis and characterization of La2Ti2O7 and La2Ti2O7/AgO films and its evaluation in Congo red degradation and as antibacterial control to Staphylococcus aureus.

In this article, we propose a simple photochemical method to synthesize pure La2Ti2O7 films and La2Ti2O7 films doped with silver at 1.0, 3.0, and 5.0 mol%. After annealing the photo-deposited films at 900 °C, XRD, SEM, and XPS analyses showed the formation of a monoclinic La2Ti2O7 phase and the presence of Ag and AgO in doped samples. Photocatalytic tests for Congo red degradation demonstrated that pure La2Ti2O7 achieved 25.4% degradation, while doped samples reached a maximum of 92.7% degradation. Moreover, increasing silver doping on La2Ti2O7 films significantly reduced the growth of Staphylococcus aureus, indicating potential antibacterial properties. The enhanced photoactivity was attributed to the formation of a type I heterojunction between La2Ti2O7 and AgO, and a degradation mechanism was proposed based on Congo red degradation.

Shape, Microstructure, and Chemical Composition of Pearls from the Freshwater Clam Diplodon chilensis Native to South America.

The capability to produce pearls is widespread in the phylum Mollusca, including bivalves of the superfamily Unionoidea. Here, we identified and characterized natural pearls formed by Diplodon chilensis, a freshwater clam native to southern South America, using samples obtained from two lakes located in the Chilean Patagonia. Pearls were studied using light and scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), Fourier transform infrared spectroscopy (FTIR), and Raman spectroscopy. Naturally formed pearls were found in both male and female D. chilensis specimens. Pearls are produced in different shapes, including spherical, ellipsoidal, buttoned, and bumpy, ranging in size from 200 µm to 1.9 mm. The internal microstructure is composed of irregular polygonal tablets, about 0.40 to 0.55 µm in thickness. EDX analysis showed that pearls are composed of calcium carbonate. FTIR and Raman spectra recorded several peaks attributable to the aragonite in pearls of this species, as has been shown in other mollusks. In addition to these results, pearls of different colors are illustrated.

Multi-monoubiquitination controls VASP-mediated actin dynamics.

The actin cytoskeleton performs multiple cellular functions, and as such, actin polymerization must be tightly regulated. We previously demonstrated that reversible, non-degradative ubiquitination regulates the function of the actin polymerase VASP in developing neurons. However, the underlying mechanism of how ubiquitination impacts VASP activity was unknown. Here we show that mimicking multi-monoubiquitination of VASP at K240 and K286 negatively regulates VASP interactions with actin. Using in vitro biochemical assays, we demonstrate the reduced ability of multi-monoubiquitinated VASP to bind, bundle, and elongate actin filaments. However, multi-monoubiquitinated VASP maintained the ability to bind and protect barbed ends from capping protein. Lastly, we demonstrate the introduction of recombinant multi-monoubiquitinated VASP protein altered cell spreading morphology. Collectively, these results suggest a mechanism in which ubiquitination controls VASP-mediated actin dynamics.

Phenotypic and molecular characterization of Staphylococcus aureus isolates conducted in nares of psoriatic patients attending a public hospital in Argentina / Caracterización fenotípica y molecular de aislamientos de Staphylococcus aureus portados en narinas de pacientes con psoriasis que concurren a un hospital público en Argentina

Abstract Although Staphylococcus aureus increases its relative abundance in psoriasis when compared with the microbiome of healthy subjects, it is not the most important microorganism underlying this disease. However, there is scant data on the role and molecular features of S. aureus strains in psoriasis; therefore, the aim of this study was to evaluate nasal carriage of this microorganism, its phenotypic and molecular characteristics as well as the impact of host factors on its carriage in psoriatic patients. The presence of S. aureus was analyzed in nasal swabs from 46 healthy volunteers and 50 psoriatic patients by conventional microbiology techniques. Nasal carriage of S. aureus was higher in psoriatic patients than in the control group (37.24% vs 22.98%, respectively), being associated to sex (male), age (adults) and severity of the disease (more frequent in moderate and severe cases). Determination of antibiotic resistance detected 12% of (-lactam resistant isolates, with variable accompanying resistance to macrolides, aminoglycosides and fluoroquinolones. No resistance to rifampicin, vancomycin, mupirocin or trimethoprim/sulfamethoxazole was found. A preliminary molecular characterization of the isolates was performed by PCR amplification of virulence genes. Molecular characterization of the strains did not reveal a predominant strain in psoriatic patients. Although we established host factors related to increased carriage of S. aureus in psoriatic patients, we could not establish the predominance of one type of strain. Genomic and transcriptomic analysis of the isolated strains would be necessary to address this point.

Resumen A pesar de que Staphylococcus aureus incrementa su abundancia relativa en la psoriasis cuando se compara con el microbioma de personas sanas, no es el microorganismo más importante subyacente a la enfermedad. Sin embargo, existen pocos datos sobre el papel y las características moleculares de las cepas de S. aureus en pacientes con psoriasis. Nuestro objetivo fue evaluar la portación nasal de este microorganismo, sus características fenotípicas y moleculares, y el impacto de factores del hospedador sobre dicha portación en estos pacientes. Se analizó la presencia de S. aureus en hisopados nasales de 46 voluntarios sanos y 50 pacientes con psoriasis mediante técnicas microbiológicas convencionales. Se encontró mayor portación en pacientes con psoriasis que en el grupo control (37,24% vs. 22,98%, respectivamente) y esta estuvo asociada al sexo (masculino), la edad (adultos) y la gravedad de la enfermedad (más frecuente en casos moderados a graves). El 12% de los aislamientos de S. aureus mostraron resistencia a betalactámicos, con resistencia acompañante a macrólidos, aminoglucósidos y fluoroquinolonas en grado variable. No se encontró resistencia a rifampicina, vancomicina, mupirocina o trimetroprima/sulfametoxazol. Se realizó una caracterización molecular preliminar de los aislamientos por amplificación de genes de virulencia mediante PCR. Si bien se identificaron factores relacionados con el hospedador que incrementan la portación nasal de S. aureus en pacientes con psoriasis, la caracterización molecular de las cepas no reveló ninguna característica genotípica predominante asociada a esta afección. Se necesitan más estudios genómicos y transcriptómicos para profundizar en esta caracterización.

Phenotypic and molecular characterization of Staphylococcus aureus isolates conducted in nares of psoriatic patients attending a public hospital in Argentina.

Although Staphylococcus aureus increases its relative abundance in psoriasis when compared with the microbiome of healthy subjects, it is not the most important microorganism underlying this disease. However, there is scant data on the role and molecular features of S. aureus strains in psoriasis; therefore, the aim of this study was to evaluate nasal carriage of this microorganism, its phenotypic and molecular characteristics as well as the impact of host factors on its carriage in psoriatic patients. The presence of S. aureus was analyzed in nasal swabs from 46 healthy volunteers and 50 psoriatic patients by conventional microbiology techniques. Nasal carriage of S. aureus was higher in psoriatic patients than in the control group (37.24% vs 22.98%, respectively), being associated to sex (male), age (adults) and severity of the disease (more frequent in moderate and severe cases). Determination of antibiotic resistance detected 12% of ß-lactam resistant isolates, with variable accompanying resistance to macrolides, aminoglycosides and fluoroquinolones. No resistance to rifampicin, vancomycin, mupirocin or trimethoprim/sulfamethoxazole was found. A preliminary molecular characterization of the isolates was performed by PCR amplification of virulence genes. Molecular characterization of the strains did not reveal a predominant strain in psoriatic patients. Although we established host factors related to increased carriage of S. aureus in psoriatic patients, we could not establish the predominance of one type of strain. Genomic and transcriptomic analysis of the isolated strains would be necessary to address this point.

Unveiling the Biosynthetic Pathway for Short Mycolic Acids in Nontuberculous Mycobacteria: Mycobacterium smegmatis MSMEG_4301 and Its Ortholog Mycobacterium abscessus MAB_1915 Are Essential for the Synthesis of α'-Mycolic Acids.

Mycolic acids, a hallmark of the genus Mycobacterium, are unique branched long-chain fatty acids produced by a complex biosynthetic pathway. Due to their essentiality and involvement in various aspects of mycobacterial pathogenesis, the synthesis of mycolic acids-and the identification of the enzymes involved-is a valuable target for drug development. Although most of the core pathway is comparable between species, subtle structure differences lead to different structures delineating the mycolic acid repertoire of tuberculous and some nontuberculous mycobacteria. We here report the characterization of an α'-mycolic acid-deficient Mycobacterium smegmatis mutant obtained by chemical mutagenesis. Whole-genome sequencing and bioinformatic analysis identified a premature stop codon in MSMEG_4301, encoding an acyl-CoA synthetase. Orthologs of MSMEG_4301 are present in all mycobacterial species containing α'-mycolic acids. Deletion of the Mycobacterium abscessus ortholog MAB_1915 abrogated synthesis of α'-mycolic acids; likewise, deletion of MSMEG_4301 in an otherwise wild-type M. smegmatis background also caused loss of these short mycolates. IMPORTANCE Mycobacterium abscessus is a nontuberculous mycobacterium responsible for an increasing number of hard-to-treat infections due to the impervious nature of its cell envelope, a natural barrier to several antibiotics. Mycolic acids are key components of that envelope; thus, their synthesis is a valuable target for drug development. Our results identify the first enzyme involved in α'-mycolic acids, a short-chain member of mycolic acids, loss of which greatly affects growth of this opportunistic pathogen.

Bioinformatic Analysis of a Set of 14 Temperate Bacteriophages Isolated from Staphylococcus aureus Strains Highlights Their Massive Genetic Diversity.

Epidemiology and virulence studies of Staphylococcus aureus showed that temperate bacteriophages are one of the most powerful drivers for its evolution not only because of their abundance but also because of the richness of their genetic payload. Here, we report the isolation, genome sequencing, and bioinformatic analysis of 14 bacteriophages induced from lysogenic S. aureus strains from human or veterinary (cattle) origin. The bacteriophages belonged to the Siphoviridae family; were of similar genome size (40 to 45 kbp); and fell into clusters B2, B3, B5, and B7 according to a recent clustering proposal. One of the phages, namely, vB_SauS_308, was the most unusual one, belonging to the sparsely populated subcluster B7 but showing differences in protein family contents compared with the rest of the members. This phage contains a type I endolysin (one catalytic domain and noncanonical cell wall domain [CBD]) and a host recognition module lacking receptor binding protein, cell wall hydrolase, and tail fiber proteins. This phage also lacked virulence genes, which is opposite to what has been reported for subcluster B6 and B7 members. None of six phages, taken as representatives of each of the four subclusters, showed activity on coagulase-negative staphylococci (excepted for two Staphylococcus hominis strains in which propagation and a very slow adsorption rate were observed) nor transducing ability. Immunity tests on S. aureus RN4220 lysogens with each of these phages showed no cross immunity. IMPORTANCE To the best of our knowledge, this set of sequenced bacteriophages is the largest one in South America. Our report describes for the first time the utilization of MultiTwin software to analyze the relationship between phage protein families. Notwithstanding the fact that most of the genetic information obtained correlated with recently published information, due to their geographical origin, the reported analysis adds up to and confirms currently available knowledge of Staphylococcus aureus temperate bacteriophages in terms of phylogeny and role in host evolution.

Actin assembly requirements of the formin Fus1 to build the fusion focus.

In formin-family proteins, actin filament nucleation and elongation activities reside in the formin homology 1 (FH1) and FH2 domains, with reaction rates that vary by at least 20-fold between formins. Each cell expresses distinct formins that assemble one or several actin structures, raising the question of what confers each formin its specificity. Here, using the formin Fus1 in Schizosaccharomyces pombe, we systematically probed the importance of formin nucleation and elongation rates in vivo. Fus1 assembles the actin fusion focus, necessary for gamete fusion to form the zygote during sexual reproduction. By constructing chimeric formins with combinations of FH1 and FH2 domains previously characterized in vitro, we establish that changes in formin nucleation and elongation rates have direct consequences on fusion focus architecture, and that Fus1 native high nucleation and low elongation rates are optimal for fusion focus assembly. We further describe a point mutant in Fus1 FH2 that preserves native nucleation and elongation rates in vitro but alters function in vivo, indicating an additional FH2 domain property. Thus, rates of actin assembly are tailored for assembly of specific actin structures.

Evaluation of factors influencing expression and extraction of recombinant bacteriophage endolysins in Escherichia coli.

BACKGROUND: Endolysins are peptidoglycan hydrolases with promising use as environment-friendly antibacterials mainly when used topically. However, in general, endolysin expression is hampered by its low solubility. Thus, a critical point in endolysin industrial production is optimizing their expression, including improvement of solubility and recovery from cell extracts. RESULTS: We report the expression of two endolysins encoded in the genome of phages infecting Staphylococcus aureus. Expression was optimized through changes in the concentration of the inducer and growth temperature during the expression. Usually, only 30-40% of the total endolysin was recovered in the soluble fraction. Co-expression of molecular chaperones (DnaK, GroEL) or N-term fusion tags endowed with increased solubility (DsbC, Trx, Sumo) failed to improve that yield substantially. Inclusion of osmolytes (NaCl, CaCl2, mannitol, glycine betaine, glycerol and trehalose) or tensioactives (Triton X-100, Tween 20, Nonidet P-40, CHAPS, N-lauroylsarcosine) in the cell disruption system (in the absence of any molecular chaperone) gave meager improvements excepted by N-lauroylsarcosine which increased recovery to 54% of the total endolysin content. CONCLUSION: This is the first attempt to systematically analyze methods for increasing yields of recombinant endolysins. We herein show that neither solubility tags nor molecular chaperones co-expression are effective to that end, while induction temperature, (His)6-tag location and lysis buffer additives (e.g. N-lauroylsarcosine), are sensible strategies to obtain higher levels of soluble S. aureus endolysins.

Evolutionarily diverse LIM domain-containing proteins bind stressed actin filaments through a conserved mechanism.

The actin cytoskeleton assembles into diverse load-bearing networks, including stress fibers (SFs), muscle sarcomeres, and the cytokinetic ring to both generate and sense mechanical forces. The LIM (Lin11, Isl- 1, and Mec-3) domain family is functionally diverse, but most members can associate with the actin cytoskeleton with apparent force sensitivity. Zyxin rapidly localizes via its LIM domains to failing SFs in cells, known as strain sites, to initiate SF repair and maintain mechanical homeostasis. The mechanism by which these LIM domains associate with stress fiber strain sites (SFSS) is not known. Additionally, it is unknown how widespread strain sensing is within the LIM protein family. We identify that the LIM domain-containing region of 18 proteins from the Zyxin, Paxillin, Tes, and Enigma proteins accumulate to SFSS. Moreover, the LIM domain region from the fission yeast protein paxillin like 1 (Pxl1) also localizes to SFSS in mammalian cells, suggesting that the strain sensing mechanism is ancient and highly conserved. We then used sequence and domain analysis to demonstrate that tandem LIM domains contribute additively, for SFSS localization. Employing in vitro reconstitution, we show that the LIM domain-containing region from mammalian zyxin and fission yeast Pxl1 binds to mechanically stressed F-actin networks but does not associate with relaxed actin filaments. We propose that tandem LIM domains recognize an F-actin conformation that is rare in the relaxed state but is enriched in the presence of mechanical stress.

Weirdo19ES is a novel singleton mycobacteriophage that selects for glycolipid deficient phage-resistant M. smegmatis mutants.

The sequencing and bioinformatics analysis of bacteriophages infecting mycobacteria has yielded a large amount of information on their evolution, including that on their environmental propagation on other genera such as Gordonia, closely related to Mycobacterium. However, little is known on mycobacteriophages cell biology such as the nature of their receptor(s) or their replication cycle. As part of our on-going screening for novel mycobacteriophages, we herein report the isolation and genome bioinformatics analysis of Weirdo19ES, a singleton Siphoviridae temperate mycobacteriophage with a 70.19% GC content. Nucleotide and protein sequence comparison to actinobacteriophage databases revealed that Weirdo19ES shows low homology to Gordonia phage Ruthy and mycobacteriophages falling in clusters Q and G and to singleton DS6A.Weirdo19ES also displays uncommon features such as a very short Lysin A gene (with only one enzymatic domain) and two putative HNH endonucleases. Mycobacterium smegmatis mutants resistant to Weirdo19ES are cross- resistant to I3. In agreement with that phenotype, analysis of cell envelope of those mutants showed that Weirdo19ES shares receptors with the transducing mycobacteriophage I3.This singleton mycobacteriophage adds up to the uncommonness of local mycobacteriophages previously isolated by our group and helps understanding the nature of mycobacteriophage receptors.

Pseudomonas aeruginosa exoenzyme Y directly bundles actin filaments.

Pseudomonas aeruginosa uses a type III secretion system (T3SS) to inject cytotoxic effector proteins into host cells. The promiscuous nucleotidyl cyclase, exoenzyme Y (ExoY), is one of the most common effectors found in clinical P. aeruginosa isolates. Recent studies have revealed that the nucleotidyl cyclase activity of ExoY is stimulated by actin filaments (F-actin) and that ExoY alters actin cytoskeleton dynamics in vitro, via an unknown mechanism. The actin cytoskeleton plays an important role in numerous key biological processes and is targeted by many pathogens to gain competitive advantages. We utilized total internal reflection fluorescence microscopy, bulk actin assays, and EM to investigate how ExoY impacts actin dynamics. We found that ExoY can directly bundle actin filaments with high affinity, comparable with eukaryotic F-actin-bundling proteins, such as fimbrin. Of note, ExoY enzymatic activity was not required for F-actin bundling. Bundling is known to require multiple actin-binding sites, yet small-angle X-ray scattering experiments revealed that ExoY is a monomer in solution, and previous data suggested that ExoY possesses only one actin-binding site. We therefore hypothesized that ExoY oligomerizes in response to F-actin binding and have used the ExoY structure to construct a dimer-based structural model for the ExoY-F-actin complex. Subsequent mutational analyses suggested that the ExoY oligomerization interface plays a crucial role in mediating F-actin bundling. Our results indicate that ExoY represents a new class of actin-binding proteins that modulate the actin cytoskeleton both directly, via F-actin bundling, and indirectly, via actin-activated nucleotidyl cyclase activity.

Chlamydomonas reinhardtii formin FOR1 and profilin PRF1 are optimized for acute rapid actin filament assembly.

The regulated assembly of multiple filamentous actin (F-actin) networks from an actin monomer pool is important for a variety of cellular processes. Chlamydomonas reinhardtii is a unicellular green alga expressing a conventional and divergent actin that is an emerging system for investigating the complex regulation of actin polymerization. One actin network that contains exclusively conventional F-actin in Chlamydomonas is the fertilization tubule, a mating structure at the apical cell surface in gametes. In addition to two actin genes, Chlamydomonas expresses a profilin (PRF1) and four formin genes (FOR1-4), one of which (FOR1) we have characterized for the first time. We found that unlike typical profilins, PRF1 prevents unwanted actin assembly by strongly inhibiting both F-actin nucleation and barbed-end elongation at equimolar concentrations to actin. However, FOR1 stimulates the assembly of rapidly elongating actin filaments from PRF1-bound actin. Furthermore, for1 and prf1-1 mutants, as well as the small molecule formin inhibitor SMIFH2, prevent fertilization tubule formation in gametes, suggesting that polymerization of F-actin for fertilization tubule formation is a primary function of FOR1. Together, these findings indicate that FOR1 and PRF1 cooperate to selectively and rapidly assemble F-actin at the right time and place.

Mechanical and kinetic factors drive sorting of F-actin cross-linkers on bundles.

In cells, actin-binding proteins (ABPs) sort to different regions to establish F-actin networks with diverse functions, including filopodia used for cell migration and contractile rings required for cell division. Recent experimental work uncovered a competition-based mechanism that may facilitate spatial localization of ABPs: binding of a short cross-linker protein to 2 actin filaments promotes the binding of other short cross-linkers and inhibits the binding of longer cross-linkers (and vice versa). We hypothesize this sorting arises because F-actin is semiflexible and cannot bend over short distances. We develop a mathematical theory and lattice models encompassing the most important physical parameters for this process and use coarse-grained simulations with explicit cross-linkers to characterize and test our predictions. Our theory and data predict an explicit dependence of cross-linker separation on bundle polymerization rate. We perform experiments that confirm this dependence, but with an unexpected cross-over in dominance of one cross-linker at high growth rates to the other at slow growth rates, and we investigate the origin of this cross-over with further simulations. The nonequilibrium mechanism that we describe can allow cells to organize molecular material to drive biological processes, and our results can guide the choice and design of cross-linkers for engineered protein-based materials.

Cooperation between tropomyosin and α-actinin inhibits fimbrin association with actin filament networks in fission yeast.

We previously discovered that competition between fission yeast actin binding proteins (ABPs) for binding F-actin facilitates their sorting to different cellular networks. Specifically, competition between endocytic actin patch ABPs fimbrin Fim1 and cofilin Adf1 enhances their activities, and prevents tropomyosin Cdc8's association with actin patches. However, these interactions do not explain how Fim1 is prevented from associating strongly with other F-actin networks such as the contractile ring. Here, we identified α-actinin Ain1, a contractile ring ABP, as another Fim1 competitor. Fim1 competes with Ain1 for association with F-actin, which is dependent upon their F-actin residence time. While Fim1 outcompetes both Ain1 and Cdc8 individually, Cdc8 enhances the F-actin bundling activity of Ain1, allowing Ain1 to generate F-actin bundles that Cdc8 can bind in the presence of Fim1. Therefore, the combination of contractile ring ABPs Ain1 and Cdc8 is capable of inhibiting Fim1's association with F-actin networks.

The Influence of Bullying and Cyberbullying in the Psychological Adjustment of Victims and Aggressors in Adolescence.

The objective of the present study was to analyze the extent to which violent peer behavior and victimization, both traditional and cybernetic, and predict certain indicators of psychological maladjustment in adolescents, such as self-concept, satisfaction with life, feeling of loneliness, depressive symptomatology, perceived stress, social anxiety, empathy, and emotional intelligence. Participants in the study were 1318 adolescents of both sexes, aged between 11 and 18 years and enrolled in Compulsory Secondary Education schools. The design of the study was cross-sectional. The results indicated that the victims generally present greater maladjustment than the aggressors. Both victims and cybervictims showed a greater decrease in all the dimensions of self-concept, compared with aggressors and cyberaggressors. However, the two types of aggressors showed a higher likelihood of presenting low levels of empathy. Feeling of loneliness, depressive symptomatology, perceived stress, and degree of life satisfaction was more probable to be present in all groups of aggressors and victims. Finally, with regard to emotional intelligence, victims had a higher probability of obtaining low scores in all the dimensions of this construct; this was the case for traditional aggressors only in the dimension of emotion regulation. These results contribute to our understanding of the consequences of harassment in the adaptation of the students involved, with relevant practical implications.