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Summary As a multifunctional hormone‐like molecule, melatonin exhibits a pleiotropic role in plant salt stress tolerance. While actin cytoskeleton is essential to plant tolerance to salt stress, it is unclear if and how actin cytoskeleton participates in the melatonin‐mediated alleviation of plant salt stress. Here, we report that melatonin alleviates salt stress damage in pigeon pea by activating a kinase‐like protein, which interacts with an actin‐depolymerizing factor. Cajanus cajan Actin‐Depolymerizing Factor 9 (CcADF9) has the function of severing actin filaments and is highly expressed under salt stress. The CcADF9 overexpression lines (CcADF9‐OE) showed a reduction of transgenic root length and an increased sensitivity to salt stress. By using CcADF9 as a bait to screen an Y2H library, we identified actin depolymerizing factor‐related phosphokinase 1 (ARP1), a novel protein kinase that interacts with CcADF9. CcARP1, induced by melatonin, promotes salt resistance of pigeon pea through phosphorylating CcADF9, inhibiting its severing activity. The CcARP1 overexpression lines (CcARP1‐OE) displayed an increased transgenic root length and resistance to salt stress, whereas CcARP1 RNA interference lines (CcARP1‐RNAi) presented the opposite phenotype. Altogether, our findings reveal that melatonin‐induced CcARP1 maintains F‐actin dynamics balance by phosphorylating CcADF9, thereby promoting root growth and enhancing salt tolerance.

Hemipteran insects that transmit plant viruses in a persistent circulative manner acquire, retain and transmit viruses for their entire life. The mechanism enabling this persistence has remained unclear for many years. Here, we determined how wheat dwarf virus (WDV) persists in its leafhopper vector Psammotettix alienus. We found that WDV caused the up‐regulation of actin‐depolymerizing factor (ADF) at the mRNA and protein levels in the midgut cells of leafhoppers after experiencing a WDV acquisition access period (AAP) of 6, 12 or 24 h. Experimental inhibition of F‐actin depolymerization by jasplakinolide and dsRNA injection led to lower virus accumulation levels and transmission efficiencies, suggesting that depolymerization of F‐actin regulated by ADF is essential for WDV invasion of midgut cells. Exogenous viral capsid protein (CP) inhibited ADF depolymerization of actin filaments in vitro and in Spodoptera frugiperda 9 (Sf9) cells because the CP competed with actin to bind ADF and then blocked actin filament disassembly. Interestingly, virions colocalized with ADF after a 24‐h AAP, just as actin polymerization occurred, indicating that the binding of CP with ADF affects the ability of ADF to depolymerize F‐actin, inhibiting WDV entry. Similarly, the luteovirus barley yellow dwarf virus also induced F‐actin depolymerization and then polymerization in the gut cells of its vector Schizaphis graminum. Thus, F‐actin dynamics are altered by nonpropagative viruses in midgut cells to enable virus persistence in vector insects. Biphasic dynamics of F‐actin mediated by the ADF–CP interaction may play an important role in controlling virus entry and the virus accumulation level required for persistent transmission while maintaining the health of the vector.

Epithelial and endothelial monolayers maintain homeostasis by adapting to physiological stimuli and injury through conversion processes that remain incompletely understood. Using human umbilical vein endothelial cell cultures (HUVECs), we elucidate how monolayer maturation and mechanotransduction‐induced remodeling are molecularly regulated. Maturation involves reduced cell perimeter leading to increased junctional VE‐cadherin that recruits junctional actin, integrins and vinculin to establish a quiescent, stable monolayer. Remarkably, we identify a previously unrecognized, rapid and reversible intermediate‐state, marked by VE‐cadherin linearization (clustering) and actomyosin relaxation via myosin light chain (MLC)‐dephosphorylation, that emerges during mechanotransduction‐induced activation, triggered by onset or shifts in shear stress‐induced mechanical load. This novel tension‐mediated intermediate state enhances junctional actin, integrin and vinculin recruitment, thereby strengthening barrier function while protecting endothelial cells from overstimulation and mechanical damage. MLC rephosphorylation dissolves junctional actin, forms stress fibers and induces the formation of “Junction‐Associated‐Intermittent‐Lamellipodia” (JAIL), enabling cell shape change and arterial phenotype remodeling. Overall, junctional VE‐cadherin concentration, together with mechanosensitive signaling that reduces actomyosin tension, governs actin recruitment, revealing a tension‐sensitive, intermediate state that protects cells and primes endothelial remodeling. The data provide a broader model for endothelial mechanotransduction and stress adaptation. This study investigates how endothelial monolayers balance cohesion and plasticity during mechanical stimulation. In HUVECs, shear stress induces a tension‐sensitive intermediate state in which junctional VE‐cadherin and actomyosin relaxation levels coordinate actin/integrin remodeling. This rapid, reversible state strengthens cell junctions, preserves barrier integrity, and prepares cells for the transition from quiescence to activation.

Spinal muscular atrophy (SMA) is a devastating motoneuron (MN) disorder caused by homozygous loss of SMN1. Rarely, SMN1 -deleted individuals are fully asymptomatic despite carrying identical SMN2 copies as their SMA III-affected siblings suggesting protection by genetic modifiers other than SMN2 . High plastin 3 (PLS3) expression has previously been found in lymphoblastoid cells but not in fibroblasts of asymptomatic compared to symptomatic siblings. To find out whether PLS3 is also upregulated in MNs of asymptomatic individuals and thus a convincing SMA protective modifier, we generated induced pluripotent stem cells (iPSCs) from fibroblasts of three asymptomatic and three SMA III-affected siblings from two families and compared these to iPSCs from a SMA I patient and control individuals. MNs were differentiated from iPSC-derived small molecule neural precursor cells (smNPCs). All four genotype classes showed similar capacity to differentiate into MNs at day 8. However, SMA I-derived MN survival was significantly decreased while SMA III- and asymptomatic-derived MN survival was moderately reduced compared to controls at day 27. SMN expression levels and concomitant gem numbers broadly matched SMN2 copy number distribution; SMA I presented the lowest levels, whereas SMA III and asymptomatic showed similar levels. In contrast, PLS3 was significantly upregulated in mixed MN cultures from asymptomatic individuals pinpointing a tissue-specific regulation. Evidence for strong PLS3 accumulation in shaft and rim of growth cones in MN cultures from asymptomatic individuals implies an important role in neuromuscular synapse formation and maintenance. These findings provide strong evidence that PLS3 is a genuine SMA protective modifier.

Homotypic or entotic cell-in-cell invasion is an integrin-independent process observed in carcinoma cells exposed during conditions of low adhesion such as in exudates of malignant disease. Although active cell-in-cell invasion depends on RhoA and actin, the precise mechanism as well as the underlying actin structures and assembly factors driving the process are unknown. Furthermore, whether specific cell surface receptors trigger entotic invasion in a signal-dependent fashion has not been investigated. In this study, we identify the G-protein-coupled LPA receptor 2 (LPAR2) as a signal transducer specifically required for the actively invading cell during entosis. We find that G12/13 and PDZ-RhoGEF are required for entotic invasion, which is driven by blebbing and a uropod-like actin structure at the rear of the invading cell. Finally, we provide evidence for an involvement of the RhoA-regulated formin Dia1 for entosis downstream of LPAR2. Thus, we delineate a signaling process that regulates actin dynamics during cell-in-cell invasion. Entosis is the invasion of one cell by another and can be observed in aggressive cancers. Although the invading cell is usually killed, the surviving cell is sometimes left with the wrong number of chromosomes. This suggests that entosis may help cancer to progress because cells with an abnormal number of chromosomes are common in cancers. For entosis to occur, the invading cell must be released from the tissue that surrounds it, so it can move towards and attach to the cell it is about to invade. Very little is currently known about the cellular and molecular events that enable these processes to occur. Purvanov et al. studied entosis in cells grown in the laboratory and observed that invading cells produce bulges and projections at their rear end for invasion. These projections contain a protein called mDia1. This protein is involved in controlling the growth of the cytoskeleton—the structure that helps cells to both maintain their shape and to move. Adding the signaling molecule lysophosphatidic acid, which is present in human serum, increased the likelihood that cells would invade others. From this, Purvanov et al. established the identities of the proteins involved in transmitting the lysophosphatidic acid signal that controls mDia1 activity during entosis. Changes to this signaling pathway have been associated with cancer and how it spreads between different organs and its involvement in entosis lends further support to the notion that there may be a link between cell-in-cell invasion and the advancement of cancer.

Cellular actin dynamics are tightly regulated by actin-binding proteins with specialized domains. Here, we identify SH3BGRL proteins as modulators of actin dynamics, characterized by their thioredoxin (Trx) fold and the absence of the canonical enzymatic site. The Trx fold is generally associated with enzymatic activity; however, in this context, it functions non-enzymatically to enhance actin nucleation, inhibit depolymerization and cap the growing pointed end of the actin filament. Our results indicate that all SH3BGRL isoforms weakly promote actin nucleation in vitro by stabilizing energetically unstable actin dimers and trimers. Using molecular dynamics simulations and assays that directly probe the pointed end of the actin filament, we show that SH3BGRL proteins efficiently inhibit actin subunit addition at the pointed end by direct association with the terminal actin subunits. However, SH3BGRL proteins are less effective at preventing subunit loss from the pointed end, but they can cooperate with tropomodulin to enhance this activity in an isoform-specific manner, indicating that all isoforms are capable of forming a tripartite complex with actin and tropomodulin. Based on our results, we propose a new and more appropriate name that reflects the function of the SH3BGRL protein family: t hioredoxin fold–containing p ointed e nd c apping proteins (TPECs).

SignificanceActin filaments assemble into ordered networks able to exert forces and shape cells. In response, filaments are exposed to mechanical stress which can potentially modulate their interactions with regulatory proteins. We developed in vitro tools to manipulate single filaments and study the impact of mechanics on the activity of actin depolymerizing factor (ADF)/cofilin, the central player in actin disassembly. While tension has almost no effect, curvature enhances severing by ADF/cofilin. We also discovered a mechanism that boosts the severing of anchored filaments: When binding to these filaments, ADF/cofilin locally increases their natural helicity, generating a torque that accelerates filament fragmentation up to 100-fold. As a consequence, interconnected filament networks are severed far more efficiently than independent filaments. Proteins of the actin depolymerizing factor (ADF)/cofilin family are the central regulators of actin filament disassembly. A key function of ADF/cofilin is to sever actin filaments. However, how it does so in a physiological context, where filaments are interconnected and under mechanical stress, remains unclear. Here, we monitor and quantify the action of ADF/cofilin in different mechanical situations by using single-molecule, single-filament, and filament network techniques, coupled to microfluidics. We find that local curvature favors severing, while tension surprisingly has no effect on cofilin binding and weakly enhances severing. Remarkably, we observe that filament segments that are held between two anchoring points, thereby constraining their twist, experience a mechanical torque upon cofilin binding. We find that this ADF/cofilin-induced torque does not hinder ADF/cofilin binding, but dramatically enhances severing. A simple model, which faithfully recapitulates our experimental observations, indicates that the ADF/cofilin-induced torque increases the severing rate constant 100-fold. A consequence of this mechanism, which we verify experimentally, is that cross-linked filament networks are severed by cofilin far more efficiently than nonconnected filaments. We propose that this mechanochemical mechanism is critical to boost ADF/cofilin’s ability to sever highly connected filament networks in cells.

The vast majority of excitatory synapses are formed on small dendritic protrusions termed dendritic spines. Dendritic spines vary in size and density that are crucial determinants of excitatory synaptic transmission. Aberrations in spine morphogenesis can compromise brain function and have been associated with neuropsychiatric disorders. Actin filaments (F-actin) are the major structural component of dendritic spines, and therefore, actin-binding proteins (ABP) that control F-actin dis-/assembly moved into the focus as critical regulators of brain function. Studies of the past decade identified the ABP cofilin1 as a key regulator of spine morphology, synaptic transmission, and behavior, and they emphasized the necessity for a tight control of cofilin1 to ensure proper brain function. Here, we report spine enrichment of cyclase-associated protein 1 (CAP1), a conserved multidomain protein with largely unknown physiological functions. Super-resolution microscopy and live cell imaging of CAP1-deficient hippocampal neurons revealed impaired synaptic F-actin organization and dynamics associated with alterations in spine morphology. Mechanistically, we found that CAP1 cooperates with cofilin1 in spines and that its helical folded domain is relevant for this interaction. Moreover, our data proved functional interdependence of CAP1 and cofilin1 in control of spine morphology. In summary, we identified CAP1 as a novel regulator of the postsynaptic actin cytoskeleton that is essential for synaptic cofilin1 activity.

Cell migration is a pervasive process in many biology systems and involves protrusive forces generated by actin polymerization, myosin dependent contractile forces, and force transmission between the cell and the substrate through adhesion sites. Here we develop a computational model for cell motion that uses the phase-field method to solve for the moving boundary with physical membrane properties. It includes a reaction-diffusion model for the actin-myosin machinery and discrete adhesion sites which can be in a \"gripping\" or \"slipping\" mode and integrates the adhesion dynamics with the dynamics of the actin filaments, modeled as a viscous network. To test this model, we apply it to fish keratocytes, fast moving cells that maintain their morphology, and show that we are able to reproduce recent experimental results on actin flow and stress patterns. Furthermore, we explore the phase diagram of cell motility by varying myosin II activity and adhesion strength. Our model suggests that the pattern of the actin flow inside the cell, the cell velocity, and the cell morphology are determined by the integration of actin polymerization, myosin contraction, adhesion forces, and membrane forces.

Srv2p/CAP1 is an essential regulator of actin turnover, but its exact function in regulating actin polymerization, particularly the contribution of its actin nucleotide exchange activity, remains incompletely understood. We found that, although Arabidopsis CAP1 is distributed uniformly in the cytoplasm, its loss of function has differential effects on the actin cytoskeleton within different regions of the pollen tube. Specifically, the F-actin level increases in the shank but decreases in the apical region of cap1 pollen tubes. The reduction in apical F-actin results mainly from impaired polymerization of membrane-originated actin within cap1 pollen tubes. The actin nucleotide exchange activity of CAP1 is involved in apical actin polymerization. CAP1 acts synergistically with pollen ADF and profilin to promote actin turnover in vitro, and it can overcome the inhibitory effects of ADF and synergize with profilin to promote actin nucleotide exchange. Consistent with its role as a shuttle molecule between ADF and profilin, the cytosolic concentration of CAP1 is much lower than that of ADF and profilin in pollen. Thus, CAP1 synergizes with ADF and profilin to drive actin turnover in pollen and promote apical actin polymerization in pollen tubes in a manner that involves its actin nucleotide exchange activity.