Intracellular actin networks assemble through the addition of ATP-actin subunits at the growing barbed ends of actin filaments. This is followed by \"aging\" of the filament via ATP hydrolysis and subsequent phosphate release. Aged ADP-actin subunits thus \"treadmill\" through the filament before being released back into the cytoplasmic monomer pool as a result of depolymerization at filament pointed ends. The necessity for aging before filament disassembly is reinforced by preferential binding of cofilin to aged ADP-actin subunits over newly-assembled ADP-Pi actin subunits in the filament. Consequently, investigations into how cofilin influences pointed-end depolymerization have, thus far, focused exclusively on aged ADP-actin filaments. Using microfluidics-assisted Total Internal Reflection Fluorescence (mf-TIRF) microscopy, we reveal that, similar to their effects on ADP filaments, cofilin and cyclase-associated protein (CAP) also promote pointed-end depolymerization of ADP-Pi filaments. Interestingly, the maximal rates of ADP-Pi filament depolymerization by CAP and cofilin together remain approximately 20-40 times lower than for ADP filaments. Further, we find that the promotion of ADP-Pi pointed-end depolymerization is conserved for all three mammalian cofilin isoforms. Taken together, the mechanisms presented here open the possibility of newly-assembled actin filaments being directly disassembled from their pointed-ends, thus bypassing the slow step of Pi release in the aging process.

Cellular actin networks display distinct assembly and disassembly dynamics resulting from multicomponent reactions occurring primarily at the two ends and the sides of actin filaments [1-3]. While barbed ends are considered the hotspot of actin assembly [4], disassembly is thought to primarily occur via reactions on filament sides and pointed ends [3, 5-11]. Cyclase-associated protein (CAP) has emerged as the main protagonist of actin disassembly and remodeling - it collaborates with cofilin to increase pointed-end depolymerization by 300-fold [6, 7], promotes filament \"coalescence\" in presence of Abp1 [12], and accelerates nucleotide exchange to regenerate monomers for new rounds of assembly [13-15]. CAP has also been reported to enhance cofilin-mediated severing [16, 17], but these claims have since been challenged [7]. Using microfluidics-assisted three-color single-molecule imaging, we now reveal that CAP also has important functions at filament barbed ends. We reveal that CAP is a processive barbed-end depolymerase capable of tracking both ends of the filament. Each CAP binding event leads to removal of about 5,175 and 620 subunits from the barbed and pointed ends respectively. We find that the WH2 domain is essential, and the CARP domain is dispensable for barbed-end depolymerization. We show that CAP co-localizes with barbed-end bound formin and capping protein, in the process increasing residence time of formin by 10-fold and promoting dissociation of CP by 4-fold. Our barbed-end observations combined with previously reported activities of CAP at pointed ends and sides, firmly establish CAP as a key player in actin dynamics.

Sex-steroid signaling, especially estrogen, has a paradoxical impact on regulating airway remodeling. In our previous studies, we demonstrated differential effects of 17β-estradiol (E2) towards estrogen receptors (ERs: α and β) in regulating airway smooth muscle (ASM) cell proliferation and extracellular matrix (ECM) production. However, the role of ERs and their signaling on ASM migration is still unexplored. In this study, we examined how ERα versus ERβ affects the mitogen (Platelet-derived growth factor, PDGF)-induced human ASM cell migration as well as the underlying mechanisms involved. We used Lionheart-FX automated microscopy and transwell assays to measure cell migration and found that activating specific ERs had differential effects on PDGF-induced ASM cell migration. Pharmacological activation of ERβ or shRNA mediated knockdown of ERα and specific activation of ERβ blunted PDGF-induced cell migration. Furthermore, specific ERβ activation showed inhibition of actin polymerization by reducing the F/G-actin ratio. Using Zeiss confocal microscopy coupled with three-dimensional algorithmic ZEN-image analysis showed an ERβ-mediated reduction in PDGF-induced expressions of neural Wiskott-Aldrich syndrome protein (N-WASP) and actin-related proteins-2/3 (Arp2/3) complex, thereby inhibiting actin-branching and lamellipodia. In addition, ERβ activation also reduces the clustering of actin-binding proteins (vinculin and paxillin) at the leading edge of ASM cells. However, cells treated with E2 or ERα agonists do not show significant changes in actin/lamellipodial dynamics. Overall, these findings unveil the significance of ERβ activation in regulating lamellipodial and focal adhesion dynamics to regulate ASM cell migration and could be a novel target to blunt airway remodeling.

Conventionally, only the normal cell membrane fluctuations have been studied and used to ascertain membrane properties like the bending rigidity. A new concept, the membrane local slope fluctuations was introduced recently (Vaippullyet al2020Soft Matter167606), which can be modelled as a gradient of the normal fluctuations. It has been found that the power spectral density (PSD) of slope fluctuations behave as (frequency)-1while the normal fluctuations yields (frequency)-5/3even on the apical cell membrane in the high frequency region. In this manuscript, we explore a different situation where the cell is applied with the drug Latrunculin-B which inhibits actin polymerization and find the effect on membrane fluctuations. We find that even as the normal fluctuations show a power law (frequency)-5/3as is the case for a free membrane, the slope fluctuations PSD remains (frequency)-1, with exactly the same coefficient as the case when the drug was not applied. Moreover, while sometimes, when the normal fluctuations at high frequency yield a power law of (frequency)-4/3, the pitch PSD still yields (frequency)-1. Thus, this presents a convenient opportunity to study membrane parameters like bending rigidity as a function of time after application of the drug, while the membrane softens. We also investigate the active athermal fluctuations of the membrane appearing in the PSD at low frequencies and find active timescales of slower than 1 s.

An active actin dynamic is a crucial feature of brain microglia. Here we report that LRRK2, a primary familial Parkinson\'s disease-associated gene, negatively regulates microglia\'s actin dynamics. LRRK2 depolymerized filamentous actin (F-actin) by directly binding to it or inhibiting microglia\'s Rac-PAK signaling. LRRK2 knockdown resulted in a reduced ruffle and enhanced lamellipodia formation of ADP-activated microglia, altering the microglia\'s physiological activity to vigorous migration toward damaged cells. These results suggest that LRRK2 is a negative regulator for the controlled actin dynamics in microglia.

Integrin-dependent adhesion of neutrophils to tissue, accompanied by the development of neutrophil-induced inflammation, occurs both in the focus of infection and in the absence of infection in metabolic disorders such as reperfusion after ischemia, diabetes mellitus, or the development of pneumonia in patients with cystic fibrosis or viral diseases. Hyaluronic acid (HA) plays an important role in the recruitment of neutrophils to tissues. 4-methylumbilliferon (4-MU), an inhibitor of HA synthesis, is used to treat inflammation, but its mechanism of action is unknown. We studied the effect of 4-MU on neutrophil adhesion and concomitant secretion using adhesion to fibronectin as a model for integrin-dependent adhesion. 4-MU reduced the spreading of neutrophils on the substrate and the concomitant secretion of granule proteins, including pro-inflammatory components. 4-MU also selectively blocked adhesion-induced release of the free amino acid hydroxylysine, a product of lysyl hydroxylase, which can influence cell invasion by modifying the extracellular matrix. Finally, 4-MU inhibited the formation of cytonemes, the extracellular membrane secretory structures containing the pro-inflammatory bactericides of the primary granules. The anti-inflammatory effect of 4-MU may be associated with the suppression of secretory processes that ensure the neutrophil invasion and initiate inflammation. We suggest that HA, due to the peculiarities of its synthesis, can promote the release of secretory carriers from the cell and 4-MU can block this process.

During infection or certain metabolic disorders, neutrophils can escape from blood vessels, invade and attach to other tissues. The invasion and adhesion of neutrophils is accompanied and maintained by their own secretion. We have previously found that adhesion of neutrophils to fibronectin dramatically and selectively stimulates the release of the free amino acid hydroxylysine. The role of hydroxylysine and lysyl hydroxylase in neutrophil adhesion has not been studied, nor have the processes that control them. Using amino acid analysis, mass spectrometry and electron microscopy, we found that the lysyl hydroxylase inhibitor minoxidil, the matrix metalloproteinase inhibitor doxycycline, the PI3K/Akt pathway inhibitors wortmannin and the Akt1/2 inhibitor and drugs that affect the actin cytoskeleton significantly and selectively block the release of hydroxylysine and partially or completely suppress spreading of neutrophils. The actin cytoskeleton effectors and the Akt 1/2 inhibitor also increase the phenylalanine release. We hypothesize that hydroxylysine release upon adhesion is the result of the activation of lysyl hydroxylase in interaction with matrix metalloproteinase, the PI3K/Akt pathway and intact actin cytoskeleton, which play important roles in the recruitment of neutrophils into tissue through extracellular matrix remodeling.

Numerous studies have assembled a complex picture, in which extracellular stimuli and intracellular signaling pathways modulate the chondrocyte phenotype. Because many diseases are mechanobiology-related, this review asked to what extent phenotype regulators control chondrocyte function through the cytoskeleton and cytoskeleton-regulating signaling processes. Such information would generate leverage for advanced articular cartilage repair. Serial passaging, pro-inflammatory cytokine signaling (TNF-α, IL-1α, IL-1β, IL-6, and IL-8), growth factors (TGF-α), and osteoarthritis not only induce dedifferentiation but also converge on RhoA/ROCK/Rac1/mDia1/mDia2/Cdc42 to promote actin polymerization/crosslinking for stress fiber (SF) formation. SF formation takes center stage in phenotype control, as both SF formation and SOX9 phosphorylation for COL2 expression are ROCK activity-dependent. Explaining how it is molecularly possible that dedifferentiation induces low COL2 expression but high SF formation, this review theorized that, in chondrocyte SOX9, phosphorylation by ROCK might effectively be sidelined in favor of other SF-promoting ROCK substrates, based on a differential ROCK affinity. In turn, actin depolymerization for redifferentiation would \"free-up\" ROCK to increase COL2 expression. Moreover, the actin cytoskeleton regulates COL1 expression, modulates COL2/aggrecan fragment generation, and mediates a fibrogenic/catabolic expression profile, highlighting that actin dynamics-regulating processes decisively control the chondrocyte phenotype. This suggests modulating the balance between actin polymerization/depolymerization for therapeutically controlling the chondrocyte phenotype.

Triple-negative breast cancers (TNBCs) are aggressive cancers, which currently do not have effective treatment options. Migration and establishment of metastatic colonies require dynamic cytoskeletal modifications characterized by polymerization and depolymerization of actin. Studies have demonstrated a direct molecular link between the integrin-focal adhesion kinase (FAK) pathway and cytoskeletal modifications. Nimbolide, a major bioactive compound present in neem leaves, shows promising anti-cancer effect on various cancers. In this study, we have demonstrated the growth and metastasis inhibitory potential of nimbolide on TNBC cells. Nimbolide inhibited cell proliferation, migratory, and invasive abilities of TNBC cells and also changed the shape of MDA-MB-231 cells, which is correlated with cytoskeletal changes including actin depolymerization. Furthermore, analysis revealed that integrins αV and β3, ILK, FAK, and PAK levels were downregulated by nimbolide. Even in cells where Rac1/Cdc42 was constitutively activated, nimbolide inhibited the formation of filopodial structures. Immunofluorescence analysis of phosphorylated p21 activated kinase (pPAK) showed reduced expression in nimbolide-treated cells. Nimbolide significantly reduced the metastatic colony formation in lung, liver, and brain of athymic nude mice. In conclusion, our data demonstrate that nimbolide inhibits TNBC by altering the integrin and FAK signaling pathway.

OBJECTIVE: The mTOR/S6K1 signaling axis, known for cell growth regulation, is hyper-activated in multiple cancers. In this study, we have examined the mechanisms for ribosomal protein p70-S6 kinase 1 (S6K1) associated transformed human hepatocyte (THH) growth regulation.
METHODS: THH were treated with p70-S6K1 inhibitor and analyzed for cell viability, cell cycle distribution, specific marker protein expression by western blot, and tumor inhibition in a xenograft mouse model. We validated our results by knockdown of p70-S6K1 using specific siRNA.
RESULTS: p70-S6K1 inhibitor treatment caused impairment of in vitro hepatocyte growth, and arrested cell cycle progression at the G1 phase. Further, p70-S6K1 inhibitor treatment exhibited a decrease in FAK and Erk activation, followed by altered integrin-β1 expression, caspase 8, and PARP cleavage appeared to be anoikis like growth inhibition. p70-S6K1 inhibitor also depolymerized actin microfilaments and diminished active Rac1/Cdc42 complex formation for loss of cellular attachment. Similar results were obtained with other transformed human hepatocyte cell lines. p70-S6K1 inhibition also resulted in a reduced phospho-EGFR, Slug and Twist; implicating an inhibition of epithelial-mesenchymal transition (EMT) state. A xenograft tumor model, generated from implanted THH in nude mice, following intraperitoneal injection of S6K1 inhibitor prevented further tumor growth.
CONCLUSIONS: Our results suggested that p70-S6K1 inhibition alters orchestration of cell cycle progression, induces cell detachment, and sensitizes hepatocyte growth impairment. Targeting p70 isoform of S6K1 by inhibitor may prove to be a promising approach together with other therapies for hepatocellular carcinoma (HCC) treatment.