The mechanism underlying the preferential and cooperative binding of cofilin and the expansion of clusters toward the pointed-end side of actin filaments remains poorly understood. To address this, we conducted a principal component analysis based on available filamentous actin (F-actin) and C-actin (cofilins were excluded from cofilactin) structures and compared to monomeric G-actin. The results strongly suggest that C-actin, rather than F-ADP-actin, represented the favourable structure for binding preference of cofilin. High-speed atomic force microscopy explored that the shortened bare half helix adjacent to the cofilin clusters on the pointed end side included fewer actin protomers than normal helices. The mean axial distance (MAD) between two adjacent actin protomers along the same long-pitch strand within shortened bare half helices was longer (5.0-6.3 nm) than the MAD within typical helices (4.3-5.6 nm). The inhibition of torsional motion during helical twisting, achieved through stronger attachment to the lipid membrane, led to more pronounced inhibition of cofilin binding and cluster formation than the presence of inorganic phosphate (Pi) in solution. F-ADP-actin exhibited more naturally supertwisted half helices than F-ADP.Pi-actin, explaining how Pi inhibits cofilin binding to F-actin with variable helical twists. We propose that protomers within the shorter bare helical twists, either influenced by thermal fluctuation or induced allosterically by cofilin clusters, exhibit characteristics of C-actin-like structures with an elongated MAD, leading to preferential and cooperative binding of cofilin.

Crosstalk between the actin and microtubule cytoskeletons is important for many cellular processes. Recent studies have shown that microtubules and F-actin can assemble to form a composite structure where F-actin occupies the microtubule lumen. Whether these cytoskeletal hybrids exist in physiological settings and how they are formed is unclear. Here, we show that the short-crossover Class I actin filament previously identified inside microtubules in human HAP1 cells is cofilin-bound F-actin. Lumenal F-actin can be reconstituted in vitro, but cofilin is not essential. Moreover, actin filaments with both cofilin-bound and canonical morphologies reside within human platelet microtubules under physiological conditions. We propose that stress placed upon the microtubule network during motor-driven microtubule looping and sliding may facilitate the incorporation of actin into microtubules.

OBJECTIVE: In this study, we investigated the mechanism of action of LIMK1 in cervical cancer progression.
METHODS: The biological role of LIMK1 in regulating the growth, invasion, and metastasis of cervical cancer was studied in SiHa, CaSki cells and nude mice tumor models. The role of LIMK1 in the growth of cervical cancer was evaluated by HE staining. The role of LIMK1 in the invasion, metastasis, and proliferation of cervical cancer was evaluated by cell scratch, Transwell, and monoclonal experiments. The interaction among LIMK1, ROS, and Src was evaluated by Western blotting. The effects of regulating ROS and p-Src expression on LIMK1 in the migration/invasion and proliferation of cervical cancer cells were evaluated through cellular functional assays.
RESULTS: Overexpression of LIMK1 promoted tumor growth in nude mice. Cell scratch, Transwell, and monoclonal experiments suggested that LIMK1 promoted the invasion, metastasis, and proliferation of cervical cancer cells. Western blotting suggested that LIMK1 can promote the expression of ROS-related proteins NOX2, NOX4, p-Src, and downstream proteins p-FAK, p-ROCK1/2, p-Cofilin-1, F-actin and inhibit the expression of p-SHP2 protein. Correction experiments showed that LIMK1 regulated the expression of p-FAK and p-Cofilin-1 proteins by regulating ROS and p-Src. Through the detection of cervical cancer cell functions, it was found that the activation of ROS and p-Src induced by LIMK1 is an early event that promotes the migration, proliferation, and invasion of cervical cancer cells.
CONCLUSIONS: LIMK1 promotes the expression of F-actin and promotes the development of cervical cancer by regulating the oxidative stress/Src-mediated p-FAK/p-ROCK1/2/p-Cofilin-1 pathway.

Cofilin, an actin-severing protein, plays key roles in muscle sarcomere addition and maintenance. Our previous work found that Drosophila cofilin (DmCFL) knockdown in muscle causes progressive deterioration of muscle structure and function and produces features seen in nemaline myopathy caused by cofilin mutations. We hypothesized that disruption of actin cytoskeleton dynamics by DmCFL knockdown would impact other aspects of muscle development, and, thus, conducted an RNA-sequencing analysis that unexpectedly revealed upregulated expression of numerous neuromuscular junction (NMJ) genes. We found that DmCFL is enriched in the muscle postsynaptic compartment and that DmCFL muscle knockdown causes F-actin disorganization in this subcellular domain prior to the sarcomere defects observed later in development. Despite NMJ gene expression changes, we found no significant changes in gross presynaptic Bruchpilot active zones or total postsynaptic glutamate receptor levels. However, DmCFL knockdown resulted in mislocalization of GluRIIA class glutamate receptors in more deteriorated muscles and strongly impaired NMJ transmission strength. These findings expand our understanding of the roles of cofilin in muscle to include NMJ structural development and suggest that NMJ defects may contribute to the pathophysiology of nemaline myopathy.

UNASSIGNED: Plasma concentration of PAI-1 (plasminogen activator inhibitor-1) correlates with arterial stiffness. Vascular smooth muscle cells (SMCs) express PAI-1, and the intrinsic stiffness of SMCs is a major determinant of total arterial stiffness. We hypothesized that PAI-1 promotes SMC stiffness by regulating the cytoskeleton and that pharmacological inhibition of PAI-1 decreases SMC and aortic stiffness.
UNASSIGNED: PAI-039, a specific inhibitor of PAI-1, and small interfering RNA were used to inhibit PAI-1 expression in cultured human SMCs. Effects of PAI-1 inhibition on SMC stiffness, F-actin (filamentous actin) content, and cytoskeleton-modulating enzymes were assessed. WT (wild-type) and PAI-1-deficient murine SMCs were used to determine PAI-039 specificity. RNA sequencing was performed to determine the effects of PAI-039 on SMC gene expression. In vivo effects of PAI-039 were assessed by aortic pulse wave velocity.
UNASSIGNED: PAI-039 significantly reduced intrinsic stiffness of human SMCs, which was accompanied by a significant decrease in cytoplasmic F-actin content. PAI-1 gene knockdown also decreased cytoplasmic F-actin. PAI-1 inhibition significantly increased the activity of cofilin, an F-actin depolymerase, in WT murine SMCs, but not in PAI-1-deficient SMCs. RNA-sequencing analysis suggested that PAI-039 upregulates AMPK (AMP-activated protein kinase) signaling in SMCs, which was confirmed by Western blotting. Inhibition of AMPK prevented activation of cofilin by PAI-039. In mice, PAI-039 significantly decreased aortic stiffness and tunica media F-actin content without altering the elastin or collagen content.
UNASSIGNED: PAI-039 decreases intrinsic SMC stiffness and cytoplasmic stress fiber content. These effects are mediated by AMPK-dependent activation of cofilin. PAI-039 also decreases aortic stiffness in vivo. These findings suggest that PAI-1 is an important regulator of the SMC cytoskeleton and that pharmacological inhibition of PAI-1 has the potential to prevent and treat cardiovascular diseases involving arterial stiffening.

Periventricular nodular heterotopia (PNH), the most common brain malformation diagnosed in adulthood, is characterized by the presence of neuronal nodules along the ventricular walls. PNH is mainly associated with mutations in the FLNA gene - encoding an actin-binding protein - and patients often develop epilepsy. However, the molecular mechanisms underlying the neuronal failure still remain elusive. It has been hypothesized that dysfunctional cortical circuitry, rather than ectopic neurons, may explain the clinical manifestations. To address this issue, we depleted FLNA from cortical pyramidal neurons of a conditional Flnaflox/flox mice by timed in utero electroporation of Cre recombinase. We found that FLNA regulates dendritogenesis and spinogenesis thus promoting an appropriate excitatory/inhibitory inputs balance. We demonstrated that FLNA modulates RAC1 and cofilin activity through its interaction with the Rho-GTPase Activating Protein 24 (ARHGAP24). Collectively, we disclose an uncharacterized role of FLNA and provide strong support for neural circuit dysfunction being a consequence of FLNA mutations.

Rabies virus (RABV) is highly lethal and triggers severe neurological symptoms. The neuropathogenic mechanism remains poorly understood. Ras-related C3 botulinum toxin substrate 1 (Rac1) is a Rho-GTPase that is involved in actin remodeling and has been reported to be closely associated with neuronal dysfunction. In this study, by means of a combination of pharmacological inhibitors, small interfering RNA, and specific dominant-negatives, we characterize the crucial roles of dynamic actin and the regulatory function of Rac1 in RABV infection, dominantly in the viral entry phase. The data show that the RABV phosphoprotein interacts with Rac1. RABV phosphoprotein suppress Rac1 activity and impedes downstream Pak1-Limk1-Cofilin1 signaling, leading to the disruption of F-actin-based structure formation. In early viral infection, the EGFR-Rac1-signaling pathway undergoes a biphasic change, which is first upregulated and subsequently downregulated, corresponding to the RABV entry-induced remodeling pattern of F-actin. Taken together, our findings demonstrate for the first time the role played by the Rac1 signaling pathway in RABV infection and may provide a clue for an explanation for the etiology of rabies neurological pathogenesis.IMPORTANCEThough neuronal dysfunction is predominant in fatal rabies, the detailed mechanism by which rabies virus (RABV) infection causes neurological symptoms remains in question. The actin cytoskeleton is involved in numerous viruses infection and plays a crucial role in maintaining neurological function. The cytoskeletal disruption is closely associated with abnormal nervous symptoms and induces neurogenic diseases. In this study, we show that RABV infection led to the rearrangement of the cytoskeleton as well as the biphasic kinetics of the Rac1 signal transduction. These results help elucidate the mechanism that causes the aberrant neuronal processes by RABV infection and may shed light on therapeutic development aimed at ameliorating neurological disorders.

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.

Peroxiredoxin 1 (PRDX1) is an important member of the peroxiredoxin family (PRDX) and is upregulated in a variety of tumors. Previous studies have found that high PRDX1 expression is closely related to the metastasis of oral squamous cell carcinoma (OSCC), but the specific molecular mechanism is elusive. To elucidate the role of PRDX1 in the metastasis process of OSCC, we evaluated the expression of PRDX1 in OSCC clinical specimens and its impact on the prognosis of OSCC patients. Then, the effect of PRDX1 on OSCC metastasis and cytoskeletal reconstruction was explored in vitro and in nude mouse tongue cancer models, and the molecular mechanisms were also investigated. PRDX1 can directly interact with the actin-binding protein Cofilin, inhibiting the phosphorylation of its Ser3 site, accelerating the depolymerization and turnover of actin, promoting OSCC cell movement, and aggravating the invasion and metastasis of OSCC. In clinical samples and mouse tongue cancer models, PRDX1 also increased lymph node metastasis of OSCC and was negatively correlated with the phosphorylation of Cofilin; PRDX1 also reduced the overall survival rate of OSCC patients. In summary, our study identified that PRDX1 may be a potential therapeutic target to inhibit OSCC metastasis.

Cofilin, a key actin-binding protein, orchestrates the dynamics of the actomyosin network through its actin-severing activity and by promoting the recycling of actin monomers. Recent experiments suggest that cofilin forms functionally distinct oligomers via thiol post-translational modifications (PTMs) that promote actin nucleation and assembly. Despite these advances, the structural conformations of cofilin oligomers that modulate actin activity remain elusive because there are combinatorial ways to oxidize thiols in cysteines to form disulfide bonds rapidly. This study employs molecular dynamics simulations to investigate human cofilin 1 as a case study for exploring cofilin dimers via disulfide bond formation. Utilizing a biasing scheme in simulations, we focus on analyzing dimer conformations conducive to disulfide bond formation. Additionally, we explore potential PTMs arising from the examined conformational ensemble. Using the free energy profiling, our simulations unveil a range of probable cofilin dimer structures not represented in current Protein Data Bank entries. These candidate dimers are characterized by their distinct population distributions and relative free energies. Of particular note is a dimer featuring an interface between cysteines 139 and 147 residues, which demonstrates stable free energy characteristics and intriguingly symmetrical geometry. In contrast, the experimentally proposed dimer structure exhibits a less stable free energy profile. We also evaluate frustration quantification based on the energy landscape theory in the protein-protein interactions at the dimer interfaces. Notably, the 39-39 dimer configuration emerges as a promising candidate for forming cofilin tetramers, as substantiated by frustration analysis. Additionally, docking simulations with actin filaments further evaluate the stability of these cofilin dimer-actin complexes. Our findings thus offer a computational framework for understanding the role of thiol PTM of cofilin proteins in regulating oligomerization, and the subsequent cofilin-mediated actin dynamics in the actomyosin network.