Inhibition Of Actin Polymerization Research Articles

TrCla4 promotes actin polymerization at the hyphal tip and mycelial growth in Trichophyton rubrum.

Dermatophytes invade and colonize host superficial tissues via hyphal growth. Although cytoskeletal reorganization and its regulation are essential for hyphal growth, the molecular mechanisms in dermatophytes and their applicability as antifungal drug targets remain poorly understood. The p21-activated kinase (PAK) is a downstream effector of the small GTPases Rac and CDC42, also known as p21, and is involved in various molecular and cellular functions, including actin polymerization and cell morphogenesis. In this study, we investigated the contribution of the PAK protein TrCla4 to morphogenesis and mycelial growth in Trichophyton rubrum, the most frequently isolated fungus in dermatophytosis (athlete's foot). The actin polymerization inhibitor, cytochalasin A inhibited actin accumulation at the hyphal tip and mycelial growth of T. rubrum, suggesting the involvement of the actin cytoskeleton in mycelial growth. In the Trcla4 knockout strain (ΔTrcla4), we observed defects in mycelial growth, hyphal branching, and the accumulation of polymerized actin at the hyphal tip. Chemical inhibitors of TrRac-dependent TrCla4 kinase activity, FRAX486 and IPA-3, also inhibited spore germination and mycelial growth. Interestingly, ΔTrcla4 showed no additional inhibition of mycelial growth when treated with these inhibitors, indicating that their inhibitory effects are primarily mediated through TrCla4. In an invertebrate dermatophyte infection model, animals infected with ΔTrcla4 had higher survival rates than those infected with the wild-type, and IPA-3 and FRAX486 treatments both significantly improved animal survival rates. These results suggest that the dermatophyte PAK promotes mycelial growth by facilitating actin polymerization at the hyphal tip, making it a potential therapeutic target for dermatophytosis. IMPORTANCE Superficial fungal infections, such as athlete's foot, affect more than 10% of the world's population and have a significant impact on quality of life. Despite the fact that treatment-resistant fungi are a concern, there are just a few antifungal drug targets accessible, as opposed to the wide range of therapeutic targets found in bacterial infections. As a result, additional alternatives are sought. In this study, we generated a PAK TrCla4 deletion strain (∆Trcla4) of Trichophyton rubrum. The ∆Trcla4 strain exhibited deficiencies in mycelial growth, hyphal morphology, and polarized actin localization at the hyphal tip. IPA-3 and FRAX486, small chemical inhibitors of mammalian PAK, were discovered to limit fungal mycelial proliferation. According to our findings, fungal PAKs are interesting therapeutic targets for the development of new antifungal medicines.

Intracellular remodeling associated with endoplasmic reticulum stress modifies biomechanical compliance of bladder cells

Bladder cells face a challenging biophysical environment: mechanical cues originating from urine flow and regular contraction to enable the filling voiding of the organ. To ensure functional adaption, bladder cells rely on high biomechanical compliance, nevertheless aging or chronic pathological conditions can modify this plasticity. Obviously the cytoskeletal network plays an essential role, however the contribution of other, closely entangled, intracellular organelles is currently underappreciated. The endoplasmic reticulum (ER) lies at a crucial crossroads, connected to both nucleus and cytoskeleton. Yet, its role in the maintenance of cell mechanical stability is less investigated. To start exploring these aspects, T24 bladder cancer cells were treated with the ER stress inducers brefeldin A (10-40nM BFA, 24 h) and thapsigargin (0.1-100nM TG, 24 h). Without impairment of cell motility and viability, BFA and TG triggered a significant subcellular redistribution of the ER; this was associated with a rearrangement of actin cytoskeleton. Additional inhibition of actin polymerization with cytochalasin D (100nM CytD) contributed to the spread of the ER toward cell periphery, and was accompanied by an increase of cellular stiffness (Young´s modulus) in the cytoplasmic compartment. Shrinking of the ER toward the nucleus (100nM TG, 2 h) was related to an increased stiffness in the nuclear and perinuclear areas. A similar short-term response profile was observed also in normal human primary bladder fibroblasts. In sum, the ER and its subcellular rearrangement seem to contribute to the mechanical properties of bladder cells opening new perspectives in the study of the related stress signaling cascades.AnQ6eoaFZ-G6Rs7Kzp4dpwVideo

Tunable hydrogel viscoelasticity modulates human neural maturation.

Human-induced pluripotent stem cells (hiPSCs) have emerged as a promising in vitro model system for studying neurodevelopment. However, current models remain limited in their ability to incorporate tunable biomechanical signaling cues imparted by the extracellular matrix (ECM). The native brain ECM is viscoelastic and stress-relaxing, exhibiting a time-dependent response to an applied force. To recapitulate the remodelability of the neural ECM, we developed a family of protein-engineered hydrogels that exhibit tunable stress relaxation rates. hiPSC-derived neural progenitor cells (NPCs) encapsulated within these gels underwent relaxation rate-dependent maturation. Specifically, NPCs within hydrogels with faster stress relaxation rates extended longer, more complex neuritic projections, exhibited decreased metabolic activity, and expressed higher levels of genes associated with neural maturation. By inhibiting actin polymerization, we observed decreased neuritic projections and a concomitant decrease in neural maturation gene expression. Together, these results suggest that microenvironmental viscoelasticity is sufficient to bias human NPC maturation.

RGMa and Neogenin control dendritic spine morphogenesis via WAVE Regulatory Complex-mediated actin remodeling.

Structural plasticity, the ability of dendritic spines to change their volume in response to synaptic stimulation, is an essential determinant of synaptic strength and long-term potentiation (LTP), the proposed cellular substrate for learning and memory. Branched actin polymerization is a major force driving spine enlargement and sustains structural plasticity. The WAVE Regulatory Complex (WRC), a pivotal branched actin regulator, controls spine morphology and therefore structural plasticity. However, the molecular mechanisms that govern WRC activation during spine enlargement are largely unknown. Here we identify a critical role for Neogenin and its ligand RGMa (Repulsive Guidance Molecule a) in promoting spine enlargement through the activation of WRC-mediated branched actin remodeling. We demonstrate that Neogenin regulates WRC activity by binding to the highly conserved Cyfip/Abi binding pocket within the WRC. We find that after Neogenin or RGMa depletion, the proportions of filopodia and immature thin spines are dramatically increased, and the number of mature mushroom spines concomitantly decreased. Wildtype Neogenin, but not Neogenin bearing mutations in the Cyfip/Abi binding motif, is able to rescue the spine enlargement defect. Furthermore, Neogenin depletion inhibits actin polymerization in the spine head, an effect that is not restored by the mutant. We conclude that RGMa and Neogenin are critical modulators of WRC-mediated branched actin polymerization promoting spine enlargement. This study also provides mechanistic insight into Neogenin's emerging role in LTP induction.

Estrogen receptors differentially modifies lamellipodial and focal adhesion dynamics in airway smooth muscle cell migration

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.

Aminopeptidase N/CD13 Crosslinking Promotes the Activation and Membrane Expression of Integrin CD11b/CD18.

The β2 integrin CD11b/CD18, also known as complement receptor 3 (CR3), and the moonlighting protein aminopeptidase N (CD13), are two myeloid immune receptors with overlapping activities: adhesion, migration, phagocytosis of opsonized particles, and respiratory burst induction. Given their common functions, shared physical location, and the fact that some receptors can activate a selection of integrins, we hypothesized that CD13 could induce CR3 activation through an inside-out signaling mechanism and possibly have an influence on its membrane expression. We revealed that crosslinking CD13 on the surface of human macrophages not only activates CR3 but also influences its membrane expression. Both phenomena are affected by inhibitors of Src, PLCγ, Syk, and actin polymerization. Additionally, after only 10 min at 37 °C, cells with crosslinked CD13 start secreting pro-inflammatory cytokines like interferons type 1 and 2, IL-12p70, and IL-17a. We integrated our data with a bioinformatic analysis to confirm the connection between these receptors and to suggest the signaling cascade linking them. Our findings expand the list of features of CD13 by adding the activation of a different receptor via inside-out signaling. This opens the possibility of studying the joint contribution of CD13 and CR3 in contexts where either receptor has a recognized role, such as the progression of some leukemias.

B-308 Focal Adhesion Kinase Activation is Involved in Tension Development During Contractile Stimulation of Mouse Detrusor Smooth Muscle to Modulate Bladder Dysfunction-Related Lower Urinary Tract Symptoms

Abstract Background The function of the bladder is to store and expel urine. Bladder dysfunction-related lower urinary tract symptoms (LUTS) is a common urological problem and negatively affects health-related quality of life. Investigation of detrusor muscle contraction mechanisms is necessary to understand its underlying pathophysiology. Voiding occurs through bladder muscle contraction, which is mediated primarily by the increased intracellular Ca2+ concentration-induced myosin regulatory light chain (MLC) phosphorylation. Recent studies showed that focal adhesion kinase (FAK)-regulated focal adhesion assembly and actin polymerization are involved in vascular and airway smooth muscle contraction. However, the role of FAK activation in visceral smooth muscle contraction is not well documented. This study aims to determine if FAK activation is involved in mouse detrusor smooth muscle contraction. Methods Normal adult male and female mouse bladders were harvested. The trigone and dome areas were cut, and the bladder body was used. The mucosa was carefully removed. Detrusor muscle strips (1.5–2 × 8–10 mm) were prepared and then suspended in a 20 mL tissue bath filled with physiological Krebs solution and bubbled with 95% O2 and 5% CO2 to obtain a pH of 7.4 at 37 °C. The isometric contraction was recorded. After establishing a stable baseline, the strips were stimulated with 120 mM potassium chloride (KCl), which was used to normalize the contractility induced by subsequent stimuli. One of the following specific inhibitors or the same volume of vehicle (DMSO) was applied and incubated for 30 min: PF-573228 (2 µM, a FAK inhibitor) or latrunculin B (1 µM, an inhibitor of actin polymerization). Then, the contractile responses to KCl (90 mM), electrical field stimulation (EFS, 2–32 Hz), or carbachol (CCh, 10−7.5–10−4.5 M ) were measured. In a separate experiment, the detrusor strips were stimulated with CCh (10 µM) after incubation with PF-573228 (2 µM) or vehicle (DMSO) for 30 min. Once the peak contraction was reached, the strips were snap-frozen for immunoblotting analysis of the phosphorylated FAK (p-FAK) and MLC (p-MLC). Statistical analysis was performed (GraphPad Prism 6). The contractile responses to KCl and EFS between vehicle and inhibitor groups were compared using a paired t-test. Nonlinear regression was used to compare CCh-induced dose-response curves. For the expression of phosphorylated proteins, one-way ANOVA, followed by Tukey’s posthoc test, was used for analysis. Results KCl-induced contractile responses decreased significantly after incubation with PF-573228 or latrunculin B compared to the corresponding vehicle group (P < 0.0001). The contractile responses induced by EFS were significantly inhibited by preincubation with PF-573228 at 8, 16, and 32 Hz (P < 0.05) or latrunculin B at 16 and 32 Hz (P < 0.01). Following the application of PF-573228 or latrunculin B, CCh-induced dose-response contractions were significantly lower than those in the corresponding vehicle-treated strips (P = 0.0021 and 0.0003, respectively). Immunoblotting analysis demonstrated that CCh stimulation caused increased expression of p-FAK and p-MLC, whereas preincubation with PF-573228 led to decreased expression of p-FAK but not p-MLC Conclusion FAK activation is involved in tension development induced by contractile stimulation in mouse detrusor muscle, probably through enhancing actin polymerization, but not MLC phosphorylation.

Mechanical Properties and Nanomotion of BT-20 and ZR-75 Breast Cancer Cells Studied by Atomic Force Microscopy and Optical Nanomotion Detection Method.

Cells of two molecular genetic types of breast cancer-hormone-dependent breast cancer (ZR-75 cell line) and triple-negative breast cancer (BT-20 cell line)-were studied using atomic force microscopy and an optical nanomotion detection method. Using the Peak Force QNM and Force Volume AFM modes, we revealed the unique patterns of the dependence of Young's modulus on the indentation depth for two cancer cell lines that correlate with the features of the spatial organization of the actin cytoskeleton. Within a 200-300 nm layer just under the cell membrane, BT-20 cells are stiffer than ZR-75 cells, whereas in deeper cell regions, Young's modulus of ZR-75 cells exceeds that of BT-20 cells. Two cancer cell lines also displayed a difference in cell nanomotion dynamics upon exposure to cytochalasin D, a potent actin polymerization inhibitor. The drug strongly modified the nanomotion pattern of BT-20 cells, whereas it had almost no effect on the ZR-75 cells. We are confident that nanomotion monitoring and measurement of the stiffness of cancer cells at various indentation depths deserve further studies to obtain effective predictive parameters for use in clinical practice.

Codependencies of mTORC1 signaling and endolysosomal actin structures.

The mechanistic target of rapamycin complex 1 (mTORC1) is part of the amino acid sensing machinery that becomes activated on the endolysosomal surface in response to nutrient cues. Branched actin generated by WASH and Arp2/3 complexes defines endolysosomal microdomains. Here, we find mTORC1 components in close proximity to endolysosomal actin microdomains. We investigated for interactors of the mTORC1 lysosomal tether, RAGC, by proteomics and identified multiple actin filament capping proteins and their modulators. Perturbation of RAGC function affected the size of endolysosomal actin, consistent with a regulation of actin filament capping by RAGC. Reciprocally, the pharmacological inhibition of actin polymerization or alteration of endolysosomal actin obtained upon silencing of WASH or Arp2/3 complexes impaired mTORC1 activity. Mechanistically, we show that actin is required for proper association of RAGC and mTOR with endolysosomes. This study reveals an unprecedented interplay between actin and mTORC1 signaling on the endolysosomal system.

Dynamic actin/septin network in megakaryocytes coordinates proplatelet elaboration.

Megakaryocytes (MK) undergo extensive cytoskeletal rearrangements as they give rise to platelets. While cortical microtubule sliding has been implicated in proplatelet formation, the role of the actin cytoskeleton in proplatelet elongation is less understood. It is assumed that actin filament reorganization is important for platelet generation given that mouse models with mutations in actin-associated proteins exhibit thrombocytopenia. However, due to the essential role of the actin network during MK development, a differential understanding of the contribution of the actin cytoskeleton on proplatelet release is lacking. Here, we reveal that inhibition of actin polymerization impairs the formation of elaborate proplatelets by hampering proplatelet extension and bead formation along the proplatelet shaft, which was mostly independent of changes in cortical microtubule sliding. We identify Cdc42 and its downstream effectors, septins, as critical regulators of intracellular actin dynamics in MK, inhibition of which, similarly to inhibition of actin polymerization, impairs proplatelet movement and beading. Super-resolution microscopy revealed a differential association of distinctive septins with the actin and microtubule cytoskeleton, respectively, which was disrupted upon septin inhibition and diminished intracellular filamentous actin dynamics. In vivo, septins, similarly to F-actin, were subject to changes in expression upon enforcing proplatelet formation through prior platelet depletion. In summary, we demonstrate that a Cdc42/septin axis is not only important for MK maturation and polarization, but is further required for intracellular actin dynamics during proplatelet formation.

Enhancing suspended cell transfection by inducing localized distribution of the membrane actin cortex before exposure to electromechanical stimulation.

During physical transfection, an electrical field or mechanical force is used to induce cell transfection. We tested if the disruption of a dense actin layer underneath the membrane of a suspended cell enhances cell transfection. A bubble generator was used to electromechanically stimulate suspended cells. To clarify the influence of the actin layer (the actin cortex) on cell transfection efficiency, we used an actin polymerization inhibitor (cytochalasin D) to disrupt the actin cortex before electromechanical stimulation. Without cytochalasin D treatment, signals from the overall actin cortex decreased after electromechanical stimulation. With cytochalasin D treatment, there was localized F-actin aggregation under static conditions. After electromechanical stimulation, there was a partial loss (localized disruption), but no overall disruption, of the actin cortex. With the pretreatment with cytochalasin D, the transfection efficiency of plasmids (4.7, 8.3, or 11 kbp) into NIH/3T3 or UMR-106 cells increased significantly after exposure to electromechanical stimulation. Localized distribution of the actin cortex before exposure to electromechanical stimulation is crucial for inducing a partial loss of the cortex, which improves transfection efficiency and large plasmid delivery.

Functional differences in human aortic valve interstitial cells from patients with varying calcific aortic valve disease.

Calcific aortic valve disease (CAVD) is characterized by progressive stiffening of aortic valve (AV) tissues, inducing stenosis and insufficiency. Bicuspid aortic valve (BAV) is a common congenital defect in which the AV has two leaflets rather than three, with BAV patients developing CAVD decades years earlier than in the general population. Current treatment for CAVD remains surgical replacement with its continued durability problems, as there are no pharmaceutical therapies or other alternative treatments available. Before such therapeutic approaches can be developed, a deeper understanding of CAVD disease mechanisms is clearly required. It is known that AV interstitial cells (AVICs) maintain the AV extracellular matrix and are typically quiescent in the normal state, transitioning into an activated, myofibroblast-like state during periods of growth or disease. One proposed mechanism of CAVD is the subsequent transition of AVICs into an osteoblast-like phenotype. A sensitive indicator of AVIC phenotypic state is enhanced basal contractility (tonus), so that AVICs from diseased AV will exhibit a higher basal tonus level. The goals of the present study were thus to assess the hypothesis that different human CAVD states lead to different biophysical AVIC states. To accomplish this, we characterized AVIC basal tonus behaviors from diseased human AV tissues embedded in 3D hydrogels. Established methods were utilized to track AVIC-induced gel displacements and shape changes after the application of Cytochalasin D (an actin polymerization inhibitor) to depolymerize the AVIC stress fibers. Results indicated that human diseased AVICs from the non-calcified region of TAVs were significantly more activated than AVICs from the corresponding calcified region. In addition, AVICs from the raphe region of BAVs were more activated than from the non-raphe region. Interestingly, we observed significantly greater basal tonus levels in females compared to males. Furthermore, the overall AVIC shape changes after Cytochalasin suggested that AVICs from TAVs and BAVs develop different stress fiber architectures. These findings are the first evidence of sex-specific differences in basal tonus state in human AVICs in varying disease states. Future studies are underway to quantify stress fiber mechanical behaviors to further elucidate CAVD disease mechanisms.

Pan-cancer analysis to identify molecules interacting with CasL-Like 2 to promote angiogenesis via mTOR/HIF1A/VEGFA pathway in hypoxia tumor environment.

e15002 Background: Cytoskeleton dynamics generate power for diverse motile and morphogenetic processes in eukaryotic cells. Aberrant actin dynamics are closely associated with pathological conditions and implicated in the occurrence of various tumors. The underlying mechanisms remain to be explored. This study aims to identify a unique redox enzyme, Molecules Interacting with CasL-Like 2 (MICALL2), as a regulator of actin filaments during tumorigenesis and to evaluate its potential value in predicting drug sensitivity to anti-angiogenesis agents. Methods: First, bioinformatic analysis was used to demonstrate MICALL2 expression level across distinct cancers and its relationship with clinical characteristics from TCGA and CPTAC databases. Functional enrichment analysis and whole-mountin situ hybridization were performed. Gene correlation analysis and IHC were used to detect the correlation between MICALL2 and HIF family. Next, chosen kidney renal clear cell carcinoma (KIRC) as a research object, the location of MICALL2 and actin was determined by immunofluorescence. IHC on MICALL2, HIF1A, VEGFA, CD31, CD34 of 44 clinical KIRC tissue samples was performed. The role of MICALL2 in proliferation, hypoxia and angiogenesis of KIRC cells was investigated using EDU staining, CCK-8 assay, ROS analysis, flow cytometry, and tubule formation assays. The effect of MICALL2 on the mTOR-related signal pathway was evaluated by western blot. Last, drug sensitivity analysis was obtained from the GDSC, CTRP, and PRISM databases to reveal the relationship between MICALL2 and anti-angiogenesis agents and cytoskeleton-regulating drugs. Results: MICALL2 expression was elevated in most cancers and correlated with poor survival outcomes. Pan-cancer analysis demonstrated that MICALL2 was co-expressed with HIF family members and strongly linked to angiogenesis pathway. Interfering the translation of MICALL2 during zebrafish embryogenesis resulted in defective caudal vein plexus morphogenesis. Additionally, KIRC patients with high MICALL2 expression displayed enhanced HIF1A expression and increased CD34-labeled neovascularization. In KIRC cells, MICALL2 was found co-located with F-actin. MICALL2 overexpressed KIRC cells exhibited more aggressive behaviors, with the tumor microenvironment remodeling to a more hypoxia condition. Up-regulation of MICALL2 promoted tumor angiogenesis via activating a novel mTOR/HIF1A/VEGFA pathway. Furthermore, tumor patients with high MICALL2 expression were more likely to be resistant to anti-angiogenesis agents and microtubule depolymerization inhibitors, but sensitive to actin polymerization inhibitors. Conclusions: Our results suggest that MICALL2 plays a crucial role in tumor angiogenesis and may be a potential predictor of anti-angiogenic agent sensitivity across multiple cancers.

Zhuidu Formula suppresses the migratory and invasive properties of triple-negative breast cancer cells via dual signaling pathways of RhoA/ROCK and CDC42/MRCK

Ethnopharmacological relevanceZhuidu Formula (ZDF) is composed of triptolide, cinobufagin and paclitaxel, which are the active ingredients of Tripterygium wilfordii Hook. F, dried toad skin and Taxus wallichiana var. chinensis (Pilg) Florin, respectively. Modern pharmacological studies show that triptolide, cinobufagin, and paclitaxel are well-known natural compounds that exert anti-tumor effects by interfering with DNA synthesis, inducing tumor cell apoptosis, and inhibiting the dynamic balance of the tubulin. However, the mechanism by which the three compounds inhibit triple-negative breast cancer (TNBC) metastasis is unknown. ObjectiveThe objective of this investigation was to examine the inhibitory essences of ZDF on the metastasis of TNBC and elucidate its potential mechanism. Materials and methodsCell viability of triptolide (TPL), cinobufagin (CBF), and paclitaxel (PTX) on MDA-MB-231 cells was assessed employing a CCK-8 assay. The drug interactions of the three drugs on MDA-MB-231 cells were determined in vitro utilizing the Chou-Talalay method. MDA-MB-231 cells were identified for migration, invasion and adhesion in vitro through the implementation of the scratch assay, transwell assay and adhesion assay, respectively. The formation of cytoskeleton protein F-actin was detected by immunofluorescence assay. The expressions of MMP-2 and MMP-9 in the supernatant of the cells were determined by ELISA analysis. The Western blot and RT-qPCR were employed to explore the protein expressions associated with the dual signaling pathways of RhoA/ROCK and CDC42/MRCK. The anti-tumor efficacy of ZDF in vivo and its preliminary mechanism were investigated in the mouse 4T1 TNBC model. ResultsThe results demonstrated that ZDF could significantly reduce the viability of the MDA-MB-231 cell, and the combination index (CI) values of actual compatibility experimental points were all less than 1, demonstrating a favorable synergistic compatibility relationship. It was found that ZDF reduces RhoA/ROCK and CDC42/MRCK dual signaling pathways, which are responsible for MDA-MB-231cell migration, invasion, and adhesion. Additionally, there has been a significant reduction in the manifestation of cytoskeleton-related proteins. Furthermore, the expression levels of RhoA, CDC42, ROCK2, and MRCKβ mRNA and protein were down-regulated. ZDF significantly decreased the protein expressions of vimentin, cytokeratin-8, Arp2 and N-WASP, and inhibited actin polymerization and actomyosin contraction. Furthermore, MMP-2 and MMP-9 levels in the high-dose ZDF group were decreased by 30% and 26%, respectively. ZDF significantly reduced the tumor volume and protein expressions of ROCK2 and MRCKβ in tumor tissues without eliciting any perceptible alterations in the physical mass of the mice, and the reduction was more pronounced than that of the BDP5290 treated group. ConclusionThe current investigation demonstrates that ZDF exhibits a proficient inhibitory impact on TNBC metastasis by regulating cytoskeletal proteins through the dual signaling pathways of RhoA/ROCK and CDC42/MRCK. Furthermore, the findings indicate that ZDF has significant anti-tumorigenic and anti-metastatic characteristics in breast cancer animal models.

Comparison of thermal and athermal dynamics of the cell membrane slope fluctuations in the presence and absence of Latrunculin-B

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 (Vaippully et al 2020 Soft Matter 16 7606), 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)−1 while the normal fluctuations yields (frequency) even 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) as 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), 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.

RHOJ controls EMT-associated resistance to chemotherapy

The resistance of cancer cells to therapy is responsible for the death of most patients with cancer1. Epithelial-to-mesenchymal transition (EMT) has been associated with resistance to therapy in different cancer cells2,3. However, the mechanisms by which EMT mediates resistance to therapy remain poorly understood. Here, using a mouse model of skin squamous cell carcinoma undergoing spontaneous EMT during tumorigenesis, we found that EMT tumour cells are highly resistant to a wide range of anti-cancer therapies both in vivo and in vitro. Using gain and loss of function studies in vitro and in vivo, we found that RHOJ—a small GTPase that is preferentially expressed in EMT cancer cells—controls resistance to therapy. Using genome-wide transcriptomic and proteomic profiling, we found that RHOJ regulates EMT-associated resistance to chemotherapy by enhancing the response to replicative stress and activating the DNA-damage response, enabling tumour cells to rapidly repair DNA lesions induced by chemotherapy. RHOJ interacts with proteins that regulate nuclear actin, and inhibition of actin polymerization sensitizes EMT tumour cells to chemotherapy-induced cell death in a RHOJ-dependent manner. Together, our study uncovers the role and the mechanisms through which RHOJ acts as a key regulator of EMT-associated resistance to chemotherapy.

Human Endonuclease ANKLE1 Localizes at the Midbody and Processes Chromatin Bridges to Prevent DNA Damage and cGAS-STING Activation.

Chromatin bridges connecting the two segregating daughter nuclei arise from chromosome fusion or unresolved interchromosomal linkage. Persistent chromatin bridges are trapped in the cleavage plane, triggering cytokinesis delay. The trapped bridges occasionally break during cytokinesis, inducing DNA damage and chromosomal rearrangements. Recently, Caenorhabditis elegans LEM-3 and human TREX1 nucleases have been shown to process chromatin bridges. Here, it is shown that ANKLE1 endonuclease, the human ortholog of LEM-3, accumulates at the bulge-like structure of the midbody via its N-terminal ankyrin repeats. Importantly, ANKLE1-/- knockout cells display an elevated level of G1-specific 53BP1 nuclear bodies, prolonged activation of the DNA damage response, and replication stress. Increased DNA damage observed in ANKLE1-/- cells is rescued by inhibiting actin polymerization or reducing actomyosin contractility. ANKLE1 does not act in conjunction with structure-selective endonucleases, GEN1 and MUS81 in resolving recombination intermediates. Instead, ANKLE1 acts on chromatin bridges by priming TREX1 nucleolytic activity and cleaving bridge DNA to prevent the formation of micronuclei and cytosolic dsDNA that activate the cGAS-STING pathway. It is therefore proposed that ANKLE1 prevents DNA damage and autoimmunity by cleaving chromatin bridges to avoid catastrophic breakage mediated by actomyosin contractile forces.

Cholesterol and Sphingomyelin Polarize at the Leading Edge of Migrating Myoblasts and Involve Their Clustering in Submicrometric Domains.

Myoblast migration is crucial for myogenesis and muscular tissue homeostasis. However, its spatiotemporal control remains elusive. Here, we explored the involvement of plasma membrane cholesterol and sphingolipids in this process. In resting C2C12 mouse myoblasts, those lipids clustered in sphingomyelin/cholesterol/GM1 ganglioside (SM/chol/GM1)- and cholesterol (chol)-enriched domains, which presented a lower stiffness than the bulk membrane. Upon migration, cholesterol and sphingomyelin polarized at the front, forming cholesterol (chol)- and sphingomyelin/cholesterol (SM/chol)-enriched domains, while GM1-enriched domains polarized at the rear. A comparison of domain proportion suggested that SM/chol- and GM1-enriched domains originated from the SM/chol/GM1-coenriched domains found at resting state. Modulation of domain proportion (through cholesterol depletion, combined or not with actin polymerization inhibition, or sphingolipid synthesis inhibition) revealed that the higher the chol- and SM/chol-enriched domains, the higher the myoblast migration. At the front, chol- and SM/chol-enriched domains were found in proximity with F-actin fibers and the lateral mobility of sphingomyelin in domains was specifically restricted in a cholesterol- and cytoskeleton-dependent manner while domain abrogation impaired F-actin and focal adhesion polarization. Altogether, we showed the polarization of cholesterol and sphingomyelin and their clustering in chol- and SM/chol-enriched domains with differential properties and roles, providing a mechanism for the spatial and functional control of myoblast migration.

Nanoscopic lipid diffusion in the live cell plasma membrane revealed by ultrafast single-molecule tracking.

Membrane organization and dynamics underlie various molecular interactions required for membrane functions. Although the importance of investigating biological membranes at the nanoscale is well recognized, resolving nanoscopic structures and dynamics is technically challenging. In this work, we employ ultrafast single-particle tracking techniques to measure single phospholipid diffusion in the plasma membrane of live cells. We attach a monovalent gold nanoparticle to the headgroup of biotinylated phospholipid and record its diffusive motion on microsecond resolution and nanometer precision by advanced optical interference microscopy. We find that both saturated and unsaturated phospholipids (biotin-PEG-DSPE and biotin-PEG-DOPE, respectively) undergo anomalous subdiffusion in the length scale below 100 nm. The unsaturated lipid diffuses only slightly more freely than the saturated lipid. Moreover, the nanoscopic lipid diffusion is highly heterogeneous in space and time. With statistical analyses, we find that the apparent dual-mobility subdiffusion can describe measured diffusion. The subdiffusion agrees well with the hop diffusion model that describes a Brownian diffuser moving in periodic and partially permeable barriers created by the cytoskeleton. Inhibition of actin polymerization by the chemical drug CK-666 reduces confinement's effective size and strength. Furthermore, we examine the effect of cholesterol on lipid subdiffusion by manipulating the cholesterol concentration. Cholesterol depletion by methyl-b-cyclodextrin leads to a more restricted lipid diffusion. We attribute the more confined diffusion to the formation of solid-like gel-phase nanodomains that move slowly and act as diffusion obstacles. Lowing the system temperature shows similar effects on lipid diffusion, suggesting that the cholesterol-dependent lipid diffusion connects to the nanoscopic phase separation. Overall, this study provides experimental evidence that lipid diffusion in the cell membrane is heterogeneous at the nanoscale and regulated by the membrane cholesterol and actin cytoskeleton.

Quantifying obscurin's impact on epithelial cell area, perimeter & localization with the cell membrane.

Obscurin is a giant signaling protein believed to be responsible for resisting applied forces on a cell by localizing to areas of high tension. We are interested to know: (1) what is the effect of obscurin on cells’ area and perimeter, and (2) where does obscurin move within the cell over time. While cell size and location of a protein can be described “by eye,” the challenge is finding a way to quantify these properties consistently. To address the first question, we used a machine learning algorithm called Weka, a plugin within the FIJI image analysis software, to accurately segment many cells at once. Once segmented, area and perimeter statistics are further analyzed in a custom Python program that compares the mean area and perimeter between experimental conditions using t-tests. We found that the presence of Blebbistatin, a drug that inhibits myosin II, significantly decreases the overall size of the cell, while the presence of Cytochalasin D, a drug that inhibits actin polymerization, significantly increases cell size. Experimentation with these drugs, known to disrupt cell mechanics, allows us to better understand obscurin's possible role in cell tension. To determine where obscurin moves within the cell over time, we utilized kymographs and colocalization plots. These techniques can help determine how much obscurin is present within the cell, and its location relative to the cell membrane. The utilization of adenovirus to increase the amount of obscurin in epithelial cells led to a wide range of florescent obscurin expression. Our localization analysis suggests that obscurin colocalizes to the cell membrane when an optimal amount of fluorescent obscurin is present. Altogether, quantifying cell shape properties and obscurin movement can help determine if obscurin is localizing to areas of high tension.