Search Results (21)

Focal adhesions (FAs) are multi-protein complexes that mediate cell attachment to the extracellular matrix. Their formation and maturation depend on intracellular tension generated by actin filaments interacting with phosphorylated myosin II. Using live-cell and confocal microscopy, we investigated how FA dynamics are regulated by actin polymerization and myosin II-driven contractility. We found that knockdown of myosin II resulted in complete and irreversible disassembly of FAs. However, partial inhibition of myosin II, through either ROCK or myosin light chain kinase (MLCK) inhibitors, leads to gradual FA shrinkage. In contrast, complete inhibition of myosin II phosphorylation causes disassembly of existing FAs, followed by the formation of new, small FAs at the cell periphery. In both cases, FAs formed after inhibition of myosin II phosphorylation exhibited significantly longer lifespans than FAs in control cells. Similarly, partial inhibition of actin polymerization using nanomolar concentrations of latrunculin B or cytochalasin D also promoted the formation of small FAs. Complete and irreversible FA disassembly occurred only when actin filaments were fully disrupted, leading to cell lamella retraction. These findings suggest that actin polymerization at the cell edge is the minimal and sufficient requirement for the assembly of small FAs. Notably, our data demonstrate for the first time that perturbation of the actin–myosin system results in stabilization and prolonged lifespan of small FAs, whereas larger FAs, formed in the presence of myosin II activity, are more dynamic. Together, these results emphasize the essential role of cortical actin organization and myosin II phosphorylation in the maintenance and turnover of FAs. Full article

In the marine red alga Pyropia yezoensis, filamentous phases of the life cycle, e.g., the conchocelis (sporophyte) and conchosporangium (conchosporophyte), proliferate by tip growth. In this study, we investigated the possible involvement of phosphoinositide turnover and actin polymerization in the spontaneous initiation and tip growth of new branches in isolated single-celled conchocelis cells using pharmacological treatments. Treatment with LY294002 and U73122, specific inhibitors of phosphoinositide-phosphate 3-kinase and phospholipase C, respectively, reduced side-branch formation and inhibited the elongation of branches. In addition, two inhibitors of the actin cytoskeleton, cytochalasin B (CCB) and latrunculin B (LAT-B), had similar effects on tip growth. However, CCB did not alter the branching rate of single-celled conchocelis, whereas LAT-B did. As CCB and LAT-B affect actin polymerization through different mechanisms, this result suggests differences in the contributions of actin polymerization to branch initiation versus tip growth. These findings demonstrate the critical and diverse functional roles played by phosphoinositide turnover and actin polymerization in the regulation of the initiation and maintenance of tip growth in the conchocelis phase of P. yezoensis. Full article

Chordomas are rare malignant tumors of the bone, originating from remnants of notochordal cells. The transcription factor brachyury, encoded by TBXT, serves as a critical diagnostic marker and is essential for tumor growth. While brachyury’s role in regulating the cytoskeleton during embryogenesis and tumorigenesis is well understood, the reverse—whether cytoskeletal alterations can influence brachyury levels—remains unclear. Despite advances in understanding chordoma biology, there are currently no approved targeted therapies, underscoring the need for novel therapeutic approaches. Three chordoma cell lines were treated with cytoskeletal inhibitors, including the actin-targeting compounds Cucurbitacin B (CuB) and Latrunculin B (LatB). Morphological changes, TBXT expression, and cell viability were analyzed. The effects of CuB were examined over time and across concentrations, with cell viability assessed via apoptosis and cytotoxicity assays. Microarray gene expression profiling of ten chordoma cell lines was performed to explore CuB-mediated transcriptional changes. Rescue experiments using a TBXT open reading frame vector and co-treatments with autophagy and proteasome inhibitors were conducted to elucidate the mechanisms of brachyury depletion. Both CuB and LatB induced significant morphological changes, but only CuB caused near-complete depletion of brachyury. This effect was time- and concentration-dependent, correlating with reduced cell viability driven primarily by apoptosis. Microarray analysis revealed that CuB treatment upregulated protein refolding pathways and downregulated protein glycosylation. Notably, TBXT transcription was only slightly suppressed, indicating that brachyury depletion was largely post-transcriptional. Rescue experiments and co-treatments implicated dysregulated protein refolding and endoplasmic reticulum (ER) stress as key mechanisms underlying CuB-mediated brachyury loss. This study demonstrates that actin cytoskeleton disruption by CuB depletes brachyury in chordoma cells, primarily through dysregulated protein refolding and ER stress rather than transcriptional repression. These findings suggest that targeting actin cytoskeleton dynamics or protein unfolding pathways may provide novel therapeutic approaches for chordoma treatment. Full article

Passion fruit (Passiflora edulis), mainly distributed in tropical and subtropical regions, is popular for its unique flavor and health benefits. The actin cytoskeleton plays a crucial role in plant growth and development, and villin is a key regulator of actin dynamics. However, the mechanism underlying the actin filament regulation of reproductive development in passion fruit remains poorly understood. Here, we characterized a villin isovariant in passion fruit, Passiflora edulis VLN4 (PeVLN4), highly and preferentially expressed in pollen. Subcellular localization analysis showed that PeVLN4 decorated distinct filamentous structures in pollen tubes. We next introduced PeVLN4 into Arabidopsis villin mutants to explore its functions on the growing pollen tubes. PeVLN4 rescued defects in the elongation of villin mutant pollen tubes. Pollen tubes expressing PeVLN4 were revealed to be less sensitive to latrunculin B, and PeVLN4 partially rescued defects in the actin filament organization of villin mutant pollen tubes. Additionally, biochemical assays revealed that PeVLN4 bundles actin filaments in vitro. Thus, PeVLN4 is an important regulator of F-actin stability and is required for normal pollen tube growth in passion fruit. This study provides a new insight into the function of the actin regulator villin involved in the reproduction development of passion fruit. Full article

Osteoclasts are bone-resorbing multinucleated giant cells formed by the fusion of monocyte/macrophage lineages. Various small GTPases are involved in the multinucleation and differentiation of osteoclasts. However, the roles of small GTPases regulatory molecules in osteoclast differentiation remain unclear. In the present study, we examined the role of Dennd2c, a putative guanine nucleotide exchange factor for Rab GTPases, in osteoclast differentiation. Knockdown of Dennd2c promoted osteoclast differentiation, resorption, and expression of osteoclast markers. Morphologically, Dennd2c knockdown induced the formation of larger osteoclasts with several protrusions. In contrast, overexpression of Dennd2c inhibited the multinucleation and differentiation of osteoclasts, bone resorption, and the expression of osteoclast markers. Dennd2c-overexpressing macrophages exhibited spindle-shaped mononuclear cells and long thin protrusions. Treatment of Dennd2c-overexpressing cells with the Cdc42 inhibitor ML-141 or the Rac1 inhibitor 6-thio-GTP prevented protrusion formation. Moreover, treatment of Dennd2c-overexpressing cells with the actin polymerization inhibitor latrunculin B restored multinucleated and TRAP-positive osteoclast formation. These results indicate that Dennd2c negatively regulates osteoclast differentiation and multinucleation by modulating protrusion formation in macrophages. Full article

The chromatin-remodeling protein ATRX, which is currently recognized as one of the key genome caretakers, plays an important role in oogenesis and early embryogenesis in mammals. ATRX distribution in the nuclei of mouse embryos developing in vivo and in vitro, including when the embryos are arrested at the two-cell stage—the so-called two-cell block in vitro—was studied using immunofluorescent labeling and FISH. In normally developing two- and four-cell embryos, ATRX was found to be closely colocalized with pericentromeric DNA sequences detected with a probe to the mouse major satellite DNA. The association of ATRX with pericentromeric heterochromatin is mediated by nuclear actin and reduced after the treatment of embryos with latrunculin B. When culturing embryos in vitro, the distribution pattern of ATRX changes, leading to a decrease in the association of this protein with major satellite DNA especially under the two-cell block in vitro. Taken together, our data suggest that the intranuclear distribution of ATRX reflects the viability of mouse embryos and their probability of successful preimplantation development. Full article

The growth of pollen tubes, which depends on actin filaments, is pivotal for plant reproduction. Pharmacological experiments showed that while oryzalin and brefeldin A treatments had no significant effect on the lipid droplets (LDs) trafficking, while 2,3-butanedione monoxime (BDM), latrunculin B, SMIFH2, and cytochalasin D treatments slowed down LDs trafficking, in such a manner that only residual wobbling was observed, suggesting that trafficking of LDs in pollen tube is related to F-actin. While the trafficking of LDs in the wild-type pollen tubes and in myo11-2, myo11b1-1, myo11c1-1, and myo11c2-1 single mutants and myo11a1-1/myo11a2-1 double mutant were normal, their trafficking slowed down in a myosin-XI double knockout (myo11c1-1/myo11c2-1) mutant. These observations suggest that Myo11C1 and Myo11C2 motors are involved in LDs movement in pollen tubes, and they share functional redundancy. Hence, LDs movement in Arabidopsis pollen tubes relies on the actomyosin system. Full article

Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal disease characterized by progressive and irreversible lung scarring associated with persistent activation of fibroblasts. Epigenetics could integrate diverse microenvironmental signals, such as stiffness, to direct persistent fibroblast activation. Histone modifications by deacetylases (HDAC) may play an essential role in the gene expression changes involved in the pathological remodeling of the lung. Particularly, HDAC3 is crucial for maintaining chromatin and regulating gene expression, but little is known about its role in IPF. In the study, control and IPF-derived fibroblasts were used to determine the influence of HDAC3 on chromatin remodeling and gene expression associated with IPF signature. Additionally, the cells were grown on hydrogels to mimic the stiffness of a fibrotic lung. Our results showed a decreased HDAC3 in the nucleus of IPF fibroblasts, which correlates with changes in nucleus size and heterochromatin loss. The inhibition of HDAC3 with a pharmacological inhibitor causes hyperacetylation of H3K9 and provokes an increased expression of Col1A1, ACTA2, and p21. Comparable results were found in hydrogels, where matrix stiffness promotes the loss of nuclear HDAC3 and increases the profibrotic signature. Finally, latrunculin b was used to confirm that changes by stiffness depend on the mechanotransduction signals. Together, these results suggest that HDAC3 could be a link between epigenetic mechanisms and the fibrotic microenvironment. Full article

Detachment of cancer cells is the first step in tumor metastasis and malignancy. However, studies on the balance of initial tumor anchoring and detachment are limited. Herein, we revealed that the regulation of cytoskeleton proteins potentiates tumor detachment. The blockage of TGF-β1 using neutralizing antibodies induced cancer cell detachment in the Boyden chamber and 3D in-gel spheroid models. Moreover, treatment with latrunculin B, an actin polymerization inhibitor, enhanced cell dissociation by abolishing actin fibers, indicating that TGF-β1 mediates the formation of actin stress fibers, and is likely responsible for the dynamics of anchoring and detachment. Indeed, latrunculin B disrupted the formation of external TGF-β1-induced actin fibers and translocation of intracellular vinculin, a focal adhesion protein, resulting in the suppression of cell adhesion. Moreover, the silencing of vimentin substantially reduced cell adhesion and enhanced cell detachment, revealing that cell adhesion and focal adhesion protein translocation stimulated by TGF-β1 require vimentin. Using the 3D in-gel spheroid model, we found that latrunculin B suppressed the cell adhesion promoted by external TGF-β1, increasing the number of cells that penetrated the Matrigel and detached from the tumor spheres. Thus, cytoskeleton remodeling maintained the balance of cell anchoring and detachment, and the TGF-β1/vimentin/focal adhesion protein assembly axis was involved in the control dynamics of initial tumor detachment. Full article

GBM is a high-grade cancer that originates from glial cells and has a poor prognosis. Although a combination of surgery, radiotherapy, and chemotherapy is prescribed to patients, GBM is highly resistant to therapies, and surviving cells show increased aggressiveness. In this study, we investigated the molecular mechanism underlying GBM progression after radiotherapy by establishing a GBM orthotopic xenograft mouse model. Based on transcriptomic analysis, we found that the expression of BEX1 and BEX4 was upregulated in GBM cells surviving radiotherapy. We also found that upregulated expression of BEX1 and BEX4 was involved in the formation of the filamentous cytoskeleton and altered mechanotransduction, which resulted in the activation of the YAP/TAZ signaling pathway. BEX1- and BEX4-mediated YAP/TAZ activation enhanced the tumor formation, growth, and radioresistance of GBM cells. Additionally, latrunculin B inhibited GBM progression after radiotherapy by suppressing actin polymerization in an orthotopic xenograft mouse model. Taken together, we suggest the involvement of cytoskeleton formation in radiation-induced GBM progression and latrunculin B as a GBM radiosensitizer. Full article

Investigation of the Red Sea sponge Negombata magnifica gave two novel alkaloids, magnificines A and B (1 and 2) and a new β-ionone derivative, (±)-negombaionone (3), together with the known latrunculin B (4) and 16-epi-latrunculin B (5). The analysis of the NMR and HRESIMS spectra supported the planar structures and the relative configurations of the compounds. The absolute configurations of magnificines A and B were determined by the analysis of the predicted and experimental ECD spectra. Magnificines A and B possess a previously unreported tetrahydrooxazolo[3,2-a]azepine-2,5(3H,6H)-dione backbone and represent the first natural compounds in this class. (±)-Negombaionone is the first β-ionone of a sponge origin. Compounds 1-3 displayed selective activity against Escherichia coli in a disk diffusion assay with inhibition zones up to 22 mm at a concentration of 50 µg/disc and with MIC values down to 8.0 µM. Latrunculin B and 16-epi-latrunculin B inhibited the growth of HeLa cells with IC₅₀ values down to 1.4 µM. Full article

The epithelial cytoskeleton encompasses actin filaments, microtubules, and keratin intermediate filaments. They are interconnected and attached to the extracellular matrix via focal adhesions and hemidesmosomes. To study their interplay, we inhibited actin and tubulin polymerization in the human keratinocyte cell line HaCaT by latrunculin B and nocodazole, respectively. Using immunocytochemistry and time-lapse imaging of living cells, we found that inhibition of actin and tubulin polymerization alone or in combination induced keratin network re-organization albeit differently in each situation. Keratin filament network retraction towards the nucleus and formation of bundled and radial keratin filaments was most pronounced in latrunculin-B treated cells but less in doubly-treated cells and not detectable in the presence of nocodazole alone. Hemidesmosomal keratin filament anchorage was maintained in each instance, whereas focal adhesions were disassembled in the absence of actin filaments. Simultaneous inhibition of actin and tubulin polymerization, therefore, allowed us to dissect hemidesmosome-specific functions for keratin network properties. These included not only anchorage of keratin filament bundles but also nucleation of keratin filaments, which was also observed in migrating cells. The findings highlight the fundamental role of hemidesmosomal adhesion for keratin network formation and organization independent of other cytoskeletal filaments pointing to a unique mechanobiological function. Full article

Plasmolysis is usually introduced to cell biology students as a tool to illustrate the plasma membrane: hypertonic solutions cause the living protoplast to shrink by osmotic water loss; hence, it detaches from the surrounding cell wall. What happens, however, with the subcellular structures in the cell cortex during this process of turgor loss? Here, we investigated the cortical endoplasmic reticulum (ER) in moss protonema cells of Physcomitrella patens in a cell line carrying a transgenic ER marker (GFP-HDEL). The plasma membrane was labelled simultaneously with the fluorescent dye FM4-64 to achieve structural separation. By placing the protonemata in a hypertonic mannitol solution (0.8 M), we were able to follow the behaviour of the cortical ER and the protoplast during plasmolysis by confocal laser scanning microscopy (CLSM). The protoplast shape and structural changes of the ER were further examined after depolymerisation of actin microfilaments with latrunculin B (1 µM). In its natural state, the cortical ER is a dynamic network of fine tubes and cisternae underneath the plasma membrane. Under acute and long-term plasmolysis (up to 45 min), changes in the protoplast form and the cortical ER, as well as the formation of Hechtian strands and Hechtian reticula, were observed. The processing of the high-resolution z-scans allowed the creation of 3D models and gave detailed insight into the ER of living protonema cells before, during and after plasmolysis. Full article

While alkaloids often exert unique pharmacological effects on animal cells, exposure of sea urchin eggs to nicotine causes polyspermy at fertilization in a dose-dependent manner. Here, we studied molecular mechanisms underlying the phenomenon. Although nicotine is an agonist of ionotropic acetylcholine receptors, we found that nicotine-induced polyspermy was neither mimicked by acetylcholine and carbachol nor inhibited by specific antagonists of nicotinic acetylcholine receptors. Unlike acetylcholine and carbachol, nicotine uniquely induced drastic rearrangement of egg cortical microfilaments in a dose-dependent way. Such cytoskeletal changes appeared to render the eggs more receptive to sperm, as judged by the significant alleviation of polyspermy by latrunculin-A and mycalolide-B. In addition, our fluorimetric assay provided the first evidence that nicotine directly accelerates polymerization kinetics of G-actin and attenuates depolymerization of preassembled F-actin. Furthermore, nicotine inhibited cofilin-induced disassembly of F-actin. Unexpectedly, our results suggest that effects of nicotine can also be mediated in some non-cholinergic pathways. Full article

Cell fusion as a rare event was observed following the co-culture of human MDA-MB-231^cherry breast cancer cells or benign neoplastic MCF10A^cherry breast epithelial cells together with different mesenchymal stroma/stem-like cells (MSC^GFP) cultures, respectively, resulting in the generation of double-fluorescing hybrid cells. Analysis of potential molecular mechanisms for the formation of cancer hybrid cells revealed cytoskeletal components, including F-actin. Thus, a sub-lethal concentration of cytochalasin D, which blocks elongation of actin filaments, was able to significantly reduce cancer hybrid cell formation. Simultaneously, cell cycle progression of the different co-cultures remained unaffected following treatment with cytochalasin D, indicating continued proliferation. Moreover, exposure to 50 nM cytochalasin D revealed little if any effect on the expression of various integrins and cell adhesion molecules in the different co-cultures. However, LC-MS proteome analysis of the different control co-cultures compared to corresponding cytochalasin-treated co-cultures demonstrated predominant differences in the expression of actin-associated cytoskeletal proteins. In addition, the requirement of structured actin to provide an appropriate cytoskeletal network for enabling subsequent fusion processes was also substantiated by the actin filament disrupting latrunculin B, which inhibits the fusion process between the breast cancer populations and mesenchymal stroma/stem-like cells (MSC). Together, these findings suggest an important role of distinct actin structures and associated cytoskeletal components during cell fusion and the formation of breast cancer hybrid cells. Full article