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Cytoskeletal filaments provide cells with mechanical stability and organization. The main key players are actin filaments and microtubules governing a cell's response to mechanical stimuli. We investigated the specific influences of these crucial components by deforming MCF-7 epithelial cells at small (≤5% deformation) and large strains (>5% deformation). To understand specific contributions of actin filaments and microtubules, we systematically studied cellular responses after treatment with cytoskeleton influencing drugs. Quantification with the microfluidic optical stretcher allowed capturing the relative deformation and relaxation of cells under different conditions. We separated distinctive deformational and relaxational contributions to cell mechanics for actin and microtubule networks for two orders of magnitude of drug dosages. Disrupting actin filaments via latrunculin A, for instance, revealed a strain-independent softening. Stabilizing these filaments by treatment with jasplakinolide yielded cell softening for small strains but showed no significant change at large strains. In contrast, cells treated with nocodazole to disrupt microtubules displayed a softening at large strains but remained unchanged at small strains. Stabilizing microtubules within the cells via paclitaxel revealed no significant changes for deformations at small strains, but concentration-dependent impact at large strains. This suggests that for suspended cells, the actin cortex is probed at small strains, while at larger strains; the whole cell is probed with a significant contribution from the microtubules.

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.

The mechanical phenotype of a cell is an inherent biophysical marker of its state and function, with many applications in basic and applied biological research. Microfluidics-based methods have enabled single-cell mechanophenotyping at throughputs comparable to those of flow cytometry. Here, we present a standardized cross-laboratory study comparing three microfluidics-based approaches for measuring cell mechanical phenotype: constriction-based deformability cytometry (cDC), shear flow deformability cytometry (sDC) and extensional flow deformability cytometry (xDC). All three methods detect cell deformability changes induced by exposure to altered osmolarity. However, a dose-dependent deformability increase upon latrunculin B-induced actin disassembly was detected only with cDC and sDC, which suggests that when exposing cells to the higher strain rate imposed by xDC, cellular components other than the actin cytoskeleton dominate the response. The direct comparison presented here furthers our understanding of the applicability of the different deformability cytometry methods and provides context for the interpretation of deformability measurements performed using different platforms. This Analysis compares microfluidics-based methods for assessing mechanical properties of cells in high throughput.

The burden of irreversible vision loss from Glaucoma continues to rise. While the disease pathogenesis is not well understood, intraocular pressure (IOP) is the only modifiable risk factor identified to prevent glaucomatous vision loss. Medical management remains the first-line of treatment in most adult glaucomas and the evolution of medical therapy for glaucoma has followed an exponential curve. This review tracks the rapid development of new medications and drug delivery systems in the recent years. Introduction of Rho kinase inhibitors with an entirely new mechanism of action from that of the currently used anti glaucoma medications has been a significant milestone. Latanoprostene Bunod is a novel, single molecule which provides two active metabolites that work through two different pathways for reducing intra ocular pressure. Bimatoprost implants and travoprost punctum plugs attempt to ease chronic medication use in glaucoma patients. Nanotechnology is an evolving route of drug delivery. Role of cannabinoids in medical management of glaucoma remain equivocal. The relatively short term effect on IOP, the risks of developing tolerance and side effects impacting patients' neurocognitive health greatly outweigh the potential benefit. Research on Latrunculin B, Adenosine receptor agonists, Specific gene silencing and Stem cell therapy are poised to make an impact on glaucoma treatment. While there is some evidence to support the role of Brimonidine in neuroprotection, further research is needed to clarify the role of Memantine and Neurotrophins. Evidence for benefit from dietary supplementation with Alpha lipoic acid, Forskolin, and Ginko Biloba is limited

The pancreas originates from two epithelial evaginations of the foregut, which consist of multipotent epithelial progenitors that organize into a complex tubular epithelial network. The trunk domain of each epithelial branch consists of bipotent pancreatic progenitors (bi-PPs) that give rise to both duct and endocrine lineages, whereas the tips give rise to acinar cells 1 . Here we identify the extrinsic and intrinsic signalling mechanisms that coordinate the fate-determining transcriptional events underlying these lineage decisions 1 , 2 . Single-cell analysis of pancreatic bipotent pancreatic progenitors derived from human embryonic stem cells reveal that cell confinement is a prerequisite for endocrine specification, whereas spreading drives the progenitors towards a ductal fate. Mechanistic studies identify the interaction of extracellular matrix (ECM) with integrin α5 as the extracellular cue that cell-autonomously, via the F-actin–YAP1–Notch mechanosignalling axis, controls the fate of bipotent pancreatic progenitors. Whereas ECM–integrin α5 signalling promotes differentiation towards the duct lineage, endocrinogenesis is stimulated when this signalling cascade is disrupted. This cascade can be disrupted pharmacologically or genetically to convert bipotent pancreatic progenitors derived from human embryonic stem cells to hormone-producing islet cells. Our findings identify the cell-extrinsic and intrinsic mechanotransduction pathway that acts as gatekeeper in the fate decisions of bipotent pancreatic progenitors in the developing pancreas. Single-cell analysis reveals that interactions with the extracellular matrix via integrin α5 and mechanotransducer YAP1 determine whether pancreatic progenitors develop along the duct or endocrine lineages.

Many organisms possess multiple and often divergent actins whose regulation and roles are not understood in detail. For example, Chlamydomonas reinhardtii has both a conventional actin (IDA5) and a highly divergent one (NAP1); only IDA5 is expressed in normal proliferating cells. We showed previously that the drug latrunculin B (LatB) causes loss of filamentous (F-) IDA5 and strong up-regulation of NAP1, which then provides essential actin function(s) by forming LatB-resistant F-NAP1. RNA-sequencing analyses now show that this up-regulation of NAP1 reflects a broad transcriptional response, much of which depends on three proteins (LAT1, LAT2, and LAT3) identified previously as essential for NAP1 transcription. Many of the LAT-regulated genes contain a putative cis-acting regulatory site, the “LRE motif.” The LatB transcriptional program appears to be activated by loss of F-IDA5 and deactivated by formation of F-NAP1, thus forming an F-actin–dependent negative-feedback loop. Multiple genes encoding proteins of the ubiquitin-proteasome system are among those induced by LatB, resulting in rapid degradation of IDA5 (but not NAP1). Our results suggest that IDA5 degradation is functionally important because nonpolymerizable LatB-bound IDA5 interferes with the formation of F-NAP1. The genes for the actin-interacting proteins cofilin and profilin are also induced. Cofilin induction may further the clearance of IDA5 by promoting the scission of F-IDA5, whereas profilin appears to function in protecting monomeric IDA5 from degradation. This multifaceted regulatory system allows rapid and quantitative turnover of F-actin in response to cytoskeletal perturbations and probably also maintains F-actin homeostasis under normal growth conditions.

Actin has emerged as a versatile regulator of gene transcription. Cytoplasmatic actin regulates mechanosensitive-signaling pathways such as MRTF–SRF and Hippo-YAP/TAZ. In the nucleus, both polymerized and monomeric actin directly interfere with transcription-associated molecular machineries. Natural actin-binding compounds are frequently used tools to study actin-related processes in cell biology. However, their influence on transcriptional regulation and intranuclear actin polymerization is poorly understood to date. Here, we analyze the effects of two representative actin-binding compounds, Miuraenamide A (polymerizing properties) and Latrunculin B (depolymerizing properties), on transcriptional regulation in primary cells. We find that actin stabilizing and destabilizing compounds inversely shift nuclear actin levels without a direct influence on polymerization state and intranuclear aspects of transcriptional regulation. Furthermore, we identify Miuraenamide A as a potent inducer of G-actin-dependent SRF target gene expression. In contrast, the F-actin-regulated Hippo-YAP/TAZ axis remains largely unaffected by compound-induced actin aggregation. This is due to the inability of AMOTp130 to bind to the amorphous actin aggregates resulting from treatment with miuraenamide. We conclude that actin-binding compounds predominantly regulate transcription via their influence on cytoplasmatic G-actin levels, while transcriptional processes relying on intranuclear actin polymerization or functional F-actin networks are not targeted by these compounds at tolerable doses.

Extracellular matrix (ECM) is widely considered to be integral to the function of skeletal muscle, providing mechanical support, transmitting force, and contributing to passive stiffness. Many functions and dysfunctions attributed to ECM are thought to stem from its mechanical properties, yet there are few data describing the mechanics of intact ECM. Such measurements require isolating intact ECM from the muscle cells it surrounds. The objectives of this study were to quantify the efficiency of three techniques for this purpose: Triton, Triton with sodium dodecyl sulfate, and latrunculin B; and to determine their impact on properties of the remaining ECM. Efficiency was quantified by DNA content and evaluation of western blot intensities for myosin and actin. The properties of ECM were quantified by collagen content and uniaxial tensile testing. We found that latrunculin B was the most efficient method for removing skeletal muscle cells, reducing DNA content to less than 10% of that seen in control muscles, and substantially reducing the myosin and actin to 15% and 23%, respectively; these changes were larger than for the competing methods. Collagen content after decellularization was not significantly different from control muscles for all methods. Only the stiffness of the muscles decellularized with latrunculin B differed significantly from control, having a Young’s modulus reduced by 47% compared to the other methods at matched stresses. Our results suggest that latrunculin B is the most efficient method for decellularizing skeletal muscle and that the remaining ECM accounts for approximately half of the stiffness in passive muscle.

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.

Mitochondria fission and mitophagy are fundamentally crucial to cellular physiology and play important roles in cancer progression. Developing a comprehensive understanding of the molecular mechanism underlying mitochondrial fission and mitophagy will provide novel strategies for cancer prevention and treatment. Actin has been shown to participate in mitochondrial fission and mitophagy regulation. Cofilin is best known as an actin-depolymerizing factor. However, the molecular mechanism by which cofilin regulates mitochondrial fission and mitophagy remains largely unknown. Here we report that knockdown of cofilin attenuates and overexpression of cofilin potentiates mitochondrial fission as well as PINK1/PARK2-dependent mitophagy induced by staurosporine (STS), etoposide (ETO), and carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Cofilin-mediated-PINK1 (PTEN-induced putative kinase 1) accumulation mainly depends on its regulation of mitochondrial proteases, including peptidase mitochondrial processing beta (MPPβ), presenilin-associated rhomboid-like protease (PARL), and ATPase family gene 3-like 2 (AFG3L2), via mitochondrial membrane potential activity. We also found that the interaction and colocalization of G-actin/F-actin with cofilin at mitochondrial fission sites undergo constriction after CCCP treatment. Pretreatment with the actin polymerization inhibitor latrunculin B (LatB) increased and actin-depolymerization inhibitor jasplakinolide (Jas) decreased mitochondrial translocation of actin induced by STS, ETO, and CCCP. Both LatB and Jas abrogated CCCP-mediated mitochondrial fission and mitophagy. Our data suggest that G-actin is the actin form that is translocated to mitochondria, and the actin-depolymerization activity regulated by cofilin at the mitochondrial fission site is crucial for inducing mitochondrial fission and mitophagy.