Methyl-β-cyclodextrin, an actin depolymerizer augments the antiproliferative potential of microtubule-targeting agents.
ABSTRACT: Methyl-β-cyclodextrin (MCD), an established pharmacological excipient, depolymerizes the actin cytoskeleton. In this work, we investigated the effect of MCD-mediated actin depolymerization on various cellular phenotypes including traction force, cell stiffness, focal adhesions, and intracellular drug accumulation. In addition to a reduction in the contractile cellular traction, MCD acutely inhibits the maturation of focal adhesions. Alteration of contractile forces and focal adhesions affects the trypsin-mediated detachment kinetics of cells. Moreover, MCD-mediated actin depolymerization increases the intracellular accumulation of microtubule-targeting agents (MTAs) by ~50% with respect to the untreated cells. As MCD treatment enhances the intracellular concentration of drugs, we hypothesized that the MCD-sensitized cancer cells could be effectively killed by low doses of MTAs. Our results in cervical, breast, hepatocellular, prostate cancer and multidrug-resistant breast cancer cells confirmed the above hypothesis. Further, the combined use of MCD and MTAs synergistically inhibits the proliferation of tumor cells. These results indicate the potential use of MCD in combination with MTAs for cancer chemotherapy and suggest that targeting both actin and microtubules simultaneously may be useful for cancer therapy. Importantly, the results provide significant insight into the crosstalk between actin and microtubules in regulating the traction force and dynamics of cell deadhesion.
Project description:Crawling cells have characteristic shapes that are a function of their cell types. How their different shapes are determined is an interesting question. Fish epithelial keratocytes are an ideal material for investigating cell shape determination, because they maintain a nearly constant fan shape during their crawling locomotion. We compared the shape and related molecular mechanisms in keratocytes from different fish species to elucidate the key mechanisms that determine cell shape. Wide keratocytes from cichlids applied large traction forces at the rear due to large focal adhesions, and showed a spatially loose gradient associated with actin retrograde flow rate, whereas round keratocytes from black tetra applied low traction forces at the rear small focal adhesions and showed a spatially steep gradient of actin retrograde flow rate. Laser ablation of stress fibers (contractile fibers connected to rear focal adhesions) in wide keratocytes from cichlids increased the actin retrograde flow rate and led to slowed leading-edge extension near the ablated region. Thus, stress fibers might play an important role in the mechanism of maintaining cell shape by regulating the actin retrograde flow rate.
Project description:Cell motility is a cornerstone of embryogenesis, tissue remodeling and repair, and cancer cell invasion. It is generally thought that migrating cells grab and exert traction force onto the extracellular matrix in order to pull the cell body forward. While previous studies have shown that myosin II deficient cells migrate efficiently, whether these cells exert traction forces during cell migration in the absence of the major contractile machinery is currently unknown. Using an array of micron-sized pillars as a force sensor and shRNA specific to each myosin II isoform (A and B), we analyzed how myosin IIA and IIB individually regulate cell migration and traction force generation. Myosin IIA and IIB localized preferentially to the leading edge where traction force was greatest, and the trailing edge, respectively. When individual myosin II isoforms were depleted by shRNA, myosin IIA deficient cells lost actin stress fibers and focal adhesions, whereas myosin IIB deficient cells maintained similar actin organization and focal adhesions as wild-type cells. Interestingly, myosin IIA deficient cells migrated faster than wild-type or myosin IIB deficient cells on both a rigid surface and a pillar array, yet myosin IIA deficient cells exerted significantly less traction force at the leading edge than wild-type or myosin IIB deficient cells. These results suggest that, in the absence of myosin IIA mediated force-generating machinery, cells move with minimal traction forces at the cell periphery, thus demonstrating the remarkable ability of cells to adapt and migrate.
Project description:Microtubule-targeting agents (MTAs) are widely used chemotherapy drugs capable of disrupting microtubule-dependent cellular functions, such as division and migration. We show that two clinically approved MTAs, paclitaxel and vinblastine, each suppress stiffness-sensitive migration and polarization characteristic of human glioma cells on compliant hydrogels. MTAs influence microtubule dynamics and cell traction forces by nearly opposite mechanisms, the latter of which can be explained by a combination of changes in myosin motor and adhesion clutch number. Our results support a microtubule-dependent signaling-based model for controlling traction forces through a motor-clutch mechanism, rather than microtubules directly relieving tension within F-actin and adhesions. Computational simulations of cell migration suggest that increasing protrusion number also impairs stiffness-sensitive migration, consistent with experimental MTA effects. These results provide a theoretical basis for the role of microtubules and mechanisms of MTAs in controlling cell migration.
Project description:BACKGROUND:The spatiotemporal regulation of adhesion to the extracellular matrix is important in metazoan cell migration and mechanosensation. Although adhesion assembly depends on intracellular and extracellular tension, the biophysical regulation of force transmission between the actin cytoskeleton and extracellular matrix during this process remains largely unknown. RESULTS:To elucidate the nature of force transmission as myosin II tension is applied to focal adhesions, we correlated the dynamics of focal adhesion proteins and the actin cytoskeleton to local traction stresses. Under low extracellular tension, newly formed adhesions near the cell periphery underwent a transient retrograde displacement preceding elongation. We found that myosin II-generated tension drives this mobility, and we determine the interface of differential motion, or "slip," to be between integrin and the ECM. The magnitude and duration of both adhesion slip and associated F-actin dynamics is strongly modulated by ECM compliance. Traction forces are generated throughout the slip period, and adhesion immobilization occurs at a constant tension. CONCLUSIONS:We have identified a tension-dependent, extracellular "clutch" between integrins and the extracellular matrix; this clutch stabilizes adhesions under myosin II driven tension. The current work elucidates a mechanism by which force transmission is modulated during focal adhesion maturation.
Project description:The contractile system of nonmuscle cells consists of interconnected actomyosin networks and bundles anchored to focal adhesions. The initiation of the contractile system assembly is poorly understood structurally and mechanistically, whereas system's maturation heavily depends on nonmuscle myosin II (NMII). Using platinum replica electron microscopy in combination with fluorescence microscopy, we characterized the structural mechanisms of the contractile system assembly and roles of NMII at early stages of this process. We show that inhibition of NMII by a specific inhibitor, blebbistatin, in addition to known effects, such as disassembly of stress fibers and mature focal adhesions, also causes transformation of lamellipodia into unattached ruffles, loss of immature focal complexes, loss of cytoskeleton-associated NMII filaments and peripheral accumulation of activated, but unpolymerized NMII. After blebbistatin washout, assembly of the contractile system begins with quick and coordinated recovery of lamellipodia and focal complexes that occurs before reappearance of NMII bipolar filaments. The initial formation of focal complexes and subsequent assembly of NMII filaments preferentially occurred in association with filopodial bundles and concave actin bundles formed by filopodial roots at the lamellipodial base. Over time, accumulating NMII filaments help to transform the precursor structures, focal complexes and associated thin bundles, into stress fibers and mature focal adhesions. However, semi-sarcomeric organization of stress fibers develops at much slower rate. Together, our data suggest that activation of NMII motor activity by light chain phosphorylation occurs at the cell edge and is uncoupled from NMII assembly into bipolar filaments. We propose that activated, but unpolymerized NMII initiates focal complexes, thus providing traction for lamellipodial protrusion. Subsequently, the mechanical resistance of focal complexes activates a load-dependent mechanism of NMII polymerization in association with attached bundles, leading to assembly of stress fibers and maturation of focal adhesions.
Project description:Cell migration over heterogeneous substrates during wound healing or morphogenetic processes leads to shape changes driven by different organizations of the actin cytoskeleton and by functional changes including lamellipodial protrusions and contractile actin cables. Cells distinguish between cell-sized positive and negative curvatures in their physical environment by forming protrusions at positive ones and actin cables at negative ones; however, the cellular mechanisms remain unclear. Here, we report that concave edges promote polarized actin structures with actin flow directed towards the cell edge, in contrast to well-documented retrograde flow at convex edges. Anterograde flow and contractility induce a tension anisotropy gradient. A polarized actin network is formed, accompanied by a local polymerization-depolymerization gradient, together with leading-edge contractile actin cables in the front. These cables extend onto non-adherent regions while still maintaining contact with the substrate through focal adhesions. The contraction and dynamic reorganization of this actin structure allows forward movements enabling cell migration over non-adherent regions on the substrate. These versatile functional structures may help cells sense and navigate their environment by adapting to external geometric and mechanical cues.
Project description:Contractile arrays of actin and myosin II filaments drive many essential processes in nonmuscle cells, including migration and adhesion. Sequential organization of actin and myosin along one dimension is followed by expansion into a two-dimensional network of parallel actomyosin fibers, in which myosin filaments are aligned to form stacks. The process of stack formation has been studied in detail. However, factors that oppose myosin stack formation have not yet been described. Here, we show that tropomyosins act as negative regulators of myosin stack formation. Knockdown of any or all tropomyosin isoforms in rat embryonic fibroblasts resulted in longer and more numerous myosin stacks and a highly ordered actomyosin organization. The molecular basis for this, we found, is the competition between tropomyosin and alpha-actinin for binding actin. Surprisingly, excessive order in the actomyosin network resulted in smaller focal adhesions, lower tension within the network, and smaller traction forces. Conversely, disordered actomyosin bundles induced by alpha-actinin knockdown led to higher than normal tension and traction forces. Thus, tropomyosin acts as a check on alpha-actinin to achieve intermediate levels of myosin stacks matching the force requirements of the cell.
Project description:Caveolin-1 (Cav1), a major Src kinase substrate phosphorylated on tyrosine-14 (Y14), contains the highly conserved membrane-proximal caveolin scaffolding domain (CSD; amino acids 82-101). Here we show, using CSD mutants (F92A/V94A) and membrane-permeable CSD-competing peptides, that Src kinase-dependent pY14Cav1 regulation of focal adhesion protein stabilization, focal adhesion tension, and cancer cell migration is CSD dependent. Quantitative proteomic analysis of Cav1-GST (amino acids 1-101) pull downs showed sixfold-increased binding of vinculin and, to a lesser extent, ?-actinin, talin, and filamin, to phosphomimetic Cav1Y14D relative to nonphosphorylatable Cav1Y14F. Consistently, pY14Cav1 enhanced CSD-dependent vinculin tension in focal adhesions, dampening force fluctuation and synchronously stabilizing cellular focal adhesions in a high-tension mode, paralleling effects of actin stabilization. This identifies pY14Cav1 as a molecular regulator of focal adhesion tension and suggests that functional interaction between Cav1 Y14 phosphorylation and the CSD promotes focal adhesion traction and, thereby, cancer cell motility.
Project description:Nonmuscle myosin II (NM-II) is an important motor protein involved in cell migration. Incorporation of NM-II into actin stress fiber provides a traction force to promote actin retrograde flow and focal adhesion assembly. However, the components involved in regulation of NM-II activity are not well understood. Here we identified a novel actin stress fiber-associated protein, LIM and calponin-homology domains 1 (LIMCH1), which regulates NM-II activity. The recruitment of LIMCH1 into contractile stress fibers revealed its localization complementary to actinin-1. LIMCH1 interacted with NM-IIA, but not NM-IIB, independent of the inhibition of myosin ATPase activity with blebbistatin. Moreover, the N-terminus of LIMCH1 binds to the head region of NM-IIA. Depletion of LIMCH1 attenuated myosin regulatory light chain (MRLC) diphosphorylation in HeLa cells, which was restored by reexpression of small interfering RNA-resistant LIMCH1. In addition, LIMCH1-depleted HeLa cells exhibited a decrease in the number of actin stress fibers and focal adhesions, leading to enhanced cell migration. Collectively, our data suggest that LIMCH1 plays a positive role in regulation of NM-II activity through effects on MRLC during cell migration.
Project description:Cells generate mechanical stresses via the action of myosin motors on the actin cytoskeleton. Although the molecular origin of force generation is well understood, we currently lack an understanding of the regulation of force transmission at cellular length scales. Here, using 3T3 fibroblasts, we experimentally decouple the effects of substrate stiffness, focal adhesion density, and cell morphology to show that the total amount of work a cell does against the substrate to which it is adhered is regulated by the cell spread area alone. Surprisingly, the number of focal adhesions and the substrate stiffness have little effect on regulating the work done on the substrate by the cell. For a given spread area, the local curvature along the cell edge regulates the distribution and magnitude of traction stresses to maintain a constant strain energy. A physical model of the adherent cell as a contractile gel under a uniform boundary tension and mechanically coupled to an elastic substrate quantitatively captures the spatial distribution and magnitude of traction stresses. With a single choice of parameters, this model accurately predicts the cell's mechanical output over a wide range of cell geometries.