Cortical Actin Network Research Articles

Simulating the Architecture of the Actin Cortex during Bleb Nucleation

Blebs are round membrane protrusions involved in important cellular processes such as cell motility, cytokinesis, and apoptosis. Recent studies have focused on the importance of cells that use blebs for cell migration in 3D fibrous environments. Blebs are initiated either by a local delamination of the actin cortex from the membrane due to loss of membrane-cortex adhesion proteins or by a local disruption of the actin cortex. In either case, the cortex degrades in the region of bleb initiation and then reassembles in the bleb. Myosin contractility has been shown to be necessary for bleb formation. It has further been hypothesized that a localized increase in myosin contractility on the cortex results in a rupture of the actin network that can lead to bleb nucleation. A minimal model consisting of actin filaments, myosin molecular motors, and actin cross-linker proteins is used to quantify conditions where holes, or defects, can develop in an initially isotropic cytoskeletal network. The model is simulated using the Cytosim computational platform. Results show that increased myosin fosters actin alignment, while increasing the concentration of actin cross-linkers decreases the pore size of the cytoskeletal network. Holes in the actin network develop at relatively high concentration of both cross-linkers and myosin. Simulation results suggest that a spatially localized defect, representing cortical actin network architecture during bleb nucleation, requires localized actin depolymerization rather than localized distributions of cross-linkers and myosin.

Dynamic multimerization of Dab2–Myosin VI complexes regulates cargo processivity while minimizing cortical actin reorganization

Myosin VI ensembles on endocytic cargo facilitate directed transport through a dense cortical actin network. Myosin VI is recruited to clathrin-coated endosomes via the cargo adaptor Dab2. Canonically, it has been assumed that the interactions between a motor and its cargo adaptor are stable. However, it has been demonstrated that the force generated by multiple stably attached motors disrupts local cytoskeletal architecture, potentially compromising transport. In this study, we demonstrate that dynamic multimerization of myosin VI-Dab2 complexes facilitates cargo processivity without significant reorganization of cortical actin networks. Specifically, we find that Dab2 myosin interacting region (MIR) binds myosin VI with a moderate affinity (184 nM) and single-molecule kinetic measurements demonstrate a high rate of turnover (1 s−1) of the Dab2 MIR–myosin VI interaction. Single-molecule motility shows that saturating Dab2-MIR concentration (2 μM) promotes myosin VI homodimerization and processivity with run lengths comparable with constitutive myosin VI dimers. Cargo-mimetic DNA origami scaffolds patterned with Dab2 MIR-myosin VI complexes are weakly processive, displaying sparse motility on single actin filaments and “stop-and-go” motion on a cellular actin network. On a minimal actin cortex assembled on lipid bilayers, unregulated processive movement by either constitutive myosin V or VI dimers results in actin remodeling and foci formation. In contrast, Dab2 MIR–myosin VI interactions preserve the integrity of a minimal cortical actin network. Taken together, our study demonstrates the importance of dynamic motor–cargo association in enabling cargo transportation without disrupting cytoskeletal organization.

Gold nanoparticles disrupt actin organization and pulmonary endothelial barriers

This study explored the impact of gold nanoparticles on the metabolic activity and morphology of human pulmonary endothelial cell monolayers. We developed a gold nanoparticle library of three different sizes and two surface chemistries that include anionic citrate and the cationic polyelectrolyte poly(allylamine hydrochloride). The nanoparticles were characterized in cell culture medium to assess how their physical properties are altered after exposure to biological fluids. A bovine serum albumin pretreatment protocol was developed to stabilize the nanoparticles in cell culture medium. Results of this study show that an 18 h exposure of human pulmonary artery endothelial cells to the different nanoparticles modestly affects cellular metabolic activity. However, nanoparticle exposure perturbs the cortical actin networks and induces the formation of intercellular gaps. In particular, exposure to the poly(allylamine hydrochloride)-coated particles reduces the area of cell–cell junctions—a change that correlates with increased leakiness of endothelial barriers. The presence of excess polyelectrolyte capping agents in the supernatant of poly(allylamine hydrochloride)-coated nanoparticles significantly impacts endothelial morphology. Pretreatment of the particle supernatant with bovine serum albumin mitigates the negative effects of free or bound polyelectrolytes on endothelial cell monolayers.

The cortical actin network regulates avidity-dependent binding of hyaluronan by the lymphatic vessel endothelial receptor LYVE-1

Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) mediates the docking and entry of dendritic cells to lymphatic vessels through selective adhesion to its ligand hyaluronan in the leukocyte surface glycocalyx. To bind hyaluronan efficiently, LYVE-1 must undergo surface clustering, a process that is induced efficiently by the large cross-linked assemblages of glycosaminoglycan present within leukocyte pericellular matrices but is induced poorly by the shorter polymer alone. These properties suggested that LYVE-1 may have limited mobility in the endothelial plasma membrane, but no biophysical investigation of these parameters has been carried out to date. Here, using super-resolution fluorescence microscopy and spectroscopy combined with biochemical analyses of the receptor in primary lymphatic endothelial cells, we provide the first evidence that LYVE-1 dynamics are indeed restricted by the submembranous actin network. We show that actin disruption not only increases LYVE-1 lateral diffusion but also enhances hyaluronan-binding activity. However, unlike the related leukocyte HA receptor CD44, which uses ERM and ankyrin motifs within its cytoplasmic tail to bind actin, LYVE-1 displays little if any direct interaction with actin, as determined by co-immunoprecipitation. Instead, as shown by super-resolution stimulated emission depletion microscopy in combination with fluorescence correlation spectroscopy, LYVE-1 diffusion is restricted by transient entrapment within submembranous actin corrals. These results point to an actin-mediated constraint on LYVE-1 clustering in lymphatic endothelium that tunes the receptor for selective engagement with hyaluronan assemblages in the glycocalyx that are large enough to cross-bridge the corral-bound LYVE-1 molecules and thereby facilitate leukocyte adhesion and transmigration.

The insulin-PI3K-Rac1 axis contributes to terminal adipocyte differentiation through regulation of actin cytoskeleton dynamics.

Adipocyte differentiation is accompanied by a pronounced change in the actin cytoskeleton characterized by the reorganization of filamentous (F)-actin stress fibers into cortical F-actin structures. We previously showed that depolymerization of F-actin stress fibers induced by inactivation of RhoA-ROCK (Rho-associated kinase) signaling acts as a trigger for adipocyte differentiation. The relevance and underlying mechanism of the formation of cortical F-actin structures from depolymerized actin during adipocyte differentiation have remained unclear, however. We have now examined the mechanistic relation between actin dynamics and adipogenic induction. Transient exposure to the actin-depolymerizing agent latrunculin A (LatA) supported the formation of adipocyte-associated cortical actin structures and the completion of terminal adipocyte differentiation in the presence of insulin, whereas long-term exposure to LatA prevented such actin reorganization as well as terminal adipogenesis. Moreover, these effects of insulin were prevented by inhibition of phosphatidylinositol 3-kinase (PI3K)-Rac1 signaling and the actin-related protein 2/3 (Arp2/3) complex which is a critical component of the cortical actin networks. Our findings thus suggest that the insulin-PI3K-Rac1 axis leads to the formation of adipocyte-associated cortical actin structures which is essential for the completion of adipocyte differentiation.

CLIC4 and CLIC1 bridge plasma membrane and cortical actin network for a successful cytokinesis.

CLIC4 and CLIC1 are members of the well-conserved chloride intracellular channel proteins (CLICs) structurally related to glutathione-S-transferases. Here, we report new roles of CLICs in cytokinesis. At the onset of cytokinesis, CLIC4 accumulates at the cleavage furrow and later localizes to the midbody in a RhoA-dependent manner. The cell cycle-dependent localization of CLIC4 is abolished when its glutathione S-transferase activity-related residues (C35A and F37D) are mutated. Ezrin, anillin, and ALIX are identified as interaction partners of CLIC4 at the cleavage furrow and midbody. Strikingly, CLIC4 facilitates the activation of ezrin at the cleavage furrow and reciprocally inhibition of ezrin activation diminishes the translocation of CLIC4 to the cleavage furrow. Furthermore, knockouts of CLIC4 and CLIC1 cause abnormal blebbing at the polar cortex and regression of the cleavage furrow at late cytokinesis leading to multinucleated cells. We conclude that CLIC4 and CLIC1 function together with ezrin where they bridge plasma membrane and actin cytoskeleton at the polar cortex and cleavage furrow to promote cortical stability and successful completion of cytokinesis in mammalian cells.

Interleukin-38 interacts with destrin/actin-depolymerizing factor in human keratinocytes.

Interleukin (IL)-38 is a member of the IL-1 family of cytokines, which was proposed to exert anti-inflammatory effects. IL-38 is constitutively expressed in the skin, where keratinocytes are the main producing cells. Little information is currently available concerning IL-38 biology. Here, we investigated the subcellular localization and interaction partners of the IL-38 protein in human keratinocytes. IL-38 expression was reduced in primary keratinocytes grown in monolayer (2D) cultures. We thus used IL-38 overexpressing immortalized normal human keratinocytes (NHK/38) to study this cytokine in cell monolayers. In parallel, differentiation of primary human keratinocytes in an in vitro reconstructed human epidermis (RHE) 3D model allowed us to restore endogenous IL-38 expression. In NHK/38 cells and in RHE, IL-38 was mainly cell-associated, rather than released into culture supernatants. Intracellular IL-38 was preferentially, although not exclusively, cytoplasmic. Similarly, in normal human skin sections, IL-38 was predominantly cytoplasmic in the epidermis and essentially excluded from keratinocyte nuclei. A yeast two-hybrid screen identified destrin/actin-depolymerizing factor (DSTN) as a potential IL-38-interacting molecule. Co-immunoprecipitation and proximity ligation assay confirmed this interaction. We further observed partial co-localization of IL-38 and DSTN in NHK/38 cells. Endogenous IL-38 and DSTN were also co-expressed in all epidermal layers in RHE and in normal human skin. Finally, IL-38 partially co-localized with F-actin in NHK/38 cells, in particular along the cortical actin network and in filopodia. In conclusion, IL-38 is found predominantly in the cytoplasm of human keratinocytes, where it interacts with DSTN. The functional relevance of this interaction remains to be investigated.

Drosophila Atg9 regulates the actin cytoskeleton via interactions with profilin and Ena.

Autophagy ensures the turnover of cytoplasm and requires the coordinated action of Atg proteins, some of which also have moonlighting functions in higher eukaryotes. Here we show that the transmembrane protein Atg9 is required for female fertility, and its loss leads to defects in actin cytoskeleton organization in the ovary and enhances filopodia formation in neurons in Drosophila. Atg9 localizes to the plasma membrane anchor points of actin cables and is also important for the integrity of the cortical actin network. Of note, such phenotypes are not seen in other Atg mutants, suggesting that these are independent of autophagy defects. Mechanistically, we identify the known actin regulators profilin and Ena/VASP as novel binding partners of Atg9 based on microscopy, biochemical, and genetic interactions. Accordingly, the localization of both profilin and Ena depends on Atg9. Taken together, our data identify a new and unexpected role for Atg9 in actin cytoskeleton regulation.

Correction to "Characterizing Large-Scale Receptor Clustering on the Single Cell Level: A Comparative Plasmon Coupling and Fluorescence Superresolution Microscopy Study".

Spatial clustering of cell membrane receptors has been indicated to play a regulatory role in signal initiation, and the distribution of receptors on the cell surface may represent a potential biomarker. To realize its potential for diagnostic purposes, scalable assays capable of mapping spatial receptor heterogeneity with high throughput are needed. In this work, we use gold nanoparticle (NP) labels with an average diameter of 72.17 ± 2.16 nm as bright markers for large-scale epidermal growth factor receptor (EGFR) clustering in hyperspectral plasmon coupling microscopy and compare the obtained clustering maps with those obtained through fluorescence superresolution microscopy (direct stochastic optical reconstruction microscopy, dSTORM). Our dSTORM experiments reveal average EGFR cluster sizes of 172 ± 99 and 150 ± 90 nm for MDA-MB-468 and HeLa, respectively. The cluster sizes decrease after EGFR activation. Hyperspectral imaging of the NP labels shows that differences in the EGFR cluster sizes are accompanied by differences in the average separations between electromagnetically coupled NPs. Because of the distance dependence of plasmon coupling, changes in the average interparticle separation result in significant spectral shifts. For the experimental conditions investigated in this work, hyperspectral plasmon coupling microscopy of NP labels identified the same trends in large-scale EGFR clustering as dSTORM, but the NP imaging approach provided the information in a fraction of the time. Both dSTORM and hyperspectral plasmon coupling microscopy confirm the cortical actin network as one structural component that determines the average size of EGFR clusters.

Filamin A mediates isotropic distribution of applied force across the actin network.

Cell sensing of externally applied mechanical strain through integrin-mediated adhesions is critical in development and physiology of muscle, lung, tendon, and arteries, among others. We examined the effects of strain on force transmission through the essential cytoskeletal linker talin. Using a fluorescence-based talin tension sensor (TS), we found that uniaxial stretch of cells on elastic substrates increased tension on talin, which was unexpectedly independent of the orientation of the focal adhesions relative to the direction of strain. High-resolution electron microscopy of the actin cytoskeleton revealed that stress fibers (SFs) are integrated into an isotropic network of cortical actin filaments in which filamin A (FlnA) localizes preferentially to points of intersection between SFs and cortical actin. Knockdown (KD) of FlnA resulted in more isolated, less integrated SFs. After FlnA KD, tension on talin was polarized in the direction of stretch, while FlnA reexpression restored tensional symmetry. These data demonstrate that a FlnA-dependent cortical actin network distributes applied forces over the entire cytoskeleton-matrix interface.

WASH phosphorylation balances endosomal versus cortical actin network integrities during epithelial morphogenesis

Filamentous actin (F-actin) networks facilitate key processes like cell shape control, division, polarization and motility. The dynamic coordination of F-actin networks and its impact on cellular activities are poorly understood. We report an antagonistic relationship between endosomal F-actin assembly and cortical actin bundle integrity during Drosophila airway maturation. Double mutants lacking receptor tyrosine phosphatases (PTP) Ptp10D and Ptp4E, clear luminal proteins and disassemble apical actin bundles prematurely. These defects are counterbalanced by reduction of endosomal trafficking and by mutations affecting the tyrosine kinase Btk29A, and the actin nucleation factor WASH. Btk29A forms protein complexes with Ptp10D and WASH, and Btk29A phosphorylates WASH. This phosphorylation activates endosomal WASH function in flies and mice. In contrast, a phospho-mimetic WASH variant induces endosomal actin accumulation, premature luminal endocytosis and cortical F-actin disassembly. We conclude that PTPs and Btk29A regulate WASH activity to balance the endosomal and cortical F-actin networks during epithelial tube maturation.

Cytoskeletal actin patterns shape mast cell activation

Activation of immune cells relies on a dynamic actin cytoskeleton. Despite detailed knowledge of molecular actin assembly, the exact processes governing actin organization during activation remain elusive. Using advanced microscopy, we here show that Rat Basophilic Leukemia (RBL) cells, a model mast cell line, employ an orchestrated series of reorganization events within the cortical actin network during activation. In response to IgE antigen-stimulation of FCε receptors (FCεR) at the RBL cell surface, we observed symmetry breaking of the F-actin network and subsequent rapid disassembly of the actin cortex. This was followed by a reassembly process that may be driven by the coordinated transformation of distinct nanoscale F-actin architectures, reminiscent of self-organizing actin patterns. Actin patterns co-localized with zones of Arp2/3 nucleation, while network reassembly was accompanied by myosin-II activity. Strikingly, cortical actin disassembly coincided with zones of granule secretion, suggesting that cytoskeletal actin patterns contribute to orchestrate RBL cell activation.

AbLIM1 constructs non-erythroid cortical actin networks to prevent mechanical tension-induced blebbing

The cell cortex is a layer of cytoskeletal networks underneath the plasma membrane, formed by filamentous actin (F-actin) and cortex proteins including spectrin, adducin, and myosin. It provides cells with proper stiffness, elasticity, and surface tension to allow morphogenesis, division, and migration. Although its architecture and formation have been widely studied in red blood cells, they are poorly understood in non-erythrocytes due to structural complexity and versatile functions. In this study, we identify the actin-binding protein abLIM1 as a novel non-erythroid cell-specific cortex organizer. Endogenous abLIM1 colocalized with cortical βII spectrin but upon overexpression redistributed to thick cortical actin bundles. abLIM1 associated with major cortex proteins such as spectrins and adducin in vivo. Depletion of abLIM1 by RNAi induced prominent blebbing during membrane protrusions of spreading or migrating RPE1 cells and impaired migration efficiency. Reducing cortical tensions by culturing the cells to confluency or inhibiting myosin activity repressed the blebbing phenotype. abLIM1-depleted RPE1 or U2OS cells lacked the dense interwoven cortical actin meshwork observed in control cells but were abundant in long cortical actin bundles along the long axis of the cells. In-vitro assays indicated that abLIM1 was able to crosslink and bundle F-actin to induce dense F-actin network formation. Therefore, abLIM1 governs the formation of dense interconnected cortical actin meshwork in non-erythroid cells to prevent mechanical tension-induced blebbing during cellular activities such as spreading and migration.

Actin Network Discussion during Mitotic Pseudo-Furrowing

Two types of cortical actin networks act during mitotic pseudocleavage furrowing in the Drosophila syncytium, but how they interact has remained elusive. In this issue of Developmental Cell, Zhang etal. (2018) show how these networks shape each other and propose that furrowing is driven by actin polymerization-derived pushing forces.

Actin Remodeling in Regulated Exocytosis: Toward a Mesoscopic View.

Cellular communication relies on fusion of secretory vesicles with the plasma membrane, following dynamic events that change the micro- and nanoscale environment of the approaching vesicles in the vicinity of docking sites. Visualization of fine cortical actin network structures and their interactions with vesicle and plasma membrane has recently been facilitated by the development of new imaging technologies. Consequently, a greater understanding is emerging of the role of the cortical actin network on controlling secretory vesicles as they undergo docking, priming, and fusion in exocytic hot spots. In this review, we propose a mechanistic framework underpinning the mesoscopic properties of the cortical actin and discuss how molecular coupling of these pleiotropic effects orchestrate every single step of regulated exocytosis.

ENA/VASP proteins regulate exocytosis by mediating myosin VI-dependent recruitment of secretory granules to the cortical actin network.

In neurosecretory cells, myosin VI associated with secretory granules (SGs) mediates their activity-dependent recruitment to the cortical actin network and is necessary to sustain exocytosis. The mechanism by which myosin VI interacts with SGs is unknown. Using a myosin VI pull-down assay and mass spectrometry we identified Mena, a member of the ENA/VASP family, as a myosin VI binding partner in PC12 cells, and confirmed that Mena colocalized with myosin VI on SGs. Using a knock-sideways approach to inactivate the ENA/VASP family members by mitochondrial relocation, we revealed a concomitant redistribution of myosin VI. This was ensued by a reduction in the association of myosin VI with SGs, a decreased SG mobility and density in proximity to the plasma membrane as well as decreased evoked exocytosis. These data demonstrate that ENA/VASP proteins regulate SG exocytosis through modulating the activity of myosin VI.

The actin binding cytoskeletal protein Moesin is involved in nuclear mRNA export

Current models imply that the evolutionarily conserved, actin-binding Ezrin-Radixin-Moesin (ERM) proteins perform their activities at the plasma membrane by anchoring membrane proteins to the cortical actin network. Here we show that beside its cytoplasmic functions, the single ERM protein of Drosophila, Moesin, has a novel role in the nucleus. The activation of transcription by heat shock or hormonal treatment increases the amount of nuclear Moesin, indicating biological function for the protein in the nucleus. The distribution of Moesin in the nucleus suggests a function in transcription and the depletion of mRNA export factors Nup98 or its interacting partner, Rae1, leads to the nuclear accumulation of Moesin, suggesting that the nuclear function of the protein is linked to mRNA export. Moesin localizes to mRNP particles through the interaction with the mRNA export factor PCID2 and knock down of Moesin leads to the accumulation of mRNA in the nucleus. Based on our results we propose that, beyond its well-known, manifold functions in the cytoplasm, the ERM protein of Drosophila is a new, functional component of the nucleus where it participates in mRNA export.

Actin cortex architecture regulates cell surface tension.

Animal cell shape is largely determined by the cortex, a thin actin network underlying the plasma membrane in which myosin-driven stresses generate contractile tension. Tension gradients result in local contractions and drive cell deformations. Previous cortical tension regulation studies have focused on myosin motors. Here, we show that cortical actin network architecture is equally important. First, we observe that actin cortex thickness and tension are inversely correlated during cell cycle progression. We then show that the actin filament length regulators CFL1, CAPZB, DIAPH1 regulate mitotic cortex thickness and find that both increasing and decreasing thickness decreases tension in mitosis. This suggests that the mitotic cortex is poised close to a tension maximum. Finally, using a computational model, we identify a physical mechanism by which maximum tension is achieved at intermediate actin filament lengths. Our results indicate that actin network architecture, alongside myosin activity, is key to cell surface tension regulation.

A role for tropomyosins in activity-dependent bulk endocytosis?

Bulk endocytosis allows stimulated neurons to take up a large portion of the presynaptic plasma membrane in order to regenerate synaptic vesicle pools. Actin, one of the most abundant proteins in eukaryotic cells, plays an important role in this process, but a detailed mechanistic understanding of the involvement of the cortical actin network is still lacking, in part due to the relatively small size of nerve terminals and the limitation of optical microscopy. We recently discovered that neurosecretory cells display a similar, albeit much larger, form of bulk endocytosis in response to secretagogue stimulation. This allowed us to identify a novel highly dynamic role for the acto-myosin II cortex in generating constricting rings that precede the fission of nascent bulk endosomes. In this review we focus on the mechanism underpinning this dramatic switch in the organization and function of the cortical actin network. We provide additional experimental data that suggest a role of tropomyosin Tpm3.1 and Tpm4.2 in this process, together with an emerging model of how actin controls bulk endocytosis.

Membrane shaping by actin and myosin during regulated exocytosis.

The cortical actin network in neurosecretory cells is a dense mesh of actin filaments underlying the plasma membrane. Interaction of actomyosin with vesicular membranes or the plasma membrane is vital for tethering, retention, transport as well as fusion and fission of exo- and endocytic membrane structures. During regulated exocytosis the cortical actin network undergoes dramatic changes in morphology to accommodate vesicle docking, fusion and replenishment. Most of these processes involve plasma membrane Phosphoinositides (PIP) and investigating the interactions between the actin cortex and secretory structures has become a hotbed for research in recent years. Actin remodelling leads to filopodia outgrowth and the creation of new fusion sites in neurosecretory cells and actin, myosin and dynamin actively shape and maintain the fusion pore of secretory vesicles. Changes in viscoelastic properties of the actin cortex can facilitate vesicular transport and lead to docking and priming of vesicle at the plasma membrane. Small GTPase actin mediators control the state of the cortical actin network and influence vesicular access to their docking and fusion sites. These changes potentially affect membrane properties such as tension and fluidity as well as the mobility of embedded proteins and could influence the processes leading to both exo- and endocytosis. Here we discuss the multitudes of actin and membrane interactions that control successive steps underpinning regulated exocytosis.