Actin Meshwork Research Articles

Probing Lipid Vesicle Transport in 3D by Teams of Myosin Va Motors at Suspended Actin Intersections in vitro

In cells, myosin Va motor teams are forced to transport vesicles through a complex three-dimensional (3D) actin meshwork. To characterize how these motors navigate this physically challenging meshwork, we created actin filament intersections adhered to a coverslip surface and confronted a team of ∼10 myosin Va HMM motors carrying a 350nm synthetic lipid vesicle. When approaching the intersection on the lower filament, the motor-vesicle complex (MVC) switched to the upper intersecting filament with a 51% probability while crossing over the intersection with only a 33% probability. If approaching the intersection on the upper filament, the MVC preferred staying on the upper filament through the intersection and only switched to the lower filament with a 31% probability. These data suggest that the extent of MVC surface contact with the target binding zone of the intersecting actin filaments dictates the directional outcome. To build complexity, actin filaments were strung between 3μm beads, creating suspended filament intersections. The 3D spatial relations between the MVC and the individual filaments were determined by super-resolution STORM imaging. As the MVC approached the intersection, the complex spiraled around the actin filament with a pitch of ∼1.3µm. If the MVC was forced to navigate through the intersection and the separation between actin filaments (<100nm) was less than the vesicle diameter, the MVC initially paused and then switched to the intersecting filament. For this to occur, motors must be free to diffuse on the fluid lipid (DOPC) vesicle surface and engage an actin filament anywhere that the vesicle surface contacts an actin filament. This 3D model system with added complexity will provide an experimental platform to understand how myosin Va motor ensembles maneuver their cargo through the complex actin cytoskeletal network.

Host cell invasion by apicomplexan parasites: the junction conundrum.

Host cell invasion by apicomplexan parasites: the junction conundrum.

Spatially coordinated changes in intracellular rheology and extracellular force exertion during mesenchymal stem cell differentiation

The mechanical properties within the cell are regulated by the organization of the actin cytoskeleton, which is linked to the extracellular environment through focal adhesion proteins that transmit force. Chemical and mechanical stimuli alter the organization of cytoskeletal actin, which results in changes in cell shape, adhesion, and differentiation. By combining particle-tracking microrheology and traction force cytometry, we can monitor the mechanical properties of the actin meshwork and determine how changes in the intracellular network contribute to force generation. In this study, we investigated the effects of chemical (differentiation factors) and mechanical (substrate rigidity) stimuli important in mesenchymal stem cell (MSC) differentiation on the intracellular mechanics and traction stress generation. We found the presence of adipogenic factors resulted in stiffening of the actin meshwork regardless of substrate rigidity. In contrast, these factors increased traction stresses on hard substrates, which was associated with increased expression of contractility genes. Furthermore, MSCs cultured on hard substrates expressed both adipogenic and osteogenic markers indicative of mixed differentiation. On hard substrates, heterogeneity in the local elastic modulus-traction stress correlation was also increased in response to adipogenic factors, indicating that these mechanical properties may be reflective of differences in the level of MSC differentiation. These results suggest intracellular rheology and traction stress generation are spatially regulated and contribute insight into how single cell mechanical forces contribute to MSC differentiation.

Synergies between Aip1p and capping protein subunits (Acp1p and Acp2p) in clathrin-mediated endocytosis and cell polarization in fission yeast.

Aip1p cooperates with actin-depolymerizing factor (ADF)/cofilin to disassemble actin filaments in vitro and in vivo, and is proposed to cap actin filament barbed ends. We address the synergies between Aip1p and the capping protein heterodimer Acp1p/Acp2p during clathrin-mediated endocytosis in fission yeast. Using quantitative microscopy and new methods we have developed for data alignment and analysis, we show that heterodimeric capping protein can replace Aip1p, but Aip1p cannot replace capping protein in endocytic patches. Our quantitative analysis reveals that the actin meshwork is organized radially and is compacted by the cross-linker fimbrin before the endocytic vesicle is released from the plasma membrane. Capping protein and Aip1p help maintain the high density of actin filaments in meshwork by keeping actin filaments close enough for cross-linking. Our experiments also reveal new cellular functions for Acp1p and Acp2p independent of their capping activity. We identified two independent pathways that control polarization of endocytic sites, one depending on acp2(+) and aip1(+) during interphase and the other independent of acp1(+), acp2(+), and aip1(+) during mitosis.

Local and global analysis of endocytic patch dynamics in fission yeast using a new "temporal superresolution" realignment method.

Quantitative microscopy is a valuable tool for inferring molecular mechanisms of cellular processes such as clathrin-mediated endocytosis, but, for quantitative microscopy to reach its potential, both data collection and analysis needed improvement. We introduce new tools to track and count endocytic patches in fission yeast to increase the quality of the data extracted from quantitative microscopy movies. We present a universal method to achieve "temporal superresolution" by aligning temporal data sets with higher temporal resolution than the measurement intervals. These methods allowed us to extract new information about endocytic actin patches in wild-type cells from measurements of the fluorescence of fimbrin-mEGFP. We show that the time course of actin assembly and disassembly varies <600 ms between patches. Actin polymerizes during vesicle formation, but we show that polymerization does not participate in vesicle movement other than to limit the complex diffusive motions of newly formed endocytic vesicles, which move faster as the surrounding actin meshwork decreases in size over time. Our methods also show that the number of patches in fission yeast is proportional to cell length and that the variability in the repartition of patches between the tips of interphase cells has been underestimated.

The immunomodulatory drug lenalidomide augments NK cell IFN-γ secretion and lowers the threshold for NK cell activation (INC6P.351)

Abstract The immunomodulatory drug lenalidomide is widely used for the treatment of multiple myeloma. NK cells can kill tumour cells in early stage myeloma but there is a decline in NK cell cytotoxicity as the disease progresses. Previous studies have shown that lenalidomide increases antibody-dependent cellular cytotoxicity, yet little is known about its effects on cytokine secretion. Here, we show that lenalidomide increases IFN-γ secretion from primary human NK cells stimulated by NK-sensitive target cells. Moreover, after ligation of NK activating receptors CD16 and NKG2D, lenalidomide caused a 2-fold increase in the proportion of primary NK cells producing IFN-γ, and a 10-fold increase in the amount of IFN-γ produced per cell. This implies that lenalidomide acts downstream from early membrane-proximal signalling, which is distinct for these two receptors. In addition, lenalidomide-treated NK cells displayed a 2-fold decrease in the amount of ligand required to reach 50% of maximal activation, establishing that lenalidomide lowers the threshold for NK cell activation. Finally, using super resolution microscopy, we found that lenalidomide treatment increases the periodicity of the actin meshwork at the immune synapse during cytokine secretion, a cytoskeletal rearrangement likely to be important for its mechanism of action. Taken together, these results establish that lenalidomide influences cytokine secretion and lowers the threshold for NK cell activation.

Axon initial segment cytoskeleton comprises a multiprotein submembranous coat containing sparse actin filaments

The axon initial segment (AIS) of differentiated neurons regulates action potential initiation and axon-dendritic polarity. The latter function depends on actin dynamics, but actin structure and functions at the AIS remain unclear. Using platinum replica electron microscopy (PREM), we have characterized the architecture of the AIS cytoskeleton in mature and developing hippocampal neurons. The AIS cytoskeleton assembly begins with bundling of microtubules and culminates in formation of a dense, fibrillar-globular coat over microtubule bundles. Immunogold PREM revealed that the coat contains a network of known AIS proteins, including ankyrin G, spectrin βIV, neurofascin, neuronal cell adhesion molecule, voltage-gated sodium channels, and actin filaments. Contrary to existing models, we find neither polarized actin arrays, nor dense actin meshworks in the AIS. Instead, the AIS contains two populations of sparse actin filaments: short, stable filaments and slightly longer dynamic filaments. We propose that stable actin filaments play a structural role for formation of the AIS diffusion barrier, whereas dynamic actin may promote AIS coat remodeling.

A contractile and counterbalancing adhesion system controls the 3D shape of crawling cells

How adherent and contractile systems coordinate to promote cell shape changes is unclear. Here, we define a counterbalanced adhesion/contraction model for cell shape control. Live-cell microscopy data showed a crucial role for a contractile meshwork at the top of the cell, which is composed of actin arcs and myosin IIA filaments. The contractile actin meshwork is organized like muscle sarcomeres, with repeating myosin II filaments separated by the actin bundling protein α-actinin, and is mechanically coupled to noncontractile dorsal actin fibers that run from top to bottom in the cell. When the meshwork contracts, it pulls the dorsal fibers away from the substrate. This pulling force is counterbalanced by the dorsal fibers' attachment to focal adhesions, causing the fibers to bend downward and flattening the cell. This model is likely to be relevant for understanding how cells configure themselves to complex surfaces, protrude into tight spaces, and generate three-dimensional forces on the growth substrate under both healthy and diseased conditions.

Novel Roles of Focal Adhesion Kinase in Cytoplasmic Entry and Replication of Influenza A Viruses

Viruses modulate cellular signaling pathways at almost every step of the infection cycle. Cellular signaling pathways activated at later times of influenza infection have previously been investigated; however, early influenza virus-host cell interactions remain understudied. Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that regulates phosphatidylinositol 3-kinase (PI3K) activation and actin reorganization, two critical processes during influenza A virus (IAV) infection in most cell types. Using 6 influenza A virus strains (A/Puerto Rico/8/1934, A/Aichi/2/1968 × A/Puerto Rico/8/1934 reassortant [X-31], A/California/04/2009, mouse-adapted A/California/04/2009, A/WSN/1933, and A/New Caledonia/20/1999), we examined the role of FAK during IAV entry. We found that influenza virus attachment induced PI3K-dependent FAK-Y397 phosphorylation. Pharmacological FAK inhibition or expression of a kinase-dead mutant of FAK led to disruption of the actin meshwork that resulted in sequestration of IAV at the cell periphery and reduced virion localization to early endosomes. Additionally, FAK inhibition impeded viral RNA replication at later times of infection and ultimately resulted in significantly reduced viral titers in both A549 and differentiated normal human bronchial epithelial (NHBE) cells. Although not all tested strains activated FAK, all of them exhibited a reduction in viral replication in response to inhibition of FAK signaling. These findings highlight novel biphasic roles of FAK activation during IAV infection and indicate that FAK serves as a central link between receptor-mediated PI3K activation and actin reorganization during IAV infection. We found that FAK links early activation of PI3K and actin reorganization, thereby regulating influenza virus entry. Surprisingly, we also found that FAK can regulate viral RNA replication independently of its role in entry. Our study addresses a knowledge gap in the understanding of signaling events triggered by influenza virus that mediate its internalization and initiation of the infection cycle. Understanding of these fundamental molecular events will be necessary to identify novel host targets, such as FAK, and development of future anti-influenza virus therapeutics.

Selective Microtubule-Based Transport of Dendritic Membrane Proteins Arises in Concert with Axon Specification

The polarized distribution of membrane proteins to axonal or somatodendritic neuronal compartments is fundamental to nearly every aspect of neuronal function. The polarity of dendritic proteins depends on selective microtubule-based transport; the vesicles that carry these proteins are transported into dendrites but do not enter the axon. We used live-cell imaging of fluorescently tagged dendritic and axonal proteins combined with immunostaining for initial segment and cytoskeletal markers to evaluate different models of dendrite-selective transport in cultured rat hippocampal neurons. In mature neurons, dendritic vesicles that entered the base of the axon stopped at the proximal edge of the axon initial segment, defined by immunostaining for ankyrinG, rather than moving into the initial segment itself. In contrast, axonal vesicles passed through the initial segment without impediment. During development, dendrite-selective transport was detected shortly after axons formed, several days before initial segment assembly, before the appearance of a dense actin meshwork in the initial segment, and before dendrites acquire microtubules of mixed polarity orientation. Indeed, some elements of selective transport were detected even before axon specification. These findings are inconsistent with models for selective transport that depend on the presence of an F-actin-based cytoplasmic filter in the initial segment or that posit that transport into dendrites is mediated by dyneins translocating along minus-end out microtubules. Instead our results suggest that selective transport involves the coordinated regulation of the different motor proteins that mediate dendritic vesicle transport and that the selectivity of motor-microtubule interactions is one facet of this process.

A lipid bound actin meshwork organizes liquid phase separation in model membranes.

The eukaryotic cell membrane is connected to a dense actin rich cortex. We present FCS and STED experiments showing that dense membrane bound actin networks have severe influence on lipid phase separation. A minimal actin cortex was bound to a supported lipid bilayer via biotinylated lipid streptavidin complexes (pinning sites). In general, actin binding to ternary membranes prevented macroscopic liquid-ordered and liquid-disordered domain formation, even at low temperature. Instead, depending on the type of pinning lipid, an actin correlated multi-domain pattern was observed. FCS measurements revealed hindered diffusion of lipids in the presence of an actin network. To explain our experimental findings, a new simulation model is proposed, in which the membrane composition, the membrane curvature, and the actin pinning sites are all coupled. Our results reveal a mechanism how cells may prevent macroscopic demixing of their membrane components, while at the same time regulate the local membrane composition. DOI: http://dx.doi.org/10.7554/eLife.01671.001.

Spire and Formin 2 Synergize and Antagonize in Regulating Actin Assembly in Meiosis by a Ping-Pong Mechanism

Author SummaryMammalian reproduction requires successful meiosis, which consists of two strongly asymmetric cell divisions. In meiosis I, movement of the spindle (the subcellular structure that segregates chromosomes during division) toward the oocyte cortex (the outer layer of the egg) is essential for fertility. This process requires that actin filaments assemble in a dynamic mesh, driven by three actin binding proteins, profilin, formin 2, and Spire. To date the molecular mechanisms by which these three proteins cooperate are not known. We now explore this in vitro by a combination of bulk solution and single actin filament assembly assays in the presence of profilin, Spire, and formin 2. Individually, Spire binds to actin filament ends to block their growth, and by itself, formin 2 associates poorly with filament ends, promoting fast processive assembly from the profilin-actin complex. However, when present together, Spire and formin 2 interact with one another (the formin 2 C-terminal binds to the N terminal Spire KIND domain), forming transient complexes at filament ends from which each binds alternately to the filament ends to regulate actin assembly by a ping-pong mechanism. Our in vitro observations are validated by injection studies in mouse oocytes. In oocytes, the additional interaction of Spire and formin 2 with Rab11a-myosin Vb vesicles couples high actin dynamics to vesicle traffic.

The Sh3-Binding Domain of Kv1.3 Channels is Required for their Cortactin-Conveyed Coupling to Actin

Kv1.3 channels of T lymphocytes play an important role in several physiological functions like activation or migration, which are accompanied by the redistribution of channels in the cell membrane. Both processes are characterized by F-actin polymerization indicating the involvement of the actin cytoskeleton in the associated channel's redistribution. Several studies reported that Kv1.x channels are linked to or co-localize with various adapter proteins which also possess actin-binding domains: hDlg1 via the PDZ-binding domain and cortactin through either the cortactin- or the SH3-binding site. In this study we investigated the molecular requirements for Kv1.3 channel lateral membrane diffusion.FLAG/EGFP-tagged Kv1.3 wild-type or mutant constructs were expressed in HEK-293 cells. We designed C-terminal truncated (without (delta1) or with (delta2) cortactin binding sequence, and both without SH3; with cortactin and SH3 binding motif (delta3)) and few-amino-acid Kv1.3 mutants (deltaSH3/deltaPDZ: SH3/PDZ domain ruptured). Biophysical properties of each phenotype were evaluated with patch-clamping. Interaction/co-localization between channel and adaptor proteins was studied with co-immunoprecipitation/confocal microscopy. Lateral mobility of Kv1.3 was assessed with FRAP and defined by the channel's mobile fraction (Mf).Parameters of channel gating for mutants were indistinguishable from those of wild-type Kv1.3. Confocal microscopy images and co-immunoprecipitation experiments demonstrated that cortactin and Kv1.3 interact in HEK-293 cells. Furthermore, wild-type, delta3 and deltaPDZ mutants have reduced Mf upon stimulation of F-actin polymerization/stabilization by jasplakinolide (unlike delta1, delta2 and deltaSH3 constructs). When cortactin was knocked down by shRNA, exposure to jasplakinolide induced no decrease in Mf of wild-type Kv1.3 channels. These findings point out that cortactin serves as a bridge between Kv1.3 and the actin meshwork via the channel's SH3-binding domain, and can regulate mobility/immobility of Kv1.3 channels. (NIH 2R01CA095286).

Atomic Force Microscopy Reveals the Structure and Dynamics of the Cell Cortex

Recent studies have shown that processes such as eukaryotic cell-cell interactions, differentiation and tissue development are controlled by mechanical signals. These mechanical stimuli come from the outside of the cells and induce remodeling of the cytoskeleton in the cell interior. Unfortunately, no dynamic data at high spatial resolution could be acquired so far on the cytoskeleton of live cells, and the mechanical heterogeneity at the subcellular level remains unknown. In particular, the cell cortex is a major determinant of the cell mechanics but its spatial arrangement is poorly understood, and the dynamic behavior of its elements could only be inferred through indirect methods. Here we demonstrate that simultaneous topography imaging and mechanical mapping of live cells under physiological conditions at high resolution and low forces is possible using atomic force microscopy. We applied our methods to perform direct imaging of the cell membrane actin cortex, reaching a resolution inferior to 100nm and a maximal 10s image acquisition rate. The cell cortex is structurally, mechanically and dynamically heterogeneous at the subcellular level, and its fastest rearrangement time was in the 10s range. Our resolution enabled direct sizing the sub-membrane actin meshwork, confirming estimates from electron microscopy and molecular diffusion studies. Furthermore, we can attribute dynamic parameters to actin meshworks of various architecture and estimate the forces that they can exert on neighboring cells.

A Lipid Bound Actin Meshwork Organizes Liquid Phase Separation in Model Membranes

The eukaryotic cell membrane is connected to a dense actin rich cortex. We present FCS and STED experiments showing that dense membrane bound actin networks have severe influence on lipid phase separation. A minimal actin cortex was bound to a supported lipid bilayer via biotinylated lipid streptavidin complexes (pinning sites). In general, actin binding to ternary membranes prevented macroscopic liquid-ordered and liquid-disordered domain formation, even at low temperature. Instead, depending on the type of pinning lipid, an actin correlated multi-domain pattern was observed. FCS measurements revealed hindered diffusion of lipids in the presence of an actin network. To explain our experimental findings, a new simulation model is proposed, in which the membrane composition, the membrane curvature, and the actin pinning sites are all coupled. Our results reveal a mechanism how cells may prevent macroscopic demixing of their membrane components, while at the same time regulate the local membrane composition.

Long-Term Live Observation of Membrane Protein Interaction with Lipid Nanodomains Show Dependence on Cell Cycle and Time After Transfection

The study of cholesterol-stabilized nanodomains in the cell membrane, aka lipid rafts, is complicated by differences in techniques, cell type, physiological parameters and possible changes of cell and membrane composition during the cell cycle and after transient transfection. We recently developed a non-destructive and non-perturbing technique combining fluorescence correlation spectroscopy (FCS) with spatially-resolved camera TIRF imaging, bimFCS. This method provides a quantification of the strength of the membrane protein - lipid nanodomain interaction, the membrane protein concentration and its diffusion coefficient. As an optical method it can be combined with other imaging methods such as we record changes in cytoskeleton structure simultaneously. bimFCS data was acquired with hourly intervals for periods longer than one day while keeping the cells growing at physiological temperature on the microscope stage. Transiently transfecting PtK2 or CHO cells with GPI-anchored membrane proteins associated with cholesterol-stabilized nanodomains, we observed a higher association with such domains in earlier stages of protein expression and slower diffusion compared to later times. Using synchronized cell cultures, we also quantify the membrane protein interaction with lipid nanodomains and record the images of membrane cytoskeletal actin bundles as function of the cell cycle. To discriminate between possible membrane ultrastructure models, we combine the experimental results with diffusion simulations of molecules interacting with such domains in the presence and absence of linear actin meshwork acting as pinpoints for the domains.

Re‐defining ERM function in lymphocyte activation and migration

Lymphocyte activation and migration involve large-scale actin cytoskeletal remodeling. The Ezrin-Radixin-Moesin (ERM) family proteins reversibly link the plasma membrane and cortical actin meshwork and mediate the dynamic nature of the membrane-cytoskeletal interface to facilitate remodeling. The reversibility of this linkage is controlled by the conformation of ERM proteins and depends on the phosphorylation of a conserved threonine residue in the actin-binding domain. Disruption of the phospho-cycling nature of ERM proteins through dominant negative and constitutively active mutants results in impaired lymphocyte migration and activation. In recent years, a novel role has emerged for ERM proteins as signaling scaffolds that can modulate B and T-cell activation through additional posttranslational modifications at tyrosine residues. Here, we highlight recent studies that have redefined the role of ERM proteins in lymphocyte activation and migration. We discuss how lymphocyte-specific knockouts of ERM proteins and high resolution imaging techniques have identified a novel function for them as rheostats that modulate the strength of antigen receptor signaling in B cells. Finally, we describe scenarios in which ERM protein function is coopted by pathogens for their own transmission and speculate on the potential of ERM proteins for regulating undesirable lymphocyte behaviors such as autoimmunity and malignancy.

Cytoskeletal function in the immune system

Cytoskeletal function in the immune system

Filopodia-like Actin Cables Position Nuclei in Association with Perinuclear Actin in Drosophila Nurse Cells

SummaryControlling the position of the nucleus is vital for a number of cellular processes from yeast to humans. In Drosophila nurse cells, nuclear positioning is crucial during dumping, when nurse cells contract and expel their contents into the oocyte. We provide evidence that in nurse cells, continuous filopodia-like actin cables, growing from the plasma membrane and extending to the nucleus, achieve nuclear positioning. These actin cables move nuclei away from ring canals. When nurse cells contract, actin cables associate laterally with the nuclei, in some cases inducing nuclear turning so that actin cables become partially wound around the nuclei. Our data suggest that a perinuclear actin meshwork connects actin cables to nuclei via actin-crosslinking proteins such as the filamin Cheerio. We provide a revised model for how actin structures position nuclei in nurse cells, employing evolutionary conserved machinery.

Involvement of the sieve element cytoskeleton in electrical responses to cold shocks.

This study dealt with the visualization of the sieve element (SE) cytoskeleton and its involvement in electrical responses to local cold shocks, exemplifying the role of the cytoskeleton in Ca(2+)-triggered signal cascades in SEs. High-affinity fluorescent phalloidin as well as immunocytochemistry using anti-actin antibodies demonstrated a fully developed parietal actin meshwork in SEs. The involvement of the cytoskeleton in electrical responses and forisome conformation changes as indicators of Ca(2+) influx was investigated by the application of cold shocks in the presence of diverse actin disruptors (latrunculin A and cytochalasin D). Under control conditions, cold shocks elicited a graded initial voltage transient, ΔV1, reduced by external La(3+) in keeping with the involvement of Ca(2+) channels, and a second voltage transient, ΔV2. Cytochalasin D had no effect on ΔV1, while ΔV1 was significantly reduced with 500 nm latrunculin A. Forisome dispersion was triggered by cold shocks of 4°C or greater, which was indicative of an all-or-none behavior. Forisome dispersion was suppressed by incubation with latrunculin A. In conclusion, the cytoskeleton controls cold shock-induced Ca(2+) influx into SEs, leading to forisome dispersion and sieve plate occlusion in fava bean (Vicia faba).