Transient F-actin Research Articles

Chemokine-mediated F-actin dynamics, polarity, and migration in B lymphocytes depend on WNK1 signaling.

Ligand-engaged chemokine receptors trigger nucleotide exchange in heterotrimeric Gαi proteins, which stimulates cytoskeletal reorganization and cell polarity changes. To better understand the signaling events responsible for these cellular changes, we focused on early changes in F-actin dynamics after engagement of the chemokine receptor CXCR5 in murine splenic B cells. Within 10 seconds of exposure to the CXCR5 ligand CXCL13, three-dimensional lamellar-like pseudopods and F-actin-rich ridges appeared. The transient F-actin increase depended on Gαi2/3 signaling, the PI3K/AKT pathway, ERK activation, phospholipase C activity, and Rac1/2 activation mediated by Dock2 (dedicator of cytokinesis 2). Immunoblot analyses identified the kinase WNK1 (with no lysine kinase 1) as a potential early AKT effector. Treating B cells with specific WNK inhibitors disrupted F-actin dynamics and impaired B cell polarity, motility, and chemotaxis. These changes were mimicked in a murine B cell line by CRISPR-Cas9 gene editing of Wnk1, which also suggested that WNK1 contributed to B cell proliferation. Administration of a single dose of a WNK inhibitor transiently reduced B cell motility and polarity in the lymph nodes of live mice. These results indicate that WNK1 signaling maintains B cell responsiveness to CXCL13 and suggest that pharmacological inhibition of WNK1, which is involved in cancer progression and blood pressure regulation, may affect humoral immunity.

PALS1 is a key regulator of the lateral distribution of tight junction proteins in renal epithelial cells.

The evolutionarily conserved apical Crumbs (CRB) complex, consisting of the core components CRB3a (an isoform of CRB3), PALS1 and PATJ, plays a key role in epithelial cell-cell contact formation and cell polarization. Recently, we observed that deletion of one Pals1 allele in mice results in functional haploinsufficiency characterized by renal cysts. Here, to address the role of PALS1 at the cellular level, we generated CRISPR/Cas9-mediated PALS1-knockout MDCKII cell lines. The loss of PALS1 resulted in increased paracellular permeability, indicating an epithelial barrier defect. This defect was associated with a redistribution of several tight junction-associated proteins from bicellular to tricellular contacts. PALS1-dependent localization of tight junction proteins at bicellular junctions required its interaction with PATJ. Importantly, reestablishment of the tight junction belt upon transient F-actin depolymerization or upon Ca2+ removal was strongly delayed in PALS1-deficient cells. Additionally, the cytoskeleton regulator RhoA was redistributed from junctions into the cytosol under PALS1 knockout. Together, our data uncover a critical role of PALS1 in the coupling of tight junction proteins to the F-actin cytoskeleton, which ensures their correct distribution along bicellular junctions and the formation of tight epithelial barrier.

Caenorhabditis elegans Flamingo FMI-1 controls dendrite self-avoidance through F-actin assembly.

Self-avoidance is a conserved mechanism that prevents crossover between sister dendrites from the same neuron, ensuring proper functioning of the neuronal circuits. Several adhesion molecules are known to be important for dendrite self-avoidance, but the underlying molecular mechanisms are incompletely defined. Here, we show that FMI-1/Flamingo, an atypical cadherin, is required autonomously for self-avoidance in the multidendritic PVD neuron of Caenorhabditis elegans The fmi-1 mutant shows increased crossover between sister PVD dendrites. Our genetic analysis suggests that FMI-1 promotes transient F-actin assembly at the tips of contacting sister dendrites to facilitate their efficient retraction during self-avoidance events, probably by interacting with WSP-1/N-WASP. Mutations of vang-1, which encodes the planar cell polarity protein Vangl2 previously shown to inhibit F-actin assembly, suppress self-avoidance defects of the fmi-1 mutant. FMI-1 downregulates VANG-1 levels probably through forming protein complexes. Our study identifies molecular links between Flamingo and the F-actin cytoskeleton that facilitate efficient dendrite self-avoidance.

F-actin flashes on phagosomes mechanically deform contents for efficient digestion in macrophages.

The mechanism and role of transient F-actin recruitment, or F-actin 'flashes', on phagosomes remains enigmatic. Here we provide a comprehensive characterization of F-actin flashing dynamics on phagosomes, including receptor and signaling involvement. F-actin flashes predominate during the integrin-driven complement receptor (CR)-mediated phagocytosis. F-actin flashes begin shortly after internalization and persist on phagosomes for approximately 3 minutes before disassembling and reassembling several times within the first hour. Strikingly, the appearance of F-actin flashes on phagosomes coincides with morphological deformation, lysis and occasional fission of internalized red blood cells. The cadence of flashes depends on particle stiffness, and the F-actin networks on phagosomes are enriched in mechanosensitive components including focal adhesion proteins, RhoA and actomyosin. Inhibiting Arp2/3 and myosin IIA activity significantly reduces the frequency at which phagosome cargo becomes deformed during transient F-actin accumulation. At later time points, post-F-actin flashing, enhanced degradation of phagosome contents is observed, compared with non-flashing phagosomes. Taken together, these data suggest that actomyosin-driven phagosome contractions serve to disrupt malleable particles physically, a process akin to mastication, to enhance later enzymatic digestion.

Arp2/3 nucleates F-actin coating of fusing insulin granules in pancreatic β cells to control insulin secretion.

F-actin dynamics are known to control insulin secretion, but the point of intersection with the stimulus-secretion cascade is unknown. Here, using multiphoton imaging of β cells isolated from Lifeact-GFP transgenic mice, we show that glucose stimulation does not cause global changes in subcortical F-actin. Instead, we observe spatially discrete and transient F-actin changes around each fusing granule. This F-actin remodelling is dependent on actin nucleation and is observed for granule fusion induced by either glucose or high potassium stimulation. Using GFP-labelled proteins, we identify local enrichment of Arp3, dynamin 2 and clathrin, all occurring after granule fusion, suggesting early recruitment of an endocytic complex to the fusing granules. Block of Arp2/3 activity with drugs or shRNA inhibits F-actin coating, traps granules at the cell membrane and reduces insulin secretion. Block of formin-mediated actin nucleation also blocks F-actin coating, but has no effect on insulin secretion. We conclude that local Arp2/3-dependent actin nucleation at the sites of granule fusion plays an important role in post-fusion granule dynamics and in the regulation of insulin secretion.

Multiple CaMKII Binding Modes to the Actin Cytoskeleton Revealed by Single-Molecule Imaging

Localization of the Ca2+/calmodulin-dependent protein kinase II (CaMKII) to dendritic spine synapses is determined in part by the actin cytoskeleton. We determined binding of GFP-tagged CaMKII to tag-RFP-labeled actin cytoskeleton within live cells using total internal reflection fluorescence microscopy and single-molecule tracking. Stepwise photobleaching showed that CaMKII formed oligomeric complexes. Photoactivation experiments demonstrated that diffusion out of the evanescent field determined the track lifetimes. Latrunculin treatment triggered a coupled loss of actin stress fibers and the colocalized, long-lived CaMKII tracks. The CaMKIIα (α) isoform, which was previously thought to lack F-actin interactions, also showed binding, but this was threefold weaker than that observed for CaMKIIβ (β). The βE′ splice variant bound more weakly than α, showing that binding by β depends critically on the interdomain linker. The mutations βT287D and αT286D, which mimic autophosphorylation states, also abolished F-actin binding. Autophosphorylation triggers autonomous CaMKII activity, but does not impair GluN2B binding, another important synaptic protein interaction of CaMKII. The CaMKII inhibitor tatCN21 or CaMKII mutations that inhibit GluN2B association by blocking binding of ATP (βK43R and αK42M) or Ca2+/calmodulin (βA303R) had no effect on the interaction with F-actin. These results provide the first rationale for the reduced synaptic spine localization of the αT286D mutant, indicating that transient F-actin binding contributes to the synaptic localization of the CaMKIIα isoform. The track lifetime distributions had a stretched exponential form consistent with a heterogeneously diffusing population. This heterogeneity suggests that CaMKII adopts different F-actin binding modes, which is most easily rationalized by multiple subunit contacts between the CaMKII dodecamer and the F-actin cytoskeleton that stabilize the initial weak (micromolar) monovalent interaction.

Perturbations of the actin cytoskeleton activate a Dictyostelium STAT signalling pathway

The Dictyostelium transcription factor STATc is tyrosine phosphorylated and accumulates in the nucleus when cells are exposed either to hyper-osmotic stress or to the prestalk-inducing polyketide DIF-1. In the case of stress STAT activation is mediated by regulated dephosphorylation; whereby two serine residues on PTP3, the tyrosine phosphatase that de-activates STATc, become phosphorylated after exposure to stress so inhibiting enzymatic activity. We now show that the more highly regulated of the two PTP3 serine residues, S747, is also phosphorylated in response to DIF-1, suggesting a common activation mechanism. Hyper-osmotic stress causes a re-distribution of F-actin to the cortex, cell rounding and shrinkage and we show that DIF-1 induces a similar but transient F-actin re-distribution and rounding response. We also find that two mechanistically distinct inhibitors of actin polymerization, latrunculin A and cytochalasin A induce phosphorylation at S747 of PTP3 and activate STATc. We suggest that PTP3 phosphorylation, and consequent STATc activation, are regulated by changes in F-actin polymerization status during stress and DIF-induced cytoskeletal remodelling.

Symmetry breaking in mouse oocytes requires transient F-actin meshwork destabilization

Female meiotic divisions are extremely asymmetric, giving rise to a large oocyte and small degenerating polar bodies, keeping the maternal stores for further embryo development. This asymmetry is achieved via off-center positioning of the division spindle. Mouse oocytes have developed a formin-2-dependent actin-based spindle positioning mechanism that allows the meiotic spindle to migrate towards the closest cortex. Using spinning disk microscopy and FRAP analysis, we studied the changes in the organization of the cytoplasmic F-actin meshwork during the first meiotic division. It is very dense in prophase I, undergoes a significant density drop upon meiosis resumption and reforms progressively later on. This meshwork remodeling correlates with endogenous formin 2 regulation. High formin 2 levels at meiosis I entry induce meshwork maintenance, leading to equal forces being exerted on the chromosomes, preventing spindle migration. Hence, the meshwork density drop at meiosis resumption is germane to the symmetry-breaking event required for successful asymmetric meiotic divisions.

Symmetry breaking in mouse oocytes requires transient F-actin meshwork destabilization

Female meiotic divisions are extremely asymmetric, giving rise to a large oocyte and small degenerating polar bodies, keeping the maternal stores for further embryo development. This asymmetry is achieved via off-center positioning of the division spindle. Mouse oocytes have developed a formin-2-dependent actin-based spindle positioning mechanism that allows the meiotic spindle to migrate towards the closest cortex. Using spinning disk microscopy and FRAP analysis, we studied the changes in the organization of the cytoplasmic F-actin meshwork during the first meiotic division. It is very dense in prophase I, undergoes a significant density drop upon meiosis resumption and reforms progressively later on. This meshwork remodeling correlates with endogenous formin 2 regulation. High formin 2 levels at meiosis I entry induce meshwork maintenance, leading to equal forces being exerted on the chromosomes, preventing spindle migration. Hence, the meshwork density drop at meiosis resumption is germane to the symmetry-breaking event required for successful asymmetric meiotic divisions.

Annexin A1 is a new functional linker between actin filaments and phagosomes during phagocytosis

Remodelling of the actin cytoskeleton plays a key role in particle internalisation and the phagosome maturation processes. Actin-binding proteins (ABPs) are the main players in actin remodelling but the precise role of these proteins in phagocytosis needs to be clarified. Annexins, a group of ABPs, are known to be present on phagosomes. Here, we identified annexin A1 as a factor that binds to isolated latex bead phagosomes (LBPs) in the presence of Ca(2+) and facilitates the F-actin-LBP interaction in vitro. In macrophages the association of endogenous annexin A1 with LBP membranes was strongly correlated with the spatial and temporal accumulation of F-actin at the LBP. Annexin A1 was found on phagocytic cups and around early phagosomes, where the F-actin was prominently concentrated. After uptake was completed, annexin A1, along with F-actin, dissociated from the nascent LBP surface. At later stages of phagocytosis annexin A1 transiently concentrated only around those LBPs that showed transient F-actin accumulation ('actin flashing'). Downregulation of annexin A1 expression resulted in impaired phagocytosis and actin flashing. These data identify annexin A1 as an important component of phagocytosis that appears to link actin accumulation to different steps of phagosome formation.

Actin regulators take the reins in Drosophila myoblast fusion

AbstractSkeletal muscle formation, growth and repair depend on myoblast fusion events. Therefore, in-depth understanding of the underlying molecular mechanisms controlling these events that ultimately lead to skeletal muscle formation may be fundamental for developing new therapies for tissue repair. To this end, the greatest advances in furthering understanding myoblast fusion has been made in Drosophila. Recent studies have shown that transient F-actin structures, so-called actin plugs or foci, are known to form at the site of contacting myoblasts. Indeed, actin regulators of the WASP family that control the activation of the Arp2/3 complex and thereby branched F-actin formation have been demonstrated to be crucial for myoblast fusion. Myoblast-specific cell adhesion molecules seem to be involved in the recruitment of WASP family members to the site of myoblast fusion and form a Fusion-Restricted Myogenic-Adhesive Structure (FuRMAS). Currently, the exact role of the FuRMAS is not completely understood. However, recent studies indicate that WASP-dependent F-actin regulation is required for fusion pore formation as well as for the correct integration of fusing myoblasts into the growing muscle. In this review, I discuss latest cellular studies, and recent genetic and biochemical analyses on actin regulation during myoblast fusion.

MAPKAPK2-mediated LSP1 phosphorylation and FMLP-induced neutrophil polarization

In neutrophils, the major substrate of MAPKAPK2 (MK2) is an F-actin binding protein LSP1. Studies using mutants of the two potential Serine phosphorylation sites in LSP1 C-terminal F-actin binding region indicated that the major phosphorylation site for MK2 is Ser243 in murine neutrophils (Ser252 in humans). Human phosphoLSP1 antibodies that recognize phosphoSer252 site were prepared and revealed fMLP-induced neutrophil LSP1 phosphorylation. The phosphorylation was inhibited by p38 MAPK (upstream kinase for MK2) inhibitor SB203580. The antibodies also detect LSP1 phosphorylation in murine neutrophils. Immunostaining revealed that in WT murine neutrophils phosphoLSP1 was localized in F-actin enriched lamellipodia and oriented toward the fMLP gradient while non-phosphoLSP1 failed to colocalize with F-actin. In suspension, WT neutrophils exhibited persistent F-actin polarization following fMLP stimulation, while MK2 −/− neutrophils exhibited transient F-actin polarization. These studies suggest that MK2-regulated LSP1 phosphorylation is involved in stabilization of F-actin polarization during neutrophil chemotaxis.

Plant and animal profilins are functionally equivalent and stabilize microfilaments in living animal cells.

We have analyzed the degree of functional similarity between birth and mammalian profilins, two members of the profilin family which show only a moderate sequence homology (22%) in living animal cells. The plant profilin, derived from birch pollen, was stably expressed in BHK-21 cells. Plant and endogenous profilin synthesis and cellular distribution were monitored by specific monoclonal antibodies. Quantitation of profilin and actin on calibrated immunoblots showed that two stable clones contained in total 1.4 and 2.0 times as much profilin as the parental cells. Using double fluorescence and confocal laser scanning microscopy, it was seen that the endogenous and the plant profilin colocalized with dynamic microfilaments, in particular with F-actin-rich foci and cortical microfilament webs of spreading cells, with dynamic microfilament bundles induced by serum deprival, and with cytochalasin D- and latrunculin-induced transient F-actin aggregates. The increase in the overall profilin concentration correlated with a significantly higher resistance of actin filaments to these drugs. Our data indicate that even profilins of highly distant evolutionary origin can functionally substitute for each other and support the hypothesis that in animal cells, profilins are engaged in regulating either the stability or the kinetic properties of actin filaments.