Maryse Bailly

Regulation of actin dynamics by annexin 2

Annexin 2 is a ubiquitous Ca2+‐binding protein that is essential for actin‐dependent vesicle transport. Here, we show that in spontaneously motile cells annexin 2 is concentrated in dynamic actin‐rich protrusions, and that depletion of annexin 2 using siRNA leads to the accumulation of stress fibres and loss of protrusive and retractile activity. Cells co‐expressing annexin 2‐CFP and actin‐YFP exhibit Ca2+‐dependent fluorescense resonance energy transfer throughout the cytoplasm and in membrane ruffles and protrusions, suggesting that annexin 2 may directly interact with actin. This notion was supported by biochemical studies, in which we show that annexin 2 reduces the polymerisation rate of actin monomers in a dose‐dependent manner. By measuring actin polymerisation rates in the presence of barbed‐end and pointed‐end cappers, we further demonstrate that annexin 2 specifically inhibits filament elongation at the barbed ends. These results show that annexin 2 has an essential role in maintaining the plasticity of the dynamic membrane‐associated actin cytoskeleton, and that its activity in this context may be at least partly explained through direct interactions with polymerised and monomeric actin.

Actin at cell-cell junctions is composed of two dynamic and functional populations

The ability of epithelial cells to polarize requires cell-cell adhesion mediated by cadherin receptors. During cell-cell contact, the mechanism via which a flat, spread cell shape is changed into a tall, cuboidal epithelial morphology is not known. We found that cadherin-dependent adhesion modulates actin dynamics by triggering changes in actin organization both locally at junctions and within the rest of the cell. Upon induction of cell-cell contacts, two spatial actin populations are distinguishable: junctional actin and peripheral thin bundles. With time, the relative position of these two populations changes and becomes indistinguishable to form a cortical actin ring that is characteristic of mature, fully polarized epithelial cells. Junctional actin and thin actin bundles differ in their actin dynamics and mechanism of formation, and interestingly, have distinct roles during epithelial polarization. Whereas junctional actin stabilizes clustered cadherin receptors at cell-cell contacts, contraction of peripheral actin bundle is essential for an increase in the maximum height at the lateral domain during polarization (cuboidal morphology). Thus, both junctional actin and thin bundles are necessary, and cooperate with each other to generate a polarized epithelial morphology.

The F-actin side binding activity of the Arp2/3 complex is essential for actin nucleation and lamellipod extension

Most eukaryotic cells rely on localized actin polymerization to generate and sustain the protrusion activity necessary for cell movement [1, 2]. Such protrusions are often in the form of a flat lamellipod with a leading edge composed of a dense network of actin filaments [3, 4]. The Arp2/3 complex localizes within that network in vivo [3, 4] and nucleates actin polymerization and generates a branched network of actin filaments in vitro [5-7]. The complex has thus been proposed to generate the actin network at the leading edge of crawling cells in vivo [3, 4, 8]. However, the relative contributions of nucleation and branching to protrusive force are still unknown. We prepared antibodies to the p34 subunit of the Arp2/3 complex that selectively inhibit side binding of the complex to F-actin. We demonstrate that side binding is required for efficient nucleation and branching by the Arp2/3 complex in vitro. However, microinjection of these antibodies into cells specifically inhibits lamellipod extension without affecting the EGF-stimulated appearance of free barbed ends in situ. These results indicate that while the side binding activity of the Arp2/3 complex is required for nucleation in vitro and for protrusive force in vivo, it is not required for EGF-stimulated increases in free barbed ends in vivo. This suggests that the branching activity of the Arp2/3 complex is essential for lamellipod extension, while the generation of nucleation sites for actin polymerization is not sufficient.

Synergistic interaction between the Arp2/3 complex and cofilin drives stimulated lamellipod extension.

Both the Arp2/3 complex and cofilin are believed to be important for the generation of protrusive force at the leading edge; however, their relative contributions have not been explored in vivo. Our results with living cells show that cofilin enters the leading edge immediately before the start of lamellipod extension, slightly earlier than Arp2/3, which begins to be recruited slightly later as the lamellipod is extended. Blocking either the Arp2/3 complex or cofilin function in cells results in failure to extend broad lamellipods and inhibits free barbed ends, suggesting that neither factor on its own can support actin polymerization-mediated protrusion in response to growth factor stimulation. High-resolution analysis of the actin network at the leading edge supports the idea that both the severing activity of cofilin and the specific branching activity of the Arp2/3 complex are essential for lamellipod protrusion. These results are the first to document the relative contributions of cofilin and Arp2/3 complex in vivo and indicate that cofilin begins to initiate the generation of free barbed ends that act in synergy with the Arp2/3 complex to create a large burst in nucleation activity.

Chemoattractant-induced lamellipod extension.

The mammary adenocarcinoma cell line MTLn3 is chemotactic towards epidermal growth factor (EGF), and this induced motility is thought to promote breast cancer invasion and metastasis. Stimulation of MTLn3 cells with EGF results in the extension of a flat, thin structure filled with filamentous actin and termed a lamellipod. Lamellipod extension is dependent on actin polymerization and is localized to the border of adherent cells. The structure of EGF‐stimulated lamellipods in MTLn3 cells is well suited to analysis of chemoattractant‐stimulated protrusion. Actin polymerization occurs within 200 nm of the extending edge of the lamellipod. Although extension of the lamellipod is not dependent upon interaction with the substratum, stabilization of the extended lamellipod is dependent on an adhesive substratum. Dorsal ruffling is suppressed during lamellipod extension. Tyrosine phosphorylation is reduced in preexisting focal contacts compared to new contacts induced by EGF stimulation. The coordination of turnover of focal contacts with lamellipod extension is proposed to result in polarized cell motility in response to gradients of chemoattractants. Microsc. Res. Tech. 43:433–443, 1998.

Cell motility: insights from the backstage

In the true spirit of Michael Abercrombies pioneering studies on cell locomotion, the Fifth Abercrombies Symposium on Cell Behaviour — held in St Catherines College at Oxford University (September 15–18, 2002) — celebrated the intricate beauty of cell motility with an explosion of new technologies that Abercrombie could only have dreamed of. Building on the complementary approaches of quantitative cell biology, biochemistry and genetics, the meeting provided new insights into the ever-growing complexity of the signal transduction pathways involved in cell movement.

Connecting cell adhesion to the actin polymerization machinery: vinculin as the missing link?

Cell migration is essential to many physiological and pathological processes such as embryogenesis, wound healing or metastasis. This complex process involves a tight coordination between three essential steps - protrusion, adhesion and retraction. Although historically protrusion and adhesion have been linked through structural protein-protein interactions, a direct functional link between the two has long eluded biologists. Recent work from the Burridge laboratory now suggests that vinculin, a cytoskeletal protein involved in the building of the adhesion scaffold, could be the missing link that connects early adhesion sites to the actin-driven protrusive machinery.

Polarised Migration: Cofilin Holds the Front

Persistent cell locomotion is a key feature of eukaryotic cells responding to diverse physiological cues. New work now directly implicates ADF/cofilin proteins as essential regulators of polarised cell migration.

Rod disc renewal occurs by evagination of the ciliary plasma membrane that makes cadherin-based contacts with the inner segment

Significance Photoreceptors of the vertebrate retina contain specialized outer segments (OSs) where phototransduction begins. Rod OSs contain stacks of ordered membranous discs that undergo a daily renewal process essential for vision. Mechanisms underlying disc renewal are unclear. The biosynthetic machinery resides in the inner segment (IS), which is connected to the OS via a connecting cilium. Here, we use electron microscopy and tomography to show that the visual pigment, rhodopsin, traffics to the OS via the ciliary plasma membrane, which evaginates to form discs that are initially extracellularly exposed and that make novel contacts with the IS. Leading edges of adjacent evaginations then fuse to form discrete discs. Tomographic analysis leads us to propose a potential mechanism underlying the evagination process. The outer segments of vertebrate rod photoreceptors are renewed every 10 d. Outer segment components are transported from the site of synthesis in the inner segment through the connecting cilium, followed by assembly of the highly ordered discs. Two models of assembly of discrete discs involving either successive fusion events between intracellular rhodopsin-bearing vesicles or the evagination of the plasma membrane followed by fusion of adjacent evaginations have been proposed. Here we use immuno-electron microscopy and electron tomography to show that rhodopsin is transported from the inner to the outer segment via the ciliary plasma membrane, subsequently forming successive evaginations that “zipper” up proximally, but at their leading edges are free to make junctions containing the protocadherin, PCDH21, with the inner segment plasma membrane. Given the physical dimensions of the evaginations, coupled with likely instability of the membrane cortex at the distal end of the connecting cilium, we propose that the evagination occurs via a process akin to blebbing and is not driven by actin polymerization. Disassembly of these junctions is accompanied by fusion of the leading edges of successive evaginations to form discrete discs. This fusion is topologically different to that mediated by the membrane fusion proteins, SNAREs, as initial fusion is between exoplasmic leaflets, and is accompanied by gain of the tetraspanin rim protein, peripherin.

Local delivery of novel MRTF/SRF inhibitors prevents scar tissue formation in a preclinical model of fibrosis

The myocardin-related transcription factor/serum response factor (MRTF/SRF) pathway represents a promising therapeutic target to prevent fibrosis. We have tested the effects of new pharmacological inhibitors of MRTF/SRF signalling in a preclinical model of fibrosis. CCG-222740, a novel MRTF/SRF inhibitor, markedly decreased SRF reporter gene activity and showed a greater inhibitory effect on MRTF/SRF target genes than the previously described MRTF-A inhibitor CCG-203971. CCG-222740 was also five times more potent, with an IC50 of 5 μM, in a fibroblast-mediated collagen contraction assay, was less cytotoxic, and a more potent inhibitor of alpha-smooth muscle actin protein expression than CCG-203971. Local delivery of CCG-222740 and CCG-203971 in a validated and clinically relevant rabbit model of scar tissue formation after glaucoma filtration surgery increased the long-term success of the surgery by 67% (P < 0.0005) and 33% (P < 0.01), respectively, and significantly decreased fibrosis and scarring histologically. Unlike mitomycin-C, neither CCG-222740 nor CCG-203971 caused any detectable epithelial toxicity or systemic side effects with very low drug levels measured in the aqueous, vitreous, and serum. We conclude that inhibitors of MRTF/SRF-regulated gene transcription such as CCG-222740, potentially represent a new therapeutic strategy to prevent scar tissue formation in the eye and other tissues.