Automated segmentation and tracking for large-scale analysis of focal adhesion dynamics.

Cell adhesion, a process mediated by the formation of discrete structures known as focal adhesions (FAs), is pivotal to many biological events including cell motility. Much is known about the molecular composition of FAs, although our knowledge of the spatio-temporal recruitment and the relative occupancy of the individual components present in the FAs is still incomplete. To fill this gap, an essential prerequisite is a highly reliable procedure for the recognition, segmentation and tracking of FAs. Although manual segmentation and tracking may provide some advantages when done by an expert, its performance is usually hampered by subjective judgement and the long time required in analysing large data sets. Here, we developed a model-based segmentation and tracking algorithm that overcomes these problems. In addition, we developed a dedicated computational approach to correct segmentation errors that may arise from the analysis of poorly defined FAs. Thus, by achieving accurate and consistent FA segmentation and tracking, our work establishes the basis for a comprehensive analysis of FA dynamics under various experimental regimes and the future development of mathematical models that simulate FA behaviour.

Evidence for a molecular complex consisting of Fyb/SLAP, SLP-76, Nck, VASP and WASP that links the actin cytoskeleton to Fcgamma receptor signalling during phagocytosis.

Phagocytosis by macrophages and neutrophils involves the spatial and temporal reorganisation of the actin-based cytoskeleton at sites of particle ingestion. Local polymerisation of actin filaments supports the protrusion of pseudopodia that eventually engulf the particle. Here we have investigated in detail the cytoskeletal events initiated upon engagement of Fc receptors in macrophages. Ena/vasodilator-stimulated phosphoprotein (VASP) proteins were recruited to phagosomes forming around opsonised particles in both primary and immortalised macrophages. Not only did the localisation of Ena/VASP proteins coincide, spatially and temporally, with the phagocytosis-induced reorganisation of actin filaments, but their recruitment to the phagocytic cup was required for the remodelling of the actin cytoskeleton, extension of pseudopodia and efficient particle internalisation. We also report that SLP-76, Vav and profilin were recruited to forming phagosomes. Upon induction of phagocytosis, a large molecular complex, consisting in part of Ena/VASP proteins, the Fyn-binding/SLP-76-associated protein (Fyb/SLAP), Src-homology-2 (SH2)-domain-containing leukocyte protein of 76 kDa (SLP-76), Nck, and the Wiskott-Aldrich syndrome protein (WASP), was formed. Our findings suggest that activation of Fcgamma receptors triggers two signalling events during phagocytosis: one through Fyb/SLAP that leads to recruitment of VASP and profilin; and another through Nck that promotes the recruitment of WASP. These converge to regulate actin polymerisation, controlling the assembly of actin structures that are essential for the process of phagocytosis.

ActA from Listeria monocytogenes can interact with up to four Ena/VASP homology 1 domains simultaneously.

The facultative intracellular human pathogenic bacterium Listeria monocytogenes actively recruits host actin to its surface to achieve motility within infected cells. The bacterial surface protein ActA is solely responsible for this process by mimicking fundamental steps of host cell actin dynamics. ActA, a modular protein, contains an N-terminal actin nucleation site and a central proline-rich motif of the 4-fold repeated consensus sequence FPPPP (FP(4)). This motif is specifically recognized by members of the Ena/VASP protein family. These proteins additionally recruit the profilin-G-actin complex increasing the local concentration of G-actin close to the bacterial surface. By using analytical ultracentrifugation, we show that a single ActA molecule can simultaneously interact with four Ena/VASP homology 1 (EVH1) domains. The four FP(4) sites have roughly equivalent affinities with dissociation constants of about 4 microm. Mutational analysis of the FP(4) motifs indicate that the phenylalanine is mandatory for ActA-EVH1 interaction, whereas in each case exchange of the third proline was tolerated. Finally, by using sedimentation equilibrium centrifugation techniques, we demonstrate that ActA is a monomeric protein. By combining these results, we formulate a stoichiometric model to describe how ActA enables Listeria to utilize efficiently resources of the host cell microfilament for its own intracellular motility.

Annexin 2 has an essential role in actin-based macropinocytic rocketing.

Annexin 2 is a Ca(2+) binding protein that binds to and aggregates secretory vesicles at physiological Ca(2+) levels [1] and that also associates Ca(2+) independently with early endosomes [2, 3]. These properties suggest roles in both exocytosis and endocytosis, but little is known of the dynamics of Annexin 2 distribution in live cells during these processes. We have used evanescent field microscopy to image Annexin 2-GFP in live, secreting rat basophilic leukemia cells and in cells performing pinocytosis. Although we found no evidence of Annexin 2 involvement in exocytosis, we observed an enrichment of Annexin 2-GFP in actin tails propeling macropinosomes. The association of Annexin 2-GFP with rocketing macropinosomes was specific because Annexin 2-GFP was absent from the actin tails of rocketing Listeria. This finding suggests that the association of Annexin 2 with macropinocytic rockets requires native pinosomal membrane. Annexin 2 is necessary for the formation of macropinocytic rockets since overexpression of a dominant-negative Annexin 2 construct abolished the formation of these structures. The same construct did not prevent the movement of Listeria in infected cells. These results show that recruitment of Annexin 2 to nascent macropinosome membranes 16656is an essential prerequisite for actin polymerization-dependent vesicle locomotion.

The actin cytoskeleton and plasma membrane connection: PtdIns(4,5)P(2) influences cytoskeletal protein activity at the plasma membrane.

The co-ordination of rearrangements of the actin cytoskeleton depends on its tight connection to the plasma membrane. Phosphatidylinositol 4,5-bisphosphate is thought to transmit signals originating at the plasma membrane to the underlying actin cytoskeleton. This lipid binds to, and influences the activity of, several actin-associated proteins in vitro that regulate the architecture of the actin cytoskeleton. Signalling intermediates in this process include focal adhesion molecules such as vinculin and members of two families of proteins, ERM and WASP. These proteins interact with phosphatidylinositol 4,5-bisphosphate and appear to be regulated by interplay between small GTPases and phosphatidylinositol 4,5-bisphosphate metabolism, and thus link the plasma membrane with cytoskeletal remodelling.

Mutations of arginine residues within the 146-KKRRK-150 motif of the ActA protein of Listeria monocytogenes abolish intracellular motility by interfering with the recruitment of the Arp2/3 complex.

The recruitment of actin to the surface of intracellular Listeria monocytogenes and subsequent tail formation is dependent on the expression of the bacterial surface protein ActA. Of the different functional domains of ActA identified thus far, the N-terminal region is absolutely required for actin filament recruitment and intracellular motility. Mutational analysis of this domain which abolished actin recruitment by intracellular Listeria monocytogenes identified two arginine residues within the 146-KKRRK-150 motif that are essential for its activity. More specifically, recruitment of the Arp2/3 complex to the bacterial surface, as assessed by immunofluorescence staining with antibodies raised against the p21-Arc protein, was not obtained in these mutants. Consistently, treatment of infected cells with latrunculin B, which abrogated actin filament formation, did not affect association of ActA with p21-Arc at the bacterial surface. Thus, the initial recruitment of the Arp2/3 complex to the bacterial surface is independent of, and precedes, actin polymerisation. Our data suggest that binding of the Arp2/3 complex is mediated by specific interactions dependent on arginine residues within the 146-KKRRK-150 motif present in ActA.

Fyn-binding protein (Fyb)/SLP-76-associated protein (SLAP), Ena/vasodilator-stimulated phosphoprotein (VASP) proteins and the Arp2/3 complex link T cell receptor (TCR) signaling to the actin cytoskeleton.

T cell receptor (TCR)-driven activation of helper T cells induces a rapid polarization of their cytoskeleton towards bound antigen presenting cells (APCs). We have identified the Fyn- and SLP-76-associated protein Fyb/SLAP as a new ligand for Ena/ vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domains. Upon TCR engagement, Fyb/SLAP localizes at the interface between T cells and anti-CD3-coated beads, where Evl, a member of the Ena/VASP family, Wiskott-Aldrich syndrome protein (WASP) and the Arp2/3 complex are also found. In addition, Fyb/SLAP is restricted to lamellipodia of spreading platelets. In activated T cells, Fyb/SLAP associates with Ena/VASP family proteins and is present within biochemical complexes containing WASP, Nck, and SLP-76. Inhibition of binding between Fyb/SLAP and Ena/VASP proteins or WASP and the Arp2/3 complex impairs TCR-dependent actin rearrangement, suggesting that these interactions play a key role in linking T cell signaling to remodeling of the actin cytoskeleton.

Accumulation of profilin II at the surface of Listeria is concomitant with the onset of motility and correlates with bacterial speed.

The spatial and temporal activity of the actin cytoskeleton is precisely regulated during cell motility by several microfilament-associated proteins of which profilin plays an essential role. We have analysed the distribution of green fluorescent protein (GFP)-tagged profilins in cultured and in Listeria-infected cells. Among the different GFP-profilin fusion proteins studied, only the construct in which the GFP moiety was fused to the carboxy terminus of profilin II (profilin II-GFP) was recruited by intracellular Listeria. The in vitro ligand-binding properties of this construct, e.g. the binding to monomeric actin, poly-L-proline and phosphatidylinositol 4,5-bisphosphate (PIP2), were unaffected by GFP. Profilin II-GFP co-localised with vinculin and Mena to the focal adhesions in REF-52 fibroblasts and was distributed as a thin line at the front of protruding lamellipodia in B16-F1 mouse melanoma cells. In Listeria-infected cells, profilin II-GFP was recruited, in an asymmetric fashion, to the surface of Listeria at the onset of motility whereas it was not detectable on non-motile bacteria. In contrast to the vasodilator-stimulated phosphoprotein (VASP), profilin II-GFP localised at the bacterial surface only on motile Listeria. Moreover, the fluorescence intensity of profilin II-GFP directly correlated with the speed of the bacteria. Thus, the use of GFP-tagged profilin II provides new insights into the role of profilins in cellular motility.

The Arp2/3 complex is essential for the actin-based motility of Listeria monocytogenes.

Actin polymerisation is thought to drive the movement of eukaryotic cells and some intracellular pathogens such as Listeria monocytogenes. The Listeria surface protein ActA synergises with recruited host proteins to induce actin polymerisation, propelling the bacterium through the host cytoplasm [1]. The Arp2/3 complex is one recruited host factor [2] [3]; it is also believed to regulate actin dynamics in lamellipodia [4] [5]. The Arp2/3 complex promotes actin filament nucleation in vitro, which is further enhanced by ActA [6] [7]. The Arp2/3 complex also interacts with members of the Wiskott-Aldrich syndrome protein (WASP) [8] family - Scar1 [9] [10] and WASP itself [11]. We interfered with the targeting of the Arp2/3 complex to Listeria by using carboxy-terminal fragments of Scar1 that bind the Arp2/3 complex [11]. These fragments completely blocked actin tail formation and motility of Listeria, both in mouse brain extract and in Ptk2 cells overexpressing Scar1 constructs. In both systems, Listeria could initiate actin cloud formation, but tail formation was blocked. Full motility in vitro was restored by adding purified Arp2/3 complex. We conclude that the Arp2/3 complex is a host-cell factor essential for the actin-based motility of L. monocytogenes, suggesting that it plays a pivotal role in regulating the actin cytoskeleton.

The isolated comet tail pseudopodium of Listeria monocytogenes: a tail of two actin filament populations, long and axial and short and random.

Listeria monocytogenes is driven through infected host cytoplasm by a comet tail of actin filaments that serves to project the bacterium out of the cell surface, in pseudopodia, to invade neighboring cells. The characteristics of pseudopodia differ according to the infected cell type. In PtK2 cells, they reach a maximum length of approximately 15 microm and can gyrate actively for several minutes before reentering the same or an adjacent cell. In contrast, the pseudopodia of the macrophage cell line DMBM5 can extend to >100 microm in length, with the bacteria at their tips moving at the same speed as when at the head of comet tails in bulk cytoplasm. We have now isolated the pseudopodia from PtK2 cells and macrophages and determined the organization of actin filaments within them. It is shown that they possess a major component of long actin filaments that are more or less splayed out in the region proximal to the bacterium and form a bundle along the remainder of the tail. This axial component of filaments is traversed by variable numbers of short, randomly arranged filaments whose number decays along the length of the pseudopodium. The tapering of the tail is attributed to a grading in length of the long, axial filaments. The exit of a comet tail from bulk cytoplasm into a pseudopodium is associated with a reduction in total F-actin, as judged by phalloidin staining, the shedding of alpha-actinin, and the accumulation of ezrin. We propose that this transition reflects the loss of a major complement of short, random filaments from the comet, and that these filaments are mainly required to maintain the bundled form of the tail when its borders are not restrained by an enveloping pseudopodium membrane. A simple model is put forward to explain the origin of the axial and randomly oriented filaments in the comet tail.