Antonio S. Sechi

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.

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.

Scar/WAVE is localised at the tips of protruding lamellipodia in living cells.

Cell motility entails the extension of cytoplasmic processes, termed lamellipodia and filopodia. Extension is driven by actin polymerisation at the tips of these processes via molecular complexes that remain to be characterised. We show here that a green fluorescent protein (GFP) fusion of the Wiskott–Aldrich syndrome protein family member Scar1/WAVE1 is specifically recruited to the tips of lamellipodia in living B16F1 melanoma cells. Scar1–GFP was recruited only to protruding lamellipodia and was absent from filopodia. The localisation of Scar was facilitated by the finding that the formerly described inhibition of lamellipodia formation by ectopical expression of Scar, could be overcome by the treatment of cells with aluminium fluoride. These findings show that Scar is strategically located at sites of actin polymerisation specifically engaged in the protrusion of lamellipodia.

A Disintegrin and Metalloproteinase 17 (ADAM17) Mediates Inflammation-induced Shedding of Syndecan-1 and -4 by Lung Epithelial Cells

Syndecans are cell surface proteoglycans that bind and modulate various proinflammatory mediators and can be proteolytically shed from the cell surface. Within the lung, syndecan-1 and -4 are expressed as transmembrane proteins on epithelial cells and released in the bronchoalveolar fluid during inflammation. We here characterize the mechanism leading to the generation of soluble syndecan-1 and -4 in cultured epithelial cells and murine lung tissue. We show that the bladder carcinoma epithelial cell line ECV304, the lung epithelial cell line A459 and primary alveolar epithelial cells express and constitutively release syndecan-1 and -4. This release involves the activity of the disintegrin-like metalloproteinase ADAM17 as demonstrated by use of specific inhibitors and lentivirally transduced shRNA. Stimulation of epithelial cells with PMA, thrombin, or proinflammatory cytokines (TNFα/IFNγ) led to the down-regulation of surface-expressed syndecan-1 and -4, which was associated with a significant increase of soluble syndecans and cell-associated cleavage fragments. The enhanced syndecan release was not related to gene induction of syndecans or ADAM17, but rather due to increased ADAM17 activity. Soluble syndecan-1 and -4 were also released into the bronchoalveolar fluid of mice. Treatment with TNFα/IFNγ increased ADAM17 activity and syndecan release in murine lungs. Both constitutive and induced syndecan shedding was prevented by the ADAM17 inhibitor. ADAM17 may therefore be an important regulator of syndecan functions on inflamed lung epithelium.

ENA/VASP proteins: multifunctional regulators of actin cytoskeleton dynamics.

The spatial and temporal regulation of the actin cytoskeleton is fundamental to several cellular processes as diverse as cell motility and immune responses. At the molecular level, the remodelling of the actin cytoskeleton depends on two key events: actin filament nucleation and elongation. Seminal studies on the actin-based intracellular motility of the bacterial pathogen Listeria monocytogenes have been instrumental for the characterisation of a class of actin filament elongating factors, the proteins of the Ena/VASP family. Ena/VASP proteins enhance actin filament elongation via the recruitment of profilin:actin complexes to sites of active actin remodelling such as the tips of spreading lamellipodia and the surface of intracellular Listeria. Moreover, Ena/VASP proteins not only enhance actin filament elongation but also influence the activity of the Arp2/3 complex and counteract the inhibition of actin polymerisation by capping proteins. These findings, taken together with the observation that Ena/VASP proteins can influence actin filament architecture by affecting the actin filament branching activity of the Arp2/3 complex, define Ena/VASP proteins as multifunctional organisers of the actin cytoskeleton.

A crucial role for profilin-actin in the intracellular motility of Listeria monocytogenes

We have examined the effect of covalently crosslinked profilin–actin (PxA), which closely matches the biochemical properties of ordinary profilin–actin and interferes with actin polymerization in vitro and in vivo, on Listeria monocytogenes motility. PxA caused a marked reduction in bacterial motility, which was accompanied by the detachment of bacterial tails. The effect of PxA was dependent on its binding to proline‐rich sequences, as shown by the inability of PH133S xA, which cannot interact with such sequences, to impair Listeria motility. PxA did not alter the motility of a Listeria mutant that is unable to recruit Ena (Enabled)/VASP (vasodilator‐stimulated phosphoprotein) proteins and profilin to its surface. Finally, PxA did not block the initiation of actin‐tail formation, indicating that profilin–actin is only required for the elongation of actin filaments at the bacterial surface. Our findings provide further evidence that profilin–actin is important for actin‐based processes, and show that it has a key function in Listeria motility.

Listeria monocytogenes exploits ERM protein functions to efficiently spread from cell to cell

Cell‐to‐cell spread is a fundamental step in the infection cycle of Listeria monocytogenes that strictly depends on the formation of bacteria‐induced protrusions. Since Listeria actin tails in the protrusions are tightly associated with the plasma membrane, we hypothesised that membrane–cytoskeleton linkers would be required for initiating and sustaining their formation and the subsequent cell‐to‐cell spread. We have found that ezrin, a member of the ezrin, radixin and moesin (ERM) family that functions as a key membrane–cytoskeleton linker, accumulates at Listeria protrusions. The ability of Listeria to induce protrusions and effectively spread between adjacent cells depends on the interaction of ERM proteins with both a membrane component such as CD44 and actin filaments. Interfering with either of these interactions or with ERM proteins phosphorylation not only reduces the number of protrusions but also alters their morphology, resulting in the formation of short and collapsed protrusions. As a consequence, Listeria cell‐to‐cell spread is severely impaired. Thus, ERM proteins are exploited by Listeria to escape the host immune response and to succeed in the development of the infection.

The role of multiple toll-like receptor signalling cascades on interactions between biomedical polymers and dendritic cells

Biomaterials are used in several health-related applications ranging from tissue regeneration to antigen-delivery systems. Yet, biomaterials often cause inflammatory reactions suggesting that they profoundly alter the homeostasis of host immune cells such as dendritic cells (DCs). Thus, there is a major need to understand how biomaterials affect the function of these cells. In this study, we have analysed the influence of chemically and physically diverse biomaterials on DCs using several murine knockouts. DCs can sense biomedical polymers through a mechanism, which involves multiple TLR/MyD88-dependent signalling pathways, in particular TLR2, TLR4 and TLR6. TLR-biomaterial interactions induce the expression of activation markers and pro-inflammatory cytokines and are sufficient to confer on DCs the ability to activate antigen-specific T cells. This happens through a direct biomaterial-DC interaction although, for degradable biomaterials, soluble polymer molecules can also alter DC function. Finally, the engagement of TLRs by biomaterials profoundly alters DC adhesive properties. Our findings could be useful for designing structure-function studies aimed at developing more bioinert materials. Moreover, they could also be exploited to generate biomaterials for studying the molecular mechanisms of TLR signalling and DC activation aiming at fine-tuning desired and pre-determined immune responses.

Primary cultures of glomerular parietal epithelial cells or podocytes with proven origin.

Parietal epithelial cells (PECs) are crucially involved in the pathogenesis of rapidly progressive glomerulonephritis (RPGN) as well as in focal and segmental glomerulosclerosis (FSGS). In this study, transgenic mouse lines were used to isolate pure, genetically tagged primary cultures of PECs or podocytes using FACsorting. By this approach, the morphology of primary glomerular epithelial cells in culture could be resolved: Primary podocytes formed either large cells with intracytoplasmatic extensions or smaller spindle shaped cells, depending on specific culture conditions. Primary PECs were small and exhibited a spindle-shaped or polygonal morphology. In the very early phases of primary culture, rapid changes in gene expression (e.g. of WT-1 and Pax-2) were observed. However, after prolonged culture primary PECs and podocytes still segregated clearly in a transcriptome analysis - demonstrating that the origin of primary cell cultures is important. Of the classical markers, synaptopodin and podoplanin expression were differentially regulated the most in primary PEC and podocyte cultures. However, no expression of any endogenous gene allowed to differentiate between the two cell types in culture. Finally, we show that the transcription factor WT1 is also expressed by PECs. In summary, genetic tagging of PECs and podocytes is a novel and necessary tool to derive pure primary cultures with proven origin. These cultures will be a powerful tool for the emerging field of parietal epithelial cell biology.

Requirements for leukocyte transmigration via the transmembrane chemokine CX3CL1.

The surface-expressed transmembrane CX3C chemokine ligand 1 (CX3CL1/fractalkine) induces firm adhesion of leukocytes expressing its receptor CX3CR1. After shedding by the disintegrins and metalloproteinases (ADAM) 10 and 17, CX3CL1 also acts as soluble leukocyte chemoattractant. Here, we demonstrate that transmembrane CX3CL1 expressed on both endothelial and epithelial cells induces leukocyte transmigration. To investigate the underlying mechanism, we generated CX3CR1 variants lacking the intracellular aspartate-arginine-tyrosine (DRY) motif or the intracellular C-terminus which led to a defect in intracellular calcium response and impaired ligand uptake, respectively. While both variants effectively mediated firm cell adhesion, they failed to induce transmigration and rather mediated retention of leukocytes on the CX3CL1-expressing cell layer. Targeting of ADAM10 led to increased adhesion but reduced transmigration in response to transmembrane CX3CL1, while transmigration towards soluble CX3CL1 was not affected. Thus, transmembrane CX3CL1 mediates leukocyte transmigration via the DRY motif and C-terminus of CX3CR1 and the activity of ADAM10.