Invadopodia: a guided tour

The controlled degradation of extracellular matrix is crucial in physiological and pathological cell invasion alike. In cultured cells, degradation occurs at specific sites where invasive cells make contact with the extracellular matrix via specialized plasma membrane protrusions termed invadopodia. Considerable progress has been made in recent years towards understanding the basic molecular components and the ultrastructural features of invadopodia. This current knowledge will be reviewed here together with some of the most important open questions in invadopodia biology. Considering the substantial interest and momentum in the field, the need for an operational framework to correctly define and identify invadopodia will also be discussed.     

Responses of antioxidants in the lichen Ramalina lacera may serve as an early-warning bioindicator system for the detection of air pollution stress

The aim of this study was to identify, in the lichen Ramalina lacera, antioxidants that could provide indications of air pollution stress, and respond earlier than traditionally used structural/physiological parameters. The pollution-sensitive lichen R. lacera was transplanted from its relatively unpolluted natural habitat to two air-polluted sites for a period of up to 6 months. The superoxide dismutase and catalase activities, total water- and lipid-soluble low-molecular-weight antioxidant capacities and chlorophyll b/chlorophyll a ratios were assessed every 6 weeks. The earliest signs of oxidative stress were detected in the activities of fungal copper/zinc-superoxide dismutase, algal iron-superoxide dismutase and water-soluble low-molecular-weight antioxidants, which increased significantly as early as 42 days after exposure to pollution. Catalase activity increased in lichens transplanted to the polluted sites after 90 days. All activities decreased towards the end of the experiment. The impact of air pollution on R. lacera, using the traditionally employed parameter of chlorophyll b/chlorophyll a ratio, was only detected after 6 months of exposure to air pollution. Our results indicate that antioxidant parameters may serve as improved early-warning indicators of air pollution stress in lichens.     

Actin dynamics at sites of extracellular matrix degradation

The degradation of extracellular matrix (ECM) by proteases is crucial in physiological and pathological cell invasion alike. In vitro, degradation occurs at specific sites where invasive cells make contact with the ECM via specialized plasma membrane protrusions termed invadopodia. Here we present an extensive morpho-functional analysis of invadopodia actively engaged in ECM degradation and show that they are actin comet-based structures, not unlike the well-known bacteria-propelling actin tails. The relative mapping of the basic molecular components of invadopodia to actin tails is also provided. Finally, a live-imaging analysis of invadopodia highlights the intrinsic long-term stability of the structures coupled to a highly dynamic actin turnover. The results offer new insight into the tight coordination between signalling, actin remodelling and trafficking activities occurring at sites of focalized ECM degradation by invadopodia. In conclusion, invadopodia-associated actin comets are a striking example of consistently arising, spontaneous expression of actin-driven propulsion events that also represent a valuable experimental paradigm.     

Functional characterization of the putative Aspergillus nidulans poly(ADP-ribose) polymerase homolog PrpA

Poly(ADP-ribose) polymerase (PARP) is a highly conserved enzyme involved in multiple aspects of animal and plant cell physiology. For example, PARP is thought to be intimately involved in the early signaling events that trigger the DNA damage response. However, the genetic dissection of PARP function has been hindered by the presence of multiple homologs in most animal and plant species. Here, we present the first functional characterization of a putative PARP homolog (PrpA) in a microbial system (Aspergillus nidulans). PrpA belongs to a group of PARP homologs that includes representatives from filamentous fungi and protists. The genetic analysis of prpA demonstrates that it is an essential gene whose role in the DNA damage response is sensitive to gene dosage. Notably, temporal patterns of prpA expression and PrpA-GFP nuclear localization suggest that PrpA acts early in the A. nidulans DNA damage response. Additional studies implicate PrpA in farnesol-induced cell death and in the initiation of asexual development. Collectively, our results provide a gateway for probing the diverse functions of PARP in a sophisticated microbial genetic system.     

Diverse UV-B Resistance of Culturable Bacterial Community from High-Altitude Wetland Water

Isolation of most ultraviolet B (UV-B)-resistant culturable bacteria that occur in the habitat of Laguna Azul, a high-altitude wetland [4554 m above sea level (asl)] from the Northwestern Argentinean Andes, was carried out by culture-based methods. Water from this environment was exposed to UV-B radiation under laboratory conditions during 36 h, at an irradiance of 4.94 W/m². It was found that the total number of bacteria in water samples decreased; however, most of the community survived long-term irradiation (312 nm) (53.3 kJ/m²). The percentage of bacteria belonging to dominant species did not vary significantly, depending on the number of UV irradiation doses. The most resistant microbes in the culturable community were Gram-positive pigmented species (Bacillus megaterium [endospores and/or vegetative cells], Staphylococcus saprophyticus, and Nocardia sp.). Only one Gram-negative bacterium could be cultivated (Acinetobacter johnsonii). Nocardia sp. that survived doses of 3201 kJ/m² were the most resistant bacteria to UV-B treatment. This study is the first report on UV-B resistance of a microbial community isolated from high-altitude extreme environments, and proposes a method for direct isolation of UV-B-resistant bacteria from extreme irradiated environments. This article is dedicated to the memory of Carolina Colin.     

Definition of the bacterial N-glycosylation site consensus sequence

The Campylobacter jejuni pgl locus encodes an N-linked protein glycosylation machinery that can be functionally transferred into Escherichia coli. In this system, we analyzed the elements in the C. jejuni N-glycoprotein AcrA required for accepting an N-glycan. We found that the eukaryotic primary consensus sequence for N-glycosylation is N terminally extended to D/E-Y-N-X-S/T (Y, X not equalP) for recognition by the bacterial oligosaccharyltransferase (OST) PglB. However, not all consensus sequences were N-glycosylated when they were either artificially introduced or when they were present in non-C. jejuni proteins. We were able to produce recombinant glycoproteins with engineered N-glycosylation sites and confirmed the requirement for a negatively charged side chain at position -2 in C. jejuni N-glycoproteins. N-glycosylation of AcrA by the eukaryotic OST in Saccharomyces cerevisiae occurred independent of the acidic residue at the -2 position. Thus, bacterial N-glycosylation site selection is more specific than the eukaryotic equivalent with respect to the polypeptide acceptor sequence.