Characterization of Neurospora crassa α-Actinin

α-Actinin, an actin-binding protein of the spectrin superfamily, is present in most eukaryotes except plants. It is composed of three domains: N-terminal CH-domains, C-terminal calcium-binding domain (with EF-hand motifs), and a central rod domain. We have cloned and expressed Neurospora crassa α-actinin as GST and GFP fusion proteins for biochemical characterization and in vivo localization, respectively. The intracellular localization pattern of α-actinin suggests that this protein is intimately associated with actin filaments and plays an important role in the processes of germination, hyphal elongation, septum formation, and conidiation. These functions were confirmed by the experiments on the effect of α-actinin gene deletion in N. crass

Overexpression of FurA in Anabaena sp. PCC 7120 Reveals New Targets for This Regulator Involved in Photosynthesis, Iron Uptake and Cellular Morphology

Previous genomic analyses of the filamentous nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 have identified three ferric uptake regulator (Fur) homologs with low sequence identities and probably different functions in the cell. FurA is a constitutive protein that shares the highest homology with Fur from heterotrophic bacteria and appears to be essential for invitro growth. In this study, we have analysed the effects of FurA overexpression on the Anabaena sp. phenotype and investigated which of the observed alterations were directly operated by FurA. Overexpression of the regulator led to changes in cellular morphology, resulting in shorter filaments with rounded cells of different sizes. The furA-overexpressing strain showed a slower photoautotrophic growth and a marked decrease in the oxygen evolution rate. Overexpression of the regulator also decreased both catalase and superoxide dismutase activities, but did not lead to an increase in the levels of intracellular reactive oxygen species. By combining phenotypic studies, reverse transcription-PCR analyses and electrophoretic mobility shift assays, we identified three novel direct targets of FurA, including genes encoding a siderophore outer membrane transporter (schT), bacterial actins (mreBCD) and the PSII reaction center protein D1 (psbA). The affinity of FurA for these novel targets was markedly affected by the absence of divalent metal ions, confirming previous evidence of a critical role for the metal co-repressor in the function of the regulator invivo. The results unravel new cellular processes modulated by FurA, supporting its role as a global transcriptional regulator in Anabaena sp. PCC 712

Bacterial nanocellulose production and biomedical applications

Bacterial nanocellulose (BNC) is a homopolymer of β-1,4 linked glycose, which is synthesized byAcetobacterusing simple culturing methods to allow inexpensive and environmentally friendly small- and large-scale production. Depending on the growth media and types of fermentation methods, ultra-pure cellulose can be obtained with different physio-chemical characteristics. Upon biosynthesis, bacterial cellulose is assembled in the medium into a nanostructured network of glucan polymers that are semitransparent, mechanically highly resistant, but soft and elastic, and with a high capacity to store water and exchange gasses. BNC, generally recognized as safe as well as one of the most biocompatible materials, has been found numerous medical applications in wound dressing, drug delivery systems, and implants of heart valves, blood vessels, tympanic membranes, bones, teeth, cartilages, cornea, and urinary tracts

Chlorotetracycline fluorescent probing of Ca²⁺ sequestration in mitochondria fronting the acidic tip of germ tubes outgrowing from conidia of <i>Neurospora crassa</i>

Fluorescent probing with chlorotetracycline of outgrowing and elongating germ tubes from Neurospora crassa conidia revealed a maximal fluorescence and therefore sequestration of Ca²⁺ ions in their subapical mitochondria

A Stringent Role of F-Actin in the Ascogonial Differentiation of <i>Neurospora crassa Fluffy</i> Mutant

Cytochalasin D, an inhibitor of actin polymerization, interferes with ascogonial differentiation in the fertile fluffy mutant of Neurospora crassa. As the total level of actin and its mRNA remain unchanged, this suggests that it is in its microfilamentous form (F-form) that actin is stringently required for female differentiation

Differential effects of anticytoskeletal compounds on the localization and chemical patterns of actin in germinating conidia of <i>Neurospora crassa</i>

Anti-actin drugs, cytochalasins A and B, inhibited both normal single, and benomyl-induced multiple, germ tube outgrowth from conidia of Neurospora crassa. Actin was cytochemically found to be concentrated in each of the benomyl-induced germ tube tips. No significant quantitative changes either in total actin or its isoforms were measured in the inhibitor-treated germlings. While intact microtubules are required for normal, monopolar axiation of the germ tube, they appear not to be necessary for benomyl-induced multipolar outgrowth which, in contrast, still requires intact actin microfilaments. Microfilaments and microtubules thus play complementary roles in the normal germination of conidia

Actin isoform synthesis and mRNA levels in quiescent and proliferating rat aortic smooth muscle cells in vivo and in vitro

The relative levels of actin isoform synthesis in rat aortic smooth muscle cells (SMC) have been studied in vivo and in culture by means of [35S]methionine incorporation. They have been compared to the functional levels of actin isoform mRNAs, assayed by translation of total cell RNA in a reticulocyte lysate or, in some cases, to the actin isoform RNA content, assayed by Northern-blot hybridization to a total actin cRNA probe. In normal media and in freshly isolated SMC, the relative levels of actin isoform synthesis and the actin mRNA translation products show a remarkable similarity, but differ from the proportions of actin isoforms present in a total cell extract (Gabbiani G, Kocher O, Bloom WS, Vandekerckhove J, Weber K: Actin expression in smooth muscle cells of rat aortic intimal thickening, human atheromatous plaque, and cultured rat aortic media. J Clin Invest 73:148, 1984); (Skalli O, Bloom WS, Ropraz P, Azzarone B, Gabbiani G: Cytoskeletal remodeling of rat aortic smooth muscle cells in vitro: relationship to culture conditions and analogies to in vivo situations. J Submicrosc Cytol, in press 1986). This suggests that different actin isoforms have different stabilities. Fifteen days after balloon induced endothelial denudation in vivo, and after being placed in culture, SMC show a decrease in the proportions of alpha-actin synthesis and alpha-actin mRNA levels with a corresponding increase in these parameters for beta- and lambda-actins. The proportions of actin isoform synthesis and actin mRNA translation products in intimal SMC revert to normal values 60 days after balloon induced endothelial denudation, when the aorta is reendothelialized; however, in culture decreased alpha-actin synthesis and mRNA level persist up to the fifth passage (P5). These changes may be helpful for the understanding of SMC adaptation mechanisms during arterial development and atheromatous plaque formation