Comparative Live-Cell Imaging Analyses of SPA-2, BUD-6 and BNI-1 in Neurospora crassa Reveal Novel Features of the Filamentous Fungal Polarisome

A key multiprotein complex involved in regulating the actin cytoskeleton and secretory machinery required for polarized growth in fungi, is the polarisome. Recognized core constituents in budding yeast are the proteins Spa2, Pea2, Aip3/Bud6, and the key effector Bni1. Multicellular fungi display a more complex polarized morphogenesis than yeasts, suggesting that the filamentous fungal polarisome might fulfill additional functions. In this study, we compared the subcellular organization and dynamics of the putative polarisome components BUD-6 and BNI-1 with those of the bona fide polarisome marker SPA-2 at various developmental stages of Neurospora crassa. All three proteins exhibited a yeast-like polarisome configuration during polarized germ tube growth, cell fusion, septal pore plugging and tip repolarization. However, the localization patterns of all three proteins showed spatiotemporally distinct characteristics during the establishment of new polar axes, septum formation and cytokinesis, and maintained hyphal tip growth. Most notably, in vegetative hyphal tips BUD-6 accumulated as a subapical cloud excluded from the Spitzenkörper (Spk), whereas BNI-1 and SPA-2 partially colocalized with the Spk and the tip apex. Novel roles during septal plugging and cytokinesis, connected to the reinitiation of tip growth upon physical injury and conidial maturation, were identified for BUD-6 and BNI-1, respectively. Phenotypic analyses of gene deletion mutants revealed additional functions for BUD-6 and BNI-1 in cell fusion regulation, and the maintenance of Spk integrity. Considered together, our findings reveal novel polarisome-independent functions of BUD-6 and BNI-1 in Neurospora, but also suggest that all three proteins cooperate at plugged septal pores, and their complex arrangement within the apical dome of mature hypha might represent a novel aspect of filamentous fungal polarisome architecture

Dinucleotide controlled null models for comparative RNA gene prediction

<p>Abstract</p> <p>Background</p> <p>Comparative prediction of RNA structures can be used to identify functional noncoding RNAs in genomic screens. It was shown recently by Babak <it>et al</it>. [BMC Bioinformatics. 8:33] that RNA gene prediction programs can be biased by the genomic dinucleotide content, in particular those programs using a thermodynamic folding model including stacking energies. As a consequence, there is need for dinucleotide-preserving control strategies to assess the significance of such predictions. While there have been randomization algorithms for single sequences for many years, the problem has remained challenging for multiple alignments and there is currently no algorithm available.</p> <p>Results</p> <p>We present a program called SISSIz that simulates multiple alignments of a given average dinucleotide content. Meeting additional requirements of an accurate null model, the randomized alignments are on average of the same sequence diversity and preserve local conservation and gap patterns. We make use of a phylogenetic substitution model that includes overlapping dependencies and site-specific rates. Using fast heuristics and a distance based approach, a tree is estimated under this model which is used to guide the simulations. The new algorithm is tested on vertebrate genomic alignments and the effect on RNA structure predictions is studied. In addition, we directly combined the new null model with the RNAalifold consensus folding algorithm giving a new variant of a thermodynamic structure based RNA gene finding program that is not biased by the dinucleotide content.</p> <p>Conclusion</p> <p>SISSIz implements an efficient algorithm to randomize multiple alignments preserving dinucleotide content. It can be used to get more accurate estimates of false positive rates of existing programs, to produce negative controls for the training of machine learning based programs, or as standalone RNA gene finding program. Other applications in comparative genomics that require randomization of multiple alignments can be considered.</p> <p>Availability</p> <p>SISSIz is available as open source C code that can be compiled for every major platform and downloaded here: <url>http://sourceforge.net/projects/sissiz</url>.</p

Cell fusion in the filmentous fungus, Neurospora crassa

Hyphal fusion occurs at different stages in the vegetative and sexual life cycle of filamentous fungi. Similar to cell fusion in other organisms, the process of hyphal fusion requires cell recognition, adhesion, and membrane merger. Analysis of the hyphal fusion process in the model organism Neurospora crassa using fluorescence and live cell imaging as well as cell and molecular biological techniques has begun to reveal its complex cellular regulation. Several genes required for hyphal fusion have been identified in recent years. While some of these genes are conserved in other eukaryotic species, other genes encode fungal-specific proteins. Analysis of fusion mutants in N. crassa has revealed that genes previously identified as having nonfusion-related functions in other systems have novel hyphal fusion functions in N. crassa. Understanding the molecular basis of cell fusion in filamentous fungi provides a paradigm for cell communication and fusion in eukaryotic organisms. Furthermore, the physiological and developmental roles of hyphal fusion are not understood in these organisms; identifying these mechanisms will provide insight into environmental adaptation

InAs/GaSb type-II superlattices for single- and dual-color focal plane arrays for the mid-infrared spectral range

Focal Plane Arrays for high-performance thermal imaging systems operating in the mid-infrared spectral range between 3-5 µm have been realized by type-II InAs/GaSb short-period superlattices. A fully operational 256 x 256 camera system showing a noise equivalent temperature difference below 10 mK is presented. The suitability of InAs/GaSb type-II superlattices for next generation thermal imagers is demonstrated with the first 288 x 384 dual color demonstrator camera which features a simultaneous and spatially coincident acquisition of two separate spectral regimes in the mid-infrared

InAs/(GaIn)Sb short-period superlattices for focal plane arrays

An infrared camera based on a 256x256 focal plane array for the Mid-IR spectral range (3-5 mu m) has been realized for the first time with InAs/GaSb short-period superlattices. The detector shows a cut-off wavelength of 5.4 mu m and reveals a quantum efficiency of 30%. The noise equivalent temperature difference (NETD) reaches 9.4 mK at 73 K with F/2 optics and 6.5 ms integration time. Excellent thermal images with low NETD values and a very good modulation transfer function are presented. Furthermore, a new method to passivate InAs/GaInSb superlattice photodiodes for the 8-10 mu m regime is demonstrated. The approach is based on the epitaxial overgrowth of wet-etched mesa diodes using lattice matched AlGaAsSb. A complete suppression of surface leakage currents in small sized test diodes with 70 mu m diameter is observed