Ernestina Castro-Longoria

Architecture and development of the Neurospora crassa hypha -- a model cell for polarized growth.

Neurospora crassa has been at the forefront of biological research from the early days of biochemical genetics to current progress being made in understanding gene and genetic network function. Here, we discuss recent developments in analysis of the fundamental form of fungal growth, development and proliferation -- the hypha. Understanding the establishment and maintenance of polarity, hyphal elongation, septation, branching and differentiation are at the core of current research. The advances in the identification and functional dissection of regulatory as well as structural components of the hypha provide an expanding basis for elucidation of fundamental attributes of the fungal cell. The availability and continuous development of various molecular and microscopic tools, as utilized by an active and co-supportive research community, promises to yield additional important new discoveries on the biology of fungi.

Biosynthesis and microscopic study of metallic nanoparticles

Nanobiotechnology, bionanotechnology, and nanobiology are terms that have emerged in reference to the combination of nanotechnology and biology. Through the convergence of these disciplines, the production of metallic nanoparticles (NPs) using biological material as reducing agents is rapidly progressing. In the near future, the application of clean, non-toxic, and eco-friendly nanostructured material will be possible in industry and/or biomedicine. Currently, there is a wide range of organisms that have been reported to be useful in producing NPs. However, the development of finer protocols and the applicability of biosynthesized nanostructures are presently under study. Silver and gold are among the most studied metals due to their potential use in medical treatment. In fact, silver NPs have been evaluated as antimicrobial agents, having been successfully used against several types of fungi and bacteria. However, the use of such material in our daily life must be carefully evaluated. This article summarizes some of the most significant results using organisms to produce metallic NPs as well as the microscopic analyses used to characterize the nanostructured material obtained, providing a valuable database for future research.

Functional Characterization and Cellular Dynamics of the CDC-42 – RAC – CDC-24 Module in Neurospora crassa

Rho-type GTPases are key regulators that control eukaryotic cell polarity, but their role in fungal morphogenesis is only beginning to emerge. In this study, we investigate the role of the CDC-42 – RAC – CDC-24 module in Neurospora crassa. rac and cdc-42 deletion mutants are viable, but generate highly compact colonies with severe morphological defects. Double mutants carrying conditional and loss of function alleles of rac and cdc-42 are lethal, indicating that both GTPases share at least one common essential function. The defects of the GTPase mutants are phenocopied by deletion and conditional alleles of the guanine exchange factor (GEF) cdc-24, and in vitro GDP-GTP exchange assays identify CDC-24 as specific GEF for both CDC-42 and RAC. In vivo confocal microscopy shows that this module is organized as membrane-associated cap that covers the hyphal apex. However, the specific localization patterns of the three proteins are distinct, indicating different functions of RAC and CDC-42 within the hyphal tip. CDC-42 localized as confined apical membrane-associated crescent, while RAC labeled a membrane-associated ring excluding the region labeled by CDC42. The GEF CDC-24 occupied a strategic position, localizing as broad apical membrane-associated crescent and in the apical cytosol excluding the Spitzenkörper. RAC and CDC-42 also display distinct localization patterns during branch initiation and germ tube formation, with CDC-42 accumulating at the plasma membrane before RAC. Together with the distinct cellular defects of rac and cdc-42 mutants, these localizations suggest that CDC-42 is more important for polarity establishment, while the primary function of RAC may be maintaining polarity. In summary, this study identifies CDC-24 as essential regulator for RAC and CDC-42 that have common and distinct functions during polarity establishment and maintenance of cell polarity in N. crassa.

SERS properties of different sized and shaped gold nanoparticles biosynthesized under different environmental conditions by Neurospora crassa extract.

Surface-enhanced Raman scattering (SERS) is a surface-sensitive technique that enhances Raman scattering by molecules adsorbed on rough metal surfaces. It is known that metal nanoparticles, especially gold and silver nanoparticles, exhibit great SERS properties, which make them very attractive for the development of biosensors and biocatalysts. On the other hand, the development of ecofriendly methods for the synthesis of metallic nanostructures has become the focus of research in several countries, and many microorganisms and plants have already been used to biosynthesize metallic nanostructures. However, the majority of these are pathogenic to plants or humans. Here, we report gold nanoparticles with good SERS properties, biosynthesized by Neurospora crassa extract under different environmental conditions, increasing Raman signals up to 40 times using methylene blue as a target molecule. Incubation of tetrachloroauric acid solution with the fungal extract at 60°C and a pH value of a) 3, b) 5.5, and c) 10 resulted in the formation of gold nanoparticles of a) different shapes like triangles, hexagons, pentagons etc. in a broad size range of about 10-200 nm, b) mostly quasi-spheres with some different shapes in a main size range of 6-23 nm, and c) only quasi-spheres of 3-12 nm. Analyses included TEM, HRTEM, and EDS in order to corroborate the shape and the elemental character of the gold nanoparticles, respectively. The results presented here show that these ‘green’ synthesized gold nanoparticles might have potential applicability in the field of biological sensing.

Ultrastructural Analysis of Candida albicans When Exposed to Silver Nanoparticles

Candida albicans is the most common fungal pathogen in humans, and recently some studies have reported the antifungal activity of silver nanoparticles (AgNPs) against some Candida species. However, ultrastructural analyses on the interaction of AgNPs with these microorganisms have not been reported. In this work we evaluated the effect of AgNPs on C. albicans, and the minimum inhibitory concentration (MIC) was found to have a fungicidal effect. The IC50 was also determined, and the use of AgNPs with fluconazole (FLC), a fungistatic drug, reduced cell proliferation. In order to understand how AgNPs interact with living cells, the ultrastructural distribution of AgNPs in this fungus was determined. Transmission electron microscopy (TEM) analysis revealed a high accumulation of AgNPs outside the cells but also smaller nanoparticles (NPs) localized throughout the cytoplasm. Energy dispersive spectroscopy (EDS) analysis confirmed the presence of intracellular silver. From our results it is assumed that AgNPs used in this study do not penetrate the cell, but instead release silver ions that infiltrate into the cell leading to the formation of NPs through reduction by organic compounds present in the cell wall and cytoplasm.

The polarisome component SPA-2 localizes at the apex of Neurospora crassa and partially colocalizes with the Spitzenkörper.

In fungal hyphae multiple protein complexes assemble at sites of apical growth to maintain cell polarity and promote nucleation of actin. Polarity allows the directional traffic of vesicles to the Spitzenkörper (Spk) prior to fusing with the plasma membrane to provide precursors and enzymes required for cell extension and nutrition. One of these complexes is the polarisome, which in Saccharomyces cerevisiae contains Spa2p, Pea2p, Bud6p/Aip3p and Bni1p. To investigate the localization and role of the polarisome during Spk establishment in Neurospora crassa we tagged SPA-2 with the green fluorescent protein (GFP) and examined growing cells by laser scanning confocal microscopy in elongating germ tubes and mature hyphae. SPA-2-GFP accumulated gradually at the apex of germ tubes, when a FM4-64 stained Spk was not still detectable. When the germlings reached about 40microm in length, a FM4-64 stained Spk started to be apparent and from this point on SPA-2-GFP was observed in the apical region of both germ tubes and mature hyphae, as a hand fan shape with a brighter spot at the base. Fusion of the N. crassa SPA-2-GFP strain with a N. crassa strain expressing chitin synthase 1 (CHS-1) labeled with mCherryFP indicated only partial colocalization of the polarisome and the Spk core. N. crassa SPA-2-GFP was also found at the apex of forming branches but not in septa, suggesting that it participates only in areas of tip growth. A Deltaspa-2 strain displayed hyphae with uneven constrictions, apices with an unstable Spk, reduced growth rate and higher number of branches than the wild type strain, indicating that SPA-2 is required for the stability, behavior and morphology of the Spk and maintenance of regular apical growth in hyphae of N. crassa, although not for polarity or Spk establishment.

Circadian rhythms in Neurospora crassa: Dynamics of the clock component frequency visualized using a fluorescent reporter

The frequency (frq) gene of Neurospora crassa has long been considered essential to the function of this organisms circadian rhythm. Increasingly, deciphering the coupling of core oscillator genes such as frq to the output pathways of the circadian rhythm has become a major focus of circadian research. To address this coupling it is critical to have a reporter of circadian activity that can deliver high resolution spatial and temporal information about the dynamics of core oscillatory proteins such as FRQ. However, due to the difficulty of studying the expression of circadian rhythm genes in aerobic N. crassa cultures, little is known about the dynamics of this gene under physiologically realistic conditions. To address these issues we report a fluorescent fusion to the frq gene using a codon optimized version of the mCherry gene. To trace the expression and accumulation of FRQ-mCherryNC (FRQ-mCh) during the circadian rhythm, growing vegetative hyphae were scanned every hour under confocal microscopy (100x). Fluorescence of FRQ-mCh was detected only at the growing edge of the colony, and located in the cytoplasm and nuclei of vegetative hyphae for a distance of approximately 150-200microm from the apices of leading hyphae. When driven by the frq promoter, apparently there was also a second FRQ entrance into the nucleus during the circadian cycle; however the second entrance had a lower accumulation level than the first entrance. Thus this fluorescent fusion protein has proven useful in tracking the spatial dynamics of the frq protein and has indicated that the dynamics of the FRQ proteins nuclear trafficking may be more complex than previously realized.

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.

CDC-42 and RAC-1 regulate opposite chemotropisms in Neurospora crassa.

ABSTRACT Cell polarization and fusion are crucial developmental processes that occur in response to intracellular and extracellular signals. Asexual spores (conidia) of the mold Neurospora crassa differentiate two types of polarized cell protrusions, germ tubes and conidial anastomosis tubes (CATs), which exhibit negative and positive chemotropism, respectively. We provide the first evidence that shared and separate functions of the Rho-type GTPases CDC-42 and RAC-1 regulate these opposite chemotropisms. We demonstrate that RAC-1 is essential for CAT formation and cell fusion, whereas CDC-42 is necessary and sufficient for normal germ tube development. Cdc42-Rac-interactive-binding (CRIB) reporters were constructed to exclusively label locally activated GTP-bound GTPases. Time course analyses showed that repositioning of these activated GTPase clusters within germ tube and CAT tip apices controls directional growth in the absence of a tip-localized vesicle supply center (Spitzenkörper). We propose a model in which the local assembly of a plasma-membrane-associated GTPase–PAK–MAPK signaling platform regulates chemoattractant perception and secretion in order to synchronize oscillatory cell–cell communication and directional CAT tip growth.

Formation of silk-gold nanocomposite fabric using grapefruit aqueous extract

Gold nanostructures were synthesized by reduction of gold ions using aqueous extract of grapefruit pulp (Citrus paradisi). This eco-friendly bioreduction method allows the formation in solution and support of gold nanostructures on silk fibers. Bioreduction techniques involve biomolecules of grapefruit extract for reducing a gold precursor to obtain different kinds of nanostructures. Carbohydrates and organic acids, present in C. paradise, are believed to be responsible for the formation of nanoparticles. Analysis of gold–silk nanocomposites by electron microscopy shows gold nanostructures with quasi-spherical, hexagonal, and triangle shapes. The evolution of functional groups in the silk fibers before and after the bioreduction process was followed by infrared spectroscopy. Diffuse reflectance spectroscopy (DRS) and laser scanning confocal microscopy (LSCM) were used to probe surface plasmon resonance and fluorescent behavior in the silk–gold composite. This simple and novel methodology for obtaining these types of nanocomposite may have important applications in the development of functional fibers.