Endocytic Recycling Pathways in Aspergillus nidulans

ungi, which dominate many ecosystems as major decomposers and pathogens, generally colonize through the formation of long, tubular cells called hyphae. Understanding hyphal growth has been of interest to cell biologists for a century, and holds the potential to provide insights for many eukaryotic systems. Hyphal growth and shape are intimately connected with membrane trafficking, which is divided into exocytosis (membrane fusion) and endocytosis (membrane fission). Both of these processes are, in turn, essential for hyphal growth. However, endocytosis works to remove membrane, and why it should be important for cell expansion is not known. Here, using genetic manipulation combined with live cell imaging, the role of endocytosis in hyphal growth was scrutinized in the fungus Aspergillus nidulans. First, I examined the localization and function of the canonical endocytic coat protein, clathrin, and discovered that it is involved in budding at the Golgi apparatus, but does not appear to play a significant role in endocytosis. Second, I looked for proteins that may traffic through an endocytic recycling pathway within the hyphal tip, which resulted in identification and characterization of the phospholipid flippases DnfA-D in A. nidulans. These proteins regulate phospholipid asymmetry in the plasma membrane and the endocytic pathway, and are predicted to be involved in linking endocytosis and exocytosis. I discovered that DnfA and DnfB are stratified within the hyphal tip, likely on different vesicles, and require endocytosis for their steady-state localization. Loss of either DnfA or DnfB function has a minor effect on hyphal shape and growth, but loss of both is lethal. Additionally, the phospholipid phosphatidylserine is normally concentrated strictly on the outside of secretory vesicles, but the localization of this phospholipid, as well as several secretory proteins, was disrupted and diffused through the cytoplasm in the absence of DnfA. These results highlight the importance of endocytic recycling in the maintenance of polarized hyphal growth, as well the complexity of the homeostatic mechanisms at work in an actively expanding hyphal cell, which involve proteins and a variety of lipids for normal function

Introduction to fungal physiology

This chapter describes some basic aspects of fungal cell physiology, focusing primarily on nutrition, growth, metabolism in unicellular yeasts and filamentous fungi, and cell death. It considers the most common growth forms, the filamentous fungi and unicellular yeasts. Fungal growth involves transport and assimilation of nutrients, followed by their integration into cellular components, followed by biomass increase and eventual cell division or septation. The physiology of vegetative reproduction and its control in fungi has been most widely studied in two model eukaryotes, the budding yeast, Saccharomyces cerevisiae, and the fission yeast, Schizosaccharomyces pombe. An understanding of the death of fungal cells is important from a fundamental viewpoint because fungi, especially yeasts, represent valuable model systems for the study of cellular aging and apoptosis (programed cell death). Recycling and redeployment of cellular material also helps drive the apical growth of filamentous fungi and the mycelium explores and extends through the environment

A Gin4-Like Protein Kinase GIL1 Involvement in Hyphal Growth, Asexual Development, and Pathogenesis in Fusarium graminearum

Fusarium graminearum is the main causal agent of Fusarium head blight (FHB) on wheat and barley. In a previous study, a GIN4-like protein kinase gene, GIL1, was found to be important for plant infection and sexual reproduction. In this study we further characterized the functions of GIL1 kinase in different developmental processes. The Δgil1 mutants were reduced in growth, conidiation, and virulence, and formed whitish and compact colonies. Although phialide formation was rarely observed in the mutants, deletion of GIL1 resulted in increased hyphal branching and increased tolerance to cell wall and cell membrane stresses. The Δgil1mutants produced straight, elongated conidia lacking of distinct foot cells and being delayed in germination. Compared with the wild type, some compartments in the vegetative hyphae of Δgil1 mutants had longer septal distances and increased number of nuclei, suggesting GIL1 is related to cytokinesis and septation. Localization of the GIL1-GFP fusion proteins to the septum and hyphal branching and fusion sites further supported its roles in septation and branching. Overall, our results indicate that GIL1 plays a role in vegetative growth and plant infection in F. graminearum, and is involved in septation and hyphal branching

Pressure cycling technology for challenging proteomic sample processing: application to barnacle adhesive.

AbstractSuccessful proteomic characterization of biological material depends on the development of robust sample processing methods. The acorn barnacle Amphibalanus amphitrite is a biofouling model for adhesive processes, but the identification of causative proteins involved has been hindered by their insoluble nature. Although effective, existing sample processing methods are labor and time intensive, slowing progress in this field. Here, a more efficient sample processing method is described which exploits pressure cycling technology (PCT) in combination with protein solvents. PCT aids in protein extraction and digestion for proteomics analysis. Barnacle adhesive proteins can be extracted and digested in the same tube using PCT, minimizing sample loss, increasing throughput to 16 concurrently processed samples, and decreasing sample processing time to under 8 hours. PCT methods produced similar proteomes in comparison to previous methods. Two solvents which were ineffective at extracting proteins from the adhesive at ambient pressure (urea and methanol) produced more protein identifications under pressure than highly polar hexafluoroisopropanol, leading to the identification and description of >40 novel proteins at the interface. Some of these have homology to proteins with elastomeric properties or domains involved with protein-protein interactions, while many have no sequence similarity to proteins in publicly available databases, highlighting the unique adherent processes evolved by barnacles. The methods described here can not only be used to further characterize barnacle adhesive to combat fouling, but may also be applied to other recalcitrant biological samples, including aggregative or fibrillar protein matrices produced during disease, where a lack of efficient sample processing methods has impeded advancement. Data are available via ProteomeXchange with identifier PXD012730

Cassiosomes are stinging-cell structures in the mucus of the upside-down jellyfish Cassiopea xamachana

This work is licensed under a Creative Commons Attribution 4.0 International License.Snorkelers in mangrove forest waters inhabited by the upside-down jellyfish Cassiopea xamachana report discomfort due to a sensation known as stinging water, the cause of which is unknown. Using a combination of histology, microscopy, microfluidics, videography, molecular biology, and mass spectrometry-based proteomics, we describe C. xamachana stinging-cell structures that we term cassiosomes. These structures are released within C. xamachana mucus and are capable of killing prey. Cassiosomes consist of an outer epithelial layer mainly composed of nematocytes surrounding a core filled by endosymbiotic dinoflagellates hosted within amoebocytes and presumptive mesoglea. Furthermore, we report cassiosome structures in four additional jellyfish species in the same taxonomic group as C. xamachana (Class Scyphozoa; Order Rhizostomeae), categorized as either motile (ciliated) or nonmotile types. This inaugural study provides a qualitative assessment of the stinging contents of C. xamachana mucus and implicates mucus containing cassiosomes and free intact nematocytes as the cause of stinging water

Interneuronal mechanisms for learning-induced switch in a sensory response that anticipates changes in behavioural outcomes

Sensory cues in the natural environment predict reward or punishment, important for survival. For example, the ability to detect attractive tastes indicating palatable food is essential for foraging while the recognition of inedible substrates prevents harm. While some of these sensory responses are innate, they can undergo fundamental changes due to prior experience associated with the stimulus. However, the mechanisms underlying such behavioral switching of an innate sensory response at the neuron and network levels require further investigation. We used the model learning system of Lymnaea stagnalis1, 2, 3 to address the question of how an anticipated aversive outcome reverses the behavioral response to a previously effective feeding stimulus, sucrose. Key to the switching mechanism is an extrinsic inhibitory interneuron of the feeding network, PlB (pleural buccal4,5), which is inhibited by sucrose to allow a feeding response. After multi-trial aversive associative conditioning, pairing sucrose with strong tactile stimuli to the head, PlB’s firing rate increases in response to sucrose application to the lips and the feeding response is suppressed; this learned response is reversed by the photoinactivation of a single PlB. A learning-induced persistent change in the cellular properties of PlB that results in an increase rather than a decrease in its firing rate in response to sucrose provides a neurophysiological mechanism for this behavioral switch. A key interneuron, PeD12 (Pedal-Dorsal 12), of the defensive withdrawal network5,6 does not mediate the conditioned suppression of feeding, but its facilitated output contributes to the sensitization of the withdrawal response

Spatially clustered resources increase male aggregation and mating duration in Drosophila melanogaster

In environments where females mate multiply, males should adjust their behaviour and physiology in response to the perceived level of sperm competition to maximize their fitness. Evidence of such plasticity has been found in several laboratory and field studies, but little is known about the cues stimulating these responses in natural populations. One way in which males appear to assess sperm competition risk is through encounter rates with conspecific males. Such encounter rates may be driven by the spatial distribution of resources required by males (i.e. food patches or potential mates), which in turn affects local density. However, explicit links between resource distribution, male encounter rates and shifts in behaviour related to sperm competition have not been demonstrated. We found that when group size of Drosophila melanogaster males was held constant, a small decrease in the distance between patches of food resources had striking effects on male behaviour. Compared to those from dispersed resources, males on clustered resources had a significantly reduced intermale distance (and hence encounter rate) and subsequently a longer noncompetitive copulation duration, previously shown to be a reliable indicator of male perception of sperm competition risk. The aggregation of resources, operating via increased encounter rate, can stimulate shifts in behaviour affecting male sperm competition performance. Given that the spatial distribution of resources is typically variable in natural populations (and often unpredictable), selection is likely to favour the evolution of plasticity in sexual behaviour where resource aggregation increases the probability of sperm competition

Proteomics Reveals Distinct Changes Associated with Increased Gamma Radiation Resistance in the Black Yeast Exophiala dermatitidis

The yeast Exophiala dermatitidis exhibits high resistance to γ-radiation in comparison to many other fungi. Several aspects of this phenotype have been characterized, including its dependence on homologous recombination for the repair of radiation-induced DNA damage, and the transcriptomic response invoked by acute γ-radiation exposure in this organism. However, these findings have yet to identify unique γ-radiation exposure survival strategies—many genes that are induced by γ-radiation exposure do not appear to be important for recovery, and the homologous recombination machinery of this organism is not unique compared to more sensitive species. To identify features associated with γ-radiation resistance, here we characterized the proteomes of two E. dermatitidis strains—the wild type and a hyper-resistant strain developed through adaptive laboratory evolution—before and after γ-radiation exposure. The results demonstrate that protein intensities do not change substantially in response to this stress. Rather, the increased resistance exhibited by the evolved strain may be due in part to increased basal levels of single-stranded binding proteins and a large increase in ribosomal content, possibly allowing for a more robust, induced response during recovery. This experiment provides evidence enabling us to focus on DNA replication, protein production, and ribosome levels for further studies into the mechanism of γ-radiation resistance in E. dermatitidis and other fungi