Welcome to IMC 2018 International Mycological Congress
Conference Calendar
Endosome-coupled mRNA transport during fungal growth
- M. Feldbrügge
Abstract
Abstract
Active transport and local translation of mRNAs ensure the appropriate spatial organization of proteins within cells. Recent work has shown that this process is intricately connected to membrane trafficking. We study the model organism Ustilago maydis. In highly polarized cells of this fungus microtubule-dependent co-transport of mRNAs and endosomes is essential for efficient polar growth. We discuss a novel concept of endosome-coupled translation that loads shuttling endosomes with septin cargo, a process important for correct septin filamentation. Key players are RNA-binding proteins containing RNA recognition motifs for mRNA binding as well as Mademoiselle domains for protein/protein interaction. Here, new insights on protein RNA as well as protein-protein interactions will be presented. Interestingly, evidence is accumulating that RNA and membrane trafficking are also tightly interwoven in higher eukaryotes suggesting that this phenomenon is a common theme and not an exception restricted to fungi.
Phospholipids markers and membrane traffic in Neurospora crassa
- R. Mouriño-Pérez
- I. Murillo-Corona
- O. Callejas-Negrete
- B. Shaw
Abstract
Abstract
In fungal cells, specialized proteins gather in specific places to break cell symmetry and produce hyphae. This organization includes the orchestration of two distinct vesicle processes, endocytosis, and exocytosis that take place in tandem in different areas of the apical compartment in growing hyphae. Part of the signals for endocytosis and endocytosis include the asymmetry of the plasma membrane phospholipid bilayer. We studied the flippases, DNF-1, DRS-2 and DNF-4 that seems to be responsible for this membrane asymmetry in Golgi, vesicles and the plasma membrane. The mutation of dnf-1 and drs-2 genes produced alterations in the maintenance and stability of the Spitzenkörper and affected the actin cytoskeleton organization in the apical compartment. Surprisingly, neither of the flippases DNF-1 and DRS-2 was present in the plasma membrane; both were localized in different layers of the Spitzenkörper, associated to different secretory vesicles. DRS-2 was associated with vesicles transporting chitin synthases. DNF-4 seemed to be present in the Golgi equivalent. Each flippase is in charge of the localization of different phospholipids, their presence in different compartments can predict which phospholipid is more abundant. These results indicate that phospholipid flippases (P4 ATPases) may be important for the polarization of secretory vesicles, Spitzenkörper integrity and thus for the localization of many tip growing proteins.
An Ultrastructural View of the Endomembrane Network in Hyphae of Neurospora crassa
- R. Roberson
Abstract
Abstract
The endomembrane network is a system of cytoplasmic membranes that partition the cell into functional and structural organelles and compartments, which together are involved in biomolecular synthesis, break down, and transport. The endomembrane network of Neurospora crassa hyphae was examined using transmission electron microscopy. All hyphae were prepared by cryofixation and freeze substitution protocols. Neurospora crassa hyphal tip cells contained three cytoplasmic regions based on content and organization. Region I corresponded to the hyphal apex and contained a well-defined Spitzenkörper composed of macro and microvesicles, plus cytoskeletal elements. Golgi equivalents (GE) and aggregations of cisternae with electron-dense lumen were present in this region. Region II extended behind region I approximately 10 to 20 µm and contained abundant vesicles, GE, and rough endoplasmic reticulum (rER). Smooth, flattened cisternae with electron-transparent contents were also present in region II. Endocytotic profiles along the plasma membrane were not common in regions I and II. The transition into region III was marked by abundant nuclei, multivesicular bodies (MVBs), vacuoles containing granular-like material, rER, GE, and flattened cisternae. Cytosolic surfaces of flattened cisternae were coated with a fibrous, electron-dense material. These coated surfaces were restricted to the edges of the flattened cisternae. Microtubules were in close proximity to GE, MVBs, and flattened cisternae. MVBs were diverse in size and shape. Observations of serial sections revealed that vacuoles and flattened cisternae were continuous
Imaging the secretory compartments involved in CHS-4 biosynthesis in Neurospora crassa
- A. Rico-Ramírez
- R. Roberson
- M. Riquelme
Abstract
Abstract
In Neurospora crassa hyphae the localization of all seven chitin synthases (CHSs) at the Spitzenkörper (Spk) and at developing septa has been well analyzed. Hitherto, the mechanisms of CHSs traffic and sorting from synthesis to delivery sites remain largely unexplored. In Saccharomyces cerevisiae exit of Chs3p from the endoplasmic reticulum (ER) requires chaperone Chs7p. Here, we analyzed the role of CSE-7, N. crassa Chs7p orthologue in the biogenesis of CHS-4 (orthologue of Chs3p). In a N. crassa Δcse-7 mutant, CHS-4-GFP no longer accumulated at the Spk and septa. Instead, fluorescence was retained in hyphal subapical regions in an extensive network of elongated cisternae (NEC) referred to previously as tubular vacuoles. In a complemented strain expressing a copy of cse-7 the localization of CHS-4-GFP at the Spk and septa was restored, providing evidence that CSE-7 is necessary for CHS-4 to exit the NEC and for its localization at hyphal tips and septa. CSE-7 was revealed at delimited regions of the ER at the immediacies of nuclei, at the NEC, and remarkably also at septa and the Spk. The organization of the NEC was dependent on the microtubule cytoskeleton. SEC-63, an extensively used ER marker, and NCA-1, a SERCA-type ATPase previously localized at the nuclear envelope, were used as markers to discern the nature of the membranes containing CSE-7. Both SEC-63 and NCA-1 were found at the nuclear envelope, but also at regions of the NEC. However, at the NEC only NCA-1 co-localized extensively with CSE-7. Observations by transmission electron microscopy revealed abundant rough ER sheets and distinct electron translucent smooth flattened cisternae, which could correspond collectively to the NEC, thorough the subapical cytoplasm. This study identifies CSE-7 as the putative ER receptor for its cognate cargo, the polytopic membrane protein CHS-4, and elucidates the complexity of the ER system in filamentous fungi.
Mitosis, nuclear migration and actin formation in Schizophyllum commune
- E. Jung
- M. Raudaskoski
- E. Kothe
Abstract
Abstract
Schizophyllum commune belongs to the white rot basidiomycetes and is relevant for wood degradation worldwide. As early colonizer after forest fire and of tree wounds, the fungus has also phytopathogenic importance. Its high competitive ability is based on the recognition of other bacteria and fungi, the production of specific extracellular metabolites and a strategy of fast growth.The fungal cytoskeleton, composed of a complex network of microtubules and actin structures, has a major impact on transport of vesicles as well as endo- and exocytosis processes. Visualization of the actin cytoskeleton in actively growing hyphae was performed with Lifeact-GFP. Thereby cortical actin patches were visualized at cell tips and clamps and as well as in subapical cells, preceded septation. The actin cytoskeleton in living hyphae during septum development shows close association with nuclear division. Clamp cell formation, typical of many model basidiomycetes including S. commune, indicated an aggregation of actin filaments to ring structures at the future site of nuclear division. Additionally, GFP-labeling of histone H2B enables visualization of nuclear movement and mitosis events in monokaryotic and dikaryotic cells. After mating events, fast nuclear exchange in anastomoses and hyphal cells were observed.
Trafficking of membrane and endocytic cargo proteins in A. nidulans
- B. Commer
- Z. Schultzhaus
- B. Shaw
Abstract
Abstract
During growth, filamentous fungi uniquely produce polarized cells called hyphae. It is generally presumed that polarization of hyphae is dependent upon a mechanism called apical recycling, which maintains a balance between the tightly coupled processes of endocytosis and exocytosis. Endocytosis predominates in an annular domain deemed the sub-apical endocytic collar, which is located in the region of plasma membrane 1-5μm distal to the Spitzenkörper (SPK). Here, a bioinformatics approach was utilized to methodically identify 42 Aspergillus nidulans proteins that are predicted to be cargo of endocytosis based on the presence of an NPFxD (or similar DPFxD) peptide motif. This motif is a necessary endocytic signal sequence first established in the model yeast Saccharomyces cerevisiae, where it marks proteins for endocytosis. The focus of this project is to examine the predicted endocytic association and function of these motif-containing proteins during hyphal growth using fluorescent markers and live-cell imaging. Many of these proteins have orthologs in budding and fission yeasts that have previously been shown to play a role in cell development and regulation of polar cellular morphology. Based on this data, we hypothesize that NPFxD or DPFxD motif-containing proteins in A. nidulans that are cargo for endocytosis will localize to at least one of three regions where cargo are characteristically observed. These predicted regions include the sub-apical collar, where cargo is actively endocytosed and internalized into the hypha, as well as the apical crescent, which lines the membrane at the apex of the hypha and terminates roughly where the sub-apical collar is predicted to begin. The third and final anticipated area of localization is the SPK, which contains two subsets of differently sized vesicles, and is considered to be the key determinant of hyphal growth and directionality. Proteins that are cargo for endocytosis can be observed in each of these areas based on the step(s) of the recycling process they are involved with during membrane turnover. At this time, we have observed localization to the predicted regions associated with hyphal growth for 9 of the 42 motif-containing proteins in A. nidulans. Mutants in these genes have varied in their ability to establish or maintain polarity and also display atypical development in various cell types, which suggests that the genes in question are involved with membrane turnover.