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Robert W. Roberson - One of the best experts on this subject based on the ideXlab platform.

  • The actin motor MYO-5 effect in the intracellular organization of Neurospora crassa.
    Fungal genetics and biology : FG & B, 2019
    Co-Authors: Arianne Ramírez-del Villar, Robert W. Roberson, Olga A. Callejas-negrete, Rosa R. Mouriño-pérez
    Abstract:

    In filamentous fungi, polarized growth is the result of vesicle secretion at the hyphal apex. Motor proteins mediate vesicle transport to target destinations on the plasma membrane via actin and microtubule cytoskeletons. Myosins are motor proteins associated with actin filaments. Specifically, class V myosins are responsible for cargo transport in eukaryotes. We studied the dynamics and localization of myosin V in wild type hyphae of Neurospora crassa and in hyphae that lacked MYO-5. In wild type hyphae, MYO-5-GFP was localized concentrated in the hyphal apex and colocalized with Spitzenkorper. Photobleaching studies showed that MYO-5-GFP was transported to the apex from subapical hyphal regions. The deletion of the class V myosin resulted in a reduced rate of hyphal growth, apical hyperbranching, and intermittent loss of hyphal polarity. MYO-5 did not participate in breaking the symmetrical growth during germination but contributed in the apical organization upon establishment of polarized growth. In the Δmyo-5 mutant, actin was organized into thick cables in the apical and subapical hyphal regions, and the number of endocytic patches was reduced. The microvesicles-chitosomes observed with CHS-1-GFP were distributed as a cloud occupying the apical dome and not in the Spitzenkorper as the WT strain. The mitochondrial movement was not associated with MYO-5, but tubular vacuole position is MYO-5-dependent. These results suggest that MYO-5 plays a role in maintaining apical organization and the integrity of the Spitzenkorper and is required for normal hyphal growth, polarity, septation, conidiation, and proper conidial germination.

  • Hyphal tip growth and cytoplasmic characters of Conidiobolus coronatus (Zoopagomycota, Entomophthoromycotina).
    Mycologia, 2018
    Co-Authors: Karen E. Fisher, David Lowry, Isobel Romberger, Phakade Shange, Robert W. Roberson
    Abstract:

    Characteristics of hyphal structure and growth can provide insights into the mechanisms of polarized growth and support investigations of fungal phylogeny. To assist with the resolution of evolutionary relationships of the zygomycetes, the authors used comparative bioimaging methods (light [LM] and transmission electron [TEM] microscopy) to describe selected subcellular characters of hyphal tips of Conidiobolus coronatus. Growing hyphae of C. coronatus contain Spitzenkorper (Spk). Spk are most commonly present in hyphae of Dikarya (Ascomycota and Basidiomycota) and are rarely reported in zygomycete hyphae, which possess an apical vesicle crescent (AVC). Such findings raise questions regarding the evolution of the Spk and its relationship with the AVC. Descriptions of additional subcellular characters (e.g., mitotic-phase spindle pole bodies, cytoplasmic behavior, organelle structure) are also presented.

  • 3 Hyphal Tip Growth in Filamentous Fungi
    Growth Differentiation and Sexuality, 2016
    Co-Authors: Meritxell Riquelme, Robert W. Roberson, Eddy Sánchez-león
    Abstract:

    Hyphae of filamentous fungi extend at the apex by a polarized mechanism that involves the highly ordered and regulated delivery of secretory vesicles. In Dikarya fungi these tip-directed vesicles accumulate temporarily at the Spitzenkorper (Spk) before being delivered to the apical plasma membrane, where they provide the machinery needed for cell wall synthesis. The organizational complexity of the Spk is known only in a few fungal taxa. Yet, it is still unclear whether the structural differences found are correlated with a specific function. Hyphal tip growth requires, in addition, the precise positioning and coordinated interactions of cytoskeleton components and key secretory organelles needed to sustain the continuous process of hyphal elongation.

  • Cytology and molecular phylogenetics of Monoblepharidomycetes provide evidence for multiple independent origins of the hyphal habit in the Fungi
    Mycologia, 2015
    Co-Authors: Jaclyn Dee, Robert W. Roberson, Marilyn R. Noyes Mollicone, Joyce E. Longcore, Mary L. Berbee
    Abstract:

    The evolution of filamentous hyphae underlies an astounding diversity of fungal form and function. We studied the cellular structure and evolutionary origins of the filamentous form in the Monoblepharidomycetes (Chytridiomycota), an early-diverging fungal lineage that displays an exceptional range of body types, from crescent-shaped single cells to sprawling hyphae. To do so, we combined light and transmission electron microscopic analyses of hyphal cytoplasm with molecular phylogenetic reconstructions. Hyphae of Monoblepharidomycetes lack a complex aggregation of secretory vesicles at the hyphal apex (i.e. Spitzenkorper), have centrosomes as primary microtubule organizing centers and have stacked Golgi cisternae instead of tubular/fenestrated Golgi equivalents. The cytoplasmic distribution of actin in Monoblepharidomycetes is comparable to the arrangement observed previously in other filamentous fungi. To discern the origins of Monoblepharidomycetes hyphae, we inferred a phylogeny of the fungi based on 1...

  • The Neurospora crassa exocyst complex tethers Spitzenkörper vesicles to the apical plasma membrane during polarized growth
    Molecular biology of the cell, 2014
    Co-Authors: Meritxell Riquelme, Robert W. Roberson, Olga A. Callejas-negrete, Erin L. Bredeweg, Sarah Ludwig, Alejandro Beltrán-aguilar, Stephan Seiler, Peter Novick, Michael Freitag
    Abstract:

    Fungal hyphae are among the most highly polarized cells. Hyphal polarized growth is supported by tip-directed transport of secretory vesicles, which accumulate temporarily in a stratified manner in an apical vesicle cluster, the Spitzenkorper. The exocyst complex is required for tethering of secretory vesicles to the apical plasma membrane. We determined that the presence of an octameric exocyst complex is required for the formation of a functional Spitzenkorper and maintenance of regular hyphal growth in Neurospora crassa. Two distinct localization patterns of exocyst subunits at the hyphal tip suggest the dynamic formation of two assemblies. The EXO-70/EXO-84 subunits are found at the peripheral part of the Spitzenkorper, which partially coincides with the outer macrovesicular layer, whereas exocyst components SEC-5, -6, -8, and -15 form a delimited crescent at the apical plasma membrane. Localization of SEC-6 and EXO-70 to the plasma membrane and the Spitzenkorper, respectively, depends on actin and microtubule cytoskeletons. The apical region of exocyst-mediated vesicle fusion, elucidated by the plasma membrane-associated exocyst subunits, indicates the presence of an exocytotic gradient with a tip-high maximum that dissipates gradually toward the subapex, confirming the earlier predictions of the vesicle supply center model for hyphal morphogenesis.

Meritxell Riquelme - One of the best experts on this subject based on the ideXlab platform.

  • Off the wall: The rhyme and reason of Neurospora crassa hyphal morphogenesis.
    Cell surface (Amsterdam Netherlands), 2019
    Co-Authors: Jorge Verdín, Eddy Sánchez-león, Rosa A. Fajardo-somera, Adriana M. Rico-ramírez, Leonora Martínez-núñez, Meritxell Riquelme
    Abstract:

    The fungal cell wall building processes are the ultimate determinants of hyphal shape. In Neurospora crassa the main cell wall components, β-1,3-glucan and chitin, are synthesized by enzymes conveyed by specialized vesicles to the hyphal tip. These vesicles follow different secretory routes, which are delicately coordinated by cargo-specific Rab GTPases until their accumulation at the Spitzenkorper. From there, the exocyst mediates the docking of secretory vesicles to the plasma membrane, where they ultimately get fused. Although significant progress has been done on the cellular mechanisms that carry cell wall synthesizing enzymes from the endoplasmic reticulum to hyphal tips, a lot of information is still missing. Here, the current knowledge on N. crassa cell wall composition and biosynthesis is presented with an emphasis on the underlying molecular and cellular secretory processes.

  • fungal morphogenesis from the polarized growth of hyphae to complex reproduction and infection structures
    Microbiology and Molecular Biology Reviews, 2018
    Co-Authors: Meritxell Riquelme, Salomon Bartnickigarcia, Jesus Aguirre, Gerhard H Braus, Michael Feldbrugge, Ursula Fleig, Wilhelm Hansberg, Alfredo Herreraestrella, Jorg Kamper, Ulrich Kuck
    Abstract:

    Filamentous fungi constitute a large group of eukaryotic microorganisms that grow by forming simple tube-like hyphae that are capable of differentiating into more-complex morphological structures and distinct cell types. Hyphae form filamentous networks by extending at their tips while branching in subapical regions. Rapid tip elongation requires massive membrane insertion and extension of the rigid chitin-containing cell wall. This process is sustained by a continuous flow of secretory vesicles that depends on the coordinated action of the microtubule and actin cytoskeletons and the corresponding motors and associated proteins. Vesicles transport cell wall-synthesizing enzymes and accumulate in a special structure, the Spitzenkorper, before traveling further and fusing with the tip membrane. The place of vesicle fusion and growth direction are enabled and defined by the position of the Spitzenkorper, the so-called cell end markers, and other proteins involved in the exocytic process. Also important for tip extension is membrane recycling by endocytosis via early endosomes, which function as multipurpose transport vehicles for mRNA, septins, ribosomes, and peroxisomes. Cell integrity, hyphal branching, and morphogenesis are all processes that are largely dependent on vesicle and cytoskeleton dynamics. When hyphae differentiate structures for asexual or sexual reproduction or to mediate interspecies interactions, the hyphal basic cellular machinery may be reprogrammed through the synthesis of new proteins and/or the modification of protein activity. Although some transcriptional networks involved in such reprogramming of hyphae are well studied in several model filamentous fungi, clear connections between these networks and known determinants of hyphal morphogenesis are yet to be established.

  • 3 Hyphal Tip Growth in Filamentous Fungi
    Growth Differentiation and Sexuality, 2016
    Co-Authors: Meritxell Riquelme, Robert W. Roberson, Eddy Sánchez-león
    Abstract:

    Hyphae of filamentous fungi extend at the apex by a polarized mechanism that involves the highly ordered and regulated delivery of secretory vesicles. In Dikarya fungi these tip-directed vesicles accumulate temporarily at the Spitzenkorper (Spk) before being delivered to the apical plasma membrane, where they provide the machinery needed for cell wall synthesis. The organizational complexity of the Spk is known only in a few fungal taxa. Yet, it is still unclear whether the structural differences found are correlated with a specific function. Hyphal tip growth requires, in addition, the precise positioning and coordinated interactions of cytoskeleton components and key secretory organelles needed to sustain the continuous process of hyphal elongation.

  • Live imaging of β-1,3-glucan synthase FKS-1 in Neurospora crassa hyphae.
    Fungal genetics and biology : FG & B, 2015
    Co-Authors: Eddy Sánchez-león, Meritxell Riquelme
    Abstract:

    The subcellular localization and dynamics of FKS-1, the putative catalytic subunit of the β-1,3-glucan synthase complex, was analyzed in growing hyphae of Neurospora crassa by live confocal microscopy. GFP-tagged FKS-1 accumulated at the outer layer of the Spitzenkorper (Spk), and at the apical plasma membrane (PM). Fluorescence recovery after photobleaching analysis revealed arrival of FKS-1-containing carriers first at the immediate surroundings of the core region of the Spk, and thereafter to the Spk most outer region. The results obtained here and previous data suggest that FKS-1 is transported to the Spk in macrovesicles.

  • The Spitzenkörper: a choreographer of fungal growth and morphogenesis.
    Current opinion in microbiology, 2014
    Co-Authors: Meritxell Riquelme, Eddy Sánchez-león
    Abstract:

    The Spitzenkorper (SPK) is a multicomponent pleomorphic structure found at hyphal apices. It is necessary to maintain hyphal growth and morphogenesis in numerous fungal species, including plant and human pathogens. At the turn of the 21st century extraordinary advances in protein tagging technology and live microscopy allowed uncovering the main molecular constituents of the SPK. Distinct layers of macrovesicles and microvesicles, each carrying different cell wall synthetic enzymes, along with the actin cytoskeleton and related proteins are some of the components that make up the SPK. One of the biggest current challenges is to decipher the functional relationship between the SPK components and macromolecular complexes, such as the polarisome and the exocyst, which partially co-localize within the hyphal dome.

Peter E. Sudbery - One of the best experts on this subject based on the ideXlab platform.

  • Fluorescent proteins illuminate the structure and function of the hyphal tip apparatus.
    Fungal genetics and biology : FG & B, 2011
    Co-Authors: Peter E. Sudbery
    Abstract:

    Fungal hyphae show extreme polarized growth at the tip. Electron microscope studies have revealed a apical body called the Spitzenkorper that is thought to drive polarized growth. Studies of polarized growth in S. cerevisiae have identified the protein components of the polarized growth machinery, that are conserved in other fungi. Fusion of these proteins to GFP and its variants has for the first time allowed the localization of these proteins in real time to the hyphal tip without the need for drastic fixation procedures. Such studies showed that vesicle-associated proteins localize to the Spitzenkorper and identified a second compartment located at the tip surface composed of exocyst and other proteins that mediate the fusion of secretory vesicles with the plasma membrane.

  • The mating projections of Saccharomyces cerevisiae and Candida albicans show key characteristics of hyphal growth
    Fungal biology, 2011
    Co-Authors: Bernardo Chapa-y-lazo, Sheu Lee, Hannah Regan, Peter E. Sudbery
    Abstract:

    Fungi can grow in a variety of growth forms: yeast, pseudohyphae and hyphae. The human fungal pathogen Candida albicans can grow in all three of these forms. In this fungus, hyphal growth is distinguished by the presence of a Spitzenkorper-like structure at the hyphal tip and a band of septin bars around the base of newly evaginated germ tubes. The budding yeast Saccharomyces cerevisiae grows as yeast and pseudohyphae, but is not normally considered to show hyphal growth. We show here that in mating projections of both C. albicans and S. cerevisiae a Spitzenkorper-like structure is present at the growing tip and a band of septin bars is present at the base. Furthermore, in S. cerevisiae mating projections, Spa2 and Bni1 form a cap to the 3-dimensional ball of FM4-64 staining, exactly as previously observed in C. albicans hyphae, suggesting that the putative Spitzenkorper may be a distinct structure from the polarisome. Taken together this work shows that mating projections of both S. cerevisiae and C. albicans show the key characteristics of hyphal growth.

  • Spitzenkorper, exocyst, and polarisome components in Candida albicans hyphae show different patterns of localization and have distinct dynamic properties.
    Eukaryotic cell, 2010
    Co-Authors: Laura A. Jones, Peter E. Sudbery
    Abstract:

    During the extreme polarized growth of fungal hyphae, secretory vesicles are thought to accumulate in a subapical region called the Spitzenkorper. The human fungal pathogen Candida albicans can grow in a budding yeast or hyphal form. When it grows as hyphae, Mlc1 accumulates in a subapical spot suggestive of a Spitzenkorper-like structure, while the polarisome components Spa2 and Bud6 localize to a surface crescent. Here we show that the vesicle-associated protein Sec4 also localizes to a spot, confirming that secretory vesicles accumulate in the putative C. albicans Spitzenkorper. In contrast, exocyst components localize to a surface crescent. Using a combination of fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) experiments and cytochalasin A to disrupt actin cables, we showed that Spitzenkorper-located proteins are highly dynamic. In contrast, exocyst and polarisome components are stably located at the cell surface. It is thought that in Saccharomyces cerevisiae exocyst components are transported to the cell surface on secretory vesicles along actin cables. If each vesicle carried its own complement of exocyst components, then it would be expected that exocyst components would be as dynamic as Sec4 and would have the same pattern of localization. This is not what we observe in C. albicans. We propose a model in which a stream of vesicles arrives at the tip and accumulates in the Spitzenkorper before onward delivery to the plasma membrane mediated by exocyst and polarisome components that are more stable residents of the cell surface.

  • Hyphal growth in Candida albicans requires the phosphorylation of Sec2 by the Cdc28-Ccn1/Hgc1 kinase.
    The EMBO journal, 2010
    Co-Authors: Amy Bishop, Bernardo Chapa-y-lazo, Rachel F. Lane, Richard Beniston, Carl Smythe, Peter E. Sudbery
    Abstract:

    Polarized growth is a fundamental property of cell growth and development. It requires the delivery of post-Golgi secretory vesicles to the site of polarized growth. This process is mediated by Rab GTPases activated by their guanine exchange factors (GEFs). The human fungal pathogen, Candida albicans, can grow in a budded yeast form or in a highly polarized hyphal form, and thus provides a model to study this phenomenon. During hyphal, but not yeast growth, secretory vesicles accumulate in an apical body called a Spitzenkorper, which acts to focus delivery of the vesicles to the tip. Post-Golgi transport of secretory vesicles is mediated by the Rab GTPase Sec4, activated by its GEF Sec2. Using a combination of deletion mapping, in vitro mutagenesis, an analogue-sensitive allele of Cdc28 and an in vitro kinase assay, we show that localization of Sec2 to the Spitzenkorper and normal hyphal development requires phosphorylation of Serine 584 by the cyclin-dependent kinase Cdc28. Thus, as well as controlling passage through the cell cycle, Cdc28 has an important function in controlling polarized secretion.

  • Candida albicans hyphae have a Spitzenkörper that is distinct from the polarisome found in yeast and pseudohyphae.
    Journal of Cell Science, 2005
    Co-Authors: Helen Crampin, Judith Berman, Helen Court, Kenneth R. Finley, Maryam Gerami-nejad, Cheryl A. Gale, Peter E. Sudbery
    Abstract:

    Fungi grow with a variety of morphologies: oval yeast cells, chains of elongated cells called pseudohyphae and long, narrow, tube-like filaments called hyphae. In filamentous fungi, hyphal growth is strongly polarised to the tip and is mediated by the Spitzenkorper, which acts as a supply centre to concentrate the delivery of secretory vesicles to the tip. In the budding yeast Saccharomyces cerevisiae, polarised growth is mediated by the polarisome, a surface cap of proteins that nucleates the formation of actin cables delivering secretory vesicles to the growing tip. The human fungal pathogen, Candida albicans, can grow in all three morphological forms. Here we show the presence of a Spitzenkorper at the tip of C. albicans hyphae as a ball-like localisation of secretory vesicles, together with the formin Bni1 and Mlc1, an ortholog of an S. cerevisiae myosin regulatory light chain. In contrast, in C. albicans yeast cells, pseudohyphae and hyphae Spa2 and Bud6, orthologs of S. cerevisiae polarisome components, as well as the master morphology regulator Cdc42, localise predominantly, but not exclusively, to a surface cap resembling the polarisome of S. cerevisiae yeast cells. A small amount of Cdc42 also localises to the Spitzenkorper. Furthermore, we show differences in the genetic and cytoskeletal requirements, and cell cycle dynamics of polarity determinants in yeast, pseudohyphae and hyphae. These results, together with the cytological differences between the cell types, suggest that the Spitzenkorper and polarisome are distinct structures, that the polarisome and Spitzenkorper coexist in hyphae, and that polarised growth in hyphae is driven by a fundamentally different mechanism to that in yeast and pseudohyphae.

Gero Steinberg - One of the best experts on this subject based on the ideXlab platform.

  • Class V chitin synthase and β(1,3)-glucan synthase co-travel in the same vesicle in Zymoseptoria tritici.
    Fungal genetics and biology : FG & B, 2019
    Co-Authors: Martin Schuster, Celia Guiu-aragones, Gero Steinberg
    Abstract:

    The fungal cell wall consists of proteins and polysaccharides, formed by the co-ordinated activity of enzymes, such as chitin or glucan synthases. These enzymes are delivered via secretory vesicles to the hyphal tip. In the ascomycete Neurospora crassa, chitin synthases and β(1,3)-glucan synthase are transported in different vesicles, whereas they co-travel along microtubules in the basidiomycete Ustilago maydis. This suggests fundamental differences in wall synthesis between taxa. Here, we visualize the class V chitin synthase ZtChs5 and the β(1,3)-glucan synthase ZtGcs1 in the ascomycete Zymoseptoria tritici. Live cell imaging demonstrate that both enzymes co-locate to the apical plasma membrane, but are not concentrated in the Spitzenkorper. Delivery involves co-transport along microtubules of the chitin and glucan synthase. Live cell imaging and electron microscopy suggest that both cell wall synthases locate in the same vesicle. Thus, microtubule-dependent co-delivery of cell wall synthases in the same vesicle is found in asco- and basidiomycetes.

  • Fluorescent markers for the Spitzenkörper and exocytosis in Zymoseptoria tritici.
    Fungal genetics and biology : FG & B, 2015
    Co-Authors: M. Guo, Sreedhar Kilaru, Martin Schuster, Meike A.c. Latz, Gero Steinberg
    Abstract:

    Fungal hyphae are highly polarized cells that invade their substrate by tip growth. In plant pathogenic fungi, hyphal growth is essential for host invasion. This makes polarity factors and secretion regulators potential new targets for novel fungicides. Polarization requires delivery of secretory vesicles to the apical Spitzenkorper, followed by polarized exocytosis at the expanding cell tip. Here, we introduce fluorescent markers to visualize the apical Spitzenkorper and the apical site of exocytosis in hyphae of the wheat pathogen Zymoseptoria tritici. We fused green fluorescent protein to the small GTPase ZtSec4, the myosin light chain ZtMlc1 and the small GTPase ZtRab11 and co-localize the fusion proteins with the dye FM4-64 in the hyphal apex, suggesting that the markers label the hyphal Spitzenkorper in Z. tritici. In addition, we localize GFP-fusions to the exocyst protein ZtExo70, the polarisome protein ZtSpa2. Consistent with results in the ascomycete Neurospora crassa, these markers did localize near the plasma membrane at the hyphal tip and only partially co-localize with FM4-64. Thus, these fluorescent markers are useful molecular tools that allow phenotypic analysis of mutants in Z. tritici. These tools will help develop new avenues of research in our quest to control STB infection in wheat.

  • Hyphal Growth: a Tale of Motors, Lipids, and the Spitzenkörper
    Eukaryotic cell, 2007
    Co-Authors: Gero Steinberg
    Abstract:

    Filamentous fungi are a large and evolutionarily successful group of organisms of enormous ecological importance ([27][1], [114][2]). Fungi also have a considerable impact on our economy because they serve as bio-factories for the industrial production of proteins ([90][3], [130][4]) and because

Brian D. Shaw - One of the best experts on this subject based on the ideXlab platform.

  • Localization of NPFxD motif-containing proteins in Aspergillus nidulans
    Fungal genetics and biology : FG & B, 2020
    Co-Authors: Blake Commer, Zachary Schultzhaus, Brian D. Shaw
    Abstract:

    Abstract During growth, filamentous fungi produce polarized cells called hyphae. It is generally presumed that polarization of hyphae is dependent upon secretion through the Spitzenkorper, as well as a mechanism called apical recycling, which maintains a balance between the tightly coupled processes of endocytosis and exocytosis. Endocytosis predominates in an annular domain called the sub-apical endocytic collar, which is located in the region of plasma membrane 1–5 μm distal to the Spitzenkorper. It has previously been proposed that one function of the sub-apical endocytic collar is to maintain the apical localization of polarization proteins. These proteins mark areas of polarization at the apices of hyphae. However, as hyphae grow, these proteins are displaced along the membrane and some must then be removed at the sub-apical endocytic collar in order to maintain the hyphoid shape. While endocytosis is fairly well characterized in yeast, comparatively little is known about the process in filamentous fungi. Here, a bioinformatics approach was utilized to identify 39 Aspergillus nidulans proteins that are predicted to be cargo of endocytosis based on the presence of an NPFxD peptide motif. This motif is a necessary endocytic signal sequence first established in Saccharomyces cerevisiae, where it marks proteins for endocytosis through an interaction with the adapter protein Sla1p. It is hypothesized that some proteins that contain this NPFxD peptide sequence in A. nidulans will be potential targets for endocytosis, and therefore will localize either to the endocytic collar or to more proximal polarized regions of the cell, e.g. the apical dome or the Spitzenkorper. To test this, a subset of the motif-containing proteins in A. nidulans was tagged with GFP and the dynamic localization was evaluated. The documented localization patterns support the hypothesis that the motif marks proteins for localization to the polarized cell apex in growing hyphae.

  • Phospholipid flippases DnfA and DnfB exhibit differential dynamics within the A. nidulans Spitzenkörper.
    Fungal genetics and biology : FG & B, 2016
    Co-Authors: Zachary Schultzhaus, Rosa R. Mouriño-pérez, Wenhui Zheng, Zonghua Wang, Brian D. Shaw
    Abstract:

    The Spitzenkorper is a structure at the apex of growing cells in many filamentous fungi. Ultrastructural studies indicate that the Spitzenkorper is an organized mass of secretory vesicles, with different types of vesicles present in outer and inner layers. Here, we used live-cell imaging to demonstrate that the phospholipid flippases DnfA and DnfB, which preferentially localize to the outer and inner layers, respectively, exhibit different dynamics in the Spitzenkorper of Aspergillus nidulans. Additionally, deletion of dnfA partially destabilized the Spitzenkorper, while the depletion of cdc50, an essential β-subunit of most flippases, had dramatic effects on hyphal tip organization and morphology.

  • Aspergillus nidulans flippase DnfA is cargo of the endocytic collar and plays complementary roles in growth and phosphatidylserine asymmetry with another flippase, DnfB.
    Molecular microbiology, 2015
    Co-Authors: Zachary Schultzhaus, Huijuan Yan, Brian D. Shaw
    Abstract:

    Summary Endocytosis and exocytosis are strictly segregated at the ends of hyphal cells of filamentous fungi, with a collar of endocytic activity encircling the growing cell tip, which elongates through directed membrane fusion. It has been proposed that this separation supports an endocytic recycling pathway that maintains polar localization of proteins at the growing apex. In a search for proteins in the filamentous fungus Aspergillus nidulans that possess an NPFxD motif, which signals for endocytosis, a Type 4 P-Type ATPase was identified and named DnfA. Interestingly, NPFxD is at a different region of DnfA than the same motif in the Saccharomyces cerevisiae ortholog, although endocytosis is dependent on this motif for both proteins. DnfA is involved in asexual sporulation and polarized growth. Additionally, it is segregated within the Spitzenkorper from another Type 4 P-type ATPase, DnfB. Next, the phosphatidylserine marker GFP::Lact-C2 was expressed in growing hyphae, which revealed that this phospholipid is enriched on the cytosolic face of secretory vesicles. This distribution is affected by deleting either dnfA or dnfB. These findings provide evidence for the spatial and temporal segregation of Type4-ATPases in filamentous fungi, and the asymmetric distribution of phosphatidylserine to the Spitzenkorper in A. nidulans.

  • Aspergillus nidulans ArfB Plays a Role in Endocytosis and Polarized Growth
    Eukaryotic cell, 2008
    Co-Authors: Soo Chan Lee, Sabrina N. Schmidtke, Lawrence J. Dangott, Brian D. Shaw
    Abstract:

    Polarized growth is a biological process observed for many different eukaryotic cell systems, including pollen tube formation (50), neuronal cell development (7), and fungal hyphal growth (5). Filamentous fungi are characterized by extensive polarized growth throughout most of their life cycles. This unique reliance on hyphal morphogenesis makes filamentous fungi an attractive system to study polarized growth. Invasive hyphal growth is an important strategy for animal and plant pathogenic fungi to infect and penetrate their hosts. Therefore, understanding hyphal growth will be critical to the development of therapeutic or antifungal agents. Girbardt reported the existence of the Spitzenkorper (apical body) at growing hyphae and proposed that the Spitzenkorper coincided with the polarized growth site (18). The Spitzenkorper consists of two populations of vesicles, apical vesicles (79 to 90 nm in diameter) and microvesicles (30 to 40 nm in diameter). Microvesicles possess chitin synthesis activity and have been called “chitosomes” (4, 6). The postulated vesicle supply center thought to be necessary to explain hyphal tip growth (19, 20) may correspond with the Spitzenkorper. This has driven the hypothesis that vesicle assembly and trafficking play key roles in polarized growth in filamentous growth. ADP ribosylation factors (Arfs) are small GTPase proteins that function in vesicle assembly and trafficking (34, 36). In Saccharomyces cerevisiae, Arf3 is involved in polarity establishment during budding and an arf3 mutant shows a random budding phenotype (16, 22). The Arf3 protein localizes to cell membranes and predominantly to sites of polarized growth (22). Arf3 also genetically interacts with actin cable and actin patch components, including profilin (Pfy1), Arp2/3 complex protein (Las17p and Vrp1), formins (Bni1 and Bnr1), Arf-GTP exchange factor (Gea1), and actin-organizing proteins (Syp1 and Bud6) (30). Another function of Arf3p in yeast is in membrane trafficking during endocytosis (9, 10). ARF6, the human homolog of S. cerevisiae Arf3p (ScArf3p), is also involved in endocytosis, cytokinesis, and actin cytoskeleton organization (reviewed in reference 17). Shaw et al. reported that a temperature-sensitive N-myristoyl transferase (NMT) mutant displayed a polarity maintenance defect in Aspergillus nidulans (43). Myristate from myristoyl-coenzyme A (CoA) is covalently attached to the secondary glycine of target proteins by NMT increasing in hydrophobicity (24). Myristoylated protein more readily associates with membranes or takes part in hydrophobic protein-protein interactions (24, 41). Many Arfs, including ScArf3p, are N myristoylated (2, 35). We hypothesized that Arf proteins may be the direct connection between N myristoylation and polarized cell morphogenesis in filamentous fungi. Indeed, in S. cerevisiae, N-myristoylated Arfs, including Arf1p, Arf2p, Arf3p, and Arl1p, have been shown to be associated with polarized budding, in which Arf1p, Arf2p, and Arl1p are involved in a secretion pathway through the Golgi network (40, 46). These secretory vesicles may be destined for the Spitzenkorper in filamentous fungi. In this study we found six Arf family proteins in A. nidulans. Three, namely, AN1126.3, AN5020.3, and AN5912.3 (ArfA, ArfB, and ArlA, respectively), are predicted to be myristoylated (31). Overexpression of ArfA has been shown to suppress the swoF myristoylation mutant (32). Here ArfB is investigated further, since it is the putative ortholog of Arf3p in S. cerevisiae associated with polarized bud site selection (22) and actin organization (11, 30). We show that ArfB is important for the establishment and maintenance of hyphal polarity, possibly by directing endocytosis in A. nidulans, and that this role requires N myristoylation of the protein.

  • Polarisome Meets Spitzenkörper: Microscopy, Genetics, and Genomics Converge
    Eukaryotic cell, 2005
    Co-Authors: Steven D. Harris, Robert W. Roberson, Brian D. Shaw, Stephan Seiler, Nick D. Read, Mike Plamann, Michelle Momany
    Abstract:

    The impact of filamentous fungi on human welfare has never been greater. Fungi are acknowledged as the most economically devastating plant pathogens ([1][1]) and are attaining increasing notoriety for their ability to cause life-threatening infections in humans ([57][2], [71][3]), and fungal