scholarly journals The Plasma Membrane Proton Pump PMA-1 Is Incorporated into Distal Parts of the Hyphae Independently of the Spitzenkörper in Neurospora crassa

2013 ◽  
Vol 12 (8) ◽  
pp. 1097-1105 ◽  
Author(s):  
Rosa A. Fajardo-Somera ◽  
Barry Bowman ◽  
Meritxell Riquelme
Keyword(s):  
Plasma Membrane ◽  
Neurospora Crassa ◽  
Nutrient Uptake ◽  
Laser Scanning ◽  
Germ Tube ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Fungal Growth ◽  
Electrochemical Gradient ◽  
Content Type

ABSTRACT Most models for fungal growth have proposed a directional traffic of secretory vesicles to the hyphal apex, where they temporarily aggregate at the Spitzenkörper before they fuse with the plasma membrane (PM). The PM H + -translocating ATPase (PMA-1) is delivered via the classical secretory pathway (endoplasmic reticulum [ER] to Golgi) to the cell surface, where it pumps H + out of the cell, generating a large electrochemical gradient that supplies energy to H + -coupled nutrient uptake systems. To characterize the traffic and delivery of PMA-1 during hyphal elongation, we have analyzed by laser scanning confocal microscopy (LSCM) strains of Neurospora crassa expressing green fluorescent protein (GFP)-tagged versions of the protein. In conidia, PMA-1-GFP was evenly distributed at the PM. During germination and germ tube elongation, PMA-1-GFP was found all around the conidial PM and extended to the germ tube PM, but fluorescence was less intense or almost absent at the tip. Together, the data indicate that the electrochemical gradient driving apical nutrient uptake is generated from early developmental stages. In mature hyphae, PMA-1-GFP localized at the PM at distal regions (>120 μm) and in completely developed septa, but not at the tip, indicative of a distinct secretory route independent of the Spitzenkörper occurring behind the apex.

scholarly journals Functional Analysis of Sterol Transporter Orthologues in the Filamentous Fungus Aspergillus nidulans

2015 ◽  
Vol 14 (9) ◽  
pp. 908-921 ◽  
Author(s):  
Nicole Bühler ◽  
Daisuke Hagiwara ◽  
Norio Takeshita
Keyword(s):  
Plasma Membrane ◽  
Aspergillus Nidulans ◽  
Filamentous Fungi ◽  
Gene Deletion ◽  
Fluorescent Protein ◽  
Fungal Growth ◽  
Hyphal Growth ◽  
Apical Plasma Membrane ◽  
Important Species ◽  
Content Type

ABSTRACT Polarized growth in filamentous fungi needs a continuous supply of proteins and lipids to the growing hyphal tip. One of the important membrane compounds in fungi is ergosterol. At the apical plasma membrane ergosterol accumulations, which are called sterol-rich plasma membrane domains (SRDs). The exact roles and formation mechanism of the SRDs remained unclear, although the importance has been recognized for hyphal growth. Transport of ergosterol to hyphal tips is thought to be important for the organization of the SRDs. Oxysterol binding proteins, which are conserved from yeast to human, are involved in nonvesicular sterol transport. In Saccharomyces cerevisiae seven oxysterol-binding protein homologues (OSH1 to -7) play a role in ergosterol distribution between closely located membranes independent of vesicle transport. We found five homologous genes ( oshA to oshE ) in the filamentous fungi Aspergillus nidulans . The functions of OshA-E were characterized by gene deletion and subcellular localization. Each gene-deletion strain showed characteristic phenotypes and different sensitivities to ergosterol-associated drugs. Green fluorescent protein-tagged Osh proteins showed specific localization in the late Golgi compartments, puncta associated with the endoplasmic reticulum, or diffusely in the cytoplasm. The genes expression and regulation were investigated in a medically important species Aspergillus fumigatus , as well as A. nidulans . Our results suggest that each Osh protein plays a role in ergosterol distribution at distinct sites and contributes to proper fungal growth.

scholarly journals Endocytic Machinery Protein SlaB Is Dispensable for Polarity Establishment but Necessary for Polarity Maintenance in Hyphal Tip Cells of Aspergillus nidulans

2010 ◽  
Vol 9 (10) ◽  
pp. 1504-1518 ◽  
Author(s):  
América Hervás-Aguilar ◽  
Miguel A. Peñalva
Keyword(s):  
Plasma Membrane ◽  
Aspergillus Nidulans ◽  
Germ Tube ◽  
Fluorescent Protein ◽  
Hyphal Growth ◽  
Gene Replacement ◽  
Actin Binding ◽  
Nitrate Medium ◽  
Content Type ◽  
Tube Emergence

ABSTRACT The Aspergillus nidulans endocytic internalization protein SlaB is essential, in agreement with the key role in apical extension attributed to endocytosis. We constructed, by gene replacement, a nitrate-inducible, ammonium-repressible slaB1 allele for conditional SlaB expression. Video microscopy showed that repressed slaB1 cells are able to establish but unable to maintain a stable polarity axis, arresting growth with budding-yeast-like morphology shortly after initially normal germ tube emergence. Using green fluorescent protein (GFP)-tagged secretory v-SNARE SynA, which continuously recycles to the plasma membrane after being efficiently endocytosed, we establish that SlaB is crucial for endocytosis, although it is dispensable for the anterograde traffic of SynA and of the t-SNARE Pep12 to the plasma and vacuolar membrane, respectively. By confocal microscopy, repressed slaB1 germlings show deep plasma membrane invaginations. Ammonium-to-nitrate medium shift experiments demonstrated reversibility of the null polarity maintenance phenotype and correlation of normal apical extension with resumption of SynA endocytosis. In contrast, SlaB downregulation in hyphae that had progressed far beyond germ tube emergence led to marked polarity maintenance defects correlating with deficient SynA endocytosis. Thus, the strict correlation between abolishment of endocytosis and disability of polarity maintenance that we report here supports the view that hyphal growth requires coupling of secretion and endocytosis. However, downregulated slaB1 cells form F-actin clumps containing the actin-binding protein AbpA, and thus F-actin misregulation cannot be completely disregarded as a possible contributor to defective apical extension. Latrunculin B treatment of SlaB-downregulated tips reduced the formation of AbpA clumps without promoting growth and revealed the formation of cortical “comets” of AbpA.

EGF-and NGF-stimulated translocation of cytohesin-1 to the plasma membrane of PC12 cells requires PI 3-kinase activation and a functional cytohesin-1 PH domain

1999 ◽  
Vol 112 (12) ◽  
pp. 1957-1965 ◽  
Author(s):  
K. Venkateswarlu ◽  
F. Gunn-Moore ◽  
J.M. Tavare ◽  
P.J. Cullen
Keyword(s):  
Plasma Membrane ◽  
Pc12 Cells ◽  
Laser Scanning ◽  
Time Course ◽  
Fluorescent Protein ◽  
Dominant Negative ◽  
Head Group ◽  
Nucleotide Exchange ◽  
Ph Domain

ADP-ribosylation factors (ARFs) are small GTP-binding proteins that function as regulators of eukaryotic vesicle trafficking. Cytohesin-1 is a member of a family of ARF guanine nucleotide-exchange factors that contain a C-terminal pleckstrin homology (PH) domain which has been proposed to bind the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3). Here we demonstrate that in vitro, recombinant cytohesin-1 binds, via its PH domain, the inositol head group of PIP3, inositol 1,3,4, 5-tetrakisphosphate (IP4), with an affinity greater than 200-fold higher than the inositol head group of either phosphatidylinositol 4, 5-bisphosphate or phosphatidylinositol 3,4-bisphosphate. Moreover, addition of glycerol or diacetylglycerol to the 1-phosphate of IP4 does not alter the ability to interact with cytohesin-1, data which is entirely consistent with cytohesin-1 functioning as a putative PIP3 receptor. To address whether cytohesin-1 binds PIP3 in vivo, we have expressed a chimera of green fluorescent protein (GFP) fused to the N terminus of cytohesin-1 in PC12 cells. Using laser scanning confocal microscopy we demonstrate that either EGF- or NGF-stimulation of transiently transfected PC12 cells results in a rapid translocation of GFP-cytohesin-1 from the cytosol to the plasma membrane. This translocation is dependent on the cytohesin-1 PH domain and occurs with a time course that parallels the rate of plasma membrane PIP3 production. Furthermore, the translocation requires the ability of either agonist to activate PI 3-kinase, since it is inhibited by wortmannin (100 nM), LY294002 (50 microM) and by coexpression with a dominant negative p85. This data therefore suggests that in vivo cytohesin-1 can interact with PIP3 via its PH domain.

scholarly journals Secretory Pathway-Dependent Localization of the Saccharomyces cerevisiae Rho GTPase-Activating Protein Rgd1p at Growth Sites

2012 ◽  
Vol 11 (5) ◽  
pp. 590-600 ◽  
Author(s):  
Fabien Lefèbvre ◽  
Valérie Prouzet-Mauléon ◽  
Michel Hugues ◽  
Marc Crouzet ◽  
Aurélie Vieillemard ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Cell Cycle Progression ◽  
Secretory Pathway ◽  
Rho Gtpase ◽  
Secretory Vesicles ◽  
Gtpase Activating Protein ◽  
Content Type

ABSTRACT Establishment and maintenance of cell polarity in eukaryotes depends upon the regulation of Rho GTPases. In Saccharomyces cerevisiae , the Rho GTPase activating protein (RhoGAP) Rgd1p stimulates the GTPase activities of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively. Consistent with the distribution of Rho3p and Rho4p, Rgd1p is found mostly in areas of polarized growth during cell cycle progression. Rgd1p was mislocalized in mutants specifically altered for Golgi apparatus-based phosphatidylinositol 4-P [PtdIns(4)P] synthesis and for PtdIns(4,5)P 2 production at the plasma membrane. Analysis of Rgd1p distribution in different membrane-trafficking mutants suggested that Rgd1p was delivered to growth sites via the secretory pathway. Rgd1p may associate with post-Golgi vesicles by binding to PtdIns(4)P and then be transported by secretory vesicles to the plasma membrane. In agreement, we show that Rgd1p coimmunoprecipitated and localized with markers specific to secretory vesicles and cofractionated with a plasma membrane marker. Moreover, in vivo imaging revealed that Rgd1p was transported in an anterograde manner from the mother cell to the daughter cell in a vectoral manner. Our data indicate that secretory vesicles are involved in the delivery of RhoGAP Rgd1p to the bud tip and bud neck.

scholarly journals Life and Death of Fungal Transporters Under the Challenge of Polarity

2020 ◽  
Author(s):  
Sofia Dimou ◽  
George Diallinas
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Cell Polarity ◽  
Secretory Pathway ◽  
Fungal Growth ◽  
Present Evidence ◽  
Life And Death ◽  
Early Endosomes ◽  
Stress Signals

Eukaryotic plasma membrane (PM) transporters face critical challenges that are not widely present in prokaryotes. The two most important issues are proper subcellular traffic and targeting to the PM, and regulated endocytosis in response to physiological, developmental or stress signals. Sorting of transporters from their site of synthesis, the Endoplasmic Reticulum (ER), to the PM has been long thought, but not formally shown, to occur via the conventional Golgi-dependent vesicular secretory pathway. Endocytosis of specific eukaryotic transporters has been studied more systematically and shown to involve ubiquitination, internalization, and sorting to early endosomes, followed by turnover in the MVB/lysosomes/vacuole system. In specific cases internalized transporters have been shown to recycle back to the PM. However, the mechanisms of transporter forward trafficking and turnover have been overturned recently through systematic work in the model fungus Aspergillus nidulans. In this review we present evidence that shows that transporter traffic to the PM takes place through Golgi-bypass and transporter endocytosis operates via a mechanism that is distinct from that of recycling membrane cargoes essential for fungal growth. We discuss these findings in relation to adaptation to challenges imposed by cell polarity in fungi as well as in other eukaryotes and provide a rationale why transporters and possibly other housekeeping membrane proteins ‘avoid’ routes of polar trafficking.

scholarly journals The Equine Herpesvirus 1 US2 Homolog Encodes a Nonessential Membrane-Associated Virion Component

1999 ◽  
Vol 73 (4) ◽  
pp. 3430-3437 ◽  
Author(s):  
Alexandra Meindl ◽  
Nikolaus Osterrieder
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Viral Envelope ◽  
Equine Herpesvirus ◽  
Equine Herpesvirus 1 ◽  
Amino Terminal ◽  
Infected Cells ◽  
Herpesvirus 1

ABSTRACT Experiments were conducted to analyze the equine herpesvirus 1 (EHV-1) gene 68 product which is encoded by the EHV-1 US2 homolog. An antiserum directed against the amino-terminal 206 amino acids of the EHV-1 US2 protein specifically detected a protein with an M r of 34,000 in cells infected with EHV-1 strain RacL11. EHV-1 strain Ab4 encodes a 44,000-M r Us2 protein, whereas vaccine strain RacH, a high-passage derivative of RacL11, encodes a 31,000-M r Us2 polypeptide. Irrespective of its size, the US2 protein was incorporated into virions. The EHV-1 US2 protein localized to membrane and nuclear fractions of RacL11-infected cells and to the envelope fraction of purified virions. To monitor intracellular trafficking of the protein, the green fluorescent protein (GFP) was fused to the carboxy terminus of the EHV-1 US2 protein or to a truncated US2 protein lacking a stretch of 16 hydrophobic amino acids at the extreme amino terminus. Both fusion proteins were detected at the plasma membrane and accumulated in the vicinity of nuclei of transfected cells. However, trafficking of either GFP fusion protein through the secretory pathway could not be demonstrated, and the EHV-1 US2 protein lacked detectable N- and O-linked carbohydrates. Consistent with the presence of the US2 protein in the viral envelope and plasma membrane of infected cells, a US2-negative RacL11 mutant (L11ΔUS2) exhibited delayed penetration kinetics and produced smaller plaques compared with either wild-type RacL11 or a US2-repaired virus. After infection of BALB/c mice with L11ΔUS2, reduced pathogenicity compared with the parental RacL11 virus and the repaired virus was observed. It is concluded that the EHV-1 US2 protein modulates virus entry and cell-to-cell spread and appears to support sustained EHV-1 replication in vivo.

scholarly journals Traffic of Chitin Synthase 1 (CHS-1) to the Spitzenkörper and Developing Septa in Hyphae of Neurospora crassa: Actin Dependence and Evidence of Distinct Microvesicle Populations

2011 ◽  
Vol 10 (5) ◽  
pp. 683-695 ◽  
Author(s):  
Eddy Sánchez-León ◽  
Jorge Verdín ◽  
Michael Freitag ◽  
Robert W. Roberson ◽  
Salomon Bartnicki-Garcia ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Neurospora Crassa ◽  
Laser Scanning ◽  
Fine Particles ◽  
Fluorescent Protein ◽  
Apical Cell ◽  
Subcellular Location ◽  
Chitin Synthase ◽  
Wide Field ◽  
Chitin Synthases

ABSTRACTWe describe the subcellular location of chitin synthase 1 (CHS-1), one of seven chitin synthases inNeurospora crassa. Laser scanning confocal microscopy of growing hyphae showed CHS-1–green fluorescent protein (GFP) localized conspicuously in regions of active wall synthesis, namely, the core of the Spitzenkörper (Spk), the apical cell surface, and developing septa. It was also present in numerous fine particles throughout the cytoplasm plus some large vacuoles in distal hyphal regions. Although the same general subcellular distribution was observed previously for CHS-3 and CHS-6, they did not fully colocalize. Dual labeling showed that the three different chitin synthases were contained in different vesicular compartments, suggesting the existence of a different subpopulation of chitosomes for each CHS. CHS-1–GFP persisted in the Spk during hyphal elongation but disappeared from the septum after its development was completed. Wide-field fluorescence microscopy and total internal reflection fluorescence microscopy revealed subapical clouds of particles, suggestive of chitosomes moving continuously toward the Spk. Benomyl had no effect on CHS-1–GFP localization, indicating that microtubules are not strictly required for CHS trafficking to the hyphal apex. Conversely, actin inhibitors caused severe mislocalization of CHS-1–GFP, indicating that actin plays a major role in the orderly traffic and localization of CHS-1 at the apex.

scholarly journals Structure and Distribution of Organelles and Cellular Location of Calcium Transporters in Neurospora crassa

2009 ◽  
Vol 8 (12) ◽  
pp. 1845-1855 ◽  
Author(s):  
Barry J. Bowman ◽  
Marija Draskovic ◽  
Michael Freitag ◽  
Emma Jean Bowman
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Neurospora Crassa ◽  
Fluorescent Protein ◽  
Protein A ◽  
Vacuolar Membrane ◽  
Red Fluorescent Protein ◽  
Animal Cells ◽  
Marker Proteins ◽  
Hyphal Tip

ABSTRACT We wanted to examine the cellular locations of four Neurospora crassa proteins that transport calcium. However, the structure and distribution of organelles in live hyphae of N. crassa have not been comprehensively described. Therefore, we made recombinant genes that generate translational fusions of putative organellar marker proteins with green or red fluorescent protein. We observed putative endoplasmic reticulum proteins, encoded by grp-78 and dpm, in the nuclear envelope and associated membranes. Proteins of the vacuolar membrane, encoded by vam-3 and vma-1, were in an interconnected network of small tubules and vesicles near the hyphal tip, while in more distal regions they were in large and small spherical vacuoles. Mitochondria, visualized with tagged ARG-4, were abundant in all regions of the hyphae. Similarly, we tagged the four N. crassa proteins that transport calcium with green or red fluorescent protein to examine their cellular locations. NCA-1 protein, a homolog of the SERCA-type Ca2+-ATPase of animal cells, colocalized with the endoplasmic reticulum markers. The NCA-2 and NCA-3 proteins are homologs of Ca2+-ATPases in the vacuolar membrane in yeast or in the plasma membrane in animal cells. They colocalized with markers in the vacuolar membrane, and they also occurred in the plasma membrane in regions of the hyphae more than 1 mm from the tip. The cax gene encodes a Ca2+/H+ exchange protein found in vacuoles. As expected, the CAX protein localized to the vacuolar compartment. We observed, approximately 50 to 100 μm from the tip, a few spherical organelles that had high amounts of tagged CAX protein and tagged subunits of the vacuolar ATPase (VMA-1 and VMA-5). We suggest that this organelle, not described previously in N. crassa, may have a role in sequestering calcium.

scholarly journals Regulation of Pathogenic Spore Germination by CgRac1 in the Fungal Plant Pathogen Colletotrichum gloeosporioides

2011 ◽  
Vol 10 (8) ◽  
pp. 1122-1130 ◽  
Author(s):  
Iris Nesher ◽  
Anna Minz ◽  
Leonie Kokkelink ◽  
Paul Tudzynski ◽  
Amir Sharon
Keyword(s):  
Cell Division ◽  
Plant Pathogen ◽  
Germ Tube ◽  
Colletotrichum Gloeosporioides ◽  
Fluorescent Protein ◽  
Nuclear Division ◽  
Simultaneous Formation ◽  
Content Type ◽  
Germ Tubes

ABSTRACT Colletotrichum gloeosporioides is a facultative plant pathogen: it can live as a saprophyte on dead organic matter or as a pathogen on a host plant. Different patterns of conidial germination have been recognized under saprophytic and pathogenic conditions, which also determine later development. Here we describe the role of CgRac1 in regulating pathogenic germination. The hallmark of pathogenic germination is unilateral formation of a single germ tube following the first cell division. However, transgenic strains expressing a constitutively active CgRac1 (CA-CgRac1) displayed simultaneous formation of two germ tubes, with nuclei continuing to divide in both cells after the first cell division. CA-CgRac1 also caused various other abnormalities, including difficulties in establishing and maintaining cell polarity, reduced conidial and hyphal adhesion, and formation of immature appressoria. Consequently, CA-CgRac1 isolates were completely nonpathogenic. Localization studies with cyan fluorescent protein (CFP)-CgRac1 fusion protein showed that the CgRac1 protein is abundant in conidia and in hyphal tips. Although the CFP signal was equally distributed in both cells of a germinating conidium, reactive oxygen species accumulated only in the cell that produced a germ tube, indicating that CgRac1 was active only in the germinating cell. Collectively, our results show that CgRac1 is a major regulator of asymmetric development and that it is involved in the regulation of both morphogenesis and nuclear division. Modification of CgRac1 activity disrupts the morphogenetic program and prevents fungal infection.

scholarly journals Neurospora crassa NKIN2, a Kinesin-3 Motor, Transports Early Endosomes and Is Required for Polarized Growth

2013 ◽  
Vol 12 (7) ◽  
pp. 1020-1032 ◽  
Author(s):  
Constanze Seidel ◽  
Sergio David Moreno-Velásquez ◽  
Meritxell Riquelme ◽  
Reinhard Fischer
Keyword(s):  
Neurospora Crassa ◽  
Fluorescent Protein ◽  
Early Endosome ◽  
Mechanical Forces ◽  
Structural Components ◽  
Content Type ◽  
Early Endosomes ◽  
Polarized Cells ◽  
Green Fluorescent

ABSTRACT Biological motors are molecular nanomachines, which convert chemical energy into mechanical forces. The combination of mechanoenzymes with structural components, such as the cytoskeleton, enables eukaryotic cells to overcome entropy, generate molecular gradients, and establish polarity. Hyphae of filamentous fungi are among the most polarized cells, and polarity defects are most obvious. Here, we studied the role of the kinesin-3 motor, NKIN2, in Neurospora crassa . We found that NKIN2 localizes as fast-moving spots in the cytoplasm of mature hyphae. To test whether the spots represented early endosomes, the Rab5 GTPase YPT52 was used as an endosomal marker. NKIN2 colocalized with YPT52. Deletion of nkin2 caused strongly reduced endosomal movement. Combined, these results confirm the involvement of NKIN2 in early endosome transport. Introduction of a rigor mutation into NKIN2 labeled with green fluorescent protein (GFP) resulted in decoration of microtubules. Interestingly, NKIN2 rigor was associated with a subpopulation of microtubules, as had been shown earlier for the Aspergillus nidulans orthologue UncA. Other kinesins did not show this specificity.

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