Coronin Is a Component of the Endocytic Collar of Hyphae of Neurospora crassa and Is Necessary for Normal Growth and Morphogenesis

Coronin plays a major role in the organization and dynamics of actin in yeast. To investigate the role of coronin in a filamentous fungus (Neurospora crassa), we examined its subcellular localization using fluorescent proteins and the phenotypic consequences of coronin gene (crn-1) deletion in hyphal morphogenesis, Spitzenkörper behavior and endocytosis. Coronin-GFP was localized in patches, forming a subapical collar near the hyphal apex; significantly, it was absent from the apex. The subapical patches of coronin colocalized with fimbrin, Arp2/3 complex, and actin, altogether comprising the endocytic collar. Deletion of crn-1 resulted in reduced hyphal growth rates, distorted hyphal morphology, uneven wall thickness, and delayed establishment of polarity during germination; it also affected growth directionality and increased branching. The Spitzenkörper of Δcrn-1 mutant was unstable; it appeared and disappeared intermittently giving rise to periods of hyphoid-like and isotropic growth respectively. Uptake of FM4-64 in Δcrn-1 mutant indicated a partial disruption in endocytosis. These observations underscore coronin as an important component of F-actin remodeling in N. crassa. Although coronin is not essential in this fungus, its deletion influenced negatively the operation of the actin cytoskeleton involved in the orderly deployment of the apical growth apparatus, thus preventing normal hyphal growth and morphogenesis

The Neurospora crassa exocyst complex tethers Spitzenkörper vesicles to the apical plasma membrane during polarized growth

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 Spitzenkörper. 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 Spitzenkörper 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 Spitzenkörper, 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 Spitzenkörper, 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.This is the publisher’s final pdf. The published article is copyrighted by the author(s) and published by the American Society for Cell Biology. The published article can be found at: http://www.molbiolcell.org/

MTB-3, a Microtubule Plus-End Tracking Protein (+TIP) of Neurospora crassa

The microtubule (MT) "plus end" constitutes the platform for the accumulation of a structurally and functionally diverse group of proteins, collectively called "MT plus-end tracking proteins" (+TIPs). +TIPs control MT dynamics and link MTs to diverse sub-cellular structures. Neurospora crassa MicroTubule Binding protein-3 (MTB-3) is the homolog of yeast EB1, a highly conserved +TIP. To address the function of MTB-3, we examined strains with mtb-3 deletions, and we tagged MTB-3 with GFP to assess its dynamic behavior. MTB-3-GFP was present as comet-like structures distributed more or less homogeneously within the hyphal cytoplasm, and moving mainly towards the apex at speeds up to 4x faster than the normal hyphal elongation rates. MTB-3-GFP comets were present in all developmental stages, but were most abundant in mature hyphae. MTB-3-GFP comets were observed moving in anterograde and retrograde direction along the hypha. Retrograde movement was also observed as originating from the apical dome. The integrity of the microtubular cytoskeleton affects the presence and dynamics of MTB-3-GFP comets, while actin does not seem to play a role. The size of MTB-3-GFP comets is affected by the absence of dynactin and conventional kinesin. We detected no obvious morphological phenotypes in Delta mtb-3 mutants but there were fewer MTs in Delta mtb-3, MTs were less bundled and less organized. Compared to WT, both MT polymerization and depolymerization rates were significantly decreased in Delta mtb-3. In summary, the lack of MTB-3 affects overall growth and morphological phenotypes of N. crassa only slightly, but deletion of mtb-3 has strong effect on MT dynamics

BredewegErinMolecularCellularBiologyNeurosporaCrassaExocyst.pdf

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 Spitzenkörper. 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 Spitzenkörper 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 Spitzenkörper, 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 Spitzenkörper, 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

BredewegErinMolecularCellularBiologyNeurosporaCrassaExocyst_SupplementalMaterials.zip

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 Spitzenkörper. 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 Spitzenkörper 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 Spitzenkörper, 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 Spitzenkörper, 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

Protein interactors of Spindle Pole Body (SPB) components and septal proteins in fungus Neurospora crassa: A mass spectrometry-based dataset

Microtubule Organizing Centers (MTOC) are subcellular structures in eukaryotic cells where nucleation of microtubules (MTs) takes place and represents the filament's minus end. Their localization depends on the species, cell type, and cell cycle stage. Along the fungal kingdom, the Spindle Pole Body (SPB) in the nucleus (an equivalent to Centrosomes in animal cells) is the principal MTOC. Other MTOCs have been identified in filamentous fungi, such as the Spitzenkörper in the hyphal tips of Schizosaccharomyces pombe or the septal pore of Aspergillus nidulans. However, in the fungal-model organism Neurospora crassa, these alternative MTOCs have not been recognized. Here, we present a Mass spectrometry-based dataset of proteins interacting with four MTOC components of N. crassa tagged with fluorescent proteins: γ-Tubulin-sGFP (main nucleator at the SPB), MZT-1-sGFP (structural SPB microprotein), APS-2-dRFP (septal protein and recognized SPB component), and SPA-10-sGFP (septal MTOC protein). A WT and a cytosolic GFP expressing strain were included as controls. The protein interactors were pulled down by Co-IP1, using GFP-Magnetic agarose that captures recombinant GFP proteins (including GFP-derivatives) in their native state. Bounded proteins were separated by SDS-PAGE and identified by nano LC-MS/MS2. The protein annotation was done using the N. crassa protein database

Two microtubule-plus-end binding proteins LIS1-1 and LIS1-2, homologues of human LIS1 in Neurospora crassa.

LIS1 is a microtubule (Mt) plus-end binding protein that interacts with the dynein/dynactin complex. In humans, LIS1 is required for proper nuclear and organelle migration during cell growth. Although gene duplication is absent from Neurospora crassa, we found two paralogues of human LIS1. We named them LIS1-1 and LIS1-2 and studied their dynamics and function by fluorescent tagging. At the protein level, LIS1-1 and LIS1-2 were very similar. Although, the characteristic coiled-coil motif was not present in LIS1-2. LIS1-1-GFP and LIS1-2-GFP showed the same cellular distribution and dynamics, but LIS1-2-GFP was less abundant. Both LIS1 proteins were found in the subapical region as single fluorescent particles traveling toward the cell apex, they accumulated in the apical dome forming prominent short filament-like structures, some of which traversed the Spitzenkörper (Spk). The fluorescent structures moved exclusively in anterograde fashion along straight paths suggesting they traveled on Mts. There was no effect in the filament behavior of LIS1-1-GFP in the Δlis1-2 mutant but the dynamics of LIS1-2-GFP was affected in the Δlis1-1 mutant. Microtubular integrity and the dynein-dynactin complex were necessary for the formation of filament-like structures of LIS1-1-GFP in the subapical and apical regions; however, conventional kinesin (KIN-1) was not. Deletion mutants showed that the lack of lis1-1 decreased cell growth by ∼75%; however, the lack of lis1-2 had no effect on growth. A Δlis1-1;Δlis1-2 double mutant showed slower growth than either single mutant. Conidia production was reduced but branching rate increased in Δlis1-1 and the Δlis1-1;Δlis1-2 double mutants. The absence of LIS1-1 had a strong effect on Mt organization and dynamics and indirectly affected nuclear and mitochondrial distribution. The absence of LIS1-1 filaments in dynein mutants (ropy mutants) or in benomyl treated hyphae indicates the strong association between this protein and the regulation of the dynein-dynactin complex and Mt organization. LIS1-1 and LIS1-2 had a high amino acid homology, nevertheless, the absence of the coiled-coil motif in LIS1-2 suggests that its function or regulation may be distinct from that of LIS1-1

Two microtubule-plus-end binding proteins LIS1-1 and LIS1-2, homologues of human LIS1 in Neurospora crassa.

LIS1 is a microtubule (Mt) plus-end binding protein that interacts with the dynein/dynactin complex. In humans, LIS1 is required for proper nuclear and organelle migration during cell growth. Although gene duplication is absent from Neurospora crassa, we found two paralogues of human LIS1. We named them LIS1-1 and LIS1-2 and studied their dynamics and function by fluorescent tagging. At the protein level, LIS1-1 and LIS1-2 were very similar. Although, the characteristic coiled-coil motif was not present in LIS1-2. LIS1-1-GFP and LIS1-2-GFP showed the same cellular distribution and dynamics, but LIS1-2-GFP was less abundant. Both LIS1 proteins were found in the subapical region as single fluorescent particles traveling toward the cell apex, they accumulated in the apical dome forming prominent short filament-like structures, some of which traversed the Spitzenkörper (Spk). The fluorescent structures moved exclusively in anterograde fashion along straight paths suggesting they traveled on Mts. There was no effect in the filament behavior of LIS1-1-GFP in the Δlis1-2 mutant but the dynamics of LIS1-2-GFP was affected in the Δlis1-1 mutant. Microtubular integrity and the dynein-dynactin complex were necessary for the formation of filament-like structures of LIS1-1-GFP in the subapical and apical regions; however, conventional kinesin (KIN-1) was not. Deletion mutants showed that the lack of lis1-1 decreased cell growth by ∼75%; however, the lack of lis1-2 had no effect on growth. A Δlis1-1;Δlis1-2 double mutant showed slower growth than either single mutant. Conidia production was reduced but branching rate increased in Δlis1-1 and the Δlis1-1;Δlis1-2 double mutants. The absence of LIS1-1 had a strong effect on Mt organization and dynamics and indirectly affected nuclear and mitochondrial distribution. The absence of LIS1-1 filaments in dynein mutants (ropy mutants) or in benomyl treated hyphae indicates the strong association between this protein and the regulation of the dynein-dynactin complex and Mt organization. LIS1-1 and LIS1-2 had a high amino acid homology, nevertheless, the absence of the coiled-coil motif in LIS1-2 suggests that its function or regulation may be distinct from that of LIS1-1