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

Effect of cytoskeleton depolymerization drugs on the localization and integrity of coronin patches.

<p>Hyphae exposed to: (A) the anti-actin drug, 1.0 µg ml⁻¹ cytochalasin A, (B) 5.0 µg ml⁻¹ cytochalasin A, and (C) the anti-tubulin drug 2.5 µg ml⁻¹ benomyl. Scale bar = 5 µm.</p

Co-expression of coronin with fimbrin, Arp2 and actin.

<p>(A–C) Colocalization of Fimbrin (FIM-GFP) and CRN-1-mChFP. (D–F) Colocalization of Arp2 (ARP-2-GFP) and CRN-1-mChFP. (G–I) Partial colocalization of the actin marker Lifeact-GFP and CRN-1-mChFP. (J–L) Co-expression of CRN-1-mChFP and Lifeact-GFP showing the lack of colocalization between coronin patches and actin cables. are depicted by. (L) Merge, not clear association of crn-1 patches is observed with actin filaments, arrowhead shows colocalization of actin patches with CRN-1-mChFP. The white arrow points a region where there is only labeling with Lifeact-GFP and the blue arrow show the patches where CRN-1-mChFP and Lifeact-GFP colocalized. Note the presence of actin in the Spk but not of patch related ABPs. The red arrows in (K) point the actin cables and the white arrowhead show the colocalization of actin and coronin in the patches subapical collar. Scale bar = 5 µm.</p

Subapical localization of coronin.

<p>(A) CRN-1-GFP forms a subapical collar along the inner perimeter of the hypha (arrows), (B) FM4-64 staining reveals the position of the Spk (arrowheads), (C) merge of CRN-1-GFP and FM4-64 staining shows the absence of CRN-1-GFP in the Spk, single confocal plane images. (D) 3D reconstruction of merged confocal z-stacks showing CRN-1-GFP and FM4-64 localization, (E) orthogonal view of the 3D reconstruction shown in (D), the yellow line indicates the position within the tip where the cross-section was taken. Scale bars = 5 µm.</p

Reconstructions of morphogenetic sequences from time lapse movies of the Δ<i>crn-1</i> mutant and the WT strain; the mutant shows a lack of directionality and temporal loss of polarized growth, the green dots show the places of endocytic patches and how they are misplaced in the mutant when the Spk is not present, the small arrows show the shifts between polarized growth and isotropic growth.

<p>Reconstructions of morphogenetic sequences from time lapse movies of the Δ<i>crn-1</i> mutant and the WT strain; the mutant shows a lack of directionality and temporal loss of polarized growth, the green dots show the places of endocytic patches and how they are misplaced in the mutant when the Spk is not present, the small arrows show the shifts between polarized growth and isotropic growth.</p

Comparison of conidial morphology and size.

<p>Conidiophores of (A) Δ<i>crn-1</i> mutant and (B) WT strain. Composite image of conidia representing the most common shapes in (C) Δ<i>crn-1</i> mutant and (D) WT strain. (E) Relative abundance of spherical and non-spherical conidia in the Δ<i>crn-1</i> mutant and WT strain. (F) Average size of conidia in the Δ<i>crn-1</i> mutant and WT strain. The error bars represent the 95% confidence interval. Scale bar = 5 µm.</p

Comparative morphogenesis and Spk behavior revealed by staining hyphae with FM4-64.

<p>(A–E) Δ<i>crn-1</i> mutant and (F–J) WT strain. Arrows show the Spk. Details of Spk dynamics in (K–O) Δ<i>crn-1</i> mutant and (P–S) WT strain. Spk trajectories plotted relative to the growth axis (abscissa). Time in min:sec. Scale bar = 10 µm.</p

Conidial germination.

<p>(A–E) Time series of the Δ<i>crn-1</i> mutant by bright field microscopy and (F–J) WT strain by phase contrast microcopy. Reconstruction of the morphological differences during conidial germination of above sequences over longer time periods (K) Δ<i>crn-1</i> mutant and (L) WT. Time in h:min. Scale bar = 5 µm.</p