Polarisome Meets Spitzenkörper: Microscopy, Genetics, and Genomics Converge

Arizona State University, Tempe, Arizona, United States
Eukaryotic Cell (Impact Factor: 3.18). 03/2005; 4(2):225-9. DOI: 10.1128/EC.4.2.225-229.2005
Source: PubMed


The impact of filamentous fungi on human welfare has never been greater. Fungi are acknowledged as the most economically devastating plant pathogens (1) and are attaining increasing notoriety for their ability to cause life-threatening infections in humans (57, 71), and fungal products sustain a billion dollar manufacturing industry (70). The tools available to study filamentous fungi are more sophisticated than ever and include the complete annotated genome sequences of multiple filamentous fungi (12), resources being made available through various functional genomics projects, and advanced bioimaging methods, including high-resolution live-cell imaging (20, 32) and electron tomography (19, 50). The increasing impact of filamentous fungi, along with the rediscovery of pseudohyphal growth in yeast (22), has focused attention on the molecular mechanisms underlying hyphal morphogenesis. Attempts to understand hyphal morphogenesis have historically followed two different lines of investigation. Microscopists have defined, with increasing detail, the subcellular organization of the hyphal tip. This led to the description of the Spitzenkö̈rper, an apical cluster of vesicles, cytoskeletal elements, and other proteins, which plays a crucial role in hyphal extension (4). Geneticists have identified gene products required for hyphal morphogenesis by characterizing morphological mutants (51, 52). Initial studies in the laboratories of Beadle, Tatum, and colleagues attempted to link morphogenesis to specific biochemical pathways. More recent screens have identified a multitude of signaling and cytoskeletal functions required for hyphal extension (62, 72). In the past few years, comparative genomics efforts have allowed fungal biologists interested in hyphal morphogenesis to exploit the wealth of knowledge about polarized growth in the yeast Saccharomyces cerevisiae. Many informative homologies between filamentous fungi and yeast have been uncovered. Notably, this includes several components of a multiprotein complex termed the polarisome (28), which regulates microfilament formation at polarized growth sites in yeast (61). Perhaps more importantly, several gene products involved in hyphal morphogenesis have been shown to have no homologue outside of the filamentous fungi. This emphasizes the potential novelty of the mechanisms underlying hyphal morphogenesis. In this review, we summarize past efforts to understand hyphal morphogenesis and pose a series of questions designed to focus future efforts in this area.

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Available from: Nick Read, May 13, 2014
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    • "Tight coordination between actin and tubulin cytoskeletons (and the corresponding molecular motors) is crucial for the delivery of wall materials [9] and thus the maintenance of hyphal tip extension (see for example 10–12). Building components are distributed to the tip by an apical body called Spitzenkörper [13] utilizing myosin motors and actin filaments [14,15]. New cell-wall components are initially contained within vesicles or endosomes that are transported from distal regions of hyphae to the apical body [5,16]. "
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    ABSTRACT: Karyopherins are transporters involved in the bidirectional, selective and active transport of macromolecules through nuclear pores. Importin-β1 is the paradigm of karyopherins and, together with its cargo-adapter importin-α, mediates the general nuclear import pathway. Here we show the existence of different cellular pools of both importin-α and -β1 homologues, KapA and KapB, in the coenocytic ascomycete Aspergillus nidulans. Fluorescence analysis of haploid and diploid strains expressing KapB::GFP and/or KapA::mRFP showed patches of both karyopherins concurrently translocating long distances in apically-growing cells. Anterograde and retrograde movements allowed those patches to reach cell tips and distal regions with an average speed in the range of μm/s. This bidirectional traffic required microtubules as well as kinesin and dynein motors, since it is blocked by benomyl and also by the inactivation of the dynein/dynactin complex through nudA1 or nudK317 mutations. Deletion of Kinesin-3 motor UncA, required for the transport through detyrosinated microtubules, strongly inhibited KapA and KapB movement along hyphae. Overall, this is the first report describing the bidirectional dynamics of the main nuclear import system in coenocytic fungi. A functional link is proposed between two key cellular machines of the filamentous fungal cell: nuclear transport and the tip-growth apparatus.
    Full-text · Article · Dec 2013 · PLoS ONE
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    • "Bright GFP-SncA signals were observed along the hyphae but were more pronounced at the hyphal tips (Fig. 1b). The highest intensity of fluorescence was visible at the very apex of growing hyphae and at newly formed branches reminiscent of the Spitzenkörper, a vesicle-rich region present at actively growing hyphal tips of filamentous fungi, also known as the vesicle supply centre (Fig. 1b) (Harris et al., 2005; Steinberg, 2007). The dynamic movement of vesicles in growing A. niger cells and the movement of the Spitzenkörper along the hyphal tip during growth were observed from four-dimensional image sets (Z-series captured over time, Supplementary video S1) as described "
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    ABSTRACT: The filamentous fungus Aspergillus niger is an industrially exploited protein expression platform, well known for its capacity to secrete high levels of proteins. To study the process of protein secretion in A. niger, we established a GFP-v-SNARE reporter strain in which the trafficking and dynamics of secretory vesicles can be followed in vivo. The biological role of putative A. niger orthologs of seven secretion-specific genes, known to function in key aspects of the protein secretion machinery in S. cerevisiae, was analyzed by constructing respective gene deletion mutants in the GFP-v-SNARE reporter strain. Comparison of the deletion phenotype of conserved proteins functioning in the secretory pathway revealed common features but also interesting differences between S. cerevisiae and A. niger. Deletion of the S. cerevisiae Sec2p ortholog in A. niger (SecB), encoding a guanine exchange factor for the GTPase Sec4p (SrgA in A. niger), did not have an obvious phenotype, while SEC2 deletion in baker's yeast is lethal. Similarly, deletion of the A. niger ortholog of the S. cerevisiae exocyst subunit Sec3p (SecC) did not result in a lethal phenotype as in S. cerevisiae, although severe growth reduction of A. niger was observed. Deletion of secA, secH and ssoA (the A. niger orthologs of S. cerevisiae Sec1p, Sec8p and Sso1/2p, respectively) showed that these genes are essential for A. niger, similarly to the situation in S. cerevisiae. These data demonstrate that the orchestration of exocyst-mediated vesicle transport is only partially conserved in S. cerevisiae and A. niger.
    Full-text · Article · Dec 2013 · Microbiology
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    • "Although the underlying reason for this observation is not yet clear, the polarized localization of stress granules suggests a spatial specificity of the posttranscriptional regulation of gene expression in A. oryzae. It is well known that filamentous fungi have a highly polarized cell structure in which secretory vesicles, cytoskeletal elements, and related components are concentrated at the hyphal tip as a well-organized cluster that determines hyphal growth and polarity [67], [68]. The localization of ribosomes at the hyphal tip supports the idea that mRNA translation actively occurs in this region [69]. "
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    ABSTRACT: Stress granules are a type of cytoplasmic messenger ribonucleoprotein (mRNP) granule formed in response to the inhibition of translation initiation, which typically occurs when cells are exposed to stress. Stress granules are conserved in eukaryotes; however, in filamentous fungi, including Aspergillus oryzae, stress granules have not yet been defined. For this reason, here we investigated the formation and localization of stress granules in A. oryzae cells exposed to various stresses using an EGFP fusion protein of AoPab1, a homolog of Saccharomyces cerevisiae Pab1p, as a stress granule marker. Localization analysis showed that AoPab1 was evenly distributed throughout the cytoplasm under normal growth conditions, and accumulated as cytoplasmic foci mainly at the hyphal tip in response to stress. AoSO, a homolog of Neurospora crassa SO, which is necessary for hyphal fusion, colocalized with stress granules in cells exposed to heat stress. The formation of cytoplasmic foci of AoSO was blocked by treatment with cycloheximide, a known inhibitor of stress granule formation. Deletion of the Aoso gene had effects on the formation and localization of stress granules in response to heat stress. Our results suggest that AoSO is a novel component of stress granules specific to filamentous fungi. The authors would specially like to thank Hiroyuki Nakano and Kei Saeki for generously providing experimental and insightful opinions.
    Full-text · Article · Aug 2013 · PLoS ONE
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