The actin module of endocytic internalization in Aspergillus nidulans : a critical role of the WISH/DIP/SPIN90 family protein Dip1
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Using FPs fused with actin or its fiduciary probes Lifeact, Chromobody and Tractin, we studied, by time-resolved microscopy, the actin module of endocytosis in Aspergillus nidulans . F-actin probes efficiently labeled endocytic patches and the Spitzenkörper (SPK), but not exocytic cables generated by the SPK-associated formin, illuminated by tropomyosin. The SPK contains actin, tropomyosin and capping protein, but not fimbrin or Arp2/3, showing that the actin mesh in this locale does not contain branched actin. F-actin and its associates Arp2/3, AbpA Abp1 and fimbrin reside in patches for 12-14 sec before disappearing. In contrast, verproline resides in patches only during the first half of the actin module, and the scaffold SlaB Sla2 arrives long before the burst of F-actin. Hyphal growth depends on endocytic recycling, which we exploited to assess the efficiency of endocytosis in the mutants. slaBΔ , arpC1Δ , cof1Δ (cofilin) and fimAΔ (fimbrin) were lethal, with germlings arresting growth shortly after establishing polarity, correlating with a complete block in actin patch progression. srv2Δ , vpr1Δ (verprolin), capAΔ (capping protein alpha) and dip1Δ were very debilitating, indicating an important role for these proteins, whereas a double abpAΔ capAΔ mutation is nearly lethal. The lifetime of actin in mutant patches correlated with the extent of growth and endocytic defects. Formin does not localize to patches and its deficiency does not affect the F-actin patch lifecycle, ruling out an endocytic role. In contrast, ablation of the WDS (WISH/DIP/SPIN90) Dip1 protein disorganizes tip-proximal patches including the endocytic collar, establishing Dip1-mediated activation of Arp2/3 as a source of seed filaments. However, dip1Δ severely impairs endocytosis but does not preclude it, indicating that Dip1-independent preformed seed filaments exist. We document a close association of endocytic patches with cortical actin filaments and abundant traffic of F-actin “worms” that, we propose, might serve as seeds for Arp2/3-mediated dendritic polymerization.
Authors’ summary
Filamentous fungi have a deep impact in our lives as friends and foes. Certain species are used as cell factories for the production of proteins or biopharmaceuticals. In contrast, phytopathogenic species, cause important losses in crops whereas those able to infect humans represent serious risks for global health. Filamentous fungi form tubular cells, denoted hyphae, that grow by apical extension. This requires the coupling between exocytosis and endocytosis in the so-called endocytic recycling pathway, which is needed, for example, to maintain the polarization of enzymes which synthesize the cell wall as growth proceeds. Remarkably, detailed studies on endocytosis in filamentous fungi are wanting. Here we report the characterization of the endocytic pathway in the genetic model Aspergillus nidulans , a filamentous ascomycete which is well-suited for genetic manipulation and in vivo fluorescence microscopy. Our study demonstrates that endocytosis is essential for filamentous fungal life and provides significant insight on how F-actin powers the internalization of endocytic vesicles, including an important physiological role of Dip1, a protein required to provide seed filaments for the formation of F-actin branching networks. Importantly, as yet unidentified Dip1-independent mechanisms that synthesize these seeds must exist, opening new avenues for future research.
