Clathrin localization and dynamics in Aspergillus nidulans

Abstract

Cell growth necessitates extensive membrane remodeling events including vesicle fusion or fission, processes that are regulated by coat proteins. The hyphal cells of filamentous fungi concentrate both exocytosis and endocytosis at the apex. This investigation focuses on clathrin in Aspergillus nidulans, with the aim of understanding its role in membrane remodeling in growing hyphae. We examined clathrin heavy chain (ClaH-GFP) which localized to three distinct subcellular structures: late Golgi (trans-Golgi equivalents of filamentous fungi), which are concentrated just behind the hyphal tip but are intermittently present throughout all hyphal cells; the region of concentrated endocytosis just behind the hyphal apex (the "endocytic collar"); and small, rapidly moving puncta that were seen trafficking long distances in nearly all hyphal compartments. ClaH localized to distinct domains on late Golgi, and these clathrin "hubs" dispersed in synchrony after the late Golgi marker PH . Although clathrin was essential for growth, ClaH did not colocalize well with the endocytic patch marker fimbrin. Tests of FM4-64 internalization and repression of ClaH corroborated the observation that clathrin does not play an important role in endocytosis in A. nidulans. A minor portion of ClaH puncta exhibited bidirectional movement, likely along microtubules, but were generally distinct from early endosomes.

The AP-2 complex has a specialized clathrin-independent role in apical endocytosis and polar growth in fungi

“…While the dispensability of ClaL in apical cargo localization is clear, the non-essential role of ClaH on flippase polarization establishment is more difficult to formally confirm because depletion of ClaH affects Golgi function and secretion, and thus polarization maintenance. Our results are in agreement with results reported in Schultzhaus et al (2017a); (2017b), which establish that ClaH is localized principally in late Golgi, only weakly in the apical collar region and is excluded from the hyphal tip. Overall, these results further showed that AP-2 and clathrin have distinct roles in cargo subcellular localization in A. nidulans.…”

“…Panel C further shows a rather weak association of ClaL with subcortical patches close to the collar endocytic region. The apparent prominent localization of ClaL in Golgi-like structures is in full agreement with results in Schultzhaus et al (2017b), who have shown that ClaH-GFP localizes principally in the late Golgi, and has only a weak association with the sub-apical collar region. In the same article, the authors have further shown that ClaH and ClaL co-localize.…”

“…This picture was obtained independently from the presence or absence of the endocytic trigger. Under the light of the recent publication of Schultzhaus et al (2017b), which showed that ClaH is principally localized in the late Golgi, but also based on our own independent results on the effect of ClaH on other apical markers, described later in this work, it seems that ab initio  knockdown of ClaH severely affects secretion of cargoes, including transporters. This leads to apparent Golgi and endosome collapse and progressive lethality, which could easily explain why we detect UapA-GFP in cytosolic structures, rather than in the PM.…”

“…In the absence of thiamine (repressor), strains expressing thiA p -claH or thiA p -claL grew similar to wild-type isogenic control strains (not shown), whereas in the presence of thiamine thiA p -claH did not grow to form colonies, while thiA p -claL formed very small compact colonies, compatible with a severe growth defect (Figure 4A). Schultzhaus et al (2017b), recently reported very similar growth phenotypes. We further examined the thiamine-repressible mutants under the microscope (Figure 4B).…”

“…An exception is a very recent report that studied clathrin dynamics in A. nidulans , following basically the subcellular localization and role in growth, of the clathrin heavy chain, ClaH (Schultzhaus et al, 2017b). This report showed that ClaH is localized mostly in late ( trans ) Golgi and secondarily to tentative endocytic sites, and although absolutely essential for growth, it does not seem to be involved in endocytosis.…”