Formation and stability of eisosomes in the filamentous fungus Ashbya gossypii

Abstract

SummaryOne hallmark of the rapid expansion of the polar surface of fungal hyphae is the spatial separation of regions of exocytosis and endocytosis at hyphal tips, as recently shown for Ashbya gossypii and Aspergillus nidulans. To determine where cortex-associated eisosomes form with respect to these two regions, we monitored fluorescently marked eisosomes in A. gossypii. Each minute, 1.6±0.5 eisosomes form within the first 30 m of each hypha and are exclusively subapical of the endocytosis region. This spatial separation of the processes of eisosome formation and endocytosis, and the much lower frequency of eisosome formation compared with that of endocytic vesicle production do not support a recently proposed role for eisosomes in endocytosis. Levels of mRNA encoding eisosome components are tenfold higher in spores than in hyphae, explaining the observed higher eisosome density at the cortex of germ bubbles. As in Saccharomyces cerevisiae, eisosomes in A. gossypii are very stable. In contrast to S. cerevisiae, however, the A. gossypii homologue of Pil1, one of the main eisosome subunits, is very important for polar growth, whereas the homologue of Nce102, which colocalizes with eisosomes, is not needed for eisosome stability. By testing partial deletions of the A. gossypii homologue of Ymr086w, another component of the eisosome, we identified a novel protein domain essential for eisosome stability. We also compare our results with recent findings about eisosomes in A. nidulans.

Microcompartments within the yeast plasma membrane

“…The latter are among the most abundant cytosolic proteins (with approximately 100 000 molecules of each per cell) and form furrow-like invaginations of the yeast plasma membrane harboring the nine marker proteins of the MCC, which, oddly enough, are especially resistant to endocytosis (Grossmann et al , 2008 ). Consistent with this observation, the position of the eisosomes in Ashbya gossypii , a filamentous fungus closely related to S. cerevisiae , did not coincide with the bulk of the endocytic traffic at the hyphal tip, but they rather accumulated in the subapical tip regions (Seger et al , 2011 ). In vivo photobleaching experiments in S. cerevisiae suggest that the organization of the MCC compartments within the yeast plasma membrane has no influence on the intracellular vesicle trafficking, also opposing a function in endocytosis (Brach et al , 2011 ).…”

Eisosome Ultrastructure and Evolution in Fungi, Microalgae, and Lichens

“…Our studies point to the following six eisosomal features that are encountered across several phyla: (i) a curved, elongated membrane indentation of uniform width (ϳ30 to 50 nm) and depth (ϳ50 nm) that fails to pinch off into vesicles; (ii) the presence of cell wall material in the furrow; (iii) a large-scale topology that can be either punctate or elongated, with the capacity to convert between the two configurations; (iv) "embossed" lipid configurations and IMP endowments that differ from the surrounding plasma membrane; (v) fracture faces that are noncomplementary; and (vi) "flat" versions of the intramembranous domains. In addition, fungal eisosomes tagged with GFP-labeled components are observed to be immobile (16,21,29,35,43), albeit they exhibit subunit turnover (87); the uniform distribution of punctate eisosomes in algae (e.g., see Fig. 7, 9, 10, and 12) suggests that this immobility feature is also conserved.…”

“…A recent review of fungal eisosomes (90) concludes: "Thus, we have a complex and strongly conserved cellular apparatus, which has even been called an organelle, without an obvious physiological function." Knockouts of eisosome-associated proteins in fungi generate a range of mutant phenotypes, from undetectable (33,35,90) to mild (14,16) to substantial (25,29,91,92). Douglas and Konopka (25) reviewed in detail the numerous functions that have been proposed for fungal eisosomes.…”

“…Additional proteins also associate with these punctate domains, in some cases in a transient fashion (17), and the MCC component is reportedly enriched in ergosterol (18) and influenced by phosphoinositide (6,19,20) and sphingolipid (14,17,(21)(22)(23)(24) levels. Hence, the current yeast model (25,26) proposes that Pil1p and Lsp1p, which contain membrane curvatureinducing BAR domains (6,27,28), together with Seg1p (29,30), form a submembrane complex (6) reportedly influenced by Pil1p phosphorylation (25); this complex then either creates or associates with the MCC domains, presumably inducing an inward curvature. The MCC protein Nce102p has also been implicated in generating curvature (5,31).…”

“…Walther et al (16) proposed the name eisosome (eis, Gr., into or portal) for the Pil1p/Lsp1p heteromer because their experiments indicated that it was associated with endocytosis, an interpretation that has since been challenged (29,(32)(33)(34)(35); but see reference 36). Formally, the full membrane/submembrane complex of yeast proteins should be denoted "MCC/eisosomes," and the invaginations should be denoted "furrows" (25).…”