Summary:
The plasma membrane is implicated in a variety of functions, whose coordination
necessitates highly dynamic organization of its constituents into domains of
distinct protein and lipid composition. Eisosomes, at least partially, mediate
this lateral plasma membrane compartmentalization. In the model filamentous
fungus Aspergillus nidulans, PilA and PilB, two homologues of the Saccharomyces
cerevisiae eisosome proteins Pil1/Lsp1, and SurG, a strict orthologue of Sur7,
are assembled and form tightly packed structures in conidiospores, products of
the asexual cycle.
Our results show that core eisosome proteins PilA, PilB and SurG are not
expressed in hulle cells or early ascospores, but are expressed in mature
ascospores. In mature but quiescent ascospores, PilA forms static punctate
structures (foci) at the plasma membrane. PilB also was observed at the
ascospore membrane as well, with higher concentration at the areas where the
two halves of ascospores are joined together. Finally, SurG was localized both
at the membrane of ascospores and perinuclearly. In germlings originating from
ascospores the punctate structures along the hypha were shown to be composed
only of PilA. PilB is diffused in the cytoplasm and SurG was located in
vacuoles, endosomes and in foci in the hyphal head. This localization is
identical to that found in germlings originated from conidiospores. In
germinated ascospores PilA foci did not colocalise with the highly mobile and
transient peripheral punctate structures of AbpA, a marker for sites of
clathrin-mediated endocytosis. Deletions of each one or all the three core
eisosomal genes do not affect viability or germination of ascospores. In the
presence of myriocin – a specific inhibitor of sphingolipid biosynthesis –
PilA-GFP foci of ascospore germlings were less numerous and their distribution
was significantly altered, suggesting a correlation between PilA foci and
sphingolipid biosynthesis.
Moreover due to the fact that the tetraspan protein Nce102 has been implicated
as part of a sensor for sphingolipid homeostasis, the role of Nce102 homologue
(AnNce102) in A.nidulans was investigated. We showed that AnNce102 forms stable
structures with low mobility that colocalizes with eisosomes and plays a
crucial role in density/number of PilA/SurG foci in the head of germlings. In
addition we demonstrate that AnNce102 and PilA negatively regulate sphingolipid
biosynthesis, since their deletions partially suppress the thermosensitivity of
basA mutant encoding sphingolipid C4-hydroxylase and the growth defects
observed upon treatment with inhibitors of sphingolipid biosynthesis, myriocin
and Aureobasidin A. Moreover, we show that YpkA repression mimics genetic or
pharmacological depletion of sphingolipids, conditions that induce the
production of Reactive Oxygen Species (ROS) and can be partially overcome by
deletion of pilA and/or annce102. Consistent with these findings, pilAΔ and
annce102Δ also show differential sensitivity to various oxidative agents, while
AnNce102 overexpression can bypass sphingolipid depletion regarding the
PilA/SurG foci number and organization, also leading to the mis-localization of
PilA to septa.
Keywords:
Membrane, Eisosomes, Sphingolipids, Ascospores, Endocytosis
