Morphodynamics of non-canonical autophagic structures in Neurospora crassa

Abstract

ABSTRACT Autophagy is a major pathway for unspooling cytoplasmic constituents and recycling them. Here, we investigate autophagy in Neurospora crassa, a close ascomycete relative to the model yeast Saccharomyces cerevisiae . High-pressure-freeze and freeze-substitution techniques proved keys to preservation of the surprising autophagic structures observed in Neurospora by transmission electron microscopy and electron tomography. Depriving Neurospora of carbon triggers two parallel processes at the plasma membrane: formation of vacuoles, initially along the plasma membrane but stuffing 70%–80% of cytoplasmic volume after several hours. The vacuoles contain material left over from digested proteins, disrupted ribosomes or glycogen clumps, plus other discrete organelle-like objects. Also, they accumulate the canonical autophagy marker Atg8, appear to be lytic from the start, and probably grow from lysosomes. Formation of diverse phagophores and autophagosomes, multi-membrane organelles manufactured de novo by the plasma membrane—a feature so far unique to Neurospora . These autophagic structures can be observed attached either to the cell membrane, to the cell wall, or free in the cytoplasm, viz. as mature organelles after detachment from the cell membrane. Although phagophores are clearly observed in carbon-replete cells, their production increases with carbon starvation—and these, too, often appear lytic. ATG1 , the canonical initiator of phagophore formation in yeast, is not required for the formation of phagophores in Neurospora . This work provides clear structural and functional evidence that Neurospora ’s autophagic organelles differ from those observed by others in yeast. IMPORTANCE Neurospora is a quintessential tip-growing organism, which is well known for packaging and longitudinal transport of tip-building blocks. Thus far, however, little attention has been paid to the co-essential process of reclamation, that is—taking apart of upstream, older structural elements, otherwise known as “autophagy”. We are not yet prepared to set out the chemistry of that elaborate process, but its morphological start alone is worthy of attention. Carbon starvation triggers significant autophagic changes, beginning with prolific vacuolation along the plasma membrane, and eventual filling of 70% (or more) of cytoplasmic volume. Additionally, the Neurospora plasma membrane elaborates a variety of phagophores which themselves often look lytic. These have either dual enclosing membranes, like the familiar autophagosomes, can be doubled and have four wrapping membranes, or can be compounded with multiple membrane layers. These reclamation processes must be accommodated by the mechanism of tip growth.