e hyphae and studied their response to glucose depletion. Similar to our results, they observed secondary growth fueled by carbon recy cling, which was morphologically characterized by the formation Nutlin-3a of hyphae with signi?cantly reduced diame ters. For A. niger and A. oryzae hyphal diam eters were shown to linearly correlate with the speci?c growth rate, hence the reduction of hyphal diameters re?ects the slow rate of secondary growth during the starvation phase. Focusing on non empty compartments, we analyzed hyphal population dynamics from statis tically valid sample sizes for di?erent cultivation time points. Our data showed that older hyphae with larger diameters grown during carbon su?cient conditions gradually became empty, giving rise to a new population of thinner hyphae.

Carbon for this secondary phase of growth might have been liberated from extra and or intracellular sources. In agreement with another study of A. niger, our secretomic data revealed that the relative contribution of lysis was very limited, even under starvation conditions. Compared to exponential growth, no relative accumulation of pro teins without predicted signal peptide sequences was observed in culture ?ltrates. However, because these results could also be explained by an equilibrium between proteolytic degradation and leakage of cytoplasmic pro teins, it still remains to be shown whether intracellular resources are endogenously recycled by neighboring com partments or ?rst leak into the culture broth where they are subsequently taken up by surviving compartments.

One process known to be important for endoge nous recycling of cytoplasmic content in eukaryotes is macroautophagy. In ?lamentous fungi, it is thought to play an GSK-3 important role in nutrient tra?cking along the hyphal network promoting foraging of substrate explor ing hyphae and conidiation. However, besides endogenous recycling of nutrients, autophagy in general is clearly associated with cell death and is discussed to have protective roles related to the degradation of e. g. damaged mitochondria or unfolded proteins. It is strongly evident from our transcriptomic data that the induction of autophagic processes is a hall mark of carbon starved aging fungal cultures. To which extend autophagic processes play a role in the protec tion against apoptotic necrotic PCD, endogenous recy cling and autophagic PCD remains to be shown in future studies.

The GO enrichment showed a joint downregulation of general protein biosynthesis and secretion pathways during Alisertib side effects carbon starvation. However, the extracellular accu mulation of certain proteins with predicted signal pep tide sequences including proteases, glycosyl hydrolases and phospholipases indicates a speci?cation of those pathways which might be related to the emergence of the second population of thin poorly branching hyphae. This phenomenum has been observed, for example, dur ing nitrogen starving surface cultures of Phanerochaete chrysosporium for which thin hyphae