Annexins to the Rescue: First Responders to Fight Loss of Membrane Integrity in Neurospora crassa

The plasma membrane forms a crucial barrier between the cytosol and the outer environment. Rupture of the plasma membrane allows mixture of intra- and extracellular components, leading to chemical imbalance within the cell and subsequent cell death. Restoration of membrane integrity is therefore essential for cell survival. Threats to membrane integrity include mechanically induced wounding — such as that occurring during cell-cell fusion — and exposure to membrane-targeting substances, including plant defense compounds. To counteract these events, cells have evolved a variety of mechanisms to mend membrane disruptions, collectively termed as membrane repair mechanisms. These processes involve Ca2+-signaling, cytoskeleton remodeling, and targeted recruitment of cellular components to the site of injury. To investigate the molecular basis of fungal membrane repair, the filamentous ascomycete Neurospora crassa was employed. This work identified the proteins PEF1, ANX14, and ANXC4 as key players in membrane repair, each with distinct but partially overlapping functions. To assess their function, gene knockout mutants were analyzed during mechanically induced damage. Deletion of anx14 or anxc4 led to increased lysis rates in fusing germling pairs compared to the wild type and the Δpef1 mutant. Notably, lysis rates doubled in the Δpef1 Δanx14 double mutant, suggesting that PEF1 and ANX14 operate through independent repair pathways. To examine repair in response to chemically induced damage, spore dilution spot assays were performed on media containing various antifungal compounds. These assays revealed that deletion of pef1, but not anx14 or anxc4, impaired colony growth under stress conditions. These findings indicate that annexins may be more critical for repairing mechanically induced membrane disruptions, while PEF1 plays a more prominent role in responding to membrane-targeting substances. Life-cell imaging and subcellular localization studies of GFP-fusion strains further support these distinctions: both ANX14 and PEF1 localize to the membrane following mechanical and chemical injury. However, ANX14 recruitment spans the entirety of the plasma membrane, while PEF1 accumulates in discrete spots at the membrane, likely corresponding to the site of injury. Interestingly, ANXC4 is not recruited under normal conditions, but in the absence of ANX14, it is partially recruited, suggesting a potential compensatory mechanism among annexins. Additional evidence implicates that the cytoskeletal protein actin plays a role in membrane repair. Actin accumulates at the membrane after exposure to antifungal substances. Inhibition of actin polymerization using latrunculin A abolishes PEF1 recruitment, while ANX14 recruitment remains unaffected. This strikingly different behavior of PEF1 and ANX14 reinforces the hypothesis that both proteins function in distinct membrane repair mechanisms. Altogether, these findings contribute to a more comprehensive model of membrane repair in N. crassa, highlighting the interplay between proteins and cytoskeletal components. Within this framework, this thesis underscores the biological significance of rapid, context specific membrane repair responses in filamentous fungi.