Time-Lapse Super-Resolution Imaging of Apical Membrane Protein Domains in Live Filamentous Fungi

Abstract

The core mechanism of the asymmetric and the polarized cell growth is a conserved process among many organisms. This process involves complex concerted interplay of dynamic rearrangements of cytoskeletons, the mobilization of proteins from intracellular pools, active transport of vesicles to fusion sites and the accumulation of “cell end marker” proteins. While many of the key components for the polarized growth are known, the detailed mechanism of these complex processes is still unclear. TeaR is one of the cell end marker protein that plays a crucial role in the initiation and the maintenance of straight-growth in a filamentous fungi, Aspergillus nidulans. While many end marker proteins show a single accumulation of the domain near the end of the cell, widefield fluorescence microscopy data shows a distribution of multiple TeaR domains. Our current colocalization studies strongly suggest that majority of TeaR domains consist of accumulated secretory vesicles that are docked near the plasma membrane. This suggests that these domains mark the exocytosis sites of the hypha. In order to elucidate the detailed architecture of TeaR domains near the hypha tip, we have performed a super-resolution microscopy imaging, photoactivated localization microscopy (PALM) in live filamentous fungi. PALM imaging of TeaR fused with a photoconvertible fluorescent protein, mEosFPthermo, shows a cluster size distribution centered around 140 nm. Time-lapse PALM imaging further reveals that these clusters in growing cells are highly dynamic. Processes resemble vesicle trafficking along the cytoskeleton, docking, accumulation of vesicles, dispersion of proteins along the membrane and the growth of the membrane has been observed. We present the affect of key deletion mutants that influence the directionality of the hypha growth on these dynamics.