Temporal and spatial in vivo optical analysis of microtubules in Neurospora crassa

Abstract

Microtubules are a crucial part of the fungal cytoskeleton and facilitate long-distance vesicle transport to the growing apex that constitutes one of the main driving forces of polarized growth. This study observed the spatial and temporal distribution of microtubules in growing <i>Neurospora crassa </i>hyphae in two and three dimensions. The fungal strain used expressed a normal growth pattern combined with the expression of green fluorescent protein (GFP) along the microtubules enabling their direct study with fluorescence and confocal microscopy. Time-lapse imaging revealed that the microtubules were dynamic, being assembled and disassembled at high rates as well as moving with the cytoplasmic flow. In the apical compartment, the filaments were arranged mostly parallel but not helical to the growth axis and appeared to determine the growth direction in close interaction with the Spitzenkörper. The microtubule distribution in subapical compartments was more random and their motility appeared to be driven by the cytoplasmic flow. However, this flow is affected by hyphal septa that act as partition walls with small connecting pores. Three-dimensional imaging showed that in order to pass through a septum, the filaments had to align parallel to the growth axis. The aim of this study was to attempt to reveal deeper insight into the role of microtubules in fungal growth thus confirming and challenging some suggestions proposed in previous literature.