The Role of Actin in Hyphal Tip Growth

This thesis investigates whether there are alternative mechanisms of tip growth in invasive and non-invasive hyphae of the fungus Neurospora crassa. The cytoskeleton protein actin is thought to play a pivotal role in hyphal tip growth, performing a multitude of tasks, one of which may be the provision of a resistive force to counter turgor pressure. An Actin depleted zone (ADZ) was the dominant feature of invasive hyphal tips, which was largely absent from non-invasive hyphae. The Spitzenkörper was slightly larger in invasive hyphae but this size difference alone was thought insufficient to account for the exclusion of filamentous actin (F-actin) from the tip. The actin nucleating protein formin was found at sites where actin nucleation is occurring, while cofilin, a protein that severs F-actin, was found to localise where F-actin disassembly was likely to be occurring. It is suggested that these proteins are likely to play a role in controlling a dynamic cytoskeleton, rearrangements of which are required for the two modes of growth. Invasive hyphae were found to generate a higher turgor than non-invasive hyphae. These results suggest that the F-actin rearrangements facilitated by cofilin give an ADZ that may play a role in invasive hyphal tip growth; possibly through a reduction of tip resistance; thus enabling the provision of a greater protrusive force by turgor

Time-lapse imaging of the surface of a porcupine quill’s cortical cell after indentation.

<p>AFM images obtained with peak force tapping mode of a cortical cell extracted from a porcupine quill, the scan size is 5 µm. The images were taken before, 30 min and 20 h after nano-indentation on a 5 by 5 grid with a maximum load of 120 µN. The indent positions are indicated by white dots. The elastic map channel (Log DMT) reveals that the cell membrane complex (CMC) between macrofibrils is stiffer than the macrofibril themselves. After indentation, the patterned formed by the boundaries was significantly disturbed and in one case (arrowhead) several lines of high stiffness radiated from an indent. After 20 h the entire elastic map reveals an increase in Young’s modulus compared to the reference map while the indents are still clearly visible.</p

Time-lapse imaging of the surface of the cortex of a human hair after indentation.

<p>AFM images obtained with peak force tapping mode of the surface of a human hair fibre with exposed cortex, the scan size is 10 µm. The images were taken before, 30 min, 1 h and 1.5 h after nano-indentation on a 10 by 10 grid with a maximum load of 90 µN. The indent positions are indicated by white dots. After 1 h the indents have completely disappeared but the elastic map channel (Log DMT) reveals an increase in elastic modulus mainly at the indents’ sites.</p

Summary of dynamic Young’s modulus measured on the hair and porcupine quill samples.

&<p>Hair fibre with exposed cortical cells.</p><p>@Single cortical cell extracted from a porcupine quill.</p

Dynamic Young’s modulus as a function of indentation depth.

<p>The Young’s moduli were extracted using the Oliver and Pharr method from force curves obtained at a velocity of 0.2 µm/s. Hair fibre with exposed cortical cells (open symbols, two different data sets), single cortical cell extracted from a porcupine quill (solid symbols, two different data sets).</p

Ultrastructure of a cortical cell.

<p>SEM image of cortical cell extracted from a human hair fibre showing the spindle-shape of the cell (inset) as well as the array of macrofibrils.</p

The cortex of a human hair stiffens after indentation.

<p>DMT modulus histograms of the elastic maps shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041814#pone-0041814-g005" target="_blank">Figure 5</a>. The DMT modulus distributions follow Poisson-like statistics. The mean and the standard deviation of the distribution both increase with time indicating a stiffening of the sample.</p