F-actin rearrangements and analysis of physical environment of invasive hyphal growth.

Invasive growth through a substrate requires a massive amount of penetrative force, and this is generated in the space of a few microns in a growing tip. This process is known to be critical in the root hair, pollen tube, rhizoids, and the topic of this thesis, hyphal growth. However defining the mechanisms underlying the tip growth remains a contentious issue. Shortcomings in control of direction and regulation of growth began to undermine early turgor-based theories, and the cytoskeletal protein actin, ubiquitous in nature and with crucial roles in structure and motility became a target for investigation. A major breakthrough came with the discovery that a characteristic actin depleted zone (ADZ) occurs at the growing tip of hyphae during invasive but not non-invasive hyphal growth. The ADZ is likely to have an important role in generating the greater protrusive force required for invasive growth. However, since its discovery, little has been determined about the characteristics of the ADZ. Uncertainty in the description of the physical environment the hyphae face adds a layer of complexity to interpretation of results. This thesis aims to address this issue, studying the impact of increasing agarose substrate concentration on the presence and dimensions of the ADZ in the oomycete A. bisexualis. Furthermore, agarose is examined by compression and imaging to compare the physical characteristics of the agar samples over the range of concentrations, and determine whether increasing agarose concentration influences agarose gel structure. Results suggest a difference in the number of ADZ observed in non-invasive compared with invasive samples, however no significant differences in the number or dimensions of ADZ were found amongst the 1-4% w/v agarose concentrations. The 0% sample showed 20.7 percent of hyphae exhibited depleted zones, while 1, 2, 3 and 4% samples showed 56.9%, 48.8%, 40.9% and 54.2% respectively. ADZ dimensions did not correlate with agarose concentration. The average ADZ area:hyphal diameter ratio was 0.634, 0.526, 0.430, 1.09, and 0.65 for 0-4% agarose concentrations respectively. Additionally, investigation of gel compression forces revealed gel strength increases with agarose concentration. The force required to compress the agarose increased from 1.85 Psi in 1% agarose to 4.85, 7.09 and 12.22 Psi in 2, 3 and 4% agarose concentrations respectively. SEM imaging, however, suggests heterogeneity of the fibrous interconnected network of agarose gels at a microscopic scale with variable porous structure at all agarose concentrations. This scale is relevant to hyphal tip growth. In combination, these results suggest F-actin depletion may be a response mechanism to provide greater force for invasive growth. Additionally, this response is not dependent on the concentration of the agarose media, possibly due to the variability encountered within the media. These results contribute another important step forward in unraveling the elusive mechanism of tip growth