We have previously shown that chitin synthases (CHS-1, CHS-3 and CHS-6) travel along actively growing hyphae of Neurospora crassa by secretory microvesicles that follow an unknown unconventional route and accumulate in the core of the Spitzenkörper. In the proposed study, we will label with either fluorescent or epitope tags the remaining CHS in Neurospora crassa (CHS-2, CHS-4, CHS-5 and CHS-7) to analyze their distribution pattern and identify putative interacting proteins. Single and multiple knock out strains will be used to analyze the role, distribution and chitin synthase (CS) activity and expression of a given CHS when specific CHS are lacking. To test which cytoskeleton is responsible for the delivery of microvesicles to the apex and their balanced maintenance at the Spk core we will image the CHS-FP fusions in N. crassa strains expressing fluorescent microtubules or fluorescent actin microfilaments. Also we will use microtubule and actin inhibitors and strains with mutations for microtubule or actin associated proteins. In addition, we will obtain cell fractions enriched with chitosomes and identify associated motor proteins. The localization of CHS will be additionally studied in nox-1 mutants and in csn mutants to investigate whether reactive oxygen species (ROS) have a role in the localization of CHS at the apex and whether CHS turnover is dependent on the COP9 signalosome function, respectively.
CHS-1 accumulates at the hyphal apex, when no intact microtubules are visible. LSCM analysis of hyphae resulting from vegetative fusion of a CHS-1-mChFP expressing strain and a tubulin-GFP expressing strain treated with 100µg/ml of the de-polymerizing microtubule agent, Benomyl (A-G). Untreated hypha (A) shows CHS-1-mChFP at the Spk (white arrow) and a great abundance of long GFP labeled microtubules along the hypha (red arrow). After adding the inhibitor, hyphal morphology was seriously affected and microtubules immediately depolymerized allowing to observe more rounded shaped nuclei (B, white arrowheads) and small fluorescent patches unevenly distributed in the affected cells (C, yellow arrows). After 28 minutes CHS-1-mChFP fluorescence re-appeared at the apical dome (E-G), without the presence of microtubules.