Polarized growth of filamentous fungi depends on the actin and the microtubule (MT) cytoskeleton along with their associated motor proteins, myosins, kinesins and dynein. A. nidulans contains eleven different kinesins. UncA belongs to the kinesin-3 family (formerly Unc-104) and transports vesicles. Surprisingly, UncA-dependent vesicle movement occurred along a subpopulation of MTs. GFP labeled UncArigor decorated a single MT, which remained intact during mitosis, while other cytoplasmic MTs were depolymerized. The observed subpopulation of MTs consists of detyrosinated alpha-tubulin. In contrast, kinesin-1 (conventional kinesin) and kinesin-7 (KipA) did not show a preference for certain MTs. This is the first example for different MT subpopulations in filamentous fungi and the first example for the preference of a kinesin-3 motor for detyrosinated MTs. The role of detyrosinated MTs, the specificity for UncA binding and the content of cargo vesicles will be analyzed. Detyrosinated MTs appear to be more stable than tyrosinated ones, but the C-terminal end appears to be modified after MTs are stabilized. To understand this stabilization, MT plus end-associated proteins will be characterized. For the understanding of the role of UncA in A. nidulans, it is essential to identify cargo proteins. Vesicles will be isolated and their content analyzed in a proteome approach.
Localization of UncArigor along a single microtubule.
(A) The colony of an uncArigor mutant (left colony) (SNZ14) shows the same phenotype as an uncA-deletion strain (SNZ9) in comparison to the wild type (right colony).
(B) GFP-UncArigor localizes to a rod-like structure in a hyphal compartment.
(C) Immunostaining of a tip compartment of a mRFP1-UncArigor strain (SNZ54) with anti-alpha-tubulin antibodies and FITC-labeled secondary antibodies. Nuclei were stained with DAPI. Upper panel: FITC fluorescence; middle panel: mRFP1 fluorescence; lower panel: overlay with the DAPI channel.