The Aspergillus nidulans kinesin-3 tail is necessary and sufficient to recognize modified microtubules.

Posttranslational microtubule modifications (PTMs) are numerous; however, the biochemical and cell biological roles of those modifications remain mostly an enigma. The Aspergillus nidulans kinesin-3 UncA uses preferably modified microtubules (MTs) as tracks for vesicle transportation. Here, we show that a positively charged region in the tail of UncA (amino acids 1316 to 1402) is necessary for the recognition of modified MTs. Chimeric proteins composed of the kinesin-1 motor domain and the UncA tail displayed the same specificity as UncA, suggesting that the UncA tail is sufficient to establish specificity. Interaction between the UncA tail and alpha-tubulin was shown using a yeast two-hybrid assay and in A. nidulans by bimolecular fluorescence complementation. This is the first demonstration of how a kinesin-3 motor protein distinguishes among different MT populations in fungal cells, and how specificity determination depends on the tail rather than the motor domain, as has been demonstrated for kinesin 1 in neuronal cells.

Interaction of the Aspergillus nidulans microtubule-organizing center (MTOC) component ApsB with gamma-tubulin and evidence for a role of a subclass of peroxisomes in the formation of septal MTOCs.

Peroxisomes are a diverse class of organelles involved in different physiological processes in eukaryotic cells. Although proteins imported into peroxisomes carry a peroxisomal targeting sequence at the C terminus (PTS1) or an alternative one close to the N terminus (PTS2), the protein content of peroxisomes varies drastically. Here we suggest a new class of peroxisomes involved in microtubule (MT) formation. Eukaryotic cells assemble MTs from distinct points in the cell. In the fungus Aspergillus nidulans, septum-associated microtubule-organizing centers (sMTOCs) are very active in addition to the spindle pole bodies (SPBs). Previously, we identified a novel MTOC-associated protein, ApsB (Schizosaccharomyces pombe mto1), whose absence affected MT formation from sMTOCs more than from SPBs, suggesting that the two protein complexes are organized differently. We show here that sMTOCs share at least two further components, gamma-tubulin and GcpC (S. pombe Alp6) with SPBs and found that ApsB interacts with gamma-tubulin. In addition, we discovered that ApsB interacts with the Woronin body protein HexA and is targeted to a subclass of peroxisomes via a PTS2 peroxisomal targeting sequence. The PTS2 motif was necessary for function but could be replaced with a PTS1 motif at the C terminus of ApsB. These results suggest a novel function for a subclass of peroxisomes in cytoskeletal organization.

The Aspergillus nidulans kinesin-3 UncA motor moves vesicles along a subpopulation of microtubules.

The extremely polarized growth form of filamentous fungi imposes a huge challenge on the cellular transport machinery, because proteins and lipids required for hyphal extension need to be continuously transported to the growing tip. Recently, it was shown that endocytosis is also important for hyphal growth. Here, we found that the Aspergillus nidulans kinesin-3 motor protein UncA transports vesicles and is required for fast hyphal extension. Most surprisingly, UncA-dependent vesicle movement occurred along a subpopulation of microtubules. Green fluorescent protein (GFP)-labeled UncA(rigor) decorated a single microtubule, which remained intact during mitosis, whereas other cytoplasmic microtubules were depolymerized. Mitotic spindles were not labeled with GFP-UncA(rigor) but reacted with a specific antibody against tyrosinated alpha-tubulin. Hence, UncA binds preferentially to detyrosinated microtubules. In contrast, kinesin-1 (conventional kinesin) and kinesin-7 (KipA) did not show a preference for certain microtubules. This is the first example for different microtubule subpopulations in filamentous fungi and the first example for the preference of a kinesin-3 motor for detyrosinated microtubules.

Polarized growth in fungi--interplay between the cytoskeleton, positional markers and membrane domains.

One kind of the most extremely polarized cells in nature are the indefinitely growing hyphae of filamentous fungi. A continuous flow of secretion vesicles from the hyphal cell body to the growing hyphal tip is essential for cell wall and membrane extension. Because microtubules (MT) and actin, together with their corresponding motor proteins, are involved in the process, the arrangement of the cytoskeleton is a crucial step to establish and maintain polarity. In Saccharomyces cerevisiae and Schizosaccharomyces pombe, actin-mediated vesicle transportation is sufficient for polar cell extension, but in S. pombe, MTs are in addition required for the establishment of polarity. The MT cytoskeleton delivers the so-called cell-end marker proteins to the cell pole, which in turn polarize the actin cytoskeleton. Latest results suggest that this scenario may principally be conserved from S. pombe to filamentous fungi. In addition, in filamentous fungi, MTs could provide the tracks for long-distance vesicle movement. In this review, we will compare the interaction of the MT and the actin cytoskeleton and their relation to the cortex between yeasts and filamentous fungi. In addition, we will discuss the role of sterol-rich membrane domains in combination with cell-end marker proteins for polarity establishment.

Aspergillus nidulans Dis1/XMAP215 protein AlpA localizes to spindle pole bodies and microtubule plus ends and contributes to growth directionality.

The dynamics of cytoplasmic microtubules (MTs) is largely controlled by a protein complex at the MT plus end. In Schizosaccharomyces pombe and in filamentous fungi, MT plus end-associated proteins also determine growth directionality. We have characterized the Dis1/XMAP215 family protein AlpA from Aspergillus nidulans and show that it determines MT dynamics as well as hyphal morphology. Green fluorescent protein-tagged AlpA localized to MT-organizing centers (centrosomes) and to MT plus ends. The latter accumulation occurred independently of conventional kinesin or the Kip2-familiy kinesin KipA. alpA deletion strains were viable and only slightly temperature sensitive. Mitosis, nuclear migration, and nuclear positioning were not affected, but hyphae grew in curves rather than straight, which appeared to be an effect of reduced MT growth and dynamics.