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Regulation of motors in bidirectional motility of early endosomes in the model pathogenic fungus Ustilago maydis

Reference	BB/F022956/1
Principal Investigator / Supervisor	Professor Gero Steinberg
Co-Investigators / Co-Supervisors
Institution	University of Exeter
Department	Biosciences
Funding type	Research
Value (£)	351,506
Status	Completed
Type	Research Grant
Start date	01/08/2008
End date	30/09/2011
Duration	38 months

Abstract

We recently demonstrated that hyphal tip growth and fungal pathogenicity involve endocytic recycling processes in Ustilago maydis (Fuchs et al. 2006, Plant Cell, 18, 2066). Recycling is based on early endosomes (EE) that rapidly move along microtubules. The underlying motor proteins are the Kinesin-3 and the counteracting dynein, which takes the EEs from the hyphal tip back to cell body (Lenz et al. 2006, EMBO J., 25, 2275). Our preliminary evidence indicates that n apical localization of EEs is not sufficient to support hyphal growth. Instead the transport process itself appears to be essential for endosome function in U. maydis. This proposal sets out to elucidate the regulation and interplay of motors in EE trafficking. The U. maydis kinesin-3, Kin3, moves EE to the hyphal tip, where it subsequently becomes a passive cargo on dynein-driven endosomes. Surprisingly, the cellular presence of Kin3 is required for full dynein activity. This suggests that that (1) both motors directly or indirectly interact on the organelle, or (2) that kinesin-3 takes dynein activators to the hyphal tip. We will address these possibilities by expressing truncated, as well as fusion proteins, in kinesin-3 null mutants and then monitor the effect on dynein-dependent EE motility. The tail of U. maydis Kin3 shares significant similarity (24.5% identity) with human Kif1A, an important motor in axonal transport. By expression of partially truncated fusion proteins in kin3 mutant strains in which EEs are tagged by GFP-Rab5a, we will map regions in the tail of kinesin-3 that are related to its role in EE trafficking. These studies will be flanked by a biochemical approach using TAP-tagged or S-tagged versions expressed in kinesin-3 null mutants to identify interacting partners of Kin3. We will also characterise mutants defective in hyphal growth and motility of EEs. and use photoactivatable GFP-Rab5a to undertake a detailed quantitative analysis of EE dynamics in living cells.

Summary

Cell polarization is a fundamental feature of eukaryotic cells and is carried to an extreme in polarized growing cells, such as animal neurons, filamentous fungi, plant pollen tubes and root hairs. In the mammalian axon, expansion of the growth cone is supported by transport of membranous organelles, such as endosomes, synaptic vesicles, proteins and RNA along the fibres of the cytoskeleton, namely microtubules and F-actin. Specialized protein machines, called molecular motors rapidly move along these 'tracks' for delivery of their cargo. Axonal transport happens mainly along microtubules and is essential for brain function and development. Consequently, defects in motor activity result in severe neuro-degenerative diseases. Despite its importance the molecular basis of long-distance transport is not well-understood. This is in part due to the lack of simple and genetically tractable model organism to study long-distance transport. This gap may be filled by filamentous fungi, which are genetically tractable and also grow as highly polarized cells called hyphae. Hyphal growth requires delivery of enzymes, membranes and cell wall-precursors to the expanding tip. Similar to axons, microtubule-based transport is required for tip growth, which involves motor proteins, such as kinesin-3 and kinesin-1, that are also found in mammals. At the growth region enzymes and wall-components are released by a process called exocytosis. Very recently, we have shown in a fungus called Ustilago maydis that endocytosis, which is the uptake of material into the cell, participates in hyphal tip growth and is necessary for the ability of this fungus to cause plant disease. We found that early endosomes (EEs), which are membrane-bound organelles that collect the up-taken material, are of crucial importance. This is most likely achieved by a supportive function of EEs in recycling of enzymes and receptors at the growth region. Interestingly, we also found that EEs rapidly move up anddown the hyphae of U. maydis. This is achieved by the two motor proteins, kinesin-3 and dynein, which move endosomes in opposite directions along microtubules. By genetic means we interfered with the balance of their activity, and this resulted in defects in endosomes motility and a block in hyphal elongation. This result strongly implies that the movement itself is essential for fungal tip growth. However, neither the cellular role of endosome motility nor the regulation of the underlying motors is currently known. U. maydis is one of the best established model systems for studying fungal pathogenicity and the role of the cytoskeleton in hyphal growth. U. maydis combines powerful technical advantages, including a published genome, and numerous genetic tools (e.g. inducible promoters) and cytological tools such as GFP, mRFP, CFP, YFP and photoactivatable GFP are established. We will make use of these technical advantages in order to address the following questions: (1) How is bi-directional EE motility regulated und how do motors balance their activity? (2) Which part other of the kinesin-3 motor binds to EEs? (3) What proteins interact with kinesin-3 and which role do these have in EE motility? The project will provide novel insight into the mechanism of hyphal tip growth by fungi. It will therefore be of fundamental interest to all aspects of fungal research, but will particularity stimulate research on fungal pathogenicity. Therefore, our work will be of benefit to the UK pharmaceutical and agricultural biotechnology industries. Of even greater potential significance, however, is that the motor proteins involved (kinesin-3 and dynein) are also important in long-distance axonal transport in neurons. Therefore, the proposed studies promise also to provide a better understanding of motor protein interplay in mammalian cells.

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	Microbiology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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