Research Topic C - Endocytosis and early endosome motility

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Research articles

Wedlich-Söldner, R., Bölker, M., Kahmann, R. & Steinberg, G. (2000) A putative endosomal t-SNARE links exo- and endocytosis in the phytopathogenic fungus Ustilago maydis. EMBO J. 19, 1974-1986.

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Summary - We undertook a genetic screen in order to find proteins required for shaping the cell. We found a SNAP25-like tSNARE, named Yup1, that localizes on the surface of motile early endosomes, where it controls entry into the organelle. Consequently, defects in Yup1 result in a block in later steps of endocytosis, but also morphogenesis, suggesting that endocytic recycling supports hyphal growth. This paper provides first evidence that endocytosis is required for hyphal growth of fungi.

Wedlich-Söldner, R., Straube, A., Friedrich, M.W. & Steinberg, G. (2002) A balance of KIF1A-like kinesin and dynein organizes early endosomes in the fungus Ustilago maydis. EMBO J, 21, 2946-2957.

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Summary - Early endosomes move rapidly along microtubules. Motility is driven by a kinesin-3 motor that counteracts cytoplasmic dynein. Bidirectional movements ensure that early endosomes become rearranged during the cell cycle of yeast-like cells, which is a perquisite for an alternating budding pattern at both poles of the elongating cell. This paper demonstrates that kinesin-3 and dynein establish a balance of force in endosome motility.

Lenz, J.H., Straube, A. & Steinberg, G. (2006) A dynein loading zone for retrograde endosome motility at microtubule plus-ends. EMBO J, 25, 2275-2286.

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Summary - In hyphae early endosomes reach the growing tip by the transport activity of kinesin-3. Dynein is concentrated at microtubule plus-ends, where it becomes activated when organelles arrive. Early endosomes are loaded onto dynein and are taken back towards subapical parts of the hypha. Kinesin-3 is travelling backwards as passive cargo of dynein, whereas dynein requires kinesin-1 for targeting to plus-ends. These findings led to the concept of an apical dynein loading zone that is established in the hyphal tip in order to ensure that organelles reach the hyphal tip before they reverse direction.

Fuchs, U., Hause, G., Schuchardt I. and Steinberg, G. (2006) Endocytosis is essential for pathogenic development in the corn smut fungus Ustilago maydis. Plant Cell, 18, 2066-2081.

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Summary - Making use of a temperature-sensitive Yup1 mutant we demonstrate that endocytosis is essential for several steps during pathogenic development of U. maydis, including cell-cell recognition and spore formation. The defect in early cell-cell recognition is due to a lack of recycling of the pheromone receptor (Figure 1), which results in depletion from the surface and makes the cell 'blind' for the mating pheromone of the partner. This is the first demonstration that endocytosis is essential for fungal pathogenicity; identification of the first molecular cargo for recycling in filamentous fungi.

Ashwin, P., Lin, C. & Steinberg, G. (2010) Queuing induced by bidirectional motor motion at the end of a microtubule. Phys. Rev. E, 82, 051907.

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Summary - This is a mathematical description of the behaviour of dynein at microtubule plus-ends. The model is assuming two lanes only and is based on quantitative analysis of dynein motility in living U. maydis cells. This approach is novel and substantially differs from other modelling approaches that are based on in vitro experiments, often combining data derived from work on several different motor proteins from various biological sources.

Lin, C., Steinberg, G. & Ashwin, P. (2011) Bidirectional transport and pulsing states in a multi-lane ASEP model. J Stat Mech., P09027.

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Summary - This is a mathematical description of the behaviour of dynein at microtubule plus-ends substantially extends the two-lane model by Ashwin et al. 2010, Phys. Rev. E, 82: 051907. This model takes into account that (a) microtubules consist of 13 protofilaments that each can serve as a "track" for motor motility, (b) that dynein can change protofilaments while "walking" towards minus-ends of microtubules, (c) that kinesin-1 (which delivers dynein) cannot change lanes, (d) that a fraction of the delivered dynein "falls off" at microtubule plus-ends. Again, this modelling approach is based on quantitative in vivo observation of dynein in U. maydis.

Schuster, M., Kilaru, S, Ashwin, P., Lin, C., Severs, N. & Steinberg, G. (2011) Stochastic and controlled dynein accumulation prevents kinesin3-delivered endosomes from falling off at the microtubule plus-ends. EMBO J., 30, 652-664.

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Summary - In this paper we address the simple question "why do early endosomes not fall off at the end of a microtubule?". Using U. maydis as a model system we show that at average 55 dynein motors accumulate at the microtubule plus-ends. This is achieved by (a) actively capturing about half of the motors via an interaction of dynein/dynactin with the plus-end binding protein EB1 and (b) a stochastic "traffic jam" that keeps the other ~25 motors at the plus end (Figure 2). Both populations show different kinetics and, together, increase the probability of stochastic binding of released dynein to the kinesin-3-delivered early endosomes. In other words, an arriving early endosome meets an increasing number of dynein motors the closer it gets to the microtubule plus-end. This increases the chances of binding of the motor to the organelle, which in turn triggers retrograde motility towards minus-ends and prevents the organelle from "falling off" at the microtubule plus-end.

Schuster, M., Lipowsky, R., Assmann, M.-A., Lenz, P. & Steinberg, G. (2011) Transient stochastic binding of dynein controls bidirectional long-range motility of early endosomes. PNAS, 108, 3618-3622.

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Summary - In this paper we visualise native levels of kinesin-3 and dynein and by establishing nuclear pores as internal calibration standards, we show that 2-5 kinesin-3 motors take early endosomes towards plus-ends at the hyphal tip, whereas single dynein motors move them back towards the minus-ends. Interestingly, kinesin-3 is constantly attached to the organelles (though we show that some turn-over exists) whereas dynein transiently binds and unbinds to the early endosomes and moves them over long distances. The moment dynein binds, transport direction reverses from plus-end to minus-end directed. Thus, this dynamic attachment/detachment of dynein determines the run-length and motility behaviour of the early endosomes. This is the first description of an "exclusionary presence" mechanism in which the presence of a motor on an organelle determines the transport direction.

Schuster, M., Treitschke, S., Molloy, J., Kilaru, S., Harmer, N. & Steinberg, G. (2011) Kinesin-3 and dynein cooperate in long-range retrograde endosome motility along a nonuniform microtubule array. Mol Biol Cell, 22, 3645-3657.

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Summary - We show here that hyphal cells of U. maydis contain a antipolar array of microtubule bundles that is reminiscent of the dendritic extensions of neurons. This microtubule arrangement allows the cooperation of kinesin-3 and dynein in long-range retrograde motility of early endosomes, with dynein moving the organelles over ~10-20 micrometers through a unipolar microtubule array, whereas kinesin-3 moves the organelles over long distances along the antipolar microtubule bundles. This is the first report of cooperation of motors of opposing transport direction, and it emphasises the importance of the organization of the microtubule array in membrane trafficking.

Overview articles

Fuchs, U. & Steinberg, G. (2005) Endocytosis in the plant pathogenic fungus Ustilago maydis. Protoplasma, 226, 75-80.

Steinberg, G. & Fuchs, U. (2004) Microtubules in cellular organisation and endocytosis in the plant pathogen Ustilago maydis. J. Microscopy, 214, 114-123.

Steinberg, G. (2007) On the move: Endosomes in fungal growth and pathogenicity. Nat. Rev. Microbiol., 5, 309-316.

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Steinberg, G. (2011) Motors in fungal morphogenesis: cooperation versus competition. Curr Opin Microbiol, 14, 660-667.

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Pheromone induced a-hyphae

Figure 1: Pheromone induced a-hyphae that express the pheromone sensing receptor (Pra1) and histone 4 (H4) that labels the nucleus. The receptor is concentrated at the growing tip, but also becomes endocytosed and is sorted to the vacuole. Image provided by U. Fuchs.

Model of the formation and function of a dynein accumulation at the ends of microtubules

Figure 2: Model of the formation and function of a dynein accumulation at the ends of microtubules. (A) Dynein is targeted to plus-ends by kinesin-1. About half of the motors are rapidly turned over (fast release) whereas the other half gets trapped at the MT plus-ends. This involves an interaction between dynactin and the plus-end binding EB1(Peb1). This active retention is implicated in the observed slow release of dynein. (B) EEs get delivered by kinesin-3. Loading of the cargo onto dynein is a stochastic process that is most likely when the dynein density reaches its maximum.This stochastic loading process prevents the arriving early endosomes from "falling off" at the end of the microtubule. Image taken from Schuster et al., 2011, EMBO J. 30: 652.