Endocytosis in filamentous fungi: Cinderella gets her reward

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Endocytosis has been the Cinderella of membrane trafficking studies in filamentous fungi until recent work involving genetically tractable models has boosted interest in the field. Endocytic internalization predominates in the hyphal tips, spatially coupled to secretion. Early endosomes (EEs) show characteristic long-distance motility, riding on microtubule motors. The fungal tip contains a region baptised the ‘dynein loading zone’ where acropetally moving endosomes reaching the tip shift from a kinesin to dynein, reversing the direction of their movement. Multivesicular body biogenesis starts from these motile EEs. Maturation of EEs into late endosomes and vacuoles appears to be essential. The similarities between fungal and mammalian endocytic trafficking suggest that conditional mutant genetic screens would yield valuable information.

Introduction

Endocytosis is the process by which eukaryotic cells internalize plasma membrane (PM; Box 1) lipids and associated proteins in vesicles that fuse with the endosomal system. Subsequent segregation into different endosomal domains determines whether a given cargo recycles to the PM, traffics to the Golgi or follows the endocytic pathway to the vacuolar lumen, thus undergoing degradation. Figure 1 shows a model of endosomal pathways, inspired by work in Saccharomyces cerevisiae [1] and based on studies covered by this review.

In spite of the key role that one fungus, the yeast S. cerevisiae, played in understanding endocytic internalization in cortical ‘actin patches’ [2, 3, 4], studies of endocytosis in filamentous fungi started only recently. The finding that the lipophilic dye FM4-64 can be used to trace the fate of endocytosed membranes in genetically amenable fungi such as Neurospora crassa and Aspergillus nidulans [5, 6] fostered interest in the field. One key finding was that FM4-64 stains the Spitzenkörper, indicating that secretory carriers reaching this structure recycle endocytosed membrane to the apex and implicating this recycling in apical extension [5]. Also seminal were studies on the Ustilago maydis endosomal SNARE Yup-1, which localizes to several endocytic compartments, including a class of rapidly moving EEs [7]. This characteristic motility of EEs in U. maydis hyphae inspired later studies in A. nidulans.

Section snippets

Endocytosis and apical extension: the subapical endocytic internalization collar

Overwhelming evidence strongly indicates that endocytic internalization, although also taking place in subapical compartments/regions, predominates in the tips. F-actin is strongly polarized in hyphae [8••, 9, 10, 11, 12]. GFP/RFP-tagging of endocytic internalization machinery components including C. albicans type I myosin Myo5 [13] and A. nidulans AbpA (actin binding protein 1, Abp1), AmpA (Rvs167), SlaB (Sla2) and FimA (fimbrin; Sac6) [14•, 15•] revealed that this polarization is largely

Involvement of endocytosis in polar growth: mutational evidence

If endocytosis is coupled to apical extension, mutations preventing endocytosis should prevent growth. In agreement, A. nidulans slaBΔ is lethal. slaBΔ conidia rescued from heterokaryons are able to establish polarity but arrest in apical extension shortly after germ-tube emergence [14]. Also null mutants of arfB encoding a human Arf6 (a GTPase regulating endocytic internalization) orthologue display markedly extended isotropic growth and polarity maintenance defects [21]. Finally, disruption

Early endosomes show bi-directional long-distance movement

A seminal finding was that amongst the endocytic structures to which U. maydis Q-SNARE Yup1 localizes is a class of endosomes showing rapid (∼3 μm/s) bi-directional movement, mediated by MT-dependent motors [7]. These were shown to be EEs, labelled with FM4-64 at early time points [7] and colocalizing with the EE marker Rab5a [29]. Subsequently, Rab5 EEs of A. nidulans were also shown to undergo bi-directional long-distance movement on MTs [6, 30••, 31••]. While it remains to be established

Maturation of early endosomes into late endosomes

After labelling motile EEs FM4-64 reaches larger static structures probably representing LEs [7, 33•]. The motile EE residents Ustilago Yup1 [7] and the two A. nidulans Rab5 paralogues, RabA and RabB, also label larger static structures, whose abundance, in the case of the Rab5s, is increased upon overexpression [30••, 39••]. Thus, it seems that maturation of EEs into LEs is accompanied by an increase in size resulting from homotypic fusion (see below), with subsequent loss of motility.

Whether

Endosomal maturation is essential

EE maturation is essential for A. nidulans. rabAΔ and rabBΔ mutations are synthetically lethal, whereas vps45Δ and vps8Δ strains, although viable, are severely sick [39••]. vps24 encoding a component of ESCRT-III is virtually essential [42•, 43] as are genes encoding ESCRT-0, ESCRT-I, ESCRT-II and other ESCRT-III proteins including the major structural component Vps32 (Ana M. Calcagno, M.A.P. and Herbert N. Arst, unpublished). Of note, C. albicans mutants lacking Vps52p involved in recycling

The MVB pathway is active from the EE stage

Thus far the MVB pathway has been studied only in A. nidulans, and even here to a limited extent. Endogenous GFP-tagging of the key, essential subunits of ESCRT-III impairs their function and thus this method cannot be used to study ESCRT-III localization. However, C-terminal GFP/RFP tags interfere with the release of ESCRT-III monomers from membrane-bound polymers but not with their endosomal localization, and thus Vps32-GFP/mRFP has been studied in cells also expressing the endogenous

Endocytic downregulation of plasma membrane transporters

Endocytosis mediates the rapid removal of transporters and cation pumps from the cell surface under inappropriate circumstances, a process usually involving their ubiquitination by the HECT ubiquitin-ligase Rsp5 [50]. The A. nidulans purine transporter UapC was the first reported filamentous fungal example of such endocytic downregulation [51]. In A. niger UapC was used to study endocytosis and to demonstrate the long-distance motility of EEs [52]. Another notable example is the uric

Conclusions and open questions

In summary, research in several filamentous fungi has underscored the importance of endocytosis in apical extension as well as the spatial coupling of endocytosis with secretion; it has uncovered the long-distance movement of early endosomes as a distinguishing feature of this class of organelles and has pointed at the key role that endocytic recycling would appear to play in hyphal fungi; it has shown that endosomal maturation appears essential. Finally, it has suggested the existence other

References and recommended reading

  • • of special interest

  • •• of outstanding interest

Acknowledgements

Work in the author's laboratory is currently supported by the Spanish Ministry of Research and Innovation (Grant BIO2009-7281 to M.A.P.) and by a Comunidad de Madrid Regional Government Networking Grant SAL/0246/2006. My work on endocytosis over the past four years was primed by the generous hospitality of Hugh Pelham and the Medical Research Council UK during a sabbatical stay at the MRC Laboratory of Molecular Biology in Cambridge, UK. I would also like to thank all past and present members

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