Endocytosis in filamentous fungi: Cinderella gets her reward
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
References (68)
Insights from yeast endosomes
Curr Opin Cell Biol
(2002)- et al.
A modular design for the clathrin- and actin-mediated endocytosis machinery
Cell
(2005) Tracing the endocytic pathway of Aspergillus nidulans with FM4-64
Fungal Genet Biol
(2005)- et al.
Identification and characterization of Aspergillus nidulans mutants defective in cytokinesis
Genetics
(1994) - et al.
Candida albicans INT1-induced filamentation in Saccharomyces cerevisiae depends on Sla2p
Mol Cell Biol
(2001) - et al.
Constitutive activation of endocytosis by mutation of myoA, the myosin I gene of Aspergillus nidulans
J Biol Chem
(1998) - et al.
Organization and dynamics of the Aspergillus nidulans Golgi during apical extension and mitosis
Mol Biol Cell
(2009) - et al.
A dynein loading zone for retrograde endosome motility at microtubule plus-ends
EMBO J
(2006) - et al.
Shape and dynamics of tip-growing cells
Curr Biol
(2009) - et al.
Transferrin receptor-like proteins control the degradation of a yeast metal transporter
EMBO J
(2006)
The Ypt/Rab family and the evolution of trafficking in fungi
Traffic
Ontogeny of the Spitzenkorper in germlings of Neurospora crassa
Fungal Genet Biol
Nuclear movement is β-tubulin-dependent in Aspergillus nidulans
Cell
Candida albicans hyphae have a Spitzenkörper that is distinct from the polarisome found in yeast and pseudohyphae
J Cell Sci
Application of electron tomography to fungal ultrastructure studies
New Phytol
Functional stratification of the Spitzenkörper of Neurospora crassa
Mol Microbiol
Harnessing actin dynamics for clathrin-mediated endocytosis
Nat Rev Mol Cell Biol
Distinct acto/myosin-I structures associate with endocytic profiles at the plasma membrane
J Cell Biol
Confocal microscopy of FM4-64 as a tool for analysing endocytosis and vesicle trafficking in living fungal hyphae
J Microsc
A putative endosomal t-SNARE links exo- and endocytosis in the phytopathogenic fungus Ustilago maydis
EMBO J
F-actin dynamics in Neurospora crassa
Eukaryot Cell
Localization of actin and characterization of its isoforms in the hyphae of Neurospora crassa
FEMS Microbiol Lett
Maximal polar growth potential depends on the polarisome component AgSpa2 in the filamentous fungus Ashbya gossypii
Mol Biol Cell
Apical localization of actin patches and vacuolar dynamics in Ashbya gossypii depend on the WASP homolog Wal1p
J Cell Sci
Myosin I is required for hypha formation in Candida albicans
Eukaryot Cell
Preferential localization of the endocytic internalization machinery to hyphal tips underlies polarization of the actin cytoskeleton in Aspergillus nidulans
Mol Microbiol
The role of actin, fimbrin and endocytosis in growth of hyphae in Aspergillus nidulans
Mol Microbiol
The tip growth apparatus of Aspergillus nidulans
Mol Biol Cell
Growth-speed-correlated localization of exocyst and polarisome components in growth zones of Ashbya gossypii hyphal tips
J Cell Sci
Visualization of F-actin localization and dynamics with live cell markers in Neurospora crassa
Fungal Genet Biol
Slow diffusion of proteins in the yeast plasma membrane allows polarity to be maintained by endocytic cycling
Curr Biol
Spontaneous cell polarization through actomyosin-based delivery of the Cdc42 GTPase
Science
Aspergillus nidulans ArfB plays a role in endocytosis and polarized growth
Eukaryot Cell
BAR domain proteins Rvs161 and Rvs167 contribute to Candida albicans endocytosis, morphogenesis, and virulence
Infect Immun
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2021, Fungal Biology ReviewsEndocytosis of nutrient transporters in fungi: The ART of connecting signaling and trafficking
2021, Computational and Structural Biotechnology JournalCitation Excerpt :Rapid and dynamic turnover of nutrient transporters by endocytosis allows cells to quickly respond and adapt to nutrient fluctuations. Endocytic processes have been thoroughly characterized in model fungi such as Saccharomyces cerevisiae or Aspergillus nidulans [2–5]. Endocytosis is usually preceded by transporter ubiquitylation (the covalent attachment of the 76-amino-acid polypeptide ubiquitin) which signals the PM transporter for internalization.