- Email: [email protected]
Does endocytosis occur in fungal hyphae?
Fungal Genetics and Biology 39 (2003) 199–203 www.elsevier.com/locate/yfgbi
Commentary
Does endocytosis occur in fungal hyphae? Nick D. Read* and Eric R. Kalkman Fungal Cell Biology Group, Institute of Cell and Molecular Biology, University of Edinburgh, Rutherford Building, Edinburgh EH9 3JH, UK Received 31 January 2003; accepted 12 March 2003
Abstract The evidence and arguments for and against the occurrence of endocytosis in fungal hyphae are summarized. The balance of evidence is in favour of the existence of endocytosis. This is supported by an analysis of the recently sequenced Neurospora genome which strongly suggests that this fungus possesses the complex protein machinery required to conduct endocytosis. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Endocytosis; FM4-64; Lanthanum; Lucifer yellow; Neurospora genome
1. Introduction Endocytosis provides a mechanism for plasma membrane proteins and lipids, and extracellular molecules, to be internalized by cells. It is generally regarded as an essential process of eukaryotic cells serving many functions including recycling membrane proteins and lipids, removal of membrane proteins and lipids for degradation, and the uptake of signal molecules (Mellman, 1996). Endocytosis has been well characterized in budding yeast cells which possess a complex endocytic machinery (Geli and Riezman, 1998), and there is a significant body of evidence for endocytosis occurring in filamentous fungi (Atkinson et al., 2002; Fischer-Parton et al., 2000; Hoffmann and Mendgen, 1998; Read and Hickey, 2001; Wedlich-S€ oldner et al., 2000). However, its existence in hyphae has been questioned in a recent controversial paper published in Fungal Genetics and Biology by Torralba and Heath (2002). The purpose here is to summarise and critically assess the evidence and arguments for and against the occurrence of endocytosis in hyphae.
* Corresponding author. Fax: +44-131-650-5392. E-mail address: [email protected] (N.D. Read).
2. Evidence and arguments favouring endocytosis occurring in filamentous fungi 1. Membrane-selective markers of endocytosis are internalized. FM1-43 and FM4-64 are membrane-selective dyes that are widely used endocytosis markers because they become incorporated into endocytic vesicle membranes (Betz et al., 1996; Fischer-Parton et al., 2000). Cells of a range of filamentous fungal species readily take up these dyes (Atkinson et al., 2002; Fischer-Parton et al., 2000; Fisher et al., 2000; Hickey et al., 2002; Hoffmann and Mendgen, 1998; Read and Hickey, 2001; Steinberg et al., 1998; Torralba and Heath, 2002; Yamashita and May, 1998; Wedlich-S€ oldner et al., 2000). 2. Markers of fluid-phase endocytosis are internalized. Lucifer Yellow and FITC-dextran normally cannot cross the plasma membrane. They are thus frequently used as markers of fluid-phase endocytosis because they become trapped within the Ôfluid-phaseÕ of endocytic vesicles (Dulic et al., 1991). Both dyes were taken up by conidial germlings of Magnaporthe (Atkinson et al., 2002) and Lucifer Yellow was internalized by protoplasts of Ustilago (Steinberg et al., 1998). 3. Internalization of endocytosis markers is active and not by diffusion. Since endocytosis is an active process driven by ATP hydrolysis, cold treatment and metabolic inhibitors such as sodium azide will strongly inhibit it (Vida and Emr, 1995). Lucifer Yellow, FITC-dextran and FM4-64 internalization by conidial germlings of
1087-1845/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S1087-1845(03)00045-8
200
N.D. Read, E.R. Kalkman / Fungal Genetics and Biology 39 (2003) 199–203
Magnaporthe was reversibly inhibited by these treatments (Atkinson et al., 2002), and FM4-64 uptake was blocked by both inhibitory treatments in Neurospora and Uromyces (Fischer-Parton et al., 2000; Hoffmann and Mendgen, 1998). 4. Internalization of FM4-64 is actin-mediated. Endocytosis in budding yeast cells is actin-mediated (Geli and Riezman, 1998). Cytochalasin D, which depolymerises F-actin, inhibited FM4-64 uptake into hyphae of Aspergillus nidulans (Yamashita and May, 1998).
5. Neurospora genes encode a complex endocytic protein machinery. The recently sequenced genome of Neurospora crassa (Galaghan et al., 2003; www.genome. wi.mit.edu/annotation/fungi/neurospora/) was searched using BLASTP (Altschul et al., 1997) for homologues of 29 of the key proteins involved in budding yeast endocytosis. Each of these yeast proteins had one or more protein homologues in the Neurospora genome with very low E-values (E 6 1e-14), indicating high homology (Table 1). This strongly suggests that Neurospora
Table 1 Proteins involved in endocytosis in Saccharomyces cerevisiae and their homologues in N. crassa Yeast protein
Type of protein and comments
Neurospora protein homologuesa
E-value
Abp1p Aps2p Ark1p Arp2p Arp3p Chc1p Clc1p Dnm1p
Arp 2/3 activating Adaptor protein subunit Protein kinase Actin-related Actin-related Clathrin heavy chain Clathrin light chain Dynamin-like
End3p Ent1p Ent2p Inp51p
Eps15-like Epsin-like Epsin-like Synaptojanin-like
Inp52p
Synaptojanin-like
Inp53p
Synaptojanin-like
Las17p Myo3p Myo5p Pan1p Pfy1 Prk1p Rvs161p Rvs167p Sla1p Sla2p Yap1801p Yap1802p Ypt51p
WASP-like Type 1 myosin Type 1 myosin Eps15-like Profilin-like Protein kinase Amphiphysin-like Amphiphysin-like Interacts with cortical actin and associated proteins Interacts with cortical actin and associated proteins Component of clathrin coat Component of clathrin coat Rab5-like
Ypt52p
Rab5-like
Ypt53p
Rab5-like
NCU10073.1 NCU07989.1 NCU06202.1 NCU07171.1 NCU01756.1 NCU02510.1 NCU04115.1 NCU09808.1 NCU04100.1 NCU01255.1 NCU06347.1 NCU04783.1 NCU04783.1 NCU03298.1 NCU03792.1 NCU03298.1 NCU00896.1 NCU01330.1 NCU03792.1 NCU08689.1 NCU03298.1 NCU03792.1 NCU00896.1 NCU01330.1 NCU08689.1 NCU07438.1 NCU02111.1 NCU02111.1 NCU06171.1 NCU06397.1 NCU06202.1 NCU01069.1 NCU04637.1 NCU02978.1 NCU01956.1 NCU02586.1 NCU02586.1 NCU06410.1 NCU00895.1 NCU01523.1 NCU08477.1 NCU06410.1 NCU00895.1 NCU06410.1 NCU00895.1 NCU01523.1 NCU06404.1
4e-14 3e-39 5e-88 1e-144 1e-148 0 6e-20 0 1e-154 1e-45 2e-42 3e-33 5e-32 2e-78 4e-35 1e-137 3e-45 3e-43 5e-40 1e-32 1e-144 2e-38 5e-38 4e-37 2e-27 1e-18 0 0 5e-90 7e-26 1e-86 2e-78 9e-85 7e-48 6e-83 2e-49 7e-42 9e-58 2e-48 8e-29 2e-27 2e-34 5e-34 2e-48 6e-43 4e-27 4e-25
a
Locus of hypothetical protein in N. crassa genome (www-genome.wi.mit.edu/annotation/fungi/neurospora/).
N.D. Read, E.R. Kalkman / Fungal Genetics and Biology 39 (2003) 199–203
201
possesses the complex endocytic protein machinery required to conduct endocytosis. 6. Endocytosis is of critical importance for the functioning of eukaryotic cells. Endocytosis plays important roles in membrane recycling, membrane degradation, and the uptake of signal molecules, as well as having numerous other functions in eukaryotic cells (Mellman, 1996). It seems unlikely that fungal hyphae should not use endocytosis for similar purposes. Endocytosis is believed to occur in other tip-growing cells such as pollen tubes (e.g., see Camacho and Malh o, 2003).
2002). Furthermore, it has been proposed that excess membrane may also be accommodated by invaginations of the plasma membrane which are commonly observed at the ultrastructural level in chemically fixed hyphae (Torralba and Heath, 2002). 7. Not all cell types (e.g., vegetative hyphae) may need to undergo endocytosis. Besides possibly not needing endocytosis to retrieve excess membrane, other processes such as the uptake of pheromones by receptormediated endocytosis may not be a process undergone by vegetative hyphae (Torralba and Heath, 2002).
3. Evidence and arguments against endocytosis occurring filamentous fungi
4. Conclusions
1. FM4-64 is not taken up by hyphae of some species. Cole et al. (1998) have reported that Pisolithus tinctorius hyphae did not internalize FM4-64. 2. Lucifer Yellow is not internalized by hyphae of many filamentous fungi. Lucifer Yellow has been reported not to be taken up by hyphae of Neurospora (Fischer-Parton et al., 2000; Torralba and Heath, 2002) and P. tinctorius (Cole et al., 1997), or by sporidia and hyphae of Ustilago (Steinberg et al., 1998). 3. La3þ is not internalized by Neurospora. La3þ is a membrane impermeant endocytosis marker that can be visualized at the ultrastructural level. Torralba and Heath (2002) were unable to show that La3þ is incorporated into endocytic vesicles of Neurospora hyphae. 4. An energy-dependent process that does not involve endocytosis could internalize FM-dyes. An alternative mechanism for FM-dye uptake and distribution amongst different organelles in hyphae has been proposed by Fischer-Parton et al. (2000). This involves the amphilic FM-dyes being ÔflippedÕ by flippases across the plasma membrane and then subsequently transported to different organelles by lipid transfer proteins. 5. Clathrin-coated vesicles have not been identified in fungal cells. Clathrin-coated vesicles and pits are commonly visualized with the electron microscope in animal and plant cells, and are often indicative of clathrinmediated endocytosis (Kirchausen, 2000). We are unaware of any convincing published observations of this class of vesicle at the ultrastructural level in cells of filamentous fungi or budding yeast. 6. Hyphae do not need to recover excess membrane because they exhibit indeterminate growth. It has been proposed that more membrane may be added to the plasma membrane during secretion than is required to maintain growth and thus membrane recycling by endocytosis could provide a mechanism to retrieve this excess membrane (Read and Hickey, 2001). However, because hyphae are continually extending it has been argued that all the membrane added to the tip might be accommodated by tip expansion (Torralba and Heath,
Overall, positive evidence supporting the existence of endocytosis in filamentous fungi is increasing and is all consistent with what is known about endocytosis in budding yeast. All of the evidence against endocytosis occurring has been based on negative data or on hypotheses yet to be verified. These negative results might actually provide important clues about the unique cell biology of filamentous fungi. Let us take a more critical look at the evidence and arguments against the occurrence of endocytosis in hyphae. First, the non-uptake of endocytic marker stains, which was only observed in some species. This may reflect problems with the permeability of the cell walls of some species or cell types. In Ustilago, for instance, it was found that walled hyphae did not internalise Lucifer Yellow whilst wall-less protoplasts did (Steinberg et al., 1998). We need to assess the uptake of these probes into protoplasts from other species such as N. crassa. Second, the possibility that FM4-64 is internalised by an alternative active mechanism to endocytosis. Although flippase activity is a key feature of membrane bilayer assembly (Menon, 1995), flippase proteins have not yet been identified in filamentous fungi although lipid transfer proteins have (Record et al., 1998). Clearly we need to analyse the kinetics of FM4-64 internalization and distribution within hyphae in mutants defective in these proteins (see Table 1). At this stage we cannot discount the possibility that flippases and lipid transfer proteins may play some role in FM4-64 internalization. Torralba and Heath (2002) also reported that FM4-64, when combined with the vacuole-selective dye DFFDA, caused alterations in the membrane organization of Neurospora hyphae. In our experience, this is a photoxicity artifact induced by irradiating cells containing both dyes. We do not observe membrane alterations when FM4-64 is used alone, and this photoxicity effect can be reduced by decreasing the level of irradiation (Kalkman, E.R. and Read, N.D., unpublished). Third, the lack of identifiable clathrin-coated vesicles. Light and heavy chain clathrin are present in the
202
N.D. Read, E.R. Kalkman / Fungal Genetics and Biology 39 (2003) 199–203
Neurospora genome (Table 1). In budding yeast, both clathrin-dependent and clathrin-independent endocytosis occur but again clathrin-coated vesicles are apparently not evident at the ultrastructural level. However, clathrin-mediated endocytosis is not essential for budding yeast and thus does not appear to have the preeminent role that it does in animal cell endocytosis (Geli and Riezman, 1998). Also, clathrin-coated vesicles are involved in other parts of the vesicle trafficking network (Kirchausen, 2000). Obviously using live-cell imaging to analyse clathrin-defective mutants could be insightful here. Immunolabelling clathrin in hyphae might also prove useful although previous attempts have failed (Caesar-Ton That et al., 1987). Fourth, the possibility that indeterminate hyphal growth may alleviate the need for endocytosis. Although difficult, we need to make careful estimates of the amount of plasma membrane material added to the hyphal tip during both hyphal growth and the secretion of enzymes not involved in hyphal tip growth (e.g., W€ osten et al., 1991). The fact that a large number of the secretory vesicles fusing with the hyphal tip may not be involved in hyphal tip growth in itself suggests that there may be a significant need for membrane recovery by endocytosis. The possibility that excess membrane might be accommodated by plasma membrane invaginations in actively growing hyphae is unlikely because these types of invaginations, which are commonly observed in chemically fixed hyphae, are probably artifactual and rarely observed when prepared by the superior technique of freeze-substitution (Hoch, 1986). Although not conclusively proven, the balance of evidence is in favour of the occurrence of endocytosis in fungal hyphae. In the future, it will be essential to look at expression patterns in hyphae and other cells of the hypothetical Neurospora proteins listed in Table 1. We also need good genetic evidence, including live-cell analysis of mutants defective in endocytosis. Analysis of the kinetics of FM4-64 uptake by hyphae of these mutants will be very important in order to determine whether dye internalization is affected in a manner consistent with endocytosis, or not. The sequencing of Neurospora genome (Galaghan et al., 2003) has clearly identified candidate endocytosis genes to mutate (Table 1).
Acknowledgments Thanks are due to the Darwin Trust for a Studentship for E.R.K., and to Dr. Kathryn Ayscough and Alex Zelter for helpful comments. We are also grateful to the Whitehead Institute/MIT Center for Genome Research (http://www-genome.wi.mit.edu) for conducting the Neurospora Sequencing Project.
References Atkinson, H.A., Daniels, A., Read, N.D., 2002. Live-cell imaging of endocytosis during conidial germination in the rice blast fungus, Magnaporthe grisea. Fungal Genet. Biol. 37, 233–244. Altschul, S.F., Madden, T.L., Sch€affer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. Betz, W.J., Mao, F., Smith, C.B., 1996. Imaging exocytosis and endocytosis. Curr. Opin. Neurobiol. 6, 365–371. Caesar-Ton That, T.C., Hoangvan, K., Turian, G., Hoch, H.C., 1987. Isolation and characterization of coated vesicles from filamentous fungi. Eur. J. Cell Biol. 43, 189–194. Camacho, L., Malh o, R., 2003. Endo/exocytosis in the pollen tube apex is differentially regulated by Ca2þ and GTPases. J. Exper. Bot. 54, 83–92. Cole, L., Hyde, G.J., Ashford, A.E., 1997. Uptake and compartmentalisation of fluorescent probes by Pisolithus tinctorius hyphae: evidence for an anion transport mechanism at the tonoplast but not for fluid-phase endocytosis. Protoplasma 199, 18–29. Cole, L., Orlovich, D.A., Ashford, A.E., 1998. Structure, function, and motility of vacuoles in filamentous fungi. Fungal Genet. Biol. 24, 86–100. Dulic, V., Egerton, M., Elgundi, I., Raths, S., Singer, B., Riezman, H., 1991. Yeast endocytosis assays. Methods Enzymol. 194, 697–710. Fischer-Parton, S., Parton, R.M., Hickey, P.C., Dijksterhuis, J., Atkinson, H.A., Read, N.D., 2000. Confocal microscopy of FM464 as a tool for analysing endocytosis and vesicle trafficking in living fungal hyphae. J. Microsc. 198, 246–259. Fisher, K.E., Lowry, D.S., Roberson, R.W., 2000. Cytoplasmic cleavage in living zoosporangia of Allomyces macrogynus. 199, 260–269. Galaghan, J., Calvo, S., Borkovich, K., Selker, E., Read, N.D., FitzHugh, W., Ma, L.-J., Smirnov, S., Purcell, S., Rehman, B., Elkins, T., Engels, R., Wang, S., Nielsen, C.B., Butler, J., Jaffe, D., Endrizzi, M., Qui, D., PIanakiev, P., Bell-Pedersen, D., Nelson, M.A., Werner-Washburne, M., Selitrennikoff, C.P., Kinsey, J.A., Braun, E.L., Zelter, A., Schulte, U., Kothe, G.O., Jedd, G., Mewes, W., Staben, C., Marcotte, E., Greenberg, D., Roy, A., Foley, K., Naylor, J., Stange-Thomann, N., Barrett, R., Gnerre, S., Kamal, M., Kamvysselis, M., Bielke, C., Rudd, S., Frishman, D., Krystofova, S., Rasmussen, C., Metzenberg, R.L., Perkins, D.D., Kroken, S., Catcheside, D., Li, W., Pratt, R.J., Osmani, S.A., DeSouza, C.P.C., Glass, L., Orbach, M.J., Berglund, J.A., Voelker, R., Yarden, O., Plamann, M., Seiler, S., Dunlap, J., Radford, A., Aramayo, R., Natvig, D.O., Alex, L.A., Mannhaupt, G., Ebbole, D.J., Freitag, M., Paulsen, I., Sachs, M.S, Lander, E.S, Nusbaum, C., Birren, B., 2003. The genome sequence of the filamentous fungus Neurospora crassa. Nature 422, 859–868. Geli, M.I., Riezman, H., 1998. Endocytic internalization in yeast and animal cells: similar and different. J. Cell Sci. 111, 1031– 1037. Hickey, P.C., Jacobson, D.J., Read, N.D., Glass, N.L., 2002. Live-cell imaging of vegetative hyphal fusion in Neurospora crassa. Fungal Genet. Biol. 37, 109–119. Hoch, C., 1986. Freeze-substitution of fungi. In: Aldrich, H.C., Todd, W.J. (Eds.), Ultrastructure Techniques for Microorganisms. Plenum Press, New York, pp. 183–212. Hoffmann, J., Mendgen, K., 1998. Endocytosis and membrane turnover in the germ tube of Uromyces fabae. Fungal Genet. Biol. 24, 77–85. Kirchausen, T., 2000. Three ways to make a vesicle. Nature Rev. Mol. Cell Biol. 1, 187–198.
N.D. Read, E.R. Kalkman / Fungal Genetics and Biology 39 (2003) 199–203 Mellman, I., 1996. Endocytosis and molecular sorting. Annu. Rev. Cell Dev. Biol. 12, 575–625. Menon, A.K., 1995. Flippases. Trends Cell Biol. 5, 355–360. Read, N.D., Hickey, P.C., 2001. The vesicle trafficking network and tip growth in fungal hyphae. In: Geitmann, A., Cresti, M., Heath, I.B. (Eds.), Cell Biology of Plant and Fungal Tip Growth. IOS Press, Amsterdam, pp. 137–146. Record, E., Asther, M., Moukha, S., Marion, D., Burlat, V., Ruel, K., 1998. Localization of a phosphatidylglycerol/phosphatidylinositol transfer protein in Aspergillus oryzae. Can. J. Microbiol. 44, 945–953. Steinberg, G., Schliwa, M., Lehmler, C., B€ olker, M., Kahmann, R., McIntosh, J.R., 1998. Kinesin from the plant pathogenic fungus Ustilago maydis is involved in vacuole formation and cytoplasmic migration. J. Cell Sci. 111, 2235–2246.
203
Torralba, S., Heath, I.B., 2002. Analysis of three separate probes suggests the absence of endocytosis in Neurospora crassa hyphae. Fungal Genet. Biol. 37, 221–232. Vida, T.A., Emr, S.D., 1995. A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J. Cell Biol. 128, 779–792. Wedlich-S€ oldner, R., B€ olker, 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. W€ osten, H.A.B., Moukha, S.M., Sitesma, J.H., Wessels, J.G.H., 1991. Localization of growth and secretion of proteins in Aspergillus niger. J. Gen. Microbiol. 137, 2017–2023. Yamashita, R.A., May, G.S., 1998. Constitutive activation of endocytosis by myoA, the Myosin 1 gene of Aspergillus nidulans. J. Biol. Chem. 273, 14644–14648.
Commentary
Does endocytosis occur in fungal hyphae? Nick D. Read* and Eric R. Kalkman Fungal Cell Biology Group, Institute of Cell and Molecular Biology, University of Edinburgh, Rutherford Building, Edinburgh EH9 3JH, UK Received 31 January 2003; accepted 12 March 2003
Abstract The evidence and arguments for and against the occurrence of endocytosis in fungal hyphae are summarized. The balance of evidence is in favour of the existence of endocytosis. This is supported by an analysis of the recently sequenced Neurospora genome which strongly suggests that this fungus possesses the complex protein machinery required to conduct endocytosis. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Endocytosis; FM4-64; Lanthanum; Lucifer yellow; Neurospora genome
1. Introduction Endocytosis provides a mechanism for plasma membrane proteins and lipids, and extracellular molecules, to be internalized by cells. It is generally regarded as an essential process of eukaryotic cells serving many functions including recycling membrane proteins and lipids, removal of membrane proteins and lipids for degradation, and the uptake of signal molecules (Mellman, 1996). Endocytosis has been well characterized in budding yeast cells which possess a complex endocytic machinery (Geli and Riezman, 1998), and there is a significant body of evidence for endocytosis occurring in filamentous fungi (Atkinson et al., 2002; Fischer-Parton et al., 2000; Hoffmann and Mendgen, 1998; Read and Hickey, 2001; Wedlich-S€ oldner et al., 2000). However, its existence in hyphae has been questioned in a recent controversial paper published in Fungal Genetics and Biology by Torralba and Heath (2002). The purpose here is to summarise and critically assess the evidence and arguments for and against the occurrence of endocytosis in hyphae.
* Corresponding author. Fax: +44-131-650-5392. E-mail address: [email protected] (N.D. Read).
2. Evidence and arguments favouring endocytosis occurring in filamentous fungi 1. Membrane-selective markers of endocytosis are internalized. FM1-43 and FM4-64 are membrane-selective dyes that are widely used endocytosis markers because they become incorporated into endocytic vesicle membranes (Betz et al., 1996; Fischer-Parton et al., 2000). Cells of a range of filamentous fungal species readily take up these dyes (Atkinson et al., 2002; Fischer-Parton et al., 2000; Fisher et al., 2000; Hickey et al., 2002; Hoffmann and Mendgen, 1998; Read and Hickey, 2001; Steinberg et al., 1998; Torralba and Heath, 2002; Yamashita and May, 1998; Wedlich-S€ oldner et al., 2000). 2. Markers of fluid-phase endocytosis are internalized. Lucifer Yellow and FITC-dextran normally cannot cross the plasma membrane. They are thus frequently used as markers of fluid-phase endocytosis because they become trapped within the Ôfluid-phaseÕ of endocytic vesicles (Dulic et al., 1991). Both dyes were taken up by conidial germlings of Magnaporthe (Atkinson et al., 2002) and Lucifer Yellow was internalized by protoplasts of Ustilago (Steinberg et al., 1998). 3. Internalization of endocytosis markers is active and not by diffusion. Since endocytosis is an active process driven by ATP hydrolysis, cold treatment and metabolic inhibitors such as sodium azide will strongly inhibit it (Vida and Emr, 1995). Lucifer Yellow, FITC-dextran and FM4-64 internalization by conidial germlings of
1087-1845/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S1087-1845(03)00045-8
200
N.D. Read, E.R. Kalkman / Fungal Genetics and Biology 39 (2003) 199–203
Magnaporthe was reversibly inhibited by these treatments (Atkinson et al., 2002), and FM4-64 uptake was blocked by both inhibitory treatments in Neurospora and Uromyces (Fischer-Parton et al., 2000; Hoffmann and Mendgen, 1998). 4. Internalization of FM4-64 is actin-mediated. Endocytosis in budding yeast cells is actin-mediated (Geli and Riezman, 1998). Cytochalasin D, which depolymerises F-actin, inhibited FM4-64 uptake into hyphae of Aspergillus nidulans (Yamashita and May, 1998).
5. Neurospora genes encode a complex endocytic protein machinery. The recently sequenced genome of Neurospora crassa (Galaghan et al., 2003; www.genome. wi.mit.edu/annotation/fungi/neurospora/) was searched using BLASTP (Altschul et al., 1997) for homologues of 29 of the key proteins involved in budding yeast endocytosis. Each of these yeast proteins had one or more protein homologues in the Neurospora genome with very low E-values (E 6 1e-14), indicating high homology (Table 1). This strongly suggests that Neurospora
Table 1 Proteins involved in endocytosis in Saccharomyces cerevisiae and their homologues in N. crassa Yeast protein
Type of protein and comments
Neurospora protein homologuesa
E-value
Abp1p Aps2p Ark1p Arp2p Arp3p Chc1p Clc1p Dnm1p
Arp 2/3 activating Adaptor protein subunit Protein kinase Actin-related Actin-related Clathrin heavy chain Clathrin light chain Dynamin-like
End3p Ent1p Ent2p Inp51p
Eps15-like Epsin-like Epsin-like Synaptojanin-like
Inp52p
Synaptojanin-like
Inp53p
Synaptojanin-like
Las17p Myo3p Myo5p Pan1p Pfy1 Prk1p Rvs161p Rvs167p Sla1p Sla2p Yap1801p Yap1802p Ypt51p
WASP-like Type 1 myosin Type 1 myosin Eps15-like Profilin-like Protein kinase Amphiphysin-like Amphiphysin-like Interacts with cortical actin and associated proteins Interacts with cortical actin and associated proteins Component of clathrin coat Component of clathrin coat Rab5-like
Ypt52p
Rab5-like
Ypt53p
Rab5-like
NCU10073.1 NCU07989.1 NCU06202.1 NCU07171.1 NCU01756.1 NCU02510.1 NCU04115.1 NCU09808.1 NCU04100.1 NCU01255.1 NCU06347.1 NCU04783.1 NCU04783.1 NCU03298.1 NCU03792.1 NCU03298.1 NCU00896.1 NCU01330.1 NCU03792.1 NCU08689.1 NCU03298.1 NCU03792.1 NCU00896.1 NCU01330.1 NCU08689.1 NCU07438.1 NCU02111.1 NCU02111.1 NCU06171.1 NCU06397.1 NCU06202.1 NCU01069.1 NCU04637.1 NCU02978.1 NCU01956.1 NCU02586.1 NCU02586.1 NCU06410.1 NCU00895.1 NCU01523.1 NCU08477.1 NCU06410.1 NCU00895.1 NCU06410.1 NCU00895.1 NCU01523.1 NCU06404.1
4e-14 3e-39 5e-88 1e-144 1e-148 0 6e-20 0 1e-154 1e-45 2e-42 3e-33 5e-32 2e-78 4e-35 1e-137 3e-45 3e-43 5e-40 1e-32 1e-144 2e-38 5e-38 4e-37 2e-27 1e-18 0 0 5e-90 7e-26 1e-86 2e-78 9e-85 7e-48 6e-83 2e-49 7e-42 9e-58 2e-48 8e-29 2e-27 2e-34 5e-34 2e-48 6e-43 4e-27 4e-25
a
Locus of hypothetical protein in N. crassa genome (www-genome.wi.mit.edu/annotation/fungi/neurospora/).
N.D. Read, E.R. Kalkman / Fungal Genetics and Biology 39 (2003) 199–203
201
possesses the complex endocytic protein machinery required to conduct endocytosis. 6. Endocytosis is of critical importance for the functioning of eukaryotic cells. Endocytosis plays important roles in membrane recycling, membrane degradation, and the uptake of signal molecules, as well as having numerous other functions in eukaryotic cells (Mellman, 1996). It seems unlikely that fungal hyphae should not use endocytosis for similar purposes. Endocytosis is believed to occur in other tip-growing cells such as pollen tubes (e.g., see Camacho and Malh o, 2003).
2002). Furthermore, it has been proposed that excess membrane may also be accommodated by invaginations of the plasma membrane which are commonly observed at the ultrastructural level in chemically fixed hyphae (Torralba and Heath, 2002). 7. Not all cell types (e.g., vegetative hyphae) may need to undergo endocytosis. Besides possibly not needing endocytosis to retrieve excess membrane, other processes such as the uptake of pheromones by receptormediated endocytosis may not be a process undergone by vegetative hyphae (Torralba and Heath, 2002).
3. Evidence and arguments against endocytosis occurring filamentous fungi
4. Conclusions
1. FM4-64 is not taken up by hyphae of some species. Cole et al. (1998) have reported that Pisolithus tinctorius hyphae did not internalize FM4-64. 2. Lucifer Yellow is not internalized by hyphae of many filamentous fungi. Lucifer Yellow has been reported not to be taken up by hyphae of Neurospora (Fischer-Parton et al., 2000; Torralba and Heath, 2002) and P. tinctorius (Cole et al., 1997), or by sporidia and hyphae of Ustilago (Steinberg et al., 1998). 3. La3þ is not internalized by Neurospora. La3þ is a membrane impermeant endocytosis marker that can be visualized at the ultrastructural level. Torralba and Heath (2002) were unable to show that La3þ is incorporated into endocytic vesicles of Neurospora hyphae. 4. An energy-dependent process that does not involve endocytosis could internalize FM-dyes. An alternative mechanism for FM-dye uptake and distribution amongst different organelles in hyphae has been proposed by Fischer-Parton et al. (2000). This involves the amphilic FM-dyes being ÔflippedÕ by flippases across the plasma membrane and then subsequently transported to different organelles by lipid transfer proteins. 5. Clathrin-coated vesicles have not been identified in fungal cells. Clathrin-coated vesicles and pits are commonly visualized with the electron microscope in animal and plant cells, and are often indicative of clathrinmediated endocytosis (Kirchausen, 2000). We are unaware of any convincing published observations of this class of vesicle at the ultrastructural level in cells of filamentous fungi or budding yeast. 6. Hyphae do not need to recover excess membrane because they exhibit indeterminate growth. It has been proposed that more membrane may be added to the plasma membrane during secretion than is required to maintain growth and thus membrane recycling by endocytosis could provide a mechanism to retrieve this excess membrane (Read and Hickey, 2001). However, because hyphae are continually extending it has been argued that all the membrane added to the tip might be accommodated by tip expansion (Torralba and Heath,
Overall, positive evidence supporting the existence of endocytosis in filamentous fungi is increasing and is all consistent with what is known about endocytosis in budding yeast. All of the evidence against endocytosis occurring has been based on negative data or on hypotheses yet to be verified. These negative results might actually provide important clues about the unique cell biology of filamentous fungi. Let us take a more critical look at the evidence and arguments against the occurrence of endocytosis in hyphae. First, the non-uptake of endocytic marker stains, which was only observed in some species. This may reflect problems with the permeability of the cell walls of some species or cell types. In Ustilago, for instance, it was found that walled hyphae did not internalise Lucifer Yellow whilst wall-less protoplasts did (Steinberg et al., 1998). We need to assess the uptake of these probes into protoplasts from other species such as N. crassa. Second, the possibility that FM4-64 is internalised by an alternative active mechanism to endocytosis. Although flippase activity is a key feature of membrane bilayer assembly (Menon, 1995), flippase proteins have not yet been identified in filamentous fungi although lipid transfer proteins have (Record et al., 1998). Clearly we need to analyse the kinetics of FM4-64 internalization and distribution within hyphae in mutants defective in these proteins (see Table 1). At this stage we cannot discount the possibility that flippases and lipid transfer proteins may play some role in FM4-64 internalization. Torralba and Heath (2002) also reported that FM4-64, when combined with the vacuole-selective dye DFFDA, caused alterations in the membrane organization of Neurospora hyphae. In our experience, this is a photoxicity artifact induced by irradiating cells containing both dyes. We do not observe membrane alterations when FM4-64 is used alone, and this photoxicity effect can be reduced by decreasing the level of irradiation (Kalkman, E.R. and Read, N.D., unpublished). Third, the lack of identifiable clathrin-coated vesicles. Light and heavy chain clathrin are present in the
202
N.D. Read, E.R. Kalkman / Fungal Genetics and Biology 39 (2003) 199–203
Neurospora genome (Table 1). In budding yeast, both clathrin-dependent and clathrin-independent endocytosis occur but again clathrin-coated vesicles are apparently not evident at the ultrastructural level. However, clathrin-mediated endocytosis is not essential for budding yeast and thus does not appear to have the preeminent role that it does in animal cell endocytosis (Geli and Riezman, 1998). Also, clathrin-coated vesicles are involved in other parts of the vesicle trafficking network (Kirchausen, 2000). Obviously using live-cell imaging to analyse clathrin-defective mutants could be insightful here. Immunolabelling clathrin in hyphae might also prove useful although previous attempts have failed (Caesar-Ton That et al., 1987). Fourth, the possibility that indeterminate hyphal growth may alleviate the need for endocytosis. Although difficult, we need to make careful estimates of the amount of plasma membrane material added to the hyphal tip during both hyphal growth and the secretion of enzymes not involved in hyphal tip growth (e.g., W€ osten et al., 1991). The fact that a large number of the secretory vesicles fusing with the hyphal tip may not be involved in hyphal tip growth in itself suggests that there may be a significant need for membrane recovery by endocytosis. The possibility that excess membrane might be accommodated by plasma membrane invaginations in actively growing hyphae is unlikely because these types of invaginations, which are commonly observed in chemically fixed hyphae, are probably artifactual and rarely observed when prepared by the superior technique of freeze-substitution (Hoch, 1986). Although not conclusively proven, the balance of evidence is in favour of the occurrence of endocytosis in fungal hyphae. In the future, it will be essential to look at expression patterns in hyphae and other cells of the hypothetical Neurospora proteins listed in Table 1. We also need good genetic evidence, including live-cell analysis of mutants defective in endocytosis. Analysis of the kinetics of FM4-64 uptake by hyphae of these mutants will be very important in order to determine whether dye internalization is affected in a manner consistent with endocytosis, or not. The sequencing of Neurospora genome (Galaghan et al., 2003) has clearly identified candidate endocytosis genes to mutate (Table 1).
Acknowledgments Thanks are due to the Darwin Trust for a Studentship for E.R.K., and to Dr. Kathryn Ayscough and Alex Zelter for helpful comments. We are also grateful to the Whitehead Institute/MIT Center for Genome Research (http://www-genome.wi.mit.edu) for conducting the Neurospora Sequencing Project.
References Atkinson, H.A., Daniels, A., Read, N.D., 2002. Live-cell imaging of endocytosis during conidial germination in the rice blast fungus, Magnaporthe grisea. Fungal Genet. Biol. 37, 233–244. Altschul, S.F., Madden, T.L., Sch€affer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. Betz, W.J., Mao, F., Smith, C.B., 1996. Imaging exocytosis and endocytosis. Curr. Opin. Neurobiol. 6, 365–371. Caesar-Ton That, T.C., Hoangvan, K., Turian, G., Hoch, H.C., 1987. Isolation and characterization of coated vesicles from filamentous fungi. Eur. J. Cell Biol. 43, 189–194. Camacho, L., Malh o, R., 2003. Endo/exocytosis in the pollen tube apex is differentially regulated by Ca2þ and GTPases. J. Exper. Bot. 54, 83–92. Cole, L., Hyde, G.J., Ashford, A.E., 1997. Uptake and compartmentalisation of fluorescent probes by Pisolithus tinctorius hyphae: evidence for an anion transport mechanism at the tonoplast but not for fluid-phase endocytosis. Protoplasma 199, 18–29. Cole, L., Orlovich, D.A., Ashford, A.E., 1998. Structure, function, and motility of vacuoles in filamentous fungi. Fungal Genet. Biol. 24, 86–100. Dulic, V., Egerton, M., Elgundi, I., Raths, S., Singer, B., Riezman, H., 1991. Yeast endocytosis assays. Methods Enzymol. 194, 697–710. Fischer-Parton, S., Parton, R.M., Hickey, P.C., Dijksterhuis, J., Atkinson, H.A., Read, N.D., 2000. Confocal microscopy of FM464 as a tool for analysing endocytosis and vesicle trafficking in living fungal hyphae. J. Microsc. 198, 246–259. Fisher, K.E., Lowry, D.S., Roberson, R.W., 2000. Cytoplasmic cleavage in living zoosporangia of Allomyces macrogynus. 199, 260–269. Galaghan, J., Calvo, S., Borkovich, K., Selker, E., Read, N.D., FitzHugh, W., Ma, L.-J., Smirnov, S., Purcell, S., Rehman, B., Elkins, T., Engels, R., Wang, S., Nielsen, C.B., Butler, J., Jaffe, D., Endrizzi, M., Qui, D., PIanakiev, P., Bell-Pedersen, D., Nelson, M.A., Werner-Washburne, M., Selitrennikoff, C.P., Kinsey, J.A., Braun, E.L., Zelter, A., Schulte, U., Kothe, G.O., Jedd, G., Mewes, W., Staben, C., Marcotte, E., Greenberg, D., Roy, A., Foley, K., Naylor, J., Stange-Thomann, N., Barrett, R., Gnerre, S., Kamal, M., Kamvysselis, M., Bielke, C., Rudd, S., Frishman, D., Krystofova, S., Rasmussen, C., Metzenberg, R.L., Perkins, D.D., Kroken, S., Catcheside, D., Li, W., Pratt, R.J., Osmani, S.A., DeSouza, C.P.C., Glass, L., Orbach, M.J., Berglund, J.A., Voelker, R., Yarden, O., Plamann, M., Seiler, S., Dunlap, J., Radford, A., Aramayo, R., Natvig, D.O., Alex, L.A., Mannhaupt, G., Ebbole, D.J., Freitag, M., Paulsen, I., Sachs, M.S, Lander, E.S, Nusbaum, C., Birren, B., 2003. The genome sequence of the filamentous fungus Neurospora crassa. Nature 422, 859–868. Geli, M.I., Riezman, H., 1998. Endocytic internalization in yeast and animal cells: similar and different. J. Cell Sci. 111, 1031– 1037. Hickey, P.C., Jacobson, D.J., Read, N.D., Glass, N.L., 2002. Live-cell imaging of vegetative hyphal fusion in Neurospora crassa. Fungal Genet. Biol. 37, 109–119. Hoch, C., 1986. Freeze-substitution of fungi. In: Aldrich, H.C., Todd, W.J. (Eds.), Ultrastructure Techniques for Microorganisms. Plenum Press, New York, pp. 183–212. Hoffmann, J., Mendgen, K., 1998. Endocytosis and membrane turnover in the germ tube of Uromyces fabae. Fungal Genet. Biol. 24, 77–85. Kirchausen, T., 2000. Three ways to make a vesicle. Nature Rev. Mol. Cell Biol. 1, 187–198.
N.D. Read, E.R. Kalkman / Fungal Genetics and Biology 39 (2003) 199–203 Mellman, I., 1996. Endocytosis and molecular sorting. Annu. Rev. Cell Dev. Biol. 12, 575–625. Menon, A.K., 1995. Flippases. Trends Cell Biol. 5, 355–360. Read, N.D., Hickey, P.C., 2001. The vesicle trafficking network and tip growth in fungal hyphae. In: Geitmann, A., Cresti, M., Heath, I.B. (Eds.), Cell Biology of Plant and Fungal Tip Growth. IOS Press, Amsterdam, pp. 137–146. Record, E., Asther, M., Moukha, S., Marion, D., Burlat, V., Ruel, K., 1998. Localization of a phosphatidylglycerol/phosphatidylinositol transfer protein in Aspergillus oryzae. Can. J. Microbiol. 44, 945–953. Steinberg, G., Schliwa, M., Lehmler, C., B€ olker, M., Kahmann, R., McIntosh, J.R., 1998. Kinesin from the plant pathogenic fungus Ustilago maydis is involved in vacuole formation and cytoplasmic migration. J. Cell Sci. 111, 2235–2246.
203
Torralba, S., Heath, I.B., 2002. Analysis of three separate probes suggests the absence of endocytosis in Neurospora crassa hyphae. Fungal Genet. Biol. 37, 221–232. Vida, T.A., Emr, S.D., 1995. A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J. Cell Biol. 128, 779–792. Wedlich-S€ oldner, R., B€ olker, 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. W€ osten, H.A.B., Moukha, S.M., Sitesma, J.H., Wessels, J.G.H., 1991. Localization of growth and secretion of proteins in Aspergillus niger. J. Gen. Microbiol. 137, 2017–2023. Yamashita, R.A., May, G.S., 1998. Constitutive activation of endocytosis by myoA, the Myosin 1 gene of Aspergillus nidulans. J. Biol. Chem. 273, 14644–14648.