Department of Biology, University College London, Gower Street, London, WC1E 6BT, UK
Author for correspondence (e-mail: j.hyams{at}massey.ac.nz)
Accepted 7 June 2005
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Summary |
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Key words: Endocytosis, FM4-64, Actin, Fission yeast
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Introduction |
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Although the role of actin in CAR assembly and contraction is becoming increasingly well understood, the role of actin patches at the cell poles and equator is less well established. As in budding yeast (Doyle and Botstein, 1996; Waddle et al., 1996
), actin patches are dynamic (Pelham and Chang, 2001
) but whether they share the molecular complexity of their budding yeast counterparts (Pruyne and Bretscher, 2000
) remains to be investigated. Cortical actin is required for the localisation of two classes of enzymes concerned with cell wall synthesis:
-glucan synthases (Katayama et al., 1999
; Win et al., 2001
) and ß-glucan synthases (Cortés et al., 2002
) but other functions almost certainly remain to be revealed. Studies in a range of eukaryotic cell types have linked the cortical actin cytoskeleton to endocytosis (Geli and Riezman, 1998
; Jeng and Welch, 2001
). However, until very recently, the structural relationship between actin and the endocytic pathway remained imprecisely defined (Qualmann et al., 2000
). Actin was thought to localise endocytosis to particular regions of the cell cortex, or to play a role in membrane internalisation and/or to move vesicles to the cell interior. Recent reports have gone some way to resolve some of these issues (Kaksonen et al., 2003
; Huckaba et al., 2004
). In the budding yeast Saccharomyces cerevisiae, actin patches are assembled at sites of endocytosis and serve to aid the internalisation of endocytic vesicles. Actin polymerisation at the patch (Kaksonen et al., 2003
) or the dynamic properties of actin cables (Huckaba et al., 2004
) then drives the vesicle into the cell. The association between patch and vesicle is transient, accounting for the short life span of actin patches (Smith et al., 2001
; Kaksonen et al., 2003
).
Here we present the first attempt to describe the endocytic pathway in fission yeast. We show that endocytosis precisely mirrors the pattern of cell growth and cytokinesis in S. pombe. We show that the internalisation step of endocytosis at the cell poles is dependent upon actin patches and that, as in budding yeast, endocytic vesicles and actin patches transiently colocalise. However, endocytosis at the cell equator additionally requires actin cables. The cytological clarity of endocytosis in fission yeast and the ability to isolate endocytosis to particular regions of the cell at different cell cycle stages suggest that S. pombe will become a valuable model system in which to dissect the mechanisms and function of this fundamental cellular process.
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Materials and Methods |
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Results |
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To further investigate the correlation between the site of cell wall deposition/actin and endocytosis we investigated situations in which the pattern of cell growth was altered. Cells lacking the kinesin-like protein Tea2 form lateral branches with growth confined to the tip of the branch (Browning et al., 2000). Staining of tea2
cells with FM4-64 once again revealed a clear correlation between the site of cell growth/actin and FM4-64 uptake (Fig. 1E). Wild-type cells starved for nitrogen accumulate in G1, prior to NETO. Although endocytosis is reduced in such cells, FM4-64 uptake was uniquely associated with the old end (identified by its bright staining with the cell wall dye Calcofluor; Fig. 1F). Finally, we examined the situation in cdc10-129 cells that were arrested in G1 by growth at the restrictive temperature and then allowed to pass NETO synchronously following return to the permissive temperature. Endocytosis changed from monopolar to bipolar in concert with the redistribution of actin as post-NETO cells switched to bipolar growth (Fig. 1G,H).
To distinguish between vacuoles and other vesicular compartments, we added FM4-64 to cells in which vacuoles were prelabelled with CDCFDA, a dye that enters vacuoles independently of the endocytic pathway. FM4-64 fluorescence was initially distinct from CDCFDA fluorescence and was associated with numerous small vesicular structures. These initially increased in number but then declined as fluorescence transferred to the vacuolar membrane (Fig. 2A,B). When CDCFDA-labelled vacuoles were enlarged by fusion prior to the addition of FM4-64, fluorescence remained associated with the small vesicular compartments (Fig. 2C,D). We tentatively identify these structures as prevacuolar compartments (PVC) although we cannot exclude possibility that they include vesicles that recycle back to the cell membrane or to other cellular compartments.
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Endocytosis in fission yeast is associated with actin
Given that FM4-64 is taken up at the poles and the equator of fission yeast cells, regions that are rich in actin, we further investigated this relationship, taking advantage of cells expressing GFP-tagged Crn1, the fission yeast coronin homologue, which is predominantly associated with actin patches (Pelham and Chang, 2001). FM4-64 labelling of Crn1-GFP cells showed that a subset of endocytic vesicles was spatially coincident with a subset of actin patches (Fig. 4A). An individual vesicle remained associated with an individual actin patch for <20 seconds, following which colocalisation was lost. When Crn1-GFP cells were treated with the actin inhibitor latrunculin B at a concentration that completely destroys the actin cytoskeleton prior to the addition of FM4-64, internalisation of the dye was largely abolished. Some coronin patches remained after this treatment (Pelham and Chang, 2001
) and FM4-64 was internalised at these residual structures but fluorescence was not transferred to internal compartments (Fig. 4B). Bright fluorescence was also detected at the membrane of dividing cells in the region of the septum (Fig. 4B). Quantification of FM4-64 uptake confirmed the inhibitory effect of latrunculin B but not the microtubule inhibitor TBZ (Fig. 4C). Neither latrunculin nor TBZ inhibited the uptake of CDCFDA (Fig. 4D). To investigate whether, in addition to an early step in endocytosis, actin was also associated with later events (transfer to prevacuolar compartments and vacuole), we prelabelled vacuoles with CDCFDA then added FM4-64. After 3 minutes the FM4-64 was washed out and either latrunculin B or TBZ added. Cells were then observed to see whether fluorescence was transferred to the vacuole in the presence of either inhibitor. FM4-64 transferred effectively to the vacuole membrane (Fig. 4E,F), hence neither disruption of the actin cytoskeleton nor depolymerisation of the microtubule network affected the later events of endocytic pathway
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To eliminate possible non-specific effects of latrunculin, we followed endocytosis in the temperature-sensitive actin mutant cps8. Even at the permissive temperature, FM4-64 uptake at the poles was markedly inhibited in this genetic background: bright patches of staining remained adjacent to the cell membrane but with little or no transfer to internal compartments (Fig. 5A,B). By contrast, bright fluorescence was associated with the membrane flanking the cytokinetic septum although transfer did not progress beyond this point. No inhibition was observed in the cold-sensitive -tubulin mutant nda2 at the restrictive temperature (Fig. 5A,C). FM4-64 uptake was also inhibited at the restrictive temperature in the mutant arp2, encoding a component of the Arp2/3 complex, in which actin patches are selectively destabilised (Morrell et al., 1999
) (Fig. 5D) but not in for3
, which has normal patches and lacks actin cables (Feierbach and Chang, 2001
) (Fig. 5E). As in cps8, patches of fluorescence were seen at the cell membrane in arp2 and bright fluorescence was seen at the cell membrane at the poles and septa.
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Endocytosis at the cell equator is associated with actin patches and filaments
A feature of endocytosis in cps8 and arp2 cells was the persistence of staining at the cell membrane at the equatorial region of dividing cells (Fig. 5B,D). Because the actin dependence of equatorial FM4-64 uptake appeared to be somewhat different to that at the poles, we re-examined the colocalisation of FM4-64 uptake and actin patches in this region using the Crn1-GFP strain. As described previously for the cell poles, endocytic vesicles again showed a clear but transient colocalisation with actin patches (Fig. 6A).
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Endocytosis at the cell equator is associated with exocytosis and septation
To further examine the relationship between equatorial endocytosis and septum formation, we followed FM4-64 uptake in the SIN mutants cdc7-24, sid1-125 and sid2-250. At the restrictive temperature, cytokinesis is inhibited in these strains, which nevertheless go through repeated nuclear divisions, becoming multinucleate. All three mutants were followed through two rounds of nuclear division in the presence of FM4-64 at the restrictive temperature. In no case was an accumulation of FM4-64 in the region of the dividing nuclei observed whereas endocytosis was still localised at the cell tips (Fig. 7A-C). Thus, the relocation of endocytosis requires the activation of the SIN pathway. We also investigated the relationship of equatorial endocytosis to the exocyst, a complex of proteins associated with exocytosis that has a specific role in cytokinesis in fission yeast (Wang et al., 2002). Exocyst components localise both to the cell poles and division plane in a F-actin-dependent manner in S. pombe but exocyst mutants are defective only for septum cleavage and cell separation. We therefore added FM4-64 to sec8-1 labelled with DAPI and scored the percentage of cells showing FM4-64 fluorescence between the dividing nuclei at both the permissive and restrictive temperatures. Equatorial endocytosis was inhibited in this mutant (Fig. 8A), although endocytosis at the cell poles was apparently normal (data not shown). A similar result was observed in cells treated with BFA, an inhibitor of the ER-to-Golgi step of the secretory pathway (Fig. 8A). Thus, endocytosis at the division plane may serve to balance exocytosis specifically at this site and at this stage of the cell cycle. To confirm that the two processes are indeed independent, we examined FM4-64 uptake at the cell equator in cells expressing Sec8-GFP to label secretory vesicles. Endocytic vesicles and secretory vesicles were distinct, with fluorescence only overlapping at the membrane flanking the septum (Fig. 8B).
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Discussion |
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Intermediate stages in the endocytic pathway appear to be easier to visualise in S. pombe than in budding yeast where they have proved difficult to capture cytologically (Munn, 2000). This may reflect differences in the pattern of cell growth or the fact that vacuolar organisation is very different in the two yeasts, S. cerevisiae possessing two or three large vacuoles (Wickner, 2002
), fission yeast up to 80 small ones (Bone et al., 1998
; Mulvihill et al., 2001
; Takegawa et al., 2003
). Both we (Mulvihill et al., 2001
), and others have noted that FM4-64 enters fission yeast cells at the cell poles and at the division site but in no case was this investigated further (Brazer et al., 2000
; Iwaki et al., 2004
). Here we show that in both wild-type cells and in mutants in which growth is restricted to a single cell pole or in which the septum is misplaced, endocytosis is restricted to regions of cell growth and, hence, is associated with concentrations of actin patches (Marks and Hyams, 1985
). Accumulating evidence from a variety of cell types links the internalisation step of endocytosis to the actin cytoskeleton (Engqvist-Goldstein and Drubin, 2003
). Most persuasive are genetic studies in budding yeast reporting that actin mutants are defective in endocytosis (Munn, 2000
; Munn, 2001
; Shaw et al., 2001
), and drug studies showing that endocytosis has an absolute requirement for a dynamic actin cytoskeleton (Ayscough, 2000
). As in fission yeast, interphase budding yeast cells contain two actin structures, patches and cables. Whereas actin cables are thought to underlie the bud-directed transport of organelles (Bretscher, 2003
), actin patches are associated with both cell wall deposition (Tang et al., 2000
; Utsigi et al., 2002) and endocytosis. Actin patches contain at least 20 proteins (Pruyne and Bretscher, 2000
) and mutations in many of these also show endocytosis defects (Munn, 2001
). Although the precise role of actin remains unknown, an important recent study from Kaksonen et al. (Kaksonen et al., 2003
) has shown that actin patches form at sites of endocytosis and are transiently associated with endocytic vesicles as they move away from the cell membrane. The latter may be coupled to the dynamic properties of actin cables (Huckaba et al., 2004
). Similar events are also thought to underlie endocytosis in mammalian cells (Merrifield, 2004
).
Our results demonstrate that endocytosis in fission yeast is also actin dependent. The evidence for this is threefold: (1) the ratio of endocytosis at the two cells ends is roughly equivalent to the ratio of actin patches at these locations; (2) endocytic vesicles and actin patches transiently colocalise at the cell poles and equator; (3) endocytosis is abolished when treated with latrunculin and in the mutant arp2, in which actin patches are disrupted (Morrell et al., 1999; Feoktistova et al., 1999
). As in budding yeast, later steps in the endocytic pathway in fission yeast are independent of actin (Kaksonen et al., 2003
). However, the integrity of Syb1 vesicles is also actin dependent, despite the fact that Syb1 vesicles at the poles and equator show differential sensitivity to latrunculin (Edamatsu and Toyoshima, 2003
). Based on the rapid appearance of FM4-64 fluorescence in these structures, Syb1 vesicles are an early component of the fission yeast endocytic pathway. However, their precise relationship to actin patches remains to be determined. Taken together, our findings indicate that the mechanism of endocytic uptake is fundamentally similar in the two yeasts. However, differences may exist. Actin patches persist for up to 3 minutes in fission yeast (Pelham and Chang, 2001
), considerably longer than has been observed in S. cerevisiae (Smith et al., 2001
; Kaksonen et al., 2003
), and although two-thirds of patches move away from the cell poles in S. pombe, consistent with a role in directing endocytic vesicles away from the cell membrane, the remaining one-third move in the opposite direction (Pelham and Chang, 2001
). In fission yeast, actin polymerisation is envisaged to drive patches along cables (Pelham and Chang, 2001
), whereas in S. cerevisiae patches are thought to use the force generated by cable polymerisation (Huckaba et al., 2004
). Our results show that endocytosis at the cell poles is insensitive to concentrations of latrunculin that depolymerise cables and, indeed, polar endocytosis is unaffected in cells lacking the formin, For3, in which these structures are absent. On the other hand, equatorial endocytosis does appear to require the presence of cables. The explanation for this difference probably lies in the type V myosin, Myo52 (Win et al., 2001
; Motegi et al., 2001
). Myo52 localises to the poles and equator of fission yeast cells and is associated with the redirection of the cell wall synthesising machinery between these two sites at cell division (D. P. Mulvihill and J.S.H., unpublished). The cell cycle redistribution of Syb1 vesicles is also dependent upon Myo52 (Edamatsu and Toyoshima, 2003
) suggesting that Myo52 has a major role in the global redistribution of the cell growth and endocytosis machinery at G2-M. Mutant for3
cells lacking actin cables have aberrant Myo52 distribution (Feirbach and Chang, 2001) show a delay in the equatorial relocation of endocytosis during M phase. Intriguingly, these cells also have a NETO defect. One daughter cell from each division grows in a monopolar manner and, hence, exhibits no NETO whereas the other grows in a bipolar manner and thus exhibits premature NETO (Feierbach and Chang, 2001
). Whether this phenotype is a consequence of the failure of endocytosis to properly relocalise to the cell equator at the previous cell division remains to be determined. Regardless, our findings emphasise the importance of membrane traffic to cytokinesis in fission yeast (Rajagopalan et al., 2003
).
A number of protein kinases are involved in the control of NETO (Bähler and Pringle, 1998; Verde et al., 1998
; Rupes et al., 1999
), whereas a signal transduction pathway consisting of several protein kinases, a GTPase and a protein phosphatase (the SIN) governs septation (McCollum and Gould, 2001
). Although both growth transitions involve the global reorganisation of the actin cytoskeleton and consequently, of both cell wall synthesis and endocytosis, the two events are in fact quite distinct. One involves the activation of growth at a pre-existing cell pole, the other the de novo creation of two cell poles following the synthesis and cleavage of the septum. It is therefore not surprising that the relationship of endocytosis to these two processes is distinct. The early steps of equatorial endocytosis persist in the actin mutants cps8 and arp2, in which endocytosis at the poles was largely arrested. It is also selectively inhibited by BFA and in the exocyst mutant sec8, both of which block the secretory pathway (Liu et al., 2002
). Thus, although the conventional explanation for endocytosis at sites of secretion, namely that the fusion of secretory vesicles with the cell membrane must be balanced by membrane internalisation by endocytosis (Gundelfinger et al., 2003
), could well be true at the cell equator, it is unlikely to be the explanation for endocytosis at the cell poles. One possibility is that equatorial endocytosis restricts the lateral diffusion of the receptors of polar determinants within the cell membrane (Snaith and Sawin, 2003
), resulting in their unequal distribution between the two daughter cells. The redirection of endocytosis to the division site requires a functional SIN. The latter conclusion was based on the inhibition of equatorial endocytosis in SIN mutants and the fact that endocytic vesicles become aligned across the equatorial region of the cell early in anaphase, the point at which SIN components become asymmetrically associated with the spindle poles (Cerutti and Simanis, 1999
). A known target of the SIN includes Cps1/Bgs1, a ß-glucan synthase involved in septum deposition (Liu et al., 2002
), and our observations add to the view that the SIN is a global regulator of cytokinetic events. More than 100 genes or 2% of the budding yeast genome is estimated to be involved in endocytosis (D'Hondt et al., 2000
; Wiederkehr et al., 2001
), emphasising its importance to yeast biology. Although we are a long way from such a detailed analysis in S. pombe, our description of the endocytic pathway suggests that fission yeast can make a useful contribution to our understanding of the molecular mechanisms of endocytic membrane transport.
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Acknowledgments |
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Footnotes |
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Present address: Institute of Molecular BioSciences, Massey University, Private Bag 11-222, Palmerston North, New Zealand
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References |
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