A dynein loading zone for retrograde endosome motility at microtubule plus-ends
2006; Springer Nature; Volume: 25; Issue: 11 Linguagem: Inglês
10.1038/sj.emboj.7601119
ISSN1460-2075
AutoresJonathan Lenz, Isabel Schuchardt, Anne Straube, Gero Steinberg,
Tópico(s)Protist diversity and phylogeny
ResumoArticle11 May 2006free access A dynein loading zone for retrograde endosome motility at microtubule plus-ends J H Lenz J H Lenz Search for more papers by this author I Schuchardt I Schuchardt Search for more papers by this author A Straube A StraubePresent address: Wellcome Trust Centre for Cell Biology, University of Edinburgh, King's Buildings, Edinburgh EH9 3JR, Scotland, UK Search for more papers by this author G Steinberg Corresponding Author G Steinberg Max-Planck-Institut für terrestrische Mikrobiologie, Marburg, Germany Search for more papers by this author J H Lenz J H Lenz Search for more papers by this author I Schuchardt I Schuchardt Search for more papers by this author A Straube A StraubePresent address: Wellcome Trust Centre for Cell Biology, University of Edinburgh, King's Buildings, Edinburgh EH9 3JR, Scotland, UK Search for more papers by this author G Steinberg Corresponding Author G Steinberg Max-Planck-Institut für terrestrische Mikrobiologie, Marburg, Germany Search for more papers by this author Author Information J H Lenz, I Schuchardt, A Straube and G Steinberg 1 1Max-Planck-Institut für terrestrische Mikrobiologie, Marburg, Germany *Corresponding author. Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch-Straße, 35043 Marburg, Germany. Tel.: +49 6421 178 530; Fax: +49 6421 599; E-mail: [email protected] The EMBO Journal (2006)25:2275-2286https://doi.org/10.1038/sj.emboj.7601119 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info In the fungus Ustilago maydis, early endosomes move bidirectionally along microtubules (MTs) and facilitate growth by local membrane recycling at the tip of the infectious hypha. Here, we set out to elucidate the molecular mechanism of this process. We show that endosomes travel by Kinesin-3 activity into the hyphal apex, where they reverse direction and move backwards in a dynein-dependent manner. Our data demonstrate that dynein, dynactin and Lis1 accumulate at MT plus-ends within the hyphal tip, where they provide a reservoir of inactive motors for retrograde endosome transport. Consistently, endosome traffic is abolished after depletion of the dynein activator Lis1 and in Kinesin-1 null mutants, which was due to a defect in targeting of dynein and dynactin to the apical MT plus-ends. Furthermore, biologically active GFP-dynein travels on endosomes in retrograde and not in anterograde direction. Surprisingly, a CLIP170 homologue was neither needed for dynein localization nor for endosome transport. These results suggest an apical dynein loading zone in the hyphal tip, which ensure that endosomes reach the expanding growth region before they reverse direction. Introduction Fungal pathogenicity is based on the ability to invade the host tissue by polarized growth of the hypha. It is thought that this process is based on motor-mediated transport of growth supplies along the cytoskeleton to the growing hyphal apex (Steinberg, 2000), where polarized exocytosis occurs (Gow, 1995). In addition, evidence exist that endosome-based membrane recycling supports hyphal tip growth (Wedlich-Söldner et al, 2000). Interestingly, in yeast-like cells and hyphae of the dimorphic plant pathogenic fungus Ustilago maydis, early endosomes (EE) show rapid microtubule (MT)-dependent motility (Wedlich-Söldner et al, 2000). In yeast-like cells, this bidirectional traffic is mediated by Kinesin-3 and cytoplasmic dynein (Wedlich-Söldner et al, 2002), and similar motors might mediate the motility of endosomes in vertebrates (Hoepfner et al, 2005). Currently, there are several concepts for the activity of kinesins and dynein in bidirectional organelle traffic. Most experimental evidence from animal cells suggests that both motors interact on the same organelle, thus controlling each other's activity (Gross et al, 2002; Welte, 2004). This notion is supported by the finding that Kinesin-1 directly binds the dynein complex (Ligon et al, 2004). However, such physical interaction could also reflect that one motor is a cargo of the other. This notion is supported by recent results showing that fungal dynein, which accumulates at the plus-ends of MTs (Han et al, 2001; Lee et al, 2003; Sheeman et al, 2003), requires Kinesin-1 for its plus-ends targeting in Aspergillus nidulans (Zhang et al, 2003). In Saccharomyces cerevisiae growing MT plus-ends take dynein to the cortex, where it becomes activated and ‘off-loaded’ in order to migrate towards the minus-ends, thereby mediating spindle positioning (Lee et al, 2003; Sheeman et al, 2003). A key component in this process is Pac1 (Sheeman et al, 2003), which is an NudF/LIS1 homologue (Xiang et al, 1999). These factors are thought to activate dynein for minus-end directed motility (Faulkner et al, 2000; Smith et al, 2000; Coquelle et al, 2002; Zhang et al, 2003; Lansbergen et al, 2004; Mesngon et al, 2006). Another factor involved in dynein regulation is dynactin (Schroer, 2004), which also affects dynein activity and its MT affinity at plus-ends (Valetti et al, 1999; Zhang et al, 2003). Moreover, together with the cytoplasmic linker protein 170 (CLIP-170), dynactin is thought to recruit endosomes to MT plus-ends (Pierre et al, 1992; Vaughan, 2005), and similar mechanisms might also account for other cellular cargoes (Mimori-Kiyosue and Tsukita, 2003). Taken together, these findings emphasize the importance of MT plus-ends for intracellular organelle motility. Based on these studies, it is tempting to speculate that plus-end located fungal dynein represents a reservoir for minus-end directed transport in interphase (Yamamoto and Hiraoka, 2003; Zhang et al, 2003). Here, we provide experimental evidence for such a concept. We show that cytoplasmic dynein and homologues of the dynein activators LIS1 and dynactin localize to plus-ends of MTs. A Kinesin-3 delivers EE to the hyphal apex, where they are loaded onto dynein for retrograde transport. This ensures that endosomes reach the hyphal tip, where they participate in cell expansion by endocytic membrane recycling. Results Dynein and a Kif1A-like kinesin support bidirectional endosome traffic in hyphae of U. maydis It was previously shown that EE move along MTs in yeast-like cells and hyphae of U. maydis (Wedlich-Söldner et al, 2000). As a first step towards an understanding of this motility, we determined the directionality of MTs in hyphae by the observation of a YFP-tagged EB1-homologue Peb1 that binds only to growing MT plus-ends (Straube et al, 2003). Therefore, any moving signal indicates the orientation of a MT. Infectious hyphae of U. maydis consist of a single tip cell of ∼120–150 μm in length and 2–3 μm width (Figure 1A), with the nucleus being located close to the center of the cell (Figure 1B [II]; chromosomal DNA labeled by CFP-histone 4 is given in blue; strain AB33Peb1Y_H4C, for all strains see Table I; see Supplementary Movie 1). Hyphae contain long MTs that often form bundles (Steinberg et al, 2001). The majority of Peb1-YFP signals that, based on their consistent signal intensity, marked individual MT plus-ends moved towards the growing apex and the basal septum, suggesting that plus-ends are directed to the cell poles (Figure 1B [I]+[III] and Figure 1C; Peb1 in red). In the middle of the cell, Peb1-YFP signals grow towards each other (Figure 1B [II], 1C) and passed the nucleus in opposite directions, indicating that MTs have an antipolar orientation in the central region of the hypha. Consistent with previous results (Wedlich-Söldner et al, 2000), individual MTs and bundles of MTs served as tracks for bidirectional motility of EE that were tagged with the specific t-SNARE Yup1-GFP (Figure 2A; strain AB33RT_YG; see Supplementary Movie 2), but which also localizes to stationary vacuoles, albeit to a minor extend (Wedlich-Söldner et al, 2000). Interestingly, most EE reached the MT ends in the hyphal tip before they reversed direction (Figure 2B). This was a first indication that factors in the hyphal apex participate in retrograde transport. In yeast-like cells, EE traffic depends on Kinesin-3 and dynein (Wedlich-Söldner et al, 2002). Consistently, deletion of Kinesin-3 in hyphae almost abolished EE motility (Figure 2C, ΔKin3; strain AB33ΔKin3_YG; for ‘control’ see Figure 2D, strain AB33YG; for Figure 2D–F; see Supplementary Movie 3 on EMBO web site) and led to clustering of EE around the nucleus in the middle of the shorter hyphae (Figure 2E), whereas EE were equally distributed in control hyphae (Figure 2D). Although these Δkin3 hyphae were bipolar (Figure 2E, Schuchardt et al, 2005), MT orientation was not altered (89% plus-ends towards tip; n=59; strain AB33ΔKin3_Peb1Y), indicating that EE are trapped at minus-ends of MTs near the cell center. In contrast, depletion of dynein shifted EE to the cell poles (Figure 2F, Dyn1↓; strain AB33rDyn1_YG), where MT plus-ends were concentrated. Unfortunately, Western analysis revealed that expression of dynein was not completely abolished in these mutants (not shown). Therefore, we analyzed EE traffic in pheromone-induced hyphae of temperature-sensitive dynein mutants (strain FB1Dyn2ts_YG). Such dynein mutant hyphae show growth defects (Fuchs et al, 2005) and contained longer MTs (not shown). However, MT orientation was not affected, as 95.5% of all Peb1-RFP signals were directed to the tip (n=40; strain FB1Dyn2tsPeb1Y). Again, EE were trapped at plus-ends in the hyphal tip (not shown) and almost all motility was abolished (Figure 2C, Dyn2ts). These observations are most consistent with the notion that a balance of Kinesin-3 and dynein activity underlies long-distance EE transport in U. maydis hyphae. Figure 1.Microtubule orientation in hyphae of U. maydis. (A) Overview of a hypha of U. maydis. Boxed regions (I–III) correspond to series given in 1B. Note that tip cells leave ‘empty’ cell wall section behind (arrow) while they grow at their tip (right end). Bar: 10 μm. (B) Peb1-YFP (given in red) marks MT plus-ends that elongate towards the basal septum (I, arrows), as well as the hyphal tip (III, arrows), indicating that the plus-ends are orientated towards both poles of the hyphal cell. Around the nucleus (II; labeled by histone 4-CFP is given in blue), Peb1-signals move in both directions (arrow and arrowhead), indicating that MTs have an antipolar orientation. Elapsed time is given in seconds. Bars: 5 μm. (C) Quantitative analysis of growing MT ends demonstrates that the plus-ends (indicated by ‘plus’) are focused to the tip (indicated by red arrows) and the subapical septum (indicated by blue arrows). An antipolar MT orientation in the middle of the cell suggests that minus-ends are located near the nucleus. Numbers indicate the percentage of Peb1-YFP signals that move in the direction indicated. Supplementary movie for Panel B is given on the EMBO web site. Download figure Download PowerPoint Figure 2.Endosome organization and motility in the Kinesin-3 and dynein mutants. (A) EE that are labeled by a fusion protein of GFP and the membrane receptor Yup1 (arrowhead, arrow; Wedlich-Söldner et al, 2000) migrate bidirectioally along MTs, which are visualized by expression of RFP-α-tubulin. Elapsed time is given in seconds. Bar: 3 μm. (B) A quantitative analysis of the behavior of individual EE confirmed that most organelles reach the MT plus-ends before they reverse direction. (C) Quantitative analysis of the EE motility frequencies at 10 μm distance to the tip of control and mutant hyphae. Almost identical numbers of vesicles move towards the tip (anterograde) and the basal nuclear region (retrograde) of control hyphae. This motility is abolished in Kinesin-3 mutants (Δkin3) and in dynein mutants (Dyn2ts). (D) In control hyphae, Yup1-GFP labeled EE are motile and equally distributed along the length of the hyphae. Occasionally, transient endosome concentrations are found in the apex. Bar: 10 μm. (E) Δkin3 mutant hyphae are usually bipolar and much shorter (Schuchardt et al, 2005). The EE cluster near the nucleus, where the minus-ends of MTs are concentrated. Note that the MT orientation in Δkin3 hyphae was confirmed by analysis of Peb1-YFP motility (not shown). Bar: 10 μm. (F) In the conditional dynein mutants, depletion of dynein strengthens anterograde EE motility and leads to accumulations of EE at the MT plus-ends near the cell poles. Bar: 10 μm. Supplementary movie for Panels A and D–F is given on the EMBO web site. Download figure Download PowerPoint Table 1. Strains and plasmids used in this study Strains/plasmid Genotype Reference AB33RT_YG a2 PnarbW2 PnarbE1, bleR/potefRFPTub1/potefYup1GFP This study AB33YG a2 PnarbW2 PnarbE1, bleR/potefYup1GFP This study AB33ΔKin3_YG a2 PnarbW2 PnarbE1 Δkin3∷hygR, bleR/potefYup1GFP This study FB1Dyn2ts_YG a1b1 dyn2ts, natR/potefYup1GFP This study AB33ΔKin1_Peb1Y a2 PnarbW2 PnarbE1 Δkin1∷hygR, Ppeb1-peb1-yfp, bleR, natR This study AB33ΔKin3_Peb1Y a2 PnarbW2 PnarbE1 Δkin3∷hygR, Ppeb1-peb1-yfp, bleR, natR This study FB1Dyn2tsPeb1Y a1b1 dyn2∷dyn2ts Ppeb1-peb1-yfp, natR, bleR This study AB33rDyn1_YG a2 PnarbW2 PnarbE1 Pcrg1-dyn1, bleR, natR/potefYup1GFP This study AB33ΔKin1_YG a2 PnarbW2 PnarbE1 Δkin1∷hygR/potefYup1GFP This study AB33Peb1Y_H4C a2 PnarbW2 PnarbE1 Ppeb1-peb1-yfp, bleR, natR/pH4CFP This study AB33G3Dyn2 a2 PnarbW2 PnarbE1 3xgfp-dyn2, bleR, natR This study AB33G3Dyn2_RT a2 PnarbW2 PnarbE1 3xgfp-dyn2, bleR, hygR/potefRFPTub1 This study AB33ΔKin1_G3D2_RT a2 PnarbW2 PnarbE1 Δkin1∷hygR 3xgfp-dyn2, natR, bleR/potefRFPTub1 This study AB33ΔKin1_G3D2_YR2 a2 PnarbW2 PnarbE1 Δkin1∷hygR 3xgfp-dyn2, natR, bleR/potefYup1RFP2 This study AB33G3Dyn2_YR2 a2 PnarbW2 PnarbE1 3xgfp-dyn2, hygR, bleR/potefYup1RFP2 This study FB2GLis1 a2b2 PLis1-gfp-lis1, hygR This study FB2GLis1_RT a2b2 PLis1-gfp-lis1, hygR/potefRFPTub1 This study FB2nGLis1 a2b2 Pnar-gfp-lis1, natR This study FB2G3Dyn2_nLis1 a2b2 Pdyn2-3xgfp-dyn2 Pnar-lis1, hygR natR This study FB2G3Dyn2 a2b2 Pdyn2-3xgfp-dyn2, hygR This study FB2G3Dyn2_nLis1_RT a2b2 Pdyn2-3xgfp-dyn2 Pnar-lis1, hygR natR/potefRFPTub1 This study FB2G3Dyn2_nLis1_YR2 a2b2 Pdyn2-3xgfp-dyn2 Pnar-lis1, hygR natR/potefYup1RFP2 This study FB2YR2 a2b2/potefYup1RFP2 This study FB2nLis1_Peb1R_GT a2b2 Pnar-lis1 Ppeb1-peb1-rfp, natR, bleR/potefGFPTub1 This study FB2G3Lis1_YR2 a2b2 Plis1-3xgfp-lis1, natR /potefYup1RFP2 This study FB2G3Dya1_RT a2b2 Pdya1-3xgfp-dya1, bleR/potefRFPTub1 This study FB2rGDya1 a2b2 Pcrg1-gfp-dya1, bleR This study FB2rDya1_G3D a2b2 Pcrg1-dya1 Pdyn2-3xgfp-dyn2, bleR, hygR This study FB2rDya1_G3D_RT a2b2 Pcrg1-dya1 Pdyn2-3xgfp-dyn2, bleR, hygR/potefRFPTub1 This study FB2rDya1_YG a2b2 Pcrg1-dya1, bleR/potefYup1GFP This study FB2rDya1_Peb1R_GT a2b2 Pcrg1-dya1 Ppeb1-peb1-rfp, natR, bleR/potefGFPTub1 This study FB2G3Dya1_YR2 a2b2 Pdya1-3xgfp-dya1, bleR/potefYup1RFP2 This study FB2YG a2b2/potefYup1GFP This study FB2ΔKin1_G3Dya1_RT a2b2 Δkin1∷hygR Pdya1-3xgfp-dya1, bleR/potefRFPTub1 This study FB2rKin1_GLis1 a2b2 Plis1-gfp-lis1 Pcrg1-kin1, hygR natR This study FB2GClip1_RT a2b2 Pclip1-gfp-clip1, hygR/potefRFPTub1 This study FB2ΔClip1 a2b2 Δclip1∷bleR This study FB2ΔClip1_YG a2b2 Δclip1∷bleR/potefYup1GFP This study FB2ΔClip1_G3Dyn2_RT a2b2 Δclip1∷bleR Pdyn2-3xgfp-dyn2, hygR/potefRFPTub1 This study FB2GT a2b2/potefGFPTub1 (Steinberg et al, 2001) FB2ΔClip1_GT a2b2 ΔClip1∷bleR/potefGFPTub1 This study FB2ΔClip1 _Peb1R_GT a2b2 Δclip1∷bleR, Ppeb1-peb1-rfp, natR/potefGFPTub1 This study RWS9 a1b1/pKin3YFP/pPX10-146CFP (Wedlich-Söldner et al, 2002) potefGFPTub1 Potef-egfp-tub1, cbxR (Steinberg et al, 2001) potefRFPTub1 Potef-mrfp-tub1, cbxR (Straube et al, 2005) potefYup1GFP Potef-yup1-sgfp, cbxR (Wedlich-Söldner et al, 2000) potefYup1RFP2 Potef-yup1-2xmrfp, cbxR This study pKin3YFP Potef-kin3-yfp, cbxR (Wedlich-Söldner et al, 2002) pPX10-146CFP Potef-yup1PX1–146-cfp, bleR (Wedlich-Söldner et al, 2002) PH4CFP Phis4-h4-cfp, hygR (Straube et al, 2005) a, b, mating type loci; Δ, deletion; P, promoter; ∷, homologous replacement; -, fusion; hphR, hygromycin resistance; bleR, phleomycin resistance; natR, nourseothricin resistance; cbxR, carboxin resistance; /, ectopically integrated; otef, constitutive promoter; nar, conditional nitrate reductase promoter; crg1, conditional arabinose-induced promoter; E1, W2, genes of the b mating type locus; gfp, enhanced green fluorescent protein; yfp, yellow-shifted fluorescent protein; mrfp, monomeric red fluorescent protein; yup1: endosomal t-SNARE; kin1, kinesin-1 family member; kin3, kinesin-3 family member; dyn1: N-terminal half of the dynein heavy chain; dyn2: C-terminal half of the dynein heavy chain; dyn2ts: temperature-sensitive allele of dyn2; tub1, α-tubulin; lis1, LIS1 homologue; clip1, CLIP-170 homologue; dya1, p150glued homologue; yup1PX1−146, localization domain of Yup1; his4, histone 4. Dynein accumulates at MT plus-ends in the hyphal tip Next, we set out to visualize dynein by fusing a triple GFP-tag to the N-terminus of the endogenous dyn2 gene (strain AB33G3Dyn2), which encodes the C-terminal half of the dynein heavy chain (Straube et al, 2001). The fusion protein did not cause any morphological phenotypes, indicating that it is fully functional. GFP3-Dyn2 formed comet-like motile structures (Figure 3A, arrowhead; see Supplementary Movie 5) and was significantly concentrated at MT ends in the tip of growing hyphae (Figure 3A, arrow and Figure 3B; strain AB33G3Dyn2_RT). From there, small dynein particles could be detected that move in retrograde direction (see below), suggesting that the dynein accumulation is a reservoir for retrograde motor proteins. Quantitative analysis of the fluorescence intensity revealed that the dynein signals in the first 3 μm of the hyphae were significantly stronger than those at MT tips in subapical regions (Figure 3C). A more detailed analysis revealed that the amount of dynein remained constant over long distances, but rapidly increased in the apical 5 μm and reached maximum values in the close proximity to the hyphal tip (Figure 3D), suggesting that dynein is actively concentrated in the apex. In vivo observation of EE in strain AB33G3Dyn2_YR2 revealed that EE reach the dynein accumulation before they reverse direction (Figure 3E, arrow; see Supplementary Movie 4). Figure 3.Dynein localization and EE movement. (A) In U. maydis hyphae dynein, visualized by a fusion of 3xGFP and the endogenous Dyn2 forms motile comet-like structures (arrowheads). The strongest signals are always found in the hyphal apex (arrow, inset). Bar: 10 μm. (B) Colocalization of GFP3Dyn2 (Dyn, green in overlay) and RFP-α-tubulin labeled MTs (red) in control hyphae. Dynein localizes to the MT plus-ends that are reaching into the hyphal apex. Bar: 5 μm. (C) A quantitative analysis of the GFP3-Dyn2 signal intensity at MT plus-ends in the first 3 μm of the hyphal tip (apical) and in at least 12 μm distance from the tip (basal) reveals that the dynein accumulations at the hyphal tip is more than twice as strong as in basal parts of the cell. Note that the signal intensity has been normalized to a single GFP. (D) The average intensity of dynein signals at MT plus-ends was measured in relation to distance to the tip, demonstrating that the amount of dynein remains constant over most of the length of the hypha, but rapidly increases at the hyphal apex. (E) Colocalization of GFP3Dyn2 (Dyn, green in overlay series) and Yup-RFP2 (EE, red in overlay series) demonstrates that EE (arrows) migrate into the dynein signal before they reverse direction (colocalization results in yellow). Elapsed time is given in seconds. Bar: 2 μm. Supplementary movie for Panel E is given on the EMBO web site. Download figure Download PowerPoint Retrograde EE movement depends on activation of dynein by an LIS1 homologue The accumulation of dynein in the hyphal tip suggested that the motor stays inactive until its cargo reaches the MT plus-ends. Therefore, we analyzed the mechanism of dynein activation and its impact on EE motility. A good candidate for dynein regulation is LIS1, which is thought to activate dynein (Faulkner et al, 2000; Smith et al, 2000; Zhang et al, 2003; Mesngon et al, 2006). The LIS1 homologue in U. maydis shares 47% amino-acid identity with its closest homologue NudF from A. nidulans (Xiang et al, 1999) and, like other LIS1-members, is predicted to contain an LisH motif, a coiled-coil region and seven WD40 domains (Figure 4A). We fused 3xGFP to the N-terminus of the endogenous lis1 gene, and this did not result in any defects in nuclear migration or growth (see below), indicating that the protein is fully functional. GFP3-Lis1 localized to MT tips (Figure 4B; strain FB2GLis1_RT), and concentrates at plus-ends in the hyphal tip (Figure 4B and C), suggesting that the putative dynein activator is part of the apical dynein complex. Next, we analyzed the role of Lis1 in dynein activation. In strain FB2G3Dyn2_nLis1, lis1 is expressed under the control of the inducible nar1-promoter (Brachmann et al, 2001). Upon growth in complete medium (CM) containing ammonium, this promoter is repressed and Lis1 is depleted (see Supplementary Figure 1 on EMBO web site), whereas it is induced in the presence of nitrate (NM). Reduction of Lis1 levels led to nuclear migration defects in yeast-like cells and longer MTs in hyphae (not shown). However, 87.5% of all plus-ends were still oriented to the hyphal tip (n=48; strain FB2nLis1_Peb1R_GT). Neither of these treatments affected dynein protein levels (see Supplementary Figure 1 on EMBO web site). However, the amount of dynein at MT plus-ends in the hyphal tip was significantly increased in the absence of Lis1, while it was slightly decreased upon overexpression of Lis1 (Figure 4D and E; Lis1↑: high protein levels, Lis1: no Lis1; FB2G3Dyn2_nLis1_RT; see Supplementary Movie 5). Consistent with the concept that EE are loaded onto dynein at the hyphal apex and that Lis1 activates dynein, EE were not able to leave the tip in the absence of Lis1 (Figure 4F; overlay of two time points, stationary organelles appear in yellow; FB2G3Dyn2_nLis1_YR2; for Figures 4F, 5F and 6E see also Supplementary Movie 1). Consequently, EE motility was significantly impaired (Figure 4G; control strain: FB2YR2; see Supplementary Movie 6). In summary, these results indicate that the LIS1-homologue Lis1 is crucial for the activation of dynein for retrograde EE transport. Figure 4.The role of a LIS1-homologue in dynein localization and endosome motility. (A) Lis1 from U. maydis (UmLis1, Accession Number: EAK84394) shares a similar domain structure with other LIS1-homologues, including those from A. nidulans (AnNudF, EAA57983), S. cerevisiae (ScPac1p, AAB00685) and human (HsLis1, NP 000421). (B) GFP-tagged endogenous Lis1 (green) localizes to plus-ends of RFP-α-tubulin containing MTs (red). Note that the tip contains the strongest signal. Bar: 5 μm. (C) Quantification of the GFP-Lis1 intensity at MT plus-ends in the apical 3 μm of the hyphal tip (apical) and in at least 12 μm distance from the tip (basal) demonstrates that Lis1 is concentrated at MT plus-ends in the tip. (D) The absence of Lis1 (Lis1↓) results in an increase of dynein at plus-ends within the hyphal tip. Overexpression of Lis1 (Lis1↑) reduces the apical dynein signal. Bar: 2 μm. (E) Quantitative analysis of GFP3Dyn2 intensity at MT plus-ends revealed that the absence of Lis1 (↓Lis1) significantly increases the amount of dynein at MT plus-ends. Consistently, Lis1 overexpression (↑Lis1) has the opposite effect. (F) Overlay of two images at different time points illustrates the motility of RFP2-tagged endosomes. Moving organelles appear in green or red, while stationary signals result in yellow color. Depletion of Lis1 led to an immobile accumulation of EE in the hyphal apex (strain FB2G3Dyn2_nLis1_YR2). Bar: 5 μm. Supplementary movies for Panel D and F are given on the EMBO web site. (G) A quantitative analysis of EE traffic reveals that overexpression of Lis1 (↑Lis1) affects EE motility only slightly, while nearly all motility is abolished in the absence of Lis1 (↓Lis1). Download figure Download PowerPoint Figure 5.The role of a p150glued homologue in dynein localization and endosome motility. (A) The p150glued homologues from U. maydis (UmDya1, Accession Number: EAK84712) shares a similar domain structure with other p150glued homologues from A. nidulans (AnNudM, EAA58707), S. cerevisiae (ScNip100p, NP 015151) and human (Hsp150, NP 004073). (B) GFP3-tagged endogenous Dya1 (green) accumulates at the plus-ends of RFP-α-tubulin labeled MTs (red). The strongest signal is present in the hyphal apex. Bar: 5 μm. (C) Fluorescence intensity measurements of GFP3Dya1 at MT plus-ends in the first 3 μm of the hyphal tip (apical) and in basal parts of the cell (in >12 μm distance from the tip) reveals that dynactin was ∼2-times more concentrated at MT plus-ends in the hyphal tip than in subapical regions. (D) Quantification of the GFP3Dyn2 signals at MT plus-ends and in the apical cytoplasm shows that high levels of Dya1 (↑Dya1) has no significant effect on dynein localization (P=0.306), whereas dya1 repression (↓Dya1) leads to a dramatic decrease of the dynein signal. (E) No dynein is present at apical MT plus-ends after depletion of Dya1 (Dya1↓). Bar: 2.5 μm. (F) Overlay of two images at different time points illustrates the motility of GFP-tagged endosomes in strain FB2rDya1_YG. Moving organelles appear in green or red, while stationary signals result in yellow. Depletion of Dya1 leads to an immobile accumulation of EE in the hyphal apex. Bar: 5 μm. Supplementary movies for Panels E and F are given on the EMBO web site. (G) The expression of Dya1 (↑Dya1) has no effect on EE transport, whereas the absence of Dya1 (↓Dya1) abolishes almost all EE motility. Download figure Download PowerPoint Figure 6.The role of a CLIP-170 homologue in dynein and endosome targeting. (A) The CLIP-170 homologues from U. maydis (UmClip1, Accession Number: EAK82811) shares a similar domain structure with other CLIP-170 members from S. cerevisiae (ScBik1p, NP 009901), Schizosaccharomyces pombe (SpTip1, P79065) and human (HsCLIP170, A43336). (B) GFP-tagged endogenous Clip1 (green) localizes to plus-ends of RFP-α-tubulin labeled MTs (red). Bar: 5 μm. (C) In contrast to dynein, dynactin and Lis1 intensities GFP-Clip1 fluorescence signals at MT plus-ends in the first 3 μm of the hyphae (apical) do not differ from those in basal parts of the cells (in >12 μm distance from tip). (D) In Δclip1 cells, the intensity of the dynein signal at MT plus-ends in the tip is not different from those of control cells. (E) In the clip1 deletion strain, GFP3-tagged dynein (green) still accumulates at plus-ends of RFP-labeled MTs (red). Bar: 2.5 μm. (F) Deletion of clip1 does not affect bidirectional EE motility. (G) In Δclip1 mutants, the organization of GFP-α-tubulin labeled MTs is not altered compared to control cells (strain FB2GT). Bar: 5 μm. Supplementary movies for Panels E and G are given on the EMBO web site. (H) Parameters of MT dynamic instability in control cells and Δclip1 mutant hyphae. Download figure Download PowerPoint Dynactin is essential for dynein plus-ends localization and retrograde EE transport In order to further characterize the regulation of dynein, we investigated the role of the dynein activator dynactin (Schroer, 2004) in EE transport. We identified dya1, a putative component of the dynactin complex that shares 19% amino acid identity with its homologue from A. nidulans. Similar to other p150glued homologues, Dya1 is predicted to contain a CAP-Gly motif, as well as numerous coiled-coil regions (Figure 5A). We fused a triple GFP-tag to the N-terminus of the endogenous dya1 gene and found that the fusion protein is functional, as it did not cause the dya1 mutant phenotype (see below). The fusion protein localized to the plus-ends of MTs (Figure 5B; strain FB2G3Dya1_RT), with most dynactin being concentrated at the apical MT ends (Figure 5B and C), which corresponds well with the data raised for dynein and Lis1 (see above). Next, we generated conditional mutants that expressed dya1 under the control of the strong crg1-promoter (Bottin et al, 1996). In the presence of arabinose, this promoter allowed expression of GFP-Dya1, whereas in the presence of glucose it was repressed and no fusion protein was detected (see Supplementary Figure on EMBO web site; strain FB2rGDya1). These growth conditions neither affected the orientation of MTs (86.2% of plus-ends grow towards the hyphal tip, n=29; strain FB2rDya1_Peb1R_GT) nor changed the levels of dynein in cell extracts of strain FB2rDya1_G3D (see Supplementary Figure on EMBO web site). Depletion of Dya1 abolished dynein accumulation in the tip (Figure 5D and E; Dya1↓: no Dya1, Dya1↑: high levels of Dya1; strain FB2rDya1_G3D_RT; see Supplementary Movie 5) and this led to an EE aggregation in the hyphal apex (Figure 5F; overlay of two time points, stationary signals appear in yellow) and a significant reduction in EE motility (Figure 5G; strain FB2rDya1_YG; see Supplementary Movie 6). These results demonstrate that the dynactin subunit Dya1 is essential for setting up a dynein reservoir at the hyphal tip, which in turn is required for retrograde EE traffic. Hence, these results add further support to our concept that organelles are loaded onto dynein in the hyphal tip. Clip1 is not necessary for dynein plus-ends binding
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