Distinct trafficking routes of polarized and non-polarized membrane cargoes in Aspergillus nidulans
- Department of Biology, National and Kapodistrian University of Athens, Panepistimioupolis, Greece
- Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, Greece
- School of Medicine, University of Crete, Greece
Figures
Figure 1
Synchronously co-expressed UapA and SynA follow distinct routes to the plasma membrane (PM) (widefield microscopy).
(A) Growth test analysis of the strain co-expressing alcAp-uapA-gfp and alcAp-mCherry-synA compared to isogenic wt and ΔuapA or ΔsynA strains. Notice that the alcAp-uapA-gfp and alcAp-mCherry-synA strain grows similar to the wt in the presence of uric acid as a N source (substrate of UapA) and fructose as a C source. On the other hand, on uric acid/glucose media the alcAp-uapA-gfp and alcAp-mCherry-synA strain resembles the ΔuapA mutant, confirming that uapA transcription is well repressed. Notice also that lack of SynA expression, either in the null mutants or in the repressible alcAp-mCherry-synA strain, does not affect growth. (B) Maximal intensity projections of deconvolved snap shots showing steady-state localization of de novo expressed UapA and SynA in germlings and hyphae after 4–6 hr derepression. UapA-GFP labels homogeneously the PM in a non-polarized or even anti-polarized manner, while mCherry-SynA marks mostly the PM of the apical region of hyphae, with some overlap with UapA-GFP, at the subapical region. (C) Dynamic localization of de novo UapA/SynA after 1 hr derepression. On their way to the PM, while both cargoes are still in cytoplasmic structures, they label distinct compartments that do not seem to overlap significantly at any stage before their accumulation to the PM. (D) Schematic representation of an A.nidulans germling depicting the subcellular organization of key components of the secretory pathway (ER, ER-Exit Sites [ERES], early-, late-Golgi, cytoskeleton). Scale bars: 5 μm.
Figure 2 with 3 supplements
Synchronously co-expressed UapA and SynA mark distinct dynamic secretory compartments on their way to the plasma membrane (PM) (high-speed spinning-disc confocal microscopy).
(A) Maximal intensity projections of deconvolved snap shots of UapA and SynA trafficking dynamics, extracted from a 7-min video (Figure 2—video 1), after synchronous derepression of transcription for 3 hr, using ultra-fast spinning-disc confocal microscopy. UapA-GFP has a non-polarized distribution to the PM, while mCherry-SynA is mostly localized in the apical area. UapA structures moving laterally toward the PM are indicated with yellow arrow heads (left bottom panel). Cytoplasmic UapA and SynA structures do not colocalize significantly (Pearson’s correlation coefficient [PCC] = 0.01, n = 52), apart from a small number of doubly labeled, mostly non-cortical puncta (0.33 ± 0.03 µm in diameter) with low-range mobility (right bottom panel). (B) Maximal intensity projections of deconvolved snap shots of UapA trafficking dynamics, extracted from a 3-min video (Figure 2—video 2), showing that UapA-GFP labels cytoplasmic oscillating thread structures decorated by pearl-like foci (0.32 ± 0.02 µm – white arrow heads) as well as a very faint vesicular/tubular network (magenta arrow heads). Pearl-like structures (yellow arrow heads) moving toward the PM are estimated to reach their destination in 12–18 s. Scale bars: 5 μm.
Figure 2—video 1
7-min video of de novo UapA-GFP versus mCherry-SynA after 3 hr of derepression.
Maximal intensity projection of deconvolved video showing that cytoplasmic UapA and SynA structures do not colocalize significantly (Pearson’s correlation coefficient [PCC] = 0.01, n = 52) apart from a small number of doubly labeled puncta (0.33 ± 0.03 µm) with low-range mobility. Scale bar: 5 μm.
Figure 2—video 2
3-min video of de novo UapA-GFP trafficking dynamics after 3 hr of derepression.
Maximal intensity projection of deconvolved video showing cytoplasmic oscillating thread structures decorated by pearl-like foci (0.32 ± 0.02 µm) and a faint vesicular/tubular network. Scale bar: 5 μm.
Figure 2—video 3
2-min video of de novo mCherry-SynA after 3 hr of derepression.
Maximal intensity projection of deconvolved video showing thread-like structures with pearls and puncta moved toward the apical area. Scale bar: 5 μm.
Figure 3 with 5 supplements
Unlike SynA, dynamically secreted UapA, does not colocalize with late-Golgi marker.
(A) Maximal intensity projections of deconvolved snap shots of deconvolved videos (Figure 3—videos 1 and 2) in young hyphae after derepression of transcription of UapA-GFP for 3 hr. Colocalization study of UapA with the ERGIC/early-Golgi marker mCherry-SedV (left panel) and with the late-Golgi/trans-Golgi network (TGN) marker PHOSBP-mRFP (right panel). Neosynthesized UapA-GFP does not colocalize significantly with any of the Golgi markers as indicated by the Pearson’s correlation coefficient (PCC) (0.01, n = 100 UapA-SedV; 0.01, n = 35 UapA-PHOSBP). A small number of colocalized structures of UapA-GFP with mCherry-SedV is observed lasting ~6 s, without being statistically significant. (B) Maximal intensity projections of deconvolved snap shots of videos (Figure 3—videos 3 and 4) in young hyphae after derepression of transcription of GFP-SynA for 3 hr. Colocalization study of SynA with the ERGIC/early-Golgi marker mCherry-SedV (left panel) and with the late-Golgi/TGN marker PHOSBP-mRFP (right panel). Unlike UapA-GFP, neosynthesized GFP-SynA colocalizes significantly with the late-Golgi/TGN marker PHOSBP (PCC = 0.74, n = 76, p < 0.0001) and very little with the ERGIC/early-Golgi marker SedV (PCC = 0.04, n = 40). Notice that after exiting the TGN, fast moving SynA puncta are directed to the apical area of the hyphae (white arrow heads – right panel). Scale bars: 5 μm.
Figure 3—figure supplement 1
Super Resolution Radial Fluctuation (SRRF) imaging of colocalization of SynA with the late-Golgi marker PHosbp in fixed cells.
Maximal intensity projection of 57 z-stacks (left panel) and a specific z-stack (z = 8, right panel) showing a significant colocalization of GFP-SynA with the late-Golgi marker mRFP-PHOSBP. Scale bars: 5 μm.
Figure 3—video 1
10-min video of mCherry-SedV versus de novo UapA-GFP after 3 hr of derepression.
Maximal intensity projection of deconvolved video showing that neosynthesized UapA does not colocalize significantly with the ERGIC/early-Golgi marker SedV (Pearson’s correlation coefficient [PCC] = 0.01, n = 100). Scale bar: 5 μm.
Figure 3—video 2
6-min video of mRFP-PHOSBP versus de novo UapA-GFP after 3 hr of derepression.
Maximal intensity projection of deconvolved video showing that neosynthesized UapA does not colocalize with the late-Golgi marker PHOSBP (Pearson’s correlation coefficient [PCC] = 0.01, n = 35). Scale bar: 5 μm.
Figure 3—video 3
5-min video of mCherry-SedV versus de novo GFP-SynA after 3 hr of derepression.
Maximal intensity projection of deconvolved video showing that neosynthesized SynA does not colocalize significantly with the ERGIC/early-Golgi marker SedV (Pearson’s correlation coefficient [PCC] = 0.04, n = 40). Scale bar: 5 μm.
Figure 3—video 4
6-min video of mRFP-PHosbp versus de novo GFP-SynA after 3 hr of derepression.
Maximal intensity projection of deconvolved video showing that neosynthesized SynA colocalizes significantly with the late-Golgi marker PHosbp (Pearson’s correlation coefficient [PCC] = 0.74, n = 76, p < 0.0001). Scale bar: 5 μm.
Figure 4
UapA and SynA sorting to the plasma membrane (PM) is differentially dependent on COPII components.
(A) Overview of COPII formation at ER-Exit Sites (ERES) (left panel). Western blot analysis of COPII-repressible alleles sarA, sec24, sec13, and sec31 (right panel). More specifically, COPII proteins were expressed under the tightly repressed thiAp in the presence and absence of thiamine in the medium. Notice that in the absence of thiamine, all COPII proteins (SarA, Sec24, Sec13, and Sec31) are expressed and detected using anti-FLAG antibody. However, when thiamine is added in the medium, thiAp is repressed and the respective proteins are not expressed to an extent not detectable in western blot analysis. Equal loading and protein steady-state levels are normalized against the amount of actin, detected with an anti-actin antibody. Sec12 detection was impossible even in the absence of thiamine. (B) Growth test analysis of strains expressing the repressible COPII components. In the absence of thiamine (−thi) all strains form colonies similar to wt, while in the presence of thiamine (+thi) strains do not grow at all (thiAp-sarA, thiAp-sec24, thiAp-sec13, and thiAp-sec31) or form dramatically reduced colonies (thiAp-sec12). (C) Maximal intensity projections of deconvolved snap shots showing the subcellular localization of de novo UapA-GFP and mCherry-SynA using the alcAp-uapA-gfp/alcAp-mCherry-synA strain in different genetic backgrounds repressible for conventional COPII formation. In wt background UapA was sorted non-polarly to the PM, while SynA was localized in the apical area of the PM. Repression of SarA, Sec24, Sec13, and Sec31 led to total retention of SynA at the ER network in 100% of cells. UapA trafficking was also totally blocked upon SarA or Sec31 repression (100% of cells), and also efficiently blocked when Sec24 was repressed (~76% of cells). Notably, UapA trafficking was not blocked upon Sec13 repression in most cells (79%). Notice also that when the trafficking of SynA is blocked, SynA partitions in the ER membranes, whereas in the fraction of cells where UapA translocation to the PM is blocked, the protein forms aggregates (e.g., in 24% of cells repressed for sec24, and 21% of cells repressed for sec13). UapA aggregation might be to the fact that it is a large homodimeric protein with 28 transmembrane segments that oligomerizes further upon translocation into the ER membrane, apparently causing ER stress and turnover. Sec12 repression led to a moderate simultaneous negative effect on the sorting of UapA and SynA to the PM (33%) The number of cells used for quantitative analysis upon repressed conditions was n = 101 for SarA, n = 158 for Sec12, n = 163 for Sec24, n = 256 for Sec13, and n = 126 for Sec31, respectively. (D) Relative 3H-xanthine transport rates of UapA in genetic backgrounds repressible for COPII components Sec12, Sec24, and Sec13 expressed as percentages of initial uptake rates compared to the wt. UapA-mediated transport was very low when Sec24 was repressed (~15% of wt) but remained relatively high when Sec13 or Sec12 was repressed (~65–72% of wt). Results are averages of three measurements for each concentration point. Scale bars: 5 μm.
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Figure 4—source data 1
Original files for western blot analysis displayed in Figure 4A.
- https://cdn.elifesciences.org/articles/103355/elife-103355-fig4-data1-v2.zip
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Figure 4—source data 2
PDF file containing original western blots for Figure 4A, indicating the relevant bands and treatments for each strain.
- https://cdn.elifesciences.org/articles/103355/elife-103355-fig4-data2-v2.pdf
Figure 5
Genetic block in COPII formation or repression of COPI activity traps UapA and SynA in distinct ER-associated aggregates.
(A) Growth test analysis of sec31ts-AP at 25 and 42°C, pH 6.8, compared to an isogenic wild-type (wt) control strain. Notice the severe growth defect of sec31ts-AP at 42°C (left panel). Strategy for achieving a synchronized accumulation/release of de novo made UapA and SynA at ER-Exit Sites (ERES) (right panel). (B) Maximal intensity projections of deconvolved z-stacks, using a sec31ts-AP strain co-expressing UapA-GFP and mCherry-SynA. At the restrictive temperature (42°C), both cargoes are retained in the ER marking distinct areas of a membranous network (merged image). Upon shifting down to the permissive temperature (42→25°C), ER export is restored and cargoes are progressively incorporated into ERES-like structures. UapA and SynA appear mostly in distinct puncta, showing a low degree of colocalization (open arrowheads, non-colocalizing dots; white arrowheads, colocalizing dots), as illustrated also by the calculation of Pearson’s correlation coefficient (PCC) on the right (PCC = 0.28 ± 0.06, n = 17). (C) Maximal intensity projections of deconvolved z-stacks, using sec31ts-AP strains co-expressing UapA-GFP/Sec16-mCherry or GFP-SynA/Sec16-mCherry upon a shift from 42 to 25°C. Both UapA and SynA exhibited significant colocalization with the ERES marker Sec16 (PCC = 0.66 ± 0.08, n = 22 UapA-Sec16; PCC = 0.58 ± 0.09, n = 37 SynA-Sec16) strongly suggesting that UapA and SynA are recruited to spatially distinct ERES. (D) Maximal intensity projections of deconvolved z-stacks, using strains co-expressing UapA-GFP and mCherry-SynA under conditions where copA or arfA transcription is repressed by addition of thiamine (+thi). De novo synthesis of cargoes takes place after full repression of CopA or ArfA is achieved (>16 hr). Repression of either CopA or ArfA abolishes the translocation of both cargoes to the plasma membrane (PM). Notice that upon CopA repression, UapA and SynA collapse in distinct internal structures, similar to those when the sec31ts-AP strain was shifted from 42 to 25°C. Quantification of colocalization (right panel) when copA or arfA transcription is repressed, by calculating PCC, shows clear non-colocalization of UapA with SynA (PCC = 0.15 ± 0.08, n = 9 in thiAp-copA and PCC = −0.14 ± 0.1, n = 10 in thiAp-arfA). Scale bars: 5 μm.
Figure 6 with 1 supplement
Golgi maturation and conventional post-Golgi vesicular secretion are not needed for UapA localization to the plasma membrane (PM).
(Α) Schematic depiction of Golgi maturation and post-Golgi secretion. (B) Growth test analysis of strains expressing early/late/post-Golgi repressible alleles sedV, geaA, rabO, hypB, rabE, or ap1σ . In the absence of thiamine (−thi) all strains form colonies similar to wt, while in the presence of thiamine (+thi) strains could not form colonies when Golgi proteins were repressed, apart from the thiAp-hypB mutant which produced a small compact colony. (C) Maximal intensity projections of deconvolved snap shots showing the subcellular localization of de novo UapA-GFP and mCherry-SynA using the alcAp-uapA-gfp/alcAp-mCherry-synA strain in different genetic backgrounds repressible for conventional Golgi-dependent secretion. In wt background SynA was localized to the apical area of the PM, while UapA was sorted non-polarly to the subapical PM. Repression of SedV, GeaA, RabO, RabE, and Ap1σ led to total retention of SynA in cytoplasmic structures, while repression of HypB abolished SynA trafficking in 56% of cells. Notice that upon repression of SedV and GeaA, SynA mostly labeled the ER network, indicating the close dynamic association of the ER with the ERGIC/early-Golgi. In contrast to SynA, UapA reached the PM when several essential ERGIC/early-Golgi and post-Golgi were repressed. More specifically, in GeaA, RabO, HypB, and Ap1σ repressible strains UapA translocated to the PM in 100% of cells. Repression of SedV and RabE had a partial negative effect on UapA trafficking. In particular, lack of SedV led to block of UapA traffic in 65% of cells, while upon RabE repression, a significant fraction of UapA could translocate to the PM in ~75% of cells, but in this case, the same cells also showed a significant number of large cytoplasmic structures. The number of cells used for quantitative analysis upon repressed conditions was n = 266 for SedV, n = 201 for GeaA, n = 104 for RabO, n = 159 for HypB, n = 219 for RabE, and n = 190 for Ap1σ, respectively. (D) Relative 3H-xanthine transport rates of UapA in genetic backgrounds repressible for SedV, HypB, RabE, and Ap1σ expressed as percentages of initial uptake rates compared to the wt. UapA-mediated transport was very high when HypB or Ap1σ were repressed (83–100% of wt), while transport activity was reduced to 27% and 19% of the wt when RabE and SedV were repressed, respectively. Results are averages of three measurements for each concentration point. (E) Subcellular localization of de novo UapA-GFP and mCherry-SynA in wt background upon treatment with the Golgi inhibitor Brefeldin A (BFA) after 90 min of derepression of cargoes. In the absence of BFA, UapA and SynA were localized properly in the PM. Addition of BFA for 60 min after derepression blocked SynA trafficking leading to its trapping into large Golgi aggregates (brefeldin bodies), while UapA could still reach the PM (upper panel). Notice that cytoplasmic SynA structures upon BFA addition are similar with those obtained when the early-Golgi protein GeaA, which is a target of BFA, was repressed (white arrow heads, lower panel). (F) Subcellular localization of de novo UapA-GFP and mCherry-SynA in wt background upon treatment with the anti-microtubule drug Benomyl for 90 min after derepression of cargoes. Notice that microtubule depolymerization is dispensable for UapA trafficking, while it is absolutely necessary for the proper sorting of SynA to the apical PM. Addition of Benomyl blocks SynA in cytoplasmic structures, while UapA translocation to the PM is unaffected. Scale bars: 5 μm.
Figure 6—figure supplement 1
UapA membrane aggregates upon RabE repression do not colocalize with early- or late-Golgi markers.
Maximal intensity projections of deconvolved z-stacks, using thiAp-rabE strains co-expressing UapA-GFP and the early-Golgi marker mCherry-SedV (left panel) or the late-Golgi marker mRFP-PHosbp (right panel). Notice the almost non-existent degree of overlap (white color in the merged image) between UapA-GFP and Golgi-markers. Scale bars: 5 μm. Quantification of colocalization by calculating Pearson’s correlation coefficient (PCC) verifies the low degree of colocalization of UapA with both early- and late-Golgi markers (PCC = 0.17 ± 0.07, n = 6 with SedV and PCC = 0.26 ± 0.08, n = 11 with PHosbp).
Figure 7
UapA translocation to the plasma membrane (PM) occurs independently of SNAREs Ykt6, Sec22, Bet1, and Bos1.
(A) Schematic illustration of the SNARE complex mediating membrane fusion of ER-derived vesicles to Golgi cisternae (upper panel). Growth phenotypes of null or repressible mutants of SNAREs involved in ER-to-Golgi trafficking (lower panels). In the absence of thiamine from the growth medium (derepressed conditions), the corresponding strains grow nearly as an isogenic wild-type control strain. In the presence of thiamine to the growth medium, thiAp-ykt6, Δsec22 thiAp-ykt6, and thiAp-Sft11 do not form colonies, while thiAp-bet1 and thiAp-bos1 form a slow-growing colony with reduced conidiospore production. Growth test analysis of Δsec22 at 37 and 42°C, pH 6.8, compared to an isogenic wild-type (wt) control strain, showing a temperature-dependent growth defect at 42°C. (B) Maximal intensity projections of deconvolved z-stacks, using ER-to-Golgi SNARE mutant strains co-expressing UapA-GFP and mCherry-SynA. UapA translocation to the PM is not affected in any of the SNARE mutants tested, even in the double mutant strain lacking both Ykt6 and Sec22. SynA trafficking to the hyphal apex is not impaired upon Bos1 or Bet1 repression or Sec22 deletion, but is abolished upon Ykt6 or Sft1 repression, and in the double mutant thiAp-ykt6 Δsec22. Scale bars: 5 μm.
Figure 8 with 1 supplement
UapA translocation to the plasma membrane (PM) requires the SsoA-Sec9 Q-SNARE complex, but not the R-SNARE SynA or the exocyst effector RabD.
(A) Schematic illustration of the SNARE complex mediating membrane fusion of secretory vesicles to the PM (upper panel). Growth phenotypes of null or repressible mutants of SNAREs involved in vesicular fusion to the PM (lower panels). Growth test analysis of ΔsynA at 25 and 37°C, at pH 6.8 and 8.0, compared to an isogenic wild-type (wt) control strain. Notice the reduced colony growth of ΔsynA at 25°C, pH 8.0. Growth test analysis of ΔrabD at 37°C, pH 6.8 compared to an isogenic wild-type (wt) control strain, showing a markedly reduced colony size resulting from rabD knock-out. Growth test analysis of the thiAp-sec9 and thiAp-ssoA strains compared to a wild-type control strain in the presence (+thi) or absence (−thi) of thiamine from the media, showing that Sec9 or SsoA repression impedes colony formation. (B) Western blot analysis of Sec9 using the anti-FLAG antibody. In the absence of thiamine (−thi) from the growth medium, Sec9 is expressed, while upon addition of thiamine (+thi) at the onset of conidiospore germination (ab initio repression), the expression of these proteins is tightly repressed. Equal loading and protein steady-state levels are normalized against the amount of actin, detected with an anti-actin antibody. (C) Maximal intensity projections of deconvolved z-stacks, using strains expressing UapA-GFP or GFP-ChsB in genetic backgrounds where exocytic SNAREs are repressed or deleted. Repression of Sec9 or SsoA abolishes the translocation of UapA and ChsB to the PM, while SynA deletion has no effect on the trafficking of both cargoes to the PM. (D) Maximal intensity projections of deconvolved z-stacks, using a ΔrabD strain co-expressing UapA-GFP and mCherry-SynA. Deletion of RabD impairs the translocation of SynA to the apical PM, which remains trapped in cytosolic membrane aggregates, while UapA trafficking to the PM is not affected. Scale bars: 5 μm.
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Figure 8—source data 1
Original files for western blot analysis displayed in Figure 8B.
- https://cdn.elifesciences.org/articles/103355/elife-103355-fig8-data1-v2.jpg
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Figure 8—source data 2
PDF file containing original western blots for Figure 8B, indicating the relevant bands and treatments for each strain.
- https://cdn.elifesciences.org/articles/103355/elife-103355-fig8-data2-v2.pdf
Figure 8—figure supplement 1
Triple R-SNARE knockout Δsec22 ΔnyvA ΔsynA does not affect UapA or ChsB trafficking to the plasma membrane (PM).
(A) Growth phenotypes of null mutants of R-SNAREs at 37 and 42°C, compared to an isogenic wild-type (wt) control strain. ΔnyvA shows no growth defect, while the triple knock-out Δsec22 ΔnyvA ΔsynA exhibits a severe growth defect at both temperatures. (B) Maximal intensity projections of deconvolved z-stacks, using strains expressing UapA-GFP or GFP-ChsB in genetic backgrounds where single or triple R-SNAREs are deleted. Deletion of either nyvA or all three R-SNAREs has no effect on the trafficking of both cargoes to the PM. Scale bars: 5 μm.
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/103355/elife-103355-mdarchecklist1-v2.docx
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Supplementary file 1
Strains used in this study.
- https://cdn.elifesciences.org/articles/103355/elife-103355-supp1-v2.docx
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Supplementary file 2
Annotation of proteins used in this study.
- https://cdn.elifesciences.org/articles/103355/elife-103355-supp2-v2.docx
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Supplementary file 3
Primers used for cloning and gene construction.
- https://cdn.elifesciences.org/articles/103355/elife-103355-supp3-v2.docx
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Distinct trafficking routes of polarized and non-polarized membrane cargoes in Aspergillus nidulans
eLife 13:e103355.
https://doi.org/10.7554/eLife.103355
