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 …
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 …
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 …
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 …
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 …
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 …
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] = …
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). …
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] = …
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 = …
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 …
-
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
-
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 …
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 . …
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 …
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 …
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 …
-
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
-
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 …
Additional files
-
MDAR checklist
- https://cdn.elifesciences.org/articles/103355/elife-103355-mdarchecklist1-v2.docx
-
Supplementary file 1
Strains used in this study.
- https://cdn.elifesciences.org/articles/103355/elife-103355-supp1-v2.docx
-
Supplementary file 2
Annotation of proteins used in this study.
- https://cdn.elifesciences.org/articles/103355/elife-103355-supp2-v2.docx
-
Supplementary file 3
Primers used for cloning and gene construction.
- https://cdn.elifesciences.org/articles/103355/elife-103355-supp3-v2.docx
