Phospholipid imbalance impairs autophagosome completion (2022)
Figure 7

Figure 7: Phospholipid balance promotes succession of autophagosome biogenesis

A. Representative images of Δypt7, Δopi3Δypt7 expressing GFP-Atg8 (z-stack projection). Cells were grown to log phase in SD-URA medium and starved in SD-N, images were taken during starvation in indicated time points by widefield microscopy (left panel). Scale bar 1 µm. Right panel- quantification of GFP-Atg8 puncta per cell in Δypt7, Δopi3Δypt7 at indicated time points. Statistical analysis was done by Anova multiple comparisons test- Sidak's (****, p≤0.0001, ns- not significant), compared to WT. Error bars represent SEM of least 3 independent experiments. Number of cells analyzed for each strain and time point, 0 hours: Δypt7 (n=322), Δopi3Δypt7 (n=194), 1.5 hours: Δypt7 (n=220), Δopi3Δypt7 (n=155), 3.5 hours Δypt7 (n=216), Δopi3Δypt7 (n=307).

B. Quantification of cup-shaped phagophores in Δypt7 and Δopi3Δypt7 cells expressing GFP-Atg8 during starvation (1-3h). Statistical analysis was done by students t-test (***, p≤0.001), error bars represent SEM from at least 3 independent experiments. Number of cells analyzed for each strain, Δypt7 (n= 347), Δopi3Δypt7 (n= 474).

C. Representative images of mNG-tagged Atg8 colocalized with mScarletI-PXVam7. WT, Δopi3, Δatg1 and Δopi3Δatg1 cells on the background of Δymr1, expressing mScarletI-PXVam7 under the CUP1 promoter from the knocked-out VPS38 locus, were grown to log phase in SD medium in the presence of 10 µM copper sulfate, and shifted to SD-N in the presence of 10 µM copper sulfate. Images were taken during SD-N (1-3 h) by widefield microscopy. Scale bar 1 µm.

D. Quantification of autophagic structure diameters in WT and Δopi3 cells on the background of Δymr1Δvps38 (C) by ImageJ. Statistical analysis was done by student's t-test, unpaired, two sided (ns- not significant), error bars represent SEM of at least three independent experiments. Number of cells analyzed for each strain, WT (n=51), Δopi3 (n=28).

E. Representative images of Δypt7, Δopi3Δypt7 cells expressing GFP-Atg8 (projection). Cells were grown to log phase in SD-URA medium and starved in SD-N, with supplementation of choline (1 mM) to SD and SD-N as indicated. Images were taken during growth and starvation by confocal microscopy (left panel). Scale bar 1 µm. Right panel- quantification of cell with more than two GFP-Atg8 puncta, in Δypt7 and Δopi3Δypt7 at growth (SD) or starvation (SD-N) in the presence (+choline: SD, SD-N) or absence (-choline) of choline (1 mM). Incidence were normalized to Δypt7 (SD-N) control. Statistical analysis was done by Anova multiple comparisons test- Sidak's (***, p≤0.001). Error bars represent SEM of least 3 independent experiments. Number of cells analyzed for each strain and condition, SD: Δypt7 (n=209), Δopi3Δypt7 (n=111), SD-N: Δypt7 (n=118), Δopi3Δypt7 (n=103), SD+Choline: Δypt7 (n=107), Δopi3Δypt7 (n=120), Δypt7 (n=84), Δopi3Δypt7 (n=74).

F. CLEM images of Δopi3Δypt7 cells expressing GFP-Atg8, grown to log phase in SD-URA medium and shifted to SD-N for 4 h, with supplementation of choline (1 mM) excluded (-choline) or present at growth and starvation (+choline SD, SD-N) as indicated. Cells were harvested, deep frozen and processed for CLEM. For each cell - whole cell (left), magnification of yellow dashed area of GFP-Atg8 positive phagophore (top right), TEM image only (middle right), schematic representation of autophagic structure (bottom right). Scale bar 0.5 µm.

Table 1 : Yeast strains

WT

GFP-Atg8

BY4741 (MATa, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0)

pRS316-GFP-Atg8

opi3∆

BY4741 opi3∆::natMX4

cho2∆

BY4741 cho2∆::natMX4

Fba1-GFP

atg1∆

BY4741 atg1∆::natMX4

opi3∆atg1∆

BY4741 atg1∆::natMX4 opi3∆::kanMX4

atg9∆

BY4741 atg9∆::natMX4

opi3∆atg9∆

BY4741 atg9∆::natMX4 opi3∆::kanMX4

ypt7∆

BY4741 ypt7∆::natMX4

opi3∆
ypt7∆

BY4741 ypt7∆::natMX4 opi3∆::kanMX4

Atg5-mNG/ sfTq2-Atg8

w303

ura3::pUAS_F_E_C_CORE1-OsTIR1

trp1::pATG8-sfTq2-Atg8-tATG8::hphNT1
ATG5-mNG::tSYNTH8::CgTRP1

ymr1∆

ymr1∆::KlURA3

w303
ura3::pUAS_F_E_C_CORE1-OsTIR1
trp1::pATG8-sfTq2-Atg8-tATG8::hphNT1
ATG5-mNG::tSYNTH8::CgTRP1 opi3∆::CgHIS3

Atg1-mNG/ sfTq2-Atg8

ura3::pUAS_F_E_C_CORE1-OsTIR1
trp1::pATG8-sfTq2-Atg8-tATG8::hphNT1
ATG1-mNG::tSYNTH8::CgTRP1

Atg9-mNG/ sfTq2-Atg8

trp1::pATG8-sfTq2-Atg8-tATG8::hphNT1
ATG9-mNG::tSYNTH8::CgTRP1

mNG-Atg8/mScarletI-PXVam7

mNG-Atg8/ mScarletI-PXVam7

opi3∆ymr1∆

ymr1∆atg1∆

opi3∆ymr1∆atg1∆

mNG-Atg8/ mScarletI-Ape1

atg3∆

trp1::pATG8-sfTq2-Atg8-tATG8::hphNT1
ATG5-mNG::tSYNTH8::CgTRP1 atg3∆::KlURA3

opi3∆atg3∆

BFP-Ape1

BY4741
trp1::pATG8-mNG-Atg8-tATG8::hphNT1

pDP105 (pRS315[pAPE1-TagBFP-Ape1-tCYC1::pCUP1-Ape1-tAPE1])

BY4741
opi3::natMX4
trp1::pATG8-mNG-Atg8-tATG8::hphNT1

Table 1: Yeast strains used in this study.

Expanded view: