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Transcript of Developmental Cell, Volume 37 Developmental Cell, Volume 37 Supplemental Information Reticulons...

  • Developmental Cell, Volume 37

    Supplemental Information

    Reticulons Regulate the ER Inheritance

    Block during ER Stress

    Francisco Javier Piña, Tinya Fleming, Kit Pogliano, and Maho Niwa

  • Inventory of Supplemental Materials

    Figure S1, related to Figure 1. Contains representative images for class the I, II, III cER

    inheritance phenotype (A) and representative images of ire1Δ (B) and slt2Δ (C) cells for

    the FRAP experiments.

    Figure S2, related to Figure 1. Contains graphs for the kar2-1-sfGFP FRAP experiments

    (A and B) and representative images of cells for the Hmg1-GFP FRAP experiments (C).

    Figure S3, related to Figure 4. Displays representative images for the ER phenotype of

    WT, Δtether and rtn1Δrtn2Δyop1Δ cells (A), representative images of cells for FRAP

    experiments with Δtether (B), rtn1Δrtn2Δyop1Δ (C), and rtn1Δrtn2Δyop1Δlnp1Δ (D)

    cells, and images of tubule formation in WT, slt2Δ, rtn1Δrtn2Δyop1Δ, and

    rtn1Δrtn2Δyop1Δlnp1Δ cells (E).

    Figure S4, related to Figure 4. FRAP analysis for Hmg1-GFP in rtn1Δrtn2Δyop1Δ cells

    (A). Shows a cartoon model depicting ER stress, tubule formation, and cER inheritance

    in WT cells vs different mutant strains used in the study (B and C).

    Supplemental Experimental Procedures

    ER inheritance assay

    CPY*-mRFP and GFP-CFTR assays

    Construction of Kar2-sfGFP strains

    Yeast strains used

    Plasmids used

    Primers used

    Supplemental References

  • Supplemental Figure 1

    B slt2Δ Kar2sfGFP bleach 18 sec 84 sec

    Tm D

    M S

    O Tm

    D M

    S O

    pre-bleach ire1Δ Kar2sfGFP bleach 18 sec 84 sec

    Tm D

    M S

    O Tm

    D M

    S O

    pre-bleach

    C

    A No ER stress (-Tm) ER stress (+Tm)

    m ed

    iu m

    la rg

    e sm

    al l

    cE R

    pn E

    R

    cE R

    pn E

    R

  • Supplemental Figure 2

    time (sec) -1 0 6 18 30 42 54 66 78

    Tm

    DMSO

    time (sec) -1 0 6 18 30 42 54 66 78

    Tm

    DMSO

    kar2-1-sfGFP

    kar2-1-sfGFP

    3 hrs

    3 hrs

    yrevoce R ecnecseroul

    F 0.0 0.2 0.4 0.6 0.8 1.0 1.2

    time (sec) -1 0 6 18 30 42 54 66 78

    Tm

    DMSO

    kar2-1-sfGFP 30 min

    A

    B

    1.2yrevoce R ecnecseroul

    F 0.0 0.2 0.4 0.6

    0.8

    1.0

    time (sec) -1 0 6 18 30 42 54 66 78

    Tm

    DMSO

    kar2-1-sfGFP 30 min

    cER

    pnER

    C Wildtype Hmg1-GFP

    pre-bleach bleach 18 sec 84 sec

    Tm D

    M S

    O Tm

    D M

    S O

    cE R

    pn E

    R

  • D M

    S O

    Tm A

    B

    Δtetherwildtype rtn1Δrtn2Δyop1Δ

    pre-bleach bleach 18 sec 84 sec Δtether Kar2sfGFP

    D M

    S O

    Tm

    rtn1Δrtn2Δyop1Δlnp1Δ Kar2sfGFP pre-bleach bleach 18 sec 84 sec

    D M

    S O

    Tm D

    M S

    O Tm

    D

    C rtn1Δrtn2Δyop1Δ Kar2sfGFP

    pre-bleach bleach 18 sec 84 sec

    D M

    S O

    Tm D

    M S

    O Tm

    Supplemental Figure 3 D

    M S

    O Tm

    slt2Δwildtype rtn1Δrtn2Δ yop1Δ

    rtn1Δrtn2Δ yop1Δlnp1Δ

    E

    cE R

    pn E

    R

    cE R

    pn E

    R cE

    R pn

    E R

  • Supplemental figure 4

    D

    slt2Δ

    ER Inheritance no Slt2 Slt2 (active)

    rtn1Δrtn2Δyop1Δ

    Slt2 (active) no

    ER Inheritance

    rtn1Δrtn2Δyop1Δlnp1Δ +Tm

    C +Tm

    Slt2 (active) noER Inheritance

    -Tm

    ER Inheritance

    A

    time (sec) -1 0 6 18 30 42 54 66 78

    Tm

    DMSO

    rtn1Δrtn2Δyop1Δ

    time (sec) -1 0 6 18 30 42 54 66 78

    Tm

    DMSO

    rtn1Δrtn2Δyop1Δ

    0.0 0.2 0.4

    0.6 0.8 1.0 1.2yrevoce

    R ecnecseroul F

    Hmg1-GFPHmg1-GFP

    cER pnER

    0.0 0.2 0.4

    0.6 0.8 1.0 1.2yrevoce

    R ecnecseroul F

    time (sec) -1 0 6 18 30 42 54 66 78

    time (sec) -1 0 6 18 30 42 54 66 78

    slt2Δ

    cER pnER slt2Δ

    B

    Tm

    DMSO

    Tm

    DMSOHmg1-GFPHmg1-GFP

  • Supplemental Figure 1. ER stress causes a block in inheritance of cER but not

    pnER. Related to Figure 1.

    (A) ER inheritance was monitored in Hmg1-GFP-expressing WT cells treated with

    DMSO (-Tm) or 1 µg/ml Tm for 3 hr. Representative cells are shown for class I, II and III

    with small, medium, and large buds, respectively.

    (B–C) Representative images of ire1Δ (B) and slt2Δ (C) cells from Kar2-sfGFP FRAP

    experiments. Cells were incubated with DMSO or 1 µg/ml Tm for 3 hr before small

    regions of the cER (orange box) or pnER (green box) were photobleached. Images were

    acquired before (pre-bleach), at the same time as (bleach), and at 18 or 84 sec after

    photobleaching. Scale bar is 2 µm.

    Supplemental Figure 2. FRAP analysis of Hmg1-GFP-expressing WT cells and

    mutant kar2-1-sfGFP-expressing cells. Related to Figure 1.

    (A and B) kar2-1-sfGFP cells (Kabani et al., 2003) were incubated with DMSO or 1 µg/ml

    Tm for 30 min or 3 hr before FRAP in the cER (B) and pnER (C) were analyzed. Graphs

    are the mean ± SD of 3 experiments, each examining ≥7 cells. Scale bar is 2 µm.

    (C) WT cells expressing Hmg1-GFP were incubated with DMSO or 1 µg/ml Tm before a

    small region of the cER (orange box) or pnER (green box) was photobleached. Images

    were acquired before (pre-bleach), at the same time as (bleach), and at 18 or 84 sec

    after photobleaching. Scale bar is 2 µm.

    Supplemental Figure 3. FRAP analysis of Kar2-sfGFP-expressing Δtether,

    rtn1Δrtn2Δyop1Δ , and rtn1Δrtn2Δyop1Δ lnp1Δ cells. Related to Figure 3 and 4.

    (A) Δtether and rtn1Δrtn2Δyop1Δ cells have defective ER morphology and distribution. In

    WT cells, the ER is juxtaposed to the PM and appears as a continuous ring around the

    periphery of the cell and the nucleus. In Δtether cells, the cER is discontinuous and is

    not in close apposition to the PM. In rtn1Δrtn2Δyop1Δ cells, the cER appears to be

    discontinuous around the periphery of the cell with invaginations displaced from the PM.

    Scale bar is 2 µm.

    (B-D) Representative images from FRAP experiments with Δtether (B), rtn1Δrtn2Δyop1Δ

    (C), and rtn1Δrtn2Δyop1Δlnp1Δ (D) cells expressing Kar2-sfGFP. Cells were treated with

    DMSO or 1 µg/ml Tm for 3 hr before FRAP in the pnER (green box) or cER (orange box)

    was assessed. Scale bars are 2 µm.

  • (E) Images of tubule formation in WT, slt2Δ, rtn1Δrtn2Δyop1Δ, and

    rtn1Δrtn2Δyop1Δlnp1Δ cells expressing Hmg1-GFP. Tubule formation from the pnER

    was examined early in the cell cycle during bud initiation in cells incubated with DMSO

    or 1 µg/ml Tm for 3 hr. ER stress reduces or causes abnormal ER tubule formation.

    Scale bar is 2 µm.

    Supplemental Figure 4. Model for Slt2 and RTN function in ER tubule formation. Related to Figure 4. (A) FRAP analysis in unstressed (DMSO, 3hr) or stressed (Tm, 3hr) cER or pnER of

    slt2Δ cells expressing Hmg1-GFP. Graphs represent the mean ± SD of 3 experiments,

    each examining ≥7 cells.

    (B) FRAP analysis in unstressed (DMSO, 3hr) or stressed (Tm, 3hr) cER or pnER of

    rtn1Δrtn2Δyop1Δ cells expressing Hmg1-GFP. Graphs represent the mean ± SD of 3

    experiments, each examining ≥7 cells.

    (C, left) Under normal growth conditions (-Tm), the cER and pnER have similar

    environments with high mobility of the ER-resident chaperone Kar2/BiP reporter, Kar2-

    sfGFP. At a specific but currently unknown point in the cell cycle, tubular ER emerges

    from the pnER orientated towards the bud. We propose that a balance in the activity or

    localization of ER shaping proteins (Rtn1/2, Yop1, Sey1, and Lnp1) at the three-way

    junction formed between tubular ER at the pnER regulates tubule formation from the

    pnER and orientation toward the new bud site. Ultimately, the tubular ER enters the

    daughter cell, becomes anchored to the bud tip, and spreads around the cortex.

    (C, right) When ER stress is induced (+Tm) in WT cells, Kar2-sfGFP mobility decreases

    in the cER, but not the pnER. Thus, the combination of (1) an unperturbed pnER

    environment, (2) a block in the emergence of tubular ER from the pnER, and (3) a

    subsequent block in cER inheritance can be described as the “WT ER stress

    phenotype.” This requires Slt2 activation, which may alter the balance between the

    Reticulons, Yop1, Lnp1 and Sey1.

    (D, left) Maintenance of an unperturbed pnER environment under conditions of ER

    stress requires Slt2 activation. Thus, in slt2Δ cells, ER stress decreases Kar2-sfGFP

    mobility to a similar extent in the cER and the pnER and both the block in tubular ER

    emergence and the block in cER inheritance are abolished, possibly due to inability to

    alter the balance of ER structural proteins. Note that the establishment of asymmetric

    cER and pnER environments is not simply due to disturbance