1. Volume 150, Issue 1, Pages 151-164 (July 2012)
Proteasomal Degradation Resolves Competition between Cell Polarization and Cellular Wound Healing 
Keiko Kono, Yasushi Saeki, Satoshi Yoshida, Keiji Tanaka, David Pellman 
Cell 
Volume 150, Issue 1, Pages (July 2012)
DOI: /j.cell
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2. Figure 1 A Local Wound Healing Response in Budding Yeast
(A–H) In this and subsequent figures, the site of laser damage is marked with the star, and the GFP signal of the indicated reporters was monitored by live-cell imaging. Numbers in the upper-left corner of each panel indicate the time after damage (min). Scale bar, 2 μm. Arrows show recruitment of GFP signals to the damage site. Graphs report the normalized average GFP signal intensity after background subtraction. Error bars represent SEM. Under these imaging conditions, there was no significant bleaching of signal from undamaged control cells within the field of view (not shown).
(I) Local activation of the chitin emergency response, as assayed by calcofluor-white (CFW) staining of chitin.
See also Figure S1 and Movie S1.
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3. Figure 2 Mechanisms Underlying Cellular Wound Healing in Budding Yeast
(A–C) Laser damage was performed, and data are presented as in Figure 1. Error bars represent SEM.
(A) Requirement of Rom2 for the recruitment of Pkc1 to the damage site.
(B) The GEF activity of Rom2 is required for the recruitment of Pkc1 to the damage site.
(C) Bnr1 is required for the appearance of a sharply focused spot of Pkc1-GFP at the damage site.
(D and E) Loss of Bni1 accelerated actin and myosin V depolarization after heat shock (39°C). Filamentous actin was visualized with Alexa-phalloidin, and the percentage of cells with polarized actin (>50% of actin patches within the daughter cell) was determined. Myosin V was visualized with Myo2-GFP.
(F) Loss of Bni1 after 39°C heat shock. (Top) Western blot to detect Bni1-13myc or α-tubulin as a loading control. (Bottom) Quantification of the western blot shown above.
(G) Degradation of Bni1 after cell wall damage. The expression of MET3::BNI1-13myc was induced for 90 min by methionine washout at 24°C. Expression was shut off, cycloheximide was added to block protein synthesis, and then the degradation of Bni1 was monitored in the presence or absence of cell wall damage (SDS).
See also Figure S2 and Movies S2–S4.
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4. Figure 3
Bni1 Degradation after Cell Wall Damage Requires Pkc1, the Proteasome, and the E3 Ligase Rsp5
(A) Rho1 and Pkc1, but not its downstream MAP kinase cascade, are required for downregulation of Bni1. GFP-Bni1 steady-state levels were detected in the indicated strains after a 3 hr shift to 37°C.
(B) Bni1 degradation after cell wall damage requires Pkc1. pkc1-2 and control cells were shifted to 37°C for 2 hr. After 2 hr at 37°C, cells have recovered from heat shock and have polarized actin. Bni1-13myc expression was then induced for 90 min.
(C) Bni1 degradation requires the proteasome. Cyclohexamide-chase experiments were performed as in (B).
(D) Vacuolar protease activity is not required for damage-induced Bni1 degradation.
(E) Bni1 accumulates in cells lacking functional Rsp5. Temperature shift of the indicated strains to 37°C was for 4 hr.
(F) Bni1 degradation after cell wall damage requires Rsp5.
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5. Figure 4
A Bni1 Domain that Is Sufficient for Pkc1- and Rsp5-Dependent Degradation
(A) (Left) Schematic diagram of Bni1. RBD, Rho-binding domain; DID, diaphanous inhibitory domain; SBD, Spa2 binding domain; FH1, formin homology 1 domain; FH2, formin homology 2 domain; BBD, bud6 binding domain. (Right) Bni myc levels, but not Bni myc levels, sharply decline after heat shock (39°C, 30 min).
(B) Inactivation of Rsp5 results in the accumulation of a slow-migrating form of Bni myc (37°C, 3 hr).
(C) Rsp5 mediates Bni1-642 ubiquitylation in vitro.
(D) Pkc1 is required for loss of Bni myc after a 3 hr shift to 37°C. Note the slow mobility of Bni myc in rsp5-1 strains; this mobility shift represents phosphorylation (see below), and this phosphorylation requires Pkc1 because it is absent in pkc1-2 strains.
(E) Bni myc is phosphorylated. rsp5-1 cells were shifted to 37°C for 4 hr. Immunoprecipitation was performed with or without lambda-phosphatase treatment, along with a phosphatase inhibitor control.
(F) Pkc1-dependent phosphorylation of full-length Bni1. Bni1-4HA was immunoprecipitated in the presence or absence of overexpressed constitutively active Pkc1 (Pkc1∗), and Bni1 was detected with either anti-HA Ab or anti-phospho-PKC/PKA consensus site Ab.
See also Figure S3.
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6. Figure 5
Bni1 Ubiquitylation and Degradation Involves a Phosphorylation-Dependent Recognition Mechanism by Rsp5
(A) The Rsp5-WW domain binds Bni Purified GST-Rsp5-WW pulls down Bni myc from yeast lysates.
(B) The interaction between Bni myc and the Rsp5-WW domain requires Pkc1-dependent phosphorylation. The Rsp5-WW domain selectively pulls down the phosphorylated form of Bni myc; binding is also lost in a pkc1-2 strain at 37°C (3 hr).
(C) Mutation of three consensus WW-binding motifs abolishes the interaction between Bni myc and the Rsp5-WW domain. Cell lysates were prepared from rsp5-1 strains expressing either Bni myc or Bni1-PA myc.
(D) The WW-binding motifs (PY motifs) in Bni1-642 are required for its damage-induced degradation.
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7. Figure 6
Functional Role of Bni1 Degradation in the Wound Healing Response
(A) Persistent steady-state levels of Bni1Δ after heat shock (39°C, 30 min).
(B) Bni1Δ is resistant to degradation after cell wall damage.
(C) Prolonged retention of Bni1Δ at the bud tip after heat shock. (Left) Representative images of cells after a 30 min shift to 39°C to visualize Bni1 or Bni1Δ (GFP fluorescence). (Right) Percentage of cells with polarized Bni1 or polarized Bni1Δ
(D) Actin depolarization is delayed in cells expressing Bni1 (Δ87–343) after heat shock. (Left) Representative images of cells after a 30 min shift to 39°C to visualize F-actin (phalloidin). (Right) Percentage of Bni1- or Bni1 (Δ87–343)-expressing cells with polarized actin.
(E) The effect of stable Bni1 on Pkc1's release from the bud tip and its recruitment to the site of laser damage. (Top) Ping-pong dynamics of Pkc1 after laser damage of cells expressing Bni1 (Δ87–343). (Bottom) Quantification of the Pkc1 signal intensity of top panels.
See also Figure S4 and Movie S5.
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8. Figure 7
The Exocyst Subunit Sec3 Is Targeted for Pkc1-Dependent Degradation after Cell Wall Damage
(A) Sec3-GFP levels decline sharply after cell wall damage. Cells expressing the indicated GFP fusion proteins were exposed to heat shock (39°C, 30 min) (top) or SDS (bottom).
(B) Pkc1 is required for loss of Sec3 at 37°C. The indicated strains were shifted to 37°C for 3 hr.
(C) The degradation of Sec3 requires Pkc1.
(D) The first 308 amino acids of Sec3 are required for the decline in its steady-state levels after heat shock (39°C, 30 min).
(E) The first 308 amino acids of Sec3 are required for its cell wall damage-induced degradation.
(F) Combinatorial effect of stable Bni1 and stable Sec3 on the Pkc1 recruitment to the laser damage site. (Left) The indicated strains were subjected to the laser damage assay. (Upper right) Average time for the Pkc1-GFP signal to fall to background levels after laser damage. Error bars indicate SEM. (Lower right) Average time for the Pkc1-GFP signal to appear at the laser damage site. Error bars indicate SEM.
(G) Combinatorial effect of stable Bni1 and stable Sec3 on viability after cell wall damage. Indicated strains were spotted on plates with SDS (0.02%) or control plates.
See also Figures S5, S6, and Movie S6.
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9. Figure S1
Large-Scale Reorganization of Polarity Factors after Laser Damage, Related to Figure 1
(A–F) Validation of Pkc1(HR1-C2)-GFP as a Rho biosensor.
(A) Expression of GTP-locked Rho1 (RHO1-Q68L) recruits Pkc1(HR1-C2)-GFP to the bud cortex. In the control, Pkc1(HR1-C2)-GFP primarily localizes to the tips of small and mid-sized buds. RHO1-Q68L expression produces diffuse cortical localization in buds of all sizes.
(B) Functional Rho1 is required for membrane localization of Pkc1(HR1-C2)-GFP. Pkc1(HR1-C2)-GFP localization is abolished in rho1-5 strains (where Pkc1 signaling is impaired) grown at 30°C, a semipermissive temperature. Bud tip localization was observed in 62/100 small budded control cells whereas localization was abolished in the rho1-5 strain (0/100).
(C) Comparable steady-state protein levels of Pkc1(HR1-C2)-GFP in RHO1 and rho1-5 strains.
(D) A point mutation in the Pkc1 HR1 domain [Pkc1(HR1-C2)(L54S)-GFP] that disrupts Rho1-binding also abolishes membrane localization (0/100).
(E) Comparable steady-state protein levels of Pkc1(HR1-C2)-GFP and Pkc1(HR1-C2)(L54S)-GFP.
(F) Pkc1(HR1-C2)-GFP binds active Rho1. Pkc1(HR1-C2)-GFP preferentially immunoprecipitates HA-Rho1(Q68L) relative to the HA-Rho1 control. In addition, an HR1 domain point mutation [Pkc1(HR1-C2)(L54S)-GFP] abolishes this interaction.
(G) The kinetics of polarity factor reorganization after laser damage.
(H) Schematic representation of (G).
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10. Figure S2
Bnr1 Is Required for Focused Rho1 Activity at the Damage Site; SDS Treatment Causes Actin Depolarization Coincident with the Loss of Bni1 Protein Steady-State Levels, Related to Figure 2
(A) The focused recruitment of an active Rho1 biosensor, Pkc1(HR1-C2)-GFP, to the laser-damage site requires Bnr1. bnr1Δ cells expressing Pkc1(HR1-C2)-GFP were subjected to the laser damage assay.
(B) Rom2 is not required for the recruitment of 3GFP-Bnr1 to the damage site. rom2Δ cells expressing 3GFP-Bnr1 were subjected to the laser damage assay.
(C and D) Loss of polarized actin after SDS treatment along with recovery after SDS wash-out. Yeast cells were exposed to SDS for 20 min, which was then washed out with fresh medium. F-actin was visualized with Alexa-phalloidin. Polarized actin was scored as in Figure 2D.
(E) Bni1 protein levels decline after SDS treatment. Expression of Bni1-13myc was monitored in cells at the indicated time points by Western blotting.
(F) Degradation of Bni1 is not a consequence of actin cable disassembly. Actin cable disassembly was induced by inactivation of tropomyosin and Bni1 steady state levels were unaffected. Note that the tropomyosin temperature-sensitive strain was shifted to 34.5°C, a temperature that causes complete actin cable disassembly in this mutant, but a temperature that is below the threshold to trigger cell wall damage (37°C).
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11. Figure S3
Domains Required for the Cell Wall Damage-Induced Loss of Bni1, Related to Figure 4
(A–C) The expression of the indicated Bni1 truncations was determined after cell wall damage by Western blotting with an anti-GFP Ab.
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12. Figure S4
Stability of Bni1 N-Terminal Truncations Cannot Be Explained by Constitutive Activation of Bni1, Related to Figure 6
(A) Bni1-V360 is an activating mutation. The indicated Bni1 constructs were overexpressed from the GAL1 promoter in a bni1Δ strain. After 3 days of growth on YPGal (upper panel) or YPD (lower panel) plates at 24°C. Activating mutations cause lethality in this assay.
(B) Cells expressing Bni1-V360 from its native promoter have increased number of actin cables like cells expressing Bni1-(Δ87-343). F-actin was visualized by Alexa-phalloidin labeling.
(C) Comparable steady state protein levels of Bni1 and Bni1-V360D after heat shock. Western blotting was performed as described in Figure 6.
(D) (Left) V360D does not affect cell wall damage-induced actin depolarization. F-actin was visualized by Alexa-phalloidin. (Right) The percentage of cells with polarized actin. Polarized actin was scored as in Figure 2D. Error bars represent SEM.
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13. Figure S5
Blocking Rsp5-Dependent Proteolysis Impairs the Reorganization of Polarity Factors after Laser Damage, Related to Figure 7
(A) Sec3 remains polarized after laser damage in strains where Bni1 and Sec3 are stabilized. (Left) The indicated strains were subjected to the laser damage assay. (Right) The signal intensity was quantified as in Figure 1. Graphs report the normalized average GFP signal intensity after background subtraction for all independent experiments. (n = 5 per strain). y axis is logarithmic scale.
(B and C) Pkc1-GFP, and Myo2-GFP remain polarized after laser damage in rsp5-1 strains at 37°C. (Left) The indicated strains were subjected to the laser damage assay. (Right) The signal intensity was quantified as in Figure 1. Graphs report the normalized average GFP signal intensity after background subtraction for all independent experiments. (n ≥ 7 per strain). y axis is logarithmic scale. Error bars represent SEM.
(D) Unlike the expression of Bni1Δ87-343, expression of Bni1-V360D did not result in cell wall damage-induced lethality in strains that also express Sec3Δ(1-308). Shown are images of plates taken after 3 days growth at the indicated temperature.
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14. Figure S6 Schematic of the Results, Related to Figure 7
Budding yeast have a wound healing response that enables the targeting of repair factors to the specific site of damage. The response is composed of at least two sequential processes: (1) disassembling polarity complexes and (2) targeted cellular wound healing. Protein kinase C promotes the destruction of the formin Bni1 and the exocyst component Sec3. This degradation is a pre-requisite for the subsequent wound healing response. Polarity regulators including Pkc1, Rho1, formin Bnr1, Exocyst Exo70, type V myosin Myo2 and cell wall synthase Chs3 are targeted to the local wound. In the right panel temporal order of recruitment is color-coordinated from red to blue (red: early, yellow: middle, and blue: late recruitment). See also Figures S1G and S1H.
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15. Cell  , DOI: ( /j.cell )
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