Journal: eLife

Article Title: Spatial quality control bypasses cell-based limitations on proteostasis to promote prion curing

doi: 10.7554/eLife.04288

Figure Lengend Snippet: ( A ) A [ PSI + ] Weak HSP104GFP culture (SY2126) was imaged over time in a microfluidics chamber at 30°C after a 30 min incubation at 30°C, 37°C, 40°C, or 37°C before 40°C. Fluorescence intensity in daughter and mother cells was quantified at the first cell division in cells that were budded (gray) or unbudded (orange) after thermal stress. Lines represent medians; boxes represent upper and lower quartiles, and whiskers represent maximum and minimum. All pairwise comparisons are significantly distinct, with a p < 0.015, except where indicated (N.S.), by unpaired t-test; n ≥ 10. ( B ) A [ PSI + ] Weak HSP104GFP WT (SY2126, gray) or BNI1 deletion strain (Δ bni1 ) (SY2486, green) was imaged over time in a microfluidics chamber at 30°C after a 30 min incubation at 40°C. Fluorescence intensity in daughter and mother cells was quantified at the first cell division. Lines represent medians; boxes represent upper and lower quartiles; and whiskers represent maximum and minimum; n ≥ 14; p = 0.0075 by unpaired t-test. ( C ) [ PSI + ] Weak WT (SLL2600) or Δ bni1 strains (SY1888), treated as described in ( B ), were plated on YPD to analyze curing by colony color phenotype. Data represent means; error bars represent standard deviations; n = 3; p < 0.0001 by unpaired t-test. ( D ) A [ PSI + ] Weak HSP104GFP strain (SY2126) was imaged over time in a microfluidics chamber at 30°C after a 30 min incubation at 40°C and with GdnHCl added before or after the 40°C incubation. Fluorescence intensity in daughter and mother cells was quantified at the first cell division. Lines represent medians; boxes represent upper and lower quartiles; and whiskers represent maximum and minimum; n > 11; *p = 0.0003, **p = 0.0026 by unpaired t-test. ( E ) A [ PSI + ] Weak strain (SLL2600) was incubated at 40°C for 30 min and plated on rich medium. Mother and daughter pairs were separated by micromanipulation and allowed to form colonies, which were then dispersed to YPD for analysis of curing by colony color phenotype. n = 15. ( F ) A [ PSI + ] Weak HSP104GFP culture (SY2126) was incubated at 30°C (dotted) or at 40°C for 30 min and allowed to recover for 30 min at 30°C (solid) before analysis of GFP fluorescence intensity by flow cytometry. Based on these intensities, cells were sorted into four fractions (orange, blue, purple, red) by FACS. ( G ) Cells collected in ( F ) were plated on YPD to analyze curing by colony color phenotype. Data represent means; error bars represent standard deviations; n = 2; *p = 0.02 by paired t-test. DOI: http://dx.doi.org/10.7554/eLife.04288.010

Article Snippet: Microfluidics experiments were performed on a Zeiss Axio Observer Z1 using a CellAsics microfluidics plate with temperature controls and media flow of 2 psi on a Y0C4 yeast perfusion plate (channel size 3.5–5 μm).

Techniques: Incubation, Fluorescence, Micromanipulation, Flow Cytometry

Journal: eLife

Article Title: Spatial quality control bypasses cell-based limitations on proteostasis to promote prion curing

doi: 10.7554/eLife.04288

Figure Lengend Snippet: ( A ) A [ psi − ] strain expressing heat-inducible untagged GFP (SY2091) was imaged over time in a microfluidics chamber at 30°C after 30 min incubation at 40°C (red) or 30°C (gray). Fluorescence intensity in daughter and mother cells was quantified at the first cell division in budded cells. Lines represent medians, boxes represent upper and lower quartiles, and whiskers represent maximum and minimum; n ≥ 11. ( B ) A [ PSI + ] Weak strain expressing a GFP-tagged endogenous Ssa1 and DsRedNLS (SY2659) was imaged after a 90 min incubation at 30°C, 37°C, 40°C, or 37°C before 40°C. Scale bar = 2 μm. ( C ) A [ PSI + ] Weak strain expressing a GFP-tagged endogenous Sis1 and DsRedNLS (SY2485) was imaged after a 90-min incubation at 30°C, 37°C, 40°C, or 37°C before 40°C. Scale bar = 2 μm. ( D ) A [ PSI + ] Weak SSA1GFP culture (SY2658) was imaged over time in a microfluidics chamber at 30°C after a 30 min incubation at 40°C (red) or 30°C (gray). Fluorescence intensity in daughter and mother cells was quantified at the first cell division in budded cells. Lines represent medians, boxes represent upper and lower quartiles, and whiskers represent maximum and minimum; n > 15. ( E ) A [ PSI + ] Weak SIS1GFP culture (SY2447) was imaged over time in a microfluidics chamber at 30°C after a 30 min incubation at 40°C (red) or 30°C (gray). Fluorescence intensity in daughter and mother cells was quantified at the first cell division in budded cells. Lines represent medians, boxes represent upper and lower quartiles, and whiskers represent maximum and minimum; n ≥ 7. ( F ) Quantitative immunoblotting for Hsp104 was performed on lysates from WT (SLL2600) or Δbni1 (SY1888) [ PSI + ] Weak cultures treated at 30°C (black) and 40°C (white) for 30 min following SDS-PAGE. Data represent means; error bars represent standard deviations; n = 3. ( G ) Lysates were isolated from WT (SLL2600) or Δbni1 (SY1888) [ PSI + ] Weak strains that were incubated at 30°C or 40°C for 30 min, and heat-induced protein aggregates were analyzed by differential centrifugation and Bradford assay. Data represent means; error bars represent standard error; n = 3. DOI: http://dx.doi.org/10.7554/eLife.04288.011

Article Snippet: Microfluidics experiments were performed on a Zeiss Axio Observer Z1 using a CellAsics microfluidics plate with temperature controls and media flow of 2 psi on a Y0C4 yeast perfusion plate (channel size 3.5–5 μm).

Techniques: Expressing, Incubation, Fluorescence, Western Blot, SDS Page, Isolation, Centrifugation, Bradford Assay

Journal: eLife

Article Title: Spatial quality control bypasses cell-based limitations on proteostasis to promote prion curing

doi: 10.7554/eLife.04288

Figure Lengend Snippet: ( A ) The number of [ PSI + ] Weak HSP104GFP (SY2126) cells containing fluorescent foci was quantified in cultures recovering at 30°C over time following a 90 min incubation at 40°C (white). Colony forming units in these cultures were quantified by plating (black). Data represent means; error bars represent standard deviations; n = 3. ( B ) [ PSI + ] Weak HSP104GFP cells (SY2126) treated for 30 min at 40°C and imaged over time in a microfluidics chamber are shown. Cells that were budded at the time of thermal stress are outlined in white, while unbudded cells are outlined in orange. Solid lines mark mothers, and dotted lines mark daughters. Scale bar = 1 µm. ( C ) A [ PSI + ] Weak HSP104GFP strain (SY2126) was imaged over time in a microfluidics at 30°C after a 30 min incubation at 40°C chamber. Budded or unbudded cells were scored at the first cell division for the presence or absence of fluorescent aggregates. Data represent means; error bars represent standard deviations; n = 3; p = 0.0005 by unpaired t-test. DOI: http://dx.doi.org/10.7554/eLife.04288.015

Article Snippet: Microfluidics experiments were performed on a Zeiss Axio Observer Z1 using a CellAsics microfluidics plate with temperature controls and media flow of 2 psi on a Y0C4 yeast perfusion plate (channel size 3.5–5 μm).

Techniques: Incubation

Journal: Nature Communications

Article Title: Management of E. coli sister chromatid cohesion in response to genotoxic stress

doi: 10.1038/ncomms14618

Figure Lengend Snippet: ( a ) Cell cycle restart pattern after a brief MMC treatment using a microfluidic platform. WT cells were introduced into the chambers, and fresh minimal medium A was perfused for 20 min; 10 μg ml −1 MMC was then perfused for 10 min and immediately washed with clean medium, and incubation and imaging were continued for over 3.5 h. Cell lineage was measured for 100 cells; each colour corresponds to a given state of the cell. ( b ) The same experiment as described in a was performed in the recN mutant, which exhibits delayed cell cycle restart. ( c ) Representative time-lapse microscopy of RecA-mCherry focus dynamics in the presence of MMC in the WT strain. Time-lapse imaging starts at 5 min after initial contact with MMC. Pictures were acquired every 3 min for 2 h on an agarose pad with MMC. ( d ) RecA-mCherry focus dynamics in the presence of MMC in the recN mutant. Experiments were performed as described for c . ( e ) Analysis of RecA focus dynamics. Kymograph and time series of cell slices for RecA-mCherry WT. The experiment was performed as described in c . ( f ) Analysis of RecA foci dynamics in the recN mutant. The experiment was performed as described in d . ( g ) The frequency of bundles was estimated as a function of the shape of the RecA mCherry signal in WT and the recN mutant. The WT and recN distribution are significantly different ( t -test P =10 −30 ). Scale bar is 1 μm.

Article Snippet: A microfluidic plate was set-up according to the Merck Millipore protocol for bacteria.

Techniques: Incubation, Imaging, Mutagenesis, Time-lapse Microscopy