A. Observation of Hsp104-GFP-containing aggregate movement in the wild-type BY4741 strain in a representative 3D time-lapse movie (Movie S1A). Image stacks were collected and shown at 20s intervals, and are shown as maximum projections.
B. Example of an aggregate (arrow) moving from bud to mother in wild type (Movie S2A). Image stacks were collected and shown at 1 min intervals, and are shown as maximum projections.
C. Example of an aggregate (arrow) moving from mother to bud in wild type (Movie S2B). Image stacks were collected at 1 min intervals, and a maximum projection is shown.
D. The frequency of B>M or M>B movement per cell in an exponentially population was calculated by counting the totally number of each type of event divided by the totally number of budded cells observed in each movie. The bar graphs show mean and standard error of the mean (SEM).
E. Percentage of cells in the population in (D) in which no trans-bud neck movement was observed during the 1 hour time lapse movies. Shown are mean and SEM. For both D and E, on average 40 cells/movie from 4–6 movies were quantified for each strain.
F. The frequency of B>M or M>B movement per cell in small budded cells was calculated by counting the totally number of each type of event divided by the totally number of small budded cells observed in each movie. Shown are mean and SEM.
G. Percentage of cells in the population in (F) in which no trans-bud neck movement was observed during the 1 hour time lapse movies. Shown are mean and SEM. For F and G, on average 14 cells/movie from 4–6 movies were quantified for each strain.

A. Comparison of diffusion coefficients calculated from aggregate tracking between the two wild type strains. Shown are mean and SEM. More than 1000 aggregates from at least 2 movies/strain were tracked. wt, RLY7110; wt*, YBD401 (Liu et al., 2010).
B. Quantification of aggregate dissolution (see Experimental Procedures) in the two strains in (A). Shown are plots of total aggregate intensity as a function of time from time-lapse movies of a field of cells (50–100 cells per field) starting from the 30 min frame when aggregates in wild type no longer grew in brightness. Each plot is an average from two movies. Black square: RLY7110; open circle: YBD401.
C. Quantification of aggregate dissolution following the same method as in (B) in wild-type (wt) cells and hsp104^Y662A cells.
D. Diffusion coefficient of each strain and condition as indicated quantified from aggregate tracking data (see Experimental Procedures). Shown are mean and SEM. *The quantification for hsp104^Y662A cells was done from the 3.5 min heat shock experiment to mimic wild-type aggregate density. For each strain or condition, more than 450 (and in many cases over 1000) aggregates from 2–4 movies were tracked and quantified.
E–F: Quantification of aggregate dissolution following the same method as in (B) for the same conditions or strains examined in (D). See also Figure S1, S2, Movie S4, S6, S7, S8.

A. An example of retention of protein aggregates in a wild-type mother cell undergoing budding. Image stacks were collected 1 min intervals; montage is shown as maximum projections every 4 min (see also Movie S10A).
B. Time-lapse images of a cell showing two different fates of aggregates that leaked into the bud from the mother during bud formation. Image stacks were collected 1 min intervals; montage is shown as maximum projections every 4 min. Arrows point to an aggregate that returned to the mother after leakage into the bud; arrowhead points to an aggregate that dissolved after leakage into the bud. See movie S10B to observe these events at a higher time resolutions.
C. Percentages of cells displaying three different types aggregate behavior during new bud formation and growth in three different genetic backgrounds as indicated. Cells either with a tiny bud initially devoid of any aggregates or giving birth to a new bud during the first 30min of 1 hr long 3D time-lapse movies were scored. Gray bar: cells with no aggregates leakage into the bud; black bar: cells in which aggregates leaked into the bud were subsequent cleared by dissolution; white bar: cells in which aggregates leaked into the bud and subsequent moved back to mother. N=108 cells (wt); 74 cells (bni1Δ), 133 cells (bnr1Δ). See also Movie S8, S9.

A. Probability density map of protein aggregates after a cell cycle period (90 min) from a 3D numerical simulation of aggregate retention assuming random walk (α = 1) (see Experimental Procedures) as a 2D sum projection (top) and lateral profiles of central plane (bottom). Each colored line corresponding to simulation with a particular neck size (diameter) as indicated. The brown line shows the initial distribution at the start of the simulation and the green line shows the 1.25 μm neck size simulation with confined diffusion (α = 0.50, as measured in aged cells).
B. Analysis of protein aggregate retention using a 1D analytical solution (Formula S12, see Supplementary Information) for D=0.0005 μm²/s (black) and D=0.001μm²/s (red). The dashed blue line denotes 90 min time point.
C. A comparison of aggregates distribution after 90min in 3D simulation for D=0.001 μm²/s and neck size of 4.25 μm (mimicking open neck, blue dashed curve) to the 1D analytical solution (Formula S7, using 20 terms, see Extended Experimental Procedures) (red solid curve). The brown curve represents the initial aggregates distribution. 0 on x axis is the position of bud neck and mother-bud orientation as in A; L1 and L2 is the length of mother and bud cell, respectively.

A. Left panel: A representative field of trajectories from tracking of protein aggregates from 1 hr time-lapse movies. For comparison, the right panel shows a field of simulated trajectories of random walk with observed distribution of trajectory lengths and diffusion coefficient.
B. A plot of MSD vs time shift, showing that aggregate movement can be characterized as random walk with a small amount of confinement (solid line). Dotted line shows plot expected for pure random walk.
C. α value distributions from 5000 simulations of aggregate movement with the diffusion coefficient and trajectory length distribution similar to those observed in yeast cells for each of the three scenarios: 1) pure random walk (blue); 2) random walk with 10% super-diffusion (green); 3) random walk with 30% sub-diffusion (red). The distribution of α values for each population was compared to the experimentally observed distribution (black), showing the observed is consistent with scenario (3).
D. Comparison of average α values (mean and SEM) from aggregates in different population of cells in 60min 3D time-lapse movie. Blue: aggregates in all the cells in the movies (N=3542); green: all aggregates in the small budded S/G2 cells (N=182); red: aggregates in the buds of the S/G2 cells (N=31). N refers to the number of aggregates. See also Movie S3.

A. Hsp104-GFP-associated aggregates show gradual clearance through dissolution. The arrow indicates an example of an aggregate dissolving; arrowheads indicate two aggregates fusing and then dissolving. Images stacks were collected at 1 min intervals; the montage shows maximum projections at 6min intervals from a 90 min movie (Movie S5).
B. Comparison of the percentage of cells where the last aggregate was cleared from the bud by dissolution (white bar) to that by bud-to-mother movement (gray bar) in the wild type. On average 47 cells/movie from 5 movies were quantified. Shown are mean and SEM. C. Aggregate dissolution depends on chaperon activity of Hsp104p. In hsp104^Y662A mutant cells, the aggregates not only did not dissolve over a representative 3hr movie (Movie S6A) but also increased in brightness over time. Montage starts from 18 min of the movie and frames (maximum projections) are shown at 18min intervals.

A. Age distribution of magnetic-beads sorted old wild-type cells determined by bud scar counting (> 170 cells were counted).
B. Representative images showing asymmetric distribution of Hsp104-associated protein aggregates in aged wild-type from the populations in (A). Shown are maximum projections of 3D image stacks.
C. Trajectory (white line in left panel) and MSD (right panel) analysis of an Hsp104-GFP-associated aggregate (arrow) in an old wild-type cell from a 60 min 3D time-lapse movie.
D. Hsp104-GFP-containing protein aggregates in old wild-type cells exhibit a similar type of movement. Shown are mean and SEM of α values calculated from long aggregate tracks. N=21 tracks for wild-type aggregates. See also Movie S11-.