Based on the analyses described above, we conclude that the clearance of protein aggregates from the bud does not depend on actin cable-mediated retrograde transport. Our conclusion is based on quantification of aggregates movement from time-lapse recording of a large number of cells. Although linear bud-to-mother movement was observed occasionally, mother-to-bud movement of aggregates was observed at a similar frequency, and thus there was no net directionality in aggregate movement along the mother-bud axis. Particle tracking and trajectory analysis showed that the movement of protein aggregates can be described as random walk with a small amount of confinement. Even though some apparently linear trajectories can be observed, these can be recapitulated by random walk simulations using the same diffusion coefficient and trajectory length. We emphasize that although our data does not support a direct role for actin-based retrograde transport in aggregate segregation, the data does not necessarily argue against an involvement for actin cytoskeleton or cell polarity proteins in this process. It is
conceivable, for example, that the abnormally wide bud neck in some of the cell polarity mutants could increase the leaking of protein aggregates into the bud, although such effect is likely to be too subtle to observe reliably in our experiments.
Disassembly of actin filaments following LatA treatment partially abrogated aggregates movement and slowed their dissolution. Although it is possible that residual actin filaments remained and were below detection in LatA treated cells, this observation suggests that actin cables, which were no longer present in LatA-treated cells, are not required for the movement or dissolution of heat-induced aggregates. The effect of LatA on aggregate movement or dissolution may also be indirect if the loss of actin filaments induces a high-stress state interfering with aggregate dissolution or motility. Regardless of the mechanism, our observed effect of LatA on aggregate motility does not help explain a positive role for actin in aggregate asymmetric segregation, as based on our model, reduced aggregate diffusive motion would predict their better retention in the mother. The inhibitory effect of actin depolymerization on the Hsp104-dependent aggregate dissolution is consistent with a functional linkage between actin and Hsp104 reported in recent studies (Erjavec et al., 2007
; Tessarz et al., 2009
We did not observe any apparent requirement for the formin protein Bni1p or Bnr1p in the motility or retention of heat-induced protein aggregates in the mother. The experiments performed previously on the retention of protein aggregates relied on quantification of nonsynchronized cell populations at different time points during a continuous incubation at high temperature (Liu et al., 2010
). Such an assay would not be sufficient for distinguishing between the possibilities of aggregate clearance by dissolution versus by movement. Because the dynamics of new aggregate formation during the prolonged heat exposure were unknown, buds displaying no aggregates might be new buds that formed after the cells had adapted to the heat stress and ceased the formation of new aggregates, rather than reflecting active aggregate clearance from the bud. Additionally, the unbudded cells observed in this assay might not be the new G1 cells after cytokinesis but instead might be arrested cells failing to adapt to the heat stress. By contrast, we directly observed retention of pre-formed aggregates during new bud formation by time-lapse movies, and we did not observe any defect in this process in the formin mutants.
Finally, based on the observed diffusion rates of protein aggregates, and borrowing the insight from a recent study explaining the asymmetric inheritance of episomal DNA during yeast nuclear division (Gehlen et al., 2011
), we propose that the limited mobility and the narrowness of the bud neck ensure that the vast majority of the proteins aggregates accumulated with ageing are retained in the mother during the limited time of a cell cycle. This idea is supported by our 3D simulation as well as a 1D analytical model. This model implies that no additional mechanism may be needed to confer the asymmetric segregation of protein aggregates during yeast cell divisions.