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Prospects & Overviews
Asymmetric damage segregation at cell
division via protein aggregate fusion and
attachment to organelles
Miguel Coelho1)! and Iva M. Tolic!2)3)!
The segregation of damaged components at cell division
determines the survival and aging of cells. In cells that
divide asymmetrically, such as Saccharomyces cerevisiae,
aggregated proteins are retained by the mother cell. Yet,
where and how aggregation occurs is not known. Recent
work by Zhou and collaborators shows that the birth of
protein aggregates, under specific stress conditions,
requires active translation, and occurs mainly at the
endoplasmic reticulum. Later, aggregates move to the
mitochondrial surface through fis1-dependent association. During replicative aging, aggregate association with
the mother-cell mitochondria contributes to the asymmetric segregation of aggregates, because mitochondria
in the daughter cell do not carry aggregates. With
increasing age of mother cells, aggregates lose their
connection to the mitochondria, and segregation is less
asymmetric. Relating these findings to other mechanisms
of aggregate segregation in different organisms, we
postulate that fusion between aggregates and their
tethering to organelles such as the vacuole, nucleus, ER, or
mitochondria are common principles that establish
asymmetric segregation during stress resistance and
aging.
.
Keywords:
asymmetric segregation; endoplasmic reticulum; fusion;
mitochondria; organelle tethering; partitioning; protein
aggregates; retention; sequestration
Introduction
How do cells cope with the errors that accumulate during cell
division? Damaged molecules that the cell was unable to repair
in the previous cell cycle will be inherited by both cells in the next
division. If cells accumulate damage faster than they can repair it
and when the accumulated damage exceeds a certain threshold,
it will interfere with cell-cycle progression and doom all cells in
the population to die. However, if a cell can asymmetrically
segregate damage to only one of its daughters, half of the cells in
the population will be born rejuvenated and damage-free at each
division. This reductionist damage segregation strategy implies
the necessity for either physical or molecular “handles”: while
the cell geometry [1], and active transport [2–4] or tethering to
sub-cellular structures [2–11] might facilitate damage retention
in the mother cell, damage fusion into a single unit ensures
completely asymmetric segregation of damage to one of the two
cells at cell division [1, 3, 8, 10, 12–17].
The segregation of damaged proteins is associated with
stress survival, aging, and cell death. This process represents a
DOI 10.1002/bies.201400224
1)
2)
3)
Department of Molecular and Cellular Biology, Harvard University,
Cambridge, MA, USA
Max Planck Institute of Molecular Cell Biology and Genetics, Dresden,
Germany
! Institute, Zagreb, Croatia
Division of Molecular Biology, Rud̵er Bo"
skovic
Abbreviations:
CHO, Chinese hamster ovary; COS-7, monkey kidney tissue; ER, endoplasmic reticulum; GFP, green fluorescent protein; HEK, human embryonic
kidney; Hsp, heat shock protein; MTOC, microtubule organizing center; N2a,
mouse neuroblastoma; SPB, spindle pole body; ts, temperature sensitive.
*Corresponding authors:
Miguel Coelho
E-mail: [email protected]
!
Iva M. Tolic
E-mail: [email protected]
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Prospects & Overviews
Aggregate formation after stress occurs
at the ER and requires active translation
To study protein aggregate segregation it is essential to follow
the aggregation process continuously during the cell cycle.
Although pioneering studies using fixed samples allowed to
detect the intracellular localization of damaged proteins and
to infer asymmetries in their segregation [3, 20, 21, 23], the
advent of live-cell imaging coupled with fluorescent protein
reporters allowed for the tracking of individual aggregates
with high spatial and temporal resolution in vivo [1, 4, 5, 8, 10,
12–15, 17, 19]. This also prompted researchers to study protein
aggregation response during genetic, chemical, or physical
perturbations, over several cell divisions, which led to the
characterization of different types of aggregates, specialized
Bioessays 37: 740–747, ! 2015 WILEY Periodicals, Inc.
compartments, and segregation mechanisms. However, it is
still unclear where the aggregates originate, and aggregate
nucleation is assumed to be a random process that occurs
around aggregation-prone proteins in the cytoplasm.
In a recent work, Zhou et al. [19] used live-cell imaging of
aggregate nucleation during stress to show that in the
presence of cycloheximide, a translation inhibitor, aggregation was severely reduced. This was also observed in a
peroxiredoxin tsa1 S. cerevisiae mutant [24] and in the muscle
cells of Caenorhabditis elegans [25]. Although stronger
stresses, such as temperatures above the range tested by
Zhou et al. [19] or higher concentration of denaturing agents,
do result in direct aggregation even in the presence of
translation inhibitors [26], the finding that aggregation
requires translation suggests that aggregates, at least under
the conditions that allow the cells to survive and grow after
stress, are more likely to form at the major source of
aggregation-prone proteins in the cell: the unfolded nascent
polypeptide chains in the ER-associated ribosomes. This result
defines the intracellular localization for aggregate genesis,
before their movement, tethering to organelles and segregation at cell division occur. It will be interesting to see similar
experiments in other cell types, which might reveal a common
role for active translation during aggregate formation.
Aggregates localize to the cytoplasm,
cytoskeleton, and mitochondria
Whether generated at the ER or elsewhere in the cell,
aggregates localize to specific sites in the cell, which to a large
extent determines their dynamics and the segregation pattern
at cell division (Fig. 1). To simplify the complex description of
different aggregate types and functional storage sites, which
might not always contain highly insoluble clusters, we will
proceed by considering two major aggregate types: natural
and stress-induced, and compare their behavior in different
model organisms. Aggregates which are present under
favorable growth conditions, here termed natural aggregates–aggresomes [2, 3], IPOD and JUNQ [5] and inclusion
bodies [12, 13, 17], can be tethered to cell components: nuclear
membrane and vacuole [5, 7, 9, 10], actin cables [4, 6, 9],
ER [15, 19], and mitochondria [19] (Fig. 1C). Stress-induced
aggregates, peripheral aggregates, Q-bodies, and stress foci [4,
10, 12, 13, 15], were observed to be free in the cytoplasm, either
due to de novo aggregate formation [12, 13, 15] or due to stressinduced release because of disruption of cytoskeletal or
organellar structures [1, 10]. More recently, stress-induced
aggregates have also been associated with ER or mitochondria [15, 19]. While nucleation and association with the ER
remains untested in S. pombe, natural aggregates do not seem
to be tethered to the following cell components [13]: MTOC
analog SPB, vacuoles, endosomes, nuclear membrane, actin
or microtubule cytoskeleton, and by inference the mitochondria, which associate with microtubules in this organism
(Fig. 1B). Stress-induced aggregates have the same localization pattern as natural aggregates, and due to their larger
size are later confined to the cell-pole region [13]. In E. coli,
natural and stress-induced aggregates localize to the
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useful model to address how cells deal with damaged
molecules they cannot repair, which therefore rapidly
accumulate over successive divisions. Damaged proteins
can be stored in granular cytoplasmic structures, or form
insoluble aggregates and inclusion structures that can
associate with organelles. For simplicity, will use the term
“aggregate” to refer to these structures ubiquitously, however
we note that they have different biochemical (protein content
and structure) and biophysical (solubility and movement)
properties. Aggregates can be visualized during cell division,
either by direct [2, 3, 5–7, 10, 15, 18, 19] or indirect [1, 4, 9, 12–
14, 17] labeling using fluorescent protein reporters. Direct
visualization is based on fluorescent-protein fusions of
aggregation-prone proteins (Htt103Q, VHL, Ubc9ts, Rnq1,
Luciferase, and a-synuclein in yeast), whereas for indirect
visualization fusions of chaperone proteins that associate with
aggregates are used (IbpA in E. coli, and Hsp16/42, Hsp70, and
Hsp104 in S. cerevisiae and S. pombe). Careful description of
the localization and movement of these aggregates during
different growth conditions and in distinct genetic backgrounds has revealed that during asymmetric cell division the
identity of the cell that inherits the damage is normally preestablished by polarity cues [4, 6, 7, 10, 15, 18, 19], while
during symmetric cell division it is established by the position
of damage relative to the division plane at the moment
of division [2, 3, 8, 11–14, 20–22]. In the budding yeast
Saccharomyces cerevisiae, asymmetric cell division results in
an aging mother cell that retains protein aggregates, and
a daughter cell that is clear of aggregates and rejuvenated
[1, 4, 5, 7, 10, 15, 19, 21, 23, 24]. The apparently contradictory
aggregate segregation mechanisms presented for S. cerevisiae
can be partly attributed to different probes used to label
aggregates and growth conditions [1, 6, 10, 15, 19] (Table 1).
Expanding the analysis to protein aggregate segregation in
two other model organisms, the fission yeast Schizosaccharomyces pombe [12, 13] and the prokaryote Escherichia coli [14,
17, 22], two cell types that divide symmetrically, may help to
reveal conserved principles of these mechanisms. To extrapolate general properties of aggregates we focus our analysis on
the comparison between S. cerevisiae and S. pombe, since in
these two distantly related organisms protein aggregation
dynamics has been studied in great detail.
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M. Coelho and I. M. Tolic
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Hsp104-GFP,
Hsp104Y662AGFP/IbpA-YFP
CFTR, GFP-250,
Dendra2-VHL,
HttQ97-103/HttQ103,
Hsp104-GFP, Ubc9ts,
VHL1, DssCPY*,
Luciferase
CFTR, GFP-250,
Dendra2-VHL/
Hsp104-GFP,
Ubc9ts-GFP/
Hsp104-GFP/
IbpA-YFP
Active
transport
Diffusion
barrier/Cell
geometry
Organelle
tethering/
Cytoskeleton
association
Fusion
Growth conditions
Normal growth,
proteasome
inhibition/normal
growth, atp depletion,
translation inhibition,
heat stress/
normal growth,
heat stress/
normal growth
Normal growth/
prolonged
heat stress
Short heat stress,
aged cells/normal
growth
Normal growth,
proteasome
inhibition/proteasome
and translation
inhibition, oxidative
and heat stress
Bar separates the observations by respective organism.
Aggregate labels
CFTR, GFP-250/
Hsp104-GFP
Mechanism
Table 1. Segregation mechanisms of protein aggregates
Cell components
Centrosome,
(aggresome), nucleus
(JUNQ), vacuole
(IPOD)/polarisome,
JUNQ, IPOD,
actin cables,
Q-bodies, ER,
mitochondria, vimentin
Encounters between
aggregates in the
cytoplasm, or
associated to
cytoskeleton or
organellar surfaces.
Centrosome
(aggresome)/
actin cables
Bud-neck/cell pole
Molecules
Dynein? or myosin,
Vimentin/Hsp42/
Hsp16/?
(factors controlling
bud aperture size/
cell shape?)
Actin-myo2,bnj1,
cmd1; IPOD/JUNQ
-Hsp42, sis1, cur1,
btn2; Q-bodies Hsp70;
mitochondria- fis1,
protein importers?
Dynein, microtubules/
myosin, actin
Organisms
HEK, CHO,
COS-7 and
N2a cells/
S. cerevisiae/
S. pombe /
E. coli
HEK, CHO,
COS-7 and
N2a cells/
S. cerevisiae
HEK, CHO and
COS-7 cells/
S. cerevisiae
S. cerevisiae/
E. coli
Fusion between
aggregates generates
a single large
aggregate
Aggregates are
transported by motors
and cytoskeleton
Aggregates diffusion
and cell geometry
dictates segregation
Aggregates associate
with organelles or
cytoskeleton and are
retained in one cell
Key observation
[2, 3, 8]/
[4, 5, 10, 15]
/[12, 13]/
[17]
[2, 3, 8]/
[4, 5, 10, 15]
[1]/[14]
[2, 3]/[4, 6]
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The degree to which each movement type
contributes to segregation at cell division
depends largely on the aggregate (natural
versus stress-induced) and cell type. As an
example, aggregates in S. cerevisiae anchored to actin cables or transported via
myosin motors along these cables [4] switch
to diffusion after heat shock probably due
to depolymerization of the actin cytoskeleton [1, 6, 15]. As a contrary example, in
S. pombe, depolymerization of the actin or
microtubule cytoskeleton does not affect
aggregate movement, which remains diffusive [13]. Diffusive
movement was also observed for protein aggregates in
E.coli [14].
It is harder to interpret the movement of aggregates
tethered to organelles: while Zhou et al. [19] show that
confinement to the mitochondrial surface restricts diffusion,
fis1, the factor responsible for this association is also involved
in mitochondrial fission, which may enhance the movement
of mitochondria and thus also of the tethered aggregates.
Knocking-out fis1 leads to a reduction of aggregate retention
in the mother, but to a smaller extent than other non-
Figure 1. Localization of protein aggregates in different cell types. A: In the rod-shaped
prokaryote E. coli, aggregates (bright green) are found in the cytoplasm, excluded from
the central region of the cell by the presence of the nucleoid (white thread). These
aggregates tend to cluster at the cell poles. B: In the rod-shaped and symmetrically
dividing eukaryote S. pombe, aggregates are in the cytoplasm, without links to organelles
or the cytoskeleton. C: In the asymmetrically dividing eukaryote S. cerevisiae, aggregates
localize to surfaces of organelles: vacuole (IPOD), nucleus (JUNQ), actin cables, ER, and
mitochondria. D: In mammalian CHO cells, aggregates are surrounded by vimentin cages
close to the centrosome (JUNQ), and present in inclusions in the cytoplasm (IPOD). In
panels C and D, IPOD and JUNQ are present under favorable growth conditions and
here referred to as natural aggregates, while peripheral aggregates that are free in the
cytoplasm are stress-induced.
nucleoid-free regions at the cell pole [14, 17, 22] although
aggregation, and possibly localization, can be influenced by
interference of aggregation-prone fluorescent proteins used
to visualize the aggregates [27] (Fig. 1A). In multicellular
organisms, natural aggregates were shown to be associated
with the centrosome [3] and vimentin cages [2, 3, 8], while
stress-induced aggregates seem to be present in multiple
regions in the cytoplasm [28] (Fig. 1D).
While in symmetrically dividing cells such as E. coli and
S. pombe aggregates are confined to cytoplasmic compartments, in the asymmetrically dividing S. cerevisiae and in
higher eukaryotic organisms aggregates are tethered to or
enclosed in cytoskeletal or organellar compartments. Stressinduced aggregates [1, 13, 15], or aggregates in aged cells [1, 12]
are more similar across different organisms, as they show a
generally decreased association with cytoskeletal or organellar compartments, which might influence their segregation
at cell division.
Aggregates move by diffusion, active
transport, or with the organelles
To understand aggregate segregation at cell division, it is
important to consider their movement during the cell cycle.
Protein aggregates were described to move by active transport
(dynein-dependent transport along microtubules [2] or a
polarisome- and tropomyosin-dependent flow of aggregates
along actin cables [4]), by confined organelle movement
(MTOCs, nucleus, vacuole, actin cables [2, 4, 5, 10] or recently
ER, and mitochondria [19]) or by diffusion (in the cytoplasm
confined by organelle and cell boundaries [1, 13, 14]) (Fig. 2).
Bioessays 37: 740–747, ! 2015 WILEY Periodicals, Inc.
Figure 2. Types of aggregate movements in S. cerevisiae. Aggregates (green) move by diffusion in the cytoplasm, retrograde
transport from the bud to the mother cell along actin cables (pink,
active transport), diffusion on the surface of organelles (purple) to
which they are tethered, or by organelle dynamics, such as fusion
and fission of mitochondria (not depicted).
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M. Coelho and I. M. Tolic
mitochondria related segregation mutants [4], which suggests
other mechanisms play a role in this process. In general,
organelle growth and remodeling, such as fusion and fission
of mitochondria, microtubule or actin polymerization, or
active transport might increase the accessibility of aggregates
to specific cellular regions.
A unifying aspect of aggregate dynamics is that aggregate
movement and tethering to sub-cellular regions both
contribute to physical encounters between aggregates that
may result in their fusion, and consequently in a decrease in
the total aggregate number (Fig. 3). The formation of unitary
aggregate compartments, rather than the specific type of
Figure 3. Aggregate segregation in different cell types depends on
fusion and tethering. A: In E. coli and S. pombe, aggregates (green),
which are not tethered but move by diffusion in the cytoplasm, fuse
with one another. Aggregate localization in the cytoplasm before cell
division (dashed line) determines in which daughter cell they will end
up. Asymmetric segregation is achieved by aggregate fusion into a
single unit, which is inherited by one daughter cell while the other
one is born clean of aggregates (see panel C). B: In S. cerevisiae
and in mammalian cells, where aggregates are tethered to organelles (purple) and move by diffusion on the organelle surface, by
organelle dynamics and by active transport (e.g. along actin cables,
pink) as well as by diffusion in the cytoplasm, each of these types of
movement may lead to aggregate fusion. Fusion, in addition to
tethering and active transport, promotes the retention of aggregates
in the mother cell. C: In general, aggregate fusion into a single unit
leads to asymmetric segregation at division, where one cell inherits
the aggregate while the other one is born clean.
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compartment itself, might be a defining feature of aggregate
segregation that is common across species.
Segregation of aggregates depends on
their fusion and tethering to cell
components
While different types of aggregate movements occur in different
cellular regions, cell-cycle phases or under different growth
conditions, these movements generally contribute to aggregate
fusion events that lower the total number of aggregates, up to a
single unit (Fig. 3A and B). The most robust way to ensure
completely asymmetric segregation of a cell component,
independently of the cell type or morphological symmetry of
division, is to have an indivisible unit that will be inherited by
only one of the two daughter cells (Fig. 3C). This seems to be the
prevalent situation for natural aggregates in E. coli [14, 17],
S. cerevisiae [1, 10], S. pombe [12, 13], and higher eukaryotes [2, 3,
8, 16, 18, 29].
In S. cerevisiae and higher eukaryotes, the fusion of
natural aggregates into a unitary component is promoted by
active transport, organelle tethering, or confinement (Fig. 3B).
In S. cerevisiae, based on the results of Zhou et al. [19], both
the ER and the mitochondria could provide a scaffold for
aggregate fusion and facilitate asymmetric segregation. A
major difference in the segregation between natural and
stress-induced aggregates in S. cerevisiae is that stressinduced aggregates are less likely to be retained in the mother,
probably due to their higher number, smaller size, and
unconstrained movement. However, Zhou et al. [19] showed
that mitochondrial tethering promotes mother retention
of both natural and stress-induced aggregates, a process
dependent on the mitochondrial fission complex (fis1-mdv1dnm1). When the mitochondria enter the bud, these
aggregates, similarly to what happens for nucleoids containing the mitochondrial genome, do not enter the bud neck. The
authors postulate that this is due to the rapid expansion of the
mitochondrial protrusions towards the bud tip, but mitochondrial quality control mechanisms might play a role.
During aging, aggregates lose their tethers to mitochondria, which leads to a more frequent entry of aggregates into
the bud and less asymmetric segregation. In addition, there
is a decrease in mitochondrial fusion during aging [30].
This could increase the number of mitochondrial fragments
that carry aggregates, contributing to the loss of asymmetry
in segregation. Moreover, a filtering process where oxidized
mitochondria are retained in the mother while the “young”
reduced mitochondria enter the bud could also influence
protein aggregate segregation [31], if aggregates preferentially localize to old mitochondria versus young. Since
mitochondria are also transported along actin cables, both
anterograde and retrograde, it is hard to distinguish whether
aggregates move on actin, vesicles associated with actin, or
indirectly bound through mitochondria, or by a combination
of these processes.
In natural aggregates of S. pombe and E. coli and stressinduced aggregates in general, the formation of a unitary
component occurs via diffusive movement (Fig. 3A) and
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M. Coelho and I. M. Tolic
Prospects & Overviews
Glue me, move me, and dump me:
Molecules for the manipulation of
aggregate dynamics in vivo
What are the molecules involved in protein aggregate (i)
formation, (ii) movement, and (iii) sorting to aggregateassociated compartments and tethering to organelles? The
answers largely define their segregation at cell division:
1. Formation of both natural and stress-induced aggregates
is promoted by Hsp42 in S. cerevisiae [9] and Hsp16 in
S. pombe [13], a small heat shock protein described to coaggregate with protein aggregates. Disruption of Ydj1 and
Hlj1, two ER-associated proteins, interferes with the
formation of stress-induced aggregates [15]. Deletion of
single Hsp40 [13] or Hsp70 chaperones (ssa1-4), with
the exception of a quadruple functional knock-out of
Hsp70 [15], does not seem to lower the number or size of
protein aggregates formed.
2. Movement of aggregates in S. cerevisiae depends on genetic
interactors of the Sir2 transcription factor: the CCT
chaperonin in actin folding, the formin Bnj1, the motor
protein Myo2, and the calmodulin protein Cmd1, all
required for the correct function of both actin and
microtubule cytoskeletons [4, 32]. In S. pombe, the
cytoskeleton does not seem to play a prevalent role in
aggregate motility [13]. The lack of the actin-biding and
aggregate sorting N-terminal domain in the Hsp16 of
S. pombe when compared to the S. cerevisiae homolog
Hsp42 [9] might underlie the differential association of
protein aggregates with the cytoskeleton in these two
model organisms.
3. Sorting of aggregated proteins to cytoplasmic compartments depends on two Hook-family proteins (Btn2 which
interacts with Hsp42 and sis1, and Cur1 which binds
sis1 [7]) while tethering to the ER might depend on the
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sec-family of proteins, kar2, and cdc48 [32]. Recently it has
been shown that fusion between aggregates decreases in
the absence of Hsp42/Hsp16 [9, 13] and in the farnesylation mutant of Ydj1 [15], indicating that association with
the ER facilitates fusion. Zhou et al. [19] showed that
tethering to the mitochondria depends not only on fis1,
a protein also involved in mitochondrial fission [19, 33],
but also on mdv1 and dnm1 which form a mitochondrial
fission complex. This presents a paradox since a decrease
in mitochondrial fusion [30], but not fission, is correlated
with the loss of mitochondrial aggregate tethering during
aging [19].
How are aggregates anchored to mitochondria? Besides
fis1, which decorates the outer mitochondrial membrane,
membrane potential, and surface charge interactions could
play a role, but a more specific hypothesis would be that
tethering results from incomplete protein import. Importers
in the mitochondrial surface could grab polypeptides
protruding from the aggregates, and unable to unfold
them, hold them for a brief period of time, which may be
sufficient to reduce diffusion at the surface. It is also
possible that functional domains in the outer mitochondrial
membrane capture aggregates to specific mitochondrial
regions, which could spatially correlate with “damaged”
mitochondria.
Furthermore, what is the difference between the aggregates bound to mitochondria and ER? A key step towards
understanding whether there are different functional classes
of aggregates is direct biochemical analysis of purified protein
aggregates, which might be facilitated by crosslinking the
molecules that tether aggregates to surfaces. Since translation
sites are likely to be protein aggregation sites, recent work
from the Weissman lab where the RNA binding profile of
ribosomes in the ER [34] and mitochondria [35] was studied
might be a useful approach to determine which aggregates
form in different cellular locations. The nature of the proteins
in these aggregates probably represents localized specific
mRNAs being translated. Also, mass-spectrometry analysis of
subcellular fractions of aggregates will identify not only other
proteins, but possibly also RNA or lipid molecules that may be
important for aggregate formation or tethering. It would also
be interesting to understand which fraction of the proteome is
aggregation-prone, and where it localizes inside the cell [36].
Such experiments will shed more light on the molecular
mechanisms that establish the asymmetry in segregation
described by Zhou et al. [19].
Conclusion and outlook
Of the many possible strategies to segregate aggregates at cell
division, organellar tethering, and fusion of aggregates with
one another seem to be most conserved across a wide range of
organisms. Tethering of aggregates to organelles facilitates
their retention in the mother cell, and may help their fusion by
keeping them close to each other. Aggregate fusion into a
single unit ensures asymmetric segregation to one of the two
offspring cells, whereas the other one is born aggregate-free
and ready to start a new life.
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factors that promote fusion after aggregates come into contact
with each other (Hsp16 and potentially IbpA or another
molecular chaperone). With different means to achieve the
same end, asymmetrically dividing cells then use differential
organelle partitioning or the geometry of division to determine
the polarity of aggregate segregation, while symmetrically
dividing cells randomly segregate a unitary aggregate to one
of the cells, which contributes to a functional difference
between the daughter cells. Therefore, S. cerevisiae might be a
more suited model to understand segregation of protein
aggregates during asymmetric cell division, such as differentiating stem cells or cells with an asymmetric morphology of
division (e.g. spatially constrained in tissues), while S. pombe
and E. coli might be relevant models for segregation during
general symmetric cell division (symmetrically dividing stem
cells, cancer cells). A better understanding of the biochemical
composition of different aggregates and the molecular players
that fine-tune asymmetric aggregate segregation are needed to
use “molecular handles” to test hypotheses on the causative
versus correlative effect of aggregate segregation during cell
death and aging.
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M. Coelho and I. M. Tolic
Aggregates are found in a wide variety of intracellular
structures. What is the function of these different structures
and how are they involved in aggregate degradation, storage,
and segregation? Following single aggregate formation and
growth during the cell cycle, with in vivo photo-activatable or
photo-switchable fluorescent tags to pulse-chase aggregates
would allow for tracking their dynamic life inside the cell.
Mass-spectrometry characterization of sub-populations of
aggregates coupled with EM-tomography will lead to a better
understanding of the individual characteristics of aggregates.
These techniques, combined with genetic perturbations to
increase or decrease the rate of aggregate formation, movement or fusion, across different cell types will allow to better
understand the impact of protein aggregation on cell
physiology, shedding light on how aggregate segregation
strategies evolved.
Although asymmetric segregation of aggregates has been
correlated with aging, it is unclear whether aggregates
accumulate as a by-product of aging or if their accumulation
causes aging. Work from the Gottschling lab in S. cerevisiae
has been pivotal in establishing a temporal order of organelle
dysfunction during replicative aging: a proton imbalance
driven by competition between membrane [37] and vacuolar [30] transporters leads to a pH increase in the vacuole,
which causes the loss of mitochondrial fusion and membrane
potential, while protein aggregate accumulation seems to
occur only later. Functional experiments where aggregate
number and size are increased or decreased either by
controlling the rate of aggregate degradation (via stimulation
of translation, proteasome, or increase in the concentration of
chaperones, or laser ablation) or by changing the symmetry of
segregation would indicate how the aggregate burden affects
the final steps of aging.
Acknowledgments
The authors’ research was supported by the Max Planck
Society. While in the Toli!c lab, M.C. received a fellowship
(SFRH/BD/37056/2007) from the Portuguese Foundation for
Science and Technology (FCT). M.C. is currently a postdoctoral fellow of the Human Frontier Science Program
(LT000694/2014-L). The authors thank Gregg Wildenberg for
" !c for the illustrations.
insightful discussions and Ivana Sari
The authors declare no conflict of interest.
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