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Cell Motility and the Cytoskeleton 37:199–210 (1997)
Mitochondrial Inheritance:
Cell Cycle and Actin Cable Dependence
of Polarized Mitochondrial Movements
in Saccharomyces cerevisiae
V.R. Simon, S.L. Karmon, and L.A. Pon*
Department of Anatomy and Cell Biology, Columbia University,
College of Physicians and Surgeons, New York, New York
Asymmetric growth and division of budding yeast requires the vectorial transport
of growth components and organelles from mother to daughter cells. Time lapse
video microscopy and vital staining were used to study motility events which result
in partitioning of mitochondria in dividing yeast. We identified four different stages
in the mitochondrial inheritance cycle: (1) mitochondria align along the motherbud axis prior to bud emergence in G1 phase, following polarization of the actin
cytoskeleton; (2) during S phase, mitochondria undergo linear, continuous and
polarized transfer from mother to bud; (3) during S and G2 phases, inherited
mitochondria accumulate in the bud tip. This event occurs concomitant with
accumulation of actin patches in this region; and (4) finally, during M phase prior
to cytokinesis, mitochondria are released from the bud tip and redistribute
throughout the bud. Previous studies showed that yeast mitochondria colocalize
with actin cables and that isolated mitochondria contain actin binding and motor
activities on their surface. We find that selective destabilization of actin cables in a
strain lacking the tropomyosin 1 gene (TPM1) has no significant effect on the
velocity of mitochondrial motor activity in vivo or in vitro. However, tpm1D
mutants display abnormal mitochondrial distribution and morphology; loss of long
distance, directional mitochondrial movement; and delayed transfer of mitochondria
from the mother cell to the bud. Thus, cell cycle-linked mitochondrial motility patterns
which lead to inheritance are strictly dependent on organized and properly oriented actin
cables. Cell Motil. Cytoskeleton 37:199–210, 1997. r 1997 Wiley-Liss, Inc.
Key words: tropomyosin; organelle motility; yeast; time lapse fluorescence microscopy
INTRODUCTION
Cellular organelles are produced by growth and
division of preexisting organelles. Therefore, organelle
inheritance, or transfer of organelles from mother to
daughter cells, is necessary for proliferation [for reviews
see Nunnari and Walter, 1996; Warren and Wickner,
1996]. Here, we focused on the mechanisms underlying
mitochondrial movement and inheritance during asymmetric cell division in the budding yeast Saccharomyces
cerevisiae. Previous studies indicate that mitochondria
are one of the first organelles to be transferred into the
bud [Stevens, 1977], and that mutations that prevent this
transfer result in cell death [McConnell et al., 1990].
r 1997 Wiley-Liss, Inc.
Abbreviations: ACT1, gene encoding yeast actin; TPM1, gene encoding tropomyosin 1 protein; Tpm1p, tropomyosin 1 protein; tpm1D,
deletion of the TPM1 gene; YPD, rich media for yeast growth
consisting of yeast extract, peptone and dextrose.
Contract grant sponsor: NRSA; Contract grant number: 5 F31
GM15644; Contract grant sponsor: Summer Undergraduate Research
Fellowship from the Department of Biological Sciences at Columbia
University; Contract grant sponsor: National Institutes of Health;
Contract grant number: RO1 GM45735.
*Correspondence to: Liza A. Pon, Department of Anatomy and Cell
Biology, Columbia University P&S 12-425, 630 West 168th Street,
New York, NY 10032. E-mail: lap5@columbia.edu
Received 3 October 1996; accepted 6 March 1997.
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Simon et al.
Thus, mitochondrial inheritance is an essential process
which begins early in the cell division cycle.
In vegetative yeast, mitochondria are resolved as
elongated organelles which are organized as individual
tubular structures and as a branching reticulum [Damsky,
1976; Stevens, 1977]. Fusion of mitochondria has been
documented during zygote formation and meiosis in yeast
[Miyakawa et al., 1984; Azpiroz and Butow, 1993; Smith
et al., 1995] and may contribute to mitochondrial plasticity in vegetative cells. Short-term, time lapse microscopy
in vegetative yeast revealed that tubular mitochondrial
structures align along the mother-bud axis and undergo
polarized movement from mother cells into developing
buds during cell division. This inheritance-associated
movement is linear and long-distance, and displays an
average velocity of 50 nm/sec [Simon et al., 1995]. Upon
transfer into the bud, newly inherited mitochondria are
observed to move towards the bud tip, where they
become immobilized [Simon et al., 1995]. This immobilization event prevents return of mitochondria into the
mother cell, effectively increasing the efficiency of mitochondrial inheritance.
Mitochondrial movement in vegetative yeast is
dependent upon the actin cytoskeleton. The actin cytoskeleton of yeast consists of two structures detected by light
and electron microscopy: actin cables and actin patches.
Actin cables are bundles of actin filaments that extend
along the mother-bud axis, and actin patches are believed
to be invaginations in the plasma membrane invested
with F-actin. Actin mutations which depolarize and
destabilize the actin cables and patches result in mitochondrial aggregation, complete inhibition of mitochondrial
movement, and impaired mitochondrial inheritance
[Drubin et al., 1993; Lazzarino et al., 1994; Simon et al.,
1995]. In addition, light and electron microscopy studies
indicate that mitochondria colocalize with actin cables
[Drubin et al., 1993; Lazzarino et al., 1994; Mulholland et
al., 1994]. Moreover, cell free assays indicate that isolated yeast mitochondria bind to the lateral surface of
actin filaments. This actin binding activity is ATPsensitive, reversible, saturable and mediated by a protein
or proteins on the mitochondrial surface [Lazzarino et al.,
1994]. Finally, an ATP-driven, actin-dependent motor
activity has been identified on yeast mitochondrial outer
membranes [Simon et al., 1995]. These observations
support a model whereby mitochondrial movement from
mother cells to buds is mediated by mitochondrial motor
driven movement using actin cables as tracks.
To test this hypothesis, we examine: (1) the temporal relations between actin and mitochondrial organization during yeast cell division, and (2) the effect of actin
cable destabilization on mitochondrial movement and
inheritance. Reorganization of the yeast actin cytoskeleton is required for the establishment of cell polarity and
vectorial transport of cellular components from mother to
daughter cells, prior to and during cell division [Lew and
Reid, 1993]. We find that cell cycle-dependent changes in
actin cytoskeletal organization correlate with inheritanceassociated mitochondrial motility events.
The role of actin cables in mitochondrial movement
and inheritance was evaluated using a strain bearing a
deletion in the tropomyosin 1 gene (TPM1) [Liu and
Bretscher, 1989, 1992; Drees et al., 1995]. Deletion of
this gene results in selective destabilization of actin
cables, without significantly affecting the abundance or
polarization of actin patches. Phenotypes associated with
this loss of actin cables are accumulation of late secretory
vesicles, defects in polarized secretion, abnormal chitin
deposition, altered cell size and shape, low growth rates,
and defects in shmoo formation and cell fusion. TPM1
encodes the only yeast actin binding protein found
exclusively in actin cables. This, together with the finding
that overexpression of Tpm1p partially restores actin
cables in a strain carrying a mutant allele of the actin gene
(act1-2), indicate that Tpm1p interacts directly with
F-actin and is required to stabilize yeast actin cables [Liu
and Bretscher, 1989]. These findings support a role for
actin cables in polarized secretion, cell growth and
mating. Our studies indicate that actin cables are also
required for cell cycle linked mitochondrial movements
leading to inheritance.
MATERIALS AND METHODS
Yeast Cell Manipulation
The following strains were used for this study:
MSY106 (MATa/a, his4-619/his4-619), CUY58
(MATa/a, ade2/ADE2, his3D200/his3D200, leu2-3, 112/
leu2-3, 112, lys2-801/LYS2, trp1-1(am)/TRP1, ura3-52/
ura3-52) and ABY320 (MATa, ade2, his3-D200, leu2-3,
112, ura3-52, tpm1D::LEU2). Yeast cell manipulations
were carried out according to Sherman [1991].
Cell Growth and Synchronization
Yeast were grown to midlog phase in lactate-based
liquid media at 30°C [Glick and Pon, 1995]. Cells were
collected by centrifugation, resuspended in lactate media
and applied to a 5–15% sorbitol gradient. Cells were then
separated on the basis of density by centrifugation at
121 3 g for 4 min [Mitchison, 1988]. The top gradient
fraction, which contained the largest number of unbudded
and small budded cells, was transferred to glucose-based
liquid media (YPD) such that the final concentration of
cells was 1 3 107 cells/ml. Aliquots of this synchronized
culture were removed at different time intervals and used
for fixation or time-lapse video microscopy.
Actin Cable Dependent Mitochondrial Movement
Fixation and Immunofluorescence
The fixation and immunofluorescence methods used
are variations of the methods described in Pringle et al.
[1989]. In brief, synchronized cells were fixed at 10-min
intervals over 2 hr. To fix samples, paraformaldehyde and
potassium phosphate buffer (pH 6.5) were added directly
to the growth medium to final concentrations of 3.7% and
100 mM, respectively. This mixture was incubated for 15
min at room temperature (RT) and cells were collected by
centrifugation. The resulting pellet was resuspended in a
second fixative solution (100 mM potassium phosphate
pH 6.5, 2 mM MgCl2 and 5% paraformaldehyde) and
incubated for 2 hr at RT. Fixed cells were then washed
three times with ‘‘wash solution’’ (20 mM potassium
phosphate, pH 7.5, 0.8 M KCl). To remove the yeast cell
wall, fixed cells were incubated in wash solution containing 10 mM DTT for 20 min followed by incubation in
wash solution containing (0.125 mg/ml) Zymolyase
20,000T (Seikagaku, Ijamsville, MD). Fixed spheroplasts
were washed with NS (20 mM Tris-HCl pH 7.6, 0.25 M
sucrose, 1 mM EDTA, 1 mM MgCl2, 0.1 mM ZnCl2, 0.1
mM CaCl2, 0.8 mM phenylmethylsulfonylfluoride, 0.05%
[v/v] 2-mercaptoethanol) and stored at 4°C in NS supplemented with 0.02% Na-azide.
Fixed spheroplasts were applied to poly-lysinecoated coverslips. Immobilized spheroplasts were then
washed in phosphate-buffered saline (PBS) and incubated
in PBT (1 3 PBS, 0.1% [v/v] Triton X-100, 0.02% [v/v]
sodium azide, (albumin) 1% [w/v] bovine serum albumin
[BSA]). Mitochondria were visualized using polyclonal
antiserum raised against total mitochondrial outer membrane proteins [Smith et al., 1995], and FITC-coupled
goat anti-rabbit IgG (Boehringer Mannheim, Indianapolis, IN). The actin cytoskeleton was stained using rhodamine phalloidin (Molecular Probes, Eugene, OR). Stained
spheroplasts were mounted on microscope slides using
mounting solution (1 µg/ml p-phenylenediamine, 90%
[w/v] glycerol, and 13 PBS) containing the DNAbinding dye 48,6-diamidino-2-phenylindole (DAPI) [Williamson and Fennell, 1975] at a final concentration of 1
µg/ml. Coverslips were then sealed onto glass microscope
slides with clear nail polish. Slides were stored at 220°C
in the dark prior to viewing.
Vital Staining
A growth chamber was prepared using a modification of methods described by Koning et al. [1993]. A
1-cm2 well was prepared on a microscope slide using a
double layer of cellophane tape. The well was filled with
YPD media containing 3% high melting point agarose
(Sigma Chemicals, St. Louis, MO), 1 µg/ml of the
membrane potential-sensing dye 3,38-dihexyloxacarbocyanineiodide (DiOC6; Molecular Probes), and oxyrase at a
concentration recommended by the manufacturer (Oxy-
201
rase Inc., Mansfield, OH). A coverslip was placed over
the filled well, and uniform force was applied to the
coverslip to compress the agar into the well. The coverslip and excess agar were removed from the well. An
aliquot of synchronized cells was resuspended in YPD
media to a concentration of 2 3 108 cells/ml. 6 3 105
cells were spread across the surface of the solidified agar
and allowed to sink into the agar. The chamber was then
covered with a coverslip and sealed on all sides with clear
nail polish.
These growth chambers support cell growth at rates
similar to that of cell growth in liquid culture for up to 5
hr. The length of the cell division cycle for wild type cells
under these conditions is approximately 90 min, and was
not significantly affected by addition of oxyrase. Therefore, oxyrase-dependent oxygen depletion has little or no
effect on cell growth during the time of our visualization
(,150 min). Finally, the DiOC6 staining conditions used
result in mitochondria-specific staining without altering
morphology or spatial organization of mitochondria.
Preparation of Actin and Mitochondria From Yeast
Actin was isolated from midlog phase yeast by
DNase1 affinity chromatography and multiple rounds of
polymerization and depolymerization as described previously [Lazzarino et al., 1994]. Highly purified mitochondria were isolated from D273-10B yeast by differential
and isopycnic centrifugation using Nycodenz gradients as
previously described [Lazzarino et al., 1994; Glick and
Pon, 1995].
In Vitro Motility Assay
The in vitro filament sliding assay is a modification
of the Kron and Spudich [1986] assay as described in
Simon et al. [1995]. The experimental flow cell consists
of a nitrocellulose-coated (Fullam Inc., Latham, NY)
coverslip placed on a microscope slide. Two parallel
strips of double-sided tape are used as spacers to create a
35–50 µl flow chamber between the coverslip and the
microscope slide. Mitochondria were suspended to a final
concentration of 1 mg/ml in complete AB buffer (25 mM
imidazole hydrochloride, pH 7.4, 25 mM KCl, 4 mM
MgCl2, 1 mM EGTA, 10 mM DTT) containing a protease
inhibitors cocktail, 1 mM phenylmethyl-sulfonyl fluoride, 10 µM benzamidine, 1 µg/ml 1,10-phenanthroline,
0.5 µg/ml antipain, 0.5 µg/ml chymostatin, 0.5 µg/ml
leupeptin, 0.5 µg/ml pepstatin, and 0.5 µg/ml aprotinin
(Sigma Chemical Co.); and oxygen scavengers, 3 mg/ml
glucose, 9.3 U/ml glucose oxidase (Sigma Chemical Co.),
18 µg/ml catalase (Calbiochem, La Jolla, CA). DiOC6
was added to a final concentration of 100 ng/ml. Mitochondria (100 µg protein) were perfused into the microscope
flow cell. After incubation for 10 min at 4°C, the flow cell
was washed with three volumes of complete AB/BSA
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Simon et al.
buffer (complete AB buffer containing 4 mg/ml BSA) and
nonspecific protein binding sites were blocked by incubation with the AB/BSA buffer for 10 min at 4°C.
Rhodamine-phalloidin-labeled yeast actin filaments
(1.5 µg/ml in complete AB buffer) were perfused into the
flow cell and incubated with immobilized mitochondria
for 10 min at RT. Microfilament sliding and release were
observed after addition of AB buffer containing 10 µM
ATP and an ATP regeneration system (10 µM creatine
phosphate and 250 µg/ml creatine phosphokinase; Sigma
Chemical Co.).
Fluorescence and Video Microscopy
Cells were viewed with a Leitz Dialux microscope
(Rockleigh, NJ). Rhodamine-phalloidin-labeled actin
structures were viewed with excitation and emission
wavelengths of 540–552 nm and 570 nm, respectively.
FITC-stained yeast mitochondria and DiOC6-stained cells
were viewed with excitation and emission wavelengths of
490–495 nm and 525 nm, respectively. DAPI images
were viewed with excitation and emission wavelengths of
340–365 nm and 450–488 nm, respectively. Images of
stained mitochondria in vivo and fluorescent mitochondria and filaments in vitro were collected for 1–2 sec at
different time intervals using a cooled CCD camera
(Star-1, Photometrics, Tucson, AZ). Light output from the
100W mercury arc lamp was controlled using a shutter
driver (Uniblitz D122, Vincent Associates, Rochester,
NY) and attenuated using neutral density filters (Omega
Optical Corporation, Brattleboro, VT). Image enhancement and analysis were performed on a Macintosh
Quadra 800 computer (Cupertino, CA) using the public
domain program NIH Image 1.55. Images were stored on
a magnetic optical disk drive (Peripheral Land Inc.,
Fremont, CA).
Velocity Measurements
For analysis of mitochondrial motility as a function
of progression through the cell division cycle, time lapse
series were obtained at 2.5- to 5-min intervals for periods
up to 120 min. These long-term imaging studies reveal
the patterns of mitochondrial motility throughout the
entire cell cycle. However, since organelles move into
and out of the plane of focus during 2.5- to 5-min
intervals, these conditions preclude tracking of an individual organelle in consecutive still frames. Therefore,
velocities of mitochondrial movement were studied in
time lapse series obtained at 20-sec intervals over 10 min
of real time. For these movements, velocities were
determined by measuring the change in position of the
end of tubular mitochondrial structures as a function of
time in a time lapse series, and motile mitochondrial ends
were defined as structures that stayed within one plane of
focus and displayed linear, detectable movement for at
least 60 sec of real time. The limit of resolution of our
system is 1 pixel/20 sec or approximately 8.0 nm/sec.
Mitochondrial movements which resulted in displacement less than 1 pixel, or which persisted for less than
three consecutive still frames, were assigned a velocity of
0 nm/sec.
The velocities of filament sliding on immobilized
mitochondria were determined by measuring the change
in position of the tip of each moving actin filament as a
function of time in time lapse series. Time lapse series for
these velocity measurements were obtained at 20-sec
intervals for 5–10 min of real time. Sliding actin filaments
were defined as filaments which: (1) colocalized with
immobilized mitochondria, (2) moved parallel to their
long axis, and (3) displayed detectable, linear motion for
60 sec of real time (three consecutive still frames).
For all velocity measurement, NIH Image V1.55
was used to determine the position (x-y coordinates) of
moving mitochondria or actin filaments. The linear
distances between successive positions of mitochondria
or microfilaments were calculated using Microsoft Excel,
and instantaneous velocities were averaged to give the
mean velocity.
RESULTS
Dynamics of Mitochondrial Inheritance
Time lapse imaging of mitochondrial inheritance in
dividing yeast revealed polarized, linear movement of
mitochondria from mother cells to developing buds
[Simon et al., 1995]. However, the methods used during
those early efforts precluded long-term imaging of living
cells. As a result, fundamental issues pertaining to the
timing and duration of mitochondrial inheritance events
were not examined. To address these issues, we developed a growth chamber and vital staining conditions
which support normal kinetics of yeast cell division, and
allow visualization of inheritance-associated mitochondrial movements for intervals equal to or longer than one
full cell division cycle (see Materials and Methods).
Analysis of mitochondrial motility patterns as a
function of progression through the cell division cycle
reveals a sequence of motility events leading to partitioning of mitochondria in mother and daughter cells. Mitochondrial tubules are oriented along the mother-bud axis
throughout cell division. This organization persists in
mother cells after completion of cell division and cell
separation (Fig. 1A). Reorganization of these organelles
Actin Cable Dependent Mitochondrial Movement
203
(Fig. 1F– H). This transfer of mitochondria is produced
by directed, linear movement of mitochondria from the
mother cell to the bud (Table I).
Newly inherited mitochondria move to the bud tip
and accumulate in this region (Fig. 1G, arrowhead). This
accumulation is first detected 30–35 min after bud
emergence (Fig. 2D), and can persist up to 55–65 min
after bud emergence. When the bud is approximately
70% of the size of the mother cell, all inherited mitochondria are immobilized at the bud tip (Fig. 2E, arrow). At
this stage, no further transfer of mitochondria from the
mother cell to the bud is observed. Mitochondria are then
released from the bud tip (Fig. 2F–H). Upon release,
mitochondria redistribute throughout the bud but do not
reenter the mother cell. Cell septation occurs within 20
min of mitochondrial release from the bud tip, 90–100
min after bud emergence.
Mitochondrial Organization and Movements
During Inheritance Correlate With Cell
Cycle-Regulated Actin Rearrangements
Fig. 1. A–H: Mitochondrial movements leading to inheritance in wild
type cells. Synchronized wild type cells (MSY106) were immobilized
on a microscope growth chamber, which contained the membrane
potential sensing dye DiOC6. Time lapse images of mitochondria in
living cells were obtained at 2.5-min intervals over a period of 102 min.
The times shown in each still frame correspond to minutes of real time,
where t 5 0 min was defined as the time of bud emergence.
Mitochondria are resolved as tubular structures (C, open arrow) which
reorganize to converge at the site of bud emergence (A, arrow), enter
the bud, and accumulate in the bud tip (G, arrowhead). Bar 5 1 µm.
occurs upon initiation of the next round of cell division,
10–15 min prior to bud emergence (Fig. 1B). During
reorganization, mitochondria converge at a new site on
the cell surface, the site of bud emergence (Fig. 1C,
arrow). A single mitochondrial tubule enters the bud
within 2–5 min after bud emergence (Fig. 1D). Mitochondria continue to move linearly into the bud as it grows
In yeast, the actin cytoskeleton undergoes a defined
sequence of cell cycle-regulated rearrangements [Adams
and Pringle, 1984; Kilmartin and Adams, 1984]. A ring of
actin patches forms at the presumptive bud site (Fig. 3A).
Shortly thereafter, actin cables align along the long axis
of the mother cell. After bud emergence, actin patches
remain within the bud, and actin cables persist along the
mother bud axis (Fig. 3D). During early stages of bud
enlargement, growth is directed towards the apical region
of the bud. At this stage, actin patches concentrate in the
bud tip (Fig. 3G). Thereafter, bud growth is isotropic and
directed to the entire surface of the bud. This apical to
isotropic switch is associated with redistribution of actin
patches from the bud tip to the entire surface of the bud
(Fig. 3J). Finally, redistribution of cortical actin structures
to the neck region occurs prior to cytokinesis (Fig. 3M)
[Lew and Reed, 1993].
We evaluated the temporal relation between mitochondrial inheritance events and cell cycle-regulated
actin rearrangements. To do so, we fixed synchronized
yeast cultures at various stages of cell division, and
determined the organization of mitochondria, actin and
nuclei. We find that accumulation of a ring of actin
patches at the presumptive bud site precedes convergence
of mitochondria at that site (Fig. 3A–C). Transfer of
mitochondria from mother cell to bud begins shortly after
bud emergence. At this stage, actin patches are localized
exclusively in the bud, and actin cables are aligned along
the mother-bud axis where they colocalize with mitochondria (Fig. 3D–F). Immobilization of mitochondria in the
204
Simon et al.
TABLE I. The Effect of TPM1 Deletion on Mitochondrial Movement and Inheritance*
Strain
MSY106 (WT)
CUY58 (TPM1)
ABY320 (tpm1D )
Motile
mitochondria (%)
Velocity
(nm/sec)
Polarized
Bud tip
Inheritance in
movement (%) accumulation (%) small buds (%)
55
54
26
49 6 21 (n 5 200)
54 6 17 (n 5 65)
36 6 12 (n 5 36)
59
55
34
65
56
23
95
90
29
*Mitochondrial movement in wild type cells (CUY58) and in cells bearing a deletion in the TPM1 gene (ABY320)
was visualized as for Figure 1. The percentage of motile mitochondria represents the mitochondria that showed a
net displacement during the time recorded. Polarized movement refers to the percentage of motile mitochondria
that showed a net displacement towards the bud. Measurements on the extent, velocity and degree of polarity of
mitochondrial movement were determined using time lapse images recorded at 20-sec intervals. Bud tip
accumulation was quantified as the percentage of medium-sized and large buds that contained mitochondria at the
bud tip. The efficiency of mitochondrial inheritance was determining by measuring the percentage of small buds
(1–1.5 µm) which contain mitochondria. n, no. of mitochondria studied.
Fig. 2. A–H: Mitochondrial accumulation at the bud tip contributes to
efficient mitochondrial inheritance. Mitochondrial movements in synchronized cells were visualized as described in Figure 1. Time lapse
images were obtained at 2.5-min intervals for a total of 95 min. The
times shown in each still frame correspond to minutes of real time,
where t 5 0 min was defined as the time of bud emergence.
Mitochondria are observed to move into the bud (C) and accumulate at
the bud tip (D and E, arrows). Release of mitochondria from the bud tip
is illustrated in F and G. Subsequent to release, mitochondria repopulate the bud, but do not reenter the mother cell. Bar 5 1 µm.
bud tip occurs concomitant with actin patch accumulation
in this region (Fig. 3G–I). Release of immobilized
mitochondria from the bud tip occurs subsequent to
redistribution of actin patches within the bud, and prior to
nuclear inheritance (Fig. 3J–L). Finally, depolarization of
actin cables and accumulation of actin patches at the site
of cell-cell separation occur concomitant with redistribution of mitochondria throughout the bud (Fig. 3M–O).
Actin Cable Dependent Mitochondrial Movement
Fig. 3. Mitochondrial inheritance events during cell division cycle.
Synchronized wild type cells (MSY106) were fixed at different time
points (see Materials and Methods). In this triple label experiment,
actin structures were visualized using rhodamine phalloidin
(A, D, G, J, M); mitochondria were visualized using an antibody
205
specific to the mitochondrial outer membrane and an FITC-coupled
secondary antibody (B, E, H, K, N), and mitochondrial and nuclear
DNA were stained using the DNA binding dye, DAPI (C, F, I, L, O).
Arrows point to examples of mitochondrial colocalization with actin
cables. m, mitochondrion, n, nuclei. Bar 5 1µm.
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Simon et al.
Selective Destabilization of Actin Cables Results
in Defects in the Mitochondrial Inheritance Cycle
To determine the relative contributions of actin
cables and actin patches in inheritance-associated mitochondrial movements, we studied mitochondrial motility
and morphology in a TPM1 deletion mutant (tpm1D).
Actin cables are not detected in tpm1D mutants at any
stage of cell division. Nonetheless, the abundance and
reorganization of actin patches in the tpm1D mutant are
similar to those observed in wild type cells. Actin patches
accumulate at the presumptive bud site, within the bud
and at the tips of growing buds in the tpm1D mutant (Figs.
4A,D and G). Actin patch redistribution in large buds
(Fig. 4J) and accumulation at the site of cell-cell separation are also evident in the tpm1D mutant (Fig. 4M).
Thus, cell cycle-dependent polarization of actin patches
is not defective in these cells.
Defects in mitochondrial morphology, inheritance
and motility are also observed in tpm1D cells (Fig. 4,
Table I). Mitochondria in tpm1D mutants are resolved as
abnormal short tubular structures or aggregates in these
cells (Fig. 4). Deletion of the TPM1 gene also results in
loss of alignment of mitochondria along the mother-bud
axis. Mitochondria are detected in the bud in tpm1D
mutants; however, we observe a significant delay in transfer
of mitochondria from mother cells to buds. Approximately
90% of small buds in a wild type strain contain mitochondria.
In contrast, only 29% of small buds in the tpm1D culture
contain mitochondria (Table I). Thus, deletion of TPM1
results in a 3-fold decrease in the efficiency of mitochondrial
inheritance during early stages of the cell division cycle.
Finally, mitochondria within the bud are aggregated and show
no significant accumulation in the bud tip.
The velocity of mitochondrial movement in the
TPM1 mutant is similar to that in wild type cells (Table I).
However, time lapse image analysis of the tpm1D strain
reveals fundamental differences in the level of mitochondrial motility and in the pattern of mitochondrial movement. In these studies, motile mitochondria were defined as
particles that undergo positional displacement over several
minutes of real time. We observe a 2-fold decrease in the
percentage of motile mitochondria in the tpm1D mutant
compared to isogenic wild type cells (Table I). Moreover,
mitochondria in tpm1D cells undergo frequent changes in
direction (Fig. 5). This is distinct from the prolonged, linear
excursions of mitochondria in wild type cells, which average
2.5 µm within an interval of 1.4 min. As a result, deletion of
TPM1 causes a 2-fold decrease in the level of bud-directed
mitochondrial movement (Table I).
Mitochondrial Motor Activity In Vitro
is not Affected by Deletion of TPM1
To investigate whether the mitochondrial motor
activity was affected by deletion of TPM1, we compared
microfilament sliding activity on mitochondria isolated
from tpm1D and wild type cells (see Materials and
Methods). Mitochondria isolated from tpm1D mutants
maintain a membrane potential, as determined by staining
with the membrane potential sensing dye, DiOC6. In
addition, mitochondria from tpm1D cells bind rhodaminephalloidin-labeled yeast actin filaments in the absence of
ATP (data not shown). Finally, mitochondria isolated
from tpm1D cells support ATP-dependent, microfilament
sliding with velocities similar to those supported by
mitochondria from wild type cells (Table II). These
experiments suggest that the defects in mitochondrial
organization, movement and inheritance observed in the
tpm1D mutant are not due to the loss of mitochondrial
motor activity.
DISCUSSION
Cell division in yeast occurs by asymmetric growth
of the developing daughter cell or bud. First, a bud site is
selected. Thereafter, reorganization of the yeast cytoskeleton results in establishment of cell polarity and vectorial
transport of organelles and other cellular components to
the bud. Recent studies document a role for Cdc28 and
cyclins in reorganization of the actin cytoskeleton in
dividing yeast, and further define the phase during cell
division in which each actin rearrangement occurs [Lew
and Reed, 1993, 1995]. Polarization of actin patches to
the site of bud emergence and alignment of actin cables
along the mother-bud axis during G1 phase are triggered
by G1 cyclin (Cln1/2)-mediated activation of Cdc28.
Subsequent depolarization of the actin patches and cables
occurs at the interface of G2 and M phases. This event is
dependent upon activation of Cdc28 by mitotic cyclins
(Clb1/2). Finally, redistribution of cortical actin structures to the site of cell-cell separation occurs at the end of
M phase in response to destruction of mitotic cyclins and
Cdc28 inactivation.
Using long-term time lapse video microscopy to
visualize mitochondrial movements through the entire
cell cycle, we define the ‘‘mitochondrial inheritance
cycle,’’ a sequence of mitochondrial motility events
which partition mitochondria between mother and daughter cells during yeast cell division. These inheritanceassociated mitochondrial motility events correlate temporally with cell cycle-regulated actin reorganization. The
hallmarks of the mitochondrial inheritance cycle are: (1)
convergence of mitochondria at the site of bud emergence
following polarization of the actin cytoskeleton in G1
phase, (2) linear, directed movement of mitochondria
from mother cells into developing buds in S phase, (3)
accumulation of newly inherited mitochondria and actin
patches at the bud tip during S and G2 phases, and (4)
release of immobilized mitochondria from the bud tip
Actin Cable Dependent Mitochondrial Movement
Fig. 4. Mitochondrial inheritance in cells lacking actin cables. Cells
bearing a deletion in the tropomyosin 1 gene (ABY320) were
synchronized, fixed at different time points and stained as for Figure 3.
Visualization of actin (A, D, G, J, M) reveals normal patterns of actin
patch localization, and absence of actin cables in the tropomyosin 1
deletion mutant. Mitochondria (B, E, H, K, N) and mitochondrial and
207
nuclear DNA (C, F, I, L, O) localization in the same cells are shown.
Small buds devoid of mitochondria are observed (G–I, and J–L)
suggesting a delay in mitochondrial inheritance. Mitochondrial morphology and organization are defective in mother cells and buds. m,
mitochondrion; n, nuclei; and b, bud. Bar 5 1 µm.
208
Simon et al.
Fig. 5. Actin cables are required for linear, long distance polarized
mitochondrial movements. Mitochondrial movement in wild type
(MSY106; A) and tpm1D cells (ABY320; B) was visualized as for
Figure 1. Tracings of the movements of individual mitochondria in
DiOC6-stained cells were made by marking the position of the tip of
motile organelles during the time in which they remained in the plane
of focus. The points denote the position of organelles at 20-sec
intervals. Tracings are shown relative to the boundary of the dividing
yeast cell. Bar 5 1 µm.
TABLE II. Mitochondria Isolated From the Tpm1D Mutant
Support Actin Filament Sliding on Their Surface*
Strain
CUY58 (TPM1)
ABY320 (tpm1D )
Velocity of filament sliding
(nm/sec)
36 6 14 (n 5 20)
33 6 16 (n 5 20)
*To evaluate motor activity, mitochondria were isolated from wild type
(CUY58) and tropomyosin mutant (ABY320) strains. The velocity of
ATP-dependent mitochondrial motor activity was determined using the
microfilament sliding assay (see Materials and Methods). n, no. of
filaments measured.
concomitant with redistribution of actin patches throughout the bud during M phase (Fig. 6).
Previous studies on mitochondrial inheritance in
budding yeast support roles for an intermediate filamentlike protein (Mdm1p), a fatty acid desaturase (Mdm2p),
and specific mitochondrial outer membrane proteins
(Mmm1p and Mdm10p) in transfer of mitochondria from
mother cells to buds [McConnell and Yaffe, 1992;
Stewart and Yaffe, 1991; Sogo and Yaffe, 1994; Burgess
et al., 1994]. Our findings indicate that the mitochondrial
inheritance cycle consists of at least four cell cycle-linked
mitochondrial motility events. Therefore, it is possible
that MDM1, MDM2, MDM10, and MMM1 may exert
direct or indirect effects on one or more of these
mitochondrial motility events.
Findings in various laboratories also implicate the
actin cytoskeleton in mitochondrial movement and inheritance [Drubin et al., 1993; Lazzarino et al., 1994; Simon
Fig. 6. The mitochondrial inheritance cycle. See text.
et al., 1995; Smith et al., 1995]. Indeed, we observe
colocalization of tubular mitochondria with actin cables
during all stages in the mitochondrial inheritance cycle in
which polarized mitochondrial movement occurs. Therefore, we used a tropomyosin 1 deletion mutation to
examine the effect of selective destabilization of actin
cables on inheritance-associated mitochondrial movement. Deletion of the TPM1 gene causes loss of actin
cables without affecting the abundance or cell cycledependent reorganization of actin patches. We find that
this mutation also produces severe defects in mitochondrial morphology, organization, motility patterns and
delayed inheritance. Mitochondria in the tpm1D mutant
are resolved as abnormal short tubular structures or
aggregates which do not align along the mother-bud axis,
and do not display linear, directional movements. Retention of mitochondria in the bud tip is also compromised in
the tpm1D mutant. Finally, we observe a delay in
mitochondrial inheritance in tpm1D mutants: deletion of
TPM1 results in a 3-fold decrease in the efficiency of
mitochondrial inheritance during the S phase of the cell
division cycle. Since mitochondria isolated from tpm1D
mutants display F-actin binding activity and normal
velocities of ATP-dependent microfilament sliding, these
defects are not due to the inability of mitochondria to bind
filamentous actin.
It is possible that the abnormal mitochondrial
phenotypes observed are due to an indirect effect of the
TPM1 deletion. For example, vesicles which are known
to accumulate in tpm1D mutants may block transfer of
mitochondria across the bud neck, or interaction of
mitochondria with actin structures. We favor an alternative interpretation. It is possible that deletion of TPM1
Actin Cable Dependent Mitochondrial Movement
destabilizes actin cables and disrupts the organization of
filamentous actin structures. Thus, mitochondria move
along the disorganized arrays of actin filaments in a
nondirected manner. Loss of directed mitochondrial movement could, in turn, decrease the efficiency of mitochondrial inheritance. This interpretation is supported by the
observations that deletion of TPM1 alters the pattern of
mitochondrial movement without affecting either the
velocity of mitochondrial movement in vivo, or the
velocity of mitochondria driven microfilament sliding in
vitro.
In summary, our findings indicate that a defined
sequence of mitochondrial reorganization, mobilization
and immobilization events results in high-efficiency,
directed transfer of mitochondria from mother cells to
developing buds, retention of newly inherited mitochondria within bud tips, and redistribution of mitochondria in
the fully formed bud. These events correlate with cell
cycle-regulated changes in organization of the actin
cytoskeleton, and are severely compromised upon deletion of TPM1 and loss of actin cables. The observed
defects are not due to TPM1 deletion-dependent loss of
mitochondrial DNA, or loss of the mitochondrial motor
activity. Rather, it appears that selective destabilization of
actin cables perturbs mitochondrial morphology and the
mitochondrial inheritance cycle. This, together with the
observations that (1) mitochondria colocalize with polarized actin cables, and (2) mitochondria contain an actindependent motor on their surface [Simon et al., 1995],
support the model that mitochondria use polarized actin
cables as tracks for alignment prior to bud emergence,
and subsequently for directed movement from the mother
cell to the developing bud. Future studies will focus on
the role of cell cycle regulators in control of mitochondrial movement and inheritance.
ACKNOWLEDGMENTS
We thank I. Boldogh, M. Smith, T. Swayne and Dr.
A.J. Silverman for support and critical comments on the
manuscript. We also thank Dr. A. Bretscher for yeast
strains.
This work was supported by a pre-doctral NRSA (5
F31 GM15644) to V.R.S., a Summer Undergraduate
Research Fellowship from the department of Biological
Sciences at Columbia University to S.L.K. and a Research Grant (RO1 GM45735) from the National Institutes of Health.
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