Microbiology(1 998), 144, 1783-1 797
Printed in Great Britain
The aberrant positioning of nuclei and the
microtubular cytoskeleton in Saccharomyces
cerevisiae due to improper actin function
Marie Kopeck6 and Miroslav Gabriel
Author for correspondence : Marie Kopecki. Tel :
e-mail : [email protected]
Department of Biology,
Faculty of Medicine,
Masaryk University,
Jortova 10,66243 Brno,
Czech Republic
+420 5 42126 266. Fax : +420 5 42126 200.
An excentric position of the nuclei, random orientation of mitoses, and
multinuclear budding cells were identified in part of a population of
temperature-sensitive (ts) Sacchammyces cerevisiae actin mutants a t the
permissive temperature of 23 "C by fluorescence and electron microscopy. The
phenotype resembled that of mutants in Ptubulin, dynein, JNMI, NUM1, ACT3,
ACTS, myosins, profilin, tropomyosin 1, SLA2 and other genes. The question
was addressed whether the cause was (i)
defects in cell polarity in some ts
actin mutants, manifested by lack of asymmetry of actin cortical patches, or
(ii)lack of cytoplasmic or astral microtubules. The results indicated that in the
cells with the nuclear defects, actin cortical patches showed the normal
asymmetric distribution typical of undisturbed polarity. Cytoplasmic, astral
and spindle microtubules were also preserved. The principal difference found
between the wild-type and actin mutant cells was in actin cables, which in the
actin mutants were developed insufficiently. It is suggested that actin cables
serve as a 'suspensory apparatus' and/or 'intracellular corridor',
predetermining: the location of the nucleus in the central position in
interphase; the axis of nuclear movement to the bud neck before mitosis; the
direction of the elongating nucleus during mitosis; and the motion of each
nucleus from an excentric to a central position during cytokinesis, in
cooperation with the above-mentioned and other gene products.
Keywords: actin, microtubular cytoskeleton, nuclear positioning, cell division cycle,
Saccharomyces cereuisiae
INTRODUCTION
Only very limited information is available about possible relations between actin and the microtubular
cytoskeleton in the cell cycle of the budding yeast
Saccharomyces cereuisiae (Kilmartin & Adams, 1984 ;
Adams & Pringle, 1984; Goldstein & Vale, 1992; Welch
et al., 1994; Schroer, 1994; Langford, 1995; Drubin &
Nelson, 1996; Winsor & Schiebel, 1997; Gavin, 1997).
Palmer et al. (1992) discovered that mis-oriented
spindles and binuclear cell bodies increased in frequency
under two different conditions: (i) if disruption of actin
function was induced in a population of temperaturesensitive (ts) actin mutants with the act2-4 allele by
Abbreviations: aMFs, actin microfilaments; DAPI, 4,6-diamidino-2phenylindole dihydrochloride; MTs, microtubules; aMTs, astral MTs; cMTs,
cytoplasmic MTs; Rhph, rhodamine-phalloidin; SPB,spindle pole body; ts,
temperature sensitive.
0002-2102 Q 1998 SGM
incubation at the restrictive temperature of 37 "Cin cells
synchronized after nuclear migration but prior to
anaphase by hydroxyurea inhibition; (ii) if disruption of
astral microtubules (aMTs) (Sullivan & Huffaker, 1992)
was induced in a cold-sensitive /I-tubulin mutant with
the tub2401 allele by incubation at 18 "C, synchronized
after nuclear migration but prior to anaphase of mitosis
by a 3 h shift to 36 "C due to the cdcl6 ts mutation in
cdcl6 tub2401 double mutants. The authors suggested
that proper spindle orientation and migration is maintained by directly or indirectly tethering the aMTs to the
actin cytoskeleton. aMTs are important for spindle
orientation and mediate force on the spindle, generating
spindle displacement and, in turn, chromosome movement (Palmer et al., 1992).
The relation of actin and MTs is further analysed in the
present paper in the nuclear behaviour of ts actin mutant
DBY 1693 actl-I (Shortle et al., 1984; Novick &
Botstein, 1985; Gabriel & Kopecka, 1995; Kopecka &
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M. K O P E C K A a n d M. GABRIEL
Fig. 7. Three micrographs of an identical group of DBY 1690 ACT1IACTl cells grown in malt extract medium at 23 "C for
24 h and fixed in this medium for 90 min. Bars, 10 pm. (a) Phasetontrast microscopy. (b) MTs, immunostained with the
anti-a-tubulin antibody TAT1 and FITC. (c) Nuclear staining with DAPI. Numbers 1-6 in (a) mark cells in six stages of the
cell cycle according to nuclear and cell morphology: 1, unbudded cell with a single nucleus in the central position; 2,
budding cell with a single nucleus in the central position; 3, budding cell with a single nucleus near the neck of the bud
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Nuclear position and actin cytoskeleton in yeast
Gabriel, 1995). Most papers dealing with ts mutants
consider as permissive a temperature that allows survival and reproduction of the cell population of the
culture. Behaviour of the individual cells in such a
population is not uniform (Novick & Botstein, 1985;
Drubin et al., 1993). Since deviations from uniformity
are usually not the subject of the paper, they are not
usually considered as a matter of great importance. A
permissive temperature of 23 "C for the ts actin mutant
DBY 1693 was established by Novick & Botstein (1985)
and confirmed by us (Gabriel & Kopeckd, 1995;
Kopecka & Gabriel, 1995). However, we noticed some
morphological and, mainly, nuclear aberrations. The
subject of the present paper is the identification of the
causes of these aberrations. (i) A study of the nuclei has
been devoted to the mapping of all types of defects that
appear in ts actin mutants DBY 1693 with the actl-1
allele at the permissive temperature of 23 "C, in contrast
to the wild-type strain DBY 1690 with the standard actin
allele A C T l . (ii) A parallel study of MTs was aimed at
verification of the hypothesis that the cause of the
orientation defects of the nuclear spindles may be a
lack of cytoplasmic-aMTs (as discovered in p-tubulin
mutants by Sullivan & Hufffaker, 1992, and Palmer et
al., 1992) in ts actin mutants due to mutated actin. (iii)A
study of the actin cytoskeleton aimed to answer the
question whether the cause of the nuclear defects may be
improper actin function due to cell polarity defects even
at the permissive temperature, manifested by the lack of
asymmetric location of the actin cortical patches. At the
restrictive temperature 37 "C, this was found by Novick
& Botstein (1985) without demonstration of the involvement of nuclei and MTs, and by us in a study of
nuclei and MTs (Gabriel & Kopecka, 1995). In other ts
actin mutants, with act2-129 to actl-136 alleles, Drubin
et al. (1993) found multinucleate cells at the restrictive
temperature, too, without study of their MTs.
We were the first to show multiple nuclei and MTs in ts
actin mutants even at the permissive temperature of
23 *C (Gabriel & Kopecka, 1992). Multinuclear cells at
the permissive temperature were mentioned by Palmer
et al. (1992) and Drubin et al. (1993), who also
showed multinuclear cells and mis-oriented mitoses at
25 "C (without MTs). Multinuclear actin mutant cells
of the fission yeast Schizosaccharomyces pombe at
the permissive temperature of 28 "C were also
reported (Ishiguro & Kobayashi, 1996). Binucleate cells
originated by inhibition of actin polymerization with
cytochalasin D in Schizosaccharomyces japonicus
(Gabriel et al., 1998).
METHODS
Yeast strain. The Saccharomyces cerevisiae strains used were
from the collection of temperature-sensitive, conditionallethal actin mutants (Novick & Botstein, 1985),which were
kindly provided by Professor D. Botstein and Dr P. Grisafi
from MIT, Cambridge, MA, USA. The strains were as
follows: DBY 1990 (MATaIMATa, ade2-2/
his4-619/ ,
ACTl IACTl), DBY 1693 (MATaIMATa, ura3-52/+ ,his4actl-l/actl-I), DBY 1691 (MATa, his#-619, actl-I),
6191
DBY 1692 (MATa, his#-619, act2-I),DBY 1694 (MATa,his#619, actl-2) and DBY 1695 (MATa, his#-629, act1 -2). Their
construction is described by Shortle et al. (1984).
+,
+
+,
Media and cell cultivation. Cells from stored cultures on 2 o/'
(w/v) malt extract agar pH 5.5 at 4 "C were inoculated into
YEPD medium (Novick & Botstein, 1985) or into malt extract
medium of pH5.5 and cultivated in a shaking waterbath
overnight, either at 23 O C or at other temperatures, as
indicated in Results. After 24 h the cells were fixed for 10 or
90 min directly in culture media using paraformaldehyde at a
final concentration of 5 o/' (v/v) in phosphate buffer pH 6.9
according to HaHek et al. (1986),as reported earlier (Gabriel&
Kopeck& 1995; Kopeckd & Gabriel, 1995).
Actin visualization. Actin was labelled with rhodaminephalloidin R-415 (Rhph) (Molecular Probes) according to
Pringle et al. (1989) and Alfa et al. (1993).Before staining the
cells were permeabilized with 1% (v/v) Triton X-100 in PBS
(Pringle et al., 1989) for 1 min, then washed twice with PBS
and stained as reported earlier (Gabrielet al., 1992; Gabriel &
Kopeckd, 1995; Kopecka & Gabriel, 1995).
MT visualization with monoclonal anti-tubulin antibody TU
01. Monoclonal anti-tubulin antibody TU 01 (Viklickjr et al.,
1982) was applied after 90 min fixation of cells as reported
earlier (Gabriel& Kopeck&1995),followingpermeabilization
of cells with 1% (v/v) Triton X-100 in PBS for 1 min. Then
cells were washed twice with PBS in the test tube by
centrifugation, and application of primary and secondary
antibodies was done as described previously (Gabriel &
Kopecka, 1995),but in test tubes.
MT visualization with monoclonal anti-a-tubulin antibody
TATl. Anti-a-tubulin monoclonal antibody TATl (Woods et
al. 1989) was applied according to Svoboda et al. (1995).After
5 min permeabilization of the cells with 1o/' (v/v) Triton X100 (the cells were fixed for 90 min and their walls partly
digested by snail enyzmes),2% (v/v) BSA in PBS was applied
for 30 min at room temperature, followed by antibody TATl
for 2 h at 37 "C,triple washing with PBS at 5 min intervals,
and treatment with the secondary antibody SWAM-FITC
(Sevac Prague) for 1.5 h at 37 OC. Finally, the preparations
were washed three times with PBS at 5 min intervals. In some
experiments, lysing enyzmes from Trichoderma harzianum
L-20065 (lot 34H0240; Sigma) were used at 1 mg ml-l for
20 min at room temperature in PBS instead of the snail
enyzmes.
(premitotic nuclear migration phase); 4, budding cell with an elongated mitotic nucleus in the neck of the bud (nucleardivision morphology); 5, budding cell with two nuclei at excentric positions (post-mitotic excentric position of nuclei); 6,
budding cell with two nuclei in the central positions (postmitotic migration from excentric to central position).
Fig. 2. Three micrographs of an identical group of DBY 1690 ACTIIACTI cells grown in malt extract medium at 23 "C for
24 h, fixed in this medium for 10 min, and stained with Rhph or DAPI. Bars, 10 pm. (a) Phasetontrast microscopy. (b)
Actin cytoskeleton, labelled with Rhph. The arrow indicates an actin ring (see text). (c) Nuclear staining with DAPI.
Numbers 1-6 refer to the stages of the cell cycle, as explained in the legend of Fig. 1.
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M. KOPECKA and M. GABRIEL
Nuclear staining. This was performed by adding 1 pg 4,6diamidino-2-phenyl-indol dihydrochloride (DAPI) ml-' (Serva
Feinbiochemica) into the mounting medium (Pringle et al.
(1989).
light microscopy. Observations were made using either
fluorescence microscopy (Jenalumar, Zeiss) with Nomarski
differential interference contrast, or fluorescence microscopy
(Laborlux S Leitz) with phase contrast. Photographs were
taken on Ilford 400 film. Exposure time was 30 s for actin, 4 s
for DAPI, 1-2s for phase-contrast and 20s for MTs. The
primary magnifications were 320 x with Jenalumar, and
375 x with Laborlux.
Freeze-etching. Replicas were prepared using living cells.
After cultivation at room temperature, the cells were centrifuged and the pellet was placed on copper grids, frozen in
Freon 22 and stored in liquid nitrogen. Replicas were prepared
after sublimation for 1 min at - 100 "C in a Balzers apparatus
(BA 360) by the method of Moor & Miihlethaler (1963).
Electron microscopy. Specimens were viewed and photographed in a Tesla BS SO0 electron microscope.
RESULTS
Control cells of the wild-type DBY 1690 with the
standard actin allele ACT1
Morphology. The appearance of the cells accorded with
the typical budding yeast cells of S. cereuisiae (Figs 1and
2) and the course of the cell cycle events proceeded in the
standard manner. For easier identification of individual
stages of the cell cycle we have marked them by numbers
1-6 in Fig. 6. The same identification of cell cycle stages
is used in Figs 1 and 2. For actin mutants, the defects of
the cell cycle stages are designated by the stage number,
followed by a hyphen and another number indicating a
given type of aberration (for example, individual types
of aberrant mitoses, that is stage 4 of the cell cycle, are
designated 4-1, 4-2,4-3, etc.) (Fig. 7).
Microtubular apparatus. Again, this corresponded to the
typical picture for S . cereuisiae. At G , phase a nucleus
with one spindle pole body (SPB) was detected, from
which cMTs emanated, usually pointing to a future cell
bud (Fig. Ib, cells 1).During the start of budding the SPB
doubles and intranuclear spindle is formed between the
two resulting SPBs. One bundle of cMTs, emanating
from the SPB, is positioned towards the developing bud
(Fig. lb, cell 2). Before M phase the nucleus moves from
the cell centre to the bud neck. The bundle of cMTs
points to a bud (Fig. lb, cell 3). During mitotic nuclear
division the budding cells have the spindle MTs
elongated along the polarity axis (Fig. 1b, cells 4) ;cMTs
seem to be anchored at the pole of the bud and mother
cell (Fig. lb, cell 4). After constriction and separation,
one nucleus remains in the mother cell, and the second
one in the daughter cell; both, however, are in excentric
positions (Figs. I b and 2c, cells 5). The nuclei move to
the centre of cells during cytokinesis (Fig. 2c, cells 6),
taking the typical central location for the GI phase. The
microtubular apparatus of the wild-type cells is shown
in Fig. l ( b ) and diagrammatically in Fig. 9.
Actin cytoskeleton. This did not differ from the typical
localization and dynamics of S. cerevisiae. It is formed
by cytoplasmic cables of actin filaments, localized under
the cell surface and proceeding through the mother cell
along the longitudinal cell axis. In the course of budding,
actin micro filament (aMF) cables extend along the
mother-bud axis (Fig. 2b, Fig. 9 ) . In stationary-phase
cultures, microfilament cables are not visible in the
cytoplasm. The second component of the actin cytoskeleton is the submembrane actin patches. They
undergo dynamic rearrangement in the course of the cell
cycle. Before budding they accumulate at the sites of
future buds, and during budding they accumulate in the
growing bud (Fig. 2b, cells 1 , 2 and 3). An asymmetry of
actin patches is still preserved during mitotic nuclear
division (Fig. 2b, cells 4), but after the nuclei have
separated, actin patches become evenly distributed in
mother and daughter cells. Actin cables connect mother
and daughter cells along their long axis (Fig. 2b, cells 5).
The actin patches accumulate in the neck area during
cytokinesis (Fig. 2c, cells 6). In the short period before
cytokinesis the third component of the actin cytoskeleton - an actin (contractile?)ring - was identified in
the area of the future septum (Fig. 2b, cell 5 in the centre,
white arrow).
Nuclear division. Nuclear division during the cell cycle of
budding yeast is presented in Fig. l(c) and Fig. 2(c), and
diagrammaticaly in Fig. 6. It is in accordance with the
dynamics of this process in Saccharomyces. The G,
nucleus (cell 1)and S phase nucleus (cell 2) were located
at the centre of the mother cell. Towards the end of
phase G,, the nucleus moved to the neck of the bud (cell
3). During M phase, the nucleus went through the
narrow neck, and elongated along the mother-bud axis
(cells 4). After separation, the nuclei were located in the
submembrane regions of the opposite poles of the double
cells, in eccentric positions (cell 5). In the course of
cytokinesis, usually before cell separation, nuclei moved
again to the centre of both cells (cell 6), Figs l(c),2(c),6,
8 and 9.
Temperature-sensitiveactin mutant DBY 1693 with
mutated actin allele actl-1 at the permissive
temperature of 23 "C
Morphology and cell cycle. Actin mutant cells multiply by
budding at the permissive temperature of 23 O C . The
population is, however, very heterogeneous (Figs 3,
4, 7 and 8). The population heterogeneity changed
depending on temperature (subrestrictive) and length of
cultivation (see below). At the permissive temperature
of 23 "C,the cultures of the ts actin mutant contained a
mixture of cells similar to the control DBY 1690, and
cells that differed. The latter cells were variable in both
size and morphology. Their size ranged from 3 pm to
25 pm, while the mean size of control cells was 7 l m . In
comparison with the wild-type, cells were mostly round.
Cells with two or more buds were also found (Fig. 4, cell
2 in the centre, black vertical arrow). 'Stationary'
cultures of actin mutants at 23 O C always contained a
mixture of cells in different stages of budding, in contrast
-
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Nuclear position and actin cytoskeleton in yeast
Figrn3. Micrographs of two identical groups (a, b, c and d, e, f) of DBY 1693 actl-7lactl-7 actin mutant cells grown in
malt extract medium a t the permissivetemperature of 23 "C for 24 h and fixed in this medium for 90 min. Bars, 10 pm. (a,
d) Phase-contrast microscopy. (b, e) MTs, immunostained with the anti-a-tubulin antibody TAT1 and FITC. (c, f) Nuclear
staining with DAPI. Numbers 1-6 in (a) refer t o the stages of the cell cycle, as explained in the legend of Fig. 1. Numbers
in (b) and (e) refer t o misoriented nuclear divisions (4-1, 4-2, 4-3, 4-4) and multinuclear cells (5-1, 5-2, 5-3, 5-4).
to wild-type cultures, which were without buds. This
indicates an error in the G, phase in Start, where the cell
cycle of actin mutants should stop. The main feature
was a difference in nuclei in comparsion with the
standard picture of the yeast cell cycle. Standard
dynamics of rearrangement of the actin cytoskeleton
was also markedly changed (see below). The cell
polymorphism of the culture is shown in Figs 3 and 4,
and diagrammatically in Figs 7 and 8.
Microtubular apparatus. MTs were well developed in cells
of the ts actin mutant in all stages of the cell cycle (Fig.
3b, cells 1-5). Cells had SPB, cMTs and mitotic spindles
with aMTs. However, nuclei were in excentric positions
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M. KOPECKA a n d M. GABRIEL
..................... ...................................
........................................... .,.................................. .................................................. ............................................* .............................
Fig. 4. Three micrographs of an identical group of DBY 1693 act7-llactl-7 actin mutant cells grown in malt extract
medium a t the permissive temperature of 23 O C for 24 h, fixed in this medium for 10 min, and stained with Rhph or DAPI.
Bars, 10 pm. (a) Nomarski differential interference contrast. (b) Actin cytoskeleton, labelled with Rhph. (c) Nuclear
staining with DAPI. Numbers 1-6 refer t o the stages of the cell cycle, as explained in legend of Fig. 1. Single arrows in (a),
cells with two buds; double arrow in (a), dividing cell with two nuclei in excentric positions; s, very small cell; g, gigantic
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Nuclear position and actin cytoskeleton in yeast
(Fig. 3b, cells 1 and 2). A different location of the M T
system coincided with deviations in the nuclear position
and nuclear division (see below). The location of the
mitotic spindle deviated from the longitudinal axis (Fig.
3b, e, cells 4-1,4-2,4-3,4-4). Cells with small buds had
cMTs that did not point to a bud. Their orientation was
random (Fig. 3b, cell 2). MTs were well developed in all
types of multinuclear cells, though there were sometimes
errors in their spatial position (Fig. 3b, e, cells 5-1, 5-2,
5-3, 5-4). However, most cells had a longitudinal
direction of spindles and their MTs pointed to the bud,
as in control cells (Fig. 3b, cells 3 , 4 and 5 ) .
Actin cytoskeleton. Longitudinally oriented actin cables
were rarely detected at 23 "C, as reported by Novick &
Botstein (1985). In contrast to wild-type cells (Fig. 2b),
actin patches were also regularly found on the mother
cells. Nevertheless, asymmetric distribution of actin
patches was a typical feature. However, in some cells,
actin patches were more prevalent on mother cells than
on buds, or were absent on the buds or neck. The
increase in the frequency of actin patches on mother
cells corresponded to their round shape, larger size and
perhaps to their continuous isodiametric growth (Fig.
4b, double white arrow). No simple relation was found
between the distribution of actin patches and the curious
location of nuclei (see below). During mitotic nuclear
division typical for the control cells the actin patches
were found mainly on the bud and then on the base of
the bud neck on the mother cell (Fig. 4b).
Nuclear division. In the course of cultivation of the actin
mutant cells for 24 h at 23 "C, nuclear division in 75%
of cells in the population proceeds similarly to the wildtype cells in phases 1-6, with some exceptions. In nonbudding cells, the GI-phase nucleus is in an excentric
position (Fig. 3c, cells 1).During the growth of the small
bud, the nucleus moves toward the cell centre (Fig. 3c,
cell 2). It moves toward the neck of the bud at the end
of G, phase (Figs 3c, 4c, cells 3). During M phase it
elongates in the direction of the mother-bud axis (Figs
3c, 4c, cells 4). The nucleus remains in the excentric
position near the cell poles not only after separation of
the daughter cell's nuclei (Figs 3c, 4c, cells 5 ) , but also
after cytokinesis (Fig. 4c, cell 6). It returns to the centre
of new cells in the course of budding in the new cell cycle
(Figs 3c, 4c, cells 2).
About 25% of the actin mutant cell population shows
different aberrations of the nuclear and cell cycle during
cultivation at 23 "C. The occurrence of multinuclear
cells (both budding and non-budding) in this culture
proves that cytokinesis and the cycle of nuclear division
are separate events (Figs 3a, c, d, f, 4a, c, 7 and 8). The
nuclei are able to divide, but unable to separate properly
into mother and daughter cells. Instead, both nuclei may
remain in the mother cell. In a proprotion of the actin
mutant cells at the permissive temperature both cell
growth and division (cytokinesis) are retarded beyond
nuclear division. In the heterogeneous population,
between 17 and 38 different types of aberrations of
nuclear and cell cycles were detected (Tables 1 and 2,
Fig. 8). From the point of view of the dissociation of
cytokinesis and nuclear division it was significant that
we detected (i) non-budding binuclear cells (3-19 OO/ of
the entire population), and (ii) budding binuclear cells,
where both nuclei remain in the mother cells (5-20 % of
the entire population). They were seen at the same
frequencies as the observations of the nuclei with the
M T apparatus which was not oriented from the SPB to
the bud (see above). Binuclear mother cells with large
anucleate buds were found. However, these buds did not
separate from the mother cells. In some binuclear cells,
where the crosswise position of two aberrant mitoses
occurred, accumulation of actin patches on the
anucleate, mononuclear or binuclear buds was normal,
similar to the wild-type cells (Figs 4a, b, c, cells 4-1,4-2,
S l d , 5-2), demonstrating undisturbed polarity of actin.
Dividing nuclei in actin mutants growing at 23 "C had
normally developed mitotic spindles. However, the
orientation of the nuclear division in the space was
random. Crosswise and mutually crossed mitoses were
detected (Figs 3a-f, 4a-q cells 4-1, 4-2, 4-3, 4-4). Both
odd and even numbers of nuclei were found in one
system of mother-bud cells. The highest number of
nuclei detected was 16. The ratio between the number of
nuclei in the mother cell and in the bud was different
(Fig. 8). The ploidy of nuclei was not identified, but the
size of the nuclei was strikingly different.
The multiplication of the nuclear equipment (1,2,4, 8,
16 nuclei) in the multinuclear cells, and occurrence of
deviations from this geometrical array in the number of
nuclei, were detected. Synchronized division of nuclei in
multinuclear cells was observed directly (Figs 3a-f, 4a-c,
cells 4-2, 4-3, 4-4). Our earlier study showed also that
multiple nuclei may arise in both a synchronized and an
asynchronized way in protoplasts of budding yeast,
while in protoplasts of fission yeast, synchronized
nuclear divisions were typical (M. Gabriel, unpublished
data). In actin mutant cells a relatively high frequency of
mitoses was found. The question is, whether this
phenomenon is not a terminal stage of surviving actin
mutant cells with the loss of vitality in this stage of
mitosis.
Influence of temperature and time of cultivation
upon nuclear and cell aberration
Major differences in size and morphology of cells,
number of nuclei and cell division were detected during
the cultivation of ts actin mutant at 23 "C. Slow bud
growth and the isodiametric growth of some cells show
that the temperature of 23 "C is not permissive for all
cells in the population. In part of the population the
cell. The single arrow in (b) indicates the asymmetric staining pattern of cortical actin dots; actin cables are almost
invisible. The double arrow shows the cell lacking the asymmetric staining pattern of the cortical actin assembly. Numbers
in (c) represent misoriented nuclear division (4-1, 4-2) or multinuclear cells (5-1, 5-ld [d, daughter], 5-2, 5-3, 5-4, 5-5).
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M. KOPECKA a n d M. G A B R I E L
Fig. 5. Electron micrographs of fractures through cells of the t s actin mutant DBY 1695 actl-2, grown at the permissive
temperature of 23 "C for 24 h. Freeze-etching. Bars, 1 pm. (a) Dividing nucleus (N) elongated between mother and bud.
(b) Misoriented dividing nucleus (N) in mother cell; vesicles visible in the cytoplasm. (c) One nucleus (N) in the bud and
two nuclei (N) in the mother cell are visible.
disruption of actin is the reason for the lack of polarized
cell growth (Fig. 4b, cells 2g-1, 5-1, about 4.5% of the
population, Fig. 8). A comparison was made between
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the proportion of cells with 'normal ' (wild-type) morphology and those with aberrant morphology for the ts
actin mutant cultivated at different temperatures. At the
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Nuclear position and actin cytoskeleton in yeast
1
n
1
2
3
4
4
5
6
1
.......................... .......,..................................................................... ....................................... ...
Fig. 6. The stages of the cell cycle of 5. cerevisiae according to
nuclear and cell morphology, as deduced from the study of
DBY 1690 cells (Figs 1 and 2). 1, The unbudded cell with a single
nucleus in the central position; 2, the budding cell with a
single nucleus in the central position; 3, the budding cell with
a single nucleus near the neck of the bud (premitotic nuclear
migration phase); 4, the budding cell with an elongated
nucleus in the neck of the bud (nuclear-division morphology);
5, the budding cell with two nuclei in excentric positions
(excentric position of nuclei a t poles of mother and daughter
cells); 6, the budding cell with two nuclei in the central
positions (postmitotic migration from excentric to central
position). Stages 1, 2, 3, 4 and 6 are reported in detail in
reviews by Hartwell (1974), Fantes (1989), Cid e t al. (1995) and
others.
Electron microscopy
The material for electron microscopy was prepared by
freeze-etching. The consequences of mutation in the
actin gene during cultivation at restrictive temperature
of 37 "C for 24 h were observed in a small surviving
population : isodiametric cell growth, formation of
aberrant cell walls, accumulation of secretory vesicles,
blockage of bud growth and further defects (Gabriel &
Kopecka, 1995). Cultivation of the actin mutant at
23 "C, usually considered as a permissive temperature
(Novick & Botstein, 1985), is for part of the population
a 'semirestrictive ' (subrestrictive) temperature. Cultivation at 23 "C led to heterogeneity in the cell ultrastructure and size. For instance, besides the standard
submicroscopic picture (Fig. 5a) we observed a higher
frequency of dividing nuclei. Some of the dividing nuclei
were aberrantly localized inside of mother cell only (Fig.
5b). Several cells had a multiplicate membrane system,
an increased number of vesicles also in the mother cell,
and were multinuclear (Fig. 5c). Electron microscopy
confirmed the findings of excentrically positioned nuclei
and of the random orientation of mitoses. The observed
multinuclear cells of ts actin mutants contained up to
two or three nuclei at the permissive temperature only
(Fig. 5c).
DISCUSSION
1-1
2-1
3-1
4-1
4-2
5-1
5-2
Fig. 7. Mechanism of misorientation of nuclear division in
budding yeast. An actin mutant cell with an excentric nucleus
in stages 1, 2 and 3 (marked as 1-1, 2-1, 3-1). Mitotic nuclear
division starts in an incorrectly localized nucleus inside the
mother cell. Two nuclei originate in the mother cell (5-1). The
next cell cycle leads to two synchronous mitoses (4-2) and a
four-nucleated cell (5-2).
same time numbers of different types of nuclear aberrations were identified after staining by DAPI. The
results are presented in Table 1. The dependence of the
number of aberrations and their different types on the
temperature is evident. The frequency of nuclear aberrations increases with increasing temperature from 11 to
37 "C. On the other hand, the frequency of aberrant cells
decreased with increasing time of cultivation. At
temperatures over 30 "C the differences in appearance of
the aberrant cells became insignificant. With increasing
temperature the effect of actin filament disruption was
accelerated under the influence of ts mutation act2 -1.
The aberrant forms stagnate at cytokinesis and they are
replaced by more vital dividing cells (Table 2).
It is not known how the position of the nucleus in
interphase eukaryotic cells is established. However, the
nucleus is seen inside a complex cytoskeleton ' basket'
(Alberts et al., 1994). Analysis of nuclear positioning in
the yeast cell may be easier than in the mammalian cell,
because the yeast cytoskeleton consists of MTs and
microfilaments only, and ts mutants allow study of the
missing functions in mutated phenotypes.
Role of MTs in nuclear migration and positioning of
mitosis
Nuclear migration to the bud neck in budding yeast
before mitosis is ascribed to the M T system (reviewed by
Drubin, 1991; Chant, 1994, 1996; Masuda, 1995;
Harold, 1995; Pringle et al., 1995; Drubin & Nelson,
1996; Winsor & Schiebel, 1997). The motor protein
involved is dynein (Eshel et al., 1993 ;Li et al., 1993; Yeh
et al., 1995; Carminati & Stearns, 1997; reviewed by
Schroer, 1994; Morris et al., 1995; Hoyt & Geiser,
1996). cMTs, radiating away from the SPB (Byers &
Goetsch, 1973), are probably tethered to actin (Palmer et
al., 1992). A cortical aMF-rich region of the plasma
membrane of buds, containing concentrated actin
patches, has been considered (Schroer, 1994; Hoyt &
Geiser, 1996). The nuclear migration does not proceed
without MTs (Jacobs et al., 1988 ;Huffaker et al., 1988).
For migration and correct orientation of the spindle,
aMTs are necessary. Their disruption blocks spindle
migration to the bud neck. After mitosis, binuclear
mother cells and anucleate buds are formed (Sullivan &
Huffaker, 1992; Palmer et al., 1992). The analogous
phenotype was found in dynein mutants (Eshel et al.,
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1791
M. K O P E C K A and M. GABRIEL
(a) DBY 1690 (wild-type)
~~
_____
~
(b) DBY 1693 (actin mutant)
Fig, 8. Diagrammatic representation of
normal and aberrant cells from one of many
experiments with (a) wild-type DBY 1690
(control) and (b) t s actin mutant DBY 1693,
cultivated for 24 h at the permissive
temperature of 23 "C. Normal cells are cells
without aberrations; aberrant cells are cells
having any aberration of the number of
nuclei or position of the spindle. Numbers
represent the percentage frequency of each
cell type in the population (about 300
control cells and about 300 actin mutant
cells were counted).
Table 7. Frequency of aberrations in the nuclear and
cell cycle in t s actin mutants in relation to the
temperature of cell cultivation after 24 h
~~
Cultivation
temp. ("C)
~
No. of different
types of
Frequency of
aberration (%)*
aberration
1lt
18
23
30
37
2t
10
17
39
11
7t
22
26
46
9 (died)
* 100o/' was about 300 counted cells.
time of cultivation was 57 h, because of the very long
t The
generation period at this low temperature.
1993; Li et al., 1993; Yeh et al., 1995; Carminati &
Stearns, 1997),in mutated proteins related to interaction
of tubulin and the submembrane region with dynein/
dynactin (JNMZ, N U M I , ACT3, ACTS; McMillan &
Tatchel, 1994; Farkasovsky & Kuntzel, 1995; Clark &
1792
Meyer, 1994; Muhua et al., 1994; McCollum et al.,
1996) or in other genes of unclear function (McMillan &
Tatchel, 1994). This paper describes an analogous
phenotype of aberrant mitoses in ts actin mutant cells
that have well-developed aMTs. The classical pattern of
aMTs and spindles directed to the bud is, however,
broken. Instead, aMTs proceed from SPBs in a random
way; spindles and dividing nuclei are randomly
positioned and oriented in the mother cell in the oblique
or cross position. While MTs are preserved in ts actin
mutant cells, the actin cytoskeleton is deficient. From
this we deduce that not MTs alone, but MTs and aMFs,
determine nuclear migration and the positioning of
mitosis.
Role of actin in nuclear movement and orientation of
mitosis in budding yeast
Palmer et al. (1992) detected defects of spindle orientation and migration after disruption of actin cytoskeleton (manifested by similar numbers of cortical
patches in mother cell and bud, and no actin cables).
This was induced by incubation at a restrictive temperature (36 "C)for 3 h as a result of mutated allele a d -
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Nuclear position and actin cytoskeleton in yeast
Table 2. Frequency of occurrence of aberrations in
nuclei and of different types of aberrations in t s actin
mutants in relation to the cultivation temperature and
time
Cultivation
Temp.
("C)
Normal cell
cycle (%)+
Time
Aberrations
Percentage" No. of
types
(h)
~
DBY 1690 (ACTIIACTI)
23
16
100
23
24
100
23
57
100
30
18
100
30
24
100
30
44
99
DBY 1693 ( ~ c t l - I / ~ ~ t l - I )
23
16
65
23
24
73
23
57
86
30
18
66
30
24
61
30
44
61
0
0
0
0
0
1
0
0
0
0
0
1
35
27
14
34
39
39
10
17
4
39
38
40
60
59
DBY 1695 ( a d - 2 )
30
24
40
* 100% was about 300 cells.
4 of the actin gene (Dunn & Shortle, 1990) and their
transfer to 23 "C. In our ts actin mutant cells with the
actl-1 allele (Shortle et al., 1984) cultivated at the
restrictive temperature (37 "C ; Novick & Botstein,
1985) similar defects were also detected (Gabriel &
Kopeck& 1992, 1995). Analogous effects were detected
in other ts actin mutants with the mutated actin alleles
actl-229 to actl-136 (Drubin et al., 1993). We conclude
that mutation in the actin gene is phenotypically expressed by multinucleate cells due to spindle disorientation. This confirms that actin is necessary for
proper spindle orientation (Palmer et al., 1992). Multinucleate cells also originate in mutants with polarity
defects due to mutations in genes encoding actinassociated proteins. These include genes for myosin
M Y O I , encoding M H C (Watts et al., 1987), myosinrelated protein M Y 0 2 (Johnston et al., 1991; Sellers et
al., 1996),profilin (Haarer et al., 1990) and tropomyosin
TPMZ (Liu & Bretscher, 1992; Bretscher et al., 1994).
Another group are affected in actin-interacting proteins,
mutants revealing synthetic lethality with actin-binding
protein 1 ( A B P I ) in SLA2 (Holtzman et al., 1993)
manifested also by defects of the actin cytoskeleton.
Based on the analysis of morphological aberrations of
nuclear division in ts actin mutants under subrestrictive
conditions, this study aimed to deduce the possible
mechanisms by which the actin cytoskeleton participates
in individual phases of nuclear division. First, we will
consider the possible consequences of dysfunction of
actin cables.
..................................................,......................................,..............................................,,.......
Fig. 9. Suggested mechanism of positioning of nucleus in the
cell cycle of budding yeast: actin cables may function as a
'suspensory apparatus' and/or 'corridor' necessary for correct
positioning of the nucleus in the cell centre, for movement of
the nucleus to the bud neck before mitosis, and for movement
after mitosis from an excentric to a central position. B, bud; M,
mother cell; N, nucleus; AC, actin cables; AP, actin patches; EN,
elongated mitotic nucleus. Rearrangement of actin patches
during the cell cycle is based on observations of our
photographs of DBY 1690. The mechanism rearrangement of
actin patches during the cell cycle was explained by Lew & Reed
(1993, 1995) by means of cyclin-dependent protein kinase
~
3 activity
4
and
~ inactivation
~
~
during
~
cell division cycle
stages. M-phase of the cell cycle (stage 4) was triggered by MPF
( ~ 3 complexed
4 ~ ~ with
~ ~mitotic cyclin; reviewed by Fantes,
1989; Nurse, 1992; Alberts et a/., 1994; Nigg, 1995; Nasmyth,
1996a, b).
Influence of the lack or the partial dysfunction of the
actin cables upon the movement of non-dividing
(interphase) nuclei
The best-developed actin cables in wild-type budding
yeast are found during budding, karyokinesis and
cytokinesis. In actl-2 actin mutant cells only faint actin
cables, difficult to record photographically, are present
at the permissive temperature. More significant occurrence of actin cables was detected at the same period
in reverting ts actin mutant cells during their transfer
from restrictive to permissive temperature (M. Gabriel
and M. Kopeck& unpublished). In act1-1 ts actin mutant
cells at the permissive and subrestrictive temperatures,
actin cables are hardly detected and nuclei remain at
excentric locations after mitosis. Therefore, we deduced
a certain missing role of the aMF cables in the backward
movement of divided daughter nuclei from excentric
regions toward the central region. We suggest that actin
cables play a role in postmitotic nuclear migration from
an excentric to a central position. In the wild-type, the
nucleus moves into the neck during the budding. In ts
actin mutants, however, cell growth (budding) and
nuclear division are not always synchronized at subrestrictive temperature. The nucleus does not reach the
bud neck from an excentric position after the previous
mitosis in time, but remains in the ' half-way ' position.
The next nuclear division proceeds within the mother
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1793
M. KOPECKA a n d M. GABRIEL
cell, resulting in a binucleate mother and an anucleate
bud. The cause of this may be a defect in the tethering of
aMTs to actin dots in the bud (Schroer, 1994), slow
polar growth, and/or slow function of aMFs in premitotic nuclear migration.
The orientation of the nuclear division axis is
influenced by disruption of the aMFs
Some actin mutant cells are round due to isodiametric
growth, polar growth being absent at 23 OC. This is the
result of aMF disruption in these cells (Novick &
Botstein, 1985 ;Gabriel & Kopecka, 1995). In these cells,
defects in the orientation of nuclear division occur. Since
the spindle orientation defects occur in the budding
cells, it can be established that the aMF cables can create
the ' intracellular corridor' or ' track ' or ' way' or
'suspensory apparatus ' for orientation and direction of
nuclei toward the buds by means of aMTs (Fig. 9). The
lack of polarity of the spherical, isodiametrically growing cells induced by disruption of the aMF cables is
characterized by randomly positioned nuclei (Gabriel &
Kopeck& 1995). A similar situation is found in isodiametrically growing protoplasts of fission and budding
yeasts, in which the aMFs are also absent and nuclear
divisions are randomly oriented (Gabriel, 1983, 1984,
1994; Gabriel et al., 1992; M. Gabriel & M. Kopecka,
unpublished). It is more difficult to explain the disorientation of nuclear division in the budding cells of
actin mutants at the permissive temperature, presented
in this work. Areas rich in actin dots on the bud mark
the active polarized growth of the bud without defects in
cell polarity, while both daughter nuclei were found
inside the mother cell. We believe that aMFs in these
cells are capable of carrying the secretory machinery
necessary for polar growth, but are so functionally
insufficient that they are not able to lead a large nucleus,
having aMTs, into the bud neck rapidly. Consequently,
mitosis proceeds in the mother cell without mitotic
nuclear position-stabilizing in the bud neck, and hence
in a random orientation. This could be regarded as
evidence for a close relation of aMFs to nuclear
movement during the cell cycle and to the orientation of
nuclear division. Very impressive are the findings of the
budding, arrangement of actin cables and correct
orientation of mitoses in the cells that after a few hours
at a restrictive temperature of 37 "C were transferred to
the permissive temperature of 23 "C.New buds start to
grow from previously blocked budding mother cells,
having a high number of actin dots. The restoration of
budding in these 'reverting' cells is marked by the
appearance of actin cables, directed toward the new
bud. The nucleus, dividing again at the permissive
temperature, is also directed towards the bud (M.
Gabriel & M. Kopeck& unpublished). These phenomena testify to the function of aMF cables (in cooperation
with the microtubular apparatus) in nuclear location,
movement and mitotic division along the polarity axis in
the course of the yeast budding cell cycle.
1794
Role of actin patches in positioning of nucleus and
mitosis
The actin patches are evenly distributed under the entire
surface of spherically growing actin mutant cells and
protoplasts in the absence of actin cables (Novick &
Botstein, 1985; Gabriel et al., 1992; Gabriel & Kopecka,
1992, 1995; KopeckA & Gabriel, 1995). Since a typical
feature of these cells is the excentric position of nuclei
and random orientation of mitoses, we believe that not
only actin dots, probably in cooperation with cMTs,
aMTs and several other proteins and motors (Schroer,
1994; Carminati & Stearns,l997), but more importantly,
actin cables are necessary for proper nuclear positioning,
mitosis, and post-mitotic nuclear migration.
Random distribution of nuclei in t s actin mutants at
subrestrictive (permissive) temperature
The permissive temperature of 23 "C was established for
ts actin mutant DBY 1693. At this temperature, cells of
the ts actin mutant multiply by budding and reveal the
asymmetry of the cortical actin cytoskeleton. However,
cells show a variable response in sensitivity to the
temperature of 23 "C. The more sensitive cells are more
retarded and may become rounded (Novick & Botstein,
1985). The culture therefore becomes heterogeneous in
size and shape of cells, increasingly so with increasing
temperature. This phenomenon is the reason for the
slow growth of some cells. Lack of budding does not
necessarily stop nuclear division (Hartwell et al., 1974;
Pringle & Hartwell, 1981; Nurse, 1992; Nigg,
1995; Nasmyth 1996a, b), as well as the slow polar
growth of some actin mutants. We did not, however,
find any law governing the relation between cell size and
number of nuclei. A large cell may have one (large,
polyploid) nucleus, while other, smaller, cells can be the
multinucleate ones. The number of cells in the culture
with these aberrations increases with increasing subrestrictive temperature. The large collection of budding
cells with nuclear aberrations revealed that the partial
defect of the actin cytoskeleton must be the reason for
the random distribution of nuclei between the mother
and daughter cells. This random pattern is conspicious
in the nuclear distribution in multinucleate cells and/or
the multinucleate cells with more buds. Where aMF
cables are deficient (due to actin gene mutation), the
dividing nucleus may not find the way to its place of
destination, i.e. into the daughter cell - bud. The analysis of ts actin mutants at different temperatures showed
that the frequency of nuclear aberrations increased with
increases in the subrestrictive temperature. Frequent
findings of synchronous nuclear divisions in the cells of
the actin mutant at subrestrictive temperature indicate
that nuclear division may be retarded, i.e. may proceed
for a longer time. The possibility that nuclear division is
halted at anaphase, while nuclei are not separated,
cannot be excluded. This is reminiscent of blocked
CDCS function (Schild & Byers, 1980) coding polo-like
kinase (Glover et al., 1996). The finding of odd numbers
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Nuclear position and actin cytoskeleton in yeast
of nuclei in cells is not important in this respect. In the
course of synchronous division the single nuclei can be
injured. During the normal cell cycle the processes are
choreographed in both time and space. This paper
demonstrates that the choreography of the cell cycle is
disturbed by the mutation of the ‘conventional actin’
gene and consequent insufficiently developed aMF
cables. The partially defective function due to
insufficient actin cables creates conditions for dissociation in both time and space of the nuclear movement and nuclear mitotic division in the cell division
cycle of budding yeast. This paper points out that the
cooperation and interaction of aMFs, MTs and possibly
several other proteins and motors (Goldstein & Vale,
1992; Kuznetsov et al., 1992; Langford, 1995; Drubin &
Nelson, 1996; Gavin, 1997; Winsor & Schiebel, 1997;
Schroer, 1994; Carminati & Stearns, 1997) are necessary
for correct positioning of the nucleus in the centre of the
cell, for pre-mitotic movement of the nucleus, for
correctly located and oriented mitosis, and for correct
distribution of the two nuclei, created by mitosis, into
the new cells. The avenues are open for research into
actin cytoskeleton function in relation to the cell nucleus.
NOTE ADDED IN PROOF
After this paper was submitted the following papers
reported similar aberrant positioning of nuclei and/or
MTs, as shown in this paper, but due to mutations in
other genes: in a kar9 mutant (Kar9p is required for
orientation of cMTs to the bud) [R. K. Miller & M.
Rose (1998).] Cell Biol 140,377-3901 ;in a kip3 mutant
(Kip3p is a kinesin-related MT-motor protein, required
for nuclear migration to the bud neck and orientation of
the spindle along the mother-bud axis) [T. M. De Zwaan
and others (1997).J Cell Bioll38,1023-1040]; in a kip3
dynl double mutant (Kip3p and Dynlp are MT-motor
proteins required for nuclear migration to the bud and
movement of the nucleus through the bud neck) [F. R.
Cottingham & M. A. Hoyt (1997). J Cell Biol 138,
1041-10.531; in spindle migration and orientation, not
only dynein, but also kinesin-related motor proteins
KIP3, KIP2 and KAR3, which work together, are
involved [reviewed by T. Stearns (1997). J Cell Biol138,
957-9601, On the basis of the above papers aberrant
positioning of nuclei and MTs in ts actin mutants (our
paper) may be caused also by missing function of KarSp,
Kip3p and Dynlp due to improper actin function. Proper
actin filament function may be involved in their delivery
to their destination. Thus, improper actin function may
be followed by improper function of MTs and motor
protein.
ACKNOWLEDGEMENTS
We thank Professor A. Svoboda for kindly reading the
manuscript, Dr J. PeGek DrSc for kind help with the
manuscript, and V. Ramikova, J. Drapalova, V. Travnickova,
J. Krobauer and M. Holubar for technical assistance.
The research was supported by grants 204/93/0667 and
204/97/1150from the Grant Agency of the Czech Republic.
This paper is dedicated to Professor Eva Vlkova, Head of
Ophthalmological Clinics of the Faculty Hospital, BrnoBohunice, for her successful operation on M. K., which
enabled her to finish the work.
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Received 15 August 1997; revised 9 February 1998; accepted 10 March
1998.
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