Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3280
Localization of dynein-green fluorescent
protein (GFP) to cytoplasmic microtubules allowed us
to obtain one of the first views of the dynamic properties of astral microtubules in live budding yeast. Several
novel aspects of microtubule function were revealed by
time-lapse, three-dimensional fluorescence microscopy.
Astral microtubules, about four to six in number for
each pole, exhibited asynchronous dynamic instability
throughout the cell cycle, growing at 0.3-1.5 µm/min
toward the cell surface then switching to shortening at
similar velocities back to the spindle pole body (SPB).
During interphase, a conical array of microtubules
trailed the SPB as the nucleus traversed the cytoplasm.
Microtubule disassembly by nocodozole inhibited these
movements, indicating that the nucleus was pushed
around the interior of the cell via dynamic astral microtubules. These forays were evident in unbudded G1
cells, as well as in late telophase cells after spindle disassembly. Nuclear movement and orientation to the
bud neck in S/G2 or G2/M was dependent on dynamic
astral microtubules growing into the bud. The SPB and
nucleus were then pulled toward the bud neck, and further microtubule growth from that SPB was mainly
oriented toward the bud. After SPB separation and
central spindle formation, a temporal delay in the acquisition of cytoplasmic dynein at one of the spindle
poles was evident. Stable microtubule interactions with
the cell cortex were rarely observed during anaphase,
and did not appear to contribute significantly to spindle
alignment or elongation into the bud. Alterations of microtubule dynamics, as observed in cells overexpressing
dynein-GFP, resulted in eventual spindle misalignment.
These studies provide the first mechanistic basis for understanding how spindle orientation and nuclear positioning are established and are indicative of a microtubule-based searching mechanism that requires dynamic
microtubules for nuclear migration into the bud.
THE ultimate developmental program of any organism relies on the proper coordination of mitosis
with cytokinesis. In many organisms, coordination
is accomplished when the mitotic apparatus signals for the
position of the cleavage plane, which later bisects the segregated chromosomes. This is graphically observed in many asymmetric cell divisions where microtubule-dependent
mechanisms align the spindle along the axis of polarity
(White and Strome, 1996 Cytoplasmic dynein and astral microtubules are required for spindle orientation along the mother/bud axis
before chromosome segregation and for timely nuclear migration into the bud during anaphase (Palmer et al., 1992 The requirement for astral microtubules and cytoplasmic dynein for proper nuclear movement into the bud, together with dynein's ability to move toward the minus end
of a microtubule lattice, has led to the notion that dynein
at the cell cortex exerts a pulling force on the astral microtubules (Eshel et al., 1993 Astral microtubules are reported to be oriented toward
the site of bud growth before bud emergence. Coimmunofluorescence of microtubules and Spa2p have indicated
that microtubules at the cell cortex preferentially localize
with proteins that mark the incipient site of bud growth
(Snyder et al., 1991 In this study we have constructed a dynein fusion with
green fluorescent protein (dynein-GFP) that allows visualization of astral microtubule dynamics throughout the cell
cycle in yeast. Time-lapse imaging indicates dramatic microtubule dynamic instability providing both the mechanical force for nuclear movement, as well as a searching
mechanism for the nascent bud. Astral microtubules remain dynamic as the spindle elongates, indicating that
transient interactions of microtubules within the bud play
a predominant role in the alignment of the mitotic spindle.
These transient interactions may also provide an inherent
mechanism for error correction in segregation of spindle
poles to mother and daughter cells.
Strains
All strains used in this study were derived from strain 15C (Mata, leu2-3,
112, ura3-52, his4-580, trp1
Construction and Expression of a Dynein-GFP
Fusion Protein
A dynein-GFP fusion protein was constructed by cloning GFP (S65T) into
the COOH terminus of the cytoplasmic dynein heavy chain (DHC1, DYN1;
see Fig. 1). The resulting protein is ~500 kD, containing the COOH-terminal 300-4,081 amino acids of cytoplasmic dynein fused to GFP. The
Ser-65-Thr GFP mutation was used for the enhanced solubility and fluorescence of the fusion protein (Heim et al., 1995 Immunofluorescence Staining
Immunofluorescence staining of yeast cells was performed essentially as
described (Pringle et al., 1989 Microscopy and Image Processing
Cells were pipetted onto slabs of 25% gelatin containing minimal media
+2% glucose as described by Yeh et al. (1995) A single background image (average of 24, 3-s exposures with no illumination) was subtracted from each GFP image. The images corresponding to a single time-lapse point were projected to a single image by using
only the brightest pixel at any one location. Registration of DIC and fluorescence images was verified by imaging of 1-µm fluorescent beads in DIC
and fluorescence modes. For presentation, dynein-GFP and corresponding DIC images were overlaid or placed side by side and contrast enhanced using MetaMorph (Universal Imaging Corp., West Chester, PA)
or Photoshop (Adobe Systems Corp., San Jose, CA). Images for publication were scaled and interpolated to 300 dots per inch.
Analysis and Quantitation
Variable levels of dynein-GFP were observed in all transformed strains
necessitating a standard procedure for the selection of cells for imaging.
The average and maximum fluorescence (arbitrary units) from a 3-s exposure of a single cell was assessed, using the quantitation tools in MetaMorph, and compared to the identical measurements made on a plastic
fluorescent reference slide (Applied Precision Inc., Issaquah, WA). Cells
having a maximum fluorescence <20% of the reference were used for further analysis and quantitation. Two image acquisition protocols were
used, one for the analysis of cells over long periods of time (>40 min) and
a second for determining rates.
For time-lapse analysis of cells (>40 min), five fluorescence exposures
of 3-s duration and a single DIC image were taken at 1-min intervals. Excitation was attenuated to between 1-10% to prevent phototoxicity. A total of 1,493 time points were compiled as two-dimensional images from 15 cells for analysis. Anaphase was observed in 11 out of 15 cells and initial
spindle pole separation was discretely followed in 4 of 15 cells.
Rates of microtubule elongation and shortening were determined by
measurement of microtubule length in time-lapse image sequences from
35 cells. Three exposures of 1-s duration were taken at 100% fluorescence
excitation at 1 µm axial steps and projected to a single plane for analysis.
The acquisition regime was repeated every 15 s for a maximum of 15 min
and represents >1,800 time points. Microtubule length was determined by
measuring individual microtubules from the center of the SPB fluorescence to the microtubule end and converting from pixels to microns using
the image of a stage micrometer.
Nocodazole Treatment
Exponentially growing cultures were treated with 1% DMSO (control) or
1% DMSO with 20 µg/ml nocodazole (Sigma Chemical Co. St. Louis,
MO). After 2 h, >80% of nocodazole-treated cells were large budded.
Dynamic Instability of Astral Microtubules
The function of a plasmid-borne, cytoplasmic dynein-GFP
fusion protein (Fig. 1) was assessed by the fidelity of nuclear segregation in individual cells whose sole source of
dynein was the GFP fusion. Anucleate and binucleate cells
were not generated in individuals expressing an average
fluorescence of <20% of a fluorescence reference (described in Materials and Methods). Orientation of the nucleus along the mother-bud axis was not affected by replacing the wild-type dynein with the dynein-GFP fusion.
The rates of spindle elongation were in accordance with previous observations (Yeh et al., 1995 The dynein-GFP fusion protein decorated both the SPB
and astral microtubules exclusively throughout the cell cycle.
In cells fixed for microtubule immunofluorescence by standard procedures in yeast (Pringle et al., 1989
Dynamic instability of astral microtubules occurred
throughout the cell cycle (Fig. 3). Individual microtubules
exhibited growth and shortening at 0.5 µm/min (range from
0.3 to 1.5 µm/min; n = 63 microtubules). These velocities
are an underestimate due to the projection of microtubules in a three-dimensional data set onto a two-dimensional plane. No statistical difference in velocities was observed from interphase to mitosis at the level of resolution
of this study. The average microtubule length was 1.58 ± 0.54 µm (n = 63). Shortening microtubules were observed to switch back to growth (rescue) (Fig. 3), although determination of rescue events close to the pole was difficult to
observe due to the high density of microtubules at the SPB.
In G1, Astral Microtubule Dynamics Push the SPB
and Nucleus
The finding that cytoplasmic dynein decorated astral microtubules, and was concentrated at the single SPB in unbudded cells (G1 stage of the cell cycle, Fig. 4), allowed us
to determine the in vivo orientation and movements of astral microtubules, the SPB, and nucleus. We examined nuclear movement by time-lapse images taken at 60-s intervals. G1 cells contained conical astral microtubule arrays
(~60° arc) undergoing constant polymerization and depolymerization (Figs. 3 and 4). These arrays apparently pushed
the nucleus in the opposite direction of microtubule growth (18 cells examined), resulting in nuclear excursions through
the cell. The direction of these excursions depended upon
the orientation of a growing microtubule, and were reflected by the nuclear movements limited to a 60° arc in
the opposite direction of the conical microtubule array.
The astral microtubules were constantly growing and
shortening during this time and no static interactions (interaction >3 min) with the cell cortex were observed. When
the astral microtubules reached the cell cortex, continued
growth was accompanied either by SPB movement away
from the cortex, or in microtubule buckling (Fig. 4). In 2 cells (of 18 cells examined), the SPB could be seen to transiently move once toward the tip of a microtubule at the
cell surface. These movements were short ( The nuclear movements were completely dependent upon
intact microtubule arrays (Fig. 5). Nocodazole treatment produced no distinct astral microtubules, barely detectable
dynein-GFP accumulation at the pole, and inhibition of SPB
and nuclear migration. These results corroborate the previous report of Yeh et al. (1995)
Nuclear Movement to the Bud Neck in S/G2 Depends
on an Astral Microtubule Search and Capture Process
Time-lapse images revealed that nuclear orientation to the
bud neck in S/G2 was dependent upon astral microtubules
penetrating the newly formed bud. Nuclear movements as
described above continued in a direction away from microtubule growth as bud emergence occurred in S/G2. There
was no discernible association between the SPB or astral microtubules and the incipient site of bud emergence (Fig.
6). Growing microtubules frequently and transiently contacted the cell cortex; however, the pattern of nuclear movement was unchanged from that of G1 cells until an astral
microtubule grew into the bud. Upon penetration, the SPB
and nucleus moved in the direction of the polymerized microtubule, toward the bud neck. Nuclear orientations occurred in this fashion with no obvious relationship to bud
size. The apparent pulling force oriented the SPB and its
astral microtubule array towards the bud so that as their
dynamic instability continued, more and more astral microtubules grew toward and into the bud, rather than into
the mother cell. Such microtubule dependent pulling forces
(sustained SPB movement in the direction of microtubule
growth) occurred only with microtubules polymerized into
the bud, not in the mother cell. Upon penetration of astral microtubules into the bud, nuclear movement was restricted to the mother-bud axis, and the nucleus ceased its
progression around the cell.
In G2/M, Dynein-GFP Accumulates on the Second SPB
After Spindle Pole Separation
SPB separation typically occurred after nuclear orientation
to the bud neck. The spatial resolution of either dynein-GFP or Nuf2p-GFP in the fluorescence images did not allow a precise temporal determination of SPB duplication.
Decoration of the second pole body with dynein-GFP
could only be observed after the SPBs separated >1 µm
(Fig. 7). It is important to note that in the few published electron micrographs of astral microtubules emanating
from duplicated but unseparated SPBs (Byers, 1981
Maintenance of Nuclear Orientation
to the Bud Neck Does Not Require Stable Astral
Microtubule-Cortical Interactions
In G2/M, the nucleus remained near the neck of the budded cell. The SPBbud and nucleus exhibited short range
movements. The SPBmother rotated about the neck site as
described previously for spindles by DIC microscopy (Yeh
et al., 1995 Spindle Elongation in Anaphase
Anaphase onset was observed as elongation of the central
spindle and nuclear envelope through the bud neck by DIC
(Kahana et al., 1995
Astral microtubules were capable of forming static interactions (>3 min) with the bud tip during anaphase, but
they were rare (n = 2 out of 18 cells recorded) (Fig. 8).
Different microtubules from SPBbud interact with the bud
cortex or undergo dynamic instability (Fig. 8). Spindles undergoing anaphase without microtubules stably attached in
the bud generally had several dynamic microtubules polymerizing into the bud and occasionally back into the
mother cell. However by the completion of anaphase, microtubules from the SPBbud were rarely seen in the mother
cell. Spindle elongation was biphasic with an initial rate of
1 µm/min followed by slower elongation into the bud at
one-third that rate regardless of apparent microtubule attachment to the cell cortex (n = 9). Cells with Nuf2-GFP-
labeled SPBs (containing wild-type dynein) exhibited the
same rate of spindle elongation (n = 4, data not shown).
These data are in accord with the rates of spindle elongation from previously published DIC (Yeh et al., 1995 Spindle Disassembly in Telophase
The astral microtubules and the nucleus resumed the behavior typified by G1 cells (above) before cytokinesis. The
nucleus and SPB moved, in either the mother or daughter
cell, in a direction opposite microtubule growth. This movement tended to push the SPB and nucleus toward and
through the center of the cell. This indicates that the stiffness and pushing forces of the central spindle in anaphase
may be much stronger than the pushing forces generated
by the growth phases of microtubule dynamic instability.
Overexpression of Dynein-GFP Results in a Defect in
Nuclear Migration
Cells expressing elevated levels of dynein-GFP were identified in the population by levels of average fluorescence
equivalent to >50% of our fluorescence reference. Gross
overexpression of dynein-GFP did not appear to inhibit
dynamic instability, per se, but resulted in elongated and
bundled microtubules preferentially in the bud during mitosis. Bundling was evidenced by occasional splaying apart
of microtubule bundles. Microtubule growth in the mother
cell often exhibited normal dynamic instability and bundling was much less frequent than in the bud. The hyperelongated astral microtubules exhibited sweeping motions
along the inner surface of the bud, movements not characteristic of short microtubule arrays in cells expressing low
levels of dynein-GFP. Mitotic cells overexpressing dynein-GFP had properly aligned spindles before anaphase (Fig.
9, row 5 column 1 and row 6 column 4). Upon anaphase onset (Fig. 9, row 6 column 5), the spindle seemed to be
prevented from entering the bud by the persistent astral
microtubule array extending into the bud. Further growth
of this microtubule bundle pushed the nucleus and spindle
towards the base of the mother cell. In a few instances,
where disassembly of the stable microtubules occurred,
nuclear translocation into the bud followed rapidly (Ozoy,
Z., C. Yang, E. Yeh, and K. Bloom, data not shown).
The ability to visualize astral microtubules with dynein-GFP
has revealed robust dynamic instability in yeast. More profound, however was the ability to integrate microtubule
dynamics with spindle and nuclear movements throughout
an entire cell cycle. The results herein indicate: (a) a fundamental role for microtubule dynamic instability in positioning nuclei and aligning spindles; (b) an extranuclear
force on the SPB arising primarily from transient interactions
of astral microtubules in the bud; and (c) a possible mechanism for assuring unity of SPB transmission into the bud.
Yeast Use Dynamic Instability and Transient
Microtubule Interactions to Position the Nucleus and
Align the Mitotic Spindle Along the Mother/bud Axis
Live-cell imaging of interphase cells dramatically illustrates
the constant polymerization and depolymerization of the
conical shaped astral microtubule arrays previously documented in budding yeast (Fig. 10). These microtubule arrays pushed the nucleus in the opposite direction of microtubule growth, resulting in nuclear excursions through the
cell. Stable attachments of microtubules to the cell cortex
and pulling forces postulated from still images did not contribute significantly to nuclear movements. The astral microtubules, in performing this action of moving the nucleus, probed the entire cell cortex during interphase.
Upon finding the emerging bud, nuclear movement was
biased toward the direction of the bud. Depolymerization
of astral microtubules with nocodazole completely inhibited both the slow progression of the nucleus and the directed movement to the bud neck, underscoring the requirement for astral microtubules and confirming earlier
works (Huffaker et al., 1988
The centrality of microtubule dynamics to the fidelity of
nuclear migration was further evidenced by analysis of cells
overexpressing dynein. Persistent and hyperelongated microtubules were observed in the bud in cells expressing excess dynein-GFP. Anaphase was completed in the mother
cell due to the inability of the bud-resident astral microtubules to depolymerize. Spindle elongation in the mother
was always accompanied by persistent microtubules in the bud, and did not result from the lack of microtubule interactions in the bud or cell cortex. Hence, eventual spindle
misalignment may result from the persistence of highly
stable microtubules that present a physical barrier to nuclear translocation into the bud.
The dramatic changes in microtubule dynamic instability in cells expressing no (Carminati and Stearns, 1997 Orientation of the Nucleus and Mitotic Spindle
Depends Primarily upon Transient Interactions of
Astral Microtubules in the Bud
Alignment of the mitotic spindle along the mother-bud
axis does not appear to be the consequence of the rigid interaction of cortical microtubules with the cell cortex in
the mother and bud, but rather due to the net sum of less
static interactions of dynamic microtubules within the bud
alone. Though we were able on two occasions to document
stable interactions of individual astral microtubules with
the bud tip during anaphase, that interaction had no measurable effect on the rate of spindle elongation or nuclear
penetration into the bud. We presume stable interactions have little to do with spindle orientation as only 2 of 11 cells imaged through anaphase exhibited any perceived
stable microtubule interaction with the bud cortex. We conclude from this analysis that the majority of information
for spindle alignment and the majority of force applied externally to the spindle do not come from stable interactions of astral microtubules with the cell cortex.
The movement of the nucleus in the direction of an astral microtubule that has penetrated the bud in S/G2 is indicative of pulling forces at this stage in the cell cycle, and
is consistent with previous work showing that SPBs can
migrate into the bud in the absence of a central spindle
(Yeh et al., 1995 These observations provide a mechanism for understanding several classic observations of asymmetric nuclear positioning in higher animals and plants. In Caenorhabditis
elegans (Hyman and White, 1987 A Proposed Mechanism for the Transmission of a
Single SPB into the Bud
Dynamic microtubules and transient interactions with sites
in the bud could account for the mechanism that ensures
unity in SPB deposition in to the bud. In G1, only one astral microtubule array is evident. Once a microtubule penetrates the bud, subsequent microtubule growth from that
SPB is biased toward the bud, largely due to the SPB being
partially imbedded in the nuclear envelope. One pole was
usually biased toward the bud, well before the second pole
was competent to acquire dynein and nucleate astral microtubule (Figs. 6 and 10). Therefore, microtubules from
the second pole (SPBmother) did not accumulate in the nascent bud, avoiding potential errors that would lead to migration of both SPBs (and the accompanying spindle and
nucleus) in the bud. Once the central spindle was assembled (1.5-2.0 µm) the inherent "stiffness" of the spindle oriented the SPBmother to face away from the bud site, and
the restriction in astral microtubule growth to one face of
the pole body further predisposed astral microtubules
from this pole away from the bud and into the mother cell.
The transient nature of astral microtubule associations
with the cortex may contribute to error correction mechanisms. If microtubule contacts in the bud were stable, then
errors from stray microtubules would be difficult to correct. However, multiple transient interactions between microtubules and sites in the bud greatly favor the SPB first
oriented to the bud to remain facing the bud. A stray microtubule from the opposite pole that does move into the
bud would rapidly disassemble and not result in reorienting the distal SPB. In one case we have observed a microtubule from the SPBbud that grew into the mother well after anaphase onset. This microtubule grew quite long (>6
µm), extending well into the mother cell before catastrophe and shortening. Microtubule growth from SPBbud to
the mother and vice versa were infrequent relative to the
total number of events. Nevertheless, such events did not
result in aberrant mitoses, and are indicative of a mechanism that does not rely on a stable microtubule-cortical interaction.
A Role for Dynein in Spindle Alignment
The interaction of dynein with dynactin, and the association of the dynactin complex with actin-related proteins
(Schroer, 1994 Dynein has been implicated in providing the directional
information for nuclear migration in yeast. However, the
finding that dynein is associated with microtubules in both
mother and bud preclude it being the determinant in generating asymmetry. Instead, there are likely to be components in the bud and bud tip that are capable of transient
interactions with dynein. The polarization of "transient
stabilization" domains to the bud may result from mechanisms that contribute to actin polarization toward the bud, and the bud site selection machinery (Pringle et al., 1995). Spindle positioning is of particular importance in the budding yeast Saccharomyces cerevisiae where the cleavage plane is established at the start
of the cell cycle independent of the mitotic apparatus. The
nucleus and its intranuclear spindle must migrate to the
bud neck and orient along the mother/bud axis to segregate chromosomes into mother and bud before cell cleavage. Though microtubules are required for nuclear positioning and spindle alignment in budding yeast (Jacobs et al.,
1988
; Sullivan and Huffaker, 1992
), the role of microtubule organization, interactions, and dynamics in these processes is not well understood.
;
Sullivan and Huffaker, 1992
; Eshel et al., 1993
; Li et al.,
1993
). In the absence of the cytoplasmic dynein heavy
chain gene, DHC1/DYN1, nuclear migration is delayed, resulting in binucleate cells, while spindle formation, initial elongation, and chromosome segregation to opposite
poles continue (Yeh et al., 1995
). A conditional, lethal mutation in
-tubulin (tub2-401) that preferentially destabilizes the astral microtubules prevents nuclear migration
into the daughter cell, and leads to bi- and anucleated cells
after cytokinesis (Sullivan and Huffaker, 1992
). The similarity in phenotypes suggests that dynein's role in nuclear
orientation and spindle alignment occurs via astral microtubules.
; Li et al., 1993
; Schroer, 1994
).
In the absence of a central spindle, ndc1, spindle pole bodies (SPBs)1 (only one of which is associated with chromosomes) are segregated to the mother and bud, indicating
the presence of forces on the SPBs independent of the
central spindle (Thomas and Botstein, 1986
; Winey et al.,
1993
). Dynein's role in the generation of such forces was
demonstrated by Yeh et al. (1995)
, who showed that only
the defective SPB without attached chromatin could be translocated into the bud in cells lacking dynein and the
central spindle (ndc1, dhc1 double mutants). These data,
together with the localization of cytoplasmic dynein to astral microtubules (Yeh et al., 1995
) indicate that dynein
plays a dominant role in pulling the SPB into the bud via
astral microtubules. Cells lacking cytoplasmic dynein and
force generators in the central spindle (Cin8p and Kip1p)
are deficient in spindle elongation (Saunders et al., 1995
),
substantiating the view that dynein exerts a pulling force.
). Similarly, in electron microscopic
studies, the SPB is reported to be oriented toward the bud
in cells with small buds (Byers and Goetsch, 1975
; Byers,
1981
). Thus the microtubule cytoskeleton has been hypothesized to orient the nucleus toward the bud site, and
then dock the nucleus to the future neck of the budded
cell. An apparent paradox between the immunofluorescence studies with fixed cells and imaging of the nucleus in
live cells is the finding that the nucleus moves with a high
degree of freedom in unbudded cells, and is restricted in
its movement only as it migrates to the neck of budded
cells and not before (Koning et al., 1993
; Yeh et al., 1995
).
Materials and Methods
, pep4-3) and MAY591 (Mat
, leu2-3,112,
lys2-801, his3-200, ura3-52) (Saunders and Hoyt, 1992
). Haploid derivatives 8d (Mata, lys2-801, trp1
, ura3-52, leu2-3,112) and 9d (Mat
, lys2-801, his3-200, ura3-52, leu2-3,112) were mated to generate the diploid
strain (8dx9d) (Mata/
, lys2/lys2, ura3/ura3, leu2/leu2, his3/+, +/trp1).
A complete deletion of the 12,276-bp dynein heavy chain was obtained
by fragment-mediated transformation into 9d
dhc (dhc1::LEU2). The
GAL1-glutathione-S-transferase (GST)-dynein-GFP-containing plasmid
(pKBY701; see Fig. 1) was introduced into 9d, 9d
dhc, or the diploid,
8dx9d by standard transformation.
Fig. 1.
Construction of the
dynein-GFP-containing plasmid, pKBY701. The parent plasmid is based on a high copy, galactose-inducible shuttle vector previously described (Baldari et
al., 1987) that contains the 2-µm origin of replication, URA3, and
Leu2-d. Mitchell et al. (1993)
modified the vector for the conditional expression of GST fusions in S. cerevisiae. We inserted the
dynein heavy chain gene (11,333-bp NheI fragment, amino acids
303-4,081) into the XbaI site of pEGKG. The resulting plasmid,
pJU1 contains a galactose-inducible (black), GST (blue)/dynein
(gray) fusion protein. The S65T derivative of GFP (green) (716 bp) was fused to the COOH terminus of dynein, at the unique
SalI site generating the plasmid pKBY701 (22.3 kbp).
[View Larger Version of this Image (10K GIF file)]
). Expression of the fusion
protein was controlled by the GAL1 promoter to allow varied induction/
repression regimes for regulation of protein levels (galactose, induction,
and glucose, repression). Cells were grown to mid-logarithmic growth
phase in glucose, followed by a short induction on galactose (~2 h), and
aliquots were removed and placed onto a 5-µm-thick gelatin slab containing glucose (Yeh et al., 1995
). Expression was repressed for the duration
of recording to restrict analysis to existent dynein.
). Cells were fixed overnight at 4°C in 3.7%
formaldehyde. Microtubules were visualized with the rat anti-
-tubulin
antibody YOL1/34 (1:200 dilution) (Accurate Chemical and Scientific Corp.,
Westbury, NY). Rabbit anti-GST antibodies (gift of E. Bi, University of
North Carolina at Chapel Hill) (1:5 dilution), were used to visualize the
dynein-GFP fusion protein (see Fig. 1). FITC-conjugated goat anti-rat
and rhodamine-conjugated goat anti-rabbit (Cappel Labs, Cochranville, PA) were used as secondary antibodies (1:200 dilution).
. Imaging was performed
using the microscope system and acquisition protocol described in Shaw
et al. (1997)
. The microscope (Salmon et al., 1994
) was modified for automated switching between fluorescence and differential interference contrast (DIC) by replacement of the camera mount with a filter wheel
(BioPoint 99B100; Ludl Electronic Products Ltd., Hawthorne, NY) containing the analyzer component of the DIC optics. A x60 1.4 numerical
aperture Plan-Apochromatic objective lens and x2 + x1.25 intermediate
projection magnified the specimen image x150 directly onto the cooled,
slow-scan CCD imaging device (C4880; Hamamatsu Photonics, Bridgewater, NJ). The computer-controlled (MetaMorph 2.5 software; Universal
Imaging Corp., West Chester, PA) microscope executed an acquisition
protocol taking fluorescence images at 1-µm axial steps and a single DIC image corresponding to the central fluorescence image. Fluorescence excitation through a 490 ± 10-nm filter was normally attenuated to 1-10% of
the available light from the 100 W mercury arc lamp. Fluorescence emission was collected through a 530 ± 15-nm band pass filter. DIC images
were made by rotating the analyzer into the light path and taking a 0.6-s
exposure.
Results
). Only a slight delay
(<5 min) was observed in the penetration of the nucleus
through the bud neck at the time of anaphase onset (see
below). Thus the fusion protein was able to complement
the spindle orientation and nuclear movement defects of
dynein null mutants (Yeh et al., 1995
).
), dynein-GFP
staining was concentrated at SPBs and distributed along the
length of the great majority of astral microtubules preserved
in cells expressing the dynein-GFP (Fig. 2). Occasionally
(23 out of 153 microtubules), a region of a microtubule or
a whole microtubule lacked detectable dynein-GFP staining (Fig. 2). The distribution of dynein-GFP staining along the microtubules in the fixed preparations was very uneven (Fig. 2 B) in comparison to the uniform linear distributions seen in the live cell images (Figs. 3 and 4). This indicates that the microtubule fixation procedures are not able
to completely preserve the native distribution of dynein-GFP along microtubules. For the live cell studies, a single
fluorescence image was inadequate to follow dynamic microtubules, which often extended in and out of the plane
of focus. To solve this problem, a two-dimensional representation of their three-dimensional distribution was obtained by the projection of five fluorescent images taken at
1-µm focal steps through the cell onto one plane. A single
DIC image was taken at the middle focal plane to provide
accurate definition of the cortex and nuclear boundaries
(Shaw et al., 1997
). At no time was any significant concentration of dynein-GFP observed at the cell cortex or associated with membranous structures. A general background
fluorescence was observed in the cytosol presumably representing unbound dynein-GFP. Dynein-GFP did not enter the nucleus at any stage, and consequently, the nucleus
was observed as a dark area within the slightly brighter cytosol. The SPB was identified as a focus of fluorescence at
the periphery of the nuclear envelope (Figs. 3 and 4). The
accompanying DIC image allowed visualization of the nucleus, and confirmation that the SPB was indeed the site of
intranuclear spindle assembly, as well as the point of origin
for the cytoplasmic astral microtubules (Fig. 4, right). Four
to six astral microtubules emanated from the SPB throughout the cell cycle, with individual microtubules splaying out in the periphery of the cytoplasm.
Fig. 2.
Immunofluorescence microscopy of microtubules and dynein-GFP in a
G1 cell. (A) Microtubules
and (B) dynein-GFP. Cells
were double labeled using a
rat anti--tubulin antibody,
YOL1/34, to label microtubules and rabbit anti-GST
antibodies to label a GST epitope in the dynein-GFP
fusion protein.
[View Larger Version of this Image (93K GIF file)]
Fig. 3.
Microtubules exhibit dynamic instability.
High resolution fluorescence
images of unbudded G1 cells.
Haploid strain 9dd lacks an
endogenous copy of dynein
and contains the GST-dynein-
GFP fusion (pKBY701). A
series of eight time points at
1-min intervals are displayed
from top to bottom on the
left and continuing on the
right. Each image displayed
represents a reconstruction
from five exposures taken at
1-µm axial steps. A single microtubule at the bottom of
each panel is shown to polymerize in the first three panels before undergoing a catastrophe and shortening in panel four. The same microtubule rescues and grows to
the cell cortex before undergoing catastrophe and shortening back toward the spindle pole. Bar, 2 µm.
[View Larger Version of this Image (77K GIF file)]
Fig. 4.
Microtubule growth in
G1 is coupled to nuclear movement. Dynamic astral microtubules from a single SPB push
against the cell cortex and propel the nucleus in the opposite
direction. High resolution DIC
with overlain fluorescence (right)
and fluorescence only (left) images of unbudded G1 cells. Haploid strain 9dd, lack an endogenous copy of dynein and
contain dynein-GFP (pKBY701). The focus of fluorescence represents the SPB, which could be
seen at the edge of the nucleus
in DIC (right, overlay). A series
of five time points at 1-min intervals are displayed from top to bottom. Nuclear movement is
from left to right over the time
course. The cytoplasmic microtubules were organized into a cone-shaped array facing away from
the nucleus. Note the SPB at the
leading edge of the nucleus, and
the cytoplasmic microtubules
growing opposite to the direction of movement. As individual
microtubule (or microtubule arrays) extended to the left, the nucleus was propelled rightward.
Bar, 2 µm.
[View Larger Version of this Image (64K GIF file)]
0.3 µm), appeared to occur after the microtubule switched from growth
to shortening, were not specific to the bud neck region,
and were not significant in comparison to the movements
of the SPB and nucleus associated with microtubule growth.
that reported that the
concentration of a Dhc1p-lacZ fusion to the SPB was also
dependent upon intact microtubules. Most likely, the very
weak SPB pattern shown in Fig. 5 reflects short microtubule arrays at the SPB, which are not able to produce nuclear movement. SPBs marked by Nuf2p-GFP (Nuf2p, a
spindle pole antigen; Kahana et al., 1995
) displayed SPB
movements in G1 cells typical of those observed in cells
expressing the dynein-GFP fusion (data not shown). These
observations confirm that movements shown in Fig. 4 are representative of wild-type nuclear motility.
Fig. 5.
Nuclear movement requires cytoplasmic microtubules.
Haploid strain 9dd (containing pKBY701) cells were treated with
nocodazole (NZ) as described in the Materials and Methods. Five
fluorescence images (from left to right) at 5-min intervals are
shown. The cell on the lower left is small budded, and the poles
have separated (note the asymmetry in intensity at the poles).
The cell on the upper right has only one fluorescent SPB. No nuclear movements were detected throughout the analysis. Thus,
nuclear motility is dependent upon intact cytoplasmic microtubules. Points of fluorescence, indicative of dynein-GFP, could be
seen at the SPBs even upon prolonged NZ treatment. Short tufts
of microtubules may be resistant to NZ treatment. Bar, 5 µm.
[View Larger Version of this Image (29K GIF file)]
Fig. 6.
Microtubule penetration in the bud precedes nuclear
movement to the neck. Montage from time-lapse experiments
demonstrating that once cytoplasmic astral microtubules penetrate the bud, the nucleus migrates to the bud neck. Unbudded
haploid cells (9dd containing pKBY701) were captured in the
process of bud emergence. A series of DIC (right) and fluorescence (left) images over the time course indicated in the lower
left (min) are shown from top to bottom, in consecutive series. In
the upper left quadrant, the unbudded cell has a single SPB (embedded in the nuclear envelope), that is positioned at approximately five o'clock. In the next sequence, the SPB has migrated
to approximately the seven o'clock position. Note that the astral
microtubules trail the SPB and form a conical array. The position
of bud emergence is six o'clock (see 47 min). The SPB migrates
around the cell periphery until the 9-10 o'clock position (47 min),
well past the emerging bud. At this time, a long microtubule (7 µm) can be seen extended toward and into the bud. The nucleus
migrates in the direction of the bud after microtubule penetration
of the bud (57-69 min). The nucleus continues toward the bud
neck, in the direction of the microtubule over the next 23 min
(69-92 min). Fluorescently labeled SPBs were apparent at 117 min. Bar, 2 µm.
[View Larger Version of this Image (111K GIF file)]
), the
microtubules originate from the bridge structure extending between the two pole bodies. Cytoplasmic dynein was
clearly localized to the single array of microtubules at this
time, a component of which had penetrated the bud. Once
the SPBs separated during spindle formation, cytoplasmic dynein remained with the pole destined for the bud (SPBbud;
Fig. 6) in three of four cells where both spindle poles could
be followed discretely. There was a delay of about 10 min
after visible separation of the SPBs before the spindle pole
destined for the mother cell (SPBmother) accumulated cytoplasmic dynein (and astral microtubules). At this time, the
SPBmother was 1-1.5 µm from the SPBbud (Fig. 7, right). We
could not distinguish the old and new pole from these
studies. However, the microtubule array the G1 cells were born with was most often the array of microtubules destined for the bud.
Fig. 7.
Dynein-GFP accumulation at the SPB. This sequence
of fluorescence stacks demonstrates the accumulation of dynein
on separated SPBs in budded cells. Cell cycle progression is from
left to right at 5-min intervals. The upper left cell has only one
visible SPB by fluorescence. In 5 min, the second SPB can be visualized, well separated from the first (~1.4 µm). Over time, the
second SPB acquired fluorescence equal in intensity to the first.
Quantitation of fluorescence accumulation over time is graphed
in the panel to the right. Bar, 2 µm.
[View Larger Version of this Image (45K GIF file)]
). SPBs decorated with Nuf2p-GFP revealed
similar short-range SPB movements (data not shown). At
this time in the cell cycle, the bipolar spindle was evident
within the nucleus by DIC microscopy with the spindle ~1.5-2 µm in length. Astral microtubules from the SPBmother
grew into the mother (30 out of 30 microtubules counted),
whereas SPBbud initiated microtubules grew mainly into
the bud (20 out of 23 microtubules counted). Stable interactions (localization >3 min) of astral microtubules with
the cortex were very rare (2 out of 53 microtubules
counted), indicating that the restriction in nuclear movements was not dependent upon stable attachments of astral microtubules in the mother or bud.
; Yeh et al., 1995
) and rapid separation
of SPBs in dynein-GFP or Nuf2-GFP fluorescence images.
The average microtubule length in anaphase was 1.98 ± 1.0 (n = 12), slightly higher than the average non-anaphase microtubule length of 1.2 ± 0.2 (n = 51). As the
spindle elongated (anaphase B) there was concordant decrease in the average length of the SPBbud microtubule array as the SPBbud migrated toward the tip of the bud (Fig.
8). In addition, astral microtubules in the mother were displaced during anaphase, and could be observed to trail the
SPB in its movement to the cortex (astral microtubules
trailing SPB in mother; Fig. 8, 15-25 min). After the SPBmother
reached the distal portion of the mother cells, the astral
microtubules were consistently short and difficult to resolve.
Fig. 8.
Microtubule attachment in the tip of budded cells in
anaphase. DIC (bottom) and fluorescence (top) images demonstrating the stabilization of a single microtubule to a bud tip and
the movement of the nucleus to that site with concomitant shortening of the microtubule. This behavior was infrequent, and is
not typical of most anaphases. Two SPBs of equal intensity can
be seen in the upper left 10 min before anaphase onset. The SPB
closest to the neck is destined for the bud. The nucleus can be
seen in DIC where the positions of the spindle poles have been
denoted by an asterisk. By 10 min, the fast portion of anaphase is
complete and by 15 min after anaphase onset, a microtubule from
SPBbud extends into the bud to the tip of the budded cell. Note
that the nucleus spans the neck at this time point (15 min after
anaphase onset) and that the spindle pole does not lead the nucleus. The microtubule in the bud shortens with spindle elongation (5-min intervals, 15-25 min). The cytoplasmic microtubules
associated with SPBmother remain short and distant from the cortex as SPBmother moves toward the base of the mother cell. The
cytoplasmic microtubules associated with SPBbud that are not in
contact with the cortex remain dynamic. By the end of anaphase,
the SPB migrates to the site where the microtubule in the bud is
apparently "attached." Bar, 2 µm.
[View Larger Version of this Image (80K GIF file)]
) and
Nuf2-GFP (Kahana et al., 1995
) studies.
Fig. 9.
Overexpression of
dynein-GFP. Cells with elevated levels of fluorescence
(>three- to fourfold over
the bulk of the population) exhibited hyperstabilized microtubules only in the bud,
and only during late G2/M or
anaphase. Two cells expressing different amounts of dynein-GFP are shown at 4-min
intervals from left to right. The cell to the left contains
low levels of dynein-GFP
and completes an entire cell
cycle, budding a second time
within the 160-min of this
time-lapse. Note in this cell
that there is no apparent attachment of astral microtubules to the bud tip during
anaphase. The cell on the
right hand side contains
three to four times more dynein-GFP fluorescence than
cells normally selected for
imaging. Observable in this
cell are very dynamic astral
microtubules from the single
SPB in G1 and from both
SPBs during the S/G2 stage. Note that the mitotic nucleus does not appear to remain near the bud neck, but
when an astral microtubule
enters the bud neck (row 4, column 3), the spindle becomes properly oriented along the mother/bud axis (row 4, column 6). The astral
microtubule array in the bud remains very dynamic and undergoes numerous rescues, never depolymerizing back to the mother cell.
Sweeping of the astral microtubule array laterally under the bud cortex can be observed (row 6, columns 1-3). Upon anaphase (row 6,
column 4), the spindle is properly aligned but is prevented from entering the bud by the stable astral microtubule array extending into the bud. Further growth of this microtubule bundle pushes the nucleus and spindle towards the base of the mother cell. These cells often
become blocked in the cell cycle at this stage.
[View Larger Version of this Image (166K GIF file)]
Discussion
; Jacobs et al., 1988
). This stochastic process of microtubules searching for the bud via
dynamic instability is reminiscent of the "search and capture" mechanisms postulated for microtubule attachment
to kinetochores during prometaphase (Mitchison et al.,
1986
; Holy and Leibler, 1994
; Hyman and Karsenti, 1996
).
Fig. 10.
Schematic representation of astral microtubule dynamics throughout the yeast cell cycle. The nucleus is indicated in
blue, astral microtubules in green, and the intranuclear spindle
microtubules in red. In unbudded cells and cells with small buds,
the astral microtubules originate from the single SPB (G1, G1/S).
The astral microtubules are organized most often into a conical
array. Individual microtubules show asynchronous dynamic instability throughout the cell cycle with growth and shortening at
0.5 µm/min. As microtubules reach the cell cortex, continued
growth results in nuclear movement in the opposite direction.
Thus, astral microtubule growth pushes against the cell cortex to
propel the nucleus around the cell interior (G1/S-S/G2). The nucleus and/or microtubules make no stable contact in unbudded
cells. Once a bud has formed, the dynamically unstable microtubules penetrate the bud (S/G2). At this point, the nucleus moves
toward the neck of the budded cell (G2/M). The second pole
body is delayed in acquiring the dynein-GFP fusion protein
(S/G2-G2/M). Only after the pole bodies have separated, is the
second pole competent to nucleate astral microtubules (G2/M). Once both SPB's are labeled with dynein-GFP, and the nucleus is at the neck, biphasic spindle elongation ensues (M). Astral microtubules that contact the bud tip, shorten as the spindle elongates (Anaphase). Finally, when central spindle disassembly occurs
(Telophase), the astral microtubule dynamic instability and the
movements of the SPB and nucleus become typical of G1 cells.
[View Larger Version of this Image (43K GIF file)]
) or
high levels of dynein reflect recent observations that Kar3p,
a minus-end-directed, microtubule-based motor protein,
also influences the assembly of cytoplasmic astral microtubules (Saunders et al., 1997
). Loss of Kar3p results in increased number and average length of astral microtubule
arrays. Whereas the mechanisms leading to the generation of hyperelongated microtubules are likely to be different
in the dynein deficiency strain versus the cells overexpressing dynein-GFP, the regulation of microtubule-based motors clearly affects the distribution of astral microtubules
in fundamental ways. Hence, the disruption of microtubule dynamics by microtubule-based motors, when mutant
or when expression levels are modified, explains, in part,
the defects observed in nuclear migration, spindle alignment, and elongation into the bud.
). In the present study, seeing that the nucleus follows the microtubules, and measurements indicating that the microtubules shortened as the distance between the SPBbud and bud tip diminished, may provide additional evidence for pulling forces in the bud. Very few
stable microtubules were observed in the mother cells, and
microtubule shortening was not associated with migration
of SPBmother during anaphase. In contrast, astral arrays associated with SPBmother were displaced upon anaphase onset and often followed the pole body as the central spindle
elongated towards the distal end of the mother cell. Therefore, microtubule-dependent forces capable of pulling the
SPB must be transient in nature and confined primarily to
the emerging bud.
; Hyman, 1989
; White and
Strome, 1996
) and Pelvetia fastigiata (Allen and Kropf,
1992
) a nuclear rotation in early development has an essential role in determining the fate of progeny cells. Migration of spindle poles close to a site in the cell surface precedes asymmetrical cell divisions in oocytes and early embryos as well (Baker et al., 1993
). These events may be
related mechanistically to the orientation of the spindle in
budding yeast. A microtubule-based process of searching
that is mediated by dynamic instability to position the
spindle with respect to the nascent bud, or other cortical
sites may be a common feature of spindle and nuclear movements in a variety of cell types.
) point toward the notion that dynein-
dynactin complex in the bud may be mobile and physically
associated with the actin cytoskeleton. One of the most
pronounced asymmetries in yeast is the polarized distribution of actin into the growing bud (Lew and Reed, 1995
).
A mechanism that couples dynein with the actin cytoskeleton would account for dynein's ability to transiently stabilize cytoplasmic microtubules and polarize one SPB toward the bud. As described above, there may not be a
"single" site for microtubule capture in the bud. Rather,
cumulative transient interactions between dynein and dynactin, the actin cytoskeleton, and dynamic astral microtubules emanating from the SPB may provide the force, as
well as the mechanism that ensures spindle alignment and
that only one SPB is deposited into the bud.
).
Interestingly, several proteins that mark the bud site are
restricted both temporally and spatially to the tip of growing buds in yeast (Amberg et al., 1997
; Evangelista et al.,
1997
), and in asymmetrically dividing blastomeres of the
germline lineages in C. elegans (Etemad-Moghadam et al.,
1995
). These transient interactions may be critical in the
capacity for differential positioning within the cell and mechanisms that ensure the distribution of one and only
pole to the progeny.
Received for publication 12 May 1997 and in revised form 11 August 1997.
Address all correspondence to K. Bloom, Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280. Tel.: (919) 962-1182. Fax: (919) 962-1625. E-mail: ksb.fordham{at}mhs.unc.eduWe thank J. Kahana and P. Silver (Harvard University, Cambridge, MA) for constructing and generously supplying the bright and reliable Nuf2-GFP clone, E. Bi (University of North Carolina at Chapel Hill) for supplying the rabbit anti-GST antibodies, and S. Inoue for laboratory space at the Marine Biological Laboratory (Woods Hole, MA) at the inception of the imaging.
This work was supported by research grants from the National Institutes of Health General Medical Science (to K. Bloom and E.D. Salmon). E. Yeh was supported by the North Carolina Employment and Security Commission.
DIC, differential interference contrast; GFP, green fluorescent protein; GST, glutathione-S-transferase; SPB, spindle pole body.
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