Download End4/Sla2 is involved in establishment of a new growth zone in

Research Article
1843
End4/Sla2 is involved in establishment of a new
growth zone in Schizosaccharomyces pombe
Stefania Castagnetti1,*, Ralf Behrens2 and Paul Nurse3
1
Cell Cycle Lab Cancer Research UK, 44 Lincoln’s Inn Field, London, WC2A 3PX, UK
Paul-Ehrlich-Institute, Paul-Ehrlich-Straße 51-59, 63225 Langen, Germany
3
Rockefeller University, 1230 York Avenue, New York, NY 10021, USA
2
*Author for correspondence (e-mail: [email protected])
Accepted 7 February 2005
Journal of Cell Science 118, 1843-1850 Published by The Company of Biologists 2005
doi:10.1242/jcs.02311
Journal of Cell Science
Summary
The rod-shaped Schizosaccharomyces pombe cell grows
in a polarized fashion from opposing ends. Correct
positioning of the growth zones is directed by the polarity
marker Tea1 located at the cell ends where actin patches
accumulate and cell growth takes place. We show that the
S. pombe homologue of Saccharomyces cerevisiae SLA2,
a protein involved in cortical actin organization and
endocytosis, provides a link between the polarity marker
and the growth machinery. In wild-type fission yeast cells,
this homologue End4/Sla2 is enriched at cell ends during
interphase and localizes to a medial ring at cell division,
mirroring the actin localization pattern throughout the cell
cycle. Proper localization relies on membrane trafficking
Introduction
Many cellular activities including motility, differentiation and
division, depend on the ability of cells to become polarized, a
process that generally requires assembly of a polarized actin
cytoskeleton to target secretion, vesicular transport and growth.
Understanding how this polarization is brought about is a key
problem in cell biology.
In the rod-shaped yeast Schizosaccharomyces pombe,
polarity is expressed in the pattern of cell growth, which is
restricted to the ends of the long axis of the cell. This controlled
spatial organization depends on both the actin and microtubule
cytoskeletons. F-actin is required for polarized growth, whilst
the microtubular cytoskeleton is required to accurately position
the growth zones at the opposite ends of the cell. Actin is
organized in cortical patches associated with the growing
surfaces of the cell and interference with the actin cytoskeleton
completely disrupts cell polarity, resulting in cells becoming
round. In contrast, disruption of the microtubule cytoskeleton
results in mispositioning of the growth zones and leads to
alterations in cell morphology (Hagan, 1998; Sawin and Nurse,
1998). During interphase, microtubules are organized in
antiparallel bundles with the plus ends facing the tips of the
cell where they deliver the polarity marker, Tea1 (Drummond
and Cross, 2000; Hagan, 1998; Mata and Nurse, 1997). Cells
lacking Tea1 still polarize growth but either fail to properly
position the growth zones, which results in bending and
branching of cells, or grow only from one end, leading to
monopolar growth (Mata and Nurse, 1997).
and is independent of both the actin and microtubule
cytoskeletons. End4/Sla2 is required for the establishment
of new polarised growth zones, and deletion of its Cterminal talin-like domain prevents the establishment of
a new growth zone after cell fission. We propose that
End4/Sla2 acts downstream of the polarity marker Tea1
and is implicated in the recruitment of the actin
cytoskeleton to bring about polarised cell growth.
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/118/9/1843/DC1
Key words: End4/Sla2, fission yeast, Tea1, cell growth
In wild-type cells the pattern of growth changes during the
cell cycle. After cell division growth is monopolar, taking place
exclusively at the ‘old’ end, which was present in the previous
cell cycle. However once a minimal cell size requisite is met
and DNA replication is completed, growth is activated at the
opposite ‘new’ end that was generated at cytokinesis. This
transition from monopolar to bipolar growth is known as new
end take off or NETO (Mitchison and Nurse, 1985), and
represents a major morphogenetic transition in the fission yeast
cell cycle. We reasoned that understanding the mechanisms
underlying NETO might reveal new links between the actin
and microtubule cytoskeletons and illuminate their roles in the
spatial organization of the cell. Hence, we looked for
homologues of known regulators of actin dynamics that might
link actin organization to new end localization.
One such regulator is Sc-SLA2, which is essential in
budding yeast for the proper assembly and function of the
cortical actin cytoskeleton and for the establishment of cell
polarity (Holtzman et al., 1993). Sc-SLA2 binds actin in vitro
through its talin-like domain and partially colocalizes with
cortical actin patches at the growing surface of the cell
(McCann and Craig, 1997; Yang et al., 1999). Loss of Sc-SLA2
activity results in complete delocalization of cortical actin
patches (Holtzman et al., 1993) as well as defects in the
internalization step of endocytosis (Kaksonen et al., 2003;
Raths et al., 1993). Homologues of Sc-SLA2 have already been
identified in humans as huntingtin interacting proteins (Hip1
and Hip1R), and it has been suggested that Sla2-related
1844
Journal of Cell Science 118 (9)
proteins link the endocytic machinery to the actin cytoskeleton
(Engqvist-Goldstein et al., 1999). By interfering with
membrane-actin cytoskeleton interactions (Kalchman et al.,
1997), the loss of the huntingtin-Hip1R interaction might
contribute to the neurodegenerative disorder observed in
Huntington patients.
Here we show that the recently identified S. pombe
orthologue of Sc-SLA2 (End4/Sla2) (Iwaki et al., 2004) is
enriched at sites of actin accumulation in fission yeast, is
required for polarized cell growth and organization of the
cortical actin cytoskeleton and its localization at the new end
depends on Tea1. We conclude that End4/Sla2 is required at
the new end to organize or activate a new growth zone.
Journal of Cell Science
Materials and Methods
Strains and media
All S. pombe strains used in this study are listed in Table 1. Standard
methods were used for growth, transformation and genetic
manipulations (Moreno et al., 1991). All experiments, unless
otherwise stated, were performed in YES (yeast extract with added
250 mg/l histidine, adenine, leucine and uridine). sla2∆, sla2-GFP
and sla2∆talin were generated using a PCR-based method (Bahler et
al., 1998) with a kanMX template and oligos with 60-80 nt long
homology to sla2. Sla2R1083G was generated using the same
approach, with an oligonucleotide bearing the point mutation. All
deletions, mutations and tagging were performed in a diploid strain
and then sporulated to produce viable haploids. Deletions and
insertions were checked by PCR for presence of a KanMX cassette at
the right locus. Point mutants were confirmed by sequencing of
genomic DNAs.
Actin and FM4-64 staining and immunofluorescence
For actin staining, cells were fixed by adding formaldehyde (with a
final concentration of 3.7%) to the medium for 25 minutes, washed
twice with PEM (100 mM Pipes, 1 mM EGTA, 1 mM MgSO4, pH
6.9), permeabilized with 1% Triton X-100, washed twice with PEM
and stained with Alexa Fluor 488-phalloidin (Molecular Probes).
FM4-64 staining was performed as described by Brazer et al.
(Brazer et al., 2000). Briefly, cells were pelleted and resuspended in
YES containing 10 nM FM4-64 (Molecular Probes). Cells were
incubated at 4°C for 30 minutes, then washed, resuspended in fresh
YES medium at a concentration of 5×106 cells/ml and grown at 25°C.
Staining was monitored every 5 minutes for up to 2 hours using an
Axioplan Zeiss microscope (546 nm).
For immunofluorescence, cells were collected by filtration and fixed
in –80°C methanol for 8 minutes and processed as described
previously (Alfa et al., 1993). For microtubule detection, TAT1 (αtubulin; a kind gift from K. Gull) antibody was used at 1:200 dilution,
and Alexa Fluor 546-linked anti-mouse (Molecular Probes) at 1:1000
was used as secondary antibody. Photographs were taken using an
Axioplan Zeiss microscope.
Latranculin A, MBC and brefeldin A treatments
Cells were grown at 25°C in YES to OD595 0.5. Methyl-2benzimidazole-carbamate (MBC) and brefeldin A (BfA) were then
added to the medium to a final concentration of 25 µg/ml and 100
µg/ml, respectively. Sla2-GFP-expressing cells were fixed with –80°C
methanol and stained for tubulin. Fluorescence from Sla2-GFP is very
strong and was still visible after fixation. For latranculin A (latA)
treatment, Sla2-GFP-expressing cells were fixed to a coverslip coated
with lectin (100 µg/ml). Medium containing 40 µM latA was flushed
under the coverslip and time-lapse images were taken every 2 seconds.
Table 1. Strains used in this study
Strain
Genotype
Source
PN611
ura4-D18/ura4-D18. leu1-32/leu1-32,
ade6M210/ade6M216
h– 972
h– sla2::kanMX leu1-32 ura4-D18 ade6-M210
h– la2-GFP-kanMX ura4D18 ade6-210 leu1-32
sla2-GFP-kanMX, tea1::ura4D-18, ura4D-18
sla2-GFP-kanMX, tea1∆(200)-kanMX
h– sla2-GFP-kanMX, cdc10-129 leu1-32
h+ sla2∆talin-kanMX leu1-32 ade6-M216
ura4-D18
h+ sla2∆talin-kanMX, cdc10-129 ura4-D18
sla2R1083G-kanMX leu1-32 ade6-M210
h– sla2∆talin-GFP:kanMX
h– tea1::ura4+ ura4-D18 ade6-M210
h– cdc10-129
h– cdc25-22
sla2∆talin-kanMX, tea1::ura4+ ura4-D18
Lab collection
PN1
PN4392
PN4516
SC36
SC38
PN4519
PN4669
SC123
SC117
SC215
PN1733
PN17
PN7
SC236
Lab collection
This study
This study
This study
This study
This study
This study
This study
This study
This study
Lab collection
Lab collection
Lab collection
This study
For block and release experiments, cdc10-129 cells were blocked
for 3 hours (unless otherwise stated) at the restrictive temperature
(36°C). Following drug addition (at appropriate concentrations as
previously stated), cells were quickly released to the permissive
temperature (25°C) and samples for immunofluorescence and Sla2GFP were taken at different time points. For latA treatment, cells were
filtered following the block and resuspended in 1/10 of the original
volume in medium containing 40 µM latA. After 10 minutes cells
were released to permissive temperature and stained after 30 minutes.
LatA pulse experiments were performed as described by Rupes et
al. (Rupes et al., 1999). Briefly, cells were blocked at restrictive
temperature for 3 hours. Aliquots were filtered and resuspended in
1/10 volume of medium containing 40 µM latA for 6 minutes at 36°C.
LatA was then washed out by filtration, cells were washed with
preconditioned medium and resuspended in fresh medium. Cells were
fixed for actin staining at 30-minute time intervals.
Results
End4/Sla2 is involved in actin organization
We identified the sla2 gene in the S. pombe genome database
by homology with the S. cerevisiae sla2 gene. S. pombe sla2
(SPAC688.11) encodes a protein with a predicted molecular
mass of 123 kDa. Sequence alignment with other Sla2-related
proteins showed that End4/Sla2 shares 36% similarity with ScSLA2 and 26% similarity with human HIP1R. End4/Sla2 has
the same domain structure and organization as the other Sla2related proteins, namely an N-terminal ENTH (epsin Nterminal homology) domain, a central coiled coil region, and
a C-terminal talin-like domain. The talin-like domain contains
the I/LWEQ-module predicted to bind to F-actin (McCann and
Craig, 1999).
sla2 was deleted in a wild-type diploid using a PCR-based
approach (Bahler et al., 1998). As in S. cerevisiae, sla2∆ cells
were temperature sensitive for growth; cells failed to grow at
36°C and at 32°C, and grew slowly at 25°C with a doubling
time of about 6 hours, more than twice that of wild-type cells
(2 hours 50 minutes). sla2∆ cells grown at 25°C were wider
than wild-type cells and showed a variety of growth patterns:
cells were often branched, swollen in their middle regions or
exhibited a complete loss of polarity (Fig. 1).
sla2∆ cells had an aberrant actin cytoskeleton. In wild-type
interphase cells, F-actin is organized in polarized cortical
patches enriched at growing tips and in thin cables running
Journal of Cell Science
End4/Sla2 and polarized growth
1845
along the main axis of the cell
(Marks and Hyams, 1985). In
contrast, about half of sla2∆
cells
at
25°C
showed
misoriented cables (data not
shown)
and
disorganized
cortical structures, with actin
patches delocalized throughout
the cell body (Fig. 1C). In the
cells, which maintained the
normal rod shape at 25°C, actin
patches were observed only at
one end (data not shown). After
shifting to 36°C for 2 hours,
sla2∆ cells became round and
swollen and the actin patches
Fig. 1. sla2∆ cells have an aberrant morphology. (A) Morphological phenotype, (B) microtubules, (C)
became completely delocalized
distribution of actin patches, (D) Tea1-GFP localization in wild type (upper panels) and sla2∆ (lower
panels) grown at 25°C. Bar, 5 µm.
around the cortex, and medial
rings could no longer be
detected (Table 2). sla2∆ cells
to F-actin (McCann and Craig, 1999). Cells bearing this point
with extreme polarity defects had microtubules curling under
mutation (Sla2R1083G) in End4/Sla2 showed NETO defects;
the cortex (data not shown). However, at 25°C cells that still
62% of cells grew in a monopolar fashion with a monopolar
retained polarity showed a normal microtubule cytoskeleton,
actin pattern (Fig. 2A).
with three to four bundles running along the main axis of the
To test if the talin-like domain plays an essential role in the
cell (Fig. 1B). In these cells, Tea1 accumulated at opposite
switch to bipolar growth, we checked whether it was possible
ends, as in wild-type cells (Fig. 1D).
to promote NETO in sla2∆talin cells by making actin
To investigate further the role of End4/Sla2 in actin
monomers available in the cell. A pool of free actin monomers
organization, we generated a talin deletion of End4/Sla2. As
could be generated with a pulse of latranculin A (latA) (Rupes
the talin-like domain is dispensable for most Sc-SLA2
et al., 1999). Cdc10-129 and cdc10-129 sla2∆talin cells were
functions in S. cerevisiae (Wesp et al., 1997), we reasoned that
blocked for 2 hours at restrictive temperature (36°C). After the
this deletion might specifically interfere with actin regulation.
block, cells of both genotypes showed a monopolar actin
A strain bearing a talin-truncated version of End4/Sla2
distribution: 89% in cdc10-129 and 98% in cdc10-129
(sla2∆talin) was viable at all temperatures and grew at rates
sla2∆talin cells (Fig. 3A). Cells were than treated for 6 minutes
similar to those of wild type. As in budding yeast (Wesp et al.,
with 40 µM latA, which completely disrupted actin localization
1997; Yang et al., 1999), no obvious difference in bulk lipid
(Fig. 3B). After washing out the drug, actin localization was
endocytosis (by up-take of the lipophilic steryl dye FM4-64)
followed for up to 120 minutes at 36°C. Within 1 hour most
was detected between wild-type and sla2∆talin cells (data not
cdc10-129 cells showed a bipolar actin distribution, whereas
shown). As shown by calcofluor staining (Fig. 2B), the only
most cells bearing the talin deletion in End4/Sla2 had
defect detected in this strain was that most cells grew from only
monopolar actin patches. As shown in Fig. 3A, 2 hours after
one end. Like wild-type cells, sla2∆talin cells had actin cables
wash out of the drug, 76% of cdc10-129 cells showed a bipolar
running along the main axis of the cell and cortical actin
actin distribution compared to 22% of cdc10-129 sla2∆talin
patches at growing ends. Unlike the wild-type population,
cells. These results suggest that End4/Sla2 talin-like domain
where only 15% of cells were in a pre-NETO state and showed
plays an essential role in the switch from monopolar to bipolar
monopolar actin, 78% of sla2∆talin cells had a monopolar
growth and in the organization of cortical actin patches at the
distribution of cortical actin patches (Fig. 2A,B). The growth
new end.
pattern of sla2∆talin cells was further analysed by time-lapse
phase-contrast microscopy. In wild-type cells after fission, both
daughter cells activate growth from the old end (growing in the
Table 2. Actin distribution in wild type and sla2∆ cells
previous cycle). In contrast, in 53% of sla2∆talin cells one
daughter cell grew from the old end and one from the new end
Wild type
sla2∆
(Fig. 2D).
25°C
36°C*
25°C
36°C*
Deletion of the talin-like domain did not affect End4/Sla2
Rings
23%
20%
19%
–
protein levels (Fig. 2C). Moreover, sla2∆talin cells showed no
Cortical
77%
80%
81%
100%
obvious defect in bulk lipid endocytosis, using the FM4-64
Monopolar†
26%
32%
36%
9%
Bipolar‡
74%
68%
8%
–
uptake assay (data not shown). Thus as in S. cerevisiae, the C§
Delocalized
–
–
56%
91%
terminal talin-like domain is dispensable for most End4/Sla2
functions. However, in S. pombe the End4/Sla2 talin-like
*Actin distribution after 2 hours at 36°C.
†
domain is required for activation of growth at the new end, and
Actin only at one end. ‡Actin at opposite cell ends. §Actin patches
possibly mediates recruitment of actin to the new end.
throughout the cell.
Cells with monopolar and bipolar actin distribution had a rod-shaped
Further support for this was obtained by mutating arginine
appearance.
400 cells were scored in two independent experiments.
1083, which abolishes binding of the isolated I/LWEQ-module
Journal of Cell Science
1846
Journal of Cell Science 118 (9)
individual Sla2-GFP patches revealed a
lifetime of 110-150 seconds. During early
mitosis, Sla2-GFP dots form a ring in the
middle of the cell, overlying the nucleus,
which persists until the end of mitosis
(Fig. 4A). Upon cell division, Sla2-GFP
localization was re-established at the old
end. Time-lapse movies (data not shown) of
single cells expressing Sla2-GFP showed
that after cell division only a few dots were
left at the new end. Shortly after division,
Sla2-GFP quickly accumulated again at cell
ends, giving rise to the bipolar interphase
pattern (data not shown). To determine
whether End4/Sla2 accumulates only at
growing cell ends, we examined the pattern
of Sla2-GFP localization in the temperaturesensitive cell cycle mutants cdc10-129 and
cdc25-22 (Mitchison and Nurse, 1985),
which block before and after activation of
bipolar growth, respectively. When shifted
to the restrictive temperature, cdc10-129
cells blocked, before NETO, with
monopolar actin and monopolar Sla2-GFP
distribution (Fig. 4D). Conversely, cdc25-22
cells, at restrictive temperature, blocked
Fig. 2. End4/Sla2 talin-like domain is required for actin organization at new growth zones.
entry into mitosis and accumulated as long
(A) Schematic organization of End4/Sla2 mutant proteins generated in these study and
rod-shaped cells with bipolar actin and
percentage of cells with monopolar growth in each strain. (B) Wild-type (left) and
bipolar Sla2-GFP (data not shown). We
sla2∆talin (right) cells were stained with calcofluor (upper panel) and for actin (lower
conclude that End4/Sla2 has a distribution
panel). (C) Western blot analysis of total extracts from wild-type, Sla2-GFP and
that mirrors that of the cortical actin
Sla2∆talin-GFP strains with α-GFP (top) and α-tubulin (bottom) antibody. (D) Patterns of
patches, accumulating at actively growing
growth of sla2∆talin, tea1∆ and sla2∆talin tea1∆ cells after division.
tips during interphase and re-localizing to a
central ring prior to mitosis. However, coEnd4/Sla2 is enriched at sites of actin accumulation
staining of Sla2-GFP and actin showed only a partial
colocalization of End4/Sla2 dots and actin patches (Fig. 4D,E).
We next determined the localization of End4/Sla2 in the cell
The proper location of new growth zones relies on the
throughout the cell cycle by using a C-terminal Sla2-GFP
polarity marker Tea1, which defines cell geometry by marking
fusion expressed from the sla2 endogenous promoter. The GFP
sites of cell growth (Mata and Nurse, 1997). Double labelling
fusion protein is functional, as Sla2-GFP fully rescued the
of Sla2-GFP-expressing cells with GFP and Tea1 antibodies
defects observed in sla2∆ cells. During interphase, Sla2-GFP
showed some colocalization of the two proteins at cell tips
accumulated in cortical dots at both cell ends (Fig. 4A) at the
(Fig. 4F). As End4/Sla2 also associates closely with the actin
site of cell growth and actin patch accumulation. Evaluation of
cytoskeleton
and
the
growth
machinery, we tested whether its
localization depends on Tea1. The
central accumulation of Sla2-GFP at
the ring was unaffected in tea1∆ cells,
whereas the end localization of Sla2GFP was abnormal. Instead of the
Fig. 3. End4/Sla2 talin-like domain is
required for establishment of a new
growth zone. (A) Schematic
representation of latA pulse experiment
(top) and scoring (bottom) of cells with
monopolar actin distribution before the
treatment (grey) and 2 hours after wash
out (black). (B) Cells before (top), during
(middle) and 2 hours after latA treatment
(bottom): cdc10-129 (left) cdc10-129
sla2∆talin (right). Bar, 5 µm.
End4/Sla2 and polarized growth
1847
Journal of Cell Science
µg/ml MBC, which depolymerizes microtubules, or 40 µM
latA, which sequesters actin monomers leading to complete
disassembly of the actin cytoskeleton. Cells were blocked at
the restrictive temperature of 36°C for 3 hours to allow most
cells to accumulate in G1 with monopolar Sla2-GFP. Cells
were then released to the permissive temperature in MBC or
latA to allow cells to proceed through the cell cycle in the
absence of microtubules or F-actin structures, respectively. As
shown in Fig. 5, neither the MBC nor the latA treatment
impaired Sla2-GFP accumulation at the new end. Bipolar
accumulation of Sla2-GFP was also observed when cells were
treated with both MBC and latA together, excluding the
possibility of a redundant dependence between the two systems
(Fig. 5). Taken together these observations suggest that
End4/Sla2 end localization does not directly require the actin
and microtubular cytoskeletons.
Fig. 4. End4/Sla2 localization in vivo. (A) Sla2-GFP accumulation
(green) at cell ends during interphase (top) and in a medial ring over
the nucleus (middle), which persists until the end of mitosis (bottom).
The nucleus is stained with DAPI (blue). (B) Central accumulation of
Sla2-GFP is unaffected in tea1∆ cells (arrow). Sla2-GFP is enriched
only at the growing end in tea1∆ and (C) in tea1∆(200) cells. (D)
Accumulation of Sla2-GFP (bottom) and actin (top) at the growing end
in cdc10-129 cells. (E) Staining of Sla2-GFP-expressing cells with αactin and (F) α-Tea1 antibody. Bar, 5 µm.
normal bipolar localization, most cells showed monopolar
Sla2-GFP accumulation at the growing end (Fig. 4B)
throughout the cell cycle, suggesting a role for Tea1 in proper
End4/Sla2 localization. However, because Tea1 influences
microtubular organization, the aberrant pattern of Sla2-GFP
localization could also be an indirect consequence of the
defective microtubular array observed in tea1∆ cells. To
distinguish between these two possibilities, we examined the
pattern of Sla2-GFP localization in tea1∆(200) cells, which
show a wild-type array of microtubules but fail to accumulate
the truncated version of Tea1 at cell ends (Behrens and
Nurse, 2002). tea1∆(200) cells showed monopolar Sla2-GFP
accumulation which occurred only at growing ends, even
though microtubules were normal in these cells (Fig. 4C).
Hence, Tea1 is necessary to recruit End4/Sla2 to the new end
where growth has not previously occurred, independently of its
effect on microtubules.
To further test if the accumulation of End4/Sla2 at cell ends
required the microtubule or actin cytoskeletons, we performed
a cdc10 block and release experiment in the presence of 25
End4/Sla2 localization depends on vesicular traffic
In fission yeast, membrane trafficking is required for proper
actin ring dynamics and affects its localization (Pardo and
Nurse, 2003). The presence of a ENTH domain, shown to be
involved in membrane binding (Ford et al., 2001; Itoh et al.,
2001) at the C terminus of End4/Sla2, and the tight association
of End4/Sla2 with the cortical membrane after latA treatment,
led us to investigate whether membrane trafficking is also
involved in localizing End4/Sla2 at cell ends.
Brefeldin A (BfA) blocks protein secretion by blocking
vesicular transfer from the endoplasmic reticulum to the Golgi
and therefore disrupting the Golgi apparatus. Treatment with
BfA does not affect Tea1 localization (Mata and Nurse, 1997),
but it caused Sla2-GFP to disappear completely from cell tips,
becoming randomly distributed throughout the cell within 5
minutes (Fig. 6Ac), suggesting that End4/Sla2 accumulation at
cell ends might be mediated by active vesicular trafficking.
Conversely, treatment with 40 µM latA (Fig. 6Ab) for up to 1
hour did not affect Sla2-GFP end localization. Similarly,
deletion of the actin binding talin-like domain has no effect on
End4/Sla2 localization. As shown in Fig. 6Ba, Sla2∆talin-GFP
accumulates at both cell ends. Co-staining of Sla2∆talin-GFP
and actin (Fig. 6C) shows accumulation of Sla2∆talin-GFP at
both growing and not-growing ends.
However, similarly to budding yeast where latA treatment
abolishes Sc-SLA2 movement (Kaksonen et al., 2003), the
rapid and apparently random movement of Sla2-GFP dots near
the cell cortex was completely blocked by addition of 40 µM
latA to the medium (Fig. 6D). BfA-dependent delocalization
of Sla2-GFP was also suppressed by the simultaneous addition
of 40 µM latA (Fig. 6Ad), as well as by the deletion of the
talin-like domain (Fig. 6Bc). Thus End4/Sla2 localization
requires active vesicular trafficking and is only maintained at
cell ends for a short time of around 5 minutes. We conclude
that rapid removal of End4/Sla2 from cell ends requires the
actin cytoskeleton and the talin-like domain.
Discussion
We have identified End4/Sla2 as a protein required to recruit
actin and the growth machinery to the new cell end in fission
yeast. Phenotypic analysis of sla2∆ mutants showed that this
gene is required for proper actin organization and cell
Journal of Cell Science
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Journal of Cell Science 118 (9)
domain of End4/Sla2 was deleted. Our analysis of the
sla2∆talin strain instead uncovered a novel function
for End4/Sla2 in establishment of new growth zones in
fission yeast. Moreover, in contrast with previous work
in S. cerevisiae (Yang et al., 1999) and in Candida
albicans (Asleson et al., 2001), which attributed only
a marginal role to the talin-like domain, our data
suggest a role for the talin-like domain in the
recruitment of actin and activation of growth at new
cell ends. Sla2∆talin cells are, in fact, NETO defective,
with monopolar actin patches. End4/Sla2 talin-like
domain mutant cells fail to activate growth at the new
end but do polarize growth at the old end and grow in
a monopolar fashion. Unlike ssp1∆ cells, which can
activate bipolar growth upon latA treatment (Rupes et
al., 1999), sla2∆talin cells have lost their intrinsic
ability to activate growth at the new end, and their
NETO defect cannot be overcome by an artificial
increase in free actin monomers. Hence End4/Sla2 has
an essential role in linking actin organization and
growth. Since the truncated protein localizes to both
the growing and the non-growing end, however, the
role played by the talin-like domain might be restricted
to establishment of bipolar growth.
As the talin-like domain binds actin both in vitro
(McCann and Craig, 1999) and in two-hybrid assays
(Yang et al., 1999), it is tempting to suggest that
End4/Sla2 acts downstream of the polarity machinery
at the interface with actin, and that the NETO defect
seen in sla2∆talin cells is due to the inability of the
truncated
protein to either polymerize or stabilize actin
Fig. 5. Localization of Sla2-GFP is independent of the actin and microtubule
at the new end despite the presence of polarity
cytoskeletons. (A) cdc10-129 cells expressing Sla2-GFP were grown at 25°C
markers. However, given that it has also been
and then transferred to 36°C for 3 hours (left). After addition of DMSO or 40
suggested that the talin-like domain is required for
µM latA or 25 µg/ml MBC, or 25 µg/ml MBC plus 40 µM latA (indicated by
an arrow in the scheme) respectively, cells were released to permissive
protein-protein interaction and regulation of Sc-SLA2
temperature (25°C) for an hour in the presence of the drug and then analysed
activity (Yang et al., 1999), an alternative explanation
for Sla2-GFP localization (right panels). (B) Quantification of cells with
could be that the truncated form is inactive or unable
bipolar actin distribution at 36°C (black bar) and 30 minutes after shift down
to interact with the growth machinery.
to 25°C (white and grey bars) in the presence of the different drugs. Bar, 5 µm.
Full deletion of Sla2/End4, which affects
endocytosis as well as actin organization, leads to
complete loss of polarity, whereas deletion of the talinmorphogenesis. Cells lacking End4/Sla2 have an aberrant
like domain, which does not interfere with bulk endocytosis,
shape and a depolarized cortical actin cytoskeleton. This
does not affect polarized growth but does influence
phenotype is reminiscent of that shown by deletion of Sc-sla2.
establishment of new sites of polarized growth. Interestingly in
In budding yeast, sla2∆ cells are spherical and have delocalized
budding yeast, sla2-6 mutant cells, which, unlike sla2∆ cells,
cortical actin structures, with aggregated actin patches
grow well at all temperatures and show polarized actin, exhibit
dispersed throughout the cytoplasm (Holtzman et al., 1993).
a bipolar budding defect (Yang et al., 1997). This suggests that
Deletion of Sc-sla2 also caused defects in endocytosis (Wesp
in both fission and budding yeast Sla2 might have two
et al., 1997). Sc-SLA2 is involved in the internalization step of
independent functions: to maintain polarized cell growth
endocytosis (Kaksonen et al., 2003; Raths et al., 1993), but
(requiring the full length protein) and to promote NETO/distal
does not require its C-terminal talin-like domain for bulk lipid
budding. Formally, however it is also possible, at least in S.
endocytosis (Baggett et al., 2003). Recent work by Iwaki et al.
pombe, that End4/Sla2 is required only for establishment of
showed that in fission yeast, End4/Sla2 is also required for
polarized growth, with activation at the new end requiring a
endocytosis (Iwaki et al., 2004). Deletion of the end4/sla2 gene
higher level of activity and therefore being more readily
inhibits delivery of FM4-64 to the vacuolar membrane,
affected by alteration of the protein, such as the deletion of the
accumulation of luciferase yellow CH and internalization of
talin-like domain. As previously suggested for Orb3, Orb8 and
the membrane protein Map3 (Iwaki et al., 2004). Although we
Orb9 (Snell and Nurse, 1994; Verde et al., 1995), End4/Sla2
cannot exclude a defect in receptor-mediated endocytosis as is
might be required to reorganize the actin cytoskeleton after a
observed in S. cerevisiae at elevated temperatures (Baggett et
major cell cycle transition, such as mitosis. Remarkably the
al., 2003), we observed no defects in fluid phase endocytosis
general loss of polarization observed in sla2∆ cells resembles
(assayed by FM4-64 uptake) when the C-terminal talin-like
that shown by orb mutants at restrictive temperature (Snell and
Journal of Cell Science
End4/Sla2 and polarized growth
Fig. 6. Sla2-GFP localization depends on vesicular transport.
(A) Sla2-GFP cells were treated with (a) ethanol and DMSO, (b) 40
µM latA (10 minutes at 25°C), (c) 100 µg/ml of brefeldin A (BfA)
(10 minutes at 25°C), or (d) 100 µg/ml BfA and 40 µM latA (10
minutes at 25°C). BfA treatment delocalizes Sla2-GFP in 5 minutes.
Sla2-GFP de-localization is abolished by co-treatment with latA.
(B) Localization of Sla2∆talin-GFP (a) and calcofluor staining (b).
Sla2∆talin-GFP cells treated with BfA at 25°C for 10 minutes (c).
(C) Staining of Sla2∆talin-GFP-expressing cells with Rhodamine
phalloidin. (D) Sla2-GFP local movement is abolished by treatment
with 40 µM latA. Arrowheads point to the same GFP dot in different
frames of a time-lapse recording (see Movie 1 in supplementary
material; +latA frames are from Movie 2). Bar, 5 µm.
Nurse, 1994; Verde et al., 1995). Interestingly most of the orb
genes identified so far encode protein kinases and Sc-SLA2 is
phosphorylated (Ficarro et al., 2002) in logarithmically growing
cells. This phosphorylation has been suggested as a potential
regulatory switch to trigger End4/Sla2-actin interaction at the
cell cortex (Yang et al., 1999) making End4/Sla2 a candidate
downstream target of the Orb morphogenetic pathway.
In the rod-shaped fission yeast it is thought that the plus ends
of microtubules define cell ends by delivering polarity markers
such as Tea1 (Mata and Nurse, 1997). End4/Sla2 in also
enriched at cell ends during interphase and is therefore a good
1849
candidate to play a direct role in the recruitment/assembly of
cortical polar actin. Consistently, End4/Sla2 end localization is
independent of microtubules but requires Tea1 to recognize the
end where growth has not previously occurred. Cells lacking
Tea1 do not accumulate End4/Sla2 at the new end and fail to
undergo NETO. Therefore our work places End4/Sla2 as a link
between microtubules and the machinery involved in sensing
overall cell space and the actin cytoskeleton, which leads to
polarized growth. In this model Tea1 would mark new sites of
cell growth and recruit End4/Sla2, which in turn could initiate
accumulation of cortical actin.
Tea1 and End4/Sla2 only partially colocalize at cell ends.
However, if Tea1 is only required for the initial recruitment of
End4/Sla2 but not for its maintenance at cell ends, their
interaction could be only transient, and no longer be necessary
once the End4/Sla2-cortex interaction was established. The
functional interaction between Tea1 and End4/Sla2 might
reflect a direct physical interaction, and consistent with this we
could detect a direct interaction between the C terminus of
Tea1 and End4/Sla2 in a two-hybrid assay (our unpublished
observation), although we could not confirm it in vitro in fission
yeast cell extracts. It is also possible that End4/Sla2 acts in
conjunction with other factors such as Bud6, a factor required
for NETO and shown to physically associate with Tea1 (Glynn
et al., 2001). Interestingly, bud6∆ and sla2∆talin show the same
growth patter (Glynn et al., 2001). Moreover, the proper
localization of both Bud6 (Jin and Amberg, 2000) and
End4/Sla2 (BfA experiment, this study) is mediated by the
secretory pathway and requires Tea1. Interestingly, it was
postulated that the secretion and endocytosis might be coupled:
the secretory pathway being required to replenish components
needed at the plasma membrane for endocytosis (Riezman,
1985). However, S. cerevisiae end3 mutant cells, which are
defective in endocytosis, still grow and therefore have an active
secretion pathway (Raths et al., 1993). Although the coupling
might not be essential, a functional link might still exist.
Localized secretion may be required to accumulate proteins at
sites of growth, and localised endocytosis could define and
delimit the growth zone to cell tips by removing excess factors
from neighbouring areas. In this scenario, depolarized
endocytosis would result in loss of cellular domains and
consequently in loss of polarity, as seen in sla2∆ cells.
Studies in other organisms have shown that, as in fission
yeast, Sla2 homologues are enriched in subcellular regions,
which show elevated membrane turnover and actin
accumulation. Hip1, the human homologue of Sla2,
colocalizes with and binds to clathrin and adaptor protein-2
(AP2) (Engqvist-Goldstein et al., 2001; Mishra et al., 2001),
both highly enriched at presynaptic nerve terminals, where
they facilitate rapid retrieval of synaptic vesicles (De Camilli
et al., 2001; Sun et al., 2002). Hip1 was identified through its
interaction with huntingtin (Kalchman et al., 1997; Wanker
et al., 1997), a protein containing a polyglutamine stretch.
Polyglutamine expansion causes a decrease in the Hip1huntingtin interaction and is associated with the onset of
Huntington’s disease (HD). Huntingtin, like Tea1, associates
with microtubules (Gutekunst et al., 1995; Hoffner et al.,
2002; Muchowski et al., 2002; Tukamoto et al., 1997), and
so Sla2/Hip1 family proteins may have a conserved role
working at the interface between the actin and microtubular
networks.
1850
Journal of Cell Science 118 (9)
We thank all members of the Nurse lab for help and discussion.
Special thanks to J. Hayles, K. Leonhard, R. Carazo-Salas and M.
Pardo for valuable comments on the manuscript. S.C. was supported
by a Marie Curie postdoctoral fellowship and by a CRUK postdoctoral
fellowship. R.B. was supported by ICRF and by a Boehringer
Ingelheim Fonds fellowship.
Journal of Cell Science
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