3226
Research Article
The exocytic gene secA is required for Dictyostelium
cell motility and osmoregulation
Roberto Zanchi, Gillian Howard, Mark S. Bretscher and Robert R. Kay*
MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH, UK
*Author for correspondence ([email protected])
Journal of Cell Science
Accepted 5 June 2010
Journal of Cell Science 123, 3226-3234
© 2010. Published by The Company of Biologists Ltd
doi:10.1242/jcs.072876
Summary
We investigated the link between cell movement and plasma membrane recycling using a fast-acting, temperature-sensitive mutant of
the Dictyostelium SecA exocytic protein. Strikingly, most mutant cells become almost paralysed within minutes at the restrictive
temperature. However, they can still sense cyclic-AMP (cAMP) gradients and polymerise actin up-gradient, but form only abortive
pseudopodia, which cannot expand. They also relay a cAMP signal normally, suggesting that cAMP is released by a non-exocytic
mechanism. To investigate why SecA is required for motility, we examined membrane trafficking in the mutant. Plasma membrane
circulation is rapidly inhibited at the restrictive temperature and the cells acquire a prominent vesicle. Organelle-specific markers show
that this is an undischarged contractile vacuole, and we found the cells are correspondingly osmo-sensitive. Electron microscopy shows
that many smaller vesicles, probably originating from the plasma membrane, also accumulate at the restrictive temperature. Consistent
with this, the surface area of mutant cells shrinks. We suggest that SecA mutant cells cannot move at the restrictive temperature because
their block in exocytosis results in a net uptake of plasma membrane, reducing its area, and so restricting pseudopodial expansion.
This demonstrates the importance of proper surface area regulation in cell movement.
Key words: Cell motility, SecA, Exocytosis, Contractile vacuole, Dictyostelium, cAMP relay
Introduction
The plasma membrane, in cooperation with the cytoskeleton, has
a vital role in cell movement (Bretscher, 1996). It receives and
processes guidance signals from the environment (Van Haastert
and Devreotes, 2004; King and Insall, 2009; Swaney et al., 2010)
and also supplies attachments to the substratum. In neutrophils,
these attachments (integrins) are essential for migration on 2D
surfaces (Lammermann et al., 2008), and their endocytosis and
return to the cell surface at the leading edge might create membrane
flow (Lawson and Maxfield, 1995), which in turn could provide
motive force for the cell or assist in its polarisation (Bretscher,
1984; Valdez-Taubas and Pelham, 2003).
The plasma membrane also represents a physical constraint to
cell movement because, although fully flexible, it is barely
extensible (Mohandas and Evans, 1994). The surface area of
moving amoeboid cells is unlikely to remain constant, as such cells
often progress by cycles of expansion and rounding up (Wessels et
al., 1998), and can extend a pseudopod without first withdrawing
their tail (Weber et al., 1995). Measurement from confocal
reconstructions confirms that the surface area of moving
Dictyostelium cells continuously fluctuates, with increases of 30%
sometimes occurring over a few minutes (Traynor and Kay, 2007);
even greater increases are found in spreading mammalian cells
(Gauthier et al., 2009). As these changes are much greater than
membrane stretching allows, it seems likely that cells have evolved
mechanisms to increase their surface area on demand, such as
membrane ‘unwrinkling’ (Hallett and Dewitt, 2007), or by
regulation of their endocytic cycle (Kay et al., 2008).
Dictyostelium cells maintain an active endocytic cycle, taking
up their surface area once every 5–10 minutes, even when starving
(Aguado-Velasco and Bretscher, 1999; Traynor and Kay, 2007). To
test the importance of the endocytic cycle in cell movement, we
previously created temperature-sensitive mutants of the essential
nsfA gene, and found that they become paralysed within 30 minutes
at the restrictive temperature (Thompson and Bretscher, 2002;
Traynor and Kay, 2007). This is in stark contrast to most mutations
of the actin cytoskeleton, which have only a minimal impact on
cell motility (Noegel and Schleicher, 2000). NSF is required for
resolving SNARE complexes after vesicle fusion, and impairment
of its function is expected to inhibit all membrane fusion events in
the cell, resulting in severe perturbation in organelles such the
Golgi and endoplasmic reticulum, as well as blocking exocytosis
(Novick et al., 1980; Malhotra et al., 1988; Sudhof and Rothman,
2009). Since these perturbations might indirectly affect cell motility,
we sought a more specific tool to better dissect the role of
exocytosis in cell movement.
SecA is the Dictyostelium homologue of the yeast Sec1p and
mammalian Munc18 proteins. These proteins interact with exocytic
SNARE proteins during vesicle docking and fusion and so are
essential for exocytosis, but they do not appear to function at other
points in the secretary pathway (Carr et al., 1999; Grote et al., 2000;
Shen et al., 2007; Sudhof and Rothman, 2009). A library of
temperature-sensitive secA mutants was previously created by
replacing the endogenous gene with randomly mutated variants
using homologous recombination, and screening for lack of growth
at the restrictive temperature (Bretscher and Clotworthy, 2007).
Many of these mutants develop poorly at the permissive temperature,
but here we have identified a mutant that does develop and becomes
chemotactic to cyclic-AMP (cAMP). This mutant shows a striking
movement defect at the restrictive temperature and has a second
defect in contractile vacuole discharge. However, cells can still
polarise in response to cAMP, and relay a cAMP signal, showing
that many other cellular functions continue unimpaired; we argue
that the movement defect is due to surface area constriction.
SecA, exocytosis and motility
3227
Results
Journal of Cell Science
A temperature-sensitive secA mutant that is
developmentally competent
The Dictyostelium homologue of the Munc18/Sec1p proteins was
identified bioinformatically (Bretscher and Clotworthy, 2007). The
SecA primary structure is very similar to its homologues in higher
eukaryotes and is devoid of the low complexity repeats found in
many Dictyostelium proteins. Temperature-sensitive mutants of
SecA were obtained previously by mutagenic replacement of the
endogenous gene using homologous recombination (Bretscher and
Clotworthy, 2007). We found that that most of these are impaired
in early development even at the permissive temperature, making
them unsuitable for investigating chemotaxis to cAMP, which is a
developmentally acquired property. By re-screening this pool, we
found one strain, which carried the secA2 allele (resulting in seven
amino acid substitutions: M250L, A296V, M427L, S435R, K505N,
V552D, H570Q), where development was relatively normal at the
permissive temperature. The secA2 allele is fast acting, with visible
effects on cell morphology within 30 minutes at the restrictive
temperature (see later) and was reconstituted in the Ax2 wild-type
background by homologous recombination.
Cells of the resulting strain (HM1325) grew normally on
suspensions of bacteria at the permissive temperature of 18°C
(mean generation times: wild type5.7±0.5 hours; mutant5.3±0.5
hours; n3, ± s.e.m.), though they did not grow in shaken axenic
culture. However, at the restrictive temperature of 27.5°C, growth
completely stopped, both on bacterial lawns and in suspension
(mean generation times: wild type4.1±0.1 hours; mutant cells die,
halving every 18.2±3.4 hours; n3, ± s.e.m.), thereby demonstrating
that the gene is essential (Fig. 1A). This growth defect was
completely rescued in parasexual diploid strains harbouring one
copy of the wild-type gene (Fig. 1A), showing that the secA2 allele
is recessive. This indicates that the effect of the mutations is
through loss of SecA function, rather than through an indirect
dominant inhibitory effect of the mutant protein on other proteins.
The mutant cells remained intact at the restrictive temperature,
but rapidly rounded up and acquired prominent vacuoles (Fig. 1B),
which we show later to be undischarged contractile vacuoles.
Crucially, they did not lose viability for at least 4 hours at 27.5°C
(Fig. 1C), which is longer than used in subsequent experiments,
indicating that the acute effects on motility are not due to cell
death, and are reversible.
Cell motility requires SecA function
We compared the motility of mutant and wild-type cells at the
permissive and restrictive temperatures (18–20°C and 27.5°C,
respectively, with a 30 minute pre-incubation). Vegetative mutant
and wild-type cells move similarly at the permissive temperature
(not shown), but movement of the mutant is strongly suppressed at
the restrictive temperature (Fig. 2A). Only around 20% of the
mutant cells move more than 20 m from their initial positions
(Fig. 2B) and, because the average diameter of a Dictyostelium cell
is approximately 10 m, this means that most mutant cells still
overlapped their original position at the end of the experiment,
contrasting starkly with the highly motile wild type. This is reflected
in the average speed distribution, which peaked at 1.8 m/minute
in the mutant, whereas the mean for the much broader wild-type
distribution was 8.5 m/minute (Fig. 2C).
Similarly, chemotaxis to cAMP was strongly impaired in the
mutant at the restrictive temperature. In these experiments, mutant
and wild-type aggregation-competent cells were placed on opposite
Fig. 1. A temperature-sensitive mutant of SecA. (A)secA is an essential
gene. The temperature-sensitive mutant strain HM1325 (ts-mutant) carrying
the secA2 allele grows normally at the permissive temperature of 18°C, but not
at the restrictive temperature of 27.5°C. A diploid strain harbouring the mutant
and wild-type alleles grows normally at both temperatures, showing that the
mutant allele is recessive. Cells were picked onto bacterial lawns and
photographed after 4 days. Scale bar: 5 mm. (B)Cell morphology at
permissive and restrictive temperatures. Mutant and wild type are similar at
18°C. After 30 minutes at 27.5°C, mutant cells become rounded and heavily
vacuolated (arrowheads). Freshly starved cells were incubated at 18°C or
27.5°C for 30 minutes before imaging (Normarski). Scale bar: 10m. (C)Cell
survival at the restrictive temperature. The wild-type (solid line) and
temperature-sensitive (HM1325; dashed line) strains were incubated at the
restrictive temperature for up to 4 hours (over twice the duration of any assay
in this study) and viability determined clonal plating on bacterial lawns at the
permissive temperature. No significant change in viability was observed (n3,
error bars represent s.e.m.).
Journal of Cell Science
3228
Journal of Cell Science 123 (19)
Fig. 2. Random motility of vegetative SecA mutant cells is almost
abolished at the restrictive temperature. (A)Tracks of wild-type and SecA
mutant cells (HM1325; ts-mutant) at 27.5°C. The tracks of 100 randomly
sampled cells are shown centred at the origin. (B)Horizon plot. The fraction of
the population moving a given distance from the origin is plotted. More than
75% of mutant cells (lower curve) remain within 2 cell diameters (~ 20m)
and less than 5% reach a distance of 55m from their origin. By contrast, over
50% of the wild-type cells (upper curve) travel at least 85m from the origin.
(C)Speed distribution. The average mean speed distribution for wild-type
cells, white bars, and for the mutant, black bars, is shown. The mean speeds
are 8.5±1.6m/minute and 1.8±0.4m/minute for the wild type and mutant,
respectively. Error bars represent s.e.m., n3; wild type212 cells, temperature
sensitive mutant348 cells. Cells were washed free of bacteria and plated in
axenic medium and incubated for 30 minutes at 27.5°C before imaging for 30
minutes at 3 frames per minute.
sides of a Dunn chamber and filmed in parallel using a motorised
stage, so that they experienced the same gradient and temperature
conditions. It was apparent that the mutant was barely motile in
comparison with the wild type (Fig. 3A,B; supplementary material
Movie 1).
Conceivably, the poor motility of mutant cells at the restrictive
temperature might be due to a failure to deploy their actin
cytoskeletons effectively. We first tested their ability to polymerise
actin in response to a uniform cAMP stimulus, using the F-actin
reporter ABP120-GFP (Pang et al., 1998). The initial actin response
was very similar in mutant and wild-type cells, with F-actin levels
robustly peaking at 5–10 seconds and then falling off. F-actin
levels peaked for a second time in the wild type at 1–2 minutes,
but this peak was much reduced in the mutant (Fig. 3C). The
second peak correlates with pseudopod projection, but in our hands
it was very variable and often barely discernable even in the wild
type (Langridge and Kay, 2006; Hoeller and Kay, 2007).
Similarly, both mutant and wild type responded to a cAMP
gradient by polymerising actin on the up-gradient side of the cell.
In the wild type, this resulted in extension of a pseudopod and
movement up the gradient, whereas the mutant generally could not
produce a distinct pseudopod or move (Fig. 3D; supplementary
Fig. 3. Cells of the SecA mutant can sense chemotactic gradients at the
restrictive temperature, but barely move. (A)Tracks of wild-type and SecA
mutant cells in a cAMP gradient at the restrictive temperature of 27.5°C.
Tracks of aggregation-competent cells in a Dunn chamber (35 wild-type and
45 mutant cells) from paired samples are shown, centred at the origin; filmed
for 30 minutes at 3 frames per minute. Representative of four independent
experiments; see supplementary material Movie S1. (B)Speed distributions of
wild-type and SecA mutant cells. Taken from four independent data sets (124
wild-type cells, white bars; 158 mutant cells, black bars). Mean speeds are
wild type, 13.4±1.4m/minute; mutant, 2.0±0.3m/minute; error bars
represent s.e.m. (C)Actin polymerisation produced by uniform cAMP
stimulus. All cells display a robust peak 5–10 seconds after stimulation and a
more variable second peak about 60 seconds thereafter, which is similar to
bulk assays. Aggregation-competent cells were pre-incubated at 27.5°C for 30
minutes and then bath-stimulated with 10M cAMP at t0. Actin
polymerisation at the cortex of individual cells was monitored using ABD120GFP as reporter for F-actin, and fluorescence quantified using QuimP. Average
from 22 wild-type and 23 mutant cells from three independent experiments.
(D)Polarised actin polymerisation induced by a cAMP gradient. Wild-type and
mutant cells respond to cAMP gradients by polarised F-actin accumulation up
the gradient. This drives expansion of a pseudopod and cell movement in the
wild type at either temperature, but the SecA mutant can only extend a
pseudopod at the permissive temperature, producing a bulge or even remaining
rounded at the restrictive temperature (the extreme cell is from the same
population). Cells expressing ABD120-GFP were made aggregation competent
and either used immediately or shifted to the restrictive temperature for 30
minutes before imaging. They were stimulated using a needle filled with
cAMP (white dot). Scale bar: 10m; see supplementary material Movies 2
and 3.
material Movies 2 and 3); in the extreme example, the mutant cell
remained rounded and could do no more than slightly bulge towards
the needle, despite appropriate actin polymerisation.
SecA, exocytosis and motility
3229
We also considered two more trivial explanations for the
movement defect: that it might be due to the presence of the
undischarged contractile vacuole (see later), or loss of adhesion to
the substratum, as a result of cell rounding. We found that placing
cells in solutions of increasing osmolarity (KK2 supplemented
with 100–200 mM sorbitol) reduced the size of the contractile
vacuole, but did not restore wild-type motility (result not shown).
Similarly, pressing cells against the substratum with an agarose
overlay (Laevsky and Knecht, 2001), which can restore motility to
at least one poorly adhesive mutant (Langridge and Kay, 2007),
also did not rescue motility of the SecA mutant (not shown).
Journal of Cell Science
cAMP relay does not require SecA function
As well as performing chemotaxis, aggregation-competent cells
respond to cAMP by producing and releasing more cAMP (Shaffer,
1975). This relay response depends on many of the same signal
transduction components as chemotaxis and we therefore used it
as a more global test for impairment of mutant cell function. Since
the mechanism of cAMP release from the cell is not known, we
also asked whether release depends on SecA function.
It is clear from the measurements at the restrictive temperature
that SecA was not required for cAMP production. More
surprisingly, it was also dispensable for cAMP release, which
might reasonably have been assumed to be by vesicle exocytosis
(Fig. 4A). We microscopically checked the integrity of the cells at
the end of the experiments and found no difference from the wild
type. To confirm these findings, the same experiment was repeated
with the NSF temperature-sensitive mutant (Thompson and
Bretscher, 2002), with the same result (Fig. 4B).
Thus, SecA is required for efficient cell motility, but is not
required to transduce a chemotactic gradient into appropriate actin
polymerisation, or for the closely related signal transduction events
required for cAMP relay.
Fig. 4. cAMP relay by aggregation-competent cells. Cells were stimulated
with 15M 2⬘-deoxy cAMP and total cAMP produced over 5 minutes
(extracellular + cytosolic) and the proportion released determined. (A)SecA
mutant (means of five independent experiments; error bars represent s.e.m.).
(B)NsfA mutant (means of three independent experiments; error bars
represent s.e.m.). No significant differences were observed.
or C-terminal fusion proteins showed similar localisation patterns,
often strongly staining the plasma membrane at the rear of moving
cells (supplementary material Fig. S2, Movie 4). These results
reveal a complex SecA localisation pattern in vegetative cells and
a variable one in chemotaxing cells, where SecA can also be at the
front or back. Neither pattern is especially informative. We next
investigated membrane trafficking.
SecA localisation
To understand the connection between SecA and cell motility, we
first attempted to localise SecA protein. An affinity-purified serum
against full-length, recombinant SecA, which specifically
recognises SecA in western blots (supplementary material Fig.
S1), stained the plasma membrane of vegetative cells and puncta
within them. Cells chemotaxing to cAMP stained in patches at the
front and rear, often in the same cell, but also independently
(supplementary material Fig. S2). Cells with a knocked in SecA Cterminal GFP fusion were viable, but did not grow in axenic
medium, showing that the fusion was only partially functional, and
unfortunately the GFP was too faint to detect. Overexpressed N-
SecA is required for normal plasma membrane circulation
Plasma membrane circulation was measured in a continuous assay
using the lipophilic dye FM1-43, which becomes intensely
fluorescent when it partitions into the membrane (Betz et al., 1996;
Traynor and Kay, 2007). The initial fluorescence produced upon
mixing dye and cells gives a measure of collective cell surface
area, and the subsequent increase a measure of exocytosis, because
previously internal membrane becomes available for binding
(endocytosed membrane retains its dye and continues to fluoresce).
From Table 1 it is apparent that at the permissive temperature, the
wild-type and SecA mutant cells circulate their plasma membrane
Table 1. Exocytic rate of wild-type and SecA temperature-sensitive cellsa
Strain
Ax2
HM1325
Ax2
HM1325
Temperature
(°C)
Exocytic rate (minutes/cell
surface area equivalent ± s.e.m.)
n
Adjusted exocytic rate
(minutes/cell surface area equivalent)
18
18
27.5
27.5
6.2±1.0
7.7±0.7
6.2±1.2
12.3±2.1
4
4
4
4
7.4
11.3
9.7
>100
a
The rate of exocytosis to the plasma membrane was calculated from the initial rate of increase in FM1-43 fluorescence, and expressed as the time required to
externalise one cell surface area. There is no significant difference in rates between the wild type, Ax2 and the SecA mutant (HM1325, secA2) at 18°C,
exocytosis is disrupted in mutant cells incubated at 27.5°C for 30 minutes (P<0.05, n=4). The rates in the presence of the metabolic poison, 10 mM sodium azide
[0.046±0.017 (n=6) fractional area/minute at 18°C, and 0.081 (n=4) fractional area/minute at 27.5°C], were subtracted, giving the adjusted values in the final
column. As the residual for the mutant at 27.5°C is very small and within experimental error, only an approximate value is given. In this assay, it is assumed that
the dye is carried into cells along with internalised membranes and continues to fluoresce, and therefore that increases in fluorescence with time represent binding
to freshly exocytosed membrane.
3230
Journal of Cell Science 123 (19)
Journal of Cell Science
at similar rates, taking approximately 10 minutes to exocytose the
equivalent of their surface area, with no statistically significant
difference between them. At the restrictive temperature, the mutant
circulates its plasma membrane more slowly than the wild type: if
no corrections are made, the rate is approximately half (P<0.05)
that of the wild type; but if allowance is made for the increase in
fluorescence that occurs even in azide-poisoned cells, then it
appears that the endocytic cycle is almost completely suppressed
in the mutant. This indicates that normal plasma membrane
circulation requires the function of SecA.
SecA is required for discharge of the contractile vacuole
The most visible perturbation of the mutant membrane system at
the restrictive temperature is the presence of one, or more, large
translucent vesicles in each cell (Fig. 1B). Cells of the temperaturesensitive NSF mutant similarly round up at the restrictive
temperature, but do not produce these characteristic vesicles
(Traynor and Kay, 2007).
We investigated the origin of these vesicles using standard
markers for subcellular compartments. They were not stained by
markers for ER, Golgi, late endosome or plasma membrane, nor
was the distribution of these markers visibly disturbed in the
mutant. The markers used were: calnexin-GFP and calreticulinGFP for the ER (Muller-Taubenberger et al., 2001); GFP-golvesin,
GFP-⌬(1–75)golvesin, ⌬(1–75,119–579)golvesin-GFP for the
Golgi (Schneider et al., 2000; Gerisch et al., 2004); vacuolinBGFP for late endosomes (Jenne et al., 1998); and ACA-YFP and
cAR1-GFP for the plasma membrane (Kriebel et al., 2003; Xiao et
al., 1997); although the latter did faintly stain the vesicle (not
shown).
The only exception was the Dajumin-GFP construct, an integral
membrane protein that strictly defines the contractile vacuole
system (Gabriel et al., 1999); this was found to strongly stain all
the large vesicles and virtually no other structure (Fig. 5A).
The contractile vacuole fills with fluid and periodically
discharges by fusion with the plasma membrane. At this point it
becomes accessible to the external medium and can be back-filled
with a small dye, such as Alexa Fluor 594. When dye was added
to mutant cells at the start of their incubation at the restrictive
temperature, the contractile vacuole was accessible to the label;
but if addition was delayed for 30 minutes, the vesicle remained
unstained (Fig. 5B), indicating that SecA activity is required for
fusion of the contractile vacuole with the plasma membrane.
As the contractile vacuole is the organelle of osmoregulation,
we tested whether the SecA mutant is osmosensitive. Cells were
transferred to distilled water before or after incubation at 27.5°C.
Wild-type cells, or the SecA mutant at the permissive temperature
were able to effectively resist this osmotic shock, but not the
mutant at the restrictive temperature, where approximately 90% of
the cells died after 30 minutes (P0.003, n3; Fig. 5C). Thus we
conclude that the large vesicles are undischarged contractile
vacuoles.
Fig. 5. Contractile vacuole discharge requires SecA activity. (A)The large
vesicles formed at the restrictive temperature are undischarged contractile
vacuoles. SecA mutant cells, expressing Dajumin-GFP as a contractile vacuole
marker, were incubated for 30 minutes at the restrictive temperature of 27.5°C.
All large vesicles visible by DIC display the marker. Scale bar: 10m. (B)The
contractile vacuole ceases discharge at the restrictive temperature. When the
contractile vacuole (Dajumin-GFP, green channel) discharges, it becomes
accessible to back-filling by small bulk-phase tracers such as Alexa Fluor 594
(red channel). Cells were incubated with dye, either for the first 30 minutes at
the restrictive temperature (27.5°C), when it has access to the contractile
vacuole (top panels), or for the next 30 minutes, when it does not (lower
panels). In both cases, dye in the medium was washed away before observing
the cells. Scale bars: 10m. (C)The SecA mutant is osmo-sensitive at the
restrictive temperature. Cells were transferred from growth medium to water,
and after incubation for 30 minutes, their viability was determined by clonal
plating on bacterial lawns. Viability of the SecA mutant (ts-mutant 27.5°C)
was reduced to less than 10% at the restrictive temperature.
Journal of Cell Science
SecA, exocytosis and motility
3231
Small vesicles accumulate, which might originate from the
plasma membrane
Loss of SecA function results in reduced plasma
membrane area
Electron microscopy reveals a second perturbation of the SecA
mutant membrane system at the restrictive temperature: an
increased number of small vesicles in the cytoplasm (Fig. 6A; the
vesicles are taken as those less than 0.5 m diameter). In central
sections of the cell – those containing at least part of the nucleus
– their number nearly doubles from 0.68±0.42 per m2 of cytoplasm
in the wild type to 1.22±0.40 m2 of cytoplasm in the mutant (n8
sections, ± s.d.; the area of the nucleus and of large vesicles is
excluded from the calculation), accompanied by a marginal increase
in average diameter (193±48 nm to 266±24 nm). The smaller
vesicles are often found in the proximity of the plasma membrane
and sometimes in close juxtaposition with it. Apart from these
vesicles, other cellular compartments were not visibly changed in
the mutant.
To investigate the origin of the small vesicles, we used a
phosphotungstic acid and chromic acid stain (Fig. 6B). Although
the basis for the selectivity of this stain is not known, it strongly
stains the plasma membrane of the wild type and other membranes
to a lesser extent, but the contractile vacuole system is not stained
at all (Quiviger et al., 1978). Staining of the mutant plasma
membrane was reduced at the restrictive temperature, whereas a
number of internal vesicles became intensely stained. The simplest
explanation for this is that components from the plasma membrane
are sequestered into these vesicles when SecA is inactivated.
Two features of the mutant phenotype seem particularly relevant
to understanding its movement defect: the cells round up at the
restrictive temperature, and the number of small vesicles, apparently
originating from the plasma membrane, increases. Both features
suggest that the plasma membrane area decreases, and we supposed
that such a constriction might physically restrict the ability of a cell
to extend pseudopodia and therefore to move. We therefore
measured the surface area of cells after transfer to the restrictive
temperature, using cAR1-GFP as a surface marker, and
reconstructing the surface from confocal stacks.
As is apparent from Fig. 7, the surface area of mutant cells
shrunk by 20–30% during incubation for 30 minutes at the
restrictive temperature, whereas wild-type cells showed a transient
increase, before returning gradually to their initial value. Mutant
cells also decreased in volume, although to a lesser extent. Since
the surface to volume ratio of a sphere increases as it shrinks (3/r),
these measurements imply that the mutant cells shrink from a
shape with a higher surface to volume ratio than a sphere towards
a more sphere-like ratio.
Discussion
There is now a powerful genetic case that a functional endocytic
cycle is required for Dictyostelium cell motility. Cells of both the
NSF mutant studied previously (Thompson and Bretscher, 2002;
Fig. 6. Ultrastructure of SecA mutant cells. (A)Electron
micrographs of representative wild-type and mutant
aggregation-competent cells at the restrictive temperature
(27.5°C). (a–c) wild-type cell morphology. This is unchanged
by the temperature shift. (d–i) Mutant cell morphology at low
power, with selected regions at higher power. Two changes in
the endomembrane system are apparent: large vacuoles (Va) –
undischarged contractile vacuoles – appear, and the number of
small vacuoles increases. Often these cluster near the plasma
membrane (g and h), sometimes in tight juxtaposition (i).
Scale bars: 1m (f, for a–f; h, for g,h), 0.2m (i).
(B)Redistribution of plasma membrane components in the
SecA mutant at the restrictive temperature detected using
phosphotungstic acid and chromic acid stain. (a–c) Permissive
temperature. The plasma membrane stains more strongly than
any other membrane and the contractile vacuole does not
stain. (d–f) After incubation for 30 minutes at the restrictive
temperature. Staining of the plasma membrane is greatly
reduced, but a subset of large multi-vesiculated compartments
become strongly stained (d and e) and the small vesicles are
finely stained, suggesting that they also receive plasma
membrane material (f). Scale bars: 1m (d, for a and d; c, for
b and c; f, for e and f). Aph, autophagosome; MVC, multivesciculated compartments; N, nucleus; PM, plasma
membrane; Va, vacuole; Ve, vesicle.
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Journal of Cell Science 123 (19)
Journal of Cell Science
Fig. 7. Changes in surface area and volume of mutant cells at the
restrictive temperature. Vegetative cells expressing the membrane marker
cAR1-GFP were imaged at 27.5°C, taking confocal stacks every 5 minutes.
The volume and surface area measurements of wild-type (open circles) and
mutant cells (closed circles) are normalised to the initial values (t0, room
temperature). Error bars represent s.e.m. A significant decrease in surface area
could be detected in the mutant after 20 minutes at 27.5°C, resulting on
average in a ~25% reduction in surface area at 30 minutes. A small decrease in
the volume of the mutant is also noticeable, but this is not statistically
significant. *P<0.05, **P<0.01, ***P<0.001, two-tailed sample student’s ttest (n6 for the wild-type sample, n7 for the mutant sample, acquired over 6
different days).
Traynor and Kay, 2007), and the SecA mutant studied here, rapidly
become essentially immotile at the restrictive temperature. Since
the appearance of the movement defect is so rapid – it is apparent
within minutes and complete in around 30 minutes – it cannot be
due to long-term changes in gene expression, nor is it probably due
to defects in trafficking of newly synthesised proteins. Rather, it
seems likely that the endocytic cycle is directly required for
movement.
In both the SecA and NSF mutants, chemotactic signalling to
the cytoskeleton appears to be normal, and both can polymerise
actin at the up-gradient side of the cell. Therefore it is unlikely that
their movement defects are caused by a failure of signal
transduction, or of cytoskeletal mobilisation.
By contrast, the endocytic cycle of both mutants is substantially
blocked and they rapidly round up at the restrictive temperature,
resulting in the significant reduction in surface area shown here for
the SecA mutant. Although NsfA and SecA have very different
roles membrane trafficking – NsfA is expected to be essential for
all membrane-fusion events, whereas SecA is specifically required
in exocytosis – neither is directly required for endocytosis, at least
in yeast (Hicke et al., 1997). We therefore suggest the following
interpretation of the NsfA and SecA phenotypes. At the restrictive
temperature, exocytosis is substantially blocked, but endocytosis
runs on for a while, resulting in a net uptake of plasma membrane
and a decrease in surface area. As the surface area decreases,
membrane tension increases, so physically restricting the extension
of pseudopodia and inhibiting cell movement. It is notable that the
few mutant cells that do move at the restrictive temperature seem
to ‘shuffle’ with greatly reduced pseudopodia, presumably reflecting
the limited slack in the plasma membrane. In the converse situation,
where membrane tension is artificially decreased, cell extension is
correspondingly stimulated (Raucher and Sheetz, 2000).
Our attempts to understand how SecA is required for cell
movement revealed two other aspects of SecA biology. First,
neither SecA nor NSF is required for cAMP release into the
medium during cAMP relay. Relay is fundamental to aggregation,
because it allows cAMP waves to propagate from signalling centres
through a field of responsive cells, and guide their inward
movement. Electron microscopy reveals that signalling cells
accumulate and release small vesicles (Maeda and Gerisch, 1977),
suggesting that cAMP is released by a conventional exocytic
mechanism. Our work contradicts this idea and is consistent with
other work showing that cAMP is completely released from cells
by lysis in conditions not expected to break small vesicles (Schoen
et al., 1989) and that there is a build-up of free cAMP in the
cytoplasm during cAMP relay (Bagorda et al., 2009). It seems
likely that cAMP is released by a non-exocytic mechanism, possibly
through a membrane transporter. The small vesicles (Maeda and
Gerisch, 1977) might have some other function, perhaps serving as
a membrane reservoir for the regulation of surface area.
Second, SecA appears to be essential for discharge of the
contractile vacuole. The contractile vacuole cycle involves filling,
fusion with the plasma membrane, and emptying (Gerisch et al.,
2002; Heuser, 2006; Du et al., 2008). When SecA function is
impaired, the cells carry a prominent, engorged contractile vacuole,
which does not empty, suggesting that fusion with the plasma
membrane requires SecA and thus resembles other exocytic
processes. Fusion is believed to be ‘kiss and run’ and is not thought
to involve intermingling of the contractile vacuole and plasma
membranes, so it is unlikely that the contractile vacuole normally
contributes to surface area regulation, except perhaps in specific
cases (Yoshida and Inouye, 2001).
This work confirms the importance of a functional endocytic
cycle for cell movement. At the very minimum, the cycle must be
regulated to provide the cell with sufficient plasma membrane area
to project pseudopodia. The nature of this regulation remains open,
but we favour a model that involves membrane tension: if tension
becomes too high, extra membrane is supplied by exocytosis,
increasing the surface area and decreasing the tension; if it is too
low, membrane is withdrawn by endocytosis. The link between
membrane tension and the endocytic cycle might involve stretchoperated receptors in the plasma membrane, or perhaps direct
inhibition of endocytosis by increasing tension. Finally, the
availability of a fast-acting temperature-sensitive mutant in the
essential secA gene will be invaluable for future studies of
exocytosis and contractile vacuole discharge.
Materials and Methods
Cell procedures
Cells were grown with bacteria on SM agar plates at 18°C (Kay, 1987), freed of
bacteria by centrifugation in KK2 (16.5 mM KH2PO4, 3.8 mM K2HPO4, 2 mM
MgCl2, 0.1 mM CaCl2) and used immediately (vegetative cells) or made aggregation
competent by pulsing shaken cells with 50–90 nM cAMP every 6 minutes for 4–5
hours, starting at 1 hour of starvation. The secA2 allele was identified from a library
of mutants (Bretscher and Clotworthy, 2007) and recreated by homologous
recombination (Knecht and Pang, 1995) in the Kay laboratory stock of Ax2 wildtype cells, which has minimal duplications (Bloomfield et al., 2008).
Three independent strains (HM1323, HM1324, HM1325) all had the same secA2
allele of the secA gene, resulting in 7 amino acid changes: M250L, A296V, M427L,
S435R, K505N, V552D, H570Q. All behaved similarly and HM1325 was studied
further. It was transformed with ABD120-GFP (Pang et al., 1998) and GFP-SecA
[GFP S65T cDNA fused via a GGRGSEFKLLE linker to the N-terminus of the full
length secA cDNA and inserted into the pDXA-3C expression vector (Manstein et
al., 1995)] using G418 selection.
Diploid strain DM246 was created by crossing HM1325 and HM2068, a G418resistant strain expressing GFP (Thompson and Kay, 2000), and diploids selected
with 10 g/ml each of blasticidin and G418. Growth was measured in shaken
suspension cultures of heat-killed bacteria in KK2 and viability by plating cells
clonally on bacterial lawns at 18°C. Osmotic shock was produced by shifting cultures
from growth medium to de-ionised water.
Membrane exocytosis was measured from the initial rate of change in fluorescence
of cells incubated with 5 M FM1-43 (Molecular Probes) in a stirred cuvette
(470±2.5 nm excitation, 570±5 nm emission), normalised to the initial value on
mixing (Traynor and Kay, 2007). cAMP production was measured with 5 mM
SecA, exocytosis and motility
dithiothreitol to inhibit breakdown (Traynor et al., 2000); total cAMP is that produced
in 5 minutes following stimulation with 15 M 2⬘-deoxy cAMP minus that present
at t0, and released cAMP is the proportion in the supernatant after the cells are
rapidly centrifuged (5–10 seconds at 13,000 g).
Live cell imaging and analysis
Cells were imaged at ~2⫻105 cells/cm2 in Lab-Tek (Nalgene) chambered coverglasses
in axenic medium (vegetative) or KK2 (aggregation competent), on a Nikon Eclipse
TE300 inverted microscope with a Bio-Rad confocal system, or an Axiovert S100
with a motorised stage. Temperature was controlled by an ASI 400 Air Stream Stage
Incubator (Nevtek) plus a heated stage on the confocal system (Linkam MC60); cells
were pre-incubated at 27.5°C for 30 minutes as necessary. Randomly moving cells
were imaged every 20 seconds for 30 minutes. Cells were stimulated uniformly with
10 M cAMP or with a microneedle containing 1 M cAMP. Dunn chambers had
1 M cAMP in the chemoattractant reservoir and two cell populations on opposite
sides of the same coverslip were imaged in parallel (1 frame/20 seconds).
Films were processed by threshold binarisation and centroid tracking with ImageJ
(NIH) and MTrack2. Only tracks lasting for the entire movie were analysed for
random motility, and only those of at least 30 frames for chemotaxis. Parameters and
trajectories were extracted using custom scripts in the R statistical language
(http://www.R-project.org). In the actin polymerisation assays, fluorescence was
compared using QuimP plugin (Dormann et al., 2002).
Stacks for 3D reconstruction were acquired on an Olympus IX71 inverted
microscope equipped with a Perkin Elmer Ultra View RS spinning disk confocal
system, using a 60⫻ 1.20 NA water objective (Olympus) and piezoelectric focusing
device, taking images at 0.5 m intervals, and thus allowing a single volume to be
imaged in 2–4 seconds. Stage temperature was regulated as above. Images were
processed and reconstructed using Volocity (Improvision).
Journal of Cell Science
Immunofluorescence
Cells were fixed with formaldehyde and picric acid (Jungbluth et al., 1994), blocked
with 0.1% BSA in phosphate saline buffer, 0.05% Tween-20, incubated with affinitypurified rabbit polyclonal antibody against SecA (1:200, 1 hour at room temperature)
followed by a secondary anti-rabbit conjugated to Alexa Fluor 488 (Molecular
Probes, 1:500, 1 hour at room temperature). Phalloidin-TRITC (Fluka) was used
1:500 (30 minutes at room temperature).
Electron microscopy
Aggregation-competent cells were prepared in NS (20 mM MES, 20 mM KCl, 20
mM NaCl, 1 mM CaCl2, 1 mM MgCl2, pH 6.2) and fixed overnight on ice with
1% glutaraldehyde in the same buffer. Cells were washed, embedded in 2% agar
and osmicated (1% in NS, for 1 hour on ice), then washed in deionised water and
dehydrated in an ascending graded ethanol series. Ethanol was replaced with
propylene oxide before embedding in low viscosity resin (Agar Scientific Ltd.).
Sections of 60-80 nm (silver to pale gold iridescence) were post-stained with
saturated aqueous uranyl acetate (30 minutes) washed in deionised water, then
with Reynolds lead citrate (3-5 minutes) and washed. Alternatively, they were
stained with phosphotungstic acid and chromic acid for 30 minutes (Ryter and de
Chastellier, 1977). Sections were viewed on a Philips EM200 transmission electron
microscope with an 80 kV beam; vesicle quantification was carried out with
ImageJ (NIH).
We would like to acknowledge all members of the Kay group for
useful discussions and Gunther Gerisch for the dajumin-GFP construct.
Core funding was from the Medical Research Council. Deposited in
PMC for release after 6 months.
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/123/19/3226/DC1
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