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Endosomal recycling controls plasma membrane
area during mitosis
Emmanuel Boucrot and Tomas Kirchhausen*
Department of Cell Biology and CBR Institute for Biomedical Research, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115
Communicated by Marc W. Kirschner, Harvard Medical School, Boston, MA, March 16, 2007 (received for review November 21, 2006)
The shape and total surface of a cell and its daughters change
during mitosis. Many cells round up during prophase and meta-
phase and reacquire their extended and flattened shape during
cytokinesis. How does the total area of plasma membrane change
to accommodate these morphological changes and by what mech-
anism is control of total membrane area achieved? Using single-cell
imaging methods, we have found that the amount of plasma
membrane in attached cells in culture decreases at the beginning
of mitosis and recovers rapidly by the end. Clathrin-based endo-
cytosis is normal throughout all phases of cell division, whereas
recycling of internalized membranes back to the cell surface slows
considerably during the rounding up period and resumes at the
time at which recovery of cell membrane begins. Interference with
either one of these processes by genetic or chemical means impairs
cell division. The total cell-membrane area recovers even in the
absence of a functional Golgi apparatus, which would be needed
for export of newly synthesized membrane lipids and proteins. We
propose a mechanism by which modulation of endosomal recycling
controls cell area and surface expression of membrane-bound
proteins during cell division.
clathrin 兩endocytosis 兩exocytosis 兩cell division 兩dynasore
Acharacteristic of eukaryotic cells, particularly when spread
on a substrate and unprotected by a cell wall, is to round up
during mitosis, becoming more compact at metaphase and
recovering during cytokinesis. By altering its size and shape, the
cell presumably acquires a mechanism for modulating spatially
segregated signaling pathways as it divides (1) and for ensuring
transfer of a similar complement of constituents to the daughter
cells. Control of membrane dynamics must therefore be linked
to progress through the stages of cell division. Simple geometric
considerations argue that transformation from an extended cell
interphase to a rounded mitotic cell should be accompanied by
a large reduction in cell surface, particularly if the cellular
volume remains approximately constant after having doubled
during the S to M phase transition (2).
One way to accommodate the apparent change in cell area
during mitosis is to maintain the total amount of membrane at
the cell surface by allowing formation of extensive folds. Imaging
by scanning electron microscopy of the surface of synchronized
mastocytoma cells grown in suspension showed an increase in
the number of microvilli between G
1
and G
2
and an apparent
doubling of the surface area; based on these observations it was
proposed (3) that ‘‘cytokinesis is a physical unfolding or stretch-
ing process’’ that provides the extra surface required for the two
daughter cells. Similar studies done with adherent CHO or
BHK21 cells also showed a larger number of microvilli, blebs,
and ruff les in mitotic rounded cells than in the completely spread
cells in interphase (4, 5). In these early studies, however, it was
recognized that rapid membrane redistribution through endo-
cytosis and recycling rather than surface stretching could also
account for the changes in cell surface (4).
Another way to accommodate the altered surface-to-volume
ratio when cells round up is to regulate membrane traffic
between the cell surface and its interior, leading to membrane
loss at the onset of mitosis and recovery afterward. Imaging
along the cell cycle by electron microscopy of neuroblastoma
cells showed the appearance of blebs at the plasma membrane
and the accumulation of single-bilayer or multilamellar vesicles
close to the blebs (6). Based on these observations, it was
proposed that fusion of these structures with the plasma mem-
brane was responsible for a substantial increase in cell surface
area particularly at late stages during mitosis. Most recent
studies on the role of membrane traffic during cell division have
focused on delivery and retrieval of membrane at the cleavage
furrow during cytokinesis (for a recent review, see ref. 7). A
number of proteins found to be essential for cytokinesis also have
established functions in endocytic, exocytic, and recycling path-
ways. Interpretation of these findings have emphasized the role
of exocytic events during the actomyosin-based constriction
responsible for cleavage furrow formation and for abscission
(separation of the two daughter cells).
A number of observations demonstrate profound changes in
membrane traffic during mitosis. The Golgi apparatus disassem-
bles thus preventing constitutive exocytosis along the biosyn-
thetic pathway (8, 9). Moreover, transferrin uptake through the
clathrin-based endocytic pathway and f luid-phase uptake both
decline sharply during metaphase and anaphase (10–13) and
recover during telophase (14).
We have capitalized on newly introduced live-cell imaging
approaches to reinvestigate the role of membrane traffic in
regulating cell surface area. In particular, we have followed
individual cells through different stages of the cell cycle, instead
of monitoring bulk properties of synchronized populations of
cells. We have monitored changes in total plasma membrane in
cells undergoing mitosis, and we have correlated these data with
the dynamics of endocytosis and exocytosis during all stages of
cell division. Three key observations have emerged from these
studies: (i) the cell surface area decreases at the onset of mitosis,
when the mother cell rounds up, and recovers starting in
anaphase, with complete recovery before abscission; (ii) endo-
cytosis is normal throughout all phases of cell division; and (iii)
recycling of internalized membranes back to the cell surface
slows considerably during the rounding-up period and reacti-
vates by a concerted fusion of endosomes with the plasma
membrane, starting in anaphase. This recovery occurs even
under conditions in which the Golgi apparatus has been ren-
dered nonfunctional. We propose a simple mechanism in which
modulation of endosomal recycling controls surface area during
cell division.
Results
Cells Change Plasma Membrane Area During Mitosis. In preparation
for cell division, interphase cells growing on a substrate round up
Author contributions: E.B. and T.K. designed research; E.B. performed research; E.B.
analyzed data; and E.B. and T.K. wrote the paper.
The authors declare no conflict of interest.
Abbreviation: VAMP, vesicle associated membrane protein.
*To whom correspondence should be addressed. E-mail: kirchhausen@crystal.harvard.edu.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0702511104/DC1.
© 2007 by The National Academy of Sciences of the USA
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0702511104 PNAS
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May 8, 2007
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vol. 104
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no. 19
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CELL BIOLOGY
(Fig. 1A), and their surface area, measured as described below,
decreases in a process that starts at the onset of prophase and
ends with metaphase (Fig. 1B). Reversal of these steps begins at
anaphase, continues through telophase, and becomes particu-
larly prominent during cytokinesis (Fig. 1 A–C). Where does the
extra membrane go at the beginning of cell division, and where
does it come from at the end? To monitor with high temporal
resolution the total plasma-membrane area throughout a com-
plete cell-division cycle, we used three-dimensional, spinning
disk confocal, live-cell imaging of single cells labeled with the
membrane-impermeant dye, FM 1-43, which becomes f luores-
cent upon binding to the outer leaflet of the plasma membrane
(15) [Fig. 1Band supporting information (SI) Fig. 7A]. The
surface f luorescence signal is a measure of exposed membrane,
because the charged head group of FM 1-43 prevents it from
flipping to the inner leaflet, and internal membranes can only be
reached by endocytic vesicular traffic (15).
Cells selected at different stages during cell division were
incubated for a brief time with FM 1-43 at 37°C, and image stacks
were acquired after 2 min, a time sufficient to achieve a stable
fluorescent signal and short enough to reduce to a minimum the
contribution from dye association with endosomal membranes
because of endocytosis (Fig. 1B, z view). Any remaining intra-
cellular fluorescence was removed from each optical section by
manual masking (see Materials and Methods). The total surface
area within a population of cells at interphase varied widely (Fig.
1C). At metaphase, when cells rounded up, we found a large
reduction in that area, about two-fold for human HeLa cells and
about six- to eight-fold for monkey BSC1 cells (Fig. 1C).
Nonadherent human Jurkat T cells imaged in the same way
display a large amount of plasma-membrane projections during
interphase, in stark contrast to the fairly smooth appearance
during metaphase (SI Fig. 7A), suggesting that the amount of
plasma membrane is also lower during metaphase. As a com-
plementary approach to determine cell surface area, we moni-
tored the fluorescence signal at the plasma membrane elicited by
a chimera of EGFP fused at its C terminus with the peptide-
sorting motif CAAX expressed in BSC1 or HeLa cells (SI Fig.
7Band C). We followed the changes in cell surface of single cells
and found that it also increases during the transition from
metaphase to cytokinesis.
Cell rounding alone cannot account for the observed decrease
in surface area, because the surface remained constant in
interphase cells that rounded upon brief treatment with trypsin
(Fig. 1D). That is, the surface of trypsinized cells must ruffle or
fold when they round up. In addition, we ruled out a decrease in
FM 1-43 dye accessibility during cell rounding by observing that
the total amount of intracellular and plasma membrane available
(determined by total f luorescence) remained the same in mi-
totic, trypsinized, or interphase cells when we gently permeabil-
ized their plasma membrane with saponin before incubation w ith
the dye (SI Fig. 8A). These data confirm that the decrease of
plasma membrane during mitosis is matched by its accumulation
inside cells.
The compensatory increase in surface area began at the onset
of anaphase and continued throughout cytokinesis, until the two
daughter cells had recovered the total area originally present in
the mother cell (Fig. 1C). Similar results were obtained from
sequential measurements of surface area from the same cells
imaged at different stages during the cell cycle, using a modified
staining protocol (SI Fig. 8B). The period of cell membrane
reduction lasts 30–45 min, the time it takes cells to round up and
reach metaphase. Recovery, which is relatively fast (15–20 min),
occurs before a perinuclear Golgi apparatus has assembled (SI
Fig. 8C).
Endocytosis Is Not Affected During Mitosis. It has been shown by
electron microscopy that cells contain plasma membrane coated
pits and vesicles regardless of their stage of cell division (16). All
endocytic plasma membrane coated pits and vesicles contain
clathrin adaptor AP-2 (17). We have now demonstrated that the
dynamics of AP-2 incorporation into plasma membrane pits and
vesicles during mitosis is the same as in interphase (Fig. 2). We
used BSC1 cells stably expressing
2-EGFP (17), part of AP-2
and imaged the surface attached to the coverslip to facilitate
data acquisition (Fig. 2 and SI Movies 1–3). Quantitative analysis
of these data (17) shows fluorescent
2-EGFP spots with
equivalent lifetimes during mitosis and interphase (SI Fig. 9 A
Fig. 1. Changes in cell shape and amount of plasma membrane during
mitosis. (A) BSC1 cells visualized at several stages during the cell cycle, using
bright field. N, nucleus. (B) BSC1 cells incubated for 2 min with FM 1-43 dye at
37°C and imaged at the same magnification at interphase, metaphase and
cytokinesis. Three-dimensional image stacks were obtained from sequential
optical sections acquired 0.25
m apart by using the spinning disk confocal
configuration. Shown is the fluorescence signal along the zaxis (Upper)atits
two dimensional projection (Lower). The integrated fluorescence, corrected
for any fluorescence signal inside the cells, represents the amount of plasma
membrane. Scale bar, 10
m. (C) Amount of plasma membrane at different
stages during the cell cycle was obtained in six experiments from 45 BSC1 and
64 HeLa cells, respectively. I, interphase; M, metaphase; C, cytokinesis. (D)
Amount of plasma membrane in BSC1 cells, rounded up immediately after
detachment by treatment with trypsin for 5 min at 37°C and then imaged as
in B. Data from two experiments from nine trypsinized cells and six untreated
control cells in interphase and spread on the coverslip.
7940
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www.pnas.org兾cgi兾doi兾10.1073兾pnas.0702511104 Boucrot and Kirchhausen
and B Left). Thus, the time needed to form a coated pit and for
it to bud as a coated vesicle is uniform throughout the cell cycle.
In addition, we could detect no change in the sizes of the coated
vesicles (SI Fig. 9B Right) or in the frequency of assembly events
per unit surface area (SI Fig. 9C). Similar results were obtained
with HeLa cells (SI Movie 4). These observations suggest that
endocytosis carried by the clathrin pathway remains normal
during mitosis.
Other forms of uptake probably also remain active during
mitosis, as the uptake of Alexa-594 labeled (10 kDa) dextran by
fluid phase endocytosis continued during mitosis (SI Fig. 10 A
and Bfor BSC1 cells and SI Fig. 10Cfor HeLa cells). As
expected, the internal membranes of rounding cells became
labeled by plasma membrane uptake when incubated steadily
with FM 1-43 during prophase and metaphase (SI Fig. 10D).
During mitosis, cells do not appear to internalize transferrin,
a ligand specifically taken up by clathrin-coated vesicles (12, 13).
These observations can be reconciled with our results, that
clathrin-based endocytosis remains uniformly active during the
cell cycle, if we recognize that significant reduction of recycling
would greatly decrease the amount of transferrin receptor
available on the cell surface. To test this hypothesis, we used
established methods (18, 19) to measure the uptake of f luores-
cently labeled transferrin and to normalize that uptake to the
number of accessible transferrin receptors with the potential to
internalize ligand (SI Fig. 11). The amount of transferrin bound
at the cell surface (Alexa488) or inside individual cells
(Alexa594) was calculated from two-dimensional projections of
three dimensional image stacks obtained by epif luorescence
microscopy at different stages during the cell cycle (SI Fig. 11 A
for BSC1 cells and SI Fig. 11Bfor HeLa cells). We observed a
substantial decrease in the amount of transferrin internalized
during prophase and metaphase (Fig. 3, Internalized and SI Fig.
11), consistent with all previous observations. We further ob-
served a marked decrease in the number of cell-sur face receptors
during prophase and metaphase followed by a very fast recovery
starting with anaphase, and becoming very prominent with
telophase and continuing into cytokinesis (Fig. 3 and SI Fig. 11,
Surface), also in agreement with previous obser vations (13). The
endocytic rate (18, 19), defined as the ratio of internalized
transferrin to transferrin bound at the cell surface (In/Sur ratio),
did not decrease during prophase and metaphase, but actually
showed a modest but significant increase compared with the rate
in cells during interphase or late stages of cytokinesis (Fig. 3).
Previous measurements were not done in a way in which this
ratio could have been measured in individual cells. Our results
show that the reduction in transferrin internalization during
mitosis does not correspond to an endocytic block, as formerly
concluded, but instead that most of the receptors remain trapped
within endosomes, most likely explained by a partial reduction in
recycling rates, which are then released back to the cell surface
as cytokinesis proceeds.
Recovery of the Plasma Membrane Correlates With Surface Bleb
Formation. Live cell imaging, using phase contrast or fluores-
cence microscopy of adherent cells incubated with FM 1-43
throughout the recovery phase, shows the appearance of a large
number of blebs at the outer surface of the cell, mostly at the
distal poles and away from the cleavage furrow (SI Fig. 12 and
SI Movies 5 and 6). These blebs appear and disappear rapidly
(seconds) (SI Fig. 12 A) until late stages of cytokinesis (SI Fig.
12B, 25 min). Bleb formation is not deleterious, because cells
that have completed cytokinesis spread and remain viable (SI
Fig. 12B, 52 min and SI Movie 6). Similar bebbling also occurs
in confluent HeLa cells (SI Fig. 12Cand SI Movie 7) and in the
nonadherent human Jurkat T and insect Sf9 cells (SI Fig. 12 D
and Eand SI Movies 8 and 9).
Recovery of the Plasma Membrane Does Not Require a Functional
Golgi Apparatus. The appearance of newly synthesized viral G
protein at the cell surface is strongly inhibited during metaphase,
anaphase, and early telophase (9). The images shown in SI Fig.
8Cindicate a diffuse intracellular distribution of the Golgi
marker, GalT-EGFP, during anaphase and telophase. Only
during cytokinesis does this marker start to appear punctate and
to localize in the perinuclear region (20, 21). Thus, a fully
reorganized Golgi apparatus is not required for recovery of
surface membrane. It is nonetheless possible that the substantial
exocytic traffic in anaphase and telophase requires a functional
postmitotic Golgi apparatus, even though a well localized GalT-
EGFP perinuclear signal is still absent (20, 21). We ruled out
involvement of a functional Golgi at this stage by observing the
appearance of blebs during anaphase and telophase and during
entrance to cytokinesis in cells treated with Brefeldin A before
entering mitosis (SI Fig. 12Fand SI Movie 10). The failure of
Brefeldin A to prevent the rapid recovery of plasma membrane
observed during anaphase and telophase shows that recovery
can occur without the contribution of newly synthesized mem-
branes from the Golgi. A similar Brefeldin A-insensitive mem-
brane deposition at the cleavage furrow of sea urchin zygotes has
been previously observed (22). However, a requirement for a
Fig. 2. Formation of clathrin coated pits and coated vesicles is not affected
by mitosis. Live cell fluorescence imaging of AP-2, containing clathrin coated
pits and coated vesicles located at the bottom surface of BSC1 cells. AP-2 was
labeled by stable expression of
2-adaptin fused to EGFP (17). Time series
collected for 6 min and at 37°C from one cell during interphase, and from
another cell sequentially imaged during metaphase and anaphase and ⬇15
min into cytokinesis. The still images (Left) correspond to Middle and Bottom
optical sections (scale bars, 10 and 5
m, respectively) acquired after 3 min of
data collection; the kymographs (Right) represent the complete time-series.
The data are representative of experiments done in triplicate and were
acquired every 2 s with 1-s exposures, using the spinning disk confocal con-
figuration.
Fig. 3. Receptor-mediated endocytosis of transferrin during cell division.
The amounts of internalized transferrin (In) and of transferrin bound to the
cell surface (Sur) were obtained by integration of their corresponding fluo-
rescence signals present in the three-dimensional image stacks. The data
(mean ⫾standard deviation) were obtained from 24, 18, 14, 5, and 20 cells
imaged at interphase (I), prophase (P), metaphase (M), anaphase (A), and late
stages of cytokinesis (C), respectively.
Boucrot and Kirchhausen PNAS
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CELL BIOLOGY
Brefeldin A-sensitive mechanism has been reported during
membrane deposition at the apex of the late cleavage furrow in
isolated, dividing C. elegans blastomeres (23) and during cell
plate during plant cytokinesis (24).
Exocytosis Controls the Recovery of Plasma Membrane. Some of the
blebs stain positively with an antibody specific for the luminal
domain of Lamp-1, a marker of late endosomes and lysosomes
normally absent from the cell surface during interphase (Fig.
4A, Interphase). Thus some blebs arise from fusion of Lamp-1
containing membranes with the plasma membrane (Fig. 4A,
Cytokinesis, control). Other blebs are labeled with the inter-
nalized transferrin receptor (Fig. 4A, Cytokinesis, control),
which accumulates even during mitosis in Lamp-1 negative
early/recycling endosomes (SI Fig. 13). Still other blebs have
neither Lamp-1 nor transferrin and may arise from additional
endosomal compartments. Presumably these are the multilayer
or single-membrane vesicles observed next to the blebs during
late mitosis (6). Formation of these blebs seems to require the
proper function of Myosin II, as their number was reduced in
cells incubated during telophase with blebbistatin, an inhibitor
of Myosin II (25) (SI Fig. 14A). Blebbistatin treatment also
decreased the reappearance of internalized Lamp-1, but barely
affected the recycling to the plasma membrane of transferrin
receptor (SI Fig. 14B). These observations suggest that during
cell division, the acto-myosin system has a role controlling
fusion, particularly of late endosomes/lysosomes with the
plasma membrane. They are consistent with the role of Myosin
II in cell membrane repair as mediated by calcium-dependent
exocytosis (26).
To further examine the relative contributions of early and late
endosomes to plasma-membrane recovery, we took advantage of
a published procedure (27, 28) to interfere with the fusion of
either type of endosome to the plasma membrane. VAMP3-DN
and VAMP7-DN are dominant negative cytosolic fragments of
v-soluble N-ethylmaleimide-sensitive factor attachment protein
receptors (V-SNAREs) specific for each path (27, 28). These
fragments were transiently overexpressed for ⬇4 – 6 h in BSC1
cells, and those cells entering mitosis were selected for imaging.
We observed that expression of VAMP3-DN substantially de-
creased the reappearance of transferrin receptor (but not of
Lamp-1) at the cell surface following anaphase (Fig. 4Aand SI
Fig. 15A); this interference coincided with an almost complete
block in the recovery of plasma membrane (SI Fig. 15B) together
with a failure to undergo cytokinesis (Fig. 4Band SI Movie 11).
Expression for longer periods was not considered, because cell
division was strongly inhibited and was accompanied by multi-
ploidy in ⬎80% of the cells (SI Fig. 15C). Likewise, short
expression of the corresponding cytosolic fragment of VAMP7
prevented Lamp-1 reappearance (but not of transferrin recep-
tor) at the cell surface (Fig. 4 Aand SI Fig. 15A), together with
a significant block in plasma membrane recovery (SI Fig. 15B).
Expression of VAMP7-DN also prevented cytokinesis (Fig. 4B
and SI Movie 12) and induced mutliploidy (SI Fig. 15D). We
conclude that both recycling and late endosomes participate in
membrane redeposition.
Clathrin-Mediated Endocytosis Is Required for Retrieval of Plasma
Membrane During Mitosis. Clathrin-mediated endocytosis is a
high capacity membrane traffic pathway. It generally carries at
least 50% of total endocytic traffic, and within 1 h it can
internalize the equivalent of the entire cell surface (29). If, as
proposed in Endocytosis Is Not Affected During Mitosis, endo-
cytosis remains active during all stages of cell division, whereas
exocytosis decreases substantially at the onset of mitosis, then
reducing endocytosis during mitosis should prevent the de-
crease in cell surface and retard or prevent mitotic roundup.
We confirmed this prediction in two ways. In one approach, we
reduced clathrin-based endocytosis by depleting, with RNA
interference in BSC1 (Fig. 5 and SI Fig. 16)orHeLa(SI Fig.
17) cells, the amount of
2-adaptin, a component required to
form the endocytic clathrin adaptor complex AP-2. As ex-
pected, 3 days of RNAi treatment was sufficient to block
transferrin uptake (Fig. 5, Overlay). All cells with an equato-
rial disposition of chromosomes (metaphase) failed to round
up; many (⬇50%) had aberrant spindles (Fig. 5, DAPI and SI
Fig. 16A, quantification). All remained flat and large, and
their plasma membrane area was the same as control cells in
interphase with normal endocytosis (SI Fig. 16B). This treat-
ment also led to a significant accumulation of multinucleated
cells because of incomplete cell division (SI Fig. 16C). In a
second and complementary approach, we inhibited clathrin-
Fig. 4. Exocytosis is required during cell division. (A) Surface distribution of
transferrin receptor and Lamp-1 during interphase and cytokinesis. HeLa cells
were processed for immunofluorescence at 4°C by incubation with antibodies
specific for the luminal domain of the transferrin receptor (green) and of
Lamp-1 (red), followed by fixation and addition of fluorescently tagged
secondary antibodies (Alexa-647 and Alexa-594, respectively). Images ac-
quired in the absence (control) or presence of overexpressed cytosolic forms of
the EGFP-labeled v-soluble N-ethylmaleimide-sensitive factor attachment
protein receptors (V-SNAREs) (VAMP3-DN or VAMP7-DN). The cells expressing
these constructs were identified by the cytosol EGFP signal (data not shown).
The control panel (cytokinesis) corresponds to two optical sections 2
m apart.
Cells in interphase contain little Lamp-1 at their cell surface and stain weakly
for transferrin receptor. Some blebs present during cytokinesis (control) score
positive for Lamp-1, whereas others are labeled with transferrin receptor; the
overall transferrin receptor signal is significantly stronger when compared
with cells in interphase. Short expression (4– 6 h) of VAMP3-DN prevents the
surface expression of transferrin receptor but not of Lamp-1 in cells under-
going telophase; in contrast, similar expression of VAMP7-DN prevents the
surface appearance of Lamp-1 but not of transferrin receptor. DNA was
labeled with DAPI (blue). Scale bar, 20
m. (B) Normal function of VAMP3 and
VAMP7 is required for completion of cytokinesis. Top panel (control): Images
acquired by bright field illumination of a BSC1 cell starting with anaphase,
continuing through cytokinesis and ending with the spreading and separation
of the two daughter cells. Middle panel (VAMP3-DN): images (from SI Movie
11) of BSC1 cell expressing VAMP3-DN for 4– 6 h before imaging; blebs are
present, ingression of the cleavage furrow occurs but cells do not separate.
Bottom panel (VAMP7-DN): images (from SI Movie 12) of BSC1 cell expressing
VAMP7-DN for 4– 6 h before imaging; blebs are absent, ingression of the
cleavage furrow occurs, but cells do not separate. Scale bar, 20
m.
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based endocytosis by acute interference with the function of
dynamin, a large GTPase essential for coated vesicle formation
(30–32). We incubated BSC1 cells for a brief interval with
dynasore, a recently discovered small molecule inhibitor of the
dynamin GTPase (33). Just as with the extended AP-2 deple-
tion, 30 min incubation with 80
M dynasore was sufficient to
block transferrin uptake, to prevent cell rounding (Fig. 5),
induce abnormal spindle formation (SI Fig. 16 A), and de-
crease cell surface area (SI Fig. 16B); 0.8% DMSO, used as
carrier, had no effect (Fig. 5).
Discussion
We have shown that the substantial decrease in total sur face area
that accompanies cell rounding is due mainly to a shutdown in
membrane recycling from endosomal compartments back to the
cell surface (Fig. 6). Most adherent cells in tissue culture round
when they divide, and cells within tissues also acquire a spherical
geometry during metaphase (34–37). This rounding appears to
be important for ensuing proper spindle formation and appro-
priate distribution of components to the daughter cells and for
establishing the correct intracellular spatial gradients for signal-
ing molecules (38). Indeed, we find that failure to round up,
induced by various forms of endocytic block, results in aberrant
spindle formation and incorrect cell division.
We have also shown that plasma membrane recovery is
essential for cell division. This rapid recovery is mediated by
massive fusion of endosomal membranes, starting at the onset
of anaphase and continuing through telophase. Our results
provide a satisfying explanation for recent observations ac-
quired in the course of RNAi screens aimed at identifying gene
products important for cell division (39 – 42). Clathrin heavy
chain, dynamin, Hsc70 (part of the uncoating machinery for
the clathrin coat), a number of plasma membrane and Golgi-
specific soluble N-ethylmaleimide-sensitive factor attachment
protein receptors (SNAREs), subunits of the Golgi COPI
complex, and regulatory small GTPases, such as Rab1 and
Rab7, are among theproteins whose depletion have a clear impact
in cell division.
The mechanisms that underlie membrane recovery studied
here are probably distinct from a number of other processes in
which new membrane is deposited. For example, specific struc-
tures such as the exocyst appear to participate in cleavage furrow
formation and abscission at cytokinesis (43), and Golgi-derived
vesicles are thought to contribute to the membranes laid down
during cellularization in Drosophila (44) and endocytic vesicles
(45). In plant cytokinesis, endocytic and Golgi-derived vesicles
are the sources of material for phragmoplast deposition (46). In
contrast, we propose here the formation of an internal mem-
brane reservoir during rounding up which stores membrane
components for subsequent release when called for later in cell
division.
In addition to the experiments described here, we have also
studied the potential role of Ca
2⫹
in regulating exocytosis during
mitosis (see SI Results). We suggest that Ca
2⫹
signaling is
essential for triggering the synchronous and coordinated fusion
with the plasma membrane of endomembranes stored inside the
cells, at a time when the Golgi apparatus has yet to assemble into
a fully functional organelle. A rise in intracellular Ca
2⫹
triggers
rapid fusion of late endosomes and lysosomes with the plasma
membrane (see ref. 47 for recent review); a similar process, also
triggered by Ca
2⫹
-influx during mechanical membrane injury,
governs wound healing. Similarly, the Ca
2⫹
-dependent mem-
brane deposition required for the formation of a phagocytic cup
(48, 49) derives, at least in part, from VAMP3 dependent
recycling endosomes (27) and from VAMP7-dependent late
endosomes (50). Perhaps the use of these pathways during cell
division is the ancestral form. In contrast, normal traffic of
recycling endosomes is not known to depend on Ca
2⫹
signaling,
at least during interphase.
It remains to be determined how modulation of these path-
ways occurs during mitosis. Control of these exocytic processes
must be linked to stages in cell division, to produce endosomal
recyling shutdown early in mitosis and to trigger abrupt reacti-
vation at anaphase.
Materials and Methods
Cell Preparation and Mitotic Stage Identification. The frequency of
adherent cells undergoing mitosis was increased from ⬇1% to
10–20% by allowing them to reach 100% conf luency for 1 day,
a condition where a large fraction of the cells are arrested at the
end of G
1
(51). Cells were then trypsinized and seeded on 25-mm
diameter glass no. 1.5 coverslips at ⬇50–70% conf luency; this
plating condition increases significantly the proportion of cells
simultaneously entering S phase at the time of seeding. Finally,
cells were imaged 12–14 h (HeLa) or 18–20 h (BSC1) after
Fig. 5. Endocytosis is required for retrieval of plasma membrane during
mitosis. BSC1 cells stably expressing EGFP-LCa were treated for 3 days with
RNAi for
2-adaptin to deplete AP-2 (center images) or for 30 min with 80
M
dynasore, a small molecule inhibitor of dynamin GTPase function (33) (right-
most image). These treatments inhibit clathrin-based endocytosis and during
mitosis prevent cell rounding and loss of cell membrane (see SI Fig. 16B). Cells
depleted of AP-2 and incubated for 5 min at 37°C with Alexa-594 transferrin
(red) display surface staining, an almost complete absence of internalized
transferrin, and the expected absence of endocytic clathrin coated pits and
vesicles (EGFP-LCa, green). Because of the relative brief incubation with
dynasore, these cells do not accumulate transferrin receptor at their surface
even though receptor endocytosis is blocked; the punctate pattern of EGFP-
LCa represents coated pits locked at the cell surface. A metaphase cell treated
with only 0.8% DMSO (Ctrl) is shown. Whereas most control mitotic cells have
normal spindles [decorated with EGFP-LCa, (53)], ⬇50% of equivalent cells
depleted of AP-2 or treated with dynasore display aberrant spindles (see SI Fig.
16A). Scale bar, 20
m.
Fig. 6. Model for membrane traffic during different stages of the cell cycle.
The schematic representations highlight the changes in balance of membrane
traffic between endocytic routes and secretory together with recycling path-
ways during key stages of the cell cycle. These pathways are balanced during
interphase. During the rounding up occurring during prophase and meta-
phase, the amount of plasma membrane decreases because of the traffic
imbalance arising from normal endocytosis combined with a considerable
decrease in secretory and recycling traffic, creating an internal ‘‘membrane
reservoir.’’ During anaphase, telophase and cytokinesis the amount of plasma
membrane increases rapidly along with the appearance of surface blebs. Most
of the plasma membrane is recovered by the rapid fusion of the previously
stored endo-membranes with the cell surface. A Ca2⫹signal is required to
trigger the rapid fusion of endomembranes with the plasma membrane in
what constitutes a form of regulated exocytosis. At this stage, the Golgi
apparatus is barely reassembled, and hence secretory traffic is still minimal.
Boucrot and Kirchhausen PNAS
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CELL BIOLOGY
plating. Imaging by phase contrast bright field illumination was
used to determine the stage of single cells along the cell cycle
(selected from the unsynchronized population) according to the
following criteria: cells in interphase appear f lat, contain un-
condensed chromosomes surrounded by a nuclear envelope;
cells in prophase contain condensed chromosomes also sur-
rounded by a nuclear envelope; cells in metaphase appear round,
contain condensed chromosomes aligned at the metaphase plate
and lack their nuclear envelope; cells in anaphase appear less
round and contain two sets of condensed chromosomes each
starting to migrate toward opposite spindle poles; cells in
telophase start to develop the furrow or invagination separating
the two daughter cells and still contain condensed chromosomes
that reached the spindle poles; during cytokinesis, (imaged 20
min after the onset of anaphase), cells generate a deeper furrow,
still display condensed chromosomes while the nuclear envelope
starts to form around each chromosomal mass until abscission.
All live cell imaging were performed as described in ref. 52 and
SI Methods.
We thank Drs. A. Yu, S. Saffarian and R. Massol and the members of
our laboratory for their help and advice, and Dr. J. Stow and J. Luzio for
providing reagents. This work was supported by National Institutes of
Health Grants GM075252 and GM62566 (to T.K.), by the Perkin Fund
to purchase part of the imaging equipment used here, and the Interna-
tional Human Frontier Science Program Organization (to E.B.).
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