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Research Article
Mammalian erythroblast enucleation requires
PI3K-dependent cell polarization
Junxia Wang1,*, Tzutzuy Ramirez1,*, Peng Ji2,§, Senthil Raja Jayapal2,3, Harvey F. Lodish2 and
Maki Murata-Hori1,4,`
1
Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, 117604, Singapore
Whitehead Institute for Biological Research, Department of Biology, Massachusetts Institute of Technology, 9 Cambridge Center, Floor 6,
Cambridge, MA 02142, USA
3
Stem Cell Group 4, Genome Institute of Singapore, #08-01, Genome, 60 Biopolis Street, Singapore 138672
4
Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543
2
*These authors contributed equally to this work
§
Present address: Department of Pathology, Feinberg School of Medicine, Northwestern University, 303 East Chicago Avenue, Ward 3-140, Chicago, IL 60611, USA
`
Author for correspondence ([email protected])
Journal of Cell Science
Accepted 11 August 2011
Journal of Cell Science 125, 340–349
2012. Published by The Company of Biologists Ltd
doi: 10.1242/jcs.088286
Summary
Enucleation, the final step in terminal differentiation of mammalian red blood cells, is an essential process in which the nucleus
surrounded by the plasma membrane is budded off from the erythroblast to form a reticulocyte. Most molecular events in enucleation
remain unclear. Here we show that enucleation requires establishment of cell polarization that is regulated by the microtubule-dependent
local activation of phosphoinositide 3-kinase (PI3K). When the nucleus becomes displaced to one side of the cell, actin becomes
restricted to the other side, where dynamic cytoplasmic contractions generate pressure that pushes the viscoelastic nucleus through a
narrow constriction in the cell surface, forming a bud. The PI3K products PtdIns(3,4)P2 and PtdIns(3,4,5)P3 are highly localized at the
cytoplasmic side of the plasma membrane. PI3K inhibition caused impaired cell polarization, leading to a severe delay in enucleation.
Depolymerization of microtubules reduced PI3K activity, resulting in impaired cell polarization and enucleation. We propose that
enucleation is regulated by microtubules and PI3K signaling in a manner mechanistically similar to directed cell locomotion.
Key words: Erythroblast enucleation, PI3-kinase, Microtubules, Cell polarization
Introduction
In the last step of erythropoiesis, mammalian erythroblasts
undergo enucleation, a process that is crucial for the formation of
mature functional red blood cells. During enucleation, the
erythroblast extrudes its nucleus tightly apposed to the plasma
membrane, forming a reticulocyte (Ihle and Gilliland, 2007;
Koury et al., 2002; Richmond et al., 2005). Pioneering studies
using electron microscopy revealed that at the earliest stage of
enucleation the erythroblast nucleus becomes located close to the
cell membrane away from the center of the cell (Simpson and
Kling, 1967; Skutelsky and Danon, 1967) and that a cytokineticlike furrow is formed in the region between the extruded nucleus
and the incipient reticulocyte (Koury et al., 1989; Skutelsky and
Danon, 1967). Actin filaments (F-actin) accumulate in the
cytokinetic-like furrow (Ji et al., 2008; Koury et al., 1989) and
disruption of F-actin (Ji et al., 2008; Koury et al., 1989; Yoshida
et al., 2005) or depletion of mDia2, a regulator of actin
polymerization (Ji et al., 2008), blocked enucleation, suggesting
that actin-based forces drive nuclear extrusion. However, many
questions remain unanswered concerning the process of
erythroblast enucleation. In particular, little is known how an
asymmetry is established within the erythroblast (i.e. how the
nucleus becomes localized to one side of the cell and the
cytoplasm to the other), although this polarized state appears to
be important for enucleation. Moreover, the detailed organization
of actin and microtubules in polarized erythroblasts is unknown.
Phosphoinositide 3-kinase (PI3K) is well known as a central
regulator of chemotaxis. In migrating Dictyostelium discoideum,
neutrophils and fibroblasts, the PI3K products PtdIns(3,4)P3 and
PtdIns(3,4,5)P3 accumulate locally at the leading edge of the
surface membrane and control cell polarization (Haugh et al.,
2000; Parent et al., 1998; Servant et al., 2000). Although
involvement of PI3K in the early stages of Epo (erythropoietin)regulated differentiation of erythroid progenitors has been
established (Ghaffari et al., 2006; Zhao et al., 2006), little is
known about its role in the much later steps of enucleation.
We investigated how erythroblasts establish cell polarization
and whether this polarization plays a role in expelling the nucleus
from the cell. We used a powerful combination of an in vitro
cell culture system that mimics normal terminal erythroid
proliferation, differentiation and enucleation (Ji et al., 2008),
combined with several microscopic imaging techniques. Our
results show that proper enucleation requires establishment and
maintenance of cell polarization mediated by PI3K in a manner
similar to that seen in migrating cells.
Results
Erythroblast enucleation is initiated through
establishment of cell polarization, followed by dynamic
cytoplasmic contractions
We first wanted to determine when the terminal erythroblast
becomes polarized and how the nucleus is extruded from the
Journal of Cell Science
Mechanics of mammalian erythroblast enucleation
erythroblast. To this end, we conducted a detailed microscopic
analysis of the enucleation process using an in vitro cell culture
system employing mouse fetal liver erythroblasts. Enucleation
begins ,35 hours after stimulation of erythroid progenitors (Ji
et al., 2008). This system employs normal primary erythroid cells
and thus does not have the obvious abnormalities associated with
virus-infected and/or transformed cell lines. Moreover, the timecourse of erythroid differentiation in this system has been well
established (Ji et al., 2008), providing us in a time-dependent
manner with erythroblasts at different stages of differentiation.
We could follow cultured erythroblast cells after they underwent
a final mitotic division (Fig. 1A, 24 minutes) and generated two
late erythroblasts in which the nuclei were located at the center of
each cell (Fig. 1A, 44 minutes). At ,3 hours after each was
generated (210±8.64 minutes, n58), these late erythroblasts
underwent significant polarization, displacing the nucleus
to one side of the cell (Fig. 1A, 115 and 153 minutes, arrows).
Around 1 hour after cell polarization, enucleation initiated
(74.7±8.64 minutes, n58) and dynamic and random contractions
occurred at the side of the cytoplasm opposite to the nucleus
(n516; Fig. 1B and supplementary material Movie 1). Strikingly,
a part of the nucleus suddenly protruded from the cell, forming a
bleb-like structure from a limited area of the cortex adjacent to the
nucleus (3.35±0.64 mm diameter; Fig. 1B, arrows). Then the
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whole nucleus was quickly squeezed out (5.98±2.73 minutes), in
the process undergoing extensive deformations into an hourglasslike shape (supplementary material Movie 1). Fixed late
erythroblasts at different stages of enucleation (Fig. 1C) were
morphologically similar to live cells (Fig. 1B).
Before late erythroblasts became polarized, F-actin formed
small particles and patch-like structures distributed throughout
the cytoplasm (Fig. 1D, Non-polarized). After cell polarization,
F-actin was mostly restricted to the side of the cytoplasm away
from the nucleus, and little was localized at the side with the
nucleus (Fig. 1D, Polarized). While the nucleus was being
expelled, a fraction of F-actin concentrated at the neck region of
the bleb (Fig. 1D, Extruding nucleus, F-actin, arrowheads) where
it was previously described as the cytokinetic-like furrow or
contractile actin ring (Ji et al., 2008; Koury et al., 1989;
Skutelsky and Danon, 1967).
Similar to F-actin, after late erythroblasts became polarized small
particles of active myosin II (myosin II regulatory light chain
phosphorylated at Ser19; pMRLC) (Matsumura et al., 1998) became
restricted to the side of the cytoplasm away from the nucleus
(Fig. 1D, pMRLC). A fraction of active myosin II also became
localized to the neck region of the bleb (Fig. 1D, pMRLC, arrows).
Although previous studies have shown that F-actin is required
for enucleation (Ji et al., 2008; Koury et al., 1989; Repasky
Fig. 1. Enucleation is initiated through
establishment of cell polarization, followed by
contractions of asymmetrically localized
cytoplasmic actomysoin. (A) Time-lapse images
of late erythroblasts undergoing cell polarization.
White dots depict the nucleus. Time elapsed in
minutes after completion of the final mitotic
division of the erythroid progenitor cell is shown.
(B) Time-lapse images of late erythroblasts
undergoing enucleation. (C) Late erythroblasts
were fixed and stained for DNA (blue). The
populations of late erythroblasts were classified
into non-polarized cells, polarized cells that show
nuclear displacement, early phase of nuclear
extrusion and late phase of nuclear extrusion.
(D) Immunofluorescence of late erythroblasts at
different stages of enucleation. Single confocal
images of F-actin (red), active myosin II
(pMRLC; green) and DNA (blue) are shown.
Note that both F-actin and active myosin II
become mostly restricted to the opposite side of
the cytoplasm after cells become polarized. A
small fraction of both F-actin and active myosin
(arrows) is found accumulated in the neck region
of the bleb-like protrusion. (E) DMSO,
cytochalasin D or blebbistatin was applied to
cells at 36 hours and then polarized late
erythroblasts were identified for time-lapse
recording. Scale bars: 5 mm.
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Journal of Cell Science 125 (2)
and Eckert, 1981; Yoshida et al., 2005), it remained
unknown whether enucleation is powered by contractions of
asymmetrically localized actomyosin. To this end, we disrupted
F-actin or inhibited myosin II function specifically in polarized
late erythroblasts. We applied specific inhibitors of actin or
myosin II to cells at 36 hours of culture, and polarized cells were
then identified for time-lapse recording. In polarized cells treated
with cytochalasin D, an agent that disrupts F-actin, dynamic
contractions were quickly and completely suppressed and
enucleation was blocked (Fig. 1E, Cytochalasin D).
Similarly, treatment of polarized cells with blebbistatin, an
inhibitor of myosin II activity (Straight et al., 2003), at a
concentration (100 mM) that completely inhibits cytokinesis
(Mukhina et al., 2007), caused the suppression of cytoplasmic
contractions and a failure of enucleation (Fig. 1E, Blebbistatin,
top). Strikingly, even in cells already extruding the nucleus,
following blebbistatin treatment the nucleus regressed into
the cell (Fig. 1E, Blebbistatin, bottom). These results suggest
that nuclear extrusion is achieved by pressure generated by
contractions of asymmetrically localized actomyosin and thus
that the establishment of cell polarization is crucial for
enucleation. Our observations also indicate that completion of
enucleation probably involves actomyosin-driven constriction of
the cortical actin ring at the neck of the bleb-like protrusion.
PI3K activity is required for proper enucleation by
regulating the establishment and maintenance of
cell polarization
How is cell polarization established and maintained during
enucleation? We speculated that PI3K, which regulates cell
polarization in migrating cells, might be involved in this process.
To test this hypothesis, we first examined the localization of the
PI3K products PtdIns(3,4)P2 and PtdIns(3,4,5)P3 by expressing
the reporter (3)Akt(PH)–GFP (the trimerized PH domain of Akt
tagged with green fluorescent protein) in late erythroblasts
(Fig. 2A) (Luo et al., 2005).
In non-polarized cells, (3)Akt(PH)–GFP was localized
throughout the entire plasma membrane (n511; Fig. 2A, Nonpolarized). Strikingly, when cells became polarized, (3)Akt(PH)–
GFP became restricted in localization to the segment of plasma
membrane facing the cytoplasm (n58; Fig. 2A, Polarized); this
localization pattern remained unchanged during nuclear extrusion
(n516; Fig. 2A, Extruding nucleus). Three-dimensional images
of (3)Akt(PH)–GFP and mCherry, used as a cytoplasmic volume
marker, showed that the asymmetric distribution of (3)Akt(PH)–
GFP in polarized cells was not caused by volume effects in
the cytoplasm because mCherry was uniformly distributed
throughout the cell (Fig. 2B). In late erythroblasts,
(3)Akt(PHR25C)–GFP, a mutant that is unable to bind to
PtdIns(3,4)P2 or PtdIns(3,4,5)P3 (Franke et al., 1997), failed to
localize to the plasma membrane and was diffusely distributed in
the cytoplasm, indicating that (3)Akt(PH)–GFP is a specific
reporter for PI3K activity (supplementary material Fig. S1).
Next, we wanted to determine whether PI3K activity is
required for cell polarization during enucleation. Because PI3K
activity is involved in the early steps of Epo-triggered cell
division and differentiation of erythroid progenitor cells (Ghaffari
et al., 2006; Zhao et al., 2006), we needed to use a specific
inhibitor of PI3K to block its activity only in late erythroblasts –
more specifically during enucleation. To this end, we applied
Fig. 2. (3)Akt(PH)–GFP is asymmetrically localized in
polarized late erythroblasts. (A) Two-dimensional and
(B) three-dimensional images of (3)Akt(PH)–GFP in late
erythroblasts that are non-polarized, polarized and extruding
its nucleus are shown. White dots depict the nucleus. The
color bar indicates the intensity of GFP signal from the lowest
(black) to highest (red). (C) Localization of (3)Akt(PH)–GFP
in cells treated with DMSO or LY294002. Each graph shows
the distribution of the GFP (green) and mCherry (red)
fluorescence intensity (y axis) in the corresponding distance
(x axis; double-headed arrows). Scale bars: 5 mm.
Mechanics of mammalian erythroblast enucleation
polarized during this time period (n536; Fig. 3A and Fig. 4). The
distance between the cellular and nuclear centroids in polarized
control cells was 1.59±0.14 mm, whereas the maximum distance
between these centroids was 0.62±0.09 mm in LY294002-treated
cells that were not able to undergo cell polarization (Fig. 3B),
suggesting that the nucleus remained localized in the cell center in
LY294002-treated cells. F-actin was found localized throughout
the cytoplasm in the LY294002-treated cells that remained nonpolarized (n523; Fig. 3C).
LY294002- and DMSO-treated (control) cells were also fixed in
order to perform quantitative analysis on the progression of
enucleation by counting the number of late erythroblasts at each
stage of enucleation per total number of late erythroblasts and
enucleated cells. We observed only a slight (although statistically
significant) decrease in the percentage of polarized cells as well
Journal of Cell Science
LY294002, a well-established specific inhibitor of PI3K, to
late erythroblasts expressing (3)Akt(PH)–GFP. Strikingly, after
,1 hour of incubation the membrane association of (3)Akt(PH)–
GFP became strongly diminished in 24 cells out of 28 (24/28)
treated with LY294002 but not in the DMSO-treated control cells
(0/17) (Fig. 2C), indicating that LY294002 effectively inhibits
PI3K activity in late erythroblasts.
To determine whether PI3K inhibition affects the establishment
of cell polarization, we applied LY294002 to cells at 31 hours in
culture, where ,80% of late erythroblasts were not yet polarized
(see supplementary material Fig. S2). After 1 hour of incubation,
non-polarized cells were identified and monitored by time-lapse
recording. The majority of control non-polarized cells underwent
cell polarization within 3 hours (80%, n540), whereas 75% of the
non-polarized cells treated with LY294002 failed to become
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Fig. 3. PI3K activity is required for
establishment of cell polarization during
enucleation. (A) Time-lapse images of nonpolarized cells treated with DMSO or
LY294002. White dots depict the nucleus.
(B) Quantification of data obtained with
cells similar to those shown in A. The
distance between the nuclear and cellular
centroids in individual cells treated with
DMSO (blue) or LY294002 (red) over time
was plotted. Four representative cells from
each treatment are shown. (C) Z-projections
of F-actin in late erythroblasts treated with
DMSO or LY294002. (D,E) Cells treated
with DMSO or LY294002 from 31 to
35 hours (D) or from 31 to 48 hours in
culture (E) were fixed and stained for Factin and DNA. We considered an
enucleated nucleus as an enucleated cell.
More than 100 cells were counted in each
experiment. Three independent experiments
for each treatment were performed; values
are means ± s.e.m. (*P,0.05). Scale bars:
5 mm.
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Journal of Cell Science 125 (2)
Fig. 4. Summary of the defects in nuclear displacement and extrusion in cells treated with LY294002 or nocodazole. Live cells were treated with 50 mM
LY294002 or 10 mM nocodazole in culture and the percentage of cells showing defects in enucleation was calculated. Control cells were treated with DMSO.
Figures in parentheses indicate the number of cells showing the phenotype out of the total number of cells examined.
as enucleated cells in cells treated with LY294002 from 31 to
35 hours in culture, compared with control cells (polarized cells,
17.56±1.84% vs 24.48±2.67%; enucleated cells, 7.54±0.68% vs
14.24±1.80%; P,0.05, Fig. 3D). Similar results were obtained
when late erythroblasts were treated with another PI3K inhibitor,
wortmannin (supplementary material Fig. S3), suggesting that the
defects in enucleation caused by LY294002 were indeed due to
inhibition of PI3K activity and that PI3K inhibition did not
completely block cell polarization. When cells were treated with
LY294002 from 31 to 48 hours in culture, the decrease in
polarization and enucleation was no longer statistically significant
(Fig. 3E). These results suggest that PI3K inhibition caused a severe
delay rather than arrest in the establishment of cell polarization.
To determine whether PI3K inhibition affects nuclear
extrusion, LY294002 was applied to cells at 35 hours in culture
and, after 1 hour of incubation, polarized cells were identified
and monitored by time-lapse video microscopy. Strikingly, 76%
of polarized cells treated with LY294002 exhibited a wide range
of delay and/or arrest in nuclear extrusion (35/46), whereas in all
control cells enucleation was completed within 25 minutes (46/
46; Fig. 5A). Some cells completed nuclear extrusion after a
delay (11/46), whereas others moved out of the field of view
before the extrusion was completed or did not complete nuclear
extrusion during time-lapse imaging (24/46, Fig. 4 and
supplementary material Fig. S4).
Cells that showed a delay in nuclear extrusion typically exhibited
one or both of the following phenotypes: (a) Nuclear extrusion was
initiated but the nucleus regressed into the cell before the extrusion
was completed; some cells repeated this ‘trial and error’ of nuclear
extrusion several times. (b) Nuclear extrusion was initiated but
failed to complete, with formation of an hourglass-shaped nucleus
(Fig. 5A, supplementary material Movies 2 and 3).
In control cells, nuclear extrusion was completed, with a
distance between the cellular and nuclear centroids of
6.11±0.40 mm (Fig. 5B, DMSO), whereas in LY294002-treated
cells that showed a delay in nuclear extrusion the distance
between these centroids was 1.59±0.32 mm and remained
relatively unchanged until extrusion was completed (Fig. 5B,
LY294002). Quantitative analysis revealed no significant
decrease in the percentage of enucleated cells among cells
treated with LY294002 from 35 to 39 hours in culture compared
with control cells (Fig. 5C), suggesting that PI3K inhibition
caused a delay rather than a complete arrest in nuclear extrusion.
These results suggest that PI3K activity is important to maintain
cell polarization. Taken together, our observations suggest that
PI3K activity is required for proper enucleation by regulating the
establishment and maintenance of cell polarization.
Microtubule-dependent local activation of PI3K is required
for proper cell polarization during enucleation
Next, we wanted to determine how PI3K activity is regulated
during enucleation. In both migrating neutrophils (Xu et al.,
2005) and macrophages during phagocytosis (Khandani et al.,
2007), localized PI3K activity is dependent on microtubules.
Thus, we speculated that microtubules might regulate PI3K
activity during enucleation. Before testing this hypothesis, it
was important to analyze the microtubule organization during
enucleation because this had not been determined, especially in
polarized late erythroblasts.
Immunofluorescence showed that, as previously described,
before cells became polarized microtubule arrays were radially
symmetric, forming a cage-like structure surrounding the
nucleus (Fig. 6A) (Koury et al., 1989). Concomitant with cell
polarization, microtubules formed an asymmetric array oriented
toward the cortex on the side of the cytoplasm opposite to the
nucleus (Fig. 6A, Polarized and Extruding nucleus). We also
found that, during enucleation, one or two foci of GFP–c-tubulin
were localized together in the cytoplasm in close proximity to the
nucleus at the side of the cytoplasm (n56; Fig. 6B).
Journal of Cell Science
Mechanics of mammalian erythroblast enucleation
Fig. 5. PI3K activity is required for maintenance of cell polarization
during enucleation. (A) Time-lapse images of polarized cells treated with
DMSO or LY294002. Time 0 indicates the onset of nuclear extrusion. White
dots depict the nucleus. (B) Quantification of data obtained with cells similar to
those shown in A. The distance between the nuclear and cellular centroids in
individual cells treated with DMSO (blue) or LY294002 (red) over time after the
onset of nuclear extrusion (time 0) was plotted. Representative cells for each
treatment (five for DMSO and seven for LY294002) are shown. (C) Cells treated
with DMSO or LY294002 from 35 to 39 hours in culture were fixed and stained
for F-actin and DNA. Three independent experiments for each treatment were
performed; values are means ± s.e.m. (*P,0.05). Scale bar: 5 mm.
Similar localization patterns of endogenous c-tubulin (n521)
and pericentrin (n511) were observed in late erythroblasts
(supplementary material Fig. S5A,B). Moreover, when late
erythroblasts were stained for EB1, a protein that binds to plusends of growing microtubules (Mimori-Kiyosue et al., 2000),
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typical comet-like structures were observed regardless of the stage
of enucleation (Fig. 6C, arrows), suggesting that microtubules
are dynamic during enucleation. These results suggest that
microtubule organization is dramatically rearranged into an
asymmetric array with the centrosome when late erythroblasts
become polarized.
To determine whether microtubules regulate PI3K activity
during enucleation, late erythroblasts expressing (3)Akt
(PH)–GFP were treated with nocodazole, a microtubuledepolymerization agent. Strikingly, in nocodazole-treated cells
where microtubules were severely disrupted (3)Akt(PH)–GFP
failed to associate with the plasma membrane and instead
was diffusely localized in the cytoplasm (11/15). By contrast,
(3)Akt(PH)–GFP was associated with the membrane in control
cells (12/12) (Fig. 6D,E), suggesting that microtubules are
important for maintaining PI3K activity.
Nocodazole treatment caused defects in enucleation similar to
those seen in cells treated with LY294002. Live-cell analysis
showed that when nocodazole was applied to cells at 32 hours in
culture only 20% of non-polarized cells were able to undergo
polarization within 3 hours (n520; Fig. 6F, Fig. 4 and
supplementary material Fig. S5C). F-actin was localized
throughout the cytoplasm in nocodazole-treated cells that
remained non-polarized (n520; supplementary material
Fig. S5D). When nocodazole was applied to cells at 36 hours
in culture, 63% of polarized cells exhibited a severe delay in
nuclear extrusion (n535; Fig. 6G, Fig. 4, supplementary material
Fig. S4, Fig. S5E and Movie 4), with phenotypes similar to those
seen in LY294002-treated cells (Fig. 5A).
Quantitative analysis revealed only a slight decrease in the
percentage of polarized and enucleated cells among cells treated
with nocodazole from 32 to 36 hours in culture, compared with
control cells (supplementary material Fig. S5F). It is noteworthy
that treatment of erythroblasts with nocodazole from 32 to
48 hours in culture resulted in severe defects in nuclear and
cellular morphology (data not shown). The decrease was not
significant when cells were treated with nocodazole from 36 to
40 hours in culture (supplementary material Fig. S5G). These
results suggest that microtubules are required for proper
enucleation by regulating the establishment and maintenance of
cell polarization. As detailed below, in large measure this
resolves past controversies about whether or not microtubules
play an essential role in enucleation (Keerthivasan et al., 2010;
Koury et al., 1989; Sonoda et al., 1998). Unlike the crucial role of
microtubules in the regulation of PI3K activity, PI3K inhibition
caused no drastic defects in microtubule organization in late
erythroblasts (Fig. 6H). Taken together, our observations suggest
that microtubule-dependent local activation of PI3K is required
for proper enucleation.
PI3K regulates cell polarization through promoting the
movement of the nucleus but not the MTOC
during enucleation
In migrating fibroblasts, reorientation of the microtubuleorganizing center (MTOC) is the initial polarization event and
occurs by the movement of the nucleus but not the MTOC
(Gomes et al., 2005; Schmoranzer et al., 2009). Our live-cell
analyses showed that when late erythroblasts became polarized
the nucleus moved to one side of the cell (Fig. 3A,B), suggesting
that cell polarization might be initiated in a way similar to that
seen in migrating cells and that PI3K might regulate this process.
Journal of Cell Science 125 (2)
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346
Fig. 6. Enucleation requires microtubule-dependent local activation of PI3K. (A) Microtubule organization in cells prior to and during enucleation. Zprojections of microtubules (green) and DNA (blue) are shown. (B) Time-lapse fluorescence images of cells expressing GFP–c tubulin (GFP-cTub) during
enucleation. Asterisks indicate the position of the nucleus. Dashed lines outline the nucleus and the cell edge. (C) Z-projections of EB1 (red) and DNA (blue) are
shown. Typical comet-like structures of EB1 are detected (arrows). (D) Localization of (3)Akt(PH)–GFP in live cells treated with DMSO or nocodazole. Each
graph shows the distribution of the GFP (green) and mCherry (red) fluorescence intensity (y axis) in the corresponding distance (x axis; double-headed arrows).
(E) Microtubule organization (red) and localization of (3)Akt(PH)–GFP (green) in fixed cells treated with DMSO (top) or nocodazole (bottom). Z-projections of
microtubules (red) are shown. (F) Time-lapse images of non-polarized cells treated with DMSO or nocodazole. Dashed lines depict the nucleus. (G) Time-lapse
images of polarized cells treated with DMSO or nocodazole. Time 0 indicates the onset of nuclear extrusion. Dashed lines depict the nucleus. (H) Microtubule
organization in late erythroblasts treated with DMSO (top) or LY294002 (bottom). Z-projections of microtubules (green) are shown. Scale bars: 5 mm.
To test this, cells treated with LY294002 from 31 to 35 hours in
culture were fixed and stained for the nucleus and c-tubulin (see
supplementary material Fig. S6). Then, we measured the distance
between the cellular centroid and the MTOC or the nuclear
centroid (Fig. 7).
In control cells (Fig. 7, DMSO), the average distance between
the MTOC and the cellular centroid in non-polarized cells
(1.75±0.06 mm) was similar to that in polarized cells
(1.71±0.08 mm), suggesting that the position of the MTOC
remains unchanged during cell polarization. By contrast, as
observed in live-cell analysis (Fig. 3A,B), the distance between
the nuclear and cellular centroids was significantly increased
when the cells became polarized (Fig. 7, DMSO). These results
indicate that during the early stages of enucleation, MTOC
reorientation occurs by the movement of the nucleus but not the
MTOC.
In non-polarized cells, LY294002 treatment had no effect on
the distances between the MTOC or the nuclear centroid and the
cellular centroid (Fig. 7, Non-polarized, LY294002). In the few
LY294002-treated cells that managed to become polarized, the
Mechanics of mammalian erythroblast enucleation
Journal of Cell Science
Fig. 7. PI3K regulates cell polarization through promoting the movement
of the nucleus but not the MTOC. Average distance between the centroids
of the nucleus (red) or the MTOC (blue) and the cell in late erythroblasts
treated with DMSO or LY294002. *P,0.05.
distance between the nuclear and the cellular centroids was
increased, whereas the position of the MTOC remained
unchanged, similar to that seen in control DMSO-treated cells
(Fig. 7, Polarized, LY294002). These results suggest that PI3K
inhibition causes a delay in nuclear repositioning. Taken
together, our observations suggest that PI3K regulates cell
polarization through promoting the movement of the nucleus
but not the MTOC during enucleation.
Discussion
One of the long-standing questions in erythropoiesis is how the
erythroblast extrudes its nucleus to form the reticulocyte that later
becomes the mature red blood cell. In particular, little is known
about how erythroblasts establish and maintain a polarized state
during enucleation. The live-cell analyses we performed in this
study have provided a deeper understanding of the mechanisms
underlying the regulation of cell polarization and its importance
in enucleation.
Early work suggested a mechanism of enucleation similar to
that used in cytokinesis of mitotic cells, where local cortical
contractions occur between the two daughter cells (Skutelsky and
Danon, 1967; Koury et al., 1989). By contrast, our observations
demonstrate that nuclear extrusion is driven by contractile forces
generated by asymmetrically distributed cytoplasmic actomyosin
in late erythroblasts. This finding reiterates the importance of
establishment and maintenance of cell polarization during
enucleation.
Although previous studies revealed that PI3K was required for
early Epo-dependent stages of erythropoiesis (Ghaffari et al.,
2006; Zhao et al., 2006), here we demonstrated a second and
novel role for PI3K in the establishment and maintenance of cell
polarization during enucleation. Moreover, our results also
showed that PI3K promotes cell polarization by repositioning
of the nucleus during enucleation. In migrating fibroblasts, cell
polarization is established by the movement of the nucleus
instead of the MTOC (Gomes et al., 2005). Our results indicate
that this is also the case in late erythroblasts during enucleation.
A possible mechanism that regulates the repositioning of the
nucleus by PI3K is discussed below.
347
The roles of microtubules in enucleation have been
controversial (Keerthivasan et al., 2010; Koury et al., 1989;
Sonoda et al., 1998). We observed different effects of
microtubule disruption agents on enucleation when cells were
treated at different stages of erythropoiesis and/or for different
time periods of incubation. Imaging and quantitative analysis of
live and fixed cells revealed that disruption of microtubules
caused a severe delay in enucleation, but did not affect the final
extent of enucleation. This indicates that microtubules are
required for, but not essential to, enucleation. Currently it is
unknown how microtubules regulate PI3K activation during
enucleation. Interestingly, biochemical analyses showed that the
regulatory subunits of PI3K can bind to tubulin (Inukai et al.,
2000; Kapeller et al., 1995), but we do not know whether this
occurs in late erythroblasts or whether this affects activation or
localization of PI3K.
Interestingly, our results show that cell polarization is initiated
through repositioning of the nucleus but not MTOC during
enucleation, similar to that seen in migrating fibroblasts (Gomes
et al., 2005; Schmoranzer et al., 2009). Nuclear repositioning
occurs concomitantly with the rearrangement of the microtubule
array that leads to asymmetric distribution of actomyosin and
thus the generation of asymmetric force (see below). This force
most probably drives the repositioning of the nucleus, somewhat
similar to that seen in migrating cells where nuclear movement
needs actin flow (Gomes et al., 2005).
In comparison with migrating cells, in late erythroblasts the
nucleus moves only a short distance (,1 mm) for the cell to
become polarized. Then why do late erythroblasts need a long
time (2–3 hours) to establish polarization? This is probably
because either rearrangement of the microtubule array or stable
association of the nucleus with the plasma membrane, or both,
might be a relatively slow event.
Our proposed model for erythroblast enucleation is
summarized in Fig. 8. The reorientation from a radial
microtubule array to an asymmetric array somehow results in
asymmetric activation of PI3K. Asymmetric accumulation of the
PI3K products in the plasma membrane then induce asymmetric
actin assembly and disassembly by stimulating their effectors,
such as a-actinin (Franke et al., 1997), profilin (Lassing and
Lindberg, 1985) or gelsolin (Janmey et al., 1987), as well as Rac1
activity (Ji et al., 2008) probably through activation of its
upstream regulator (Han et al., 1998; Kunisaki et al., 2006). This
asymmetric actin assembly and disassembly and actomyosin
contractions promote nuclear displacement as well as nuclear
extrusion.
In cells defective in this microtubule-regulated PI3Kdependent polarization mechanism, enucleation could be
achieved by random cytoplasmic contractions alone but in a
highly ineffective and slow manner. It is likely that such defects
in enucleation cause some proteins to be wrongly sorted to the
reticulocytes or the extruded nucleus, leading to the formation of
abnormal reticulocytes.
Our results suggest that enucleation is carried out in a way
mechanistically similar to directed cell migration. Some
migrating cells form a polarized bleb-like protrusion in order to
move forward (Charras and Paluch, 2008; Haston and Shields,
1984; Paluch et al., 2006), and the difference between the role of
the bleb-like protrusion in cell migration and enucleation
is probably influenced by differences in their cytoskeletal
organization. The unexpected findings in the present study thus
348
Journal of Cell Science 125 (2)
Fig. 8. Proposed model for PI3K-mediated regulation of erythroblast enucleation. The reorientation from a radial microtubule array to an asymmetric array
results in localized activation of PI3K and thereby promotes asymmetric actin assembly. Enucleation is triggered and continued by a pressure generated by
contractions of cytoplasmic actomyosin (large arrow) and is probably completed by contractions of concentrated actomyosin at the bleb neck (small arrows).
begin to elucidate the regulatory and mechanistic mechanisms
underlying mammalian erythroblast enucleation.
Materials and Methods
Cell culture and infection
Journal of Cell Science
Pregnant C57 BL/6J mice were purchased from the Genome Institute of Singapore
and the Center for Animal Resources (National University of Singapore). The
purification and culturing of mouse fetal liver erythroblast precursors (CFU-e;
TER119-negative cells) were performed as previously described (Ji et al., 2008;
Zhang et al., 2003). For retroviral gene transfer experiments, we used the murine
stem cell retroviral vector system (MSCV; Clontech, BD Biosciences). Infection of
primary cells with mouse-specific retroviruses encoding fusion proteins of
(3)Akt(PH)–GFP (Luo et al., 2005), (3)Akt(PHR25C)–GFP, mCherry, RFP or
GFP–c-tubulin was performed as previously described (Ji et al., 2008). All animal
experiments were performed according to the relevant regulatory standards.
Microscopy and image processing
For phase-contrast and bright-field time-lapse live-cell imaging, purified TER119negative cells grown on fibronectin-coated glass chamber dishes (McKenna and
Wang, 1989) were maintained at 37 ˚C in a custom-made incubator built on top of
an Axiovert 135 or Axiovert 200M inverted microscope (Carl Zeiss) and viewed
with a 1006, Plan Apo NA 1.25 or 1006, NA 1.30, Plan-NEOFLUAR lens.
Images were acquired with a video camera (Mintron) or a cooled charge-coupled
device camera (CoolSNAPHQ, Roper Scientific) and processed with MetaView
(Universal Imaging) or Image-Pro Plus software. Images of cells expressing
(3)Akt(PH)–GFP, cells expressing (3)Akt(PH)–GFP and mCherry or RFP, cells
expressing (3)Akt(PHR25C)–GFP and cells expressing GFP–c-tubulin were
acquired using an Axiovert 200M inverted microscope (Carl Zeiss) equipped
with 1006, NA 1.4 Plan-Apochromat lens, a spinning disk confocal (Yokogawa
CSU-21) scan head and Hamamatsu Orca-ER cooled CCD camera. Bright field
images (Fig. 1B,D) were uniformly filtered to reduce noise using ImageJ (NIH,
Bethesda, MD).
For imaging of F-actin, stacks of images acquired using a LSM 510 Meta
confocal microscope system (1006, NA 1.4 Plan-Apochromat lens) were
collected at a vertical interval of 0.75 mm and processed with IMARIS
(Bitplane, Scientific Solutions).
For imaging of microtubules, Z-stacks of wide-field images were collected at
intervals of 0.3 mm, were deconvoluted (for Fig. 3A) using AutoQuant (Media
Cybernetics) and processed with a pattern recognition program that detects linear
structures as described previously (Wang, 2003). For imaging of EB1 and
pericentrin, stacks of wide-field images were collected at vertical intervals of
0.3 mm and projected with a maximum-intensity algorithm using ImageJ. For ctubulin, single wide-field images were collected.
Immunofluorescence
Immunofluorescence staining was performed according to the methods previously
described (Wheatley and Wang, 1998). We used primary antibodies against rabbit
Ser19-phosphorylated myosin light chain II (1:50, Cell Signaling), mouse anti-btubulin (1:1000, AbCam), mouse anti-EB1 (1:50, BD Biosciences, Pharmingen),
mouse anti-c-tubulin (1:1000, Sigma); rabbit anti-pericentrin (1:700, Abcam).
Secondary antibodies Alexa-Fluor-488-conjugated goat anti-rabbit IgG, AlexaFluor-488-conjugated goat anti-mouse IgG and Alexa-Fluor-546-conjugated goat
anti-mouse IgG (Molecular Probes), all diluted 1:100. To observe F-actin,
Rhodamine–phalloidin (Molecular Probes) was used at 1:300 dilution. Hoechst
33258 (1 mg/ml) was used to locate the nucleus.
Drug treatment
Cytochalasin D (Sigma), blebbistatin (Toronto Research Chemicals), nocodazole
(Sigma), LY294002 (Invitrogen) and wortmannin (Sigma) were dissolved in
DMSO to make stock solutions of 25 mM, 100 mM, 3.3 mM, 50 mM and 1 mM,
respectively, and applied to a culture dish at final concentration of 2 mM, 100 mM,
10 mM, 50 mM and 1 mM, respectively. Note that .10 mM nocodazole was
required to completely depolymerize microtubules in late erythroblasts.
Data analysis
To measure the size of the nuclear area, the area of the ellipse corresponding to the
shape of the nucleus was calculated using ImageJ. To track the distance between
the centroids of the cell and the centroids of the nucleus or the MTOC, each
perimeter was drawn and the centroid corresponding to the nucleus and the cell
was calculated using ImageJ. The distance between these centroids was calculated.
Acknowledgements
We thank TLL Microscopy and Animal Care facilities and Shazmina
Rafee for their technical assistance, Koichi Okumura (Cancer
Science Institute of Singapore) for helpful suggestions, and G.
Wright and J. Brocher for advice on image processing. We thank all
members of the Murata-Hori laboratory for discussions and/or
critical reading of the manuscript.
Funding
This study was supported by intramural funds from the Temasek Life
Sciences Laboratory to M.M.-H. and by the National Institutes of
Health [grant number P01 HL 32262] to H.F.L. Deposited in PMC
for release after 12 months.
Supplementary material available online at
http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.088286/-/DC1
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