mammalian erythroblast enucleation requires pi3k-dependent ...mammalian erythroblast enucleation...

Mammalian erythroblast enucleation requiresPI3K-dependent cell polarization

Junxia Wang1,*, Tzutzuy Ramirez1,*, Peng Ji2,§, Senthil Raja Jayapal2,3, Harvey F. Lodish2 andMaki Murata-Hori1,4,`

1Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, 117604, Singapore2Whitehead Institute for Biological Research, Department of Biology, Massachusetts Institute of Technology, 9 Cambridge Center, Floor 6,Cambridge, MA 02142, USA3Stem Cell Group 4, Genome Institute of Singapore, #08-01, Genome, 60 Biopolis Street, Singapore 1386724Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543

*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])

Accepted 11 August 2011Journal of Cell Science 125, 340–349� 2012. Published by The Company of Biologists Ltddoi: 10.1242/jcs.088286

SummaryEnucleation, the final step in terminal differentiation of mammalian red blood cells, is an essential process in which the nucleussurrounded by the plasma membrane is budded off from the erythroblast to form a reticulocyte. Most molecular events in enucleationremain 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 becomesrestricted to the other side, where dynamic cytoplasmic contractions generate pressure that pushes the viscoelastic nucleus through anarrow 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 thatenucleation 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

IntroductionIn 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 cytokinetic-

like 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.

ResultsErythroblast 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

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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 (Jiet 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 time-

course 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 twolate erythroblasts in which the nuclei were located at the center of

each cell (Fig. 1A, 44 minutes). At ,3 hours after each wasgenerated (210±8.64 minutes, n58), these late erythroblastsunderwent 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 contractionsoccurred 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

whole nucleus was quickly squeezed out (5.98±2.73 minutes), in

the process undergoing extensive deformations into an hourglass-

like 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.

Mechanics of mammalian erythroblast enucleation 341

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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, Non-polarized). 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); thislocalization pattern remained unchanged during nuclear extrusion

(n516; Fig. 2A, Extruding nucleus). Three-dimensional imagesof (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.

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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 thenon-polarized cells treated with LY294002 failed to become

polarized during this time period (n536; Fig. 3A and Fig. 4). Thedistance between the cellular and nuclear centroids in polarized

control cells was 1.59±0.14 mm, whereas the maximum distancebetween these centroids was 0.62±0.09 mm in LY294002-treatedcells 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 non-

polarized (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

Fig. 3. PI3K activity is required for

establishment of cell polarization during

enucleation. (A) Time-lapse images of non-

polarized 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 F-

actin 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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as enucleated cells in cells treated with LY294002 from 31 to35 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 obtainedwhen 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 toinhibition of PI3K activity and that PI3K inhibition did notcompletely block cell polarization. When cells were treated with

LY294002 from 31 to 48 hours in culture, the decrease inpolarization and enucleation was no longer statistically significant(Fig. 3E). These results suggest that PI3K inhibition caused a severedelay rather than arrest in the establishment of cell polarization.

To determine whether PI3K inhibition affects nuclearextrusion, LY294002 was applied to cells at 35 hours in cultureand, 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 rangeof 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 adelay (11/46), whereas others moved out of the field of view

before the extrusion was completed or did not complete nuclearextrusion during time-lapse imaging (24/46, Fig. 4 andsupplementary material Fig. S4).

Cells that showed a delay in nuclear extrusion typically exhibited

one or both of the following phenotypes: (a) Nuclear extrusion wasinitiated but the nucleus regressed into the cell before the extrusionwas completed; some cells repeated this ‘trial and error’ of nuclear

extrusion several times. (b) Nuclear extrusion was initiated butfailed 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 adistance between the cellular and nuclear centroids of6.11±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 remainedrelatively 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-tubulinwere localized together in the cytoplasm in close proximity to the

nucleus at the side of the cytoplasm (n56; Fig. 6B).

Fig. 4. Summary of the defects in nuclear displacement and extrusion in cells treated with LY294002 or nocodazole. Live cells were treated with 50 mMLY294002 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.


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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 plus-

ends of growing microtubules (Mimori-Kiyosue et al., 2000),

typical comet-like structures were observed regardless of the stageof enucleation (Fig. 6C, arrows), suggesting that microtubules

are dynamic during enucleation. These results suggest thatmicrotubule organization is dramatically rearranged into anasymmetric array with the centrosome when late erythroblasts

become polarized.

To determine whether microtubules regulate PI3K activityduring enucleation, late erythroblasts expressing (3)Akt

(PH)–GFP were treated with nocodazole, a microtubule-depolymerization agent. Strikingly, in nocodazole-treated cellswhere microtubules were severely disrupted (3)Akt(PH)–GFP

failed to associate with the plasma membrane and insteadwas diffusely localized in the cytoplasm (11/15). By contrast,(3)Akt(PH)–GFP was associated with the membrane in controlcells (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 analysisshowed that when nocodazole was applied to cells at 32 hours inculture only 20% of non-polarized cells were able to undergopolarization within 3 hours (n520; Fig. 6F, Fig. 4 andsupplementary material Fig. S5C). F-actin was localizedthroughout the cytoplasm in nocodazole-treated cells thatremained non-polarized (n520; supplementary materialFig. S5D). When nocodazole was applied to cells at 36 hoursin culture, 63% of polarized cells exhibited a severe delay innuclear extrusion (n535; Fig. 6G, Fig. 4, supplementary materialFig. S4, Fig. S5E and Movie 4), with phenotypes similar to thoseseen in LY294002-treated cells (Fig. 5A).

Quantitative analysis revealed only a slight decrease in the

percentage of polarized and enucleated cells among cells treatedwith nocodazole from 32 to 36 hours in culture, compared withcontrol cells (supplementary material Fig. S5F). It is noteworthy

that treatment of erythroblasts with nocodazole from 32 to48 hours in culture resulted in severe defects in nuclear andcellular morphology (data not shown). The decrease was notsignificant when cells were treated with nocodazole from 36 to

40 hours in culture (supplementary material Fig. S5G). Theseresults suggest that microtubules are required for properenucleation by regulating the establishment and maintenance of

cell polarization. As detailed below, in large measure thisresolves past controversies about whether or not microtubulesplay an essential role in enucleation (Keerthivasan et al., 2010;

Koury et al., 1989; Sonoda et al., 1998). Unlike the crucial role ofmicrotubules in the regulation of PI3K activity, PI3K inhibitioncaused no drastic defects in microtubule organization in late

erythroblasts (Fig. 6H). Taken together, our observations suggestthat microtubule-dependent local activation of PI3K is requiredfor proper enucleation.

PI3K regulates cell polarization through promoting themovement of the nucleus but not the MTOCduring enucleation

In migrating fibroblasts, reorientation of the microtubule-organizing 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-cellanalyses showed that when late erythroblasts became polarized

the nucleus moved to one side of the cell (Fig. 3A,B), suggestingthat cell polarization might be initiated in a way similar to thatseen in migrating cells and that PI3K might regulate this process.

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.


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To test this, cells treated with LY294002 from 31 to 35 hours in

culture were fixed and stained for the nucleus and c-tubulin (seesupplementary 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 MTOCremains 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

Fig. 6. Enucleation requires microtubule-dependent local activation of PI3K. (A) Microtubule organization in cells prior to and during enucleation. Z-

projections 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.


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distance between the nuclear and the cellular centroids wasincreased, 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 PI3Kinhibition causes a delay in nuclear repositioning. Taken

together, our observations suggest that PI3K regulates cellpolarization through promoting the movement of the nucleusbut not the MTOC during enucleation.

DiscussionOne of the long-standing questions in erythropoiesis is how theerythroblast extrudes its nucleus to form the reticulocyte that laterbecomes the mature red blood cell. In particular, little is known

about how erythroblasts establish and maintain a polarized stateduring enucleation. The live-cell analyses we performed in thisstudy have provided a deeper understanding of the mechanisms

underlying the regulation of cell polarization and its importancein enucleation.

Early work suggested a mechanism of enucleation similar tothat used in cytokinesis of mitotic cells, where local cortical

contractions occur between the two daughter cells (Skutelsky andDanon, 1967; Koury et al., 1989). By contrast, our observationsdemonstrate that nuclear extrusion is driven by contractile forces

generated by asymmetrically distributed cytoplasmic actomyosinin late erythroblasts. This finding reiterates the importance ofestablishment and maintenance of cell polarization during

enucleation.

Although previous studies revealed that PI3K was required forearly 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 cellpolarization during enucleation. Moreover, our results alsoshowed that PI3K promotes cell polarization by repositioning

of the nucleus during enucleation. In migrating fibroblasts, cellpolarization is established by the movement of the nucleusinstead 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 thenucleus by PI3K is discussed below.

The roles of microtubules in enucleation have beencontroversial (Keerthivasan et al., 2010; Koury et al., 1989;

Sonoda et al., 1998). We observed different effects ofmicrotubule disruption agents on enucleation when cells weretreated at different stages of erythropoiesis and/or for different

time periods of incubation. Imaging and quantitative analysis oflive and fixed cells revealed that disruption of microtubulescaused a severe delay in enucleation, but did not affect the finalextent of enucleation. This indicates that microtubules are

required for, but not essential to, enucleation. Currently it isunknown how microtubules regulate PI3K activation duringenucleation. 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 thisoccurs in late erythroblasts or whether this affects activation or

localization of PI3K.

Interestingly, our results show that cell polarization is initiatedthrough repositioning of the nucleus but not MTOC during

enucleation, similar to that seen in migrating fibroblasts (Gomeset al., 2005; Schmoranzer et al., 2009). Nuclear repositioningoccurs concomitantly with the rearrangement of the microtubule

array that leads to asymmetric distribution of actomyosin andthus the generation of asymmetric force (see below). This forcemost probably drives the repositioning of the nucleus, somewhatsimilar 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 tobecome polarized. Then why do late erythroblasts need a longtime (2–3 hours) to establish polarization? This is probablybecause 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 issummarized in Fig. 8. The reorientation from a radialmicrotubule array to an asymmetric array somehow results inasymmetric activation of PI3K. Asymmetric accumulation of the

PI3K products in the plasma membrane then induce asymmetricactin assembly and disassembly by stimulating their effectors,such as a-actinin (Franke et al., 1997), profilin (Lassing andLindberg, 1985) or gelsolin (Janmey et al., 1987), as well as Rac1activity (Ji et al., 2008) probably through activation of itsupstream regulator (Han et al., 1998; Kunisaki et al., 2006). This

asymmetric actin assembly and disassembly and actomyosincontractions promote nuclear displacement as well as nuclearextrusion.

In cells defective in this microtubule-regulated PI3K-dependent polarization mechanism, enucleation could beachieved by random cytoplasmic contractions alone but in a

highly ineffective and slow manner. It is likely that such defectsin enucleation cause some proteins to be wrongly sorted to thereticulocytes or the extruded nucleus, leading to the formation of

abnormal reticulocytes.

Our results suggest that enucleation is carried out in a waymechanistically similar to directed cell migration. Some

migrating cells form a polarized bleb-like protrusion in order tomove 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 enucleationis probably influenced by differences in their cytoskeletalorganization. The unexpected findings in the present study thus

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.


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begin to elucidate the regulatory and mechanistic mechanisms

underlying mammalian erythroblast enucleation.

Materials and MethodsCell culture and infection

Pregnant C57 BL/6J mice were purchased from the Genome Institute of Singaporeand the Center for Animal Resources (National University of Singapore). Thepurification 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 murinestem cell retroviral vector system (MSCV; Clontech, BD Biosciences). Infection ofprimary cells with mouse-specific retroviruses encoding fusion proteins of(3)Akt(PH)–GFP (Luo et al., 2005), (3)Akt(PHR25C)–GFP, mCherry, RFP orGFP–c-tubulin was performed as previously described (Ji et al., 2008). All animalexperiments were performed according to the relevant regulatory standards.

Microscopy and image processing

For phase-contrast and bright-field time-lapse live-cell imaging, purified TER119-negative cells grown on fibronectin-coated glass chamber dishes (McKenna andWang, 1989) were maintained at 37 C̊ in a custom-made incubator built on top ofan Axiovert 135 or Axiovert 200M inverted microscope (Carl Zeiss) and viewedwith 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-coupleddevice 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, cellsexpressing (3)Akt(PHR25C)–GFP and cells expressing GFP–c-tubulin wereacquired using an Axiovert 200M inverted microscope (Carl Zeiss) equippedwith 1006, NA 1.4 Plan-Apochromat lens, a spinning disk confocal (YokogawaCSU-21) scan head and Hamamatsu Orca-ER cooled CCD camera. Bright fieldimages (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 Metaconfocal microscope system (1006, NA 1.4 Plan-Apochromat lens) werecollected 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 atintervals of 0.3 mm, were deconvoluted (for Fig. 3A) using AutoQuant (MediaCybernetics) and processed with a pattern recognition program that detects linearstructures as described previously (Wang, 2003). For imaging of EB1 andpericentrin, stacks of wide-field images were collected at vertical intervals of0.3 mm and projected with a maximum-intensity algorithm using ImageJ. For c-tubulin, single wide-field images were collected.

Immunofluorescence

Immunofluorescence staining was performed according to the methods previouslydescribed (Wheatley and Wang, 1998). We used primary antibodies against rabbitSer19-phosphorylated myosin light chain II (1:50, Cell Signaling), mouse anti-b-tubulin (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, Alexa-Fluor-488-conjugated goat anti-mouse IgG and Alexa-Fluor-546-conjugated goatanti-mouse IgG (Molecular Probes), all diluted 1:100. To observe F-actin,Rhodamine–phalloidin (Molecular Probes) was used at 1:300 dilution. Hoechst33258 (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 inDMSO 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 wasrequired to completely depolymerize microtubules in late erythroblasts.

Data analysis

To measure the size of the nuclear area, the area of the ellipse corresponding to theshape of the nucleus was calculated using ImageJ. To track the distance betweenthe centroids of the cell and the centroids of the nucleus or the MTOC, eachperimeter was drawn and the centroid corresponding to the nucleus and the cellwas calculated using ImageJ. The distance between these centroids was calculated.

AcknowledgementsWe thank TLL Microscopy and Animal Care facilities and ShazminaRafee for their technical assistance, Koichi Okumura (CancerScience Institute of Singapore) for helpful suggestions, and G.Wright and J. Brocher for advice on image processing. We thank allmembers of the Murata-Hori laboratory for discussions and/orcritical reading of the manuscript.

FundingThis study was supported by intramural funds from the Temasek LifeSciences Laboratory to M.M.-H. and by the National Institutes ofHealth [grant number P01 HL 32262] to H.F.L. Deposited in PMCfor 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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mammalian erythroblast enucleation requires pi3k-dependent ...mammalian erythroblast enucleation...

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