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SNX9, SNX18 and SNX33 are required for progressionthrough and completion of mitosis

Maggie P. C. Ma and Megan Chircop*Children’s Medical Research Institute, The University of Sydney, 214 Hawkesbury Road, Westmead, NSW 2145, Australia

*Author for correspondence (mchircop@cmri.org.au)

Accepted 24 May 2012Journal of Cell Science 125, 4372–4382� 2012. Published by The Company of Biologists Ltddoi: 10.1242/jcs.105981

SummaryMitosis involves considerable membrane remodelling and vesicular trafficking to generate two independent cells. Consequently,endocytosis and endocytic proteins are required for efficient mitotic progression and completion. Several endocytic proteins also

participate in mitosis in an endocytosis-independent manner. Here, we report that the sorting nexin 9 (SNX9) subfamily members –SNX9, SNX18 and SNX33 – are required for progression and completion of mitosis. Depletion of any one of these proteins using siRNAinduces multinucleation, an indicator of cytokinesis failure, as well as an accumulation of cytokinetic cells. Time-lapse microscopy on

siRNA-treated cells revealed a role for SNX9 subfamily members in progression through the ingression and abscission stages ofcytokinesis. Depletion of these three proteins disrupted MRLCS19 localization during ingression and recruitment of Rab11-positiverecycling endosomes to the intracellular bridge between nascent daughter cells. SNX9 depletion also disrupted the localization of Golgi

during cytokinesis. Endocytosis of transferrin was blocked during cytokinesis by depletion of the SNX9 subfamily members, suggestingthat these proteins participate in cytokinesis in an endocytosis-dependent manner. In contrast, depletion of SNX9 did not blocktransferrin uptake during metaphase but did delay chromosome alignment and segregation, suggesting that SNX9 plays an additional

non-endocytic role at early mitotic stages. We conclude that SNX9 subfamily members are required for mitosis through bothendocytosis-dependent and -independent processes.

Key words: SNX9, SNX18, SNX33, Mitosis, Vesicle trafficking, Abscission

IntroductionEndocytosis is thought to shut down during mitosis then resume

during the final stage, cytokinesis (Schweitzer et al., 2005). In supportof this idea, several endocytic proteins including disabled-2 (Dab2),dynamin II (dynII), epsin, eps15, a-adaptin and amphiphysin II, areinactivated by Cdk1-mediated phosphorylation during mitosis (Chen

et al., 1999; Chircop et al., 2010; Dephoure et al., 2008; He et al.,2003; Kariya et al., 2000). Furthermore, clathrin, epsin and cyclin-G-associated kinase (GAK) have been shown to play non-endocytic

roles in chromosome alignment and segregation during mitosis (Liuand Zheng, 2009; Royle et al., 2005; Shimizu et al., 2009).

Cytokinesis results in the generation of two independent

daughter cells and can be divided into two stages: (1) membraneingression to generate a cleavage furrow between segregatedchromosomes followed by (2) membrane abscission at a site along

the intracellular bridge (ICB) connecting the two nascent daughtercells (Glotzer, 2005). Vesicles derived from internal organelles,including the Golgi, lysosomes, early endosomes and recyclingendosomes, traffic from both daughter cells to the ICB. These

vesicles accumulate asymmetrically on one side of the centrallylocated midbody ring (MR) and are required for abscission (Gossand Toomre, 2008). However, the role of these vesicles in

abscission is unknown.

During cytokinesis, endocytosis contributes to the generation ofrecycling endosomes that are delivered to the ICB. Depletion or

functional inhibition of dynII and Arf6 results in multinucleatecells, an indicator of cytokinesis failure (Chircop et al., 2011a;Fielding et al., 2005; Joshi et al., 2010). Arf6 depletion blocks

trafficking of Rab11-positive recycling endosomes to the ICB

(Fielding et al., 2005). In contrast, dynII and clathrin localise to themidbody (Chircop et al., 2010; Gromley et al., 2005; Low et al.,

2003; Warner et al., 2006), suggesting that they participate moredirectly in the abscission process. The identification of all

endocytic proteins required for mitosis and their roles during thisprocess are yet to be revealed.

Sorting nexin 9 (SNX9) is one of the major binding partners fordynII in endocytosis (Lundmark and Carlsson, 2004). SNX9 belongs

to the sorting nexin (SNX) superfamily of proteins that consistsof 33 proteins in mammals (Seet and Hong, 2006). All members

contain a SNX–Phox–homology (PX) domain and associate with

phosphotidylinositol-3-monophosphate-enriched elements of theearly endocytic network via their PX domain (Yarar et al., 2008).

The SNXs function in diverse processes, including endocytosis,endosomal sorting and endosomal signalling (Carlton et al., 2005;

Seet and Hong, 2006). Together with SNX18 and SNX33, SNX9belongs to the SNX9 subfamily due to the presence of a Bin–

Amphiphysin–Rvs (BAR) domain that allows dimerization and

contributes to modulation and shaping of membrane curvature(Carlton et al., 2004), as well as a Src-homology 3 (SH3) domain that

allows interaction with a wide range of proline-rich PXXP motifcontaining proteins (Alto et al., 2007; Mayer, 2001). Of the three

SNX9 subfamily members, SNX9 is the best characterized. It is amajor binding partner of a number of proteins involved in clathrin-

mediated endocytosis (CME), such as clathrin, cargo adaptor protein-

2 (AP-2), dynamin, synaptojanin, actin-related protein-2/3 (ARP2/3)complex activator, Wiskott-Aldrich syndrome protein (WASp) and

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Cdc42-associated kinase (ACK) (Badour et al., 2007; Lundmark and

Carlsson, 2002; Lundmark and Carlsson, 2004; Shin et al., 2008;

Soulet et al., 2005; Yarar et al., 2007; Yeow-Fong et al., 2005). SNX9

is also essential for dorsal-ruffle formation and clathrin-independent,

actin-dependent fluid phase endocytosis (Yarar et al., 2007).

Although the three SNX9 subfamily members share highly similar

structures, they appear to have non-overlapping functions and it

remains unclear if they can form heteromeric complexes (Dislich

et al., 2011; Haberg et al., 2008). SNX18 functions in clathrin-

independent endosomal trafficking that is dependent on AP1 and the

retrograde trafficking protein PACS1 (Haberg et al., 2008). SNX18

also plays a redundant role to SNX9 in CME (Park et al., 2010).

SNX33 has been implicated in dynamin-dependent endocytosis by

modulating amyloid precursor protein (APP) endocytosis and a-

secretase cleavage (Schobel et al., 2008) and the formation of prion

protein (Heiseke et al., 2008). Of the three SNX9 subfamily members,

only SNX33 has been shown to play a role in mitosis, as its

overexpression results in micronucleate cells (Zhang et al., 2009). Its

role in mitosis is thought to be linked to its ability to regulate actin

polymerization via its association with WASp (Zhang et al., 2009).

Thus it is proposed to be a modulator of the actinomyosin II

contractile ring for membrane ingression during cytokinesis.

In this study we aimed to determine the roles of all three SNX9

subfamily members in mitosis. We show that all are required for

successful completion of both the ingression and abscission

stages of cytokinesis and for the delivery of a subset of vesicles to

the ICB during this process. We also show that SNX9 plays an

additional role during metaphase that is not dependent on its

endocytic function.

ResultsDepletion of SNX9, SNX18 or SNX33 causes

multinucleation and delays mitotic exit

SNX33 is required for successful completion of mitosis (Zhang

et al., 2009). Therefore, we aimed to determine whether SNX9

and SNX18 are also required for mitosis by assessing the mitotic

phenotype of HeLa cells depleted of these proteins using siRNA.

Two siRNAs were targeting each SNX9 subfamily member

was assessed. At 72 h post-transfection, immunoblot analyses

revealed satisfactory reduction in the expression level of SNX9,

SNX18 and SNX33 by their specific siRNAs compared to

untransfected and luciferase (Luc) siRNA controls (Fig. 1A–C).

A similar reduction in the protein level of these proteins was also

observed by immunofluorescence microscopy (supplementary

Fig. 1. SNX9, SNX18 or SNX33 depletion

causes cytokinesis failure. HeLa cells were

either untreated (–), or transfected with luciferase

siRNA (Luc) or siRNA targeting SNX9 (A),

SNX18 (B) or SNX33 (C). Two siRNA per

SNX9 subfamily member were assessed. At 72 h

post-transfection, protein lysates (200 mg) were

immunoblotted with anti-SNX9, anti-SNX18 or

anti-SNX33 antibodies. c-tubulin served as a

loading control. Duplicates of each siRNA

treatment were immunoblotted. The separate

sections of blots shown in A–C were all from the

same blot. Cells were transfected as described in

A–C and synchronized at the G2–M boundary

using RO-3306. Upon RO-3306 wash out cells

were released for 6 hours, fixed and

immunostained for a-tubulin and scored for

multinuclei (D) and cytokinesis (E).

(F–H) Rescue experiment. Untreated and SNX-

siRNA-treated HeLa cells were transfected with

either GFP-SNX9, HA-SNX18 or GFP-SNX33.

Prepared protein lysates (200 mg) were

immunoblotted with anti-SNX9 (F), anti-SNX18

(G), anti-SNX33 (H), anti-HA (G) and anti-GFP

(F and H). In parallel, cells were immunostained

for a-tubulin and scored for multinuclei

(F–H, bottom panels). The graphs show the

means 6 s.e.m. from three independent

experiments where n.200 cells were scored for

each sample in each experiment. *P,0.05;

**P,0.01; ***P,0.001 (one-way ANOVA).

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material Fig. S1). The siRNAs were highly specific to each SNX

protein as depletion of any one of the three SNX proteins by their

specific siRNA did not affect expression of the other two SNX9

subfamily members (Fig. 1A–C). Depletion of any one of these

proteins caused a significant increase in the percentage of

multinucleate cells, an indicator of cytokinesis failure (Fig. 1D).

Specifically, depletion of SNX9 resulted in a fourfold increase in

multinucleation (untreated: 3.860.9%; Luc siRNA: 3.460.7%;

SNX9-1 siRNA: 11.963.2%; SNX9-2 siRNA: 12.161%;

Fig. 1D), whereas depletion of SNX18 or SNX33 caused a

sevenfold increase in multinucleation (SNX18-1 siRNA:

20.561.7%; SNX18-2 siRNA: 30.460.5%; SNX33-1 siRNA:

17.662.3%; SNX33-2 siRNA: 30.563%; Fig. 1D). At 6 h post-

mitotic entry, there was a significant increase in cells undergoing

cytokinesis following depletion of SNX9 subfamily members

(untreated: 2.060.52%; Luc siRNA: 2.860.4%; SNX9-1 siRNA:

8.263.2%; SNX9-2 siRNA: 10.962.8%; SNX18-1 siRNA:

10.960.9%; SNX18-2 siRNA: 13.661.5%; SNX33-1 siRNA:

15.461.5%; SNX33-2 siRNA: 19.361.5%; Fig. 1E), suggesting

that depletion of any one of these proteins delays mitotic exit.

Since both siRNAs assessed per protein induced similar

phenotypes, SNX9-1, SNX18-1 and SNX33-1 siRNAs were used

in all subsequent experiments. Depletion of all three SNX proteins

produced similar phenotypes in U2OS human osteosarcoma cells

(supplementary material Fig. S2). We next aimed to rescue the

multinucleation phenotype induced by SNX9, SNX18 or SNX33

depletion by overexpressing GFP–SNX9 (Fig. 1F), HA–SNX18

(Fig. 1G) and GFP–SNX33 (Fig. 1H), respectively. Indeed,

ectopic expression of these proteins completely rescued the

multinucleation phenotype (Fig. 1F–H). We conclude that all

three SNX9 subfamily members are required for successful

completion of mitosis.

To further characterise the role of the SNX9 subfamily in

mitosis we carried out time-lapse video microscopy to measure the

time spent by individual cells in mitosis (Fig. 2A). Consistent with

our fixed cell analysis (Fig. 1D,E), an increased number of SNX9-

subfamily-depleted cells failed mitosis and either became

binucleate or did not complete mitosis within the 20 h

experimental period (Fig. 2B). Regardless of whether or not

these depleted cells completed or failed mitosis they spent a longer

period of time in mitosis than cells treated with Luc siRNA

(Fig. 2C,D). Specifically, cells depleted of SNX9, SNX18 and

Fig. 2. SNX9 subfamily proteins are

required for efficient mitotic progression.

(A) Schematic diagram of the phases of

mitosis with a plan of the time-lapse

experiment shown above. Pro, prophase; Ana,

anaphase; Comp, completion; Multi,

multinucleation. (B,C) HeLa cells were

treated with siRNA targeting Luc, SNX9,

SNX18 or SNX33 and visualized by time-

lapse microscopy. (B) The graph shows the

percentage of cells that entered but did not

complete mitosis or were multinucleate by the

end of the 20 h experimental period (n.80

cells per sample). (C) The graph shows the

time each individual cell took to undergo

mitosis (n.80 per sample). Depletion of any

one of the SNX9 subfamily members prolongs

the time cells spend in mitosis. The unbroken

black line represents the median time.

(D) Selected frames from representative time-

lapse movies of Luc-, SNX9-, SNX18- and

SNX33-siRNA-treated cells. Data shown in

B–D are from one representative experiment.

Similar results were obtained in at least two

independent experiments.

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SNX33 spent a median time of 210, 220 and 270 min in mitosis,

respectively, compared to 120 min for Luc siRNA control cells(Fig. 2C,D). Thus, the function of these three proteins is associated

with efficient mitotic progression and completion.

SNX9, 18 and 33 have multiple points of actionthroughout mitosis

To determine the point of action of the three SNX9 subfamilymembers during mitosis, we determined the time siRNA-treated

cells took to progress through the following four mitotic stages:

prophase to anaphase, and anaphase to completion of cytokinesis,

either successfully or unsuccessfully generating a multinucleatecell (Fig. 2A). Neither SNX18 nor SNX33 appear to play a role at

early mitotic stages from prophase to anaphase since cells

depleted of these proteins completed this transition with similarkinetics to Luc siRNA control cells (Fig. 3A). In contrast,

completion of this mitotic transition was delayed in SNX9-

depleted cells, with a median time of 60 min vs 50 min for Luc

siRNA control cells (Fig. 3A). Depletion of all three proteinscaused a significant delay at the anaphase to completion or

multinucleation transition (Fig. 3B). These findings indicate that

all three SNX9 subfamily members are required for efficientprogression through cytokinesis and that SNX9 may have an

Fig. 3. SNX9 subfamily members have multiple points of action

throughout mitosis. (A,B) Those HeLa cells that were analysed in Fig. 2B,C

were further characterized by quantifying the time they took to complete the

following mitotic transitions: prophase–anaphase (A), and anaphase to mitotic

completion or multinucleate formation (B). Pro, prophase; Ana, anaphase;

Comp, completion; Multi, multinucleation; n.80 cells per sample. The

median time per sample is represented by a solid black line. Data shown in

graphs are from one representative experiment. Similar results were obtained

in at least two independent experiments.

Fig. 4. Localization of SNX9, SNX18 and SNX33 in HeLa

cells during mitosis. (A–C) Asynchronously growing HeLa

cells were fixed and stained for SNX9 (A), SNX18 (B), or

SNX33 (C) and co-stained for a-tubulin (red). DNA was

stained with DAPI (blue). Representative microscopy images

illustrating the localization of the three SNX9 subfamily

members at the indicated mitotic stages are shown. Int,

interphase; Promet, prometaphase; Met, metaphase; Ana,

anaphase; Tel, telophase; Cyto, cytokinesis.

(D) Representative microscopy images of cytokinetic HeLa

cells co-stained for the indicated SNX9 subfamily protein

(green) and the midbody ring marker, c-tubulin (red). SNX9

and SNX33 colocalize with c-tubulin.

(E) Representative microscopy images of cytokinetic HeLa

cells ectopically expressing GFP–SNX9 or stained for SNX18

or SNX33 (green) and co-stained with the flanking midbody

ring marker, dynIIS764 (red). SNX9 localizes to the flanking

midbody rings. GFP–SNX9 was ectopically expressed in this

experiment due to antibody cross-reactivity. Scale bars: 10 mm

(A–C); 1 mm (D,E).

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additional role in ensuring efficient completion of chromosomealignment and segregation in early mitosis.

We next examined the subcellular localizations of SNX9subfamily members during mitosis. In interphase, all threeproteins were observed in the cytoplasm in punctate structures

(Fig. 4A–C), consistent with previous observations (Haberg et al.,2008). We also observed SNX18 and SNX33 located in thenucleus. Upon mitotic entry (prometaphase), SNX9, but not

SNX18 and SNX33, accumulated at the spindle poles (Promet;Fig. 4A). SNX9 dissociated from the spindle poles uponchromosome alignment (Met; Fig. 4A) and segregation (Ana;

Fig. 4A), then accumulated at the ingressing furrow during earlystages of cytokinesis (Tel; Fig. 4A). In contrast, SNX18 and 33were localized to puncta in the cytoplasm throughout mitosis(Fig. 4B,C). Towards the end of the abscission stage, a small

amount of SNX9 and SNX33, but not SNX18, located to themidbody region within the ICB (Cyto; Fig. 4A,C). The ICBcontains a three ring structure: the centrally located MR and twin

flanking midbody rings (FMRs) that reside on either side of the MR(Chircop et al., 2010; Julian et al., 1993). Co-immunofluorescencemicroscopy revealed that SNX9 and SNX33 localize to a region

that overlaps all three rings as indicated by their colocalization withc-tubulin (a MR marker; Fig. 4D) and phospho-dynII (a FMRmarker; Fig. 4E). SNX18 did not localize to any of the ICBrings (Fig. 4D,E). The specificity of these cell cycle specific

localizations were confirmed as they were not observed in cellsdepleted of the relevant SNX protein (supplementary material Fig.S1). The distinct mitotic localizations of SNX9, SNX18 and

SNX33 suggest that they have different mitotic roles.

Effects of the depletion of SNX9 subfamily members onendocytosis during interphase and mitosis

We next sought to determine if the mitotic role(s) of SNX9,

SNX18 and SNX33 are dependent on their endocytic functions.We used the cellular uptake of Transferrin (Tfn) conjugated toTexas Red as a marker for endocytic function. We found that Tfnuptake was significantly reduced during metaphase compared to

during interphase and this resumed during cytokinesis (Fig. 5), aspreviously reported (Schweitzer et al., 2005). Depletion of SNX9blocked endocytosis of Tfn by .77% in interphase cells (Fig. 5),

as previously reported (Lundmark and Carlsson, 2003; Soulet et al.,2005). An analogous reduction in Tfn uptake in interphase cellswere also observed in SNX18 and SNX33 depleted cells (Fig. 5).

This is presumably an indirect effect, due to a block in endosomaltrafficking (Park et al., 2010; Schobel et al., 2008). Tfn uptake wasalso blocked by the depletion of these proteins in cytokinetic cells

(Fig. 5). In contrast, although Tfn uptake was already low incontrol metaphase cells, there was no further reduction in uptake inSNX9-subfamily-depleted metaphase cells (Fig. 5). This finding isconsistent with previous reports illustrating that endocytic proteins

are inactivated during mitosis. We conclude that all SNX9subfamily proteins are required for efficient endocytosis duringinterphase and cytokinesis but not during metaphase.

SNX9 participates in chromosome alignment andsegregation

Mitotic stages from prophase to anaphase involve chromosomecondensation and alignment at the metaphase plate followed by

their equal segregation. Mitotic progression through these earliermitotic stages is slower in SNX9-depleted cells (Fig. 3A). To gaininsight into the specific role of SNX9 during the earlier stages of

mitosis, we again examined the progression of SNX9-depleted

cells from prophase to anaphase by time-lapse video microscopy

but in this case HeLa cells stably expressing GFP–H2B were

analysed to clearly define each mitotic stage. Moreover, cells were

observed at 2 min intervals (instead of 10 min) to more accurately

assess the point of mitosis that is delayed. Similar to the results

shown in Fig. 3A, HeLa-GFP–H2B cells depleted of SNX9 also

spent a significantly longer period of time in the prophase to

anaphase transition with a median time of 75 min (SNX9-1

siRNA) and 71 min (SNX9-2 siRNA) vs 38 min in untreated cells

(Fig. 6A). We next separated this mitotic transition into distinct

events: chromosome alignment (prophase–metaphase) and

chromosome segregation (metaphase–anaphase). The results

indicated that SNX9 plays a role in both mitotic events as HeLa-

GFP–H2B cells depleted of SNX9 were delayed in their mitotic

progression through both transitions [median time of 34 min

Fig. 5. Endocytosis of transferrin (Tfn) is blocked by depletion of SNX9

members during interphase and cytokinesis. Untreated and SNX9-,

SNX18- and SNX33-depleted HeLa cells at the indicated mitotic stage were

subject to an endocytosis assay to observe the cellular uptake of Texas-Red-

conjugated Tfn. (A) Representative microscopy images at each mitotic stage.

DNA is shown in blue. (B) Graph showing the quantification of Tfn uptake

during interphase, metaphase and cytokinesis. Values are means 6 s.e.m.

from three independent experiments. n510–30 cells per sample in each

experiment. ns, not significant. *P,0.05; **P,0.01; ***P,0.001 (one-way

ANOVA). Scale bars: 10 mm.

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(SNX9-1 siRNA) and 38 min (SNX9-2 siRNA) vs 20 min for

untreated cells during prophase–metaphase (Fig. 6B); median time

of 31 min (SNX9-1 siRNA) and 34 min (SNX9-2 siRNA) vs

22 min for untreated cells during metaphase–anaphase; Fig. 6C].

Again, consistent with our findings in HeLa cells (Fig. 3), depletion

of SNX18 or SNX33 did not affect mitotic progression of HeLa-

GFP–H2B cells through these mitotic phases (Fig. 6A–C).

We next investigated the requirement of SNX9 in regulating

chromosome dynamics. The width of the centre of the metaphase

plate was significantly wider in metaphase HeLa-GFP–H2B cells

that were depleted of SNX9 compared to untreated cells

(Fig. 6D,G). Consistent with the time-lapse data indicating that

SNX18 and SNX33 are not required for progression from prophase

to anaphase, the width of the metaphase plate was not affected in

HeLa-GFP–H2B cells – depleted of these proteins (Fig. 6E–G).

These findings indicate that the metaphase function of SNX9

contributes to chromosome congression and alignment.

Role of SNX9, SNX18 and SNX33 during the ingression and

abscission phases of cytokinesis

Cleavage furrow formation during the ingression phase of

cytokinesis is driven by the activity of the actinomyosin II

contractile ring, which assembles at the cell equator between

segregated chromosomes. Myosin regulatory light chain (MRLC)

is a component of the myosin II filaments, and its phosphorylation

on T18 and S19 drives the assembly of the actinomyosin II

contractile ring (Ikebe et al., 1988) and activity of the myosin II

motor (Komatsu et al., 2000; Yamakita et al., 1994).

Phosphorylation of the major site, S19, allows myosin II to

interact with actin to assemble an actin–myosin II complex and

initiate contraction (Scholey et al., 1980). Given that all three SNX9

subfamily members are required for cytokinesis (Fig. 3B), we

assessed the activity of the myosin II motor using an antibody that

specifically recognises MRLC phosphorylated at S19. MRLCS19

localization was disrupted in SNX9-subfamily-depleted cells

Fig. 6. SNX9 functions during metaphase for chromosome

alignment. (A–C) HeLa-GFP–H2B cells were either

untreated or treated with siRNA targeting Luc, SNX9, SNX18

or SNX33, and visualized by time-lapse microscopy; images

were captured at 2 min intervals for 6 h. The graphs show the

time taken for each individual cell to complete the following

mitotic transitions: prophase to anaphase (A), prophase to

metaphase (B) and metaphase to anaphase (C). Pro, prophase;

Met, metaphase; Ana, anaphase. n56–10 cells. Solid black

line represents the median time. (D–F) Representative

microscopy images of metaphase cells showing the

chromosomes (GFP–H2B) and indicated endogenous SNX9

subfamily members (red) in untreated and SNX-depleted

HeLa-GFP–H2B cells. (G) Graph showing the width of the

metaphase plate, which was quantified by measuring the width

of the centre of the metaphase plate of HeLa-GFP–H2B cells

in metaphase that were treated with the indicated siRNAs.

Values are means 6 s.e.m. from three independent

experiments; n520–30 cells per sample in each experiment.

ns, not significant; ***P,0.001 (one-way ANOVA). Scale

bars: 10 mm.

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during anaphase and telophase (Fig. 7A). In contrast to control cells

where MRLCS19 was concentrated at the ingressing furrow during

anaphase and telophase, it was diffusely distributed in the

cytoplasm in cells depleted of any one of the SNX9 subfamily

proteins (Fig. 7A). The ratio of the fluorescence intensity of

MRLCS19 staining at the ingressing furrow or cleavage furrow:the

fluorescence intensity of MRLCS19 at the polar region was used to

quantitate this effect (Fig. 7A, right panel). A significant reduction

of .40% in MRLCS19 fluorescence signal at the ingressing furrow

and cleavage furrow was detected (Fig. 7A–C). This was not due to

an overall loss of protein as MRLCS19 levels were not significantly

different in depleted cells compared to untreated controls cells

(supplementary material Fig. S3). These findings indicate that all

SNX9 subfamily members contribute to the efficient accumulation

of active myosin II at the cleavage furrow during ingression.

All three SNX9 members are required for endocytosis (Fig. 5)

and vesicle trafficking is required for the abscission phase of

cytokinesis. Therefore, we next assessed the role of these proteins

in regulating vesicle trafficking from the Golgi, recycling

endosomes (RE), lysosomes and endoplasmic reticulum (ER) to

the ICB. In untreated cells, GM130 (a Golgi marker) was observed

as a network of punctate structures distributed throughout the

cytoplasm in metaphase (Fig. 8A, Met), consistent with the early

stages of Golgi reformation (Sutterlin and Colanzi, 2010). During

cytokinesis, GM130 accumulated in two pools in each of the nascent

daughter cells (Fig. 8A, Cyto), as previously reported (Gaietta et al.,

2006). Depletion of SNX9 resulted in an accumulation of GM130 at

the inner pool, as indicated by an increase in the ratio of the

fluorescence intensity of the inner pool:fluorescence intensity of the

outer pool in each nascent daughter cell (Fig. 8A, bottom panel;Fig. 8B). The overall protein level of GM130 was unaffected by thedepletion of SNX9 during metaphase (supplementary material Fig.

S4A) or cytokinesis (supplementary material Fig. S4B), suggestingthat SNX9 is involved in vesicle trafficking from the Golgi to theICB. Depletion of SNX18 and SNX33 had no effect on thedistribution and the level of GM130 during metaphase and

cytokinesis (Fig. 8A,B; supplementary material Fig. S4).

We next examined the recycling endosomes (RE). The REmarker, Rab11, localized at the spindle poles and the cleavage

furrow during telophase and along the ICB during cytokinesis inuntreated cells (Fig. 8C), as previously observed (Wilson et al.,2005). Depletion of any one of the SNX9 members disruptedRab11 localization at both mitotic stages (Fig. 8C–E) but did not

affect the overall level of Rab11 in telophase (supplementarymaterial Fig. S5A) or cytokinesis (supplementary material Fig.S5B). In contrast, the level and localization of PDI (an ER

marker) and Lamp1 (a lysosome marker) were unaffected by thedepletion of these proteins (supplementary material Figs S6, S7).We conclude that all three SNX9 members are required for

localization of the recycling endosomes to the cleavage furrowand ICB during cytokinesis. SNX9 plays an additional role in theaccumulation of Golgi at the ICB during abscission.

DiscussionIn this study, we confirmed the requirement of SNX33 for mitoticprogression and completion. In addition, our findings show that its

two closest-related SNX members, SNX9 and SNX18, also playimportant roles in mitosis. Collectively, these three SNX9subfamily members are essential for efficient progression andcompletion of cytokinesis. These proteins are also required for

endocytosis during these mitotic phases, suggesting that theycontribute to vesicle trafficking during cytokinesis. Indeed, allthree proteins are required for the correct localization of recycling

endosomes during cytokinesis. SNX9 also contributes to thelocalization of Golgi to the ICB. Our additional major findingindicates that SNX9 is also required for chromosome alignment

and segregation during metaphase and anaphase. SNX9 does notparticipate in endocytosis during metaphase, suggesting that itsrole in this process is independent of its endocytic function. Thesefindings demonstrate that SNX9 subfamily proteins participate in

multiple stages throughout mitosis in both endocytic-dependentand -independent manners.

Only SNX9 was shown to have a role during metaphase. The

increased width of the metaphase plate observed in SNX9-depletedcells supports a role for SNX9 in chromosome alignment.Consistent with this role, it is found to be enriched at the spindlepoles during prometaphase. Clathrin is a major binding partner for

SNX9 in endocytosis (Lundmark and Carlsson, 2003) and is alsorequired for chromosome alignment and segregation (Royle et al.,2005). Like SNX9, its role in this process is independent of its role

in endocytosis (Royle et al., 2005). During metaphase, clathrinlocalizes to the mitotic spindle and spindle poles (Royle et al.,2005). Depletion or mutation of clathrin leads to destabilization of

the kinetochore fibres, which results in a defect in chromosomealignment and delay in chromosome segregation (Royle et al.,2005). This results in persistent activation of the spindle assembly

checkpoint. It will be interesting to determine if SNX9 is alsoinvolved in stabilizing kinetochore fibres and if it co-operates withclathrin at the mitotic spindle for chromosome alignment and

Fig. 7. SNX9 subfamily members regulate active myosin II localization

during anaphase and telophase. (A) Representative microscopy images of

MRLCS19 (red) localization in untreated and SNX9-subfamily-depleted HeLa

cells during anaphase and telophase. Schematic diagrams (right) show the

regions selected for fluorescence intensity quantification in B and C. DNA is

shown in blue. (B,C) Ratio of fluorescence intensity of MRLCS19 at the

ingressing furrow/polar region during anaphase (B) and telophase (C). Values

are means 6 s.e.m. from two independent experiments. n55–10 cells per

sample in each experiment. ns, not significant. *P,0.05; **P,0.01;

***P,0.001 (one-way ANOVA); Scale bars: 10 mm.

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segregation, thus providing further mechanistic insight into the

non-endocytic mitotic function(s) of these proteins.

All SNX9 subfamily members participate in cytokinesis. Our

findings suggest that these proteins function in an endocytic-

dependent manner during this mitotic phase. However, they may

also participate in cytokinesis in an endocytic-independent

manner. Consistent with this idea, depletion of any of the three

SNX9 members prevented accumulation of active myosin II at the

cleavage furrow and delayed progression through the ingression

phase. SNX33 has previously been shown to be required for

cytokinesis and its role in this process was hypothesized to be due

to its ability to regulate actin polymerization via interaction with

WASp (Badour et al., 2007; Lundmark and Carlsson, 2003; Zhang

et al., 2009). SNX9 and SNX18 also bind WASp and regulate actin

dynamics (Badour et al., 2007). During clathrin-mediated

endocytosis, SNX9 is thought to recruit N-WASP to the plasma

membrane, which in turns stimulates actin recruitment and

polymerization (Cullen, 2008). Thus, it is possible that SNX9

and its subfamily members may contribute to the recruitment of

WASp and actin polymerization for efficient formation and

activity of the actinomyosin II contractile ring at the ingressing

cleavage furrow. In line with this idea, depletion of any one of the

three SNX9 members resulted in disruption of MRLCS19 cleavage

furrow localization, which is predicted to disrupt contractile ring

function.

The role of SNX9 members during cytokinesis appears to be

dependent on their endocytic activity. Supporting this idea, we

have shown that depletion of any one of these proteins delays or

blocks cytokinesis and also blocks Tfn uptake during this

process. Endocytosis is thought to contribute to the generation

of recycling endosomes that are delivered to the ICB for

abscission. We show that Rab11-positive recycling endosomes do

not accumulate at the cleavage furrow and ICB in cytokinetic

cells depleted of the SNX9 subfamily members. This could be

due to a block in (i) the generation of recycling endosomes, (ii)

endocytic activity at internalized endosomes or (iii) recruitment

of vesicles derived from recycling endosomes to the cleavage

furrow and ICB.

Fig. 8. SNX9 subfamily members regulate membrane

trafficking during cytokinesis. (A,B) Untreated and SNX9-,

SNX18- and SNX33-depleted HeLa cells at the indicated

mitotic phases were stained for the Golgi marker GM130

(red). Representative microscopy images of GM130

localization in untreated and SNX9-subfamily-depleted cells

during metaphase and cytokinesis are shown in A. The graph

in B shows the means 6 s.e.m. (from two independent

experiments) of the amount of GM130 in each sample,

expressed as ratio of fluorescence intensity at the inner pool to

that at the outer pool in each nascent daughter cell as

illustrated in the schematic diagram (bottom panel in A).

n.34 per sample. (C) Representative microscopy images of

Rab11 localization (red) in untreated HeLa cells and in HeLa

cells depleted of SNX9, SNX18 or SNX33 during telophase

and cytokinesis. DNA is shown in blue. (D,E) Graphs show

the localization of Rab11 during telophase (D) and cytokinesis

(E) in cells analysed in C. Values are means 6 s.e.m. from

two independent experiments.; n.20 per sample. ns, not

significant; ***P,0.001 (one-way ANOVA). Scale bars:

10 mm.

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SNX9 and SNX33 may also participate in abscission more

directly. We show that these proteins localise to the FMRs and

MR within the ICB. We have recently shown that dynII locates to

the FMRs (Chircop et al., 2010) and functions here in an

endocytic-independent manner for abscission (Chircop et al.,

2011b). Numerous proteins that locate to the MR are involved in

protein and vesicular recruitment. Some of these include the

endocytic proteins, Arf6, and components of the ESCRT

machinery. Thus, it is possible that SNX9 and SNX33 are

involved in recruiting key proteins and/or vesicles to the

abscission site. Not only did the depletion of all three SNX9

members prevent recruitment of Rab11-positive recycling

endosomes to the ICB, but depletion of SNX9 also caused an

accumulation of Golgi at the edge of the ICB. We hypothesize

that SNX9 is required for the recruitment of Golgi-derived

vesicles to the ICB. This was highly specific as the localization of

vesicles derived from lysosomes or the ER was unaffected. The

exact role of vesicles at the ICB is unclear, however they are

proposed to (i) provide extra total cell surface area, an increase of

at least 25% is required to complete division (Boucrot and

Kirchhausen, 2007), (ii) deliver critical cytokinetic proteins to the

abscission site (Low et al., 2003), and/or (iii) be directly involved

in compound fusion, whereby numerous vesicles fuse with the

plasma membrane during abscission to separate the daughter

cells (Goss and Toomre, 2008; Gromley et al., 2005; Low et al.,

2003; Prekeris and Gould, 2008). The role of SNX9 subfamily

members in this process is critical to understand as cytokinesis

failure leads to aneuploidy.

Overall, our findings revealed that the three SNX9 subfamily

proteins are involved in ensuring efficient progression and

completion of mitosis at several distinct stages. We have shown

that all three SNX9 subfamily members most likely function

during cytokinesis in an endocytic-dependent manner. We have

further revealed a non-endocytic function for SNX9, specifically

in chromosome alignment.

Materials and MethodsCell culture and transfection

HeLa human cervical carcinoma cells and HeLa cells stably expressing GFP–H2Bwere maintained in RPMI 1640 medium. U2OS human osteosarcoma cells weremaintained in DMEM medium. All medium were supplemented with 10% foetalbovine serum (FBS) and cells were grown at 37 C in a humidified 5% CO2

atmosphere. Cells were seeded at 50–60% confluence (16105 cells per 10 cm dish,0.56105 cells per well of a 6-well plate; 0.26105 cells per well of a 12-well plate).For siRNA analyses, cells were transfected with 1000 pmol of siRNA (per 10 cmdish for immunoblotting), 200 pmol of siRNA (per well of a 6-well plate forimmunofluorescence and time-lapse microscopy experiments) or 100 pmol (perwell of a 12-well plate for immunofluorescence and time-lapse microscopyexperiments). For DNA transfections, 1.5 mg of the indicated plasmid DNA wasused in each well of a 6-well plate. In both cases, cells were transfected withLipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.

Cell synchronization and treatment

HeLa, HeLa-GFP-H2B and U2OS cells grown on glass coverslips weresynchronized at the G2–M border by treatment with the selective Cdk1 small-molecule inhibitor RO-3306 (9 mM) for at least 18 h. Cells were allowed to progressthrough mitosis upon RO-3306 wash out. Following RO-3306 wash out, cells wereincubated at 37 C/5% CO2 for 80 min (metaphase), 2.5 h (cytokinesis) or 6 h(multinucleation and cytokinesis accumulation scoring).

Plasmid constructions

GFP-SNX9 was a gift from Sandra Schmid (Soulet et al., 2005). HA-SNX18 andGFP-SNX33 constructs were kindly provided by Stefan F. Lichtenthaler (Dislichet al., 2011). The siRNA target sequences in the sense orientation for the followingproteins are: SNX9-1: 59-AACCUACUAACACUAAUCGAU-39 (Lundmark andCarlsson, 2004); SNX9-2: 59-AACAGUCGUGCUAGUUCCUCA-39 (Shin et al.,2008); SNX18-1: 59-CGUCAUGGACCUAUUAGCGCUGUAU-39; SNX18-2: 59-

CACCGACGAGAAAGCCUGGAAUU-39 (Qiagen); SNX33-1: 59-CAAGAUCG-CUGAGACAUACUCCA-39; SNX33-2: 59-CACACGGGCCGUACCUAUUGA-A-39; Luciferase: 59-CGUACGCGGAAUACUUCGA-39.

Rescue experiment

HeLa cells were transfected with siRNA targeted the indicated SNX9 subfamilymember using Neon Transfection System as per manufacturer’s instruction(Invitrogen). On the next day, the indicated plasmid construct (GFP-SNX9, HA-SNX18 and GFP-SNX33) were transfected using the Neon Transfection System(Invitrogen) to overexpress the relevant SNX9 member in the appropriate siRNA-depleted cells. For western blot analyses, cells were collected 24 h post-transfection.For multinucleation analyses, cells were synchronized with RO-3306 for 18 hfollowed by RO-3306 wash out for 6 h.

Time-lapse microscopy analyses

Immediately following release into the cell cycle G2–M synchronized HeLa orHeLa-GFP–H2B cells were viewed with an Olympus IX81 inverted microscopeand a time-lapse series was acquired using a fully motorized stage, 106objective,and Metamorph software using the Time-lapse modules as previously described(Chircop et al., 2010; Joshi et al., 2010). Temperature control was achieved usingthe Incubator XL, providing a humidified atmosphere with 5% CO2. Imaging wasperformed for 20 h with a lapse time of 2 min or 10 min as indicated.

Immunofluorescence microscopy

Cells were fixed in ice-cold 100% methanol for 3 min at 220 C and blocked in 3%bovine serum albumin (BSA)/PBS for 45 min prior to incubation with the primaryantibody. For membrane trafficking studies, cells were fixed in ice-cold 4% PFA inPBS for 20 min and then permeabilised with 0.2% Triton X-100 in PBS for 15 minbefore blocking in 3% BSA/PBS for 45 min prior to primary antibody incubation.The following antibodies were used: anti-SH3PX1 (anti-SNX9, NB100-2813,Novus Biological), anti-SNX18 (GTX106319, GeneTex), anti-SH3PX3 (anti-SNX33, H00257364-D01P, Abnova), anti-Lamp1 (ab25630, Abcam), anti-PDI(ab2792, Abcam), anti-Rab11 (610657, BD Transduction), anti-phospho-myosinlight chain S19 (3675S, Cell Signaling), anti-GM130 (610822, BD Transduction),anti-dynI phospho-S778 which detects dynII phospho-S764 (Anggono et al.,2006), anti-a-tubulin (clone DM1A; Sigma) and anti-c-tubulin (GTU88; Sigma).Fluorescein- or Texas-Red-conjugated AffiniPure secondary antibodies (JacksonImmunoResearch Laboratories, Inc.) were then applied. Cell nuclei werecounterstained with DAPI (4969-diamidino-2-phenylindole; Sigma). Cells werewashed three times with PBS between each step except for after blocking. Cellswere viewed and scored with a fluorescence microscope (Olympus IX80).Fluorescence images were captured under an Olympus IX81 inverted microscopeusing 606or 1006oil immersion lenses and deconvolved using AutoDeblur v9.3(AutoQuant Imaging, Watervliet, NY). Integrated fluorescence intensity wasmeasured using Metamorph software (Version 7.7.0.0.).

Immunoblotting

Cellular extracts were prepared as described previously (Fabbro et al., 2004). Inbrief, cells were collected by centrifugation, washed with PBS, then resuspended inice-cold lysis buffer for sonication [25 mM Tris-HCl pH 7.4, 150 mM NaCl,1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 1% Triton X-100, and EDTA-freeComplete protease inhibitor cocktail (Roche)] followed by incubation on ice for30 min. The supernatant was collected following centrifugation at 13,000 rpmfor 30 min at 4 C. Cell lysates (200 mg) were fractionated by SDS-PAGE forimmunoblot analysis with the following antibodies: anti-SNX9 (Santa Cruz), anti-SNX18 (GeneTex), anti-SNX33 [a gift from Stefan F. Lichtenthaler (Dislich et al.,2011)], anti-GFP (Clonetech), anti-HA.11 (Covance) and anti-c-tubulin (Sigma).Antibody bound to the indicated protein was detected by incubation with ahorseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearchLaboratories, Inc.). Blotted proteins were visualized using the ECL detectionsystem (Pierce).

Endocytosis assay

Quantitative high-throughput receptor-mediated endocytosis (RME) assays wereperformed as previously described (Hill et al., 2009; Odell et al., 2010) usingTransferrin (Tfn) conjugated to Texas Red in untreated and siRNA-transfected HeLacells for 10 min. Cells were then washed with ice-cold PBS and acid washed (0.2 Macetic acid/0.5 M NaCl, pH 2.8) for 15 min on ice. Cells were then fixed with ice-cold 4% PFA in PBS for 15 min and processed for immunofluorescence microscopyanalysis as described above. Cells were imaged on an ImageXpressmicro platereader(Molecular Devices) and Tfn uptake was analysed using MetaXpress software(V3.1.0.79, Molecular Devices) by calculating the cell:vesicle integrated intensity.

AcknowledgementsWe thank Sandra Schmid for providing the GFP-SNX9 constructs andStefan F. Lichtenthaler for providing the anti-SNX33 antibody, as well

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as HA-SNX18 and GFP-SNX33 constructs. Scott L. Page is thankedfor his technical assistance. We also thank Phillip J. Robinson, RoseBoutros and Scott L. Page for critic reading of the manuscript.

FundingThis work was supported by grants from the National Health andMedical Research Council of Australia [grant number 477102 toM.C.] and the National Health and Medical Research CouncilBiomedical Career Development Award Fellowship [grant number477104 to M.C.].

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.105981/-/DC1

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Journal of Cell Science 125 (18)4382

Fig. S1. Validating the specificity of SNX9, SNX18 and SNX33 antibodies by immunofluorescence microscopy. HeLa cells depleted of the indicated SNX9 subfamily member were stained with anti-SNX9, anti-SNX18 and anti-SNX33 antibodies (green). No detectable staining was observed in cells at the indicated cell cycle stage illustrating that these antibodies have no detectable non-specific cross-reactivity. DNA shown in blue. Scale bars: 10 mm.

Fig. S2. SNX9, SNX18 and SNX33 cause cytokinesis failure in U2OS cells. (A-C) U2OS cells were either untreated (-), or transfected with luciferase siRNA (Luc) or siRNA targeting SNX9 (A), SNX18 (B) and SNX33 (C). At 72 h post-transfection, protein lysates (200 mg) were immunoblotted with anti-SNX9, anti-SNX18 or anti-SNX33 antibodies respectively. g-tubulin served as a loading control. (D,E) Cells were transfected as described in A-C followed by synchronization at the G2/M boundary using RO-3306. Upon RO-3306 wash out cells were released for 6 h, fixed and immunostained for a-tubulin and scored for multinucleated cells (D) and cytokinetic cells (E). The graphs show the mean ± s.e.m. from three independent experiments where n>200 cells were scored for each sample in each experiment. *P<0.05; **P<0.01; ***P<0.001 (one-way ANOVA).

Fig. S3. Expression level of MRLCS19 in SNX9 subfamily-depleted HeLa cells. Quantification of the total MRLCS19 fluorescence within the whole cell as a measure of its expression level in untreated and siRNA-treated HeLa cells during anaphase (A) and telophase (B). Graphs represent the mean ± s.e.m. from two independent experiments. n=5-10 cells per sample in each experiment. ns, not significant. (one-way ANOVA).

Fig. S4. Effect of depletion of SNX9 subfamily members on the expression level of GM130 in HeLa cells during mitosis. (A,B) Graphs represent the mean fluorescence of GM130 in metaphase (A) or cytokinesis (B). Values represent the means ± s.e.m. from two independent experiments. n>30 per sample. ns, not significant (one-way ANOVA).

Fig. S5. Effect of depletion of SNX9 subfamily members on the expression level of Rab11 in HeLa cells during mitosis. (A,B) Graphs represent the mean fluorescence of Rab11 in telophase (A) or cytokinesis (B). Values represent the means ± s.e.m. from two independent experiments. n>20 per sample. ns, not significant (one-way ANOVA).

Fig. S6. Effect of depletion of SNX9 subfamily members on the distribution of lyosomes during cytokinesis. (A) Representative microscopy images of Lamp1 localization in untreated and SNX9 subfamily-depleted cells during telophase and cytokinesis. DNA is shown in blue. (B,C) The graphs represent the distribution of Lamp1 in HeLa cells as shown in A, expressed as a ratio of fluorescence intensity of the inner pool compared to the outer pool in each nascent daughter cell during telophase (B) and cytokinesis (C). (D,E) Graphs illustrate the overall fluorescence intensity of Lamp1 within the whole cell in HeLa cells depleted of SNX9 subfamily members during telophase (D) and cytokinesis (E). Scale bars: 10 mm. Values represent the mean ± s.e.m. from two independent experiments. n>6 per sample. ns, not significant (one-way ANOVA).

Fig. S7. The expression level and localization of endoplasmic reticulum in SNX9 subfamily-depleted HeLa cells during cytokinesis. (A) Representative microscopy images of PDI localization (red) in untreated HeLa cells and in HeLa cells depleted of the indicated SNX9 subfamily member during telophase and cytokinesis. DNA is shown in blue. (B,C) Graphs show the overall fluorescence intensity of PDI within the whole cell in HeLa cells depleted of SNX9 subfamily members during telophase (B) and cytokinesis (C). Scale bars: 10 mm. Values represent the mean ± s.e.m. from two independent experiments. n>10 per sample. ns, not significant (one-way ANOVA).