Linking cytoplasmic dynein and transport of Rab8vesicles to the midbody during cytokinesis by thedoublecortin domain-containing 5 protein

Anna Kaplan and Orly Reiner*Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

*Author for correspondence (orly.reiner@weizmann.ac.il)

Accepted 4 July 2011Journal of Cell Science 124, 3989–4000� 2011. Published by The Company of Biologists Ltddoi: 10.1242/jcs.085407

SummaryCompletion of mitosis requires microtubule-dependent transport of membranes to the midbody. Here, we identified a role in cytokinesisfor doublecortin domain-containing protein 5 (DCDC5), a member of the doublecortin protein superfamily. DCDC5 is a microtubule-

associated protein expressed in both specific and dynamic fashions during mitosis. We show that DCDC5 interacts with cytoplasmicdynein and Rab8 (also known as Ras-related protein Rab-8A), as well as with the Rab8 nucleotide exchange factor Rabin8 (also knownas Rab-3A-interacting protein). Following DCDC5 knockdown, the durations of the metaphase to anaphase transition and cytokinesis,and the proportion of multinucleated cells increases, whereas cell viability decreases. Furthermore, knockdown of DCDC5 or addition of

a dynein inhibitor impairs the entry of Golgi-complex-derived Rab8-positive vesicles to the midbody. These findings suggest thatDCDC5 plays an important role in mediating dynein-dependent transport of Rab8-positive vesicles and in coordinating late cytokinesis.

Key words: Rab8 vesicles, Cytokinesis, Cytoplasmic dynein, Doublecortin domain-containing protein 5 (DCDC5), Midbody

IntroductionCytokinesis is the final step of mitosis, whereby one parent cell is

split into two daughter cells in a process that requires generation

of new membrane barriers. This process consists of several

morphological stages: specifying and positioning the cleavage site,

cleavage-furrow formation and constriction, spindle midzone

formation, and cell separation or abscission. Although it is

crucial for successfully completing mitosis, cytokinesis has been

relatively less investigated than earlier mitosis-related events. The

slow progress in this area might be because many proteins play

dual roles in early and late mitotic events, and cellular analyses

have traditionally focused on the early mitotic roles. Likewise,

cytoplasmic dynein, the main retrograde motor, as well as its many

subunits and accessory and interacting proteins have been

implicated in regulating several stages of mitosis (reviewed by

Sharp et al., 2000c). When mitosis is initiated, dynein is involved

in regulating nuclear envelope breakdown (NEBD), where it acts

together with interacting proteins such as LIS1 and NDEL1, and

microtubules (MTs) (Hebbar et al., 2008; Salina et al., 2002).

Dynein has been also implicated in spindle formation and mitosis

(Echeverri et al., 1996; Gaetz and Kapoor, 2004; Gaglio et al.,

1997; Goshima and Vale, 2003; Vaisberg et al., 1993). Following

NEBD, the correct positioning of dynein and its interacting

proteins LIS1, NDE1 and NDEL1 on kinetochores, astral MTs and

cortical sites assists in moving polarized chromosomes toward the

minus ends of spindle MTs, as well as moving the spindle poles to

position of the spindle within the cell (Busson et al., 1998;

Coquelle et al., 2002; Cottingham and Hoyt, 1997; Faulkner et al.,

2000; Gonczy et al., 1999; Grishchuk et al., 2007; O’Connell and

Wang, 2000; Pfarr et al., 1990; Sharp et al., 2000a; Sharp et al.,

2000b; Yan et al., 2003; Yingling et al., 2008). The role of dynein

in later stages of mitosis is implied by the phenotypes induced

following suppression of several subunits of dynein. Knockdown

of the LIC2, Roadblock 1 and LC8 subunits impaired the

completion of cytokinesis, at least in part by affecting endosome

distribution (Palmer et al., 2009) (reviewed by Eggert et al., 2006).

The role of dynein during cytokinesis is probably similar to that of

other mitotic motors at earlier stages of mitosis. Mitotic motors

play at least three distinct roles: (1) crossbridging and sliding

MTs relative to adjacent MTs or other structures; (2) transporting

specific mitotic cargoes along the surface lattice of spindle (or

midbody) MTs; and (3) regulating MT assembly dynamics and

coupling movements with regard to MT growth and shrinkage

(reviewed by Sharp et al., 2000c).

The final step of cytokinesis, namely, abscission, requires

serial dynamic events involving ingression of the contractile ring.

This process depends on actin and its associated motor myosin,

and the formation of an intracellular bridge (also termed

midbody) filled with antiparallel arrays of microtubules

(reviewed by Guizetti and Gerlich, 2010). Proper abscission

requires adding a new plasma membrane during closure of the

cleavage furrow (Maiato et al., 2004). Vesicle trafficking is also

needed for cleavage furrow ingression. Two types of vesicles are

known to participate in the abscission: Golgi-complex- and

recycling-endosome-derived vesicles. Vesicles of Golgi-complex

origin are characterized by the presence of the small GTPase

proteins Rab6 or Rab8. By contrast, Rab11 is found in recycling

endosome-derived vesicles, and its inactivation leads to a

prolonged midbody stage and eventually to furrow regression

(Wilson et al., 2005; Yu et al., 2007).

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Our research focuses on elucidating the role of doublecortindomain-containing protein 5 (DCDC5), a member of the

doublecortin (DCX) superfamily of proteins (Coquelle et al.,2006; Reiner et al., 2006). Mutations in the X-linked gene DCX orits interacting protein LIS1 result in lissencephaly or subcorticalband heterotopia (SBH) (Caspi et al., 2000; des Portes et al.,

1998; Gleeson et al., 1998; Reiner et al., 1993). DCX is aneuronal MT-associated protein (MAP) (Horesh et al., 1999) thatbinds to MTs in a unique position in between the protofilaments

through a defined DCX domain (Moores et al., 2004; Sapir et al.,2000; Taylor et al., 2000). Furthermore, both LIS1 and DCXwere found to complex with dynein and to regulate centrosomal–

nuclear coupling in migrating neurons by a dynein-dependentpathway (Shu et al., 2004; Tanaka et al., 2004; Tsai et al., 2007;Tsai et al., 2005). DCX domains are conserved in evolution anddefine a superfamily of proteins of which eleven members are

present in mammals (Coquelle et al., 2006; Reiner et al., 2006;Sapir et al., 2000). Several members of the DCX superfamilyplay important roles during mitosis. DCX knockout mice exhibit

deficits in proliferation of neuronal progenitors (Pramparo et al.,2010). Doublecortin-like kinase regulates the formation of themitotic spindle and the progression of the cell cycle in developing

neuroblasts (Shu et al., 2006). In addition, mutations in the wormdoublecortin-like kinase ortholog, Zyg-8, affect anaphase spindlepositioning (Gonczy et al., 2001). Our results suggest a mitotic

role for DCDC5, a member of the DCX superfamily. DCDC5 isspecifically expressed during mitosis. The reduced DCDC5expression levels compromised cell viability and increased thenumber of multinucleated cells. In particular, the relative position

of the midbody was affected, cytokinesis duration was extendedand Rab8-positive vesicles failed to enter the midbody.Importantly, DCDC5 directly interacts with cytoplasmic dynein

and Rab8. Therefore, we propose that DCDC5 assists in dynein-dependent transport of Rab8 vesicles to the midbody duringcytokinesis.

ResultsDCDC5 is a mitotic protein

The subcellular localization of endogenous DCDC5 was studied

by immunostaining HeLa cells with DCDC5-specific antibodies.Whereas no immunostaining was noted in interphase cells(Fig. 1A–A0), DCDC5 exhibited a dynamic expression pattern

during mitosis (Fig. 1B–K0; supplementary material Movies 1–3of three-dimensional mitotic images). During prophase, punctateDCDC5 immunostaining appeared (Fig. 1B–B0), which

condensed around centrosomal areas. During metaphase,DCDC5 was highly enriched on a subset of spindle MTs anddecorated cortical sites (Fig. 1C–C0), but it was absent in astralMTs. When anaphase began, DCDC5 was present on a subset of

MTs and part of the protein was distributed in a punctate manner(Fig. 1D–D0). During telophase, the punctate pattern was stillvisible and DCDC5 was highly concentrated along midbody-

forming MTs (Fig. 1E–E0). By contrast, during late cytokinesis,DCDC5 was distributed more uniformly; however, someenrichment was observed along the midbody (Fig. 1G–G0).

DCDC5 colocalized preferentially with stable MTs, asindicated by co-immunostaining with anti-acetylated tubulinantibodies (Fig. 1H–H0). We used Pearson’s correlation

coefficient (PCC) to evaluate the colocalization betweenDCDC5 and acetylated tubulin. The colocalization was high(Fig. 1I–I0, PCC50.77±0.02), whereas its colocalization with

a-tubulin was moderate (Fig. 1J–J0, PCC50.65±0.01). Following

methanol fixation or live imaging of GFP-tagged DCDC5(supplementary material Fig. S1A–A0 and B) DCDC5 was alsodetected in the Flemming body, which is the contractile ring

within the midbody. In mitotic HeLa cells a similar dynamicexpression pattern of GFP-tagged DCDC5 was evident: duringmetaphase DCDC5 decorated the spindle and later thefluorescent protein was highly enriched in the midbody

(supplementary material Movie 4). The specificity of DCDC5antibodies was examined during metaphase. DCDC5 antibodiesthat were preincubated with the antigen used to generate them

practically abolished DCDC5 immunoreactivity (supplementarymaterial Fig. S2A–D). In addition, we showed by massspectrometry (MS) analysis that these antibodies specifically

immunoprecipitated DCDC5 (data not shown). Moreover, themitosis-specific expression of DCDC5 was corroborated bywestern blot analysis using synchronized HeLa cells collected at

different stages of the cell cycle (Fig. 1K), and the subsequentdetection of enriched amounts of DCDC5 immunoprecipitatedfrom mitotic HeLa cell extracts (Fig. 1L). Furthermore, a short-hairpin RNA (shRNA) sequence specific for DCDC5 reduced

the apparent DCDC5 immunostaining in mitotic cells by 65%(supplementary material Fig. S2F–H). DCDC5 cDNA wasreadily amplified only from control mitotic cells using semi-

quantitative RT-PCR, whereas a weak signal was obtained fromcontrol interphase cells, suggesting that the mitotic expression ofDCDC5 is regulated at the mRNA level. No PCR product was

obtained from interphase or mitotic HeLa cell lines expressingDCDC5 shRNA sequences (supplementary material Fig. S2E).These results indicate that DCDC5 is a mitotic specific protein.

DCDC5 is a microtubule-associated protein (MAP)

DCDC5 is a member of the doublecortin superfamily ofproteins. The doublecortin (DCX) domains bind to and activelypolymerize MTs (Horesh et al., 1999; Moores et al., 2004; Sapir

et al., 2000), and serve as scaffolds for several proteininteractions (Coquelle et al., 2006; Reiner et al., 2006).Because DCDC5 has two DCX domains, we determined

whether DCDC5 is also a microtubule-associated protein(MAP). Recombinant DCDC5 co-assembled with polymerizedMTs was enriched in the pellet in the presence of Taxol

(Fig. 2A). Following nocodazole treatment, which results mainlyin disassembled tubulin dimers, both DCDC5 and tubulin werefound in the supernatant. The interaction between DCDC5 and

tubulin was further examined by expressing biotin-taggedDCDC5 in transfected cells (Fig. 2B). A prominent tubulinsignal was found with affinity-purified biotinylated DCDC5 butnot with affinity-purified biotinylated GFP (Fig. 2C). These

results suggest that DCDC5 can interact both with polymerizedMTs and with tubulin subunits. Consistent with this, wepreviously demonstrated that one DCX domain of DCDC5 was

sufficient to promote MT polymerization (Coquelle et al., 2006).Collectively, these data and the above colocalization studiessuggest that DCDC5 is a mitotic MAP.

DCDC5 knockdown increased apoptosis and the numberof multinucleated cells

DCDC5 expression levels were reduced in HeLa cells using

stable or transient transfections of DCDC5 shRNA. The reducedlevels of DCDC5 increased cellular apoptosis, as manifestedby immunostaining with anti-activated caspase-3 antibodies

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(compare Fig. 3A with B). The proportion of activated caspase-3-

positive cells after DCDC5 shRNA treatment, increased by more

than 77% (supplementary material Table S1). These results were

corroborated using non-synchronized cells sorted according to

their DNA content (Fig. 3C). The proportion of cells in the

subG1 fraction within a population of DCDC5-shRNA-treated

cells increased by 2.85-fold (from 6±0.8% to 16.95±3.1%;

P,0.01). In addition, there was a modest decrease in the

proportion of cells in the G1/G0 phases (P,0.05). These findings

were supported by a significant decrease (32%) in cell viability,

Fig. 1. DCDC5 expression is specific to mitosis. (A–J0) HeLa cells were immunostained with anti-DCDC5 antibodies (green) and either anti-a-tubulin

antibodies (A9–G9) or anti-acetylated a-tubulin antibodies (H9–J9). DAPI staining of DNA (blue) was used to stage the cells during the cell cycle. (A0–H0) Merged

images. (A–A0) Interphase. (B–B0) Prophase. (C–C0) Metaphase. (D–D0) Anaphase. (E–E0) Telophase I. (F–F0) Telophase II. (G–G0) Cytokinesis.

(H–J0) Metaphase. (I–J0) The colocalization channel (yellow) for DCDC5 and either acetylated tubulin (I0) or a-tubulin (J0) was generated using a JACoP

colocalization plugin of ImageJ. White scale bars: 30 mm (A–A0 and C–G0); yellow scale bars: 15 mm. The PCC was calculated for three different images and the

mean value ± s.e.m. is given. (K) HeLa cells were arrested at G1/S phase by thymidine, released, and then re-arrested by nocodazole at prometaphase. Cells were

harvested at the indicated time points following nocodazole release, and equal amounts of the cell extracts were immunoblotted with anti-DCDC5 (upper panel)

and anti-cyclin B1 (lower panel) antibodies. (L) DCDC5 was immunoprecipitated by anti-DCDC5 antibodies from non-synchronized (NS) or metaphase (M) cell

lysates (left panel). The cell lysates were immunoblotted with anti-DCDC5 antibodies (right panel). The DCDC5 expected molecular mass is 77 kDa.

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determined using the MTT assay (supplementary material

Table S1). Furthermore, the percentage of multinucleated cells

almost doubled following DCDC5 knockdown (supplementary

material Table S1). A similar increase in the relative numbers of

multinucleated cells was also observed during live imaging of

control and treated cells (from 8.6±0.6%, n525, to 25.6±10%,

n528, respectively). Collectively, these data suggest that

DCDC5 plays an essential role in cytokinesis.

DCDC5 knockdown affects midbody positioning andmitosis progression

Next, we investigated the relative position of the midbody and

the cell body during cytokinesis by immunostaining (Fig. 3D–F).

Whereas in control cells the midbody is typically found in the

middle of the dividing cells, the position of the midbody changed

following reduction of DCDC5 (Fig. 3D–D0 compared with E–

E0; summarized in the graph in F), revealing a significant

deviation from the midline (P,0.0001 using the Mann–Whitney

t-test; DCDC5-shRNA-treated cells 3.5±0.29 mm versus the

control 1±0.13 mm, n552 and 41, respectively). This striking

finding prompted us to investigate the progression of DCDC5-

shRNA-treated cells throughout mitosis using time-lapse

microscopy (Fig. 3G–I; supplementary material Movies 5, 6, 7,

8). To this end, HeLa cells were transfected with a fluorescent

histone marker, H1E–GFP, to enable us to clearly evaluate

mitosis progression. Cells treated with DCDC5 shRNA exhibited

a significant delay in the progression from metaphase to anaphase

in comparison with controls (41±4 minutes, n580 versus

22±2 minutes, n576; DCDC5 shRNA and control,

respectively, P50.0007 using an unpaired t-test with Welch’s

correction; Fig. 3G–H9, summarized in the graph in I). In

addition, cytokinesis completion differed between the control and

shRNA-treated cells. Whereas the control cells divided properly,

giving rise to two daughter cells (Fig. 3M), DCDC5-shRNA-

treated cells either fused to form multinucleated cells (Fig. 3Q)

or separated after an extended time (Fig. 3U). The observed cell

fusion events might explain the increase in multinucleated cells

following DCDC5 reduction. DCDC5-shRNA-treated cells spent

approximately 50% more time in cytokinesis compared with

control cells (211±16 minutes versus 141±4 minutes, n523, 30,

respectively, P,0.0001, unpaired t-test with Welch’s correction;

summarized in Fig. 3U9). To determine whether the shift

in midbody position from the centre of the cell causes

mislocalization of known midbody proteins, we examined the

localization of Polo kinase-1 (Plk1). However, no change in the

localization of Plk1 within the contractile ring of the midbody

was detected following DCDC5 reduction (supplementary

material Fig. S3). These results suggest that DCDC5 is required

for cells to progress through mitosis because it promotes proper

localization of the midbody during cytokinesis.

Fig. 2. DCDC5 is a microtubule-associated protein (MAP).

(A) Recombinant His-tagged DCDC5 was incubated with purified bovine

tubulin in the presence of either Taxol or nocodazole. Following

centrifugation, the supernatant (S) and pellet (P) fractions were analyzed by

immunoblotting with anti-His antibodies (upper panel); Ponceau red staining

shows the amounts of tubulin in the different fractions (lower panel).

(B) Scheme of biotinylated DCDC5. A DCDC5 expression construct

containing an N-terminal biotinylatable peptide tag (on lysine; K*) and GFP

was co-transfected with E. coli Bir A biotin ligase. (C) Proteins bound to

biotinylated DCDC5 were purified by streptavidin. Tubulin (indicated by an

arrow) interacted with biotinylated GFP–DCDC5 (left top panel) but not with

biotinylated GFP (left lower panel). Arrows indicate the positions of

biotinylated GFP–DCDC5 and biotinylated GFP detected by anti-GFP

antibodies. Right upper and lower panels: total cell extracts were

immunoblotted with anti-GFP antibodies, arrows indicate the positions of

biotinylated GFP–DCDC5 and biotinylated GFP (top), and anti-tubulin

antibodies, the position of tubulin is indicated by an arrow (bottom).

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Fig. 3. See next page for legend.

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DCDC5 and membrane transport

Abscission depends on a proper supply of vesicles to the

midbody, which allows for membrane sealing before cell

separation (Fielding et al., 2005; Montagnac et al., 2008; Pohl

and Jentsch, 2008; Skop et al., 2001; Skop et al., 2004; Wilson

et al., 2005). The involvement of Rab small GTP-binding

proteins in the regulation of membrane trafficking is well

documented (Balch, 1990). Therefore, we investigated the

localization of several Rab-positive vesicles during cytokinesis.

First, we visualized Rab11, which marks recycling endosome

vesicles. No difference in the localization of Rab11–GFP vesicles

was detected in HeLa cells expressing DCDC5 shRNA

(supplementary material Fig. S4A–D9) and no interaction with

DCDC5 was observed (supplementary material Fig. S4E). Next,

we examined Golgi-complex-derived secretory vesicles using

Rab8. Analysis of fixed cells expressing DCDC5 shRNA

immunostained with anti-Rab8 antibodies revealed a significant

reduction in Rab8-positive vesicles in the midbody [75% in

control (n535) versus 33.3% in shRNA-expressing cells (n530);

Fig. 4A–C9]. Similar results were observed for Rab8–GFP-

transfected cells. These results were strengthened by analysis of

time-lapse movies of cells expressing DCDC5 shRNA or control

shRNA that were transfected with Rab8–GFP. Relative to control

cells, the persistence of Rab8–GFP vesicles, which enter the

midbody during cytokinesis, was significantly reduced in

DCDC5-shRNA-treated cells (supplementary material Movies 9

and 10, respectively). Quantification of this effect revealed that in

DCDC5-shRNA-treated cells, Rab8–GFP vesicles entered the

midbody for only a fifth of the duration of cytokinesis, whereas in

control cells it was present in the midbody for almost half of the

period of cytokinesis (P,0.005, unpaired t-test, 22.04±5.799%

versus 48.10±5.766%, n513).

Next, we investigated whether Rab8 binds to DCDC5. To this

end, we performed co-immunoprecipitation (co-IP) experimentswith the recombinant proteins. DCDC5 was found to bind boththe active (GTP-bound) and inactive (GDP-bound) forms of Rab8

(Fig. 4D). Rabin8 (also known as Rab-3A-interacting protein) isa guanine nucleotide exchange factor (GEF) that participates inthe polarized delivery of Rab8 vesicles to the cell surface(Hattula et al., 2002). Therefore, a possible interaction between

Rabin8 and DCDC5 was also investigated (Fig. 4E). Myc-taggedDCDC5 and Rabin8–GFP co-immunoprecipitated reciprocally,confirming that DCDC5 interacts with this protein.

DCDC5 interacts with the molecular motor cytoplasmicdynein and the interacting proteins

DCX and DCLK have been shown to co-immunoprecipitate with

cytoplasmic dynein (Shu et al., 2006; Tanaka et al., 2004).Moreover, several motor proteins, including cytoplasmic dyneinwere detected in the midbody, suggesting that they might be

involved in cell separation (Karki et al., 1998; Nislow et al., 1990;Vernos et al., 1995). Therefore, we postulated that DCDC5 mightinteract with the main retrograde motor, cytoplasmic dynein.Indeed, recombinant DCDC5 interacted directly with the

recombinant dynein intermediate chain (DIC). His-taggedDCDC5 or His-tagged Ndel1, a known dynein interactingprotein (Niethammer et al., 2000; Sasaki et al., 2000), was

successfully pulled-down with GST–DIC but not with GST(Fig. 5A). Furthermore, the endogenous proteins interactedin synchronized G2/M HeLa cell extracts. DIC co-

immunoprecipitated with DCDC5 antibodies and DCDC5reciprocally co-immunoprecipitated with anti-DIC antibodies(Fig. 5B). DCDC5 immunoprecipitated two additional dynein-

interacting proteins, NudC and LIS1, with the strongest signaloccurring in the mitotic cell extract (Fig. 5C). Furthermore,DCDC5 interacted with p150Glued, a core subunit of dynactin,which is another major dynein interacting protein (Fig. 5D).

Bidirectional co-immunoprecipitation occurred; myc-taggedDCDC5 co-immunoprecipitated GFP–dynactin (p150Glued) andGFP–dynactin (p150Glued) co-immunoprecipitated myc-tagged

DCDC5. We concluded that DCDC5 can bind to cytoplasmicdynein and its interacting proteins.

DCDC5 links cytoplasmic dynein and Rab8

Our knockdown and physical interaction studies of DCDC5 led tothe hypothesis that cytoplasmic dynein might be involved in thetrafficking of Rab8-positive vesicles to the midbody during

the final stages of mitosis, and that this is mediated by theirinteraction with DCDC5. We therefore investigated a possiblecolocalization of these proteins in the midbody region duringcytokinesis using cells transfected with GFP–dynactin

(p150Glued) and immunostained with anti-DCDC5 and anti-Rab8 antibodies. To this end, we analyzed super-resolutionimages of endogenous Rab8, DCDC5 and MTs in the midbodies

using the OMX system (Applied Precision, GE Healthcare;Fig. 6A–D0) and found high colocalization between DCDC5 andMTs (PCC50.77±0.08) and moderate colocalization between

DCDC5 and Rab8 (PCC50.56±0.07). In addition, overexpressedGFP–dynactin (p150Glued) and endogenous DCDC5 colocalizedin the midbody (supplementary material Fig. S5). Furthermore,

the role of cytoplasmic dynein in regulating the transport ofRab8-positive vesicles to the midbody was probed by adding aspecific inhibitor of dynein, EHNA, after which time-lapse

Fig. 3. DCDC5 knockdown in HeLa cells. (A–C) HeLa cells treated with

DCDC5 shRNA are more susceptible to apoptosis. (A,B) Representative

images of HeLa cells stably expressing a control vector (A) or DCDC5 shRNA

(B) immunostained with anti-a-tubulin antibodies (green), anti-activated

caspase-3 antibodies (red) and DAPI. Scale bar: 40 mm. (C) HeLa cells stably

expressing DCDC5 shRNA or a control vector were subjected to flow

cytometry analysis following propidium iodide staining; the left bar in each pair

represents control cells and the right one represents DCDC5-shRNA-expressing

cells. (D–S) Mitosis is affected in HeLa cells treated with DCDC5 shRNA.

(D–E0) Representative images of cytokinetic HeLa control (D–D0) and DCDC5

shRNA (E–E0) cells, immunostained with anti-DCDC5 (red) and anti-a-tubulin

(green) antibodies. Scale bar: 15 mm. The arrows indicates the midbodies; the

line in D0,E0 was used for measuring the distance of the midbody from the cell

median. (F) Quantification for the midbody position in DCDC5-shRNA-treated

(3.5±0.29 mm; n552) and control-shRNA-treated (1±0.13 mm; n541) cells.

(G–H9) Representative images from time-lapse movies of control (G–G9) and

DCDC5 shRNA cells transfected with H1A–GFP (H–H9). The selected

differential interphase contrast (DIC) images and fluorescence images were

taken at the indicated time points: (G,H; metaphase, G9,H9; anaphase). Scale

bar: 10 mm. (I) Quantification of the anaphase-to-metaphase transition duration

in DCDC5-shRNA-treated (41±4 minutes; n580) and control-shRNA-treated

(22±2 minutes; n576) cells. (J–U) Selected frames from time-lapse sequences

of control shRNA (J–M) or DCDC5 shRNA (N–U)-treated cells at different

stages of the cell cycle. Note the distinct stages of the abscission process

following different treatments of cells (M,Q,U). Scale bar: 5 mm. (U9)

Quantification of anaphase-to-abscission duration, in DCDC5-shRNA-treated

(211±16 minutes; n523) and control-shRNA-treated (l141±4 minutes:

n530) cells. ***P,0.0001 (considered extremely significant); *P,0.05

(considered significant).

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microscopy was used to monitor the position of GFP-taggedRab8 vesicles. Adding EHNA resulted in a complete lack of

Rab8–GFP-positive vesicles in the midbody (Fig. 6F–H9). With

this treatment abscission failed and cells remained interconnected

with the midbody bridge (in 16 out of 19 movies) or collided to

form multinucleated cells (in 3 out of 19 movies).

DiscussionDCDC5 is a mitotic MAP and is dynamically expressedduring cell division

The expression of DCDC5, as well as its subcellular localization,

is cell cycle dependent. DCDC5 is the first member of the DCX

superfamily of proteins to be identified as being expressed in a

mitotic-specific manner. However, two other members of theDCX family, DCX and DCLK, have been previously shown to

play a role in cell division (Couillard-Despres et al., 2004;

Gonczy et al., 2001; Santra et al., 2010; Shu et al., 2006;

Vreugdenhil et al., 2007). DCDC5 is associated with MTs, in

particular, with stabilized MTs of the mitotic spindle during

metaphase and with midbody MTs during cytokinesis. As

expected from a member of the DCX superfamily consisting oftwo DCX domains, DCDC5 was found to bind directly to

polymerized MTs in vitro experiments, again supporting the

previous notion that DCDC5 is a MAP.

Knockdown of DCDC5 interferes with the final stageof mitosis

Cells with reduced levels of DCDC5 exhibited prolonged

cytokinesis, as well as impairment in the abscission process, thusresulting in cell fusion events. The consequences of these events

were decreased cell viability and an increased proportion of

apoptotic and multinucleated cells among the DCDC5 knockdown

cell population. Multinucleated cells can arise either from early

mitotic aberrations, for instance, centrosomal amplification

(Brinkley, 2001; Yun et al., 2004) or from defects in cytokinesis

(Sagona and Stenmark, 2010; Zhao et al., 2006). Our data areconsistent with the interpretation that the multinucleated cells

resulted from a failure to complete cytokinesis.

DCDC5 mediates the delivery of Rab8-positive vesicles tothe midbody through a cytoplasmic dyneinmolecular motor

It has been established that both the midbody structure and

membrane fusion are essential for proper cytokinesis (Albertson

et al., 2005; Otegui et al., 2005). However, how different vesicles

manage to reach the midbody and contribute to proper abscissionis presently poorly understood. In plant cells, the mechanism by

which the secretory pathway contributes to cytokinesis is well

established (reviewed by Jurgens, 2005; Van Damme et al.,

Fig. 4. DCDC5 affects the transport of Rab8 to the midbody, and the two proteins interact. (A–C9) HeLa cells were immunostained with anti-Rab8 and anti-

DCDC5 antibodies. Scale bar: 15 mm. DCDC5 knockdown reduced Rab8 localization in the midbody (A9–C9). The percentage of Rab8-positive midbodies is

given in the lower right corner of C and C9 (n5number of cells analyzed). (D) Direct interaction of recombinant proteins (upper panel). Recombinant 6xHis-

tagged DCDC5 was pulled down in the presence of either GTPcS or GDP by GST-tagged Rab8 using glutathione beads. GST (negative control) did not pull down

His-tagged DCDC5. The input levels of the proteins were revealed by immunoblotting; His–DCDC5 in the supernatants (lower panel, anti-His antibodies) and

GST–Rab8 (middle panel, anti-Rab8 antibodies). (E) DCDC5 and Rabin8 interact. Myc-tagged DCDC5 immunoprecipitated Rabin8–GFP (detected with anti-

GFP antibodies; left panel), Rabin8–GFP was immunoprecipitated by anti-GFP antibodies, but not by anti-myc antibodies in the absence of myc–DCDC5.

Rabin8–GFP immunoprecipitated myc–DCDC5 using anti-GFP antibodies, anti-myc antibodies immunoprecipitated myc–DCDC5; no myc–DCDC5 was

immunoprecipitated by anti-GFP antibodies in the absence of Rabin8–GFP (right panel).

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2008). However, the role of post-Golgi complex secretory

vesicles in regulating cytokinesis and abscission of animal cells

is not as clear. Rab GTPases define particular routes within the

Golgi-complex-derived secretory pathway by controlling vesicle

formation, motility, docking, and fusion events (Zerial and

McBride, 2001).

We hypothesized that DCDC5 is involved in the trafficking of

Rab-dependent membrane vesicles to the midbody. The role of

the small GTPase Rab8, which marks the Golgi-complex-derived

secretory vesicles in cytokinesis, is less established; it has

been mainly implicated in basolateral membrane transport, cell

polarity and ciliogenesis (Ang et al., 2003; Hattula et al., 2002;

Nachury et al., 2007) However, it has been shown that Rab8

endosomes are transported along MTs to the midbody ring,

suggesting their potential contribution to membrane dynamics

during cytokinesis (Pohl and Jentsch, 2008b). Importantly, Rab8

was identified in a large-scale RNA interference (RNAi) screen

aimed at identifying all proteins that are required for mitosis in

human cells (MitoCheck, http://www.mitocheck.org/cgi-bin/

mtc?query5MCG_0004801). The reported phenotypes include

multinucleated cells, nuclei that stay together, and cell death;

phenotypes similar to those observed following DCDC5 knock-

down. Importantly, we showed that DCDC5 binds directly to

both activated GTP-bound and inactivated GDP-bound forms of

Rab8 and is also found in an immunocomplex with the Rab8

exchange factor Rabin8.

Rab GTPases have been implicated in motor-protein-

dependent movement of vesicles on cytoskeletal tracks from

Fig. 5. DCDC5 and interacting partners. (A) DCDC5 interacts directly with dynein intermediate chain (DIC). Recombinant 6xHis-tagged DCDC5 (left panel)

as well as 6-His-tagged Ndel1 (positive control, right panel) were pulled down by purified GST-tagged DIC but not by the GST control using glutathione beads.

The levels of input proteins are indicated by Ponceau red staining (lower panels) (B) Co-immunoprecipitation of DCDC5 and DIC. Proteins were extracted from

non-synchronized (NS) HeLa cells or collected at different stages of the cell cycle: Pr, prophase/S phase; M, metaphase; Cyt, cytokinesis. DIC was

immunoprecipitated by anti-DCDC5 (upper panel) and DCDC5 was immunoprecipiated by anti-DIC antibodies (lower panel); anti-Myc antibodies or A/G beads

were negative controls. (C) DCDC5 immunoprecipitated NudC (upper panel) and LIS1 (upper band in the lower panel) during mitosis, when the expression level

of DCDC5 is high compared with non-synchronized (NS) or post-mitotic cells (240 and 480 minutes after nocodazole release). (D) DCDC5 interacts with

p150Glued, a core subunit of dynactin. Co-immunoprecipitation of DCDC5 and p150Glued: HEK-293 cells were transfected with either myc-tagged DCDC5, GFP-

tagged p150Glued or both. DCDC5-myc was immunoprecipitated by anti-GFP (upper panel) and p150Glued–GFP was immunoprecipiated by anti-myc antibodies

(lower panel).

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sites of their formation to sites of fusion (Echard et al., 1998a).

Among possible candidate motor proteins that might directly

interact with DCDC5, the obvious choice was the main

retrograde motor protein, cytoplasmic dynein, because it has

been previously demonstrated that two DCDC5 family members,

DCX and DCLK, interact with this motor complex (Shu et al.,

2006; Tanaka et al., 2004a). Our experiments showed that

DCDC5 directly binds the dynein intermediate chain. Moreover,

DCDC5 co-immunoprecipitates with additional members of the

dynein-dynactin protein complex, including p150Glued, LIS1 and

NudC. It was reported that the dynein motor is more efficiently

recruited to acetylated MTs (Dompierre et al., 2007; Friedman

et al., 2010), suggesting that enhancing MT acetylation stimulates

retrograde transport. Because DCDC5 is a MAP and it

colocalizes with acetylated MTs during mitosis, it might serve

as a target protein for capturing Rab8-positive vesicles and for

ensuring their spatial proximity to stabilized MT tracks and the

molecular motor dynein delivery to the midbody.

Consistent with this proposal, we found that following DCDC5

shRNA treatment, as well as specific inhibition of the dynein

motor, the delivery of Rab8-positive vesicles to the midbody was

significantly reduced. Therefore, our results corroborate the

role of Golgi-complex-derived secretory vesicles in regulating

cytokinesis in animal cells. However, to date, mainly the

plus-end-directed kinesin motors and kinesin-like proteins have

been identified as being required for abscission in C. elegans

(Raich et al., 1998), Drosophila embryos (Adams et al., 1998)

and mammalian cells (Carleton et al., 2006). The dynein–

dynactin complex also participates in retrograde membrane

trafficking during cytokinesis (Montagnac et al., 2009; Palmer

et al., 2009). The orientation of microtubules in the midbody

directs membrane trafficking to the cleavage plane. Cryo-electron

tomography analysis of midbodies supports the presence of

minus-end-directed trafficking by revealing a bundle of MTs thattraverse the midbody with their plus ends positioned away from

the center of the midbody (Elad et al., 2011). These specificcontinuous MTs persist to the verge of abscission, and they couldprovide tracks for dynein-induced trafficking of Rab8 to the

midbody for proper completion of cytokinesis.

DCDC5 and apoptosis

DCDC5 knockdown induces, on the one hand, prolongedcytokinesis and, on the other hand, an increased number ofapoptotic cells. Several proteins are known to be crucial for cell

cycle events as well for protecting cells against apoptosis(Verhagen et al., 2001), including BIR domain proteins (BIRPs)such as survivin and cIAP1 (Li et al., 1999; Samuel et al., 2005).

Another member of the BIRP family, BRUCE, plays an importantrole in delivering membrane vesicles to the site of abscission andmaintains the integrity of the midbody (Pohl and Jentsch, 2008).

The depletion of BRUCE in cultured cells sensitizes againstapoptotic stimuli and finally leads to cell death (Hao et al., 2004;Ren et al., 2005). Interestingly, BRUCE localizes to Rab8 tubularendosomes (Pohl and Jentsch, 2008). Decreased delivery of Rab8-

positive vesicles to the midbody in DCDC5 knockdown cellsmight affect proper BRUCE activity in this region and therebyimpair its function as an apoptotic inhibitor.

We describe here a novel retrograde motor-dependentmechanism of specific vesicle trafficking involved in the finalstages of mitosis. To date, the majority of the studies conducted

in the field of vesicular trafficking have focused on theimportance of membrane traffic, delivery and fusion with theplasma membrane (Straight and Field, 2000; Finger and White,

2002), whereas less attention has been placed on the potentialcargo inside the vesicles that are delivered to the surface ofdaughter cells. The contents of the vesicles and their membrane

Fig. 6. DCDC5 colocalizes with Rab8 and links

it to dynein during cytokinesis. (A,B) HeLa

cells were immunostained with anti-DCDC5 and

either anti-Rab8 (A) or anti-a-tubulin antibodies

(B) and analyzed using the DeltaVision OMXHsuper-resolution imaging system. The images are

maximal projections of eight 0.5 mm z-sections.

The colocalization channel (yellow) was

generated using the JACoP colocalization plugin

of ImageJ. Scale bar: 5 mm. PCCs were calculated

for three (in A) and four (in B) different images

and the mean values ± s.e.m. are given.

(C–E0) Selected frames of Rab8–GFP-transfected

HeLa cells without (C) or with (D–E0) the dynein

inhibitor EHNA. Time-lapse imaging was

initiated immediately after adding EHNA. In

contrast to control cells (C), Rab8–GFP was not

detected in the midbodies of EHNA-treated cells

during late telophase (E9) or during cytokinesis

(E0). Scale bar: 30 mm.

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protein components might be important for stabilizing newly

formed membranes in the terminal phase of cytokinesis. Thus,

one of the most intriguing future objectives arising from our

study would be to investigate the content of Rab8-positive

vesicles that is required to enter the midbody for proper cell

separation. Resolving this mystery will provide further valuable

information about the mechanism underlying cytokinesis

completion.

Materials and MethodsPlasmids and antibodies

A full-length cDNA clone of human DCDC5 was obtained from the NITEBiological Research Center, Kisarazu, Japan (clone TESTI4001348). The codingsequence of the cDNA was amplified by PCR using the Proofstar DNA polymerase(Qiagen, Valencia, CA), digested by XhoI or SalI and subcloned in-frame into themammalian expression vector pEGFP-N1 (Clontech Laboratories, Montain View,CA); Biotin–GFP was a gift from Niels Galjart, Erasmus University MedicalCenter, the Netherlands (de Boer et al., 2003).

The following primers were used for the N-terminus: 59-TCCGCTCGAGCAATGAAGAAACCCATCTGTAA-39 and for the C-terminus:59-ACGGGGTACCTAAGAAGAATTGCCATCGTC-39 or 59-CGGGTCGACTG-GCCAAAATTCTGTCCTTTTTTA-39. C-terminal myc-tagged DCDC5 wasgenerated by PCR with the following primers: 59-ATAGTTTAGCGGC-CGCTCACAGGTCCTCGGAGATCAGCTTCTGCTCGCAGCCAAAATTCT-GTC-39 and 59-TCCGCTCGAGCAATGAAGAAACCCATCTGTAA-39. Thefragment was cloned into a pCAGGS vector between XhoI and NotI. His-taggedDCDC5–ricin domain fusion protein was obtained by amplifying nucleotides2186–2660 subcloned into pRSET-A (Invitrogen, Carlsbad, CA) using 59-TCCGCTCGAGCAGGGCTCTTTGAAAATGTGGA-39 and 59-ACGGGGTAC-CAGATGATGTACAGACACCAGCA-39. His-DCDC5 (nucleotides 1443-3488)was generated similarly.

Rab8–GFP and Rabin8–GFP were a gift from Johan Peranen, University ofHelsinki, Finland. Rab8–GST was generated by PCR amplification of the Rab8open reading frame, followed by inserting the fragment into EcoRI–XhoI sites ofpGEX-4T1. p150Glued–GFP was kindly provided by Alexander Bershadsky(Weizmann Institute of Science, Rehovot, Israel). pH1E–GFP (Gerlitz et al.,2007) was used as a histone marker in live imaging experiments. Anti-DCDC5antibodies were generated in rabbits using purified His-tagged DCDC5–ricindomain fusion protein as an antigen.

Monoclonal anti-b-tubulin (DM1A) and anti-acetylated tubulin were obtainedfrom Sigma (Rehovot, Israel). Monoclonal anti-dynein intermediate chain (DIC),monoclonal anti-cyclin B1 and polyclonal anti-caspase3 antibodies wereobtained from Santa Cruz (CA, USA); polyclonal phospho-histone 3 waspurchased from Upstate Biotechnology (Waltham, MA) and monoclonal anti-Rab8 from BD Transduction Laboratories (Franklin Lakes, NJ). Monoclonalanti-NudC clone c15 was a gift from Junken Aoki, Tokyo University, Japan, andpolyclonal anti-GFP was obtained from Jan Faix, Hannover Medical School,Germany. The anti-LIS1 antibody was monoclonal clone 210 (Sapir et al., 1997).Rhodamine-conjugated affipure goat anti-mouse and Cy3-conjugated affipuregoat anti-rabbit IgG (H+L) were from Jackson Immunoresearch (West Grove,PA). Alexa FluorH 488 goat anti-mouse IgG (H+L) was from Molecular Probes(Invitrogen).

Cell culture, synchronization and transfections

HeLa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM)(Biological Industries, Kibbutz Beit Haemek, Israel) supplemented with 8% fetalbovine serum, 4 mM glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin.HeLa cells were synchronized using a thymidine–nocodazole block; cells weretreated with 2.0 mM thymidine (Sigma) for 18 hours, followed by a wash and theaddition of 40 mg of nocodazole (Sigma) for 4 hours after their release for12 hours. Cells at prometaphase were subjected to mitotic shake off and eithercollected for further biochemical analysis or washed and released into the cellcycle for 40 minutes in fresh pre-warmed medium in order to obtain cells intelophase to cytokinesis phases. HeLa cells were transfected using the JetPEITM

transfection reagent (Polyplus-transfection, NY).

RNA interference

DCDC5 small interfering RNAs were generated in pSUPER-derived expressionvectors using the following primers: shRNA1, 59-gatccccGCATAAACC-TGTAGCAATTttcaagagaAATTGCTACAGGTTTATGCtttttggaaa-39 and shRNA2,59-gatccccGTTTGTGGCTATCCAGTTAttcaagagaTAACTGGATAGCCACAAAC-tttttggaaa-39. Lowercase letters indicate spacer sequences, whereas uppercase lettersindicate DCDC5 mRNA sequence.

Flow cytometry

For each sample, 16106 cells were collected by trypsinization, fixed in 70%ethanol, washed in PBS, resuspended in 1 ml PBS containing 1 mg/ml RNase and

40 mg/ml propidium iodide (Sigma), incubated in the dark for 30 minutes at roomtemperature, and finally analyzed using fluorescence-activated cell sorting (FACS)at ,200 cells/second on a LSRII flow cytometer system (BD Biosciences).

MTT assay

HeLa cells were plated at a density of 56103 cells per 100 ml. MTT reagent [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; 0.5 mg/ml; Sigma]was added to the wells 48 hours after plating. The plate was incubated for 2 hoursat 37 C, after which, the medium were removed and 170 ml of DMSO was added

to each well to dissolve the formazan crystals that had formed. The absorbance at550 nm was determined using an ELISA (enzyme-linked immunosorbent assay)plate reader.

Immunostaining, time-lapse sequences and image collection

HeLa cells were plated on 13-mm-thick cover slips (Menzel-Glaser,Braunschweig, Germany). Forty-eight hours after transfection, cells were fixedfor 20 minutes at room temperature with 4% paraformaldehyde in PHEM buffer(60 mM PIPES, 25 mM HEPES, 10 mM EGTA and 4 mM MgSO4, pH 6.9)buffer and treated as previously described (Coquelle et al., 2006). For methanolfixation, the coverslips were incubated in cold methanol for 5 minutes at 220 C.Immunostained cells were visualized using wide-field microscopy (DeltaVision,

Applied Precision, CA). As indicated in the legends, a number of 200-nm z-sections were deconvolved and projected to a maximal intensity projection imageusing Delta Vision software. Where indicated, we used DeltaVision OMX, athree-dimensional structured illumination microscopy system that provides super-resolution imaging. DeltaVision OMX surpasses the 250-nm resolution limit ofconventional microscopy by a factor of two and enables imaging beyond thesurface of the coverslip with multiple probes. For time-lapse sequences, HeLacells were grown on 35-mm-diameter glass-bottom microwell dishes (MatTek,Ashland, MA) overnight and were visualized using the DeltaVision system(Applied Precision, CA). Each recording session lasted from 3 to 6 hours at 37 C,and frames were taken every 2.5 minutes. Eight 500-nm z-sections were

deconvolved and projected to obtain a maximal intensity projection image foreach time point using DeltaVision software. The Pearson correlation coefficient(PCC) was used to quantify the degree of colocalization between fluorophores.We used the Java constraint solver tool JACoP to analyze the PCCs (Bolte et al.,2006). The dynein inhibitor, 1 mM EHNA [erythro-9-(2-hydroxy-3-nonyl)adenine; Sigma, St. Louis, MO] was added before beginning the time-lapse sequences. Images and time-lapse sequences were processed using theDeltaVision system package, or Imaris software (for 3D images; Bitplane).Statistical analysis was conducted using Prism 4 for Macintosh (GraphPadSoftware, Inc.).

The microtubule binding assay was conducted as previously described (Horeshet al., 1999).

Immunoblots and immunoprecipitation

Adherent cultured cells were washed in cold PBS and collected using cell scrapers.Mitotic synchronized cells were collected by the shake off method and cell pelletswere obtained. Cell pellets were either flash frozen in liquid nitrogen and stored at280 C for later lysis or were lysed directly by resuspension in lysis buffercontaining 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 20 mM b-glycerophosphate,1 mM sodium vanadate supplemented with protease inhibitor cocktail (Sigma).For immunoblots, 40 mg protein was mixed with SDS sample buffer and separatedby SDS-PAGE. The protein lysates were incubated with the appropriate antibodies

for 2 hours at 4 C. Thereafter, 15 ml (bed volume) of protein A/G agarose (SantaCruz) pre-blocked in lysis buffer supplemented with 10 mg/ml BSA (Sigma) wasadded to each sample for another 2 hours. Immunoprecipitated proteins werepelleted by centrifugation and washed three times in lysis buffer. The proteinswere eluted from the beads by adding SDS sample buffer, then boiled for3 minutes, separated on 10% SDS-PAGE, and finally subjected to western blotanalysis with the indicated antibodies.

Semiquantitative RT-PCR

RNA from confluent cells in 10-cm dishes of non-synchronized or synchronizedHeLa cells was isolated using TRI REAGENT (Sigma). Total RNA (5 mg) wasreverse transcribed using the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen), and 1/25 of the reverse-transcribed material was used in PCR

reactions. The DCDC5 ricin domain transcript was amplified using the followingprimers: F1 59-TCCGCTCGAGCAGGGCTCTTTGAAAATGTGGA-39 and R159-ACGGGGTACCAGATGATGTACAGACACCAGCA-39. The GAPDH transcriptwas amplified using primers F1 59-ATCTGGCACCACACCTTCTACAAT-GAGCTGCG-39 and R1 59-CGTCATACTCCTGCTTGCTGATCCACATCTGC-39.

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AcknowledgementsWe thank the current and previous lab members for theircontribution, especially Michal Segal and Talia Levy for technicalassistance; Johan Peranen, University of Helsinki; Finland, BrunoGoud, Curie Institute, France; Alexander Bershadsky, WeizmannInstitute of Science, Israel; Niels Galjart, Anna Akhmanova,Erasmus University Medical Center, the Netherlands; Junken Aoki,Tokyo University, Japan; Jan Faix, Hannover Medical School,Germany; and Angelika Barnekow, University of Munster, Germany,for providing useful reagents. We thank Applied Precision forgenerating the OMX images.

FundingThis research was supported by the Israel Science Foundation [grantnumbers 270/04, 47/10 to O.R.]; the Legacy Heritage BiomedicalProgram of the Israel Science Foundation [grant number 1062/08 toO.R.]; the Israel Cancer Association [grant number 20090073 toO.R.]; the Minerva Foundation with funding from the FederalGerman Ministry for Education and Research; the Benoziyo Centerfor Neurological Diseases; the Helen and Martin Kimmel Stem CellResearch Institute; and the David and Fela Shapell Family Center forGenetic Disorders Research. O.R. is an incumbent of the Berstein-Mason Professorial Chair of Neurochemistry.

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.085407/-/DC1

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