Annexin B9 binds to bH-spectrin and is required formultivesicular body function in Drosophila

Monika Tjota*, Seung-Kyu Lee*, Juan Wu, Janice A. Williams`, Mansi R. Khanna and Graham H. Thomas§

Departments of Biology and of Biochemistry and Molecluar Biology, 208 Mueller Laboratory, The Pennsylvania State University, University Park,PA 16802, USA

*These authors contributed equally to this work`Present address: Vanderbilt University Medical Center, Surgical Research, 2213 Garland Avenue, 10435-H MRBIV, Nashville, TN 37232-0443, USA§Author for correspondence (gxt5@psu.edu)

Accepted 4 May 2011Journal of Cell Science 124, 2914–2926� 2011. Published by The Company of Biologists Ltddoi: 10.1242/jcs.078667

SummaryThe role of the cytoskeleton in protein trafficking is still being defined. Here, we describe a relationship between the small Ca2+-dependent membrane-binding protein Annexin B9 (AnxB9), apical bHeavy-spectrin (bH) and the multivesicular body (MVB) in

Drosophila. AnxB9 binds to a subset of bH spliceoforms, and loss of AnxB9 results in an increase in basolateral bH and its appearanceon cytoplasmic vesicles that overlap with the MVB markers Hrs, Vps16 and EPS15. Similar colocalizations are seen when bH-positiveendosomes are generated either by upregulation of bH in pak mutants or through the expression of the dominant-negative version of bH.In common with other mutations disrupting the MVB, we also show that there is an accumulation of ubiquitylated proteins and elevated

EGFR signaling in the absence of AnxB9 or bH. Loss of AnxB9 or bH function also causes the redistribution of the DE-Cadherin(encoded by shotgun) to endosomal vesicles, suggesting a rationale for the previously documented destabilization of the zonula adherensin karst (which encodes bH) mutants. Reduction of AnxB9 results in degradation of the apical–lateral boundary and the appearance of

the basolateral proteins Coracle and Dlg on internal vesicles adjacent to bH. These results indicate that AnxB9 and bH are intimatelyinvolved in endosomal trafficking to the MVB and play a role in maintaining high-fidelity segregation of the apical and lateral domains.

Key words: Annexin, Spectrin, Drosophila, endosome, Multivesicular body, Protein trafficking

IntroductionThe spectrin-based membrane skeleton (SBMS) is a ubiquitous

membrane-associated cytoskeletal network (Bennett and Baines,

2001). Roles for the SBMS have been demonstrated in multiple

organisms and tissues including: in neuronal structure, function

and membrane organization (Hammarlund et al., 2007;

Hulsmeier et al., 2007; Ikeda et al., 2006; Lacas-Gervais et al.,

2004; Pielage et al., 2006); in epithelial structure and stability

(Lee et al., 2010; Lee et al., 1997; Thomas et al., 1998); and in

muscle function (Bennett and Healy, 2008; Mohler et al., 2005).

Widely regarded as a static structural element, emerging results

have indicated that these proteins actually have dynamic roles

in transport processes including: trans-Golgi network to plasma

membrane (Kizhatil et al., 2007a); ER to Golgi (Stabach et al.,

2008; Stankewich et al., 2010); at the early endosome (Phillips

and Thomas, 2006); and endosome to lysosome transport

(Johansson et al., 2007). Some of these roles arise through its

interaction with the dynactin complex (Johansson et al., 2007;

Lorenzo et al., 2010; Muresan et al., 2001), and together these

data suggest that regulation of trafficking is another core

function of the spectrins. The SBMS is involved in both apical

and basolateral membrane organization, and growth in response

to polarity cues (Johnson et al., 2002; Kizhatil et al., 2007b;

Pellikka et al., 2002). Precisely how spectrin makes these

contributions remains unknown.

bHeavy-spectrin (bH) is apically restricted in most tissues

(Thomas and Kiehart, 1994) and is required for epithelial

morphogenesis (Lee et al., 2010; Thomas et al., 1998; Zarnescu

and Thomas, 1999). In primary epithelia, bH is recruited by the

apical polarity determinant Crumbs (Medina et al., 2002; Pellikka

et al., 2002) through the FERM-binding motif in the Crumbs

cytoplasmic domain (Medina et al., 2002). This motif is required

to stabilize the Cadherin-based zonula adherens (ZA) (Klebes and

Knust, 2000), and loss-of-function mutations in karst (which

encodes bH) result in a mild disruption of the ZA (Zarnescu and

Thomas, 1999) and of the Ig-CAM, Roughest (Lee et al., 2010).

This disruption probably results in the morphogenetic defects

seen in karst mutants; however, the specific role played by bH in

stabilizing these junctions remains unknown. A primary role for

bH in protein and membrane trafficking is indicated by the

observations that karst mutant cells exhibit endosomal defects

(Phillips and Thomas, 2006), that overexpression of the C-

terminus of bH causes membrane expansion (Williams et al.,

2004) and that bH collaborates with Crumbs to regulate apical

membrane size (Johnson et al., 2002; Pellikka et al., 2002). These

data lead to the hypothesis that the karst phenotype arises from

defects in the trafficking of multiple cargoes in the

endomembrane system, including the adhesion molecules DE-

Cadherin (encoded by shotgun) and Roughest.

In a genome-wide yeast two-hybrid screen, Giot and

colleagues previously reported an interaction between bH and

Annexin B9 (AnxB9, also known as AnnIX) (Giot et al., 2003).

We have pursued this interaction and demonstrate that AnxB9

binds to specific bH isoforms and is responsible for

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intermembrane adhesion generated by expression of the bH C-

terminus, and that reduction in the level of AnxB9 causes an

elevation of basolateral bH and the appearance of elevated levels

of multivesicular body (MVB) markers that overlap with bH on

cytoplasmic vesicles. bH can also be driven into these structures

either by upregulation, as found in pak mutants, or through the

use of a dominant-negative bH protein. Reduction in AnxB9 or

bH results in the accumulation of cargoes in cytoplasmic vesicles

and elevated EGFR signaling, consistent with a defect in cargo

progression through the MVB. We also show that reduction in

AnxB9 degrades the apical–lateral domain boundary. Taken

together, our data support a model in which AnxB9 is required

for efficient cargo progression through the MVB and high-

fidelity segregation of the apical and lateral domain. By contrast,

bH has an earlier role in cargo trafficking that is disrupted by the

absence of AnxB9.

ResultsAnxB9 physically interacts with the C-terminus of bH

In a genome-wide yeast two-hybrid (Y2H) screen, Giot and

colleagues previously identified a protein interaction between bH

and AnxB9 (AnxB9 bait #CT17989) (Giot et al., 2003). We

pursued this interaction because overexpression of the 33rd

segment of bH (bH33) results in the production of intermembrane

‘junctions’ that are similar to those produced by vertebrate

annexins [compare Williams et al. (Williams et al., 2004) with

Lambert et al. (Lambert et al., 1997)]. Although Curagen was no

longer able to supply the bH clone originally used in the study by

Giot et al., they were able to supply a short sequence indicating

that it included the C-terminal portion of the protein. We

therefore began by extending the previous Y2H result. FlyBase

predicts four splice variants in bH33 (Fig. 1A; http://www.

flybase.org), confirmed by RT-PCR and sequencing (Fig. 1B).

Y2H mapping revealed that AnxB9 binds to the bH-C and bH-D

spliceoforms (Fig. 1C). Mapping experiments with AnxB10 and

AnxB11 indicated that this binding is specific for AnxB9

(supplementary material Fig. S1A,B).

Antibody #182Y, raised against AnxB9, recognized both

AnxB9 and AnxB10 (Fig. 1D); however, AnxB9-specific

reactivity could be achieved by subtraction of cross-reacting

antibodies using an AnxB10 fusion protein (Fig. 1D, antibody

‘B9’). AnxB9 showed a punctate cytoplasmic distribution during

embryogenesis, with occasional cortical concentrations

(Fig. 1E,F), and does not generally colocalize with bH

(Fig. 1E–F0). This suggests that the wild-type in vivo

interaction between AnxB9 and bH is transient, and it might be

associated with an internal membrane compartment.

We have previously shown that expression of bH33 causes

membrane expansion and intermembrane adhesions

(‘bimembranes’) (Williams et al., 2004) that bear a striking

resemblance to the intermembrane junctions induced by some

vertebrate annexins (Lambert et al., 1997). Co-staining for bH33

and AnxB9 in the salivary gland of the developing embryo

showed that the two proteins colocalize in bimembranes

(Fig. 1H–H0) indicating a close relationship between the two

proteins independently from the Y2H assay. Interestingly, the

minimal region that will recruit AnxB9 into bimembranes is

amino acids 3560–3920 [the construct bHPH+5-3 in (Williams

et al., 2004), extending to the arrowhead in Fig. 1A, ‘splicing

33’], which does not contain the AnxB9-binding site, so there

must be both a direct and an indirect mechanism for AnxB9 tobind to bH33.

AnxB9 is required for bH33-induced bimembranes

To test the role of AnxB9 in bH function and bimembrane

formation we used an inducible RNAi line (UAS-AnxB9RNAi,hereafter AnxB9RNAi) to knockdown expression of this protein.We chose to examine the role of AnxB9 in the salivary gland

because this tissue does not express AnxB10 (see below),eliminating any ambiguity due to antibody or RNAi cross-reaction, and because the large size of these cells permits thevisualization of cytoplasmic compartments. The AB1-GAL4

(hereafter AB1) and 185Y-GAL4 drivers used initiate expressionshortly after gland invagination and persist throughout larvallife. Immunoblot analysis of third-instar glands from

AB1.AnxB9RNAi individuals indicated that we could achievesubstantial knockdown of AnxB9 using this construct (Fig. 2A).RT-PCR analysis on all fly Annexins, indicated that AnxB10 was

not expressed in the third-instar gland and that no knockdown ofAnxB11 was observed (Fig. 2B).

We next tested to see whether AnxB9RNAi expression wouldmodify bH33-induced bimembranes. In 185Y-GAL4.

AnxB9RNAi + bH33 embryonic salivary glands, bimembranesappeared rapidly but subsequently faded away with time,presumably owing to the eventual knockdown and turnover ofAnxB9 (Fig. 2C–C0). This suggests a role for AnxB9 in the bH33–

membrane interaction. Examination of late-stage 185Y-GAL4.

AnxB9RNAi + bH33 glands by transmission electron microscopy(TEM), showed that reducing AnxB9 resulted in the loss of all

bimembranes (Fig. 2D,E). This suggests that AnxB9 is responsiblefor the intramembrane adhesion induced by bH33 expression andthat bimembranes are an exaggerated manifestation of a normal

functional partnership.

AnxB9 knockdown perturbs bH localization

Because no mutations are available in AnxB9, we stained for bH

in glands where AnxB9 was knocked down, to test the role ofAnxB9 in bH localization. Whereas bH is exclusively apical in

most epithelia (Thomas and Kiehart, 1994), it also exhibitedweak lateral and basal staining in wild-type glands, where it wasconfined to below the septate junctions (SJ; Fig. 2F). bH was also

seen on inward membrane folds on the basal surface (Fig. 2F,G).In AB1.AnxB9RNAi glands bH was still seen at the apicalmembrane but was increased on the basolateral surfaces

(Fig. 2H). In addition, bH was present on a number of internalvesicular structures in AB1.AnxB9RNAi glands (arrows inFig. 2H). These varied in morphology from small puncta orvesicles to larger more complex structures (broken line in Fig. 2I)

that often appeared to have connections to the lateral or basalmembranes (arrows in Fig. 2I). The overall levels of bH in thegland were not detectably perturbed by the reduction in AnxB9

(Fig. 2J), and these effects were not seen when we knocked downAxnB11 using any of three different lines available from theVienna stock center (data not shown), indicating that these effects

are specific to AnxB9.

a-Spectrin and basolateral b-spectrin were also present on theinternalized structures (Fig. 2K–M0). Antibody incompatabilityprevents co-staining for bH and b-spectrin; however, co-staining

for each b-chain with a-spectrin showed that a-spectrin andbH are predominantly in separate domains (Fig. 2K–K0;supplementary material Fig. S2A,B,D), whereas a-spectrin and

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b-spectrin colocalization is fairly precise (Fig. 2M–M0;

supplementary material Fig. S2C,E). Thus, bH might not be

associated with a-spectrin on these structures, and segregation of

the SBMS is occurring into a+b and bH-only domains. These

results indicate that AnxB9 has a role in the apical restriction of

bH and that a subpopulation of bH accumulates on internal

vesicles and tubules in the absence of AnxB9.

AnxB9 knockdown perturbs the endosomal system

Previous data has suggested a role for bH in the endosome

pathway (Phillips and Thomas, 2006), and vertebrate annexins

are required for endosome function and organization (Futter and

White, 2007). We therefore stained the AB1.AnxB9RNAi glands

for various endosomal compartments. Four markers for the

multivesicular body (MVB)-late endosome pathway exhibited

Fig. 1. bH isoforms C and D bind specifically to

Annexin B9. (A) The top diagram illustrates bH

protein segments: 1, actin binding; 2,3, dimer

nucleation site; 7, SH3 domain, 32, tetramerization

domain; 2–6 and 8–31 are all spectrin repeats; 33,

segment 33, amino acids 3592–4097 [see Thomas et

al. (Thomas et al., 1997) for other segment

boundaries]. The middle diagram (splicing 33) is a

splicing map of the bH pre-mRNA in the segment 33

region. PH, pleckstrin homology encoding region.

The bottom diagram (C-terminal variants) shows the

alternative bH C-termini produced by transcripts

RA–RD. (B) RT-PCR analysis of bH33 splicing.

Sequencing confirmed the RA–RD sequences

predicted by FlyBase. *Nonspecific products.

(C) Yeast two-hybrid interaction analysis between

bH33 derivatives and full-length AnxB9. –L –T

(viability control) and –L –T –H –A (interaction-

dependent growth); the dropout media lack leucine

(–L), tryptophan (–T), histidine (–H) and adenine

(–A), as indicated. Labels indicate bait/prey

combinations: empty, no insert controls verifying no

self-activation is occurring; bH33, bH amino acids

3560–4097 from the RA transcript (see A); RB, RC

and RD, alternatively spliced exons after amino acid

3969 in RA; Cy and Dg, regions common to RC and

RD or unique to RD, respectively; AnxB9, full-

length DNA-binding domain::AnxB9 fusion. AnxB9

interacts specifically with RC and RD, but can

interact with either the Cy (red in A) or Dg (blue in

A) region. swi1/swi4 is the positive control. No

interaction with any bH spliceoform is seen using

AnxB10 or AnxB11 baits (supplementary material

Fig. S1). (D) Characterization of our chicken anti-

AnxB9 antibody. Im, immune IgY detecting a strong

band with an apparent molecular mass of 32 kDa.

Pre, preimmune IgY. This band can be resolved into

two proteins by two-dimensional electrophoresis

(upper right-hand panel). The slightly more acidic

pH of the lower species (arrowhead) suggested this

was AnxB10. Subtracting out AnxB10 cross-reacting

antibodies gives a monospecific IgY (B9; right

bottom). (E–F" ) AnxB9 and bH co-staining in the

embryonic ectoderm (E) and salivary gland (F) using

B9 for AnxB9. AnxB9 exhibits a particulate

distribution that does not generally colocalize with

bH. The bracket in F0 indicates the lumen.

(G–G" ) Preimmune IgY and bH co-staining in the

embryonic salivary gland. No signal is seen with

preimmune IgY. (H–H" ) bH33 and AnxB9

costaining in the embryonic salivary gland (Fkh-

Gal4.bH33). Images show salivary gland cells that

have failed to internalize (marked by bH33).

Expression of bH33 sequesters AnxB9 in large

membrane structures termed ‘bimembranes’

[arrowheads (see also Williams et al., 2004)].

Elsewhere AnxB9 is unperturbed. Scale bars: 20 mm.

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altered distributions: Hrs, which functions during MVB

formation as part of the endosomal sorting complex required

for transport-0 (ESCRT-0) (Lloyd et al., 2002); EPS15, a

molecular partner of Hrs with functions both at MVBs and

during endocytosis (Roxrud et al., 2008); and Vps16, as well as

Rab7, which controls endosome to lysosome trafficking

(Pulipparacharuvil et al., 2005; Nickerson et al., 2009). Other

compartment markers, such as Rab4 and Rab5 (early endosome),

and Rab11 (recycling endosome), were not visibly perturbed

(data not shown). Although previous data linked bH to the Golgi-

resident protein Lava lamp (Sisson et al., 2000), the distribution

of this protein was also unaffected by AnxB9RNAi (supplementary

material Fig. S3A–A9).

Hrs was normally present in peripheral puncta (Fig. 3A–A999)

where it colocalized with Vps16 (supplementary material Fig.

S3B–B0). In AB1.AnxB9RNAi glands, larger Hrs vesicles were

seen throughout the cytoplasm, with smaller puncta seen in

discontinuous circular patterns (Fig. 3B–B0), suggesting that Hrs

is present on subdomains of larger structures. Hrs was also

present on structures that resemble bH internalizations (see

below). Vps16 exhibited a similar perturbation upon AnxB9

depletion (compare Fig. 3C with 3D) and continued to be

Fig. 2. AnxB9 knockdown causes bimembrane disassembly

and alters the distribution of bH. (A) Knockdown of AnxB9 in

AB1.AnxB9RNAi third-instar salivary glands. Upper panel:

AnxB9 detected with B9 IgY. Lower panel: actin-loading control.

kd, AnxB9RNAi for two lines; wt, wild-type. (B) RT-PCR to verify

the specificity of AnxB9RNAi. Primers detect: Rp49, ribosomal

protein 49 (loading control); B9, B10, B11, AnxB9, AnxB10 and

AnxB11, respectively; wt and kd, wild-type and knockdown

glands as the cDNA source, respectively; B10-g, AnxB10 primers

used on genomic DNA. Markers (top to bottom) are 1.0, 0.8, 0.6,

0.4 and 0.2 kb (left); and 2.5, 2.0, 1.5, 1.0, 0.8, 0.6 and 0.4 kb

(right). (C) Staining for bH33 in Fkh-Gal4.bH33 + AnxB9RNAi

embryonic salivary gland tissue. In older embryos bH33

disappears. (st, embryonic stage) (D) Transmission electron

micrograph showing bimembranes induced by bH33 expression

(arrows). (E) Transmission electron micrograph showing no

bimembranes in a late-stage embryo coexpressing AnxB9RNAi and

bH33. (F,G) Wild-type staining for bH in the third-instar salivary

gland. (F) Sagittal section. The bracket indicates a facing view of

lateral membrane. The arrows with bar indicate the position of SJ.

The arrow shows bH on an inward fold of the basal membrane.

(G) A series of images showing linear folds in the basal membrane

extending up to 4 mm (e.g. arrowhead). (H,I) Staining for bH in

AB1.AnxB9RNAi salivary glands. (H) Sagittal section. The

bracket indicates a facing view of lateral membrane. Arrows

indicate internal bH structures. (I) Tangential section. Arrows

show apparent connections to the membrane. The broken line

outlines a more diffuse network of bH staining in the cytoplasm.

(J) Levels of bH in AB1.AnxB9RNAi glands. Each lane

represents ten glands. Labeling is as in A. 220, 220 kDa size

marker. (K) Staining for a-spectrin (K, red in K9) and bH (K0,

green in K9) in AB1.AnxB9 glands. Arrows indicate internalized

spectrin; arrowheads indicate cell boundaries. (L) A three-

dimensional rendering of a spectrin internalization (a-spectrin and

bH) where a-spectrin is blue at the plasma membrane and red on

the internal structure. bH is in green. (M) Staining for a-spectrin

(M, red in M9) and b-spectrin (M0, green in M9) in AB1.AnxB9

glands. The arrow indicates internalized spectrin, arrowheads

indicate cell boundaries. Scale bars: 1 mm (D,E); 20 mm (F–K, M);

8 mm (L).

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associated with Hrs (supplementary material Fig. S3C–C0).

Vps16 was also present in the nuclear region in a single

elongated structure that was often branched and appeared to

protrude into the nucleoplasm (Fig. 3C9). This was largely

unperturbed in AB1.AnxB9RNAi cells (Fig. 3D9). In wild-type

cells, Rab7 was present in puncta that were concentrated at the

cell periphery (Fig. 3E). In AB1.AnxB9RNAi glands, puncta

were more commonly seen deep within the cytoplasm and the

signal was also seen on larger circular structures, although not

with the frequency of Hrs or Vps16 (Fig. 3E9). EPS15 exhibited a

slightly different response to AnxB9 knockdown. In wild-type

cells, EPS15 was present in peripheral puncta and faintly at the

apical surface (Fig. 3F,G). In AB1.AnxB9RNAi cells these

puncta were found throughout the cytoplasm but not in the

distinctive circular groupings seen with Hrs and Vps16 (Fig. 3H).

However, EPS15 was also present on basal and lateral structures

like bH, Hrs and Vps16 (arrowheads in Fig. 3H).

Taken together, these data indicate that reducing the levels of

AnxB9 causes the abnormal accumulation of vesicular structureslabeled with MVB markers, suggesting a role for AnxB9 in thecreation and progression of this compartment.

bH is also associated with endosomal compartments

The karst endosome phenotype (Phillips and Thomas, 2006) andthe appearance of cytoplasmic bH in cells expressing AnxB9RNAi

(Fig. 2), suggests that AnxB9 interacts with bH during proteinrecycling and/or degradation. Co-staining for bH and Hrs or EPS15in AB1.AnxB9RNAi glands reveals that both Hrs- and EPS15-

positive structures overlap with cytoplasmic bH (Fig. 4B–C0). Thelack of detectable cytoplasmic bH in wild-type cells (Fig. 4A)suggests that bH is recruited de novo to these vesicles upon AnxB9

knockdown or that the equilibrium level of bH on such structures inwild-type cells is low or hard to fix. We therefore investigatedother methods to probe the role of bH in these processes.

Fig. 3. The late endosome markers

Hrs and Vps16 are perturbed by

AnxB9RNAi. (A–A’’’) Hrs in wild-type

cells. (A) A low magnification sagittal

view showing small and large puncta

(arrowheads) and faint apical staining

(arrow). (A9) Small Hrs puncta just

below the basal membrane.

(A0) deeper (2 mm) Hrs puncta are

larger. (A999) deep (20 mm) Hrs puncta

are near cell boundaries. (B–B" ) Hrs

in AB1.AnxB9RNAi glands.

(B) Sagittal section. (B9) Tangential

section (B0) Higher magnification

tangential section. Hrs-labeled

structures are present throughout the

cytoplasm (e.g. arrowheads) and

generally circular in morphology.

(C,C’) Tangential section showing

wild-type Vps16 puncta near the basal

surface (C) and a nuclear Vps16

structure (C9). (D,D’) Tangential

section showing Vps16 staining in

AB1.AnxB9RNAi glands.

(D) Circular Vps16 structures

resembling Hrs; (D9) Nuclear Vps16

structure. (E,E’) Rab7 staining in

wild-type (E) and AB1.AnxB9RNAi

glands (E9). Arrowheads indicate

examples of circular vesicular

staining. (F,G) Wild-type Eps15

staining in sagittal and tangential

sections, respectively. (H) Eps15

staining in AB1-Gal4.AnxB9RNAi

glands. The puncta are larger and

more numerous. Arrowheads–

complex basal and lateral structures.

Scale bars: 20 mm.

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The Rac effector Pak is a negative regulator of bH and the

viable genotype pak6/pak11 causes an increase in apical bH levels

(Conder et al., 2007). Upregulation of bH in this genotype

revealed bH to be present on conspicuous vesicular structures,

where it colocalized with Hrs (Fig. 4D–D0). Further evidence for

an association of bH with endosomal structures comes from the

analysis of the dominant-negative bH construct Minikarst, a bH

derivative that lacks only segments 14–28 and is tagged with

mCherry (Fig. 4E). The Minikarst transgene encodes all four C-

terminal spliceoforms, but cannot rescue the karst mutation and

results in lethality or a variety of visible phenotypes when

expressed in a wild-type background (data not shown). In the

salivary gland (AB1.Minikarst), Minikarst protein accumulated

in distinct patches at the membrane (arrows in Fig. 4F; a facing

view of a lateral membrane, created by projection, is shown in

Fig. 4G) and also on vesicular structures in the cytoplasm

(arrowhead in Fig. 4F). Minikarst vesicles also labeled with Hrs

(arrowhead in Fig. 4F–F0) and with Vps16 (Fig. 4H–H0), again

suggesting that they are endosomal in origin and that this protein

causes a block in the endosomal pathway at a similar step to

AnxB9 knockdown.

These data represent three diverse and independent treatments,

all of which result in bH accumulation on cytoplasmic structures

positive for MVB markers. Taken together, this evidence

suggests that bH has a normal role in the endosomal system but

is present transiently or at levels that are too low to detect with

our current reagents. The normal role for AnxB9 might be to

release bH from such compartments.

Fig. 4. Several mutant backgrounds cause bH to

accumulate in endosomal structures. (A–A" ) bH

and Hrs co-staining in wild-type cells. (B–B" ) bH

and Hrs co-staining in AnxB9RNAi cells.

Cytoplasmic bH is closely associated with a subset

of Hrs positive structures (e.g. in the areas outlined

by broken lines). (C–C" ) bH and Eps15 co-staining

in AnxB9RNAi cells. Cytoplasmic bH is closely

associated with Eps15 positive structures (e.g.

broken line). (D–D" ) bH and Hrs co-staining in

pak6/pak11 mutant cells. bH is internalized and

colocalizes with Hrs (e.g. arrowhead).

(E) Schematic of the Minikarst protein, which lacks

repeats 14–28 and is mCherry tagged. Minikarst

retains the actin-binding domain, SH3 domain,

dimer nucleation site, tetramerization site and all

spiceoforms in segment 33 illustrated in Fig. 1.

(F–F" ) Staining for Hrs in Minikarst (Minikst)-

expressing cells. Minikarst localizes to the plasma

membrane (e.g. between arrows) and colocalizes

with Hrs on internal structures (e.g. arrowheads).

(G) Maximum projection of a short stack of

Minikarst images, which provides a view of the

distribution at the lateral membrane. There are

distinct domains of this protein at the membrane.

(H–H" ) Staining for Vps16 in Minikarst-expressing

cells. Internal Minikarst overlaps with Vps16

tubules or tubular extensions (e.g. bracket). Scale

bars: 20 mm.

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Reduction of AnxB9 or bH causes the accumulation of

ubiquitylated proteins

The accumulation of vesicles labeled with MVB markers

suggests that AnxB9 is involved in MVB function. Perturbation

of MVB formation by mutations in the ESCRT 0–III complexes

causes large accumulations of specific cargoes and

overproliferation of imaginal tissue (Vaccari et al., 2009). We

did not detect overproliferation in any tissue examined upon

AnxB9 knockdown (data not shown), and we did not see large

accumulations of specific cargoes (see below). This suggests that

the roles for bH and AnxB9 at the MVB are modulatory rather

than as a key part of the core machinery, leading to milder

defects. Nonetheless, if AnxB9 does modulate traffic through the

MVB, expression of AnxB9RNAi should have a detectable affect

on many cargoes. We therefore stained for Ubiquitin as a proxy

for proteins entering endosomes; because Ubiquitin is removed

during intralumenal vesicle formation at the MVB, its

accumulation is a sensitive indicator of MVB defects. In wild-

type glands Ubiquitin was present in a few dispersed puncta and

at the apical surface in both the anterior and posterior part of the

gland (Fig. 5A–A999). Ubiquitin was also concentrated in puncta

in the perinuclear region. In AB1.AnxB9RNAi glands, Ubiquitin

puncta were elevated in number and size in both the anterior and

posterior gland (Fig. 5B,C). In the posterior gland, Ubiquitin

puncta were concentrated in the apical cytoplasm (Fig. 5B),

whereas in anterior regions the puncta were concentrated in the

central cytoplasm (Fig. 5C). These results are consistent with the

hypothesis that AnxB9 is required for cargoes to progress to the

de-ubiquitylation step at the MVB.

If AnxB9 partners with bH in the endosome system, a

reduction in the levels of bH should also cause accumulation of

Ubiquitin. In glands expressing bHRNAi, we indeed observed

accumulation of Ubiquitin, consistent with a role for bH in cargo

progression to and through the MVB (Fig. 5D). However, the

pattern was strikingly different from that in cells expressing

AnxB9RNAi, as Ubiquitin was concentrated in large prominences

at or near the apical membrane. The distinct nature of these

results permitted a simple epitstasis test; in AB1.AnxB9RNAi +

bHRNA glands Ubiquitin accumulated at the apical membrane

(Fig. 5E), formally putting bH upstream of AnxB9.

These observations are consistent with the hypothesis that

MVB function is decreased when AnxB9 is reduced, causing the

accumulation of ubiquitylated cargoes. Our observations also

demonstrate that bH probably has an earlier role in cargo

movement than AnxB9.

bH loss-of-function or AnxB9 knockdown increases EGFReceptor signaling

An increase in receptor signaling is a hallmark of mutations in

endosome and MVB functions because receptors signaling from

endosomes do not progress promptly to the MVB for final

inactivation (Vaccari and Bilder, 2009). The EGF receptor

(EGFR) is one such receptor (Vaccari et al., 2009); hence, we

examined changes in EGFR activity when bH or AnxB9 were

reduced. Because we believe that bH and AnxB9 do not provide

core MVB functions, and they lead to mild MVB phenotypes, we

looked for increased EGFR activity in the sensitized background

provided by the rhomboidve (rhove) allele. rhove is a regulatory

mutation that reduces the production of the primary EGFR

ligand, Spitz (Sturtevant et al., 1993), and homozygous rhove flies

exhibit truncated wing veins (Fig. 6B) because EGFR plays an

essential role in wing vein formation (Shilo, 2005). Introduction

of one copy of the kst1 allele into this genetic background results

in complete suppression of the vein defect for L2–L4

(Fig. 6C9,D9) and partial suppression for L5 (Fig. 6F–F0). The

kst2 allele exhibited a similar, but milder suppression (Fig. 6D0).

This suggests that reduction in the amount of bH results in an

increase in EGFR activity. Similarly, if we reduce the levels of

AnxB9 in the wing blade (MS1096-Gal4.AnxB9RNAi) in a rhove

background, vein formation is also restored (Fig. 6E9).

To specifically link wing vein restoration to MVB formation,

we took advantage of the fact that kst1 does not fully suppress the

formation of vein L5, and introduced representative alleles in

genes encoding endosomal and MVB functions to the rhove kst1/

Fig. 5. Ubiquitinated proteins accumulate in

AnxB9RNAi cells. (A) Staining for Ubiquitin (Ubi)

in the anterior region of a wild-type gland. The

bracket indicates the lumen. (A–A’’’) Staining for

Ubiquitin in the posterior region of a wild-type

gland. (A9) Sagittal section (apical to the left).

(A0) Maximum projection of four confocal

sections. The bracket indicates the lumen.

(A999) A more extensive projection of sections.

(B) Staining for Ubiquitin in the posterior region of

an AB1.AnxB9RNAi gland. Maximum projection

of confocal sections through half of a gland

diameter. Brackets indicate an apical concentration

of puncta. (C) Staining for Ubiquitin in

AB1.AnxB9RNAi cells in the anterior region.

Large puncta accumulate in the central cytoplasm.

(D) Staining for Ubiquitin in AB1.bHRNAi cells in

the anterior region. Ubiquitin accumulates in

prominent apical protrusions. (E) Staining for

Ubiquitin in AB1.AnxB9RNAi + bHRNAi cells in

the anterior region. Ubiquitin accumulation

resembles that upon expression of bHRNAi alone.

Scale bars: 20 mm.

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rhove + flies to see whether further suppression would be

achieved. Whereas ,20% of L5 veins were suppressed by kst1

alone, alleles at several loci resulted in complete restoration of L5

in a large majority of flies. None of these alleles caused any

visible suppression of rhove when present in the absence of a

karst allele (data not shown). This synergy strongly suggests that

the suppression of the rhove phenotype by karst alleles is as a

result of a role for bH in endosome progression through the MVB.

AnxB9 knockdown, bH loss-of-function and Minikarst

expression all perturb DE-Cadherin trafficking

bH generally colocalizes with the ZA and can also be present on

the apical surface (Thomas and Kiehart, 1994; Thomas et al.,

1998), and karst (bH) mutations cause a mild variable disruption

of the ZA (Zarnescu and Thomas, 1999; unpublished results).

Given the emerging role for bH in protein recycling, this

phenotype might arise from inappropriate trafficking of DE-

Cadherin in the absence of bH. We therefore examined the effects

of AnxB9RNAi and bH mutations on the distribution of DE-

Cadherin. Because this aspect of the karst phenotype is relatively

weak and variable, we sensitized the system to bH and AnxB9

defects by overexpressing DE-Cadherin.

Imaging of DE-Cadherin–GFP in AB1.DE-Cadherin–GFP

glands revealed a lateral and basolateral accumulation of this

protein and caused a distinct bulging of the basal surface

(Fig. 7A–A0). In such glands, bH exhibited extensive

colocalization with DE-Cadherin–GFP in the basolateral

domain, and is more highly concentrated where there is more

DE-Cadherin–GFP (Fig. 7A–B0). However, bH on the apical

surface retained its independence.

In AB1.AnxB9RNAi + DE-Cadherin–GFP glands there was a

striking accumulation of DE-Cadherin–GFP on internal vesicles

(Fig. 7C–D0). Some of these structures were very large, Hrs-

negative vesicles (e.g. arrowhead in Fig. 7D9), which we have not

yet been able to identify. A second population is much smaller

and is Hrs-positive, identifying them as endosomal (insets

Fig. 7D–D0). We interpret this result to indicate that DE-

Cadherin–GFP recycling and/or degradation is being slowed

owing to the loss of AnxB9, leading to its accumulation to high

levels in intermediate compartments.

We next reduced the levels of bH by introducing one copy of

various karst alleles or bHRNAi into an AB1.DE-Cadherin–GFP

background. AB1.DE-Cadherin–GFP; kst1/+ glands exhibited a

striking accumulation of small punctate DE-Cadherin–GFP

signal in the subapical cytoplasm (Fig. 7E), and AB1.DE-

Cadherin–GFP + bHRNAi glands, where bH is substantially

eliminated (data not shown), accumulated larger DE-Cadherin–

GFP vesicles (Fig. 7F). The effect of reducing bH is thus very

similar to loss of AnxB9 and is consistent with the notion that

these two proteins collaborate in DE-Cadherin trafficking.

Finally, we tested the effects of Minikarst on DE-Cadherin–

GFP distribution. In AB1-Gal4.DE-Cadherin–GFP + Minikarst

glands, there was a striking suppression of the basal bulging

Fig. 6. Reduction of bH and AnxB9 leads to elevated EGF Receptor

signaling. (A) Wild-type wing. Veins L2–L5 are labeled. L1 runs along the

anterior margin (top). (B) Homozygous rhove wing. Veins L2–L5 do not reach

the wing margin (arrowheads). (C–C" ) Introduction of a heterozygous kst1

(C9) or kst2 (C0) allele into a rhove background (C). kst1 fully restores L2–L4,

whereas L5 is usually incomplete (see F and G). kst2 only has a marginal

effect in this background. (D–D" ) Introduction of a heterozygous kst1 (D9) or

kst2 (D0) allele into a rhove/Df(3L)ru-22 (RU) background (D). kst1 fully

restores L2–L4, whereas L5 is usually incomplete. kst2 suppresses L2 and L3

but not L4 or L5. Identical results are seen in a rhove/Df(3L)Ar14-8

background (data not shown). (E–E" ) AnxB9 knockdown

(MS1096.AnxB9RNAi) restores wing veins L2 and L3 (E9) in a rhove/rhove

background (E). The MS1096 driver alone has no impact on the rhove

phenotype (E0). (F–F" ) Wild-type L5 veins reach the posterior wing margin

(F). In rhove kst1/rhove + wings, vein L5 can be complete (F9) or incomplete

(F0). Arrowheads indicate a suppressed region (compare with B).

(G) Frequency of L5 vein completion in various genotypes. rho, in rhove/rhove

and rhove/Df(3L)ru-22 L5 is never complete; rho kst/+, L5 completion in

rhove kst1/rhove + (black bar) and rhove kst1/Df(3L)ru-22 (gray bar) wings. In

all other columns ‘+’ indicates these same two genotypes with one loss-of-

function allele in the indicated gene. For example, +Rab5 is rab52/+; rhove

kst1/rhove + (black bar) and rab52/+; rhove kst1/Df(3L)ru-22 (gray bar). In

parentheses, EE indicates early endosome function, 0–III indicate ESCRT

0–III components. n>30 wings for all genotypes.

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phenotype and a distended lumen was often observed (Fig. 7G).

Again DE-Cadherin–GFP is present in two classes of vesicular

structures: large DE-Cadherin–GFP vesicles were Minikarst

negative (Fig. 7H9), whereas the smaller ones (often associated

with basal invaginations) were Minikarst positive (inset in

Fig. 7H–H0) and endosomal (see Fig. 4 for Minikarst and Hrs

colocalization). These data are also consistent with the hypothesis

that bH and AnxB9 both act to modulate DE-Cadherin

trafficking. Interestingly, the Minikarst interaction was

somewhat distinct from AnxB9 and karst or kstRNAi, in that the

basal domain had approximately the same level of DE-Cadherin–

GFP as the lateral membranes and the bulging phenotype was

suppressed. This suggests that bH has more than one role in

conjunction with DE-Cadherin and that Minikarst disrupts more

than one of these roles.

AnxB9 knockdown degrades apicobasal polarity

Vertebrate AnxA2 has a major role in apical domain

development in MDCK cells (Martin-Belmonte et al., 2007).

Cells expressing AnxB9RNAi remain polarized and retain a lumen

despite lowering AnxB9 to undetectable levels. However, the

elevation of bH in the basolateral domains along with the

appearance of bH-positive endosomal structures associated with

those domains, and the effects on basolateral DE-Cadherin–GFP

trafficking, all point to a role for AnxB9 in basolateral trafficking

despite being identified as a partner of an overtly apical protein

(i.e. bH). We therefore wondered whether AnxB9 might be

important for maintaining the basolateral restriction of polarity

markers.

Co-staining for bH and the basolateral group protein Coracle,

showed that Coracle was concentrated at the SJ, which lacks bH

(Fig. 8A–A0). On lateral membranes below the SJ, both bH and

Coracle were present (Fig. 8B–B0). Coracle was also present on

small cytoplasmic puncta (Fig. 8B0), and bH was concentrated at

the basal edge of the SJ (arrows in Fig. 8A–A0). The two proteins

exhibited a precise segregation at the apical–lateral margin

(Fig. 8C–C0). In AB1.AnxB9RNAi cells, bH and Coracle still

resided in separate domains, but staining at the apical–lateral

boundary showed some intermingling (Fig. 8D–D0). In addition,

Coracle now showed overlapping staining with internal bH

structures (Fig. 8E–E0). A similar blurring of the apical–lateral

boundary was seen with Discs large protein (Dlg; Fig. 8F,G), and

Dlg was also seen on basolateral internalizations (Fig. 8I).

Taken together, these results suggest that AnxB9 has a role in

maintaining not only the apical bias of the bH network, but in the

segregation of the apical and lateral domains. Furthermore, the

appearance of both bH and basolateral markers in a close

proximity on internal compartments, suggests that a role of

Fig. 7. AnxB9RNAi, karst loss-of-function and

Minikarst affect DE-Cadherin trafficking.

(A–A" ) Staining for bH in DE-Cadherin–GFP

(Cad)-expressing wild-type glands. The curved

arrow indicates DE-Cadherin–GFP-induced

bulging of the basal surface. (B–B" ) Staining for

bH in DE-Cadherin–GFP-expressing wild-type

glands. Arrows, bH and DE-Cadherin–GFP

concentrate at sites of cell contact. (C) Staining for

bH in DE-Cadherin–GFP-expressing AnxB9RNAi

glands. (D–D" ) Staining for Hrs in DE-Cadherin–

GFP-expressing glands. Hrs is only on small DE-

Cadherin–GFP-positive structures. DE-Cadherin–

GFP signal is enhanced in insets. (E,E’) DE-

Cadherin–GFP in live kst1/+ glands. Brackets in E9

indicate the subapical DE-Cadherin–GFP-positive

vesicles. (F) Imaging of DE-Cadherin–GFP in live

bHRNAi glands. (G) Coexpression of Minikarst

(Minikst) with DE-Cadherin–GFP.

(H–H" ) Coexpression of Minikarst with DE-

Cadherin–GFP. The inset shows the colocalization

of Minikarst and DE-Cadherin–GFP on membrane

invaginations and small vesicles. Scale bars: 50 mm

(A–B0, E, G); 20 mm (C–D0, F, H–H0).

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AnxB9 is to facilitate their segregation at or in conjunction with a

compartment where apical and basal proteins are normally sorted

from one another.

DiscussionHere, we describe a physical and genetic relationship between

AnxB9, bH and markers of MVBs. bH is primarily apical, and

loss of AnxB9 results in an increase in basolateral bH and its

appearance on cytoplasmic structures that overlap with the MVB

markers. Similar colocalizations are seen when bH internalization

is generated either in pak mutants or through the expression of a

dominant-negative version of bH. We also show that there is an

accumulation of ubiquitylated proteins in the absence of AnxB9,

and that loss of AnxB9 or bH causes elevated EGFR signaling

and the redistribution of DE-Cadherin to endosomal vesicles. We

also demonstrate that reduction of AnxB9 results in degradation

of the apical–lateral boundary and the appearance of the

basolateral proteins Coracle and Dlg on vesicles adjacent to

bH-spectrin.

Annexins have been widely associated with the endomembrane

system (Futter and White, 2007; Gerke et al., 2005; Grewal and

Enrich, 2009), where AnxA1 is required for inwards vesiculation

of intralumenal vesicles at the MVB (White et al., 2006). AnxA6

participates in late endosome to lysosome transport (Grewal et al.,

2000; Grewal et al., 2010; Pons et al., 2001), binds to spectrin and

associates with this protein on endosomes (Grewal et al., 2000;

Kamal et al., 1998; Watanabe et al., 1994). In the absence of

AnxB9 in Drosophila, we see accumulation of MVB protein

markers and a failure to de-ubiquitylate protein cargoes, showing

that these are conserved functions. However, it is not possible to

say which vertebrate annexin(s) are true orthologs of AnxB9

becuase the annexins underwent independent expansions in

different phyla (Fernandez and Morgan, 2003; Moss and Morgan,

2004). In addition, the presence of 12 annexins in vertebrates, but

only three in the fly, suggests that each fly protein might have

functions that overlap with multiple vertebrate isoforms. The

observation that reducing the levels of AnxB9 does not lead to

overproliferation or to large elevations in the level of apical

proteins, such as Crumbs and EGFR, as seen with mutations in

core endosome and MVB functions (Chanut-Delalande et al.,

2010; Lu and Bilder, 2005; Vaccari et al., 2009), suggests a

modulatory role for this protein in MVB biology. The loss of

Fig. 8. AnxB9RNAi degrades the apical, lateral

boundary. (A–E" ) Staining for bH and Coracle (Cor).

(F–I) Staining for Dlg. (A–A0) Wild-type section with

the apical surface visible. Arrowheads, a concentrated

signal for bH just below the SJ (bracket). Arrow, basal

fold staining for bH. (B–B0) Tangential section.

(C–C0) Apical surface of a wild-type gland. Coracle is

present as a ragged line of staining (arrowheads in C0)

that is distinct from the subapical lateral membrane,

which is sharp (arrow in C0). A gap in the bH staining is

seen at cell boundaries (e.g. arrowheads in C).

(D–D0) Apical surface in an AnxB9RNAi gland.

Boundaries are blurred in the bH stain, and Coracle

staining is much broader (arrowheads in D0). The inset

shows a schematic interpretation of the spread of

Coracle in the absence of AnxB9. (E–E0) Lateral

membranes near the basal surface in an AnxB9RNAi

gland. bH-positive cytoplasmic structures overlap with

Coracle. (F–I0) Dlg staining at the apical margin is

tighter in wild-type cells than in AnxB9RNAi

(arrowheads, compare F with G). Basolateral Dlg is

only faintly present in wild-type cells (H), but

accumulates internally in the same way as Coracle in

AnxB9RNAi cells (I). Scale bars: 20 mm.

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annexins from the Saccharomyces cerivisiae genome (Fernandez

and Morgan, 2003) and the viability of AnxA6-knockout mice

(Hawkins et al., 1999) also suggest that these proteins are not part

of the core MVB machinery.

The precise role for AnxB9 in MVB formation and function

remains to be elucidated. The trapping of elevated levels of a-, b-

and bH-spectrins on MVB-related structures with conspicuous

connections to the plasma membrane in the absence of AnxB9,

suggests that spectrin has traveled in from the plasma membrane

during internalization and that AnxB9 is required to release it

from endosomal structures. In support of this hypothesis, quick-

freeze deep-etch images have shown that spectrin remains

associated with freshly internalized vesicles for some distance

below the plasma membrane (Hirokawa et al., 1983), and AnxA6

has been suggested to induce spectrin proteolysis to facilitate

clathrin-coated pit release (Kamal et al., 1998). We speculate that

AnxB9 similarly triggers spectrin proteolysis to release it from

endosomes during MVB formation (Fig. 9). In addition, our

observation that AnxB9 is responsible for intermembrane

adhesion on the cytoplasmic leaflet (see Fig. 2) (Williams et al.,

2004) suggests that it could also participate in inter-endosome

adhesion and fusion, or directly in intralumenal vesicle

formation, as suggested for other annexins (Fig. 9) (Futter and

White, 2007). The epistasis test between AnxB9 and bH

knockdown suggests that bH acts upstream of AnxB9 in the

endosome pathway and is fully consistent with the model

proposed in Fig. 9. Future work with appropriate transport assays

will permit direct mechanistic testing of this model.

Historically, the SBMS has been seen to modulate protein

endocytosis as a physical barrier to coat protein assembly (e.g.

Marshall et al., 1984) or as an anchor to increase protein half-life

at the membrane (e.g. Hammerton et al., 1991). Although these

roles undoubtedly exist (.50 proteins bind to the SBMS) (De

Matteis and Morrow, 2000), these mechanisms are largely

passive and inspired by the omnipresent erythrocyte model.

Hints of a more dynamic life were obtained through visualizationof spectrin bound to endocytic vesicles in the terminal web(Hirokawa et al., 1983) and the identification of bIII spectrin as

the anchor for the dynactin complex (Holleran et al., 2001;Muresan et al., 2001). However, spectrin is now emerging as asignificant modulator of trafficking processes. Spectrin binding is

an essential step for Rab7-stimulated dynein activation duringtransport to lysosomes (Johansson et al., 2007) and for thelocalization of dynactin to costameres in muscle (Ayalon et al.,

2011), suggesting that spectrin is an integral part of the dynein-dynactin system. Furthermore, expression of mutant b-spectrinisoforms in the fly leads to dynein-dynactin-based axonaltransport defects (Lorenzo et al., 2010). Finally, b2 spectrin is

required for trans-Golgi network to lateral membrane transport inHBE cells (Kizhatil et al., 2007a), a pathway that is sufficientlyvigorous that its disruption leads to a significant shortening of the

lateral domain (Kizhatil et al., 2007b). Our data significantlyadds to the view that spectrin represents an important interfacebetween the actin cytoskeleton and endomembrane transport

processes.

bH is conspicuously associated with the ZA (Lee et al., 2010;Thomas and Kiehart, 1994; Thomas et al., 1998; Zarnescu andThomas, 1999) and is required for its integrity (Zarnescu andThomas, 1999). Given the growing association of bH with protein

recycling (Phillips and Thomas, 2006; Williams et al., 2004) (thispaper), we hypothesize that this phenotype arises from defectiveor inappropriate trafficking of DE-Cadherin. The observation that

DE-Cadherin relocates to vesicular structures when levels of bH

are reduced supports this idea. In addition, the karst ZAphenotype is quite variable, and the requirement for AnxB9 for

stress resistance suggests that this variability arises because DE-Cadherin recycling is less robust in the absence of these proteins.The degradation of the apical–lateral boundary in AnxB9RNAi

cells might also reflect a problem with this lateral diffusionbarrier.

The observations that the apical–lateral margin is degradedwhen levels of AnxB9 are reduced, and that cytoplasmic spectrinin AnxB9RNAi cells segregates into apical (bH) and basolateral (a-

spectrin, b-spectrin Coracle, Dlg) domains, suggests that theannexin-mediated process we have uncovered has somerelationship to apical–lateral sorting. We note that reduction of

PATJ, another apical Crumbs partner, has also been shown tocause mislocalization of lateral TJ proteins when it is knockeddown in CACO2 cells (Michel et al., 2005) and that there is an

increasing realization of the significance of protein endocytosisand recycling in apicobasal polarity maintenance (Harris andTepass, 2010). It will be interesting to clarify this relationship infuture work.

In conclusion, we have shown that AnxB9 is a molecular

partner of bH-spectrin that is required for efficient proteintrafficking through the MVB and for robust segregation of apicaland lateral proteins in Drosophila. Our results also provide a

strong rationale for the stabilization of the ZA throughmodulation of protein trafficking by bH and strengthen anaccelerating body of evidence that spectrin is intimately involved

in protein trafficking events.

Materials and MethodsAntibodies and immunoblotting

To produce an antibody to AnxB9, exon 2 was amplified from genomic DNAusing the primers 59-cggaattctctagagcCGTCGAGGATGCGGCTATTCTGC-39

and 59-ccatcctcgaggctctagacGCCGCTAAACTCCCGCTTGATGG-39. This

Fig. 9. Model for the role of bH and AnxB9 in endosome to MVB

maturation. The simple model shown accounts for all our data. We suggest

that bH (green) at the plasma membrane (PM) remains associated with freshly

internalized vesicles carrying ubiquitylated (Ub) integral membrane proteins

(yellow) until MVB formation is initiated (green arrow). At this point AnxB9

is required to release spectrin from the endosomes and might additionally

participate in intralumenal vesicle formation (large red arrow).

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fragment was expressed as a GST fusion protein in pGex-4T1, purified by standardmethods and used to immunize a leghorn chicken. IgY antibodies (#182Y) werepurified from egg yolks and recognize AnxB9 and AnxB10.

To specifically detect AnxB9 we subtracted antibodies that cross-reacted withAnxB10. To generate the AnxB10 fusion protein, exons 2–4 were amplified byRT-PCR using the primers 59-cgtcccccgggGCCCACGGTTAAGGACGCAG-39

and 59-gcccgctcgagCAGGGCCCGCTTGTAGTCA-39. This fragment wasexpressed as a GST fusion protein in pGEX-4T1, purified, immobilized ontonitrocellulose and used to remove all detectable cross-reaction with AnxB10.

Other antibodies used were: rabbit anti-bH antibody, which was affinity purifiedby standard methods using the immunogen for serum #243 (Thomas and Kiehart,1994) (1:100); mouse anti-Actin antibody (#C4; 1:25,000), which was obtainedcommercially; mouse anti-Crumbs antibody (#Cq4; 1:25), which was obtainedfrom Elisabeth Knust (Max Planck Institute of Molecular Cell Biology andGenetics, Dresden, Germany) (Tepass et al., 1990); mouse monoclonal anti-Mycantibody (1:100; Oncogene Research Products, San Diego, CA); mouse anti-Ubiquitin antibody (1:1000) was obtained from Enzo Life Sciences (PlymouthMeeting, PA); Guinea pig anti-Hrs antibody (1:800) and anti-EPS15 antibody wereobtained from Hugo Bellen (Baylor College of Medicine, Houston, TX); Chickenanti-Avalanche antibody (1:500) was obtained from David Bilder (University ofCalifornia Berkeley, Berkelely, CA); mouse anti-Coracle antibody (1:50) wasobtained from Richard Fehon (University of Chicago, Chicago, IL); pabbit anti-Lava-lamp antibody (1:5000) was obtained from John Sisson (University of Texas,Austin, TX); rabbit anti-dVps16A antibody (1:1000) was obtained from HelmutKramer (UT Southwestern Medical Center at Dallas, Dallas, TX); rabbit anti-Rab7antibody (1:100) was obtained from Patrick Dolph (Dartmouth College, Hanover,NY); Alexa-Fluor-labeled secondary antibodies were obtained from Invitrogen andwere used at 1:250 following pre-adsorbtion against fixed wild-type embryos.

One- and two-dimensional PAGE followed standard protocols. All blotting wasonto nitrocellulose filters which, were blocked and probed in Tris-buffered salinecontaining 5% dried milk powder and 0.1% Tween 20. Immunoblot detectionutilized horseradish-peroxidase-conjugated secondary antibodies from JacksonImmunoresearch and chemiluminescent substrates from Pierce Biotechnology.

Immunostaining and microscopy

For embryo immunostaining, appropriate collections of embryos were fixed using4% paraformaldehyde (PFA) for 20 minutes with shaking, as previously described(Thomas and Kiehart, 1994).

Third-instar salivary glands were dissected and chilled as rapidly as possiblewithout separating glands from other organs until after fixation. Thus, dissection inPBS and transfer to ice-cold PBS was performed in 2–5 seconds per larvae.Fixation was performed on ice in 4% (w/v) PFA in PEM buffer on ice for 30 or 60minutes with gentle agitation, followed by five PBS rinses, and blocking,extraction, staining and washing in incubation solution (10% normal goat serum,0.2% Saponin, 0.3% deoxycholate and 0.3% Triton X-100 in PBS). For Crumbsstaining, samples were post-fixed in 100% methanol for 10 minutes and rehydratedthrough a methanol-PBT (PBS with 0.1% Tween 20) series before staining asabove.

Embryos were imaged using a Zeiss LSM 510 META confocal. Salivary glandswere imaged on a CARV II spinning disc confocal (BD Biosystems). Embryoswere prepared for electron micrcoscopy according to Tepass and Hartenstein(Tepass and Hartenstein, 1994) and imaged on a JEOL JEM 1200 EXIItransmission electron microscope.

Fly stocks

Oregon-R or the transformation host yellow white were used as control lines. karststocks were as described previously (Thomas et al., 1998; Zarnescu and Thomas,1999). Stocks carrying the mutant alleles rab52, rabenosyn40-3, vps28B9, vps25A3,vps20I3, and vps32G5 were obtained from David Bilder (University of California,Berkeley, CA). hrsy28 was obtained from Hugo Bellen (Baylor College ofMedicine, Houston, TX). eps15E75 (#24900), Df(3L)ru-22 (#4214; uncoveringrhomboid) and Df(3L)Ar14-8 (#439; uncovering rhomboid), as well as the driverlines AB1-Gal4 (#1824), 185Y-Gal4 (#3731) and MS1096-Gal4 (#8860) wereobtained from the Bloomington Stock Center (Bloomington, IN). RNAiknockdown lines specific for karst (#37074 and #37075), AnxB9 (#27493 and#106867) and AnxB11 (#29693, #36186 and #101313) were obtained from theVienna Drosophila RNAi Center (Dietzl et al., 2007). In addition, we also madeour own AnxB9 RNAi line by cloning two copies of the above fragment, inopposition, into a modified pUAST vector (Brand and Perrimon, 1993), containingan intron from OAMB (a gift from Kyung-An Han, University of Texas at El Paso,TX). The intron separates the two inserts and promotes stability of the clone.Transformed lines (UAS-AnxB9RNAi) were produced by standard methods (Rubinand Spradling, 1982). RNAi specificity was confirmed by semi-quantitativeRT-PCR for AnxB9, AnxB10 and AnxB11 using the primer pairs: 59-CAAAATG-AGTTCCGCTGAGT-39 and 59-AATGGTCTTGATGCCGTAGTT-39; 59-CGGC-ACCGACGAGCAGGAAATC-39 and 59-GGGCCCGCTTGTAGTCACCAGAGG-39; and 59-CCAACGAGCAGCGCCAGGAGAT-39 and 59-CGCAGTTCGCC-GGCTTTCAGTAG-39, respectively (all pairs span introns to detect DNA

contamination). Primers 59-TACAGGCCCAAGATGGTGAA-39 and 59-ACGT-TGTGCACCAGGAACTT-39 were used to detect ribosomal protein Rps49, as acontrol.

Minikarst, a dominant-negative bH construct, was made by deleting segments14–28 (see Thomas et al., 1997). Minikarst was built from a cDNA segmentencoding amino acids 1–1605 and a genomic clone starting at amino acid 3200through to the normal stop codon. To facilitate cloning, two amino acids (Ser-Arg)were inserted at the joint between these two segments. The Minikarst transgenetherefore lacks repeats 14–28 but retains the actin-binding domain, the SH3domain, the dimer nucleation site and the tetramerization site and encodes allspiceoforms in segment 33. The stop codon was deleted and the protein fused tomCherry (Shu et al., 2006). This construct was cloned into pUASTattB andtransformed lines (UAS-Minikarst) were produced by integration to thechromosome 3R attP landing site at 99F8 (stock #BL24867) by RainbowTransgenic Flies (Newbury Park, CA).

We thank many investigators for supplying antibodies, as well asthe Bloomington and Vienna Stock Centers. We also thank MissyHazen and Ruth Haldeman for electron microscope training andassistance, Richard Cyr and Simon Gilroy for use of their confocalearly in this study, David Gilmour for the rp49 primers, Curagen forsupplying their AnxB9 clone and Scott Selleck for the use of Imaris.This work was funded by NSF grant #0644691 to G.H.T.

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.078667/-/DC1

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