A Golgi-Localized Pool of th

Current Biology 24, 2687–2692, November 17, 2014 ª2014 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.cub.2014.09.052

Reporte Mitotic

Checkpoint Component Mad1 ControlsIntegrin Secretion and Cell Migration

Jun Wan,1,2 Fen Zhu,1 Lauren M. Zasadil,1,3 Jiaquan Yu,4

Lei Wang,5 Adam Johnson,5 Erwin Berthier,4

David J. Beebe,4,6 Anjon Audhya,5,6 and Beth A. Weaver1,6,*1Department of Cell and Regenerative Biology2Physiology Training Program3Molecular and Cellular Pharmacology Training Program4Department of Biomedical Engineering5Department of Biomolecular Chemistry6Carbone Cancer CenterUniversity of Wisconsin, Madison, WI 53705, USA

Summary

Mitotic arrest deficient 1 (Mad1) plays a well-characterized

role in the major cell-cycle checkpoint that regulates chro-mosome segregation during mitosis, the mitotic checkpoint

(also known as the spindle assembly checkpoint). Duringmitosis, Mad1 recruits Mad2 to unattached kinetochores

[1, 2], where Mad2 is converted into an inhibitor of theanaphase-promoting complex/cyclosome bound to its spec-

ificity factor, Cdc20 [1, 3–6]. During interphase, Mad1 re-mains tightly bound to Mad2 [2, 3, 7, 8], and both proteins

localize to the nucleus and nuclear pores [9, 10], wherethey interact with Tpr (translocated promoter region).

Recently, it has been shown that interaction with Tpr stabi-lizes both proteins [11] and that Mad1 binding to Tpr permits

Mad2 to associate with Cdc20 [12]. However, interphase

functions of Mad1 that do not directly affect the mitoticcheckpoint have remained largely undefined. Here we iden-

tify a previously unrecognized interphase distribution ofMad1 at the Golgi apparatus. Mad1 colocalizes with multiple

Golgi markers and cosediments with Golgi membranes.Although Mad1 has previously been thought to constitu-

tively bind Mad2, Golgi-associated Mad1 is Mad2 indepen-dent. Depletion of Mad1 impairs secretion of a5 integrin

and results in defects in cellular attachment, adhesion, andFAK activation. Additionally, reduction of Mad1 impedes

cell motility, while its overexpression accelerates directedcell migration. These results reveal an unexpected role for

a mitotic checkpoint protein in secretion, adhesion, andmotility. More generally, they demonstrate that, in addition

to generating aneuploidy, manipulation of mitotic check-point genes can have unexpected interphase effects that in-

fluence tumor phenotypes.

Results and Discussion

An unexpected, perinuclear localization of Mad1 (Figures S1Aand S1B available online) was identified in interphase HeLacells after immunofluorescence using an affinity purified rabbitanti-Mad1 antibody, which produces a single band on immu-noblots (Figure S1C) [13]. A similar perinuclear localizationwas observed in primary murine embryonic fibroblasts(MEFs) and the breast cancer cell line MDA-MB-231 (Figures

*Correspondence: baweaver@wisc.edu

S1A and S1B). To biochemically confirm the existence of acytoplasmic pool of Mad1, we performed a fractionationexperiment to separate nuclear from cytoplasmic extract.Three nuclear markers, histone H3, lamin A, and lamin C, aswell as a cytoplasmic marker (tubulin), were used to confirmthat appropriate fractionation was achieved. HeLa cells,MEFs, MDA-MB-231 cells, and an additional breast cancercell line, Cal51, all contained a cytoplasmic pool of Mad1 (Fig-ures S1D and S1E).Multiple experiments were performed to test the specificity

of anti-Mad1 antibodies. First, Mad1 was transiently depletedin HeLa cells using small interfering RNA (siRNA). Fractionationfollowed by immunoblotting using the rabbit anti-Mad1 anti-body revealed that total, nuclear, and cytoplasmic pools ofMad1 were depleted (Figure S1F). Second, an additional anti-body [14] was used to confirm the identity ofMad1. Thismousemonoclonal antibody also recognizes a single band of roughly85 kDa by immunoblotting (Figure S1C) that is reduced aftersiRNA-mediated depletion of Mad1 (Figure S1F). Third, stableHeLa cell lines in which Mad1 expression was knocked downconstitutively (to be referred to hereafter as Mad1-KD) weregenerated by retroviral infection of three distinct short hairpinRNA (shRNA) sequences followed by antibiotic selection.Mad1-KD cell lines grew at rates comparable to control cellsand did not have obvious delays in any stage of the cell cycle(Figures S1G–S1I). Mad1 levels were diminished, but notabsent, in all three cell lines (Figure S1J). In Mad1-KD celllines, the cytoplasmic pool of Mad1 became undetectable byimmunofluorescence (Figure S1K). Fourth, fractionation exper-iments in parental andMad1-KD cell lines 1–3 showed a reduc-tion in the cytoplasmic pool of Mad1 (Figure S1L) as detectedby both Mad1 antibodies. Fifth, both Mad1 antibodies showeda cytoplasmic fraction of YFP-tagged Mad1 (Figure S1M). Weconclude that interphase cells contain a cytoplasmic pool ofthe mitotic checkpoint protein Mad1.

Cytoplasmic Mad1 Is Localized to the GolgiPerinuclear Mad1 was coincident with the centrosome (Fig-ures 1A and 1B), as is the Golgi apparatus [15]. Golgi organiza-tion is dependent on polymerized microtubules. Microtubuledepolymerization with 12 hr (Figures 1C and 1D) or 4 hr (Fig-ure S2A) of vinblastine treatment caused the Golgi to disas-semble and perinuclear Mad1 to disperse.The fungal metabolite Brefeldin A (BFA) causes rapid disas-

sembly of theGolgi without gross perturbation ofmicrotubules[16–18]. BFA treatment also resulted in dispersal of the perinu-clear Mad1 signal (Figures 1C and 1D), strongly suggestingthat it was localized to the Golgi. To confirm this, we testedMad1 for colocalization with several Golgi antigens. Cyto-plasmic Mad1 exhibited a similar distribution as three Golgimarkers, alpha-mannosidase II (MAN-II), GM130, and Golgin97 (Figures 1E–1H). Colocalization of cytoplasmic Mad1 withGolgi markers was essentially complete when all three Golgiantigens were visualized in the same cell (Figure S2B).Mad1 did not colocalize with early or late endosomes

labeled with Rab5A and Rab7A, respectively (Figures S2Cand S2D). Nor did Mad1 colocalize with RINT-1, which cyclesbetween the Golgi and the ER (Figure S2E). However, partial

Figure 1. The Cytoplasmic Pool of Mad1 Is Localized to the Golgi

(A) Cytoplasmic Mad1 in HeLa cells colocalizes with the centrosome, identified with centrin. The boxed region is enlarged in the far-right panel.

(B) Quantification (6SD )of Mad1 colocalization with centrosomes. n = 200 cells from each of three independent experiments.

(C) Perinuclear cytoplasmic Mad1 staining in HeLa cells is dispersed in response to 12 hr of 100 ng/ml vinblastine (VBL) or 30 min of 10 mg/ml BFA, as are

Golgi proteins. The boxed regions are enlarged in the insets.

(D) Quantification (6SD) of cells with perinuclear cytoplasmic Mad1 after treatment with vinblastine (VBL) or BFA. n = 200 cells from each of three indepen-

dent experiments.

(E–G) Cytoplasmic Mad1 colocalizes with the Golgi proteins MAN-II (E), GM130 (F), and Golgin 97 (G). Like MAN-II, Mad1 disperses in response to treatment

with the microtubule destabilizing drug vinblastine (E). The boxed regions are enlarged in the far-right panels.

(H) Quantification of colocalization of Mad1 with the Golgi proteins MAN-II, GM130, and Golgin 97 in HeLa cells. Quantification (6SD) indicates the percent-

age of cells in which Mad1 colocalized with the indicated Golgi protein at 403 magnification. n = 200 cells from each of three independent experiments.

(I) Cytoplasmic Mad1 cofractionates with the Golgi in an Optiprep gradient. HeLa cell extracts were fractionated as described in the Experimental Proce-

dures. Mad1, as detected using both mouse monoclonal and rabbit polyclonal antibodies, cosedimented with the Golgi protein GM130 and is largely sepa-

rated from the ER (Ribophorin I) and the Mad1 binding partner Mad2.

(J) The Mad1 binding partner Mad2 does not colocalize with the Golgi (marked by GM130) in HeLa cells. The boxed region is enlarged in the far-right panel.

See also Figure S1.

Current Biology Vol 24 No 222688

colocalization was observed between Mad1 and Rab6A (Fig-ure S2F), which accumulates in both the trans-Golgi networkand endosomes.

To further establish the Golgi localization of Mad1, we frac-tionated cytoplasmic extract over an OptiPrep densitygradient. Mad1 was enriched in fractions containing the Golgimarker GM130 but was largely absent from fractions contain-ing the ER marker Ribophorin I (Figure 1I). These data supportthe conclusion that the perinuclear cytoplasmic pool of Mad1localizes to the Golgi apparatus.

A Mad2-free Pool of Mad1

Mad1 and Mad2 are thought to interact throughout the cell cy-cle. Mad2 is expressed in excess of Mad1 in yeast, Xenopus

extracts, and mouse and human cells [19–22]. Gel filtrationanalysis has revealed that Mad1 elutes in a single peak thatalso contains Mad2. However, Mad2 also fractionates into asecond peak of the expected size for monomeric Mad2[7, 19, 23]. Depletion of Mad2 removes all detectable Mad1 innumerous systems, whereas substantial Mad2 remains afterimmunoprecipitationwithMad1 [2, 13, 19]. These data suggestthat the entire pool of Mad1 is bound to Mad2 throughout thecell cycle. Interestingly, however, we found that Mad2 did notcofractionate with Mad1 after sedimentation on an Optiprepgradient (Figure 1I). Nor did Mad2 colocalize with Golgimarkers by immunofluorescence (Figure 1J). Thus, unlikekinetochore- and nuclear-pore-bound pools of Mad1, Golgi-localized Mad1 appears free of Mad2.

Figure 2. Golgi LocalizedMad1 Participates in a5

Integrin Secretion and Cell Adhesion

(A) Perinuclear accumulation of a5 integrin in

Mad1-KD HeLa cell lines 1–3, indicated by arrow-

heads.

(B) Quantification (6SD) of perinuclear accumu-

lation of a5 integrin, as depicted in (A). n = 200

cells from each of three independent experi-

ments.

(C) Perinuclear a5 integrin in Mad1-KD1

HeLa cells accumulates in the Golgi, as shown

by colocalization with GM130. Arrowheads

indicate Golgi accumulation of a5 integrin.

The boxed regions are enlarged in the far-right

panels.

(D) Flow-cytometry analysis demonstrating that

surface expression of an extracellular epitope

of a5 integrin is reduced on Mad1-KD cell lines

1–3 as compared to wild-type HeLa cells.

(E) Quantification (6SD) of flow cytometry mea-

surements in (D). n = 10,000 events from each

of two independent experiments.

(F) Reduced expression of Mad1 results in defec-

tive cellular attachment. Wild-type and Mad1-KD

HeLa cell lines 1–3 were plated on 5 mg/ml fibro-

nectin and incubated at 37�C for 30 min before

removal of unattached cells. The average number

of attached cells per 103 field 6SD is shown.

n = 3.

(G) Mad1-mediated a5 integrin secretion pro-

motes cell adhesion. Cell-surface proteins were

cleaved with 500 mg/ml proteinase K for 30 min

on ice. After addition of PMSF, cells were washed

and permitted to recover at 37�C for the indicated

amounts of time before 200,000 cells were plated

and allowed to adhere to fibronectin-coated

dishes for 30 min. The average number of attached cells per 103 field 6SD (normalized to the number of wild-type cells at 0.5 hr) from three independent

experiments is shown.

*p < 0.05, **p < 0.001. See also Figures S2 and S3 and Movie S1.

A Novel Role for Mad1 in Secretion and Migration2689

Mad1 Functions in the Secretion of a5 Integrin

To determinewhetherMad1 functions in protein trafficking, weanalyzed secretion of a variety of proteins. Plasma membraneaccumulation of the epidermal growth factor receptor (EGFR)was not affected in Mad1-KD cells (Figures S2G and S2H).Nor was accumulation of the vesicular stomatitis virus enve-lope glycoprotein VSVG at the plasma membrane delayeddue to reduction of Mad1 (Figures S2I and S2J and MovieS1). These data suggest that there is no global defect in proteinsecretion after Mad1 depletion. However, an intracellularepitope of a5 integrin coalesced near the nucleus in Mad1-KD cell lines 1–3 after fixation and permeabilization (Figure 2A,arrowheads, and Figure 2B). To confirm that this antibody wasindeed recognizing a5 integrin, we imaged single z planes fromthe bottom of the cell. These demonstrated the expected cell-surface staining, which was coincident with the focal adhesionmarker paxillin (Figure S3A). Costaining with GM130 revealedthat the perinuclear pool of a5 integrin was localized to theGolgi (Figure 2C, arrowheads).

To further examine whether a5 integrin secretion was in-hibited in cells depleted ofMad1,we labeled nonpermeabilizedcells with an antibody recognizing an extracellular epitope ofa5 integrin. Flow cytometry revealed that substantially lessa5 integrin localized to the cell surface in Mad1-KD cell lines1–3 as compared to wild-type cells (Figures 2D and 2E).

Secretion of other integrin subunits was then examined.Subtle perturbations in the trafficking of a1, a2, and b1 integ-rins were also observed in Mad1-KD cells (Figures S3B–S3F)

but were less pronounced than effects on a5. mRNA levels ofall four integrin subunits tested were unaffected in Mad1-KDcells (Figure S3G), as were protein levels of a5 (Figure S3H).a and b integrin subunits associate in the ER before transit-

ing the Golgi en route to the cell surface [24]. a5 subunitsexclusively pair with b1, but b1 subunits dimerize withnumerous alpha chains, including a1, a2, a3, a4, and a6 [25].Mad1 participation in the secretion of only a subset of alphasubunits may explain why a5 integrin dramatically accumu-lated in the Golgi after Mad1 depletion, but effects on its bind-ing partner b1 integrin were less conspicuous.To further examine the role of Mad1 in a5 integrin secretion,

we performed coimmunoprecipitation experiments in cellsexpressing a5 integrin-3xFLAG and GFP-tagged Mad1.Wild-type Mad1-GFP coimmunoprecipitated with a5 integrin-3xFLAG, as did a Mad1 mutant that cannot bind Mad2(K541A/L543A) [26] and a cytoplasmic version that cannotlocalize to the nucleus or nuclear pores (aa 180–718 [12]; Fig-ure S3I), suggesting that a complex containingMad1 and a5 in-tegrin facilitates a5 integrin secretion.Two cleavage events are required to produce the mature a5

integrin on the cell surface. First, the 44 aa signal peptide iscleaved from the N terminus. The second cleavage event pro-duces mature products of 98 and 18 kD and occurs in eitherthe trans-Golgi network or during exocytosis [27]. To deter-mine whether maturation of a5 integrin was affected inMad1-depleted cells, we introduced a5 integrin-3xFLAG intocontrol and Mad1-KD1 cells. Conversion of the precursor

Figure 3. Mad1 Depletion Impairs Cellular

Spreading and FAK-Tyr 397 Phosphorylation

(A) Mad1-KD cell lines 1–3 are substantially less

well spread than wild-type HeLa cells 1 and

12 hr after plating on fibronectin.

(B) Quantification of cellular phenotype 1 and

12 hr after plating on fibronectin-coated dishes.

n > 300 cells from each of three independent ex-

periments. Top: wild-type and Mad1-KD HeLa

cell lines 1–3. Bottom: wild-type and Mad1-KD1

cells transfected with the indicated shRNA-resis-

tant rescue construct. ‘‘Mad2 mut’’ indicates

Mad1 containing the mutations K541A/L543A,

which is unable to bind Mad2 [26]. ‘‘Cytopl mut’’

indicates a cytoplasmic Mad1 mutant (aa 180–

718) lacking the nuclear import signal [12]. All

rescue contructs were GFP tagged.

(C) Still images from time-lapse analysis of wild-

type and Mad1-KD1 HeLa cells showing that

wild-type cells begin spreading by 3 hr, whereas

Mad1-KD cells remain rounded 12 hr after plating

on fibronectin. See also Movie S2.

(D) Reduction of Mad1 results in reduced phos-

phorylation of FAK on Tyr 397 4 hr after plating

on fibronectin-coated coverslips.

(E) Quantification (6SD) of fluorescence intensity

of phospho-FAK (Tyr 397) in wild-type andMad1-

KD1 cells, as pictured in (D). n = 40 cells from

each of three independent experiments. **p <

0.001.

See also Figure S4 and Movie S2.

Current Biology Vol 24 No 222690

into the 18 kD mature form was notably impaired in Mad1-KDcells (Figure S3J), further supporting the conclusion that Mad1participates in a5 integrin trafficking.

Reduction of Mad1 Impairs Cellular Attachment andSpreading on Fibronectin

a5b1 heterodimers anchor cells to the extracellular matrix(ECM) by binding fibronectin [28]. The accumulation of a5 in-tegrin in the Golgi in Mad1-KD cells suggested that depletionof Mad1 may cause deficits in cell adhesion. To test thishypothesis, we plated wild-type and Mad1-KD cell lines 1–3and permitted them to attach to fibronectin-coated dishes.Significantly fewer Mad1-KD cells were adherent after 30 minas compared to control cells (Figure 2F). To determine whetherdecreased secretion was responsible for the attachmentdefect, we removed cell-surface proteins with proteinase Kbefore plating them on fibronectin. Mad1-KD1 cells again ex-hibited a defect in attachment (Figure 2G), confirming thatimpaired biosynthetic transport of newly translated a5 integrinleads to delays in cellular adhesion. In contrast, attachment tocollagen, which is mediated by a variety of integrins, includinga1b1 and a2b1, was not affected by reduction of Mad1(Figure S3K).

To determine effects of Mad1 depletion on cell spreading,we plated wild-type andMad1-KD HeLa cell lines 1–3 on fibro-nectin-coated coverslips. Within 1 hr after plating, most wild-

type cells had attached to the substrateand begun spreading, whereas manymore Mad1-KD cells remained rounded(Figures 3A and 3B). After 12 hr, 85% ofwild-type HeLa cells had elongated, ascompared to 8%, 5%, and 3% ofMad1-KD cell lines 1–3, respectively(Figures 3A and 3B). To further

characterize this defect, we plated wild-type and Mad1-KD1cells on fibronectin-coated coverslips and observed them us-ing phase-contrast time-lapse microscopy (Figure 3C andMovie S2). Wild-type cells blebbed and began extending andretracting filopodia-like protrusions almost immediately.Mad1-KD1 cells blebbed but did not extend protrusions.Wild-type cells were noticeably flattened and elongated at3 hr, whereasMad1-KD1 cells remained rounded even at 12 hr.ECM binding by integrins results in recruitment and activa-

tion of signaling molecules such as focal adhesion kinase(FAK), which becomes phosphorylated at tyrosine 397 whenactive [29]. Phospho-FAK levels were significantly diminishedin Mad1-KD1 cells (Figures 3D and 3E), consistent with adeficit in integrin-mediated signaling.

Mad1 Functions in Secretion and Attachment AreIndependent of Mad2

To test whether mitotic defects could cause deficits in cellattachment and spreading, we examined cells expressingreduced levels of the mitotic checkpoint components BubR1and Mad2. In both cases, a5 integrin did not accumulate inthe Golgi (Figures S4A–S4D). Stable reduction of Mad2 alsocaused no delay in cell spreading (Figure S4E). Together, thesedata support the conclusions that (1) Mad1 facilitates secre-tion of a5 integrin during interphase and (2) this function ofMad1 is independent of Mad2.

Figure 4. Mad1 Promotes Directional Cell Migration

(A and B) Mad1 orients with the Golgi (GM130; A) and centrosomes (centrin;

B), toward the direction of cell migration, 6 hr after wounding confluent HeLa

cells. The yellow lines in the overlaid images indicate the location of the

wound edge.

(C–F) Reduction of Mad1 inhibits directed cell migration.

(C) Still images from time-lapse analysis showing delayed wound healing in

Mad1-KD1 HeLa cells as compared to wild-type cells. Yellow lines indicate

wound edges at 0 hr. See also Movie S3.

(D) Quantification of the rate of wound healing in wild-type and Mad1-KD1

HeLa cells. The ratio of the wound size in Mad1-KD1 versus wild-type cells

at the indicated times is shown. Data indicate the mean6 SD from three in-

dependent experiments.

(E) Transwell assay showing reduced migration of Mad1-KD1 HeLa cells as

compared to wild-type cells.

(F) Quantification of the number of cells that migrated in the transwell assay

shown in (E). Error bars indicate the SD of three independent experiments.

(G–J) Overexpression of Mad1 accelerates directed cell migration.

(G) Still images from time-lapse analysis showing control (2tet) or Mad1-

YFP-expressing (+tet) MDA-MB-231 cells 0, 16, and 34 hr after wounding.

Yellow lines indicate wound edges at 0 hr. See also Movie S4.

A Novel Role for Mad1 in Secretion and Migration2691

To further test a possible role of Mad2 in integrin secre-tion, we reconstituted Mad1-KD cells with shRNA-resistantMad1 constructs. Expression of wild-type Mad1 preventedaccumulation of a5 integrin in the Golgi (Figures S4F andS4G) and permitted Mad1-KD cells to attach and spreadon fibronectin-coated dishes with normal kinetics (Figure 3B).Importantly, the Mad1 mutant that cannot bind Mad2 alsorescued a5 integrin secretion and cellular spreading, as didthe cytoplasmic Mad1 mutant (Figures 3B, S4F, and S4G).These data demonstrate that the defects in a5 integrinsecretion in Mad1-KD cells are a consequence of an inter-phase, cytoplasmic function of Mad1 that is independentof Mad2.

Mad1 Promotes Directional Cell MotilityDuring directional cell migration, the centrosome orients to-ward the leading edge, as does the Golgi [30, 31]. After wound-ing, Mad1 immunoreactivity was observed on the side of thecell adjacent to the wound edge in a pattern strikingly similarto that of the Golgi and the centrosome (Figures 4A and 4B).To directly test the role of Mad1 in cell migration, we

observed control and Mad1-KD1 cells by time-lapse micro-scopy after wound formation. Wound closure was substan-tially delayed in Mad1-KD cells relative to wild-type HeLa cells(Figures 4C–4D and Movie S3). Consistent with these results,transwell experiments revealed that Mad1-KD cells weresignificantly impaired in their migratory ability as comparedto wild-type cells (Figures 4E and 4F).We were unable to generate HeLa cells uniformly over-

expressing Mad1. Therefore, MDA-MB-231 cells stably ex-pressing Mad1-YFP in response to tetracycline were used toperform the converse experiments. Cells with elevated levelsof Mad1 repaired wounds more rapidly than control cells (Fig-ures 4G–4H and Movie S4). Similarly, in transwell assays,Mad1-YFP-expressing cells exhibited enhanced migratoryability as compared to controls (Figures 4I and 4J). These re-sults demonstrate that the level of Mad1 is directly related tothe rate of cell motility.We then examined the motility of single cells. Mad1-KD1

cells exhibited shortened migrational distance (Figures 4Kand 4L), lower directional velocity (Figure 4M), and reducedoverall speed (Figure 4N) as compared to controls. Thus, indi-vidual cells exhibit impairedmigration after reduction of Mad1.

(H) Quantification of the rate of wound closure in (G). The ratio of the size of

the wound in Mad-overexpressing cells to the size of the wound in control

cells is shown. Data indicate the mean6 SD from three independent exper-

iments.

(I) Increased migration of Mad1-YFP-expressing cells in a transwell assay.

(J) Quantification of cell migration in the transwell assay depicted in (I),

normalized to the control. Error bars indicate the SD of three independent

experiments.

(K–N) Wild-type and Mad1-KD1 HeLa cells were plated in one side of a mi-

crofluidics channel in media containing 0.1% serum. Twelve hours later,

20% fetal bovine serum was added to the opposite side of the chamber.

Cells were imaged every 5 min for 14 hr, and single-cell motility was tracked

as in [32]. n = 60 cells per condition.

(K) Tracks of individual cells.

(L) Migrational distance 6 SEM.

(M) Velocity (displacement toward final destination / time)6 SEM of individ-

ual cells.

(N) Speed (total distance / time) 6 SEM of individual cells.

(O) Model depicting Mad1 localizing to the Golgi where it facilitates secre-

tion of a5 integrin, which is required for cellular attachment, adhesion, and

directed cell motility.

*p < 0.05, **p < 0.001. See also Movies S3 and S4.

Current Biology Vol 24 No 222692

Prior to this study, interphase roles of Mad1 remainedlargely unknown. Here, we identify a novel interphase functionof Mad1 that does not involve Mad2. Endogenous Mad1 local-izes to the Golgi in multiple mammalian cell types. The pool ofMad1 in the Golgi facilitates secretion of a5 integrin, whichpromotes cellular adhesion, spreading, and motility (Fig-ure 4O). These functions are compromised in cells withreduced levels of Mad1. Heightened expression of Mad1 iscommon in breast tumors, where it serves as a marker ofpoor prognosis [13]. It will now be of interest to determinewhether increased Mad1 in cancer cells potentiates migrationand metastasis.

Supplemental Information

Supplemental Information includes Supplemental Experimental Proce-

dures, four figures, and four movies and can be found with this article online

at http://dx.doi.org/10.1016/j.cub.2014.09.052.

Acknowledgments

We thank Andrea Musacchio and Patricia Keely for antibodies, Amber

Schuh for assistance with Golgi purification, and Eric Britigan and Scott

Nelson for assistance with microscopy. This work was supported in part

by the NIH through R01CA140458 (B.A.W.), R01GM088151 (A.A.), T32

CA009135 (L.M.Z.) and R01EB010039 (D.J.B.). D.J.B. holds equity in Bell-

brook Labs, Tasso, and Salus Discovery. E.B. holds equity in Tasso, and

Salus Discovery. J.W. holds equity in Shenzhen Bionanotech.

Received: March 27, 2014

Revised: August 28, 2014

Accepted: September 17, 2014

Published: October 23, 2014

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