the interplay between the rab27a effectors slp4-a and ... dossi… · vascular biology the...

doi:10.1182/blood-2012-05-429936Prepublished online August 16, 2012;2012 120: 2757-2767

Meli, Marlene Rose, Matthew J. Hannah and Tom CarterRuben Bierings, Nicola Hellen, Nikolai Kiskin, Laura Knipe, Ana-Violeta Fonseca, Bijal Patel, Athina hormone-evoked Weibel-Palade body exocytosisThe interplay between the Rab27A effectors Slp4-a and MyRIP controls

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VASCULAR BIOLOGY

The interplay between the Rab27A effectors Slp4-a and MyRIP controlshormone-evoked Weibel-Palade body exocytosis*Ruben Bierings,1 *Nicola Hellen,1 Nikolai Kiskin,1 Laura Knipe,1 Ana-Violeta Fonseca,1 Bijal Patel,1 Athina Meli,2

Marlene Rose,2 Matthew J. Hannah,1 and Tom Carter1

1Medical Research Council National Institute for Medical Research, The Ridgeway, London, United Kingdom; and 2Faculty of Medicine, Imperial CollegeLondon, London, United Kingdom

Weibel-Palade body (WPB) exocytosis un-derlies hormone-evoked VWF secretionfrom endothelial cells (ECs). We identifynew endogenous components of theWPB: Rab3B, Rab3D, and the Rab27A/Rab3 effector Slp4-a (granuphilin), anddetermine their role in WPB exocytosis.We show that Rab3B, Rab3D, and Rab27Acontribute to Slp4-a localization to WPBs.siRNA knockdown of Slp4-a, MyRIP,Rab3B, Rab3D, Rab27A, or Rab3B/Rab27A, or overexpression of EGFP-

Slp4-a or EGFP-MyRIP showed that Slp4-ais a positive and MyRIP a negative regula-tor of WPB exocytosis and that Rab27Aalone mediates these effects. We foundthat ECs maintain a constant amount ofcellular Rab27A irrespective of the WPBpool size and that Rab27A (and Rab3s)cycle between WPBs and a cytosolic pool.The dynamic redistribution of Rab pro-teins markedly decreased the Rab27Aconcentration on individual WPBs withincreasing WPB number per cell. Despite

this, the probability of WPB release wasindependent of WPB pool size showingthat WPB exocytosis is not determinedsimply by the absolute amount of Rab27Aand its effectors on WPBs. Instead, wepropose that the probability of release isdetermined by the fractional occupancyof WPB-Rab27A by Slp4-a and MyRIP,with the balance favoring exocytosis.(Blood. 2012;120(13):2757-2767)

Introduction

Hormone-evoked VWF secretion from endothelial cells (ECs) ismediated by exocytosis of specialized secretory granules (SGs)called Weibel-Palade bodies (WPBs).1 WPB exocytosis is triggeredby increases in intracellular free Ca2� or cAMP concentrations, andinvolves a number of molecular components, including the N-ethylmaleimide–sensitive factor, VAMP3, SNAP23, syntaxin 4,RalA, the annexin A2/S100A10 complex, and phospholipase D.2-7

In addition, Rab proteins also regulate WPB exocytosis. Asubset of Rab proteins, including Rab3A-3D, Rab27A/B, andRab37, is associated with SGs in different cell types where theyregulate SG biogenesis, trafficking, and exocytosis.8 Secretorycells often express a mixture of these “secretory” Rabs, which mayhave overlapping or distinct functions. Human ECs are reported toexpress mRNA for Rab3A, Rab3D, and Rab37,3,9,10 Rab3B pro-tein,11 and Rab27A mRNA and protein.12,13 To date, Rab27A is theonly endogenous EC Rab protein that has been detected on WPBs.Through its effector MyRIP and Myosin Va, Rab27A is proposed tonegatively regulate WPB exocytosis.13,14 Rab27A can interact withdifferent effector molecules, and many secretory cells express amixture of these effectors.8 In these cases, SG exocytosis probablydepends on the balance of Rab27A interactions with the comple-ment of Rab effectors in the cell.

In addition to MyRIP, ECs contain mRNA for the Rab27Aeffector Slp4-a (granuphilin).13 Slp4-a links SGs to the plasmamembrane (PM) through SG-associated Rab proteins (principallyRab27A), PM-associated syntaxins (1a, 2, or 3) and solubleMunc18 isoforms.15-19 Syntaxins exist in open and closed conforma-

tions that determine their participation in SNARE complex forma-tion and membrane fusion. Slp4-a binds the closed conformation ofsyntaxin,16,19 which selectively interacts with Munc18 proteins tohold SGs in a fusion-incompetent state at the PM. Overexpressionof Slp4-a increased numbers of PM-docked SGs but inhibitedsecretion, whereas knockout or knockdown (KD) studies showedthe opposite.20 Unlike MyRIP, Slp4-a interacts with several differ-ent Rab proteins, including Rab3A-3D and Rab8,15,21 and withmyosin Va,22 a point of convergence with MyRIP.

In this study, we identified Slp4-a, Rab3B, and Rab3D as newcomponents of WPBs and investigated their roles in regulatinghormone-evoked WPB exocytosis and VWF secretion. We foundthat, in contrast to other secretory cells, Slp4-a acts as a positiveregulator of WPB exocytosis, and its interplay with the negativeregulator MyRIP on Rab27A bound to WPBs (WPB-Rab27A) setsthe probability of WPB exocytosis.

Methods

Tissue culture, transfection, secretion assays, antibodies, DNAconstructs, and reagents

Primary HUVECs were purchased, cultured and transfected as previouslydescribed.23 Human aortic endothelial cells (HAECs) were cultured aspreviously described.24 Secreted VWF and EGFP were assayed by ELISAas previously described.25 Primary antibodies (Abs) along with the dilutionsfor immunofluorescence and Western blotting are given in supplementalTable 1A (available on the Blood Web site; see the Supplemental Materials

Submitted May 14, 2012; accepted July 31, 2012. Prepublished online as BloodFirst Edition paper, August 16, 2012; DOI 10.1182/blood-2012-05-429936.

*R.B. and N.H. contributed equally to this study.

The online version of this article contains a data supplement.

The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked ‘‘advertisement’’ in accordance with 18 USC section 1734.

© 2012 by The American Society of Hematology

2757BLOOD, 27 SEPTEMBER 2012 � VOLUME 120, NUMBER 13




link at the top of the online article) with sources of secondary Absappended. Production of an anti-Rab27A Ab is described in supplementalFigure 1A. DNA constructs are described in the supplemental Appendix 1.Primers for amplification of full-length Rab3 isoforms, MyRIP and Slp4-a,are listed in supplemental Table 1B. All reagents were from Sigma-Aldrichunless otherwise stated. Fura-2/AM was from Invitrogen.

Immunoblotting

Cells were rinsed with PBS and lysed in 2� sample buffer12 containing�-mercaptoethanol and protease inhibitors (Sigma-Aldrich: P8340) or inPBS containing 1% Triton X-114, 2mM EDTA, and protease inhibitors. Toconcentrate hydrophobic proteins, Triton X-114 partitioning of the lysatewas performed as described.12 Protein in the detergent phase was precipi-tated with methanol-chloroform using 0.25 �g BSA as carrier. Proteinswere separated on a 12% SDS-PAGE gel, transferred to 0.2 �m nitrocellu-lose membrane, and probed with primary Ab, followed by infrareddye-coupled secondaries. Membranes were scanned with the LI-COROdyssey Infrared Imaging system (LI-COR Biosciences).

RNAi

Pools of 4 siRNA oligo duplexes (ON-TARGETplus SMARTpool, Dharma-con RNA Technologies) were used to deplete HUVECs of the mRNAs ofRab27A (#L004667), Rab3B (#L008825), Rab3D (#L010822), Slp4-a(#L007111), and MyRIP (#L013964). Oligo sequences are in supplementalTable 1C. A pool of 4 nontargeting siRNAs (ON-TARGETplus Non-targeting pool, #D001810) was used as a control (siCTRL). A total of1000 pmol of siRNA duplexes was transfected by nucleofection into7.5 � 106 HUVECs. ECs were plated at confluent density (� 0.8 � 105

cells/cm2) in gelatin-coated wells of 6- or 12-well plates or on gelatin-coated glass coverslips and cultured in HUVEC growth medium for48 hours. Depletion of Rab27A, Rab3B, Rab3D, Slp4-a, and MyRIP wasassessed by quantitative PCR, immunocytochemistry, or immunoblotting.

RT-PCR and quantitative PCR analysis

RNA was extracted using RNeasy Mini Kit (QIAGEN) and integrityverified on a 2100 Bioanalyzer (Agilent Technologies). cDNA was synthe-sized using a QuantiTect Reverse Transcription kit (QIAGEN) from 500 ngof RNA. RT-PCR primers for Rab3A to Rab3D are in supplemental Table1D. The cycling protocol was: initial 95°C for 30 seconds followed by30 cycles of 95°C for 15 seconds, 52°C for 30 seconds, 68°C for 15 seconds, final5 minutes at 72°C and products run on 2% agarose gel. Alternative primers andcycling conditions for Rab3A were also used.9 Quantitative PCR primers forreference genes (RGs: ACTB, GAPDH, B2M, UBC, YWHAZ, SF3A1, CYC1,EIF4A2, SDHA [housekeeping gene detection kit]), Rab27A, Slp4-a, MyRIP,Rab3B, and Rab3D were designed and synthesized by PrimerDesign LTD(supplemental Table 1D). Amplification was detected using Sensimix (Bioline)and analyzed on a Rotor-gene 6000 (Corbett Life Science) using 95°C for10 minutes, then 40-45 cycles of 10 seconds at 95°C, 15 seconds at 60°C, and20 seconds at 72°C, followed by a 5-minute melt sequence to verify singleamplicons. Single products were further verified on 2% agarose gels andextracted (GenElute gel extraction kit) for standard curve generation. Absolutecopy number was determined and normalized to �-actin (ACTB) expression. ForKD experiments, cDNA samples were first screened for RG expression levels,and geNorm Version 3.4 software (PrimerDesign LTD) was used to determinenormalization factors before normalization. Quantification was calculated usingthe ��CT method.26

Immunocytochemistry and quantification of antigenimmunoreactivity on WPBs

Immunostaining and imaging of fixed cells were performed as previouslydescribed.25 For both confocal (40� objective) and wide-field (100�objective) intensity measurements, exposures at each wavelength were firstset to ensure no detector saturation on the brightest sample and then keptconstant for all images. Images were processed in ImageJ (http://rsbweb.nih.gov/ij/) and numbers of WPBs quantified as previously de-scribed.25 Mean WPB fluorescence intensities (FIs) for endogenous Rab27A,

MyRIP, Slp4-a, Rab3B immunoreactivity (IR) or for EGFP-Rab27A,-MyRIP or -Slp4-a were determined from background-subtracted regions ofinterest (ROI) enclosing individual WPBs. Two (Slp4-a, MyRIP) or3 (Rab27A) independent experiments were performed using 6-12 (confocal)or � 50 (wide-field) fields of view in which � 12 WPBs/per cell were analyzedin cells from a minimum of 10 (confocal) or 1 or 2 (wide-field) cells.

Live cell imaging

Exocytosis of Proregion-EGFP-containing WPBs was determined as previ-ously described.23,25 The moment of WPB fusion for EGFP-Rab27A– orEGFP-Slp4-a–expressing cells was determined by an abrupt decrease inWPB fluorescence on fusion (illustrated for Rab27A in supplemental Figure1B). The probability of WPB exocytosis, Pr, was determined as the meanpercentage of degranulation of fluorescent WPBs after cell stimulation. Themean WPB-EGFP FI for EGFP-Rab27A was determined as described in“Immunocytochemistry and quantification of antigen immunoreactivity onWPBs.” Rab acquisition by immature WPBs was determined by time-lapseimaging of ECs expressing VWF-tdTomato (VWF-tdT) and EGFP-Rab27Aor mEGFP-Rab3B (16 hours after nucleofection). Microscope and experi-mental details are in the legend to supplemental Video 1. Mean WPB-EGFPand WPB-tdT FIs were determined from background-subtracted images asdescribed in the previous section.

Whole-WPB fluorescence recovery analysis of Rabs andeffectors cycling on WPBs

Leica TCS SP2 or Leica TCS SP5 confocal microscopes equipped with PLAPO 100 � 0.7-1.4NA (SP2) or HCX PL APO CS 100 � 1.46NA (SP5)objectives were used to study fluorescence recovery after whole WPBbleaching (FRAP) at 37°C. Imaging and bleaching settings were aspreviously described.27 For FRAP, 1-2 pulses at full laser power wereapplied to a bleaching ROI encompassing the entire WPB, and the WPB FIfollowed to monitor recovery. For whole-cell inverse FRAP (iFRAP28) a PLAPO 63 � 0.6-1.4NA objective was used to image at least 2 fluorescentcells at a time. All but a small region of 1 cell (containing a few WPBs) wasbleached using full laser power pulses at 1- to 2-second intervals overan � 5-minute period. The decline in FI in the unbleached region of the testcell was followed at 1- to 10-minute intervals. For analysis, ROIs weredefined by thresholding low-pass filtered images above the background toinclude just the bleached WPB (FRAP) or unbleached (iFRAP) WPBs inthe cell. The average FI within the ROIs was scaled by the decline becauseof bleaching in adjacent unbleached control cells/WPBs in the image.Exponential fitting was performed in Origin Version 8.5 (OriginLab)without constraints on offset or final level.

Statistical analysis

Data were plotted in Origin Version 7.5 or greater or GraphPad PrismVersion 5.0. Statistical analysis was by nonparametric t test (except whereindicated) using GraphPad Prism Version 5.0. Significance values areshown on the figures or in figure legends. Data are shown as mean � SEM.

Results

Slp4-a is localized to WPBs

Identification of Slp4-a mRNA in HUVECs13 led us to examinewhether this Rab27A effector was associated with WPBs andplayed a role in regulating WPB exocytosis. Endogenous Slp4-a-IRwas readily detectable on WPBs in HUVECs (Figure 1A; supple-mental Figure 2A) and adult HAECs (supplemental Figure 2B).Furthermore, expression of EGFP-Slp4-a specifically labeled WPBs(eg, see Figure 4Ci). However, several observations indicate thatRab27A is not solely responsible for recruiting Slp4-a to WPBs.First, HUVECs with few WPBs (eg, * in Figure 1B) consistently

2758 BIERINGS et al BLOOD, 27 SEPTEMBER 2012 � VOLUME 120, NUMBER 13




had strong WPB-Rab27A-IR, whereas HUVECs with large num-bers of WPBs (**) had almost undetectable WPB-Rab27A-IR (seealso supplemental Figure 3A-B; quantified in Figure 6A). Controlexperiments showed the intensity of WPB-Rab27A-IR was propor-tional to WPB-Rab27A-antigen concentration (supplemental Fig-ure 3C), so the reduction in WPB-Rab27A-IR reflects a reductionin WPB-Rab27A concentration. This pattern was mirrored byendogenous WPB-MyRIP-IR but not by WPB-Slp4-a-IR, whichremained prominent irrespective of WPB number, and the same

was seen in HAECs (Figure 1B; supplemental Figures 2 and 3B,D).Second, KD of Rab27A in HUVECs resulted in loss of bothWPB-Rab27A- and WPB-MyRIP-IR, but not of WPB-Slp4-a-IR(supplemental Figure 3E). Finally, we observed a subpopulation ofRab27A-negative WPBs displaying Slp4-a-IR (Figure 2D). Be-cause Slp4-a can also bind Rab8 and Rab3A-3D15,21 (and supple-mental Figure 4A), we next looked for the expression and WPBlocalization of these Rab proteins. Although HUVECs expressRab8,11,29 we detected neither endogenous nor epitope-tagged Rab8

Figure 1. Endogenous Slp4-a, Rab3B, and Rab3D are ex-pressed in HUVECs and localize to WPBs. (A) HUVECsimmunolabeled for endogenous VWF (red) and Slp4-a (green).The intensity levels of the red (VWF) channel were adjusted from0-255 to 0-130 intensity units. Scale bar represents 200 �m.Grayscale images are from the region indicated by the white box(scale bar represents 20 �m). (B) Left: HUVECs immunolabeledfor endogenous Rab27A (red), Slp4-a (green), and MyRIP (blue).Scale bar represents 20 �m. Dashed white lines indicate approxi-mate cell boundaries and a cell with few (*) and a cell with many(**) WPBs are indicated. Right top: Grayscale images are shownfor each channel as indicated and the subregions i and ii shownon expanded scales below. (C) HUVECs immunolabeled forendogenous Rab3B (mouse Ab, green) or Rab3D (mouse Ab,green) and VWF (rabbit Ab, red). Top: Endogenous Rab3B-IR islocalized to WPBs. Middle: � 95% of cells had no WPB-Rab3D-IR (see also supplemental Figure 4Civ). Bottom: 5% ofcells had weak WPB-Rab3D-IR (arrowheads). Scale bars repre-sent 10 �m. Immunofluorescence images of fixed cells here andin all subsequent figures were taken at room temperature usingLeica SP1 or SP2 confocal microscopes and software (Version2.61, build 1537) equipped with 40� and 100� objectives (SP1;PL APO40 � 1.25-0.75NA, PL APO100 � 1.4NA, SP2; HCX PLAPO40 � 1.2 NA, PL APO100 � 1.4NA).

Rab27A EFFECTORS AND VWF SECRETION 2759BLOOD, 27 SEPTEMBER 2012 � VOLUME 120, NUMBER 13




on WPBs. We therefore focused on Rab3 isoforms (specificities ofthe Rab3 reagents are shown in supplemental Figure 4Bi-vi).

Rab3B and Rab3D are expressed in ECs and localized to WPBs

Expression of mEGFP-Rab3A-3D labeled WPBs in HUVECs(supplemental Figure 4Bii-vi); however, immunostaining showedthat only endogenous Rab3B-IR was clearly detectable on WPBs inHUVECs (and HAECs), whereas WPB-Rab3D-IR was weaklydetectable in only � 5% of cells (Figure 1C; supplemental Figure4C-D). RT-PCR and Western blot analysis confirmed that onlyRab3B and Rab3D were expressed in HUVECs (supplementalFigure 4E). No Rab3A mRNA was found despite using primers andconditions reported to detect this Rab in HUVECs9 (supplementalFigure 4E). Quantitative PCR quantification showed that Rab3Band Rab3D transcripts together were � 10 times more abundantthan Rab27A transcripts.

Unlike WPB-Rab27A-IR, WPB-Rab3B-IR was clearly ob-served irrespective of WPB pool size and matched closely thepattern for endogenous Slp4-a-IR (Figure 2A). Similar to Rab27A,12

Rab3B-IR was absent from most newly formed WPBs at thetrans-Golgi network (Figure 2B); however, we observed a subpopu-lation of immature Rab27A-negative WPBs that were Rab3B-positive (Figure 2C) and displayed clear Slp4-a-IR (Figure 2D).Together, the data show that Rab27A is not the sole Slp4-a-interacting Rab protein on WPBs and that the WPB localization ofRab3B matches closely that of Slp4-a.

Rab3B is recruited slowly to immature WPBs but cycles rapidlyon mature WPBs where it recruits Slp4-a

Because differences in the kinetics of recruitment of Rab3B andRab27A to immature WPBs might account for Rab3B-IR onperinuclear Rab27A-negative WPBs, we next determined the timeconstants of recruitment (recr) of mEGFP-Rab3B and EGFP-Rab27A to immature VWF-tdT–expressing WPBs in live cells (eg,

supplemental Video 1). recr for mEGFP-Rab3B and EGFP-Rab27A were not significantly different (Figure 3A), indicatingthat other factors may account for this phenomenon (see“Discussion”).

To investigate whether Rab3B or Rab3D might contribute toWPB recruitment of Slp4-a, we performed WPB bleaching experi-ments. The rationale for FRAP experiments was that Rab proteinsand their effectors exchange (cycle) between their target mem-branes and the cytosol, and that this process, for both Rabs andeffectors, is linked to the Rab GTP-GDP cycle.30 Where an effector(Slp4-a) has access to a mixture of different Rabs, as is potentiallythe case here, the cycling kinetics will reflect a composite functionof the kinetics of the individual Rabs of the mixture. Thus, bydetermining the association time constants (ass) for individualWPB-Rabs and comparing ass for Slp4-a cycling on WPBsexpressing exogenous Rab27A, Rab3B, or Rab3D with Slp4-acycling in the absence of expressed WPB-Rabs, we might identifywith which WPB-Rab Slp4-a interacts.

We quantified ass for EGFP-tagged Rab27A, Rab3B, or Rab3Dafter whole WPB bleaching. EGFP-Rab27A recovered very slowlyand often incompletely during the observation period (Figure 3B).mRFP-Rab27A behaved like EGFP-Rab27A. mEGFP-Rab3B and-Rab3D recovered significantly faster than EGFP-Rab27A (Figure3B,D). Dissociation time constants (diss) for EGFP-Rabs, deter-mined by iFRAP, were of similar magnitude to ass (Figure 3B-C),both probably reflecting the off-rates of Rab unbinding from theWPB.31 For mEGFP-Rab3B, both ass and diss were significantlyfaster than for EGFP-Rab27A.

ass for EGFP-Slp4a to WPBs coexpressing exogenous mRFP-tagged Rab27A, Rab3B, or Rab3D were, in each case, similar tothose of the Rab protein alone (Figure 3D-E). Notably, EGFP-Slp4-a when expressed alone recovered fast, with a ass similar tothat seen in the presence of exogenous Rab3B (or Rab3D; Figure

Figure 2. Rab3B is detectable on WPBs beforeRab27A and colocalizes with Slp4-a. (A) HUVECsimmunolabeled for endogenous Rab3B (mouse Ab,green), Slp4-a (rabbit Ab, red), and VWF (sheep Ab,blue). Here and in other figures, grayscale images arefrom regions indicated by white boxes. (B) HUVECsexpressing Proregion-EGFP (Pro-EGFP, green, 24 hoursafter nucleofection) immunolabeled for endogenousRab3B (mouse Ab, red). Grayscale images representregions with perinuclear immature (right) or peripheralmature WPBs (bottom). (C) HUVECs immunolabeled forRab3B (mouse Ab, red), Rab27A (rabbit Ab, green), andVWF (sheep Ab, blue). Insets: right, arrowheads point toRab3B-positive, Rab27A-negative perinuclear immatureWPBs; bottom, Rab3B- and Rab27A-positive matureWPBs. (D) HUVECs immunolabeled for Rab3B (goat Ab;red), Rab27A (mouse Ab, green), and Slp4-a (rabbit Ab,blue). Inset: right, Rab3B-positive, Rab27A-negative,Slp4-a-positve perinuclear immature WPBs; bottom, ma-ture WPBs positive for all markers. Scale bars represent10 �m.





3E). This indicates that Slp4-a can interact with endogenous Rab3Band/or Rab3D on WPBs.

Analysis of Slp4-a, MyRIP, and WPB-Rab function in WPBexocytosis

Slp4-a is a positive and MyRIP a negative regulator of WPBexocytosis. To assess the function of Slp4-a in hormone-evokedWPB exocytosis, we depleted endogenous Slp4-a by siRNA KDand for comparison analyzed the effect of MyRIP KD. Slp4-a KD(Figure 4A) substantially decreased while MyRIP KD (Figure 4B)increased histamine-evoked VWF secretion. To examine the effectof overexpression of EGFP-Slp4-a or EGFP-MyRIP on Ca2�-driven WPB exocytosis, we performed live cell experiments. Bothconstructs labeled all WPBs, with the exception of immatureorganelles at the trans-Golgi network (Figure 4Ci). Figure 4Cii-iiicompares the kinetics and extent of WPB exocytosis in controlcells expressing the WPB-specific protein Proregion-EGFP23 (black)with that in EGFP-Slp4-a–expressing cells (red). Cumulative plotsof WPB fusion events (Figure 4Cii bottom) are aligned to theincrease in intracellular Ca2� (top in Figure 4Cii). The mean delay,maximal rate, and probability of WPB fusion (Pr), are summarizedin Figure 4Ciii. In EGFP-Slp4-a–expressing cells, WPB exocytosiswas initially slowed, although later the rate increased and reached

control values in Proregion-EGFP cells. Surprisingly, Pr wassignificantly increased (Figure 4Ciii). In marked contrast, expres-sion of EGFP-MyRIP completely blocked Ca2�-driven WPBexocytosis.

Quantification of the intensity of endogenous WPB-MyRIP-IRas a function of the FI of WPB-EGFP-Slp4-a showed thatoverexpression of EGFP-Slp4-a reduced endogenous WPB-MyRIP-IR (Figure 4Civ black symbols). In contrast, EGFP-MyRIPoverexpression had only a small effect on endogenous WPB-Slp4-a-IR (Figure 4Civ gray symbols), presumably because Slp4-a,unlike MyRIP, interacts with GTP-Rab3B, GTP-Rab3D, andGDP-Rab27A on WPBs. The data show that Slp4-a and MyRIPexert opposing effects on WPB exocytosis and VWF secretion andthat Slp4-a competes with MyRIP for its WPB-specific bindingpartner Rab27A.

Rab27A, but not Rab3B or Rab3D, regulates VWF secretion.Having established that Slp4-a acts as a positive regulator ofhistamine-evoked VWF secretion and that it can interact withWPB-Rab3s, we next determined the relative contributions ofRab3B, Rab3D, and Rab27A to hormone-evoked VWF secretion.To do this, we performed KDs of each Rab individually and ofRab27A and Rab3B together (Figure 5). KDs profoundly depletedeach Rab, with endogenous WPB-Rab3B- and WPB-Rab27A-IR

Figure 3. Rab recruitment to and cycling on WPBs:evidence for association of EGFP-Slp4-a with Rab3son mature WPBs. (A) Left: Images from time-lapsevideos showing Rab acquisition by immature VWF-tdTcontaining WPBs in HUVECs coexpressing eithermEGFP-Rab3B (top) or EGFP-Rab27A (bottom). Times areindicated below the panels. Right: Example time courses ofmEGFP-Rab3B (E, recr 709 seconds) or EGFP-Rab27A (Œ,recr 1070 seconds) recruitment to VWF-tdT–labeled WPBs.Bar plot represents the mean values of recr for WPB-mEGFP-Rab3B (1730 � 355 seconds, n � 7) and WPB-EGFP-Rab27A (2260 � 365 seconds, n � 9) were not significantlydifferent (P � .35). (B) Left: Images from FRAP experimentswith WPB-EGFP-Rab27A (top; scale bar represents 2 �m)and WPB-mEGFP-Rab3B (bottom; scale bar represents1 �m) show FI recovery after complete WPB bleaching(FRAP, t � 0 seconds). Times are indicated below frames.Right: Average WPB mEGFP-Rab3B (E) and EGFP-Rab27A (Œ) FIs normalized to the prebleach levels. Graphswere fitted with exponential association functionsA � [1 � exp(�t/ass)]. Unconstrained fits to fluorescencerecovery reached 0.80 for Rab3B and 0.63 for Rab27A.(C) Left: Images from iFRAP experiments with EGFP-Rab27A (top) and mEGFP-Rab3B (bottom). Scale barsrepresent 10 �m. Regions indicated by boxes were continu-ously bleached for 5 minutes, then FI of unbleached WPBswas measured. Fluorescence of neighboring cells was usedfor control. Times after bleaching are indicated below frames.Right: Average FIs of WPB-mEGFP-Rab3B (E) and WPB-EGFP-Rab27A (Œ) were normalized to the initial postbleachlevels. Graphs were fitted with exponential decay functionsA � exp(�t/diss). (D) Average cycling time constantsdetermined for each EGFP-Rab by FRAP (ass, gray)and iFRAP (diss, white) were as follows: EGFP-Rab27A, 1970 � 550 seconds (n � 8) and 4020 � 1090seconds (n � 6); mEGFP-Rab3B, 196 � 18 seconds (n � 6)and 468 � 211 seconds (n � 5); mEGFP-Rab3D, 496 � 79seconds (n � 8) and 1300 � 296 seconds (n � 8), respec-tively. The ass (FRAP) for mEGFP-Rab3B and Rab3D weresignificantly faster than for EGFP-Rab27A (P � .0007 andP � .002, respectively). The diss (iFRAP) for mEGFP-Rab3B was also significantly faster than for EGFP-Rab27A(P � .017). (E) Average ass for EGFP-Slp4-a coexpressedwith RFP-tagged Rab27A (n � 8), Rab3B (n � 7), Rab3D(n � 8), or alone (n � 14) as indicated. ass for EGFP-Slp4-acoexpressed with Rab3B or Rab3D were not different fromthat for EGFP-Slp4-a alone (P � .31 and P � .43, respec-tively), whereas they were significantly faster than ass withmRFP-Rab27Acoexpression.





greatly diminished in both single and double KDs (Figure 5Ai-iii).KD of Rab27A also depleted WPB-MyRIP- but not WPB-Slp4-a-IR (supplemental Figure 3E). Substantial depletion of WPB-Slp4-a-IR was observed only after Rab3B/Rab27A double KD (Figure5Aiv), and not after Rab3B or Rab27A KDs (supplemental Figures3Eii and 5).

KD of Rab27A, but not of Rab3B or Rab3D, significantlyreduced histamine-evoked VWF secretion (Figure 5Bi-ii), and nofurther significant decrease was seen after Rab3B/Rab27A KD.Together, the data show that Rab27A and not Rab3B or Rab3Dregulate histamine-evoked VWF secretion and suggest that theinfluence on exocytosis of their common effector, Slp4-a, ismediated via Rab27A alone.

The probability of WPB exocytosis is unaffected by reductionsin WPB-Rab27A concentration with increasing WPB pool size.The inhibition of histamine-evoked VWF secretion by Rab27A KDled us to ask whether the natural reduction in WPB-Rab27Aconcentration with increasing WPB pool size (Figure 1B; supple-mental Figure 3) was mirrored by a decrease in Pr. The reduction in

WPB-Rab27A concentration with increasing WPB pool size issummarized in Figure 6A (along with the corresponding variationin WPB-MyRIP and WPB-Slp4-a levels). As the same relationshipwas seen in Proregion-EGFP–expressing HUVECs (supplementalFigure 6A), we could assess directly how Pr varied with WPB poolsize in live cells. Figure 6B shows Pr as a function of the meannumber of fluorescent WPBs per cell in the same ranges as inFigure 6A. Cells were stimulated with low or high concentrationsof histamine or a maximally effective concentration of ionomycin.In each case, Pr was independent of WPB pool size. We nextaddressed the cellular mechanism for the reduction in WPB-Rab27A levels with increased WPB pool size.

We hypothesized that, if total cellular Rab27A levels remainunchanged with increasing WPB numbers, then Rab27A cyclingbetween WPBs would account for the dilution in WPB-Rab27Aconcentration. To test this hypothesis, we took advantage of the factthat VWF content and WPB numbers in HUVECs increase withtime in culture.32 We determined how cellular levels of theWPB-specific marker Proregion and of Rab27A change with time

Figure 4. Slp4-a and MyRIP have opposing effects onWPB exocytosis and VWF secretion. Quantification ofmRNA for Slp4-a (Ai) and MyRIP (Bi) after nucleofectionwith either control (siCTRL) or specific KD oligos asindicated. mRNA was depleted by 65% � 3% (Ai) or67% � 3% (Bi) in KD compared with control. Data werepooled from 3 independent experiments. (Aii,Bii) Immuno-blots for Slp4-a or MyRIP, respectively, after nucleofec-tion with either control (siCTRL) or specific KD oligos asindicated. Data are representative of 3 independentexperiments. Position of molecular weight markers (kDa)are shown to the left and -tubulin used as loadingcontrol. (Aiii,Biii) Immunostaining of HUVECs for Slp4-a(rabbit Ab, green) and VWF (sheep Ab, red; Aiii) or MyRIP(goat Ab; green) and VWF (rabbit Ab, red; Biii) in controlor after KD as indicated. Scale bars represent 10 �m.(Aiv,Biv) Histamine-evoked VWF secretion, quantified byELISA, from HUVECs nucleofected with control, Slp4-a(Aiv) or MyRIP (Biv) siRNA oligos as indicated. White andgray bars represent unstimulated (vehicle alone) andhistamine-stimulated conditions, respectively. Data werepooled from 3 independent experiments. A small butsignificant decrease in basal release was seen withSlp4-a KD (siCTRL, 0.9% � 0.08% vs siSlp4-a,0.57% � 0.1% of total VWF, P � .01) and a trend to anincrease in basal secretion for MyRIP KD (siCTRL,0.27% � 0.07% vs siMyRIP, 0.43% � 0.15% of totalVWF), although the latter did not reach significance(P � .4). (Ci) HUVECs expressing EGFP-Slp4-a (top,green) or EGFP-MyRIP (bottom, green) 24 hours afternucleofection and immunolabeled for VWF (sheep Ab;red). Grayscale images are from regions indicated bywhite boxes. Scale bars represent 20 �m. (Cii). Top:Fura-2 fluorescence ratio showing intracellular Ca2� rise.Bottom: Cumulative plot of WPB fusion event times,normalized by their total number, in Proregion-EGFP-(black, 46 cells, 2151 fusion events), EGFP-Slp4-a– (red,15 cells, 849 fusion events), and EGFP-MyRIP–express-ing cells (blue, 21 cells, total absence of fusion events).Inset: The initial period after stimulation on an expandedtime scale. (Ciii) Mean delays (seconds), maximal ratesof exocytosis (WPBs/second), and probabilities of WPBexocytosis, Pr, (percent, note broken Y scale) forProregion-EGFP (black) or EGFP-Slp4-a–expressing cells(red). (Civ) Relationship between the average intensity ofendogenous WPB-Slp4-a-IR (gray) or endogenous WPB-MyRIP-IR (black) and WPB-EGFP FI (determined forindividual WPBs) in cells expressing EGFP-MyRIP orEGFP-Slp4-a, respectively. In each cell (n � 32, EGFP-MyRIP; n � 30, EGFP-Slp4-a), at least 15 WPBs werequantified to determine the intensity of WPB-IR andEGFP fluorescence. WPB-IRs were scaled to the meanintensity of IR for each antigen in nonexpressing cells(n � 40) within the same experiment.





in culture. HUVEC numbers and total protein content increasedwith time, reaching a plateau by 96 hours (supplemental Figure6B). As expected, levels of Proregion, adjusted for cell number,increased progressively, whereas those of Rab27A and housekeep-ing proteins remained unchanged (Figures 6C). Thus, HUVECsmaintain a relatively constant intracellular pool of Rab27A in theface of large increases in the WPB pool size, consistent withRab27A cycling underlying the dilution of individual WPB-Rab27A levels.

Finally, we examined how Pr changed with time after WPBformation. We performed a morphologic pulse-chase usingProregion-EGFP, and at specific times after nucleofection cellswere either fixed and stained for endogenous Slp4-a or MyRIP, orused in live secretion experiments. Figure 6Di-iii shows theincrease in mean number of fluorescent WPBs per cell with timeafter nucleofection (Figure 6Di), the mean percentage of thesefluorescent WPBs that were positive for Slp4-a- (F) or MyRIP-IR(E; Figure 6Dii), and Pr (Figure 6Diii; F). The fraction of WPBspositive for Slp4-a- or MyRIP-IR was similar at each time,

consistent with the time-dependent recruitment of Rab27A.13 Atearly times, when most WPBs lacked Slp4-a- or MyRIP-IR, Pr

was low but increased to close to maximal levels as the fractionof WPBs positive for both effectors increased. Figure 6Diii alsoshows Pr values recalculated for the fraction of Slp4-a-positiveWPBs at each approximate time point (E). The data suggest thatPr is closely linked to the WPB recruitment of both Slp4-a andMyRIP.

Discussion

Rab proteins on WPBs: composition and recruitment

WPBs in HUVECs and HAECs recruit Rab3B and Rab3D inaddition to Rab27A. In a small number of perinuclear Rab27A-negative (immature) WPBs, we observed detectable Rab3B-IR(Figure 2C). Rabs are delivered to their target membranes by1 of 2 routes: as newly synthesized Rabs in association with Rab

Figure 5. KD of Rab27A, but not of Rab3B or Rab3D,decreases hormone-evoked VWF secretion.(Ai-ii) Quantification of mRNA and protein after nucleofec-tion with either control (siCTRL) or specific KD oligos asindicated. mRNA KD were Rab3B (89% � 2%), Rab27A(92% � 0.4%), Rab3B/Rab27A (81% � 1%/87% � 2%),and Rab3D (87% � 2.6%). Data were pooled from 3-5independent experiments. Representative immunoblotsare shown. Position of molecular weight markers (kDa)are shown to the right; -tubulin (Ai) or transferrinreceptor (TfR, Aii) were used as loading controls.(Aiii) Four left panels: Immunostainings of HUVECs forendogenous Rab3B (mouse Ab, green) or Rab27A(mouse Ab, green) and VWF (sheep Ab, red) in control orKDs as indicated. Two right panels: Immunostainings ofHUVECs for Rab3B (mouse Ab, green), Rab27A (rabbitAb, blue), and VWF (sheep Ab, red). Grayscale imagesare from regions indicated by white boxes. Scale barsrepresent 20 �m. (Aiv) Immunostainings of HUVECs forSlp4-a (rabbit Ab, green in color merge on left) and VWF(sheep Ab, red in color merge on left) in control (top) orRab3B/Rab27AKD (bottom). Scale bars represent 20 �m.(B) Histamine-evoked VWF secretion from HUVECsnucleofected with control or specific siRNA oligos forRab3B, Rab27A, or Rab3B/Rab27A (Bi), or Rab3D(Bii) as indicated. Open bars represent control (vehiclealone); and gray bars, histamine stimulation. Data werepooled from 3 independent experiments. There was nosignificant difference in VWF secretion between Rab27AKD and Rab3B/Rab27A KD (P � .26). Compared withsiCTRL, basal secretion of VWF was significantly lower inRab27A (P � .02) or Rab27A/Rab3B (P � .015) KDexperiments.





Escort Protein (REP),33,34 or during subsequent Rab cycling, inassociation with guanine-nucleotide dissociation inhibitor (GDI).35

The key step for Rab delivery by either route is membrane insertionthat requires Rab GDP-GTP exchange catalyzed by a guanine-nucleotide exchange factor (RabGEF, and possibly other as yetunidentified molecules).36 Comparison of Rab3B recr with Rab3Bass or diss (Figure 3) showed that the initial recruitment is muchslower than Rab cycling on mature WPBs, suggesting thatimmature WPBs may preferentially recruit newly synthesizedRabs. Because newly synthesized Rab3B, Rab3D, and Rab27Ashare many common molecular components for delivery totarget membranes,33,34 including a RabGEF (RabGEP36), it is notsurprising that recr for mEGFP-Rab3B and EGFP-Rab27A toimmature WPBs were similar. However, if endogenous Rab3Bwere more abundant than Rab27A at the protein level, as seen atthe transcript level, then competition between Rab3B andRab27A for shared molecular components could account forRab3B-positive Rab27A-negative WPBs. Indeed, a very lowGTPase activity for Rab27A may lend an additional competitiveadvantage to Rab3B in accessing REP.37,38 Direct evidence forcompetition between secretory Rabs in HUVECs came fromobservations that EGFP-Rab27A or mEGFP-Rab3s’ expressiondepleted endogenous WPB-Rab3B- or Rab27A-IR, respectively(supplemental Figure 7A-B). The primary point of competitionbetween these Rabs is probably membrane insertion becauseneither cytosolic EGFP nor YFP-Rab1A, which uses distinctGEFs (and GTPase activating proteins),39 displaced Rab3B fromWPBs (supplemental Figure 7C).

Slp4-a recruitment to WPBs

We show that WPB recruitment of Slp4-a involves not onlyRab27A but also Rab3B (and potentially Rab3D; Figures 3E and5Aiv), accounting for several otherwise unexpected observations:the failure of Rab27A KD to remove Slp4-a from WPBs (supple-mental Figure 3Eii), the presence of Slp4-a-IR on a population ofendogenous Rab27A-negative WPBs, and retention of Slp4-a-IRon WPBs after displacement of endogenous Rab27A-IR (andMyRIP-IR) by overexpressed EGFP-Rab3s (eg, supplementalFigure 7D).

Photobleaching studies showed fast Rab3s cycling on indi-vidual WPBs, consistent with previous bulk granule bleachingstudies in PC12 cells.40 Because Slp4-a binds to Rab3s only in theGTP form (supplemental Figure 4A),15,41 the fast ass of EGFP-Slp4-a with mRFP-Rab3–expressing WPBs (Figure 3E) indicatesthat Slp4-a cycling is strongly influenced by the underlying cyclingof Rab3s. Rab27A also cycled, but its ass was very slow, consistentwith studies in PC12 cells.40 Slow Rab27A cycling could reflect thefast constitutive GDP-GTP exchange and slow GTP hydrolysisrates reported to hold Rab27A predominantly as GTP-Rab27A onSGs.37 Unlike for Rab3s, conversion to GDP-Rab27A is notassociated with loss from membranes.37 Whether this arisesbecause Rab27A is a poor substrate for GDI or because access toGDI is hindered by binding to effectors in the GDP-state (eg,Slp4-a, coronin-3)41,42 remains to be established. The cycling ofEGFP-Slp4-a on mRFP-Rab27A–expressing WPBs mirrored thevery slow cycling of Rab27A itself, presumably because Slp4-a,

Figure 6. The probability of WPB exocytosis is main-tained over a wide range of WPB-Rab27A concentra-tions. (A) Mean intensities of WPB-IR for endogenousRab27A(squares), MyRIP (triangles), and Slp4-a (circles)as a function of WPB numbers per cell. Data scaled to themean intensity for the 50 WPBs per cell. Numbers ofcells analyzed: Rab27A, 124-218; MyRIP, 17-73; Slp4-a,16-39. For all cells with � 50 WPBs, Rab27A and MyRIPintensities of WPB-IR were significantly lower than forcells with 50 WPBs (P .0001 in all cases). ForSlp4-a, corresponding WPB-Slp4-a-IR differences werenot significant (P � .14). (B) Probabilities of WPB exocy-tosis, Pr, for ionomycin or histamine stimulation asindicated, plotted against the mean number of fluores-cent Proregion-EGFP–containing WPBs, binned simi-larly to panel A. Data from 17-51 (ionomycin), 15-32(100�M histamine), and 3-10 (1�M histamine) cells.(C) Quantification of Rab27A levels in HUVECs seededat 1.0 � 105 cells per 3.5-cm dish and cultured in HUVECgrowth medium for 24-120 hours. Growth media wasreplaced after 72 hours. At indicated times, the cells werecounted, lysates made, and protein content determined(supplemental Figure 6B top). The intracellular content ofProregion (red), Rab27A (blue), -tubulin (light gray), andactin (dark gray) at each time point was determined byimmunoblotting, band intensities quantified in ImageJ,and corrected for cell number at corresponding timepoints. Cellular protein levels are presented as a percent-age of those at 24 hours (data from 3 experiments, eachperformed in triplicate). (D) Changes in exocytosis ofProregion-EGFP–positive WPBs with time after nucleo-fection, same abscissa in subpanels i through iii.(Di) Increase in mean number of fluorescent WPBs percell. (Dii) Mean percentage of Proregion-EGFP–positiveWPBs displaying endogenous WPB-Slp4-a-IR (F) orWPB-MyRIP-IR (E) at the times indicated after nucleofec-tion. (Diii) Probability of WPB exocytosis Pr for 100�Mhistamine stimulus (F). (E), Pr values scaled by the meanfraction of Slp4-a–positive WPBs (from Figure 6Dii, � 7,12, 24, and 48 hours). Sixteen to 29 cells were analyzedat each time point.





unlike other Rabs, binds to both GTP and GDP-bound states ofRab27A.41 The fact that EGFP-Slp4-a alone cycled as fast as in thepresence of exogenous Rab3B or Rab3D indicates that Slp4-aassociates with endogenous WPB-Rab3s in living HUVECs.

Slp4-a and MyRIP have opposite effects on WPB exocytosis

Our data show that Slp4-a acts as a positive regulator of hormone-evoked WPB exocytosis and confirm that MyRIP is a negativeregulator of this process.14 The latter is also consistent withMyRIP’s reported role in phorbol 12-myristate 13-acetate (PMA)–stimulated secretion of VWF.13 The results for Slp4-a are particu-larly interesting because this molecule is reported to be inhibitoryfor SG exocytosis in other secretory cells.20,43 This inhibitory effectis thought to arise through an increased tendency to form nonpro-ductive docked complexes with PM syntaxins, although theunderlying kinetics of exocytosis remain unchanged. In contrast,we found that Slp4-a expression increased Pr in response toionomycin and slowed the initial phase of WPB fusion. The lattercould involve the aforementioned processes,20,43 but this remains tobe established.

A striking consequence of EGFP-Slp4-a overexpression was areduction in the intensity of endogenous WPB-MyRIP-IR, showingthat these effectors compete for their common binding partner,Rab27A. This raises the intriguing possibility that the increase in Pr

observed on EGFP-Slp4-a expression might arise simply from thecompetitive displacement of endogeous MyRIP from GTP-Rab27A on WPBs. Indeed, the relatively modest effect of EGFP-Slp4-a expression (20%-25% increase in WPB exocytosis), wasreminiscent of the effect of MyRIP KD (� 15%-25% increase inVWF secretion). Conversely, EGFP-MyRIP expression shoulddisplace endogenous Slp4-a from GTP-Rab27A bound to WPBs,shifting the occupancy of GTP-Rab27A toward MyRIP and thus

inhibiting WPB exocytosis. Indeed, we observed a completeinhibition of WPB exocytosis, although depletion of endogenousSlp4-a-IR from WPBs was masked by the capacity of WPB-Rab3sto recruit Slp4-a to WPBs. Compared with the relatively smalleffect of MyRIP depletion from WPBs, it indicates that endogenousWPB-MyRIP levels exert a relatively weak brake on WPBexocytosis.

Rab27A regulates VWF secretion

Rab27A KD caused a significant inhibition of both unstimulatedand stimulated VWF secretion, whereas Rab3B or Rab3Ddepletion had no effect (Figure 5B). A reduction in unstimulatedsecretion in other KD conditions (eg, Slp4-a) also mirrored theeffect on stimulated secretion. Because the majority of unstimu-lated VWF secretion arises from basal release of WPBs,44 thisindicates that the same molecular machinery influences stimu-lated and basal VWF secretion. Our Rab27A KD data differfrom a previous study reporting a substantial increase inPMA-stimulated VWF secretion,13 presumably reflecting differ-ences in the mechanisms by which PMA and hormone triggerVWF secretion. An increase in histamine-evoked VWF secre-tion was also reported in that study13; however, those resultswere equivocal because of a 3-fold increase in unstimulatedVWF release caused by Rab27A KD.

Overexpression of epitope-tagged Rab3D has also been re-ported to inhibit hormone-evoked VWF secretion,3 and we ob-served the same result (supplemental Figure 7E). However, wefound that Rab3D overexpression was accompanied by loss ofendogenous WPB-Rab27A-IR and MyRIP-IR (supplemental Fig-ure 7Bii), although WPB-Slp4-a-IR was still observed, consistentwith Slp4-a binding to WPB-Rab3s (supplemental Figure 7D). Thedata suggest that the effect of Rab3D overexpression arises through

Figure 7. Slp4-a and MyRIP acting via Rab27A controlthe probability of WPB exocytosis independently ofWPB pool size. (A) Rab27A cycles between the cytosoland the WPB membrane, a process thought to bedependent on GDP-dissociation inhibitor (GDI; rectangu-lar insert, cellular components are not drawn to scale).Rab cycling distributes Rab27A approximately evenlybetween all WPBs within the cell. Because the cellularcontent of Rab27A remains constant in culture, increasesin WPB pool size (gray gradient) over a “Physiologicrange” (beige bar) result in a decrease in the amount ofRab27A on individual WPBs (purple gradient). The prob-ability of WPB release (Pr, dashed line) remains constantover this “Physiologic range” of WPB pool sizes, indicat-ing that the control of WPB exocytosis cannot solelydepend on the absolute amount of WPB-Rab27A. Aslong as the cellular content of Rab27A (and its effectors)remains constant, the steady-state fractional occupancyof Rab27A by its effectors will also be constant, irrespec-tive of the absolute Rab27A concentration on individualWPBs. This raises the possibility that the fractionaloccupancy of Rab27A by its effectors, rather than just theabsolute amount of these molecules, sets the Pr. Substan-tial depletion (by KD, extreme right of panel A) or highoverexpression (OE, extreme left in panel A; data notshown) of Rab27A results in a reduction in Pr. (B) WPBexocytosis is determined by the balance of occupancy ofWPB-Rab27A by negative (MyRIP that binds only GTP-Rab27A) and positive (Slp4-a that binds both GTP- andGDP-Rab27A) effectors. Manipulations that alter eithereffector concentration or cellular Rab27A levels canperturb the fractional occupancy of WPB-Rab27A by itseffectors, resulting in changes (small vertical arrows) inthe probability of WPB exocytosis.





competitive displacement of Rab27A from WPBs and that, despiterecruiting Slp4-a to WPBs, Rab3s do not support the positive actionof Slp4-a on VWF secretion. The latter idea is consistent withprevious data in PC12 cells.21 Slp4-a binds to GTP- and GDP-Rab27A states,41 whereas MyRIP binds to GTP-Rab27A only.45 IfSlp4-a and MyRIP compete for WPB-Rab27A, as suggested bydata in Figure 4Civ, then the capacity of Slp4-a to bind GDP-Rab27A could confer a competitive advantage over MyRIP in apositive drive toward exocytosis.

KD of Rab3B (and Rab3D) suggests little or no role for theseRabs in controlling WPB exocytosis, raising the intriguing questionas to their function on the WPBs. Rab3B and Rab3D are implicatedin processes not directly related to control of exocytosis, includingthe regulation of catecholamine uptake into SGs,46 receptor transcy-tosis,47 SG-microtubule interactions,48 and SG maturation.49 Thefunction of Rab3s on WPBs remains to be established.

The probability of WPB exocytosis does not depend solely onthe absolute WPB-Rab27A concentration over the endogenousrange

Despite marked intercellular variations in the amount of endoge-nous Rab27A (and MyRIP) on WPBs because of increasing WPBpool size (Figures 1B and 6A; supplemental Figure 3A-B), Pr wasconstant for a given stimulus strength (Figure 6B). This suggeststhat Pr is not determined solely by the absolute amount of Rab27Aand its effectors on WPBs, although some finite amount of eachmust be required. Instead, some other parameter(s) must beimportant in determining Pr. We found that the total cellularcontent of Rab27A remains essentially constant with time inculture, and our preliminary results indicate that the same is true forMyRIP and Slp4-a (and Rab3B). In this case, the steady-statefractional occupancy of Rab27A by its effectors should also remainconstant, irrespective of the absolute amounts of Rab27A on anyindividual organelle. This raises the possibility that the fractions ofWPB-Rab27A molecules occupied by MyRIP and Slp4-a are thecrucial factor in determining Pr. Most newly formed WPBs lackendogenous Slp4-a-IR, MyRIP-IR (Figure 6Dii), Rab27A-IR12

(and Rab3B-IR, Figure 2B), and for these organelles Pr was verylow (Figure 6Diii). The close correlation between the increase in Pr

(Figure 6Diii) and increase in the fraction of WPBs with detectableSlp4-a- and MyRIP-IR (Figure 6Dii) shows that the capacity toundergo exocytosis is closely linked to acquisition of both effectormolecules. By assuming that WPBs completely lacking these

components are not fusion-competent, we found that newly formedWPBs (7 hours after nucleofection) with detectable Slp4-a-IR andMyRIP-IR had a Pr not much lower than that seen for much olderorganelles (Figure 6Diii, E). The slightly higher Pr seen for olderWPBs may reflect their predominantly peripheral localization andcloser apposition to the PM. Manipulations that alter the steady-state fractional occupancy of Rab27A (eg, overexpression or KD ofSlp4-a or MyRIP) change Pr and VWF secretion. Substantialreductions in WPB-Rab27A levels after Rab27A KD simulate areturn toward the “immature state” where Rab27A, MyRIP, andSlp4-a are missing, rendering WPBs less responsive to stimulation.

In conclusion, WPBs recruit multiple Rabs; however, our datasuggest that Slp4-a and MyRIP acting via Rab27A regulate WPBexocytosis and VWF secretion. Our data indicate that the fractionaloccupancy of WPB-Rab27A by its effectors rather than just theabsolute amount of these molecules on WPBs is an important factorin determining Pr. Thus, by simply maintaining the cellular contentof Rab27A and its effectors at approximately constant levels, ECsmaintain Pr constant irrespective of WPB pool size (Figure 7). Thisprovides a simple mechanism to ensure a coordinated and propor-tionate response of the EC population to external stimuli of varyingintensity.

Acknowledgments

M.J.H. and T.C. were supported by the United Kingdom MedicalResearch Council (reference codes U117570589 and U117573808).

Authorship

Contribution: R.B., N.H., N.K., L.K., B.P., A.M., A.-V.F., and T.C.performed research and analyzed data; A.M. and M.R. contributedvital reagents; and R.B., N.H., N.K., A.-V.F., M.J.H., and T.C.designed the research and wrote the paper.

Conflict-of-interest disclosure: The authors declare no compet-ing financial interests.

The current affiliation for M.J.H. is Microbiology ServicesColindale, Health Protection Agency, London, United Kingdom.

Correspondence: Tom Carter, Department of Physical Bio-chemistry, National Institute for Medical Research, The Ridge-way, Mill Hill, London NW7 1AA, United Kingdom; e-mail:[email protected].

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Supplementary Figures and Tables

Supplementary Appendix 1

DNA constructs

The VWF propolypeptide fused to enhanced green fluorescent protein (Proregion-EGFP), EGFP-

Rab27A, myc-Rab27A and Rab27A fused to monomeric red fluorescent protein (mRFP) have been

described previously 1,2. tdTomato-N1 was made as previously described 3 and VWF-tdTomato

(VWF-tdT) made by transferring the VWF cDNA from VWF-EGFP 4 into tdTomato-N1 as an

EcoRI/AgeI fragment. Myc-C1 was derived from EGFP-C1 (Clontech) using the strategy and oligos

described previously 2. Fusions of monomeric EGFP (mEGFP) with Rab3A-Rab3D were produced

using ligation-independent cloning into the mEGFP-LIC vector (see the next section). Primers for

amplification of full-length Rab3 isoforms, MyRIP and Slp4-a are listed in Supplementary Table

S1B. Rab3B and Rab3D were amplified from HUVEC cDNA, and Rab3A and Rab3C from cDNA

image clones (3A: Missouri S&T cDNA Resource Center, Rolla, USA; 3C: IMAGE clone 3534915,

Source Bioscience, Cambridge, UK). MyRIP was amplified from Origene Trueclone clone

(SC100943 NM_015460) using primers MyRIP-f and MyRIP-r (Supplementary Table S1B) and

cloned into EGFP-LIC. Rab27A, Rab3B and Rab3D constitutive GTP (Rab27A: Q78L, Rab3B/D:

Q81L) and GDP mutants (Rab27A: T23N, Rab3B/D: T36N) were constructed using PCR

mutagenesis with the primers listed in Supplementary Table S1B. TagRFP versions of wild-type

and mutant Rab3B and Rab3D were made by transferring AscI-NotI fragments to TagRFP-LIC.

mCherry- and EGFP-Slp4-a were made by cloning a PCR product made by amplification of full-

length Slp4-a from IMAGE clone 390837 (Source Bioscience, Cambridge, UK) with primers Slp4-a-

f and Slp4-a-r between the XhoI and BamHI sites of mCherry-C1 and EGFP-C1. All constructs

were sequence verified.

Ligation-independent cloning into the mEGFP-LIC vector

Sequences for all primers used are shown in Supplementary Table S1B. GEX-C3 was made by

PCR amplification of the multiple cloning site (MCS) of EGFP-C1 using MCS-f and MCS-r as

forward and reverse primers, respectively. The 83 bp amplicon was BglII/NotI digested and cloned

into BamHI/NotI-digested GEX-6P-1. The resultant vector was sequence verified. Ligation-

independent cloning (LIC) vectors were produced as follows: a 43 bp BsrGI-AscI-SacII-NotI linker

was formed by annealing BASN-linker-f and BASN-linker-r and this was inserted between the

BsrGI and NotI sites of EGFP-C1, resulting in LIC-int1. A 64 bp NheI-SbfI-XmaI-FseI-AgeI-BsrGI

1

linker formed by annealing NSXFAB-linker-f and NSXFAB-linker-r and was inserted between the

NheI and BsrGI sites of LIC-int1, resulting in LIC-int2. A third 37 bp adaptor, which introduces three

consecutive-frame stop codons, was formed by annealing 3XSTOP-f and 3XSTOP-r and was

inserted between the SacII and NotI sites of LIC-int2, yielding LIC-int3. EGFP-LIC, mEGFP-LIC,

mEYFP-LIC, mECFP-LIC and mCherry-LIC were made by inserting AgeI/BsrGI fragments of

EGFP-C1, mEGFP-C1, mEYFP-C1, mECFP-C1 and mCherry-C1 respectively into pLIC-int3. Myc-

LIC was made by insertion of a 48 bp adaptor formed by annealing MYC-LIC-f and MYC-LIC-r

between the AgeI and BsrGI sites of LIC-int3. TagRFP-LIC was made by amplification of TagRFP

from TagRFP-actin (Evrogen, Moscow, Russia) using TagRFP-LIC-f and TagRFP-LIC-r as forward

and reverse primers respectively. The 739 bp amplicon was AgeI/BsiWI-digested and was inserted

between the AgeI and BsrGI sites of LIC-int3. All expression vectors were sequence verified. For

construction of N-terminal-tagged expression constructs the LIC vectors were SacII-digested,

column purified and then treated with T4 DNA Polymerase (New England Biolabs, Ipswich, USA) in

the presence of dATP. Forward primers to amplify cDNAs to be N-terminal-tagged were designed

to include the first 20-25 bases of the coding sequence (including the ATG), preceded by the

sequence 5’-gggcgcgcctggtggggcc. Reverse primers were designed to include the last 20-25

bases (including the stop codon) of the coding sequence, preceded by 5’-gcggccgcctgctcgtcca.

PCR products were column purified and treated with T4 DNA Polymerase in the presence of dTTP.

For construction of C-terminal-tagged expression constructs the LIC vectors were XmaI digested,

column purified and then treated with T4 DNA Polymerase in the presence of dTTP. Forward

primers to amplify cDNAs to be C-terminal-tagged were designed to include the first 20-25 bases

of the coding sequence (including the ATG), preceded by the sequence 5’-

gcaggggcgcaacagaccccggt. Reverse primers were designed to include the last 20-25 bases

(without the stop codon) of the coding sequence, preceded by 5’-ccaccaggccggccagcacccggtcc.

PCR products were column purified and treated with T4 DNA Polymerase in the presence of dATP.

T4 DNA treated PCR products were cloned into T4 DNA treated LIC-vectors essentially as

described in 5. To facilitate swapping of cDNAs between LIC vectors, AscI and NotI restriction sites

can be used to excise and insert N-terminal-tagged cDNAs. For C-terminal-tagged cDNAs, SbfI

and FseI can be used.

2

Supplementary Table S1A. Antibody reagents

Species Manufacturer Clone / catalogue number Dilutions used for IF

Dilutions used for WB

Rab3A Rabbit pAb Synaptic systems 107102 1:100 See Figure S4B for details

Rab3B Mouse mAb

Abnova H00005865-M01 1:1000 See Figure S4B for details

Rab3B Goat pAb Santa Cruz N-15 1:100 N/A Rab3C Rabbit pAb Synaptic systems 107203 1:300 See Figure S4B for

details Rab3D Mouse

pAb Novus biologicals H00009545-A01 1:100 1:100 (Fig 5Aii)

See Figure S4Bi and S4Bvii-vii for details

Pan-Rab3 Rabbit pAb Synaptic systems 107003 N/A See Figure S4B for details

Rab27A rabbit pAb By Harlan UK Ltd, in this study

See Materials and Methods

1:100 1:200-1:500

Rab27A Mouse mAb

BD Transduction Laboratories

R52320 1:100 N/A

MyRIP Goat pAb Abcam ab10149 1:200 1:500 Slp4-a Rabbit pAb Atlas Antibodies HPA001475 1:5000 1:1000 VWF Rabbit pAb DAKO A0082 1:10000 N/A VWF Sheep pAb Serotec AHP062 1:10000 N/A Proregion Chicken

pAb Previously described See References 6,7 1:250 N/A

Proregion Rabbit pAb

Previously described See References 6,7 N/A 1:5000

α-tubulin Mouse mAb

Sigma-Aldrich DM1A N/A 1:5000

Transferrin receptor

Mouse mAb

Zymed H68.4 N/A 1:500

Actin Rabbit pAb Sigma-Aldrich A5060 N/A 1:1000 GFP Mouse

mAb Roche clones 7.1 and 13.1 1:500 1:1000

RFP Rabbit pAb Abcam ab62341 N/A 1:200

Fluorophore- or horseradish peroxidase–coupled secondary Abs were from Jackson ImmunoResearch Europe (Newmarket, UK). Infrared dye secondary Ab were from LI-COR Biosciences UK Ltd (Cambridge, UK).

3

Supplementary Table S1B. Primers used for cloning and mutagenesis

Primer Sequence Rab3A-f 5’-GGGCGCGCCTGGTGGGGCCatggcatcggccacagactc-3’ Rab3A-r 5’-GCGGCCGCCTGCTCGTCCAtcagcaggcgcagtcctggtg-3’ Rab3B-f 5’-GGGCGCGCCTGGTGGGGCCatggcttcagtgacagatgg-3’ Rab3B-r 5’-GCGGCCGCCTGCTCGTCCActagcatgagcagttctgctg-3’ Rab3C-f 5’-GGGCGCGCCTGGTGGGGCCatgagacacgaagcgcccatg-3’ Rab3C-r 5’-GCGGCCGCCTGCTCGTCCActagcaggcacagttgggctg-3’ Rab3D-f 5’-GGGCGCGCC TGGTGGGGCCatggcatcagctggagacac-3’ Rab3D-r 5’-GCGGCCGCCTGCTCGTCCActagcagctgcagctgctgg-3’ MyRIP-f 5’-GGCGCCTGGTGGGGCCatggggaggaagctggacct-3’ MyRIP-r 5’-GTCGCGGCCGCCTGCTCGTCCAgtcagtacatcacagctgactcc-3’ Slp4-a-f 5’-AGAGTCTCGAGCTatgtcggagttactggac-3’ Slp4-a-r 5’-GGTGGATCCtcataaacccagctt-3’ Rab3B-T36N-f 5’-TATAGAATtccttcctcttccgctatgctgatg-3’ Rab3B-T36N-r 5’-TAT AGAATTcttgccaacactgctgttgccaatg-3’ Rab3B-Q81L-f 5’-TATAAACGTTaccggaccatcac aacagcctattac-3’ Rab3B-Q81L-r 5’-TATAAACGTTCTAGcccagctgtgtcccagatctgcag-3’ Rab3D-T36N-f 5’-TATAGAATtccttcctgttccgatacgcggacg-3’ Rab3D-T36N-r 5’-TATAGAATTcttgcccacactgctgttgcctatc-3’ Rab3D-Q81L-f 5’-TATAAACGTTaccgcaccatcaccacgg cctactac-3’ Rab3D-Q81L-r 5’-TATAAACGTTCTAGgcccgctgtgtcccagatctgcag-3’ Rab27A-T23N-f 5’-ATATGAATTCtgtactttacca atat-3’ Rab27A-T23N-r 5’-ATATGAATtcttccctacaccagagtc-3’ Rab27A-Q78L-f 5’-ATATCCGCGGGGCTCgagaggtttcgtagc-3’ Rab27A-Q78L-r 5’-ATATCCGCGgtgtcccataactgcag-3’ MCS-f 5’-gtcataagatctaggcctcgagctcaagcttc-3’ MCS-r 5’- tatgacgcggccgctggatcccgggcccgcggtacc-3’ BASN-linker-f 5’-gtacaagggcgcgcctggtggggccgcggacgagcaggc-3’ BASN-linker-r 5’-ggccgcctgctcgtccgcggccccaccaggcgcgccctt-3’ NSXFAB-linker-f 5’-ctagccctgcaggggcgcaacagaccccgggtgctggccggcctggtggaccggttatat-3’ NSXFAB-linker-r 5’-gtacatataaccggtccaccaggccggccagcacccggggtctgttgcgcccctgcaggg-3’ 3XSTOP-f 5’-ggacgagcaggcggccgctaaatagttaaga-3’ 3XSTOP-r 5’-ggcctcttaactatttagcggccgcctgctcgtccgc-3’ MYC-LIC-f 5’-ccggtcgccaccatggagcagaagctgatctcagaggaggacct-3’ MYC-LIC-r 5’-gtacaggtcctcctctgagatcagcttctgctccatggtggcga-3’ TagRFP-LIC-f 5’-TATAACCGGTCgccaccatggtgtctaagggcgaagag-3’ TagRFP-LIC-r 5’- TATAcgtacggattaagtttgtgccccagtttgc-3’

4

Supplementary Table S1C. siRNA oligos

ON-TARGETplus SMARTpool Oligo ID sequence J-008825-08 UUAAACUGCUUAUCAUUGG J-008825-07 CAAAGGAGAACAUCAGUGU J-008825-06 CUACUCAGAUCAAGACCUA

Rab3B L008825

J-008825-05 GGACACAGACCCGUCGAUG J-010822-09 GGACGAACGUGUUGUGCCU J-010822-08 UGACAUCGCCAAUCAGGAA J-010822-07 GUACUGUGGGCAUCGAUUU

Rab3D L010822

J-010822-06 GUUCAAACUGCUACUGAUA J-004667-05 CCAUAGCACUCGCAGAGAA J-004667-06 CAGGAGAGGUUUCGUAGCU J-004667-07 UCACAACAGUGGGCAUUGA

Rab27A L004667

J-004667-08 CUACGGAUCAGUUAAGUGA J-007111-09 GAACAAGGCCAGUAAACGU J-007111-10 GAGAUGAAGAGGUCCGGAA J-007111-11 UGUUGGAGGUGACCGGAAA

Slp4-a L007111

J-007111-12 GCGACUAAAGAAUGAGUUA J-013964-08 GGAAUGGUUCUACAAUAAU J-013964-07 GUACUGACACCAUGGAAUU J-013964-06 GAUAUUGAGAGCCGGAUUU

MyRIP L013964

J-013964-05 GAUGAGAUGGGCUCCGAUA

5

Supplementary Table S1D. RT-PCR and qPCR primers

Primer Sequence Amplicon (bp) RT-PCR Rab3A Rab3A[191]-f 5’-atcaccaccgcatactaccg-3’ Rab3A[191]-r 5’-cacgttctgatgacaccac-3’

191

Rab3A * Rab3A[656]-f 5’-atggcatccgccacagactcg-3’ Rab3A[656]-r 5’-gcgcagtcctggtgcggtggcac-3’

656

Rab3B Rab3B[162]-f 5’-tactcctgggacaatgcaca-3’ Rab3B[162]-r 5’-aaaggcctgccttacactga-3’

162

Rab3C Rab3C[160]-f 5’-gcagtgtggggaaaacatct-3’ Rab3C[160]-r 5’-ctagcaggcacagttgggct-3’

160

Rab3D Rab3D[195]-f 5’-tatgacatcgccaatcagga-3’ Rab3D[195]-r 5’-ggcactggcttcaaagaact-3’

195

qPCR Rab3B Rab3B[110]-f 5’-gatgacagcacaaggcaaagt-3’ Rab3B[110]-r 5’-aggagtggcagtggagttag-3’

110

Rab3D Rab3D[128]-f 5’-gaggaggtaggattgagtaacttg-3’ Rab3D[128]-r 5’-gaccccagcttcagagact-3’

128

Rab27A Rab27A[130]-f 5’-gatgcttctggacctgataatga-3’ Rab27A[130]-r 5’-tgcccctttctccttttcttc-3’

130

Slp4-a Slp4-a[109]-f 5’-atgtgcctggaactgactgt-3’ Slp4-a[109]-r 5’-ccaccacttccccattactga-3’

109

MyRIP MyRIP[93]-f 5’-gagacatcttcagtgactaccat-3’ MyRIP[93]-r 5’-ggtgcctttcctttccttagt-3’

93

*Silva et al, 2005 previously published primers

6

Figure S1A

Supplementary Figure S1A. Characterisation of rabbit polyclonal antibody raised against

recombinant full-length human Rab27A. Lysate of HEK293 cells expressing EGFP-Rab27A was

used to screen pre-immune sera and 1st, 2nd, 3rd and terminal bleeds of 2 rabbits (B2423 and

B2424) immunized with mcKLH-coupled Rab27A. Mouse monoclonal anti-GFP and a rabbit

polyclonal antibody against Rab27A (kindly provided by Dr. Miguel Seabra, Imperial College,

London, UK; asterisk in lane 2) were included for comparison. Primary antibodies were detected

with infrared (800nm) dye-coupled anti-rabbit IgG (Ai) and infrared (700nm) dye-coupled anti-

mouse IgG (Aii). Fluorescent blots were imaged using the Odyssey and the two channels are

shown in grayscale. (Aiii) Antigen displacement of anti-Rab27A antibodies from endogenous

7

Rab27A using GST-Rab27A. The membrane proteins in the detergent phase of Triton X-114-

partitioned HUVEC lysate were precipitated, subjected to SDS-PAGE, transferred onto

nitrocellulose and probed with serial dilutions (as indicated) of anti-Rab27A (B2423) antiserum

alone (-) or following preincubation with Protein-A-Sepharose (p), GST-Rab27A immobilised on

Glutathione-Sepharose (r) or empty Glutathione-Sepharose (g). Molecular weights of protein

ladder marker bands in kDa are shown on the left.

Anti-Rab27A antibody production was as follows: Rab27A cDNA (HindIII/XmaI fragment from

EGFP-Rab27A) was transferred into GEX-C3, a modification of pGEX-6P-1 (GE Healthcare,

Hertfordshire, UK) containing a pEGFP-C3 (Clontech)-compatible multiple cloning site downstream

of the PreScission protease cleavage site. Rab27A fused to the C-terminus of Glutathione-S-

transferase (GST) (separated by a PreScission protease cleavage site) was purified using

Glutathione Sepharose 4B (GE Healthcare) from IPTG-induced BL21-Star E. coli transformed with

GEX-Rab27A. The GST-tag was removed by GST-coupled PreScission protease at 4°C for 20h

and Rab27A concentrated using a Vivaspin column (MWCO 10,000). Concentrated Rab27A was

coupled to mcKLH Imject (Thermo Scientific, Loughborough, UK). Polyclonal anti-sera to mcKLH-

Rab27A was raised in rabbits (B2423 and B2424) using standard immunization protocols (Harlan

UK Ltd, Bicester, UK) and affinity purified using recombinant Rab27A immobilized on an

Aminolink™ column according to manufacturers instructions (Thermo Scientific, Loughborough).

8

Figure S1B

Supplementary Figure S1B

Loss of mRFP-Rab27A coincides with WPB fusion pore formation. (Bi) A montage of images

from a live cell video of a single WPB coexpressing Proregion-EGFP and mRFP-Rab27A during

exocytosis evoked by 1 µM ionomycin. Top panel (green in color merge), EGFP-fluorescence;

middle panel (red in color merge), mRFP fluorescence. Images were acquired at 30 frames/sec

and every 10th frame is shown. Scale bar 2 μm. (Bii) time course for dispersal of Proregion-EGFP

(black traces) and mRFP-Rab27A (gray traces). Data plot is a 3-frame average of the raw data.

9

EGFP fluorescence intensity was normalized to the peak increase in fluorescence due to fusion.

mRFP fluorescence was normalized to the mean level prior to fusion. Asterisk at peak of EGFP

fluorescence increase in (Bii) corresponds approximately to the third frame of montage in (A).

WPB fusion pore formation was signalled by an abrupt increase in EGFP fluorescence due to

alkalinization of the WPB lumen (indicated by vertical dashed line) as previously described 4,8. As

can be seen, coincident with fusion pore formation there is a morphological change in the WPB

and an abrupt decrease in mRFP-Rab27A fluorescence.

10

Figure S2

Supplementary Figure S2. Endogenous Slp4-a localizes to WPBs in HUVEC and HAEC. (A)

Low zoom image of HUVEC immunolabeled for endogenous VWF-IR (red) and Slp4-a-IR (green).

(B) Low zoom image of HAEC immunolabeled for endogenous VWF-IR (red) and Slp4-a-IR

(green). Grayscale images are from the regions indicated by the white boxes.

11

Figure S3A

Supplementary Figure S3A. Inverse relationship between intensity of WPB-Rab27A-IR and

WPB number per cell.

Low zoom field of HUVEC immunolabeled for endogenous Rab27A-IR (green; mouse Ab, see

Supplementary Table S1A) and VWF-IR (red; sheep Ab). Regions (i-iii), indicated by white boxes,

show cells with few (i), intermediate (ii) and high (iii) number of WPBs. Grayscale images of these

regions are shown surrounding the colour image. Arrowheads point to WPBs in both channels.

Images taken from a single confocal section. Bottom right, bar graph quantifies the mean WPB-

Rab27A-IR in cells with low (<50), intermediate (51-100) and high (>100) number of WPBs as

described in Materials and Methods. 12 cells in each category were analyzed using 6-8 separate

low zoom fields taken with the same confocal settings. Data are representative of two separate

experiments.

12

Figure S3B

13

Supplementary Figure S3B. Loss of WPB-Rab27A- and MyRIP- but not of Slp4-a-IR with

increasing WPB numbers per cell.

(Bi) Representative triple immunolabeling of endogenous Proregion (top), Rab27A (middle) and

MyRIP (bottom) in HUVEC with low (<50), intermediate (51-100) or high (>100) number of WPBs

as indicated. Regions indicated by the dashed boxes are shown in top right corner insets on an

expanded scale. Scale bars 10μm. (Bii) Representative double immunolabeling of endogenous

Rab27A (top) and Slp4-a (bottom) in HUVECs with indicated as above in (Bi) number of WPBs.

Regions indicated by the white boxes are shown in insets on an expanded (150%) scale. Scale

bars 20μm.

14

Figure S3C

Supplementary Figure S3C. Expression of EGFP-Rab27A results in supra-physiological

levels of Rab27A on WPB and reveals that the intensity of WPB-Rab27A-IR is proportional

to WPB-Rab27A antigen concentration

The data were derived from images of HUVEC 24 hours after mock Nucleofection or Nucleofection

with EGFP-Rab27A. The cells were fixed and stained for endogenous VWF-IR (sheep Ab) to

identify WPBs and for WPB-Rab27A-IR (mouse Ab). The mean fluorescence intensities (FI) of

WPB-Rab27A-IR and WPB-EGFP-Rab27A in expressing cells were determined as described in

Materials and Methods. The green symbol shows the intensity of endogenous WPB-Rab27A-IR in

mock-transfected cells with <50 WPBs per cell (n=11, these cells had the most intense WPB-

Rab27A-IR, suggesting the highest concentrations of this antigen). Circles, the mean WPB-EGFP

15

fluorescence for individual EGFP-Rab27A-expressing cells (n=26 cells) plotted against their mean

WPB-Rab27A-IR. Data were fitted by linear regression (red line) with no constraints on intercept

(R2 = 0.70, intercept 309±94 a.u.). The intensity of Rab27A-IR obtained by immunolabeling was

proportional to EGFP-Rab27A concentration (fluorescence) on WPBs. Thus the intensity of

Rab27A staining reports differences in antigen concentration on the organelles. The data also

show that overexpression of EGFP-Rab27A results in levels of Rab27A antigen on WPBs higher

than even the highest WPB-Rab27A-IR levels seen in mock-transfected cells (green symbol). The

non-zero intercept of the linear regression was consistent with endogenous Rab27A-IR in non-

expressing cells.

16

Figure S3D

17

Supplementary Figure S3D. Loss of WPB-Rab27A- and MyRIP- but not of Slp4-a-IR with

increasing WPB numbers per cell in HAEC.

Representative immunolabeling of endogenous VWF-IR (red) and either Rab27A-IR (i) or MyRIP-

IR (ii) (green) in HAEC with few (left), or many (right) WPBs. (iii) Representative immunolabeling

of endogenous VWF-IR (red), Slp4-a-IR (green) and Rab27A-IR (blue) in HAEC with few (left), or

many WPBs (right). All images are single confocal optical sections taken with the same

microscope settings. Regions indicated by the white boxes are shown in insets. Scale bars 10 μm.

18

Figure S3E

Supplementary Figure S3E. Loss of WPB-Rab27A- and MyRIP- but not of Slp4-a-IR after

Rab27A KD.

(Ei) Left, representative triple immunolabeling for endogenous Proregion (left), Rab27A (middle)

and MyRIP (right) in HUVEC 48 hours after nucleofection with siCTRL or siRab27A oligos as

indicated. Scale bars 10 μm. Right, quantification of the mean WPB-Rab27A- (upper plot) and

WPB-MyRIP-IR (lower plot) in individual cells triple-stained for Proregion, Rab27A and MyRIP (like

in images shown) plotted against the number of WPBs per cell. Data points represent individual

cells from siCTRL- (black; n=29 cells) or siRab27A- treated cells (gray; n=25 cells). Inset in the

plots are the average fluorescence IR counts binned for cells with low (<50), medium (51-100) and

high (>100) number of WPBs in siCTRL (black) or siRab27A- treated cells (gray). (Eii)

Representative triple immunolabeling for endogenous Proregion (left), Slp4-a (middle) and MyRIP

19

(right) in HUVEC 48 hours after nucleofection with siCTRL or siRab27A oligos as indicated.

Regions indicated by the colored boxes in Dii are shown in grayscale to the left (red box, cell with

many WPBs) or right (green box, cell with few WPBs) for each antigen. Images are from the same

field. Scale bars 10 μm. As can be seen, Rab27A KD results in loss of WPB-Rab27A-IR in cells

irrespective of the number of WPBs present. This was mirrored by a loss of WPB-MyRIP-IR (Ei-ii)

but not of WPB-Slp4-a-IR (Eii).

20

Figure S4A

21

Supplementary Figure S4A. Interaction between Slp4-a and wild-type, constitutively active

or dominant negative Rab27A (Q78L / T23N), Rab3B (Q81L / T36N) and Rab3D (Q81L / T36N).

HEK293 cells were cotransfected with mCherry-tagged full-length Slp4-a and EGFP-tagged

Rab27A, Rab3B or Rab3D, either wild-type (WT), constitutive GTP (Q78L/Q81L) or constitutive

GDP (T23N/T36N), or with cytosolic EGFP alone. (i) Sample buffer lysates, from cells expressing

these constructs (as indicated above the lanes), were separated by SDS-PAGE and Western blot

analysis was performed using rabbit anti-RFP (IB: RFP) and mouse anti-GFP Abs (IB: GFP). (ii)

Lysates were incubated with Protein-A Dynabeads coupled with sheep anti-GFP Abs (GFP) or

naïve sheep IgG (sIgG) to pull down GFP-tagged Rab proteins. Immunoprecipitates were

redissolved in sample buffer, separated by SDS-PAGE and immunoblotted using anti-RFP (IB:

RFP) and mouse anti-GFP Abs (IB: GFP). The primary rabbit Ab (RFP) was detected with Infrared

Dye-800-coupled donkey anti-rabbit IgG and the primary mouse Ab (GFP) by infrared Dye-700-

coupled donkey anti-mouse IgG. Fluorescent blots were imaged using the Odyssey, and the two

channels are shown in grayscale. Molecular weights of protein marker bands are shown on the

right.

22

Figure S4Bi

Supplementary Figure 4Bi. Validation of isoform specificity of anti-Rab3 Abs by Western

blotting and immunocytochemistry.

23

HEK293 cells were transiently transfected with mEGFP-tagged Rab3A, Rab3B, Rab3C or Rab3D.

Sample buffer lysates were separated by SDS-PAGE and transferred to nitrocellulose membranes.

Western blotting for all Rab3 isoforms and a pan-Rab3 antibody (dilutions indicated above lanes)

was performed using the Miniblotter system, which enables multiple antigens to be probed

simultaneously on a single membrane. Mouse anti-GFP was used as a control to indicate the size

of the mEGFP-Rab3 fusion proteins. Primary antibodies were detected with infrared Dye-800-

coupled anti-rabbit IgG (a) and infrared Dye-700-coupled anti-mouse IgG (b) added together.

Fluorescent blots were imaged using the Odyssey and the two channels are shown in grayscale.

Molecular weights (kDa) of protein marker bands are shown on the left.

24

Figure S4Bii-iii

25

Figure S4Biv-v

26

Supplementary Figure S4Bii-v. Expression of mEGFP-Rab3A-3D specifically labels WPBs in

HUVEC.

Left panels show high zoom (100x) confocal images of HUVEC expressing mEGFP-Rab3A (Bii),

mEGFP-Rab3B (Biii), mEGFP-Rab3C (Biv), mEGFP-Rab3D (Bv). After fixation the cells were

stained with specific Abs for Rab3A-IR (Bii-v top, middle panels), Rab3C-IR (Bii-v middle, middle

panels) and Rab3D-IR (Bii-v bottom, middle panels) as indicated. All cells were also

immunostained for VWF-IR (right panels, sheep Ab). Scale bars 10 µm.

27

Figure S4Bvi

Supplementary Figure 4Bvi. A Rab3B specific Ab recognizes expressed mEGFP-Rab3B. Left

column, colour-merged images of HUVEC expressing mEGFP-Rab3B (green) and immunolabeled

for Rab3B-IR (red; mouse Ab). Middle and right, the grayscale images as indicated. Scale bars

10 µm.

28

Figure S4C HUVEC

Figure S4D HAEC

29

Supplementary Figure S4C-D. Rab3B and 3D are localized to WPBs in HUVEC and HAEC.

Low zoom confocal images (40x) of HUVEC (C) or HAEC (D) double immunolabeled for

endogenous Rab3A-IR (Ci, Di), Rab3B-IR (Cii, Dii), Rab3C-IR (Ciii, Diii) or Rab3D-IR (Civ, Div)

(green), and all for VWF-IR (red, sheep Ab). Scale bars 50 µm. Grayscale images on expanded

scales are from the regions indicated by the white boxes.

30

Figure S4E

Supplementary Figure S4E RT-PCR and Western Blot analysis of Rab3 isoform expression

in HUVEC.

(i) RT-PCR analysis of Rab3 isoforms in HUVEC. Primer sequences and predicted product sizes

are summarized in Supplementary Table S1D. (+) HUVEC cDNA, (-) negative (H2O) control.

Products for Rab3B (162bp) and Rab3D (195bp) but not Rab3A and Rab3C were detected. (ii)

Immunoblot analysis for Rab3 isoforms in HUVEC using isoform-specific antibodies

(Supplementary Table S1A). Western blotting was performed using a Miniblotter system. Dilutions

of anti-Rab3 Abs were (1) 1:50 (2) 1:100. Note: approximately 7 times more cellular material was

required for detection of Rab3D protein compared to Rab3B. (iii) RT-PCR analysis of Rab3A

primers from Silva et al 2005 9 using mEGFP-Rab3A-3D plasmid templates or HUVEC cDNA as

indicated. (-) shows negative (no cDNA) control.

31

Figure S5

Supplementary Figure S5. Slp4-a is detectable on WPBs after siRNA KD of Rab3B.

Images of HUVEC immunolabeled for endogenous VWF-IR (left) and Slp4-a-IR (right) 48 hours

after nucleofection with siControl (top) or siRab3B-specific oligos (bottom, see Supplementary

Table S1C). Scale bars 20 µm.

32

Figure S6

33

Supplementary Figure S6

A. Endogenous Rab27A-IR in Proregion-EGFP-expressing HUVEC. Confocal images of

HUVEC 48 hours after nucleofection with Proregion-EGFP (Pro-EGFP, Left) and stained for

endogenous Rab27A-IR (right). Note the inverse relation between WPB number and WPB-

Rab27A-IR. Scale bars 10 µm.

B. Changes in cell number, total protein and levels of Proregion, Rab27A, α-tubulin and

actin content in HUVEC with time in culture.

HUVEC were seeded at 1.0x105 cells per 3.5 cm dish and cultured in HGM for 24-120 hours.

Growth media were replaced after 72 hours. At 24, 48, 72, 88, 96 and 120 hours cells were

counted (), lysates were made and total protein content determined (, upper panel). The data

shown are from 3 independent experiments performed in triplicate. Lower panel, representative

immunoblots of all proteins tested as indicated. Molecular weights of size standard bands are

indicated on the left in kDa.

34

Figure S7

35

Supplementary Figure S7. Characterization of the effect of overexpression of epitope-

tagged Rab proteins on endogenous WPB-Rab-IR.

(Ai) Expression of EGFP-Rab27A, but not of cytosilic EGFP, decreases endogenous WPB-Rab3B-IR. Mean endogenous WPB-Rab3B-IR in HUVEC expressing EGFP-Rab27A 24 hours post-Nucleofection is plotted against the mean FI of the WPB-EGFP-Rab27A in the ranges <500 a.u.(n=46 cells), 500-1000 a.u. (n=15 cells), >1000 a.u. (n=8 cells) as indicated (black circles). The Rab3B IR values were normalized to the mean value of IR in the n=19 mock-transfected cells (left open circle). Gray circles, the mean intensity of cytosolic EGFP expressed in HUVEC, also plotted against endogenous WPB-Rab3B-IR in similar intensity bands (<500 a.u., n=17 cells, 500-1000 a.u., n=3 cells, >1000 a.u., n=5 cells). All data were acquired using a wide-field microscope with an EMCCD camera and 100x lens under identical imaging conditions (see Material and Methods). (Aii) HUVEC were either mock-transfected (left column) or nucleofected with EGFP-Rab27A (center and right columns) and 24 hours later fluorescent confocal images of EGFP in the cells were obtained at identical microscope settings (middle raw). Cells were immunostained for endogenous Rab3B-IR (red, top row). Left column, mock nucleofection. Middle and right columns, EGFP-Rab27A fluorescence (green) in cells with a low (middle) or high (right) level of WPB-EGFP-Rab27A FI. Bottom row shows color-merged images. Regions indicated by the white boxes here and below are shown in insets on an expanded scale. The intensity levels for all grayscale inserts were adjusted from 0-255 to 4-120 to account for the large differences in the EGFP signal for mock-nucleofected, low and high EGFP-Rab27A-expressing cells. Scale bars 10 μm. (Bi-Bii) Expressed Rab3A or Rab3D decrease WPB-Rab27A-IR and WPB-MyRIP-IR. HUVEC double-immunostained for endogenous Rab27A-IR (red) and MyRIP-IR (blue) after mock transfection (a, top panels in Bi and Bii) or nucleofection with mEGFP-Rab3A (Bi) or mEGFP-Rab3D (Bii). Middle (b) and lower (c) panels in Bi-ii show cells expressing low or high levels of these Rabs respectively. Scale bars 10 μm. (C) Expression of Rab1A does not affect endogenous WPB-Rab3B-IR. Top, image of HUVEC stained for endogenous Rab3B-IR (red) and VWF-IR (blue) 24 hours after mock transfection. Bottom, HUVEC stained for endogenous Rab3B-IR (red) and VWF-IR (blue) 24 hours after nucleofection with YFP-Rab1A (green). Note that endogenous WPB-Rab3B-IR is readily detectable. Scale bars 20 μm. (D) Rab3D expression depletes WPB-Rab27A-IR but not WPB-Slp4-a-IR. Experiment as in Bii, but the cells were immunostained for endogenous Slp4-a-IR (red) and Rab27A-IR (blue). Scale bars 10 μm. (E) Measured by specific ELISA for EGFP (as described in 2) effect of expression of epitope-tagged Rab1A or Rab3D on histamine-evoked secretion of Proregion-EGFP from HUVEC 48 hours after nucleofection. For comparison all data were scaled to the mean response to stimulus (+) in Proregion-EGFP/Rab1A transfected cells. Data from 4 separate experiments.

36

Supplementary Video 1. EGFP-Rab27A acquisition by an

immature VWF-tdT-labeled WPB in a living HUVEC.

Example of time-lapse imaging of a single HUVEC expressing VWF-tdT (left grayscale image and

red in color merge) and EGFP-Rab27A (middle grayscale image and green in color merge).

HUVEC transfected with VWF-tdT (and EGFP-Rab27A) start making morphologically identifiable

VWF-tdT-positive WPBs by about 5-7 hours post-transfection and continue to make new

fluorescent WPBs for up to ~24 hours after nucleofection. We chose 16 hours post-nucleofection

as a convenient time point for experiments as fluorescent WPBs lacking detectable EGFP-Rab27A

could be readily identified and imaged. Images were acquired using a Perkin-Elmer ULTRAVIEW

Spinning Disk confocal equipped with PLANAPO 100x 1.45NA objective. Emitted light was

detected using a Hamamatsu ORCA-ER CCD camera (EGFP: 525±20nm; tdT: 605±25nm). The

cell was imaged in HGM (37۫°C, 5% CO2) with sequential laser excitation at 488 nm and 561 nm at

0.2 frames/sec. White arrow indicates the WPB that will acquire EGFP-Rab27A during the

experiment. Elapsed time is indicated on the video. Scale bar is 10μm. For experiments with

mEGFP-Rab3B (not shown here) a Leica TCS SP5 confocal microscope equipped with a HCX PL

APO CS 100x 1.46NA objective was used. In that cases HUVEC were imaged in HGM (37 ۫°C, 5%

CO2) with sequential laser excitation at 488 nm and 561 nm at 3 frames/sec. Emitted light was

detected using nondescan PMT detectors (GFP;497-555nm, tdT; 569-695nm).

37

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