Short Report 3733 Binding of dynein intermediate chain 2 to paxillin controls focal adhesion dynamics and migration

Carine Rosse1,*,`, Katrina Boeckeler1, Mark Linch1, Simone Radtke1, David Frith2, Karin Barnouin2, Ali Sayed Morsi3, Majid Hafezparast3, Michael Howell4 and Peter J. Parker1,5,` 1Protein Phosphorylation Laboratory, Cancer Research UK, London Research Institute, London WC2A 3PX, UK 2Protein Analysis Laboratories, Cancer Research UK, London Research Institute, South Mimms EN6 3LD, UK 3Department of Biochemistry, School of Life Sciences, John Maynard Smith Building, University of Sussex, Brighton BN1 9RQ, UK 4High-Throughput Screening Laboratory, Cancer Research UK, London Research Institute, London WC2A 3LY, UK 5Division of Cancer Studies KCL, New Hunt’s House, Guy’s Campus, London WC2A 3LY, UK *Present address: CNRS UMR144, Institut Curie, 75248 Paris Cedex 05, France `Authors for correspondence ([email protected]; [email protected])

Accepted 7 April 2012 Journal of Cell Science 125, 3733–3738 2012. Published by The Company of Biologists Ltd doi: 10.1242/jcs.089557

Summary In migrating NRK cells, aPKCs control the dynamics of turnover of paxillin-containing focal adhesions (FA) determining migration rate. Using a proteomic approach (two-dimensional fluorescence difference gel electrophoresis), dynein intermediate chain 2 (dynein IC2) was identified as a protein that is phosphorylated inducibly during cell migration in a PKC-regulated manner. By gene silencing and co- immunoprecipitation studies, we show that dynein IC2 regulates the speed of cell migration through its interaction with paxillin. This interaction is controlled by serine 84 phosphorylation, which lies on the aPKC pathway. The evidence presented thus links aPKC control of migration to the dynein control of FA turnover through paxillin.

Key words: aPKCs, Dynein, Microtubules, Migration, Paxillin

Introduction contain distinct dynein complexes distinguished by different In order for cells to achieve efficient migration, cell adhesion and combinations of subunit isoforms and probably make possible detachment must be properly coordinated. Cells attach to the specific regulation of dynein motor activity and/or cargo binding

Journal of Cell Science extracellular matrix via focal contacts, which can mature into (Ha et al., 2008). Here we show that dynein intermediate chain 2 focal adhesions (Pollard and Borisy, 2003; Schwartz and (dynein IC2) interacts in an aPKC-regulated fashion with Horwitz, 2006; Vicente-Manzanares et al., 2007; Zaidel-Bar paxillin, controlling FA disassembly. et al., 2004). The atypical protein kinase C (PKC) isoforms (aPKCf/i) can Results and Discussion be activated through Par6/cdc42 (Yamanaka et al., 2001). Dynein IC2 is phosphorylated and required during NRK Interactions with the Par6/Par3 complex have implicated aPKC cell migration isoforms in a number of polarity (Suzuki and Ohno, 2006) and Through a 2D-DIGE screen, dynein IC2 was identified as a migratory (Nishimura and Kaibuchi, 2007; Rosse et al., 2009) phosphoprotein induced by migration in a manner sensitive to models. Recently, we demonstrated that aPKC interacts via the PKC inhibition (Fig. 1A–C). We determined the requirement for scaffold protein Kibra with the Exocyst complex, a complex of dynein IC2 in NRK cell migration by knock down of dynein IC2 eight proteins which facilitates regulated exocytosis to regions of using a smart pool (Fig. 2C); this resulted in a cell migration membrane activity (Guo and Novick, 2004; Munson and Novick, defect of ,30%, compared to the luciferase control, as assessed 2006; Rosse et al., 2009; Sugihara et al., 2002; TerBush et al., in a monolayer wound healing model (Fig. 2A,B; supplementary 1996; Zuo et al., 2006). There is a mutual dependence of aPKC material Movie 1). The defect is about 60% as assessed in and Exocyst for their behaviour in migratory cells and this is migration across porous membranes (Fig. 2D,E) using either a associated with their mutually dependent regulation of the short-interfering RNA (siRNA) smart pool or an individual delivery of signals to the leading edge of migrating cells. Key siRNA (Fig. 2C). This effect was observed without affecting the regulatory processes under the control of these local aPKC/ persistence of the cells (Fig. 2B). The effect of the depletion of Exocyst-dependent signals have been identified. In particular dynein IC2 on cell migration is also the consequence of the focal adhesion (FA) stability represents a critical migratory impairment of the dynein complex, the knock down of dynein IC2 output of the aPKC/Exocyst pathway (Rosse´ et al., 2006; Rosse triggers the depletion of the dynein heavy chain (supplementary et al., 2009). Interestingly, Par3 was shown to be associated with material Fig. S2C) without affecting the dynactin subunit p150 the dynein complex via light intermediate chain 2 (LIC2) and glued (supplementary material Fig. S2D). To address the contributes to the local regulation of microtubule (MT) dynamics specificity of siRNAs against dynein IC2 and to exclude any at NIH 3T3 cell–cell contacts and also the proper positioning of possible off-target effect of the siRNAs, we tested whether the the centrosome at the cell centre (Schmoranzer et al., 2009). Cells effects of dynein IC2 siRNAs on cell migration could be rescued Journal of Cell Science bevda diieefc ncl irto ihtedpeinof cell depletion the optimal we with migration Interestingly for cell migration. on required effect of additive indeed an speed observed is the can influencing IC2 the we GFP– migration, thus, dynein and of or specificity that material siRNA depletion GFP conclude the (supplementary validates expressed whether This The S1A). ectopically similar Fig. IC2. was was IC1 dynein dynein IC2 of dynein down endogenous knock migration normal control displayed despite to contrast cells IC1-positive in of GFP–dynein S1B, cells, consequences Fig. As material expressed. supplementary the IC1 post in GFP–dynein shown hours or directly GFP 48 with examine depletion (and IC2 transfection dynein to siRNA transfection), post hours DNA 72 NRK in used assays detected wound-healing was not of imaging is Real-time shown). and not neuronal (data cells mainly This IC1). is dynein dynein i.e. of IC2, dynein isoform of homolog closest resorted the we testing hence was to efficiency, transfection IC2 of dynein terms of targeting in expression sufficient siRNAs ectopic not the the that to note (we resistant IC2 is dynein which IC1 GFP–dynein with migration, no or migration h 3 Go migration, without h or spot [6 with unphosphorylated conditions each the six or the (B) under spot (C) phosphorylated the ( for assay. experiments cells wound NRK scratch migrating from a migration. proteins from cell of during pattern IC2 electrophoresis gel dynein dimensional of Phosphorylation 1. Fig. 3734 6h] ( h)]. (6 D oeaeo yenI2ppie rsn nmgaigcells. migrating in present peptides IC2 dynein of Coverage ) ora fCl cec 2 (16) 125 Science Cell of Journal 68 t5 at ¨6983 B , C rpsrpeetn h eno four of mean the representing Graphs ) m o h uaino h experiment the of duration the for M ( A Two- ) C h hshppieEAEALLQSMGLTTDSPIVPPPMSP phosphopeptide the IC2 of pathway. target PKCs promigratory downstream a a is on the IC2 This aPKCs dynein 2F). that all (Fig. idea does migration the cell IC2 reinforces when result of dynein inhibition of the Whereas increase depletion further the not 2F). inhibited, Fig. are aPKCs BIM1; including (using aPKCs the inhibitor except PKCs all the of inhibition the and IC2 dynein irsoyadtkn nocnieaintelclsto fthe (TIRF) of localisation fluorescence the consideration reflection into cell of taking internal during depletion and FAs total microscopy the at paxillin of using of effect rate migration the disassembly are the analysed on 2010), we aPKCs IC2 zyxin–paxillin al., dynein 2009), et the of al., (Boeckeler FAs et number Because of (Rosse the destabilisation 3D). the (Fig. IC2, for important increased dynein inhibition, FAs of aPKC of containing absence effects the we the with 2010), paxillin Consistent in and 3D). al., zyxin (Fig. on et FAs depletion FA IC2 at (Boeckeler the dynein of zyxin in effects increase the marker, analysed an another triggers of turnover. aPKCs FA content of of inhibition control aPKC the dynein its Because delivering phenocopying with in complex) action turnover, (dynein–dynactin aPKC IC2 linking FA hence and controlling IC2 down dynein knock in edge leading positive involved the a to as cells delivery is serves on RPE1 sec5 that of appears model, It depletion Fig. control. the cell material Here second S2B). (supplementary Fig. cells) a S3A,B, epithelium in very pigment are obtained results (retinal paxillin those These shown). of to not number (data similar intensity the their increased if and S2A) cells tested NRK by we down Go in Knock spots Here FAs. IC2 with controlling patches. dynein of treatment property of paxillin this or shares of IC2 aPKC dynein number against the at 2007; paxillin siRNAs increased of Kaibuchi, dynamics the sites; and controls aPKC FA (Nishimura that 2009) al., previously et Rosse described was aPKC It of turnover FA control controls the and under paxillin with interacts IC2 Dynein o hw) ec yenI2srn 4(nbl n underlined: and bold (in 84 serine IC2 EAEALLQSMGLTTDSPIVPPPMSPS Go dynein occupied with Hence treated shown). cells compared not migrating one migration or this cell cells during non-migrating the times three to 2001); just about of indicates increased phosphopeptide is quantification al., this it that of contain Label-free abundance et ion 1D). spectrometric mass (Fig. to (Vaughan site previously phosphorylation appeared described phosphopeptide also as espopoyaino eie8 fdni C uigcell were GFP–dynein during IC1-S83E GFP–dynein alone, observed IC2 and IC1-S83A GFP we dynein GFP–dynein this migration, IC1-WT, if aPKCs, of investigate influenced To of 84 S2F). phosphorylation Fig. absence serine material (supplementary the of migration In phosphorylation a generated. less in were phosphorylated serine site the was IC1) Go dynein in site equivalent eurmn o yenI1S3popoyain(n by (and phosphorylation IC1-S83 migration. during dynein S84) IC2 for dynein inference of significant indicative requirement is a behaviour have This a alone. not GFP migration with did comparison IC1-S83E in cell effect GFP–dynein and NRK IC1 dynein inhibited significantly ( IC1-S83A GFP– the constructs of around IC dynein overexpression dynein is The the S1C). for transfection Fig. 40% material around of (supplementary and alone efficiency GFP The the for cells. 80% NRK in expressed P 68-estv anr nioisaantti phosphorylation this against Antibodies manner. ¨6983-sensitive , efudrcretyi assetoer nlsso dynein of analyses spectrometry mass in recurrently found We .0;splmnaymtra i.SD,weesGFP– whereas S1D), Fig. material supplementary 0.001; , 0 seFg AB upeetr aeilFig. material supplementary 3A,B; Fig. (see 40% S ;srn 3frthe for 83 serine K; 68 (data ¨6983 ¨6983 SSK, Journal of Cell Science opee lsial soitdwt ailn oa adhesion focal paxillin, with FA the associated of components classically with To interacts complexes S2E). also IC2 Fig. aPKC dynein material if more (supplementary of determine IC2 co-immunoprecipated absence dynein paxillin with down, the strongly When knocked approach. in siRNA are a increased using aPKCs result this between paxillin in confirmed interaction We and activity. significantly the that increased IC2 implies further dynein result be This between cells. not cells; migrating interaction no could migrating non of had in interaction prominent increase nPKC) more the is This and IC2 dynein c- shown). and paxillin inhibits not (which (data BIMI effect while Go 4A,B) cells Fig. when with 40%, about from treated by recovered increased paxillin were significantly and IC2 be IC2 to dynein dynein found between was with interaction associated The was cells. more Paxillin migrating 4A,B). be paxillin (Fig. and to conditions IC2 found migratory dynein of or assessment association control interface, possible under the cell–substrate of the made at was paxillin of migration. dynamics cell during FAs in internal peripheral specifically the the on involved of is effect IC2 down, disassembly significant dynein the no that knocked shows is result there is This decreased FAs. whereas significantly IC2 40% is FAs about dynein peripheral by the for When of than rate speed disassembly FAs. disassembly the the double internal than that more is observed the FAs we peripheral supplementary the First, for 3C; 3). rate Fig. 2, FA; Movies internal material versus FA (peripheral FAs oivsiaetebsso h feto yenI2o the on IC2 dynein of effect the of basis the investigate To 68 wihihbt - -adaPKC; and n- c-, inhibits (which ¨6983 oto fpxli yaisb PCaddni C 3735 IC2 dynein and aPKC by dynamics paxillin of Control aeo hsitrcin etse o-hshrltbeand To non-phosphorylatable tested formation). might we not 84 interaction, complex does Serine this IC2 of on of phosphorylation dynein have influence assessment of what transfection determine the of that (note efficiency blot compromise western poor by detected assay, was the wound paxillin antibody and scratch an GFP with hour against immunoprecipitated three was IC2-WT a GFP–dynein with After interacted paxillin. IC2-WT established endogenous GFP–dynein first we expressed behaviour, ectopically this in that involved was on IC2 dependent dynein of is edge leading the at traffic. that aPKCs membrane showing of 2010) al., localisation et is (Osmani This the data juncture. recent this and the at action with FA aPKC consistent the with is consistent interaction of be that would endocytosis/disassembly this suggests by This 4C). controlled (Fig. probably compared control treatment DMSO paxillin inhibitor) the and to (dynamin IC Dynasore dynein on between increased regulated interaction be The could endocytosis. IC whether dynein by determined and we paxillin 2009), between al., interaction et the Ezratty 2005; al., 2001). et (Ezratty al., this et 4A); and (Vaughan Fig. studies IC2 complex; previous dynein dynactin with between the consistent to interaction is binding the (i.e. absence is Glued the as p150 in so activity dynein more aPKC and and migration of FAK with cell between during associate increased interaction to is The IC2 found 4A). indeed (Fig. and IC2 dynein analysed was (FAK) kinase odsetwehr()K oto fst-pcfcphosphorylation site-specific of control (a)PKC whether dissect To dynamin2 by mediated be could disassembly FA Because fPC sn niios[I niiigcKsadnPKCs, and cPKCs inhibiting [BIM Go inhibitors inhibition using with PKCs depletion of IC2 examine dynein of to consequences post-siRNA-transfection the h directly 72 used was assays otiig05 eu.Atr2 ,teclue eefxdand fixed were cultures the h, chambers 20 Boyden After in serum. place 0.5% were containing cells siRNAs, with transfection antibodies. ( indicated by the analysed with were immunoblotting extracts Whole-cell IC2 cells. dynein NRK of effects ( the down. cells and knock (40 experiments) migration independent of two h in 8 during persistence and speed migration natrs ( by asterisk indicated an are independent differences three significant of Statistically the means experiments. indicate the bars from as Error deviations (D) Methods. standard phalloidin and with Materials staining in by described assessed was migration cell h ape hw r ersnaieo he independent three of ( post-wounding. representative experiments. h are 8 shown is samples T8 time. The and over (T0), followed post-transfection was h healing wound 60 wounded migration. were cell siRNA indicated NRK for required is ( IC2 Dynein 2. 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NRK GFP–dynein in expressed were paxillin, mCherry–paxillin IC2 dynein with cell the in interacts where address To paxillin. with did IC2- associate IC2-S84D GFP–dynein GFP–dynein not phosphomimetic than the and paxillin 4D) with (Fig. associated WT more be to was found IC2-S84A GFP–dynein mutants. IC2 dynein phosphomimetic 3736 hr sa paetdsonc ewe nrae paxillin– increased between disconnect apparent an is There ora fCl cec 2 (16) 125 Science Cell of Journal fteFsb otoln h noyi ae yenI2was IC2 receptors dynein were transferrin paxillin of rate, accumulation and An receptors 4F). endocytic transferrin (Fig. immunostained and the cells controlling NRK turnover in by the depleted control FAs could IC2 the dynein and that of sites idea the FA further at interaction. test paxillin this To destabilizes with S84 serine that part and on phosphorylation in suggest the to least that results at recruitment These interacts for process. IC2 dynein dynamic available nor this is complex, in paxillin but a engagement in phosphorylated, neither is is which IC2 that dynein there of that pool implicit the is a to It is compartment. due FA phosphorylation this state in steady presence increased The overall components) in (recycling increase formation. consistent complex an be the and complex of would dissociation promote the IC2 both the dynein would with aspect controls this of that is phosphorylation aPKC it function subsequent and that edge its model leading the of migratory to cargo/signals this of delivery with work from evident is our it However formation. complex suppress to appears l h mgswr ae ihtesm microscope same 5 the bar: with Scale taken settings. were images the paxillin. 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F12: in inflicted DMEM–Ham’s were cultivated in Wounds cultivated were serum. were cells cells RPE1 (NRK) serum. kidney rat assay Normal wound and culture Cell Methods and Materials fdni C,dsuto ftepxli neato n hence and interaction paxillin S4). Fig. the The material supplementary of (see cells. components these disruption of the migrating recycling IC2, facilitates of phosphorylation dynein IC2 aPKC-dependent edge of dynein ensuing with and leading FAs of paxillin the endocytosis between at there of FAs interaction accumulation dynein–dynactin to the the model delivers a complex complex triggers with aPKC–Exocyst consistent the are IC2 data which The in dynein FAs. endocytosis the of at by receptor transferrin controlled depletion also the is of turnover and interaction the The with associated adhesions. is focal and fashion aPKC-regulated an in cace ntepeec rasneo h PCinhibitor aPKC the of absence Go or presence extensively the was in cells scratched NRK confluent of monolayer ltig()adqatfe sn mgJsfwr B.M, ( cells. (B). non-migrating software –, ImageJ cells; western migrating using by quantified detected and were (A) Glued blotting p150 and FAK paxillin, aPKC. and endocytosis with of colocalised ( is control and the with under interacts paxillin, IC Dynein 4. Fig. rsneo bec fteDnsr (80 Dynasore the the of in absence scratched or extensively presence was cells NRK confluent C-8Di o rsn nteFssts cl a:5 bar: Scale sites. FAs the in present not GFP–dynein is whereas IC2-S84D patches, paxillin extensively with FA more associated at is paxillin IC2-S84A with GFP–dynein associated sites, partially GFP– microscopy. is TIRF IC2-WT in by dynein imaged h were 24 Cells for cells. expressed NRK were mCherry–paxillin and -S84D ( blotting. western detected by was was paxillin IC2 endogenous GFP–dynein and and immunoprecipitated C in extensively as was scratched IC2-S84D GFP–dynein or GFP–dynein IC2-S84A IC2-WT, GFP–dynein overexpressing for cells input the of ( cell 10% immunoprecipitation. the was of panel) loading (upper The extracts panel). by (lower detected blotting was western paxillin and immunoprecipitated was hc eete nuae ihFT–rnfri.Scale FITC–transferrin. 5 with bar: incubated then were which ( A F 68 (5 ¨6983 Cer–ailnwsepesdfr2 nNKcells, NRK in h 24 for expressed was mCherry–paxillin ) , B speiul ecie (Rosse described previously As ) m m. m ) yenI a muorcpttdand immunoprecipitated was IC dynein M), E F–yenI2W r-8Aor -S84A or IC2-WT GFP–dynein ) D ooae fcnletNRK confluent of monolayer A ) ´ ta. 06,a 2006), al., et C m ooae of monolayer A ) ) yenIC2 Dynein M). m m. Journal of Cell Science io mgn etea h ntttCreCR.W r very are We Curie-CNRS. the Institut and help the their at for Centre Laboratory Microscopy Imaging Light Nikon the thank We Acknowledgements Akgu by described as Performed mass and electrophoresis spectrometry gel difference fluorescence 2010). Two-dimensional al., the et from (Boeckeler determined (max/minutes)] Metamorph. were ln rates [disassembly: using The graphs time. these time of of function slopes plotted over a and as value measured plots maximal semilogarithmic the to in was normalized were intensity adhesions fluorescence fluorescence the in disassembly, Changes individual and nm. TIRF assembly to of 107.5 adhesion to Time-lapse focal corresponds intensity dishes of pixel rates One migration. nm. the MatTek quantify 150–300 cell To of fibronectin-coated al., depth et penetration (Montagnac induce previously a in described with as to 2011) setup custom grown a wounded on performed were was were imaging lines Cells rate cell confluence. disassembly FAs NRK the of Stable analysis and Statistical microscopy 2009). Software. TIRF Prism al., with et and performed II (Rosse were Scan employed factors) Array 2 were an (ANOVA quantified, program analyses were analysis fibres Cellomics stress actin the fibres and stress spots actin paxillin and Where spots paxillin of Analysis were: sequences siRNA (Qiagen). Hyperfect with 5 nM 20 at transfected were siRNAs constructs plasmid and siRNAs 7.4), (pH sample Tris-HCl Laemmli MgCl mM hot 20 mM in 1 in plates lysed NaCl, on mM were directly 100 cells lysed immunoprecipitation, were For cells buffer. extracts, whole-cell For antibodies and immunodetection Immunoprecipitation, 8- with chambers transwell using performed were assays chamber Boyden assay D chamber 3 Boyden do to section. allows single microscope a This represents HQ2. image CoolSNAP Each Objective Camera deconvolution. Flexure CCD were Piezo a images objective and a 4D,E), immersion (Nikon) Scanner (Fig. or oil Microscope the Upright Plan-Apochromat projection 90i For with a Eclipse legend. equipped represents Applications figure Techniques image the a Cy5 Each using in and acquired laser. indicated laser HeNe HeNe as 543-nm section 633-nm a a single with a excited with a using was excited Cy3 the with laser, with acquired was equipped argon excited an was Inc.) of were 488 line Zeiss Alexa 488-nm objective. Images Carl immersion oil (LSM510, Probes). Plan-Apochromat microscope 63x/1.4 and scanning (Molecular X-100 Triton laser 1% Prolong confocal in permeabilised using paraformaldehyde, 4% mounted in fixed were Cells 2 Immunofluorescence (ANOVA Software. analysis surrounding the Prism Statistical area using readout, software. the performed Metamorph alternative in were using change factors) an time, the over As measuring wound by software. the monitored Mathematica was speed the migration to thanks performed 3738 eefxdadsandwt hlodn ahcniinwspromdi triplicate. in 10 performed a was using condition counted Each were phalloidin. Cells with cells migrating stained and and swab, fixed cotton a were with removed were cells Non-migrated chambers. nioiswr bandfo at rz(PKC Cruz Santa from (PKC obtained ImageJ.Biosciences using were performed were immunoprecipitates Antibodies the of Quantification glycerol). eeisre no2-elcluepae otiigcluemdu ih0.5% with medium culture containing plates chambers (10 culture The cells The Dickinson). 24-well serum. Becton into Inserts; inserted Culture were Cell (Biocoat membranes size UCAUAAdTdT-3 eeotie rmClice.Peaain fcl xrcsadco- and extracts (Rosse cell procedures of published to Preparations according Calbiochem. performed were from immunoprecipitations obtained were ta. 2006). al., et a oewt nIvtoe i sn h lgs CcS4 es:5 sense: IC2c-S84D oligos: the using kit Invitrogen an with done was Thermofisher. 3 tctccatccgacaagtcggtgagcacgc-3 ggtgagca-3 CcS3 es:5 sense: IC1c-S83D es:5 sense: 9 9 -GCAAGCUGCUUGUCCAUAAdTdT-3 T) Dni C-PadH-yenI2aesatposfo Dharmacon from pools smart are IC2 Hs-dynein and IC2-SP rDynein UTR). h uaeei nEF-agdmuedni Ccadmuedni IC1c dynein mouse and IC2c dynein mouse EGFP-tagged on mutagenesis The 9 -cccaacccctatgtctccctctgcgaaatcagtgag-3 9 CcS3 es:5 sense: IC1c-S83E ; ora fCl cec 2 (16) 125 Science Cell of Journal i 9 n Sc)o yatcSse Zxn.Tedfeetinhibitors different The (Zyxin). System Synaptic or rSec8) and (rPKC 6 9 -caacccctatgtctccctctgacaaatcagtgagcactcccag-3 10 4 eelae noteuprcmatet fteBoyden the of compartments upper the into loaded were ) i 1,5 -1), 2 6 . Mdtitrio,1 rtnX10 n 10% and X-100, Triton 1% dithiothreitol, mM 0.1 , 9 9 le l (Akgu al. et ¨l betv;9fed e hme eeanalysed. were chamber per fields 9 objective; CcS4 es:5 sense: IC2c-S84A ; -GCCGUAGCCUUAUCGAGUtt-3 9 -acccctatgtctccctctgagaaatcagtgagcactcc-3 9 (rPKC le l,2009). al., et ¨l 9 . f f 1,5 -1), / i ailn yenI) BD IC), dynein paxillin, , 9 -cctatgtctccatccgccaagtc- 9 -GCAAACUGCUGGU- 9 9 IC1c-S83A ; ryenIC2 (rdynein 9 -ctcctatg- m m-pore- 9 ´ ; u,X,Zag . hn,Y,Hu .C,Zo,D n u,W. Guo, and D. Zhou, C., S. Hsu, Y., Zhang, J., Zhang, B. X., Zuo, Geiger, and L. Addadi, M., Cohen, R., Zaidel-Bar, cwrz .A n owt,A R. A. Horwitz, and A. M. 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