for in utero electroporation , timed pregnant e13.5 cd1 mice were used ( the morning of the vaginal plug was e0.5 ) .
one l of purified dna ( 1 - 2g/l ) mixed with 0.005% fast green was injected in utero into the lateral ventricle and electroporated into the neocortical ventricular zone according to published protocols26 .
brains were pfa - fixed and vibratome - sectioned for analyses by confocal microscopy . in order to determine the spatial interaction between pten and myosinva
, pc12 cells were transfected with p - mcherry - mvag and/or pten - egfp - c2 , differentiated for 2 days with ngf ( 50 ng / ml ) and fixed for flim experiments to measure frster resonance energy transfer fret .
time - domain flim experiments and data analysis were performed as described previously16 . the globular domain from chick myosinva ( mvag )
was amplified by pcr ( proofstart;qiagen ) ( primers : 5-gccaccatgagtctgcagcatgagatcacc-3 and 5-gacacgtgatatgaaacccag-3 ) and cloned into pca-ires - egfpm5-grip , p - mcherry n1 ( clontech ) or p3xflag - cmv-7.1 ( sigma ) .
similarly , mouse cdna was used to clone the globular domain of myosinvb ( primers : 5- atgtccaccatcaacggcattaag-3 and ttgacttcattgagaaactccag-3 ) and myosinvc ( primers : 5-atgagtgctattaacggcatcaagc-3 and 5- ttcagtctccggagaaagccg-3 ) into p3xflag - cmv-7.1 .
site directed mutagenesis of parental flag - mvag or mvag - iresgfp vectors were performed using the quikchange ii xl mutagenesis kit ( stratagene ) according to the manufacturer s protocols .
the primers used were 5-gggtgaaaccaacagggctgcaaaacataacatccagcattgctgatga-3 and 5-tcatcagcaatgctggatgttatgttttgcagccctgttggtttcaccc-3 for the mva rkrqni mutation , and 5-gctgcacagttgttgcaagtgcaaaacataacagatgaagatgca-3 and 5-gcttctgcatcttcatctgttatgttttgcacttgcaacaactgt-3 for the mva
cortices or livers from e18 rat embryos were homogenized in lysis buffer ( 50 mm tris ph 7.5 ; 150 mm nacl ; 1% triton x-100 ; 2 m naov and 500 m naf ) containing protease inhibitors ( roche ) .
immunoprecipitations were performed overnight ( 4c ) using 5g of anti - pten antibody ( a2b1 , santa cruz ) or mouse control antibody and protein - g beads ( pierce ) .
after washing , beads were boiled in loading buffer and separated on 10% sds - polyacrylamide gels , which were fixed and stained using brilliant blue g - colloidal concentrate ( sigma ) according to manufacturer s instructions .
selected protein bands were isolated , processed and sequenced by proteome sciences plc . in short , after in gel trypsin digestion , samples were analysed by lc / ms / ms , the mass spectral data was processed into peak lists and searched against the swiss prot and ncbi non - redundant databases using mascot software ( matrix science , uk ) .
for co - immunoprecipitation experiments , hek293 cells were transfected with different constructs using lipofectamine 2000 and anti - flag m2 affinity gel ( sigma ) was used for isolating the 3xflag - mvag complex .
western blot analysis of immunoprecipitates was performed using anti - flag ( sigma ) , anti - gfp ( abcam ) or anti - pten antibodies ( santa cruz or cell signaling ) .
cortices from e15.5 dilute lethal and wildtype littermates were used to isolate total rna ( qiagen rneasy mini kit ) , which was reversed transcribed into random primed cdna ( reverse transcription kit , promega ) and served as template for all qpcr experiments .
myosinva , myosinvb , myosinvc and pten mrna levels were measured relative to gapdh , pgk1 and hprt .
primers for qpcr analysis of the different mrnas were ordered from qiagen ( quantitect primer assays ) , quantifast sybr green ( qiagen ) was used as a reporter for detection with the light cycler1.0 ( roche ) .
for in utero electroporation , timed pregnant e13.5 cd1 mice were used ( the morning of the vaginal plug was e0.5 ) .
one l of purified dna ( 1 - 2g/l ) mixed with 0.005% fast green was injected in utero into the lateral ventricle and electroporated into the neocortical ventricular zone according to published protocols26 .
in order to determine the spatial interaction between pten and myosinva , pc12 cells were transfected with p - mcherry - mvag and/or pten - egfp - c2 , differentiated for 2 days with ngf ( 50 ng / ml ) and fixed for flim experiments to measure frster resonance energy transfer fret .
the globular domain from chick myosinva ( mvag ) was amplified by pcr ( proofstart;qiagen ) ( primers : 5-gccaccatgagtctgcagcatgagatcacc-3 and 5-gacacgtgatatgaaacccag-3 ) and cloned into pca-ires - egfpm5-grip , p - mcherry n1 ( clontech ) or p3xflag - cmv-7.1 ( sigma ) .
similarly , mouse cdna was used to clone the globular domain of myosinvb ( primers : 5- atgtccaccatcaacggcattaag-3 and ttgacttcattgagaaactccag-3 ) and myosinvc ( primers : 5-atgagtgctattaacggcatcaagc-3 and 5- ttcagtctccggagaaagccg-3 ) into p3xflag - cmv-7.1 .
site directed mutagenesis of parental flag - mvag or mvag - iresgfp vectors were performed using the quikchange ii xl mutagenesis kit ( stratagene ) according to the manufacturer s protocols .
the primers used were 5-gggtgaaaccaacagggctgcaaaacataacatccagcattgctgatga-3 and 5-tcatcagcaatgctggatgttatgttttgcagccctgttggtttcaccc-3 for the mva rkrqni mutation , and 5-gctgcacagttgttgcaagtgcaaaacataacagatgaagatgca-3 and 5-gcttctgcatcttcatctgttatgttttgcacttgcaacaactgt-3 for the mva
cortices or livers from e18 rat embryos were homogenized in lysis buffer ( 50 mm tris ph 7.5 ; 150 mm nacl ; 1% triton x-100 ; 2 m naov and 500 m naf ) containing protease inhibitors ( roche ) .
immunoprecipitations were performed overnight ( 4c ) using 5g of anti - pten antibody ( a2b1 , santa cruz ) or mouse control antibody and protein - g beads ( pierce ) .
after washing , beads were boiled in loading buffer and separated on 10% sds - polyacrylamide gels , which were fixed and stained using brilliant blue g - colloidal concentrate ( sigma ) according to manufacturer s instructions .
selected protein bands were isolated , processed and sequenced by proteome sciences plc . in short , after in gel trypsin digestion , samples were analysed by lc / ms / ms , the mass spectral data was processed into peak lists and searched against the swiss prot and ncbi non - redundant databases using mascot software ( matrix science , uk ) . for co - immunoprecipitation experiments , hek293 cells were transfected with different constructs using lipofectamine 2000 and anti - flag m2 affinity gel ( sigma ) was used for isolating the 3xflag - mvag complex .
western blot analysis of immunoprecipitates was performed using anti - flag ( sigma ) , anti - gfp ( abcam ) or anti - pten antibodies ( santa cruz or cell signaling ) .
cortices from e15.5 dilute lethal and wildtype littermates were used to isolate total rna ( qiagen rneasy mini kit ) , which was reversed transcribed into random primed cdna ( reverse transcription kit , promega ) and served as template for all qpcr experiments .
myosinva , myosinvb , myosinvc and pten mrna levels were measured relative to gapdh , pgk1 and hprt .
primers for qpcr analysis of the different mrnas were ordered from qiagen ( quantitect primer assays ) , quantifast sybr green ( qiagen ) was used as a reporter for detection with the light cycler1.0 ( roche ) .
 the tumour suppressor pten can inhibit proliferation and migration as well as control cell growth in different cell types1 . 
 pten functions predominately as a lipid phsophatase , converting pi(3,4,5)p3 to pi(4,5)p2 , thereby antagonizing pi3k ( phosphoinositide 3-kinase ) and its established downstream effector pathways2 . 
 however , much is unclear concerning the mechanisms that regulate pten movement to the cell membrane necessary for pten s activity towards pi(3,4,5)p33 - 5.here we show a requirement for functional motor proteins in the control of pi3k signalling , involving a previously unknown association between pten and myosinv . 
 fret measurements revealed that pten interacts directly with myosinv , dependent on pten phosphorylation mediated by ck2 and/or gsk3 . 
 inactivation of myosinv - transport function in neurons increased cell size , which  in line with known attributes of pten - loss6 , 7 - required pi3k and mtor . 
 our data demonstrate a myosin - based transport mechanism regulating pten function , providing new insights into the signalling networks regulating cell growth . 
Methods
In utero electroporation
FRET measurements by Fluorescence Lifetime Imaging Microscopy (FLIM)
Cloning
Immunoprecipitations and tandem mass spectrometry
Real time quantitative PCR (qPCR)
Supplementary Material