phosphatidylinositol 3-kinases ( pi3ks ) are lipid kinases involved in a large set of biological processes , including membrane receptor signaling , cytoskeletal organization , and endocytic trafficking ( ghigo et  al . , 2010 ; vanhaesebroeck et  al . , mammals possess eight pi3k genes , which are divided into three classes on the basis of structural homology and substrate specificity ( class i , ii , and iii ) . all pi3ks phosphorylate the d3 position of the inositol ring of phosphatidylinositols ( ptdins ) , lipids involved in signal transduction as well as in membrane specification and dynamics ( di paolo and de camilli , 2006 ) . of the different 3-phosphorylated ptdins species , ptdins3p is the only  product that can be directly or indirectly generated by all  pi3k classes in  vivo ( jean and kiger , 2012 ) . for example , class i pi3ks ( pi3k , pi3k , pi3k , and pi3k ) produce ptdins(3,4,5)p3 that can be converted into ptdins3p by phosphatases acting on endocytic vesicles ( shin et  al . , 2005 ) . the unique member of class iii , vps34 , is responsible for a major fraction of ptdins3p produced on endocytic vesicles , where it controls the generation of autophagosomes ( backer , 2008 ) as well as docking and fusion of endosomes ( christoforidis et  al . , 1999 ) . pi3k - c2 , pi3kc2 , and pi3kc2 ) produce ptdins3p as well ( falasca et  al . , 2007 ; maffucci et  al . , 2003 ) and are involved in intracellular membrane trafficking , endocytosis , exocytosis ( falasca and maffucci , 2012 ) , and autophagy ( devereaux et  al . , 2013 ) . however , the precise function of class ii pi3k - produced ptdins3p remains partially obscure . in flies , the only class ii homolog , pi3k68d , is required for endosomal sorting from the endocytic compartment to the plasma membrane , likely via regulation of ptdins3p levels ( jean et  al . mammalian pi3k - c2 has been proposed to play a similar role in endothelial cells , where it promotes endosomal trafficking via rhoa activation and regulation of ptdins3p levels . this process is required for the targeting of vascular endothelial ( ve)-cadherin to tight junctions and consequent endothelial cell maturation and vessel integrity ( yoshioka et  al . , 2012 ) . in agreement with pi3k - c2 playing multiple roles in different membrane compartments , this lipid is crucial for clathrin - coated pit maturation and clathrin - mediated endocytosis ( posor et  al . , 2013 ) . interestingly , vesicular trafficking and metabolism of phosphorylated ptdins converge in the organization and functional maintenance of the primary cilium ( bielas et  al . , 2009 ; primary cilia are versatile organelles that , in most mammalian cells , function as motile propellers or sensorial antennas to regulate cell proliferation , polarity , differentiation , and tissue organization . primary cilia provide a separate highly regulated compartment and , for their assembly , they require influx of proteins and membranes from the cytosol ( pedersen and rosenbaum , 2008 ) . targeting of components to the cilium involves polarized trafficking of vesicles originating from the golgi and the endocytic recycling compartment ( follit et  al . , 2006 ; hsiao et  al . , 2012 ; biochemical and genetic approaches indicate that delivery and docking of secretory vesicles at the base of the cilium is regulated by a cascade of events involving the activation of specific small gtpases of the rab family ( yoshimura et  al . , 2007 ) . rab8 and its activator , rabin8 , are essential for the entry of protein cargoes into the ciliary compartment , and impaired rabin8 localization at the base of the cilium as well as defective rabin8-dependent rab8 activation reduces ciliary elongation ( das and guo , 2011 ; nachury et  al . , 2007 ) . targeting of rabin8 to the ciliary base is regulated by another rab gtpase , rab11 ( westlake et  al . , 2011 ) , a known coordinator of endosome recycling to the plasma membrane ( grant and donaldson , 2009 ) . at the ciliary base , rab11 directly associates with rabin8 and stimulates its guanine nucleotide exchange factor activity toward rab8 ( feng et  al . , 2012 ) ; consequently , loss of rab11 causes defective rab8 activation and results in impaired ciliary elongation ( kndler et  al . , 2010 ) . transport of components to and from the ciliary shaft is deeply interconnected with primary cilium function in signal transduction . for example , a well - organized and fully functional primary cilium is necessary for the cellular response to sonic hedgehog ( shh ) , a morphogen critically involved in vertebrate embryonic development ( goetz and anderson , 2010 ) . consistently , loss of proteins involved in intraflagellar transport prevents correct primary cilium formation and has a dramatic impact on shh signaling and mouse development ( huangfu and anderson , 2005 ; huangfu et  al . , 2003 ; liem et  al . , 2012 ; ocbina et  al . , 2011 ; tran et  al . , shh effectors , such as the smoothened transmembrane protein and the gli transcription factors , accumulate in cilia after pathway activation ( corbit et  al . , 2005 ; ocbina et  al . , 2011 ) . translocation of these factors to the ciliary compartment is required for downstream steps of signal transduction , and alterations of this trafficking cause impairment of shh signaling even in the presence of a fully elongated cilium ( keady et  al . , 2012 ) . genetic deletion of smo in mouse embryos completely disrupts hedgehog ( hh ) signaling , thus impairing turning , left - right axis development , and neural tube patterning and leading to early embryonic lethality ( zhang et  al . , 2001 ) . loss of the pik3c2a gene in mouse has been reported to cause early embryonic lethality , initially ascribed to defective vasculogenesis ( yoshioka et  al . , 2012 ) . here , we show that in addition to abnormal angiogenesis , the lack of pi3k - c2 causes defective primary cilium organization as well as reduced shh signaling . we report that this phenotype is linked to the ability of pi3k - c2 to control the production of ptdins3p at the endocytic recycling compartment located at the base of the primary cilium . this specific pool of ptdins3p was found to be required to activate the rab11/rab8 axis and promote smo translocation to the ciliary shaft . thus , pi3k - c2 integrates lipid signaling and rab11 activation necessary for shh signaling . to explore the intracellular distribution of pi3k - c2 , a gfp - tagged form of the protein ( gfp - pi3k - c2 ) was transfected into mouse embryonic fibroblasts ( mefs ) and localization was assessed by fluorescence microscopy . during interphase , gfp - pi3k - c2 was enriched at the perinuclear / pericentriolar area ( figure  1a ) . in g0 cells , the centrosome constitutes the cilium basal body ; thus , endogenous pi3k - c2 was stained together with a centrosomal marker ( -tubulin ) and a ciliary marker ( acetyl - tubulin ) to analyze relative positioning . this staining failed to show any signal along the ciliary shaft but underlined a specific accumulation of pi3k - c2 in vesicular structures surrounding the ciliary basal body ( figure  1b ) , a region involved in primary cilium biogenesis ( kim et  al . , 2010 ) . enrichment of the protein in proximity of the base of the primary cilium was confirmed in a cell line widely used in cilium studies : the inner medullary collecting duct 3 ( imcd3 ) cell line . downmodulation of pik3c2a through infection with specific small hairpin rna ( shrna ) sequences ( sh1 and sh2 ) severely reduced the immunofluorescence signal , thus confirming antibody specificity ( figures s1a and s1b available online ) . the pericentriolar enrichment of pi3k - c2 in primary mefs was found to colocalize with markers of the recycling compartment such as rab11 and the transferrin receptor ( tfr ) ( figure  1c , top and bottom panels , respectively ) . in further agreement , cell fractionation experiments revealed the presence of pi3k - c2 in rab11 + endosomes and not in rab7 + late endosomes ( figure  1d ) . altogether , immunofluorescence and fractionation experiments reveal enrichment of pi3k - c2 in the pericentriolar recycling endocytic compartment ( pre ) . to explore the function of pi3k - c2 in the pre , pi3k - c2-deficient ( pik3c2a ) mefs were obtained from animals genetically modified by gene targeting in the mouse germline and showing complete ablation of pi3k - c2 ( figures s2a  s2d ) . first , localization of ptdins3p , a phosphoinositide mainly involved in vesicular trafficking and produced by pi3k - c2 ( falasca et  al . , 2007 ; yoshioka et  al . , 2012 ) , was analyzed in primary mefs . interestingly , wild - type cells stained either with a ptdins3p - selective gfp - fyve fluorescent probe ( figure  2a ) or with anti - ptdins3p antibodies ( figure  s2e ) showed abundant labeling of ptdins3p around the base of the cilium . by contrast , in pik3c2a mefs , the pool of ptdins3p around the ciliary base was reduced , whereas ptdins3p detected in the rest of the cell did not show  significant changes ( figures 2a and 2b ; figure  s2e ) . a small but significant reduction in total ptdins3p cellular levels ( 21% ) was also measured by high - pressure liquid chromatography ( hplc ) analysis after metabolic labeling of starved pik3c2a mefs ( figures 2c and 2d ) , in line with the notion that a restricted pool of ptdins3p is specifically produced by pi3k - c2. the reduction of ptdins3p staining in the pericentriolar area suggested that the lack of pi3k - c2 altered the organization of the pre compartment around the ciliary base . in agreement with this hypothesis , markers of the pre such as rab11 and tfr appeared dispersed and mislocalized in pik3c2a mefs ( figures 3a and 3b , lower panels ) , whereas they were enriched at the expected pericentriolar location in wild - type cells . this was not due to a reduction of protein levels , because the total amount of rab11 and tfr in pik3c2a mefs was comparable to wild - type controls ( figures s3a and s3b ) . this effect was limited to recycling markers , because the early endosomal compartment was unaffected in cells lacking pi3k - c2 ( figure  s3c ) . to explore whether rab11 mislocalization was related to its functional state , we analyzed rab11 activity using a pull - down assay based on the ability of gtp - bound active rab11 to associate with a fragment of its effector fip3 ( eathiraj et  al . the specificity of this assay was validated using a constitutively active rab11 mutant ( rab11 q70l ) as well as rab5 mutants as negative controls ( figure  s3d ) . given that pik3c2a primary mefs scarcely proliferated in culture , rab11 pull - down assays were performed in nih 3t3 mouse fibroblasts infected with shrna sequences able to significantly knock down pi3k - c2 ( sh1 and sh2 - 3t3 ; figure  3c ) . as shown in figure  3c , reduction of pi3k - c2 expression levels significantly impaired rab11 activation . this effect was not dependent on the cell line , because the same result could be repeated in imcd3 ( sh1 and sh2-imcd3 ) and hela cells ( sh1 hela ; figure  s3e ) . overall , these data show a specific involvement of pi3k - c2 upstream of rab11 localization and activation . to better characterize the role of pi3k - c2 in the pre compartment , pik3c2a mefs were engineered to express either a kinase - inactive form of pi3k - c2 ( pi3k - c2 ) or a pi3k - c2 mutant ( pi3k - c2 ) that can produce ptdins3p , but not ptdins(3,4)p2 ( posor et  al . , 2013 ) . expression of either wild - type or pi3k - c2 mutant restored accumulation of rab11 at the pericentriolar compartment , whereas pi3k - c2 did not produce any rescue ( figure  4a ) . these experiments similarly restored tfr perinuclear localization only in the presence of the  pi3k - c2 mutant ( figure  s4 ) . these data indicate that pi3k - c2-derived ptdins3p is required to properly localize rab11 + and tfr+ recycling vesicles to the pericentriolar area . to assess the involvement of pi3k - c2 catalytic activity in triggering rab11 activation , add - back experiments were performed by transfection of shrna - resistant pi3k - c2 mutants in sh1 - 3t3 cells . although the wild - type enzyme restored rab11 activity , a kinase - dead mutant was unable to revert rab11 inactivation induced by pi3k - c2 downregulation ( figure  4b ) . similar to rescue of rab11 localization , expression of the pi3k - c2 mutant was sufficient to fully restore rab11 activity ( figure  4b ) . these results indicate that pi3k - c2 is required and sufficient for production of a pool of ptdins3p critically needed for rab11 localization and activation at the pre . at the ciliary base , active rab11 promotes a cascade of signaling events , including the activation of rab8 and its localization along the ciliary axoneme , where it controls cilium elongation ( westlake et  al . , 2011 ) . to test if rab11 mislocalization and inactivation induced by the loss of pi3k - c2 affected rab8 function , red fluorescent protein ( rfp)-tagged rab8 was transfected into wild - type and pik3c2a mefs to assess rab8 localization 24  hr poststarvation , concomitantly with primary cilium formation . although rab8 localized along the ciliary axoneme in 70% of wild - type cilia , the percentage of rab8-positive cilia was severely reduced in pik3c2a mefs , thus confirming that pi3k - c2 promotes rab8 function ( figures 5a and 5b ) . given the role of the rab11/rab8 axis in primary cilium formation ( kndler et  al . , 2010 ) , the length of primary cilia was measured in wild - type , pik3c2a , and pik3c2a mefs . ciliary length was significantly reduced in both pik3c2a and pik3c2a mefs ( figures 5c and 5d ) , with a more pronounced shortening in pik3c2a ( 60% ) compared to heterozygous mefs ( 23% ) , indicating a dose - response effect . in agreement , pik3c2a downmodulation in imcd3 cells produced a 60% and 70% reduction in  rab8-positive cilia using sh1 and sh2 , respectively ( figures s5a and s5b ) , accompanied by a 50% and 60% reduction in ciliary length ( figure  s5c ) , further demonstrating that pi3k - c2 is involved in rab11/rab8-mediated cilium elongation . next , rescue of ciliary length was attempted by transfecting pik3c2a mefs with pi3k - c2 and its mutant variants . although the add back of a wild - type pi3k - c2 completely restored ciliary length , the kinase - dead mutant did not ( figure  5e ) , indicating a crucial role for pi3k - c2 catalytic activity in ciliary elongation . more importantly , reintroduction of the pi3k - c2 mutant fully restored ciliary length ( figure  5e ) , indicating that the pi3k - c2-dependent production of ptdins3p is critical for this specific process . furthermore , to test the involvement of rab11 in this process , pik3c2a mefs were transfected with either wild - type or constitutively active rab11 . the q70l constitutively active rab11 mutant was able to rescue ciliary length in pik3c2a mefs , but neither the wild - type rab11 nor the constitutively active mutant of a different rab , rab5 ( rab5 ) , produced any effect ( figure  5f ) . given the well - established role of primary cilia in embryonic development ( goetz and anderson , 2010 ) , the embryonic - lethal phenotype induced by the loss of pi3k - c2 ( yoshioka et  al . , 2012 ) was re - evaluated in light of a cilium - related dysfunction . in line with a role in almost ubiquitous primary cilia , detection of the lacz marker inserted into the mutant allele showed ubiquitous expression in pik3c2a embryos at all developmental stages analyzed ( figure  s6a ) . similarly to what previously reported ( yoshioka et  al . , 2012 ) , homozygous inactivation of the pik3c2a locus determined evident growth defects at embryonic day 8.5 ( e8.5 ; figure  s6b ) and lethality between e10.5 and e11.5 ( table s1 ) . consistent with ciliary defects observed in cultured cells , scanning electron microscopy , as well as immunofluorescence , revealed that cilia of the ventral node were frequently bent and swollen and with a 30% and 15% shorter shaft in pik3c2a and pik3c2a embryos , respectively ( figures 6a and 6b ; figures s6c and s6d ) . thus , loss of pi3k - c2 is compatible with ciliogenesis but impairs primary cilium elongation in  vivo . analysis of pik3c2a embryos at e9.5/e10.5 showed that turning and cardiac tube looping were severely disturbed ( figures 6c and 6d ; figures s6e and s6f ; table s1 ) . furthermore , in  situ hybridization failed to detect any expression of lefty1/2 and nodal in pik3c2a embryos at the 1- to 5-somite stage , whereas these genes were correctly expressed in the ventral node and left lateral plate mesoderm of wild - type embryos at the same developmental stage ( figures 6e and 6f ; table s1 ) , indicating that loss of pi3k - c2 impairs left - right patterning . embryonic lethality and laterality defects detected in pik3c2a embryos resembled the loss of the hh signal transducer smo , which depends on membrane traffic to the primary cilium for its function . in the node of 1- to 5-somite pik3c2a embryos , expression of the shh mrna was normally detected ( figure  7a ) . however , the shh - dependent translocation of smo to the shaft of ventral node cilia was severely reduced ( figure  7b ; figure  s7a ) . similarly , the number of smo - positive cilia after hh pathway stimulation was significantly decreased in pik3c2a - silenced nih 3t3 cells compared to control cells ( figure  7c ; figure  s7b ) . a reduction of smo ciliary localization was also obtained by transfecting in wild - type cells a dominant - negative ( gfp - rab11 ) , but not a wild - type , rab11 ( figure  s7c ) . conversely , transfection of the constitutively active mutant of rab11 ( gfp - rab11 ) ( figures 7d and 7e ) , as well as the rab11-activating pi3k - c2 mutant ( figure  7e ) , normalized smo ciliary localization upon hh - pathway stimulation in pi3k - c2-deficient cells , demonstrating that the pi3k - c2/rab11 axis is required for efficient ciliary translocation of smo . this suggested that the lack of pi3k - c2 impairs one of the earliest events in hh signaling , potentially blocking downstream signaling events in the hh pathway in  vivo . in agreement , loss of pi3k - c2 was found to alter the processing of the gli3 transcription factor , which in 1- to 5-somite pik3c2a embryos was present in a significantly higher amount as a  repressor ( r ) short form than as a full - length transcriptional activator form ( figure  7f ; figure  s7d ) . furthermore , transcripts for shh downstream targets ptch1 and gli1 were reduced by 53% and 57% in pik3c2a 1- to 5-somite embryos compared to wild - type controls , whereas the hh unrelated gene fgf8 was  equally expressed in both genotypes ( figure  7 g ) . in further agreement with a reduced hh signaling , shh - mediated dorsoventral patterning of the neural tube in particular , expression of shh - dependent markers of ventral neural cell types , such as shh , foxa2 , and nkx2.2 , was absent , whereas expression of the shh - inhibited pax6 expanded to ventral cells ( figure  s7e ) . in mutant mefs , analysis of shh - induced target gene expression accordingly revealed that the upregulation of ptch1 and gli1 after 24  hr stimulation with 100  nm shh was significantly lower than that of wild - type controls ( figure  7h ) . a significant reduction in shh - mediated target gene transcription was also confirmed in sh1 - 3t3 cells ( figure  7i ) . this experimental system was next used to test the role of pi3k - c2 catalytic activity and of rab11 in shh pathway activation . expression of an shrna - resistant pi3k - c2 mutant showed a significant rescue of both gli1 and ptch1 shh - mediated transcriptional upregulation , indicating that ptdins3p produced by pi3k - c2 is critically required for shh signaling pathway activation . similarly , a constitutively active rab11 mutant , but not a wild - type rab11 , was able to fully restore sensitivity to shh ( figure  7i ) . collectively , these results establish a critical requirement for pi3k - c2-mediated production of ptdins3p for the organization of the perinuclear recycling compartment and for shh signaling , both in  vivo and in cultured cells . four distinct pi3ks ( the three class ii  ,  ,  enzymes and the class iii pi3k ) can in principle generate ptdins3p in  vivo ( vanhaesebroeck et  al . , 2010 ) , but whether each of these enzymes contributes only to a specific pool of ptdins3p with distinct intracellular function has long remained obscure . our results help to elucidate the localization and role of a distinct ptdins3p pool by demonstrating that ptdins3p selectively produced by pi3k - c2 is concentrated at the pre and promotes the correct pericentriolar localization of rab11 + vesicles as well as the activation of rab11 . this triggers the rab8-dependent pathway as well as the translocation of smo to the primary cilium , a process involved in the activation of shh signal transduction cascade . our experiments with fluorescently labeled ptdins3p probes and anti - ptdins3p antibodies as well as hplc analysis showed that in the absence of pi3k - c2 , a small , highly localized ptdins3p pool was missing . the finding that loss of pi3k - c2 causes reduced levels of ptdins3p matches what has been reported using an independently generated pi3k - c2-deficient mouse strain ( yoshioka et  al . , 2012 ) as well as silenced cells ( falasca et  al . , 2007 ) . the small but significant reduction in ptdins3p detected in the absence of pi3k - c2 was found to reflect a specific enrichment of this enzyme at the pre . this observation links pi3k - c2 to the recycling compartment and suggests that this enzyme plays a specific function at this subcellular location . the finding that pi3k - c2-derived ptdins3p controls the rab11/rab8 axis further supports this hypothesis . our results thus indicate that pi3k - c2 has multiple functions at different subcellular locations and that its role at the pre is distinct from that played , for example , at the plasma membrane ( posor et  al . , 2013 ) . pi3k - c2 is located in clathrin - coated pits , where it specifically generates ptdins(3,4)p2 . this lipid then favors snx9 recruitment , followed by dynamin association and cleavage of the membrane neck required to form a free vesicle . as a consequence , loss of pi3k - c2 causes a severe impairment of clathrin - dependent endocytosis . a pi3k - c2 mutant ( pi3k - c2 ciii ) that can only generate ptdins3p does not rescue this defect ( posor et  al . , 2013 ) but does , unexpectedly , fully restore rab11 localization and activation as well as primary cilium - dependent shh signaling . this demonstrates that the activity of pi3k - c2 at the pre is in principle independent from the endocytic defect . ( 2013 ) , our findings suggest that the substrate specificity of pi3k - c2 in  vivo may be determined in a compartment - specific manner , e.g. , by substrate availability . according to this model , ptdins serves as a preferential pi3k - c2 substrate at recycling endosomes , which likely contain little ptdinsp4 ( krauss and haucke , 2007 ) , in contrast to the plasma membrane ( hammond et  al . , however , we currently can not rule out the possibility that pi3k - c2 produces a pool of ptdins(3,4)p2 that is then rapidly converted into ptdins3p by 4p phosphatases such as inpp4b ( gewinner et  al . , 2009 ) . the inability to specifically target such phosphatases to the pre prevented us from examining this possible mechanism of action in detail . remarkably , the pool of ptdins3p generated by pi3k - c2 and associated with the pre was found to specifically control rab11 localization and activity , whereas it did not affect localization of  the endosomal marker rab5 . pi3k - c2 and rab11 were found to colocalize but could not be coimmunoprecipitated , suggesting that pi3k - c2 does not directly associate with rab11 . the loss of pi3k - c2-dependent ptdins3p generation might thus impair rab11 activation through other , possibly indirect mechanisms that require further investigation . nonetheless , our results clearly point to  an unexpected epistatic interaction placing pi3k - c2-dependent ptdins3p production upstream of rab11 activation . in the endosomal compartment , class iii pi3k - dependent ptdins3p synthesis leads to late endosome maturation ( backer , 2008 ; stein et  al . , 2003 ) , whereas our results suggest that pi3k - c2-dependent ptdins3p is involved in endosomal recycling . consistently , the drosophila homolog of class ii pi3ks , pi3k68d , produces ptdins3p required for the exit of vesicles from the endocytic compartment , sorting toward the plasma membrane , and cell protrusion formation ( jean et  al . , 2012 ; velichkova et  al . , furthermore , a previous report indicates that the loss of pi3k - c2 in endothelial cells leads to defective delivery of ve - cadherin to cell junctions , thereby causing impaired assembly of endothelial junctions and disrupted vessel integrity in pik3c2a mouse mutants ( yoshioka et  al . , 2012 ) . considering the established role of rab11 in the control of cadherin traffic ( lock and stow , 2005 ) , and in light of the results we present here , this phenotype is conceivably linked to the loss of rab11 activity , a possibility that needs further assessment . on the other hand , our data clearly show that the epistatic interaction between pi3k - c2-dependent ptdins3p production and rab11 activation at the pre is required at earlier developmental stages and is critical for rab8 and smo translocation to the plasma membrane and , ultimately , for primary cilium signaling functions . in line with this view , the phenotype of pik3c2a - null embryos indicates that pi3k - c2 is a major player in cargo delivery to the primary cilium . this is particularly evident at the ventral node , where smo did not reach the ciliary shaft . our results with the pi3k - c2 ciii and rab11q70l mutants restoring the response to shh stimulation demonstrate a critical role for rab11/pre - dependent traffic in smo translocation to the cilium . although part of the ciliary smo can derive from the endocytic recycling compartment ( kim et  al . , 2010 ) , another source is provided by lateral transport on the plasma membrane ( milenkovic et  al . , 2009 ) . given the rab11 delocalization / deactivation induced by the loss of pi3k - c2-derived ptdins3p , it is possible that the trafficking dysfunction affects the pool of smo at the plasma membrane as well . in line with this hypothesis , shh signaling was fully restored in pi3k - c2-deficient cells by either the pi3k - c2 ciii mutant or the constitutively active form of rab11 . in agreement with the profound effect on smo localization and shh signaling , embryos lacking pi3k - c2 showed a series of developmental abnormalities typically detected in the loss of hh signaling . although a previous report suggested that the loss of pi3k - c2 causes lethality because of abnormal endothelial cell function ( yoshioka et  al . , 2012 ) phenotypes detected in pik3c2a embryos do not match those detected in conditions where the primary cilium is absent that show , for example , bilateral expression of nodal and lefty and consequent randomization of situs ( huangfu et  al . , 2003 ; murcia et  al . , 2000 ; nonaka et  al . , 1998 ) as well as defective cleavage of gli3 ( huangfu and anderson , 2005 ) . consistent with this observation , the loss of pi3k - c2 did not abolish cilia , which are yet shorter and swollen , likely as a consequence of defective trafficking of ciliary components . on the contrary , phenotypes of pik3c2a embryos largely overlapped with those of embryos lacking smo or showing defective hh signal transduction activation . these embryos , for example , show smaller size , no turning , no cardiac looping , complete loss of expression of nodal and lefty , and increased gli3 cleavage ( huangfu and anderson , 2005 ; zhang et  al . , 2001 ) . these observations suggest that loss of pi3k - c2 does not affect gli3 cleavage but blocks ptdins3p as well as rab11-dependent ciliary targeting of smo , thus impairing hh signal activation . these findings place pi3k - c2 in an epistatic interaction with  the hh signaling machinery and explain a large set of the developmental abnormalities found in pik3c2a embryos . our data can not rule out that the multifaceted functions of pi3k - c2 at the plasma membrane ( posor et  al . , 2013 ) and at adherens junctions ( yoshioka et  al . nonetheless , we provide genetic evidence that pi3k - c2 crucially regulates a ptdins3p - dependent membrane traffic at the pre that acts upstream of rab11 localization / activation and promotes smo ciliary targeting and shh signaling . a lacz ( bacterial -galactosidase)-neor cassette was inserted in - frame with the atg start codon of pik3c2a via bacterial recombination . mice were backcrossed for eight generations in the c57bl/6j ; wild - type littermates from heterozygous crosses were used as controls . the experiments involving mutant and wild - type mice were carried out according to european union ( 86/609/cee , ce off j nl358 , 18 december 1986 ) and institutional animal welfare guidelines and legislation and approved by the local ethics committee . primary cultures of mefs were generated from individual e11.5 littermates by trypsinization of eviscerated embryonic bodies and expanded and maintained in dulbecco s modified eagle s medium supplemented with 10% fetal  bovine serum . mefs at 80% confluency were transfected on coverslips in 24-well plates with 0.5  g of plasmid dna using the effectene transfection reagent ( qiagen ) . for shh experiments , mefs and nih 3t3 cells were plated at near - confluent densities and serum starved for 48  hr prior to treatment to allow ciliation . rm - shh - n ( r&d systems ) stimulation lasted 24  hr . other procedures are reported in supplemental experimental procedures . plasmids containing shrna sequences ( arrest gipz lentiviral shrnamir ) against mm pik3c2a were purchased from thermo scientific and used to generate lentiviral particles to stably infect nih 3t3 and imcd3 cells as described in supplemental experimental procedures . the sh1 ( v2lmm_73461 ) target sequence was 5-ggcaagatatgttagcttt-3 , and the sh2 ( v2lmm_66190 ) target sequence was 5-caaagtttctttaactct-3. cells at early passages after infection ( second to third ) were used for experiments . sh1-resistant wild - type , kinase - inactive ( kd ) , and class iii ( ciii ) pi3kc2 were generated by creating three silent mutations in the human pegfp pi3kc2a with the quikchange site - directed mutagenesis kit ( stratagene ) using the following primer : 5-gtttaaggttggtgaagatcttcgccaggacatgttagctttacaga-3 and transfected using the effectene transfection reagent . the c - terminal portion of the rab11 effector fip3 ( rab11 binding domain ) was cloned in pgex 4t2 vector . recombinant protein was produced in bacteria ( 4  hr induction at room temperature ) , purified ( elution 10  mm glutathione , pbs ) , dialyzed , frozen in liquid nitrogen , and stocked ( 50% glycerol in tris - hcl 50  mm 5  mm mgcl2 , 100  mm nacl ) at 80c . cells were washed in ice - cold pbs and lysed in 1  ml of mlb buffer ( 25  mm hepes [ ph 7.5 ] , 150  mm nacl , 1% igepal ca-630 , 10% glycerol , 25  mm naf , 10  mm mgcl2 , 1  mm edta , 1 mm sodium orthovanadate , and protease inhibitor cocktail ) . a total of 500  g of protein extract was incubated with 30  g of recombinant protein coupled with glutathione s - transferase agarose . endogenous content of total rab11 in cell lysates was measured by loading 50  g of total extracts in a different gel followed by immunoblot and used to normalize measurements of active rab11 . for quantification analysis , pictures were taken ensuring that intensity was within the linear range and the quantity one 1-d analysis software ( bio - rad ) was used . one , two , and three asterisks in all the figures indicate significance with a p value < 0.05 , < 0.01 , and < 0.001 , respectively .
summarymultiple phosphatidylinositol ( ptdins ) 3-kinases ( pi3ks ) can produce ptdins3p to control endocytic trafficking , but whether enzyme specialization occurs in defined subcellular locations is unclear . here 
 , we report that pi3k - c2 is enriched in the pericentriolar recycling endocytic compartment ( pre ) at the base of the primary cilium , where it regulates production of a specific pool of ptdins3p . 
 loss of pi3k - c2-derived ptdins3p leads to mislocalization of pre markers such as tfr and rab11 , reduces rab11 activation , and blocks accumulation of rab8 at the primary cilium . 
 these changes in turn cause defects in primary cilium elongation , smo ciliary translocation , and sonic hedgehog ( shh ) signaling and ultimately impair embryonic development . 
 selective reconstitution of ptdins3p levels in cells lacking pi3k - c2 rescues rab11 activation , primary cilium length , and shh pathway induction . 
 thus , pi3k - c2 regulates the formation of a ptdins3p pool at the pre required for rab11 and shh pathway activation .