A role of OCRL in clathrin-coated pit dynamics and uncoating revealed by studies of Lowe syndrome cells
Reversible phosphorylation of the inositol ring of phosphatidylinositol at the 3 , 4 and 5 position generates seven phosphoinositide species , which play critical regulatory roles in cell physiology via their property to control interactions at the cytosolic surface of membranes . Phosphoinositide levels are dynamically and spatially regulated by kinases and phosphatases , establishing a code of membrane identity ( Di Paolo and De Camilli , 2006; Vicinanza et al . , 2008; Balla , 2013 ) . The human genome contains 10 genes encoding inositol 5-phosphatases , a group of enzymes that dephosphorylate the inositol ring at the 5 position , nine of which act on inositol phospholipids ( Dyson et al . , 2012; Pirruccello and De Camilli , 2012 ) . Mutations in one such gene , OCRL , give rise to Oculo-Cerebro-Renal syndrome of Lowe ( Lowe syndrome ) and type 2 Dent's disease , two X-linked diseases ( Attree et al . , 1992; Hoopes et al . , 2005 ) . Lowe syndrome is characterized by congenital cataracts , renal proximal tubule dysfunction , cognitive disabilities and developmental delay ( Delleman et al . , 1977; Kenworthy and Charnas , 1995; Böckenhauer et al . , 2008 ) . Patients with Dent's disease exhibit similar proximal tubule defects but no , or only mild , additional clinical defects ( Bökenkamp et al . , 2009; Shrimpton et al . , 2009 ) . OCRL has a modular structure that is comprised of an N-terminal PH domain ( Mao et al . , 2009 ) followed in sequence by an unstructured amino acid stretch , the 5-phosphatase domain , an ASPM-SPD2-Hydin ( ASH ) domain and a catalytically-inactive RhoGAP-like domain ( Ponting , 2006; Erdmann et al . , 2007 ) . OCRL is expressed in all tissues and has a broad subcellular distribution , which is mediated by interactions with proteins that control membrane traffic and the actin cytoskeleton . These include clathrin-heavy chain , the clathrin adaptor AP-2 , Rab and Rho family GTPases and the endosomal adaptors APPL1 and Ses1/2 ( Ungewickell et al . , 2004; Choudhury et al . , 2005 , 2009; Shin et al . , 2005; Hyvola et al . , 2006; Lichter-Konecki et al . , 2006; Erdmann et al . , 2007; Fukuda et al . , 2008; Swan et al . , 2010; Dambournet et al . , 2011; Noakes et al . , 2011; Pirruccello et al . , 2011 ) . Consistent with such interactions , OCRL has been localized to the Golgi complex , to a variety of endosomal compartments as well as to clathrin-coated pits ( Olivos-Glander et al . , 1995; Dressman et al . , 2000; Ungewickell et al . , 2004; Choudhury et al . , 2005; Faucherre et al . , 2005; Hyvola et al . , 2006; Erdmann et al . , 2007 ) . INPP5B , a 5-phosphatase very similar to OCRL , has many of the same properties , but does not bind clathrin and AP-2 , and correspondingly is not localized at endocytic clathrin-coated pits ( Erdmann et al . , 2007; Williams et al . , 2007 ) . PI ( 4 , 5 ) P2 and PI ( 3 , 4 , 5 ) P3 , the two preferred substrates of OCRL , are primarily concentrated in the plasma membrane , where they help define its identity . The broad intracellular localization of OCRL may reflect its importance in preventing the build-up of ectopic intracellular pools of PI ( 4 , 5 ) P2 and PI ( 3 , 4 , 5 ) P3 . Alternatively , OCRL may act on small yet physiologically relevant pools of these phosphoinositides on intracellular membranes . The pathological manifestations of Lowe syndrome and Dent's disease are thought to arise from abnormal protein sorting and membrane recycling ( Erdmann et al . , 2007; Swan et al . , 2010; Vicinanza et al . , 2011; Mehta et al . , 2014 ) , as loss of OCRL would disrupt the appropriate coordination between membrane progression and changes in phosphoinositide composition . One open question is the physiological significance of the localization of OCRL at endocytic clathrin-coated pits . Nucleation and expansion of endocytic clathrin coats requires PI ( 4 , 5 ) P2 at the plasma membrane . PI ( 4 , 5 ) P2 represents a critical co-receptor for all the clathrin adaptors ( Di Paolo and De Camilli , 2006; Mettlen et al . , 2009; Sun et al . , 2007; Traub , 2009 ) and its levels ( controlled by dynamic turnover ) impact the lifetime of clathrin-coated pits ( Zoncu et al . , 2007; Nakatsu et al . , 2010; Antonescu et al . , 2011 ) . Conversely , coat shedding ( uncoating ) after the endocytic reaction requires PI ( 4 , 5 ) P2 dephosphorylation , as first revealed by studies of clathrin-mediated endocytosis in axon terminals ( Cremona et al . , 1999 ) . Absence of synaptojanin 1 , the most abundant 5-phosphatase in the synapse , results in a striking accumulation of clathrin-coated vesicles ( Cremona et al . , 1999; Harris et al . , 2000; Verstreken et al . , 2003; Milosevic et al . , 2011 ) . There is also evidence that PI ( 4 , 5 ) P2 dephosphorylation may assist in the fission reaction of endocytosis ( Stefan et al . , 2002; Rusk et al . , 2003; Mani et al . , 2007; Sun et al . , 2007; Liu et al . , 2009; Chang-Ileto et al . , 2011 ) . While it has been shown that OCRL has a role in preventing the accumulation of PI ( 4 , 5 ) P2 on post-endocytic membranes ( Vicinanza et al . , 2011 ) , there is no evidence to date that the absence of OCRL , or defects in OCRL function , impairs clathrin-mediated endocytosis . The initial goal of this study was to gain new insights into the function of OCRL through a global analysis of the OCRL interactome . Such an analysis corroborated evidence for a primary role of OCRL at endocytic clathrin-coated pits as it confirmed clathrin and other endocytic adaptors as OCRL binding partners . In addition , it revealed that SNX9 , a clathrin-binding protein associated with late-stage endocytic clathrin-coated pits , directly binds OCRL . Prompted by these findings , we examined the contribution of OCRL to clathrin-mediated endocytosis in Lowe syndrome patient fibroblasts . We found that the absence of OCRL in patient cells impairs endocytic clathrin coat dynamics and results in an endocytic defect . These cells accumulate clathrin-coated vesicles that fail to loose their coat . Such vesicles nucleate at least a cohort of the intracellular actin comets previously described in Lowe syndrome patient cells ( Suchy and Nussbaum , 2002; Allen , 2003; Hayes et al . , 2009; Cui et al . , 2010; Dambournet et al . , 2011; Vicinanza et al . , 2011 ) . Our findings suggest that OCRL participates in uncoating and thus facilitates the recycling of endocytic factors , a process required for the normal progression of endocytosis . Defects in clathrin-mediated endocytosis resulting from the lack of OCRL may play a role in the clinical manifestations of Lowe syndrome and Dent's disease .
The binding partners of an enzyme define its sites of action and regulatory mechanisms . Towards an improved understanding of the function of OCRL , we carried out a global analysis of its protein interactome . OCRL immunoprecipitates were analyzed through quantitative label-free interaction proteomics by mass spectrometry . This technique , in contrast to other methods such as yeast two-hybrids or pull-downs with purified proteins , allows the preferential capture of physiologically occurring protein complexes ( Vermeulen et al . , 2008 ) . To this aim , HeLa M cell lines stably expressing either GFP-OCRL or GFP alone were generated ( Figure 1A–C ) so that OCRL specific interactions could be identified by analysis of proteins selectively recovered in anti-GFP immunoprecipitates from cells expressing GFP-OCRL . While GFP alone had a diffuse cytosolic distribution , GFP-OCRL had a punctate localization characteristic of endogeneous OCRL immunoreactivity ( Figure 1A–C ) , thus validating GFP-OCRL expressing cells as an appropriate model for our experiments . Two cell lines were used for further analysis , which expressed GFP-OCRL at near endogenous levels ( 1X ) or at about five times the endogenous level ( 5X ) , as revealed by western blotting against OCRL ( Figure 1D ) . 10 . 7554/eLife . 02975 . 003Figure 1 . The OCRL interactome strongly links this protein to clathrin-dependent membrane traffic . ( A and B ) GFP fluorescence in HeLa M cells stably expressing GFP alone ( A ) or GFP-OCRL ( B ) . Scale bar: 10 µm . ( C ) Endogenous OCRL immunoreactivity in HeLa M cells . Scale bar: 10 µm . ( D ) Anti-OCRL western blot of HeLa M cells stably expressing GFP-OCRL at near endogenous ( End . ) levels ( left ) and at five times ( 5X ) the endogenous levels ( right ) . ( E ) Coomasie Blue stained SDS-PAGE of proteins immunoprecipitated from control and GFP-OCRL stably expressing HeLa M cells using magnetic beads coupled to anti-GFP antibody . ( F ) Immunoblot analysis of the material shown in ( E ) with antibodies directed against OCRL ( red ) and clathrin ( green ) , revealing the strong recovery of clathrin along with OCRL in the anti-GFP immunoprecipitates ( IP ) . ( G ) Western blot analysis for OCRL interactors identified by mass spectrometry in anti-GFP immunoprecipitates from cells expressing GFP alone or GFP-OCRL . ( H ) Domain structure of OCRL indicating binding motifs for clathrin and AP-2 . In the OCRLX3 mutant all three binding motifs are abolished ( Mao et al . , 2009 ) . ( I ) Comparative immunoprecipitation from HeLa M cells expressing GFP-OCRLWT or GFP-OCRLX3 , followed by immunoblot analysis for the indicated proteins . DOI: http://dx . doi . org/10 . 7554/eLife . 02975 . 00310 . 7554/eLife . 02975 . 004Figure 1—figure supplement 1 . OCRL interactors identified by label-free quantitative proteomics in immunoprecipitates generated from cell lines expressing GFP-OCRL approximately at endogenous levels ( 1X ) and 5-times ( 5X ) higher levels . ( A and B ) Volcano plots illustrating results of the label-free pull-downs of OCRL from HeLa cells stably expressing GFP-OCRL at 1X ( A ) or 5X levels ( B ) . The logarithmic ratio of protein intensities in the GFP-OCRL/GFP-alone pulldowns was plotted against negative logarithmic p-values of the t test performed from triplicates . A hyperbolic curve ( red dotted line ) separates specific OCRL-interacting proteins from background . Known OCRL interactors such as clathrin subunits , AP-2 subunits , Rabs and Ses1/2 , as well as novel interactors such as SNX9 , show a significant ratio in combination with high reproducibility ( positive log2 ratios ) . DOI: http://dx . doi . org/10 . 7554/eLife . 02975 . 004 Inspection of anti-GFP immunoprecipitates generated from these cells by SDS-PAGE gels prior to mass spectrometry revealed a very robust specific enrichment of GFP-OCRL in samples from GFP-OCRL expressing cells ( Figure 1E ) . As expected , a major band was detected at 170 KD ( Figure 1E ) , shown to be clathrin heavy chain by western blotting ( Figure 1F; Ungewickell et al . , 2004; Choudhury et al . , 2005 ) . In addition , there were numerous other bands selectively observed in GFP-OCRL samples . Mass spectrometry further confirmed clathrin heavy and light chain as major hits in the immunoprecipitates and revealed additional specific OCRL interactors ( Figure 1—figure supplement 1; Supplementary file 1A , B ) . These included known binding partners of OCRL , such as subunits of the endocytic clathrin adaptor AP-2 , Rab proteins and Ses1/2 but , surprisingly , not APPL1 . Possibly APPL1 was outcompeted by Ses1/2 , which bind to the same site in OCRL but with a higher affinity ( Swan et al . , 2010; Noakes et al . , 2011; Pirruccello et al . , 2011 ) . Specific interactors also included numerous proteins implicated in membrane trafficking , primarily along the endocytic pathway and vesicular transport steps between endosomes and the Golgi complex ( Supplementary file 1A , B ) . Proteins that participate in clathrin-dependent transport steps featured particularly prominently in the OCRL interactome . The enrichment of these proteins in the GFP-OCRL immunoprecipitate relative to the homogenate was further assessed by western blot analysis . In addition to clathrin , interactors such as AP-2 , EpsinR ( CLINT1 ) , SNX9 and PI3KcIIα showed the highest enrichment by western blotting ( Figure 1G ) . The cation-independent mannose 6-phosphate receptor ( CI-M6PR ) , a cargo protein for clathrin-coated pits whose transport was previously shown to be affected by OCRL knockdown ( Vicinanza et al . , 2011 ) , was also confirmed by western blotting , but was not as enriched ( Figure 1G ) . Clathrin and AP-2 are known to bind OCRL directly , while EpsinR , SNX9 and PI3KcIIα are all known clathrin interactors ( Gaidarov et al . , 2001; Lundmark and Carlsson , 2003; Mills et al . , 2003 ) . These findings raised the possibility that at least some of these proteins may bind OCRL indirectly through clathrin . In fact , a substantial overlap was observed between major OCRL interactors revealed by our experiments and proteins previously identified in a similar mass spectrometry analysis of clathrin binding partners ( Hubner et al . , 2010 ) as well as in a global analysis of clathrin-coated vesicles ( Blondeau et al . , 2004; Borner et al . , 2006 ) . Thus , to determine the contribution of clathrin to the interactions of OCRL , anti-GFP immunoprecipitations were repeated with cells expressing a GFP-OCRL construct lacking clathrin-binding sites . Anti-GFP immunoprecipitations were performed on extracts from cells expressing either GFP-OCRLWT ( wild type OCRL ) or GFP-OCRLX3 ( an OCRL mutant in which both clathrin boxes as well as the AP-2-binding motif are mutated [Mao et al . , 2009] ) ( Figure 1H ) . Western blot analysis revealed that loss of clathrin and AP-2 binding was accompanied by the loss of CI-M6PR , EpsinR and PI3KcIIα in the anti-GFP-OCRLX3 immunoprecipitates ( Figure 1I ) . In contrast , the interaction of OCRL with SNX9 , which also contains binding sites for clathrin and for AP-2 ( Lundmark and Carlsson , 2003 ) , was not abolished by the three mutations in OCRL ( Figure 1I ) . This prompted us to explore the possibility of a direct interaction between OCRL and SNX9 . SNX9 binds other proteins implicated in the late stages of clathrin-mediated endocytosis , such as dynamin , synaptojanin and N-WASP , through an N-terminal SH3 domain that recognizes proline-rich motifs with the canonical PxxP sequence ( Figure 2A; Badour et al . , 2007; Lundmark and Carlsson , 2003; Mayer , 2001; Yeow-Fong et al . , 2005 ) . OCRL contains a PxxP site ( 175REPPPPP181 ) in the predicted unfolded region that connects its N-terminal PH domain to its central inositol 5-phosphatase domain ( Figure 2A ) . Anti-GFP immunoprecipitation of cell extracts expressing GFP-OCRLWT co-enriched endogenous SNX9 , while a GFP-OCRL mutant in which three prolines ( P177 , 178 , and 181 ) of the PxxP motif were mutated to alanine ( GFP-OCRLP3 ) , failed to co-precipitate SNX9 ( Figure 2B ) . Conversely , GFP-SNX9WT but not a GFP-SNX9 construct lacking the SH3 domain ( GFP-SNX9ΔSH3 ) co-precipitated endogenous OCRL ( Figure 2C ) . Therefore , we concluded that the PxxP motif in OCRL is necessary for the interaction with SNX9 . A close paralogue of SNX9 with partially overlapping functions is SNX18 ( Park et al . , 2010 ) . Anti-GFP immunoprecipitations from cells expressing a GFP fusion of SNX18 also recovered endogenous OCRL ( Figure 2—figure supplement 1 ) . 10 . 7554/eLife . 02975 . 005Figure 2 . SNX9 is a novel direct interactor of OCRL at clathrin-coated pits . ( A ) Domain structure of OCRL and SNX9 . The location of the PxxP site in OCRL is indicated . ( B ) Western blot showing the recovery of SNX9 in immunoprecipitates generated from HEK293T cells expressing full length GFP-OCRLWT or a GFP-OCRL mutant in which the PxxP site was mutated ( GFP-OCRLP3 ) . Endogenous SNX9 was recovered only in the immunoprecipitates of GFP-OCRLWT . Actin is shown as a control . ( C ) Western blot showing the recovery of endogenous OCRL in immunoprecipitates from cells expressing GFP-SNX9WT but not from cells expressing GFP-SNX9 mutant missing the SH3 domain ( GFP-SNX9ΔSH3 ) . Note that binding of SNX9 to clathrin , which binds SNX9 outside the SH3 domain , is not lost . ( D ) ( Top ) Schematic representation of GST-OCRL constructs comprising the PH domain and portions of the predicted unstructured flanking region including ( GST-OCRL217 ) or excluding ( GST-OCRL176 ) the PxxP motif . Both constructs include the clathrin-box found inside the PH domain . ( Bottom ) GST pulldowns from a mouse brain extract using GST alone or GST fusions of OCRL as baits . Subsequent western blot analysis of the bound material shows that both constructs recover clathrin , but only GST-OCRL217 recovers SNX9 . ( E ) Confocal microscopy of wild type human fibroblasts expressing GFP-SNX9 and mCherry-OCRL . Most SNX9 spots colocalize with OCRL ( yellow arrowheads ) and nearly all SNX9 spots acquire OCRL during the acquisition . Scale bar: 2 µm . ( F ) Sequential confocal images ( 8 s apart ) of an SNX9 spot that acquires OCRL . Scale bar: 0 . 3 µm . ( G ) Average time course of fluorescence intensity for SNX9 and OCRL at individual pits . The peak of SNX9 fluorescence was defined as time zero ( mean ± SEM , n = 150 events , 10 different cells ) . DOI: http://dx . doi . org/10 . 7554/eLife . 02975 . 00510 . 7554/eLife . 02975 . 006Figure 2—figure supplement 1 . SNX18 interacts with OCRL . ( A ) Domain structure of SNX9 and SNX18 . ( B ) Percent identity of the different domains in SNX9 and SNX18 following alignment with ClustalW . ( C ) Western blot for the proteins indicated on the left , showing the recovery of endogenous OCRL in anti-GFP immunoprecipitates from cells expressing GFP-SNX18 compared to cells expressing GFP alone . Actin is shown as a loading control . Inp = input cell extract used for the immunoprecipitation . DOI: http://dx . doi . org/10 . 7554/eLife . 02975 . 006 To determine whether the PxxP motif of OCRL was sufficient to bind SNX9 , GST fusions of N-terminal fragments of OCRL comprising the PH domain and portions of its flanking unfolded region , including ( GST-OCRL217 ) or excluding ( GST-OCRL176 ) the PxxP motif , were tested in pull-down experiments from mouse brain lysates ( Figure 2D ) . Both fragments contained a previously described clathrin box ( 73LIDIA77 ) ( Mao et al . , 2009 ) and each recovered clathrin , as expected ( Figure 2D ) . However , only the OCRL217 fragment containing the PxxP motif pulled down SNX9 ( Figure 2D ) . Collectively , these results indicate that SNX9 directly binds OCRL via an interaction of its SH3 domain with the PxxP site in OCRL . A direct binding of SNX9 to OCRL is consistent with the localization of both proteins at late-stage endocytic clathrin-coated pits , as shown by the analysis of these two proteins expressed independently ( Soulet et al . , 2005; Erdmann et al . , 2007; Yarar et al . , 2007; Taylor et al . , 2011 ) . Based on a systematic analysis of the peak of endocytic protein recruitment at pits relative to the closure of their neck , Taylor et al . ( 2011 ) further reported that OCRL peaks slightly after SNX9 . To directly compare the spatiotemporal relationship between two proteins at clathrin-coated pits , time-lapse confocal microscopy was performed on human wild type fibroblasts co-expressing GFP-SNX9 and mCherry-OCRL . Inspection of these cells revealed rapidly turning over puncta of both OCRL and SNX9 uniformly distributed on the plasma membrane , as expected for late stage clathrin-coated pits ( Figure 2E ) . In the great majority of cases ( 93 . 18 ± 1 . 25%; n = 150 events , 3 independent experiments ) , the peak of OCRL fluorescence lagged slightly behind that of SNX9 , suggesting a very late function of OCRL in clathrin-mediated budding ( Figure 2E–G; Video 1 ) . 10 . 7554/eLife . 02975 . 007Video 1 . Sequential recruitment of SNX9 and OCRL at clathrin-coated pits . Wild-type skin fibroblast co-transfected with mCherry-OCRL and GFP-SNX9 showing the punctate localization of SNX9 and OCRL at clathrin-coated pits and the sequential recruitment of SNX9 followed by OCRL at the pit . DOI: http://dx . doi . org/10 . 7554/eLife . 02975 . 007 These findings support a role of OCRL in the very late stages of clathrin-mediated endocytosis . However , evidence from loss-of-function studies confirming this role is still missing . To investigate this possibility , we examined endocytic clathrin-coated pits and their dynamics in patient cells that lack OCRL . Dermal fibroblasts derived from a Lowe syndrome patient and from a control subject were used for these studies . Patient cells lack OCRL , as indeed confirmed by immunofluorescence ( Figure 3A , B ) and western blotting ( Figure 3C ) . Accordingly , a near 1 . 5-fold increase in the steady state levels of PI ( 4 , 5 ) P2 was also observed in these cells as expected ( Figure 3D; Zhang et al . , 1998; Wenk et al . , 2003 ) . 10 . 7554/eLife . 02975 . 008Figure 3 . Abnormal distribution of endocytic factors in Lowe syndrome fibroblasts . ( A and B ) Immunofluorescence staining for OCRL in control ( A ) and Lowe syndrome fibroblasts ( B ) , revealing the lack of OCRL in patient cells . Insets show higher magnifications of the boxed regions . Scale bar full size: 10 µm; inset: 1 µm . ( C ) Immunoblot analysis using a monoclonal antibody directed against the C-terminal region of human OCRL demonstrates the absence of the protein in patient cells . Actin is shown as a loading control . ( D ) High pressure liquid chromatography ( HPLC ) analysis of cell extracts showing an increase in PI ( 4 , 5 ) P2 levels in patient cells ( Student's t test , *p = 0 . 01 ) . ( E–J ) Immunofluorescence staining of control ( left ) and Lowe syndrome fibroblasts ( right ) for clathrin ( E–F ) , AP-2 ( G–H ) and SNX9 ( I–J ) revealing the enhanced punctate immunoreactivity for endocytic factors in patient cells . Insets show higher magnifications of the boxed regions . Scale bar full size: 10 µm; inset: 1 µm . ( K–L′ ) Rescue of the clathrin phenotype ( K ) and ( L ) in patient cells following expression of GFP-OCRLWT ( K′ ) but not in cells expressing the catalytically inactive GFP-OCRLD523G ( L′ ) . Insets show higher magnifications of the boxed regions . Region and inset framed in white depict cells expressing the transfected construct ( either GFP-OCRLWT ( K–K′ ) or OCRLD523G ( L–L′ ) ) . Region and inset framed in yellow show cells that did not express the GFP-tagged constructs . Scale bar full size: 10 µm; inset: 1 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 02975 . 008 Immunofluorescence for endogenous clathrin and AP-2 showed a robust increase of their punctate immunoreactivity in patient cells ( Figure 3E–H ) . An even more striking increase was observed for SNX9 puncta ( Figure 3I–J ) ( see also below ) . Additionally , clathrin-coated pits showed a clustered distribution . Exogenous overexpression of GFP-OCRLWT rescued this phenotype ( Figure 3K–K′ ) , while expression of a catalytically inactive OCRL ( GFP-OCRLD523G ) failed to do so ( Figure 3L–L′ ) . Such phenotypes were reminiscent of the accumulation of puncta positive for endocytic clathrin coat components observed in cells where the progression of late stage clathrin-coated pits to free vesicles is impaired ( Ferguson et al . , 2009; Posor et al . , 2013 ) , strongly suggesting that lack of OCRL affects the completion of the endocytic reaction . Thus far , defects in clathrin-mediated endocytosis have not been reported in Lowe syndrome fibroblasts , and therefore we next analyzed clathrin-coated pit dynamics in patient and control cells by time-lapse microscopy . The dynamics of endocytic clathrin-coated pits in human fibroblasts were assessed by monitoring the fluorescence of GFP-tagged µ2 subunit of the clathrin adaptor AP-2 ( µ2-GFP ) by live Total Internal Reflection Fluorescence ( TIRF ) microscopy , followed by an automated analysis of µ2 puncta to determine their global lifetime distributions ( Liang et al . , 2014 ) . Using expectation-maximization algorithms , a Gaussian mixture model was then fitted to each population of puncta , grouping events into three distinct cohorts: short , medium and long with mean lifetimes of 40 s ( short ) , 108 s ( medium ) and 244 s ( long ) , respectively . As expected , µ2 puncta exhibited dynamic turnover in control cells , with only a small fraction of long-lived pits ( short: 36 . 8% , medium: 46 . 6% , long: 16 . 6% ) ( Figure 4A ) . In patient cells , the turnover of clathrin-coated pits was reduced , as revealed by a significant increase in the number of long-lived pits and a reduction of short-lived ones ( short: 22 . 3% , medium: 42 . 3% , long: 35 . 4% ) ( Figure 4B ) . This phenotype was rescued by the re-expression of wild type OCRL ( short: 33 . 8% , medium: 49 . 9% , long: 16 . 3% ) ( Figure 4C ) . 10 . 7554/eLife . 02975 . 009Figure 4 . Defects in clathrin-mediated endocytosis in Lowe syndrome fibroblasts . ( A–C ) Global lifetime distribution of AP-2 subunit µ2-GFP spots ( reflecting endocytic clathrin-coated pits ) , as assessed by TIRF microscopy in control ( A ) , patient cells ( B ) and patient cells rescued with mCherry-OCRLWT ( C ) ( Ordinate represents the relative frequency spots at each time point ) . Histograms were fitted with a Gaussian mixture model identifying three subpopulations of endocytic clathrin-coated pits: short- ( mean 40 s , blue ) , medium- ( mean 108 s , green ) and long-lived ( mean 244 s , red ) . Note the increase in size of the long-lived subpopulation ( red ) in patient cells ( B ) , which is rescued by the re-expression of mCherry-OCRLWT ( C ) , ( n of events = 8657 , 9605 , and 6930 respectively; n of cells examined = 12 , 12 or 7 , respectively ) . ( D–F ) Spinning disk analysis of endocytic clathrin-coated pits in control and patient fibroblasts expressing RFP-CLC and GFP-OCRLWT or GFP-catalytically dead OCRL ( GFP-OCRLD523G ) . Representative images are shown in the left panels , kymographs in the middle panels and tracings of the average time courses of fluorescence in the right panels ( mean ± SEM , n = 45 events per condition , 3 separate experiments ) . In control cells ( D ) , there is little overlap between red ( clathrin ) and predominantly green ( OCRL ) spots , indicating OCRL recruitment when clathrin fluorescence has started to dim . The signal for OCRL then rapidly disappears as the free vesicle moves away from the imaging plane . The same pattern can be observed in patients cells rescued with OCRLWT ( F ) , while in patient cells expressing OCRLD523G , clathrin lingers for longer times even after the recruitment of OCRL , leading to a substantial overlap of red and green spots ( E ) . Scale bar full size: 2 µm; kymograph: 1 min ( red: clathrin; green: OCRL ) . ( G ) Colocalization of clathrin and OCRL fluorescence on individual spots from images such as those shown in the left fields of ( D–F ) as determined by a colocalization plugin on ImageJ ( ‘Materials and methods’ ) and reported as Pearson's correlation coefficient ( mean ± SEM , n = 7 different cells , 4 separate experiments; two tailed student's t test , ** denotes p = 0 . 0014 and *** denotes p = 0 . 0001 ) . ( H ) Representative images ( left ) and quantification ( right ) of different stages of clathrin-mediated endocytosis showing a striking increase in the number of U-shaped pits and coated vesicles per μm of cell perimeter ( n = 60 cells; two tailed student's t test , ** denotes p = 0 . 001 , *** denotes p = 0 . 0001 and **** denotes p = 0 . 00001 ) . ( I and J ) A mid-plane confocal image of control ( I ) and patient ( J ) cells showing the internalization of Alexa594-labeled transferrin ( Tf ) that occurs in 2 min . Note that in patient cells transferrin remains mostly at the cell surface . Scale bar: 10 µm . ( K ) Time course of biotinylated-transferrin internalization in control and patient cells measured by an ELISA-based assay . Uptake is represented as percent of total surface bound biotin-transferrin at 4°C . ( n = 3 experiments; two tailed student's t test , ** denotes p = 0 . 001 , and **** denotes p = 0 . 00001 ) . ( L ) Internal and surface exposed Tf receptor in control and patient cells as revealed by a biotinylation assay , demonstrating the increase of the surface pool of receptor in patient cells . DOI: http://dx . doi . org/10 . 7554/eLife . 02975 . 009 Confocal microscopy was also used to examine control and patient cells transfected with mRFP-tagged clathrin light chain ( mRFP-CLC ) and either wild type or a catalytically inactive GFP-tagged OCRL ( GFP-OCRLWT or GFP-OCRLD523G ) . In control cells , GFP-OCRLWT was recruited to clathrin spots as the clathrin fluorescence started to diminish ( Figure 4D; Video 2 ) , in agreement with previous observations ( Erdmann et al . , 2007 ) . The signal for OCRL then rapidly disappeared as the free vesicle moved away from the imaging plane . In patient cells , the dynamics of the pits were disrupted , as demonstrated by the longer persistence of the clathrin signal ( Figure 4E; Video 3 ) . GFP-OCRLWT restored normal clathrin turnover in these cells ( Figure 4F; Video 4 ) , while GFP -OCRLD523G failed to do so despite being recruited to the pits and remaining associated with them ( Figure 4E; Video 3 ) . Delayed turnover in patient cells resulted in an increase in the number of clathrin puncta ( mRFP-tagged CLC ) that were positive for GFP-OCRLD523G at any given time ( Figure 4G ) . 10 . 7554/eLife . 02975 . 010Video 2 . Clathrin and OCRL dynamics in wild type fibroblasts . Wild-type skin fibroblast co-transfected with GFP-OCRLWT and RFP-CLC showing the recruitment of OCRL to clathrin-coated pits at the end of their lifetime . DOI: http://dx . doi . org/10 . 7554/eLife . 02975 . 01010 . 7554/eLife . 02975 . 011Video 3 . Clathrin and OCRL dynamics in patient fibroblasts . Lowe syndrome patient fibroblast co-transfected with the catalytically inactive GFP-OCRLD523G and RFP-CLC showing the presence of OCRL at almost every pit and the lack of dynamic turnover of clathrin and OCRL . DOI: http://dx . doi . org/10 . 7554/eLife . 02975 . 01110 . 7554/eLife . 02975 . 012Video 4 . Expression of wild type OCRL rescues clathrin dynamics in patient fibroblasts . Lowe syndrome patient fibroblast co-transfected with the GFP-OCRLWT and RFP-CLC showing the rescue of clathrin and OCRL dynamics at the pit in the presence of exogenously expressed wild type OCRL . DOI: http://dx . doi . org/10 . 7554/eLife . 02975 . 012 To assess the stage at which endocytic clathrin-coated pits were stalled in patient fibroblasts , electron microscopy was performed ( Figure 4H ) . This analysis confirmed the global increase in the number of clathrin-coated endocytic profiles and , in particular , a prominent increase of wide-neck ( primarily U-shaped ) pits and of free clathrin-coated vesicles in the cortical region of the cell ( less than 500 nm from the plasma membrane , that is clathrin-coated vesicles most likely of endocytic origin ) ( Figure 4H ) . These results suggest that the loss of OCRL impairs uncoating , a finding consistent with its very late recruitment at pits . Its absence also delays late steps in clathrin-mediated endocytosis , possibly as a result of sequestration of endocytic factors on uncoated vesicles . Such findings prompted us to assess the potential occurrence in patient cells of a defect in the internalization of transferrin , a well established cargo of clathrin-mediated endocytosis . Confocal microscopy of cells pre-incubated with fluorescent transferrin revealed a delay in the shift of fluorescence from the cell surface to deeper positions in the cytoplasm in patient cells compared to control fibroblasts ( Figure 4I–J ) . This defect was quantified and confirmed biochemically by measuring the uptake of biotinylated transferrin at various time points during a 20-min time course ( Figure 4K ) . In addition , biotin labeling of the endogenous surface pool of the transferrin receptor showed its accumulation at the plasma membrane in patient cells compared to controls ( Figure 4L ) . From these results , we conclude OCRL is required for normal dynamics of clathrin-mediated endocytosis . A reported phenotype of Lowe syndrome patient fibroblasts is an abnormal organization of actin , with the presence of numerous actin comets ( Suchy and Nussbaum , 2002; Allen , 2003; Hayes et al . , 2009; Cui et al . , 2010; Dambournet et al . , 2011; Vicinanza et al . , 2011 ) . Accordingly , analysis of F-actin in control and patient cells using either phalloidin ( in fixed cells ) or mCherry-CHUTR ( in live cells ) ( Burkel et al . , 2007 ) revealed a reduction in the number of actin stress fibers , with the prominent accumulation of peripheral actin foci and actin comets ( also referred to as actin tails ) in patient cells ( Figure 5A–B; Video 5 ) . These comets , which are dependent on N-WASP and the Arp2/3 complex ( Figure 5C , Figure 5—figure supplement 1A–F ) , are thought to reflect the nucleation of actin on intracellular vesicles that ectopically accumulate PI ( 4 , 5 ) P2 due to the lack of OCRL ( Vicinanza et al . , 2011 ) . Consistent with this possibility , expression of GFP-OCRLWT suppressed the comets ( Figure 5—figure supplement 1G–I′ ) , while expression of catalytically-inactive GFP-OCRLD523G localized to the head of the comets but failed to rescue this phenotype ( Figure 5C , Figure 5—figure supplement 1J–J′; Video 6 ) . 10 . 7554/eLife . 02975 . 013Figure 5 . Accumulation of endocytic clathrin coat components on intracellular vesicles pushed by actin comets . ( A and B ) Fluorescence of mCherry-CHUtrophin ( calponin homology domain of Utrophin , an F-actin probe ) in control ( A ) and patient ( B ) cells revealing the loss of stress fibers and accumulation of actin tails in Lowe syndrome fibroblasts . Scale bar: 10 µm . ( C ) Spinning disk confocal images of Lowe syndrome fibroblast co-expressing mCherry-CHUtrophin and the proteins indicated , which are all at the tips of the comets . Scale bar: 1 µm . ( D ) Quantification of the percentage of comets positive for the indicated proteins at their tip ( n = 500 comets , 5 different cells ) . ( E ) Immunofluorescence staining of patient cells for phalloidin ( an F-actin marker ) and clathrin or SNX9 , showing the endogenous presence of these proteins the tips of the comets . Scale bar: 1 µm . ( F and G ) Western blot analysis of control and patient cells for the indicated proteins showing increased levels of SNX9 in patient cells . Quantification of SNX9 is shown in ( G ) ( mean ± SEM , n = 4 experiments ) . ( H and I ) Total particulate [P] and cytosolic [C] fractions derived from control or patient cell homogenates [H] followed by immunoblot analysis ( H ) show a virtual disappearance of the cytosolic pool of unassembled clathrin in patient cells ( I ) . ( [H]: homogenate; [P]: particulate; [C]: cytosolic ) . DOI: http://dx . doi . org/10 . 7554/eLife . 02975 . 01310 . 7554/eLife . 02975 . 014Figure 5—figure supplement 1 . Perturbation of the actin cytoskeleton in patient cells . ( A and B ) Immunofluorescence of the endogenous p34 subunit of the Arp2/3 complex in control ( A ) or patient fibroblasts ( B ) . Scale bar full size: 10 µm; inset: 5 µm . ( C–F ) Time-lapse confocal microscopy of a patient fibroblast transfected with mCherry-CHUtrophin before ( E ) and after ( F ) the application the Arp2/3 inhibitor CK-666 , as well as 1 min ( G ) and 7 min ( H ) after washing out the inhibitor . ( G–H′ ) Confocal microscopy images of a control ( G ) and a patient ( H ) fibroblast ( B ) transfected with mCherry-CHUtrophin but no GFP construct . ( I–J′ ) Patient fibroblasts co-transfected with mCherry-CHUtrophin and GFP-OCRLWT ( I and I′ ) or catalytic dead GFP-OCRLD523G ( J and J′ ) showing that OCRLWT , but not GFP-OCRLD523G can rescue the comets observed in patient cells . ( K and L ) Patient cells co-transfected with mCherry-CHUtrophin and either Rab5 or 2X-FYVEHRS showing that neither Rab5 nor FYVEHRS ( a PI3P reporter ) are present at comet heads . DOI: http://dx . doi . org/10 . 7554/eLife . 02975 . 01410 . 7554/eLife . 02975 . 015Figure 5—figure supplement 2 . SNX9 knockdown in Lowe syndrome fibroblasts . ( A ) Western blot analysis of patient and control cells that were treated with either siRNA targeting SNX9 and SNX18 or a negative control siRNA . Duplicates of each set of siRNA treated cells were immunobloted . Tubulin is included as a loading control . Note the efficient knockdown of SNX9 , which is present at higher levels in patient cells under basal conditions . ( B and C ) Immunofluorescence staining for SNX9 and phalloidin ( F-actin ) in patient cells treated with negative control siRNA ( B ) or with siRNAs targeting SNX9 and SNX18 , revealing the loss of comets in cells depleted of SNX9 ( cells 1 and 2 , yellow numbers ) but not in a cell still expressing significant levels of SNX9 ( cell 3 ) . Examples of comets are shown with yellow arrowheads . Inset shows an actin comet capped by SNX9 that remains after SNX9knockdown . In these cells , most of the SNX9 left was found at the tips of comets . ( D ) Confocal microscopy images of patient fibroblasts expressing mRFP-CLC and GFP-OCRLP3 , showing the localization of this SNX9-binding mutant OCRL construct to clathrin-coated pits ( blue arrowheads ) . Scale bar: 2 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 02975 . 01510 . 7554/eLife . 02975 . 016Video 5 . Comets in Lowe syndrome patient fibroblasts . Control and Lowe syndrome patient fibroblast transfected with the F-actin probe mCherry-CHUtrophin . The control cell shows the characteristic stress fibers and small actin puncta typically associated with sites of clathrin-mediated endocytosis . Stress fibers are lost in patient cells , which instead exhibit actin comets that rocket through the cytosol . DOI: http://dx . doi . org/10 . 7554/eLife . 02975 . 01610 . 7554/eLife . 02975 . 017Video 6 . Catalytically inactive OCRL localizes to the tips of actin comets . Lowe syndrome patient fibroblast co-transfected with the F-actin probe mCherry-CHUtrophin and the catalytic dead GFP-OCRLD523G showing that the machinery necessary to recruit OCRL is present on the vesicles at the tip of the comets . DOI: http://dx . doi . org/10 . 7554/eLife . 02975 . 017 Further analysis of proteins at the head of the comets revealed the presence of late-stage endocytic clathrin coat components and their accessory factors , including clathrin , AP-2 , SNX9 , SNX18 , and PI3KcIIα , as well as transferrin , a known cargo of clathrin-mediated endocytosis ( Figure 5C; Video 7 ) . Nearly every actin tail propelled vesicles positive for these proteins ( Figure 5D ) . This was also the case for endogenous proteins , as revealed by the presence of SNX9 and clathrin light chain immunoreactivity at the tips of comets labeled by phalloidin ( an F-actin marker ) ( Figure 5E ) . The tips of these comets however , were mostly devoid of endosomal markers , such as Rab5 or the FYVE domain of HRS ( Figure 5—figure supplement 1K–L ) . 10 . 7554/eLife . 02975 . 018Video 7 . SNX9 is found on vesicles pushed by actin comets . Lowe syndrome patient fibroblast co-transfected with the F-actin probe mCherry-CHUtrophin and GFP-SNX9 revealing the presence of this endocytic factor on the vesicles at the tip of the comets . DOI: http://dx . doi . org/10 . 7554/eLife . 02975 . 018 The levels of SNX9 were strikingly increased in patient cells ( Figure 5F–G ) , possibly due to its accumulation and thus sequestration at comet tips . This may also explain the great increase of SNX9 punctate immunoreactivity in patient cells previously mentioned ( Figure 3I–J ) . In contrast , the overall cellular levels of clathrin and AP-2 were similar in control and patient cells ( Figure 5F ) . However , an increase in the pool of assembled clathrin coats in patient cells was demonstrated biochemically ( Figure 5H–I ) . Homogenization of cells under conditions known to preserve assembled clathrin , followed by high-speed centrifugation to yield cytosol ( C ) and total particulate ( P ) fractions , revealed an increased fraction of clathrin heavy chain in the particulate fraction of patient cells ( Figure 5H–I ) . Thus , the coat and cargo of the intracellular actin-propelled vesicles are very similar to those of endocytic clathrin-coated pits . This supports the idea that vesicles at comet tips are endocytic clathrin-coated vesicles whose coat failed to shed due to the lack of OCRL catalytic activity . A small subset of vesicles driving the comets were larger than would be expected from endocytic clathrin-coated vesicles and may represent micropinosomes . Given the striking increase of SNX9 levels in patient cells and its known role in coordinating clathrin-coated pits maturation with actin nucleation ( Yarar et al . , 2007 ) , we explored its importance in the formation of actin tails . Knockdown of SNX9 followed by immunostaining with phalloidin revealed a decrease in the number of endogenous comets observed in patient cells ( Figure 5—figure supplement 2A–C ) . There was noticeable cell-to-cell variability however , roughly correlating with residual SNX9 levels in knockdown cells ( generally higher in patient cells given the elevated basal level of this protein ) . Importantly , most of the remaining SNX9 puncta observed in cells after knockdown associated with the tips of the comets ( Figure 5—figure supplement 2C ) . These results suggest that the interaction between SNX9 and OCRL may help coordinate actin polymerization at late stage clathrin-coated pits with the turnover of PI ( 4 , 5 ) P2 . The recruitment of OCRL to clathrin-coated pits was not dependent on its interaction with SNX9 , however . This was evidenced by the expression of an SNX9-binding mutant construct of OCRL ( GFP-OCRLP3 ) that still localized to clathrin-coated pits in patient cells ( Figure 5—figure supplement 2D ) . Given the interactions of OCRL with several prominent endocytic factors such as clathrin and AP-2 , it is highly likely that multiple binding partners regulate its recruitment to clathrin-coated pits . If actin comets are in fact nucleated by plasma membrane-derived PI ( 4 , 5 ) P2 still present on uncoated endocytic vesicles due to the absence of OCRL , depletion of plasma membrane PI ( 4 , 5 ) P2 should result in their progressive disappearance . Alternatively , if comets are generated by the synthesis of PI ( 4 , 5 ) P2 on intracellular structures , depletion of plasma membrane PI ( 4 , 5 ) P2 would not affect them . In order to discern between these two scenarios , we selectively depleted PI ( 4 , 5 ) P2 at the plasma membrane via the activation of a PLC-coupled receptor , the M1 muscarinic acetylcholine receptor ( M1R ) . Patient cells co-expressing untagged M1R , the PI ( 4 , 5 ) P2 probe GFP-PHPLCδ and the F-actin reporter mCherry-CHUTR were analyzed by dual-color time-lapse confocal microscopy while an agonist ( OxoM ) , and subsequently an antagonist ( atropine ) , were added during live acquisition . Prior to OxoM addition , GFP-PHPLCδ was selectively localized at the plasma membrane , as typically observed for this PI ( 4 , 5 ) P2 biosensor in resting cells ( Figure 6A ) . The addition of OxoM triggered PLC-mediated PI ( 4 , 5 ) P2 hydrolysis into IP3 and diacyl glycerol , which resulted in the relocation of GFP-PHPLCδ to the cytosol , indicating PI ( 4 , 5 ) P2 depletion at the plasma membrane ( Figure 6A ) . This change correlated with a transient increase of cytosolic Ca2+ , as expected ( Figure 6C; Horowitz et al . , 2005 ) . In turn , this led to a massive transient depolymerization of actin , including actin comets , and a sharp increase in the diffuse cytosolic fluorescence of the actin reporter , mCherry-CHUTR ( Figure 6B–D ) . Subsequently , as Ca2+ levels reverted to the baseline level ( Figure 6C ) , actin comets reformed ( Figure 6B–D ) , often reemerging at sites where they had previously disappeared ( Figure 6—figure supplement 1 ) . This suggested that vesicles driving the comets had retained actin-nucleating properties during the transient actin depolymerization . 10 . 7554/eLife . 02975 . 019Figure 6 . Vesicles propelled by actin tails originate from the plasma membrane . Patient fibroblasts co-expressing the PI ( 4 , 5 ) P2 probe GFP-PHPLCδ , mCherry-CHUtrophin and untagged muscarinic ( M1R ) receptor . ( A and B ) Sequential confocal images of GFP-PHPLCδ and mCherry-CHUtrophin fluorescence ( fluorescence signal is reversed to appear dark ) . 10 µM OxoM was added at time zero and 50 µM atropine ( an M1R antagonist ) 344 s later . The PI ( 4 , 5 ) P2 reporter ( GFP-PHPLCδ ) shifts reversibly from the cell periphery to the cytosol in response to OxoM . mCherry-CHUtrophin foci represent actin comets that transiently disassemble during the Ca2+ peak , reassemble , and then gradually disappear as PI ( 4 , 5 ) P2 is depleted leading to impaired endocytosis . Scale bar: 10 µm . ( C ) Fluorescence of GCaMP3 , a Ca2+ sensor , showing a transient spike in cytosolic Ca2+ upon plasma membrane PI ( 4 , 5 ) P2 hydrolysis in response to the M1R agonist OxoM , which triggers PLC activity . ( D ) Quantification of the number of comets from thresholded mCherry-CHUtrophin fluorescence during the application of OxoM and atropine to patient cells ( mean ± SEM , n = 5 experiments ) . Encircled numbers correspond to the different five stages represented in ( B ) . DOI: http://dx . doi . org/10 . 7554/eLife . 02975 . 01910 . 7554/eLife . 02975 . 020Figure 6—figure supplement 1 . Comets reemerge from the same sites following transient actin depolymerization . ( A–C ) Frames from a patient cell co-expressing mCherry-CHUtrophin and untagged M1R and exposed to OxoM at time zero . Frames were taken 12 s before the peak of actin depolymerization induced by the application of OxoM ( A ) , at the peak ( B ) and 12 s after ( C ) the peak . ( D ) Merge of the frames shown in ( A ) and ( C ) revealing that actin foci reemerge in the same place ( white arrowhead ) or in extremely close proximity ( yellow arrowhead ) to where comets were located before actin depolymerization . This indicates that the nucleating vesicles are still present and do not move significant distances during the Ca2+-induced actin depolymerization , and are capable of re-nucleating actin comets once Ca2+ levels in the cytosol return to baseline . DOI: http://dx . doi . org/10 . 7554/eLife . 02975 . 020 In the continued presence of OxoM ( conditions where plasma membrane PI ( 4 , 5 ) P2 remains depleted ) , reappearance of the comets after the Ca2+-induced actin depolymerization was followed by their progressive loss ( Figure 6B , D; Video 8 ) . Termination of the OxoM signal by addition of its antagonist , atropine , led to the rapid reformation of PI ( 4 , 5 ) P2 at the plasma membrane ( Figure 6A ) and to a striking progressive recovery of the number of new comets rocketing throughout the cytosol ( Figure 6B , D; Video 8 ) . These results are consistent with the idea that the bulk of the vesicles found at the tip of actin comets are newly formed endocytic vesicles originating from the plasma membrane . 10 . 7554/eLife . 02975 . 021Video 8 . Selective depletion of plasma membrane PI ( 4 , 5 ) P2 abolishes the comets . Lowe syndrome patient fibroblast co-transfected with the F-actin probe mCherry-CHUtrophin , GFP-PHPLCδ ( not shown ) and the untagged muscarinic receptor M1R . The cell is treated with 10 µM OxoM at 1m44s , which stays in the imaging media for the duration of the acquisition , followed by 50 µM atropine at 7m24s . Upon the addition of OxoM there's a rapid depolymerization of F-actin due to a rise in cytosolic calcium followed by the rapid reformation of the comets . Upon the continued presence of OxoM however , the comets gradually disappear due to the continued depletion of plasma membrane PI ( 4 , 5 ) P2 . This effect is reversed upon the addition of the antagonist Atropine , which allows the reformation of PI ( 4 , 5 ) P2 at the plasma membrane , leading to the resurgence of the actin comets . DOI: http://dx . doi . org/10 . 7554/eLife . 02975 . 021 The observations described thus far suggest a scenario in which the lack of OCRL-mediated PI ( 4 , 5 ) P2 hydrolysis results in a delay of endocytosis and impaired uncoating of endocytic clathrin-coated vesicles . They further suggest that coated PI ( 4 , 5 ) P2-rich vesicles drive actin nucleation . This hypothesis , however , contrasts with the very infrequent detection of the PI ( 4 , 5 ) P2 probe , GFP-PHPLCδ , at the tip of comets ( Figure 7A , inset ) . This was particularly evident in images through the middle of the cell revealing the robust presence of PI ( 4 , 5 ) P2 at the plasma membrane but not intracellularly ( Figure 7A ) . Even a higher sensitivity probe , consisting of two tandem PH domains of PLCδ ( GFP-2XPHPLCδ ) yielded similar results . The difference in GFP-PHPLCδ labeling of comet tips and of the plasma membrane was even more striking on images of the bottom plane of a cell showing intracellular comets that encountered the plasma membrane and evaginated it into filopodia-like structures , akin to those generated by Listeria-driven comets ( Figure 7B , D; Video 9 ) . In this case , the heavy labeling of the plasma membrane of filopodia encapsulating the actin comet stood in striking contrast to the lack of labeling ( or very weak labeling ) of the vesicle at the tip of the comet . 10 . 7554/eLife . 02975 . 022Figure 7 . PI ( 4 , 5 ) P2 detection on intracellular vesicles . ( A–B ) Confocal images of patient fibroblasts co-expressing GFP-PHPLCδ and mCherry-CHUtrophin at two different focal planes , midsection and bottom surface , are shown in ( A ) and ( B ) respectively . The midsection image ( A ) shows robust presence of the PI ( 4 , 5 ) P2 probe at the plasma membrane but only occasional presence at the tip of the comets . One example is shown in the inset . The bottom surface ( B ) image shows numerous PI ( 4 , 5 ) P2 positive cell evaginations resulting from comets pushing into the plasma membrane . One example is shown in the inset . As the comet pushes into the plasma membrane it becomes enveloped in PHPLCδ-positive plasma membrane . Scale bar full size: 10 µm; inset: 1 µm . ( C ) Sequential confocal images of a patient cell expressing untagged M1R , GFP-PHPLCδ and mCherry-CHUtrophin showing that in response to plasma membrane PI ( 4 , 5 ) P2 hydrolysis and increase in cytosolic Ca2+ induced by OxoM , actin comet disassemble and the PI ( 4 , 5 ) P2 probe relocates to intracellular vesicles . As actin reassembles , mCherry-CHUtrophin displaces the PI ( 4 , 5 ) P2 probe . ( D ) Model depicting the impact of the lack of OCRL in Lowe syndrome patients on clathrin-mediated endocytosis . Absence of the 5-phosphatase activity of OCRL delays endocytosis by stalling late-stage pits , impairs uncoating and triggers the nucleation of comets from clathrin vesicles that retain PI ( 4 , 5 ) P2 and fail to uncoat . DOI: http://dx . doi . org/10 . 7554/eLife . 02975 . 02210 . 7554/eLife . 02975 . 023Figure 7—figure supplement 1 . Overexpression of PIP5K1γ ( a PI4P 5-kinase ) in wild type fibroblasts induces actin comets . ( A ) Confocal microscopy image of a control wild type fibroblast co-transfected with mCherry-CHUtrophin , GFP-PHPLCδ and untagged PIP5K1γ showing comets similar to those found in patient cells . Note the presence of actin comets negative for GFP-PHPLCδ moving through the cytosol while at the same time one comet has evaginated the plasma membrane and is encapsulated in GFP-PHPLCδ-marked plasma membrane . ( B ) Example of a comet positive for GFP-PHPLCδ at its head in a wild type cell expressing mCherry-CHUtrophin , GFP-PHPLCδ and untagged PIP5K1γ . ( C ) Sequential images of a comet negative for GFP-PHPLCδ that pushes against the GFP-PHPLCδ-positive plasma membrane and becomes enveloped by it as it deforms the membrane into a filopodium-like structure . Note the hollow tip of the comet , which is not labeled by GFP-PHPLCδ , even though this probe robustly labels the plasma membrane encapsulating the comet . DOI: http://dx . doi . org/10 . 7554/eLife . 02975 . 02310 . 7554/eLife . 02975 . 027Video 9 . Actin comets come into contact with and evaginate the plasma membrane . Lowe syndrome patient fibroblast co-transfected with the F-actin probe mCherry-CHUtrophin and GFP-PHPLCδ showing the presence of comets that come into contact with the plasma membrane and evaginate it to create filopodia-like structures , akin to those observed upon Lysteria infection . Note that comets are not positive for GFP-PHPLCδ until they come into contact with the GFP-PHPLCδ positive plasma membrane . At that point the comet pushes out the plasma membrane and is enveloped by it ( see cartoon of Figure 7 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 02975 . 027 Since these results brought into question the presence of PI ( 4 , 5 ) P2 at the tips of comets , we examined the localization of the PI ( 4 , 5 ) P2 probe in an additional cellular model of PI ( 4 , 5 ) P2-dependent actin comet formation , the overexpression of a PI4P 5-kinase . Overexpression of one such enzyme , PIPkinase type-1-gamma ( PIPK1γ ) , along with GFP-PHPLCδ and mCherry-CHUTR in control human fibroblasts cells led to a robust formation of actin comets ( Figure 7—figure supplement 1A–A″ ) . Even in this case , where the intracellular pool of PI ( 4 , 5 ) P2 was dramatically expanded , detection of GFP-PHPLCδ labeling at the tips of the comets ( Figure 7—figure supplement 1B ) was inconsistent ( although more frequently observed than in Lowe syndrome patient cells ) , in contrast to the robust labeling of the plasma membrane ( Figure 7—figure supplement 1C–C‴ ) . This finding could have three non-mutually exclusive explanations: ( 1 ) PI ( 4 , 5 ) P2 may be present at a much lower concentration on the vesicles than on the plasma membrane , but these low levels may be sufficient to nucleate actin , ( 2 ) GFP-PHPLCδ may be a better reporter of plasma membrane PI ( 4 , 5 ) P2 given the presence of a yet unidentified PHPLCδ co-receptor only on this membrane; ( 3 ) PI ( 4 , 5 ) P2 present on the vesicles may be shielded and possibly sequestered by actin and actin regulatory proteins . One observation favoring the idea that actin and actin regulatory proteins may shield/sequester PI ( 4 , 5 ) P2 came from patient cells overexpressing M1R and treated with OxoM . In such cells , several vesicles became positive for GFP-PHPLCδ during the period of actin depolymerization that accompanied the transient rise in cytosolic Ca2+ ( Figure 7C ) . However , GFP-PHPLCδ was rapidly displaced by mCherry-CHUTR as actin comets reformed , providing further evidence for the presence of PI ( 4 , 5 ) P2 on internal vesicles in Lowe syndrome patient cells ( Figure 7C ) . Overall , these findings support a model in which OCRL acts at late-stage clathrin-coated pits to dephosphorylate PI ( 4 , 5 ) P2 and facilitates the shedding of endocytic factors ( Figure 7D ) . In the absence of OCRL , as in Lowe syndrome , the accumulation of PI ( 4 , 5 ) P2-rich vesicles that fail to lose their clathrin coat leads to an endocytic defect resulting from the sequestration of endocytic factors on these vesicles ( Figure 7D ) .
Enzymes containing a 5-phosphatase domain have been implicated at multiple stages of clathrin-mediated endocytosis , from clathrin coat nucleation to uncoating ( Pirruccello and De Camilli , 2012 ) . While OCRL , which directly interacts with clathrin , had been detected at late stage clathrin-coated pits ( Erdmann et al . , 2007; Mao et al . , 2009 ) , so far there has been no evidence from loss-of-function studies for a direct impact of this enzyme on the dynamics of endocytic clathrin-mediated budding . Data concerning the action of OCRL in membrane traffic , and the impact of its loss in patient cells , pointed primarily to a post-endocytic role , including intracellular clathrin-dependent transport reactions ( Dressman et al . , 2000; Ungewickell et al . , 2004; Choudhury et al . , 2005; Hyvola et al . , 2006; Swan et al . , 2010; Vicinanza et al . , 2011 ) . Additional roles for OCRL in clathrin-independent forms of endocytosis ( micropinocytosis and phagocytosis ) had also been reported ( Bohdanowicz et al . , 2012; Kuhbacher et al . , 2012; Marion et al . , 2012 ) . Our present results demonstrate that the absence of OCRL in Lowe syndrome patient cells results in an impairment of clathrin-mediated endocytosis . These findings provide new insights into the basic mechanisms of endocytosis as well as into the molecular pathogenesis of Lowe syndrome and Dent's disease . A role of PI ( 4 , 5 ) P2 dephosphorylation in the normal progression of clathrin-mediated endocytosis was first described at neuronal synapses , where studies of synaptojanin 1 emphasized an uncoating function of this protein ( Cremona et al . , 1999 ) . Subsequent work in a variety of model systems supported this idea but also suggested a role of this phosphoinositide in the late stages of endocytosis . Dephosphorylation of PI ( 4 , 5 ) P2 in the membranes of the budding vesicles was proposed to act as a mechanism to generate a line tension at the vesicle neck ( Stefan et al . , 2002; Rusk et al . , 2003; Mani et al . , 2007; Sun et al . , 2007; Liu et al . , 2009; Chang-Ileto et al . , 2011 ) . Progression through the late stages of endocytosis could be also affected by the sequestration of endocytic proteins on PI ( 4 , 5 ) P2-rich uncoated vesicles , as postulated for synapses lacking synaptojanin 1 or all three endophilins ( Mani et al . , 2007; Milosevic et al . , 2011 ) . The present study demonstrates a defect of endocytosis in Lowe syndrome patient fibroblasts . This defect is characterized by an accumulation of late-stage ( primarily U-shaped ) endocytic clathrin-coated pits and by a delay in the internalization of transferrin , a clathrin-coated pit cargo . Our results also point to a defect in clathrin uncoating , as in the case of synaptojanin 1 KO synapses . More specifically , electron microscopy demonstrated a robust increase in the number of clathrin-coated vesicles in patient cells relative to control fibroblasts . Additionally , immunofluorescence and live microscopy analysis revealed an abundance of intracellular vesicles positive for both coat and cargo proteins of endocytic origin including , besides clathrin , the adaptor complex AP-2 , SNX9 , PI3KcIIα and transferrin . These vesicles were at the tips of actin comets , which are known to be nucleated by PI ( 4 , 5 ) P2-positive organelles . The dramatic decrease in the number of these comets upon the selective , pharmacologically-induced depletion of PI ( 4 , 5 ) P2 at the plasma membrane demonstrates that at least a large cohort of these comets are nucleated by endocytic vesicles . The prominent impact of the lack of OCRL on total cellular PI ( 4 , 5 ) P2 levels in patient cells is reminiscent of the robust increase in brain levels of this phosphoinositide observed in the absence of synaptojanin 1 ( Cremona et al . , 1999 ) . We suggest that synaptojanin 1 and OCRL have partially overlapping functions , but with predominant roles at synapses and in non-neuronal cells , respectively . The newly discovered direct interaction of OCRL with SNX9 , a protein implicated in the transition from late stage clathrin-coated pits to free clathrin-coated vesicles , is of significant interest as it provides a new link between OCRL function and late-stage endocytic clathrin-coated pit nucleation . Interestingly , SNX9 also binds synaptojanin 1 and 2 , consistent with a partial overlap in the function of these 5-phosphatases ( Yeow-Fong et al . , 2005 ) . SNX9 is a member of a family of BAR domain-containing endocytic adaptors that help coordinate changes in bilayer curvature with reactions that occur as membranes progress along the endocytic pathway . Specifically , SNX9 is thought to have an important role in coupling clathrin coat maturation to actin nucleation ( Yarar et al . , 2007 ) . Our observation that the knockdown of SNX9 decreases comet formation in patient cells supports a role for this protein in actin nucleation at clathrin-coated pits . It also suggests a functional link between SNX9-regulated actin polymerization and the turnover of PI ( 4 , 5 ) P2 controlled by OCRL . The modular structure of SNX9 is ideally suited to mediate this coordination . SNX9 binds the plasma membrane with a PX-BAR domain module , which exhibits a preference for curved membranes enriched in PI ( 4 , 5 ) P2 and 3-phosphorylated phosphoinositides ( Gallop et al . , 2013 ) , clathrin via a ‘clathrin-box’ motif and AP-2 via an α-adaptin-binding motif ( Lundmark and Carlsson , 2003 ) . Through its SH3 domain , SNX9 binds and thus helps to activate both N-WASP to trigger actin nucleation , and dynamin to mediate fission ( Yarar et al . , 2007 ) . The SH3 domain of SNX9 binds synaptojanin and also OCRL , as we have demonstrated in this study . Given that SNX9 is a protein dimer that is present in multiple copies at endocytic sites , SNX9 is an attractive candidate to help bring together all these factors . Recently , SNX9 was shown to act as an effector of PI ( 3 , 4 ) P2 , which is generated at endocytic clathrin-coated pits by PI3KcIIα ( Posor et al . , 2013 ) . In principle , 5-phosphatases such as OCRL and synaptojanin could directly contribute to PI ( 3 , 4 ) P2 production by dephosphorylation of PI ( 3 , 4 , 5 ) P3 , or indirectly by generating PI4P , the PI3KcIIα substrate , from PI ( 4 , 5 ) P2 dephosphorylation ( Posor et al . , 2013; Schmid and Mettlen , 2013 ) . These models would place OCRL upstream of SNX9 . However , SNX9 is recruited to coated pits prior to OCRL , and also in its absence . Instead , the recruitment of OCRL at endocytic clathrin-coated pits coincides with the temporal recruitment signature of GAK ( Taylor et al . , 2011 ) , which , like its homologue auxilin , is a cofactor for Hsc70 and thus a key player in the disassembly of the clathrin lattice after fission ( Fotin et al . , 2004; Massol et al . , 2006 ) . Both OCRL and GAK peak after the clathrin fluorescence has started to diminish , as expected for proteins that mediate the shedding of the clathrin coat ( GAK ) and its adaptors ( OCRL ) . Interestingly , the lack of GAK or auxilin results in endocytic defects in addition to the accumulation of clathrin-coated vesicles and empty clathrin cages ( Lee et al . , 2005 , Yim et al . , 2005 ) . This impairment of endocytosis is thought to result from the sequestration of coat components on assembled lattices . Similarly , a defect in uncoating in the absence of OCRL may impair the recycling of endocytic factors and affect the maturation of clathrin-coated pits . As OCRL binds multiple proteins present in endocytic clathrin coats , an important question for future studies will be to determine the precise mechanisms responsible for the timing of OCRL recruitment . The presence in Lowe syndrome fibroblasts of numerous actin comets driven by clathrin-positive vesicles adds further support to a close relationship between endocytic clathrin coats and actin , a topic of intense debate , although most recent studies support such a link at least for a major subset of clathrin-dependent endocytic events ( Fujimoto et al . , 2000; Merrifield et al . , 2002 , 2005; Boucrot et al . , 2006; Kaksonen et al . , 2006; Tsujita et al . , 2006; Ferguson et al . , 2009; Boulant et al . , 2011 ) . Actin has been implicated in both the invagination of clathrin-coated pits as well as in the propulsion of endocytic vesicles away from endocytic sites . The occurrence of comets in Lowe syndrome cells suggests that failure to dephosphorylate PI ( 4 , 5 ) P2 not only delays coat shedding , but also disrupts the release of actin nucleating factors associated with endocytic clathrin coats . The collective information emerging from this and previous studies suggests that the function of OCRL is needed , either directly or indirectly , at a multiplicity of sites . The 5-phosphatase activity of OCRL is required to complete endocytosis and to prevent the ectopic accumulation of PI ( 4 , 5 ) P2 on endosomes and membranes in the Golgi complex area ( this work; Choudhury et al . , 2005; Dressman et al . , 2000; Erdmann et al . , 2007; Hyvola et al . , 2006; Swan et al . , 2010; Ungewickell et al . , 2004; Vicinanza et al . , 2011 ) . This multiplicity of actions is supported by its numerous interactors , which comprise proteins implicated in membrane traffic , signaling and actin regulation in a variety of cellular compartments ( Pirruccello and De Camilli , 2012 ) . As shown here , proteins implicated in clathrin-mediated endocytosis are prominently featured in the OCRL ‘interactome’ , which also comprises numerous proteins that participate in the clathrin-dependent traffic between endosomes and the Golgi complex . These include the cation-independent mannose 6-phosphate receptor ( CI-M6PR ) and the clathrin adaptor EpsinR , implicating OCRL in these trafficking stations as well . It remains unclear whether the function of OCRL at these intracellular sites is to prevent the buildup of PI ( 4 , 5 ) P2 or to control small yet physiologically relevant PI ( 4 , 5 ) P2 pools . A surprising result of our study was the unexpected absence of a robust signal for the PI ( 4 , 5 ) P2 probe , PHPLCδ , at intracellular sites in Lowe syndrome fibroblasts . The presence of clathrin , AP-2 and actin nucleating proteins on intracellular vesicles , however , strongly suggests the presence of PI ( 4 , 5 ) P2 on these vesicles . Sequestration of PI ( 4 , 5 ) P2 on such vesicles by clathrin adaptors and actin nucleating factors may be one reason explaining absent or inefficient PHPLCδ labeling , as we have shown that transient depolymerization of actin results in the appearance of the PI ( 4 , 5 ) P2 reporter on intracellular vesicles . OCRL has a close homologue , INPP5B , and these two proteins share many common interactors including endosomal proteins and Rabs ( Shin et al . , 2005; Hyvola et al . , 2006; Erdmann et al . , 2007; Fukuda et al . , 2008 ) . INPP5B however , does not contain either clathrin or AP-2 binding sites ( Ungewickell et al . , 2004; Choudhury et al . , 2005 ) . Correspondingly , OCRL and INPP5B share similar localizations on endosomes and in the Golgi complex area but INPP5B is not present at endocytic clathrin-coated pits ( Erdmann et al . , 2007; Williams et al . , 2007; Mao et al . , 2009 ) . Thus , a major functional difference between OCRL and INPP5B seems to be the involvement of OCRL , but not of INPP5B , in clathrin-mediated endocytosis . One important open question is why the lack of OCRL produces major phenotypes in humans but not in mice , in spite of the conservation of all known properties of OCRL in mice and humans , including the SNX9 binding site reported here . As INPP5B does not participate in endocytic clathrin-coated dynamics , differences in the expression or splicing of INPP5B in mice and humans cannot fully account for this difference . Elucidating mechanisms underlying this difference represents an important goal of future studies . Previous studies had attributed the proximal tubule reabsorption defects occurring in Lowe syndrome and Dent's disease primarily to abnormal endosomal recycling , such as for example the recycling of megalin , the scavenger receptor that mediates low molecular weight protein endocytosis , and CI-M6PR ( Erdmann et al . , 2007; Vicinanza et al . , 2011; Mehta et al . , 2014 ) . Our findings suggest that endocytosis itself may also be affected , as shown previously for CLC5 mutations that produce Dent's disease ( Novarino et al . , 2010 ) although they do not exclude the previously suggested intracellular sorting abnormalities ( Lowe , 2005; Vicinanza et al . , 2011 ) . Rather , they suggest that defects in endocytosis and in the fate of newly formed endocytic vesicles ultimately contribute to abnormal cargo sorting , as exemplified by the accumulation of clathrin-coated vesicles at the tip of actin comets . Impairments in endocytosis may also contribute to the cognitive deficiencies observed in Lowe syndrome patients . While synaptojanin 1 is the key player in clathrin-mediated endocytosis at synapses , OCRL may function at clathrin-coated pits that participate in a housekeeping form of endocytosis in neurons .
GFP-Rab5 , mRFP-clathrin light chain ( mRFP-CLC ) , GFP-N-Wasp , GFP-OCRL , catalytically inactive GFP-OCRLD523G , deletion fragments GST-OCRL176 and GST-OCRL217 and clathrin-binding mutant GFP-OCRLX3 have been described previously ( Perera et al . , 2006; Erdmann et al . , 2007; Ferguson et al . , 2009; Mao et al . , 2009; Idevall-Hagren et al . , 2012 ) . mCherry-tagged OCRL was generated by excising the OCRL coding sequence from GFP‐OCRL and ligating it into the pmCherry‐C1 vector ( Clontech Laboratories , Inc . , Mountain View , CA ) using XhoI and BamHI . Three prolines in OCRL ( P177 , P178 and P181 ) were mutated to alanine using site-directed mutagenesis ( QuickChange II-XL; Agilent Technology , Santa Clara , CA ) and GFP-OCRL as the original template to generate GFP-OCRLP3 . The following primers were used: OCRL-P177 , 78 , 81A_S 5′-GGGATTCATCGGGAAGCCGCACCTCCAGCCTTTTCAGT-3′ and OCRL-P177 , 78 , 81A_AS 5′-ACTGAAAAGGCTGGAGGTGCGGCTTCCCGATGAATCCC-3′ . The following plasmids were kind gifts: GFP-SNX9 ( Kai Erdmann , University of Sheffield , United Kingdom ) , mCherry-CHUtrophin ( William Bement , University of Wisconsin , Madison , WI ) , GFP-PHPLCδ ( Antonella De Matteis , Telethon Institute of Genetics and Medicine , Naples , Italy ) , μ2-GFP ( Alexander Sorkin , University of Pittsburgh , Pittsburgh , PA ) , GFP-PI3KcIIα ( Jan Domin , University of Bedfordshire , Luton , UK ) , GFP-2X-FYVEHRS ( Harald Stenmark , University of Oslo , Oslo , Norway ) , M1 muscarinic acetylcholine receptor ( M1R ) ( Bertil Hille , University of Washington , Seattle , WA ) , and pCAG-GCaMP5 ( Loren Looger , Howard Hughes Medical Institute , Janelia Farm Research Campus , Ashburn , VA ) . Transferrin-Alexa 488 and 594 as well as biotinylated transferrin were purchased from Molecular Probes ( Life Technologies , Carlsbad , CA ) . Avidin , biocytin , OxoM and Atropine were obtained from Sigma ( Sigma-Aldrich , St . Louis , MO ) and the Arp2/3 inhibitor CK-666 was purchased from Calbiochem ( EMD Millipore Corporation , Billerica , MA ) . Antibodies for immunofluorescence and immunoblotting were obtained from commercial sources: rabbit anti-OCRL , rabbit anti-PI3KC2A , rabbit anti-CI-M6PR/IGF2R , mouse anti-tubulin , mouse anti-beta actin , ( Sigma-Aldrich , St . Louis , MO ) , rabbit anti-clathrin light chain 4045 ( EMD Millipore Corporation , Billerica , MA ) , mouse anti-clathrin heavy chain TD1 ( American Type Culture Collection , Manassas , VA ) , mouse anti-alpha adaptin AP6 ( Affinity BioReagents , Golden , CO ) , rabbit anti-EpsinR/CLINT1 ( Bethyl Laboratories , Montgomery , TX ) , mouse anti-GAPDH ( Santa Cruz Biotechnology , Dallas , TX ) , rabbit anti-GFP ( Abcam , Cambridge , MA ) , rabbit anti-p34 ( EMD Millipore Corporation , Billerica , MA ) , mouse anti-Transferrin receptor , goat anti-rabbit IgG Alexa Fluor 488 , goat anti-mouse IgG Alexa Fluor 594 and Alex Fluor 594-phalloidin ( Life Technologies , Calsbad , CA ) . Rabbit anti-SNX9 for immunoblotting was generated in our lab , while rabbit anti-SNX9 for immunofluorescence were kind gifts from Sven Carlsson ( Umeå University , Umeå , Sweden ) and Sandra Schmid ( UT Southwestern , Dallas , TX ) . De-identified human dermal fibroblasts derived from punch biopsies as well as HeLa M and HEK293 cells were cultured at 37°C in 10% CO2 in DMEM supplemented with 10% FBS and 100 mg/ml penicillin/streptomycin ( Life Technologies , Carlsbad , CA ) . For imaging , HeLa M and human wild type or patient fibroblasts were electroporated with Amaxa Nucleofector method using solution R/program A-24 ( Lonza , Basel , Switzerland ) . Cells were plated at subconfluent densities into 35 mm glass bottom ( thickness = 0 . 17 mm ) dishes ( Mattek , Ashland , MA ) and cultured for 12 to 24 hr prior to imaging . Control or patient cells seeded at day 0 were transfected with siRNAs using RNAiMax ( Life Technologies ) according to the manufacturer's instructions , expanded on day 1 , and used for the experiment on day 3 . All siRNAs sequences were used as a 27-mers DsiRNAs ( IDT , Coralville , IA ) . The SNX9 sequence used was: 5′-AACAGTCGTGCTAGTTCCTCATCCA-3′ . For silencing SNX18 , the sequence used was: 5′-GCACCGACGAGAAAGCCTGGAAGCA-3′ . The negative control was a previously validated sequence provided by IDT DNA: 5′-CGTTAATCGCGTATAATACGCGTAT-3′ . HeLa M cells were transfected with either GFP-OCRL or pEGFP-C1 ( Clontech Laboratories , Inc . , Mountain View , CA ) using Lipofectamine 2000 ( Life Technologies , Carlsbad , CA ) . 24 hr post-transfection , cells were plated into selection medium containing 750 µg/ml G418 Sulphate ( Gibco BRL , Life Technologies , Carlsbad , CA ) . Following antibiotic selection , high and low GFP-expressing cells were isolated by fluorescence‐activated cell sorting ( FACS ) and plated at low density to facilitate clonal colony formation . Individual colonies were picked , passaged directly into glass-bottom 35 mm dishes ( Mattek Corporation , Ashland , MA ) and imaged by SDC microscopy 15–24 hr later to assess the expression level , localization and dynamics of GFP-OCRL . Selected colonies were cultured and samples were then subjected to SDS-PAGE followed by Odyssey quantitative western blotting ( LI-COR Biotechnology , Lincoln , NE ) . Rabbit anti-OCRL antibody ( Sigma-Aldrich , St . Louis , MO ) was used to assess the ratio of exogenous GFP-OCRL to endogenous OCRL , which was then normalized to tubulin levels using mouse anti-tubulin antibody ( Sigma-Aldrich , St . Louis , MO ) . Cells were fixed in 2% glutaraldehyde-0 . 1 M sodium cacodylate . They were post-fixed with 1% OsO4 in 1 . 5% K4Fe ( CN ) 6 and 0 . 1 M sodium cacodylate , en bloc stained with 0 . 5% uranyl magnesium acetate , dehydrated and embedded in Embed 812 . Electron microscopy reagents were purchased from Electron Microscopy Sciences ( Hatfield , PA ) . For morphometric analysis , cells whose entire perimeter was visible in the EM sections were selected and all clathrin-coated structures , including clathrin-coated vesicular profiles within 500 nm of the plasma membrane , were counted . The total number of coated structures per unit length of the plasma membrane of each cell was calculated and averages of 30 control and patient cells were obtained . Cells were grown on 5 µg/ml fibronectin ( Millipore ) coated glass coverslips and fixed with 4% formaldehyde in 0 . 1 M sodium phosphate , pH 7 . 2 . Coverslips were washed with 50 mM NH4Cl , pH 7 . 2 , blocked and permeabilized with PBS + 3% bovine serum albumin + 0 . 1% Triton X-100 . Subsequent primary and secondary antibody incubations were also performed in this buffer . Coverslips were finally mounted in the Prolong Gold antifade reagent ( Life Technologies , Carlsbad , CA ) . Samples were imaged by spinning disk confocal microscopy . Multicolor images were acquired sequentially through a 60× oil objective ( 1 . 4 NA , CFI Plan Apochromat VC ) at 1-min intervals , and at least 10 frames were acquired before the addition of compound . All imaging experiments were performed on a spinning-disc confocal ( SDC ) microscope , using the Improvision UltraVIEW VoX system ( PerkinElmer , Waltham , MA ) built around a Nikon Ti-E Eclipse microscope equipped with Perfect Focus , 14-bit electron-multiplying charge-coupled device camera ( C9100-50; Hamamatsu Photonics , Hamamatsu , SZK , Japan ) , and spinning disc-confocal scan head ( CSU-X1; Yokogawa Corporation of America , Sugar Land , TX ) controlled by Volocity software ( PerkinElmer , Waltham , MA ) . Green fluorescence was excited with a 488-nm/50-mW diode laser ( Coherent ) and collected by a band pass ( BP ) 527/55-nm filter . Red fluorescence was excited with a 561-nm/50-mW diode laser ( Cobolt ) and collected by a BP 615/70-nm filter . Total internal fluorescence microscopy was performed using a Nikon Ti-E Eclipse inverted microscope fitted with an Apo TIRF 100 × N . A . 1 . 49 oil objective and driven by Andor iQ software ( Andor Technologies , Belfast , Ireland ) . Images were acquired with a back-illuminated Andor iXon 897 EMCCD camera ( 512 × 512 , 14 bit; Andor Technologies ) . Cells were imaged by sequential excitation at 0 . 25 Hz . Post-acquisition image analysis was performed in ImageJ software ( 1 . 46j , NIH ) and GraphPad Prism . Fluorescent particle detection , lifetime tracking , and lifetime analysis of clathrin-coated pits in control and patient cells expressing GFP-tagged µ2 subunit of AP2 ( µ2-GFP ) was performed as previously described ( Liang et al . , 2014 ) . Patient cells were rescued by expressing mCherry-OCRLWT . For this analysis all images were acquired using TIRF microscopy . 12 control cells , 12 patient cells and 7 rescued patient cells were used and an average of 809 events per cell were analyzed . Only endocytic pits that appeared during the acquisition and with a lifetime of at least 12 s were selected . An automated tracking analysis was used to determine the lifetime distributions of µ2 puncta for each cell . Using expectation-maximization algorithms , a Gaussian mixture model was then fitted to each lifetime histogram independently , identifying three subpopulations ( short , medium and long lifetimes ) . The mean lifetimes of the three subpopulations across different cells were calculated ( 40 s , 108 s , and 244 s respectively ) , fixed and then Gaussian models using these means for each subpopulation were re-fitted to the histograms . Particle counting was conducted in ImageJ ( National Institutes Of Health , Bethesda , MD ) by thresholding and using the analyze particles plug-in . To examine the sequential recruitment of proteins at the pit , raw images were analyzed with ImageJ to generate intensity plots of areas of interest and to quantitate particles as described previously ( Perera et al . , 2006; Zoncu et al . , 2007 ) . Briefly , average fluorescence/time plots were generated from individual fluorescent spots , which were subsequently time‐aligned by the conversion of one fluorescent marker to another . For each time point , the fluorescence intensity was normalized to the peak fluorescence intensity of that pit . For these analyses , clathrin-coated pits were manually selected at random . Statistical significance was calculated by two-tailed Student's t test . Uptake of biotinylated transferrin was performed as previously described ( Yarar , et al . , 2005 ) with slight modifications . Briefly , control and patient cells were starved for 1 . 5 hs , then chilled on ice for 30 min and finally incubated with biotinylated transferrin ( 10 µg/ml ) in ice-cold DMEM on ice for 45 min . Cells were then washed with cold PBS and incubated with pre-warmed culture media at 37°C for the times indicated . Internalization was stopped by placing the cells on ice and washing them three times with cold PBS . Cells were then incubated on ice with avidin ( 0 . 05 mg/ml ) for 1 hr followed by incubation with biocytin ( 0 . 05 mg/ml ) for 15 min . Cells were then washed three times with PBS and lysed ( 1% TX-100 , 0 . 1% SDS , 0 . 2% BSA , 50 mM NaCl , 1 mM Tris , pH 7 . 4 ) . Cell lysates were then added to ELISA plates coated with anti-human transferrin antibody ( Abcam ) and assayed for detectable biotinylated transferrin using chromogen-conjugated streptavidin as indicated in the manufacturer's protocol . In Figure 4K internalized biotinylated transferrin was expressed as the percent of total surface-bound at 4°C , which was not incubated with avidin or biocytin . Uptake of fluorescently label transferrin was performed in a very similar fashion , as previously described ( Ritter et al . , 2013 ) . Cells were incubated with transferrin-Alex 594 instead , placed on ice after internalization , washed three times with cold PBS , and surface bound transferrin was removed by a quick acid wash ( 0 . 2 M acetic acid , 0 . 5 M NaCl ) . Cells were then fixed with 4% PFA and imaged by spinning disk confocal microscopy . Cell were rinsed with PBS , labeled on ice for 60 min with 1 mg/ml EZ-link Sulfo-NHS- SS-Biotin ( Thermo Scientific , Rockford , IL ) , rinsed with PBS and lysed in PBS containing 1% TX100 and 0 . 1%SDS and protease inhibitor cocktail ( Roche , Indianapolis , IN ) . Biotinylated proteins were recovered on neutravidin beads ( Thermo Scientific ) and eluted by reduction with 2-mercaptoethanol containing SDS-PAGE sample buffer . Evaluation of transferrin receptor levels in starting material , biotinylated ( cell surface ) and non-biotinylated ( intracellular ) fractions was assessed by immunoblotting following SDS-PAGE . Actin immunoblotting ( mouse anti-actin antibody; Sigma ) was used as a negative control to ensure that the assay specifically distinguished between cell surface and intracellular proteins . Particulate and cytosolic fractions were purified from control and patient cells using ice-cold buffer A ( 100 mM MES , pH 6 . 5 , 1 mM EGTA , and 0 . 5 mM MgCl2 ) . Cells were harvested , pelleted , washed in PBS and repelleted . The cell pellet was resuspended in buffer A , homogenized in a glass Teflon homogenizer , passed through a 25G , 5/8 in needle and centrifuged for 5 min at 800×g in a tabletop centrifuge at 4°C . The supernatant was collected and centrifuged for 1 hr at 60 , 000×g in a TLA 100 . 2 rotor at 4°C . The resulting supernatant ( cytosol ) and pellet ( particulate fraction ) were analyzed by SDS-PAGE and Western blotting . Recombinant NH2-terminal OCRL fragments were expressed in Escherichia coli BL21 as GST-tagged fusions as previously described ( Mao et al . , 2009 ) . Fusion proteins were purified on glutathione Sepharose beads according to standard protocols . Adult mouse brain extracts were prepared by homogenization in lysis buffer ( PBS , 0 . 5% Triton [vol/vol] , protease inhibitor mixture [Roche] ) followed by ultracentrifugation ( 100 , 000×g , 60 min , 4°C ) to remove insoluble material . Cleared lysates were then incubated with GST-fusion proteins on beads for 2 hr at 4°C . After extensive wash , bound proteins were analyzed by SDS–PAGE . For co-immunoprecipitations , HEK 293 cells transiently expressing GFP-tagged constructs or HeLa cells stably expressing GFP alone or GFP-OCRL were collected and lysed in lysis buffer ( 150 mM NaCl , 10 mM Tris , pH 7 . 5 , 1% Triton X-100 , 1 mM EDTA , 200 U benzonase [Merck , Darmstadt , Germany] and cOmplete protease inhibitor cocktail tablet [Roche Diagnostics , Indianapolis , IN] ) . Cell extracts were incubated on ice for 15 min followed by centrifugation at 4000×g and 4°C for 15 min . Cleared lysates ( 2–4 mg ) were incubated under rotation for 30 min at 4°C with 50 µl magnetic beads or 25 µl sepharose beads coupled to monoclonal mouse anti‐GFP antibody ( Miltenyi Biotec Inc . , Auburn , CA ) . Following centrifugation and several washes in lysis buffer , proteins recovered on beads were eluted with SDS sample buffer and separated by SDS-PAGE . Analysis of phosphoinositides was performed by HPLC separation of glycerol-inositol phosphates after phospholipid deacylation and analysis of the mono- and bis-phosphate species by conductivity detection ( Nasuhoglu et al . , 2002 ) . Given that the bulk of cellular PIP2 and PIP are represented by PI ( 4 , 5 ) P2 and PI ( 4 ) P respectively , the two peaks closely reflect the levels of these two phosphoinositides .
Mutations in the inositol 5-phosphatase OCRL cause Lowe syndrome and Dent's disease . Although OCRL , a direct clathrin interactor , is recruited to late-stage clathrin-coated pits , clinical manifestations have been primarily attributed to intracellular sorting defects . Here we show that OCRL loss in Lowe syndrome patient fibroblasts impacts clathrin-mediated endocytosis and results in an endocytic defect . These cells exhibit an accumulation of clathrin-coated vesicles and an increase in U-shaped clathrin-coated pits , which may result from sequestration of coat components on uncoated vesicles . Endocytic vesicles that fail to lose their coat nucleate the majority of the numerous actin comets present in patient cells . SNX9 , an adaptor that couples late-stage endocytic coated pits to actin polymerization and which we found to bind OCRL directly , remains associated with such vesicles . These results indicate that OCRL acts as an uncoating factor and that defects in clathrin-mediated endocytosis likely contribute to pathology in patients with OCRL mutations .
Oculo-Cerebro-Renal syndrome of Lowe ( Lowe syndrome ) is a rare genetic disorder that can cause cataracts , mental disabilities and kidney dysfunction . It is caused by mutations in the gene encoding OCRL , a protein that modifies a membrane lipid and that is found on membranes transporting molecules ( cargo ) into cells by a process known as endocytosis . During endocytosis , the cell outer membrane is deformed into a pit that engulfs the cargo to be taken up by the cell . The pit then pinches off from the outer membrane to form a vesicle—a bubble-like compartment—inside the cell that transports the cargo to its destination . In one type of endocytosis , this process is mediated by a basket-like coat primarily made up from the protein clathrin that assembles at the membrane patch to be internalized . After the vesicle is released from the cell membrane , the clathrin coat is broken apart and its components are shed and recycled for use by new budding endocytic vesicles . The OCRL protein had previously been observed associated to newly forming clathrin-coated vesicles , but the significance of this was not known . Now , Nández et al . have used a range of imaging and analytical techniques to further investigate the properties of OCRL , taking advantage of cells from patients with Lowe syndrome . These cells lack OCRL , and so allow the effect of OCRL's absence on cell function to be deduced . OCRL destroys the membrane lipid that helps to connect the clathrin coat to the membrane , and Nández et al . show that without OCRL the newly formed vesicle moves into the cell but fails to efficiently shed its clathrin coat . Thus , a large fraction of clathrin coat components remain trapped on the vesicles , reducing the amount of such components available to help new pits develop into vesicles . As a consequence , the cell has difficulty internalizing molecules . Collectively , the findings of Nández et al . outline that OCRL plays a role in the regulation of endocytosis in addition to its previously reported actions in the control of intracellular membrane traffic . The results also help to explain some of the symptoms seen in Lowe syndrome patients .