A novel Cep120-dependent mechanism inhibits centriole maturation in quiescent cells
The centrosome is the major microtubule-organizing center ( MTOC ) in animal cells , which helps regulate the assembly of the interphase microtubule array and mitotic spindle . The centrosome in a quiescent cell is composed of a pair of centrioles surrounded by a proteinaceous matrix referred to as pericentriolar material ( PCM ) , involved in microtubule nucleation and anchoring . The two centrioles in quiescent cells differ in age , structure and function . The older of the two was assembled at least two cell cycles ago and is commonly called the ‘mother’ centriole ( MC ) . The younger one , which was assembled in the previous cell cycle , is called the ‘daughter’ centriole ( DC ) . Most activities at the centrosome are regulated and mediated by the mother centriole . For example , the MC acts as the template for the assembly of the primary cilium , an essential chemosensory organelle ( Malicki and Johnson , 2017 ) . The MC possesses distinct sets of projections called distal appendages that confer the ability to dock at the plasma membrane and initiate ciliogenesis , and subdistal appendages that help organize the PCM , nucleate and anchor microtubules ( Garcia and Reiter , 2016 ) . In contrast , the DC lacks distal and subdistal appendages , does not form a cilium , and possesses significantly less PCM and thus MTOC activity . How and why the DC is prohibited from recruiting these protein complexes remains a mystery . The predominant theory is that the DC undergoes structural and biochemical modifications during the cell cycle that confers the ability to acquire these traits , a multi-step process classically referred to as ‘maturation’ ( Hoyer-Fender , 2010 ) . However , recent studies suggest that this model may be too simplistic ( Kong et al . , 2014; Wang et al . , 2011 ) . So , how is the DC prohibited from recruiting these protein complexes during quiescence ? We previously identified Cep120 as a centrosomal protein that is preferentially enriched on the DC of quiescent cells ( Mahjoub et al . , 2010 ) . This asymmetric localization between the mother and daughter centriole was relieved coincident with new procentriole assembly in S-phase , where Cep120 became enriched on the procentrioles ( [Mahjoub et al . , 2010]; summarized in Figure 1A ) . Thus , Cep120 associates with the youngest generation of centrioles at all stages of the cell cycle , although some of the protein remains on the MC ( [Mahjoub et al . , 2010]; Figure 1A ) . To determine the functional significance of Cep120 enrichment on procentrioles , we depleted the protein and analyzed the consequences on centriole assembly . We found that Cep120 was required for procentriole formation in S-phase of cycling cells , and for nascent centriole assembly in multiciliated cells ( Mahjoub et al . , 2010 ) , revealing a conserved role for Cep120 in centriole formation . Subsequent studies helped to define the role of Cep120 in the molecular pathways regulating procentriole assembly ( Comartin et al . , 2013; Lin et al . , 2013 ) . More recently , mutations in Cep120 were identified in patients with ciliopathy phenotypes , namely Joubert syndrome and Jeune Asphyxiating Thoracic Dystrophy ( Roosing et al . , 2016; Shaheen et al . , 2015 ) , further highlighting a critical role in centriole-centrosome-cilia biology . However , one major question remains unanswered: what is the functional significance of the enrichment of Cep120 on the DC of quiescent cells , at a stage of the cell cycle when centriole duplication is not occurring ?
To address this question , we established an assay whereby we could deplete Cep120 while simultaneously arresting cells in G0 and maintaining them in a quiescent state . This is critical because loss of Cep120 disrupts procentriole assembly in S-phase , resulting in cells that lack centrioles after a few cell divisions ( Mahjoub et al . , 2010 ) . Thus , we could not utilize genome-editing approaches such as the CRISPR/Cas9 system , which entails isolation and screening of individual clones of cells , an approach that requires multiple passages and would thus disrupt centriole duplication in the absence of Cep120 . To overcome this issue , we made use of siRNA oligos that are known to specifically and robustly deplete Cep120 ( Comartin et al . , 2013 ) . Mouse embryonic fibroblasts ( MEF ) were seeded at high density , transfected with either control ( non-targeting ) or Cep120-specific siRNA , and immediately incubated in low serum-containing medium for 24–48 hr to arrest cells in G0 . Immunoblot analysis of whole-cell lysates showed this approach results in near complete depletion of Cep120 ( Figure 1B ) . Consistent with this , there was loss of endogenous Cep120 signal from the DC , as evidenced by immunofluorescence staining of fixed cells ( Figure 1C–D ) . Next , we analyzed the proportion of cells in each phase of the cell cycle by FACS , to ensure that Cep120-depleted cells are indeed arrested in G0 . Both control and Cep120-depleted MEF showed an increase in the G0-G1 population upon serum-starvation , with no significant difference in cell cycle profiles otherwise ( Figure 1E ) . Finally , we immunostained cycling and serum-starved cells for Ki-67 , a marker of cell proliferation expressed at all cell-cycle stages except G0 ( Sobecki et al . , 2017 ) . Both control and Cep120-depleted cells showed loss of Ki-67 signal upon serum removal ( Figure 1F ) . Collectively , these data indicate that our experimental scheme results in robust depletion of Cep120 in quiescent cells , which still maintain their original mother and daughter centrioles . We measured the abundance of asymmetrically localized proteins at the centrosome using quantitative single-cell immunofluorescence microscopy ( described in detail in Methods ) , beginning with components of the PCM . Pericentrin is a core scaffolding protein that attaches to the centriolar microtubules and forms the foundation upon which other PCM components are assembled ( Doxsey et al . , 1994; Mennella et al . , 2014 ) . In MEF cells transfected with control siRNA , pericentrin showed preferential enrichment on the MC ( Figure 2A ) . Depletion of Cep120 resulted in a roughly 2 . 5-fold increase in total centrosomal pericentrin ( Figure 2A–B ) . Quantification of the relative levels of pericentrin on mother versus daughter centrioles showed that most of this increase occurred on the DC ( Figure 2B ) . Cdk5Rap2 is recruited by pericentrin to help form the PCM matrix ( Fong et al . , 2008; Mennella et al . , 2014 ) , and is similarly enriched on the MC ( Figure 2C ) . Loss of Cep120 caused a near 6-fold increase in total Cdk5Rap2 levels at the centrosome , with the majority of the increase seen at the DC ( Figure 2C–D ) . Ninein is a PCM protein that localizes to the proximal ends of centrioles in addition to the subdistal appendages of the MC ( Bouckson-Castaing et al . , 1996; Mazo et al . , 2016 ) , and is thus enriched on the MC ( Figure 2E ) . Cep120 depletion resulted in greater than 4-fold increase in centrosomal ninein , with a large increase occurring on the DC ( Figure 2E–F ) . Importantly , a significant amount of the accumulation occurred around the MC , which indicates that the small pool of Cep120 there also contains some inhibitory function ( Figure 2E–F ) . Cep170 is recruited by ninein and shows a similar localization pattern in cells ( Guarguaglini et al . , 2005; Mazo et al . , 2016 ) , with preferential association with the MC ( Figure 2G ) . Loss of Cep120 caused a 4-fold increase in total centrosomal Cep170 levels ( Figure 2G–H ) . Similar to ninein , the majority of these gains occurred on the MC , in addition to the significant increase observed on the DC ( Figure 2G–H ) . Finally , we measured the relative abundance of γ-tubulin , the core component of the γ-tubulin ring complex involved in microtubule nucleation from the PCM ( Lin et al . , 2015 ) . Intriguingly , we found that loss of Cep120 had no significant effect on the abundance of γ-tubulin at the centrosome , or on mother versus daughter centrioles ( Figure 2I–J ) . This suggests that not all components of the PCM may be disrupted upon Cep120 loss . Immunoblot analysis of whole-cell lysates indicated that the increases in centrosomal PCM in Cep120-depleted cells were not due to increased protein expression , but likely changes in localization of the cytosolic pool ( Figure 3 ) . To verify the specificity of the siRNA-mediated phenotypes , we generated an expression plasmid expressing an siRNA-resistant version of Cep120 , by making six silent mutations in the siRNA-recognition site ( GFP-Cep120siRt ) . Transfection of GFP-Cep120siRt into Cep120-depleted cells rescued the centriole duplication defect in cycling cells ( Figure 2—figure supplement 1A ) . Importantly , GFP-Cep120siRt also rescued the increase in pericentrin levels in Cep120-depleted quiescent cells ( Figure 2—figure supplement 1B–C ) . We next sought to confirm that the observed changes are specifically due to Cep120 loss in G0 , and not an indirect consequence of potentially disrupting centriole duplication . To address this we depleted Sas6 , which is typically absent in quiescent cells and is expressed in G1/S-phase where it localizes to , and is critical for , procentriole assembly ( [Dammermann et al . , 2004]; Figure 2—figure supplement 2A ) . Analysis of Sas6-depleted quiescent cells showed no change in the levels of pericentrin , Cdk5Rap2 , or Cep170 ( Figure 2—figure supplement 2B–E ) . Cep120 and Sas6-depleted cells display loss of centrioles 24 hr after siRNA transfection , resulting in a significant number of cells containing one original parental centriole ( Figure 2—figure supplement 2A ) . This allowed us to test whether Cep120 that remains associated with the single daughter centriole in interphase still contained inhibitory activity . We performed this analysis in non-serum starved cells in G1 , since this is a stage when Cep120 is still enriched on the daughter centriole; in S-G2-M stages Cep120 enrichment is lost from the daughter centriole ( summarized in Figure 1A ) . We compared pericentrin levels in cells treated with Sas6 siRNA ( which still maintain Cep120 on the single daughter centriole ) to cells transfected with Cep120 siRNA ( which have lost Cep120 on the single daughter centriole ) . We noted a roughly 2-fold increase in pericentrin levels on the single daughter centriole when Cep120 was absent ( Figure 2—figure supplement 2F ) . This result is consistent with the hypothesis that the enrichment of Cep120 on the daughter centriole helps to limit PCM recruitment , and that the removal of Cep120 at G1/S is likely mediating accumulation of PCM on that centriole . Next , we confirmed the inhibitory function of Cep120 by over-expressing exogenous Myc-Cep120 , and noted a dose-dependent decrease in centrosomal pericentrin and Cdk5Rap2 ( Figure 4A–B ) . Finally , we wanted to identify functionally important domains of Cep120 that are critical for this inhibitory function . Cep120 contains an N-terminal microtubule-binding domain that is essential for directly binding microtubules in vitro ( Lin et al . , 2013; Sharma et al . , 2018 ) , a CPAP-binding domain essential for centriole duplication ( Comartin et al . , 2013; Lin et al . , 2013 ) , and a C-terminal coiled-coil domain required for directing localization to centrosomes ( Mahjoub et al . , 2010 ) ( Figure 4C ) . Using a series of deletion constructs that we previously established ( Mahjoub et al . , 2010 ) , we over-expressed truncated versions of the protein and assessed the levels of PCM at the centrosome . Loss of the C-terminal coiled-coil domain failed to inhibit pericentrin levels at the centrosome ( Figure 4C–D ) , indicating that localization to centrosomes is important for limiting PCM accumulation . Similarly , deletion of the microtubule-binding domain abrogated Cep120 inhibitory function ( Figure 4C–D ) . Collectively , these results indicate that Cep120 limits the levels of PCM proteins at the centrosome ( with relatively higher inhibitory activity at the DC ) , and that both microtubule-binding and coiled-coiled domains are essential for this function . To test whether loss of Cep120 affected other asymmetrically associated-centriolar proteins , we analyzed the distribution of distal appendage ( DAP ) and subdistal appendage ( sDAP ) proteins , which are only found on the MC during quiescence ( Garcia and Reiter , 2016 ) . Cep164 and Fbf-1 are two core components of the DAP ( Graser et al . , 2007; Mazo et al . , 2016 ) , and play a critical role in facilitating the attachment of the MC to ciliary vesicles or the plasma membrane , an essential step in cilia formation . There was no difference in Cep164 or Fbf-1 localization between control and Cep120-depleted cells ( Figure 5A–B ) . Odf-2 localizes to the sDAP of the MC and is critical for their formation and function ( Ishikawa et al . , 2005; Mazo et al . , 2016 ) . The asymmetric distribution of Odf-2 was also unchanged upon Cep120 loss ( Figure 5C ) . Since ninein and Cep170 are found at the sDAP , in addition to their PCM localization at the proximal region of centrioles ( Mazo et al . , 2016 ) , we sought to determine whether the observed increases in ninein and Cep170 were occurring at the sDAP , PCM or both . 3D-Structured Illumination Microcopy ( 3D-SIM ) of Cep120-depleted cells showed that the increase in ninein and Cep170 on both centrioles was only occurring at the proximal end ( Figure 5D–E ) . Together , these results indicate that loss of Cep120 does not affect the asymmetric distribution and localization of DAP and sDAP components , but specifically the PCM . To determine whether the increased PCM in Cep120-depleted cells affects centrosome function during quiescence , we analyzed microtubule assembly post-depolymerization . Microtubules were disassembled by a combination of nocodazole and cold treatment , and nucleation assessed at different time points during re-assembly by immunostaining for α-tubulin . There were higher levels of microtubule re-growth in Cep120-depleted quiescent cells , as evidenced by larger microtubule asters ( Figure 6A–B ) . Moreover , staining for the plus-end tracking protein EB1 showed more abundant EB1-positive comets focused around the centrosome ( Figure 6C ) , indicating a higher number of nucleated microtubules from the centrosome . Next , we tested whether microtubule-dependent centrosomal trafficking was affected . Centriolar satellites ( CS ) are non-membranous complexes found in the vicinity of the centrosome in mammalian cells ( Hori and Toda , 2017 ) . They play pivotal roles in centrosome assembly , primary cilium formation and function , via the delivery of components from the cytoplasm to the centrosome . These trafficking functions of CS are dependent on an intact , organized microtubule array ( Hori and Toda , 2017 ) . Remarkably , we discovered that multiple components of CS were dispersed in Cep120-depleted cells , including the core scaffolding protein PCM-1 , as well as regulators of ciliogenesis Azi-1 and Ccdc11 ( [Kubo et al . , 1999; Silva et al . , 2016; Staples et al . , 2012]; Figure 6D–F ) . Similarly , overexpression of exogenous Cep120-GFP , which leads to a decrease in pericentrin levels ( Figure 4D ) also resulted in dispersal of PCM-1 ( Figure 6G ) , indicating that PCM homeostasis is key for satellite localization and distribution . To understand why these CS were mislocalized , we immunostained cells for a component of the cytoplasmic dynein-dynactin motor complex p150Glued , which regulates CS localization and centrosome-directed movement ( Kim et al . , 2004 ) . In control cells , p150Glued was predominantly localized to the MC ( Figure 6H ) , as previously shown ( Kodani et al . , 2010 ) . We observed a ~ 1 . 6 fold increase in centrosomal p150Glued in Cep120-depleted cells , with the protein accumulating on the DC , which is rarely seen in control cells ( Figure 6H–J ) . We reasoned that the satellite dispersal phenotype might be caused by the increased ninein levels , which recruits p150Glued to the centrosome ( Mazo et al . , 2016 ) . Indeed , overexpression of exogenous Myc-ninein caused dispersal of PCM-1 and Azi-1 ( Figure 6K ) . Collectively , these results suggest that loss of Cep120 disrupts the localization of CS , due to changes in PCM abundance , microtubule organization , and dynein-dependent trafficking of centrosomal proteins . Since satellites play essential roles in ciliogenesis , we hypothesized that dispersal of CS upon Cep120 loss would disrupt the formation and/or function of cilia during quiescence . Indeed , Cep120-depleted cells exhibit a > 60% decrease in cilia formation compared with controls ( Figure 7A–B ) . Moreover , the fraction of cells that did assemble/retain cilia formed ones that were aberrant in length ( Figure 7C–D ) , another well-established indicator of defective ciliary protein trafficking . Once again , these defects were specific to loss of Cep120 , as control Sas6-depleted cells displayed normal CS localization ( Figure 7—figure supplement 1A ) and were competent to form cilia ( Figure 7—figure supplement 1B ) . Next , we assessed whether ciliary signaling was affected in the fraction of Cep120-depleted cells that still formed cilia . We focused on the transmembrane protein smoothened ( Smo ) , which translocates into the cilium in response to sonic hedgehog ( Shh ) ligand ( Corbit et al . , 2005 ) . The amount of Smo per unit length cilium was significantly reduced in Cep120-depleted cells ( Figure 7C–E ) . This suggests that in addition to assembly , the signaling functions of cilia may be attenuated upon Cep120 loss . The defects in ciliogenesis were not due to mislocalization of intraflagellar transport ( Jurczyk et al . , 2004 ) machinery ( Figure 7—figure supplement 1C–D ) , nor the transition zone protein Cep290 ( Figure 7—figure supplement 1E–F ) , which is at CS then localizes to the transition zone upon ciliogenesis ( Kim et al . , 2008 ) . Finally , we examined the localization of essential ciliogenesis factors that are dependent on CS for their centrosomal localization . CP110 is a centriolar distal end capping protein that interacts with CS and antagonizes ciliogenesis; its removal from the MC is a critical step in ciliogenesis ( Spektor et al . , 2007 ) . Quiescent cells that form cilia contain CP110 only on the DC , as it is removed from the MC ( Figure 7F ) . Intriguingly , we noted a significant increase in the fraction of cells that still contained CP110 on both the MC and DC ( Figure 7F ) . Finally , we examined the localization of the ciliopathy protein Ofd1 , which is essential for ciliogenesis , interacts with PCM-1 at satellites , and localizes to the distal end of both centrioles ( [Lopes et al . , 2011]; Figure 7G ) . Cep120-depleted cells showed a decrease in the number of Ofd1 foci at centrioles , indicating a defect in centriolar targeting of the protein ( Figure 7G ) . Together , these results show that loss of Cep120 during quiescence is detrimental to ciliary assembly and function , likely due to aberrant CS-dependent trafficking of proteins to the centrosome .
In this study , we have demonstrated that Cep120 plays an important inhibitory role at the centrosome , by regulating PCM levels during quiescence . Loss of Cep120 initiates a series of changes in protein localization and abundance that ultimately result in defective ciliary assembly and function ( Figure 7H ) . The first step involves changes in the maturation state of the daughter centriole , on which Cep120 is preferentially enriched . Centriole maturation is a multi-step process that includes accumulation of PCM material , assembly of appendage structures , recruitment of proteins essential for ciliary assembly , and elimination of inhibitory/licensing factors ( such as CP110 ) of ciliogenesis . Although most of these components are asymmetrically localized on mother versus daughter centrioles , our results show that Cep120 mainly controls the PCM stage of the maturation process . Removal of Cep120 did not result in complete maturation of the daughter centriole , nor enhance its ability to build a primary cilium . This suggests that the centriole maturation pathway regulated by Cep120 during quiescence is different than the one controlled by Plk1 , a kinase that triggers centrosome maturation as cells enter mitosis ( Mahen and Venkitaraman , 2012 ) . Indeed , over-expression of Plk1 in G1 can fully induce daughter centriole maturation independently of Cep120 loss , resulting in the assembly of appendages and the formation of a secondary primary cilium ( Kong et al . , 2014 ) . An alternative possibility is that Cep120 may be one of many targets of Plk1 and thus its depletion would not be expected to phenocopy Plk1 overexpression . However , inducing expression of Plk1 in G1 did not cause removal of Cep120 from the maturing daughter centriole ( Kong et al . , 2014 ) , suggesting that cells may have evolved an alternative pathway by which they can regulate centriole maturation during quiescence , controlled by protein ( s ) that localize to the daughter centriole . Depletion of Cep120 resulted in preferential accumulation of PCM components on the daughter centriole . However some PCM proteins , such as ninein and Cep170 , showed higher levels of accumulation on the mother centriole . This indicates that the small pool of Cep120 that remains associated with the mother centriole also contains some inhibitory function , and suggests that the levels of certain PCM proteins on the mother centriole are also ‘fine-tuned’ during quiescence . One intriguing reason for this type of control may be to ensure that the mother centriole ( and the centrosome in general ) assembles a microtubule network that specifically helps target ciliary proteins to the mother centriole ( Figure 7H ) . Indeed , we found that the elevated PCM negatively impacts centrosome homeostasis by disrupting the nucleation and organization of the microtubule cytoskeleton , potentially causing a ‘traffic jam’ that interrupts dynein-dependent movement of CS complexes . It is well established that the organization , localization and trafficking functions of CS are particularly sensitive to defects in the microtubule cytoskeleton ( Hori and Toda , 2017 ) . These changes in turn disrupted ciliary assembly and signaling , a process that requires CS-dependent delivery of essential factors to the centrosome . How does Cep120 regulate the asymmetric distribution of PCM proteins at the centrosome ? Using immuno-electron microscopy , we previously showed that Cep120 resides on the outer wall of the centriolar microtubules ( Mahjoub et al . , 2010 ) . Subsequent studies using superresolution microscopy also positioned Cep120 near the centriolar microtubules , placing it in a layer proximal to the centriole wall and surrounded by the PCM proteins ( Lawo et al . , 2012; Mennella et al . , 2014 ) . Moreover , Cep120 contains an N-terminal microtubule-binding domain that was shown to be essential for directly binding microtubules in vitro ( Lin et al . , 2013 ) . Intriguingly , mutations in this microtubule-binding domain are pathogenic , and cause ciliopathies such as Jeune and Joubert syndrome ( Roosing et al . , 2016; Shaheen et al . , 2015 ) . Thus , one interesting possibility is that Cep120 binds to and occupies sites on the surface of the centriolar microtubule wall , which could inhibit the association of PCM proteins in those locations . The loss of Cep120 from the daughter centriole at G1/S could potentially ‘free’ these sites and permit the accumulation of PCM proteins . The mechanism ( s ) that establish the asymmetric localization of Cep120 on mother and daughter centrioles remain unknown , and will be the focus of our future studies . In sum , our results highlight a new function for daughter centriole-associated proteins that is independent of their role in centriole duplication and assembly . Furthermore , these data illuminate the potential role ( s ) of other daughter centriole-enriched proteins , such as centrobin and Neurl4 ( Li et al . , 2012; Zou et al . , 2005 ) , in quiescent cells . Finally , our findings establish the important and exquisitely sensitive control of centrosome PCM homeostasis and its relationship to primary cilium assembly and signaling , and uncover a potentially new type of molecular defect that may underlie the pathogenesis of ciliopathies such as Jeune and Joubert syndrome .
Mammalian cells used in this study were mouse embryonic fibroblasts ( MEF ) , hTERT-immortalized human retinal pigment epithelial cells ( RPE-1 ) , NIH3T3 fibroblasts and NIH3T3 cells stably expressing Cep120-GFP ( Mahjoub et al . , 2010 ) . The cells were originally obtained from the ATCC and their identity authenticated . We have tested for mycoplasma contamination using commercially available kits . Cells were grown in DMEM medium ( Corning , cat . # 10–013-CV ) supplemented with 10% FBS ( Atlanta Biologicals , cat . # S11150 ) and 1% penicillin-streptomycin solution ( Gibco , cat . # 15140–122 ) . For serum-starvation experiments , cells were incubated for 24–48 hr in DMEM supplemented with 0 . 5% FBS . Cep120 constructs used in this study have been previously described ( Mahjoub et al . , 2010 ) : pCS2-Myc-Cep120 ( full-length , aa 1–988 ) , pCS2-Cep120-GFP ( full-length; FL ) , pCS2-Cep120-GFP ΔCC ( aa 1–700 ) , pCS2-Cep120-GFP ΔCC/CPAP-BD ( aa 1–450 ) , pCS2-Cep120-GFP ΔMT-BD ( aa 101–988 ) , and pCS2-Cep120-GFP ΔMT-BD/CPAP-BD ( aa 700–988 ) . For experiments requiring overexpression of ninein , a Myc-ninein plasmid that was previously generated was used ( Firat-Karalar et al . , 2014a ) . MEF cells were transfected with 1–5 μg of the appropriate plasmid DNA using 10 μL of Lipofectamine 2000 ( Life Technologies , cat . # 11668–027 ) according to the manufacturer’s protocol . Medium was changed 24 hr after transfection , and quiescence induced by incubating cells in low FBS ( 0 . 5% ) for another 24 hr . For experiments using siRNA-mediated depletion of Cep120 , a previously characterized ( Comartin et al . , 2013 ) synthetic oligonucleotide targeting the C-terminal sequence of the human and mouse Cep120 gene was obtained from GE Healthcare Dharmacon Inc . ( siGENOME collection , cat . # D-016493–01 ) . The siRNA sequence is: 5’- GAUGAGAACGGGUGUGUAU-3’ . For depletion of Sas6 , the previously characterized synthetic oligonucleotide siRNA#3 ( Leidel et al . , 2005 ) targeting the C-terminal domain of human Sas6 was obtained ( Dharmacon , siGENOME collection , cat . # D-019156–03 ) . The siRNA sequence is: 5’-GCACGUUAAUCAGCUACAA-3’ . A Silencer Select Negative Control siRNA ( Life Technologies , cat . # 4390844 ) was used as control . MEF or RPE-1 cells were seeded at 50–80% confluence and transfected with 100–200 nM of siRNA oligonucleotide using Lipofectamine RNAiMAX ( Life Technologies , cat . # 13778–075 ) according to manufacturer’s protocol . Depletion of Cep120 or Sas6 protein was observed 48 hr post-transfection by immunofluorescence and by immunoblotting . Ciliogenesis was induced at 24 hr post-depletion by shifting cells from high ( 10% ) to low ( 0 . 5% ) serum-containing medium for another 24 hr . For rescue experiments , we generated a siRNA-resistant GFP-Cep120 expression plasmid ( pLVX-Puro-GFP-Cep120siRt ) that contains six silent mutations introduced in the siRNA-targeting sequence . The changes to the siRNA-targeting sequence are ( altered sites marked in bold ) : 5’-AATGAGGACAGGAGTATAC-3’ . MEF cells were transfected with 5 μg of pLVX-Puro-GFP-Cep120siRt plasmid using 10 μL of Lipofectamine 2000 24 hr after siRNA transfection , and quiescence was induced by incubating cells in low-serum medium FBS for a further 24 hr . To generate a rat polyclonal Cep120 antibody , a C-terminal fragment of the mouse Cep120 gene ( amino acids 860–988 ) was PCR amplified and subcloned into pET-21a ( + ) vector ( Novagen , cat . # 69740–3 ) at unique NdeI and XhoI sites . Soluble recombinant Cep120-His6 protein was expressed in BL21-CodonPlus ( De3 ) -RIL competent cells ( Agilent Technologies , cat . # 230280 ) and purified on HisPurTM Cobalt Resin ( Pierce , cat . # 89964 ) following manufacturer’s protocols . 8 mg of antigen was used to inject three rats ( PrimmBiotech , Inc . ) . Polyclonal anti-Cep120 antibody from two of the animals showed specificity for Cep120 by immunoblot analysis of whole cell lysates ( used at a dilution of 1:5 , 000-1:10 , 000; Figure 1—figure supplement 1 ) and by immunofluorescence microcopy ( used at 1:2 , 000; Figures 1–7 ) . For indirect immunofluorescence , cells were grown on glass coverslips ( Electron Microscopy Sciences , cat . # 72230–01 ) coated with 1 mg/mL poly-L-Lysine ( Sigma , cat . # P1274 ) , fixed in either ice-cold methanol at −20°C for 10 min or with 4% paraformaldehyde ( Electron Microscopy Sciences , cat . # 15710 ) in PBS at room temperature for 10 min . Cells were rinsed twice with PBS , permeabilized with 0 . 1% Triton X-100 in PBS ( PBS-T ) , and blocked for 1 hr at room temperature with 3% BSA ( Sigma , cat . # A2153 ) in PBS-T . Samples were incubated with primary antibodies for 1–2 hr at room temperature ( see Supplementary file 1 for complete list of antibodies used ) . Alexa Fluor dye–conjugated secondary antibodies ( Life Sciences ) were used at a dilution of 1:500 at room temperature for 1 hr . Nuclei were stained with DAPI , and mounted in Mowiol ( Sigma , cat . # 81381 ) containing N-propyl gallate ( Sigma , cat . # 02370 ) . Images were captured using a Nikon Eclipse Ti-E inverted confocal microscope equipped with a 60X ( 1 . 4 NA ) or 100X ( 1 . 45 NA ) Plan Fluor oil immersion objective lens ( Nikon , Melville , NY ) . Three-dimensional super-resolution microscopy ( 3D-SIM ) images were captured on an inverted Nikon Ti-E microscope using 100X oil objective . Optical z-sections were separated by 0 . 216 µm , and 3D-SIM images were processed and reconstructed using the Nikon Elements AR 4 Software . Quantification of fluorescence intensity of the various centrosomal markers ( Pericentrin , Cdk5Rap2 , ninein , γ-tubulin , Cep170 ) was performed by measuring the total fluorescent intensity of a given marker within a 2–5 μm circular region of interest ( ROI ) centered on the centrosome , using Nikon Elements AR 4 . 20 Software . Background fluorescence values were measured using an ROI of identical size in the near proximity of each centrosome and subtracted from the centrosomal measurements . For comparison of signal intensity at mother versus daughter centrioles , two separate ROIs of 0 . 5–1 . 0 μm were drawn around each centriole . Background fluorescence subtraction was performed as described above . In both cases , the fluorescence signal of centrin was simultaneously measured and used as an internal control , and the fluorescent values of each marker normalized to the unchanged centrin signal . Centriolar satellite localization and dispersal was determined by measuring the fluorescence intensity of PCM-1 , Azi-1 , Ccdc11 and p150Glued within a 2–16 μm circular region of interest ( depending on the protein ) as described . The relative localization of various centriolar/centrosomal proteins on mother versus daughter centrioles was determined by comparing control and Cep120-depleted cells for each marker individually . These include Cep164 , Fbf-1 ( distal appendages ) , Cep170 , Odf-2 , ( subdistal appendages ) , Cep290 and Ofd1 ( transition zone ) , Cp110 ( centriole cap ) , and IFT88 ( intraflagellar transport protein ) . For overexpression of exogenous Cep120 , we compared the ratio of exogenous and endogenous protein at the centrosome by immunostaining cells with anti-Myc and anti-Cep120 antibodies . We consider 1-2X the relative amount of exogenous Cep120 ( compared to endogenous protein ) ‘low’ overexpression , while ‘high’ overexpression ranged from 2-3X . Primary cilium assembly was determined by counting the percentage of control and Cep120-depleted cells that formed a cilium following serum-starvation . To stimulate sonic hedgehog ( Shh ) signaling , control and Cep120-depleted MEFs were treated with SAG ( 500 nM , Enzo Life Sciences , cat # ALX-270–426 M001 ) in low-serum medium for 28 hr . Cells were fixed in 2% PFA for 10 min at room temperature , washed and post-fixed in ice-cold methanol for 10 min . Samples were incubated with anti-Smoothened overnight at 4°C . Cells were washed and stained the next day for centrin ( to mark centrioles ) and acetylated α-tubulin ( to mark primary cilia ) . Ciliary length was determined using acetylated α-tubulin as marker , and smoothened fluorescence intensity was measured by selecting a region encompassing the length of each cilium . The ratio of smoothened per unit length cilium was determined using Excel ( Microsoft Office ) , and graphs generated with GraphPad Prism 7 . 02 Software ( GraphPad Software Inc . , La Jolla , CA , USA ) . More than 130 cilia were scored per experiment , and averages determined from two independent experiments . For analysis of microtubule density during regrowth , serum-starved MEFs were treated with 0 . 1 μg/mL nocodazole ( SIGMA , cat # M1404 ) for 1 hr on ice to depolymerize microtubules . Coverslips were washed three times , incubated with 37°C low-serum growth medium to allow microtubule regrowth , and then fixed at 0 , 2 and 4 min intervals . Cells were stained with anti-α-tubulin to visualize microtubule asters , centrin to visualize centrioles , and EB-1 to count microtubule +tips . The average diameter/radius of growing microtubule asters was determined by drawing a circular ROI centered on the centrosome , and the distal end of the longest microtubule tip in each cell . In parallel , the number of EB-1-positive foci emanating from the growing microtubule aster was determined . More than 150 cells were scored per experiment , and the average values quantified from three independent experiments . To obtain whole-cell extracts for immunoblotting , quiescent cells were washed in PBS and lysed by the addition of SDS sample buffer . Proteins were resolved on 6–8% SDS-PAGE gels , depending on protein size . Immunoblots were incubated with primary antibodies overnight at 4°C ( see Supplementary File 1 ) . Species-specific horseradish peroxidase-conjugated secondary antibodies were: mouse anti-Rabbit-HRP ( 1:10 , 000; Sigma , cat # R3155 ) , donkey anti-Mouse-HRP ( 1:10 , 000 , Jackson Immuno Research , cat # 715-035-150 ) , donkey anti-Guinea pig-HRP ( 1:10 , 000; Jackson Immuno Research , cat # 706-035-148 ) and AffiniPure donkey anti-Rat-HRP ( 1:10 , 000; Jackson Immuno Research , cat # 712-035-153 ) . Blots were imaged on a C-DiGit Blot Scanner ( LI-COR , cat # 3600–000 ) , and signal intensities for each protein quantified and normalized to actin . Two independent assays were utilized to establish the cell cycle state of control and Cep120-depleted cells: ( 1 ) quantitation of DNA content was performed by flow cytometry . MEF cells were transfected with Cep120 or control siRNA , and cycling cells harvested after 24 hr . Alternatively , siRNA-transfected cells were incubated in low-serum medium for 48 hr ( quiescence stage ) . Cells were pelleted , resuspended in 0 . 5 ml DPBS , and permeabilized with 70% ice-cold ethanol for 2 hr . Samples were washed in cold PBS and resuspended in 0 . 5 mL of 20 μg/ml Propidium Iodide ( Life Technologies , cat # P3566 ) supplemented with 0 . 1% Triton X-100 and 2 μg/ml RNase A ( Sigma , cat # R4875 ) , and incubated for 30 min . at RT . Before FACS analysis , cell aggregates were removed by passaging through a filter ( CellTrics , 50 µm strainer , cat # 04-004-2327 ) . At least 10 , 000 cells/sample were analyzed on a digitally modified DxP FACScan ( BD Biosciences , San Jose , CA and Cytek Development , Fremont , CA ) and the data analyzed using FlowJo software version 10 . 2 ( FlowJo , LLC , Ashland , OR ) ; ( 2 ) Control and Cep120-depleted MEFs were serum-starved for 48 hr to induce quiescence , fixed and stained for the proliferation marker Ki-67 . The fraction of Ki-67-positive cells was determined by counting more than 100 cells per experiment , and average values determined from three independent experiments . Data from fluorescence intensity measurements , number of centriolar foci , centriolar satellite localization and ciliogenesis assays were examined by unpaired two-tailed Student’s t-test ( p<0 . 05 ) . Data from Shh assays were examined by one-way analysis of variance ( ANOVA ) for significance ( p<0 . 05 ) . When significant differences were detected , treatment means were further analyzed by Tukey’s honest significant difference . All graphs were created using either Excel or GraphPad Prism 7 . 02 software . Data are reported as mean ± SEM .
The two centrioles of the centrosome in quiescent cells are inherently asymmetric structures that differ in age , morphology and function . How these asymmetric properties are established and maintained during quiescence remains unknown . Here , we show that a daughter centriole-associated ciliopathy protein , Cep120 , plays a critical inhibitory role at daughter centrioles . Depletion of Cep120 in quiescent mouse and human cells causes accumulation of pericentriolar material ( PCM ) components including pericentrin , Cdk5Rap2 , ninein and Cep170 . The elevated PCM levels result in increased microtubule-nucleation activity at the centrosome . Consequently , loss of Cep120 leads to aberrant dynein-dependent trafficking of centrosomal proteins , dispersal of centriolar satellites , and defective ciliary assembly and signaling . Our results indicate that Cep120 helps to maintain centrosome homeostasis by inhibiting untimely maturation of the daughter centriole , and defines a potentially new molecular defect underlying the pathogenesis of ciliopathies such as Jeune Asphyxiating Thoracic Dystrophy and Joubert syndrome .
Among the countless components of an animal cell , microtubules perform many important roles . These hollow filaments support the cell’s shape and help to transport different materials around within it . They also form a hair-like projection on the cell surface called the primary cilium , which helps the cell sense its environment . Most microtubules in an animal cell are organized by a structure called the centrosome , which has two smaller cylindrical structures called centrioles at its core . In cells that are not dividing , these two centrioles are different in age . The older of the two centrioles was assembled at least two cell divisions ago and is commonly called the “mother” centriole . The younger one , which was assembled the previous time the cell divided , is called the “daughter” centriole . Most activities at the centrosome are controlled by the mother centriole . For example , the mother centriole contains protein complexes called appendages that allow it to dock at the cell surface and build the cilium . The mother centriole also contains a complex of proteins called the pericentriolar material , which helps it assemble microtubules and anchor them in place . In contrast , the daughter centriole lacks appendages , does not form a cilium , has less pericentriolar material and so assembles fewer microtubules . Why the daughter centriole cannot recruit these protein complexes remains a mystery . One possibly important difference between mother and daughter centrioles is that daughter centrioles in non-dividing cells have much higher levels of a protein called Cep120 . Now , Betleja et al . have studied the role of this protein in more detail . Experiments with mouse and human cells show that Cep120 plays an important inhibitory role at the daughter centriole . When the production of Cep120 was blocked , more pericentriolar material associated with the daughter centriole , and more microtubules were assembled by the centrosome . This interfered with the movement of other proteins to the centrosome , which ultimately disrupted both the centrosome’s ability to assemble cilia and the cell’s ability to sense its environment . The findings of Betleja et al . show that a Cep120-dependent mechanism actively regulates the centrosome’s function in non-dividing cells . These experiments uncover a potentially new type of molecular defect that may be responsible for diseases caused by faulty cilia , such as Joubert Syndrome and Jeune Asphyxiating Thoracic Dystrophy . The next challenge will be to understand how Cep120 inhibits the levels of pericentriolar material only at the daughter centriole but not the mother centriole .