Honors & Awards
Early Career Life Scientist Award, American Society for Cell Biology (2011)
Searle Scholar, Searle Scholar Program (2010-2013)
Fellowship Award in the Neurosciences, The Esther A. & Joseph Klingenstein Fund (2009-2012)
Scientist Development Award, American Heart Association (2009-2012)
Basil O'Connor Award, March of Dimes (2009-2011)
Career Development Award, AACR-Pancreatic Cancer Action Network (2009-2011)
Sloan Research Fellowship, Alfred P. Sloan Foundation (2009-2011)
Frederick E. Terman Fellow, Stanford University (2008-2011)
Postdoctoral Fellowship, Damon Runyon Cancer Research Foundation (2003-2005)
Ph.D. Fellowship, Boehringer Ingelheim Foundation (1998-2001)
Postdoc, Genentech, Cell Biology and Biochemistry (2007)
Ph.D., UC Berkeley and Université Paris-Orsay, Molecular and Cellular Biology (2001)
B.Sc., Ecole Normale Supérieure (Paris), Biology and Biochemistry (1997)
Current Research and Scholarly Interests
Our laboratory investigates the molecular principles of primary cilium assembly and function. Shaped like an antenna projecting out of the cell, the primary cilium exposes receptors for diverse stimuli (like platelet-derived growth factors, Hedgehog morphogens and olfactory cues) and concentrates their downstream signaling machinery to send specific molecular responses back into the cell. Despite these fascinating properties, the cilium has historically been the least studied cellular organelle and most of the fundamental questions concerning cilium biogenesis and function remain unanswered. Nowhere is our knowledge gap more striking than for the ciliopathies, a class of inherited disorders of the cilium whose central features include obesity, kidney malformations and retinal degeneration. We thus intend to uncover the molecular machines that build cilia and convey information into and out of cilia by applying proteomics, cellular imaging and in vitro reconstitution assays to entry points provided by human genetics.
Independent Studies (4)
- Directed Reading in Molecular and Cellular Physiology
MCP 299 (Aut, Win, Spr, Sum)
- Graduate Research
MCP 399 (Aut, Win, Spr, Sum)
- Medical Scholars Research
MCP 370 (Aut, Win, Spr, Sum)
- Undergraduate Research
MCP 199 (Aut, Spr, Sum)
- Directed Reading in Molecular and Cellular Physiology
Graduate and Fellowship Programs
Proteomics of Primary Cilia by Proximity Labeling
2015; 35 (4): 497-512
While cilia are recognized as important signaling organelles, the extent of ciliary functions remains unknown because of difficulties in cataloguing proteins from mammalian primary cilia. We present a method that readily captures rapid snapshots of the ciliary proteome by selectively biotinylating ciliary proteins using a cilia-targeted proximity labeling enzyme (cilia-APEX). Besides identifying known ciliary proteins, cilia-APEX uncovered several ciliary signaling molecules. The kinases PKA, AMPK, and LKB1 were validated as bona fide ciliary proteins and PKA was found to regulate Hedgehog signaling in primary cilia. Furthermore, proteomics profiling of Ift27/Bbs19 mutant cilia correctly detected BBSome accumulation inside Ift27(-/-) cilia and revealed that β-arrestin 2 and the viral receptor CAR are candidate cargoes of the BBSome. This work demonstrates that proximity labeling can be applied to proteomics of non-membrane-enclosed organelles and suggests that proteomics profiling of cilia will enable a rapid and powerful characterization of ciliopathies.
View details for DOI 10.1016/j.devcel.2015.10.015
View details for Web of Science ID 000365099300013
View details for PubMedID 26585297
- The Intraflagellar Transport Protein IFT27 Promotes BBSome Exit from Cilia through the GTPase ARL6/BBS3 DEVELOPMENTAL CELL 2014; 31 (3): 265-278
The oral-facial-digital syndrome gene C2CD3 encodes a positive regulator of centriole elongation
2014; 46 (8): 905-911
Centrioles are microtubule-based, barrel-shaped structures that initiate the assembly of centrosomes and cilia. How centriole length is precisely set remains elusive. The microcephaly protein CPAP (also known as MCPH6) promotes procentriole growth, whereas the oral-facial-digital (OFD) syndrome protein OFD1 represses centriole elongation. Here we uncover a new subtype of OFD with severe microcephaly and cerebral malformations and identify distinct mutations in two affected families in the evolutionarily conserved C2CD3 gene. Concordant with the clinical overlap, C2CD3 colocalizes with OFD1 at the distal end of centrioles, and C2CD3 physically associates with OFD1. However, whereas OFD1 deletion leads to centriole hyperelongation, loss of C2CD3 results in short centrioles without subdistal and distal appendages. Because C2CD3 overexpression triggers centriole hyperelongation and OFD1 antagonizes this activity, we propose that C2CD3 directly promotes centriole elongation and that OFD1 acts as a negative regulator of C2CD3. Our results identify regulation of centriole length as an emerging pathogenic mechanism in ciliopathies.
View details for DOI 10.1038/ng.3031
View details for Web of Science ID 000339704400023
An in vitro assay for entry into cilia reveals unique properties of the soluble diffusion barrier
JOURNAL OF CELL BIOLOGY
2013; 203 (1): 129-147
Specific proteins are concentrated within primary cilia, whereas others remain excluded. To understand the mechanistic basis of entry into cilia, we developed an in vitro assay using cells in which the plasma membrane was permeabilized, but the ciliary membrane was left intact. Using a diffusion-to-capture system and quantitative analysis, we find that proteins >9 nm in diameter (∼100 kD) are restricted from entering cilia, and we confirm these findings in vivo. Interference with the nuclear pore complex (NPC) or the actin cytoskeleton in permeabilized cells demonstrated that the ciliary diffusion barrier is mechanistically distinct from those of the NPC or the axon initial segment. Moreover, applying a mass transport model to this system revealed diffusion coefficients for soluble and membrane proteins within cilia that are compatible with rapid exploration of the ciliary space in the absence of active transport. Our results indicate that large proteins require active transport for entry into cilia but not necessarily for movement inside cilia.
View details for DOI 10.1083/jcb.201212024
View details for Web of Science ID 000325742200013
View details for PubMedID 24100294
Single molecule imaging reveals a major role for diffusion in the exploration of ciliary space by signaling receptors.
The dynamic organization of signaling cascades inside primary cilia is key to signal propagation. Yet little is known about the dynamics of ciliary membrane proteins besides a possible role for motor-driven Intraflagellar Transport (IFT). To characterize these dynamics, we imaged single molecules of Somatostatin Receptor 3 (SSTR3, a GPCR) and Smoothened (Smo, a Hedgehog signal transducer) in the ciliary membrane. While IFT trains moved processively from one end of the cilium to the other, single SSTR3 and Smo underwent mostly diffusive behavior interspersed with short periods of directional movements. Statistical subtraction of instant velocities revealed that SSTR3 and Smo spent less than a third of their time undergoing active transport. Finally, SSTR3 and IFT movements could be uncoupled by perturbing either membrane protein diffusion or active transport. Thus ciliary membrane proteins move predominantly by diffusion, and attachment to IFT trains is transient and stochastic rather than processive or spatially determined. DOI:http://dx.doi.org/10.7554/eLife.00654.001.
View details for DOI 10.7554/eLife.00654
View details for PubMedID 23930224
The Conserved Bardet-Biedl Syndrome Proteins Assemble a Coat that Traffics Membrane Proteins to Cilia
2010; 141 (7): 1208-U198
The BBSome is a complex of Bardet-Biedl Syndrome (BBS) proteins that shares common structural elements with COPI, COPII, and clathrin coats. Here, we show that the BBSome constitutes a coat complex that sorts membrane proteins to primary cilia. The BBSome is the major effector of the Arf-like GTPase Arl6/BBS3, and the BBSome and GTP-bound Arl6 colocalize at ciliary punctae in an interdependent manner. Strikingly, Arl6(GTP)-mediated recruitment of the BBSome to synthetic liposomes produces distinct patches of polymerized coat apposed onto the lipid bilayer. Finally, the ciliary targeting signal of somatostatin receptor 3 needs to be directly recognized by the BBSome in order to mediate targeting of membrane proteins to cilia. Thus, we propose that trafficking of BBSome cargoes to cilia entails the coupling of BBSome coat polymerization to the recognition of sorting signals by the BBSome.
View details for DOI 10.1016/j.cell.2010.05.015
View details for Web of Science ID 000279148100017
View details for PubMedID 20603001
A core complex of BBS proteins cooperates with the GTPase Rab8 to promote ciliary membrane biogenesis
2007; 129 (6): 1201-1213
Primary cilium dysfunction underlies the pathogenesis of Bardet-Biedl syndrome (BBS), a genetic disorder whose symptoms include obesity, retinal degeneration, and nephropathy. However, despite the identification of 12 BBS genes, the molecular basis of BBS remains elusive. Here we identify a complex composed of seven highly conserved BBS proteins. This complex, the BBSome, localizes to nonmembranous centriolar satellites in the cytoplasm but also to the membrane of the cilium. Interestingly, the BBSome is required for ciliogenesis but is dispensable for centriolar satellite function. This ciliogenic function is mediated in part by the Rab8 GDP/GTP exchange factor, which localizes to the basal body and contacts the BBSome. Strikingly, Rab8(GTP) enters the primary cilium and promotes extension of the ciliary membrane. Conversely, preventing Rab8(GTP) production blocks ciliation in cells and yields characteristic BBS phenotypes in zebrafish. Our data reveal that BBS may be caused by defects in vesicular transport to the cilium.
View details for DOI 10.1016/j.cell.2007.03.053
View details for Web of Science ID 000247390400022
View details for PubMedID 17574030
- Loss of the BBSome perturbs endocytic trafficking and disrupts virulence of Trypanosoma brucei PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 2016; 113 (3): 632-637
- Cytoplasmic Dynein Antagonists with Improved Potency and Isoform Selectivity ACS CHEMICAL BIOLOGY 2016; 11 (1): 53-60
Structural basis for Notch1 engagement of Delta-like 4
2015; 347 (6224): 847-853
Notch receptors guide mammalian cell fate decisions by engaging the proteins Jagged and Delta-like (DLL). The 2.3 angstrom resolution crystal structure of the interacting regions of the Notch1-DLL4 complex reveals a two-site, antiparallel binding orientation assisted by Notch1 O-linked glycosylation. Notch1 epidermal growth factor-like repeats 11 and 12 interact with the DLL4 Delta/Serrate/Lag-2 (DSL) domain and module at the N-terminus of Notch ligands (MNNL) domains, respectively. Threonine and serine residues on Notch1 are functionalized with O-fucose and O-glucose, which act as surrogate amino acids by making specific, and essential, contacts to residues on DLL4. The elucidation of a direct chemical role for O-glycans in Notch1 ligand engagement demonstrates how, by relying on posttranslational modifications of their ligand binding sites, Notch proteins have linked their functional capacity to developmentally regulated biosynthetic pathways.
View details for DOI 10.1126/science.1261093
View details for Web of Science ID 000349761100037
Analysis of soluble protein entry into primary cilia using semipermeabilized cells.
Methods in cell biology
2015; 127: 203-221
The primary cilium is a protrusion from the cell surface that serves as a specialized compartment for signal transduction. Many signaling factors are known to be dynamically concentrated within cilia and to require cilia for their function. Yet protein entry into primary cilia remains poorly understood. To enable a mechanistic analysis of soluble protein entry into cilia, we developed a method for semipermeabilization of mammalian cells in which the plasma membrane is permeabilized while the ciliary membrane remains intact. Using semipermeabilized cells as the basis for an in vitro diffusion-to-capture assay, we uncovered a size-dependent diffusion barrier that restricts soluble protein exchange between the cytosol and the cilium. The manipulability of this in vitro system enabled an extensive characterization of the ciliary diffusion barrier and led us to show that the barrier is mechanistically distinct from those at the axon initial segment and the nuclear pore complex. Because semipermeabilized cells enable a range of experimental perturbations that would not be easily feasible in intact cells, we believe this methodology will provide a unique resource for investigating primary cilium function in development and disease.
View details for DOI 10.1016/bs.mcb.2014.12.006
View details for PubMedID 25837393
Structural basis for membrane targeting of the BBSome by ARL6.
Nature structural & molecular biology
2014; 21 (12): 1035-1041
The BBSome is a coat-like ciliary trafficking complex composed of proteins mutated in Bardet-Biedl syndrome (BBS). A critical step in BBSome-mediated sorting is recruitment of the BBSome to membranes by the GTP-bound Arf-like GTPase ARL6. We have determined crystal structures of Chlamydomonas reinhardtii ARL6-GDP, ARL6-GTP and the ARL6-GTP-BBS1 complex. The structures demonstrate how ARL6-GTP binds the BBS1 β-propeller at blades 1 and 7 and explain why GTP- but not GDP-bound ARL6 can recruit the BBSome to membranes. Single point mutations in the ARL6-GTP-BBS1 interface abolish the interaction of ARL6 with the BBSome and prevent the import of BBSomes into cilia. Furthermore, we show that BBS1 with the M390R mutation, responsible for 30% of all reported BBS disease cases, fails to interact with ARL6-GTP, thus providing a molecular rationale for patient pathologies.
View details for DOI 10.1038/nsmb.2920
View details for PubMedID 25402481
How do cilia organize signalling cascades?
PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY B-BIOLOGICAL SCIENCES
2014; 369 (1650)
Cilia and flagella are closely related centriole-nucleated protrusions of the cell with roles in motility and signal transduction. Two of the best-studied signalling pathways organized by cilia are the transduction cascade for the morphogen Hedgehog in vertebrates and the mating pathway that initiates gamete fusion in the unicellular green alga Chlamydomonas reinhardtii. What is the role of cilia in these signalling transduction cascades? In both Hedgehog and mating pathways, all signalling intermediates have been found to localize to cilia, and, for some signalling factors, ciliary localization is regulated by pathway activation. Given a concentration factor of three orders of magnitude provided by translocating a protein into the cilium, the compartment model proposes that cilia act as miniaturized reaction tubes bringing signalling factors and processing enzymes in close proximity. On the other hand, the scaffolding model views the intraflagellar transport machinery, whose primary function is to build cilia and flagella, as a molecular scaffold for the mating transduction cascade at the flagellar membrane. While these models may coexist, it is hoped that a precise understanding of the mechanisms that govern signalling inside cilia will provide a satisfying answer to the question 'how do cilia organize signalling?'. This review covers the evidence supporting each model of signalling and outlines future directions that may address which model applies in given biological settings.
View details for DOI 10.1098/rstb.2013.0465
View details for Web of Science ID 000339646500015
View details for PubMedID 25047619
alpha-Tubulin K40 acetylation is required for contact inhibition of proliferation and cell-substrate adhesion
MOLECULAR BIOLOGY OF THE CELL
2014; 25 (12): 1854-1866
Acetylation of α-tubulin on lysine 40 marks long-lived microtubules in structures such as axons and cilia, and yet the physiological role of α-tubulin K40 acetylation is elusive. Although genetic ablation of the α-tubulin K40 acetyltransferase αTat1 in mice did not lead to detectable phenotypes in the developing animals, contact inhibition of proliferation and cell-substrate adhesion were significantly compromised in cultured αTat1(-/-) fibroblasts. First, αTat1(-/-) fibroblasts kept proliferating beyond the confluent monolayer stage. Congruently, αTat1(-/-) cells failed to activate Hippo signaling in response to increased cell density, and the microtubule association of the Hippo regulator Merlin was disrupted. Second, αTat1(-/-) cells contained very few focal adhesions, and their ability to adhere to growth surfaces was greatly impaired. Whereas the catalytic activity of αTAT1 was dispensable for monolayer formation, it was necessary for cell adhesion and restrained cell proliferation and activation of the Hippo pathway at elevated cell density. Because α-tubulin K40 acetylation is largely eliminated by deletion of αTAT1, we propose that acetylated microtubules regulate contact inhibition of proliferation through the Hippo pathway.
View details for DOI 10.1091/mbc.E13-10-0609
View details for Web of Science ID 000339651500005
View details for PubMedID 24743598
Exome sequencing of Bardet-Biedl syndrome patient identifies a null mutation in the BBSome subunit BBIP1 (BBS18)
JOURNAL OF MEDICAL GENETICS
2014; 51 (2): 132-136
Bardet-Biedl syndrome (BBS) is a recessive and genetically heterogeneous ciliopathy characterised by retinitis pigmentosa, obesity, kidney dysfunction, postaxial polydactyly, behavioural dysfunction and hypogonadism. 7 of the 17 BBS gene products identified to date assemble together with the protein BBIP1/BBIP10 into the BBSome, a protein complex that ferries signalling receptors to and from cilia.Exome sequencing performed on a sporadic BBS case revealed for the first time a homozygous stop mutation (NM_001195306: c.173T>G, p.Leu58*) in the BBIP1 gene. This mutation is pathogenic since no BBIP1 protein could be detected in fibroblasts from the patient, and BBIP1[Leu58*] is unable to associate with the BBSome subunit BBS4.These findings identify BBIP1 as the 18th BBS gene (BBS18) and suggest that BBSome assembly may represent a unifying pathomechanism for BBS.
View details for DOI 10.1136/jmedgenet-2013-101785
View details for Web of Science ID 000331191300009
View details for PubMedID 24026985
Effects of tubulin acetylation and tubulin acetyltransferase binding on microtubule structure
MOLECULAR BIOLOGY OF THE CELL
2014; 25 (2): 257-266
Tubulin undergoes posttranslational modifications proposed to specify microtubule subpopulations for particular functions. Most of these modifications occur on the C-termini of tubulin and may directly affect the binding of microtubule-associated proteins (MAPs) or motors. Acetylation of Lys-40 on α-tubulin is unique in that it is located on the luminal surface of microtubules, away from the interaction sites of most MAPs and motors. We investigate whether acetylation alters the architecture of microtubules or the conformation of tubulin, using cryo-electron microscopy (cryo-EM). No significant changes are observed based on protofilament distributions or microtubule helical lattice parameters. Furthermore, no clear differences in tubulin structure are detected between cryo-EM reconstructions of maximally deacetylated or acetylated microtubules. Our results indicate that the effect of acetylation must be highly localized and affect interaction with proteins that bind directly to the lumen of the microtubule. We also investigate the interaction of the tubulin acetyltransferase, αTAT1, with microtubules and find that αTAT1 is able to interact with the outside of the microtubule, at least partly through the tubulin C-termini. Binding to the outside surface of the microtubule could facilitate access of αTAT1 to its luminal site of action if microtubules undergo lateral opening between protofilaments.
View details for DOI 10.1091/mbc.E13-07-0387
View details for Web of Science ID 000330022900005
View details for PubMedID 24227885
Cilia grow by taking a bite out of the cell.
2013; 27 (2): 126-127
Autophagy and primary cilium assembly have long been known to be induced by the same conditions in cultured cells. Two recent studies in Nature-Tang et al. (2013) and Pampliega et al. (2013)-link the two processes, suggesting that a specialized autophagy pathway near the basal body regulates cilium assembly.
View details for DOI 10.1016/j.devcel.2013.10.013
View details for PubMedID 24176638
alpha TAT1 catalyses microtubule acetylation at clathrin-coated pits
2013; 502 (7472): 567-?
In most eukaryotic cells microtubules undergo post-translational modifications such as acetylation of α-tubulin on lysine 40, a widespread modification restricted to a subset of microtubules that turns over slowly. This subset of stable microtubules accumulates in cell protrusions and regulates cell polarization, migration and invasion. However, mechanisms restricting acetylation to these microtubules are unknown. Here we report that clathrin-coated pits (CCPs) control microtubule acetylation through a direct interaction of the α-tubulin acetyltransferase αTAT1 (refs 8, 9) with the clathrin adaptor AP2. We observe that about one-third of growing microtubule ends contact and pause at CCPs and that loss of CCPs decreases lysine 40 acetylation levels. We show that αTAT1 localizes to CCPs through a direct interaction with AP2 that is required for microtubule acetylation. In migrating cells, the polarized orientation of acetylated microtubules correlates with CCP accumulation at the leading edge, and interaction of αTAT1 with AP2 is required for directional migration. We conclude that microtubules contacting CCPs become acetylated by αTAT1. In migrating cells, this mechanism ensures the acetylation of microtubules oriented towards the leading edge, thus promoting directional cell locomotion and chemotaxis.
View details for DOI 10.1038/nature12571
View details for Web of Science ID 000325988400058
View details for PubMedID 24097348
Structure of the alpha-tubulin acetyltransferase, alpha TAT1, and implications for tubulin-specific acetylation
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2012; 109 (48): 19655-19660
Protein acetylation is an important posttranslational modification with the recent identification of new substrates and enzymes, new links to disease, and modulators of protein acetylation for therapy. ?-Tubulin acetyltransferase (?TAT1) is the major ?-tubulin lysine-40 (K40) acetyltransferase in mammals, nematodes, and protozoa, and its activity plays a conserved role in several microtubule-based processes. Here, we present the X-ray crystal structure of the human ?TAT1/acetyl-CoA complex. Together with structure-based mutagenesis, enzymatic analysis, and functional studies in cells, we elucidate the catalytic mechanism and mode of tubulin-specific acetylation. We find that ?TAT1 has an overall fold similar to the Gcn5 histone acetyltransferase but contains a relatively wide substrate binding groove and unique structural elements that play important roles in ?-tubulin-specific acetylation. Conserved aspartic acid and cysteine residues play important catalytic roles through a ternary complex mechanism. ?TAT1 mutations have analogous effects on tubulin acetylation in vitro and in cells, demonstrating that it is the central determining factor of ?-tubulin K40 acetylation levels in vivo. Together, these studies provide general insights into distinguishing features between histone and tubulin acetyltransferases, and they have specific implications for understanding the molecular basis of tubulin acetylation and for developing small molecule modulators of microtubule acetylation for therapy.
View details for DOI 10.1073/pnas.1209357109
View details for Web of Science ID 000312313900037
View details for PubMedID 23071314
A Novel Protein LZTFL1 Regulates Ciliary Trafficking of the BBSome and Smoothened
2011; 7 (11)
Many signaling proteins including G protein-coupled receptors localize to primary cilia, regulating cellular processes including differentiation, proliferation, organogenesis, and tumorigenesis. Bardet-Biedl Syndrome (BBS) proteins are involved in maintaining ciliary function by mediating protein trafficking to the cilia. However, the mechanisms governing ciliary trafficking by BBS proteins are not well understood. Here, we show that a novel protein, Leucine-zipper transcription factor-like 1 (LZTFL1), interacts with a BBS protein complex known as the BBSome and regulates ciliary trafficking of this complex. We also show that all BBSome subunits and BBS3 (also known as ARL6) are required for BBSome ciliary entry and that reduction of LZTFL1 restores BBSome trafficking to cilia in BBS3 and BBS5 depleted cells. Finally, we found that BBS proteins and LZTFL1 regulate ciliary trafficking of hedgehog signal transducer, Smoothened. Our findings suggest that LZTFL1 is an important regulator of BBSome ciliary trafficking and hedgehog signaling.
View details for DOI 10.1371/journal.pgen.1002358
View details for Web of Science ID 000297264500013
View details for PubMedID 22072986
Give chance a chance
MOLECULAR BIOLOGY OF THE CELL
2011; 22 (21): 3919-3920
How did I get to become a cell biologist? Or, more generally, why do things happen the way they do? The answer provided by the philosopher Democritus and later adopted by Jacques Monod is "everything existing in the universe is the fruit of chance and necessity." While I read Monod's book Chance and Necessity as an undergraduate student, little did I appreciate the accuracy of this citation and how much of my scientific trajectory would be guided by chance.
View details for DOI 10.1091/mbc.E11-05-0453
View details for Web of Science ID 000296603300002
View details for PubMedID 22039061
Primary Cilia: How to Keep the Riff-Raff in the Plasma Membrane
2011; 21 (11): R434-R436
A recent report suggests that plasma membrane proteins are excluded from primary cilia via anchoring to the cortical actin cytoskeleton. These findings challenge the existence of a diffusion barrier at the base of the cilium.
View details for DOI 10.1016/j.cub.2011.04.039
View details for Web of Science ID 000291668100011
View details for PubMedID 21640903
Primary cilia membrane assembly is initiated by Rab11 and transport protein particle II (TRAPPII) complex-dependent trafficking of Rabin8 to the centrosome
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2011; 108 (7): 2759-2764
Sensory and signaling pathways are exquisitely organized in primary cilia. Bardet-Biedl syndrome (BBS) patients have compromised cilia and signaling. BBS proteins form the BBSome, which binds Rabin8, a guanine nucleotide exchange factor (GEF) activating the Rab8 GTPase, required for ciliary assembly. We now describe serum-regulated upstream vesicular transport events leading to centrosomal Rab8 activation and ciliary membrane formation. Using live microscopy imaging, we show that upon serum withdrawal Rab8 is observed to assemble the ciliary membrane in ?100 min. Rab8-dependent ciliary assembly is initiated by the relocalization of Rabin8 to Rab11-positive vesicles that are transported to the centrosome. After ciliogenesis, Rab8 ciliary transport is strongly reduced, and this reduction appears to be associated with decreased Rabin8 centrosomal accumulation. Rab11-GTP associates with the Rabin8 COOH-terminal region and is required for Rabin8 preciliary membrane trafficking to the centrosome and for ciliogenesis. Using zebrafish as a model organism, we show that Rabin8 and Rab11 are associated with the BBS pathway. Finally, using tandem affinity purification and mass spectrometry, we determined that the transport protein particle (TRAPP) II complex associates with the Rabin8 NH(2)-terminal domain and show that TRAPP II subunits colocalize with centrosomal Rabin8 and are required for Rabin8 preciliary targeting and ciliogenesis.
View details for DOI 10.1073/pnas.1018823108
View details for Web of Science ID 000287377000029
View details for PubMedID 21273506
The major alpha-tubulin K40 acetyltransferase alpha TAT1 promotes rapid ciliogenesis and efficient mechanosensation
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2010; 107 (50): 21517-21522
Long-lived microtubules found in ciliary axonemes, neuronal processes, and migrating cells are marked by ?-tubulin acetylation on lysine 40, a modification that takes place inside the microtubule lumen. The physiological importance of microtubule acetylation remains elusive. Here, we identify a BBSome-associated protein that we name ?TAT1, with a highly specific ?-tubulin K40 acetyltransferase activity and a catalytic preference for microtubules over free tubulin. In mammalian cells, the catalytic activity of ?TAT1 is necessary and sufficient for ?-tubulin K40 acetylation. Remarkably, ?TAT1 is universally and exclusively conserved in ciliated organisms, and is required for the acetylation of axonemal microtubules and for the normal kinetics of primary cilium assembly. In Caenorhabditis elegans, microtubule acetylation is most prominent in touch receptor neurons (TRNs) and MEC-17, a homolog of ?TAT1, and its paralog ?TAT-2 are required for ?-tubulin acetylation and for two distinct types of touch sensation. Furthermore, in animals lacking MEC-17, ?TAT-2, and the sole C. elegans K40?-tubulin MEC-12, touch sensation can be restored by expression of an acetyl-mimic MEC-12[K40Q]. We conclude that ?TAT1 is the major and possibly the sole ?-tubulin K40 acetyltransferase in mammals and nematodes, and that tubulin acetylation plays a conserved role in several microtubule-based processes.
View details for DOI 10.1073/pnas.1013728107
View details for Web of Science ID 000285521500055
View details for PubMedID 21068373
- Maxence Nachury: a transporting view of the primary cilium. Interview by Ben Short. journal of cell biology 2010; 191 (3): 436-437
A Septin Diffusion Barrier at the Base of the Primary Cilium Maintains Ciliary Membrane Protein Distribution
2010; 329 (5990): 436-439
In animal cells, the primary cilium transduces extracellular signals through signaling receptors localized in the ciliary membrane, but how these ciliary membrane proteins are retained in the cilium is unknown. We found that ciliary membrane proteins were highly mobile, but their diffusion was impeded at the base of the cilium by a diffusion barrier. Septin 2 (SEPT2), a member of the septin family of guanosine triphosphatases that form a diffusion barrier in budding yeast, localized at the base of the ciliary membrane. SEPT2 depletion resulted in loss of ciliary membrane protein localization and Sonic hedgehog signal transduction, and inhibited ciliogenesis. Thus, SEPT2 is part of a diffusion barrier at the base of the ciliary membrane and is essential for retaining receptor-signaling pathways in the primary cilium.
View details for DOI 10.1126/science.1191054
View details for Web of Science ID 000280196500036
View details for PubMedID 20558667
The perennial organelle: assembly and disassembly of the primary cilium
JOURNAL OF CELL SCIENCE
2010; 123 (4): 511-518
Primary cilia contain signaling receptors of diverse classes, and ciliary dysfunction results in a variety of developmental defects. Thus, primary cilia are thought to have an important role in sensing and transducing cellular signals. Although there is clear evidence demonstrating that these organelles are assembled and disassembled dynamically as cells progress through the cell cycle, the mechanisms by which the cell cycle controls the assembly and disassembly of the primary cilium remain poorly understood. In this Commentary, we review the basic cellular mechanisms that underlie the early stages of cilium assembly and discuss how the cell cycle communicates with the ciliation program. A commonly held view is that ciliation occurs exclusively in cells that have exited the cell cycle and entered quiescence or differentiation. However, this concept is at odds with the finding that, during development, many actively proliferating cells require cilia-mediated signaling pathways to instruct their developmental fate. Here, we reassess the quiescence-centric view of ciliation by reviewing historic and current literature. We discuss ample evidence that cilia are in fact present on many proliferating cells, and that a transient peak of ciliation before the G1-S transition might be tightly coupled to entry into the DNA replication phase. Finally, we touch on the relationship between the ciliation and cell-division cycles and the tissue distribution of primary cilia in order to highlight potential roles for the primary cilium in restraining cells from the hyperproliferative state that contributes to cancer.
View details for DOI 10.1242/jcs.061093
View details for Web of Science ID 000274594100003
View details for PubMedID 20144999
Trafficking to the Ciliary Membrane: How to Get Across the Periciliary Diffusion Barrier?
ANNUAL REVIEW OF CELL AND DEVELOPMENTAL BIOLOGY, VOL 26
2010; 26: 59-87
The primary cilium organizes numerous signal transduction cascades, and an understanding of signaling receptor trafficking to cilia is now emerging. A defining feature of cilia is the periciliary diffusion barrier that separates the ciliary and plasma membranes. Although lateral transport through this barrier may take place, polarized exocytosis to the base of the cilium has been the prevailing model for delivering membrane proteins to cilia. Key players for this polarized exocytosis model include the GTPases Rab8 and Rab11, the exocyst, and possibly the intraflagellar tranport machinery. In turn, the sorting of membrane proteins to cilia critically relies on the recognition of ciliary targeting signals by sorting machines such as the BBSome coat complex or the GTPase Arf4. Finally, some proteins need to exit from cilia, and ubiquitination may regulate this step. The stage is now set to dissect the interplay between signaling and regulated trafficking to and from cilia.
View details for DOI 10.1146/annurev.cellbio.042308.113337
View details for Web of Science ID 000284856700003
View details for PubMedID 19575670
- The BBSome CURRENT BIOLOGY 2009; 19 (12): R472-R473
Constructing and Deconstructing Roles for the Primary Cilium in Tissue Architecture and Cancer
2009; 94: 299-313
Primary cilia are exquisitely designed sensory machines that have evolved at least three distinct sensory modalities to monitor the extracellular environment. The presence and activation of growth factor, morphogen, and hormone receptors within the confines of the ciliary membrane, the intrinsic physical relationship between the ciliary axoneme and the centriole, and the preferential assembly of primary cilia on the apical surfaces of tissue epithelia highlight the importance of this organelle in the establishment and maintenance of tissue architecture and homeostasis. Accordingly, recent studies begin to suggest roles for these organelles in oncogenesis and tumor suppression. Here, we review the sensory properties of primary cilia, assess the "history" of the primary cilium in cancer, and draw upon recent findings in a discussion of how the primary cilium may influence tissue architecture and neoplasia.
View details for DOI 10.1016/S0091-679X(08)94015-2
View details for Web of Science ID 000272975500016
View details for PubMedID 20362097
A BBSome Subunit Links Ciliogenesis, Microtubule Stability, and Acetylation
2008; 15 (6): 854-865
Primary cilium dysfunction affects the development and homeostasis of many organs in Bardet-Biedl syndrome (BBS). We recently showed that seven highly conserved BBS proteins form a stable complex, the BBSome, that functions in membrane trafficking to and inside the primary cilium. We have now discovered a BBSome subunit that we named BBIP10. Similar to other BBSome subunits, BBIP10 localizes to the primary cilium, BBIP10 is present exclusively in ciliated organisms, and depletion of BBIP10 yields characteristic BBS phenotypes in zebrafish. Unexpectedly, BBIP10 is required for cytoplasmic microtubule polymerization and acetylation, two functions not shared with any other BBSome subunits. Strikingly, inhibition of the tubulin deacetylase HDAC6 restores microtubule acetylation in BBIP10-depleted cells, and BBIP10 physically interacts with HDAC6. BBSome-bound BBIP10 may therefore function to couple acetylation of axonemal microtubules and ciliary membrane growth.
View details for DOI 10.1016/j.devcel.2008.11.001
View details for Web of Science ID 000261631500009
View details for PubMedID 19081074
Tandem affinity purification of the BBSome, a critical regulator of Rab8 in ciliogenesis
SMALL GTPASES IN DISEASE, PT B
2008; 439: 501-?
Bardet-Biedl syndrome (BBS) is a hereditary disorder whose symptoms include obesity, retinal degeneration, and kidney cysts. Intriguingly, the cellular culprit of BBS seems to lie in the primary cilium, a "cellular antenna" used by a number of signaling pathways. Yet, despite the identification of 12 BBS genes, a consistent molecular pathway for BBS had so far remained elusive. The recent discovery of a stable complex of seven BBS proteins (the BBSome) considerably simplifies the apparent molecular complexity of BBS and provides a clear insight into the molecular basis of BBS. Most tellingly, the BBSome associates with Rabin8, the guanine nucleotide exchange factor for the small GTPase Rab8, and Rab8-GTP enters the primary cilium to promote extension of the ciliary membrane. Thus, BBS is likely caused by defects in vesicular transport to the primary cilium. This chapter describes methods used to purify the BBSome using a tandem affinity purification method and presents a variation of this technique to demonstrate the existence of a stable complex of BBS proteins by sucrose gradient fractionation. When combined with state-of-the art mass spectrometry, these methods can provide a nearly complete BBSome interactome containing factors such as Rabin8.
View details for DOI 10.1016/S0076-6879(07)00434-X
View details for Web of Science ID 000255021300034
View details for PubMedID 18374185
- A Rae1-containing ribonucleoprotein complex is required for mitotic spindle assembly Cell 2005; 121 (2): 223-234
Xenopus Cdc14 alpha/beta are localized to the nucleolus and centrosome and are required for embryonic cell division
BMC CELL BIOLOGY
The dual specificity phosphatase Cdc14 has been shown to be a critical regulator of late mitotic events in several eukaryotes, including S. cerevisiae, S. pombe. C. elegans and H. sapiens. However, Cdc14 homologs have clearly evolved to regulate distinct cellular processes and to respond to regulatory signals important for these processes. The human paralogs hCdc14A and B are the only vertebrate Cdc14 homologues studied to date, but their functions are not well understood. Therefore, it is of great interest to examine the function Cdc14 homologs in other vertebrate species.We identified two open reading frames from Xenopus laevis closely related to human Cdc14A, called XCdc14alpha and XCdc14beta, although no obvious paralog of the hCdc14B was found. To begin a functional characterization of Xcdc14alpha and XCdc14beta, we raised polyclonal antibodies against a conserved region. These antibodies stained both the nucleolus and centrosome in interphase Xenopus tissue culture cells, and the mitotic centrosomes. GFP-tagged version of XCdc14alpha localized to the nucleulus and GFP-XCdc14beta localized to the centrosome, although not exclusively. XCdc14alpha was also both meiotically and mitotically phosphorylated. Injection of antibodies raised against a conserved region of XCdc14/beta into Xenopus embryos at the two-cell stage blocked division of the injected blastomeres, suggesting that activities of XCdc14alpha/beta are required for normal cell division.These results provide evidence that XCdc14alpha/beta are required for normal cellular division and are regulated by at least two mechanisms, subcellular localization and possibly phosphorylation. Due to the high sequence conservation between Xcdc14alpha and hCdc14A, it seems likely that both mechanisms will contribute to regulation of Cdc14 homologs in vertebrates.
View details for DOI 10.1186/1471-2121-5-27
View details for Web of Science ID 000222850900001
View details for PubMedID 15251038
Importin beta is a mitotic target of the small GTPase ran in spindle assembly
2001; 104 (1): 95-106
The GTPase Ran has recently been shown to stimulate microtubule polymerization in mitotic extracts, but its mode of action is not understood. Here we show that the mitotic role of Ran is largely mediated by the nuclear transport factor importin beta. Importin beta inhibits spindle formation in vitro and in vivo and sequesters an aster promoting activity (APA) that consists of multiple, independent factors. One component of APA is the microtubule-associated protein NuMA. NuMA and other APA components are discharged from importin beta by RanGTP and induce spindle-like structures in the absence of centrosomes, chromatin, or Ran. We propose that RanGTP functions in mitosis as in interphase by locally releasing cargoes from transport factors. In mitosis, this promotes spindle assembly by organizing microtubules in the vicinity of chromosomes.
View details for Web of Science ID 000166882300010
View details for PubMedID 11163243
The direction of transport through the nuclear pore can be inverted
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1999; 96 (17): 9622-9627
Transport of macromolecules across the nuclear envelope is an active process that depends on soluble factors including the GTPase Ran. Ran-GTP is predominantly located in the nucleus and has been shown to regulate cargo binding and release of import and export receptors in their respective target compartments. Recently, it was shown that transport of receptor-cargo complexes across the nuclear pore complex (NPC) does not depend on GTP-hydrolysis by Ran; however, the mechanism of translocation is still poorly understood. Here, we show that the direction of transport through the NPC can be inverted in the presence of high concentrations of cytoplasmic Ran-GTP. Under these conditions, two different classes of export cargoes are transported into the nucleus in the absence of GTP hydrolysis. The inverted transport is very rapid and can be blocked by known inhibitors of nuclear protein export. These results suggest that the NPC functions as a facilitated transport channel, allowing the selective translocation of receptor-cargo complexes. We conclude that the directionality of nucleocytoplasmic transport is determined mainly by the compartmentalized distribution of Ran-GTP.
View details for Web of Science ID 000082098500034
View details for PubMedID 10449743
Cloning and characterization of hSRP1 gamma, a tissue-specific nuclear transport factor
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1998; 95 (2): 582-587
Nuclear import of proteins containing a nuclear localization signal (NLS) is dependent on the presence of a cytoplasmic NLS receptor, the GTPase Ran, and p10/ NTF2. The NLS receptor is a heterodimeric proteins consisting of subunits of approximately 60 and 97 kDa, which have been termed importin alpha/beta, karyopherin alpha/beta, or PTAC 58/ 97. Members of the 60-kDa/importin alpha subunit family directly bind to the NLS motif and have been shown to function as adaptors that tether NLS-containing proteins to the p97/ importin beta subunit and to the downstream transport machinery. Herein we report the identification and characterization of hSRP1 gamma, a human importin alpha homologue. The hSRP1 gamma protein is around 45% identical to the previously identified human importin alpha homologues hSRP1 alpha/Rch1 and NPI/ hSRP1. hSRP1 gamma can form a complex with importin beta and is able to mediate import of a BSA-NLS substrate in an in vitro nuclear import system. Interestingly, hSRP1 gamma shows a very selective expression pattern and is most abundantly expressed in skeletal muscle, representing more than 1% of the total protein in this tissue. A potential role for hSRP1 gamma in tissue-specific transport events is discussed.
View details for Web of Science ID 000071606000028
View details for PubMedID 9435235