Honors & Awards
Fellowship Award, Damon Runyon Cancer Research Fund (2006)
Howard Temin Pathway to Independance Award (K99/R00), NCI/NIH (2007)
Young Investigator Award, American Society for Clinical Oncology (2007)
Josephine Q. Berry Faculty Scholar in Cancer Research, Stanford University (2009)
Martin D. Abeloff Scholar, V Foundation for Cancer Research (2009-2011)
Distinguished Scientist Award, Sontag Foundation (2010)
Basil O' Connor Starter Scholar Award, March of Dimes Foundation (2010-2012)
Stand Up To Cancer Innovation Research Grant, American Association for Cancer Research (2010-2013)
NIH Director's New Innovator Award, NIH (2012)
Maximizing Investigators' Research Award (MIRA), NIGMS/NIH (2016)
Fellowship, Stanford Hospital, Medical Oncology (2008)
Residency, Stanford Hospital, Internal Medicine (2004)
Ph.D., Harvard Medical School, Cell Biology (2002)
M.D., Harvard Medical School (2002)
A.B., Harvard University, Biochemical Sciences (1994)
Current Research and Scholarly Interests
Areas of research in the Rohatgi Lab:
1. The Hedgehog and WNT pathways, two cell-cell communication systems that regulate the formation of most tissues during development. These same pathways play central roles in tissue stem-cell function and organ regeneration in adults. Defects in these systems are associated with degenerative conditions and cancer.
2. Signal transduction at the primary cilium and the mechanism of cilia-associated human diseases. Primary cilia are solitary hair-like projections found on most cells in our bodies that function as critical hubs for signal transduction pathways (such as Hedgehog). Over fifty human genetic diseases, called “ciliopathies,” are caused by defects in cilia. Patients with ciliopathies can show phenotypes in nearly all organ systems, suffering from abnormalities ranging from birth defects to obesity.
3. Regulation of signaling pathways by endogenous lipids. The landscape of endogenous small-molecules and their biological functions remains a terra incognita, one that provides many opportunities to discover new regulatory layers in signaling pathways.
4. Phase separation in signal transduction. The formation of reversible, membrane-less compartments in cells by the segregation of proteins into liquid phases, hydrogels or amyloid-like assemblies is an emerging principle of cellular organization, with broad implications for areas that include signaling at the cell surface, stress response pathways, and neuro-degeneration.
5. Cellular responses to osmolar stresses. Maintaining a stable concentration of intracellular macromolecules and ions in a fluctuating environment is a universal challenge to homeostasis faced by all cells. In our own bodies, cells of the kidney and cells in inflammatory environments face tissue osmolality levels that are 3-fold higher than blood!
1. CRISPR/Cas9-based genome-wide, loss-of-function screens targeting signaling pathways.
•Enhancer and suppressor screens to comprehensively identify pathway components.
•Synthetic screens to identify the genetic vulnerabilities of cells carrying mutations in human oncogenes and tumor suppressor genes.
•Screens based on complex, physiological read-outs of signaling, such as differentiation.
2. Protein biochemistry: proteomics, structure-guided analysis, activity-based purification and cell-free reconstitution of signaling reactions in extracts and using purified components.
3. Chemical Biology: new probes to assay the interactions between proteins and small molecules.
4. Imaging: Live-cell imaging with innovative optical probes and genetically-encoded reporters to monitor the temporal and spatial progression of signaling, the quantitative phase separation behavior of proteins, and the dynamic, signal-regulated trafficking of proteins.
5. Collaborations: With experts in structural biology (Christian Siebold, Oxford, Elife 2013, 2016 and Nature 2016), genome-wide screening (Jan Carette, Stanford, Elife and Cancer Research 2016), protein and genome evolution (L. Aravind, NIH, Dev Cell 2014 and 2018), and developmental biology (James Briscoe, Francis Crick Institute, Dev Cell 2018).
Identification of Circulating Tumor Cells in the Peripheral Blood of Lung Cancer Patients
The primary aim of this study is to determine whether we can identify human lung cancer tumor cells in the peripheral blood of lung cancer patients.
Molecular Analysis of Thoracic Malignancies
A research study to learn about the biologic features of cancer development, growth, and spread. We are studying components of blood, tumor tissue, normal tissue, and other fluids, such as urine, cerebrospinal fluid, abdominal or chest fluid in patients with cancer. Our analyses of blood, tissue, and/or fluids may lead to improved diagnosis and treatment of cancer by the identification of markers that predict clinical outcome, markers that predict response to specific therapies, and the identification of targets for new therapies.
Erlotinib in Patients With Resected, Early Stage NSCLC With Confirmed Mutations in the EGFR
In this research study erlotinib will be given to eligible participants whose lung cancer has been removed by surgery. Eligible patients have adenocarcinoma, a type of non-small lung cancer, and must have 1 or more of the following characteristics: be female, be of Asian or Pacific Rim descent and/or be a never smoker. The potential participant's tumor will be examined for Epidermal growth factor (EGFR) mutations. EGFR is a protein that is overexpressed in most non-small cell lung cancers. Some EGFR has been found to have specific mutations and the participant must have one of these mutations in his tumor. Erlotinib blocks this protein and may control tumor growth and increase survival. Previous research has shown that erlotinib is most effective for people who have these specific mutations in the EGFR.
Stanford is currently not accepting patients for this trial. For more information, please contact Lei Shura, 650-723-2312.
Erlotinib Plus Tivantinib (ARQ 197) Versus Single Agent Chemotherapy in Locally Advanced or Metastatic Non-Small Cell Lung Cancer
The purpose of this study is to evaluate progression-free survival among subjects with KRAS mutation positive Non-Small Cell Lung Cancer (NSCLC) treated with erlotinib plus tivantinib (ARQ 197) compared to single agent chemotherapy.
Stanford is currently not accepting patients for this trial. For more information, please contact Lei Shura, 650-723-2312.
Erlotinib With or Without Hydroxychloroquine in Chemo-Naive Advanced NSCLC and (EGFR) Mutations
The purpose of this research study is to learn if adding hydroxychloroquine (HCQ) to erlotinib helps treat non-small cell lung cancer (NSCLC). Another goal of this research study is to learn more about NSCLC and how it may respond to study treatment. Erlotinib (Tarceva) is a type of drug called a tyrosine kinase inhibitor (TKI). TKIs block a protein called the epidermal growth factor receptor (EGFR). EGFR may control tumor growth and tumor cell survival. However, although TKI drugs can work for some lung cancer patients for a period of time, eventually the tumor finds a way to resist or counteract the TKI treatment and it begins to grow again. Hydroxychloroquine (HCQ) is a drug approved by the FDA for treating malaria, rheumatoid arthritis, and several other diseases. Laboratory research suggests that when HCQ is given with a TKI, it may help delay or prevent TKI resistance from developing.
Stanford is currently not accepting patients for this trial. For more information, please contact Zeina Babetty, (650) 723 - 2983.
- Advanced Cell Biology
BIO 214, BIOC 224, MCP 221 (Win)
- Development of Thesis Research
BIOC 350 (Aut)
Independent Studies (14)
- Directed Reading in Biochemistry
BIOC 299 (Aut, Win, Spr, Sum)
- Directed Reading in Cancer Biology
CBIO 299 (Aut, Win, Spr)
- Directed Reading in Medicine
MED 299 (Aut, Sum)
- Early Clinical Experience in Medicine
MED 280 (Aut, Sum)
- Graduate Research
CBIO 399 (Aut, Win, Spr)
- Graduate Research
MED 399 (Aut, Sum)
- Graduate Research and Special Advanced Work
BIOC 399 (Aut, Win, Spr, Sum)
- Medical Scholars Research
BIOC 370 (Aut, Win, Spr, Sum)
- Medical Scholars Research
MED 370 (Aut, Sum)
- Out-of-Department Advanced Research Laboratory in Experimental Biology
BIO 199X (Aut, Win, Spr)
- Teaching in Cancer Biology
CBIO 260 (Spr)
- The Teaching of Biochemistry
BIOC 221 (Aut, Win, Spr, Sum)
- Undergraduate Research
BIOC 199 (Aut, Win, Spr, Sum)
- Undergraduate Research
MED 199 (Aut, Sum)
- Directed Reading in Biochemistry
Prior Year Courses
- Advanced Cell Biology
BIO 214, BIOC 224, MCP 221 (Win)
- Biochemistry Bootcamp
BIOC 202 (Aut)
- Development of Thesis Research
BIOC 350 (Aut)
- Advanced Cell Biology
Postdoctoral Faculty Sponsor
G protein-coupled receptors control the sensitivity of cells to the morphogen Sonic Hedgehog.
2018; 11 (516)
The morphogen Sonic Hedgehog (SHH) patterns tissues during development by directing cell fates in a concentration-dependent manner. The SHH signal is transmitted across the membrane of target cells by the heptahelical transmembrane protein Smoothened (SMO), which activates the GLI family of transcription factors through a mechanism that is undefined in vertebrates. Using CRISPR-edited null alleles and small-molecule inhibitors, we systematically analyzed the epistatic interactions between SMO and three proteins implicated in SMO signaling: the heterotrimeric G protein subunit GαS, the G protein-coupled receptor kinase 2 (GRK2), and the GαS-coupled receptor GPR161. Our experiments uncovered a signaling mechanism that modifies the sensitivity of target cells to SHH and consequently changes the shape of the SHH dose-response curve. In both fibroblasts and spinal neural progenitors, the loss of GPR161, previously implicated as an inhibitor of basal SHH signaling, increased the sensitivity of target cells across the entire spectrum of SHH concentrations. Even in cells lacking GPR161, GRK2 was required for SHH signaling, and Gαs, which promotes the activation of protein Kinase A (PKA), antagonized SHH signaling. We propose that the sensitivity of target cells to Hedgehog morphogens, and the consequent effects on gene expression and differentiation outcomes, can be controlled by signals from G protein-coupled receptors that converge on Gαsand PKA.
View details for DOI 10.1126/scisignal.aao5749
View details for PubMedID 29438014
View details for PubMedCentralID PMC5828112
CRISPR Screens Uncover Genes that Regulate Target Cell Sensitivity to the Morphogen Sonic Hedgehog.
2018; 44 (1): 113–29.e8
To uncover regulatory mechanisms in Hedgehog (Hh) signaling, we conducted genome-wide screens to identify positive and negative pathway components and validated top hits using multiple signaling and differentiation assays in two different cell types. Most positive regulators identified in our screens, including Rab34, Pdcl, and Tubd1, were involved in ciliary functions, confirming the central role for primary cilia in Hh signaling. Negative regulators identified included Megf8, Mgrn1, and an unannotated gene encoding a tetraspan protein we named Atthog. The function of these negative regulators converged on Smoothened (SMO), an oncoprotein that transduces the Hh signal across the membrane. In the absence of Atthog, SMO was stabilized at the cell surface and concentrated in the ciliary membrane, boosting cell sensitivity to the ligand Sonic Hedgehog (SHH) and consequently altering SHH-guided neural cell-fate decisions. Thus, we uncovered genes that modify the interpretation of morphogen signals by regulating protein-trafficking events in target cells.
View details for DOI 10.1016/j.devcel.2017.12.003
View details for PubMedID 29290584
View details for PubMedCentralID PMC5792066
R-spondins can potentiate WNT signaling without LGRs.
The WNT signaling pathway regulates patterning and morphogenesis during development and promotes tissue renewal and regeneration in adults. The R-spondin (RSPO) family of four secreted proteins, RSPO1-4, amplifies target cell sensitivity to WNT ligands by increasing WNT receptor levels. Leucine-rich repeat-containing G-protein coupled receptors (LGRs) 4-6 are considered obligate high-affinity receptors for RSPOs. We discovered that RSPO2 and RSPO3, but not RSPO1 or RSPO4, can potentiate WNT/β-catenin signaling in the absence of all three LGRs. By mapping the domains on RSPO3 that are necessary and sufficient for this activity, we show that the requirement for LGRs is dictated by the interaction between RSPOs and the ZNRF3/RNF43 E3 ubiquitin ligases and that LGR-independent signaling depends on heparan sulfate proteoglycans (HSPGs). We propose that RSPOs can potentiate WNT signals through distinct mechanisms that differ in their use of either LGRs or HSPGs, with implications for understanding their biological functions.
View details for DOI 10.7554/eLife.33126
View details for PubMedID 29405118
View details for PubMedCentralID PMC5800842
Comparative genetic screens in human cells reveal new regulatory mechanisms in WNT signaling
The comprehensive understanding of cellular signaling pathways remains a challenge due to multiple layers of regulation that may become evident only when the pathway is probed at different levels or critical nodes are eliminated. To discover regulatory mechanisms in canonical WNT signaling, we conducted a systematic forward genetic analysis through reporter-based screens in haploid human cells. Comparison of screens for negative, attenuating and positive regulators of WNT signaling, mediators of R-spondin-dependent signaling and suppressors of constitutive signaling induced by loss of the tumor suppressor adenomatous polyposis coli or casein kinase 1α uncovered new regulatory features at most levels of the pathway. These include a requirement for the transcription factor AP-4, a role for the DAX domain of AXIN2 in controlling β-catenin transcriptional activity, a contribution of glycophosphatidylinositol anchor biosynthesis and glypicans to R-spondin-potentiated WNT signaling, and two different mechanisms that regulate signaling when distinct components of the β-catenin destruction complex are lost. The conceptual and methodological framework we describe should enable the comprehensive understanding of other signaling systems.
View details for DOI 10.7554/eLife.21459
View details for Web of Science ID 000393384400001
View details for PubMedID 27996937
View details for PubMedCentralID PMC5257257
Cholesterol activates the G-protein coupled receptor Smoothened to promote morphogenetic signaling.
Cholesterol is necessary for the function of many G-protein coupled receptors (GPCRs). We find that cholesterol is not just necessary but also sufficient to activate signaling by the Hedgehog (Hh) pathway, a prominent cell-cell communication system in development. Cholesterol influences Hh signaling by directly activating Smoothened (SMO), an orphan GPCR that transmits the Hh signal across the membrane in all animals. Unlike many GPCRs, which are regulated by cholesterol through their heptahelical transmembrane domains, SMO is activated by cholesterol through its extracellular cysteine-rich domain (CRD). Residues shown to mediate cholesterol binding to the CRD in a recent structural analysis also dictate SMO activation, both in response to cholesterol and to native Hh ligands. Our results show that cholesterol can initiate signaling from the cell surface by engaging the extracellular domain of a GPCR and suggest that SMO activity may be regulated by local changes in cholesterol abundance or accessibility.
View details for DOI 10.7554/eLife.20304
View details for PubMedID 27705744
View details for PubMedCentralID PMC5123864
In Vivo Formation of Vacuolated Multi-phase Compartments Lacking Membranes.
2016; 16 (5): 1228-1236
Eukaryotic cells contain membrane-less organelles, including nucleoli and stress granules, that behave like liquid droplets. Such endogenous condensates often have internal substructure, but how this is established in the absence of membrane encapsulation remains unclear. We find that the N- and C-terminal domains of TDP43, a heterogeneous nuclear ribonucleoprotein implicated in neurodegenerative diseases, are capable of driving the formation of sub-structured liquid droplets in vivo. These droplets contain dynamic internal "bubbles" of nucleoplasm, reminiscent of membrane-based multi-vesicular endosomes. A conserved sequence embedded within the intrinsically disordered region (IDR) of TDP43 promotes the formation of these multi-phase assemblies. Disease-causing point mutations in the IDR can change the propensity to form bubbles, protein dynamics within the phase, or phase-environment exchange rates. Our results show that a single IDR-containing protein can nucleate the assembly of compartmentalized liquid droplets approximating the morphological complexity of membrane-bound organelles.
View details for DOI 10.1016/j.celrep.2016.06.088
View details for PubMedID 27452472
Structural basis of Smoothened regulation by its extracellular domains.
2016; 535 (7613): 517-522
Developmental signals of the Hedgehog (Hh) and Wnt families are transduced across the membrane by Frizzledclass G-protein-coupled receptors (GPCRs) composed of both a heptahelical transmembrane domain (TMD) and an extracellular cysteine-rich domain (CRD). How the large extracellular domains of GPCRs regulate signalling by the TMD is unknown. We present crystal structures of the Hh signal transducer and oncoprotein Smoothened, a GPCR that contains two distinct ligand-binding sites: one in its TMD and one in the CRD. The CRD is stacked a top the TMD, separated by an intervening wedge-like linker domain. Structure-guided mutations show that the interface between the CRD, linker domain and TMD stabilizes the inactive state of Smoothened. Unexpectedly, we find a cholesterol molecule bound to Smoothened in the CRD binding site. Mutations predicted to prevent cholesterol binding impair the ability of Smoothened to transmit native Hh signals. Binding of a clinically used antagonist, vismodegib, to the TMD induces a conformational change that is propagated to the CRD, resulting in loss of cholesterol from the CRD-linker domain-TMD interface. Our results clarify the structural mechanism by which the activity of a GPCR is controlled by ligand-regulated interactions between its extracellular and transmembrane domains.
View details for PubMedID 27437577
View details for PubMedCentralID PMC4970916
EFCAB7 and IQCE Regulate Hedgehog Signaling by Tethering the EVC-EVC2 Complex to the Base of Primary Cilia
2014; 28 (5): 483-496
The Hedgehog (Hh) pathway depends on primary cilia in vertebrates, but the signaling machinery within cilia remains incompletely defined. We report the identification of a complex between two ciliary proteins, EFCAB7 and IQCE, which positively regulates the Hh pathway. The EFCAB7-IQCE module anchors the EVC-EVC2 complex in a signaling microdomain at the base of cilia. EVC and EVC2 genes are mutated in Ellis van Creveld and Weyers syndromes, characterized by impaired Hh signaling in skeletal, cardiac, and orofacial tissues. EFCAB7 binds to a C-terminal disordered region in EVC2 that is deleted in Weyers patients. EFCAB7 depletion mimics the Weyers cellular phenotype-the mislocalization of EVC-EVC2 within cilia and impaired activation of the transcription factor GLI2. Evolutionary analysis suggests that emergence of these complexes might have been important for adaptation of an ancient organelle, the cilium, for an animal-specific signaling network.
View details for DOI 10.1016/j.devcel.2014.01.021
View details for Web of Science ID 000333405600004
View details for PubMedID 24582806
View details for PubMedCentralID PMC4027042
Gli protein activity is controlled by multisite phosphorylation in vertebrate hedgehog signaling.
2014; 6 (1): 168-181
Gli proteins are transcriptional effectors of the Hedgehog (Hh) pathway in both normal development and cancer. We describe a program of multisite phosphorylation that regulates the conversion of Gli proteins into transcriptional activators. In the absence of Hh ligands, Gli activity is restrained by the direct phosphorylation of six conserved serine residues by protein kinase A (PKA), a master negative regulator of the Hh pathway. Activation of signaling leads to a global remodeling of the Gli phosphorylation landscape: the PKA target sites become dephosphorylated, while a second cluster of sites undergoes phosphorylation. The pattern of Gli phosphorylation can regulate Gli transcriptional activity in a graded fashion, suggesting a phosphorylation-based mechanism for how a gradient of Hh signaling in a morphogenetic field can be converted into a gradient of transcriptional activity.
View details for DOI 10.1016/j.celrep.2013.12.003
View details for PubMedID 24373970
- Structure and function of the Smoothened extracellular domain in vertebrate Hedgehog signaling. eLife 2013; 2
A Smoothened-Evc2 Complex Transduces the Hedgehog Signal at Primary Cilia
2012; 23 (4): 823-835
Vertebrate Hedgehog (Hh) signaling is initiated at primary cilia by the ligand-triggered accumulation of Smoothened (Smo) in the ciliary membrane. The underlying biochemical mechanisms remain unknown. We find that Hh agonists promote the association between Smo and Evc2, a ciliary protein that is defective in two human ciliopathies. The formation of the Smo-Evc2 complex is under strict spatial control, being restricted to a distinct ciliary compartment, the EvC zone. Mutant Evc2 proteins that localize in cilia but are displaced from the EvC zone are dominant inhibitors of Hh signaling. Disabling Evc2 function blocks Hh signaling at a specific step between Smo and the downstream regulators protein kinase A and Suppressor of Fused, preventing activation of the Gli transcription factors. Our data suggest that the Smo-Evc2 signaling complex at the EvC zone is required for Hh signal transmission and elucidate the molecular basis of two human ciliopathies.
View details for DOI 10.1016/j.devcel.2012.07.004
View details for Web of Science ID 000310036200017
View details for PubMedID 22981989
View details for PubMedCentralID PMC3586260
Oxysterols are allosteric activators of the oncoprotein Smoothened
NATURE CHEMICAL BIOLOGY
2012; 8 (2): 211-220
Oxysterols are a class of endogenous signaling molecules that can activate the Hedgehog pathway, which has critical roles in development, regeneration and cancer. However, it has been unclear how oxysterols influence Hedgehog signaling, including whether their effects are mediated through a protein target or indirectly through effects on membrane properties. To answer this question, we synthesized the enantiomer and an epimer of the most potent oxysterol, 20(S)-hydroxycholesterol. Using these molecules, we show that the effects of oxysterols on Hedgehog signaling are exquisitely stereoselective, consistent with the hypothesis that they function through a specific protein target. We present several lines of evidence that this protein target is the seven-pass transmembrane protein Smoothened, a major drug target in oncology. Our work suggests that these enigmatic sterols, which have multiple effects on cell physiology, may act as ligands for signaling receptors and provides a generally applicable framework for probing sterol signaling mechanisms.
View details for DOI 10.1038/nchembio.765
View details for Web of Science ID 000299323200014
View details for PubMedID 22231273
The output of Hedgehog signaling is controlled by the dynamic association between Suppressor of Fused and the Gli proteins
GENES & DEVELOPMENT
2010; 24 (7): 670-682
The transcriptional program orchestrated by Hedgehog signaling depends on the Gli family of transcription factors. Gli proteins can be converted to either transcriptional activators or truncated transcriptional repressors. We show that the interaction between Gli3 and Suppressor of Fused (Sufu) regulates the formation of either repressor or activator forms of Gli3. In the absence of signaling, Sufu restrains Gli3 in the cytoplasm, promoting its processing into a repressor. Initiation of signaling triggers the dissociation of Sufu from Gli3. This event prevents formation of the repressor and instead allows Gli3 to enter the nucleus, where it is converted into a labile, differentially phosphorylated transcriptional activator. This key dissociation event depends on Kif3a, a kinesin motor required for the function of primary cilia. We propose that the Sufu-Gli3 interaction is a major control point in the Hedgehog pathway, a pathway that plays important roles in both development and cancer.
View details for DOI 10.1101/gad.1902910
View details for Web of Science ID 000276235400006
View details for PubMedID 20360384
View details for PubMedCentralID PMC2849124
A single N-terminal phosphomimic disrupts TDP-43 polymerization, phase separation, and RNA splicing.
The EMBO journal
2018; 37 (5)
TDP-43 is an RNA-binding protein active in splicing that concentrates into membraneless ribonucleoprotein granules and forms aggregates in amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. Although best known for its predominantly disordered C-terminal domain which mediates ALS inclusions, TDP-43 has a globular N-terminal domain (NTD). Here, we show that TDP-43 NTD assembles into head-to-tail linear chains and that phosphomimetic substitution at S48 disrupts TDP-43 polymeric assembly, discourages liquid-liquid phase separation (LLPS)in vitro, fluidizes liquid-liquid phase separated nuclear TDP-43 reporter constructs in cells, and disrupts RNA splicing activity. Finally, we present the solution NMR structure of a head-to-tail NTD dimer comprised of two engineered variants that allow saturation of the native polymerization interface while disrupting higher-order polymerization. These data provide structural detail for the established mechanistic role of the well-folded TDP-43 NTD in splicing and link this function to LLPS. In addition, the fusion-tag solubilized, recombinant form of TDP-43 full-length protein developed here will enable future phase separation andin vitrobiochemical assays on TDP-43 function and interactions that have been hampered in the past by TDP-43 aggregation.
View details for DOI 10.15252/embj.201797452
View details for PubMedID 29438978
View details for PubMedCentralID PMC5830921
Dynamic Remodeling of Membrane Composition Drives Cell Cycle through Primary Cilia Excision.
2017; 168 (1-2): 264-279 e15
The life cycle of a primary cilium begins in quiescence and ends prior to mitosis. In quiescent cells, the primary cilium insulates itself from contiguous dynamic membrane processes on the cell surface to function as a stable signaling apparatus. Here, we demonstrate that basal restriction of ciliary structure dynamics is established by the cilia-enriched phosphoinositide 5-phosphatase, Inpp5e. Growth induction displaces ciliary Inpp5e and accumulates phosphatidylinositol 4,5-bisphosphate in distal cilia. This change triggers otherwise-forbidden actin polymerization in primary cilia, which excises cilia tips in a process we call cilia decapitation. While cilia disassembly is traditionally thought to occur solely through resorption, we show that an acute loss of IFT-B through cilia decapitation precedes resorption. Finally, we propose that cilia decapitation induces mitogenic signaling and constitutes a molecular link between the cilia life cycle and cell-division cycle. This newly defined ciliary mechanism may find significance in cell proliferation control during normal development and cancer.
View details for DOI 10.1016/j.cell.2016.12.032
View details for PubMedID 28086093
Multiple ligand binding sites regulate the Hedgehog signal transducer Smoothened in vertebrates.
Current opinion in cell biology
2017; 51: 81–88
The Hedgehog (Hh) pathway plays a central role in the development of multicellular organisms, guiding cell differentiation, proliferation and survival. While many components of the vertebrate pathway were discovered two decades ago, the mechanism by which the Hh signal is transmitted across the plasma membrane remains mysterious. This fundamental task in signalling is carried out by Smoothened (SMO), a human oncoprotein and validated cancer drug target that is a member of the G-protein coupled receptor protein family. Recent structural and functional studies have advanced our mechanistic understanding of SMO activation, revealing its unique regulation by two separable but allosterically-linked ligand-binding sites. Unexpectedly, these studies have nominated cellular cholesterol as having an instructive role in SMO signalling.
View details for DOI 10.1016/j.ceb.2017.10.004
View details for PubMedID 29268141
Chromatin-Remodeling Complex SWI/SNF Controls Multidrug Resistance by Transcriptionally Regulating the Drug Efflux Pump ABCB1
2016; 76 (19): 5810-5821
Anthracyclines are among the most effective yet most toxic drugs used in the oncology clinic. The nucleosome-remodeling SWI/SNF complex, a potent tumor suppressor, is thought to promote sensitivity to anthracyclines by recruiting topoisomerase IIa (TOP2A) to DNA and increasing double-strand breaks. In this study, we discovered a novel mechanism through which SWI/SNF influences resistance to the widely used anthracycline doxorubicin based on the use of a forward genetic screen in haploid human cells, followed by a rigorous single and double-mutant epistasis analysis using CRISPR/Cas9-mediated engineering. Doxorubicin resistance conferred by loss of the SMARCB1 subunit of the SWI/SNF complex was caused by transcriptional upregulation of a single gene, encoding the multidrug resistance pump ABCB1. Remarkably, both ABCB1 upregulation and doxorubicin resistance caused by SMARCB1 loss were dependent on the function of SMARCA4, a catalytic subunit of the SWI/SNF complex. We propose that residual SWI/SNF complexes lacking SMARCB1 are vital determinants of drug sensitivity, not just to TOP2A-targeted agents, but to the much broader range of cancer drugs effluxed by ABCB1. Cancer Res; 76(19); 5810-21. ©2016 AACR.
View details for DOI 10.1158/0008-5472.CAN-16-0716
View details for Web of Science ID 000385625500025
View details for PubMedID 27503929
View details for PubMedCentralID PMC5050136
An essential role for Grk2 in Hedgehog signalling downstream of Smoothened
2016; 17 (5): 739-752
The G-protein-coupled receptor kinase 2 (adrbk2/GRK2) has been implicated in vertebrate Hedgehog (Hh) signalling based on the effects of its transient knock-down in mammalian cells and zebrafish embryos. Here, we show that the response to Hh signalling is effectively abolished in the absence of Grk2 activity. Zebrafish embryos lacking all Grk2 activity are refractory to both Sonic hedgehog (Shh) and oncogenic Smoothened (Smo) activity, but remain responsive to inhibition of cAMP-dependent protein kinase (PKA) activity. Mutation of the kinase domain abrogates the rescuing activity of grk2 mRNA, suggesting that Grk2 acts in a kinase-dependent manner to regulate the response to Hh. Previous studies have suggested that Grk2 potentiates Smo activity by phosphorylating its C-terminal tail (CTT). In the zebrafish embryo, however, phosphomimetic Smo does not display constitutive activity, whereas phospho-null mutants retain activity, implying phosphorylation is neither sufficient nor necessary for Smo function. Since Grk2 rescuing activity requires the integrity of domains essential for its interaction with GPCRs, we speculate that Grk2 may regulate Hh pathway activity by downregulation of a GPCR.
View details for Web of Science ID 000375617400015
View details for PubMedID 27113758
View details for PubMedCentralID PMC5341524
Functional Divergence in the Role of N-Linked Glycosylation in Smoothened Signaling.
2015; 11 (8)
The G protein-coupled receptor (GPCR) Smoothened (Smo) is the requisite signal transducer of the evolutionarily conserved Hedgehog (Hh) pathway. Although aspects of Smo signaling are conserved from Drosophila to vertebrates, significant differences have evolved. These include changes in its active sub-cellular localization, and the ability of vertebrate Smo to induce distinct G protein-dependent and independent signals in response to ligand. Whereas the canonical Smo signal to Gli transcriptional effectors occurs in a G protein-independent manner, its non-canonical signal employs Gαi. Whether vertebrate Smo can selectively bias its signal between these routes is not yet known. N-linked glycosylation is a post-translational modification that can influence GPCR trafficking, ligand responsiveness and signal output. Smo proteins in Drosophila and vertebrate systems harbor N-linked glycans, but their role in Smo signaling has not been established. Herein, we present a comprehensive analysis of Drosophila and murine Smo glycosylation that supports a functional divergence in the contribution of N-linked glycans to signaling. Of the seven predicted glycan acceptor sites in Drosophila Smo, one is essential. Loss of N-glycosylation at this site disrupted Smo trafficking and attenuated its signaling capability. In stark contrast, we found that all four predicted N-glycosylation sites on murine Smo were dispensable for proper trafficking, agonist binding and canonical signal induction. However, the under-glycosylated protein was compromised in its ability to induce a non-canonical signal through Gαi, providing for the first time evidence that Smo can bias its signal and that a post-translational modification can impact this process. As such, we postulate a profound shift in N-glycan function from affecting Smo ER exit in flies to influencing its signal output in mice.
View details for DOI 10.1371/journal.pgen.1005473
View details for PubMedID 26291458
Notch Activity Modulates the Responsiveness of Neural Progenitors to Sonic Hedgehog Signaling
2015; 33 (4): 373-387
Throughout the developing nervous system, neural stem and progenitor cells give rise to diverse classes of neurons and glia in a spatially and temporally coordinated manner. In the ventral spinal cord, much of this diversity emerges through the morphogen actions of Sonic hedgehog (Shh). Interpretation of the Shh gradient depends on both the amount of ligand and duration of exposure, but the mechanisms permitting prolonged responses to Shh are not well understood. We demonstrate that Notch signaling plays an essential role in this process, enabling neural progenitors to attain sufficiently high levels of Shh pathway activity needed to direct the ventral-most cell fates. Notch activity regulates subcellular localization of the Shh receptor Patched1, gating the translocation of the key effector Smoothened to primary cilia and its downstream signaling activities. These data reveal an unexpected role for Notch shaping the interpretation of the Shh morphogen gradient and influencing cell fate determination.
View details for DOI 10.1016/j.devcel.2015.03.005
View details for Web of Science ID 000355151900004
View details for PubMedID 25936505
- Location, location, and location: compartmentalization of Hedgehog signaling at primary cilia. EMBO journal 2014; 33 (17): 1852-1854
G-protein-coupled receptors, Hedgehog signaling and primary cilia.
Seminars in cell & developmental biology
2014; 33: 63-72
The Hedgehog (Hh) pathway has become an important model to study the cell biology of primary cilia, and reciprocally, the study of ciliary processes provides an opportunity to solve longstanding mysteries in the mechanism of vertebrate Hh signal transduction. The cilium is emerging as an unique compartment for G-protein-coupled receptor (GPCR) signaling in many systems. Two members of the GPCR family, Smoothened and Gpr161, play important roles in the Hh pathway. We review the current understanding of how these proteins may function to regulate Hh signaling and also highlight some of the critical unanswered questions being tackled by the field. Uncovering GPCR-regulated mechanisms important in Hh signaling may provide therapeutic strategies against the Hh pathway that plays important roles in development, regeneration and cancer.
View details for DOI 10.1016/j.semcdb.2014.05.002
View details for PubMedID 24845016
- G-protein-coupled receptors, Hedgehog signaling and primary cilia SEMINARS IN CELL & DEVELOPMENTAL BIOLOGY 2014; 33: 63-72
A Novel Osteogenic Oxysterol Compound for Therapeutic Development to Promote Bone Growth: Activation of Hedgehog Signaling and Osteogenesis Through Smoothened Binding
JOURNAL OF BONE AND MINERAL RESEARCH
2014; 29 (8): 1872-1885
Osteogenic factors are often used in orthopedics to promote bone growth, improve fracture healing, and induce spine fusion. Osteogenic oxysterols are naturally occurring molecules that were shown to induce osteogenic differentiation in vitro and promote spine fusion in vivo. The purpose of this study was to identify an osteogenic oxysterol more suitable for clinical development than those previously reported, and evaluate its ability to promote osteogenesis in vitro and spine fusion in rats in vivo. Among more than 100 oxysterol analogues synthesized, Oxy133 induced significant expression of osteogenic markers Runx2, osterix (OSX), alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OCN) in C3H10T1/2 mouse embryonic fibroblasts and in M2-10B4 mouse marrow stromal cells. Oxy133-induced activation of an 8X-Gli luciferase reporter, its direct binding to Smoothened, and the inhibition of Oxy133-induced osteogenic effects by the Hedgehog (Hh) pathway inhibitor, cyclopamine, demonstrated the role of Hh pathway in mediating osteogenic responses to Oxy133. Oxy133 did not stimulate osteogenesis via BMP or Wnt signaling. Oxy133 induced the expression of OSX, BSP, and OCN, and stimulated robust mineralization in primary human mesenchymal stem cells. In vivo, bilateral spine fusion occurred through endochondral ossification and was observed in animals treated with Oxy133 at the fusion site on X-ray after 4 weeks and confirmed with manual assessment, micro-CT (µCT), and histology after 8 weeks, with equal efficiency to recombinant human bone morphogenetic protein-2 (rhBMP-2). Unlike rhBMP-2, Oxy133 did not induce adipogenesis in the fusion mass and resulted in denser bone evidenced by greater bone volume/tissue volume (BV/TV) ratio and smaller trabecular separation. Findings here suggest that Oxy133 has significant potential as an osteogenic molecule with greater ease of synthesis and improved time to fusion compared to previously studied oxysterols. Small molecule osteogenic oxysterols may serve as the next generation of bone anabolic agents for therapeutic development.
View details for DOI 10.1002/jbmr.2213
View details for Web of Science ID 000340243900019
View details for PubMedID 24591126
Tracking the Subcellular Fate of 20( S)-Hydroxycholesterol with Click Chemistry Reveals a Transport Pathway to the Golgi
JOURNAL OF BIOLOGICAL CHEMISTRY
2014; 289 (16): 11095-11110
Oxysterols, oxidized metabolites of cholesterol, are endogenous small molecules that regulate lipid metabolism, immune function, and developmental signaling. Although the cell biology of cholesterol has been intensively studied, fundamental questions about oxysterols, such as their subcellular distribution and trafficking pathways, remain unanswered. We have therefore developed a useful method to image intracellular 20(S)-hydroxycholesterol with both high sensitivity and spatial resolution using click chemistry and fluorescence microscopy. The metabolic labeling of cells with an alkynyl derivative of 20(S)-hydroxycholesterol has allowed us to directly visualize this oxysterol by attaching an azide fluorophore through cyclo-addition. Unexpectedly, we found that this oxysterol selectively accumulates in the Golgi membrane using a pathway that is sensitive to ATP levels, temperature, and lysosome function. Although previous models have proposed nonvesicular pathways for the rapid equilibration of oxysterols between membranes, direct imaging of oxysterols suggests that a vesicular pathway is responsible for differential accumulation of oxysterols in organelle membranes. More broadly, clickable alkynyl sterols may represent useful tools for sterol cell biology, both to investigate the functions of these important lipids and to decipher the pathways that determine their cellular itineraries.
View details for DOI 10.1074/jbc.M113.540351
View details for Web of Science ID 000334638500014
View details for PubMedID 24596093
View details for PubMedCentralID PMC4036249
- Isolation and mutational analysis of circulating tumor cells from lung cancer patients with magnetic sifters and biochips LAB ON A CHIP 2014; 14 (1): 78-88
Isolation and mutational analysis of circulating tumor cells from lung cancer patients with magnetic sifters and biochips.
Lab on a chip
2013; 14 (1): 78-88
Detection and characterization of circulating tumor cells (CTCs) may reveal insights into the diagnosis and treatment of malignant disease. Technologies for isolating CTCs developed thus far suffer from one or more limitations, such as low throughput, inability to release captured cells, and reliance on expensive instrumentation for enrichment or subsequent characterization. We report a continuing development of a magnetic separation device, the magnetic sifter, which is a miniature microfluidic chip with a dense array of magnetic pores. It offers high efficiency capture of tumor cells, labeled with magnetic nanoparticles, from whole blood with high throughput and efficient release of captured cells. For subsequent characterization of CTCs, an assay, using a protein chip with giant magnetoresistive nanosensors, has been implemented for mutational analysis of CTCs enriched with the magnetic sifter. The use of these magnetic technologies, which are separate devices, may lead the way to routine preparation and characterization of "liquid biopsies" from cancer patients.
View details for DOI 10.1039/c3lc50580d
View details for PubMedID 23969419
Cancer risk after use of recombinant bone morphogenetic protein-2 for spinal arthrodesis.
journal of bone and joint surgery. American volume
2013; 95 (17): 1537-1545
Recombinant human bone morphogenetic protein-2 (rhBMP-2) is a growth factor known to have in vitro effects on the growth and invasiveness of cancer. It has been approved by the U.S. Food and Drug Administration in limited doses for single-level anterior spinal arthrodesis, but it is commonly used off-label and at high doses. The effect of rhBMP-2 on the risk of cancer has been a concern. We sought to evaluate the risk of new cancers in patients receiving high-dose rhBMP-2.We used publicly available data from a pivotal, multicenter, randomized controlled trial of patients with degenerative lumbar spine conditions who underwent a single-level instrumented posterolateral arthrodesis with either high-dose rhBMP-2 in a compression-resistant matrix (CRM) (rhBMP-2/CRM; n = 239) or autogenous bone graft (control group; n = 224). We compared the risks of new cancers in the rhBMP-2/CRM and control groups at two and five years after surgery.At two years, with 86% follow-up, there were fifteen new cancer events in eleven patients in the rhBMP-2/CRM group compared with two new cancer events in two patients in the control group treated with autogenous bone graft. The incidence rate of new cancer events per 100 person-years was 3.37 (95% confidence interval [CI], 1.89 to 5.56) in the rhBMP-2/CRM group at two years compared with 0.50 (95% CI, 0.06 to 1.80) in the control group. The incidence rate ratio was 6.75 (95% CI, 1.57 to 60.83; p = 0.0026) at two years. Calculated in terms of the number of patients with one or more cancer events two years after the surgery, the incidence rate per 100 person-years was 2.54 (95% CI, 1.27 to 4.54) in the rhBMP-2/CRM group compared with 0.50 (95% CI, 0.06 to 1.82) in the control group at two years; the incidence rate ratio was 5.04 (95% CI, 1.10 to 46.82; p = 0.0194). At five years, there was a 37% loss of follow-up, but a significantly greater incidence of cancer events was still observed in the rhBMP-2/CRM group.A high dose of 40 mg of rhBMP-2/CRM in lumbar spinal arthrodesis was associated with an increased risk of new cancer.Therapeutic Level II. See Instructions for Authors for a complete description of levels of evidence.
View details for DOI 10.2106/JBJS.L.01483
View details for PubMedID 24005193
- Cancer Risk After Use of Recombinant Bone Morphogenetic Protein-2 for Spinal Arthrodesis JOURNAL OF BONE AND JOINT SURGERY-AMERICAN VOLUME 2013; 95A (17): 1537-1545
Chemically inducible diffusion trap at cilia reveals molecular sieve-like barrier.
Nature chemical biology
2013; 9 (7): 437-443
Primary cilia function as specialized compartments for signal transduction. The stereotyped structure and signaling function of cilia inextricably depend on the selective segregation of molecules in cilia. However, the fundamental principles governing the access of soluble proteins to primary cilia remain unresolved. We developed a methodology termed 'chemically inducible diffusion trap at cilia' to visualize the diffusion process of a series of fluorescent proteins ranging in size from 3.2 nm to 7.9 nm into primary cilia. We found that the interior of the cilium was accessible to proteins as large as 7.9 nm. The kinetics of ciliary accumulation of this panel of proteins was exponentially limited by their Stokes radii. Quantitative modeling suggests that the diffusion barrier operates as a molecular sieve at the base of cilia. Our study presents a set of powerful, generally applicable tools for the quantitative monitoring of ciliary protein diffusion under both physiological and pathological conditions.
View details for DOI 10.1038/nchembio.1252
View details for PubMedID 23666116
Singapore signalling: the 2012 hedgehog pathway cocktail
2012; 13 (7): 580-583
The 'Hedgehog Signalling in Development Evolution and Disease' conference took place in Biopolis, Singapore, in March 2012. Organized by Phil Ingham with help from Xinhua Lin, Mary-Ann Price and Fred de Sauvage, it brought leading researchers together to discuss the latest findings and exchange ideas on every aspect of hedgehog signalling.
View details for DOI 10.1038/embor.2012.79
View details for Web of Science ID 000306076700004
View details for PubMedID 22688966
Cilia 2010: The Surprise Organelle of the Decade
2011; 4 (155)
Presentations at the 2010 Conference on the Biology of Cilia and Flagella revealed new insights into the functions and assembly of cilia and highlighted their ever-expanding roles in development and disease.
View details for DOI 10.1126/scisignal.4155mr1
View details for Web of Science ID 000286102200004
View details for PubMedID 21224442
The ciliary membrane
CURRENT OPINION IN CELL BIOLOGY
2010; 22 (4): 541-546
Cilia and flagella are important organizing centers for signaling in both development and disease. A key to their function is a poorly characterized barrier at their base that allows the protein and lipid composition of the ciliary membrane to be distinct from that of the plasma membrane. We review current models of ciliary membrane biogenesis, highlighting several structures, including the ciliary necklace and ciliary pocket, that appear during biogenesis and that likely contribute to the barrier. The regulated movement of membrane proteins and lipids across this barrier is central to the sensory function of these organelles.
View details for DOI 10.1016/j.ceb.2010.03.010
View details for Web of Science ID 000280945500017
View details for PubMedID 20399632
Role of Lipid Metabolism in Smoothened Derepression in Hedgehog Signaling
2010; 19 (1): 54-65
The binding of Hedgehog (Hh) to its receptor Patched causes derepression of Smoothened (Smo), resulting in the activation of the Hh pathway. Here, we show that Smo activation is dependent on the levels of the phospholipid phosphatidylinositol-4 phosphate (PI4P). Loss of STT4 kinase, which is required for the generation of PI4P, exhibits hh loss-of-function phenotypes, whereas loss of Sac1 phosphatase, which is required for the degradation of PI4P, results in hh gain-of-function phenotypes in multiple settings during Drosophila development. Furthermore, loss of Ptc function, which results in the activation of Hh pathway, also causes an increase in PI4P levels. Sac1 functions downstream of STT4 and Ptc in the regulation of Smo membrane localization and Hh pathway activation. Taken together, our results suggest a model in which Ptc directly or indirectly functions to suppress the accumulation of PI4P. Binding of Hh to Ptc derepresses the levels of PI4P, which, in turn, promotes Smo activation.
View details for DOI 10.1016/j.devcel.2010.06.007
View details for Web of Science ID 000280469100010
View details for PubMedID 20643350
Lateral transport of Smoothened from the plasma membrane to the membrane of the cilium
JOURNAL OF CELL BIOLOGY
2009; 187 (3): 365-374
The function of primary cilia depends critically on the localization of specific proteins in the ciliary membrane. A major challenge in the field is to understand protein trafficking to cilia. The Hedgehog (Hh) pathway protein Smoothened (Smo), a 7-pass transmembrane protein, moves to cilia when a ligand is received. Using microscopy-based pulse-chase analysis, we find that Smo moves through a lateral transport pathway from the plasma membrane to the ciliary membrane. Lateral movement, either via diffusion or active transport, is quite distinct from currently studied pathways of ciliary protein transport in mammals, which emphasize directed trafficking of Golgi-derived vesicles to the base of the cilium. We anticipate that this alternative route will be used by other signaling proteins that function at cilia. The path taken by Smo may allow novel strategies for modulation of Hh signaling in cancer and regeneration.
View details for DOI 10.1083/jcb.200907126
View details for Web of Science ID 000271374200008
View details for PubMedID 19948480
- Hedgehog signal transduction by smoothened: pharmacological evidence for a two-step activation process. Proceedings of the National Academy of Sciences USA 2009; 106: 3196-3201
- Arrestin? Movement in Cilia. Science 2008; 320 (5884): 1777-1781
- Patching the gaps in Hedgehog signaling. Nat Cell Bio 2007; 9 (9): 1005-1009
- Patched1 regulates Hedgehog signaling at the primary cilium. Science 2007; 317 (5836): 372-376
- In vitro reconstitution of cdc42-mediated actin assembly using purified components. Methods in Enzymology 2006; 406: 174-190
- Loss-of-function Analysis of EphA Receptors in Retinotectal mapping. Journal of Neuroscience 2004; 24 (10): 2542-2550
- Toca-1 Mediates Cdc42- Dependent Actin Nucleation by Activating the N-WASP-WIP Complex. Cell 2004; 118 (2): 203-216
- The Mechanism of Regulation of WAVE1-induced Actin Nucleation by Rac1 and Nck. Nature 2002; 418 (6899): 790-793
- Nck and Phosphatidylinositol 4,5 Bisphosphate Synergistically Activate Actin Polymerization Through the N-WASP-Arp2/3 Pathway. Journal of Biological Chemistry 2001; 276 (28): 26448-26452
- CR16 Forms a Complex with N-WASP in Brain and is a Novel Member of a Conserved Proline-Rich Actin-Binding Protein Family. Proceedings of the National Academy of Sciences USA 2001; 98 (20): 11306-11311
- WIP Regulates N-WASP-Mediated Actin Polymerization and Filopodium Formation. Nature Cell Biology 2001; 3 (5): 484-491
- Mechanism of N-WASP Activation by CDC42 and Phosphatidylinositol 4, 5-Bisphosphate. Journal of Cell Biology 2000; 150 (6): 1299-1310
- The Interaction Between N-WASP and the Arp2/3 Complex Links Cdc42-Dependent Signals to Actin Assembly. Cell 1999; 97 (2): 221-231
- The Arp2/3 Complex Mediates Actin Polymerization Induced by the Small GTP-Binding Protein Cdc42. Proceedings of the National Academy of Sciences USA 1998; 95 (26): 15362-15367
- Non-Enzymatic, Template-Directed Ligation of Oligoribonucleotides is Highly Regioselective for the Formation of 3'-5'-Phosphodiester Bonds Journal of the American Chemical Society 1996; 118 (14): 3340-3344
- Kinetic and Mechanistic Analysis of Non-Enzymatic, Template-Directed Oligoribonucleotide Ligation. Journal of the American Chemical Society 1996; 118 (14): 3332-3339