Rajat Rohatgi

All Publications

• Regulated N-glycosylation controls chaperone function and receptor trafficking. Science (New York, N.Y.) Ma, M., Dubey, R., Jen, A., Pusapati, G. V., Singal, B., Shishkova, E., Overmyer, K. A., Cormier-Daire, V., Fedry, J., Aravind, L., Coon, J. J., Rohatgi, R. 2024; 386 (6722): 667-672

  Abstract

  One-fifth of human proteins are N-glycosylated in the endoplasmic reticulum (ER) by two oligosaccharyltransferases, OST-A and OST-B. Contrary to the prevailing view of N-glycosylation as a housekeeping function, we identified an ER pathway that modulates the activity of OST-A. Genetic analyses linked OST-A to HSP90B1, an ER chaperone for membrane receptors, and CCDC134, an ER luminal protein. During its translocation into the ER, an N-terminal peptide in HSP90B1 templates the assembly of a translocon complex containing CCDC134 and OST-A that protects HSP90B1 during folding, preventing its hyperglycosylation and degradation. Disruption of this pathway impairs WNT and IGF1R signaling and causes the bone developmental disorder osteogenesis imperfecta. Thus, N-glycosylation can be regulated by specificity factors in the ER to control cell surface receptor signaling and tissue development.

  View details for DOI 10.1126/science.adp7201

  View details for PubMedID 39509507

• Direct ionic stress sensing and mitigation by the transcription factor NFAT5. bioRxiv : the preprint server for biology Khandwala, C. B., Sarkar, P., Schmidt, H. B., Ma, M., Kinnebrew, M., Pusapati, G. V., Patel, B. B., Tillo, D., Lebensohn, A. M., Rohatgi, R. 2023

  Abstract

  Homeostatic control of intracellular ionic strength is essential for protein, organelle and genome function, yet mechanisms that sense and enable adaptation to ionic stress remain poorly understood in animals. We find that the transcription factor NFAT5 directly senses solution ionic strength using a C-terminal intrinsically disordered region. Both in intact cells and in a purified system, NFAT5 forms dynamic, reversible biomolecular condensates in response to increasing ionic strength. This self-associative property, conserved from insects to mammals, allows NFAT5 to accumulate in the nucleus and activate genes that restore cellular ion content. Mutations that reduce condensation or those that promote aggregation both reduce NFAT5 activity, highlighting the importance of optimally tuned associative interactions. Remarkably, human NFAT5 alone is sufficient to reconstitute a mammalian transcriptional response to ionic or hypertonic stress in yeast. Thus NFAT5 is both the sensor and effector of a cell-autonomous ionic stress response pathway in animal cells.

  View details for DOI 10.1101/2023.09.23.559074

  View details for PubMedID 37886503

• The Inseparable Relationship Between Cholesterol and Hedgehog Signaling. Annual review of biochemistry Siebold, C., Rohatgi, R. 2023

  Abstract

  Ligands of the Hedgehog (HH) pathway are paracrine signaling molecules that coordinate tissue development in metazoans. A remarkable feature of HH signaling is the repeated use of cholesterol in steps spanning ligand biogenesis, secretion, dispersal, and reception on target cells. A cholesterol molecule covalently attached to HH ligands is used as a molecular baton by transfer proteins to guide their secretion, spread, and reception. On target cells, a signaling circuit composed of a cholesterol transporter and sensor regulates transmission of HH signals across the plasma membrane to the cytoplasm. The repeated use of cholesterol in signaling supports the view that the HH pathway likely evolved by coopting ancient systems to regulate the abundance or organization of sterol-like lipids in membranes. Expected final online publication date for the Annual Review of Biochemistry, Volume 92 is June 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

  View details for DOI 10.1146/annurev-biochem-052521-040313

  View details for PubMedID 37001135

• Oxaliplatin disrupts nucleolar function through biophysical disintegration. Cell reports Schmidt, H. B., Jaafar, Z. A., Wulff, B. E., Rodencal, J. J., Hong, K., Aziz-Zanjani, M. O., Jackson, P. K., Leonetti, M. D., Dixon, S. J., Rohatgi, R., Brandman, O. 2022; 41 (6): 111629

  Abstract

  Platinum (Pt) compounds such as oxaliplatin are among the most commonly prescribed anti-cancer drugs. Despite their considerable clinical impact, the molecular basis of platinum cytotoxicity and cancer specificity remain unclear. Here we show that oxaliplatin, a backbone for the treatment of colorectal cancer, causes liquid-liquid demixing of nucleoli at clinically relevant concentrations. Our data suggest that this biophysical defect leads to cell-cycle arrest, shutdown of Pol I-mediated transcription, and ultimately cell death. We propose that instead of targeting a single molecule, oxaliplatin preferentially partitions into nucleoli, where it modifies nucleolar RNA and proteins. This mechanism provides a general approach for drugging the increasing number of cellular processes linked to biomolecular condensates.

  View details for DOI 10.1016/j.celrep.2022.111629

  View details for PubMedID 36351392

• Patched 1 regulates Smoothened by controlling sterol binding to its extracellular cysteine-rich domain. Science advances Kinnebrew, M., Woolley, R. E., Ansell, T. B., Byrne, E. F., Frigui, S., Luchetti, G., Sircar, R., Nachtergaele, S., Mydock-McGrane, L., Krishnan, K., Newstead, S., Sansom, M. S., Covey, D. F., Siebold, C., Rohatgi, R. 2022; 8 (22): eabm5563

  Abstract

  Smoothened (SMO) transduces the Hedgehog (Hh) signal across the plasma membrane in response to accessible cholesterol. Cholesterol binds SMO at two sites: one in the extracellular cysteine-rich domain (CRD) and a second in the transmembrane domain (TMD). How these two sterol-binding sites mediate SMO activation in response to the ligand Sonic Hedgehog (SHH) remains unknown. We find that mutations in the CRD (but not the TMD) reduce the fold increase in SMO activity triggered by SHH. SHH also promotes the photocrosslinking of a sterol analog to the CRD in intact cells. In contrast, sterol binding to the TMD site boosts SMO activity regardless of SHH exposure. Mutational and computational analyses show that these sites are in allosteric communication despite being 45 angstroms apart. Hence, sterols function as both SHH-regulated orthosteric ligands at the CRD and allosteric ligands at the TMD to regulate SMO activity and Hh signaling.

  View details for DOI 10.1126/sciadv.abm5563

  View details for PubMedID 35658032

• Patched 1 reduces the accessibility of cholesterol in the outer leaflet of membranes ELIFE Kinnebrew, M., Luchetti, G., Sircar, R., Frigui, S., Viti, L., Naito, T., Beckert, F., Saheki, Y., Siebold, C., Radhakrishnan, A., Rohatgi, R. 2021; 10

  View details for DOI 10.7554/eLife.70504.sa2

  View details for Web of Science ID 000729482600001

• Gene-teratogen interactions influence the penetrance of birth defects by altering Hedgehog signaling strength. Development (Cambridge, England) Kong, J. H., Young, C. B., Pusapati, G. V., Espinoza, F. H., Patel, C. B., Beckert, F., Ho, S., Patel, B. B., Gabriel, G. C., Aravind, L., Bazan, J. F., Gunn, T. M., Lo, C. W., Rohatgi, R. 2021

  Abstract

  Birth defects result from interactions between genetic and environmental factors, but the mechanisms remain poorly understood. We find that mutations and teratogens interact in predictable ways to cause birth defects by changing target cell sensitivity to Hedgehog (Hh) ligands. These interactions converge on a membrane protein complex, the MMM complex, that promotes degradation of the Hh transducer Smoothened (SMO). Deficiency of the MMM component MOSMO results in elevated SMO and increased Hh signaling, causing multiple birth defects. In utero exposure to a teratogen that directly inhibits SMO reduces the penetrance and expressivity of birth defects in Mosmo-/- embryos. Additionally, tissues that develop normally in Mosmo-/- embryos are refractory to the teratogen. Thus, changes in the abundance of the protein target of a teratogen can change birth defect outcomes by quantitative shifts in Hh signaling. Consequently, small molecules that re-calibrate signaling strength could be harnessed to rescue structural birth defects.

  View details for DOI 10.1242/dev.199867

  View details for PubMedID 34486668

• Cholesterol access in cellular membranes controls Hedgehog signaling. Nature chemical biology Radhakrishnan, A., Rohatgi, R., Siebold, C. 2020; 16 (12): 1303–13

  Abstract

  The Hedgehog (Hh) signaling pathway coordinates cell-cell communication in development and regeneration. Defects in this pathway underlie diseases ranging from birth defects to cancer. Hh signals are transmitted across the plasma membrane by two proteins, Patched 1 (PTCH1) and Smoothened (SMO). PTCH1, a transporter-like tumor-suppressor protein, binds to Hh ligands, but SMO, a G-protein-coupled-receptor family oncoprotein, transmits the Hh signal across the membrane. Recent structural, biochemical and cell-biological studies have converged at the surprising model that a specific pool of plasma membrane cholesterol, termed accessible cholesterol, functions as a second messenger that conveys the signal between PTCH1 and SMO. Beyond solving a central puzzle in Hh signaling, these studies are revealing new principles in membrane biology: how proteins respond to and remodel cholesterol accessibility in membranes and how the cholesterol composition of organelle membranes is used to regulate protein function.

  View details for DOI 10.1038/s41589-020-00678-2

  View details for PubMedID 33199907

• Lipid droplets can promote drug accumulation and activation. Nature chemical biology Dubey, R., Stivala, C. E., Nguyen, H. Q., Goo, Y., Paul, A., Carette, J. E., Trost, B. M., Rohatgi, R. 2020

  Abstract

  Genetic screens in cultured human cells represent a powerful unbiased strategy to identify cellular pathways that determine drug efficacy, providing critical information for clinical development. We used insertional mutagenesis-based screens in haploid cells to identify genes required for the sensitivity to lasonolide A (LasA), a macrolide derived from a marine sponge that kills certain types of cancer cells at low nanomolar concentrations. Our screens converged on a single gene, LDAH, encoding a member of the metabolite serine hydrolase family that is localized on the surface of lipid droplets. Mechanistic studies revealed that LasA accumulates in lipid droplets, where it is cleaved into a toxic metabolite by LDAH. We suggest that selective partitioning of hydrophobic drugs into the oil phase of lipid droplets can influence their activation and eventual toxicity to cells.

  View details for DOI 10.1038/s41589-019-0447-7

  View details for PubMedID 31932720

• R-spondins engage heparan sulfate proteoglycans to potentiate WNT signaling. eLife Dubey, R. n., van Kerkhof, P. n., Jordens, I. n., Malinauskas, T. n., Pusapati, G. V., McKenna, J. K., Li, D. n., Carette, J. E., Ho, M. n., Siebold, C. n., Maurice, M. n., Lebensohn, A. M., Rohatgi, R. n. 2020; 9

  Abstract

  R-spondins (RSPOs) amplify WNT signaling during development and regenerative responses. We previously demonstrated that RSPOs 2 and 3 potentiate WNT/β-catenin signaling in cells lacking leucine-rich repeat-containing G-protein coupled receptors (LGRs) 4, 5 and 6 (Lebensohn and Rohatgi, 2018). We now show that heparan sulfate proteoglycans (HSPGs) act as alternative co-receptors for RSPO3 using a combination of ligand mutagenesis and ligand engineering. Mutations in RSPO3 residues predicted to contact HSPGs impair its signaling capacity. Conversely, the HSPG-binding domains of RSPO3 can be entirely replaced with an antibody that recognizes heparan sulfate (HS) chains attached to multiple HSPGs without diminishing WNT-potentiating activity in cultured cells and intestinal organoids. A genome-wide screen for mediators of RSPO3 signaling in cells lacking LGRs 4, 5 and 6 failed to reveal other receptors. We conclude that HSPGs are RSPO co-receptors that potentiate WNT signaling in the presence and absence of LGRs.

  View details for DOI 10.7554/eLife.54469

  View details for PubMedID 32432544

• A Membrane-Tethered Ubiquitination Pathway Regulates Hedgehog Signaling and Heart Development. Developmental cell Kong, J. H., Young, C. B., Pusapati, G. V., Patel, C. B., Ho, S. n., Krishnan, A. n., Lin, J. I., Devine, W. n., Moreau de Bellaing, A. n., Athni, T. S., Aravind, L. n., Gunn, T. M., Lo, C. W., Rohatgi, R. n. 2020

  Abstract

  The etiology of congenital heart defects (CHDs), which are among the most common human birth defects, is poorly understood because of its complex genetic architecture. Here, we show that two genes implicated in CHDs, Megf8 and Mgrn1, interact genetically and biochemically to regulate the strength of Hedgehog signaling in target cells. MEGF8, a transmembrane protein, and MGRN1, a RING superfamily E3 ligase, assemble to form a receptor-like ubiquitin ligase complex that catalyzes the ubiquitination and degradation of the Hedgehog pathway transducer Smoothened. Homozygous Megf8 and Mgrn1 mutations increased Smoothened abundance and elevated sensitivity to Hedgehog ligands. While mice heterozygous for loss-of-function Megf8 or Mgrn1 mutations were normal, double heterozygous embryos exhibited an incompletely penetrant syndrome of CHDs with heterotaxy. Thus, genetic interactions can arise from biochemical mechanisms that calibrate morphogen signaling strength, a conclusion broadly relevant for the many human diseases in which oligogenic inheritance is emerging as a mechanism for heritability.

  View details for DOI 10.1016/j.devcel.2020.08.012

  View details for PubMedID 32966817

• Phase separation-deficient TDP43 remains functional in splicing. Nature communications Schmidt, H. B., Barreau, A., Rohatgi, R. 2019; 10 (1): 4890

  Abstract

  Intrinsically disordered regions (IDRs) areoften fast-evolving protein domains of low sequence complexity that can drive phase transitions and are commonly found in many proteins associated with neurodegenerative diseases, including the RNA processing factor TDP43. Yet, how phase separation contributes to the physiological functions of TDP43 in cells remains enigmatic. Here, we combine systematic mutagenesis guided by evolutionary sequence analysis with a live-cell reporter assay of TDP43 phase dynamics to identify regularly-spaced hydrophobic motifs separated by flexible, hydrophilic segments in the IDR as a key determinant of TDP43 phase properties. This heuristic framework allows customization of the material properties of TDP43 condensates to determine effects on splicing function. Remarkably, even a mutant that fails to phase-separate at physiological concentrations can still efficiently mediate the splicing of a quantitative, single-cell splicing reporter and endogenous targets. This suggests that the ability of TDP43 to phase-separate is not essential for its splicing function.

  View details for DOI 10.1038/s41467-019-12740-2

  View details for PubMedID 31653829

• Biochemical mechanisms of vertebrate hedgehog signaling. Development (Cambridge, England) Kong, J. H., Siebold, C. n., Rohatgi, R. n. 2019; 146 (10)

  Abstract

  Signaling pathways that mediate cell-cell communication are essential for collective cell behaviors in multicellular systems. The hedgehog (HH) pathway, first discovered and elucidated in Drosophila, is one of these iconic signaling systems that plays many roles during embryogenesis and in adults; abnormal HH signaling can lead to birth defects and cancer. We review recent structural and biochemical studies that have advanced our understanding of the vertebrate HH pathway, focusing on the mechanisms by which the HH signal is received by patched on target cells, transduced across the cell membrane by smoothened, and transmitted to the nucleus by GLI proteins to influence gene-expression programs.

  View details for PubMedID 31092502

• Cholesterol accessibility at the ciliary membrane controls Hedgehog signaling. eLife Kinnebrew, M. n., Iverson, E. J., Patel, B. B., Pusapati, G. V., Kong, J. H., Johnson, K. A., Luchetti, G. n., Eckert, K. M., McDonald, J. G., Covey, D. F., Siebold, C. n., Radhakrishnan, A. n., Rohatgi, R. n. 2019; 8

  Abstract

  Previously we proposed that transmission of the Hedgehog signal across the plasma membrane by Smoothened is triggered by its interaction with cholesterol (Luchetti et al., 2016). But how is cholesterol, an abundant lipid, regulated tightly enough to control a signaling system that can cause birth defects and cancer? Using toxin-based sensors that distinguish between distinct pools of cholesterol, we find that Smoothened activation and Hedgehog signaling are driven by a biochemically-defined, small fraction of membrane cholesterol, termed accessible cholesterol. Increasing cholesterol accessibility by depletion of sphingomyelin, which sequesters cholesterol in complexes, amplifies Hedgehog signaling. Hedgehog ligands increase cholesterol accessibility in the membrane of the primary cilium by inactivating the transporter-like protein Patched 1. Trapping this accessible cholesterol blocks Hedgehog signal transmission across the membrane. Our work shows that the organization of cholesterol in the ciliary membrane can be modified by extracellular ligands to control the activity of cilia-localized signaling proteins.

  View details for DOI 10.7554/eLife.50051

  View details for PubMedID 31657721

• The morphogen Sonic hedgehog inhibits its receptor Patched by a pincer grasp mechanism. Nature chemical biology Rudolf, A. F., Kinnebrew, M. n., Kowatsch, C. n., Ansell, T. B., El Omari, K. n., Bishop, B. n., Pardon, E. n., Schwab, R. A., Malinauskas, T. n., Qian, M. n., Duman, R. n., Covey, D. F., Steyaert, J. n., Wagner, A. n., Sansom, M. S., Rohatgi, R. n., Siebold, C. n. 2019

  Abstract

  Hedgehog (HH) ligands, classical morphogens that pattern embryonic tissues in all animals, are covalently coupled to two lipids-a palmitoyl group at the N terminus and a cholesteroyl group at the C terminus. While the palmitoyl group binds and inactivates Patched 1 (PTCH1), the main receptor for HH ligands, the function of the cholesterol modification has remained mysterious. Using structural and biochemical studies, along with reassessment of previous cryo-electron microscopy structures, we find that the C-terminal cholesterol attached to Sonic hedgehog (Shh) binds the first extracellular domain of PTCH1 and promotes its inactivation, thus triggering HH signaling. Molecular dynamics simulations show that this interaction leads to the closure of a tunnel through PTCH1 that serves as the putative conduit for sterol transport. Thus, Shh inactivates PTCH1 by grasping its extracellular domain with two lipidic pincers, the N-terminal palmitate and the C-terminal cholesterol, which are both inserted into the PTCH1 protein core.

  View details for DOI 10.1038/s41589-019-0370-y

  View details for PubMedID 31548691

• CRISPR Screens Uncover Genes that Regulate Target Cell Sensitivity to the Morphogen Sonic Hedgehog. Developmental cell Pusapati, G. V., Kong, J. H., Patel, B. B., Krishnan, A. n., Sagner, A. n., Kinnebrew, M. n., Briscoe, J. n., Aravind, L. n., Rohatgi, R. n. 2018; 44 (1): 113–29.e8

  Abstract

  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 PubMedID 29290584

  View details for PubMedCentralID PMC5792066

• R-spondins can potentiate WNT signaling without LGRs. eLife Lebensohn, A. M., Rohatgi, R. n. 2018; 7

  Abstract

  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 PubMedID 29405118

  View details for PubMedCentralID PMC5800842

• Comparative genetic screens in human cells reveal new regulatory mechanisms in WNT signaling ELIFE Lebensohn, A. M., Dubey, R., Neitzel, L. R., Tacchelly-Benites, O., Yang, E., Marceau, C. D., Davis, E. M., Patel, B. B., Bahrami-Nejad, Z., Travaglini, K. J., Ahmed, Y., Lee, E., Carette, J. E., Rohatgi, R. 2016; 5

  Abstract

  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 PubMedID 27996937

• Cholesterol activates the G-protein coupled receptor Smoothened to promote morphogenetic signaling. eLife Luchetti, G., Sircar, R., Kong, J. H., Nachtergaele, S., Sagner, A., Byrne, E. F., Covey, D. F., Siebold, C., Rohatgi, R. 2016; 5

  Abstract

  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. Cell reports Schmidt, H. B., Rohatgi, R. 2016; 16 (5): 1228-1236

  Abstract

  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

  View details for PubMedCentralID PMC4972689

• Structural basis of Smoothened regulation by its extracellular domains. Nature Byrne, E. F., Sircar, R., Miller, P. S., Hedger, G., Luchetti, G., Nachtergaele, S., Tully, M. D., Mydock-McGrane, L., Covey, D. F., Rambo, R. P., Sansom, M. S., Newstead, S., Rohatgi, R., Siebold, C. 2016; 535 (7613): 517-522

  Abstract

  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

• A cholesterol-binding bacterial toxin provides a strategy for identifying a specific Scap inhibitor that blocks lipid synthesis in animal cells. Proceedings of the National Academy of Sciences of the United States of America Xu, S., Smothers, J. C., Rye, D., Endapally, S., Chen, H., Li, S., Liang, G., Kinnebrew, M., Rohatgi, R., Posner, B. A., Radhakrishnan, A. 2024; 121 (7): e2318024121

  Abstract

  Lipid synthesis is regulated by the actions of Scap, a polytopic membrane protein that binds cholesterol in membranes of the endoplasmic reticulum (ER). When ER cholesterol levels are low, Scap activates SREBPs, transcription factors that upregulate genes for synthesis of cholesterol, fatty acids, and triglycerides. When ER cholesterol levels rise, the sterol binds to Scap, triggering conformational changes that prevent activation of SREBPs and halting synthesis of lipids. To achieve a molecular understanding of how cholesterol regulates the Scap/SREBP machine and to identify therapeutics for dysregulated lipid metabolism, cholesterol-mimetic compounds that specifically bind and inhibit Scap are needed. To accomplish this goal, we focused on Anthrolysin O (ALO), a pore-forming bacterial toxin that binds cholesterol with a specificity and sensitivity that is uncannily similar to Scap. We reasoned that a small molecule that would bind and inhibit ALO might also inhibit Scap. High-throughput screening of a ~300,000-compound library for ALO-binding unearthed one molecule, termed UT-59, which binds to Scap's cholesterol-binding site. Upon binding, UT-59 triggers the same conformation changes in Scap as those induced by cholesterol and blocks activation of SREBPs and lipogenesis in cultured cells. UT-59 also inhibits SREBP activation in the mouse liver. Unlike five previously reported inhibitors of SREBP activation, UT-59 is the only one that acts specifically by binding to Scap's cholesterol-binding site. Our approach to identify specific Scap inhibitors such as UT-59 holds great promise in developing therapeutic leads for human diseases stemming from elevated SREBP activation, such as fatty liver and certain cancers.

  View details for DOI 10.1073/pnas.2318024121

  View details for PubMedID 38330014

• The USP46 deubiquitylase complex increases Wingless/Wnt signaling strength by stabilizing Arrow/LRP6. Nature communications Spencer, Z. T., Ng, V. H., Benchabane, H., Siddiqui, G. S., Duwadi, D., Maines, B., Bryant, J. M., Schwarzkopf, A., Yuan, K., Kassel, S. N., Mishra, A., Pimentel, A., Lebensohn, A. M., Rohatgi, R., Gerber, S. A., Robbins, D. J., Lee, E., Ahmed, Y. 2023; 14 (1): 6174

  Abstract

  The control of Wnt receptor abundance is critical for animal development and to prevent tumorigenesis, but the mechanisms that mediate receptor stabilization remain uncertain. We demonstrate that stabilization of the essential Wingless/Wnt receptor Arrow/LRP6 by the evolutionarily conserved Usp46-Uaf1-Wdr20 deubiquitylase complex controls signaling strength in Drosophila. By reducing Arrow ubiquitylation and turnover, the Usp46 complex increases cell surface levels of Arrow and enhances the sensitivity of target cells to stimulation by the Wingless morphogen, thereby increasing the amplitude and spatial range of signaling responses. Usp46 inactivation in Wingless-responding cells destabilizes Arrow, reduces cytoplasmic accumulation of the transcriptional coactivator Armadillo/β-catenin, and attenuates or abolishes Wingless target gene activation, which prevents the concentration-dependent regulation of signaling strength. Consequently, Wingless-dependent developmental patterning and tissue homeostasis are disrupted. These results reveal an evolutionarily conserved mechanism that mediates Wnt/Wingless receptor stabilization and underlies the precise activation of signaling throughout the spatial range of the morphogen gradient.

  View details for DOI 10.1038/s41467-023-41843-0

  View details for PubMedID 37798281

  View details for PubMedCentralID 6086663

• The USP46 complex deubiquitylates LRP6 to promote Wnt/β-catenin signaling. Nature communications Ng, V. H., Spencer, Z., Neitzel, L. R., Nayak, A., Loberg, M. A., Shen, C., Kassel, S. N., Kroh, H. K., An, Z., Anthony, C. C., Bryant, J. M., Lawson, A., Goldsmith, L., Benchabane, H., Hansen, A. G., Li, J., D'Souza, S., Lebensohn, A. M., Rohatgi, R., Weiss, W. A., Weiss, V. L., Williams, C., Hong, C. C., Robbins, D. J., Ahmed, Y., Lee, E. 2023; 14 (1): 6173

  Abstract

  The relative abundance of Wnt receptors plays a crucial role in controlling Wnt signaling in tissue homeostasis and human disease. While the ubiquitin ligases that ubiquitylate Wnt receptors are well-characterized, the deubiquitylase that reverses these reactions remains unclear. Herein, we identify USP46, UAF1, and WDR20 (USP46 complex) as positive regulators of Wnt signaling in cultured human cells. We find that the USP46 complex is similarly required for Wnt signaling in Xenopus and zebrafish embryos. We demonstrate that Wnt signaling promotes the association between the USP46 complex and cell surface Wnt coreceptor, LRP6. Knockdown of USP46 decreases steady-state levels of LRP6 and increases the level of ubiquitylated LRP6. In contrast, overexpression of the USP46 complex blocks ubiquitylation of LRP6 by the ubiquitin ligases RNF43 and ZNFR3. Size exclusion chromatography studies suggest that the size of the USP46 cytoplasmic complex increases upon Wnt stimulation. Finally, we show that USP46 is essential for Wnt-dependent intestinal organoid viability, likely via its role in LRP6 receptor homeostasis. We propose a model in which the USP46 complex increases the steady-state level of cell surface LRP6 and facilitates the assembly of LRP6 into signalosomes via a pruning mechanism that removes sterically hindering ubiquitin chains.

  View details for DOI 10.1038/s41467-023-41836-z

  View details for PubMedID 37798301

  View details for PubMedCentralID 26871

• The energetics and ion coupling of cholesterol transport through Patched1. Science advances Ansell, T. B., Corey, R. A., Viti, L. V., Kinnebrew, M., Rohatgi, R., Siebold, C., Sansom, M. S. 2023; 9 (34): eadh1609

  Abstract

  Patched1 (PTCH1) is a tumor suppressor protein of the mammalian Hedgehog (HH) signaling pathway, implicated in embryogenesis and tissue homeostasis. PTCH1 inhibits the G protein-coupled receptor Smoothened (SMO) via a debated mechanism involving modulating ciliary cholesterol accessibility. Using extensive molecular dynamics simulations and free energy calculations to evaluate cholesterol transport through PTCH1, we find an energetic barrier of ~15 to 20 kilojoule per mole for cholesterol export. In silico data are coupled to in vivo biochemical assays of PTCH1 mutants to probe coupling between cation binding sites, transmembrane motions, and PTCH1 activity. Using complementary simulations of Dispatched1, we find that transition between "inward-open" and solvent "occluded" states is accompanied by Na+-induced pinching of intracellular helical segments. Thus, our findings illuminate the energetics and ion coupling stoichiometries of PTCH1 transport mechanisms, whereby one to three Na+ or two to three K+ couple to cholesterol export, and provide the first molecular description of transitions between distinct transport states.

  View details for DOI 10.1126/sciadv.adh1609

  View details for PubMedID 37611095

  View details for PubMedCentralID PMC10446486

• The Energetics and Ion Coupling of Cholesterol Transport Through Patched1. bioRxiv : the preprint server for biology Ansell, T. B., Corey, R. A., Viti, L. V., Kinnebrew, M., Rohatgi, R., Siebold, C., Sansom, M. S. 2023

  Abstract

  Patched1 (PTCH1) is the principal tumour suppressor protein of the mammalian Hedgehog (HH) signalling pathway, implicated in embryogenesis and tissue homeostasis. PTCH1 inhibits the Class F G protein-coupled receptor Smoothened (SMO) via a debated mechanism involving modulating accessible cholesterol levels within ciliary membranes. Using extensive molecular dynamics (MD) simulations and free energy calculations to evaluate cholesterol transport through PTCH1, we find an energetic barrier of ~15-20 kJ mol -1 for cholesterol export. In simulations we identify cation binding sites within the PTCH1 transmembrane domain (TMD) which may provide the energetic impetus for cholesterol transport. In silico data are coupled to in vivo biochemical assays of PTCH1 mutants to probe coupling between transmembrane motions and PTCH1 activity. Using complementary simulations of Dispatched1 (DISP1) we find that transition between 'inward-open' and solvent 'occluded' states is accompanied by Na + induced pinching of intracellular helical segments. Thus, our findings illuminate the energetics and ion-coupling stoichiometries of PTCH1 transport mechanisms, whereby 1-3 Na + or 2-3 K + couple to cholesterol export, and provide the first molecular description of transitions between distinct transport states.

  View details for DOI 10.1101/2023.02.14.528445

  View details for PubMedID 36824746

  View details for PubMedCentralID PMC9949057

• Receptor control by membrane-tethered ubiquitin ligases in development and tissue homeostasis. Current topics in developmental biology Lebensohn, A. M., Bazan, J. F., Rohatgi, R. 2022; 150: 25-89

  Abstract

  Paracrine cell-cell communication is central to all developmental processes, ranging from cell diversification to patterning and morphogenesis. Precise calibration of signaling strength is essential for the fidelity of tissue formation during embryogenesis and tissue maintenance in adults. Membrane-tethered ubiquitin ligases can control the sensitivity of target cells to secreted ligands by regulating the abundance of signaling receptors at the cell surface. We discuss two examples of this emerging concept in signaling: (1) the transmembrane ubiquitin ligases ZNRF3 and RNF43 that regulate WNT and bone morphogenetic protein receptor abundance in response to R-spondin ligands and (2) the membrane-recruited ubiquitin ligase MGRN1 that controls Hedgehog and melanocortin receptor abundance. We focus on the mechanistic logic of these systems, illustrated by structural and protein interaction models enabled by AlphaFold. We suggest that membrane-tethered ubiquitin ligases play a widespread role in remodeling the cell surface proteome to control responses to extracellular ligands in diverse biological processes.

  View details for DOI 10.1016/bs.ctdb.2022.03.003

  View details for PubMedID 35817504

• Measuring and Manipulating Membrane Cholesterol for the Study of Hedgehog Signaling. Methods in molecular biology (Clifton, N.J.) Kinnebrew, M., Johnson, K. A., Radhakrishnan, A., Rohatgi, R. 2022; 2374: 73-87

  Abstract

  Cholesterol is an abundant lipid in mammalian plasma membranes that regulates the reception of the Hedgehog (Hh) signal in target cells. In vertebrates, cell-surface organelles called primary cilia function as compartments for the propagation of Hh signals. Recent structural, biochemical, and cell-biological studies have led to the model that Patched-1 (PTCH1), the receptor for Hh ligands, uses its transporter-like activity to lower cholesterol accessibility in the membrane surrounding primary cilia. Cholesterol restriction at cilia may represent the long-sought-after mechanism by which PTCH1 inhibits Smoothened (SMO), a cholesterol-responsive transmembrane protein of the G protein-coupled receptor superfamily that transmits the Hh signal across the membrane.Protein probes based on microbial cholesterol-binding proteins revealed that PTCH1 controls only a subset of the total cholesterol molecules, a biochemically defined fraction called accessible cholesterol. The accessible cholesterol pool coexists (and exchanges) with a pool of sequestered cholesterol, which is bound to phospholipids like sphingomyelin. In this chapter, we describe how to measure the accessible and sequestered cholesterol pools in live cells with protein-based probes. We discuss how to purify and fluorescently label these probes for use in flow cytometry and microscopy-based measurements of the cholesterol pools. Additionally, we describe how to modulate accessible cholesterol levels to determine if this pool regulates Hh signaling (or any other cellular process of interest).

  View details for DOI 10.1007/978-1-0716-1701-4_7

  View details for PubMedID 34562244

• Hedgehog-Interacting Protein is a multimodal antagonist of Hedgehog signalling. Nature communications Griffiths, S. C., Schwab, R. A., El Omari, K., Bishop, B., Iverson, E. J., Malinauskas, T., Dubey, R., Qian, M., Covey, D. F., Gilbert, R. J., Rohatgi, R., Siebold, C. 2021; 12 (1): 7171

  Abstract

  Hedgehog (HH) morphogen signalling, crucial for cell growth and tissue patterning in animals, is initiated by the binding of dually lipidated HH ligands to cell surface receptors. Hedgehog-Interacting Protein (HHIP), the only reported secreted inhibitor of Sonic Hedgehog (SHH) signalling, binds directly to SHH with high nanomolar affinity, sequestering SHH. Here, we report the structure of the HHIP N-terminal domain (HHIP-N) in complex with a glycosaminoglycan (GAG). HHIP-N displays a unique bipartite fold with a GAG-binding domain alongside a Cysteine Rich Domain (CRD). We show that HHIP-N is required to convey full HHIP inhibitory function, likely by interacting with the cholesterol moiety covalently linked to HH ligands, thereby preventing this SHH-attached cholesterol from binding to the HH receptor Patched (PTCH1). We also present the structure of the HHIP C-terminal domain in complex with the GAG heparin. Heparin can bind to both HHIP-N and HHIP-C, thereby inducing clustering at the cell surface and generating a high-avidity platform for SHH sequestration and inhibition. Our data suggest a multimodal mechanism, in which HHIP can bind two specific sites on the SHH morphogen, alongside multiple GAG interactions, to inhibit SHH signalling.

  View details for DOI 10.1038/s41467-021-27475-2

  View details for PubMedID 34887403

• Human-chimpanzee fused cells reveal cis-regulatory divergence underlying skeletal evolution. Nature genetics Gokhman, D. n., Agoglia, R. M., Kinnebrew, M. n., Gordon, W. n., Sun, D. n., Bajpai, V. K., Naqvi, S. n., Chen, C. n., Chan, A. n., Chen, C. n., Petrov, D. A., Ahituv, N. n., Zhang, H. n., Mishina, Y. n., Wysocka, J. n., Rohatgi, R. n., Fraser, H. B. 2021

  Abstract

  Gene regulatory divergence is thought to play a central role in determining human-specific traits. However, our ability to link divergent regulation to divergent phenotypes is limited. Here, we utilized human-chimpanzee hybrid induced pluripotent stem cells to study gene expression separating these species. The tetraploid hybrid cells allowed us to separate cis- from trans-regulatory effects, and to control for nongenetic confounding factors. We differentiated these cells into cranial neural crest cells, the primary cell type giving rise to the face. We discovered evidence of lineage-specific selection on the hedgehog signaling pathway, including a human-specific sixfold down-regulation of EVC2 (LIMBIN), a key hedgehog gene. Inducing a similar down-regulation of EVC2 substantially reduced hedgehog signaling output. Mice and humans lacking functional EVC2 show striking phenotypic parallels to human-chimpanzee craniofacial differences, suggesting that the regulatory divergence of hedgehog signaling may have contributed to the unique craniofacial morphology of humans.

  View details for DOI 10.1038/s41588-021-00804-3

  View details for PubMedID 33731941

• Mutations in GRK2 cause Jeune syndrome by impairing Hedgehog and canonical Wnt signaling EMBO MOLECULAR MEDICINE Bosakova, M., Abraham, S. P., Nita, A., Hruba, E., Buchtova, M., Taylor, S., Duran, I., Martin, J., Svozilova, K., Barta, T., Varecha, M., Balek, L., Kohoutek, J., Radaszkiewicz, T., Pusapati, G., Bryja, V., Rush, E. T., Thiffault, I., Nickerson, D. A., Bamshad, M. J., Rohatgi, R., Cohn, D. H., Krakow, D., Krejci, P. 2020

  View details for DOI 10.15252/emmm.201911739

  View details for Web of Science ID 000577616000001

• High-throughput Flow Cytometry Assay to Investigate TDP43 Splicing Function BIO-PROTOCOL Schmidt, H., Rohatgi, R. 2020; 10 (8)

  View details for DOI 10.21769/BioProtoc.3594

  View details for Web of Science ID 000583098900012

• High-throughput Flow Cytometry Assay to Investigate TDP43 Splicing Function. Bio-protocol Schmidt, H. B., Rohatgi, R. 2020; 10 (8): e3594

  Abstract

  Mutations in RNA-binding proteins (RBPs) such as TDP43 are associated with transcriptome-wide splicing defects and cause severe neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The impact of RBP mutations on splicing function is routinely studied using PCR-based bulk measurements. However, the qualitative and low-throughput nature of this assay make quantitative and systematic analyses, as well as screening approaches, difficult to implement. To overcome this hurdle, we have developed a quantitative, high-throughput flow cytometry assay to investigate TDP43 splicing function on a single-cell level.

  View details for DOI 10.21769/BioProtoc.3594

  View details for PubMedID 33659560

  View details for PubMedCentralID PMC7842676

• Flow Homogenization Enables a Massively Parallel Fluidic Design for High-Throughput and Multiplexed Cell Isolation ADVANCED MATERIALS TECHNOLOGIES Ooi, C., Earhart, C. M., Hughes, C. E., Lee, J., Wong, D. J., Wilson, R. J., Rohatgi, R., Wang, S. X. 2020

  View details for DOI 10.1002/admt.201900960

  View details for Web of Science ID 000520259400001

• TDP-43 alpha-helical structure tunes liquid-liquid phase separation and function. Proceedings of the National Academy of Sciences of the United States of America Conicella, A. E., Dignon, G. L., Zerze, G. H., Schmidt, H. B., D'Ordine, A. M., Kim, Y. C., Rohatgi, R., Ayala, Y. M., Mittal, J., Fawzi, N. L. 2020

  Abstract

  Liquid-liquid phase separation (LLPS) is involved in the formation of membraneless organelles (MLOs) associated with RNA processing. The RNA-binding protein TDP-43 is present in several MLOs, undergoes LLPS, and has been linked to the pathogenesis of amyotrophic lateral sclerosis (ALS). While some ALS-associated mutations in TDP-43 disrupt self-interaction and function, here we show that designed single mutations can enhance TDP-43 assembly and function via modulating helical structure. Using molecular simulation and NMR spectroscopy, we observe large structural changes upon dimerization of TDP-43. Two conserved glycine residues (G335 and G338) are potent inhibitors of helical extension and helix-helix interaction, which are removed in part by variants at these positions, including the ALS-associated G335D. Substitution to helix-enhancing alanine at either of these positions dramatically enhances phase separation in vitro and decreases fluidity of phase-separated TDP-43 reporter compartments in cells. Furthermore, G335A increases TDP-43 splicing function in a minigene assay. Therefore, the TDP-43 helical region serves as a short but uniquely tunable module where application of biophysical principles can precisely control assembly and function in cellular and synthetic biology applications of LLPS.

  View details for DOI 10.1073/pnas.1912055117

  View details for PubMedID 32132204

• Mutations in GRK2 cause Jeune syndrome by impairing Hedgehog and canonical Wnt signaling. EMBO molecular medicine Bosakova, M. n., Abraham, S. P., Nita, A. n., Hruba, E. n., Buchtova, M. n., Taylor, S. P., Duran, I. n., Martin, J. n., Svozilova, K. n., Barta, T. n., Varecha, M. n., Balek, L. n., Kohoutek, J. n., Radaszkiewicz, T. n., Pusapati, G. V., Bryja, V. n., Rush, E. T., Thiffault, I. n., Nickerson, D. A., Bamshad, M. J., Rohatgi, R. n., Cohn, D. H., Krakow, D. n., Krejci, P. n. 2020; 12 (11): e11739

  Abstract

  Mutations in genes affecting primary cilia cause ciliopathies, a diverse group of disorders often affecting skeletal development. This includes Jeune syndrome or asphyxiating thoracic dystrophy (ATD), an autosomal recessive skeletal disorder. Unraveling the responsible molecular pathology helps illuminate mechanisms responsible for functional primary cilia. We identified two families with ATD caused by loss-of-function mutations in the gene encoding adrenergic receptor kinase 1 (ADRBK1 or GRK2). GRK2 cells from an affected individual homozygous for the p.R158* mutation resulted in loss of GRK2, and disrupted chondrocyte growth and differentiation in the cartilage growth plate. GRK2 null cells displayed normal cilia morphology, yet loss of GRK2 compromised cilia-based signaling of Hedgehog (Hh) pathway. Canonical Wnt signaling was also impaired, manifested as a failure to respond to Wnt ligand due to impaired phosphorylation of the Wnt co-receptor LRP6. We have identified GRK2 as an essential regulator of skeletogenesis and demonstrate how both Hh and Wnt signaling mechanistically contribute to skeletal ciliopathies.

  View details for DOI 10.15252/emmm.201911739

  View details for PubMedID 33200460

  View details for PubMedCentralID PMC7645380

• Bile Acid Biosynthesis in Smith-Lemli-Opitz Syndrome Bypassing Cholesterol: Potential Importance of Pathway Intermediates. The Journal of steroid biochemistry and molecular biology Abdel-Khalik, J. n., Hearn, T. n., Dickson, A. L., Crick, P. J., Yutuc, E. n., Austin-Muttit, K. n., Bigger, B. W., Morris, A. A., Shackleton, C. H., Clayton, P. T., Iida, T. n., Sircar, R. n., Rohatgi, R. n., Marschall, H. U., Sjövall, J. n., Björkhem, I. n., Mullins, J. G., Griffiths, W. J., Wang, Y. n. 2020: 105794

  Abstract

  Bile acids are the end products of cholesterol metabolism secreted into bile. They are essential for the absorption of lipids and lipid soluble compounds from the intestine. Here we have identified a series of unusual Δ5-unsaturated bile acids in plasma and urine of patients with Smith-Lemli-Opitz syndrome (SLOS), a defect in cholesterol biosynthesis resulting in elevated levels of 7-dehydrocholesterol (7-DHC), an immediate precursor of cholesterol. Using liquid chromatography - mass spectrometry (LC-MS) we have uncovered a pathway of bile acid biosynthesis in SLOS avoiding cholesterol starting with 7-DHC and proceeding through 7-oxo and 7β-hydroxy intermediates. This pathway also occurs to a minor extent in healthy humans, but elevated levels of pathway intermediates could be responsible for some of the features SLOS. The pathway is also active in SLOS affected pregnancies as revealed by analysis of amniotic fluid. Importantly, intermediates in the pathway, 25-hydroxy-7-oxocholesterol, (25R)26-hydroxy-7-oxocholesterol, 3β-hydroxy-7-oxocholest-5-en-(25R)26-oic acid and the analogous 7β-hydroxysterols are modulators of the activity of Smoothened (Smo), an oncoprotein that mediates Hedgehog (Hh) signalling across membranes during embryogenesis and in the regeneration of postembryonic tissue. Computational docking of the 7-oxo and 7β-hydroxy compounds to the extracellular cysteine rich domain of Smo reveals that they bind in the same groove as both 20S-hydroxycholesterol and cholesterol, known activators of the Hh pathway.

  View details for DOI 10.1016/j.jsbmb.2020.105794

  View details for PubMedID 33246156

• Mechanism, physiological and therapeutic implications of LGR-independent potentiation of WNT signaling by R-spondins Lebensohn, A. M., Rohatgi, R. AMER ASSOC CANCER RESEARCH. 2019

  View details for DOI 10.1158/1538-7445.AM2019-3445

  View details for Web of Science ID 000488279402377

• Dynamic Remodeling of Membrane Composition Drives Cell Cycle through Primary Cilia Excision CELL Phua, S., Chiba, S., Suzuki, M., Su, E., Roberson, E. C., Pusapati, G. V., Schurmans, S., Setou, M., Rohatgi, R., Reiter, J. F., Ikegami, K., Inoue, T. 2019; 178 (1): 261

  View details for DOI 10.1016/j.cell.2019.06.015

  View details for Web of Science ID 000473002700022

  View details for PubMedID 31251914

• Cholesterol Interaction Sites on the Transmembrane Domain of the Hedgehog Signal Transducer and Class F G Protein-Coupled Receptor Smoothened STRUCTURE Hedger, G., Koldso, H., Chavent, M., Siebold, C., Rohatgi, R., Sansom, M. P. 2019; 27 (3): 549-+

  View details for DOI 10.1016/j.str.2018.11.003

  View details for Web of Science ID 000460258100017

• Discovery of gene regulatory elements through a new bioinformatics analysis of haploid genetic screens PLOS ONE Patel, B. B., Lebensohn, A. M., Pusapati, G. V., Carette, J. E., Salzman, J., Rohatgi, R. 2019; 14 (1)

  View details for DOI 10.1371/journal.pone.0198463

  View details for Web of Science ID 000457046400001

• Discovery of gene regulatory elements through a new bioinformatics analysis of haploid genetic screens. PloS one Patel, B. B., Lebensohn, A. M., Pusapati, G. V., Carette, J. E., Salzman, J. n., Rohatgi, R. n. 2019; 14 (1): e0198463

  Abstract

  The systematic identification of regulatory elements that control gene expression remains a challenge. Genetic screens that use untargeted mutagenesis have the potential to identify protein-coding genes, non-coding RNAs and regulatory elements, but their analysis has mainly focused on identifying the former two. To identify regulatory elements, we conducted a new bioinformatics analysis of insertional mutagenesis screens interrogating WNT signaling in haploid human cells. We searched for specific patterns of retroviral gene trap integrations (used as mutagens in haploid screens) in short genomic intervals overlapping with introns and regions upstream of genes. We uncovered atypical patterns of gene trap insertions that were not predicted to disrupt coding sequences, but caused changes in the expression of two key regulators of WNT signaling, suggesting the presence of cis-regulatory elements. Our methodology extends the scope of haploid genetic screens by enabling the identification of regulatory elements that control gene expression.

  View details for PubMedID 30695034

• Structures of vertebrate Patched and Smoothened reveal intimate links between cholesterol and Hedgehog signalling. Current opinion in structural biology Kowatsch, C. n., Woolley, R. E., Kinnebrew, M. n., Rohatgi, R. n., Siebold, C. n. 2019; 57: 204–14

  Abstract

  The Hedgehog (HH) signalling pathway is a cell-cell communication system that controls the patterning of multiple tissues during embryogenesis in metazoans. In adults, HH signals regulate tissue stem cells and regenerative responses. Abnormal signalling can cause birth defects and cancer. The HH signal is received on target cells by Patched (PTCH1), the receptor for HH ligands, and then transmitted across the plasma membrane by Smoothened (SMO). Recent structural and biochemical studies have pointed to a sterol lipid, likely cholesterol itself, as the elusive second messenger that communicates the HH signal between PTCH1 and SMO, thus linking ligand reception to transmembrane signalling.

  View details for DOI 10.1016/j.sbi.2019.05.015

  View details for PubMedID 31247512

• Cholesterol Interaction Sites on the Transmembrane Domain of the Hedgehog Signal Transducer and Class F G Protein-Coupled Receptor Smoothened. Structure (London, England : 1993) Hedger, G., Koldso, H., Chavent, M., Siebold, C., Rohatgi, R., Sansom, M. S. 2018

  Abstract

  Transduction of Hedgehog signals across the plasma membrane is facilitated by the class F G-protein-coupled-receptor (GPCR) Smoothened (SMO). Recent studies suggest that SMO is modulated via interactions of its transmembrane (TM) domain with cholesterol. We apply molecular dynamics simulations of SMO embedded in cholesterol containing lipid bilayers, revealing a direct interaction of cholesterol with the TM domain at regions distinct from those observed in class A GPCRs. In particular the extracellular tips of helices TM2 and TM3 form a well-defined cholesterol interaction site. Potential of mean force calculations yield a free energy landscape for cholesterol binding. Alongside analysis of equilibrium cholesterol occupancy, this reveals the existence of a dynamic "greasy patch" interaction with the TM domain of SMO, which may be compared with previously identified lipid interaction sites on other membrane proteins. These predictions provide molecular-level insights into cholesterol interactions with a class F GPCR, suggesting potential druggable sites.

  View details for PubMedID 30595453

• R-spondins can potentiate WNT signaling without LGRs ELIFE Lebensohn, A. M., Rohatgi, R. 2018; 7

  View details for DOI 10.7554/eLife.33126

  View details for Web of Science ID 000424216300001

• G protein-coupled receptors control the sensitivity of cells to the morphogen Sonic Hedgehog. Science signaling Pusapati, G. V., Kong, J. H., Patel, B. B., Gouti, M. n., Sagner, A. n., Sircar, R. n., Luchetti, G. n., Ingham, P. W., Briscoe, J. n., Rohatgi, R. n. 2018; 11 (516)

  Abstract

  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 PubMedID 29438014

  View details for PubMedCentralID PMC5828112

• Spatiotemporal manipulation of ciliary glutamylation reveals its roles in intraciliary trafficking and Hedgehog signaling. Nature communications Hong, S. R., Wang, C. L., Huang, Y. S., Chang, Y. C., Chang, Y. C., Pusapati, G. V., Lin, C. Y., Hsu, N. n., Cheng, H. C., Chiang, Y. C., Huang, W. E., Shaner, N. C., Rohatgi, R. n., Inoue, T. n., Lin, Y. C. 2018; 9 (1): 1732

  Abstract

  Tubulin post-translational modifications (PTMs) occur spatiotemporally throughout cells and are suggested to be involved in a wide range of cellular activities. However, the complexity and dynamic distribution of tubulin PTMs within cells have hindered the understanding of their physiological roles in specific subcellular compartments. Here, we develop a method to rapidly deplete tubulin glutamylation inside the primary cilia, a microtubule-based sensory organelle protruding on the cell surface, by targeting an engineered deglutamylase to the cilia in minutes. This rapid deglutamylation quickly leads to altered ciliary functions such as kinesin-2-mediated anterograde intraflagellar transport and Hedgehog signaling, along with no apparent crosstalk to other PTMs such as acetylation and detyrosination. Our study offers a feasible approach to spatiotemporally manipulate tubulin PTMs in living cells. Future expansion of the repertoire of actuators that regulate PTMs may facilitate a comprehensive understanding of how diverse tubulin PTMs encode ciliary as well as cellular functions.

  View details for PubMedID 29712905

  View details for PubMedCentralID PMC5928066

• A single N-terminal phosphomimic disrupts TDP-43 polymerization, phase separation, and RNA splicing. The EMBO journal Wang, A. n., Conicella, A. E., Schmidt, H. B., Martin, E. W., Rhoads, S. N., Reeb, A. N., Nourse, A. n., Ramirez Montero, D. n., Ryan, V. H., Rohatgi, R. n., Shewmaker, F. n., Naik, M. T., Mittag, T. n., Ayala, Y. M., Fawzi, N. L. 2018; 37 (5)

  Abstract

  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 PubMedID 29438978

• Dynamic Remodeling of Membrane Composition Drives Cell Cycle through Primary Cilia Excision. Cell Phua, S. C., Chiba, S., Suzuki, M., Su, E., Roberson, E. C., Pusapati, G. V., Setou, M., Rohatgi, R., Reiter, J. F., Ikegami, K., Inoue, T. 2017; 168 (1-2): 264-279 e15

  Abstract

  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 Byrne, E. F., Luchetti, G. n., Rohatgi, R. n., Siebold, C. n. 2017; 51: 81–88

  Abstract

  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 PubMedID 29268141

• Chromatin-Remodeling Complex SWI/SNF Controls Multidrug Resistance by Transcriptionally Regulating the Drug Efflux Pump ABCB1 CANCER RESEARCH Dubey, R., Lebensohn, A. M., Bahrami-Nejad, Z., Marceau, C., Champion, M., Gevaert, O., Sikic, B. I., Carette, J. E., Rohatgi, R. 2016; 76 (19): 5810-5821

  Abstract

  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 EMBO REPORTS Zhao, Z., Lee, R. T., Pusapati, G. V., Iyu, A., Rohatgi, R., Ingham, P. W. 2016; 17 (5): 739-752

  Abstract

  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

• Functional Divergence in the Role of N-Linked Glycosylation in Smoothened Signaling. PLoS genetics Marada, S., Navarro, G., Truong, A., Stewart, D. P., Arensdorf, A. M., Nachtergaele, S., Angelats, E., Opferman, J. T., Rohatgi, R., McCormick, P. J., Ogden, S. K. 2015; 11 (8)

  Abstract

  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

  View details for PubMedCentralID PMC4546403

• Notch Activity Modulates the Responsiveness of Neural Progenitors to Sonic Hedgehog Signaling DEVELOPMENTAL CELL Kong, J. H., Yang, L., Dessaud, E., Chuang, K., Moore, D. M., Rohatgi, R., Briscoe, J., Novitch, B. G. 2015; 33 (4): 373-387

  Abstract

  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

  View details for PubMedCentralID PMC4449290

• Measuring Gli2 Phosphorylation by Selected Reaction Monitoring Mass Spectrometry. Methods in molecular biology (Clifton, N.J.) Ahrends, R., Niewiadomski, P., Teruel, M. N., Rohatgi, R. 2015; 1322: 105-123

  Abstract

  Phosphorylation is an important mechanism by which Gli proteins are regulated. When the Hedgehog (Hh) pathway is activated, multiple serine and threonine residues of Gli2 are dephosphorylated, while at least one residue undergoes phosphorylation. These changes in phosphorylation have functional relevance for the transcriptional activity of Gli proteins, as shown by in vitro and in vivo assays on Gli mutants lacking the phosphorylated residues. Here, we describe a method of quantitatively monitoring the phosphorylation of Gli proteins by triple quadrupole mass spectrometry of Gli2 immunoprecipitated from cell lysates. This method is broadly applicable to the monitoring of phosphorylation changes of immunoprecipitated Gli proteins when the putative phosphosites are known.

  View details for DOI 10.1007/978-1-4939-2772-2_10

  View details for PubMedID 26179043

• Measuring Expression Levels of Endogenous Gli Genes by Immunoblotting and Real-Time PCR HEDGEHOG SIGNALING PROTOCOLS, 2ND EDITION Niewiadomski, P., Rohatgi, R., Riobo, N. A. 2015; 1322: 81-92

  Abstract

  Gli proteins are transcription factors that mediate the transcriptional effects of Hedgehog signaling in vertebrates. The activities of Gli2 and Gli3 are regulated primarily by posttranslational modifications, while Gli1 is mostly regulated at the transcriptional level. Detection of endogenous Gli proteins had been hampered by lack of good antibodies, but this problem has been mostly resolved in recent years. In this chapter we describe methods of detecting expression of endogenous Gli genes in whole-cell lysates and in subcellular fractions and also provide protocols for the measurement of Gli mRNA levels by quantitative real-time reverse transcriptase PCR (qPCR).

  View details for DOI 10.1007/978-1-4939-2772-2_8

  View details for Web of Science ID 000376514700009

  View details for PubMedID 26179041

• Rapid Screening of Gli2/3 Mutants Using the Flp-In System HEDGEHOG SIGNALING PROTOCOLS, 2ND EDITION Niewiadomski, P., Rohatgi, R., Riobo, N. A. 2015; 1322: 125-130

  Abstract

  Gli2 and Gli3 respond to the Hedgehog (Hh) signal in mammals by undergoing posttranslational modifications and moving to the nucleus. The study of Gli proteins has been hampered by the fact that their overexpression in cells prevents their proper regulation. To address this issue, we have developed a method of rapid generation of stable cell lines expressing near-endogenous and approximately equal levels of wild-type and mutant Gli proteins. This method is applicable to the study of effects of various mutations on Gli protein modifications and activity.

  View details for DOI 10.1007/978-1-4939-2772-2_11

  View details for Web of Science ID 000376514700012

  View details for PubMedID 26179044

• Location, location, and location: compartmentalization of Hedgehog signaling at primary cilia. EMBO journal Pusapati, G. V., Rohatgi, R. 2014; 33 (17): 1852-1854

  View details for DOI 10.15252/embj.201489294

  View details for PubMedID 25037564

• Frontiers in hedgehog signal transduction SEMINARS IN CELL & DEVELOPMENTAL BIOLOGY Guerrero, I., Rohatgi, R. 2014; 33: 50-51

  View details for DOI 10.1016/j.semcdb.2014.06.009

  View details for Web of Science ID 000341477700008

  View details for PubMedID 24946961

• G-protein-coupled receptors, Hedgehog signaling and primary cilia. Seminars in cell & developmental biology Mukhopadhyay, S., Rohatgi, R. 2014; 33: 63-72

  Abstract

  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

  View details for PubMedCentralID PMC4130902

• G-protein-coupled receptors, Hedgehog signaling and primary cilia SEMINARS IN CELL & DEVELOPMENTAL BIOLOGY Mukhopadhyay, S., Rohatgi, R. 2014; 33: 63-72

  Abstract

  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 Web of Science ID 000341477700010

  View details for PubMedCentralID PMC4130902

• 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 Montgomery, S. R., Nargizyan, T., Meliton, V., Nachtergaele, S., Rohatgi, R., Stappenbeck, F., Jung, M. E., Johnson, J. S., Aghdasi, B., Tian, H., Weintraub, G., Inoue, H., Atti, E., Tetradis, S., Pereira, R. C., Hokugo, A., Alobaidaan, R., Tan, Y., Hahn, T. J., Wang, J. C., Parhami, F. 2014; 29 (8): 1872-1885

  Abstract

  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 Peyrot, S. M., Nachtergaele, S., Luchetti, G., Mydock-McGrane, L. K., Fujiwara, H., Scherrer, D., Jallouk, A., Schlesinger, P. H., Ory, D. S., Covey, D. F., Rohatgi, R. 2014; 289 (16): 11095-11110

  Abstract

  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

• EFCAB7 and IQCE Regulate Hedgehog Signaling by Tethering the EVC-EVC2 Complex to the Base of Primary Cilia DEVELOPMENTAL CELL Pusapati, G. V., Hughes, C. E., Dorn, K. V., Zhang, D., Sugianto, P., Aravind, L., Rohatgi, R. 2014; 28 (5): 483-496

  Abstract

  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. Cell reports Niewiadomski, P., Kong, J. H., Ahrends, R., Ma, Y., Humke, E. W., Khan, S., Teruel, M. N., Novitch, B. G., Rohatgi, R. 2014; 6 (1): 168-181

  Abstract

  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

  View details for PubMedCentralID PMC3915062

• Isolation and mutational analysis of circulating tumor cells from lung cancer patients with magnetic sifters and biochips LAB ON A CHIP Earhart, C. M., Hughes, C. E., Gaster, R. S., Ooi, C. C., Wilson, R. J., Zhou, L. Y., Humke, E. W., Xu, L., Wong, D. J., Willingham, S. B., Schwartz, E. J., Weissman, I. L., Jeffrey, S. S., Neal, J. W., Rohatgi, R., Wakeleebe, H. A., Wang, S. X. 2014; 14 (1): 78-88

  View details for DOI 10.1039/c3lc50580d

  View details for Web of Science ID 000327669000008

• Isolation and mutational analysis of circulating tumor cells from lung cancer patients with magnetic sifters and biochips. Lab on a chip Earhart, C. M., Hughes, C. E., Gaster, R. S., Ooi, C. C., Wilson, R. J., Zhou, L. Y., Humke, E. W., Xu, L., Wong, D. J., Willingham, S. B., Schwartz, E. J., Weissman, I. L., Jeffrey, S. S., Neal, J. W., Rohatgi, R., Wakelee, H. A., Wang, S. X. 2013; 14 (1): 78-88

  Abstract

  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 Carragee, E. J., Chu, G., Rohatgi, R., Hurwitz, E. L., Weiner, B. K., Yoon, S. T., Comer, G., Kopjar, B. 2013; 95 (17): 1537-1545

  Abstract

  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 Carragee, E. J., Chu, G., Rohatgi, R., Hurwitz, E. L., Weiner, B. K., Yoon, S. T., Comer, G., Kopjar, B. 2013; 95A (17): 1537-1545

  Abstract

  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 Web of Science ID 000323863300001

• Chemically inducible diffusion trap at cilia reveals molecular sieve-like barrier. Nature chemical biology Lin, Y., Niewiadomski, P., Lin, B., Nakamura, H., Phua, S. C., Jiao, J., Levchenko, A., Inoue, T., Rohatgi, R., Inoue, T. 2013; 9 (7): 437-443

  Abstract

  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

• Genome-wide screens for Wnt signaling in human haploid cells Lebensohn, A., Hughes, C., Marceau, C., Rohatgi, R., Carette, J. AMER ASSOC CANCER RESEARCH. 2013

  View details for DOI 10.1158/1535-7163.PMS-PR03

  View details for Web of Science ID 000209496700072

• Illuminating Passive Permeability Barrier of Primary Cilia using Novel Diffusion Trap Technique 57th Annual Meeting of the Biophysical-Society Inoue, T., Lin, Y., Lin, B., Phua, S. C., Jiao, J., Levchenko, A., Niewiadomski, P., Rohatgi, R., Nakamura, H., Inoue, T. CELL PRESS. 2013: 31A–32A

  View details for Web of Science ID 000316074300163

• Structure and function of the Smoothened extracellular domain in vertebrate Hedgehog signaling. eLife Nachtergaele, S., Whalen, D. M., Mydock, L. K., Zhao, Z., Malinauskas, T., Krishnan, K., Ingham, P. W., Covey, D. F., Siebold, C., Rohatgi, R. 2013; 2: e01340

  Abstract

  The Hedgehog (Hh) signal is transduced across the membrane by the heptahelical protein Smoothened (Smo), a developmental regulator, oncoprotein and drug target in oncology. We present the 2.3 Å crystal structure of the extracellular cysteine rich domain (CRD) of vertebrate Smo and show that it binds to oxysterols, endogenous lipids that activate Hh signaling. The oxysterol-binding groove in the Smo CRD is analogous to that used by Frizzled 8 to bind to the palmitoleyl group of Wnt ligands and to similar pockets used by other Frizzled-like CRDs to bind hydrophobic ligands. The CRD is required for signaling in response to native Hh ligands, showing that it is an important regulatory module for Smo activation. Indeed, targeting of the Smo CRD by oxysterol-inspired small molecules can block signaling by all known classes of Hh activators and by clinically relevant Smo mutants. DOI:http://dx.doi.org/10.7554/eLife.01340.001.

  View details for DOI 10.7554/eLife.01340

  View details for PubMedID 24171105

  View details for PubMedCentralID PMC3809587

• A Smoothened-Evc2 Complex Transduces the Hedgehog Signal at Primary Cilia DEVELOPMENTAL CELL Dorn, K. V., Hughes, C. E., Rohatgi, R. 2012; 23 (4): 823-835

  Abstract

  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

• Singapore signalling: the 2012 hedgehog pathway cocktail EMBO REPORTS Briscoe, J., Rohatgi, R. 2012; 13 (7): 580-583

  Abstract

  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

  View details for PubMedCentralID PMC3388788

• Oxysterols are allosteric activators of the oncoprotein Smoothened NATURE CHEMICAL BIOLOGY Nachtergaele, S., Mydock, L. K., Krishnan, K., Rammohan, J., Schlesinger, P. H., Covey, D. F., Rohatgi, R. 2012; 8 (2): 211-220

  Abstract

  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

  View details for PubMedCentralID PMC3262054

• Regulation of signaling pathways by endogenous small molecules Nachtergaele, S., Mydock, L., Krishnan, K., Covey, D., Rohatgi, R. AMER ASSOC CANCER RESEARCH. 2011

  View details for DOI 10.1158/1538-7445.AM2011-LB-238

  View details for Web of Science ID 000209701306106

• Cilia 2010: The Surprise Organelle of the Decade SCIENCE SIGNALING Smith, E. F., Rohatgi, R. 2011; 4 (155)

  Abstract

  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 Rohatgi, R., Snell, W. J. 2010; 22 (4): 541-546

  Abstract

  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

  View details for PubMedCentralID PMC2910237

• Role of Lipid Metabolism in Smoothened Derepression in Hedgehog Signaling DEVELOPMENTAL CELL Yavan, A., Nagaraj, R., Owusu-Ansah, E., Folick, A., Ngo, K., Hillman, T., Call, G., Rohatgi, R., Scott, M. P., Banerjee, U. 2010; 19 (1): 54-65

  Abstract

  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

  View details for PubMedCentralID PMC2945252

• The output of Hedgehog signaling is controlled by the dynamic association between Suppressor of Fused and the Gli proteins GENES & DEVELOPMENT Humke, E. W., Dorn, K. V., Milenkovic, L., Scott, M. P., Rohatgi, R. 2010; 24 (7): 670-682

  Abstract

  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

• Lateral transport of Smoothened from the plasma membrane to the membrane of the cilium JOURNAL OF CELL BIOLOGY Milenkovic, L., Scott, M. P., Rohatgi, R. 2009; 187 (3): 365-374

  Abstract

  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

  View details for PubMedCentralID PMC2779247

• The Hedgehog signaling pathway in lung cancer: moving beyond inhibition of cell growth Rohatgi, R. LIPPINCOTT WILLIAMS & WILKINS. 2009: S82

  View details for Web of Science ID 000269496000079

• Hedgehog signal transduction by smoothened: pharmacological evidence for a two-step activation process. Proceedings of the National Academy of Sciences USA Rohatgi R, M., Corcoran RB, Scott MP 2009; 106: 3196-3201
• Arrestin? Movement in Cilia. Science Rohatgi R, Scott MP 2008; 320 (5884): 1777-1781
• Patched1 regulates Hedgehog signaling at the primary cilium. Science Rohatgi R, M., Scott MP 2007; 317 (5836): 372-376
• Patching the gaps in Hedgehog signaling. Nat Cell Bio Rohatgi R, Scott MP 2007; 9 (9): 1005-1009
• In vitro reconstitution of cdc42-mediated actin assembly using purified components. Methods in Enzymology Ho HY, Rohatgi R, Lebensohn A, Kirschner MW 2006; 406: 174-190
• Loss-of-function Analysis of EphA Receptors in Retinotectal mapping. Journal of Neuroscience Feldheim DA, Nakamoto M, Osterfield M, Gale NW, DeChiara TM, Rohatgi R, Yancopoulos GD, Flanagan JG 2004; 24 (10): 2542-2550
• Toca-1 Mediates Cdc42- Dependent Actin Nucleation by Activating the N-WASP-WIP Complex. Cell Ho HY, R., Lebensohn A, Ma L, Li L, Gygi SP, Kirschner MW 2004; 118 (2): 203-216
• The Mechanism of Regulation of WAVE1-induced Actin Nucleation by Rac1 and Nck. Nature Eden S, Rohatgi R, Podtelejnikov AV, Mann M, Kirschner MW 2002; 418 (6899): 790-793
• 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 Ho HY, R., Ma L, Kirschner MW 2001; 98 (20): 11306-11311
• WIP Regulates N-WASP-Mediated Actin Polymerization and Filopodium Formation. Nature Cell Biology Martinez-Quiles N, Rohatgi R, Anton IM, Medina M, Saville SP, Miki H, Yamaguchi H, Takenawa T, Hartwig JH, Geha RS, Ramesh N 2001; 3 (5): 484-491
• Nck and Phosphatidylinositol 4,5 Bisphosphate Synergistically Activate Actin Polymerization Through the N-WASP-Arp2/3 Pathway. Journal of Biological Chemistry Rohatgi R, Nollau P, Ho HY, Kirschner MW, Mayer BJ 2001; 276 (28): 26448-26452
• Mechanism of N-WASP Activation by CDC42 and Phosphatidylinositol 4, 5-Bisphosphate. Journal of Cell Biology Rohatgi R, H., Kirschner MW 2000; 150 (6): 1299-1310
• The Interaction Between N-WASP and the Arp2/3 Complex Links Cdc42-Dependent Signals to Actin Assembly. Cell Rohatgi R, M., Miki H, Lopez M, Kirchhausen T, Takenawa T, Kirschner MW 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 Ma L, Rohatgi R, Kirschner MW 1998; 95 (26): 15362-15367
• Kinetic and Mechanistic Analysis of Non-Enzymatic, Template-Directed Oligoribonucleotide Ligation. Journal of the American Chemical Society Rohatgi R, Bartel DP, Szostak JW 1996; 118 (14): 3332-3339
• Non-Enzymatic, Template-Directed Ligation of Oligoribonucleotides is Highly Regioselective for the Formation of 3'-5'-Phosphodiester Bonds Journal of the American Chemical Society Rohatgi R, Bartel DP, Szostak JW 1996; 118 (14): 3340-3344