scholarly journals Patched 1 reduces the accessibility of cholesterol in the outer leaflet of membranes

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Maia Kinnebrew ◽  
Giovanni Luchetti ◽  
Ria Sircar ◽  
Sara Frigui ◽  
Lucrezia Vittoria Viti ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Sonic Hedgehog ◽  
Hedgehog Signaling ◽  
Live Cells ◽  
Transport Activity ◽  
Outer Leaflet ◽  
Cholesterol Levels ◽  
Patched 1 ◽  
Potassium Gradient ◽  
Kinetics Of

A long-standing mystery in vertebrate Hedgehog signaling is how Patched 1 (PTCH1), the receptor for Hedgehog ligands, inhibits the activity of Smoothened, the protein that transmits the signal across the membrane. We previously proposed (Kinnebrew et al., 2019) that PTCH1 inhibits Smoothened by depleting accessible cholesterol from the ciliary membrane. To directly test the effect of PTCH1 on accessible cholesterol, we measured the transport activity of PTCH1 using an imaging-based assay to follow the kinetics of cholesterol extraction from the plasma membrane of live cells by methyl-β-cyclodextrin. PTCH1 depletes accessible cholesterol in the outer leaflet of the membrane in a manner regulated by its ligand Sonic Hedgehog and the transmembrane potassium gradient. We propose that PTCH1 moves cholesterol from the outer to the inner leaflet of the membrane in exchange for potassium ion export. Our results show that proteins can change accessible cholesterol levels in membranes to regulate signaling reaction.

scholarly journals C24 sphingolipids play a surprising and central role in governing cholesterol and lateral organization of the live cell plasma membrane

2017 ◽  
Author(s):  
K. C. Courtney ◽  
W Pezeshkian ◽  
R Raghupathy ◽  
C Zhang ◽  
A Darbyson ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Acyl Chain ◽  
Live Cells ◽  
Membrane Microdomains ◽  
Giant Unilamellar Vesicles ◽  
Unilamellar Vesicles ◽  
Outer Leaflet ◽  
Acyl Chains ◽  
Lateral Organization

AbstractMammalian cell sphingolipids, primarily with C24 and C16 acyl chains, reside in the outer leaflet of the plasma membrane. Curiously, little is known how C24 sphingolipids impact cholesterol and membrane microdomains. Here, we generated giant unilamellar vesicles and live mammalian cells with C24 or C16 sphingomyelin exclusively in the outer leaflet and compared microdomain formation. In giant unilamellar vesicles, we observed that asymmetrically placed C24 sphingomyelin suppresses microdomains. Conversely, C16 sphingomyelin facilitates microdomains. Replacing endogenous sphingolipids with C24 or C16 sphingomyelin in live HeLa cells has a similar impact on microdomains, characterized by FRET between GPI-anchored proteins: C24, but not C16, sphingomyelin suppresses submicron domains in the plasma membrane. Molecular dynamics simulations indicated that, when in the outer leaflet, the acyl chain of C24 sphingomyelin interdigitates into the opposing leaflet, thereby favouring cholesterol in the inner leaflet. We indeed found that cholesterol prefers the inner over the outer leaflet of asymmetric unilamellar vesicles (80/20) when C24 sphingomyelin is in the outer leaflet. However, when C16 sphingomyelin is in the outer leaflet, cholesterol is evenly partitioned between leaflets (50/50). Interestingly, when a mixture of C24/C16 sphingomyelin is in the outer leaflet of unilamellar vesicles, cholesterol still prefers the inner leaflet (80/20). Indeed, in human erythrocyte plasma membrane, where a mixture of C24 and C16 sphingolipids are naturally in the outer leaflet, cholesterol prefers the cytoplasmic leaflet (80/20). Therefore, C24 sphingomyelin uniquely interacts with cholesterol and governs the lateral organization in asymmetric membranes, including the plasma membrane, potentially by generating cholesterol asymmetry.Statement of SignificanceThe plasma membrane bilayer of mammalian cells has distinct phospholipids between the outer and inner leaflet, with sphingolipids exclusively in the outer leaflet. A large portion of mammalian sphingolipids have very long acyl chains (C24). Little is known how C24 sphingolipids function in the outer leaflet. Mutations in the ceramide synthase 2 gene is found to decrease C24. This severely perturbs homeostasis in mice and humans. Here, we investigated unilamellar vesicles and mammalian cells with C24 sphingomyelin exclusively in the outer leaflet. We provide evidence that outer leaflet C24 sphingomyelin suppresses microdomains in model membranes and live cells by partitioning cholesterol into the inner leaflet. We propose that C24 sphingolipids are critical to the function of the plasma membrane.

scholarly journals Unique functions of Sonic hedgehog signaling during external genitalia development

Development ◽  
2001 ◽  
Vol 128 (21) ◽  
pp. 4241-4250 ◽  
Author(s):  
Ryuma Haraguchi ◽  
Rong Mo ◽  
Chi-chung Hui ◽  
Jun Motoyama ◽  
Shigeru Makino ◽  
...  
Keyword(s):  
Gene Expression ◽  
Sonic Hedgehog ◽  
Hedgehog Signaling ◽  
External Genitalia ◽  
Sonic Hedgehog Signaling ◽  
Urethral Plate ◽  
Morphogenetic Protein ◽  
Fibroblast Growth Factor 10 ◽  
Dramatic Shift ◽  
Patched 1

Coordinated growth and differentiation of external genitalia generates a proximodistally elongated structure suitable for copulation and efficient fertilization. The differentiation of external genitalia incorporates a unique process, i.e. the formation of the urethral plate and the urethral tube. Despite significant progress in molecular embryology, few attempts have been made to elucidate the molecular developmental processes for external genitalia. The sonic hedgehog (Shh) gene and its signaling genes have been found to be dynamically expressed during murine external genitalia development. Functional analysis by organ culture revealed that Shh could regulate mesenchymally expressed genes, patched 1 (Ptch1), bone morphogenetic protein 4 (Bmp4), Hoxd13 and fibroblast growth factor 10 (Fgf10), in the anlage: the genital tubercle (GT). Activities of Shh for both GT outgrowth and differentiation were also demonstrated. Shh–/– mice displayed complete GT agenesis, which is compatible with such observations. Furthermore, the regulation of apoptosis during GT formation was revealed for the first time. Increased cell death and reduced cell proliferation of the Shh–/– mice GT were shown. A search for alterations of Shh downstream gene expression identified a dramatic shift of Bmp4 gene expression from the mesenchyme to the epithelium of the Shh mutant before GT outgrowth. Regulation of mesenchymal Fgf10 gene expression by the epithelial Shh was indicated during late GT development. These results suggest a dual mode of Shh function, first by the regulation of initiating GT outgrowth, and second, by subsequent GT differentiation.

scholarly journals Lateral cerebellum is preferentially sensitive to high sonic hedgehog signaling and medulloblastoma formation

2018 ◽  
Vol 115 (13) ◽  
pp. 3392-3397 ◽  
Author(s):  
I-Li Tan ◽  
Alexandre Wojcinski ◽  
Harikrishna Rallapalli ◽  
Zhimin Lao ◽  
Reeti M. Sanghrajka ◽  
...  
Keyword(s):  
Sonic Hedgehog ◽  
Hedgehog Signaling ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Cell Of Origin ◽  
Sonic Hedgehog Signaling ◽  
Patched 1 ◽  
High Level ◽  
Opposing Effects ◽  
Developing Cerebellum

The main cell of origin of the Sonic hedgehog (SHH) subgroup of medulloblastoma (MB) is granule cell precursors (GCPs), a SHH-dependent transient amplifying population in the developing cerebellum. SHH-MBs can be further subdivided based on molecular and clinical parameters, as well as location because SHH-MBs occur preferentially in the lateral cerebellum (hemispheres). Our analysis of adult patient data suggests that tumors with Smoothened (SMO) mutations form more specifically in the hemispheres than those with Patched 1 (PTCH1) mutations. Using sporadic mouse models of SHH-MB with the two mutations commonly seen in adult MB, constitutive activation of Smo (SmoM2) or loss-of-Ptch1, we found that regardless of timing of induction or type of mutation, tumors developed primarily in the hemispheres, with SmoM2-mutants indeed showing a stronger specificity. We further uncovered that GCPs in the hemispheres are more susceptible to high-level SHH signaling compared with GCPs in the medial cerebellum (vermis), as more SmoM2 or Ptch1-mutant hemisphere cells remain undifferentiated and show increased tumorigenicity when transplanted. Finally, we identified location-specific GCP gene-expression profiles, and found that deletion of the genes most highly expressed in the hemispheres (Nr2f2) or vermis (Engrailed1) showed opposing effects on GCP differentiation. Our studies thus provide insights into intrinsic differences within GCPs that impact on SHH-MB progression.

scholarly journals Antiandrogen Exposure in Utero Disrupts Expression of Desert Hedgehog and Insulin-Like Factor 3 in the Developing Fetal Rat Testis

Endocrinology ◽  
2008 ◽  
Vol 150 (1) ◽  
pp. 445-451 ◽  
Author(s):  
Leon J. S. Brokken ◽  
Annika Adamsson ◽  
Jorma Paranko ◽  
Jorma Toppari
Keyword(s):  
Gene Expression ◽  
Sonic Hedgehog ◽  
Sexual Differentiation ◽  
Hedgehog Signaling ◽  
In Utero ◽  
Testicular Development ◽  
Expression Levels ◽  
Desert Hedgehog ◽  
Patched 1 ◽  
Gene Expression Levels

Testicular development is an androgen-dependent process, and fetal exposure to antiandrogens disrupts male sexual differentiation. A variety of testicular disorders may result from impaired development of fetal Leydig and Sertoli cells. We hypothesized that antiandrogenic exposure during fetal development interferes with desert hedgehog (Dhh) signaling in the testis and results in impaired Leydig cell differentiation. Fetal rats were exposed in utero to the antiandrogen flutamide from 10.5 d post conception (dpc) until they were killed or delivery. Fetal testes were isolated at different time points during gestation and gene expression levels of Dhh, patched-1 (Ptc1), steroidogenic factor 1 (Sf1), cytochrome P450 side-chain cleavage (P450scc), 3β-hydroxysteroid dehydrogenase type 1 (Hsd3b1), and insulin-like factor 3 (Insl3) were analyzed. To study direct effects of hedgehog signaling on testicular development, testes from 14.5 dpc fetuses were cultured for 3 d in the presence of cyclopamine, sonic hedgehog, or vehicle, and gene expression levels and testosterone secretion were analyzed. Organ cultures were also analyzed histologically, and cleaved-caspase 3 immunohistochemistry was performed to assess apoptosis. In utero exposure to flutamide decreased expression levels of Dhh, Ptc1, Sf1, P450scc, Hsd3b1, and Insl3, particularly from 17.5 dpc onward. Inhibition of hedgehog signaling in testis cultures resulted in similar effects on gene expression levels. Apoptosis in Wolffian ducts was increased by cyclopamine compared with sonic hedgehog- or vehicle-treated cultures. We conclude that exposure to the antiandrogen flutamide interferes with Dhh signaling resulting in an impaired differentiation of the fetal Leydig cells and subsequently leading to abnormal testicular development and sexual differentiation. Antiandrogenic exposure of rat fetuses in utero affects genes that are involved in sexual differentiation of the testis and suppresses sexual maturation.

scholarly journals GLUT4 trafficking in insulin-stimulated rat adipose cells: evidence that heterotrimeric GTP-binding proteins regulate the fusion of docked GLUT4-containing vesicles

1999 ◽  
Vol 343 (3) ◽  
pp. 571-577 ◽  
Author(s):  
Cynthia M. FERRARA ◽  
Samuel W. CUSHMAN
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Adenosine Deaminase ◽  
Glucose Transport ◽  
Time Course ◽  
Transport Activity ◽  
Experimental Conditions ◽  
Distinct Sequence ◽  
Kinetics Of ◽  
Adipose Cells

Agents that activate the G-protein Gi (e.g. adenosine) increase, and agents that activate Gs [e.g. isoprenaline (isoproterenol)] decrease, steady-state insulin-stimulated glucose transport activity and cell-surface GLUT4 in isolated rat adipose cells without changing plasma membrane GLUT4 content. Here we have further examined the effects of RsGs and RiGi ligands (in which Rs and Ri are Gs- and Gi-coupled receptors respectively) on insulin-stimulated cell-surface GLUT4 and the kinetics of GLUT4 trafficking in these same cells. Rat adipose cells were preincubated for 2 min with or without isoprenaline (200 nM) and adenosine deaminase (1 unit/ml), to stimulate Gs and decrease the stimulation of Gi respectively, followed by 0-20 min with insulin (670 nM). Treatment with isoprenaline and adenosine deaminase decreased insulin-stimulated glucose transport activity by 58%. Treatment with isoprenaline and adenosine deaminase also resulted in similar decreases in insulin-stimulated cell-surface GLUT4 as assessed by both bis-mannose photolabelling of the substrate-binding site and biotinylation of the extracellular carbohydrate moiety when evaluated under similar experimental conditions. After stimulation with insulin in the absence of Gs and the presence of Gi agents, a distinct sequence of plasma membrane events took place, starting with an increase in immunodetectable GLUT4, then an increase in the accessibility of GLUT4 to bis-mannose photolabel, and finally an increase in glucose transport activity. Pretreatment with isoprenaline and adenosine deaminase before stimulation with insulin did not affect the time course of the increase in immunodetectable GLUT4 in the plasma membrane, but did delay both the increase in accessibility of GLUT4 to photolabel and the increase in glucose transport activity. These results suggest that RsGs and RiGi modulate insulin-stimulated glucose transport by influencing the extent to which GLUT4 is associated with occluded vesicles attached to the plasma membrane during exocytosis, perhaps by regulating the fusion process through which the GLUT4 in docked vesicles becomes exposed on the cell surface.

scholarly journals Cholesterol accessibility at the ciliary membrane controls hedgehog signaling

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Maia Kinnebrew ◽  
Ellen J Iverson ◽  
Bhaven B Patel ◽  
Ganesh V Pusapati ◽  
Jennifer H Kong ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Birth Defects ◽  
Hedgehog Signaling ◽  
Signal Transmission ◽  
Membrane Cholesterol ◽  
Signaling Proteins ◽  
Signaling System ◽  
Ciliary Membrane ◽  
Patched 1 ◽  
Hedgehog Signal

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.

scholarly journals Creating Supported Plasma Membrane Bilayers Using Acoustic Pressure

Membranes ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 30
Author(s):  
Erdinc Sezgin ◽  
Dario Carugo ◽  
Ilya Levental ◽  
Eleanor Stride ◽  
Christian Eggeling
Keyword(s):  
Plasma Membrane ◽  
Membrane Vesicles ◽  
Membrane Dynamics ◽  
Model Membrane ◽  
Live Cells ◽  
Membrane Systems ◽  
Plasma Membrane Vesicles ◽  
Outer Leaflet ◽  
Membrane Surfaces ◽  
Membrane Bilayers

Model membrane systems are essential tools for the study of biological processes in a simplified setting to reveal the underlying physicochemical principles. As cell-derived membrane systems, giant plasma membrane vesicles (GPMVs) constitute an intermediate model between live cells and fully artificial structures. Certain applications, however, require planar membrane surfaces. Here, we report a new approach for creating supported plasma membrane bilayers (SPMBs) by bursting cell-derived GPMVs using ultrasound within a microfluidic device. We show that the mobility of outer leaflet molecules is preserved in SPMBs, suggesting that they are accessible on the surface of the bilayers. Such model membrane systems are potentially useful in many applications requiring detailed characterization of plasma membrane dynamics.

scholarly journals Analysing plasma membrane asymmetry of lipid organisation by fluorescence lifetime and correlation spectroscopy

2019 ◽  
Author(s):  
Anjali Gupta ◽  
Thomas Korte ◽  
Andreas Herrmann ◽  
Thorsten Wohland
Keyword(s):  
Plasma Membrane ◽  
Fluorescence Lifetime ◽  
Mammalian Cells ◽  
Correlation Spectroscopy ◽  
Live Cells ◽  
Fluorescence Lifetime Imaging ◽  
Outer Leaflet ◽  
Liquid Ordered ◽  
Fluorescent Lipid Analogues ◽  
Fluorescent Lipid

ABSTRACTA fundamental feature of a eukaryotic cell membrane is the asymmetric arrangement of lipids in the two leaflets. A cell invests significant energy to maintain this asymmetry and utilizes it to regulate important biological processes such as apoptosis and vesiculation. Here, we employ fluorescence lifetime imaging microscopy (FLIM) and imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS) to differentiate the dynamics and organization of the exofacial and cytoplasmic leaflet of live mammalian cells. We characterize the biophysical properties of fluorescent analogues of phosphatidylcholine (PC), sphingomyelin (SM) and phosphatidylserine (PS) in two mammalian cell membranes. Due to their specific transverse membrane distribution, these probes allow leaflet specific investigation of the plasma membrane. We compare the results with regard to the different temporal and spatial resolution of the methods. Fluorescence lifetimes of fluorescent lipid analogues were found to be in a characteristic range for the liquid ordered phase in the outer leaflet and liquid disordered phase in the inner leaflet. The observation of a more fluid inner leaflet is supported by free diffusion in the inner leaflet with high average diffusion coefficients. The liquid ordered phase in the outer leaflet is accompanied by slower diffusion and diffusion with intermittent transient trapping. Our results show that the combination of FLIM and ITIR-FCS with specific fluorescent lipid analogues provides a powerful tool to investigate lateral and trans-bilayer characteristics of plasma membrane in live cells.Abstract Figure

scholarly journals Sphingomyelin suppresses Hedgehog signaling by restricting cholesterol accessibility at the ciliary membrane

2019 ◽  
Author(s):  
Maia Kinnebrew ◽  
Ellen J. Iverson ◽  
Bhaven B. Patel ◽  
Ganesh V. Pusapati ◽  
Jennifer H. Kong ◽  
...  
Keyword(s):  
Hedgehog Signaling ◽  
Direct Interaction ◽  
Subcellular Location ◽  
Signaling System ◽  
Ciliary Membrane ◽  
Cellular Processes ◽  
Cholesterol Levels ◽  
Collateral Effects ◽  
Patched 1 ◽  
Hedgehog Signal

AbstractTransmission of the Hedgehog signal across the plasma membrane by Smoothened is proposed to be triggered by its direct interaction with cholesterol. 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 here that Smoothened activation and Hedgehog signaling are driven by a biochemically defined fraction of membrane cholesterol, termed accessible cholesterol. Increasing accessible cholesterol levels by depletion of sphingomyelin, which sequesters cholesterol in complexes, potentiates Hedgehog signaling. By inactivating the transporter-like protein Patched 1, Hedgehog ligands trigger an increase in cholesterol accessibility in the ciliary membrane, the subcellular location for Smoothened signaling. Thus, compartmentalization of Hedgehog signaling in the primary cilium may allow cholesterol accessibility to be used as a second messenger to mediate the communication between Patched 1 and Smoothened, without causing collateral effects on other cellular processes.

Regulation of pancreas development by hedgehog signaling

Development ◽  
2000 ◽  
Vol 127 (22) ◽  
pp. 4905-4913 ◽  
Author(s):  
M. Hebrok ◽  
S.K. Kim ◽  
B. St Jacques ◽  
A.P. McMahon ◽  
D.A. Melton
Keyword(s):  
Sonic Hedgehog ◽  
Hedgehog Signaling ◽  
Signaling Molecules ◽  
Pancreatic Tissue ◽  
Negative Regulator ◽  
Pancreas Development ◽  
Fourfold Increase ◽  
Embryonic Pancreas ◽  
Patched 1 ◽  
Targeted Inactivation

Pancreas organogenesis is regulated by the interaction of distinct signaling pathways that promote or restrict morphogenesis and cell differentiation. Previous work has shown that activin, a TGF(beta+) signaling molecule, permits pancreas development by repressing expression of Sonic hedgehog (Shh), a member of the hedgehog family of signaling molecules that antagonize pancreas development. Here we show that Indian hedgehog (Ihh), another hedgehog family member, and Patched 1 (Ptc1), a receptor and negative regulator of hedgehog activity, are expressed in pancreatic tissue. Targeted inactivation of Ihh in mice allows ectopic branching of ventral pancreatic tissue resulting in an annulus that encircles the duodenum, a phenotype frequently observed in humans suffering from a rare disorder known as annular pancreas. Shh(−)(/)(−) and Shh(−)(/)(−) Ihh(+/)(−) mutants have a threefold increase in pancreas mass, and a fourfold increase in pancreatic endocrine cell numbers. In contrast, mutations in Ptc1 reduce pancreas gene expression and impair glucose homeostasis. Thus, islet cell, pancreatic mass and pancreatic morphogenesis are regulated by hedgehog signaling molecules expressed within and adjacent to the embryonic pancreas. Defects in hedgehog signaling may lead to congenital pancreatic malformations and glucose intolerance.

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