scholarly journals Growth factor stimulation promotes multivesicular endosome biogenesis by prolonging recruitment of the late-acting ESCRT machinery

2019 ◽  
Vol 116 (14) ◽  
pp. 6858-6867 ◽  
Author(s):  
Kyle B. Quinney ◽  
Elisa B. Frankel ◽  
Raakhee Shankar ◽  
William Kasberg ◽  
Peter Luong ◽  
...  
Keyword(s):  
Growth Factor ◽  
Mammalian Cells ◽  
Light Sheet ◽  
Growth Conditions ◽  
Receptor Activation ◽  
Spatiotemporal Pattern ◽  
Growth Factor Signaling ◽  
Light Sheet Microscopy ◽  
Escrt Machinery ◽  
Escrt Complex

The formation of multivesicular endosomes (MVEs) mediates the turnover of numerous integral membrane proteins and has been implicated in the down-regulation of growth factor signaling, thereby exhibiting properties of a tumor suppressor. The endosomal sorting complex required for transport (ESCRT) machinery plays a key role in MVE biogenesis, enabling cargo selection and intralumenal vesicle (ILV) budding. However, the spatiotemporal pattern of endogenous ESCRT complex assembly and disassembly in mammalian cells remains poorly defined. By combining CRISPR/Cas9-mediated genome editing and live cell imaging using lattice light sheet microscopy (LLSM), we determined the native dynamics of both early- and late-acting ESCRT components at MVEs under multiple growth conditions. Specifically, our data indicate that ESCRT-0 accumulates quickly on endosomes, typically in less than 30 seconds, and its levels oscillate in a manner dependent on the downstream recruitment of ESCRT-I. Similarly, levels of the ESCRT-I complex also fluctuate on endosomes, but its average residency time is more than fivefold shorter compared with ESCRT-0. Vps4 accumulation is the most transient, however, suggesting that the completion of ILV formation occurs rapidly. Upon addition of epidermal growth factor (EGF), both ESCRT-I and Vps4 are retained at endosomes for dramatically extended periods of time, while ESCRT-0 dynamics are only modestly affected. Our findings are consistent with a model in which growth factor stimulation stabilizes late-acting components of the ESCRT machinery at endosomes to accelerate the rate of ILV biogenesis and attenuate signal transduction initiated by receptor activation.

scholarly journals Role of Secretoglobin+ (club cell) NFκB/RelA-TGFβ signaling in aero-allergen-induced epithelial plasticity and subepithelial myofibroblast transdifferentiation

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Melissa E. Skibba ◽  
Xiaofang Xu ◽  
Kurt Weiss ◽  
Jan Huisken ◽  
Allan R. Brasier
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Airway Remodeling ◽  
Light Sheet ◽  
Mucosal Injury ◽  
Small Airway ◽  
Tgfβ Signaling ◽  
Club Cell ◽  
Light Sheet Microscopy ◽  
Epithelial Plasticity

AbstractRepetitive aeroallergen exposure is linked to sensitization and airway remodeling through incompletely understood mechanisms. In this study, we examine the dynamic mucosal response to cat dander extract (CDE), a ubiquitous aero-allergen linked to remodeling, sensitization and asthma. We find that daily exposure of CDE in naïve C57BL/6 mice activates innate neutrophilic inflammation followed by transition to a lymphocytic response associated with waves of mucosal transforming growth factor (TGF) isoform expression. In parallel, enhanced bronchiolar Smad3 expression and accumulation of phospho-SMAD3 was observed, indicating paracrine activation of canonical TGFβR signaling. CDE exposure similarly triggered epithelial cell plasticity, associated with expression of mesenchymal regulatory factors (Snai1 and Zeb1), reduction of epithelial markers (Cdh1) and activation of the NFκB/RelA transcriptional activator. To determine whether NFκB functionally mediates CDE-induced growth factor response, mice were stimulated with CDE in the absence or presence of a selective IKK inhibitor. IKK inhibition substantially reduced the level of CDE-induced TGFβ1 expression, pSMAD3 accumulation, Snai1 and Zeb1 expression. Activation of epithelial plasticity was demonstrated by flow cytometry in whole lung homogenates, where CDE induces accumulation of SMA+Epcam+ population. Club cells are important sources of cytokine and growth factor production. To determine whether Club cell innate signaling through NFκB/RelA mediated CDE induced TGFβ signaling, we depleted RelA in Secretoglobin (Scgb1a1)-expressing bronchiolar cells. Immunofluorescence-optical clearing light sheet microscopy showed a punctate distribution of Scgb1a1 progenitors throughout the small airway. We found that RelA depletion in Secretoglobin+ cells results in inhibition of the mucosal TGFβ response, blockade of EMT and reduced subepithelial myofibroblast expansion. We conclude that the Secretoglobin—derived bronchiolar cell is central to coordinating the innate response required for mucosal TGFβ1 response, EMT and myofibroblast expansion. These data have important mechanistic implications for how aero-allergens trigger mucosal injury response and remodeling in the small airway.

scholarly journals Epidermal Growth Factor Signaling and Mitogenesis inPlcg1 Null Mouse Embryonic Fibroblasts

1998 ◽  
Vol 9 (4) ◽  
pp. 749-757 ◽  
Author(s):  
Qun-sheng Ji ◽  
Sandra Ermini ◽  
Josep Baulida ◽  
Feng-lei Sun ◽  
Graham Carpenter
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Growth Factor Receptor ◽  
Receptor Activation ◽  
Mitogen Activated Protein Kinase ◽  
Null Mouse ◽  
Growth Factor Signaling ◽  
Mitogen Activated Protein ◽  
Epidermal Growth ◽  
Early Mouse

Gene targeting techniques and early mouse embryos have been used to produce immortalized fibroblasts genetically deficient in phospholipase C (PLC)-γ1, a ubiquitous tyrosine kinase substrate.Plcg1 −/− embryos die at embryonic day 9; however, cells derived from these embryos proliferate as well as cells from Plcg1 +/+ embryos. The null cells do grow to a higher saturation density in serum-containing media, as their capacity to spread out is decreased compared with that of wild-type cells. In terms of epidermal growth factor receptor activation and internalization, or growth factor induction of mitogen-activated protein kinase, c-fos, or DNA synthesis in quiescent cells, PLcg1 −/− cells respond equivalently to PLcg1 +/+ cells. Also, null cells are able to migrate effectively in a wounded monolayer. Therefore, immortalized fibroblasts do not require PLC-γ1 for many responses to growth factors.

scholarly journals Application of β-Galactosidase Enzyme Complementation Technology as a High Throughput Screening Format for Antagonists of the Epidermal Growth Factor Receptor

2001 ◽  
Vol 6 (6) ◽  
pp. 401-411
Author(s):  
Debbie L. Graham ◽  
Nicola Bevan ◽  
Peter N. Lowe ◽  
Michelle Palmer ◽  
Stephen Rees
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
High Throughput ◽  
False Positive ◽  
Mammalian Cells ◽  
Egf Receptor ◽  
Receptor Activation ◽  
C2c12 Cells ◽  
Hit Rate ◽  
Epidermal Growth

We have applied enzyme complementation technology to develop a screen for antagonists of the epidermal growth factor (EGF) receptor. Chimeric proteins containing two weakly complementing deletion mutants of Escherichia coli β-galactosidase (β-gal), each fused to the EGF receptor extracellular and transmembrane domains, have been stably expressed in C2C12 cells. In this cell line, formation of active β-gal is dependent on agonist-stimulated dimerization of the EGF receptor. We have developed a homogenous 384-well assay protocol and have applied this to characterize the pharmacology of the receptor and to develop a high throughput screen (HTS) for EGF receptor antagonists. The assay is tolerant to DMSO concentrations of up to 2% and, across 21 passages in culture, exhibits an EC50 for EGF of 5.4 ± 3.6 ng/ml (n = 11) and a Z' of 0.55 ± 0.13 (n = 11). A random set of 1,280 compounds was screened in duplicate at 11 μM to examine the robustness of enzyme complementation technology and to characterize the false-positive hit rate in the assay. Using a cutoff of 40% inhibition of EGF-promoted β-gal activity, the hit rate on day 1 was 2.5% and on day 2 was 1.9%. After retesting the active compounds, the hit rate was reduced to 0.4%, of which one of the compounds was identified as a β-gal inhibitor and the remainder appeared to be nonspecific inhibitors in the assay. This technology is amenable to automated screen workstations, there are highly sensitive chemiluminescent and fluorescent β-gal assay reagents amenable to detection in miniaturized plate formats, and the assay benefits from a low false-positive hit rate. Enzyme complementation technology may have wide application within the HTS environment for the detection of modulators of receptor activation or inhibitors of protein-protein interactions in mammalian cells.

scholarly journals Phosphoprotein Enriched in Astrocytes 15 kDa (PEA-15) Reprograms Growth Factor Signaling by Inhibiting Threonine Phosphorylation of Fibroblast Receptor Substrate 2α

2010 ◽  
Vol 21 (4) ◽  
pp. 664-673 ◽  
Author(s):  
Jacob R. Haling ◽  
Fen Wang ◽  
Mark H. Ginsberg
Keyword(s):  
Growth Factor ◽  
Map Kinase ◽  
Tyrosine Phosphorylation ◽  
Growth Factor Receptor ◽  
Receptor Activation ◽  
Growth Factor Signaling ◽  
Fgf Receptor ◽  
Cellular Expression ◽  
Map Kinase Pathway ◽  
Kinase Pathway

Changes in cellular expression of phosphoprotein enriched in astrocytes of 15 kDa (PEA-15) are linked to insulin resistance, tumor cell invasion, and cellular senescence; these changes alter the activation of the extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein (MAP) kinase pathway. Here, we define the mechanism whereby increased PEA-15 expression promotes and sustains ERK1/2 activation. PEA-15 binding prevented ERK1/2 membrane recruitment and threonine phosphorylation of fibroblast receptor substrate 2α (FRS2α), a key link in fibroblast growth factor (FGF) receptor activation of ERK1/2. This reduced threonine phosphorylation led to increased FGF-induced tyrosine phosphorylation of FRS2α, thereby enhancing downstream signaling. Conversely, short hairpin RNA-mediated depletion of endogenous PEA-15 led to reduced FRS2α tyrosine phosphorylation. Thus, PEA-15 interrupts a negative feedback loop that terminates growth factor receptor signaling downstream of FRS2α. This is the dominant mechanism by which PEA-15 activates ERK1/2 because genetic deletion of FRS2α blocked the capacity of PEA-15 to activate the MAP kinase pathway. Thus, PEA-15 prevents ERK1/2 localization to the plasma membrane, thereby inhibiting ERK1/2-dependent threonine phosphorylation of FRS2α to promote activation of the ERK1/2 MAP kinase pathway.

scholarly journals Recruitment dynamics of ESCRT-III and Vps4 to endosomes and implications for reverse membrane budding

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Manuel Alonso Y Adell ◽  
Simona M Migliano ◽  
Srigokul Upadhyayula ◽  
Yury S Bykov ◽  
Simon Sprenger ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Atpase Activity ◽  
Light Sheet ◽  
Light Sheet Microscopy ◽  
Escrt Machinery ◽  
Steady Growth ◽  
Bud Neck ◽  
Membrane Scission ◽  
Membrane Budding ◽  
Quantitative Fluorescence

The ESCRT machinery mediates reverse membrane scission. By quantitative fluorescence lattice light-sheet microscopy, we have shown that ESCRT-III subunits polymerize rapidly on yeast endosomes, together with the recruitment of at least two Vps4 hexamers. During their 3–45 s lifetimes, the ESCRT-III assemblies accumulated 75–200 Snf7 and 15–50 Vps24 molecules. Productive budding events required at least two additional Vps4 hexamers. Membrane budding was associated with continuous, stochastic exchange of Vps4 and ESCRT-III components, rather than steady growth of fixed assemblies, and depended on Vps4 ATPase activity. An all-or-none step led to final release of ESCRT-III and Vps4. Tomographic electron microscopy demonstrated that acute disruption of Vps4 recruitment stalled membrane budding. We propose a model in which multiple Vps4 hexamers (four or more) draw together several ESCRT-III filaments. This process induces cargo crowding and inward membrane buckling, followed by constriction of the nascent bud neck and ultimately ILV generation by vesicle fission.

scholarly journals Dual clathrin and adhesion signaling systems regulate growth factor receptor activation

2020 ◽  
Author(s):  
Marco A. Alfonzo-Mendez ◽  
Kem A. Sochacki ◽  
Marie-Paule Strub ◽  
Justin W. Taraska
Keyword(s):  
Growth Factor ◽  
Cell Growth ◽  
Growth Factor Receptor ◽  
Receptor Activation ◽  
Growth Factor Signaling ◽  
Cell Growth And Migration ◽  
Reciprocal Regulation ◽  
Signaling Systems ◽  
Fluorescence And Electron Microscopy ◽  
And Migration

ABSTRACTThe crosstalk between growth factor and adhesion receptors is key for cell growth and migration. In pathological settings, these receptors are drivers of cancer. Yet, how growth and adhesion signals are spatially organized and integrated is poorly understood. Here we use quantitative fluorescence and electron microscopy to reveal a mechanism where flat clathrin lattices partition and activate growth factor signals via a coordinated response that involves crosstalk between epidermal growth factor receptor (EGFR) and the adhesion receptor β5-integrin. We show that ligand-activated EGFR, Grb2, Src, and β5-integrin are captured by clathrin coated-structures at the plasma membrane. Clathrin structures dramatically grow in response to ligand activation into large flat plaques and provide a signaling platform that link EGFR and β5-integrin through Src-mediated phosphorylation. Disrupting this EGFR/Src/β5-integrin axis prevents both clathrin plaque growth and receptor signaling. Our study reveals a reciprocal regulation of clathrin lattices and two different receptor systems to enhance cell growth factor signaling. These findings have broad implications for the control of growth factor receptors, mechanotransduction, and endocytosis.

scholarly journals A Fyn biosensor reveals pulsatile, spatially localized kinase activity and signaling crosstalk in live mammalian cells

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Ananya Mukherjee ◽  
Randhir Singh ◽  
Sreeram Udayan ◽  
Sayan Biswas ◽  
Pothula Purushotham Reddy ◽  
...  
Keyword(s):  
Growth Factor ◽  
Cell Fate ◽  
Mammalian Cells ◽  
Kinase Activity ◽  
Live Cells ◽  
Structural Basis ◽  
Cell Physiology ◽  
Growth Factor Signaling ◽  
Protein Activity ◽  
Signaling Crosstalk

Cell behavior is controlled through spatio-temporally localized protein activity. Despite unique and often contradictory roles played by Src-family-kinases (SFKs) in regulating cell physiology, activity patterns of individual SFKs have remained elusive. Here, we report a biosensor for specifically visualizing active conformation of SFK-Fyn in live cells. We deployed combinatorial library screening to isolate a binding-protein (F29) targeting activated Fyn. Nuclear-magnetic-resonance (NMR) analysis provides the structural basis of F29 specificity for Fyn over homologous SFKs. Using F29, we engineered a sensitive, minimally-perturbing fluorescence-resonance-energy-transfer (FRET) biosensor (FynSensor) that reveals cellular Fyn activity to be spatially localized, pulsatile and sensitive to adhesion/integrin signaling. Strikingly, growth factor stimulation further enhanced Fyn activity in pre-activated intracellular zones. However, inhibition of focal-adhesion-kinase activity not only attenuates Fyn activity, but abolishes growth-factor modulation. FynSensor imaging uncovers spatially organized, sensitized signaling clusters, direct crosstalk between integrin and growth-factor-signaling, and clarifies how compartmentalized Src-kinase activity may drive cell fate.

scholarly journals Control of Bro1-Domain Protein Rim20 Localization by External pH, ESCRT Machinery, and the Saccharomyces cerevisiae Rim101 Pathway

2006 ◽  
Vol 17 (3) ◽  
pp. 1344-1353 ◽  
Author(s):  
Jacob H. Boysen ◽  
Aaron P. Mitchell
Keyword(s):  
Surface Protein ◽  
Growth Conditions ◽  
Aaa Atpase ◽  
Escrt Machinery ◽  
Alkaline Conditions ◽  
A Subunit ◽  
Acidic Conditions ◽  
Domain Protein ◽  
Escrt Complex ◽  
External Ph

Bro1-domain proteins such as yeast Bro1 and mammalian AIP1/Alix are well-established participants in endosome metabolism. The Bro1-domain interacts with endosomal surface protein Snf7/Vps32 in yeast, a subunit of the ESCRT complex. Yeast Bro1-domain protein Rim20 has no role in endosome function, but is required for alkaline pH-stimulated cleavage of transcription factor Rim101. Rim20-GFP is cytoplasmic under acidic conditions but concentrated in punctate foci under alkaline conditions. Bro1-GFP also accumulates in foci, but they are more numerous under acidic than alkaline conditions. Colocalization experiments indicate that some Rim20-GFP foci correspond to Bro1-RFP foci, whereas others do not. Rim8, Rim9, Rim21, Dfg16, Snf7, Vps20, Vps23, and Vps25, which are required for Rim101 cleavage, are required for appearance of Rim20-GFP foci. ESCRT complexes accumulate on endosome-derived compartments in cells that lack the AAA-ATPase Vps4. We find that Rim20-GFP foci accumulate in a vps4 mutant background independently of external pH, Rim101 pathway-specific genes, and most ESCRT subunit genes except for SNF7. Rim20-GFP foci seem to represent endosomes, because they colocalize with Snf7-RFP and because they correspond to a perivacuolar compartment in the vps4 strain. We propose that alkaline growth conditions alter the endosomal surface to favor Rim20-Snf7 interaction and Rim101 cleavage. Our findings raise the possibility that Bro1-domain proteins may be differentially regulated in the same cell, thereby coupling endosome metabolism to signaling.

scholarly journals 3D single-molecule super-resolution microscopy with a tilted light sheet

2017 ◽  
Author(s):  
Anna-Karin Gustavsson ◽  
Petar N. Petrov ◽  
Maurice Y. Lee ◽  
Yoav Shechtman ◽  
W. E. Moerner
Keyword(s):  
Single Molecule ◽  
Mammalian Cells ◽  
Double Helix ◽  
Super Resolution ◽  
Nuclear Lamina ◽  
Light Sheet ◽  
Point Spread Functions ◽  
Light Sheet Microscopy ◽  
Point Spread ◽  
Resolution Imaging

Tilted light sheet microscopy with 3D point spread functions (TILT3D) combines a novel, tilted light sheet illumination strategy with long axial range point spread functions (PSFs) for low-background, 3D super-localization of single molecules as well as 3D super-resolution imaging in thick cells. Because the axial positions of the single emitters are encoded in the shape of each single-molecule image rather than in the position or thickness of the light sheet, the light sheet need not be extremely thin. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The result is simple and flexible 3D super-resolution imaging with tens of nm localization precision throughout thick mammalian cells. We validated TILT3D for 3D super-resolution imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed Tetrapod PSFs for fiducial bead tracking and live axial drift correction.

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