Single event visualization of unconventional secretion of FGF2

FGF2 is exported from cells by an unconventional secretory mechanism. Here, we directly visualized individual FGF2 membrane translocation events at the plasma membrane using live cell TIRF microscopy. This process was dependent on both PI(4,5)P2–mediated recruitment of FGF2 at the inner leaflet and heparan sulfates capturing FGF2 at the outer plasma membrane leaflet. By simultaneous imaging of both FGF2 membrane recruitment and the appearance of FGF2 at the cell surface, we revealed the kinetics of FGF2 membrane translocation in living cells with an average duration of ∼200 ms. Furthermore, we directly demonstrated FGF2 oligomers at the inner leaflet of living cells with a FGF2 dimer being the most prominent species. We propose this dimer to represent a key intermediate in the formation of higher FGF2 oligomers that form membrane pores and put forward a kinetic model explaining the mechanism by which membrane-inserted FGF2 oligomers serve as dynamic translocation intermediates during unconventional secretion of FGF2.

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Activation Kinetics of RAF Protein in the Ternary Complex of RAF, RAS-GTP, and Kinase on the Plasma Membrane of Living Cells

Journal of Biological Chemistry ◽

10.1074/jbc.m111.262675 ◽

2011 ◽

Vol 286 (42) ◽

pp. 36460-36468 ◽

Cited By ~ 26

Author(s):

Kayo Hibino ◽

Tatsuo Shibata ◽

Toshio Yanagida ◽

Yasushi Sako

Keyword(s):

Plasma Membrane ◽

Ternary Complex ◽

Living Cells ◽

Activation Kinetics ◽

Kinetics Of

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The α1 subunit of the Na,K-ATPase acts upstream of PI(4,5)P2 facilitating unconventional secretion of Fibroblast Growth Factor 2 from tumor cells

10.1101/827691 ◽

2019 ◽

Author(s):

Cyril Legrand ◽

Roberto Saleppico ◽

Jana Sticht ◽

Fabio Lolicato ◽

Hans-Michael Müller ◽

...

Keyword(s):

Plasma Membrane ◽

Growth Factor ◽

Cell Surface ◽

Fibroblast Growth Factor ◽

Tumor Cells ◽

Fibroblast Growth Factor 2 ◽

Fibroblast Growth ◽

Membrane Translocation ◽

Unconventional Secretion ◽

Α1 Subunit

SummaryFibroblast Growth Factor 2 (FGF2) is a tumor cell survival factor that is exported from cells by an unconventional secretory pathway. This process is based on direct translocation of FGF2 across the plasma membrane. FGF2 membrane translocation depends on PI(4,5)P2-induced formation of membrane-inserted FGF2 oligomers followed by extracellular trapping of FGF2 at the outer leaflet mediated by cell surface heparan sulfate proteoglycans. Beyond the well-characterized core mechanism of FGF2 membrane translocation, the Na,K-ATPase has been proposed to play a so far unknown role in unconventional secretion of FGF2. Here, we define a direct physical interaction of FGF2 with a subdomain of the cytoplasmic part of the α1 subunit of the Na,K-ATPase. Employing NMR spectroscopy and molecular dynamics simulations, we identified two lysine residues on the molecular surface of FGF2 that are shown to be essential for its interaction with α1. In intact cells, the corresponding lysine-to-glutamate variants of FGF2 were characterized by inefficient secretion and reduced recruitment to the inner plasma membrane leaflet as shown by single molecule TIRF microscopy. Our findings suggest that α1 acts upstream of PI(4,5)P2 facilitating efficient membrane translocation of FGF2 to the cell surface of tumor cells.

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Key steps in unconventional secretion of fibroblast growth factor 2 reconstituted with purified components

eLife ◽

10.7554/elife.28985 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 25

Author(s):

Julia P Steringer ◽

Sascha Lange ◽

Sabína Čujová ◽

Radek Šachl ◽

Chetan Poojari ◽

...

Keyword(s):

Secretory Pathway ◽

Protein Translocation ◽

Membrane Translocation ◽

Unconventional Secretion ◽

Molecular Machinery ◽

A Subunit ◽

Heparan Sulfates ◽

Dynamics Simulations ◽

Key Steps ◽

Inside Out

FGF2 is secreted from cells by an unconventional secretory pathway. This process is mediated by direct translocation across the plasma membrane. Here, we define the minimal molecular machinery required for FGF2 membrane translocation in a fully reconstituted inside-out vesicle system. FGF2 membrane translocation is thermodynamically driven by PI(4,5)P2-induced membrane insertion of FGF2 oligomers. The latter serve as dynamic translocation intermediates of FGF2 with a subunit number in the range of 8-12 FGF2 molecules. Vectorial translocation of FGF2 across the membrane is governed by sequential and mutually exclusive interactions with PI(4,5)P2 and heparan sulfates on opposing sides of the membrane. Based on atomistic molecular dynamics simulations, we propose a mechanism that drives PI(4,5)P2 dependent oligomerization of FGF2. Our combined findings establish a novel type of self-sustained protein translocation across membranes revealing the molecular basis of the unconventional secretory pathway of FGF2.

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Green Fluorescent Protein (GFP)-tagged Cysteine-rich Domains from Protein Kinase C as Fluorescent Indicators for Diacylglycerol Signaling in Living Cells

The Journal of Cell Biology ◽

10.1083/jcb.140.3.485 ◽

1998 ◽

Vol 140 (3) ◽

pp. 485-498 ◽

Cited By ~ 248

Author(s):

Elena Oancea ◽

Mary N. Teruel ◽

Andrew F.G. Quest ◽

Tobias Meyer

Keyword(s):

Plasma Membrane ◽

Protein Kinase C ◽

Green Fluorescent Protein ◽

Phorbol Ester ◽

Lipid Membranes ◽

Fluorescent Protein ◽

Living Cells ◽

Membrane Translocation ◽

Kinase C ◽

Green Fluorescent

Cysteine-rich domains (Cys-domains) are ∼50–amino acid–long protein domains that complex two zinc ions and include a consensus sequence with six cysteine and two histidine residues. In vitro studies have shown that Cys-domains from several protein kinase C (PKC) isoforms and a number of other signaling proteins bind lipid membranes in the presence of diacylglycerol or phorbol ester. Here we examine the second messenger functions of diacylglycerol in living cells by monitoring the membrane translocation of the green fluorescent protein (GFP)-tagged first Cys-domain of PKC-γ (Cys1–GFP). Strikingly, stimulation of G-protein or tyrosine kinase–coupled receptors induced a transient translocation of cytosolic Cys1–GFP to the plasma membrane. The plasma membrane translocation was mimicked by addition of the diacylglycerol analogue DiC8 or the phorbol ester, phorbol myristate acetate (PMA). Photobleaching recovery studies showed that PMA nearly immobilized Cys1–GFP in the membrane, whereas DiC8 left Cys1–GFP diffusible within the membrane. Addition of a smaller and more hydrophilic phorbol ester, phorbol dibuterate (PDBu), localized Cys1–GFP preferentially to the plasma and nuclear membranes. This selective membrane localization was lost in the presence of arachidonic acid. GFP-tagged Cys1Cys2-domains and full-length PKC-γ also translocated from the cytosol to the plasma membrane in response to receptor or PMA stimuli, whereas significant plasma membrane translocation of Cys2–GFP was only observed in response to PMA addition. These studies introduce GFP-tagged Cys-domains as fluorescent diacylglycerol indicators and show that in living cells the individual Cys-domains can trigger a diacylglycerol or phorbol ester–mediated translocation of proteins to selective lipid membranes.

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Cholesterol promotes both head group visibility and clustering of PI(4,5)P2 driving unconventional secretion of Fibroblast Growth Factor 2

10.1101/2021.04.16.440132 ◽

2021 ◽

Author(s):

Fabio Lolicato ◽

Roberto Saleppico ◽

Alessandra Griffo ◽

Bianca Pokrandt ◽

Hans-Michael Müller ◽

...

Keyword(s):

Plasma Membrane ◽

Growth Factor ◽

Fibroblast Growth Factor ◽

Lipid Bilayers ◽

Fibroblast Growth Factor 2 ◽

Head Group ◽

Fibroblast Growth ◽

Membrane Tension ◽

Membrane Translocation ◽

Unconventional Secretion

Fibroblast Growth Factor 2 (FGF2) is a cell survival factor involved in tumor-induced angiogenesis. FGF2 is secreted through an unconventional secretory pathway based upon direct protein translocation across the plasma membrane. Here we demonstrate that both PI(4,5)P2-dependent FGF2 recruitment at the inner plasma membrane leaflet and FGF2 membrane translocation into the extracellular space are positively modulated by cholesterol in living cells. We further reveal cholesterol to enhance FGF2 binding to PI(4,5)P2-containing lipid bilayers in a fully reconstituted system. Based on extensive atomistic molecular dynamics simulations and membrane tension experiments, we propose cholesterol to modulate FGF2 binding to PI(4,5)P2 by (i) increasing head group visibility of PI(4,5)P2 on the membrane surface, (ii) increasing avidity by cholesterol-induced clustering of PI(4,5)P2 molecules triggering FGF2 oligomerization and (iii) increasing membrane tension facilitating the formation of lipidic membrane pores. Our findings have general implications for phosphoinositide-dependent protein recruitment to membranes and explain the highly selective targeting of FGF2 towards the plasma membrane, the subcellular site of FGF2 membrane translocation during unconventional secretion of FGF2.

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A direct gateway into the extracellular space: Unconventional secretion of FGF2 through self-sustained plasma membrane pores

Seminars in Cell and Developmental Biology ◽

10.1016/j.semcdb.2018.02.010 ◽

2018 ◽

Vol 83 ◽

pp. 3-7 ◽

Cited By ~ 20

Author(s):

Julia P. Steringer ◽

Walter Nickel

Keyword(s):

Plasma Membrane ◽

Extracellular Space ◽

Membrane Pores ◽

Unconventional Secretion

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An Ultrastructural Study of Pericytes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010010007x ◽

1968 ◽

Vol 26 ◽

pp. 66-67

Author(s):

T. M. Murad ◽

E. von Haam

Keyword(s):

Plasma Membrane ◽

Endothelial Cell ◽

Basement Membrane ◽

Blood Vessel ◽

Vascular Endothelial Cells ◽

Myoepithelial Cells ◽

Capillary Blood ◽

The Body ◽

Capillary Blood Vessel ◽

Outer Plasma Membrane

Pericytes are vascular satellites present around capillary blood vessels and small venules. They have been observed in almost every tissue of the body and are thought to be related to vascular smooth muscle cells. Morphologically pericytes have great similarity to vascular endothelial cells and also slightly resemble myoepithelial cells.The present study describes the ultrastructural morphology of pericytes in normal breast tissue and in benign tumor of the breast. The study showed that pericytes are ovoid or elongated cells separated from the endothelial cell of the capillary blood vessel by the basement membrane of endothelial cell. The nuclei of pericytes are often very distinctive. Although some are round, oval, or elongated, others show marked irregularity and infolding of the nuclear membrane. The cytoplasm shows mono-or bipolar extension in which the cytoplasmic organelles are located (Fig. 1). These cytoplasmic extensions embrace the capillary blood vessel incompletely. The plasma membrane exhibits multiple areas of focal condensation called hemidesmosomes (Fig. 2, arrow). A variable number of pinocytotic vesicles are frequently seen lining the outer plasma membrane. Normally pericytes are surrounded by a basement membrane which is found more consistently on the outer plasma membrane separating the pericytes from the stromal connective tissue.

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Microtubule Dynamics and Organelle Transport in Reticulomyxa

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100158510 ◽

1990 ◽

Vol 48 (3) ◽

pp. 194-195

Author(s):

Yih-Tai Chen ◽

Ursula Euteneuer ◽

Ken B. Johnson ◽

Michael P. Koonce ◽

Manfred Schliwa

Keyword(s):

Plasma Membrane ◽

Light Microscopy ◽

Cytoplasmic Dynein ◽

Living Cells ◽

Microtubule Dynamics ◽

Model Systems ◽

Organelle Transport ◽

Biochemical Characteristics ◽

Dependent Transport

The application of video techniques to light microscopy and the development of motility assays in reactivated or reconstituted model systems rapidly advanced our understanding of the mechanism of organelle transport and microtubule dynamics in living cells. Two microtubule-based motors have been identified that are good candidates for motors that drive organelle transport: kinesin, a plus end-directed motor, and cytoplasmic dynein, which is minus end-directed. However, the evidence that they do in fact function as organelle motors is still indirect.We are studying microtubule-dependent transport and dynamics in the giant amoeba, Reticulomyxa. This cell extends filamentous strands backed by an extensive array of microtubules along which organelles move bidirectionally at up to 20 μm/sec (Fig. 1). Following removal of the plasma membrane with a mild detergent, organelle transport can be reactivated by the addition of ATP (1). The physiological, pharmacological and biochemical characteristics show the motor to be a cytoplasmic form of dynein (2).

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