scholarly journals The effects of Mitogenic and Metabolic cues on clathrin-mediated endocytosis

2021 ◽  
Author(s):  
Ralph Christian Delos Santos
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Energy Levels ◽  
Surface Proteins ◽  
Coated Pits ◽  
Calcium Signals ◽  
Cellular Energy ◽  
Software Analysis ◽  
Cellular Processes ◽  
Automated Software

The cell ‘surfaceome’ collectively describes proteins found on the plasma membrane (PM), which functions in fundamental cellular processes including growth and proliferation. The surfaceome undergoes dynamic remodeling via the addition/removal of surface proteins in response to changing environmental conditions. In mammalian cells, surfaceome remodeling is predominantly facilitated by clathrin-mediated endocytosis (CME) which involves the invagination and internalization of PM regions via clathrin-coated structures—removing proteins from the cell surface. As a major regulator of the surfaceome, it is important to understand the underlying mechanisms governing CME, given that its dysregulation has been implicated in human pathologies including neurologic and oncogenic disorders. Cellular cues including mitogenic (e.g. growth factors) and metabolic signals (e.g. cellular energy levels) induce diverse cellular processes (e.g. growth and proliferation) requiring surfaceome/PM remodeling. Precisely how mitogenic and metabolic signals may induce surfaceome remodeling however, is under-examined. As a major regulator of the surfaceome, CME is a likely mechanism through which cellular cues may remodel the PM. Poorly understood, I thus sought to investigate how mitogenic and metabolic signals may regulate CME. Mitogenic signaling by the epidermal growth factor receptor (EGFR) triggers PLCγ1-calcium signals, which I found a requirement for CME of EGFR—likely via calcium control of the Sjn1 protein. Consistently, using TIRF-M imaging coupled to automated software analysis, I demonstrate that inhibition of PLCγ1-calcium signals impairs the formation/assembly of GFR-containing clathrin structures. In addition, I hypothesize that PLCγ1-calcium signals also regulates the CME of other surface proteins given its robust control of Sjn1—which localizes broadly amongst clathrin-coated structures. AMPK is a cellular energy sensor activated by metabolic stress (e.g. starvation). Using TIRF-M imaging coupled to automated software analysis, I found that AMPK activation broadly reduces the formation/assembly of bona fide clathrin-coated pits, without impairing the internalization rates of CME cargoes (e.g. EGFR, TfR and β1-integrin). Furthermore, I found that AMPK may regulate CME throughcontrol of the Arf6 protein. Collectively, my findings uncover and provide novel mechanisms by which mitogenic (via EGFR-induced calcium signals) and metabolic signals (via AMPK control of Arf6) may induce regulation of CME—in eliciting global reorganization of the plasma membrane. 1,2,11–13,3–10

scholarly journals The effects of Mitogenic and Metabolic cues on clathrin-mediated endocytosis

2021 ◽  
Author(s):  
Ralph Christian Delos Santos
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Energy Levels ◽  
Surface Proteins ◽  
Coated Pits ◽  
Calcium Signals ◽  
Cellular Energy ◽  
Software Analysis ◽  
Cellular Processes ◽  
Automated Software

The cell ‘surfaceome’ collectively describes proteins found on the plasma membrane (PM), which functions in fundamental cellular processes including growth and proliferation. The surfaceome undergoes dynamic remodeling via the addition/removal of surface proteins in response to changing environmental conditions. In mammalian cells, surfaceome remodeling is predominantly facilitated by clathrin-mediated endocytosis (CME) which involves the invagination and internalization of PM regions via clathrin-coated structures—removing proteins from the cell surface. As a major regulator of the surfaceome, it is important to understand the underlying mechanisms governing CME, given that its dysregulation has been implicated in human pathologies including neurologic and oncogenic disorders. Cellular cues including mitogenic (e.g. growth factors) and metabolic signals (e.g. cellular energy levels) induce diverse cellular processes (e.g. growth and proliferation) requiring surfaceome/PM remodeling. Precisely how mitogenic and metabolic signals may induce surfaceome remodeling however, is under-examined. As a major regulator of the surfaceome, CME is a likely mechanism through which cellular cues may remodel the PM. Poorly understood, I thus sought to investigate how mitogenic and metabolic signals may regulate CME. Mitogenic signaling by the epidermal growth factor receptor (EGFR) triggers PLCγ1-calcium signals, which I found a requirement for CME of EGFR—likely via calcium control of the Sjn1 protein. Consistently, using TIRF-M imaging coupled to automated software analysis, I demonstrate that inhibition of PLCγ1-calcium signals impairs the formation/assembly of GFR-containing clathrin structures. In addition, I hypothesize that PLCγ1-calcium signals also regulates the CME of other surface proteins given its robust control of Sjn1—which localizes broadly amongst clathrin-coated structures. AMPK is a cellular energy sensor activated by metabolic stress (e.g. starvation). Using TIRF-M imaging coupled to automated software analysis, I found that AMPK activation broadly reduces the formation/assembly of bona fide clathrin-coated pits, without impairing the internalization rates of CME cargoes (e.g. EGFR, TfR and β1-integrin). Furthermore, I found that AMPK may regulate CME throughcontrol of the Arf6 protein. Collectively, my findings uncover and provide novel mechanisms by which mitogenic (via EGFR-induced calcium signals) and metabolic signals (via AMPK control of Arf6) may induce regulation of CME—in eliciting global reorganization of the plasma membrane. 1,2,11–13,3–10

scholarly journals p180, a novel recycling transmembrane glycoprotein with restricted cell type expression.

1990 ◽  
Vol 10 (6) ◽  
pp. 2606-2618 ◽  
Author(s):  
C M Isacke ◽  
P van der Geer ◽  
T Hunter ◽  
I S Trowbridge
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Biochemical Characterization ◽  
Surface Proteins ◽  
Sequence Information ◽  
Osteosarcoma Cell ◽  
Binding Studies ◽  
Coated Pits ◽  
Transmembrane Glycoprotein ◽  
Morphological Studies

A 180-kilodalton (kDa) protein (p180) was identified among the antigens for a panel of monoclonal antibodies raised against human fibroblast cell surface proteins. Binding studies with 125I-Fab' fragments of an anti-p180 monoclonal antibody demonstrated that 10 to 30% of p180 was located on the plasma membrane and that the remaining 70 to 90% was on intracellular membranes. p180 was rapidly internalized from the cell surface at 37 degrees C, and kinetic analyses indicated that this was a constitutive process followed by the recycling of p180 back to the plasma membrane. Morphological studies demonstrated that on the cell surface p180 was concentrated in coated pits, whereas inside the cell it was found in endosomes as suggested by its colocalization with the transferrin receptor. Immunoblot analysis with a polyclonal antiserum raised against purified human protein showed that p180 has a restricted distribution with expression at high levels in fibroblast cultures and in tissues containing cells of mesodermal origin. A biochemical characterization of p180 showed it to be a transmembrane glycoprotein with an extracellular domain, which consists of approximately 30 kDa of complex oligosaccharides attached to at least 45 kDa of the protein core. The cytoplasmic domain of p180 was found to contain a serine residue(s) that was phosphorylated both in vivo and in vitro by activated protein kinase C. p180 was purified by subjecting solubilized membrane proteins from a human osteosarcoma cell line to immunoaffinity chromatography and gel filtration. The N-terminal sequence information obtained from the purified protein showed no homology to other known proteins. It was concluded that p180 may be a novel recycling receptor which is highly restricted in its expression to fibroblastlike cells.

Endosomal system of Paramecium: coated pits to early endosomes

1992 ◽  
Vol 101 (2) ◽  
pp. 449-461 ◽  
Author(s):  
R.D. Allen ◽  
C.C. Schroeder ◽  
A.K. Fok
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Fluid Phase ◽  
Early Endosome ◽  
Coated Vesicles ◽  
Striking Similarity ◽  
Coated Pits ◽  
Early Endosomes ◽  
Membrane Components ◽  
Endosomal System

A detailed morphological and tracer study of endocytosis via coated pits in Paramecium multimicronucleatum was undertaken to compare endocytic processes in a free-living protozoon with similar processes in higher organisms. Permanent pits at the cell surface enlarge, become coated and give rise to coated vesicles (188 +/− 41 nm in diameter) that enclose fluid-phase markers such as horseradish peroxidase (HRP). Both the pits and vesicles are labeled by the immunogold technique when a monoclonal antibody (mAb) raised against the plasma membrane of this cell is applied to cryosections. The HRP is delivered to an early endosome compartment, which also shares the plasma membrane antigen. The early endosome, as shown in quick-freeze deep-etch replicas of chemically unfixed cells, is a definitive non-reticular compartment composed of many individual flattened cisternal units of 0.2 to 0.7 microns diameter, each potentially bearing one or more approximately 80-nm-wide coated evaginations. These coated evaginations on the early endosomes contain HRP but are not labeled by the mAb. The coated evaginations pinch off to form a second group of coated vesicles (90 +/− 17 nm in diameter), which can be differentiated from those formed from coated pits by their smaller size, absence of plasma membrane antigen and their location somewhat deeper into the cytoplasm. This study shows a striking similarity between protozoons and mammalian cells in their overall early endosomal machinery and in the ability of early endosomes to sort cargo from plasma membrane components. The vesicles identified in this study form two distinct populations of putative shuttle vesicles, pre-endosomal (large) and early endosome-derived vesicles (small), which facilitate incoming and outgoing traffic from the early endosomes.

Cloning of Drosophila beta-adaptin and its localization on expression in mammalian cells

1994 ◽  
Vol 107 (3) ◽  
pp. 709-718
Author(s):  
D.R. Camidge ◽  
B.M. Pearse
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Beta Subunits ◽  
Specific Interactions ◽  
Coated Pits ◽  
Cos Cells ◽  
Amino Terminal ◽  
Differential Interaction ◽  
Expression In Mammalian Cells ◽  
Choice Of Partner

A Drosophila cDNA (BAD1) encoding a structural and assembly-competent homologue of the mammalian coated pit beta-adaptins (beta and beta') has been cloned and sequenced. In its amino-terminal region (residues 1–575), the BAD1 sequence appears intermediate between that of the mammalian beta-adaptin and a predicted sequence, from cDNA 105a, which appears to code for a version of beta'-adaptin. To test its functional characteristics, a ‘myc’-tagged version of BAD1 was expressed in Cos cells. The BAD1 protein was detected most clearly in plasma membrane coated pits, where it colocalized with alpha-adaptin, although other coated pits were noted which apparently did not contain alpha-adaptin. However, these are probably gamma-adaptin containing pits, as BAD1 was also found colocalized with gamma-adaptin in Golgi coated pits in which, typically, alpha-adaptin is absent. Immunoprecipitation experiments confirmed that the BAD1 protein was present in both types of adaptor complex, unlike beta-adaptin which complexes with alpha-adaptin and beta'-adaptin which partners gamma-adaptin exclusively. In spite of this, BAD1 expression does not appear to mix alpha-adaptin and gamma-adaptin distribution amongst all the coated pits: thus the location of these adaptor complexes in mammalian cells does not depend on the differences between beta subunits but rather on membrane-specific interactions of other adaptor polypeptides. The differential interaction of beta with alpha-adaptin and beta' with gamma-adaptin in mammalian cells is likely to depend on the few non-conservative differences between their respective sequences and BAD1. Four of these (one with respect to beta and three versus 105a) are clustered in a particular region (residues 155 to 305), which may therefore represent a domain that influences the choice of partner adaptin.

scholarly journals Clostridium septicum Alpha-Toxin Is Active against the Parasitic Protozoan Toxoplasma gondii and Targets Members of the SAG Family of Glycosylphosphatidylinositol-Anchored Surface Proteins

2002 ◽  
Vol 70 (8) ◽  
pp. 4353-4361 ◽  
Author(s):  
Michael J. Wichroski ◽  
Jody A. Melton ◽  
Carolyn G. Donahue ◽  
Rodney K. Tweten ◽  
Gary E. Ward
Keyword(s):  
Plasma Membrane ◽  
Toxoplasma Gondii ◽  
Protein Trafficking ◽  
Mammalian Cells ◽  
Molecular Genetic ◽  
Molecular Genetic Analysis ◽  
Surface Proteins ◽  
Alpha Toxin ◽  
Clostridium Septicum ◽  
Ultrastructural Studies

ABSTRACT As is the case with many other protozoan parasites, glycosylphosphatidylinositol (GPI)-anchored proteins dominate the surface of Toxoplasma gondii tachyzoites. The mechanisms by which T. gondii GPI-anchored proteins are synthesized and transported through the unusual triple-membrane structure of the parasite pellicle to the plasma membrane remain largely unknown. As a first step in developing tools to study these processes, we show here that Clostridium septicum alpha-toxin, a pore-forming toxin that targets GPI-anchored protein receptors on the surface of mammalian cells, is active against T. gondii tachyzoites (50% effective concentration, 0.2 nM). Ultrastructural studies reveal that a tight physical connection between the plasma membrane and the underlying membranes of the inner membrane complex is locally disrupted by toxin treatment, resulting in a massive outward extension of the plasma membrane and ultimately lysis of the parasite. Toxin treatment also causes swelling of the parasite endoplasmic reticulum, providing the first direct evidence that alpha-toxin is a vacuolating toxin. Alpha-toxin binds to several parasite GPI-anchored proteins, including surface antigen 3 (SAG3) and SAG1. Interestingly, differences in the toxin-binding profiles between the virulent RH and avirulent P strain were observed. Alpha-toxin may prove to be a powerful experimental tool for molecular genetic analysis of GPI anchor biosynthesis and GPI-anchored protein trafficking in T. gondii and other susceptible protozoa.

scholarly journals Plasma membrane domains enriched in cortical endoplasmic reticulum function as membrane protein trafficking hubs

2013 ◽  
Vol 24 (17) ◽  
pp. 2703-2713 ◽  
Author(s):  
Philip D. Fox ◽  
Christopher J. Haberkorn ◽  
Aubrey V. Weigel ◽  
Jenny L. Higgins ◽  
Elizabeth J. Akin ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Membrane Protein ◽  
Membrane Trafficking ◽  
Protein Trafficking ◽  
Mammalian Cells ◽  
Transmembrane Protein ◽  
Surface Proteins ◽  
Hek 293 ◽  
Cortical Endoplasmic Reticulum

In mammalian cells, the cortical endoplasmic reticulum (cER) is a network of tubules and cisterns that lie in close apposition to the plasma membrane (PM). We provide evidence that PM domains enriched in underlying cER function as trafficking hubs for insertion and removal of PM proteins in HEK 293 cells. By simultaneously visualizing cER and various transmembrane protein cargoes with total internal reflectance fluorescence microscopy, we demonstrate that the majority of exocytotic delivery events for a recycled membrane protein or for a membrane protein being delivered to the PM for the first time occur at regions enriched in cER. Likewise, we observed recurring clathrin clusters and functional endocytosis of PM proteins preferentially at the cER-enriched regions. Thus the cER network serves to organize the molecular machinery for both insertion and removal of cell surface proteins, highlighting a novel role for these unique cellular microdomains in membrane trafficking.

scholarly journals Exploring Stem Cell Differentiation From A Mechanobiological Perspective: Insights From Neural Precursor Cells And Beyond

2020 ◽  
Vol 6 (5) ◽  
pp. 1-4
Author(s):  
Bruno Pontes ◽  
Keyword(s):  
Stem Cell ◽  
Plasma Membrane ◽  
Mammalian Cells ◽  
Shape Control ◽  
Stem Cell Differentiation ◽  
Neural Precursor Cells ◽  
Neural Precursor ◽  
Cellular Processes ◽  
Membrane Cytoskeleton ◽  
Actomyosin Cytoskeleton

The surface of mammalian cells consists of the plasma membrane lined by an underneath cortical actomyosin cytoskeleton. This pair of structures forms the Membrane-Cytoskeleton Complex (MCC), a known regulator of cellular processes, ranging from shape control and cell migration to molecule presentation and signaling [1-3].

The delivery of endocytosed cargo to lysosomes

2009 ◽  
Vol 37 (5) ◽  
pp. 1019-1021 ◽  
Author(s):  
J. Paul Luzio ◽  
Michael D.J. Parkinson ◽  
Sally R. Gray ◽  
Nicholas A. Bright
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Late Endosome ◽  
Snare Complex ◽  
Coated Pits ◽  
Free System ◽  
Endosomal Sorting ◽  
Protein Receptor ◽  
Late Endosomes ◽  
Early Endosomes

In mammalian cells, endocytosed cargo that is internalized through clathrin-coated pits/vesicles passes through early endosomes and then to late endosomes, before delivery to lysosomes for degradation by proteases. Late endosomes are MVBs (multivesicular bodies) with ubiquitinated membrane proteins destined for lysosomal degradation being sorted into their luminal vesicles by the ESCRT (endosomal sorting complex required for transport) machinery. Cargo is delivered from late endosomes to lysosomes by kissing and direct fusion. These processes have been studied in live cell experiments and a cell-free system. Late endosome–lysosome fusion is preceded by tethering that probably requires mammalian orthologues of the yeast HOPS (homotypic fusion and vacuole protein sorting) complex. Heterotypic late endosome–lysosome membrane fusion is mediated by a trans-SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) complex comprising Syntaxin7, Vti1b, Syntaxin8 and VAMP7 (vesicle-associated membrane protein 7). This differs from the trans-SNARE complex required for homotypic late endosome fusion in which VAMP8 replaces VAMP7. VAMP7 is also required for lysosome fusion with the plasma membrane and its retrieval from the plasma membrane to lysosomes is mediated by its folded N-terminal longin domain. Co-ordinated interaction of the ESCRT, HOPS and SNARE complexes is required for cargo delivery to lysosomes.

p180, a novel recycling transmembrane glycoprotein with restricted cell type expression

1990 ◽  
Vol 10 (6) ◽  
pp. 2606-2618
Author(s):  
C M Isacke ◽  
P van der Geer ◽  
T Hunter ◽  
I S Trowbridge
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Biochemical Characterization ◽  
Surface Proteins ◽  
Sequence Information ◽  
Osteosarcoma Cell ◽  
Binding Studies ◽  
Coated Pits ◽  
Transmembrane Glycoprotein ◽  
Morphological Studies

A 180-kilodalton (kDa) protein (p180) was identified among the antigens for a panel of monoclonal antibodies raised against human fibroblast cell surface proteins. Binding studies with 125I-Fab' fragments of an anti-p180 monoclonal antibody demonstrated that 10 to 30% of p180 was located on the plasma membrane and that the remaining 70 to 90% was on intracellular membranes. p180 was rapidly internalized from the cell surface at 37 degrees C, and kinetic analyses indicated that this was a constitutive process followed by the recycling of p180 back to the plasma membrane. Morphological studies demonstrated that on the cell surface p180 was concentrated in coated pits, whereas inside the cell it was found in endosomes as suggested by its colocalization with the transferrin receptor. Immunoblot analysis with a polyclonal antiserum raised against purified human protein showed that p180 has a restricted distribution with expression at high levels in fibroblast cultures and in tissues containing cells of mesodermal origin. A biochemical characterization of p180 showed it to be a transmembrane glycoprotein with an extracellular domain, which consists of approximately 30 kDa of complex oligosaccharides attached to at least 45 kDa of the protein core. The cytoplasmic domain of p180 was found to contain a serine residue(s) that was phosphorylated both in vivo and in vitro by activated protein kinase C. p180 was purified by subjecting solubilized membrane proteins from a human osteosarcoma cell line to immunoaffinity chromatography and gel filtration. The N-terminal sequence information obtained from the purified protein showed no homology to other known proteins. It was concluded that p180 may be a novel recycling receptor which is highly restricted in its expression to fibroblastlike cells.

scholarly journals Flat-to-curved transition during clathrin-mediated endocytosis correlates with a change in clathrin-adaptor ratio and is regulated by membrane tension

2017 ◽  
Author(s):  
Delia Bucher ◽  
Felix Frey ◽  
Kem A. Sochacki ◽  
Susann Kummer ◽  
Jan-Philip Bergeest ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Adaptor Protein ◽  
Membrane Tension ◽  
Coated Pits ◽  
Lattice Curvature ◽  
Cellular Processes ◽  
Molecular Events ◽  
Electron And Light Microscopy ◽  
Mechanical Factors ◽  
Clathrin Adaptor

AbstractAlthough essential for many cellular processes, the sequence of structural and molecular events during clathrin-mediated endocytosis remains elusive. While it was believed that clathrin-coated pits grow with a constant curvature, it was recently suggested that clathrin first assembles to form a flat structure and then bends while maintaining a constant surface area. Here, we combine correlative electron and light microscopy and mathematical modelling to quantify the sequence of ultrastructural rearrangements of the clathrin coat during endocytosis in mammalian cells. We confirm that clathrin-coated structures can initially grow flat and that lattice curvature does not show a direct correlation with clathrin coat assembly. We demonstrate that curvature begins when 70% of the final clathrin content is acquired. We find that this transition is marked by a change in the clathrin to clathrin-adaptor protein AP2 ratio and that membrane tension suppresses this transition. Our results support the model that mammalian cells dynamically regulate the flat-to-curved transition in clathrin-mediated endocytosis by both biochemical and mechanical factors.

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