Role of cell-surface carbohydrates and plasma membrane components in the internalization of cell-penetrating peptides

Cell-penetrating peptides (CPPs) have been widely used to deliver nanomaterials and other types of macromolecules into mammalian cells for therapeutic and diagnostic use. Cationic CPPs that bind to heparan sulfate (HS) proteoglycans on the cell surface induce potent endocytosis; however, the role of other surface receptors in this process is unclear. We describe the convergence of an HS-dependent pathway with the C-end rule (CendR) mechanism that enables peptide ligation with neuropilin-1 (NRP1), a cell surface receptor known to be involved in angiogenesis and vascular permeability. NRP1 binds peptides carrying a positive residue at the carboxyl terminus, a feature that is compatible with cationic CPPs, either intact or after proteolytic processing. We used CPP and CendR peptides, as well as HS- and NRP1-binding motifs from semaphorins, to explore the commonalities and differences of the HS and NRP1 pathways. We show that the CendR-NRP1 interaction determines the ability of CPPs to induce vascular permeability. We also show at the ultrastructural level, using a novel cell entry synchronization method, that both the HS and NRP1 pathways can initiate a macropinocytosis-like process and visualize these CPP-cargo complexes going through various endosomal compartments. Our results provide new insights into how CPPs exploit multiple surface receptor pathways for intracellular delivery.

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Phospholipase D2 Mediates Acute Aldosterone Secretion in Response to Angiotensin II in Adrenal Glomerulosa Cells

Endocrinology ◽

10.1210/en.2009-1159 ◽

2010 ◽

Vol 151 (5) ◽

pp. 2162-2170 ◽

Cited By ~ 15

Author(s):

Haixia Qin ◽

Michael A. Frohman ◽

Wendy B. Bollag

Keyword(s):

Plasma Membrane ◽

Angiotensin Ii ◽

Cell Surface ◽

Phosphatidic Acid ◽

Phospholipase D ◽

Cell Interior ◽

Aldosterone Secretion ◽

Phospholipase D2 ◽

Glomerulosa Cells

In primary bovine adrenal glomerulosa cells, the signaling enzyme phospholipase D (PLD) is suggested to mediate priming, the enhancement of aldosterone secretion after pretreatment with and removal of angiotensin II (AngII), via the formation of persistently elevated diacylglycerol (DAG). To further explore PLD’s role in priming, glomerulosa cells were pretreated with an exogenous bacterial PLD. Using this approach, phosphatidic acid (PA) is generated on the outer, rather than the inner, leaflet of the plasma membrane. Although PA is not readily internalized, the PA is nonetheless rapidly hydrolyzed by cell-surface PA phosphatases to DAG, which efficiently flips to the inner leaflet and accesses the cell interior. Pretreatment with bacterial PLD resulted in priming upon subsequent AngII exposure, supporting a role of DAG in this process, because the increase in DAG persisted after exogenous PLD removal. To determine the PLD isoform mediating aldosterone secretion, and presumably priming, primary glomerulosa cells were infected with adenoviruses expressing GFP, PLD1, PLD2, or lipase-inactive mutants. Overexpressed PLD2 increased aldosterone secretion by approximately 3-fold over the GFP-infected control under basal conditions, with a significant enhancement to about 16-fold over the basal value upon AngII stimulation. PLD activity was also increased basally and upon stimulation with AngII. In contrast, PLD1 overexpression had little effect on aldosterone secretion, despite the fact that PLD activity was enhanced. In both cases, the lipase-inactive PLD mutants showed essentially no effect on PLD activity or aldosterone secretion. Our results suggest that PLD2 is the isoform that mediates aldosterone secretion and likely priming.

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Mechanism of protein sorting during erythroblast enucleation: role of cytoskeletal connectivity

Blood ◽

10.1182/blood-2003-03-0928 ◽

2004 ◽

Vol 103 (5) ◽

pp. 1912-1919 ◽

Cited By ~ 68

Author(s):

James C.-M. Lee ◽

J. Aura Gimm ◽

Annie J. Lo ◽

Mark J. Koury ◽

Sharon W. Krauss ◽

...

Keyword(s):

Plasma Membrane ◽

Molecular Mechanisms ◽

Immunofluorescence Microscopy ◽

Protein Sorting ◽

Membrane Cytoskeleton ◽

Skeletal Protein ◽

Regulate Protein ◽

Membrane Components ◽

Cytoskeletal Elements

AbstractDuring erythroblast enucleation, nuclei surrounded by plasma membrane separate from erythroblast cytoplasm. A key aspect of this process is sorting of erythroblast plasma membrane components to reticulocytes and expelled nuclei. Although it is known that cytoskeletal elements actin and spectrin partition to reticulocytes, little is understood about molecular mechanisms governing plasma membrane protein sorting. We chose glycophorin A (GPA) as a model integral protein to begin investigating protein-sorting mechanisms. Using immunofluorescence microscopy and Western blotting we found that GPA sorted predominantly to reticulocytes. We hypothesized that the degree of skeletal linkage might control the sorting pattern of transmembrane proteins. To explore this hypothesis, we quantified the extent of GPA association to the cytoskeleton in erythroblasts, young reticulocytes, and mature erythrocytes using fluorescence imaged microdeformation (FIMD) and observed that GPA underwent dramatic reorganization during terminal differentiation. We discovered that GPA was more connected to the membrane cytoskeleton, either directly or indirectly, in erythroblasts and young reticulocytes than in mature cells. We conclude that skeletal protein association can regulate protein sorting during enucleation. Further, we suggest that the enhanced rigidity of reticulocyte membranes observed in earlier investigations results, at least in part, from increased connectivity of GPA with the spectrin-based skeleton.

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Surface redistribution and release of antibody-induced caps in entamoebae.

Journal of Experimental Medicine ◽

10.1084/jem.151.1.184 ◽

1980 ◽

Vol 151 (1) ◽

pp. 184-193 ◽

Cited By ~ 41

Author(s):

J Calderón ◽

M de Lourdes Muñoz ◽

H M Acosta

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Surface Segregation ◽

Surface Membrane ◽

Single Layer ◽

Surface Antigens ◽

Spontaneous Release ◽

Plasma Membrane Regeneration ◽

Membrane Components ◽

Radiolabeled Antibodies

Polyspecific antibodies bound to Entamoeba induced surface redistribution of membrane components toward the uroid region. Capping of surface antigens was obtained with a single layer of antibodies in E. histolytica and E. invadens. This surface segregation progressed to a large accumulation of folded plasma membrane that extruded as a defined vesicular cap. A spontaneous release of the cap at the end of the capping process took place. These released caps contained most of the antibodies that originally bound to the whole cell surface. Two-thirds of radiolabeled antibodies bound to the surface of E. histolytica were released into the medium in 2 h. Successive capping induced by repeated exposure of E. invadens to antibodies produced conglomerates of folded surface membrane, visualized as stacked caps, in proportion to the number of antibody exposures. These results indicate the remarkable ability of Entamoeba to rapidly regenerate substantial amounts of plasma membbrane. The properties of surface redistribution, liberation of caps, and plasma membrane regeneration, may contribute to the survival of the parasite in the host during infection.

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Neisseria gonorrhoeae Porin P1.B Induces Endosome Exocytosis and a Redistribution of Lamp1 to the Plasma Membrane

Infection and Immunity ◽

10.1128/iai.70.11.5965-5971.2002 ◽

2002 ◽

Vol 70 (11) ◽

pp. 5965-5971 ◽

Cited By ~ 16

Author(s):

Patricia Ayala ◽

Brandi Vasquez ◽

Lee Wetzler ◽

Magdalene So

Keyword(s):

Plasma Membrane ◽

Epithelial Cells ◽

Cell Surface ◽

Neisseria Gonorrhoeae ◽

Bacterial Infection ◽

Immunoglobulin A ◽

Late Endosomes ◽

A Cell ◽

Iga Protease

ABSTRACT The immunoglobulin A (IgA) protease secreted by pathogenic Neisseria spp. cleaves Lamp1, thereby altering lysosomes in a cell and promoting bacterial intracellular survival. We sought to determine how the IgA protease gains access to cellular Lamp1 in order to better understand the role of this cleavage event in bacterial infection. In a previous report, we demonstrated that the pilus-induced Ca2+ transient triggers lysosome exocytosis in human epithelial cells. This, in turn, increases the level of Lamp1 at the plasma membrane, where it can be cleaved by IgA protease. Here, we show that porin also induces a Ca2+ flux in epithelial cells. This transient is similar in nature to that observed in phagocytes exposed to porin. In contrast to the pilus-induced Ca2+ transient, the porin-induced event does not trigger lysosome exocytosis. Instead, it stimulates exocytosis of early and late endosomes and increases Lamp1 on the cell surface. These results indicate that Neisseria pili and porin perturb Lamp1 trafficking in epithelial cells by triggering separate and distinct Ca2+-dependent exocytic events, bringing Lamp1 to the cell surface, where it can be cleaved by IgA protease.

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Lipid order and molecular assemblies in the plasma membrane of eukaryotic cells

Biochemical Society Transactions ◽

10.1042/bst0371056 ◽

2009 ◽

Vol 37 (5) ◽

pp. 1056-1060 ◽

Cited By ~ 9

Author(s):

Marek Cebecauer ◽

Dylan M. Owen ◽

Anna Markiewicz ◽

Anthony I. Magee

Keyword(s):

Plasma Membrane ◽

Physical Properties ◽

Live Cell Imaging ◽

Cell Imaging ◽

Live Cell ◽

Eukaryotic Cells ◽

Dynamic Nature ◽

Membrane Components ◽

Technological Improvements

Multimolecular assemblies on the plasma membrane exhibit dynamic nature and are often generated during the activation of eukaryotic cells. The role of lipids and their physical properties in helping to control the existence of these structures is discussed. Technological improvements for live cell imaging of membrane components are also reviewed.

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