Coated pits with pinocytosis in Tetrahymena

1983 ◽  
Vol 63 (1) ◽  
pp. 209-222 ◽  
Author(s):  
J.R. Nilsson ◽  
B. van Deurs
Keyword(s):  
Cell Surface ◽  
Growth Temperature ◽  
Mammalian Cells ◽  
Inorganic Salt ◽  
Normal Growth ◽  
Cationized Ferritin ◽  
Salt Medium ◽  
Coated Pits ◽  
Food Vacuoles ◽  
Section Analysis

The possible role in pinocytosis of coated pits at the parasomal sacs of Tetrahymena has been studied using cationized ferritin (CF; pI = 8.5) as a marker of membrane and content. It is shown that CF binds evenly to the surface, including the coated pits, of Tetrahymena in an inorganic salt medium (to avoid formation of food vacuoles) at the normal growth temperature. Moreover, CF is internalized by coated vesicles (shown to be truly free by thin serial-section analysis) and delivered initially (1-5 min of incubation) to cisterna near the cell surface. Later (5-10 min) CF occurs also in autophagic vacuoles, formed as a result of starvation, and eventually (15-90 min) it is present in preformed (old) food vacuoles. These observations indicate that the coated pits at the parasomal sacs of Tetrahymena function in adsorptive pinocytosis in much the same manner as coated pits at the surface of mammalian cells.

scholarly journals A Myosin-7B dependent endocytosis pathway mediates cellular entry of α-Synuclein fibrils and polycation-bearing cargos

2020 ◽  
Author(s):  
Qi Zhang ◽  
Yue Xu ◽  
Juhyung Lee ◽  
Michal Jarnik ◽  
Xufeng Wu ◽  
...  
Keyword(s):  
Cell Surface ◽  
Heparan Sulfate ◽  
Actin Filaments ◽  
Mammalian Cells ◽  
Imaging Techniques ◽  
Protein Aggregates ◽  
Membrane Dynamics ◽  
Coated Pits ◽  
Endocytosis Pathway ◽  
Underlying Mechanisms

Cell-to-cell transmission of misfolding-prone α-Synuclein (α-Syn) has emerged as a key pathological event in Parkinson’s disease. This process is initiated when α-Syn-bearing fibrils enter cells via clathrin-mediated endocytosis, but the underlying mechanisms are unclear. Using a CRISPR-mediated knockout screen, we identify SLC35B2 and Myosin-7B (MYO7B) as critical endocytosis regulators for α-Syn preformed fibrils (PFF). We show that SLC35B2, as a key regulator of heparan sulfate proteoglycan (HSPG) biosynthesis, is essential for recruiting α-Syn PFF to the cell surface because this process is mediated by interactions between negatively charged sugar moieties of HSPGs and clustered K-T-K motifs in α-Syn PFF. By contrast, MYO7B regulates α-Syn PFF entry by maintaining a plasma-membrane-associated actin network that controls membrane dynamics. Without MYO7B or actin filaments, many clathrin-coated pits fail to be severed from the membrane, causing accumulation of large clathrin-containing ‘scars’ on the cell surface. Intriguingly, the requirement for MYO7B in endocytosis is only restricted to α-Syn PFF and other cargos that enter cells via HSPGs. Thus, by identifying new regulatory factors for α-Syn PFF endocytosis, our study defines a mechanistically distinct clathrin-mediated endocytosis pathway that requires additional force generated by MYO7B and actin filaments.SignificanceThe spreading of misfolded protein aggregates such as α-Synuclein preformed fibrils (α-Syn PFF) from cell to cell is a pathologic hallmark associated with the progression of many neurodegenerative diseases, but it is unclear how mammalian cells take up these large protein aggregates to initiate this prion-like protein transmission process. Here we define the mechanism of α-Syn PFF endocytosis using a combination of genetic, biochemical, and live-cell imaging techniques. Our study reveals how α-Syn PFF binds to the cell surface heparan sulfate proteoglycans using two lysine-bearing motifs and then enters cells following a Myosin-7B- and actin-dependent endocytosis mechanism that is specifically tailored for polycation-bearing cargos.

scholarly journals Serial-section analysis of coated pits and vesicles involved in adsorptive pinocytosis in cultured fibroblasts.

1983 ◽  
Vol 96 (1) ◽  
pp. 277-281 ◽  
Author(s):  
O W Petersen ◽  
B van Deurs
Keyword(s):  
Coated Vesicles ◽  
Skin Fibroblasts ◽  
Cationized Ferritin ◽  
Serial Sections ◽  
Human Skin Fibroblasts ◽  
Coated Pits ◽  
Cultured Fibroblasts ◽  
Thin Sections ◽  
20 Nm ◽  
Section Analysis

We have examined, by analyzing thin (15-20 nm) serial sections, whether coated pits involved in adsorptive pinocytosis in cultured fibroblasts give rise to free coated vesicles or represent permanently surface-associated structures from the neck of which uncoated receptosomes pinch off and carry ligand into the cell. Human skin fibroblasts and mouse L-929 fibroblasts were incubated with cationized ferritin (CF), a ligand known to bind to coated pit regions, at 37 degrees C before fixation. In thin sections, CF was found in coated vesicular profiles within the cytoplasm. Serial sections revealed that whereas many of these coated profiles communicated with the cell surface, thus representing pits, about 10% in L-cells and 36% in skin fibroblasts were actually free coated vesicles. Moreover, evidence for uncoated vesicular structures (receptosomes) budding off from the coated pits was not obtained. We therefore conclude that coated pits do pinch off from the plasma membrane to form free, coated vesicles (pinosomes).

What do nanometer molecular trajectories tell us about heterogeneous cell surface domaines?

1995 ◽  
Vol 53 ◽  
pp. 786-787
Author(s):  
Watt W. Webb
Keyword(s):  
Cell Surface ◽  
Lipid Membranes ◽  
Surface Proteins ◽  
Protein Diffusion ◽  
Coated Pits ◽  
Cell Surface Proteins ◽  
Membrane Heterogeneity ◽  
Fluorescence Photobleaching Recovery ◽  
Photobleaching Recovery ◽  
Plasma Membrane Heterogeneity

Plasma membrane heterogeneity is implicit in the existence of specialized cell surface organelles which are necessary for cellular function; coated pits, post and pre-synaptic terminals, microvillae, caveolae, tight junctions, focal contacts and endothelial polarization are examples. The persistence of these discrete molecular aggregates depends on localized restraint of the constituent molecules within specific domaines in the cell surface by strong intermolecular bonds and/or anchorage to extended cytoskeleton. The observed plasticity of many of organelles and the dynamical modulation of domaines induced by cellular signaling evidence evanescent intermolecular interactions even in conspicuous aggregates. There is also strong evidence that universal restraints on the mobility of cell surface proteins persist virtually everywhere in cell surfaces, not only in the discrete organelles. Diffusion of cell surface proteins is slowed by several orders of magnitude relative to corresponding protein diffusion coefficients in isolated lipid membranes as has been determined by various ensemble average methods of measurement such as fluorescence photobleaching recovery(FPR).

scholarly journals Functional Entry of Baculovirus into Insect and Mammalian Cells Is Dependent on Clathrin-Mediated Endocytosis

2006 ◽  
Vol 80 (17) ◽  
pp. 8830-8833 ◽  
Author(s):  
Gang Long ◽  
Xiaoyu Pan ◽  
Richard Kormelink ◽  
Just M. Vlak
Keyword(s):  
Electron Microscopy ◽  
Mammalian Cells ◽  
Low Ph ◽  
Coated Pits ◽  
Immuno Electron Microscopy ◽  
Endocytosis Pathway ◽  
Ph Dependent ◽  
Budded Virus

ABSTRACT Entry of the budded virus form of baculoviruses into insect and mammalian cells is generally thought to occur through a low-pH-dependent endocytosis pathway, possibly through clathrin-coated pits. This insight is primarily based on (immuno)electron microscopy studies but requires biochemical support to exclude the use of other pathways. Here, we demonstrate using various inhibitors that functional entry of baculoviruses into insect and mammalian cells is primarily dependent on clathrin-mediated endocytosis. Our results further suggest that caveolae are somehow involved in baculovirus entry in mammalian cells. A caveolar endocytosis inhibitor, genistein, enhances baculovirus transduction in these cells considerably.

scholarly journals Receptor-mediated endocytosis of alpha 2-macroglobulin in cultured fibroblasts.

1980 ◽  
Vol 28 (8) ◽  
pp. 818-823 ◽  
Author(s):  
M C Willingham ◽  
F R Maxfield ◽  
I Pastan
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Coated Pits ◽  
Cultured Fibroblasts ◽  
Receptor Mediated Endocytosis ◽  
Cytoplasmic Vesicles

Alpha 2-macroglobulin is internalized into cultured fibroblasts by receptor-mediated endocytosis. This ligand binds initially to diffusely distributed receptors on the cell surface which cluster rapidly into bristle-coated pits. Within a few minutes at 37 degrees C, these complexes are internalized into uncoated cytoplasmic vesicles, called receptosomes, which move about in the cell by saltatory motion. These vesicles interact with the Golgi-endoplasmic reticulum-lysosome system in the cell to deliver the ligand to newly formed lysosomes within 30--60 min.

scholarly journals Phosphatidylinositol-(4,5)-bisphosphate regulates clathrin-coated pit initiation, stabilization, and size

2011 ◽  
Vol 22 (14) ◽  
pp. 2588-2600 ◽  
Author(s):  
Costin N. Antonescu ◽  
François Aguet ◽  
Gaudenz Danuser ◽  
Sandra L. Schmid
Keyword(s):  
Mammalian Cells ◽  
Lipid Binding ◽  
Coated Pits ◽  
Inositol Phosphatase ◽  
Major Mechanism ◽  
Synaptojanin 1 ◽  
Phosphatidylinositol 4 ◽  
Sirna Knockdown ◽  
Interfering Rna ◽  
Pit Initiation

Clathrin-mediated endocytosis (CME) is the major mechanism for internalization in mammalian cells. CME initiates by recruitment of adaptors and clathrin to form clathrin-coated pits (CCPs). Nearly half of nascent CCPs abort, whereas others are stabilized by unknown mechanisms and undergo further maturation before pinching off to form clathrin-coated vesicles (CCVs). Phosphatidylinositol-(4,5)-bisphosphate (PIP2), the main lipid binding partner of endocytic proteins, is required for CCP assembly, but little is currently known about its contribution(s) to later events in CCV formation. Using small interfering RNA (siRNA) knockdown and overexpression, we have analyzed the effects of manipulating PIP2 synthesis and turnover on CME by quantitative total internal reflection fluorescence microscopy and computational analysis. Phosphatidylinositol-4-phosphate-5-kinase cannot be detected within CCPs but functions in initiation and controls the rate and extent of CCP growth. In contrast, the 5′-inositol phosphatase synaptojanin 1 localizes to CCPs and controls early stabilization and maturation efficiency. Together these results suggest that the balance of PIP2 synthesis in the bulk plasma membrane and its local turnover within CCPs control multiple stages of CCV formation.

scholarly journals Endocytic Delivery of Vancomycin Mediated by a Synthetic Cell Surface Receptor:  Rescue of Bacterially Infected Mammalian Cells and Tissue Targeting In Vivo

2007 ◽  
Vol 129 (2) ◽  
pp. 268-269 ◽  
Author(s):  
Siwarutt Boonyarattanakalin ◽  
Jianfang Hu ◽  
Sheryl A. Dykstra-Rummel ◽  
Avery August ◽  
Blake R. Peterson
Keyword(s):  
Cell Surface ◽  
Mammalian Cells ◽  
Cell Surface Receptor ◽  
Synthetic Cell ◽  
Tissue Targeting ◽  
Surface Receptor

Dependence of the superficial layers of Spirillum putridiconchylium on Ca2+ or Sr2+

1976 ◽  
Vol 22 (9) ◽  
pp. 1233-1244 ◽  
Author(s):  
T. J. Beveridge ◽  
R. G. E. Murray
Keyword(s):  
Cell Surface ◽  
Outer Membrane ◽  
Growth Medium ◽  
Chelating Agents ◽  
Membrane Surface ◽  
Superficial Layer ◽  
Cationized Ferritin ◽  
Membrane Component ◽  
Casamino Acids ◽  
Amorphous Precipitate

Chelating agents disrupted the superficial layers on Spirillum putridiconchylium and adsorption of cationized ferritin indicated that both upper and lower surfaces of superficial layer fragments, as well as the outer membrane surface, possessed areas which were negatively charged. Growth of the bacterium in 1% casamino acids (vitamin free) resulted in cells which were devoid of the superficial layers, and negative staining of these cells revealed an amorphous precipitate together with a vesicular outer membrane component extruding from their surfaces into the medium. Addition of either 1 mM Ca2+ or 1 mM Sr2+ to the growth medium produced the typical regularly structured cell surface, whereas addition of equal concentrations of Li+, Na+, K+, Mg2+, Ba2+, Mn2+, Fe3+, or three polyamines produced the structureless surface.

scholarly journals Surface charge distribution on the endothelial cell of liver sinusoids.

1984 ◽  
Vol 99 (2) ◽  
pp. 639-647 ◽  
Author(s):  
L Ghitescu ◽  
A Fixman
Keyword(s):  
Endothelial Cell ◽  
Cell Surface ◽  
Liver Sinusoid ◽  
Electron Microscopic ◽  
Coated Pits ◽  
Endothelial Cell Surface ◽  
Liver Sinusoids ◽  
Surface Charge Distribution ◽  
Cationic Residues

The topography of the charged residues on the endothelial cell surface of liver sinusoid capillaries was investigated by using electron microscopic tracers of different size and charge. The tracers used were native ferritin (pl 4.2-4.7) and its cationized (pl 8.4) and anionized (pl 3.7) derivatives, BSA coupled to colloidal gold (pl of the complex 5.1), hemeundecapeptide (pl 4.85), and alcian blue (pl greater than 10). The tracers were either injected in vivo or perfused in situ through the portal vein of the mouse liver. In some experiments, two tracers of opposite charge were sequentially perfused with extensive washing in between. The liver was processed for electron microscopy and the binding pattern of the injected markers was recorded. The electrostatic nature of the tracer binding was assessed by perfusion with high ionic strength solutions, by aldehyde quenching of the plasma membrane basic residues, and by substituting the cell surface acidic moieties with positively charged groups. Results indicate that the endothelial cells of the liver sinusoids expose on their surface both cationic and anionic residues. The density distribution of these charged groups on the cell surface is different. While the negative charge is randomly and patchily scattered all over the membrane, the cationic residues seem to be accumulated in coated pits. The charged groups co-exist in the same coated pit and bind the opposite charged macromolecule. It appears that the fixed positive and negative charges of the coated pit glycocalyx are mainly segregated in space. The layer of basic residues is located at 20-30-nm distance of the membrane, while most of the negative charges lie close to the external leaflet of the plasmalemma.

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