v-Src induces constitutive macropinocytosis in rat fibroblasts

1996 ◽  
Vol 109 (8) ◽  
pp. 2005-2012 ◽  
Author(s):  
A. Veithen ◽  
P. Cupers ◽  
P. Baudhuin ◽  
P.J. Courtoy
Keyword(s):  
Light And Electron Microscopy ◽  
Fluid Phase ◽  
Transformed Cells ◽  
Permissive Temperature ◽  
Coated Pits ◽  
Regulatory Machinery ◽  
Rat Fibroblasts ◽  
Pinocytic Vesicles ◽  
Stimulation Of

The role of v-Src as regulator of fluid-phase pinocytosis was investigated in Rat-1 cells expressing a stable (Rat-1/BB16) or a thermosensitive (Rat-1/tsLA29) v-Src protein. In the second cell line, this protein is inactive when cells are cultured at 40 degrees C but recovers its tyrosine kinase activity upon transfer to 34 degrees C, resulting into a transformed phenotype. The rate of fluid-phase pinocytosis of the tracer horseradish peroxidase was 2-fold higher in v-Src-transformed fibroblasts (Rat-1/BB16, Rat-1/tsLA29 cultured at 34 degrees C) as compared to non-transformed cells (Rat-1, Rat-1/tsLA29 kept at 40 degrees C). In contrast, receptor-mediated endocytosis of transferrin was poorly affected, suggesting that structures distinct from clathrin-coated pits are involved in pinocytosis stimulation. By light and electron microscopy, transformed cells frequently contained large peroxidase-labeled pinocytic vesicles located near to membrane ruffles, demonstrating that stimulation of pinocytosis corresponds to induction of constitutive macropinocytosis. Stimulation of pinocytosis occurred more than 8 hours after transfer to the permissive temperature, whereas transfer to the non-permissive temperature partially reversed the stimulation within 2 hours. Protein synthesis inhibition for 6 hours abrogated pinocytosis stimulation in transformed cells, indicating that constitutive macropinocytosis induced by v-Src depends on continuous synthesis of a short-lived regulatory machinery.

Stimulation of P(i) transport in opossum kidney cells by hyposmotic media

1993 ◽  
Vol 264 (4) ◽  
pp. F585-F592
Author(s):  
M. Loghman-Adham ◽  
G. T. Motock
Keyword(s):  
Fluid Phase ◽  
Kinetic Studies ◽  
Uptake Kinetic ◽  
Opossum Kidney ◽  
Pi Transport ◽  
Opossum Kidney Cells ◽  
Fluid Phase Endocytosis ◽  
Peroxidase Uptake ◽  
Stimulation Of

Exposure of various cells to hyposmotic media (Hypo) results in a rapid inhibition of both receptor-mediated and fluid-phase endocytosis. We used this maneuver to investigate the role of endocytosis in regulation of Pi transport in opossum kidney (OK) cells. Following exposure to Hypo, Na(+)-dependent Pi uptake increased rapidly, reaching a maximum within 5 min, and remained elevated up to 30 min. This was associated with a simultaneous reduction of horseradish peroxidase uptake. Kinetic studies showed increased apparent Vmax for Pi (9.38 +/- 0.93 vs. 13.08 +/- 1.04 nmol.mg-1.5 min-1 for control and Hypo, respectively; P < 0.05, n = 6) with no change in apparent Km. The effect was specific for Pi with no change in the Na(+)-dependent or -independent uptake of L-proline, L-glutamine, or methyl-alpha-D-glucopyranoside. Stimulation of Pi transport persisted when control and Hypo had identical ionic compositions. Stimulation of Pi transport was rapidly reversed when cells were returned to an isosmotic medium. Preincubation with Hypo at 4 degrees C had no effect on Pi transport. Addition of cycloheximide or actinomycin D did not prevent the increased Pi uptake after exposure to Hypo. The effect also persisted after protein kinase C downregulation. Stimulation of Pi transport by Hypo is consistent with reduced endocytic retrieval of Na(+)-Pi cotransporters from brush-border membrane (BBM), resulting in an increase in their number on the BBM.

scholarly journals The structure of syncytia induced by the phytoparasitic nematode Nacobbus aberrans in tomato roots, and the possible role of plasmodesmata in their nutrition

1977 ◽  
Vol 23 (1) ◽  
pp. 299-313
Author(s):  
M.G. Jones ◽  
H.L. Payne
Keyword(s):  
Light And Electron Microscopy ◽  
Transformed Cells ◽  
Root Knot Nematode ◽  
Sieve Elements ◽  
Tomato Roots ◽  
Solute Movement ◽  
Nacobbus Aberrans ◽  
Symplastic Pathway ◽  
Massive Accumulation

The structure of syncytia induced within galls in tomato roots by the false root-knot nematode Nacobbus aberrans has been examined by light and electron microscopy. A syncytium develops by breakdown or individual cell walls, which allows movement of cytoplasmic contents between transformed cells. The wall breakdown takes place at pit fields, where the plasmodesmata may be protected from digestion until the surrounding wall is removed. Numerous sieve elements differentiate in the cells outside the syncytium. These sieve elements, and also plasmodesmata in pit fields, are demonstrated by fluorescence microscopy. The possibility of a symplastic pathway of solute movement from the phloem to the syncytium is suggested. A massive accumulation of starch occurs in the gall cells and syncytial cells, which may be related to the proliferation of phloem. Wall ingrowths typical of transfer cells are absent, and a comparative survey of the structure and mode of solute entry into nematode-transformed cells in which ingrowths are present or absent is presented.

Structural Studies of Dynamin Tubular Crystals by Cryo-Electron Microscopy

1999 ◽  
Vol 5 (S2) ◽  
pp. 1024-1025
Author(s):  
Peijun Zhang ◽  
Jenny E. Hinshaw
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Lipid Vesicles ◽  
Coated Vesicle ◽  
Permissive Temperature ◽  
Coated Pits ◽  
Unique Role ◽  
Cryo Electron Microscopy ◽  
Tubular Crystals

Dynamin is a 100 kD GTPase that plays an essential role in clathrin-coated vesicle formation during receptor mediated endocytosis, and in caveolae internalization and may play a role in intracellular membrane trafficking (1). It shares an extensive sequence homology (70% identity) to shibiregene product in Drosophila(2,3). The shibiretsmutants exhibit a rapid and reversible paralysis at non-permissive temperature due to a depletion of synaptic vesicles in their nerve termini which is believed to be caused by a block in endocytosis since there is an accumulation of “collared” clathrin-coated pits at the plasma membrane (4). Synaptosomes treated with GTPγs produces elongated necks surrounded by dynamin (6). Purified recombinant dynamin itself can assemble to form spirals and bind to lipid vesicles to form tubes, which resemble the “collar” at the necks of coated pits (5). These dynamin tubes vesiculate upon GTP treatment (7), suggesting a unique role of dynamin acting as a mechanoenzyme which causes clathrin-coated vesicles to be pinched off plasma membrane.

PKC-ε regulates basolateral endocytosis in human T84 intestinal epithelia: role of F-actin and MARCKS

1999 ◽  
Vol 277 (6) ◽  
pp. C1239-C1249 ◽  
Author(s):  
Jaekyung Cecilia Song ◽  
Bruce J. Hrnjez ◽  
Omid C. Farokhzad ◽  
Jeffrey B. Matthews
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Basolateral Membrane ◽  
Phosphorylation Site ◽  
Fluid Phase ◽  
Cytochalasin D ◽  
Pkc Inhibitor ◽  
Intestinal Epithelia ◽  
Stimulation Of

Protein kinase C (PKC) and the actin cytoskeleton are critical effectors of membrane trafficking in mammalian cells. In polarized epithelia, the role of these factors in endocytic events at either the apical or basolateral membrane is poorly defined. In the present study, phorbol 12-myristate 13-acetate (PMA) and other activators of PKC selectively enhanced basolateral but not apical fluid-phase endocytosis in human T84 intestinal epithelia. Stimulation of basolateral endocytosis was blocked by the conventional and novel PKC inhibitor Gö-6850, but not the conventional PKC inhibitor Gö-6976, and correlated with translocation of the novel PKC isoform PKC-ε. PMA treatment induced remodeling of basolateral F-actin. The actin disassembler cytochalasin D stimulated basolateral endocytosis and enhanced stimulation of endocytosis by PMA, whereas PMA-stimulated endocytosis was blocked by the F-actin stabilizers phalloidin and jasplakinolide. PMA induced membrane-to-cytosol redistribution of the F-actin cross-linking protein myristoylated alanine-rich C kinase substrate (MARCKS). Cytochalasin D also induced MARCKS translocation and enhanced PMA-stimulated translocation of MARCKS. A myristoylated peptide corresponding to the phosphorylation site domain of MARCKS inhibited both MARCKS translocation and PMA stimulation of endocytosis. MARCKS translocation was inhibited by Gö-6850 but not Gö-6976. The results suggest that a novel PKC isoform, likely PKC-ε, stimulates basolateral endocytosis in model epithelia by a mechanism that involves F-actin and MARCKS.

scholarly journals Clathrin heavy chain is required for pinocytosis, the presence of large vacuoles, and development in Dictyostelium.

1992 ◽  
Vol 118 (6) ◽  
pp. 1371-1377 ◽  
Author(s):  
T J O'Halloran ◽  
R G Anderson
Keyword(s):  
Heavy Chain ◽  
Antisense Rna ◽  
Fluid Phase ◽  
Solid Particles ◽  
Endocytic Pathway ◽  
Coated Pits ◽  
Clathrin Heavy Chain ◽  
Development Cycle ◽  
Mutant Cells

To investigate the intracellular role of the clathrin heavy chain in living cells, we have used "antisense" RNA to engineer mutant Dictyostelium discoideum cells that are severely deficient in clathrin heavy chain expression. Immunoblots stained with an anti-clathrin heavy chain antiserum revealed that mutant cells contained undetectable amounts of clathrin heavy chain protein. Similarly, Northern blots showed an absence of clathrin heavy chain mRNA. Clathrin heavy chain-deficient Dictyostelium cells were viable, but exhibited growth rates twofold slower than parental cells. Whereas many morphological features of the mutant cells were normal, mutant cells lacked coated pits and coated vesicles. Clathrin-deficient cells were also missing large translucent vacuoles that serve as endosomes and contractile vacuoles. In the absence of clathrin heavy chain, mutant cells displayed three distinct functional defects: (a) impairment in endocytosis of fluid phase markers, but competence in another endocytic pathway, the phagocytosis of solid particles; (b) defects in osmoregulation; and (c) inability to complete the starvation-induced development cycle.

The Role of Radiation Quality in the Stimulation of Intercellular Induction of Apoptosis in Transformed Cells at Very Low Doses

2011 ◽  
Vol 176 (3) ◽  
pp. 346 ◽  
Author(s):  
Abdelrazek B. Abdelrazzak ◽  
David L. Stevens ◽  
Georg Bauer ◽  
Peter O'Neill ◽  
Mark A. Hill
Keyword(s):  
Transformed Cells ◽  
Low Doses ◽  
Radiation Quality ◽  
Induction Of Apoptosis ◽  
Stimulation Of

scholarly journals Sequential Actions of Rab5 and Rab7 Regulate Endocytosis in the Xenopus Oocyte

1997 ◽  
Vol 136 (6) ◽  
pp. 1227-1237 ◽  
Author(s):  
Amitabha Mukhopadhyay ◽  
Alejandro M. Barbieri ◽  
Kouichi Funato ◽  
Richard Roberts ◽  
Philip D. Stahl
Keyword(s):  
Xenopus Oocytes ◽  
Fluid Phase ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Aluminum Fluoride ◽  
General Role ◽  
Sensitive Factor ◽  
Rate Limiting ◽  
Stimulation Of

To explore the role of GTPases in endocytosis, we developed an assay using Xenopus oocytes injected with recombinant proteins to follow the uptake of the fluid phase marker HRP. HRP uptake was inhibited in cells injected with GTPγS or incubated with aluminum fluoride, suggesting a general role for GTPases in endocytosis. Injection of Rab5 into oocytes, as well as Rab5:Q79L, a mutant with decreased GTPase activity, increased HRP uptake. Injection of Rab5:S34N, the dominant-negative mutant, inhibited HRP uptake. Injection of N-ethylmaleimide–sensitive factor (NSF) stimulated HRP uptake, and ATPase-defective NSF mutants inhibited HRP uptake when coinjected with Rab5:Q79L, confirming a requirement for NSF in endocytosis. Surprisingly, injection of Rab7:WT stimulated both uptake and degradation/activation of HRP. The latter appears to be due to enhanced transport to a late endosomal/prelysosomal degradative compartment that is monensin sensitive. Enhancement of uptake by Rab7 appears to function via an Rab5-sensitive pathway in oocytes since the stimulatory effect of Rab7 was blocked by coinjection of Rab5:S34N. Stimulation of uptake by Rab5 was blocked by Rab5:S34N but not by Rab7:T22N. Our results suggest that Rab7, while functioning downstream of Rab5, may be rate limiting for endocytosis in oocytes.

scholarly journals Cell to substratum adhesion-promoting activity released by normal and virus-transformed cells in culture.

1976 ◽  
Vol 70 (3) ◽  
pp. 634-647 ◽  
Author(s):  
E G Moore
Keyword(s):  
Conditioned Media ◽  
Transformed Cells ◽  
Cultured Fibroblasts ◽  
Rous Sarcoma ◽  
Sensitive Factor ◽  
Sarcoma Virus ◽  
Rat Fibroblasts ◽  
Avian Sarcoma ◽  
Plastic Substratum

It is demonstrated here that cultured fibroblasts release into their medium a nondialyzable, protease-sensitive factor(s) capable of promoting the adhesion and spreading of virus-transformed rat fibroblasts on a plastic substratum. A relatively sensitive biological assay is described for quantitation of the adhesion-promoting factor (APF) activity in serum-free, conditioned medium harvested from the cultures. Evidence is presented which indicates that the primary mode of action of the APF is by binding to and modifying the properties of the substratum. Conditioned media harvested after 24 h of incubation in similarly populated cultures of normal fibroblasts of diverse animal species exhibited similar levels of APF activity. However, conditioned media obtained from Rous sarcoma virus (Prague strain)-transformed and avian sarcoma virus B77-transformed rat fibroblasts exhibited three- to sixfold lower levels of APF activity than media conditioned in parallel cultures of heterologous or homologous normal fibroblasts. Cultivation of B77 virus-transformed rat cells in the presence of dibutyryl cyclic AMP and theophylline led to as much as a sevenfold increase in the level of APF activity appearing in the culture medium, with a concomitant increase in the adhesiveness of the cells to the culture substratum. The results support the role of extracellular macromolecules in cell to substratum adhesion. It is postulated that the reduced adhesiveness of transformed cells to a substratum may be at least partially owing to a deficiency in the production and/or release of APF-like macromolecules.

The Role of Mitochondria in the Genesis of Neuroaxonal Dystrophy in the Brains of Aging Mice

1975 ◽  
Vol 33 ◽  
pp. 372-373
Author(s):  
J.E. Johnson
Keyword(s):  
Electron Microscopy ◽  
Present Report ◽  
Light And Electron Microscopy ◽  
Dorsal Column ◽  
Neuroaxonal Dystrophy ◽  
Nucleus Gracilis ◽  
Dystrophic Axons ◽  
The Brain ◽  
Nucleus Cuneatus

Although neuroaxonal dystrophy (NAD) has been examined by light and electron microscopy for years, the nature of the components in the dystrophic axons is not well understood. The present report examines nucleus gracilis and cuneatus (the dorsal column nuclei) in the brain stem of aging mice.Mice (C57BL/6J) were sacrificed by aldehyde perfusion at ages ranging from 3 months to 23 months. Several brain areas and parts of other organs were processed for electron microscopy.At 3 months of age, very little evidence of NAD can be discerned by light microscopy. At the EM level, a few axons are found to contain dystrophic material. By 23 months of age, the entire nucleus gracilis is filled with dystrophic axons. Much less NAD is seen in nucleus cuneatus by comparison. The most recurrent pattern of NAD is an enlarged profile, in the center of which is a mass of reticulated material (reticulated portion; or RP).

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