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.

Opposing effects of PKCα and PKCε on basolateral membrane dynamics in intestinal epithelia

2002 ◽  
Vol 283 (5) ◽  
pp. C1548-C1556 ◽  
Author(s):  
Jaekyung Cecilia Song ◽  
Patangi K. Rangachari ◽  
Jeffrey B. Matthews
Keyword(s):  
Time Course ◽  
Basolateral Membrane ◽  
Membrane Dynamics ◽  
T84 Cells ◽  
Intestinal Epithelia ◽  
Early Activation ◽  
Pkc Isoforms ◽  
Opposing Effects ◽  
Stimulation Of

PKC is a critical effector of plasma membrane dynamics, yet the mechanism and isoform-specific role of PKC are poorly understood. We recently showed that the phorbol ester PMA (100 nM) induces prompt activation of the novel isoform PKCε followed by late activation of the conventional isoform PKCα in T84 intestinal epithelia. PMA also elicited biphasic effects on endocytosis, characterized by an initial stimulatory phase followed by an inhibitory phase. Activation of PKCε was shown to be responsible for stimulation of basolateral endocytosis, but the role of PKCα was not defined. Here, we used detailed time-course analysis as well as selective activators and inhibitors of PKC isoforms to infer the action of PKCα on basolateral endocytosis. Inhibition of PKCα by the selective conventional PKC inhibitor Gö-6976 (5 μM) completely blocked the late inhibitory phase and markedly prolonged the stimulatory phase of endocytosis measured by FITC-dextran uptake. The PKCε-selective agonist carbachol (100 μM) induced prolonged stimulation of endocytosis devoid of an inhibitory phase. Actin disassembly caused by PMA was completely blocked by Gö-6850 but not by Gö-6976, implicating PKCε as the key isoform responsible for actin disruption. The Ca2+ agonist thapsigargin (5 μM) induced early activation of PKCα when added simultaneously with PMA. This early activation of PKCα blocked the ability of PMA to remodel basolateral F-actin and abolished the stimulatory phase of basolateral endocytosis. Activation of PKCα stabilizes F-actin and thereby opposes the effect of PKCε on membrane remodeling in T84 cells.

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 Identification of TFII-I as the Endoplasmic Reticulum Stress Response Element Binding Factor ERSF: Its Autoregulation by Stress and Interaction with ATF6

2001 ◽  
Vol 21 (9) ◽  
pp. 3220-3233 ◽  
Author(s):  
Ronald Parker ◽  
Trevor Phan ◽  
Peter Baumeister ◽  
Binayak Roy ◽  
Venugopalan Cheriyath ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Stress Response ◽  
Mammalian Cells ◽  
Phosphorylation Site ◽  
Endoplasmic Reticulum Stress Response ◽  
Sequence Motif ◽  
Response Element ◽  
Potent Inducer ◽  
Stressed Cells ◽  
Stimulation Of

ABSTRACT When mammalian cells are subjected to stress targeted to the endoplasmic reticulum (ER), such as depletion of the ER Ca2+ store, the transcription of a family of glucose-regulated protein (GRP) genes encoding ER chaperones is induced. The GRP promoters contain multiple copies of the ER stress response element (ERSE), consisting of a unique tripartite structure, CCAAT(N9)CCACG. Within a subset of mammalian ERSEs, N9 represents a GC-rich sequence of 9 bp that is conserved across species. A novel complex (termed ERSF) exhibits enhanced binding to the ERSE of the grp78 and ERp72 promoters using HeLa nuclear extracts prepared from ER-stressed cells. Optimal binding of ERSF to ERSE and maximal ERSE-mediated stress inducibility require the conserved GGC motif within the 9-bp region. Through chromatographic purification and subsequent microsequencing, we have identified ERSF as TFII-I. Whereas TFII-I remains predominantly nuclear in both nontreated NIH 3T3 cells and cells treated with thapsigargin (Tg), a potent inducer of the GRP stress response through depletion of the ER Ca2+ store, the level of TFII-I transcript was elevated in Tg-stressed cells, correlating with an increase in TFII-I protein level in the nuclei of Tg-stressed cells. Purified recombinant TFII-I isoforms bind directly to the ERSEs of grp78 and ERp72 promoters. The stimulation of ERSE-mediated transcription by TFII-I requires the consensus tyrosine phosphorylation site of TFII-I and the GGC sequence motif of the ERSE. We further discovered that TFII-I is an interactive protein partner of ATF6 and that optimal stimulation of ERSE by ATF6 requires TFII-I.

scholarly journals Association of Myosin I Alpha with Endosomes and Lysosomes in Mammalian Cells

1999 ◽  
Vol 10 (5) ◽  
pp. 1477-1494 ◽  
Author(s):  
Graça Raposo ◽  
Marie-Neige Cordonnier ◽  
Danièle Tenza ◽  
Bernadette Menichi ◽  
Antoine Dürrbach ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Brush Border ◽  
Mammalian Cells ◽  
Fluid Phase ◽  
Ultrastructural Level ◽  
Myosin I ◽  
Key Players ◽  
Intracellular Vesicles ◽  
Endocytic Compartments

Myosin Is, which constitute a ubiquitous monomeric subclass of myosins with actin-based motor properties, are associated with plasma membrane and intracellular vesicles. Myosin Is have been proposed as key players for membrane trafficking in endocytosis or exocytosis. In the present paper we provide biochemical and immunoelectron microscopic evidence indicating that a pool of myosin I alpha (MMIα) is associated with endosomes and lysosomes. We show that the overproduction of MMIα or the production of nonfunctional truncated MMIα affects the distribution of the endocytic compartments. We also show that truncated brush border myosin I proteins, myosin Is that share 78% homology with MMIα, promote the dissociation of MMIα from vesicular membranes derived from endocytic compartments. The analysis at the ultrastructural level of cells producing these brush border myosin I truncated proteins shows that the delivery of the fluid phase markers from endosomes to lysosomes is impaired. MMIα might therefore be involved in membrane trafficking occurring between endosomes and lysosomes.

hDectin-1 Is Involved in the Uptake of Apoptotic and Infected Cells and Mediates Cross-Presentation of Engulfed Antigens.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3086-3086
Author(s):  
Markus M. Weck ◽  
Frank Grünebach ◽  
Daniela Werth ◽  
Lothar Kanz ◽  
Christian Sinzger ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Mammalian Cells ◽  
Specific Binding ◽  
Human Tumor ◽  
Extracellular Domain ◽  
Poly I:C ◽  
Cross Presentation ◽  
Infected Cells ◽  
Stimulation Of

Abstract Dectin-1 is a member of the c-type-lectin-like receptor family that was shown to be the major receptor for fungal beta-glucans and to play an important role in the cellular responses mediated by these carbohydrates. In the present study we identified the hDectin-1b splice variant as the major isoform expressed in in vitro generated mDC using a quantitative RT-PCR and western blot analysis. Interestingly, stimulation of immature DC with the toll like receptor ligands Poly I:C (TLR3) or LPS (TLR4) but not with TLR ligands 2 and 7 or TNF-a led to a dramatic down regulation of hDectin-1 protein expression. To further analyze the possible involvement of Dectin-1 in the phagocytosis of cellular material we recombinantly expressed the extracellular domain (ECD) of hDectin-1b and used it to stain tumor cells. The recombinant ECD showed a specific binding to several human tumor cell lines that could be increased by induction of apoptosis in malignant cells and inhibited by incubation of the extracellular domain with zymosan, a crude cell wall extract of saccharomyces cerevisiae. Furthermore, uptake of tumor cells by immature mDC was reduced by zymosan or the presence of the recombinant Dectin-1 in phagocytosis assays suggesting that Dectin-1 is involved in the engulfement of tumor cells by DC. In line with the expression analysis of hDectin-1, we found that phagocytosis of apoptotic cells was dramatically reduced upon stimulation of DCs with Poly I:C or LPS as compared to immature DC or DC activated with TLR2 or 7 ligands. We next analysed the role of Dectin-1 in the cross-presentation of cell derived antigens and used DC that were incubated with CMV infected fibroblasts and an autologous CTL line specific for the HLA-A2 binding pp65 peptide. Stimulation of CMV peptide specific CTL in ELIspot assays by DC that were incubated with CMV infected fibroblasts efficiently stimulated IFN-gamma secretion that could be inhibited by zymosan and the extracellular domain of Dectin-1. In line with the results from phagocytosis assays stimulation of CMV specific CTL was reduced by stimulation of DC with TLR2 and 4 ligands. Our results identify hDectin-1 as a new receptor for endogenous ligands on mammalian cells and indicate an important role of this molecule in the clearing of apoptotic and infected cells and cross-presentation of cell derived antigens..

Establishment and analysis of conditional Rab1 and Rab5 knockout cells by using the auxin-inducible degron system

2021 ◽  
Author(s):  
Yuki Hatoyama ◽  
Yuta Homma ◽  
Shu Hiragi ◽  
Mitsunori Fukuda
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Fluid Phase ◽  
Small Gtpases ◽  
Golgi Stack ◽  
Protein Depletion ◽  
Intracellular Signals ◽  
Early Endosomes ◽  
Inducible Protein ◽  
The Impact

Two small GTPases, Rab1 and Rab5, are key membrane trafficking regulators that are conserved in all eukaryotes. Since they have recently been shown to be essential for cell survival and/or growth in cultured mammalian cells, thereby precluding the establishment of Rab1-knockout (KO) and Rab5-KO cells, it has been extremely difficult to assess the impact of complete Rab1 or Rab5 protein depletion on cellular functions. Here, we generated and analyzed conditional KO (CKO) cells for Rab1 (Rab1A/1B) and Rab5 (Rab5A/5B/5C) by using the auxin-inducible protein degradation system. Rab1-CKO and Rab5-CKO led to eventual cell death &gt;18 h and &gt;48 h, respectively, after auxin exposure. After acute Rab1 protein depletion, the Golgi stack and ribbon structures were completely disrupted, and ER-to-Golgi trafficking was severely inhibited. Moreover, we discovered a novel Rab1-depletion phenotype: perinuclear clustering of early endosomes and delayed transferrin recycling. In contrast, acute Rab5 protein depletion resulted in loss of early endosomes and late endosomes, but lysosomes appeared to be normal. We also observed a dramatic reduction in the intracellular signals of endocytic cargos via receptor-mediated or fluid-phase endocytosis in Rab5-depleted cells.

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.

Hypoxia induces basolateral membrane remodeling in T84 intestinal epithelia: Role of PKCe and PLCГ

2000 ◽  
Vol 118 (4) ◽  
pp. A613
Author(s):  
Edward C. Mun ◽  
Jaekyung C. Song ◽  
Celina M. Hanson ◽  
Jeffrey B. Matthews
Keyword(s):  
Basolateral Membrane ◽  
Membrane Remodeling ◽  
Intestinal Epithelia

scholarly journals Intracellular Ca2+ oscillations generated via the Ca2+-sensing receptor are mediated by negative feedback by PKCα at Thr888

2014 ◽  
Vol 306 (3) ◽  
pp. C298-C306 ◽  
Author(s):  
Steven H. Young ◽  
Osvaldo Rey ◽  
James Sinnett-Smith ◽  
Enrique Rozengurt
Keyword(s):  
Negative Feedback ◽  
Small Interfering Rna ◽  
Phosphorylation Site ◽  
Hek 293 ◽  
Pkc Inhibitor ◽  
Identical Response ◽  
293 Cells ◽  
Intracellular Ca ◽  
Interfering Rna

To clarify the mechanism(s) underlying intracellular Ca2+ concentration ([Ca2+]i) oscillations induced by an elevation in extracellular Ca2+ concentration ([Ca2+]e) via the extracellular Ca2+-sensing receptor (CaR), we analyzed the pattern of [Ca2+]i response in multiple (2,303) individual HEK-293 cells transfected with the human CaR. An increase in the [Ca2+]e from 1.5 to 3 mM produced oscillatory fluctuations in [Ca2+]i in 70% of the cell population. To determine the role of PKC in the generation of [Ca2+]i oscillations, cells were exposed to increasing concentrations (0.5–5 μM) of the preferential PKC inhibitor Ro-31-8220 before stimulation by extracellular Ca2+. Ro-31-8220 at 3–5 μM completely eliminated the [Ca2+]e-evoked [Ca2+]i oscillations and transformed the pattern to a peak and sustained plateau response. Treatment with other broad PKC inhibitors, including GFI or Gö6983, produced an identical response. Similarly, treatment with Ro-31-8220 or GFI eliminated [Ca2+]e-evoked [Ca2+]i oscillations in colon-derived SW-480 cells expressing the CaR. Treatment with inhibitors targeting classic PKCs, including Gö6976 and Ro-32-0432 as well as small interfering RNA-mediated knockdown of PKCα, strikingly reduced the proportion of cell displaying [Ca2+]e-evoked [Ca2+]i oscillations. Furthermore, none of the cells analyzed expressing a CaR mutant in which the major PKC phosphorylation site Thr888 was converted to alanine (CaRT888A) showed [Ca2+]i oscillations after CaR activation. Our results show that [Ca2+]i oscillations induced by activation of the CaR in response to an increase in extracellular Ca2+ or exposure to the calcimimetic R-568 result from negative feedback involving PKCα-mediated phosphorylation of the CaR at Thr888.

scholarly journals Galectin-4 and sulfatides in apical membrane trafficking in enterocyte-like cells

2005 ◽  
Vol 169 (3) ◽  
pp. 491-501 ◽  
Author(s):  
Delphine Delacour ◽  
Valérie Gouyer ◽  
Jean-Pierre Zanetta ◽  
Hervé Drobecq ◽  
Emmanuelle Leteurtre ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Functional Role ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Affinity Ligands ◽  
Membrane Markers ◽  
Polarized Trafficking ◽  
Hydroxylated Fatty Acids ◽  
Ht 29

We have previously reported that 1-benzyl-2-acetamido-2-deoxy-α-d-galactopyranoside (GalNAcα-O-bn), an inhibitor of glycosylation, perturbed apical biosynthetic trafficking in polarized HT-29 cells suggesting an involvement of a lectin-based mechanism. Here, we have identified galectin-4 as one of the major components of detergent-resistant membranes (DRMs) isolated from HT-29 5M12 cells. Galectin-4 was also found in post-Golgi carrier vesicles. The functional role of galectin-4 in polarized trafficking in HT-29 5M12 cells was studied by using a retrovirus-mediated RNA interference. In galectin-4–depleted HT-29 5M12 cells apical membrane markers accumulated intracellularly. In contrast, basolateral membrane markers were not affected. Moreover, galectin-4 depletion altered the DRM association characteristics of apical proteins. Sulfatides with long chain-hydroxylated fatty acids, which were also enriched in DRMs, were identified as high-affinity ligands for galectin-4. Together, our data propose that interaction between galectin-4 and sulfatides plays a functional role in the clustering of lipid rafts for apical delivery.

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