Gelsolin — evidence for a role in turnover of junction-related actin filaments in Sertoli cells

2002 ◽  
Vol 115 (3) ◽  
pp. 499-505 ◽  
Author(s):  
Julian A. Guttman ◽  
Paul Janmey ◽  
A. Wayne Vogl
Keyword(s):  
Endoplasmic Reticulum ◽  
Phospholipase C ◽  
Sertoli Cells ◽  
Actin Filaments ◽  
Synthetic Peptide ◽  
Inositol Triphosphate ◽  
Binding Region ◽  
Phosphoinositide Pathway ◽  
Hydrolysis Of ◽  
Adhesion Complex

The gelsolin-phosphoinositide pathway may be part of the normal mechanism by which Sertoli cells regulate sperm release and turnover of the blood-testis barrier. The intercellular adhesion complexes (ectoplasmic specializations)involved with these two processes are tripartite structures consisting of the plasma membrane, a layer of actin filaments and a cistern of endoplasmic reticulum. Gelsolin is concentrated in these adhesion complexes. In addition,phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and phosphoinositide-specific phospholipase C are found in the structures. Treatment of isolated spermatid/junction complexes with exogenous phosphoinositide-specific phospholipase C, or with a synthetic peptide consisting of the PtdIns(4,5)P2 binding region of gelsolin, results in the release of gelsolin and loss of actin from the adhesion complexes. We present a model for the disassembly of the actin layer of the adhesion complex that involves the hydrolysis of PtdIns(4,5)P2 resulting in the release of gelsolin within the plaque. Further, we speculate that the hydrolysis of PtdIns(4,5)P2 may result in a local Ca2+ surge via the action of inositol triphosphate on junctional endoplasmic reticulum. This Ca2+ surge would facilitate the actin severing function of gelsolin within the adhesion complex.

scholarly journals Phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis as a probe for the distribution of phosphatidylinositol in hepatocyte membranes

1989 ◽  
Vol 259 (3) ◽  
pp. 913-916 ◽  
Author(s):  
J A Higgins ◽  
B W Hitchin ◽  
M G Low
Keyword(s):  
Endoplasmic Reticulum ◽  
Bacillus Thuringiensis ◽  
Phospholipase C ◽  
Sodium Carbonate ◽  
Plasma Membranes ◽  
Secretory Proteins ◽  
Membrane Phospholipids ◽  
Golgi Membranes ◽  
Liver Membranes ◽  
Hydrolysis Of

Phosphatidylinositol-specific phospholipase C (PI-PLC) produced by Bacillus thuringiensis has been used as a probe for the distribution of phosphatidylinositol in hepatocyte membranes. Approx. 50% of this phospholipid was hydrolysed in microsomal vesicles (endoplasmic reticulum) with no significant hydrolysis of the remaining membrane phospholipids. Latency of mannose-6-phosphatase was retained during treatment indicating that the vesicles remained impermeable. Stripping of the ribosomes did not increase hydrolysis of phosphatidylinositol; however, when the vesicles were opened using dilute sodium carbonate, hydrolysis increased to greater than 90%. Hydrolysis of phosphatidylinositol of Golgi membranes was 35% and of plasma membranes was 50%. After treatment with PI-PLC, radiolabelled secretory proteins were retained in Golgi membranes and trapped lactate dehydrogenase was retained in plasma-membrane preparations indicating that the vesicles remained closed. Hydrolysis of phosphatidylinositol increased to greater than 90% when the membranes were opened by treatment with dilute sodium carbonate. These observations indicate that PI-PLC of Bacillus thuringiensis is a suitable probe for the distribution of phosphatidylinositol in membranes, and that in liver membranes this phospholipid occurs on each side of the bilayer, a topography consistent with its diverse roles.

scholarly journals Follicle-Stimulating Hormone-Induced Gαh/Phospholipase C-δ1 Signaling Mediating a Noncapacitative Ca2+ Influx through T-Type Ca2+ Channels in Rat Sertoli Cells

Endocrinology ◽  
2007 ◽  
Vol 149 (3) ◽  
pp. 1031-1037 ◽  
Author(s):  
Tsung-Hsuan Lai ◽  
Yuan-Feng Lin ◽  
Feng-Chang Wu ◽  
Yu-Hui Tsai
Keyword(s):  
Phospholipase C ◽  
Sertoli Cells ◽  
Synthetic Peptide ◽  
Calcium Release ◽  
Channel Blocker ◽  
Follicle Stimulating Hormone ◽  
Fold Increase ◽  
Selective Inhibitor ◽  
Free Media ◽  
Ca2 Channels

Our previous study demonstrated that FSH-induced immediate Ca2+ influx in rat Sertoli cells (SCs) is mediated by the Gαh/phospholipase C-δ1 (PLC-δ1) signaling pathway. As to which Ca2+ channel is responsible for such Ca2+ influx was not understood. In this study, thapsigargin triggered an in-store calcium release and evoked a 1.5-fold elevation of intracellular Ca2+ in Ca2+-free media, whereas FSH exhibited no effect. The readdition of CaCl2 (2.5 mm) to FSH-pretreated or thapsigargin-sensitized SCs in Ca2+-free media immediately elicited a rapid Ca2+ influx or a 2-fold increase of second intracellular Ca2+ elevation, respectively. The addition of Ca2+ chelator EGTA (0.2 mm) reduced the FSH-induced elevation of intracellular Ca2+ in SCs incubated with CaCl2. However, pretreatment with dantrolene (25 μM), which inhibits in-store calcium release, did not affect the FSH-induced elevation of intracellular Ca2+. NiCl2 (10 μM), a T-type calcium channel blocker, abolished the FSH-induced SC Ca2+ influx. Furthermore, mibefradil (10 and 100 μm), another specific blocker for T-type Ca2+ channels, dose-dependently suppressed the FSH-induced Ca2+ influx. In contrast, nifedipine (10 and 50 μm) or ω-conotoxin GVIA (100 and 500 nm), blocker of L- or N-type Ca2+ channels, respectively, did not affect the FSH-induced SC Ca2+ influx. On the other hand, FSH-induced Ca2+ influx was significantly reduced by pretreatment of SCs with myristoylated synthetic peptide (0.1 and 1 μm) of PLC-δ1 fragment TIPWNSLKQGYRHVHLL but not affected by 2′,5′-dideoxyadenosine (3 and 15 μm), a selective inhibitor of adenylate cyclase. In conclusion, the FSH-induced Gαh/PLC-δ1 pathway-dependent Ca2+ influx of rat SCs is mediated by T-type Ca2+ channels and independent of in-store calcium release.

Biosynthesis of Mitochondrial Phospholipids Using Endogenously Generated Diglycerides

1975 ◽  
Vol 53 (7) ◽  
pp. 784-795 ◽  
Author(s):  
W. C. McMurray
Keyword(s):  
Endoplasmic Reticulum ◽  
Phosphatidic Acid ◽  
Phospholipase C ◽  
Rat Liver ◽  
De Novo ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Isolated Mitochondria ◽  
Hydrolysis Of ◽  
Stimulation Of

When isolated mitochondria or microsomes from rat liver were treated with phospholipase C, the incorporation of radioactive phospholipid precursors was markedly enhanced, presumably as a result of production of diglycerides by hydrolysis of endogenous phospholipids. Incorporation of CDP[14C]choline into lecithin in rat liver or BHK-21 mitochondria could be attributed to residual contamination from elements of the endoplasmic reticulum, with added diglycerides or with endogenous diglycerides produced by the phospholipase C treatment. A similar stimulation of [γ32P]ATP incorporation into phospholipids was observed with exogenous or endogenous diglycerides, but the mitochondrial diglyceride kinase in either case was also related to the degree of microsomal contaminants. It was concluded that previous studies showing negligible capacity of mitochondria for lecithin biosynthesis de novo were not explainable on the basis of limited accessibility of added diglycerides, and that formation of phosphatidic acid by diglyceride kinase was not of significance in rat liver mitochondria.

Phototransduction in Drosophila melanogaster

2001 ◽  
Vol 204 (20) ◽  
pp. 3403-3409 ◽  
Author(s):  
Roger C. Hardie
Keyword(s):  
Drosophila Melanogaster ◽  
Phospholipase C ◽  
G Protein ◽  
Phosphatidyl Inositol ◽  
Single Photons ◽  
Inositol 1,4,5 Trisphosphate ◽  
Positive And Negative Feedback ◽  
Phosphoinositide Pathway ◽  
G Protein Coupled ◽  
Hydrolysis Of

SUMMARY As in most invertebrate microvillar photoreceptors, phototransduction in Drosophila melanogaster uses a G-protein-coupled phosphoinositide pathway, whereby hydrolysis of phosphatidyl inositol 4,5-bisphosphate (PIP2) by phospholipase C generates inositol 1,4,5-trisphosphate (InsP3) and diacyl glycerol (DAG), leading to activation of two classes of Ca2+-permeable light-sensitive channel, encoded by the trp and trpl genes. In some invertebrate photoreceptors, excitation is mediated by release of Ca2+ from intracellular stores by InsP3; however, in Drosophila melanogaster, recent evidence suggests instead that a lipid messenger, such as DAG, its metabolites and/or the reduction in PIP2 levels, may mediate excitation. Like vertebrate rods, Drosophila melanogaster photoreceptors generate quantum bumps in response to single photons, but their kinetics is approximately 10–100 times faster, and this reflects a fundamentally different strategy incorporating a threshold, positive and negative feedback by Ca2+ acting downstream of phospholipase C and a refractory period.

scholarly journals Resynthesis of phosphatidylinositol in permeabilized neutrophils following phospholipase Cβ activation: transport of the intermediate, phosphatidic acid, from the plasma membrane to the endoplasmic reticulum for phosphatidylinositol resynthesis is not dependent on soluble lipid carriers or vesicular transport

1999 ◽  
Vol 341 (2) ◽  
pp. 435-444 ◽  
Author(s):  
Jacqueline WHATMORE ◽  
Claudia WIEDEMANN ◽  
Pennti SOMERHARJU ◽  
Philip SWIGART ◽  
Shamshad COCKCROFT
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Vesicular Transport ◽  
Human Neutrophils ◽  
Permeabilized Cells ◽  
Transfer Protein ◽  
Membrane Contact ◽  
Phosphatidylinositol Transfer ◽  
Pi Hydrolysis ◽  
Hydrolysis Of

Receptor-mediated phospholipase C (PLC) hydrolysis of phosphoinositides is accompanied by the resynthesis of phosphatidylinositol (PI). Hydrolysis of phosphoinositides occurs at the plasma membrane, and the resulting diacylglycerol (DG) is converted into phosphatidate (PA). Two enzymes located at the endoplasmic reticulum (ER) function sequentially to convert PA back into PI. We have established an assay whereby the resynthesis of PI could be followed in permeabilized cells. In the presence of [γ-32P]ATP, DG generated by PLC activation accumulates label when converted into PA. The 32P-labelled PA is subsequently converted into labelled PI. The formation of labelled PI reports the arrival of labelled PA from the plasma membrane to the ER. Cytosol-depleted, permeabilized human neutrophils are capable of PI resynthesis following stimulation of PLCβ (in the presence of phosphatidylinositol-transfer protein), provided that CTP and inositol are also present. We also found that wortmannin, an inhibitor of endocytosis, or cooling the cells to 15 °C did not stop PI resynthesis. We conclude that PI resynthesis is dependent neither on vesicular transport mechanisms nor on freely diffusible, soluble transport proteins. Phosphatidylcholine-derived PA generated by the ADP-ribosylation-factor-stimulated phospholipase D pathway was found to accumulate label, reflecting the rapid cycling of PA to DG, and back. This labelled PA was not converted into PI. We conclude that PA derived from the PLC pathway is selected for PI resynthesis, and its transfer to the ER could be membrane-protein-mediated at sites of close membrane contact.

scholarly journals Effect of Phospholipase C Hydrolysis of Membrane Phospholipids on Membranous Enzymes

1972 ◽  
Vol 247 (9) ◽  
pp. 2835-2841 ◽  
Author(s):  
Richard D. Mavis ◽  
Robert M. Bell ◽  
P. Roy Vagelos
Keyword(s):  
Phospholipase C ◽  
Membrane Phospholipids ◽  
Hydrolysis Of

scholarly journals Myosin Motors and Not Actin Comets Are Mediators of the Actin-based Golgi-to-Endoplasmic Reticulum Protein Transport

2003 ◽  
Vol 14 (2) ◽  
pp. 445-459 ◽  
Author(s):  
Juan M. Durán ◽  
Ferran Valderrama ◽  
Susana Castel ◽  
Juana Magdalena ◽  
Mónica Tomás ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Light Chain ◽  
Shiga Toxin ◽  
Actin Filaments ◽  
Protein Transport ◽  
Myosin Ii ◽  
Nonmuscle Myosin ◽  
Nonmuscle Myosin Ii ◽  
Myosin Motors

We have previously reported that actin filaments are involved in protein transport from the Golgi complex to the endoplasmic reticulum. Herein, we examined whether myosin motors or actin comets mediate this transport. To address this issue we have used, on one hand, a combination of specific inhibitors such as 2,3-butanedione monoxime (BDM) and 1-[5-isoquinoline sulfonyl]-2-methyl piperazine (ML7), which inhibit myosin and the phosphorylation of myosin II by the myosin light chain kinase, respectively; and a mutant of the nonmuscle myosin II regulatory light chain, which cannot be phosphorylated (MRLC2AA). On the other hand, actin comet tails were induced by the overexpression of phosphatidylinositol phosphate 5-kinase. Cells treated with BDM/ML7 or those that express the MRLC2AA mutant revealed a significant reduction in the brefeldin A (BFA)-induced fusion of Golgi enzymes with the endoplasmic reticulum (ER). This delay was not caused by an alteration in the formation of the BFA-induced tubules from the Golgi complex. In addition, the Shiga toxin fragment B transport from the Golgi complex to the ER was also altered. This impairment in the retrograde protein transport was not due to depletion of intracellular calcium stores or to the activation of Rho kinase. Neither the reassembly of the Golgi complex after BFA removal nor VSV-G transport from ER to the Golgi was altered in cells treated with BDM/ML7 or expressing MRLC2AA. Finally, transport carriers containing Shiga toxin did not move into the cytosol at the tips of comet tails of polymerizing actin. Collectively, the results indicate that 1) myosin motors move to transport carriers from the Golgi complex to the ER along actin filaments; 2) nonmuscle myosin II mediates in this process; and 3) actin comets are not involved in retrograde transport.

scholarly journals Immunocytochemical localization of Mullerian inhibiting substance in the rough endoplasmic reticulum and Golgi apparatus in Sertoli cells of the neonatal calf testis using a monoclonal antibody.

1984 ◽  
Vol 32 (6) ◽  
pp. 649-654 ◽  
Author(s):  
M Hayashi ◽  
H Shima ◽  
K Hayashi ◽  
R L Trelstad ◽  
P K Donahoe
Keyword(s):  
Monoclonal Antibody ◽  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Rough Endoplasmic Reticulum ◽  
Sertoli Cells ◽  
Neonatal Calf ◽  
Müllerian Inhibiting Substance ◽  
Pap Method ◽  
Unlabeled Antibody ◽  
Mullerian Inhibiting Substance

Mullerian Inhibiting Substance (MIS) has been localized in the Sertoli cells of the neonatal calf testis using preembedding immunoperoxidase techniques and a monoclonal antibody which almost completely blocks the biological activity of MIS. Both the peroxidase-labeled antibody method using a peroxidase-conjugated F(ab')2 fragment of IgG as a second antibody and the unlabeled antibody peroxidase-antiperoxidase (PAP) method using Fab fragments of the PAP complex were employed. With both methods, MIS was demonstrated within the cisternae of the rough endoplasmic reticulum (RER) and the Golgi apparatus. In the Golgi, MIS was concentrated in the transmost cisternae especially at their peripheral expansions. This study indicates that MIS is synthesized in the RER and transported to the Golgi apparatus, presumably for glycosidation, before secretion from Golgi derived vacuoles.

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