scholarly journals Rab5 regulates macropinosome closure through recruitment of the inositol 5-phosphatases OCRL/Inpp5b and the hydrolysis of PtdIns(4,5)P2

2020 ◽  
Author(s):  
Michelle E. Maxson ◽  
Helen Sarantis ◽  
Allen Volchuk ◽  
John H. Brumell ◽  
Sergio Grinstein
Keyword(s):  
Plasma Membrane ◽  
Mode Of Action ◽  
Dominant Negative ◽  
Circular Membrane ◽  
Fusion Event ◽  
Phosphatidylinositol 4 ◽  
Hydrolysis Of

AbstractRab5 is required for macropinosome formation, but its site and mode of action remain unknown. We report that Rab5 acts at the plasma membrane, downstream of ruffling, to promote macropinosome sealing and scission. Dominant-negative Rab5, which obliterates macropinocytosis, had no effect on the development of membrane ruffles. However, Rab5-containing vesicles were recruited to circular membrane ruffles, and SNARE-dependent endomembrane fusion was necessary for completion of macropinocytosis. This fusion event coincided with the disappearance of PtdIns(4,5)P2 that accompanies macropinosome closure. Counteracting the depletion of PtdIns(4,5)P2 by expression of phosphatidylinositol-4-phosphate 5-kinase impaired macropinosome formation. Importantly, we found that removal of PtdIns(4,5)P2 is dependent on Rab5, through the Rab5-mediated recruitment of the inositol 5-phosphatases OCRL and Inpp5b, via APPL1. Knockdown of OCRL and Inpp5b, or APPL1 prevented macropinosome closure, without affecting ruffling. We therefore propose that Rab5 is essential for the clearance of PtdIns(4,5)P2 needed to complete macropinosome scission from the plasmalemma.

scholarly journals Rab5 regulates macropinocytosis by recruiting the inositol 5-phosphatases OCRL and Inpp5b that hydrolyse PtdIns(4,5)P2

2021 ◽  
Vol 134 (7) ◽  
Author(s):  
Michelle E. Maxson ◽  
Helen Sarantis ◽  
Allen Volchuk ◽  
John H. Brumell ◽  
Sergio Grinstein
Keyword(s):  
Plasma Membrane ◽  
Mode Of Action ◽  
Dominant Negative ◽  
Circular Membrane ◽  
Fusion Event ◽  
Attachment Protein ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Phosphatidylinositol 4 ◽  
Factor Attachment Protein Receptor

ABSTRACT Rab5 is required for macropinosome formation, but its site and mode of action remain unknown. We report that Rab5 acts at the plasma membrane, downstream of ruffling, to promote macropinosome sealing and scission. Dominant-negative Rab5, which obliterates macropinocytosis, had no effect on the development of membrane ruffles. However, Rab5-containing vesicles were recruited to circular membrane ruffles, and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent endomembrane fusion was necessary for the completion of macropinocytosis. This fusion event coincided with the disappearance of PtdIns(4,5)P2 that accompanies macropinosome closure. Counteracting the depletion of PtdIns(4,5)P2 by expression of phosphatidylinositol-4-phosphate 5-kinase impaired macropinosome formation. Importantly, we found that the removal of PtdIns(4,5)P2 is dependent on Rab5, through the Rab5-mediated recruitment of the inositol 5-phosphatases OCRL and Inpp5b, via APPL1. Knockdown of OCRL and Inpp5b, or APPL1, prevented macropinosome closure without affecting ruffling. We therefore propose that Rab5 is essential for the clearance of PtdIns(4,5)P2 needed to complete the scission of macropinosomes or to prevent their back-fusion with the plasmalemma.

scholarly journals PI(4,5)P2 controls slit diaphragm formation and endocytosis in Drosophila nephrocytes

2021 ◽  
Author(s):  
Max Gass ◽  
Sarah Borkowsky ◽  
Marie-Luise Lotz ◽  
Rita Schroeter ◽  
Pavel Nedvetsky ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Model System ◽  
Dominant Negative ◽  
Slit Diaphragm ◽  
Phosphatidylinositol 3 Kinase ◽  
Ectopic Activation ◽  
Phosphatidylinositol 4 ◽  
Asymmetrically Distributed ◽  
Phosphatidylinositol 3

Drosophila nephrocytes are an emerging model system for mammalian podocytes and podocyte-associated diseases. Like podocytes, nephrocytes exhibit characteristics of epithelial cells, but the role of phospholipids in polarization of these cells is yet unclear. In epithelia phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) and phosphatidylinositol(3,4,5)-trisphosphate (PI(3,4,5)P3) are asymmetrically distributed in the plasma membrane and determine apical-basal polarity. Here we demonstrate that both phospholipids are present in the plasma membrane of nephrocytes, but only PI(4,5)P2 accumulates at slit diaphragms. Knockdown of Skittles, a phosphatidylinositol(4)phosphate 5-kinase, which produces PI(4,5)P2, abolished slit diaphragm formation and led to strongly reduced endocytosis. Notably, reduction in PI(3,4,5)P3 by overexpression of PTEN or expression of a dominant-negative phosphatidylinositol-3-Kinase did not affect nephrocyte function, whereas enhanced formation of PI(3,4,5)P3 by constitutively active phosphatidylinositol-3-Kinase resulted in strong slit diaphragm and endocytosis defects by ectopic activation of the Akt/mTOR pathway. Thus, PI(4,5)P2 but not PI(3,4,5)P3 is essential for slit diaphragm formation and nephrocyte function. However, PI(3,4,5)P3 has to be tightly controlled to ensure nephrocyte development.

scholarly journals PI(4,5)P2 Controls Slit Diaphragm Formation and Endocytosis in Drosophila Nephrocytes

2021 ◽  
Author(s):  
Maximilian Gass ◽  
Sarah Borkowsky ◽  
Marie-Luise Lotz ◽  
Rita Schröter ◽  
Pavel Nedvetsky ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Model System ◽  
Dominant Negative ◽  
Slit Diaphragm ◽  
Phosphatidylinositol 3 Kinase ◽  
Ectopic Activation ◽  
Phosphatidylinositol 4 ◽  
Asymmetrically Distributed ◽  
Phosphatidylinositol 3

Abstract Drosophila nephrocytes are an emerging model system for mammalian podocytes and podocyte-associated diseases. Like podocytes, nephrocytes exhibit characteristics of epithelial cells, but the role of phospholipids in polarization of these cells is yet unclear. In epithelia phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) and phosphatidylinositol(3,4,5)-trisphosphate (PI(3,4,5)P3) are asymmetrically distributed in the plasma membrane and determine apical-basal polarity. Here we demonstrate that both phospholipids are present in the plasma membrane of nephrocytes, but only PI(4,5)P2 accumulates at slit diaphragms. Knockdown of Skittles, a phosphatidylinositol(4)phosphate 5-kinase, which produces PI(4,5)P2, abolished slit diaphragm formation and led to strongly reduced endocytosis. Notably, reduction in PI(3,4,5)P3 by overexpression of PTEN or expression of a dominant-negative phosphatidylinositol-3-Kinase did not affect nephrocyte function, whereas enhanced formation of PI(3,4,5)P3 by constitutively active phosphatidylinositol-3-Kinase resulted in strong slit diaphragm and endocytosis defects by ectopic activation of the Akt/mTOR pathway. Thus, PI(4,5)P2 but not PI(3,4,5)P3 is essential for slit diaphragm formation and nephrocyte function. However, PI(3,4,5)P3 has to be tightly controlled to ensure nephrocyte development.

scholarly journals Enforced expression of phosphatidylinositol 4-phosphate 5-kinase homolog alters PtdIns(4,5)P2 distribution and the localization of small G-proteins

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Yanbo Yang ◽  
Miriam Park ◽  
Masashi Maekawa ◽  
Gregory D. Fairn
Keyword(s):  
Plasma Membrane ◽  
Surface Charge ◽  
Electrostatic Interactions ◽  
Negative Charge ◽  
Anionic Charge ◽  
Negative Surface ◽  
Negative Surface Charge ◽  
Polybasic Domains ◽  
Phosphatidylinositol 4 ◽  
Hydrolysis Of

Abstract The generation of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) by phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) is essential for many functions including control of the cytoskeleton, signal transduction, and endocytosis. Due to its presence in the plasma membrane and anionic charge, PtdIns(4,5)P2, together with phosphatidylserine, provide the inner leaflet of the plasma membrane with a negative surface charge. This negative charge helps to define the identity of the plasma membrane, as it serves to recruit or regulate a multitude of peripheral and membrane proteins that contain polybasic domains or patches. Here, we determine that the phosphatidylinositol 4-phosphate 5-kinase homolog (PIPKH) alters the subcellular distribution of PtdIns(4,5)P2 by re-localizing the three PIP5Ks to endomembranes. We find a redistribution of the PIP5K family members to endomembrane structures upon PIPKH overexpression that is accompanied by accumulation of PtdIns(4,5)P2 and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3). PIP5Ks are targeted to membranes in part due to electrostatic interactions; however, the interaction between PIPKH and PIP5K is maintained following hydrolysis of PtdIns(4,5)P2. Expression of PIPKH did not impair bulk endocytosis as monitored by FM4-64 uptake but did result in clustering of FM4-64 positive endosomes. Finally, we demonstrate that accumulation of polyphosphoinositides increases the negative surface charge of endosomes and in turn, leads to relocalization of surface charge probes as well as the polycationic proteins K-Ras and Rac1.

scholarly journals Phosphatidylinositol and phosphatidic acid transport between the ER and plasma membrane during PLC activation requires the Nir2 protein

2016 ◽  
Vol 44 (1) ◽  
pp. 197-201 ◽  
Author(s):  
Yeun Ju Kim ◽  
Maria Luisa Guzman-Hernandez ◽  
Eva Wisniewski ◽  
Nicolas Echeverria ◽  
Tamas Balla
Keyword(s):  
Plasma Membrane ◽  
Phosphatidic Acid ◽  
Lipid Transfer ◽  
Acid Transport ◽  
Contact Zones ◽  
Quiescent Cells ◽  
Associated Proteins ◽  
Phosphatidylinositol 4 ◽  
Hydrolysis Of ◽  
Lateral Sclerosis

Phospholipase C (PLC)-mediated hydrolysis of the limited pool of plasma membrane (PM) phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] requires replenishment from a larger pool of phosphatidylinositol (PtdIns) via sequential phosphorylation by PtdIns 4-kinases and phosphatidylinositol 4-phosphate (PtdIns4P) 5-kinases. Since PtdIns is synthesized in the endoplasmic reticulum (ER) and PtdIns(4,5)P2 is generated in the PM, it has been postulated that PtdIns transfer proteins (PITPs) provide the means for this lipid transfer function. Recent studies identified the large PITP protein, Nir2 as important for PtdIns transfer from the ER to the PM. It was also found that Nir2 was required for the transfer of phosphatidic acid (PtdOH) from the PM to the ER. In Nir2-depleted cells, activation of PLC leads to PtdOH accumulation in the PM and PtdIns synthesis becomes severely impaired. In quiescent cells, Nir2 is localized to the ER via interaction of its FFAT domain with ER-bound VAMP-associated proteins VAP-A and–B. After PLC activation, Nir2 also binds to the PM via interaction of its C-terminal domains with diacylglycerol (DAG) and PtdOH. Through these interactions, Nir2 functions in ER–PM contact zones. Mutations in VAP-B that have been identified in familial forms of amyotrophic lateral sclerosis (ALS or Lou-Gehrig's disease) cause aggregation of the VAP-B protein, which then impairs its binding to several proteins, including Nir2. These findings have shed new lights on the importance of non-vesicular lipid transfer of PtdIns and PtdOH in ER–PM contact zones with a possible link to a devastating human disease.

scholarly journals Study of the mode of action of a polygalacturonase from the phytopathogen Burkholderia cepacia

2007 ◽  
Vol 407 (2) ◽  
pp. 207-217 ◽  
Author(s):  
Claudia Massa ◽  
Mads H. Clausen ◽  
Jure Stojan ◽  
Doriano Lamba ◽  
Cristiana Campa
Keyword(s):  
Mode Of Action ◽  
Burkholderia Cepacia ◽  
Time Course ◽  
Glycoside Hydrolase Family ◽  
Enzymatic Mechanism ◽  
Processive Enzyme ◽  
Chain Lengths ◽  
Methylation Patterns ◽  
Hydrolysis Of ◽  
Hydrolase Family

We have recently isolated and heterologously expressed BcPeh28A, an endopolygalacturonase from the phytopathogenic Gram-negative bacterium Burkholderia cepacia. Endopolygalacturonases belong to glycoside hydrolase family 28 and are responsible for the hydrolysis of the non-esterified regions of pectins. The mode of action of BcPeh28A on different substrates has been investigated and its enzymatic mechanism elucidated. The hydrolysis of polygalacturonate indicates that BcPeh28A is a non-processive enzyme that releases oligomers with chain lengths ranging from two to eight. By inspection of product progression curves, a kinetic model has been generated and extensively tested. It has been used to derive the kinetic parameters that describe the time course of the formation of six predominant products. Moreover, an investigation of the enzymatic activity on shorter substrates that differ in their overall length and methylation patterns sheds light on the architecture of the BcPeh28A active site. Specifically the tolerance of individual sites towards methylated saccharide units was rationalized on the basis of the hydrolysis of hexagalacturonides with different methylation patterns.

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.

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