scholarly journals Localized PtdIns 3,5-P2 synthesis to regulate early endosome dynamics and fusion

2006 ◽  
Vol 291 (2) ◽  
pp. C393-C404 ◽  
Author(s):  
Ognian C. Ikonomov ◽  
Diego Sbrissa ◽  
Assia Shisheva
Keyword(s):  
Morphological Changes ◽  
Degradation Pathway ◽  
Dominant Negative ◽  
Functional Requirements ◽  
Cos Cells ◽  
Cytoplasmic Vacuoles ◽  
Early Endosomes ◽  
Gradual Development ◽  
Early Expression

Perturbations in the intracellular PtdIns 3,5-P2 pool or the downstream transmission of PtdIns 3,5-P2 signals often result in a gradual development of gross morphological changes in the pleiomorphic multivesicular endosomes, culminating with the appearance of cytoplasmic vacuoles. To identify the onset of PtdIns 3,5-P2 functional requirements along the endocytic system, in this study we characterized the morphological changes associated with early expression of the dominant-negative kinase-deficient form (K1831E) of the PtdIns 3,5-P2-producing kinase PIKfyve, before the formation of cytoplasmic vacuoles in transfected COS cells. Enlarged PIKfyveK1831E-positive vesicles co-localizing with dilated EEA1- and Rab5aWT-positive perinuclear endosomes were observed (WT, wild type). This was dependent on the presence of active forms of Rab5 and the generation of PtdIns 3-P-enriched platforms on early endosomess. Because PIKfyveWT did not substantially colocalize with EEA1- or Rab5-positive endosomes in COS cells, the dynamic PIKfyve-catalyzed PtdIns 3-to-PtdIns 3,5-P2 switch was suggested to drive away PIKfyveWT from early endosomes toward later compartments. Late endosomes/lysosomes marked by LAMP1 or Rab7 were dislocated from their typical perinuclear position upon PIKfyveK1831E early expression. Cytosols derived from cells stably expressing PIKfyveK1831E stimulated endosome fusion in vitro, whereas PIKfyveWT-enriched cytosols had the opposite effect, consistent with PtdIns 3,5-P2 production negatively regulating the endosome fusion. Together, our data indicate that PtdIns 3,5-P2 defines specific endosome platforms at the onset of the degradation pathway to regulate the complex process of membrane remodeling and dynamics.

scholarly journals Adenovirus RIDα regulates endosome maturation by mimicking GTP-Rab7

2007 ◽  
Vol 179 (5) ◽  
pp. 965-980 ◽  
Author(s):  
Ankur H. Shah ◽  
Nicholas L. Cianciola ◽  
Jeffrey L. Mills ◽  
Frank D. Sönnichsen ◽  
Cathleen Carlin
Keyword(s):  
Growth Factor Receptor ◽  
Dominant Negative ◽  
Receptor Internalization ◽  
Site Directed Mutagenesis ◽  
Oxysterol Binding Protein ◽  
Early Endosomes ◽  
Catalytically Active ◽  
Guanosine Triphosphatase ◽  
Endocytic Vesicles

The small guanosine triphosphatase Rab7 regulates late endocytic trafficking. Rab7-interacting lysosomal protein (RILP) and oxysterol-binding protein–related protein 1L (ORP1L) are guanosine triphosphate (GTP)–Rab7 effectors that instigate minus end–directed microtubule transport. We demonstrate that RILP and ORP1L both interact with the group C adenovirus protein known as receptor internalization and degradation α (RIDα), which was previously shown to clear the cell surface of several membrane proteins, including the epidermal growth factor receptor and Fas (Carlin, C.R., A.E. Tollefson, H.A. Brady, B.L. Hoffman, and W.S. Wold. 1989. Cell. 57:135–144; Shisler, J., C. Yang, B. Walter, C.F. Ware, and L.R. Gooding. 1997. J. Virol. 71:8299–8306). RIDα localizes to endocytic vesicles but is not homologous to Rab7 and is not catalytically active. We show that RIDα compensates for reduced Rab7 or dominant-negative (DN) Rab7(T22N) expression. In vitro, Cu2+ binding to RIDα residues His75 and His76 facilitates the RILP interaction. Site-directed mutagenesis of these His residues results in the loss of RIDα–RILP interaction and RIDα activity in cells. Additionally, expression of the RILP DN C-terminal region hinders RIDα activity during an acute adenovirus infection. We conclude that RIDα coordinates recruitment of these GTP-Rab7 effectors to compartments that would ordinarily be perceived as early endosomes, thereby promoting the degradation of selected cargo.

scholarly journals Elevated Endosomal Cholesterol Levels in Niemann-Pick Cells Inhibit Rab4 and Perturb Membrane Recycling

2004 ◽  
Vol 15 (10) ◽  
pp. 4500-4511 ◽  
Author(s):  
Amit Choudhury ◽  
Deepak K. Sharma ◽  
David L. Marks ◽  
Richard E. Pagano
Keyword(s):  
Dominant Negative ◽  
Cholesterol Depletion ◽  
Membrane Recycling ◽  
Normal Human Skin ◽  
Lipid Storage Disease ◽  
Cholesterol Levels ◽  
Early Endosomes ◽  
Niemann Pick ◽  
Gdp Dissociation Inhibitor

In normal human skin fibroblasts (HSFs), fluorescent glycosphingolipid analogues are endocytosed and sorted into two pools, one that is recycled to the plasma membrane and one that is transported to the Golgi complex. Here, we investigated glycosphingolipid recycling in Niemann-Pick type A and C lipid storage disease fibroblasts (NPFs). Cells were incubated with a fluorescent analogue of lactosylceramide (LacCer) at 16°C to label early endosomes (EEs), shifted to 37°C, and lipid recycling was quantified. Using dominant negative rabs, we showed that, in normal HSFs, LacCer recycling was rapid (t1/2 ∼8 min) and mainly rab4-dependent. In NPFs, LacCer recycling was delayed (t1/2 ∼30–40 min), and rab4-dependent recycling was absent, whereas rab11-dependent recycling predominated. Transferrin recycling via the rab4 pathway was similarly perturbed in NPFs. Compared with normal HSFs, EEs in NPFs showed high cholesterol levels and an altered organization of rab4. In vitro extraction of rab4 (but not rab11) with GDP dissociation inhibitor was severely attenuated in NPF endosomal fractions. This impairment was reversed with cholesterol depletion of isolated endosomes or with high-salt treatment of endosomes. These data suggest that abnormal membrane recycling in NPFs results from specific inhibition of rab4 function by excess cholesterol in EEs.

Abstract 223: Parkin is Critical for the Clearance of Damaged Mitochondria via an Autophagy-independent Pathway

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Babette C Hammerling ◽  
Melissa Q Cortez ◽  
Rita H Najor ◽  
Åsa B Gustafsson
Keyword(s):  
Cell Death ◽  
Ischemia Reperfusion ◽  
Degradation Pathway ◽  
Dominant Negative ◽  
Upstream Regulator ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Response To Stress ◽  
Autophagy Pathway ◽  
Endosomal Pathway

Functional mitochondria are essential for highly metabolic organs such as the heart. When mitochondria are damaged they can release pro-death factors and reactive oxygen species which in turn can result in cell death. The E3 ubiquitin ligase Parkin plays an important role in clearing damaged mitochondria via the autophagy pathway to protect cells against unnecessary cell death. Interestingly, we have found that Parkin can mediate clearance of damaged mitochondria via an autophagy-independent pathway. In fact, Parkin promotes clearance of depolarized mitochondria at the same rate in both wild-type (WT) and autophagy deficient Atg5-/- mouse embryonic fibroblasts (MEFs) in response to the mitochondrial uncoupler FCCP. We also found that Parkin-mediated ubiquitination is critical for this process as disease associated mutants of Parkin were incapable of inducing mitochondrial clearance in Atg5-/- MEFs. Upon further investigation, we observed a significant increase in the number of Rab5+ early- and Rab7+ late endosomes in both WT and Atg5-/- MEFs after depolarization of mitochondria with FCCP or valinomycin, indicating activation of the endosomal-lysosomal degradation pathway. We did not observe activation of the endosomal pathway after exposure to actinomycin D, an inhibitor RNA synthesis and activator of apoptosis, confirming that mitochondrial damage specifically activates the endosomal degradation pathway. We also observed activation of the endosomal pathway in neonatal myocytes in response to FCCP treatment or after exposure to simulated ischemia/reperfusion (sI/R). Overexpression of the dominant negative Rab5S34N significantly enhanced sI/R-mediated cell death, suggesting that this is a protective pathway activated by cells in response to stress. Moreover, Beclin1 is well known to regulate activation of autophagy. Here, we found that knockdown of Beclin1 inhibited both the number of Rab5+ early endosomes and their colocalization with mitochondria in response to either FCCP or sI/R in myocytes, suggesting that Beclin1 is a critical upstream regulator of the endosomal degradation pathway. Thus, our data suggest that Parkin mediates clearance of damaged mitochondria via both the autophagy and endosomal pathways in cells.

scholarly journals RhoA-Binding Kinase α Translocation Is Facilitated by the Collapse of the Vimentin Intermediate Filament Network

1998 ◽  
Vol 18 (11) ◽  
pp. 6325-6339 ◽  
Author(s):  
Wun-Chey Sin ◽  
Xiang-Qun Chen ◽  
Thomas Leung ◽  
Louis Lim
Keyword(s):  
Structural Integrity ◽  
Morphological Changes ◽  
Dominant Negative ◽  
Cell Periphery ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Complex Process ◽  
Rho Effectors ◽  
Filament Network

ABSTRACT The regulation of morphological changes in eukaryotic cells is a complex process involving major components of the cytoskeleton including actin microfilaments, microtubules, and intermediate filaments (IFs). The putative effector of RhoA, RhoA-binding kinase α (ROKα), is a serine/threonine kinase that has been implicated in the reorganization of actin filaments and in myosin contractility. Here, we show that ROKα also directly affects the structural integrity of IFs. Overexpression of active ROKα, like that of RhoA, caused the collapse of filamentous vimentin, a type III IF. A RhoA-binding-deficient, kinase-inactive ROKα inhibited the collapse of vimentin IFs induced by RhoA in HeLa cells. In vitro, ROKα bound and phosphorylated vimentin at its head-rod domain, thereby inhibiting the assembly of vimentin. ROKα colocalized predominantly with the filamentous vimentin network, which remained intact in serum-starved cells. Treatment of cells with vinblastine, a microtubule-disrupting agent, also resulted in filamentous vimentin collapse and concomitant ROKα translocation to the cell periphery. ROKα translocation did not occur when the vimentin network remained intact in vinblastine-treated cells at 4°C or in the presence of the dominant-negative RhoAN19 mutant. Transient translocation of ROKα was also observed in cells subjected to heat shock, which caused the disassembly of the vimentin network. Thus, the translocation of ROKα to the cell periphery upon overexpression of RhoAV14 or growth factor treatment is associated with disassembly of vimentin IFs. These results indicate that Rho effectors known to act on microfilaments may be involved in regulating the assembly of IFs. Vimentin when phosphorylated also exhibits reduced affinity for the inactive ROKα. The translocation of ROKα from IFs to the cell periphery upon action by activated RhoA and ROKα suggests that ROKα may initiate its own cascade of activation.

Phorbol ester promotes endocytosis by activating a factor involved in endosome fusion

1999 ◽  
Vol 112 (15) ◽  
pp. 2549-2557 ◽  
Author(s):  
A. Aballay ◽  
P.D. Stahl ◽  
L.S. Mayorga
Keyword(s):  
Phorbol Ester ◽  
Fluid Phase ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Early Endosomes ◽  
Zinc Depletion ◽  
Fluid Phase Endocytosis ◽  
Endocytic Vesicles

Previous studies indicate that a zinc- and phorbol ester-binding factor is necessary for in vitro endosome fusion and for the effect of Rab5 on endosome fusion. Rab5 is a small GTPase that regulates membrane fusion between early endosomes derived from either receptor-mediated endocytosis or fluid-phase endocytosis. In its GTP-bound form, Rab5 promotes endocytosis and enhances fusion among early endosomes. To determine if PMA stimulates endocytosis by activating a factor required for endosome fusion, we overexpressed wild-type Rab5, a dominant negative mutant (Rab5:S34N), and a GTPase deficient mutant (Rab5:Q79L) in BHK-21 cells. The phorbol ester PMA stimulates endocytosis and increases the number and the size of endocytic vesicles, even in the presence of Rab5:S34N. Zinc depletion with N,N,N',N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) and addition of calphostin C (CPC), an inhibitor of PKC that interacts with zinc and phorbol ester binding motifs, inhibited both basal and Rab5-stimulated fluid phase endocytosis. These two reagents also inhibited the size and number of endocytic vesicles promoted by Rab5. These results suggest that PMA stimulates endocytosis by regulating the dynamics of the early endosome compartment.

scholarly journals The GTPase-activating protein n-chimaerin cooperates with Rac1 and Cdc42Hs to induce the formation of lamellipodia and filopodia.

1996 ◽  
Vol 16 (9) ◽  
pp. 5069-5080 ◽  
Author(s):  
R Kozma ◽  
S Ahmed ◽  
A Best ◽  
L Lim
Keyword(s):  
Mammalian Cells ◽  
Phorbol Esters ◽  
Morphological Changes ◽  
Neuroblastoma Cells ◽  
Leading Edge ◽  
Dominant Negative ◽  
Gtpase Activating Protein ◽  
Simultaneous Formation ◽  
Gdp Dissociation Inhibitor

n-Chimaerin is a GTPase-activating protein (GAP) mainly for Rac1 and less so for Cdc42Hs in vitro. The GAP activity of n-chimaerin is regulated by phospholipids and phorbol esters. Microinjection of Rac1 and Cdc42Hs into mammalian cells induces formation of the actin-based structures lamellipodia and filopodia, respectively, with the former being prevented by coinjection of the chimaerin GAP domain. Strikingly, microinjection of the full-length n-chimaerin into fibroblasts and neuroblastoma cells induces the simultaneous formation of lamellipodia and filopodia. These structures undergo cycles of dissolution and formation, resembling natural morphological events occurring at the leading edge of fibroblasts and neuronal growth cones. The effects of n-chimaerin on formation of lamellipodia and filopodia were inhibited by dominant negative Rac1(T17N) and Cdc42Hs(T17N), respectively. n-Chimaerin's effects were also inhibited by coinjection with Rho GDP dissociation inhibitor or by treatment with phorbol ester. A mutant n-chimaerin with no GAP activity and impaired p21 binding was ineffective in inducing morphological changes, while a mutant lacking GAP activity alone was effective. Microinjected n-chimaerin colocalized in situ with F-actin. Taken together, these results suggest that n-chimaerin acts synergistically with Rac1 and Cdc42Hs to induce actin-based morphological changes and that this action involves Rac1 and Cdc42Hs binding but not GAP activity. Thus, GAPs may have morphological functions in addition to downregulation of GTPases.

scholarly journals In Vitro Budding of Intralumenal Vesicles into Late Endosomes Is Regulated by Alix and Tsg101

2008 ◽  
Vol 19 (11) ◽  
pp. 4942-4955 ◽  
Author(s):  
Thomas Falguières ◽  
Pierre-Philippe Luyet ◽  
Christin Bissig ◽  
Cameron C. Scott ◽  
Marie-Claire Velluz ◽  
...  
Keyword(s):  
Egf Receptor ◽  
Light And Electron Microscopy ◽  
Degradation Pathway ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Vesicle Budding ◽  
Late Endosomes ◽  
Electron Microscopy Analysis ◽  
Membrane Components

Endosomes along the degradation pathway leading to lysosomes accumulate membranes in their lumen and thus exhibit a characteristic multivesicular appearance. These lumenal membranes typically incorporate down-regulated EGF receptor destined for degradation, but the mechanisms that control their formation remain poorly characterized. Here, we describe a novel quantitative biochemical assay that reconstitutes the formation of lumenal vesicles within late endosomes in vitro. Vesicle budding into the endosome lumen was time-, temperature-, pH-, and energy-dependent and required cytosolic factors and endosome membrane components. Our light and electron microscopy analysis showed that the compartment supporting the budding process was accessible to endocytosed bulk tracers and EGF receptor. We also found that the EGF receptor became protected against trypsin in our assay, indicating that it was sorted into the intraendosomal vesicles that were formed in vitro. Our data show that the formation of intralumenal vesicles is ESCRT-dependent, because the process was inhibited by the K173Q dominant negative mutant of hVps4. Moreover, we find that the ESCRT-I subunit Tsg101 and its partner Alix control intralumenal vesicle formation, by acting as positive and negative regulators, respectively. We conclude that budding of the limiting membrane toward the late endosome lumen, which leads to the formation of intraendosomal vesicles, is controlled by the positive and negative functions of Tsg101 and Alix, respectively.

scholarly journals Functional interaction between E-cadherin and alphav-containing integrins in carcinoma cells

2000 ◽  
Vol 113 (3) ◽  
pp. 425-437 ◽  
Author(s):  
M. von Schlippe ◽  
J.F. Marshall ◽  
P. Perry ◽  
M. Stone ◽  
A.J. Zhu ◽  
...  
Keyword(s):  
Morphological Changes ◽  
Surface Expression ◽  
Collagen Type I ◽  
Carcinoma Cells ◽  
Dominant Negative ◽  
Type I ◽  
Beta Catenin ◽  
Receptor Aggregation ◽  
E Cadherin

We have demonstrated the possibility of cross-talk between E-cadherin and alphav integrins in breast carcinoma cells. Using the function-blocking anti-alphav monoclonal antibody 17E6, applied to monolayer cultures of breast cancer lines, it was found that treatment of cells possessing detergent-insoluble (implying attachment to the actin cytoskeleton) E-cadherin resulted in the adoption of a spheroid configuration of cell growth. This effect was dependent upon not just alphav occupancy but also receptor aggregation. Thus in vitro alphav-dependent adhesion suppresses E-cadherin-mediated morphological changes. To investigate whether manipulation of E-cadherin would, conversely, modulate integrin activity we introduced a dominant-negative E-cadherin construct into one of the lines, ZR75-1, giving rise to the cell line ZR-E2R1. Surface expression of endogenous E-cadherin was downregulated (by around 25%), whereas beta-catenin levels were increased two- to threefold in ZR-E2R1 cells. There was also a highly significant increase in migration of ZR-E2R1 cells (relative to control cells) toward vitronectin (P<0.001), but not toward collagen type I, fibronectin or laminin. Such increased migration could be abrogated totally by antibody blockade of alphavbeta5 and alphavbeta1 integrins. There was no detectable change in alphav integrin levels. These data suggest that the introduction of a dominant-negative E-cadherin mutant into ZR75-1, in addition to a loss of cohesion, generates a signal (or signals) which increases migration towards vitronectin through increased activity of alphav integrins.

scholarly journals F-Box Protein RcyA Controls Turnover of the Kinesin-7 Motor KipA in Aspergillus nidulans

2014 ◽  
Vol 13 (8) ◽  
pp. 1085-1094 ◽  
Author(s):  
Saturnino Herrero ◽  
Norio Takeshita ◽  
Reinhard Fischer
Keyword(s):  
Aspergillus Nidulans ◽  
Morphological Changes ◽  
Degradation Pathway ◽  
Bimolecular Fluorescence Complementation ◽  
Snare Protein ◽  
Content Type ◽  
C Terminus ◽  
Early Endosomes ◽  
F Box Protein ◽  
Endocytic Vesicles

ABSTRACT Fungal filamentous growth depends on continuous membrane insertion at the tip, the delivery of membrane-bound positional markers, and the secretion of enzymes for cell wall biosynthesis. This is achieved through exocytosis. At the same time, polarized growth requires membrane and protein recycling through endocytosis. Endocytic vesicles are thought to enter the protein degradation pathway or recycle their content to the cell surface. In Saccharomyces cerevisiae , the Rcy1 F-box protein is involved in the recycling process of a v-SNARE protein. We identified a Rcy1 orthologue, RcyA, in the filamentous fungus Aspergillus nidulans as a protein interacting with the KipA kinesin-7 motor protein in a yeast two-hybrid screen. The interaction was confirmed through bimolecular fluorescence complementation. RcyA possesses an F-box domain at the N terminus and a prenylation (CaaX) motif at the C terminus. RcyA shows also similarity to Sec10, a component of the exocyst complex. The RcyA protein localized to the hyphal tip and forming septa, likely through transportation on secretory vesicles and partially on early endosomes, but independently of KipA. Deletion of rcyA did not cause severe morphological changes but caused partial defects in the recycling of the SynA v-SNARE protein and the positioning of the cell end markers TeaA and TeaR. In addition, deletion of rcyA led to increased concentrations of the KipA protein, whereas the transcript concentration was unaffected. These results suggest that RcyA probably labels KipA for degradation and thereby controls the protein amount of KipA.

scholarly journals Protein Tyrosine Phosphatase φ Regulates Paxillin Tyrosine Phosphorylation and Mediates Colony-Stimulating Factor 1-Induced Morphological Changes in Macrophages

2001 ◽  
Vol 21 (5) ◽  
pp. 1795-1809 ◽  
Author(s):  
Fiona J. Pixley ◽  
Pierre S. W. Lee ◽  
John S. Condeelis ◽  
E. Richard Stanley
Keyword(s):  
Protein Tyrosine Phosphatase ◽  
Catalytic Domain ◽  
Morphological Changes ◽  
Tyrosine Phosphatase ◽  
Dominant Negative ◽  
Colony Stimulating Factor ◽  
Macrophage Cell ◽  
Protein Tyrosine ◽  
Stimulating Factor

ABSTRACT Removal of colony-stimulating factor 1 (CSF-1) causes macrophages to round up and to increase their expression of protein tyrosine phosphatase φ (PTPφ). This is accompanied by the disruption of focal complexes and the formation of ruffles. Here we have overexpressed wild-type (WT) PTPφ and a phosphatase-inactive (C325S) mutant in a macrophage cell line in the presence and absence of CSF-1. In the presence of CSF-1, WT PTPφ induces cell rounding and ruffle formation, while C325S PTPφ has no effect. In contrast, in CSF-1-starved cells, C325S PTPφ behaves in a dominant negative fashion, preventing rounding and ruffling. Furthermore, C325S PTPφ increases adhesion in cycling cells, while WT PTPφ enhances motility. In WT PTPφ-overexpressing cells, the focal contact protein paxillin is selectively depleted from focal complexes and specifically dephosphorylated on tyrosine. In contrast, paxillin is hyperphosphorylated in C325S PTPφ-expressing cells. Moreover, a complex containing PTPφ, paxillin, and a paxillin-associated tyrosine kinase, Pyk2, can be immunoprecipitated from macrophage lysates, and the catalytic domain of PTPφ selectively binds paxillin and Pyk2 in vitro. Although PTPφ and Pyk2 do not colocalize with paxillin in focal complexes, all three proteins are colocalized in dorsal ruffles. The results suggest that paxillin is dephosphorylated by PTPφ in dorsal ruffles, using Pyk2 as a bridging molecule, resulting in a reduced pool of tyrosine-phosphorylated paxillin available for incorporation into focal complexes, thereby mediating CSF-1 regulation of macrophage morphology, adhesion, and motility.

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