scholarly journals Detection of the Endosomal Sorting Complex Required for Transport inEntamoeba histolyticaand Characterization of the EhVps4 Protein

2010 ◽  
Vol 2010 ◽  
pp. 1-15 ◽  
Author(s):  
Israel López-Reyes ◽  
Guillermina García-Rivera ◽  
Cecilia Bañuelos ◽  
Silvia Herranz ◽  
Olivier Vincent ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Entamoeba Histolytica ◽  
Hepatic Damage ◽  
Multivesicular Bodies ◽  
Protein Overexpression ◽  
Specific Antibodies ◽  
Endosomal Sorting ◽  
Biochemical Studies

Eukaryotic endocytosis involves multivesicular bodies formation, which is driven by endosomal sorting complexes required for transport (ESCRT). Here, we showed the presence and expression of homologous ESCRT genes inEntamoeba histolytica. We cloned and expressed theEhvps4gene, an ESCRT member, to obtain the recombinant EhVps4 and generate specific antibodies, which immunodetected EhVps4 in cytoplasm of trophozoites. Bioinformatics and biochemical studies evidenced that rEhVps4 is an ATPase, whose activity depends on the conserved E211 residue. Next, we generated trophozoites overexpressing EhVps4 and mutant EhVps4-E211Q FLAG-tagged proteins. The EhVps4-FLAG was located in cytosol and at plasma membrane, whereas the EhVps4-E211Q-FLAG was detected as abundant cytoplasmic dots in trophozoites. Erythrophagocytosis, cytopathic activity, and hepatic damage in hamsters were not improved in trophozoites overexpressing EhVps4-FLAG. In contrast, EhVps4-E211Q-FLAG protein overexpression impaired these properties. The localization of EhVps4-FLAG around ingested erythrocytes, together with our previous results, strengthens the role for EhVps4 inE. histolyticaphagocytosis and virulence.

scholarly journals Plasma membrane deformation by circular arrays of ESCRT-III protein filaments

2008 ◽  
Vol 180 (2) ◽  
pp. 389-402 ◽  
Author(s):  
Phyllis I. Hanson ◽  
Robyn Roth ◽  
Yuan Lin ◽  
John E. Heuser
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Negative Curvature ◽  
Multivesicular Body ◽  
Multivesicular Bodies ◽  
Deficient Mutant ◽  
Membrane Deformation ◽  
Endosomal Sorting ◽  
Viral Budding ◽  
Circular Arrays

Endosomal sorting complex required for transport III (ESCRT-III) proteins function in multivesicular body biogenesis and viral budding. They are recruited from the cytoplasm to the membrane, where they assemble into large complexes. We used “deep-etch” electron microscopy to examine polymers formed by the ESCRT-III proteins hSnf7-1 (CHMP4A) and hSnf7-2 (CHMP4B). When overexpressed, these proteins target to endosomes and the plasma membrane. Both hSnf7 proteins assemble into regular approximately 5-nm filaments that curve and self-associate to create circular arrays. Binding to a coexpressed adenosine triphosphate hydrolysis–deficient mutant of VPS4B draws these filaments together into tight circular scaffolds that bend the membrane away from the cytoplasm to form buds and tubules protruding from the cell surface. Similar buds develop in the absence of mutant VPS4B when hSnf7-1 is expressed without its regulatory C-terminal domain. We demonstrate that hSnf7 proteins form novel membrane-attached filaments that can promote or stabilize negative curvature and outward budding. We suggest that ESCRT-III polymers delineate and help generate the luminal vesicles of multivesicular bodies.

scholarly journals Ear1p and Ssh4p Are New Adaptors of the Ubiquitin Ligase Rsp5p for Cargo Ubiquitylation and Sorting at Multivesicular Bodies

2008 ◽  
Vol 19 (6) ◽  
pp. 2379-2388 ◽  
Author(s):  
Sébastien Léon ◽  
Zoi Erpapazoglou ◽  
Rosine Haguenauer-Tsapis
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Ubiquitin Ligase ◽  
Multivesicular Bodies ◽  
Endosomal Sorting ◽  
Frame Fusion ◽  
Vacuolar Targeting

The ubiquitylation of membrane proteins destined for the vacuole/lysosome is essential for their recognition by the endosomal sorting machinery and their internalization into vesicles of multivesicular bodies (MVBs). In yeast, this process requires Rsp5p, an essential ubiquitin ligase of the Nedd4 family. We describe here two redundant proteins, Ear1p and Ssh4p, required for the vacuolar targeting of several cargoes originating from the Golgi or the plasma membrane. Ear1p is an endosomal protein that interacts with Rsp5p through its PPxY motifs, and it is required for the ubiquitylation of selected cargoes before their MVB sorting. In-frame fusion of cargo to ubiquitin overcomes the need for Ear1p/Ssh4p, confirming a role for these proteins in cargo ubiquitylation. Interestingly, Ear1p is itself ubiquitylated by Rsp5p and targeted to the vacuole. Finally, Ear1p overexpression leads to Rsp5p accumulation at endosomes, interfering with some of its functions in trafficking. Therefore, Ear1p/Ssh4p recruit Rsp5p and assist it in its function at MVBs by directing the ubiquitylation of specific cargoes.

scholarly journals EhLimA, a Novel LIM Protein, Localizes to the Plasma Membrane in Entamoeba histolytica

2007 ◽  
Vol 6 (9) ◽  
pp. 1646-1655 ◽  
Author(s):  
Nomy Wender ◽  
Eduardo Villalobo ◽  
David Mirelman
Keyword(s):  
Plasma Membrane ◽  
Entamoeba Histolytica ◽  
Sucrose Gradient ◽  
Molecular System ◽  
Membrane Rafts ◽  
Lim Domain ◽  
Lim Protein ◽  
Cytoskeletal Elements ◽  
Host Tissues

ABSTRACT The parasitic protozoan Entamoeba histolytica relies on a very dynamic cytoskeleton in order to invade and survive in host tissues. Identification of cytoskeletal elements is key to understanding these processes. Here we present the characterization of EhLimA, the first LIM protein of E. histolytica. EhLimA consists of a single LIM domain at its N terminus and exhibits the highest degree of homology with DdLimE from Dictyostelium discoideum. Immunofluorescence localization of EhLimA using anti-EhLimA antibodies revealed that EhLimA is highly concentrated at the plasma membrane of cells. Silencing or overexpression of the EhLimA gene did not have a significant effect on the growth or morphology of the parasite. EhLimA associates with the cytoskeleton as demonstrated by the enrichment of the protein in cytoskeleton fractions as well as in pull-down assays that revealed that cytoskeleton association involves interaction with actin. EhLimA binding to actin was shown to be dependent on the N-terminal LIM domain of EhLimA, as removal of even half of the LIM domain resulted in almost complete inhibition of the binding to actin. We also found that a portion of EhLimA floats to the lower-density regions of a sucrose gradient together with portions of the Gal-lectin light subunit and actin. Treatment of cells with the cholesterol-sequestering agent digitonin resulted in increased solubility of EhLimA. These results indicate that in addition to cytoskeletal association, EhLimA may also associate with lipid rafts in the parasite plasma membrane and suggest that EhLimA may be part of the molecular system connecting the actin cytoskeleton to membrane rafts.

scholarly journals Identification and functional characterization of EhClC-A, an Entamoeba histolytica ClC chloride channel located at plasma membrane

2006 ◽  
Vol 59 (4) ◽  
pp. 1249-1261 ◽  
Author(s):  
Andres Salas-Casas ◽  
Arturo Ponce-Balderas ◽  
Rosa Maria Garcia-Perez ◽  
Pedro Cortes-Reynosa ◽  
Gerardo Gamba ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Entamoeba Histolytica ◽  
Chloride Channel ◽  
Functional Characterization ◽  
Clc Chloride Channel

Characterization of Plasma Membrane-Associated Antigens of Diagnostic and Prophylactic Importance in Entamoeba histolytica

2000 ◽  
Vol 31 (4) ◽  
pp. S21-S22
Author(s):  
Partha Mukhopadhyay ◽  
Sadhak Sengupta ◽  
Ali Akbar ◽  
Krishanu Sengupta ◽  
Sanjib Bhattacharya ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Entamoeba Histolytica

scholarly journals EhADH112 Is a Bro1 Domain-Containing Protein Involved in theEntamoeba histolyticaMultivesicular Bodies Pathway

2012 ◽  
Vol 2012 ◽  
pp. 1-15 ◽  
Author(s):  
Cecilia Bañuelos ◽  
Guillermina García-Rivera ◽  
Israel López-Reyes ◽  
Leobardo Mendoza ◽  
Arturo González-Robles ◽  
...  
Keyword(s):  
Amino Acids ◽  
Entamoeba Histolytica ◽  
Protein Trafficking ◽  
Multivesicular Bodies ◽  
Accessory Proteins ◽  
Endosomal Sorting ◽  
Trafficking Pathway ◽  
Cytoplasmic Vesicles ◽  
Late Stages

EhADH112 is anEntamoeba histolyticaBro1 domain-containing protein, structurally related to mammalian ALIX and yeast BRO1, both involved in the Endosomal Sorting Complexes Required for Transport (ESCRT)-mediated multivesicular bodies (MVB) biogenesis. Here, we investigated an alternative role for EhADH112 in the MVB protein trafficking pathway by overexpressing 166 amino acids of its N-terminal Bro1 domain in trophozoites. Trophozoites displayed diminished phagocytosis rates and accumulated exogenous Bro1 at cytoplasmic vesicles which aggregated into aberrant complexes at late stages of phagocytosis, probably preventing EhADH112 function. Additionally, the existence of a putativeE. histolyticaESCRT-III subunit (EhVps32) presumably interacting with EhADH112, led us to perform pull-down experiments with GST-EhVps32 and [35S]-labeled EhADH112 or EhADH112 derivatives, confirming EhVps32 binding to EhADH112 through its Bro1 domain. Our overall results define EhADH112 as a novel member of ESCRT-accessory proteins transiently present at cellular surface and endosomal compartments, probably contributing to MVB formation during phagocytosis.

Role of Exosomes in the Exchange of Spermatozoa after Leaving the Seminiferous Tubule: A Review

2020 ◽  
Vol 21 (5) ◽  
pp. 330-338
Author(s):  
Luming Wu ◽  
Yuan Ding ◽  
Shiqiang Han ◽  
Yiqing Wang
Keyword(s):  
Drug Delivery ◽  
Plasma Membrane ◽  
Seminiferous Tubule ◽  
Metabolic Diseases ◽  
Inflammatory Diseases ◽  
Multivesicular Bodies ◽  
Extensive Search ◽  
Neurological Research ◽  
The One

Background: Exosomes are extracellular vesicles (EVs) released from cells upon fusion of an intermediate endocytic compartment with the plasma membrane. They refer to the intraluminal vesicles released from the fusion of multivesicular bodies with the plasma membrane. The contents and number of exosomes are related to diseases such as metabolic diseases, cancer and inflammatory diseases. Exosomes have been used in neurological research as a drug delivery tool and also as biomarkers for diseases. Recently, exosomes were observed in the seminal plasma of the one who is asthenozoospermia, which can affect sperm motility and capacitation. Objective: The main objective of this review is to deeply discuss the role of exosomes in spermatozoa after leaving the seminiferous tubule. Methods: We conducted an extensive search of the literature available on relationships between exosomes and exosomes in spermatozoa on the bibliographic database. Conclusion: : This review thoroughly discussed the role that exosomes play in the exchange of spermatozoa after leaving the seminiferous tubule and its potential as a drug delivery tool and biomarkers for diseases as well.

scholarly journals Fluorescence Fluctuation Spectroscopy enables quantification of potassium channel subunit dynamics and stoichiometry

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Giulia Tedeschi ◽  
Lorenzo Scipioni ◽  
Maria Papanikolaou ◽  
Geoffrey W. Abbott ◽  
Michelle A. Digman
Keyword(s):  
Plasma Membrane ◽  
Protein Diffusion ◽  
Ion Diffusion ◽  
Fluorescence Fluctuation Spectroscopy ◽  
Biophysical Characterization ◽  
Kv Channels ◽  
Subunit Stoichiometry ◽  
Spatio Temporal ◽  
Voltage Sensing Domain

AbstractVoltage-gated potassium (Kv) channels are a family of membrane proteins that facilitate K+ ion diffusion across the plasma membrane, regulating both resting and action potentials. Kv channels comprise four pore-forming α subunits, each with a voltage sensing domain, and they are regulated by interaction with β subunits such as those belonging to the KCNE family. Here we conducted a comprehensive biophysical characterization of stoichiometry and protein diffusion across the plasma membrane of the epithelial KCNQ1-KCNE2 complex, combining total internal reflection fluorescence (TIRF) microscopy and a series of complementary Fluorescence Fluctuation Spectroscopy (FFS) techniques. Using this approach, we found that KCNQ1-KCNE2 has a predominant 4:4 stoichiometry, while non-bound KCNE2 subunits are mostly present as dimers in the plasma membrane. At the same time, we identified unique spatio-temporal diffusion modalities and nano-environment organization for each channel subunit. These findings improve our understanding of KCNQ1-KCNE2 channel function and suggest strategies for elucidating the subunit stoichiometry and forces directing localization and diffusion of ion channel complexes in general.

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