Mechanism of protein sorting during erythroblast enucleation: role of cytoskeletal connectivity

Blood ◽  
2004 ◽  
Vol 103 (5) ◽  
pp. 1912-1919 ◽  
Author(s):  
James C.-M. Lee ◽  
J. Aura Gimm ◽  
Annie J. Lo ◽  
Mark J. Koury ◽  
Sharon W. Krauss ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Molecular Mechanisms ◽  
Immunofluorescence Microscopy ◽  
Protein Sorting ◽  
Membrane Cytoskeleton ◽  
Skeletal Protein ◽  
Regulate Protein ◽  
Membrane Components ◽  
Cytoskeletal Elements

AbstractDuring erythroblast enucleation, nuclei surrounded by plasma membrane separate from erythroblast cytoplasm. A key aspect of this process is sorting of erythroblast plasma membrane components to reticulocytes and expelled nuclei. Although it is known that cytoskeletal elements actin and spectrin partition to reticulocytes, little is understood about molecular mechanisms governing plasma membrane protein sorting. We chose glycophorin A (GPA) as a model integral protein to begin investigating protein-sorting mechanisms. Using immunofluorescence microscopy and Western blotting we found that GPA sorted predominantly to reticulocytes. We hypothesized that the degree of skeletal linkage might control the sorting pattern of transmembrane proteins. To explore this hypothesis, we quantified the extent of GPA association to the cytoskeleton in erythroblasts, young reticulocytes, and mature erythrocytes using fluorescence imaged microdeformation (FIMD) and observed that GPA underwent dramatic reorganization during terminal differentiation. We discovered that GPA was more connected to the membrane cytoskeleton, either directly or indirectly, in erythroblasts and young reticulocytes than in mature cells. We conclude that skeletal protein association can regulate protein sorting during enucleation. Further, we suggest that the enhanced rigidity of reticulocyte membranes observed in earlier investigations results, at least in part, from increased connectivity of GPA with the spectrin-based skeleton.

Aberrant Protein Sorting to the Nucleus during Erythroblast Enucleation: Mechanistic Basis for Membrane Protein Loss in Hereditary Elliptocytosis and Spherocytosis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1559-1559
Author(s):  
Marcela A. Salomao ◽  
Sarah Short ◽  
Gloria Lee ◽  
Xiuli An ◽  
Mohandas Narla ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Plasma Membranes ◽  
Molecular Mechanisms ◽  
Protein Sorting ◽  
Transmembrane Proteins ◽  
Hereditary Elliptocytosis ◽  
Skeletal Protein ◽  
Aberrant Protein ◽  
Protein 4.1R

Abstract During erythroblast enucleation, nuclei surrounded by plasma membrane separate from erythroblast cytoplasm. A key aspect of this process is sorting of membrane components to plasma membranes surrounding expelled nuclei and young reticulocytes. This protein partitioning performs a crucial role in regulating the protein content of reticulocyte plasma membranes. Although it is known that cytoskeletal actin, spectrin and protein 4.1R distribute to reticulocytes, little is known about the sorting patterns of erythroblast transmembrane proteins. In hereditary spherocytosis (HS) and hereditary elliptocytosis (HE), erythrocytes contain well-described deficiencies of various transmembrane proteins, in addition to those encoded by the mutant genes. For example, elliptocytic human and murine erythrocytes resulting from mutations in the 4.1R gene lack not only protein 4.1R but also transmembrane protein glycophorin C (GPC), known to be a 4.1R binding partner with a key role in linking cytoskeleton to bilayer. Similarly, in HS resulting from mutations in the ankyrin gene, deficiencies of band 3, Rh and GPA have been documented. Various molecular mechanisms could explain deficiencies of membrane proteins in HS and HE erythrocytes including: perturbed trafficking to the erythroblast membrane; aberrant protein sorting during erythroblast enucleation; and selective loss during reticulocyte membrane remodeling. We explored whether aberrant protein sorting during enucleation might be responsible for GPC deficiency in HE. First we performed immunochemical analysis of the sorting pattern of GPC using highly purified extruded nuclei and immature reticulocytes derived from terminally differentiated murine erythroblast cultures. Proteins from equivalent numbers of expelled nuclei and reticulocytes were analyzed by Western blotting. Using antibodies specific for GPC we determined that 90% of GPC sorted to reticulocyte plasma membranes. To validate these results we used live cell, three-color immunofluorescent microscopy and analyzed enucleating erythroblasts, reticulocytes and extruded nuclei from freshly harvested murine wild type bone marrow. Independently confirming the Western blot data, we found that GPC sorted almost exclusively to reticulocytes with little or no GPC in association with nuclear plasma membrane. Strikingly, in 4.1R null erythroblasts GPC was distributed exclusively to expelled nuclei. These findings unequivocally establish that skeletal protein 4.1R is critical for normal sorting of GPC to young reticulocytes and provide clear evidence that specific skeletal protein associations can regulate protein sorting during enucleation. Moreover, our data provide a molecular explanation for the underlying basis of GPC deficiency observed in 4.1R-deficient individuals with HE. We speculate that aberrant protein sorting may be a prevalent mechanism for the deficiencies of various membrane proteins in HS and HE and that their differential loss could contribute to the variable phenotypic expression of these hemolytic disorders.

Binding of the alpha-fodrin SH3 domain to the leading lamellae of locomoting chicken fibroblasts

1993 ◽  
Vol 105 (3) ◽  
pp. 647-654
Author(s):  
J. Merilainen ◽  
R. Palovuori ◽  
R. Sormunen ◽  
V.M. Wasenius ◽  
V.P. Lehto
Keyword(s):  
Plasma Membrane ◽  
Cell Polarization ◽  
Glutathione S Transferase ◽  
Src Homology 3 ◽  
Membrane Cytoskeleton ◽  
Cytoplasmic Face ◽  
Small Domain ◽  
Skeletal Protein ◽  
Src Homology ◽  
Membrane Components

Fodrin (nonerythroid spectrin) is a membrane skeletal protein that plays an important role in the establishment and maintenance of the cell shape and polarity. We have identified in alpha-fodrin an src homology 3 (SH3)-related region, a small domain that is present in a large number of proteins that are involved in signal transduction, cell polarization and membrane-cytoskeleton interactions. In this study we have explored the function of the alpha-fodrin SH3 by incubating fixed and permeabilized cultured chicken fibroblasts with the alpha-fodrin SH3 peptide, expressed in bacteria as a fusion protein with glutathione S-transferase. Immunofluorescence and immunoelectron microscopy showed that alpha-fodrin SH3 binds to the cytoplasmic face of the plasma membrane in the leading lamellae and the pseudopodial lobes of the spreading and locomoting cells. No, or only minimal, binding was seen in immotile cells, or in the stationary trailing ends of the locomoting cells. SH3 binding was also seen in cytochalasin-D-treated cells, suggesting that actin filaments are not responsible for the binding. These findings suggest that alpha-fodrin SH3 interacts with plasma membrane components that are present in the leading lamellae exclusively or are modulated in a manner specific to the leading lamellae.

scholarly journals ESCRT-dependent targeting of plasma membrane localized KCa3.1 to the lysosomes

2010 ◽  
Vol 299 (5) ◽  
pp. C1015-C1027 ◽  
Author(s):  
Corina M. Balut ◽  
Yajuan Gao ◽  
Sandra A. Murray ◽  
Patrick H. Thibodeau ◽  
Daniel C. Devor
Keyword(s):  
Plasma Membrane ◽  
Molecular Mechanisms ◽  
Close Association ◽  
Significant Inhibition ◽  
Dominant Negative ◽  
Human Embryonic Kidney Cells ◽  
Endosomal Sorting ◽  
Escrt Machinery ◽  
Interfering Rna

The number of intermediate-conductance, Ca2+-activated K+ channels (KCa3.1) present at the plasma membrane is deterministic in any physiological response. However, the mechanisms by which KCa3.1 channels are removed from the plasma membrane and targeted for degradation are poorly understood. Recently, we demonstrated that KCa3.1 is rapidly internalized from the plasma membrane, having a short half-life in both human embryonic kidney cells (HEK293) and human microvascular endothelial cells (HMEC-1). In this study, we investigate the molecular mechanisms controlling the degradation of KCa3.1 heterologously expressed in HEK and HMEC-1 cells. Using immunofluorescence and electron microscopy, as well as quantitative biochemical analysis, we demonstrate that membrane KCa3.1 is targeted to the lysosomes for degradation. Furthermore, we demonstrate that either overexpressing a dominant negative Rab7 or short interfering RNA-mediated knockdown of Rab7 results in a significant inhibition of channel degradation rate. Coimmunoprecipitation confirmed a close association between Rab7 and KCa3.1. On the basis of these findings, we assessed the role of the ESCRT machinery in the degradation of heterologously expressed KCa3.1, including TSG101 [endosomal sorting complex required for transport (ESCRT)-I] and CHMP4 (ESCRT-III) as well as VPS4, a protein involved in the disassembly of the ESCRT machinery. We demonstrate that TSG101 is closely associated with KCa3.1 via coimmunoprecipitation and that a dominant negative TSG101 inhibits KCa3.1 degradation. In addition, both dominant negative CHMP4 and VPS4 significantly decrease the rate of membrane KCa3.1 degradation, compared with wild-type controls. These results are the first to demonstrate that plasma membrane-associated KCa3.1 is targeted for lysosomal degradation via a Rab7 and ESCRT-dependent pathway.

scholarly journals Silencing of Plasma Membrane Ca2+-ATPase Isoforms 2 and 3 Impairs Energy Metabolism in Differentiating PC12 Cells

2014 ◽  
Vol 2014 ◽  
pp. 1-13 ◽  
Author(s):  
Tomasz Boczek ◽  
Malwina Lisek ◽  
Bozena Ferenc ◽  
Antoni Kowalski ◽  
Magdalena Wiktorska ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Pc12 Cells ◽  
Molecular Mechanisms ◽  
Atp Synthesis ◽  
Mitochondrial Activity ◽  
Excitable Cells ◽  
Close Link ◽  
Atp Level ◽  
Atp Generation

A close link between Ca2+, ATP level, and neurogenesis is apparent; however, the molecular mechanisms of this relationship have not been completely elucidated. Transient elevations of cytosolic Ca2+may boost ATP synthesis, but ATP is also consumed by ion pumps to maintain a low Ca2+in cytosol. In differentiation process plasma membrane Ca2+ATPase (PMCA) is considered as one of the major players for Ca2+homeostasis. From four PMCA isoforms, the fastest PMCA2 and PMCA3 are expressed predominantly in excitable cells. In the present study we assessed whether PMCA isoform composition may affect energy balance in differentiating PC12 cells. We found that PMCA2-downregulated cells showed higher basal O2consumption, lower NAD(P)H level, and increased activity of ETC. These changes associated with higher[Ca2+]cresulted in elevated ATP level. Since PMCA2-reduced cells demonstrated greatest sensitivity to ETC inhibition, we suppose that the main source of energy for PMCA isoforms 1, 3, and 4 was oxidative phosphorylation. Contrary, cells with unchanged PMCA2 expression exhibited prevalence of glycolysis in ATP generation. Our results with PMCA2- or PMCA3-downregulated lines provide an evidence of a novel role of PMCA isoforms in regulation of bioenergetic pathways, and mitochondrial activity and maintenance of ATP level during PC12 cells differentiation.

Lipid order and molecular assemblies in the plasma membrane of eukaryotic cells

2009 ◽  
Vol 37 (5) ◽  
pp. 1056-1060 ◽  
Author(s):  
Marek Cebecauer ◽  
Dylan M. Owen ◽  
Anna Markiewicz ◽  
Anthony I. Magee
Keyword(s):  
Plasma Membrane ◽  
Physical Properties ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Eukaryotic Cells ◽  
Dynamic Nature ◽  
Membrane Components ◽  
Technological Improvements

Multimolecular assemblies on the plasma membrane exhibit dynamic nature and are often generated during the activation of eukaryotic cells. The role of lipids and their physical properties in helping to control the existence of these structures is discussed. Technological improvements for live cell imaging of membrane components are also reviewed.

scholarly journals The Role of mVps18p in Clustering, Fusion, and Intracellular Localization of Late Endocytic Organelles

2003 ◽  
Vol 14 (10) ◽  
pp. 4015-4027 ◽  
Author(s):  
Viviane Poupon ◽  
Abigail Stewart ◽  
Sally R. Gray ◽  
Robert C. Piper ◽  
J. Paul Luzio
Keyword(s):  
Molecular Mechanisms ◽  
Protein Sorting ◽  
Intracellular Localization ◽  
Critical Role ◽  
Vacuole Fusion ◽  
Late Endosomes ◽  
The Reformation ◽  
Lysosomal Protein ◽  
Direct Fusion

Delivery of endocytosed macromolecules to mammalian cell lysosomes occurs by direct fusion of late endosomes with lysosomes, resulting in the formation of hybrid organelles from which lysosomes are reformed. The molecular mechanisms of this fusion are analogous to those of homotypic vacuole fusion in Saccharomyces cerevisiae. We report herein the major roles of the mammalian homolog of yeast Vps18p (mVps18p), a member of the homotypic fusion and vacuole protein sorting complex. When overexpressed, mVps18p caused the clustering of late endosomes/lysosomes and the recruitment of other mammalian homologs of the homotypic fusion and vacuole protein sorting complex, plus Rab7-interacting lysosomal protein. The clusters were surrounded by components of the actin cytoskeleton, including actin, ezrin, and specific unconventional myosins. Overexpression of mVps18p also overcame the effect of wortmannin treatment, which inhibits membrane traffic out of late endocytic organelles and causes their swelling. Reduction of mVps18p by RNA interference caused lysosomes to disperse away from their juxtanuclear location. Thus, mVps18p plays a critical role in endosome/lysosome tethering, fusion, intracellular localization and in the reformation of lysosomes from hybrid organelles.

Role of cell-surface carbohydrates and plasma membrane components in the internalization of cell-penetrating peptides

2010 ◽  
Vol 15 (23-24) ◽  
pp. 1101-1101
Author(s):  
Chérine Bechara ◽  
Chen-Yu Jiao ◽  
Fabienne Burlina ◽  
Isabel D. Alves ◽  
Gérard Chassaing ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Cell Penetrating Peptides ◽  
Cell Penetrating ◽  
Membrane Components ◽  
Surface Carbohydrates

scholarly journals Into the breach: how cells cope with wounds

Open Biology ◽  
2018 ◽  
Vol 8 (10) ◽  
pp. 180135 ◽  
Author(s):  
Mitsutoshi Nakamura ◽  
Andrew N. M. Dominguez ◽  
Jacob R. Decker ◽  
Alexander J. Hull ◽  
Jeffrey M. Verboon ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Wound Repair ◽  
Molecular Mechanisms ◽  
Cancer Metastasis ◽  
Repair Process ◽  
Molecular Techniques ◽  
Membrane Cytoskeleton ◽  
Type Size ◽  
Cortical Cytoskeleton ◽  
Repair Models

Repair of wounds to individual cells is crucial for organisms to survive daily physiological or environmental stresses, as well as pathogen assaults, which disrupt the plasma membrane. Sensing wounds, resealing membranes, closing wounds and remodelling plasma membrane/cortical cytoskeleton are four major steps that are essential to return cells to their pre-wounded states. This process relies on dynamic changes of the membrane/cytoskeleton that are indispensable for carrying out the repairs within tens of minutes. Studies from different cell wound repair models over the last two decades have revealed that the molecular mechanisms of single cell wound repair are very diverse and dependent on wound type, size, and/or species. Interestingly, different repair models have been shown to use similar proteins to achieve the same end result, albeit sometimes by distinctive mechanisms. Recent studies using cutting edge microscopy and molecular techniques are shedding new light on the molecular mechanisms during cellular wound repair. Here, we describe what is currently known about the mechanisms underlying this repair process. In addition, we discuss how the study of cellular wound repair—a powerful and inducible model—can contribute to our understanding of other fundamental biological processes such as cytokinesis, cell migration, cancer metastasis and human diseases.

Involvement of the Ca2+-ATPase PAT1 and the contractile vacuole in calcium regulation in Dictyostelium discoideum

1999 ◽  
Vol 112 (3) ◽  
pp. 405-414 ◽  
Author(s):  
J. Moniakis ◽  
M.B. Coukell ◽  
A. Janiec
Keyword(s):  
Protein Synthesis ◽  
Plasma Membrane ◽  
Dictyostelium Discoideum ◽  
Growth And Development ◽  
Molecular Mechanisms ◽  
Calcium Regulation ◽  
Contractile Vacuole ◽  
Rich Medium ◽  
Immunofluorescence Analysis

In Dictyostelium discoideum, the Ca2+-ATPase, PAT1, is localized to membranes of the contractile vacuole and its expression is upregulated substantially when the cells are grown in Ca2+-rich medium. In this study, we have analyzed the cellular/molecular mechanisms regulating PAT1 expression and examined the role of PAT1 and the contractile vacuole in Ca2+ regulation. During both growth and development, Dictyostelium cells respond to low millimolar concentrations of extracellular Ca2+ and upregulate PAT1 in a few hours. This process is dependent on protein synthesis and the serine/threonine phosphatase, calcineurin. Immunofluorescence analysis indicates that the upregulated PAT1 is associated mainly with the contractile vacuole, but it is also on the plasma membrane. This latter finding suggests that the contractile vacuole fuses with the plasma membrane to eliminate excess intracellular Ca2+. In support of this idea, it was observed that conditions which impair contractile vacuolar function reduce the rate of Ca2+ secretion. It was also found that cells deficient in PAT1, due to the expression of antisense patA RNA or to the presence of calcineurin antagonists, grow normally in low Ca2+ medium but poorly or not at all in high Ca2+ medium. Together, these results suggest that PAT1 and the contractile vacuole are components of a Ca2+ sequestration and excretion pathway, which functions to help maintain Ca2+ homeostasis, especially under conditions of Ca2+ stress.

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