Formation, distribution and dissociation of intercellular junctions in the lens

1987 ◽  
Vol 88 (3) ◽  
pp. 351-359
Author(s):  
W.T. Gruijters ◽  
J. Kistler ◽  
S. Bullivant
Keyword(s):  
Membrane Protein ◽  
Immunofluorescence Microscopy ◽  
De Novo ◽  
Intercellular Junctions ◽  
Polar Regions ◽  
Dynamic Aspect ◽  
Outer Cortex ◽  
Specific Component ◽  
Fibre Bundles ◽  
Fibre Width

A 70,000 Mr membrane protein (MP70) has previously been identified as a specific component of lens intercellular junctions. In this paper we use anti-MP70 immunofluorescence microscopy of dissected fibre bundles to study the formation, distribution and dissociation of junctional plaques in the outer cortex region of the sheep lens. Abundant, small junctional plaques are assembled de novo in the broad sides of the elongating fibres near the equatorial lens periphery. In fully elongated, pole-to-pole fibres, junctional plaques are generally larger, and while dispersed on the broad sides of the fibres in the equatorial lens plane, these junctions line up in the middle of the broad and narrow sides of the fibres in the lens polar regions. This precisely defined positioning is independent of junction size and hence cannot solely be explained by the constraints of fibre width. Junctional plaques fragment to smaller sizes and MP70 is cleaved to MP38 in mature, enucleated fibres located in the deeper portions of the lens outer cortex. These results demonstrate a dynamic aspect of lens intercellular junctions and show that they are positioned in a precise fashion, possibly in association with other membrane or cytoskeletal components.

scholarly journals Identification of a 70,000-D protein in lens membrane junctional domains.

1985 ◽  
Vol 101 (1) ◽  
pp. 28-35 ◽  
Author(s):  
J Kistler ◽  
B Kirkland ◽  
S Bullivant
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibodies ◽  
Membrane Protein ◽  
High Frequency ◽  
Immunofluorescence Microscopy ◽  
Eye Lens ◽  
Vertebrate Species ◽  
Outer Cortex ◽  
Lens Fibers ◽  
Sds Page

A 70,000-D membrane protein (MP70), which is restricted to the eye lens fibers and is present in immunologically homologous form in many vertebrate species, has been identified. By use of anti-MP70 monoclonal antibodies for immunofluorescence microscopy and electron microscopy, this polypeptide was localized in lens membrane junctional domains. Both immunofluorescence microscopy and SDS PAGE reveal an abundance of MP70 in the lens outer cortex that coincides with a high frequency of fiber gap junctions in the same region.

scholarly journals Protein structure prediction and design in a biologically-realistic implicit membrane

2019 ◽  
Author(s):  
Rebecca F. Alford ◽  
Patrick J. Fleming ◽  
Karen G. Fleming ◽  
Jeffrey J. Gray
Keyword(s):  
Protein Structure ◽  
Amino Acid ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Structure Prediction ◽  
Protein Design ◽  
Structure Prediction ◽  
De Novo ◽  
Computational Design ◽  
Amino Acid Distribution

ABSTRACTProtein design is a powerful tool for elucidating mechanisms of function and engineering new therapeutics and nanotechnologies. While soluble protein design has advanced, membrane protein design remains challenging due to difficulties in modeling the lipid bilayer. In this work, we developed an implicit approach that captures the anisotropic structure, shape of water-filled pores, and nanoscale dimensions of membranes with different lipid compositions. The model improves performance in computational bench-marks against experimental targets including prediction of protein orientations in the bilayer, ΔΔG calculations, native structure dis-crimination, and native sequence recovery. When applied to de novo protein design, this approach designs sequences with an amino acid distribution near the native amino acid distribution in membrane proteins, overcoming a critical flaw in previous membrane models that were prone to generating leucine-rich designs. Further, the proteins designed in the new membrane model exhibit native-like features including interfacial aromatic side chains, hydrophobic lengths compatible with bilayer thickness, and polar pores. Our method advances high-resolution membrane protein structure prediction and design toward tackling key biological questions and engineering challenges.Significance StatementMembrane proteins participate in many life processes including transport, signaling, and catalysis. They constitute over 30% of all proteins and are targets for over 60% of pharmaceuticals. Computational design tools for membrane proteins will transform the interrogation of basic science questions such as membrane protein thermodynamics and the pipeline for engineering new therapeutics and nanotechnologies. Existing tools are either too expensive to compute or rely on manual design strategies. In this work, we developed a fast and accurate method for membrane protein design. The tool is available to the public and will accelerate the experimental design pipeline for membrane proteins.

scholarly journals Treponema pallidum Disrupts VE-Cadherin Intercellular Junctions and Traverses Endothelial Barriers Using a Cholesterol-Dependent Mechanism

2021 ◽  
Vol 12 ◽  
Author(s):  
Karen V. Lithgow ◽  
Emily Tsao ◽  
Ethan Schovanek ◽  
Alloysius Gomez ◽  
Leigh Anne Swayne ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Lipid Raft ◽  
Vascular Endothelium ◽  
Immunofluorescence Microscopy ◽  
Treponema Pallidum ◽  
Intercellular Junctions ◽  
Endothelial Barriers ◽  
Junctional Protein ◽  
Gain Access ◽  
Dependent Mechanism

Treponema pallidum subspecies pallidum, the causative agent of syphilis, traverses the vascular endothelium to gain access to underlying tissue sites. Herein, we investigate the mechanisms associated with T. pallidum traversal of endothelial barriers. Immunofluorescence microscopy reveals that a subpopulation of T. pallidum localizes to intercellular junctions and that viable T. pallidum, as well as a T. pallidum vascular adhesin (Tp0751), disrupts the architecture of the main endothelial junctional protein VE-cadherin. Intriguingly, in this study we show that T. pallidum traverses endothelial barriers with no disruption in barrier permeability. Furthermore, barrier traversal by T. pallidum is reduced by pretreatment of endothelial cells with filipin, an inhibitor that blocks cholesterol-mediated endocytosis. Collectively, these results suggest that T. pallidum can use a cholesterol-dependent, lipid raft-mediated endocytosis mechanism to traverse endothelial barriers. Further, treponemal localization to, and disruption of, intercellular junctions suggests that a paracellular route may also be utilized, a dual traversal strategy that has also been observed to occur for leukocytes and other invasive bacteria.

scholarly journals Autophagosome formation in relation to the endoplasmic reticulum

2020 ◽  
Vol 27 (1) ◽  
Author(s):  
Yo-hei Yamamoto ◽  
Takeshi Noda
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Protein ◽  
De Novo ◽  
Transmembrane Protein ◽  
Lipid Transfer ◽  
Membrane Structures ◽  
Autophagosome Formation ◽  
Selective Autophagy ◽  
The Relationship ◽  
Er Membrane

Abstract Autophagy is a process in which a myriad membrane structures called autophagosomes are formed de novo in a single cell, which deliver the engulfed substrates into lysosomes for degradation. The size of the autophagosomes is relatively uniform in non-selective autophagy and variable in selective autophagy. It has been recently established that autophagosome formation occurs near the endoplasmic reticulum (ER). In this review, we have discussed recent advances in the relationship between autophagosome formation and endoplasmic reticulum. Autophagosome formation occurs near the ER subdomain enriched with phospholipid synthesizing enzymes like phosphatidylinositol synthase (PIS)/CDP-diacylglycerol-inositol 3-phosphatidyltransferase (CDIPT) and choline/ethanolamine phosphotransferase 1 (CEPT1). Autophagy-related protein 2 (Atg2), which is involved in autophagosome formation has a lipid transfer capacity and is proposed to directly transfer the lipid molecules from the ER to form autophagosomes. Vacuole membrane protein 1 (VMP1) and transmembrane protein 41b (TMEM41b) are ER membrane proteins that are associated with the formation of the subdomain. Recently, we have reported that an uncharacterized ER membrane protein possessing the DNAJ domain, called ERdj8/DNAJC16, is associated with the regulation of the size of autophagosomes. The localization of ERdj8/DNAJC16 partially overlaps with the PIS-enriched ER subdomain, thereby implying its association with autophagosome size determination.

scholarly journals Centrosomal protein centrin is not detectable during early pre-implantation development but reappears during late blastocyst stage in porcine embryos

Reproduction ◽  
2006 ◽  
Vol 132 (3) ◽  
pp. 423-434 ◽  
Author(s):  
G Manandhar ◽  
D Feng ◽  
Y-J Yi ◽  
L Lai ◽  
J Letko ◽  
...  
Keyword(s):  
Western Blotting ◽  
Immunofluorescence Microscopy ◽  
Cultured Cells ◽  
De Novo ◽  
Germinal Vesicle ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Embryonic Cells ◽  
Perinuclear Area

Centrin is an evolutionarily conserved 20 kDa, Ca+2-binding, calmodulin-related protein associated with centrioles and basal bodies of phylogenetically diverse eukaryotic cells. Earlier studies have shown that residual centrosomes of non-rodent mammalian spermatozoa retain centrin and, in theory, could contribute this protein for the reconstruction of the zygotic centrosome after fertilization. The present work shows that CEN2 and CEN3 mRNA were detected in germinal vesicle-stage (GV) oocytes, MII oocytes, and pre-implantation embryos from the two-cell through the blastocyst stage, but not in spermatozoa. Boar ejaculated spermatozoa possess centrin as revealed by immunofluorescence microscopy and western blotting. Immature, GV oocytes possess speckles of centrin particles in the perinuclear area, visualized by immunofluorescence microscopy and exhibit a 19 kDa band revealed by western blotting. Mature MII stage oocytes lacked centrin that could be detected by immunofluorescence or western blotting. The sperm centrin was lost in zygotes afterin vitrofertilization. It was not detectable in embryos by immunofluorescence microscopy until the late blastocyst stage. Embryonic centrin first appeared as fine speckles in the perinuclear area of some interphase blastocyst cells and as putative centrosomes of the spindle poles of dividing cells. The cells of the hatched blastocysts developed centrin spots comparable with those of the cultured cells. Some blastomeres displayed undefined curved plate-like centrin-labeled structures. Anti-centrin antibody labeled interphase centrosomes of cultured pig embryonic fibroblast cells as distinct spots in the juxtanuclear area. Enucleated pig oocytes reconstructed by electrofusion with pig fibroblasts displayed centrin of the donor cell during the early stages of nuclear decondensation but became undetectable in the late pronuclear or cleavage stages. These observations suggest that porcine zygotes and pre-blastocyst embryonic cells lack centrin and do not retain exogenously incorporated centrin. The early embryonic centrosomes function without centrin. Centrin in the blastocyst stage embryos is likely a result ofde novosynthesis at the onset of differentiation of the pluripotent blastomeres.

De novo synthesis of a plasmid containing the Chlamydia abortus outer membrane protein A (OMP A) gene

2017 ◽  
pp. 21-24
Author(s):  
A.T Zhumabek . ◽  
◽  
А.А. Zhylkibayev ◽  
А.D. Kairzhanova ◽  
S.Z. Eskendirova ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
De Novo ◽  
Protein A ◽  
De Novo Synthesis ◽  
Chlamydia Abortus ◽  
Outer Membrane Protein A

scholarly journals Design of self-assembling transmembrane helical bundles to elucidate principles required for membrane protein folding and ion transport

2017 ◽  
Vol 372 (1726) ◽  
pp. 20160214 ◽  
Author(s):  
Nathan H. Joh ◽  
Gevorg Grigoryan ◽  
Yibing Wu ◽  
William F. DeGrado
Keyword(s):  
Protein Folding ◽  
Membrane Protein ◽  
Protein Design ◽  
De Novo ◽  
Cell Signalling ◽  
Membrane Protein Folding ◽  
Cellular Processes ◽  
Membrane Pores ◽  
Self Assembling ◽  
And Function

Ion transporters and channels are able to identify and act on specific substrates among myriads of ions and molecules critical to cellular processes, such as homeostasis, cell signalling, nutrient influx and drug efflux. Recently, we designed Rocker, a minimalist model for Zn 2+ /H + co-transport. The success of this effort suggests that de novo membrane protein design has now come of age so as to serve a key approach towards probing the determinants of membrane protein folding, assembly and function. Here, we review general principles that can be used to design membrane proteins, with particular reference to helical assemblies with transport function. We also provide new functional and NMR data that probe the dynamic mechanism of conduction through Rocker. This article is part of the themed issue ‘Membrane pores: from structure and assembly, to medicine and technology’.

scholarly journals In situ serial crystallography for rapid de novo membrane protein structure determination

2018 ◽  
Vol 1 (1) ◽  
Author(s):  
Chia-Ying Huang ◽  
Vincent Olieric ◽  
Nicole Howe ◽  
Rangana Warshamanage ◽  
Tobias Weinert ◽  
...  
Keyword(s):  
Protein Structure ◽  
Membrane Protein ◽  
Structure Determination ◽  
De Novo ◽  
Protein Structure Determination ◽  
Membrane Protein Structure ◽  
Membrane Protein Structure Determination ◽  
Serial Crystallography

scholarly journals Immunolocalization of MP70 in lens fiber 16-17-nm intercellular junctions.

1987 ◽  
Vol 104 (3) ◽  
pp. 565-572 ◽  
Author(s):  
W T Gruijters ◽  
J Kistler ◽  
S Bullivant ◽  
D A Goodenough
Keyword(s):  
Freeze Fracture ◽  
Intercellular Junctions ◽  
Membrane Structures ◽  
Outer Cortex ◽  
Double Membrane ◽  
Fiber Cells ◽  
Thin Section Electron Microscopy ◽  
Different Types ◽  
Section Electron ◽  
Section Electron Microscopy

Thin section electron microscopy reveals two different types of membrane interactions between the fiber cells of bovine lens. Monoclonal antibodies against lens membrane protein MP70 (Kistler et al., 1985, J. Cell Biol., 101:28-35) bound exclusively to the 16-17-nm intercellular junctions. MP70 localization was most dramatic in the lens outer cortex and strongly reduced deeper in the lens. In contrast, the 12-nm double membrane structures and single membranes were consistently unlabeled. In freeze-fracture replicas with adherent cortical fiber membranes, MP70 was immunolocalized in the junctional plaques which closely resemble the gap junctions in other tissues. MP70 is thus likely to be associated with intercellular communication in the lens.

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