scholarly journals Intracellular localization of p40, a protein identified in a preparation of lysosomal membranes

2006 ◽  
Vol 395 (1) ◽  
pp. 39-47 ◽  
Author(s):  
Marielle Boonen ◽  
Isabelle Hamer ◽  
Muriel Boussac ◽  
Anne-Françoise Delsaute ◽  
Bruno Flamion ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Intracellular Localization ◽  
Subcellular Fractionation ◽  
Lysosomal Membrane ◽  
Knowing That ◽  
Lysosomal Membrane Proteins ◽  
Green Fluorescent ◽  
Rat Liver Lysosomes ◽  
Lysosome Membrane

Unlike lysosomal soluble proteins, few lysosomal membrane proteins have been identified. Rat liver lysosomes were purified by centrifugation on a Nycodenz density gradient. The most hydrophobic proteins were extracted from the lysosome membrane preparation and were identified by MS. We focused our attention on a protein of approx. 40 kDa, p40, which contains seven to ten putative transmembrane domains and four lysosomal consensus sorting motifs in its sequence. Knowing that preparations of lysosomes obtained by centrifugation always contain contaminant membranes, we combined biochemical and morphological methods to analyse the subcellular localization of p40. The results of subcellular fractionation of mouse liver homogenates validate the lysosomal residence of p40. In particular, a density shift of lysosomes induced by Triton WR-1339 similarly affected the distributions of p40 and β-galactosidase, a lysosomal marker protein. We confirmed by fluorescence microscopy on eukaryotic cells transfected with p40 or p40–GFP (green fluorescent protein) constructs that p40 is localized in lysosomes. A first molecular characterization of p40 in transfected Cos-7 cells revealed that it is an unglycosylated protein tightly associated with membranes. Taken together, our results strongly support the hypothesis that p40 is an authentic lysosomal membrane protein.

Abstract 23: Characterization of MitoTimer, a Novel Tool for Monitoring Mitochondrial Turnover

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Christine A Thornton ◽  
Allen M Andres ◽  
Genaro Hernandez ◽  
Jon Sin ◽  
Roberta Gottlieb
Keyword(s):  
Protein Import ◽  
Spatial Information ◽  
Fluorescent Protein ◽  
Mitochondrial Dynamics ◽  
Inducible Promoter ◽  
Subcellular Fractionation ◽  
Fluorescent Reporter ◽  
Hek 293 ◽  
Mitochondrial Turnover

Fluorescent Timer, or DsRed1-E5, is a mutant of the red fluorescent protein, dsRed, developed by Terskikh and colleagues. Its fluorescence shifts over time from green to red as the protein matures. This molecular clock gives temporal and spatial information on protein turnover. To visualize mitochondrial turnover, we targeted Timer to the mitochondrial matrix with a mitochondrial targeting sequence (coined “MitoTimer”) and cloned it into a tetracycline-inducible promoter construct to regulate its expression. Here we report characterization of this novel fluorescent reporter for mitochondrial dynamics. Tet-On HEK 293 cells were transfected with pTRE-tight-MitoTimer and induced production with doxycycline. Mitochondrial distribution was demonstrated by fluorescence microscopy and verified by subcellular fractionation and western blot analysis. Doxycycline addition for as little as 1hr was sufficient to label mitochondria. MitoTimer was detected as early as 4hr following doxycycline addition, and persisted in mitochondria for at least 72hr. The color-specific conformation of MitoTimer was stable after fixation with 4% paraformaldehyde. MitoTimer matured to red fluorescence within 48hr, at which time a second pulse of doxycycline induced expression of green (immature) MitoTimer which was selectively incorporated into a subset of mitochondria actively engaged in protein import. The extent of new protein incorporation during a second pulse was increased under conditions of mito-biogenesis and reduced if mitochondrial membrane potential was dissipated. We conclude that MitoTimer can be used to monitor mitophagy and biogenesis.

Automatic Counting of Intra-Cellular Ribonucleo-Protein Aggregates in Saccharomyces cerevisiae Using a Textural Approach

2019 ◽  
Vol 25 (1) ◽  
pp. 164-179
Author(s):  
Ambroise Marin ◽  
Emmanuel Denimal ◽  
Lucie Bertheau ◽  
Stéphane Guyot ◽  
Ludovic Journaux ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fluorescent Protein ◽  
Protein Aggregates ◽  
Point Of View ◽  
Zernike Moment ◽  
Two Photon ◽  
Haralick Features ◽  
Green Fluorescent ◽  
Two Photon Fluorescence

AbstractIn the context of microbiology, recent studies show the importance of ribonucleo-protein aggregates (RNPs) for the understanding of mechanisms involved in cell responses to specific environmental conditions. The assembly and disassembly of aggregates is a dynamic process, the characterization of the stage of their evolution can be performed by the evaluation of their number. The aim of this study is to propose a method to automatically determine the count of RNPs. We show that the determination of a precise count is an issue by itself and hence, we propose three textural approaches: a classical point of view using Haralick features, a frequency point of view with generalized Fourier descriptors, and a structural point of view with Zernike moment descriptors (ZMD). These parameters are then used as inputs for a supervised classification in order to determine the most relevant. An experiment using a specific Saccharomyces cerevisiae strain presenting a fusion between a protein found in RNPs (PAB1) and the green fluorescent protein was performed to benchmark this approach. The fluorescence was observed with two-photon fluorescence microscopy. Results show that the textural approach, by mixing ZMD with Haralick features, allows for the characterization of the number of RNPs.

scholarly journals A dileucine signal situated in the C-terminal tail of the lysosomal membrane protein p40 is responsible for its targeting to lysosomes

2008 ◽  
Vol 414 (3) ◽  
pp. 431-440 ◽  
Author(s):  
Marielle Boonen ◽  
Roberta Rezende de Castro ◽  
Gaëlle Cuvelier ◽  
Isabelle Hamer ◽  
Michel Jadot
Keyword(s):  
Subcellular Fractionation ◽  
Lysosomal Membrane ◽  
Consensus Sequences ◽  
Cell Surface Biotinylation ◽  
Dileucine Motif ◽  
Lysosomal Sorting ◽  
Critical Residues ◽  
Lysosomal Membrane Proteins ◽  
Golgi Network

Transport of newly synthesized lysosomal membrane proteins from the TGN (trans-Golgi network) to the lysosomes is due to the presence of specific signals in their cytoplasmic domains that are recognized by cytosolic adaptors. p40, a hypothetical transporter of 372 amino acids localized in the lysosomal membrane, contains four putative lysosomal sorting motifs in its sequence: three of the YXXϕ-type (Y6QLF, Y106VAL, Y333NGL) and one of the [D/E]XXXL[L/I]-type (EQERL360L361). To test the role of these motifs in the biosynthetic transport of p40, we replaced the most critical residues of these consensus sequences, the tyrosine residue or the leucine–leucine pair, by alanine or alanine–valine respectively. We analysed the subcellular localization of the mutated p40 proteins in transfected HeLa cells by confocal microscopy and by biochemical approaches (subcellular fractionation on self-forming Percoll density gradients and cell surface biotinylation). The results of the present study show that p40 is mistargeted to the plasma membrane when its dileucine motif is disrupted. No role of the tyrosine motifs could be put forward. Taken together, our results provide evidence that the sorting of p40 from the TGN to the lysosomes is directed by the dileucine EQERL360L361 motif situated in its C-terminal tail.

scholarly journals Derivation and Characterization of EGFP-Labeled Rabbit Limbal Mesenchymal Stem Cells and Their Potential for Research in Regenerative Ophthalmology

Biomedicines ◽  
2021 ◽  
Vol 9 (9) ◽  
pp. 1134
Author(s):  
Julia I. Khorolskaya ◽  
Daria A. Perepletchikova ◽  
Daniel V. Kachkin ◽  
Kirill E. Zhurenkov ◽  
Elga I. Alexander-Sinkler ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Fluorescent Protein ◽  
Rabbit Model ◽  
Stem Cell Markers ◽  
Epithelial Stem Cells ◽  
Limbal Stem Cell ◽  
Green Fluorescent

The development of cell-based approaches to the treatment of various cornea pathologies, including limbal stem cell deficiency (LSCD), is an area of current interest in regenerative biomedicine. In this context, the shortage of donor material is urgent, and limbal mesenchymal stem cells (L-MSCs) may become a promising cell source for the development of these novel approaches, being established mainly within the rabbit model. In this study, we obtained and characterized rabbit L-MSCs and modified them with lentiviral transduction to express the green fluorescent protein EGFP (L-MSCs-EGFP). L-MSCs and L-MSCs-EGFP express not only stem cell markers specific for mesenchymal stem cells but also ABCG2, ABCB5, ALDH3A1, PAX6, and p63a specific for limbal epithelial stem cells (LESCs), as well as various cytokeratins (3/12, 15, 19). L-MSCs-EGFP have been proven to differentiate into adipogenic, osteogenic, and chondrogenic directions, as well as to transdifferentiate into epithelial cells. The possibility of using L-MSCs-EGFP to study the biocompatibility of various scaffolds developed to treat corneal pathologies was demonstrated. L-MSCs-EGFP may become a useful tool for studying regenerative processes occurring during the treatment of various corneal pathologies, including LSCD, with the use of cell-based technologies.

scholarly journals Dynamin-related Protein Drp1 Is Required for Mitochondrial Division in Mammalian Cells

2001 ◽  
Vol 12 (8) ◽  
pp. 2245-2256 ◽  
Author(s):  
Elena Smirnova ◽  
Lorena Griparic ◽  
Dixie-Lee Shurland ◽  
Alexander M. van der Bliek
Keyword(s):  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Subcellular Fractionation ◽  
Related Protein ◽  
Direct Role ◽  
Mitochondrial Division ◽  
Transfected Cells ◽  
Green Fluorescent ◽  
Time Lapse Photography

Mutations in the human dynamin-related protein Drp1 cause mitochondria to form perinuclear clusters. We show here that these mitochondrial clusters consist of highly interconnected mitochondrial tubules. The increased connectivity between mitochondria indicates that the balance between mitochondrial division and fusion is shifted toward fusion. Such a shift is consistent with a block in mitochondrial division. Immunofluorescence and subcellular fractionation show that endogenous Drp1 is localized to mitochondria, which is also consistent with a role in mitochondrial division. A direct role in mitochondrial division is suggested by time-lapse photography of transfected cells, in which green fluorescent protein fused to Drp1 is concentrated in spots that mark actual mitochondrial division events. We find that purified human Drp1 can self-assemble into multimeric ring-like structures with dimensions similar to those of dynamin multimers. The structural and functional similarities between dynamin and Drp1 suggest that Drp1 wraps around the constriction points of dividing mitochondria, analogous to dynamin collars at the necks of budding vesicles. We conclude that Drp1 contributes to mitochondrial division in mammalian cells.

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