Faculty Opinions recommendation of Redistribution and differential extraction of soluble proteins in permeabilized cultured cells. Implications for immunofluorescence microscopy.

2012 ◽  
Author(s):  
Michael Klymkowsky
Keyword(s):  
Immunofluorescence Microscopy ◽  
Cultured Cells ◽  
Soluble Proteins ◽  
Differential Extraction

Redistribution and differential extraction of soluble proteins in permeabilized cultured cells. Implications for immunofluorescence microscopy

1992 ◽  
Vol 101 (4) ◽  
pp. 731-743 ◽  
Author(s):  
M.A. Melan ◽  
G. Sluder
Keyword(s):  
Immunofluorescence Microscopy ◽  
Cultured Cells ◽  
Fluorescent Proteins ◽  
Protein S ◽  
Soluble Proteins ◽  
Differential Extraction ◽  
Preferential Localization ◽  
Net Charges ◽  
Triton X

Immunofluorescence microscopy is widely used to characterize the cellular distribution of both soluble and structural proteins. Control experiments generally address only the specificity of the antibodies used. The permeabilization/fixation conditions used to prepare cells for antibody application are assumed to preserve faithfully the in vivo distributions of the protein(s) being examined. We systematically tested the extent to which soluble proteins are redistributed into inappropriate locations and are differentially extracted from native locations during the permeabilization and fixation of the cells before antibody application. We separately introduce six soluble FITC-conjugated proteins of different net charges and sizes into living cultured cells. The labeled proteins do not adhere to the external surfaces of living cells and are evenly distributed throughout the cytoplasm with the larger proteins being excluded from the nucleus. The cells are then prepared as if for immunofluorescence using several conditions that encompass many of the methods commonly used for this purpose. Cells permeabilized with 0.1-0.2% Triton X-100 before fixation with 3.7% paraformaldehyde show a striking localization of all but one of the test proteins to the nucleus and/or nucleoli of 60–80% of labeled cells. Punctate cytoplasmic labeling and cytoskeletal-like arrays of labeled protein are also observed. Extraction with 1% detergent prior to fixation removes most but not always all of the exogenous proteins from the cell remnants. Permeabilization of cells with 0.1% detergent after paraformaldehyde fixation leaves a reticular, uneven cytoplasmic distribution of the labeled proteins, and some of the larger proteins are redistributed to the nuclei. Direct fixation/permeabilization with -20 degrees C methanol largely preserves the in vivo distributions of fluorescent proteins with some preferential localization of these proteins to nuclei, nucleoli and the perinuclear region. These results show that misleading apparent localizations of soluble proteins can result from their redistribution and/or differential extraction during the preparation of cells for primary antibody application.

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.

Macromolecular specializations that mediate lateral interactions between microfilaments and the membrane at focal contacts

1992 ◽  
Vol 50 (1) ◽  
pp. 724-725
Author(s):  
Steven J. Samuelsson ◽  
Paul W. Luther ◽  
David W. Pumplin ◽  
Robert J. Bloch
Keyword(s):  
Immunofluorescence Microscopy ◽  
Cultured Cells ◽  
Stress Fiber ◽  
Stress Fibers ◽  
Focal Contact ◽  
Lateral Interactions ◽  
Matrix Assembly ◽  
Cytoplasmic Surface ◽  
Focal Contacts ◽  
Specific Proteins

Focal contacts are membrane specializations of cultured cells where stress fibers terminate and where the cell is most closely applied to the substrate. The organization of this cytoskeletal-membrane-extracellular matrix assembly has been well characterized. Immunofluorescence microscopy has shown that two focal contact-specific proteins, vinculin and talin, colocalize with microfilaments for several microns before the stress fiber terminates. This result raises the question of whether microfilament-membrane interactions are limited to the ends of microfilaments, or if lateral interactions also occur. We addressed this question by examining the cytoplasmic surface of isolated focal contacts in detail.

scholarly journals Association of microtubule-associated protein 2 (MAP 2) with microtubules and intermediate filaments in cultured brain cells.

1983 ◽  
Vol 96 (6) ◽  
pp. 1523-1531 ◽  
Author(s):  
G S Bloom ◽  
R B Vallee
Keyword(s):  
Intermediate Filaments ◽  
Immunofluorescence Microscopy ◽  
Cultured Cells ◽  
Primary Cultures ◽  
Immune Serum ◽  
Brain Cells ◽  
Intermediate Filament Protein ◽  
Antibody Staining ◽  
Filament Protein

The classification of MAP 2 as a microtubule-associated protein is based on its affinity for microtubules in vitro and its filamentous distribution in cultured cells. We sought to determine whether MAP 2 is also able to bind in situ to organelles other than microtubules. For this purpose, primary cultures of rat brain cells were stained for immunofluorescence microscopy with a rabbit anti-MAP 2 antibody prepared in our laboratory, as well as with antibodies to vimentin, an intermediate filament protein, and to tubulin, the major subunit of microtubules. MAP 2 was present on cytoplasmic fibers in neurons and in a subpopulation of the flat cells present in the cultures. Our observations were concentrated on the flat cells because of their suitability for high-resolution immunofluorescence microscopy. Double antibody staining revealed co-localization of MAP 2 with both tubulin and vimentin in the flat cells. Pretreatment of the cultures with vinblastine resulted in the redistribution of MAP 2 into perinuclear cables that contained vimentin. Tubulin paracrystals were not stained by anti-MAP 2. In cells extracted with digitonin, the normal fibrillar distribution of MAP 2 was resistant to several treatments (PIPES buffer plus 10 mM Ca++, phosphate buffer at pH 7 or 9) that induced depolymerization of microtubules, but not intermediate filaments. Staining of the primary brain cells was not observed with preimmune serum nor with immune serum adsorbed prior to use with pure MAP 2. We detected MAP 2 on intermediate filaments not only with anti-MAP 2 serum, but also with affinity purified anti-MAP 2 and with a monoclonal anti-MAP 2 prepared in another laboratory. We conclude from these experiments that material recognized by anti-MAP 2 antibodies associates with both microtubules and intermediate filaments. We propose that one function of MAP 2 is to cross-link the two types of cellular filaments.

scholarly journals Identification of EBP50: A PDZ-containing Phosphoprotein that Associates with Members of the Ezrin-Radixin-Moesin Family

1997 ◽  
Vol 139 (1) ◽  
pp. 169-179 ◽  
Author(s):  
David Reczek ◽  
Mark Berryman ◽  
Anthony Bretscher
Keyword(s):  
Immunofluorescence Microscopy ◽  
Cultured Cells ◽  
Bovine Brain ◽  
Pdz Domains ◽  
Binding Partners ◽  
Phosphatase Treatment ◽  
Membrane Attachment ◽  
Polarized Epithelia ◽  
The Brain ◽  
Terminal Domains

Members of the ezrin-radixin-moesin (ERM) family of membrane–cytoskeletal linking proteins have NH2- and COOH-terminal domains that associate with the plasma membrane and the actin cytoskeleton, respectively. To search for ERM binding partners potentially involved in membrane association, tissue lysates were subjected to affinity chromatography on the immobilized NH2-terminal domains of ezrin and moesin, which comprise the ezrin-radixin-moesin–association domain (N-ERMAD). A collection of polypeptides at 50–53 kD from human placenta and at 58-59 kD from bovine brain bound directly to both N-ERMADs. The 50–53-kD placental proteins migrated as a major 50-kD species after phosphatase treatment, indicating that the heterogeneity is due to different phosphorylation states. We refer to these polypeptides as ERM-binding phosphoprotein 50 (EBP50). Sequence analysis of human EBP50 was used to identify an ∼2-kb human cDNA that encodes a 357-residue polypeptide. Recombinant EBP50 binds tightly to the N-ERMADs of ezrin and moesin. Peptide sequences from the brain candidate indicated that it is closely related to EBP50. EBP50 has two PSD-95/DlgA/ZO-1–like (PDZ) domains and is most likely a homologue of rabbit protein cofactor, which is involved in the protein kinase A regulation of the renal brush border Na+/H+ exchanger. EBP50 is widely distributed in tissues, and is particularly enriched in those containing polarized epithelia. Immunofluorescence microscopy of cultured cells and tissues revealed that EBP50 colocalizes with actin and ezrin in the apical microvilli of epithelial cells, and immunoelectron microscopy demonstrated that it is specifically associated with the microvilli of the placental syncytiotrophoblast. Moreover, EBP50 and ezrin can be coimmunoprecipitated as a complex from isolated human placental microvilli. These findings show that EBP50 is a physiologically relevant ezrin binding protein. Since PDZ domains are known to mediate associations with integral membrane proteins, one mode of membrane attachment of ezrin is likely to be mediated through EBP50.

Immunofluorescence detection of ezrin/radixin/moesin (ERM) proteins with their carboxyl-terminal threonine phosphorylated in cultured cells and tissues

1999 ◽  
Vol 112 (8) ◽  
pp. 1149-1158 ◽  
Author(s):  
K. Hayashi ◽  
S. Yonemura ◽  
T. Matsui ◽  
S. Tsukita
Keyword(s):  
Plasma Membranes ◽  
Immunofluorescence Microscopy ◽  
Cultured Cells ◽  
Tissue Type ◽  
Cross Linking ◽  
Dependent Manner ◽  
Cortical Actin ◽  
Erm Proteins

Ezrin/radixin/moesin (ERM) proteins are thought to play an important role in organizing cortical actin-based cytoskeletons through cross-linkage of actin filaments with integral membrane proteins. Recent in vitro biochemical studies have revealed that ERM proteins phosphorylated on their COOH-terminal threonine residue (CPERMs) are active in their cross-linking activity, but this has not yet been evaluated in vivo. To immunofluorescently visualize CPERMs in cultured cells as well as tissues using a mAb specific for CPERMs, we developed a new fixation protocol using trichloroacetic acid (TCA) as a fixative. Immunoblotting analyses in combination with immunofluorescence microscopy showed that TCA effectively inactivated soluble phosphatases, which maintained the phosphorylation level of CPERMs during sample processing for immunofluorescence staining. Immunofluorescence microscopy with TCA-fixed samples revealed that CPERMs were exclusively associated with plasma membranes in a variety of cells and tissues, whereas total ERM proteins were distributed in both the cytoplasm and plasma membranes. Furthermore, the amounts of CPERMs were shown to be regulated in a cell and tissue type-dependent manner. These findings favored the notion that phosphorylation of the COOH-terminal threonine plays a key role in the regulation of the cross-linking activity of ERM proteins in vivo.

Syncytioskeletons in choriocarcinoma in culture

1984 ◽  
Vol 66 (1) ◽  
pp. 1-20
Author(s):  
C.D. Ockleford ◽  
L. Dearden ◽  
R.A. Badley
Keyword(s):  
Indirect Immunofluorescence ◽  
Immunofluorescence Microscopy ◽  
Cultured Cells ◽  
Cortical Layers ◽  
Dense Population ◽  
Bewo Cells ◽  
Indirect Immunofluorescence Microscopy ◽  
Microtubule Organizing Centres ◽  
Transmission Electron ◽  
Cytoskeletal Elements

Indirect immunofluorescence microscopy using anti-actin serum has been used to investigate the distribution of actin-containing polymers in BeWo cells. This cell line, derived from a human choriocarcinoma, contains tissue that, like its tissue of origin, is partly syncytial. The syncytial nature has been inferred from study of Nomarski optical sections and from transmission electron microscopy. The multinucleated plaques of tissue possess a syncytioskeleton with a number of actin-containing features characteristic of cultured cells. These include stress fibres, cortical layers and ruffled membranes. Other actin-containing structures are more typical of the related non-pathological syncytiotrophoblast. These include a dense population of microvilli. The overall organization of the actin syncytioskeletons bears no obvious relationship to the number or position of nuclei in the syncytium. Indirect immunofluorescence microscopy has also been employed to localize the protein tubulin in BeWo cells. The microtubules do not appear to be spatially organized by a particular nucleus. Rather, there are numerous microtubule-organizing centres (MTOCs) that exist in the cytoplasm and do not have the expected numerical and positional relationship to nuclei. From these data it appears that polymeric cytoskeletal elements in these syncytia are organized in a manner not immediately subordinate to syncytial nuclei.

scholarly journals Rapid Dissociation of HIV-1 from Cultured Cells Severely Limits Infectivity Assays, Causes the Inactivation Ascribed to Entry Inhibitors, and Masks the Inherently High Level of Infectivity of Virions

2009 ◽  
Vol 84 (6) ◽  
pp. 3106-3110 ◽  
Author(s):  
Emily J. Platt ◽  
Susan L. Kozak ◽  
James P. Durnin ◽  
Thomas J. Hope ◽  
David Kabat
Keyword(s):  
Immunofluorescence Microscopy ◽  
Cultured Cells ◽  
Cell Entry ◽  
Inhibitory Effects ◽  
Entry Inhibitors ◽  
Ccr5 Antagonists ◽  
High Level ◽  
Hiv 1

ABSTRACT By using immunofluorescence microscopy to observe and analyze freshly made HIV-1 virions adsorbed onto cells, we found that they are inherently highly infectious, rather than predominantly defective as previously suggested. Surprisingly, polycations enhance titers 20- to 30-fold by stabilizing adsorption and preventing a previously undescribed process of rapid dissociation, strongly implying that infectivity assays for many viruses are limited not only by inefficient virus diffusion onto cells but also by a postattachment race between entry and dissociation. This kinetic competition underlies inhibitory effects of CCR5 antagonists and explains why adaptive HIV-1 mutations overcome many cell entry limitations by accelerating entry.

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