脂筏与精子膜信号传导

脂筏是细胞膜内由特殊脂质与蛋白质构成的微域。小窝是脂筏的一种形式,小窝标记蛋白有小窝蛋白和小窝舟蛋白。脂筏或小窝与生物信号传导、细胞蛋白转运和胆固醇平衡有关。最近实验证实哺乳动物精子膜具有脂筏结构,脂筏与膜胆固醇外逸对于启动受精的信号传导具有重要作用。     

Variable-angle epifluorescence microscopy: a new way to look at protein dynamics in the plant cell cortex

Live-cell microscopy imaging of fluorescent-tagged fusion proteins is an essential tool for cell biologists. Total internal reflection fluorescence microscopy (TIRFM) has joined confocal microscopy as a complementary system for the imaging of cell surface protein dynamics in mammalian and yeast systems because of its high temporal and spatial resolution. Here we present an alternative to TIRFM, termed variable-angle epifluorescence microscopy (VAEM), for the visualization of protein dynamics at or near the plasma membrane of plant epidermal cells and root hairs in whole, intact seedlings that provides high-signal, low-background and near real-time imaging. VAEM uses highly oblique subcritical incident angles to decrease background fluorophore excitation. We discuss the utilities and advantages of VAEM for imaging of fluorescent fusion-tagged marker proteins in studying cortical cytoskeletal and membrane proteins. We believe that the application of VAEM will be an invaluable imaging tool for plant cell biologists.     

Genotype-independent transmission of transgenic fluorophore protein by boar spermatozoa

Recently, we generated transposon-transgenic boars (Sus scrofa), which carry three monomeric copies of a fluorophore marker gene. Amazingly, a ubiquitous fluorophore expression in somatic, as well as in germ cells was found. Here, we characterized the prominent fluorophore load in mature spermatozoa of these animals. Sperm samples were analyzed for general fertility parameters, sorted according to X and Y chromosome-bearing sperm fractions, assessed for potential detrimental effects of the reporter, and used for inseminations into estrous sows. Independent of their genotype, all spermatozoa were uniformly fluorescent with a subcellular compartmentalization of the fluorophore protein in postacrosomal sheath, mid piece and tail. Transmission of the fluorophore protein to fertilized oocytes was shown by confocal microscopic analysis of zygotes. The monomeric copies of the transgene segregated during meiosis, rendering a certain fraction of the spermatozoa non-transgenic (about 10% based on analysis of 74 F1 offspring). The genotype-independent transmission of the fluorophore protein by spermatozoa to oocytes represents a non-genetic contribution to the mammalian embryo.     

A fluorescent cholesterol analogue for observation of free cholesterol in the plasma membrane of live cells

Free cholesterol in mammalian cells resides mostly in the plasma membrane, where it plays an important role in cellular homeostasis. We synthesized a new fluorescent cholesterol analogue that retained an intact alkyl chain and the sterane backbone of cholesterol. The hydroxyl group of cholesterol was converted into an amino group that was covalently linked to the fluorophore tetramethylrhodamine to retain the ability to form hydrogen bonds with adjacent molecules. Incubating live MDCK (Madin–Darby canine kidney) cells with our fluorescent cholesterol analogue resulted in the generation of intense signals that were detected by microscopy at the plasma membrane. Incubation with the analogue exerted minimal, if any, influence on cell growth, indicating that it could serve as a useful tool for analyzing free cholesterol at the plasma membrane.     

Latent fluorophores based on a self-immolative linker strategy and suitable for protease sensing

The self-immolative spacer para-aminobenzyl alcohol (PABA) was used as a key component in the design of new protease-sensitive fluorogenic probes whose parent phenol-based fluorophore is released through an enzyme-initiated domino reaction. First, the conjugation of the phenylacetyl moiety to 7-hydroxycoumarin (umbelliferone) and 7-hydroxy-9 H-(9,9-dimethylacridin-2-one) (DAO) by means of the heterobifunctional PABA linker has led to pro-fluorophores 6a and 6d whose enzyme activation by penicillin amidase was demonstrated. The second part of this study was devoted to the extension of this latent fluorophore strategy to the caspase-3 protease, a key mediator of apoptosis in mammalian cells. Fluorogenic caspase-3 substrates 11 and 13 derived from umbelliferone and DAO, respectively, were prepared. It was demonstrated that pro-fluorophore 11 is a sensitive fluorimetric reagent for the detection of this cysteine protease. Furthermore, in vitro assays with fluorogenic probe 13 showed a deleterious effect of biological thiols on fluorescence of the released acridinone fluorophore DAO that, to our knowledge, had not been reported until now.     

Tracking the dynamic interplay between bacterial and host factors during pathogen‐induced vacuole rupture in real time

Escape into the host cell cytosol following invasion of mammalian cells is a common strategy used by invasive pathogens. This requires membrane rupture of the vesicular or vacuolar compartment formed around the bacteria after uptake into the host cell. The mechanism of pathogen‐induced disassembly of the vacuolar membrane is poorly understood. We established a novel, robust and sensitive fluorescence microscopy method that tracks the precise time point of vacuole rupture upon uptake of Gram‐negative bacteria. This revealed that the enteroinvasive pathogen Shigella flexneri escapes rapidly, in less than 10 min, from the vacuole. Our method demonstrated the recruitment of host factors, such as RhoA, to the bacterial entry site and their continued presence at the point of vacuole rupture. We found a novel host marker for ruptured vacuoles, galectin‐3, which appears instantly in the proximity of bacteria after escape into the cytosol. Furthermore, we show that the Salmonella effector proteins, SifA and PipB2, stabilize the vacuole membrane inhibiting bacterial escape from the vacuole. Our novel approach to track vacuole rupture is ideally suited for high‐content and high‐throughput approaches to identify the molecular and cellular mechanisms of membrane rupture during invasion by pathogens such as viruses, bacteria and parasites.     

Midbody and primary cilium of neural progenitors release extracellular membrane particles enriched in the stem cell marker prominin-1

Expansion of the neocortex requires symmetric divisions of neuroepithelial cells, the primary progenitor cells of the developing mammalian central nervous system. Symmetrically dividing neuroepithelial cells are known to form a midbody at their apical (rather than lateral) surface. We show that apical midbodies of neuroepithelial cells concentrate prominin-1 (CD133), a somatic stem cell marker and defining constituent of a specific plasma membrane microdomain. Moreover, these apical midbodies are released, as a whole or in part, into the extracellular space, yielding the prominin-1-enriched membrane particles found in the neural tube fluid. The primary cilium of neuroepithelial cells also concentrates prominin-1 and appears to be a second source of the prominin-1-bearing extracellular membrane particles. Our data reveal novel origins of extracellular membrane traffic that enable neural stem and progenitor cells to avoid the asymmetric inheritance of the midbody observed for other cells and, by releasing a stem cell membrane microdomain, to potentially influence the balance of their proliferation versus differentiation.     

An RNAi screening platform to identify secretion machinery in mammalian cells

Integrative approaches to study protein function in a cellular context are a vital aspect of understanding human disease. Genome sequencing projects provide the basic catalogue of information with which to unravel gene function, but more systematic applications of this resource are now necessary. Here, we describe and test a platform with which it is possible to rapidly use RNA interference in cultured mammalian cells to probe for proteins involved in constitutive protein secretion. Synthetic small interfering RNA molecules are arrayed in chambered slides, then incubated with cells and an assay for secretion performed. Automated microscopy is used to acquire images from the experiments, and automated single-cell analysis rapidly provides reliable quantitative data. In test arrays of 92 siRNA spots targeting 37 prospective membrane traffic proteins, our approach identifies 7 of these as being important for the correct delivery of a secretion marker to the cell surface. Correlating these findings with other screens and bioinformatic information makes these candidates highly likely to be novel membrane traffic machinery components.     

Membrane Cholesterol Strongly Influences Confined Diffusion of Prestin

Prestin is the membrane motor protein that drives outer hair cell (OHC) electromotility, a process that is essential for mammalian hearing. Prestin function is sensitive to membrane cholesterol levels, and numerous studies have suggested that prestin localizes in cholesterol-rich membrane microdomains. Previously, fluorescence recovery after photobleaching experiments were performed in HEK cells expressing prestin-GFP after cholesterol manipulations, and revealed evidence of transient confinement. To further characterize this apparent confined diffusion of prestin, we conjugated prestin to a photostable fluorophore (tetramethylrhodamine) and performed single-molecule fluorescence microscopy. Using single-particle tracking, we determined the microscopic diffusion coefficient from the full time course of the mean-squared deviation. Our results indicate that prestin undergoes diffusion in confinement regions, and that depletion of membrane cholesterol increases confinement size and decreases confinement strength. By interpreting the data in terms of a mathematical model of hop-diffusion, we quantified these cholesterol-induced changes in membrane organization. A complementary analysis of the distribution of squared displacements confirmed that cholesterol depletion reduces prestin confinement. These findings support the hypothesis that prestin function is intimately linked to membrane organization, and further promote a regulatory role for cholesterol in OHC and auditory function.     

A fluorescent sphingolipid binding domain peptide probe interacts with sphingolipids and cholesterol-dependent raft domains

We have designed a tagged probe [sphingolipid binding domain (SBD)] to facilitate the tracking of intracellular movements of sphingolipids in living neuronal cells. SBD is a small peptide consisting of the SBD of the amyloid precursor protein. It can be conjugated to a fluorophore of choice and exogenously applied to cells, thus allowing for in vivo imaging. Here, we present evidence to describe the characteristics of the SBD association with the plasma membrane. Our experiments demonstrate that SBD binds to isolated raft fractions from human neuroblastomas and insect neuronal cells. In protein-lipid overlay experiments, SBD interacts with a subset of glycosphingolipids and sphingomyelin, consistent with its raft association in neurons. We also provide evidence that SBD is taken up by neuronal cells in a cholesterol- and sphingolipid-dependent manner via detergent-resistant microdomains. Furthermore, using fluorescence correlation spectroscopy to assay the mobility of SBD in live cells, we show that SBD's behavior at the plasma membrane is similar to that of the previously described raft marker cholera toxin B, displaying both a fast and a slow component. Our data suggest that fluorescently tagged SBD can be used to investigate the dynamic nature of glycosphingolipid-rich detergent-resistant microdomains that are cholesterol-dependent.     

Protein transduction domain of HIV-1 Tat protein promotes efficient delivery of DNA into mammalian cells

The plasma membrane of mammalian cells is one of the tight barriers against gene transfer by synthetic delivery systems. Various agents have been used to facilitate gene transfer by destabilizing the endosomal membrane under acidic conditions, but their utility is limited, especially for gene transfer in vivo. In this article, we report that the protein transduction domain of human immunodeficiency virus type 1 Tat protein (Tat peptide) greatly facilitates gene transfer via membrane destabilization. We constructed recombinant lambda phage particles displaying Tat peptide on their surfaces and carrying mammalian marker genes as part of their genomes (Tat-phage). We demonstrate that, when animal cells are briefly exposed to Tat-phage, significant expression of phage marker genes is induced with no harmful effects to the cells. In contrast, recombinant phage displaying other functional peptides, such as the integrin-binding domain or a nuclear localization signal, could not induce detectable marker gene expression. The expression of marker genes induced by Tat-phage is not affected by endosomotropic agents but is partially impaired by inhibitors of caveolae formation. These data suggest that Tat peptide will become a useful component of synthetic delivery vehicles that promote gene transfer independently of the classical endocytic pathway.     

Existence of catalase-less peroxisomes in Sf21 insect cells

Catalase activity, a peroxisomal marker enzyme, was not detectable in any of the subcellular fractions of Spodoptera frugiperda (Sf) 21 insect cells, although marker enzymes in other organelles were distributed in the fractions in a manner similar to that seen in mammalian cells. When a green fluorescent protein fused with peroxisome targeting signal 1 at the C-terminal (GFP-SKL) was expressed in Sf21 cells, punctate fluorescent dots were observed in the cytoplasm. The fraction where GFP-SKL was concentrated exhibited long-chain and very-long-chain fatty acid beta-oxidation activities in the presence of KCN and the density of this fraction was slightly higher than that of mitochondria. Immunoelectron microscopy studies with anti-SKL antibody demonstrated that Sf21 cells have immunoreactive peroxisome-like organelles which are structurally distinct from mitochondria, endoplasmic reticulum, and lysosomes. In contrast to peroxisomal matrix proteins, adrenoleukodystrophy protein, a peroxisomal membrane protein, was not located to peroxisomes. This suggests that the targeting signal for PMP in insect cells is distinct from that in mammalian cells. These results demonstrate that Sf21 insect cells have unique catalase-less peroxisomes capable of beta-oxidation of fatty acids.     

Coordinate analysis of murine immune cell surface markers and intracellular phosphoproteins by flow cytometry

Recently, phosphospecific flow cytometry has emerged as a powerful tool to analyze intracellular signaling events in complex populations of cells because of its ability to simultaneously discriminate cell types based on surface marker expression and measure levels of intracellular phosphoproteins. This has provided novel insights into the cell- and pathway-specific nature of immune signaling. However, we and others have found that the fixation and permeabilization steps necessary for phosphoprotein analysis often negatively affect the resolution of cell types based on surface marker analysis and light scatter characteristics. Therefore, we performed a comprehensive profile of >35 different murine surface marker Abs to understand the effects of fixation and permeabilization on surface Ag staining. Fortuitously, approximately 80% of the Abs tested resolved cell populations of interest, although with decreased separation between positive and negative populations and at very different titers than those used on live cells. The other 20% showed either complete loss of separation between populations or loss of intermediately staining populations. We were able to rescue staining of several of these Ags by performing staining after fixation, but before permeabilization, although with limited fluorophore choices. Scatter characteristics of lymphocytes were well retained, but changed dramatically for monocyte and neutrophil populations. These results compile a comprehensive resource for researchers interested in applying phosphospecific flow cytometry to complex populations of cells while outlining steps necessary to successfully apply new surface marker Abs to this platform.     

Design of a specific colonic mucus marker using a human commensal bacterium cell surface domain

Imaging living cells and organs requires innovative, specific, efficient, and well tolerated fluorescent markers targeting cellular components. Such tools will allow proceeding to the dynamic analysis of cells and the adaptation of tissues to environmental cues. In this study, we have identified and synthesized a novel non-toxic fluorescent marker allowing a specific fluorescent staining of the human colonic mucus. Our strategy to identify a molecule able to specifically bind to the human colonic mucus was on the basis of the mucus adhesion properties of commensal bacteria. We identified and characterized the mucus-binding property of a 70-amino acid domain (MUB(70)) expressed on the surface of Lactobacillus strains. The chemical synthesis of MUB(70) was achieved using the human commensal bacterium Lactobacillus reuteri AF120104 protein as a template. The synthesized Cy5-conjugated MUB(70) marker specifically stained the colonic mucus on fixed human, rabbit, and guinea pig tissues. Interestingly, murine tissue was not stained, suggesting significant differences in the composition of the murine colonic mucus. In addition, this marker stained the mucus of living cultured human colonic cells (HT29-MTX) and human colonic tissue explants. Using a biotinylated derivative of MUB(70), we demonstrated that this peptide binds specifically to Muc2, the most abundant secreted mucin, through its glycosylated moieties. Hence, Cy5-MUB(70) is a novel and specific fluorescent marker for mammalian colonic mucus. It may be used for live imaging analysis but also, as demonstrated in this study, as a marker for the diagnosis and the prognosis of colonic mucinous carcinomas.     

Proteomics analyses of microvesicles released by Drosophila Kc167 and S2 cells

Distinct types of vesicles are formed in eukaryotic cells that conduct a variable set of functions depending on their origin. One subtype designated circulating microvesicles (MVs) provides a novel form of intercellular communication and recent work suggested the release and uptake of morphogens in vesicles by Drosophila cells. In this study, we have examined cells of the hemocyte-like cell lines Kc167 and S2 and identified secreted vesicles in the culture supernatant. The vesicles were isolated and found to have characteristics comparable to exosomes and plasma membrane MVs released by mammalian cells. In wingless-transfected cells, the full-length protein was detected in the vesicle isolates. Proteomics analyses of the vesicles identified 269 proteins that include various orthologs of marker proteins and proteins with putative functions in vesicle formation and release. Analogous to their mammalian counterparts, the subcellular origin of the vesicular constituents of both cell lines is dominated by membrane-associated and cytosolic proteins with functions that are consistent with their localization in MVs. The analyses revealed a significant overlap of the Kc167 and S2 vesicle proteomes and confirmed a close correlation with non-mammalian and mammalian exosomes.     

Antibodies immobilized as arrays to profile protein post-translational modifications in mammalian cells

Previously, we demonstrated that antibodies printed on a solid support were able to detect protein-protein interaction in mammalian cells. Here we further developed the antibody array system for detecting proteins with various post-translational modifications in mammalian cells. In this novel approach, immunoprecipitated proteins were labeled with fluorescent dye followed by incubation over antibody arrays. Targeted proteins, captured by the antibodies immobilized on PVDF membrane or glass slide, were detected by means of near infrared fluorescent scanner or fluorescent microscopy. To demonstrate the application of the antibody arrays in protein post-translational modifications, we profiled protein tyrosine phosphorylation, ubiquitination, and acetylation in mammalian cells under different conditions. Our results indicate that antibody array technology can provide a powerful means of profiling a large number of proteins with different post-translational modifications in cells.     

Effects of Mutations in Proline 345 on Insertion of Diphtheria Toxin into Model Membranes

Translocation of the catalytic domain of diphtheria toxin (DT) across the endosomal membrane to the cytoplasm of mammalian cells requires the low-pH-dependent insertion of a hydrophobic helical hairpin (TH8-TH9) that is buried within the T domain of the native protein. Mutations of Pro345, which terminates helix TH8, have been reported to block toxicity for Vero cells. We found that mutant toxins in which Pro345 had been replaced by Cys, Glu, or Gly were profoundly defective at low pH in forming channels in planar phospholipid bilayers and in permeabilizing phospholipid vesicles to entrapped fluorophores. Experiments with isolated T domain containing a polarity-sensitive fluorophore attached to Cys at position 332 suggest that the P345E mutation blocks membrane insertion. None of the Pro345 mutations shifted the pH-dependence of binding in solution of the hydrophobic fluorophore, 2-p-toluidinyl-naphthalene 7-sulfonate. The results indicate that proline at position 345 is required for the T domain to insert into phospholipid bilayers or to adopt a functional conformation within the bilayer. Received: 23 July 1998/Revised: 19 October 1998     

Leukemia virus infection of mammalian cells: effect on two "transformation-associated" surface properties.

We demonstrated that the productive infection of three different mammalian cell lines with two separate leukemia viruses is sufficient to induce a change in surface architecture that may be detected as enhanced agglutinability with two different plant lectins. Subsequent transformation of one of these cell lines with a chemical carcinogen did not further modify the agglutinability of the cell lines. Using a polyoma virus-transformed derivative of one of the parental lines, we have demonstrated that the LETS protein (whose absence from the surface membrane has been considered a marker of the transformed phenotype) may be present in cells displaying the capacity to plate in soft agar.     

A subcellular prefractionation protocol for minute amounts of mammalian cell cultures and tissue

Subcellular localization represents an essential, albeit often neglected, aspect of proteome analysis. Generally, the subcellular location of proteins determines the function of cells and tissues. Here we present a robust and versatile prefractionation protocol for mammalian cells and tissues which is appropriate for minute sample amounts. The protocol yields three fractions: a nuclear, a cytoplasmic, and a combined membrane and organelle fraction. The subcellular specificity and the composition of the fractions were demonstrated by immunoblot analysis of five marker proteins and analysis of 43 proteins by two-dimensional gel electrophoresis and mass spectrometry. To cover all protein species, both conventional two-dimensional and benzyldimethyl-n-hexadecyl ammonium chloride-sodium dodecyl sulfate (16-BAC-SDS) gel electrophoresis were performed. Integral membrane proteins and strongly basic nuclear histones were detected only in the 16-BAC-SDS gel electrophoresis system, confirming its usefulness for proteome analysis. All but one protein complied to the respective subcellular composition of the analyzed fractions. Taken together, the data make our subcellular prefractionation protocol an attractive alternative to other prefractionation methods which are based on less physiological protein properties.     

Gene transfer to mammalian cells using genetically targeted filamentous bacteriophage.

We have genetically modified filamentous bacteriophage to deliver genes to mammalian cells. In previous studies we showed that noncovalently attached fibroblast growth factor (FGF2) can target bacteriophage to COS-1 cells, resulting in receptor-mediated transduction with a reporter gene. Thus, bacteriophage, which normally lack tropism for mammalian cells, can be adapted for mammalian cell gene transfer. To determine the potential of using phage-mediated gene transfer as a novel display phage screening strategy, we transfected COS-1 cells with phage that were engineered to display FGF2 on their surface coat as a fusion to the minor coat protein, pIII. Immunoblot and ELISA analysis confirmed the presence of FGF2 on the phage coat. Significant transduction was obtained in COS-1 cells with the targeted FGF2-phage compared with the nontargeted parent phage. Specificity was demonstrated by successful inhibition of transduction in the presence of excess free FGF2. Having demonstrated mammalian cell transduction by phage displaying a known gene targeting ligand, it is now feasible to apply phage-mediated transduction as a screen for discovering novel ligands.