Altering the biochemical state of individual cultured cells and organelles with ultramicroelectrodes

We describe an efficient technique for the selective chemical and biological manipulation of the contents of individual cells. This technique is based on the electric-field-induced permeabilization (electroporation) in biological membranes using a low-voltage pulse generator and microelectrodes. A spatially highly focused electric field allows introduction of polar cell-impermeant solutes such as fluorescent dyes, fluorogenic reagents, and DNA into single cells. The high spatial resolution of the technique allows for design of, for example, cellular network constructions in which cells in close contact with each other can be made to possess different biochemical, biophysical, and morphological properties. Fluorescein, and fluo-3 (a calcium-sensitive fluorophore), are electroporated into the soma of cultured single progenitor cells derived from adult rat hippocampus. Fluo-3 also is introduced into individual submicrometer diameter processes of thapsigargin-treated progenitor cells, and a plasmid vector cDNA construct (pRAY 1), expressing the green fluorescent protein, is electroporated into cultured single COS 7 cells. At high electric field strengths, observations of dye-transfer into organelles are proposed.

The V domain of herpesvirus Ig-like receptor (HIgR) contains a major functional region in herpes simplex virus-1 entry into cells and interacts physically with the viral glycoprotein D

The herpesvirus entry mediator C (HveC), previously known as poliovirus receptor-related protein 1 (PRR1), and the herpesvirus Ig-like receptor (HIgR) are the bona fide receptors employed by herpes simplex virus-1 and -2 (HSV-1 and -2) for entry into the human cell lines most frequently used in HSV studies. They share an identical ectodomain made of one V and two C2 domains and differ in transmembrane and cytoplasmic regions. Expression of their mRNA in the human nervous system suggests possible usage of these receptors in humans in the path of neuron infection by HSV. Glycoprotein D (gD) is the virion component that mediates HSV-1 entry into cells by interaction with cellular receptors. We report on the identification of the V domain of HIgR/PRR1 as a major functional region in HSV-1 entry by several approaches. First, the epitope recognized by mAb R1.302 to HIgR/PRR1, capable of inhibiting infection, was mapped to the V domain. Second, a soluble form of HIgR/PRR1 consisting of the single V domain competed with cell-bound full-length receptor and blocked virion infectivity. Third, the V domain was sufficient to mediate HSV entry, as an engineered form of PRR1 in which the two C2 domains were deleted and the V domain was retained and fused to its transmembrane and cytoplasmic regions was still able to confer susceptibility, although at reduced efficiency relative to full-length receptor. Consistently, transfer of the V domain of HIgR/PRR1 to a functionally inactive structural homologue generated a chimeric receptor with virus-entry activity. Finally, the single V domain was sufficient for in vitro physical interaction with gD. The in vitro binding was specific as it was competed both by antibodies to the receptor and by a mAb to gD with potent neutralizing activity for HSV-1 infectivity.

Formation of stable adducts and absence of depurinating DNA adducts in cells and DNA treated with the potent carcinogen dibenzo[a,l]pyrene or its diol epoxides

Polycyclic aromatic hydrocarbons (PAH) are widespread environmental contaminants, and some are potent carcinogens in rodents. Carcinogenic PAH are activated in cells to metabolites that react with DNA to form stable covalent DNA adducts. It has been proposed [Cavalieri, E. L. & Roger, E. G. (1995) Xenobiotica 25, 677–688] that unstable DNA adducts are also formed and that apurinic sites in the DNA resulting from unstable PAH adducts play a key role in the initiation of cancer. The potent carcinogen dibenzo[a,l]pyrene (DB[a,l]P) is activated in cells to (+)-syn- and (−)-anti-DB[a,l]P-11,12-diol-13,14-epoxide (DB[a,l]PDE), which have been shown to form stable adducts with DNA. To evaluate the importance of unstable PAH adducts, we compared stable adduct formation to apurinic site formation. Stable DB[a,l]PDE adducts were determined by 33P-postlabeling and HPLC. To measure apurinic sites they were converted to strand breaks, and these were monitored by examining the integrity of a particular restriction fragment of the dihydrofolate reductase gene. The method easily detected apurinic sites resulting from methylation by treatment of cells or DNA with dimethyl sulfate or from reaction of DNA with DB[a,l]P in the presence of horseradish peroxidase. We estimate the method could detect 0.1 apurinic site in the 14-kb fragment examined. However, apurinic sites were below our limit of detection in DNA treated directly with (+)-syn- or (−)-anti-DB[a,l]PDE or in DNA from Chinese hamster ovary B11 cells so treated, although in these samples the frequency of stable adducts ranged from 3 to 10 per 14 kb. We also treated the human mammary carcinoma cell line MCF-7 with DB[a,l]P and again could not detect significant amounts of unstable adducts. These results indicate that the proportion of stable adducts formed by DB[a,l]P activated in cells and its diol epoxides is greater than 99% and suggest a predominant role for stable DNA adducts in the carcinogenic activity of DB[a,l]P.

Mitochondrial mutational spectra in human cells and tissues

We have found that human organs such as colon, lung, and muscle, as well as their derived tumors, share nearly all mitochondrial hotspot point mutations. Seventeen hotspots, primarily G → A and A → G transitions, have been identified in the mitochondrial sequence of base pairs 10,030–10,130. Mutant fractions increase with the number of cell generations in a human B cell line, TK6, indicating that they are heritable changes. The mitochondrial point mutation rate appears to be more than two orders of magnitude higher than the nuclear point mutation rate in TK6 cells and in human tissues. The similarity of the hotspot sets in vivo and in vitro leads us to conclude that human mitochondrial point mutations in the sequence studied are primarily spontaneous in origin and arise either from DNA replication error or reactions of DNA with endogenous metabolites. The predominance of transition mutations and the high number of hotspots in this short sequence resembles spectra produced by DNA polymerases in vitro.

On the spurious endoproteolytic processing of the presenilin proteins in cultured cells and tissues

It has been widely reported that the presenilin proteins PS-1 and PS-2 in extracts derived from a variety of cultured cells and from tissues are fragmented extensively by endoproteolytic processing events. It generally has been presumed that this endoproteolysis is a physiologically normal intracellular event following presenilin expression, which might play an important role in the still unknown functions of these molecules in connection with Alzheimer disease. We demonstrate herein, however, that, if a variety of cultured cells and several mouse tissues are examined under conditions minimizing cell trauma, the presenilin molecules in the extracts are found to be intact but that, if the cells and tissues are prepared under somewhat more stressful conditions, the endoproteolytic fragments are then observed. We conclude that these particular endoproteolytic events are not the result of physiologically normal processing of the presenilins but are rather artifacts occurring during the common procedures of specimen preparation.

An efficient system for conditional gene expression in embryonic stem cells and in their in vitro and in vivo differentiated derivatives

We have developed a universally applicable system for conditional gene expression in embryonic stem (ES) cells that relies on tamoxifen-dependent Cre recombinase-loxP site-mediated recombination and bicistronic gene-trap expression vectors that allow transgene expression from endogenous cellular promoters. Two vectors were introduced into the genome of recipient ES cells, successively: (i) a bicistronic gene-trap vector encoding the β-galactosidase/neoR fusion protein and the Cre-ERT2 (Cre recombinase fused to a mutated ligand-binding domain of the human estrogen receptor) and (ii) a bicistronic gene-trap vector encoding the hygroR protein and the human alkaline phosphatase (hAP), the expression of which is prevented by tandemly repeated stop-of-transcription sequences flanked by loxP sites. In selected clones, hAP expression was shown to be regulated accurately by 4′hydroxy-tamoxifen. Strict hormone-dependent expression of hAP was achieved (i) in vitro in undifferentiated ES cells and embryoid bodies, (ii) in vivo in virtually all the tissues of the 10-day-old chimeric fetus (after injection of 4′hydroxy-tamoxifen to foster mothers), and (iii) ex vivo in primary embryonic fibroblasts isolated from chimeric fetuses. Therefore, this approach can be applied to drive conditional expression of virtually any transgene in a large variety of cell types, both in vitro and in vivo.

Pendrin, encoded by the Pendred syndrome gene, resides in the apical region of renal intercalated cells and mediates bicarbonate secretion

Pendrin is an anion transporter encoded by the PDS/Pds gene. In humans, mutations in PDS cause the genetic disorder Pendred syndrome, which is associated with deafness and goiter. Previous studies have shown that this gene has a relatively restricted pattern of expression, with PDS/Pds mRNA detected only in the thyroid, inner ear, and kidney. The present study examined the distribution and function of pendrin in the mammalian kidney. Immunolocalization studies were performed using anti-pendrin polyclonal and monoclonal antibodies. Labeling was detected on the apical surface of a subpopulation of cells within the cortical collecting ducts (CCDs) that also express the H+-ATPase but not aquaporin-2, indicating that pendrin is present in intercalated cells of the CCD. Furthermore, pendrin was detected exclusively within the subpopulation of intercalated cells that express the H+-ATPase but not the anion exchanger 1 (AE1) and that are thought to mediate bicarbonate secretion. The same distribution of pendrin was observed in mouse, rat, and human kidney. However, pendrin was not detected in kidneys from a Pds-knockout mouse. Perfused CCD tubules isolated from alkali-loaded wild-type mice secreted bicarbonate, whereas tubules from alkali-loaded Pds-knockout mice failed to secrete bicarbonate. Together, these studies indicate that pendrin is an apical anion transporter in intercalated cells of CCDs and has an essential role in renal bicarbonate secretion.

Subcellular Distribution and Tissue Expression of Phospholipase Dα, Dβ, and Dγ in Arabidopsis1

Three phospholipase Ds (PLDs; EC 3.1.4.4) have been cloned from Arabidopsis, and they exhibit two distinct types of activities: polyphosphoinositide-requiring PLDβ and PLDγ, and polyphosphoinositide-independent PLDα. In subcellular fractions of Arabidopsis leaves, PLDα and PLDγ were both present in the plasma membrane, intracellular membranes, mitochondria, and clathrin-coated vesicles, but their relative levels differed in these fractions. In addition, PLDγ was detected in the nuclear fraction. In contrast, PLDβ was not detectable in any of the subcellular fractions. PLDα activity was higher in the metabolically more active organs such as flowers, siliques, and roots than in dry seeds and mature leaves, whereas the polyphosphoinositide-dependent PLD activity was greater in older, senescing leaves than in other organs. PLDβ mRNA accumulated at a lower level than the PLDα and PLDγ transcripts in most organs, and the expression pattern of the PLDβ mRNA also differed from that of PLDα and PLDγ in different organs. Collectively, these data demonstrated that PLDα, PLDβ, and PLDγ have different patterns of subcellular distribution and tissue expression in Arabidopsis. The present study also provides evidence for the presence of an additional PLD that is structurally more closely related to PLDγ than to the other two PLDs.

Structure, Properties, and Tissue Localization of Apoplastic α-Glucosidase in Crucifers1

Apoplastic α-glucosidases occur widely in plants but their function is unknown because appropriate substrates in the apoplast have not been identified. Arabidopsis contains at least three α-glucosidase genes; Aglu-1 and Aglu-3 are sequenced and Aglu-2 is known from six expressed sequence tags. Antibodies raised to a portion of Aglu-1 expressed in Escherichia coli recognize two proteins of 96 and 81 kD, respectively, in vegetative tissues of Arabidopsis, broccoli (Brassica oleracea L.), and mustard (Brassica napus L.). The acidic α-glucosidase activity from broccoli flower buds was purified using concanavalin A and ion-exchange chromatography. Two active fractions were resolved and both contained a 96-kD immunoreactive polypeptide. The N-terminal sequence from the 96-kD broccoli α-glucosidase indicated that it corresponds to the Arabidopsis Aglu-2 gene and that approximately 15 kD of the predicted N terminus was cleaved. The 81-kD protein was more abundant than the 96-kD protein, but it was not active with 4-methylumbelliferyl-α-d-glucopyranoside as the substrate and it did not bind to concanavalin A. In situ activity staining using 5-bromo-4-chloro-3-indolyl-α-d-glucopyranoside revealed that the acidic α-glucosidase activity is predominantly located in the outer cortex of broccoli stems and in vascular tissue, especially in leaf traces.

Golgi Complex Reorganization during Muscle Differentiation: Visualization in Living Cells and Mechanism

During skeletal muscle differentiation, the Golgi complex (GC) undergoes a dramatic reorganization. We have now visualized the differentiation and fusion of living myoblasts of the mouse muscle cell line C2, permanently expressing a mannosidase-green fluorescent protein (GFP) construct. These experiments reveal that the reorganization of the GC is progressive (1–2 h) and is completed before the cells start fusing. Fluorescence recovery after photobleaching (FRAP), immunofluorescence, and immunogold electron microscopy demonstrate that the GC is fragmented into elements localized near the endoplasmic reticulum (ER) exit sites. FRAP analysis and the ER relocation of endogenous GC proteins by phospholipase A2 inhibitors demonstrate that Golgi-ER cycling of resident GC proteins takes place in both myoblasts and myotubes. All results support a model in which the GC reorganization in muscle reflects changes in the Golgi-ER cycling. The mechanism is similar to that leading to the dispersal of the GC caused, in all mammalian cells, by microtubule-disrupting drugs. We propose that the trigger for the dispersal results, in muscle, from combined changes in microtubule nucleation and ER exit site localization, which place the ER exit sites near microtubule minus ends. Thus, changes in GC organization that initially appear specific to muscle cells, in fact use pathways common to all mammalian cells.