Nanoparticles of compacted DNA transfect postmitotic cells

Charge-neutral DNA nanoparticles have been developed in which single molecules of DNA are compacted to their minimal possible size. We speculated that the small size of these DNA nanoparticles may facilitate gene transfer in postmitotic cells, permitting nuclear uptake across the 25-nm nuclear membrane pore. To determine whether DNA nanoparticles can transfect nondividing cells, growth-arrested neuroblastoma and hepatoma cells were transfected with DNA/liposome mixtures encoding luciferase. In both models, growth-arrested cells were robustly transfected by compacted DNA (6,900-360-fold more than naked DNA). To evaluate mechanisms responsible for enhanced transfection, HuH-7 cells were microinjected with naked or compacted plasmids encoding enhanced green fluorescent protein. Cytoplasmic microinjection of DNA nanoparticles generated a approximately 10-fold improvement in transgene expression as compared with naked DNA; this enhancement was reversed by the nuclear pore inhibitor, wheat germ agglutinin. To determine the upper size limit for gene transfer, DNA nanoparticles of various sizes were microinjected into the cytoplasm. A marked decrease in transgene expression was observed as the minor ellipsoidal diameter approached 25 nm. In summary, suitably sized DNA nanoparticles productively transfect growth arrested cells by traversing the nuclear membrane pore.     

Sample Preparation and Imaging Conditions Affect mEos3.2 Photophysics in Fission Yeast Cells

Photoconvertible fluorescent proteins (PCFPs) are widely used in super-resolution microscopy and studies of cellular dynamics. However, our understanding of their photophysics is still limited, hampering their quantitative application. For example, we do not know the optimal sample preparation methods or imaging conditions to count protein molecules fused to PCFPs by single-molecule localization microscopy in live and fixed cells. We also do not know how the behavior of PCFPs in live cells compares with fixed cells. Therefore, we investigated how formaldehyde fixation influences the photophysical properties of the popular green-to-red PCFP mEos3.2 in fission yeast cells under a wide range of imaging conditions. We estimated photophysical parameters by fitting a three-state model of photoconversion and photobleaching to the time course of fluorescence signal per yeast cell expressing mEos3.2. We discovered that formaldehyde fixation makes the fluorescence signal, photoconversion rate, and photobleaching rate of mEos3.2 sensitive to the buffer conditions likely by permeabilizing the yeast cell membrane. Under some imaging conditions, the time-integrated mEos3.2 signal per yeast cell is similar in live cells and fixed cells imaged in buffer at pH 8.5 with 1 mM DTT, indicating that light chemical fixation does not destroy mEos3.2 molecules. We also discovered that 405-nm irradiation drove some red-state mEos3.2 molecules to enter an intermediate dark state, which can be converted back to the red fluorescent state by 561-nm illumination. Our findings provide a guide to quantitatively compare conditions for imaging mEos3.2-tagged molecules in yeast cells. Our imaging assay and mathematical model are easy to implement and provide a simple quantitative approach to measure the time-integrated signal and the photoconversion and photobleaching rates of fluorescent proteins in cells.     

ELCS in ice: cryo-electron microscopy of nuclear envelope-limited chromatin sheets

Nuclear envelope-limited chromatin sheets (ELCS) form during excessive interphase nuclear envelope growth in a variety of cells. ELCS appear as extended sheets within the cytoplasm connecting distant nuclear lobes. Cross-section stained images of ELCS, viewed by transmission electron microscopy, resemble a sandwich of apposed nuclear envelopes separated by ～30 nm, containing a layer of parallel chromatin fibers. In this study, the ultrastructure of ELCS was compared by three different methods: (1) aldehyde fixation/dehydration/plastic embedding/sectioning and staining, (2) high-pressure freezing/freeze substitution into plastic/sectioning and staining, and (3) high-pressure freezing/cryo-sectioning/cryo-electron microscopy. ELCS could be clearly visualized by all three methods and, consequently, must exist in vivo and are not fixation artifacts. The ～30-nm chromatin fibers could only be observed following aldehyde fixation; none were seen in cryo-sections. Electron microscopic tomography tangential views of aldehyde-fixed ELCS suggested an ordering of the separate chromatin fibers adjacent to the nuclear envelope. Possible mechanisms of this chromatin ordering are discussed.     

Characteristics of light-induced 515-nm absorbance change in spinach chloroplasts at lower temperatures I. Participation of structural changes of thylakoid membranes in light-induced 515-nm absorbance change

Light-induced absorbance change at 515 nm in spinach chloroplastswas studied in the temperature range from –2?C to 27?C.Lowering of temperature had no marked effect on the extentsof initial "light-on" spike and the steady-state change overthe temperature range examined, whereas the rate of recoveryof the 515-nm change was significantly reduced at lower temperatures.Above 15?C, recovery of the 515-nm change after continuous illuminationshowed a first-order kinetics. In contrast, the recovery wascomposed of a fast and a slow phases at lower temperatures. The fast phase of the recovery of the 515-nm change was acceleratedby carbonyl cyanide m-chlorophenylhydrazone, valinomycin plusK⁺ or sodium tetraphenylboron, while the slow phase was completelyeliminated in glutaraldehyde-fixed chloroplasts. Light-inducedchange in absorbance at 546 nm, an indicator of structural changesof membrane, showed almost the same dependency on temperatureas the slow phase of the recovery of the 515-nm change. Theseresults suggest that not only electric field formation acrossthe thylakoid membrane but also structural or conformationalchanges in the membrane participate in the 515-nm absorbancechange observed under steady illumination. (Received July 5, 1976; )     

The lengths of DNA fragments light-induced in the presence of a photosensitizer localized at the nuclear membrane of human cells

DNA damage was apparently introduced selectively in the parts of DNA localized close to the nuclear membrane of human NHIK 3025 cells. This was obtained by illumination of the cells in the presence of a hydrophobic photosensitizer, Photofrin II, which was located in membrane structures but not in the nucleus. Photofrin II sensitizes DNA to light mainly via singlet oxygen, which diffuses about 0.1 microns in its intracellular lifetime. By measuring DNA unwinding in alkali after illumination or X-irradiation of cells, the distribution of the number of DNA bases between two adjacent, photodamaged DNA sites was estimated. The average length of these DNA fragments was found to be 155 kilobases (kb).     

Acetylcholine receptor organization in membrane domains in muscle cells: evidence for rapsyn-independent and rapsyn-dependent mechanisms

Nicotinic acetylcholine receptors (nAChR) in muscle fibers are densely packed in the postsynaptic region at the neuromuscular junction. Rapsyn plays a central role in directing and clustering nAChR during cellular differentiation and neuromuscular junction formation; however, it has not been demonstrated whether rapsyn is the only cause of receptor immobilization. Here, we used single-molecule tracking methods to investigate nAChR mobility in plasma membranes of myoblast cells during their differentiation to myotubes in the presence and absence of rapsyn. We found that in myoblasts the majority of nAChR were immobile and that ～20% of the receptors showed restricted diffusion in small domains of ～50 nm. In myoblasts devoid of rapsyn, the fraction of mobile nAChR was considerably increased, accompanied by a 3-fold decrease in the immobile population of nAChR with respect to rapsyn-expressing cells. Half of the mobile receptors were confined to domains of ～120 nm. Measurements performed in heterologously transfected HEK cells confirmed the direct immobilization of nAChR by rapsyn. However, irrespective of the presence of rapsyn, about one-third of nAChR were confined in 300-nm domains. Our results show (i) that rapsyn efficiently immobilizes nAChR independently of other postsynaptic scaffold components; (ii) nAChR is constrained in confined membrane domains independently of rapsyn; and (iii) in the presence of rapsyn, the size of these domains is strongly reduced.     

Cytochemical demonstration of actin filaments in myoepithelial cells of the human parotid gland

Ultrastructurally, myoepithelial cells were shown to contain numerous fine filaments in their cytoplasm and resembled smooth muscle cells. The myoepithelial cell of the salivary gland has been considered to play an important role in the secretion of saliva. The present study showed that all the thin filaments (actin filaments) in the myoepithelial cell of the human parotid gland bound heavy meromyosin (HMM) and formed characteristic arrowhead structures. These filaments ran in two opposite directions with the poles at different ends. On the other hand, there was no binding of HMM with thicker filaments (10-nm filaments), plasma membrane, nuclear membrane, collagen fibrils, basement membrane or other cytoplasmic organelles. The present results strongly suggest that myoepithelial cells possess a contractile function parallel to the long axis of the cell for supporting the secretion of saliva in the parotid gland.     

Photoinactivation of Acinetobacter baumannii and Escherichia coli B by a Cationic Hydrophilic Porphyrin at Various Light Wavelengths

Photodynamic treatment by the cationic TMPyP photosensitizer was undertaken on the multiple antibiotic-resistant bacteria Acinetobacter baumannii and Escherichia coli. Total eradication of the bacterial cultures was determined immediately after initiation of illumination when these bacteria were treated with 5, 10, 15, 20-tetra (4-N methylpyridyl)porphine (TMPyP) at a concentration of 29.4 μmol/L and illuminated by blue, green, or red light. Total eradication of both bacteria was obtained also after treatment of bacterial cultures with 3.7 μmol/L TMPyP and illumination with blue light (400–450 nm). On the other hand, an 8- or 16- to 20-fold higher light intensity, respectively, was required for total eradication upon illumination with green (480–550 nm) or red light (600–700 nm). A 407-nm blue light only 7 and 9 joules/cm², respectively, was needed for total eradication of both bacteria even at a concentration of 3.7 μmol/L TMPyP. X-ray-linked microanalysis demonstrated loss of potassium and a flood of sodium and chloride into the cells, indicating serious damage to the cytoplasmic membrane. Transmission electron microscopy (TEM) revealed structural changes and damage to the membrane of treated E. coli. In A. baumannii-treated cells, mesosomes and black dots that resemble aggregation of polyphosphate polymers could be seen. DNA breakage appeared only after a long period of illumination, when the bacterial cell was no longer viable. It can be concluded that cytoplasmic membrane damage and not DNA breakage is the major cause for bacterial death upon photosensitization. Received: 13 October 2000 / Accepted: 17 November 2000     

Ultrastructural localization of herpes simplex virus RNA by in situ hybridization

We studied the subcellular localization of virally encoded RNA by pre-embedding in situ hybridization, using colloidal gold as an electron-dense marker. Fibroblasts infected with Herpes simplex virus (HSV) were fixed, permeabilized, then hybridized with a biotinylated HSV DNA probe under conditions favoring DNA-RNA hybrid formation. HSV probe was localized with 5-nm streptavidin-gold conjugates. Transmission electron microscopy revealed 5-nm gold in clusters and singlets within HSV-infected cells. Formalin-fixed cells contained a mean of 4.6 clusters per cytoplasmic profile and 13.2 clusters per nuclear profile. Combined formalin-glutaraldehyde fixation increased the mean number of clusters per cytoplasmic and nuclear profile to 7.2 (57% increase) and 17.5 (33% increase), respectively. Gold clusters were frequently located in regions adjacent to the nuclear envelope but were not bound to viral nucleocapsids or endoplasmic reticulum. Labeling was unaffected by pre-hybridization DNAse treatment of cells. RNAse eliminated 87% of cytoplasmic and 97% of nuclear clusters. These findings indicate that clustered gold particles labeled viral RNA, with probable binding of multiple DNA probe molecules and/or gold particles to RNA strands. This novel pre-embedding technique may be a useful tool for ultrastructural evaluation of virus-host cell interactions.     

Induction of size reduction in Escherichia coli by near-ultraviolet light

Escherichia coli AB1157 cells, growing exponentially at 37 degrees C in 63B1 medium (supplemented with glucose and casamino acids) with a doubling time of 50 min, were subjected to continuous illumination with 366-nm light at a fluence of 1.5 kJ . m-2 X min-1. Under these conditions, the growth rate decreased and after 1 h of illumination, a new stable exponential mode was reached with a doubling-time of 73 min. This reduction in growth rate occurred without any change in the rate of cell division for two generations after the beginning of illumination. Survival was unaffected, implying that cell size must have decreased. This was confirmed with size distribution curves of control and illuminated cells obtained with a Coulter counter. Furthermore electron micrographs of negatively stained cells indicated that illumination results in a 30-40% decrease in cell length, the diameter increasing by 8%. Hence 366-nm light uncouples growth and division rates. Illumination under the above conditions triggered the accumulation in vivo of 8-13-linked tRNA. The stationary level of the 8-13 link, 80% of the maximal level, was reached precisely when the growth rate reached its new stable value. Furthermore, no reduction in growth rate occurred in a nuv- cell lacking 4-thiouridine in its tRNAs. Hence we conclude that the 366-nm photons trigger partial tRNA inactivation with consequent slowing down of protein synthesis and accordingly of the cell growth rate. In addition, the stringent response has at most a minor effect. In conclusion, near-ultraviolet light is able to decrease the rate of cell growth by restricting the availability of charged tRNAs, and this occurs without affecting the cell division rate.     

Plateau distributions of DNA fragment lengths produced by extended light exposure of extranuclear photosensitizers in human cells.

We have exploited properties of photosensitizers to study an aspect of the packing of chromatin in the cell nucleus. The fluorescent photosensitizers mesotetra(3-hydroxyphenyl) porphyrin and Photofrin II were both localized in the nuclear membrane and other membrane structures, but could not be found inside the nuclei. Light exposure of cells at 1 degrees C in the presence of the sensitizers induced DNA double-strand breaks. The length distributions of DNA fragments were determined by pulsed field gel electrophoresis. Because DNA damage is produced mainly via singlet oxygen diffusing less than 0.1 microns from the sensitizer, DNA double-strand breaks were supposedly produced within this distance of the nuclear membrane. Consistent with this, with prolonged illumination and with increasing concentrations of sensitizer the distribution of DNA fragment lengths reached a plateau level. In contrast, with the hydrophilic, intranuclear sensitizer meso-tetra(4-sulphonatophenyl)porphyrin, no such plateau level was found. The plateau distributions of DNA fragment lengths of different cell types had the same general shape with average fragment lengths ranging from 174 to 194 kilobasepairs. Particular genes, c-myc, fos and p53, were found on broad distributions of photocleaved fragment lengths. The results indicate that on each side of the genes the locus of the chromatin fibre situated close to the nuclear membrane, varied randomly.     

Towards an atlas of mammalian cell ultrastructure by cryo soft X-ray tomography

We provide a catalog of 3D cryo soft X-ray tomography (cryo-SXT) images obtained from ～6 to 12μm thick mouse adenocarcinoma cells. Included are multiple representative images of nuclei, nucleoli, nuclear membrane, nuclear membrane channels, mitochondria, lysosomes, endoplasmic reticulum, filaments and plasma membrane, plus three structures not previously described by cryo-SXT, namely Golgi, microvilli and nuclear-membrane blebs. Sections from the 3D cryo-SXT tomograms for all the preceding structures closely resemble those seen by thin-section transmission electron microscopy (TEM). Some structures such as nuclear-membrane channels and nuclear-membrane blebs are more easily detected by cryo-SXT than TEM most likely due to their better contrast and cellular preservation in cryo-SXT combined with the ability to rapidly locate these structures within a full 3D image. We identify and discuss two current limitations in cryo-SXT: variability in image quality and difficulties in detecting weaker contrast structures such as chromatin and various nuclear bodies. Progress on these points is likely to come from the solution of several technical problems in image acquisition, plus the implementation of advanced cryo soft X-ray microscopy approaches such as phase contrast or optical sectioning.     

Involvement of microtubules and 10-nm filaments in the movement and positioning of nuclei in syncytia

Previous studies (Holmes, K.V., and P.W. Choppin. J. Exp. Med. 124:501- 520; J. Cell Biol. 39:526-543) showed that infection of baby hamster kidney (BHK21-F) cells with the parainfluenza virus SV5 causes extensive cell fusion, that nuclei migrate in the syncytial cytoplasm and align in tightly-packed rows, and that microtubules are involved in nuclear movement and alignment. The role of microtubules, 10-nm filaments, and actin-containing microfilaments in this process has been investigated by immunofluorescence microscopy using specific antisera, time-lapse cinematography, and electron microscopy. During cell fusion, micro tubules and 10-nm filaments from many cells form large bundles which are localized between rows of nuclei. No organized bundles of actin fibers were detected in these areas, although actin fibers were observed in regions away from the aligned nuclei. Although colchicine disrupts microtubules and inhibits nuclear movement, cytochalasin B (CB; 20-50 microgram/ml) does not inhibit cell fusion or nuclear movement. However, CB alters the shape of the syncytium, resulting in long filamentous processes extending from a central region. When these processes from neighboring cells make contact, fusion occurs, and nuclei migrate through the channels which are formed. Electron and immunofluorescence microscopy reveal bundles of microtubules and 10-nm filaments in parallel arrays within these processes, but no bundles of microfilaments were detected. The effect of CB on the structural integrity of microfilaments at this high concentration (20 microgram/ml) was demonstrated by the disappearance of filaments interacting with heavy meromyosin. Cycloheximide (20 microgram/ml) inhibits protein synthesis but does not affect cell fusion, the formation of microtubules and 10-nm filament bundles, or nuclear migration and alignment; thus, continued protein synthesis is not required. The association of microtubules and 10-nm filaments with nuclear migration and alignment suggests that microtubules and 10-nm filaments are two components in a system which serves both cytoskeletal and force-generating functions in intracellular movement and position of nuclei.     

Two distinct attachment sites for vimentin along the plasma membrane and the nuclear envelope in avian erythrocytes: a basis for a vectorial assembly of intermediate filaments

In vitro binding studies with isolated bovine lens vimentin and avian erythrocyte membranes reveal the existence of two functionally distinct sets of intermediate filament attachment sites. One population of such receptors is located along the nuclear envelope and comprises polypeptides recognizing the carboxy-terminal tail domain of vimentin. Vimentin associates with these nuclear surface receptors in a cooperative manner and forms extensive 10-nm filaments in a concentration-dependent fashion. Conversely, the plasma membrane contains binding sites that interact in a noncooperative, saturable fashion with vimentin, recognizing its amino-terminal head domain. The functional dichotomy of the vimentin-binding sites under in vitro conditions may reflect a vectorial assembly process whereby 10-nm filaments, although structurally apolar, acquire polar features brought about by the differential attachment to specific receptors arranged along the plasma membrane and the nuclear envelope.     

INTRANUCLEAR NETWORK OF 3–5nm AND 8–10nm FIBERS IN EL-4 LYMPHOMA CELLS

While much evidence indicates a high degree of spatial organization in the nucleus, the underlying molecular structures that support it remain poorly characterized. By extracting with high concentrations of RNase A in a modification of the sequential extraction protocol of Penman, we have identified a novel intranuclear network in the mouse lymphoma cell line, EL-4. Micrographs of embedment-free sections of extracted cells reveal anastomosing filaments of two different diameters: 3–5nm and 8–10nm. The 3–5-nm filaments are interconnected in many junctions and appear to blend smoothly into each other. The 8–10-nm fibers frequently split into two 3–5-nm filaments. Some 3–5-nm fibers appear to be connected at 90° angles with the 8–10-nm fibers. All junctions are smooth with no apparent junction protein. Flow cytometric analysis of RNase A- (and DNase I-) extracted nuclear matrices indicates that they do not contain significant amounts of protein that react with anti-actin and anti-vimentin monoclonal antibodies. Extraction of EL-4 nuclear matrices with high salt does not reveal 8–10-nm core filaments described after similar treatment of tumor cell lines of cervical and mammary origin. The novel characteristics of the core filaments in EL-4 lymphoma cells may reflect cell-type specificity of the nuclear matrix.     

Urothelial plaque formation in post-Golgi compartments

Urothelial plaques are specialized membrane domains in urothelial superficial (umbrella) cells, composed of highly ordered uroplakin particles. We investigated membrane compartments involved in the formation of urothelial plaques in mouse umbrella cells. The Golgi apparatus did not contain uroplakins organized into plaques. In the post-Golgi region, three distinct membrane compartments containing uroplakins were characterized: i) Small rounded vesicles, located close to the Golgi apparatus, were labelled weakly with anti-uroplakin antibodies and they possessed no plaques; we termed them "uroplakin-positive transporting vesicles" (UPTVs). ii) Spherical-to-flattened vesicles, termed "immature fusiform vesicles" (iFVs), were uroplakin-positive in their central regions and contained small urothelial plaques. iii) Flattened "mature fusiform vesicles" (mFVs) contained large plaques, which were densely labelled with anti-uroplakin antibodies. Endoytotic marker horseradish peroxidase was not found in these post-Golgi compartments. We propose a detailed model of de novo urothelial plaque formation in post-Golgi compartments: UPTVs carrying individual 16-nm particles detach from the Golgi apparatus and subsequently fuse into iFV. Concentration of 16-nm particles into plaques and removal of uroplakin-negative membranes takes place in iFVs. With additional fusions and buddings, iFVs mature into mFVs, each carrying two urothelial plaques toward the apical surface of the umbrella cell.     

Acceleration of the Decay of Membrane Potential after Energization of Chloroplasts in Intact Zea mays Leaves

The relationship between dissipation of the flash-induced membranepotential across the thylakoid membrane and the high energystate was studied in Zea mays leaves. The dark decay of theflash-induced 515-nm absorbance change was accelerated by shortpreillumination of the leaf. No acceleration of the decay bypreillumination was observed when leaves were incubated in argonor CO² gas or treated with DCMU. These effects of preilluminationand incubation were reversible. The delayed fluorescence from chlorophyll a was reversibly decreasedby incubating leaves in argon or CO² gas, though the modes ofdepression were somewhat different from each other. In leavesincubated in argon or CO² gas, the phase of slow decrease ofthe intensity of prompt fluorescence during illumination reversiblydisappeared. The results suggested that the dissipation of membrane potentialgenerated by a flash was accelerated after the energizationof chloroplasts in leaves, probably by increased H^– permeabilityof the thylakoid membrane. O2 was important in maintaining (indarkness) and forming (under illumination) the high energy statein chloroplasts in intact leaves. (Received October 1, 1980; Accepted December 15, 1980)     

Chromatin and nuclear envelope of freeze-fractured, neuronal interphase nuclei, resolved by scanning electron microscopy

High-resolution scanning electron microscopy (SEM) has previously been used to study intracellular detail, including chromatin. It has, however, been commonly carried out either on cellular subfractions or following extraction methods to visualize detail. In the work presented here, intracellular detail of neurons of the dorsal root was visualized in situ by viewing freeze-fracture faces obtained after hypotonic expansion. This procedure permits the detailed resolution, by SEM, of juxtanuclear and intranuclear detail to a degree impossible without hypotonic dispersal. In agreement with work previously reported, nuclear chromatin of these interphase cells presents largely as 30-nm fibers, with a next higher hierarchical structure imparted by swelling in magnesium chloride. Detailed analyses showed that particles as small as 10-nm nucleosomes comprising the 30-nm chromatin fiber could be resolved, with "end-on" views of such fibers showing 5 nucleosomes per helical turn of the fiber. Chromatin fibers positioned subjacent to nuclear pores, or associated with "nuclear spaces" communicating with nuclear pores, were frequently found to be resolved as clusters, in an apparently more decondensed conformation, rather than tightly coiled into the 30-nm fiber. In addition, details of the nuclear envelope, including nuclear pores and perinuclear filaments as well as membranes of the endoplasmic reticulum, decorated with ribosomes, were clearly resolved.     

Spermiogenesis and chromatin condensation in the common tree shrew, <Emphasis Type="Italic">Tupaia glis</Emphasis>

We have investigated the cellular characteristics, especially chromatin condensation and the basic nuclear protein profile, during spermiogenesis in the common tree shrew, Tupaia glis. Spermatids could be classified into Golgi phase, cap phase, acrosome phase, and maturation phase. During the Golgi phase, chromatin was composed of 10-nm and 30-nm fibers with few 50-nm to 60-nm knobby fibers. The latter were then transformed into 70-nm knobby fibers during the cap phase. In the acrosome phase, all fibers were packed into the highest-order knobby fibers, each about 80–100 nm in width. These chromatin fibers became tightly packed in the maturation phase. In a mature spermatozoon, the discoid-shaped head was occupied by the acrosome and completely condensed chromatin. H3, the core histone, was detected by immunostaining in all nuclei of germ cell stages, except in spermatid steps 15–16 and spermatozoa. Protamine, the basic nuclear protein causing the tight packing of sperm chromatin, was detected by immunofluorescence in the nuclei of spermatids at steps 12–16 and spermatozoa. Cross-immunoreactivity of T. glis H3 and protamine to those of primates suggests the evolutionary resemblance of these nuclear basic proteins in primate germ cells. This work was supported by the Thailand Research Fund (Senior Research Fellowship to Prof. Prasert Sobhon).