A method for incorporating macromolecules into adherent cells

We describe a simple method for loading exogenous macromolecules into the cytoplasm of mammalian cells adherent to tissue culture dishes. Culture medium was replaced with a thin layer of fluorescently labeled macromolecules, the cells were harvested from the substrate by scraping with a rubber policeman, transferred immediately to ice cold media, washed, and then replated for culture. We refer to the method as "scrape-loading." Viability of cells was 50-60% immediately after scrape-loading and was 90% for those cells remaining after 24 h of culture. About 40% of adherent, well-spread fibroblasts contained fluorescent molecules 18 h after scrape-loading of labeled dextrans, ovalbumin, or immunoglobulin-G. On average, 10(7) dextran molecules (70,000-mol wt) were incorporated into each fibroblast by scrape-loading in 10 mg/ml dextran. The extent of loading depended on the concentration and molecular weight of the dextrans used. A fluorescent analog of actin could also be loaded into fibroblasts where it labeled stress fibers. HeLa cells, a macrophage-like cell line, 1774A.1, and human neutrophils were all successfully loaded with dextran by scraping. The method of scrape-loading should be applicable to a broad range of adherent cell types, and useful for loading of diverse kinds of macromolecules.     

Macropinosome maturation and fusion with tubular lysosomes in macrophages

Macropinosomes formed by addition of recombinant macrophage colony- stimulating factor (rM-CSF) to mouse macrophages migrate centripetally and shrink, remaining detectable by phase microscopy for up to 15 min. This longevity allowed us to study how macropinosomes age. Macropinosomes were pulse labeled for 1 min with fixable fluorescein dextran (FDx10f), a probe for fluid phase pinocytosis, and chased for various times. To quantify changes in their antigenic profile, pulse- labeled macropinosomes of different ages were fixed and stained for immunofluorescence with a panel of antibodies specific for the transferrin receptor (TfR), the late endosome-specific, GTP-binding protein rab 7 or lysosomal glycoprotein A (lgp-A), and the percentage of antibody positive, FDx10f-labeled macropinosomes was scored. Some newly formed macropinosomes were positive for TfR, but few were rab 7 or lgp-A-positive. With intermediate chase times (2-4 min), staining for rab 7 and lgp-A increased to > 60%, while TfR staining declined. After a long chase (9-12 min), rab 7 staining returned to low levels while lgp-A staining remained at a high level. Thus, macropinosomes matured by progressive acquisition and loss of characteristic endocytic vesicle markers. However, unlike a maturation process, their merger with the tubular lysosomal compartment more nearly resembled the incorporation of a transient vesicle into a pre-existing, stable compartment. Shortly after their formation, FDx10f-labeled macropinosomes contacted and merged with Texas red dextran (TRDx10)- labeled tubular lysosomes. This occurred in two steps: macropinosomes acquired lgp-A first, and then several minutes later the cation- independent mannose-6-phosphate receptor (CI-MPR) and markers of lysosomal content (cathepsin L or pre-loaded TRDx10), all apparently derived from tubular lysosomes. Thus, macropinosome progress through macrophages showed features of both the maturation and vesicle shuttle models of endocytosis, beginning with a maturation process and ending by merger into a stable, resident lysosomal compartment.     

Evidence for mediated protein uptake by amphibian oocyte nuclei

The objective of this investigation was to determine whether there is mediated transport of endogenous proteins across the nuclear envelope. For this purpose, we studied the nuclear uptake of a 148,000-dalton Rana oocyte polypeptide (RN1) and compared its actual uptake rate with the rate that would be expected if RN1 crossed the envelope by simple diffusion through the nuclear pores. Nuclear uptake was studied in two ways: first, oocytes were incubated in L-[3H]leucine for 1 h and, at various intervals after labeling, the amount of 3H-RN1 present in the nucleoplasm was determined. Second, L-[3H]leucine-labeled nuclear extracts, containing RN1, were microinjected into the cytoplasm of nonlabeled cells, and the proportion of 3H-RN1 that subsequently entered the nucleus was measured. It was found that RN1 can readily penetrate the nuclear envelope; for example, after 6 h, approximately 36% of the newly synthesized RN1 and 17% of the injected RN1 had entered the nucleus. The diffusion rate through pores having a radius of 45 A was calculated for several possible molecular configurations of RN1. Using axial ratios of 34, 7.5, 2, and 1, the estimated times required to reach 63% of diffusion equilibrium are 757, 468, 6,940 h, and infinity, respectively. Even assuming an axial ratio of 7.5 (the most diffusive configuration) and an equilibrium distribution of 45, simple diffusion through the pores could account for only approximately 1/20 the observed nuclear uptake of RN1. This and other comparisons indicate that some form of mediated transport is involved in the nucleocytoplasmic exchange of this polypeptide.     

Opening of size-selective pores in endosomes during human rhinovirus serotype 2 in vivo uncoating monitored by single-organelle flow analysis

The effect of virus uncoating on endosome integrity during the early steps in viral infection was investigated. Using fluid-phase uptake of 10- and 70-kDa dextrans labeled with a pH-dependent fluorophore (fluorescein isothiocyanate [FITC]) and a pH-independent fluorophore (cyanine 5 [Cy5]), we determined the pHs of labeled compartments in intact HeLa cells by fluorescence-activated cell sorting analysis. Subsequently, the number and pH of fluorescent endosomes in cell homogenates were determined by single-organelle flow analysis. Cointernalization of adenovirus and 70-kDa FITC- and Cy5-labeled dextran (FITC/Cy5-dextran) led to virus-induced endosomal rupture, resulting in the release of the marker from the low-pH environment into the neutral cytosol. Consequently, in the presence of adenovirus, the number of fluorescent endosomes was reduced by 40% compared to that in the control. When human rhinovirus serotype 2 (HRV2) was cointernalized with 10-and 70-kDa FITC/Cy5-dextrans, the 10-kDa dextran was released, whereas the 70-kDa dextran remained within the endosomes, which also maintained their low pH. These data demonstrate that pores are generated in the membrane during HRV2 uncoating and RNA penetration into the cytosol without gross damage of the endosomes; 10-kDa dextran can access the cytosol through these pores. Whereas rhinovirus-mediated pore formation was prevented by the vacuolar ATPase inhibitor bafilomycin A1, adenovirus-mediated endosomal rupture also occurred in the presence of the inhibitor. This finding is in keeping with the low-pH requirement of HRV2 infection; for adenovirus, no pH dependence for endosomal escape was found with this drug.     

Antiport mediated rounding and endocytosis are enhanced by sulphate

Rapid cell detachment concomitant with the flat-to-round (FTR) change that is mediated by an upshifted Na+/H+ antiporter via HCO3(-)-dependent H+ pumping, is significantly enhanced by the addition of Na2SO4 (FTR + SO4): (1) a faster and greater reduction in cell surface area and perimeter, and (2) a higher level of macromolecular internalization which is also amiloride sensitive. At a fixed 1 mg/ml extracellular FITC-dextran (FDx) concentration, the intracellular FDx load is similar irrespective of the particle size, in the range from 4400 to 2 million mol.wt which is a 455-fold diversity. This is inconsistent with entry via limited sized portals which would discriminate against the larger molecular weight species, such as the 2 million mol.wt species that measures up to 5 microns in width. Two million mol.wt FDx loads linearly in direct proportion to the extracellular FDx concentration, simulating simple diffusion. Large-channel endocytosis is considered to be a characteristic of specialized cell types such as phagocytes and macrophages. However, the antiporter mediated endocytosis (AME) shown here is demonstrated in two different cell types which are not known for their endocytic prowess, viz. epitheloid human Chang liver cells (ATCC CCL 13) and human lung fibroblasts (ATCC CCL 202). The rounded cells with internalized FDx start reverting back to their flat and protracted form upon flooding with warm growth medium, a round-to-flat (RTF) change. However the cell surface reversion is not associated with efflux of FDx which are sorted out into 'granular patches', the later stage endosomes without membrane outlines in AME. FDx-loaded cells grow as well as trypsinized cells without FDx loaing and they maintain a significant FDx load even after nearly 4 cell divisions. Toad sperms internalized into Chang cells via antiporter activation are also sorted into granular patches. AME provides (a) distinctive access to large particles, simulating small ion influx, and (b) an alternate membrane recycling capability where granular patches are instrumental in sorting. It appears to be not a simple endocytosis-exocytosis pathway.     

Characterization of collagenous meshworks by volume exclusion of dextrans.

The volumes from which 3H-labelled dextrans are excluded by dermal collagenous fibres were calculated by dilution of dextran probes. Five dextrans, of average Stokes' radii 1.72, 2.53, 3.92, 4.54 and 14.24nm, were investigated at concentrations between 0.1 and 3% (w/w). The excluded volume was dependent on dextran concentration only for the two smaller probes. The largest dextran was shown not to bind to the fibres. A plot of the square root of excluded volume against Stokes' radius was linear for the four smallest dextrans, corresponding to the predictions of Ogston's [(1958) Trans. Faraday Soc. 54, 1754--1757] rod-and-sphere model of fibrous exclusion, and suggesting that dextrans of Stokes' radius between 1.72 and 4.54 nm were excluded by a cylindrical solid fibre of radius 2.90 +/- 0.72 nm. Larger molecules were excluded by a structure of much greater size, since the volume exclusion for the largest dextran was only slightly greater than that of the dextran less than one-third its radius. The excluded volume of 3H2O fell slightly below the line describing the dextran data, indicating that water had access to most of the volume not occupied by the collagenous fibres.     

Large Plasma Membrane Disruptions Are Rapidly Resealed by Ca2+-dependent Vesicle–Vesicle Fusion Events

A microneedle puncture of the fibroblast or sea urchin egg surface rapidly evokes a localized exocytotic reaction that may be required for the rapid resealing that follows this breach in plasma membrane integrity (Steinhardt, R.A,. G. Bi, and J.M. Alderton. 1994. Science (Wash. DC). 263:390–393). How this exocytotic reaction facilitates the resealing process is unknown. We found that starfish oocytes and sea urchin eggs rapidly reseal much larger disruptions than those produced with a microneedle. When an ~40 by 10 μm surface patch was torn off, entry of fluorescein stachyose (FS; 1,000 mol wt) or fluorescein dextran (FDx; 10,000 mol wt) from extracellular sea water (SW) was not detected by confocal microscopy. Moreover, only a brief (~5–10 s) rise in cytosolic Ca²⁺ was detected at the wound site. Several lines of evidence indicate that intracellular membranes are the primary source of the membrane recruited for this massive resealing event. When we injected FS-containing SW deep into the cells, a vesicle formed immediately, entrapping within its confines most of the FS. DiI staining and EM confirmed that the barrier delimiting injected SW was a membrane bilayer. The threshold for vesicle formation was ~3 mM Ca²⁺ (SW is ~10 mM Ca²⁺). The capacity of intracellular membranes for sealing off SW was further demonstrated by extruding egg cytoplasm from a micropipet into SW. A boundary immediately formed around such cytoplasm, entrapping FDx or FS dissolved in it. This entrapment did not occur in Ca²⁺-free SW (CFSW). When egg cytoplasm stratified by centrifugation was exposed to SW, only the yolk platelet–rich domain formed a membrane, suggesting that the yolk platelet is a critical element in this response and that the ER is not required. We propose that plasma membrane disruption evokes Ca²⁺ regulated vesicle–vesicle (including endocytic compartments but possibly excluding ER) fusion reactions. The function in resealing of this cytoplasmic fusion reaction is to form a replacement bilayer patch. This patch is added to the discontinuous surface bilayer by exocytotic fusion events.     

In vitro stimulation of human endothelial cells by derivatized dextrans

Summary Derivatized dextrans exert a stimulatory effect on the in vitro growth of human umbilical vein endothelial cells (HUVEC). Measurements of growth were monitored by [³H]thymidine uptake and cell numbers. Our results show that some derivatized dextrans at 4 μg/ml (88 nM) increase the [³H]thymidine incorporation, whereas starting dextran (40 000 Da), dextran sulfate, and carboxymethyl dextran have no effect. In addition, heparin under similar experimental conditions shows a slight inhibitory effect on the HUVEC growth. The stimulatory effect of derivatized dextrans was also found when HUVEC grew during 7 days in medium containing 2% fetal bovine serum. We also observed that derivatized dextrans had no effect on the mitogenic activity of acidic fibroblast growth factor, a mitogenic factor for several cell types including HUVEC. By assessment of [³H]thymidine uptake at 48 h without serum, we concluded that the exogenous growth factors were not involved in the proliferative activity of these components. The stimulatory effects are related to the chemical nature and the proportion of substituents on the synthetic polysaccharides. The data indicate that benzylamide sulfonated groups play a key role in the stimulation of HUVEC growth. Neither carboxyl nor sulfate groups alone exhibit this effect. Thus, the stimulatory capacity of dextran derivatives depends strongly on the respective ratios of the functional groups.     

Nucleocytoplasmic protein traffic in single mammalian cells studied by fluorescence microphotolysis

Fluorescence microphotolysis was employed to measure in single living cells the kinetics of nucleocytoplasmic transport and the coefficients of intracellular diffusional mobility for the nuclear non-chromosomal protein nucleoplasmin. Nucleoplasmin was isolated from Xenopus ovary and labeled fluorescently. By injection into Xenopus oocytes it was ascertained that fluorescent labeling did not interfere with normal nuclear accumulation. Upon injection into the cytoplasm of various mammalian cell types nucleoplasmin was rapidly taken up by the nucleus. In rat hepatoma cells the half-time of nuclear uptake was approx. 5 min at 37 degrees C; the nucleocytoplasmic equilibrium concentration ratio had a maximum of 6.5 +/- 1.4 and depended on the injected amount. Upon co-injection of ATPases or reduction of temperature to 10 degrees C a nucleocytoplasmic equilization but no nuclear accumulation was observed. Equilization was fast (time constant 65 s at 23 degrees C), similar to that of 10-kDa dextran permeating the nuclear envelope by simple diffusion through functional pores. Nucleoplasmin (160 kDa), however, is too large to permeate passively the nuclear envelope, which is apparent from the fact that its tryptic 'core' fragment (100 kDa) could not permeate the nuclear envelope. On the other hand, a large fluorescent protein, phycoerythrin (240 kDa), was targeted to the nucleus by conjugation with nucleoplasmin. In the nucleus-to-cytoplasm direction the nuclear envelope was completely impermeable to nucleoplasmin, independently of temperature or ATP depletion. Nucleoplasmin, its core fragment, phycoerythrin and the phycoerythrin-nucleoplasmin conjugate were mobile in both cytoplasm and nucleus.     

Caspase-2 can trigger cytochrome C release and apoptosis from the nucleus

The cysteine proteases specific for aspartic residues, known as caspases, are localized in different subcellular compartments and play specific roles during the regulative and the executive phase of the cell death process. Here we investigated the subcellular localization of caspase-2 in healthy cells and during the execution of the apoptotic program. We have found that caspase-2 is a nuclear resident protein and that its import into the nucleus is regulated by two different nuclear localization signals. We have shown that in an early phase of apoptosis caspase-2 can trigger mitochondrial dysfunction from the nucleus without relocalizing into the cytoplasm. Release of cytochrome c occurs in the absence of overt alteration of the nuclear pores and changes of the nuclear/cytoplasmic barrier. Addition of leptomycin B, an inhibitor of nuclear export, did not interfere with the ability of caspase-2 to trigger cytochrome c release. Only during the late phase of the apoptotic process can caspase-2 relocalize in the cytoplasm, as consequence of an increase in the diffusion limits of the nuclear pores. Taken together these data indicate the existence of a nuclear/mitochondrial apoptotic pathway elicited by caspase-2.     

Visual and functional demonstration of growing Bax-induced pores in mitochondrial outer membranes

Bax induces mitochondrial outer membrane permeabilization (MOMP), a critical step in apoptosis in which proteins are released into the cytoplasm. To resolve aspects of the mechanism, we used cryo-electron microscopy (cryo-EM) to visualize Bax-induced pores in purified mitochondrial outer membranes (MOMs). We observed solitary pores that exhibited negative curvature at their edges. Over time, the pores grew to ∼100–160 nm in diameter after 60–90 min, with some pores measuring more than 300 nm. We confirmed these results using flow cytometry, which we used to monitor the release of fluorescent dextrans from isolated MOM vesicles. The dextran molecules were released gradually, in a manner constrained by pore size. However, the release rates were consistent over a range of dextran sizes (10–500 kDa). We concluded that the pores were not static but widened dramatically to release molecules of different sizes. Taken together, the data from cryo-EM and flow cytometry argue that Bax promotes MOMP by inducing the formation of large, growing pores through a mechanism involving membrane-curvature stress.     

Nucleo-cytoplasmic flux and intracellular mobility in single hepatocytes measured by fluorescence microphotolysis.

Fluorescence microphotolysis was used to measure nucleocytoplasmic flux in single rat hepatocytes for a series of dextrans ranging in molecular mass from 3 to 150 kd. The cytoplasmic translational diffusion coefficient DC and the nucleoplasmic diffusion coefficient DN of a 62-kd dextran were also determined. DC was approximately 2 X 10(-8) and DN approximately 3 X 10(-8) cm2/s, i.e., 1/20-1/15 of the value in free solution. The mobile fraction amounted to 0.7-0.8 in measurements of both intracellular diffusion and nucleo-cytoplasmic flux. The flux of dextrans from cytoplasm to nucleus depended inversely on molecular mass with an exclusion limit between 17 and 41 kd suggesting that the nuclear envelope has functions of a molecular sieve. Employing the Pappenheimer-Renkin equations, a functional pore radius of 50-56 A was derived. By comparison with recent measurements on isolated liver cell nuclei, large quantitative differences between the intracellularly located and the isolated nucleus were revealed.     

Intracellular macromolecular mobility measured by fluorescence recovery after photobleaching with confocal laser scanning microscopes

Fluorescence recovery after photobleaching (FRAP) is a widely used tool for estimating mobility parameters of fluorescently tagged molecules in cells. Despite the widespread use of confocal laser scanning microscopes (CLSMs) to perform photobleaching experiments, quantitative data analysis has been limited by lack of appropriate practical models. Here, we present a new approximate FRAP model for use on any standard CLSM. The main novelty of the method is that it takes into account diffusion of highly mobile molecules during the bleach phase. In fact, we show that by the time the first postbleach image is acquired in a CLSM a significant fluorescence recovery of fast-moving molecules has already taken place. The model was tested by generating simulated FRAP recovery curves for a wide range of diffusion coefficients and immobile fractions. The method was further validated by an experimental determination of the diffusion coefficient of fluorescent dextrans and green fluorescent protein. The new FRAP method was used to compare the mobility rates of fluorescent dextrans of 20, 40, 70, and 500 kDa in aqueous solution and in the nucleus of living HeLa cells. Diffusion coefficients were lower in the nucleoplasm, particularly for higher molecular weight dextrans. This is most likely caused by a sterical hindrance effect imposed by nuclear components. Decreasing the temperature from 37 to 22 degrees C reduces the dextran diffusion rates by approximately 30% in aqueous solution but has little effect on mobility in the nucleoplasm. This suggests that spatial constraints to diffusion of dextrans inside the nucleus are insensitive to temperature.     

Rheology of native dextrans in relation to their primary structure

High molecular weight dextrans were synthesized at five temperatures (3, 10, 20, 25 and 30°C) using an in-vitro enzymatic method. The rheological properties of these dextrans in aqueous solution were assessed through their flow behaviour and their viscoelastic characteristics. The results were interpreted in relation to their primary structure and particularly to their branching.

It was shown that the relatively expanded conformation of the dextrans synthesized at 3, 10 and 20°C gives to these dextrans comparable properties which are not too different from those described in literature for random-coil linear polysaccharides. Dextran synthesized at 30°C exhibited flow properties which are typical of particle suspensions in dilute and semi-dilute solution. In the concentrated domain, this dextran yielded structured systems with properties typical of weak gels. This unexpected behaviour could be related to the highly-ramified structure of this dextran in comparison with the dextrans synthesized between 3 and 20°C. On the other hand, the dextran synthesized at 25°C displayed rheological behaviour which could also be related to an intermediate primary structure between those of dextran synthesized at 20°C and dextran synthesized at 30°C.     

The size of the nucleus increases as yeast cells grow

It is not known how the volume of the cell nucleus is set, nor how the ratio of nuclear volume to cell volume (N/C) is determined. Here, we have measured the size of the nucleus in growing cells of the budding yeast Saccharomyces cerevisiae. Analysis of mutant yeast strains spanning a range of cell sizes revealed that the ratio of average nuclear volume to average cell volume was quite consistent, with nuclear volume being approximately 7% that of cell volume. At the single cell level, nuclear and cell size were strongly correlated in growing wild-type cells, as determined by three different microscopic approaches. Even in G1-phase, nuclear volume grew, although it did not grow quite as fast as overall cell volume. DNA content did not appear to have any immediate, direct influence on nuclear size, in that nuclear size did not increase sharply during S-phase. The maintenance of nuclear size did not require continuous growth or ribosome biogenesis, as starvation and rapamycin treatment had little immediate impact on nuclear size. Blocking the nuclear export of new ribosomal subunits, among other proteins and RNAs, with leptomycin B also had no obvious effect on nuclear size. Nuclear expansion must now be factored into conceptual and mathematical models of budding yeast growth and division. These results raise questions as to the unknown force(s) that expand the nucleus as yeast cells grow.     

High-efficiency intracellular magnetic labeling with novel superparamagnetic-Tat peptide conjugates

A biocompatible, dextran coated superparamagnetic iron oxide particle was derivatized with a peptide sequence from the HIV-tat protein to improve intracellular magnetic labeling of different target cells. The conjugate had a mean particle size of 41 nm and contained an average of 6.7 tat peptides. Derivatized particles were internalized into lymphocytes over 100-fold more efficiently than nonmodified particles, resulting in up to 12.7 x 10(6) particles/cell. Internalized particles localized in cytoplasm and nuclear compartments as demonstrated by fluorescence microscopy and immunohistochemistry. Labeled cells were highly magnetic, were detectable by NMR imaging, and could be retained on magnetic separation columns. The described method has potential applications for in vivo tracking of magnetically labeled cells by MR imaging and for recovering intracellularly labeled cells from organs.     

Diphtheria Toxin Forms Pores of Different Sizes Depending on Its Concentration in Membranes: Probable Relationship to Oligomerization

Diphtheria toxin forms pores in biological and model membranes upon exposure to low pH. These pores may play a critical role in the translocation of the A chain of the toxin into the cytoplasm. The effect of protein concentration on diphtheria toxin pore formation in model membrane systems was assayed by using a new fluorescence quenching method. In this method, the movement of Cascade Blue labeled dextrans of various sizes across membranes is detected by antibodies which quench Cascade Blue fluorescence. It was found that at low pH the toxin makes pores in phosphatidylcholine/phosphatidylglycerol vesicles with a size that depends on protein concentration. At the lowest toxin concentrations only the entrapped free fluorophore (MW 538) could be released from model membranes. At intermediate toxin concentrations, a 3 kD dextran could be released. At the highest toxin concentration, a 10 kD dextran could be released, but not a 70 kD dextran. Similar pore properties were found using vesicles lacking phosphatidylglycerol or containing 30% cholesterol. However, larger pores formed at lower protein concentrations in the presence of cholesterol. The dependence of pore size on toxin concentration suggests that toxin oligomerization regulates pore size. This behavior may explain some of the conflicting data on the size of the pores formed by diphtheria toxin. The formation of oligomers by membrane-inserted toxin is consistent with the results of chemical crosslinking and measurements of the self-quenching of rhodamine-labeled toxin. Based on these experiments we propose diphtheria toxin forms oligomers with a variable stoichiometry, and that pore size depends on the oligomerization state. Reasons why oligomerization could assist proper membrane insertion of the toxin and other proteins that convert from soluble to membrane-inserted states are discussed. Received: 10 March 1999/Revised: 22 June 1999     

Nuclear pore composition and gating in herpes simplex virus-infected cells

The mechanism by which herpes simplex virus (HSV) exits the nucleus remains a matter of controversy. The generally accepted route proposes that capsids exit via primary envelopment at the inner nuclear membrane and subsequent fusion of this primary particle with the outer nuclear membrane to gain capsid entry to the cytoplasm. However, recent observations indicate that HSV may induce gross morphological alterations of nuclear pores, resulting in the loss of normal pores and the appearance of dilated gaps in the nuclear membrane of up to several 100 nm. On this basis, it was proposed that a main route of capsid exit from the nucleus is directly through these altered pores. Here, we examine the biochemical composition of some of the major nuclear pore components in uninfected and HSV-infected cells. We show that total levels of major nucleoporins and their sedimentation patterns in density gradients remain largely unchanged up to 18 h after HSV infection. Some alteration in modification of one nucleoporin, Nup358/RanBP2, was observed during enrichment with anti-nucleoporin antibody and probing for O glycosylation. In addition, we examine functional gating within the nucleus in live cells, using microinjection of labeled dextran beads and a recombinant virus expressing GFP-VP16 to track the progress of infection. The nuclear permeability barrier for molecules bigger than 70 kDa remained intact throughout infection. Thus, in a functional assay in live cells, we find no evidence for gross perturbation to the gating of nuclear pores, although this might not exclude a small population of modified pores.     

An immunochemical study of the combining site specificities of C57BL/6J monoclonal antibodies to alpha (1----6)-linked dextran B512

This is the first report of an immunochemical study of the combining site specificities of a set of monoclonal antibodies to dextran B512 from C57BL/6J mice. The results confirm previous observations on antidextran combining sites and reveal specificities not seen earlier extending the observed repertoire of antibody combining sites to the single alpha (1----6)-linked glucosyl antigenic determinant. Eight C57BL/6J anti-dextran B512 hybridomas, four IgM,kappa and four IgA,kappa, were produced by PEG fusion of immune spleen cells with the nonproducer myeloma cell line P3X63Ag8 6.5.3. Antibody combining site specificities were determined by quantitative precipitin assays with 14 dextrans. Native dextrans with high percentages of linear alpha (1----6)-linked glucoses, similar to the immunogen B512, were the best precipitinogens; dextrans with alternating alpha (1----3), alpha (1----6) linkages, and highly branched dextrans were less effective. All antibodies precipitated with a synthetic, unbranched alpha (1----6)-linked dextran, suggesting their combining sites were "groove-like" and directed toward internal sequences of alpha (1----6)-linked residues, rather than "cavity-like" and directed toward a nonreducing terminal glucose. Two of the IgA hybridomas gave biphasic precipitin curves with dextran B512; this was shown to be due to differences in the precipitability of IgA monomers and polymers. Differences were observed in the reactivities of several dextrans considered previously to be structurally similar, and a newly proposed structural model of dextran B1299S was assessed. Quantitative precipitin inhibition studies with alpha (1----6)-linked isomaltosyl (IM) oligosaccharides, IM2 to IM9, showed that maximum inhibition was reached with IM6 or IM7, consistent with earlier estimates of the upper limit for the sizes of anti-B512 combining sites. Two IgM hybridomas showed a unique pattern, with inhibition being obtained only with IM5 or larger IM oligosaccharides. Association constants of the antidextrans for dextran B512 and for IM7, determined by affinity gel electrophoresis, ranged from 10(2) to 10(4) ml/g, comparable to earlier findings with antidextrans and other anticarbohydrate antibodies.