Search PubMed⌕ Search

Biomedical subjects

C M Feldherr

Publications and source records attributed to C M Feldherr.

At least 37 records · Page 2Linked to original sources
PubMed1988

Translocation of RNA-coated gold particles through the nuclear pores of oocytes.

In the present study, various sized gold particles coated with tRNA, 5S RNA, or poly(A) were used to localize and characterize the pathways for RNA translocation to the cytoplasm. RNA-coated gold particles were microinjected into the nucleus of Xenopus oocytes. The cells were fixed after 15, 60 min, or 6 h, and the particle distribution was later observed by electron microscopy. Similar results were obtained with all classes of RNA used. After nuclear injection, particles ranging from 20-230 A in diameter were observed within central channels of the nuclear pores and in the cytoplasm immediately adjacent to the pores. Particles of this size would not be expected to diffuse through the pores, suggesting that some form of mediated transport occurred. In addition, it was found that the translocation process is saturable. At least 97% of the pores analyzed appeared to be involved in the translocation process. Gold coated with nonphysiological polynucleotides (poly[I] or poly[dA]) were also translocated. When nuclei were injected with either BSA-, ovalbumin-, polyglutamic acid-, or PVP-coated gold, the particles were essentially excluded from the pores. These results indicate that the accumulation of RNA-gold within the pores and adjacent cytoplasm was not due to non-specific effects. We conclude that the translocation sites for gold particles coated with different classes of RNA are located in the centers of the nuclear pores and that particles at least 230 A in diameter can cross the envelope. Tracer particles injected into the cytoplasm were observed within the nuclear pores in areas near the site of injection. However, only a small percentage of the particles actually entered the nucleus. It was also determined, by performing double injection experiments, that individual pores are bifunctional, that is, capable of transporting both proteins and RNA.

Animals↗
PubMed1988

The effects of variations in the number and sequence of targeting signals on nuclear uptake.

To determine if the number of targeting signals affects the transport of proteins into the nucleus, Xenopus oocytes were injected with colloidal gold particles, ranging in diameter from 20 to 280 A, that were coated with BSA cross-linked with synthetic peptides containing the SV-40 large T-antigen nuclear transport signal. Three BSA conjugate preparations were used; they had an average of 5, 8, and 11 signals per molecule of carrier protein. In addition, large T-antigen, which contains one signal per monomer, was used as a coating agent. The cells were fixed at various times after injection and subsequently analyzed by electron microscopy. Gold particles coated with proteins containing the SV-40 signal entered the nucleus through central channels located within the nuclear pores. Analysis of the intracellular distribution and size of the tracers that entered the nucleus indicated that the number of signals per molecule affect both the relative uptake of particles and the functional size of the channels available for translocation. In control experiments, gold particles coated with BSA or BSA conjugated with inactive peptides similar to the SV-40 transport signal were virtually excluded from the nucleus. Gold particles coated with nucleoplasmin, an endogenous karyophilic protein that contains five targeting signals per molecule, was transported through the nuclear pores more effectively than any of the BSA-peptide conjugates. Based on a correlation between the peri-envelope density of gold particles and their relative uptake, it is suggested that the differences in the activity of the two targeting signals is related to their binding affinity for envelope receptors. It was also determined, by performing coinjection experiments, that individual pores are capable of recognizing and transporting proteins that contain different nuclear targeting signals.

Amino Acid Sequence↗
PubMed1985

Nucleocytoplasmic RNA transport.

A number of closely related post-transcriptional facets of RNA metabolism show nuclear compartmentation, including capping, methylation, splicing reactions, and packaging in ribonucleoprotein particles (RNP). These nuclear 'processing' events are followed by the translocation of the finished product across the nuclear envelope. Due to the inherent complexity of these interrelated events, in vitro systems have been designed to examine the processes separately, particularly so with regard to translocation. A few studies have utilized nuclear transplantation/microinjection techniques and specialized systems to show that RNA transport occurs as a regulated phenomenon. While isolated nuclei swell in aqueous media and dramatic loss of nuclear protein is associated with this swelling, loss of RNA is not substantial, and most studies on RNA translocation have employed isolated nuclei. The quantity of RNA transported from isolated nuclei is related to hydrolysis of high-energy phosphate bonds in nucleotide additives. The RNA is released predominantly in RNP: messenger-like RNA is released in RNP which have buoyant density and polypeptide composition similar to cytoplasmic messenger RNP, but which have distinctly different composition from those in heterogeneous nuclear RNP. Mature 18 and 28S ribosomal RNA is released in 40 and 60S RNP which represent mature ribosomal subunits. RNA transport proceeds with characteristics of an energy-requiring process, and proceeds independently of the presence or state of fluidity of nuclear membranes. The energy for transport appears to be utilized by a nucleoside triphosphatase (NTPase) which is distributed mainly within heterochromatin at the peripheral lamina. Photoaffinity labeling has identified the pertinent NTPase as a 46 kD polypeptide which is associated with nuclear envelope and matrix preparations. The NTPase does not appear to be modulated via direct phosphorylation or to reflect kinase-phosphatase activities. A large number of additives (including RNA and insulin) produce parallel effects upon RNA transport and nuclear envelope NTPase, strengthening the correlative relationship between these activities. Of particular interest has been the finding that carcinogens induce specific, long-lasting increases in nuclear envelope (and matrix) NTPase; this derangement may underlie the alterations in RNA transport associated with cancer and carcinogenesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1984

Movement of a karyophilic protein through the nuclear pores of oocytes.

It has recently been shown that large karyophilic proteins are transported across the nuclear envelope in amphibian oocytes. In consideration of this, the present experiments were performed to identify the specific sites within the envelope through which transport occurs and determine if molecular size is a limiting factor in the transport process. The following experimental procedure was employed: Colloidal gold particles, varying in size from approximately 20 to 170 A in diameter were coated with nucleoplasmin, a 165,000-mol-wt karyophilic protein, which is known to be transported through the envelope. The coated gold particles were microinjected into the cytoplasm of Xenopus oocytes, and the cells were fixed 15 min and 1 h later. The intracellular localization of the gold was then determined with the electron microscope. It was found that nucleoplasmin-coated particles readily enter the nucleus. On the basis of the distribution of the particles associated with the envelope, we concluded that transport occurs through the nuclear pores. Furthermore, the size distributions of the gold particles present in the nucleus and cytoplasm were not significantly different, indicating that the envelope does not discriminate among particles with diameters ranging from 50 to 200 A (the dimensions including the nucleoplasmin coat). Colloidal gold coated with trypsin-digested nucleoplasmin (which lacks the polypeptide domain required for transport) or exogenous polyvinylpyrrolidone were largely excluded from the nucleus and showed no evidence of transport.

Animals↗
PubMed1983

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.

Animals↗
PubMed1980

Ribosomal RNA synthesis and transport following disruption of the nuclear envelope.

Experiments were designed to determine if the synthesis, processing and transport of rRNA are affected by changes in nuclear permeability. RNA was labeled by microinjecting [3H]GTP into the cytoplasm of defolliculated oocytes. After injection, the nuclear envelopes were disrupted by puncturing the cells with glass needles. It has been shown that this procedure significantly alters the physical properties of the nuclear envelope. At appropriate intervals after puncturing, the oocytes were manually enucleated and RNA was extracted from both nuclear and cytoplasmic fractions. The extracts were analyzed on 2.5% polyacrylamide gels. The results indicate that over a period of 4 1/2 h neither the production nor the nucleocytoplasmic distribution of rRNA are affected by altering the permeability characteristics of the nuclear envelope.

Animals↗
PubMed1980

Mechanism for the selection of nuclear polypeptides in Xenopus oocytes. II. Two-dimensional gel analysis.

The role of the nuclear envelope in controlling intracellular protein exchanges was investigated in vivo, by determining the effect of altering nuclear permeability on (a) the protein composition of the nucleoplasm and (b) the nuclear uptake rates of specific endogenous proteins. The nuclear envelopes were disrupted by puncturing oocytes in the region of the germinal vesicle by use of glass needles. Nuclear proteins were analyzed in punctured and control cells by two-dimensional gel electrophoresis, fluorography, and double-labeling techniques. Over 300 nuclear polypeptides were identified in the fluorographs. Of this number, only approximately 10-15 were found to vary between punctured and control nuclei; furthermore, different polypeptides varied in each experiment. These qualitative studies indicate that specific binding within the nucleoplasm, and not selection by the envelope, is the main factor in maintaining the protein composition of the nucleus. The nuclear uptake rates of five individual polypeptides, ranging in molecular weight from 43,000 to 100,000, were analyzed by use of double-labeling procedures. Only one of the polypeptides (actin) entered the nuclei more rapidly after disruption of the envelope. That the nuclear uptake of certain endogenous proteins is unaffected by puncturing demonstrates that passage across the envelope is not a rate-limiting step in the nucleocytoplasmic exchange of these molecules.

Animals↗
PubMed1978

Mechanism for the selection of nuclear polypeptides in Xenopus oocytes.

The function of the nuclear envelope in regulating the cellular distribution of proteins was studied by experimentally altering nuclear permeability and determing the effect of the procedure on the incorporation of exogenous and endogenous polypeptides into the nucleoplasm. Using fine glass needles, nuclear envelopes were disrupted by puncturing oocytes in that region of the animal pole occupied by the germinal vesicle. This resulted in a highly significant increase in the nuclear uptake of cytoplasmically injected [125I]-bovine serum albumin ([125I]BSA), deomonstrating that the envelopes had lost their capacity to act as effective barriers to the diffusion of macromolecules. Endogenous proteins were labeled by incubating oocytes in L-[3H]lecuine. After appropriate intervals, nuclei were isolated from punctured and control cells and analyzed for tritiated polypeptides. Both total precipitable counts and the proportion of label in different size classes of polypeptides were compared. The results showed that puncturing the oocytes had no apparent quantitative or qualitative effects on the uptake of endogenous polypeptides by the nuclei. It can be concluded that the accumulation of specific nuclear proteins is not controlled by the envelope but rather by selective binding within the nucleoplasm.

Animals↗
PubMed1975

Basic principles of nuclear organization.

The preceding discussion is summarized in Table 1. It is clear that the organization of the genetic material and the ability to transmit information to the cytoplasm involve a number of different components that must maintain specific chemical and physical relationships to one another. An alteration of any of these relationships could affect a variety of cellular processes, including development.

Animals↗