The Cellular Level of PR500, a Protein Complex Related to the 19S Regulatory Particle of the Proteasome, Is Regulated in Response to Stresses in Plants

In Arabidopsis seedlings and cauliflower florets, Rpn6 (a proteasome non-ATPase regulatory subunit) was found in two distinct protein complexes of ∼800 and 500 kDa, respectively. The large complex likely represents the proteasome 19S regulator particle (RP) because it displays the expected subunit composition and all characteristics. The small complex, designated PR500, shares at least three subunits with the “lid” subcomplex of 19S RP and is loosely associated with an hsp70 protein. In Arabidopsis COP9 signalosome mutants, PR500 was specifically absent or reduced to an extent that correlates with the severity of the mutations. Furthermore, PR500 was also diminished in response to potential protein-misfolding stresses caused by the heat shock and canavanine treatment. Immunofluorescence studies suggest that PR500 has a distinct localization pattern and is enriched in specific nuclear foci. We propose that PR500 may be evolved in higher plants to cope with the frequently encountered environmental stresses.

Replication-dependent Histone Gene Expression Is Related to Cajal Body (CB) Association but Does Not Require Sustained CB Contact

Interactions between Cajal bodies (CBs) and replication-dependent histone loci occur more frequently than for other mRNA-encoding genes, but such interactions are not seen with all alleles at a given time. Because CBs contain factors required for transcriptional regulation and 3′ end processing of nonpolyadenylated replication-dependent histone transcripts, we investigated whether interaction with CBs is related to metabolism of these transcripts, known to vary during the cell cycle. Our experiments revealed that a locus containing a cell cycle-independent, replacement histone gene that produces polyadenylated transcripts does not preferentially associate with CBs. Furthermore, modest but significant changes in association levels of CBs with replication-dependent histone loci mimic their cell cycle modulations in transcription and 3′ end processing rates. By simultaneously visualizing replication-dependent histone genes and their nuclear transcripts for the first time, we surprisingly find that the vast majority of loci producing detectable RNA foci do not contact CBs. These studies suggest some link between CB association and unusual features of replication-dependent histone gene expression. However, sustained CB contact is not a requirement for their expression, consistent with our observations of U7 snRNP distributions. The modest correlation to gene expression instead may reflect transient gene signaling or the nucleation of small CBs at gene loci.

F-spondin is a contact-repellent molecule for embryonic motor neurons

The floor plate plays a key role in patterning axonal trajectory in the embryonic spinal cord by providing both long-range and local guidance cues that promote or inhibit axonal growth toward and across the ventral midline of the spinal cord, thus acting as an intermediate target for a number of crossing (commissural) and noncrossing (motor) axons. F-spondin, a secreted adhesion molecule expressed in the embryonic floor plate and the caudal somite of birds, plays a dual role in patterning the nervous system. It promotes adhesion and outgrowth of commissural axons and inhibits adhesion of neural crest cells. In the current study, we demonstrate that outgrowth of embryonic motor axons also is inhibited by F-spondin protein in a contact-repulsion fashion. Three independent lines of evidence support our hypothesis: substrate-attached F-spondin inhibits outgrowth of dissociated motor neurons in an outgrowth assay; F-spondin elicits acute growth cone collapse when applied to cultured motor neurons; and challenging ventral spinal cord explants with aggregates of HEK 293 cells expressing F-spondin, causes contact-repulsion of motor neurites. Structural–functional studies demonstrate that the processed carboxyl-half protein that contains the thrombospondin type 1 repeats is more prominent in inhibiting outgrowth, suggesting that the processing of F-spondin is important for enhancing its inhibitory activity.

Mutations Affecting Induction of Glycolytic and Fermentative Genes during Germination and Environmental Stresses in Arabidopsis1

Expression of the alcohol dehydrogenase gene (ADH) of Arabidopsis is known to be induced by environmental stresses and regulated developmentally. We used a negative-selection approach to isolate mutants that were defective in regulating the expression of the ADH gene during seed germination; we then characterized three recessive mutants, aar1–1, aar1–2, and aar2–1, which belong to two complementation groups. In addition to their defects during seed germination, mutations in the AAR1 and AAR2 genes also affected anoxic and hypoxic induction of ADH and other glycolytic genes in mature plants. The aar1 and aar2 mutants were also defective in responding to cold and osmotic stress. The two allelic mutants aar1–1and aar1–2 exhibited different phenotypes under cold and osmotic stresses. Based on our results we propose that these mutants are defective in a late step of the signaling pathways that lead to increased expression of the ADH gene and glycolytic genes.

Mapping of contact sites in complex formation between transducin and light-activated rhodopsin by covalent crosslinking: Use of a photoactivatable reagent

Interaction of light-activated rhodopsin with transducin (T) is the first event in visual signal transduction. We use covalent crosslinking approaches to map the contact sites in interaction between the two proteins. Here we use a photoactivatable reagent, N-[(2-pyridyldithio)-ethyl], 4-azido salicylamide. The reagent is attached to the SH group of cytoplasmic monocysteine rhodopsin mutants by a disulfide-exchange reaction with the pyridylthio group, and the derivatized rhodopsin then is complexed with T by illumination at λ >495 nm. Subsequent irradiation of the complex at λ310 nm generates covalent crosslinks between the two proteins. Crosslinking was demonstrated between T and a number of single cysteine rhodopsin mutants. However, sites of crosslinks were investigated in detail only between T and the rhodopsin mutant S240C (cytoplasmic loop V-VI). Crosslinking occurred predominantly with Tα. For identification of the sites of crosslinks in Tα, the strategy used involved: (i) derivatization of all of the free cysteines in the crosslinked proteins with N-ethylmaleimide; (ii) reduction of the disulfide bond linking the two proteins and isolation of all of the Tα species carrying the crosslinked moiety with a free SH group; (iii) adduct formation of the latter with the N-maleimide moiety of the reagent, maleimido-butyryl-biocytin, containing a biotinyl group; (iv) trypsin degradation of the resulting Tα derivatives and isolation of Tα peptides carrying maleimido-butyryl-biocytin by avidin-agarose chromatography; and (v) identification of the isolated peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. We found that crosslinking occurred mainly to two C-terminal peptides in Tα containing the amino acid sequences 310–313 and 342–345.

Mapping of contact sites in complex formation between light-activated rhodopsin and transducin by covalent crosslinking: Use of a chemically preactivated reagent

Contact sites in interaction between light-activated rhodopsin and transducin (T) have been investigated by using a chemically preactivated crosslinking reagent, N-succinimidyl 3-(2-pyridyldithio)propionate. The 3 propionyl-N-succinimidyl group in the reagent was attached by a disulfide exchange reaction to rhodopsin mutants containing single reactive cysteine groups in the cytoplasmic loops. Complex formation between the derivatized rhodopsin mutants and T was carried out by illumination at λ > 495 nm. Subsequent increase in pH (from 6 to 7.5 or higher) of the complex resulted in crosslinking of rhodopsin to the Tα subunit. Crosslinking to Tα was demonstrated for the rhodopsin mutants K141C, S240C, and K248C, and the crosslinked sites in Tα were identified for the rhodopsin mutant S240C. The peptides carrying the crosslinking moiety were isolated from the trypsin-digested peptide mixture, and their identification was carried out by matrix-assisted laser desorption ionization–time of flight mass spectrometry. The main site of crosslinking is within the peptide sequence, Leu-19–Arg-28 at the N-terminal region of Tα. The total results show that both the N and the C termini of Tα are in close vicinity to the third cytoplasmic loop of rhodopsin in the complex between rhodopsin and T.

Designing surfaces that kill bacteria on contact

Poly(4-vinyl-N-alkylpyridinium bromide) was covalently attached to glass slides to create a surface that kills airborne bacteria on contact. The antibacterial properties were assessed by spraying aqueous suspensions of bacterial cells on the surface, followed by air drying and counting the number of cells remaining viable (i.e., capable of growing colonies). Amino glass slides were acylated with acryloyl chloride, copolymerized with 4-vinylpyridine, and N-alkylated with different alkyl bromides (from propyl to hexadecyl). The resultant surfaces, depending on the alkyl group, were able to kill up to 94 ± 4% of Staphylococcus aureus cells sprayed on them. A surface alternatively created by attaching poly(4-vinylpyridine) to a glass slide and alkylating it with hexyl bromide killed 94 ± 3% of the deposited S. aureus cells. On surfaces modified with N-hexylated poly(4-vinylpyridine), the numbers of viable cells of another Gram-positive bacterium, Staphylococcus epidermidis, as well as of the Gram-negative bacteria Pseudomonas aeruginosa and Escherichia coli, dropped more than 100-fold compared with the original amino glass. In contrast, the number of viable bacterial cells did not decline significantly after spraying on such common materials as ceramics, plastics, metals, and wood.

Chemotropic and Contact Responses of Phytophthora sojae Hyphae to Soybean Isoflavonoids and Artificial Substrates1

We have investigated the role of the isoflavones daidzein and genistein on the chemotropic behavior of germinating cysts of Phytophthora sojae. Hyphal germlings were shown to respond chemotropically to daidzein and genistein, suggesting that hyphal tips from zoospores that have encysted adjacent to the root may use specific host isoflavones to locate their host. Observations of the contact response of hyphal germlings were made on several different substrates in the presence and absence of isoflavones. Hyphal tips of germlings detected and penetrated pores in membranes and produced multiple appressoria on smooth, impenetrable surfaces. Hyphae that successfully penetrated the synthetic membrane were observed to grow away from the membrane surface. The presence of isoflavones in the medium surrounding the hyphal germlings did not appear to alter any of those habits. Daidzein and genistein did not inhibit germination or initial hyphal growth at concentrations up to 20 μm.

Contact interactions between epitheliocytes and fibroblasts: Formation of heterotypic cadherin-containing adhesion sites is accompanied by local cytoskeletal reorganization

Contact interactions between different cell types play a number of important roles in development, for example in cell sorting, tissue organization, and ordered migration of cells. The nature of such heterocellular interactions, in contrast to interactions between cells of the same type, remains largely unknown. In this report, we present experimental data examining the dynamics of heterocellular interactions between epitheliocytes and fibroblasts, which express different cadherin cell adhesion molecules and possess different actin cytoskeletal organizations. Our analysis revealed two striking features of heterocellular contact. First, the active free edge of an epitheliocyte reorganizes its actin cytoskeleton after making contact with a fibroblast. Upon contact with the leading edge of a fibroblast, epitheliocytes disassemble their marginal bundle of actin filaments and reassemble actin filaments into a geometric organization more typical of a fibroblast lamella. Second, epitheliocytes and fibroblasts form cell–cell adhesion structures that have an irregular organization and are associated with components of cell adhesion complexes. The structural organization of these adhesions is more closely related to the type of contacts formed between fibroblasts rather than to those between epitheliocytes. Heterotypic epithelio-fibroblastic contacts, like homotypic contacts between fibroblasts, are transient and do not lead to formation of stable contact interactions. We suggest that heterocellular contact interactions in culture may be regarded as models of how tissue systems consisting of epithelia and mesenchyme interact and become organized in vivo.

Diffusion-limited contact formation in unfolded cytochrome c: estimating the maximum rate of protein folding.

How fast can a protein fold? The rate of polypeptide collapse to a compact state sets an upper limit to the rate of folding. Collapse may in turn be limited by the rate of intrachain diffusion. To address this question, we have determined the rate at which two regions of an unfolded protein are brought into contact by diffusion. Our nanosecond-resolved spectroscopy shows that under strongly denaturing conditions, regions of unfolded cytochrome separated by approximately 50 residues diffuse together in 35-40 microseconds. This result leads to an estimate of approximately (1 microsecond)-1 as the upper limit for the rate of protein folding.

Translocation of PKN from the cytosol to the nucleus induced by stresses.

Effects of environmental stresses on the subcellular localization of PKN were investigated in NIH 3T3, BALB/c 3T3, and Rat-1 cells. The immunofluorescence of PKN resided prominently in the cytoplasmic region in nonstressed cells. When these cells were treated at 42 degrees C, there was a time-dependent decrease of the immunofluorescence of PKN in the cytoplasmic region that correlated with an increase within the nucleus as observed by confocal microscope. After incubation at 37 degrees C following beat shock, the immunofluorescence of PKN returned to the perinuclear and cytoplasmic regions from the nucleus. The nuclear translocation of PKN by heat shock was supported by the biochemical subcellular fractionation and immunoblotting. The nuclear localization of PKN was also observed when the cells were exposed to other stresses such as sodium arsenite and serum starvation. These results raise the possibility that there is a pathway mediating stress signals from the cytosol to the nucleus through PKN.

Identification of a receptor/G-protein contact site critical for signaling specificity and G-protein activation.

Each G protein-coupled receptor recognizes only a distinct subset of the many structurally closely related G proteins expressed within a cell. How this selectively is achieved at a molecular level is not well understood, particularly since no specific point-to-point contact sites between a receptor and its cognate G protein(s) have been identified. In this study, we demonstrate that a 4-aa epitope on the m2 muscarinic acetylcholine receptor, a prototypical Gi/o-coupled receptor, can specifically recognize the C-terminal 5 aa of alpha subunits of the Gi/o protein family. The m2 receptor residues involved in this interaction are predicted to be located on one side of an alpha-helical receptor region present at the junction between the third intracellular loop and the sixth transmembrane domain. Coexpression studies with hybrid m2/m3 muscarinic receptors and mutant G-protein alpha q subunits showed that the receptor/G-protein contact site identified in this study is essential for coupling specificity and G-protein activation.

CD4+ blood lymphocytes are rapidly killed in vitro by contact with autologous human immunodeficiency virus-infected cells.

We have investigated the ability of human immunodeficiency virus (HIV)-infected cells to kill uninfected CD4+ lymphocytes. Infected peripheral blood mononuclear cells were cocultured with autologous 51Cr-labeled uninfected cells. Rapid death of the normal CD4-expressing target population was observed following a brief incubation. Death of blood CD4+ lymphocytes occurred before syncytium formation could be detected or productive viral infection established in the normal target cells. Cytolysis could not be induced by free virus, was dependent on gp120-CD4 binding, and occurred in resting, as well as activated, lymphocytes. CD8+ cells were not involved in this phenomenon, since HIV-infected CEMT4 cells (CD4+, CD8- cells) mediated the cytolysis of uninfected targets. Reciprocal isotope-labeling experiments demonstrated that infected CEMT4 cells did not die in parallel with their targets. The uninfected target cells manifested DNA fragmentation, followed by the release of the 51Cr label. Thus, in HIV patients, infected lymphocytes may cause the depletion of the much larger population of uninfected CD4+ cells without actually infecting them, by triggering an apoptotic death.