Cell cycle regulation and cell type-specific localization of the FtsZ division initiation protein in Caulobacter.

Many genes involved in cell division and DNA replication and their protein products have been identified in bacteria; however, little is known about the cell cycle regulation of the intracellular concentration of these proteins. It has been shown that the level of the tubulin-like GTPase FtsZ is critical for the initiation of cell division in bacteria. We show that the concentration of FtsZ varies dramatically during the cell cycle of Caulobacter crescentus. Caulobacter produce two different cell types at each cell division: (i) a sessile stalked cell that can initiate DNA replication immediately after cell division and (ii) a motile swarmer cell in which DNA replication is blocked. After cell division, only the stalked cell contains FtsZ. FtsZ is synthesized slightly before the swarmer cells differentiate into stalked cells and the intracellular concentration of FtsZ is maximal at the beginning of cell division. Late in the cell cycle, after the completion of chromosome replication, the level of FtsZ decreases dramatically. This decrease is probably mostly due to the degradation of FtsZ in the swarmer compartment of the predivisional cell. Thus, the variation of FtsZ concentration parallels the pattern of DNA synthesis. Constitutive expression of FtsZ leads to defects in stalk biosynthesis suggesting a role for FtsZ in this developmental process in addition to its role in cell division.

Cellular and subcellular localization of the vasopressin- regulated urea transporter in rat kidney.

The renal urea transporter (RUT) is responsible for urea accumulation in the renal medulla, and consequently plays a central role in the urinary concentrating mechanism. To study its cellular and subcellular localization, we prepared affinity-purified, peptide-derived polyclonal antibodies against rat RUT based on the cloned cDNA sequence. Immunoblots using membrane fractions from rat renal inner medulla revealed a solitary 97-kDa band. Immunocytochemistry demonstrated RUT labeling of the apical and subapical regions of inner medullary collecting duct (IMCD) cells, with no labeling of outer medullary or cortical collecting ducts. Immunoelectron microscopy directly demonstrated labeling of the apical plasma membrane and of subapical intracellular vesicles of IMCD cells, but no labeling of the basolateral plasma membrane. Immunoblots demonstrated RUT labeling in both plasma membrane and intracellular vesicle-enriched membrane fractions from inner medulla, a subcellular distribution similar to that of the vasopressin-regulated water channel, aquaporin-2. In the outer medulla, RUT labeling was seen in terminal portions of short-loop descending thin limbs. Aside from IMCD and descending thin limbs, no other structures were labeled in the kidney. These results suggest that: (i) the RUT provides the apical pathway for rapid, vasopressin-regulated urea transport in the IMCD, (ii) collecting duct urea transport may be increased by vasopressin by stimulation of trafficking of RUT-containing vesicles to the apical plasma membrane, and (iii) the rat urea transporter may provide a pathway for urea entry into the descending limbs of short-loop nephrons.

Molecular mechanism of transmembrane signaling by the aspartate receptor: a model.

The aspartate receptor of bacterial chemotaxis is representative of a large class of membrane-spanning receptors found in prokaryotic and eukaryotic organisms. These receptors, which regulate histidine kinase pathways and possess two putative transmembrane helices per subunit, appear to control a wide variety of cellular processes. The best characterized subgroup of the two-helix receptor class is the homologous family of chemosensory receptors from Escherichia coli and Salmonella typhimurium, including the aspartate receptor. This receptor binds aspartate, an attractant, in the periplasmic compartment and undergoes an intramolecular, transmembrane conformational change, thereby modulating the autophosphorylation rate of a bound histidine kinase in the cytoplasm. Here, we analyze recent results from x-ray crystallographic, solution 19F NMR, and engineered disulfide studies probing the aspartate-induced structural change within the periplasmic and transmembrane regions of the receptor. Together, these approaches provide evidence that aspartate binding triggers a "swinging-piston" displacement of the second membrane-spanning helix, which is proposed to communicate the signal across the bilayer.

Measurement of muscle protein synthesis by positron emission tomography with L-[methyl-11C]methionine.

Positron emission tomography (PET) with L-[methyl-11C]methionine was explored as an in vivo, noninvasive, quantitative method for measuring the protein synthesis rate (PSR) in paraspinal and hind limb muscles of anesthetized dogs. Approximately 25 mCi (1 Ci = 37 GBq) of L-[methyl-11C]methionine was injected intravenously, and serial images and arterial blood samples were acquired over 90 min. Data analysis was performed by fitting tissue- and metabolite-corrected arterial blood time-activity curves to a three-compartment model and assuming insignificant transamination and transmethylation in this tissue. PSR was calculated from fitted parameter values and plasma methionine concentrations. PSRs measured by PET were compared with arterio-venous (A-V) difference measurements across the hind limb during primed constant infusion (5-6 h) of L-[1-13C, methyl-2H3]methionine. Results of PET measurements demonstrated similar PSRs for paraspinal and hind limb muscles: 0.172 +/- 0.062 vs. 0.208 +/- 0.048 nmol-1.min-1.(g of muscle)-1 (P = not significant). PSR determined by the stable isotope technique was 0.27 +/- 0.050 nmol-1.min-1.(g of leg tissue)-1 (P < 0.07 from PET) and indicated that the contribution of transmethylation to total hind limb methionine utilization was approximately 10%. High levels of L-[methyl-11C]methionine utilization by bone marrow were observed. We conclude that muscle PSR can be measured in vivo by PET and that this approach offers promise for application in human metabolic studies.

Cell cycling and patterned cell proliferation in the wing primordium of Drosophila.

The pattern of cell proliferation in the Drosophila imaginal wing primordium is spatially and temporally heterogeneous. Direct visualization of cells in S, G2, and mitosis phases of the cell cycle reveals several features invariant throughout development. The fraction of cells in the disc in the different cell cycle stages is constant, the majority remaining in G1. Cells in the different phases of the cell cycle mainly appear in small synchronic clusters that are nonclonally derived but result from changing local cell-cell interactions. Cluster synchronization occurs before S and in the G2/M phases. Rates of cell division are neither constant nor clonal features. Cell cycle progression is linear rather than concentric. Clusters appear throughout the disc but with symmetries related to presumptive wing patterns, compartment boundaries, and vein clonal restrictions.

Signal-induced degradation of I kappa B alpha requires site-specific ubiquitination.

The inhibitor protein I kappa B alpha controls the nuclear import of the transcription factor NF-kappa B. The inhibitory activity of I kappa B alpha is regulated from the cytoplasmic compartment by signal-induced proteolysis. Previous studies have shown that signal-dependent phosphorylation of serine residues 32 and 36 targets I kappa B alpha to the ubiquitin-proteasome pathway. Here we provide evidence that lysine residues 21 and 22 serve as the primary sites for signal-induced ubiquitination of I kappa B alpha. Conservative Lys-->Arg substitutions at both Lys-21 and Lys-22 produce dominant-negative mutants of I kappa B alpha in vivo. These constitutive inhibitors are appropriately phosphorylated but fail to release NF-kappa B in response to multiple inducers, including viral proteins, cytokines, and agents that mimic antigenic stimulation through the T-cell receptor. Moreover, these Lys-->Arg mutations prevent signal-dependent degradation of I kappa B alpha in vivo and ubiquitin conjugation in vitro. We conclude that site-specific ubiquitination of phosphorylated I kappa B alpha at Lys-21 and/or Lys-22 is an obligatory step in the activation of NF-kappa B.

Shared functions in vivo of a glycosyl-phosphatidylinositol-linked aspartyl protease, Mkc7, and the proprotein processing protease Kex2 in yeast.

The MKC7 gene was isolated as a multicopy suppressor of the cold-sensitive growth phenotype of a yeast kex2 mutant, which lacks the protease that cleaves pro-alpha-factor and other secretory proproteins at pairs of basic residues in a late Golgi compartment in yeast. MKC7 encodes an aspartyl protease most closely related to product of the YAP3 gene, a previously isolated multicopy suppressor of the pro-alpha-factor processing defect of a kex2 null. Multicopy MKC7 suppressed the alpha-specific mating defect of a kex2 null as well as multicopy YAP3 did, but multicopy YAP3 was a relatively weak suppressor of kex2 cold sensitivity. Overexpression of MKC7 resulted in production of a membrane-associated proteolytic activity that cleaved an internally quenched fluorogenic peptide substrate on the carboxyl side of a Lys-Arg site. Treatment with phosphatidylinositol-specific phospholipase C shifted Mkc7 activity from the detergent to the aqueous phase in a Triton X-114 phase separation, indicating that membrane attachment of Mkc7 is mediated by a glycosyl-phosphatidylinositol anchor. Although disruption of MKC7 or YAP3 alone resulted in no observable phenotype, mkc7 yap3 double disruptants exhibited impaired growth at 37 degrees C. Disruption of MKC7 and YAP3 in a kex2 null mutant resulted in profound temperature sensitivity and more generalized cold sensitivity. The synergism of mkc7, yap3, and kex2 null mutations argues that Mkc7 and Yap3 are authentic processing enzymes whose functions overlap those of Kex2 in vivo.

Identification of an additional gene required for eukaryotic nonsense mRNA turnover.

Loss of function of any one of three UPF genes prevents the accelerated decay of nonsense mRNAs in Saccharomyces cerevisiae. We report the identification and DNA sequence of UPF3, which is present in one nonessential copy on chromosome VII. Upf3 contains three putative nuclear localization signal sequences, suggesting that it may be located in a different compartment than the cytoplasmic Upf1 protein. Epitope-tagged Upf3 (FLAG-Upf3) does not cofractionate with polyribosomes or 80S ribosomal particles. Double disruptions of UPF1 and UPF3 affect nonsense mRNA decay in a manner indistinguishable from single disruptions. These results suggest that the Upf proteins perform related functions in a common pathway.

Two redundant systems maintain levels of resident proteins within the yeast endoplasmic reticulum.

The Saccharomyces cerevisiae gene ERD2 is responsible for the retrieval of lumenal resident proteins of the endoplasmic reticulum (ER) lost to the next secretory compartment. Previous studies have suggested that the retrieval of proteins by ERD2 is not essential. Here, we find that ERD2-mediated retrieval is not an essential process only because, on its failure, a second inducible system acts to maintain levels of ER proteins. The second system is controlled by the ER membrane-bound kinase encoded by IRE1. We conclude that IRE1 and ERD2 together maintain normal concentrations of resident proteins within the ER.

Structural features of the invariant chain fragment CLIP controlling rapid release from HLA-DR molecules and inhibition of peptide binding.

The invariant chain (Ii) prevents binding of ligands to major histocompatibility complex (MHC) class II molecules in the endoplasmic reticulum and during intracellular transport. Stepwise removal of the Ii in a trans-Golgi compartment renders MHC class II molecules accessible for peptide loading, with CLIP (class II-associated Ii peptides) as the final fragment to be released. Here we show that CLIP can be subdivided into distinct functional regions. The C-terminal segment (residues 92-105) of the CLIP-(81-105) fragment mediates inhibition of self- and antigenic peptide binding to HLA-DR2 molecules. In contrast, the N-terminal segment CLIP-(81-98) binds to the Staphylococcus aureus enterotoxin B contact site outside the peptide-binding groove on the alpha 1 domain and does not interfere with peptide binding. Its functional significance appears to lie in the contribution to CLIP removal: the dissociation of CLIP-(81-105) is characterized by a fast off-rate, which is accelerated at endosomal pH, whereas in the absence of the N-terminal CLIP-(81-91), the off-rate of C-terminal CLIP-(92-105) is slow and remains unaltered at low pH. Mechanistically, the N-terminal segment of CLIP seems to prevent tight interactions of CLIP side chains with specificity pockets in the peptide-binding groove that normally occurs during maturation of long-lived class II-peptide complexes.

A splice variant of alpha 6 integrin is associated with malignant conversion in mouse skin tumorigenesis.

The epithelial-specific integrin alpha 6 beta 4 is suprabasally expressed in benign skin tumors (papillomas) and is diffusely expressed in carcinomas associated with an increase in the proliferating compartment. Analysis of RNA samples by reverse transcriptase-PCR and DNA sequencing revealed that chemically or oncogenically induced papillomas (n = 8) expressed a single transcript of the alpha 6 subunit, identified as the alpha 6 A splice variant. In contrast, carcinomas (n = 13) expressed both alpha 6A and an alternatively spliced form, alpha 6B. Primary keratinocytes and a number of keratinocyte cell lines that vary in biological potential from normal skin, to benign papillomas, to well-differentiated slowly growing carcinomas exclusively expressed alpha 6A. However, I7, an oncogene-induced cell line that produces highly invasive carcinomas, expressed both alpha 6A and alpha 6B transcript and protein. The expression of alpha 6B in I7 cells was associated with increased attachment to a laminin matrix compared to cell lines exclusively expressing alpha 6A. Furthermore, introduction of an alpha 6B expression vector into a papilloma cell line expressing alpha 6A increased laminin attachment. When a papilloma cell line was converted to an invasive carcinoma by introduction of the v-fos oncogene, the malignant cells expressed both alpha 6A and alpha 6B, while the parent cell line and cells transduced with v-jun or c-myc, which retained the papilloma phenotype, expressed only alpha 6A. Comparative analysis of alpha 6B expression in cell lines and their derived tumors indicate that alpha 6B transcripts are more abundant in tumors than cell lines, and alpha 6B is expressed to a greater extent in poorly differentiated tumors. These results establish a link between malignant conversion and invasion of squamous tumor cells and the regulation of transcript processing of the alpha 6 beta 4 integrin.

Multiple loci govern the bone marrow-derived immunoregulatory mechanism controlling dominant resistance to autoimmune orchitis.

The existence of immunoregulatory genes conferring dominant resistance to autoimmunity is well documented. In an effort to better understand the nature and mechanisms of action of these genes, we utilized the murine model of autoimmune orchitis as a prototype. When the orchitis-resistant strain DBA/2J is crossed with the orchitis-susceptible strain BALB/cByJ, the F1 hybrid is completely resistant to the disease. By using reciprocal radiation bone marrow chimeras, the functional component mediating this resistance was mapped to the bone marrow-derived compartment. Resistance is not a function of either low-dose irradiation- or cyclophosphamide (20 mg/kg)-sensitive immunoregulatory cells, but can be adoptively transferred by primed splenocytes. Genome exclusion mapping identified three loci controlling the resistant phenotype. Orch3 maps to chromosome 11, whereas Orch4 and Orch5 map to the telomeric and centromeric regions of chromosome 1, respectively. All three genes are linked to a number of immunologically relevant candidate loci. Most significant, however, is the linkage of Orch3 to Idd4 and Orch5 to Idd5, two susceptibility genes which play a role in autoimmune insulin-dependent type 1 diabetes mellitus in the nonobese diabetic mouse.

Blockade of T- and B-lymphocyte development by antibody to the gamma c subunit of the receptors for interleukins 2, 4, and 7.

Cytokines are important regulators of hematopoesis. Mutations in gamma c, which is a subunit shared by the receptors for interleukin (IL) 2, IL-4, and IL-7, have been causally associated with human X chromosome-linked severe combined immunodeficiency disease. This finding indicates a mandatory role for cytokine receptor signaling at one or more stages of lymphocyte development. To evaluate the cellular level at which gamma c is critical for lymphopoiesis, the effect of monoclonal antibodies to gamma c on the capacity of syngeneic bone marrow cells to reconstitute the hematopoietic compartment of lethally irradiated recipient mice was examined. We show that monoclonal antibody to gamma c blocked lymphocyte development at or before the appearance of pro-B cells and prior to or at the seeding of the thymus by precursor cells while erythromyeloid cell development was normal. These results suggest that one level of lymphocyte development that requires gamma c is a point in hematopoietic cell differentiation near the divergence of lymphopoiesis and erythromyelopoesis.

Lipid metabolism in Chlamydia trachomatis-infected cells: directed trafficking of Golgi-derived sphingolipids to the chlamydial inclusion.

Chlamydia trachomatis undergoes its entire life cycle within an uncharacterized intracellular vesicle that does not fuse with lysosomes. We used a fluorescent Golgi-specific probe, (N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]) aminocaproylsphingosine (C6-NBD-Cer), in conjunction with conventional fluorescence or confocal microscopy to identify interactions between the Golgi apparatus and the chlamydial inclusion. We observed not only a close physical association between the Golgi apparatus and the chlamydial inclusion but the eventual presence of a metabolite of this fluorescent probe associated with the chlamydiae themselves. Sphingomyelin, endogenously synthesized from C6-NBD-Cer, was specifically transported to the inclusion and incorporated into the cell wall of the intracellular chlamydiae. Incorporation of the fluorescent sphingolipid by chlamydiae was inhibited by brefeldin A. Chlamydiae therefore occupy a vesicle distal to the Golgi apparatus that receives anterograde vesicular traffic from the Golgi normally bound for the plasma membrane. Collectively, the data suggest that the chlamydial inclusion may represent a unique compartment within the trans-Golgi network.

The kappa-opioid receptor is primarily postsynaptic: combined immunohistochemical localization of the receptor and endogenous opioids.

Antisera were raised against a synthetic peptide corresponding to the carboxyl terminus of the kappa-opioid receptor (KOR1). Specificity of the antisera was verified by staining of COS-7 cells transfected with KOR1 and epitope-tagged KOR1 cDNAs, by recognition by the antisera of proteins on Western blots of both transfected cells and brain tissue, by the absence of staining of both brain tissue and transfected cells after preabsorption of the antisera with the cognate peptide, and on the strong correlation between the distribution of KOR1 immunoreactivity and that of earlier ligand binding and in situ hybridization studies. Results indicate that KOR1 in neurons is targeted into both the axonal and somatodendritic compartments, but the majority of immunostaining was seen in the somatodendritic compartment. In sections from rat and guinea pig brain, prominent KOR1 staining was seen in the ventral forebrain, hypothalamus, thalamus, posterior pituitary, and midbrain. While the staining pattern was similar in both species, distinct differences were also observed. The distribution of preprodynorphin and KOR1 immunoreactivity was complementary in many brain regions, suggesting that KOR1 is poised to mediate the physiological actions of dynorphin. However, the distribution of KOR1 and enkephalin immunoreactivity was complementary in some regions as well. These results suggest that the KOR1 protein is primarily, but not exclusively, deployed to postsynaptic membranes where it mediates the effects of products of preprodynorphin and possibly preproenkephalin.