unc-83 encodes a novel component of the nuclear envelope and is essential for proper nuclear migration.

Nuclear migration plays an essential role in the growth and development of a wide variety of eukaryotes. Mutations in unc-84, which encodes a conserved component of the nuclear envelope, have been shown to disrupt nuclear migration in two C. elegans tissues. We show that mutations in unc-83 disrupt nuclear migration in a similar manner in migrating P cells, hyp7 precursors and the intestinal primordium, but have no obvious defects in the association of centrosomes with nuclei or the structure of the nuclear lamina of migrating nuclei. We also show that unc-83 encodes a novel transmembrane protein. We identified three unc-83 transcripts that are expressed in a tissue-specific manner. Antibodies against UNC-83 co-localized to the nuclear envelope with lamin and UNC-84. Unlike UNC-84, UNC-83 localized to only specific nuclei, many of which were migratory. UNC-83 failed to localize to the nuclear envelope in unc-84 mutants with lesions in the conserved SUN domain of UNC-84, and UNC-83 interacted with the SUN domain of UNC-84 in vitro, suggesting that these two proteins function together during nuclear migration. We favor a model in which UNC-84 directly recruits UNC-83 to the nuclear envelope where they help transfer force between the cytoskeleton and the nucleus.

Suppressors of mdm20 in yeast identify new alleles of ACT1 and TPM1 predicted to enhance actin-tropomyosin interactions.

The actin cytoskeleton is required for many aspects of cell division in yeast, including mitochondrial partitioning into growing buds (mitochondrial inheritance). Yeast cells lacking MDM20 function display defects in both mitochondrial inheritance and actin organization, specifically, a lack of visible actin cables and enhanced sensitivity to Latrunculin A. mdm20 mutants also exhibit a temperature-sensitive growth phenotype, which we exploited to isolate second-site suppressor mutations. Nine dominant suppressors selected in an mdm20/mdm20 background rescue temperature-sensitive growth defects and mitochondrial inheritance defects and partially restore actin cables in haploid and diploid mdm20 strains. The suppressor mutations define new alleles of ACT1 and TPM1, which encode actin and the major form of tropomyosin in yeast, respectively. The ACT1 mutations cluster in a region of the actin protein predicted to contact tropomyosin, suggesting that they stabilize actin cables by enhancing actin-tropomyosin interactions. The characteristics of the mutant ACT1 and TPM1 alleles and their potential effects on protein structure and binding are discussed.

Left-right asymmetry in C. elegans intestine organogenesis involves a LIN-12/Notch signaling pathway.

The C. elegans intestine is a simple tube consisting of a monolayer of epithelial cells. During embryogenesis, cells in the anterior of the intestinal primordium undergo reproducible movements that lead to an invariant, asymmetrical 'twist' in the intestine. We have analyzed the development of twist to determine how left-right and anterior-posterior asymmetries are generated within the intestinal primordium. The twist requires the LIN-12/Notch-like signaling pathway of C. elegans. All cells within the intestinal primordium initially express LIN-12, a receptor related to Notch; however, only cells in the left half of the primordium contact external, nonintestinal cells that express LAG-2, a ligand related to delta. LIN-12 and LAG-2 mediated interactions result in the left primordial cells expressing lower levels of LIN-12 than the right primordial cells. We propose that this asymmetrical pattern of LIN-12 expression is the basis for asymmetry in later cell-cell interactions within the primordium that lead directly to intestinal twist. Like the interactions that initially establish LIN-12 asymmetry, the later interactions are mediated by LIN-12. The later interactions, however, involve a different ligand related to delta, called APX-1. We show that the anterior-posterior asymmetry in intestinal twist involves the kinase LIT-1, which is part of a signaling pathway in early embryogenesis that generates anterior-posterior differences between sister cells.

Organogenesis of the Caenorhabditis elegans intestine.

The intestine of Caenorhabditis elegans is an epithelial tube consisting of only 20 cells and is derived clonally from a single embryonic blastomere called E. We describe the cellular events that shape the intestine. These events include cytoplasmic polarization of cells in the intestinal primordium, the intercalation of specific sets of cells, the generation of an extracellular cavity within the primordium, and adherens junction formation. The polarization of the intestinal primordium is associated with the generation of an asymmetric microtubule cytoskeleton, and microtubule function plays a role in subsequent cell polarity. We show that an isolated E blastomere is capable of generating polarized intestinal cells, indicating that some of the major events in intestinal organogenesis do not depend upon interactions with surrounding tissues. We compare and contrast intestinal organogenesis with some of the basic steps in development of a second epithelial organ, the pharynx, and suggest how these differences lead to organs with distinct shapes.

The dynamin-related GTPase, Dnm1p, controls mitochondrial morphology in yeast.

The Saccharomyces cerevisiae Dnm1 protein is structurally related to dynamin, a GTPase required for membrane scission during endocytosis. Here we show that Dnm1p is essential for the maintenance of mitochondrial morphology. Disruption of the DNM1 gene causes the wild-type network of tubular mitochondrial membranes to collapse to one side of the cell but does not affect the morphology or distribution of other cytoplasmic organelles. Dnm1 proteins containing point mutations in the predicted GTP-binding domain or completely lacking the GTP-binding domain fail to rescue mitochondrial morphology defects in a dnm1 mutant and induce dominant mitochondrial morphology defects in wild-type cells. Indirect immunofluorescence reveals that Dnm1p is distributed in punctate structures at the cell cortex that colocalize with the mitochondrial compartment. These Dnm1p-containing structures remain associated with the spherical mitochondria found in an mdm10 mutant strain. In addition, a portion of Dnm1p cofractionates with mitochondrial membranes during differential sedimentation and sucrose gradient fractionation of wild-type cells. Our results demonstrate that Dnm1p is required for the cortical distribution of the mitochondrial network in yeast, a novel function for a dynamin-related protein.

Mitochondrial fusion in yeast requires the transmembrane GTPase Fzo1p.

Membrane fusion is required to establish the morphology and cellular distribution of the mitochondrial compartment. In Drosophila, mutations in the fuzzy onions (fzo) GTPase block a developmentally regulated mitochondrial fusion event during spermatogenesis. Here we report that the yeast orthologue of fuzzy onions, Fzo1p, plays a direct and conserved role in mitochondrial fusion. A conditional fzo1 mutation causes the mitochondrial reticulum to fragment and blocks mitochondrial fusion during yeast mating. Fzo1p is a mitochondrial integral membrane protein with its GTPase domain exposed to the cytoplasm. Point mutations that alter conserved residues in the GTPase domain do not affect Fzo1p localization but disrupt mitochondrial fusion. Suborganellar fractionation suggests that Fzo1p spans the outer and is tightly associated with the inner mitochondrial membrane. This topology may be required to coordinate the behavior of the two mitochondrial membranes during the fusion reaction. We propose that the fuzzy onions family of transmembrane GTPases act as molecular switches to regulate a key step in mitochondrial membrane docking and/or fusion.

Mitochondrial inheritance is delayed in Saccharomyces cerevisiae cells lacking the serine/threonine phosphatase PTC1.

In wild-type yeast mitochondrial inheritance occurs early in the cell cycle concomitant with bud emergence. Cells lacking the PTC1 gene initially produce buds without a mitochondrial compartment; however, these buds later receive part of the mitochondrial network from the mother cell. Thus, the loss of PTC1 causes a delay, but not a complete block, in mitochondrial transport. PTC1 encodes a serine/threonine phosphatase in the high-osmolarity glycerol response (HOG) pathway. The mitochondrial inheritance delay in the ptc1 mutant is not attributable to changes in intracellular glycerol concentrations or defects in the organization of the actin cytoskeleton. Moreover, epistasis experiments with ptc1delta and mutations in HOG pathway kinases reveal that PTC1 is not acting through the HOG pathway to control the timing of mitochondrial inheritance. Instead, PTC1 may be acting either directly or through a different signaling pathway to affect the mitochondrial transport machinery in the cell. These studies indicate that the timing of mitochondrial transport in wild-type cells is genetically controlled and provide new evidence that mitochondrial inheritance does not depend on a physical link between the mitochondrial network and the incipient bud site.

Mitochondrial dynamics in yeast.

Proteins that control mitochondrial dynamics in yeast are being identified at a rapid pace. These proteins include cytoskeletal elements that regulate organelle distribution and inheritance and several outer membrane proteins that are required to maintain the branched, mitochondrial reticulum. Interestingly, three of the high molecular weight GTPases encoded by the yeast genome are required for mitochondrial integrity and are potential regulators of mitochondrial branching, distribution, and membrane fusion. The recent finding that mtDNA mixing is restricted in the mitochondrial matrix has stimulated the hunt for the molecular machinery that anchors mitochondrial nucleoids in the organelle. Considering that many aspects of mitochondrial structure and behavior are strikingly similar in different cell types, the functional analyses of these yeast proteins should provide general insights into the mechanisms governing mitochondrial dynamics in all eukaryotes.

The yeast gene, MDM20, is necessary for mitochondrial inheritance and organization of the actin cytoskeleton.

In Saccharomyces cerevisiae, the growing bud inherits a portion of the mitochondrial network from the mother cell soon after it emerges. Although this polarized transport of mitochondria is thought to require functions of the cytoskeleton, there are conflicting reports concerning the nature of the cytoskeletal element involved. Here we report the isolation of a yeast mutant, mdm20, in which both mitochondrial inheritance and actin cables (bundles of actin filaments) are disrupted. The MDM20 gene encodes a 93-kD polypeptide with no homology to other characterized proteins. Extra copies of TPM1, a gene encoding the actin filament-binding protein tropomyosin, suppress mitochondrial inheritance defects and partially restore actin cables in mdm20 delta cells. Synthetic lethality is also observed between mdm20 and tpm1 mutant strains. Overexpression of a second yeast tropomyosin, Tpm2p, rescues mutant phenotypes in the mdm20 strain to a lesser extent. Together, these results provide compelling evidence that mitochondrial inheritance in yeast is an actin-mediated process. MDM20 and TPM1 also exhibit the same pattern of genetic interactions; mutations in MDM20 are synthetically lethal with mutations in BEM2 and MYO2 but not SAC6. Although MDM20 and TPM1 are both required for the formation and/or stabilization of actin cables, mutations in these genes disrupt mitochondrial inheritance and nuclear segregation to different extents. Thus, Mdm20p and Tpm1p may act in vivo to establish molecular and functional heterogeneity of the actin cytoskeleton.

The effect of pizotifen, a serotonin antagonist, and of pirenzepine, a muscarinic antagonist, on hormonal responses to metoclopramide in healthy subjects.

To examine whether and how muscarinic or serotoninergic properties might contribute to the hormone stimulating action of the dopamine antagonist metoclopramide, the effect of oral single-dose pretreatment with pirenzepine (Gastrozepin) 50 mg, a muscarinic antagonist, pizotifen (Sandomigran) 1 mg, a serotonin antagonist, or placebo on the responses of prolactin, aldosterone, plasma renin activity (PRA), cortisol (hydrocortisone) and human growth hormone (HGH) to the i.v. injection of metoclopramide 10 mg was studied in 6 young healthy volunteers. For comparison, a mere placebo study was added. Whereas aldosterone response to metoclopramide and PRA remained unchanged, prolactin response was decreased by pizotifen from 43 +/- 6.5 to 24 +/- 9 ng/ml, p less than 0.05. Pizotifen decreased base-line cortisol from 10.5 +/- 0.5 to 6.6 +/- 0.6 microgram/dl and prevented the increase in cortisol after metoclopramide in responsive subjects. Both pizotifen and pirenzepine decreased HGH responsiveness to metoclopramide. It is concluded that cortisol and, partly, prolactin stimulation by metoclopramide are due to its serotoninergic properties and that HGH responsiveness to metoclopramide becomes explainable by both the serotoninergic and muscarinic potencies of the drug.

Phase variation of Andrewes in Salmonella enteritidis bioserotype paratyphi-A.

The natural occurrence of a strain of Salmonella enteritidis bioserotype Paratyphi-A is reported, in which the flagellar antigens segregated readily into normal phase 2 antigens and mixtures of normal phase 1 and phase 2 antigens, and in which phase variation of Andrewes was demonstrated with ease.

R-B enteric differential system for identification of Enterobacteriaceae.

The R/B Enteric Differential System for identifying enteric bacteria has been evaluated with 451 "unknown" cultures from the stock culture collection of the Center for Disease Control. An average of 89.6% of these cultures were correctly identified by the R/B system, when used as recommended by the manufacturer but without the assistance of serology. This percentage ranged, however, from 47% for Klebsiella to 100% for Serratia and Providencia. Of 11 groups or genera of Enterobacteriaceae tested, only three (Enterobacter, Serratia, and Providencia) were identified with 95% or better accuracy. Four groups (Arizona, Citrobacter, Escherichia, and Salmonella) attained 90 to 95% accuracy of identification, and three groups (Edwardsiella, Proteus, and Shigella) scored between 85 and 90% accuracy. We recommend the R/B system as a screening device which is reasonably successful in grouping bacteria but not as a substitute for more exacting conventional procedures.

Preliminary report of a new system for typing Salmonella typhimurium in the United States.

A new system is described for the bacteriophage typing of Salmonella typhimurium cultures isolated in the United States. The system is based upon one phage adapted to different S. typhimurium strains.