Role of the Rab GTP-binding protein Ypt3 in the fission yeast exocytic pathway and its connection to calcineurin function

A genetic screen for mutations synthetically lethal with fission yeast calcineurin deletion led to the identification of Ypt3, a homolog of mammalian Rab11 GTP-binding protein. A mutant with the temperature-sensitive ypt3-i5 allele showed pleiotropic phenotypes such as defects in cytokinesis, cell wall integrity, and vacuole fusion, and these were exacerbated by FK506-treatment, a specific inhibitor of calcineurin. Green fluorescent protein (GFP)-tagged Ypt3 showed cytoplasmic staining that was concentrated at growth sites, and this polarized localization required the actin cytoskeleton. It was also detected as a punctate staining in an actin-independent manner. Electron microscopy revealed that ypt3-i5 mutants accumulated aberrant Golgi-like structures and putative post-Golgi vesicles, which increased remarkably at the restrictive temperature. Consistently, the secretion of GFP fused with the pho1(+) leader peptide (SPL-GFP) was abolished at the restrictive temperature in ypt3-i5 mutants. FK506-treatment accentuated the accumulation of aberrant Golgi-like structures and caused a significant decrease of SPL-GFP secretion at a permissive temperature. These results suggest that Ypt3 is required at multiple steps of the exocytic pathway and its mutation affects diverse cellular processes and that calcineurin is functionally connected to these cellular processes.     

Electrotonic Coupling between Adjacent Internodal Cells of Chara braunii: Transmission of Action Potentials beyond the Node

Many nodal cells are interposed between two internodal cellsof Chara braunii. When an action potential conducted in an internodereached the node, no electrical activation in the nodal cellscould be found, although an area of the membrane bordering thenodal cells in this internode was partially activated (end-membraneaction potential). When the action potential approached thenode along the stimulated internode, an electrotonic potentialchange (depolarization) was produced in the other internode.This depolarization was greatly depressed by the end-membraneaction potential of the stimulated internode, so that hardlyany transmission took place. The ratio of the potential changein the surface membrane of the adjoining ("postsynaptic") internode(cell b) to that of the stimulated one (cell a), the couplingratio, e_b/e_a, can be estimated from a simple equivalent circuitof the nodal region composed of two surface-membrane resistances(R_a, R_b) and intercellular resistance (R_n). If R_n remains thesame, a higher ratio should be produced with a larger R_b, butthe ratio does not depend on any change in R_a, which could beproved experimentally. Transmission of the action potentialbeyond the node was frequent when the coupling ratio was increasedand when the threshold that elicits the action potential waslowered by immersing the node in a K or Na salt solution. ¹ Present address: Department of Physiology, Tohoku UniversitySchool of Dentistry, Sendai 980, Japan. (Received December 1, 1980; Accepted January 23, 1981)     

Ephaptic Transmission and Conduction Velocity of an Action Potential in Chara Internodal Cells Placed in Parallel and in Contact with One Another

When two separated Chara internodal cells were kept in contactover a length of 14 mm or more in moist air, an action potentialof one cell could be transmitted to the other cell in about40% of cases (ephaptic transmission). The action potential ofthe former cell was sometimes eliminated transiently when anew action potential was elicited in the latter cell. The newaction potential reactivated the former cell. The conductionvelocity of the action potential was reduced from 0.30?0.11cm/s (mean?SD) to 0.15?0.05 cm/s by ephaptic transmission ofthe action potential. Substitution of an artificial pond waterfor moist air or contact over a shorter length (4 mm insteadof 14 mm or more) between the two cells reduced the couplingratio, the ratio of the change in membrane potential of onecell to that of the other cell, and no transmission was observed.After ephaptic transmission, the action potentials of the twocells were conducted at almost the same velocity along the cells.The velocity was increased to 0.68?0.06 cm/s by simultaneousconduction of the action potentials in the two adjacent cells.When the simultaneously conducted action potentials reachedthe node of one cell, at the point at which another cell madecontact, they were able very frequently to trigger a new actionpotential in the adjoining cell. (Received September 28, 1989; Accepted April 5, 1990)     

Laying workers in queenless honeybee (Apis mellifera L.) colonies have physiological states similar to that of nurse bees but opposite that of foragers

Honeybee workers shift their labors from nursing their brood to foraging according to their age after eclosion. When the queen is lost from the colony, however, some workers become 'laying workers' whose ovaries develop to lay eggs. Here we investigated whether the physiological state of laying workers is more similar to that of nurse bees or foragers by examining the hypopharyngeal gland (HPG) and hemolymph vitellogenin titers. In a normal colony, nurse bees have well-developed HPGs that synthesize 'major royal jelly proteins' and high hemolymph vitellogenin titers, whereas foragers have shrunken HPGs that synthesize 70-kDa alpha-glucosidase and low hemolymph vitellogenin titers. In queenless colonies, however, workers with developed ovaries (laying workers) tended to have more developed HPGs and to synthesize major royal jelly proteins, whereas workers with shrunken HPGs tended to synthesize alpha-glucosidase and to have undeveloped ovaries. Furthermore, the workers with developed ovaries had higher vitellogenin titers than nurse bees, whereas those with undeveloped ovaries had lower vitellogenin titers. These findings indicate that the physiological state of laying workers is similar to that of nurse bees, but opposite that of foragers.     

Role of MAPK phosphatase-1 in the induction of monocyte chemoattractant protein-1 during the course of adipocyte hypertrophy

Monocyte chemoattractant protein-1 (MCP-1), an important chemokine whose expression is increased during the course of obesity, plays a role in macrophage infiltration into obese adipose tissue. This study was designed to elucidate the role of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) in the induction of MCP-1 during the course of adipocyte hypertrophy. We examined the time course of MKP-1 and MCP-1 mRNA expression and extracellular signal-regulated kinase (ERK) phosphorylation in the adipose tissue from mice rendered mildly obese by a short term high fat diet. We also studied the role of MKP-1 in the induction of MCP-1 in 3T3-L1 adipocytes during the course of adipocyte hypertrophy. MCP-1 mRNA expression was increased, followed by ERK activation and down-regulation of MKP-1, an inducible dual specificity phosphatase to inactivate ERK, in the adipose tissue at the early stage of obesity induced by a short term high fat diet, when macrophages are not infiltrated. Down-regulation of MKP-1 preceded ERK activation and increased production of MCP-1 in 3T3-L1 adipocytes in vitro during the course of adipocyte hypertrophy. Adenovirus-mediated restoration of MKP-1 in hypertrophied 3T3-L1 adipocytes reduced the otherwise increased ERK phosphorylation, thereby leading to the significant reduction of MCP-1 mRNA expression. This study provides evidence that the down-regulation of MKP-1 is critical for increased production of MCP-1 during the course of adipocyte hypertrophy.     

A Novel Glial Growth Inhibitory Factor, Gliostatin, Derived from Neurofibroma

Neurofibroma tissue was investigated for the presence of glial growth modulators that would suppress the proliferation of glial cells. A novel endogenous polypeptide inhibitor of proliferation and DNA synthesis in glial cells, gliostatin, was purified from the extracts of neurofibroma by a procedure comprising dye and anion-exchange column chromatography, and HPLC. A monoclonal antibody raised against partially purified gliostatin showed no cross-reactivity with known cytokines, but adsorbed the growth inhibitory activity of gliostatin and immunochemically visualized the putative gliostatin bands on western blot analyses. Although the product showed an apparent M(r) of 100,000 accompanied by an inhibitory activity on gel filtration column chromatography, it migrated at a lower apparent M(r) of 50,000 under the reducing conditions on western blotting, indicating that a homodimeric structure of native gliostatin consisted of 50-kDa subcomponents. Gliostatin was a potent growth inhibitor acting at nanomolar concentrations against all glial tumor cells and glia maturation factor-stimulated astroblasts, but not neuronal cells.     

Photoperiodic response of serotonin- and galanin-immunoreactive neurons of the paraventricular organ and infundibular nucleus in Japanese quail, Coturnix coturnix japonica

We investigated the photoperiodic response of serotonin- and galanin (GA)- immunoreactive (ir) cells in the paraventricular organ (PVO) and infundibular nucleus (IF) of the Japanese quail and the interaction of these cells with gonadotropin-releasing hormone (GnRH)-ir neurons in the hypothalamus. Serotonin-ir cells were located in series from the PVO to the IF, and were connected with each other. The number of serotonin-ir cells differed significantly between light and dark phases on the short days (SD), but did not differ between light and dark phases on long days (LD). GA-ir cells were also found in the PVO and IF. The number of GA-ir cells under SD conditions was significantly greater than under LD conditions but did not change diurnally. Both serotonin-ir and GA-ir fibers ran along the GnRH-ir cells in the nucleus commissurae pallii. Serotonin-ir and GA-ir fibers were connected with the GnRH-ir fibers in the external layer of the median eminence (ME). We confirmed that GA-ir fibers were closely associated with serotonin-ir neurons in the PVO and IF. GA-ir neurons have at least 2 routes of regulating GnRH neurons directly, and indirectly via the serotonin-ir cells in the PVO and IF.