Polarized secretory trafficking directs cargo for asymmetric dendrite growth and morphogenesis

Proper growth of dendrites is critical to the formation of neuronal circuits, but the cellular machinery that directs the addition of membrane components to generate dendritic architecture remains obscure. Here, we demonstrate that post-Golgi membrane trafficking is polarized toward longer dendrites of hippocampal pyramidal neurons in vitro and toward apical dendrites in vivo. Small Golgi outposts partition selectively into longer dendrites and are excluded from axons. In dendrites, Golgi outposts concentrate at branchpoints where they engage in post-Golgi trafficking. Within the cell body, the Golgi apparatus orients toward the longest dendrite, and this Golgi polarity precedes asymmetric dendrite growth. Manipulations that selectively block post-Golgi trafficking halt dendrite growth in developing neurons and cause a shrinkage of dendrites in mature pyramidal neurons. Further, disruption of Golgi polarity produces neurons with symmetric dendritic arbors lacking a single longest principal dendrite. These results define a novel polarized organization of neuronal secretory trafficking and demonstrate a mechanistic link between directed membrane trafficking and asymmetric dendrite growth.     

Isolation and characterization of phosphatases from vascular smooth muscle

Actomyosin preparations of the carotid arteries of cattle contain a soluble phosphatase activity, which can be removed from the contractile proteins by repeated washings. This enzyme activity is lowered by high ionic strength, potassium fluoride, zinc acetate, ammonium molybdate, and vanadate. Magnesium ions enhance the enzyme activity. The phosphatase activity shows a maximum between pH 5.5 and 6.0 and a plateau of pH 7-9. By means of gel filtration on Sepharose 6B the phosphate activity is separated into three peaks, which are characterized with respect to their inhibition by potassium fluoride, ammonium molybate, and vanadate and their dependence on pH.     

Modulation of juxtamembrane cleavage ("shedding") of angiotensin-converting enzyme by stalk glycosylation: evidence for an alternative shedding protease.

The role of juxtamembrane stalk glycosylation in modulating stalk cleavage and shedding of membrane proteins remains unresolved, despite reports that proteins expressed in glycosylation-deficient cells undergo accelerated proteolysis. We have constructed stalk glycosylation mutants of angiotensin-converting enzyme (ACE), a type I ectoprotein that is vigorously shed when expressed in Chinese hamster ovary cells. Surprisingly, stalk glycosylation did not significantly inhibit release. Introduction of an N-linked glycan directly adjacent to the native stalk cleavage site resulted in a 13-residue, proximal displacement of the cleavage site, from the Arg-626/Ser-627 to the Phe-640/Leu-641 bond. Substitution of the wild-type stalk with a Ser-/Thr-rich sequence known to be heavily O-glycosylated produced a mutant (ACE-JGL) in which this chimeric stalk was partially O-glycosylated; incomplete glycosylation may have been due to membrane proximity. Relative to levels of cell-associated ACE-JGL, rates of basal, unstimulated release of ACE-JGL were enhanced compared with wild-type ACE. ACE-JGL was cleaved at an Ala/Thr bond, 14 residues from the membrane. Notably, phorbol ester stimulation and TAPI (a peptide hydroxamate) inhibition of release-universal characteristics of regulated ectodomain shedding-were significantly blunted for ACE-JGL, as was a formerly undescribed transient stimulation of ACE release by 3, 4-dichloroisocoumarin. These data indicate that (1) stalk glycosylation modulates but does not inhibit ectodomain shedding; and (2) a Ser-/Thr-rich, O-glycosylated stalk directs cleavage, at least in part, by an alternative shedding protease, which may resemble an activity recently described in TNF-alpha convertase null cells [Buxbaum, J. D., et al. (1998) J. Biol. Chem. 273, 27765-27767].     

The formation of symplasmic domains by plugging of plasmodesmata: a general event in plant morphogenesis?

Summary The plasmodesmal network was examined in multicellular protoplast-derived calluses of the dicotyledonSolanum nigrum which had not yet formed any visible adventitious organs and in globular proembryogenic structures developed from scutellar calluses of the monocotyledonMolinia caerulea. Electron microscopical analyses revealed that both calluses and proembryos consisted of small, undifferentiated cells. The interconnecting plasmodesmata at many cell interfaces were structurally inconspicuous in both systems; in particular cell walls, however, all plasmodesmata were occluded with an osmiophilic, dense material. As the blocking material was obviously located in the microchannels of the plasmodesmal cytoplasmic sleeves, the plugged plasmodesmata can be assumed to be nonfunctional. Thus, selective occlusion of all the plasmodesmata in specific cell walls resulted in the symplasmic disconnection of particular adjacent cells. Complex patterns of symplasmic continuity and discontinuity were established within the developing tissues. Some cells or groups of cells were entirely symplasmically disconnected from the surrounding cells by plugged plasmodesmata and might function as independent domains. However, blockage of plasmodesmata was achieved by the surrounding cells rather than by those cells belonging to the isolated domains. The demarcation of symplasmic domains might be a general prerequisite for differential morphogenesis, since they were found to be established very early in the course of morphogenetic processes.     

Two new subspecies of Photorhabdus luminescens, isolated from Heterorhabditis bacteriophora (Nematoda: Heterorhabditidae): Photorhabdus luminescens subsp. kayaii subsp. nov. and Photorhabdus luminescens subsp. thracensis subsp. nov

Bacterial isolates from nematodes from Turkish soil samples were initially characterized by molecular methods and seven members of the genus Photorhabdus identified to the species level, using riboprint analyses and metabolic properties. Strain 07-5 (DSM 15195) was highly related to the type strain of Photorhabdus luminescens subsp. laumondii DSM 15139T, and was regarded a strain of this subspecies. Strains 1121T (DSM 15194T), 68-3 (DSM 15198) and 47-10 (DSM 15197) formed one, strain 39-8T (DSM 15199T), 39-7 (DSM 15196) and 01-12 (DSM 15193) formed a second cluster that branched intermediate the three subspecies of Photorhabdus luminescens. Based upon moderate 16S rRNA gene sequence similarities and differences in metabolic properties among themselves and with type strains of the three subspecies we consider the two clusters to represent two new subspecies of Photorhabdus luminescens for which the names Photorhabdus luminescens subsp. kayaii, type strain 1121T (DSM 15194T, NCIMB 13951T), and Photorhabdus luminescens subsp. thracensis subsp. nov., type strain 39-8T (DSM 15199T, NCIMB 13952T) are proposed.     

The Active and the Inactive Form of the hAT1 Receptor Have an Identical Ligand-Binding Environment: An MPA Study on a Constitutively Active Angiotensin II Receptor Mutant

Several models of activation mechanisms were proposed for G protein-coupled receptors (GPCRs), yet no direct methods exist for their elucidation. The availability of constitutively active mutants has given an opportunity to study active receptor conformations within acceptable limits using models such as the angiotensin II type 1 (AT₁)¹ receptor mutant N111G-hAT₁ which displays an important constitutive activity. Recently, by using methionine proximity assay, we showed for the hAT₁ receptor that TMD III, VI, and VII form the ligand-binding pocket of the C-terminal amino acid of an antagonistic AngII analogue. In the present contribution, we investigated whether the same residues would also constitute the ligand-binding contacts in constitutively activated mutant (CAM) receptors. For this purpose, the same Met mutagenesis strategy was carried out on the N111G double mutants. Analysis of 43 receptors mutants in the N111G-hAT₁ series, photolabeled and CNBr digested, showed that there were only subtle structural changes between the wt-receptor and its constitutively active form.