Isolation of a matrix that binds medial Golgi enzymes

Rat liver Golgi stacks were extracted with Triton X-100 at neutral pH. After centrifugation the low speed pellet contained two medial-Golgi enzymes, N-acetylglucosaminyltransferase I and mannosidase II, but no enzymes or markers from other parts of the Golgi apparatus. Both were present in the same structures which appeared, by electron microscopy, to be small remnants of cisternal membranes. The enzymes could be removed by treatment with low salt, leaving behind a salt pellet, which we term the matrix. Removal of salt caused specific re-binding of both enzymes to the matrix, with an apparent dissociation constant of 3 nM for mannosidase II. Re-binding was abolished by pretreatment of intact Golgi stacks with proteinase K, suggesting that the matrix was present between the cisternae.     

Kin recognition in rattlesnakes

Snakes are often regarded as the least social of all vertebrate groups, but this assumption stems from the fact that they are secretive and difficult to observe in nature, rather than direct evidence. Recent studies have revealed a surprising degree of social complexity in snakes. Here, I examine the ability of captive-raised timber rattlesnakes (Crotalus horridus) to recognize siblings by measuring the mean separation distance and frequency of contact between pairs of individuals housed together. The results show that female siblings associate more closely with each other than nonsibling pairs. Previous studies have shown that timber rattlesnakes occupying the same hibernacula have higher relatedness than snakes using neighbouring hibernacula, and frequently form social aggregations. Rattlesnakes exhibit other characteristics consistent with advanced sociality, including group defence, conspecific alarm signals and maternal defence of young. These findings reinforce the notion that, rather than being solitary and asocial, some snake species may form family groups.     

Fragmentation and partitioning of the Golgi apparatus during mitosis in HeLa cells.

Osmium impregnation was used to determine the number of Golgi apparatus in both interphase and mitotic HeLa cells. The number was found to increase substantially during mitosis to the point where random partitioning alone would explain the nearly equal numbers found in each daughter cell.     

A mitotic form of the Golgi apparatus in HeLa cells

Galactosyltransferase, a marker for trans-Golgi cisternae in interphase cells, was localized in mitotic HeLa cells embedded in Lowicryl K4M by immunoelectron microscopy. Specific labeling was found only over multivesicular structures that we term Golgi clusters. Unlike Golgi stacks in interphase cells, these clusters lacked elongated cisternae and ordered stacking of their components but did comprise two distinct regions, one containing electron-lucent vesicles and the other, smaller, vesiculo-tubular structures. Labeling for galactosyltransferase was found predominantly over the latter region. Both structures were embedded in a dense matrix that excluded ribosomes and the cluster was often bounded by cisternae of the rough endoplasmic reticulum, sometimes on all sides. Clusters were present at all stages of mitosis examined, which included prometaphase, metaphase, and telophase. They were also identified in conventionally processed mitotic cells and shown to contain another trans-Golgi marker, thiamine pyrophosphatase. Serial sectioning showed that clusters were discrete and globular and multiple copies appeared to be dispersed in the cytoplasm. Their possible role in the division of the Golgi apparatus is discussed.     

Kin recognition in social bees

Kin recognition in social insects has become a central issue in sociobiology because studies of the recognition abilities of social insects provide a test of kin selection theory. W.D. Hamilton(1) formalized kin selection theory by showing how individuals can gain fitness by increasing the reproductive output of relatives (kin). The social interactions of individuals, or groups, should be influenced by the genetic structure of the population. The ability to recognize kin can increase the adaptive value of social behavior by modulating it according to genetic relationship. From this, the specific prediction emerges: if individuals can distinguish among others with which they interact on the basis of the degree to which they are related, then behavior should be biased preferentially toward more closely related reproductive individuals.     

Kin recognition in embryonic damselfishes

Predator‐induced mortality rates are highest in early life stages; therefore, early recognition of threats can greatly increase survival chances. Some species of coral reef fishes have been frequently found to recruit back to their natal reefs; in this instance, there is a high chance of juveniles encountering their siblings, among other kin, after hatching. Kin recognition plays an important ecological role in that it allows individuals to protect genetically similar relatives, and hence increase their inclusive fitness. By observing changes in heart rates, we demonstrated that embryos of two damselfish species, Acanthochromis polyacanthus and Amphiprion melanopus, not only possess recognition of kin and damage‐released alarm odours, but also react to them in a graded manner. Such refined olfactory capabilities in embryonic stage organisms (seven and eleven days after fertilisation) suggest identification of threats may provide survival advantages post‐hatching, such as the informed choice of low risk habitats at settlement. To our knowledge this is the first time that kin recognition has been identified in embryos of any species.     

Evidence that globular Golgi clusters in mitotic HeLa cells are clustered tubular endosomes.

By conventional electron microscopy we observed in mitotic HeLa cells the structures termed Golgi clusters by Lucocq et al. (J. Cell Biol. 104, 865-874 (1987)) and interpreted by them as clusters of vesicular remnants of the Golgi apparatus. Golgi clusters consist of tubular and vesicular profiles about 50 nm in diameter, sometimes associated with larger 250 nm vesicles. When cultures of HeLa cells were incubated for 60 min or 120 min with medium containing high specific activity horseradish peroxidase (HRP) at 10 mg/ml we found that the membrane-bound compartments in the Golgi clusters in mitotic cells contained heavy deposits of HRP reaction product. Neither interphase nor mitotic HeLa cells contain an endogenous peroxidase activity. We concluded that Golgi clusters are an endocytic compartment and confirmed this by showing that Golgi clusters could be labeled with two other endocytic tracers--bovine serum albumin conjugated to colloidal gold and transferrin conjugated to HRP. When cultures were incubated with HRP for only 15 min most of the Golgi clusters in the mitotic cells were either unlabeled or consisted of a mixture of HRP-labeled and unlabeled profiles. Since during mitosis endocytosis is inhibited this was the expected result. When interphase HeLa cells were incubated with Brefeldin A (BFA), the Golgi apparatus disassembled and immunofluorescence microscopy showed that 1,4 beta galactosyltransferase had relocated to the endoplasmic reticulum. When cells in the presence of BFA and lacking the Golgi apparatus were allowed to endocytose HRP and then entered mitosis, typical HRP-labeled Golgi clusters were seen in the mitotic cells. It is therefore highly unlikely that these structures contain membrane derived from the Golgi cisternae that are sensitive to BFA, including in HeLa cells those containing galactosyltransferase. Finally, we found that interphase HeLa cells incubated with okadaic acid contain structures that are morphologically indistinguishable from Golgi clusters but can be labeled by endocytic tracer. Taken together, this evidence indicates that most, if not all, of the membrane-bound compartments in Golgi clusters are tubular early endosomes.     

Kin recognition in a social spider

Social spiders accept immigrant spiders into their kin-based groups, suggesting that spiders cannot recognise kin and may lose inclusive fitness benefits. A field and two laboratory experiments on Diaea ergandros, a social crab spider, demonstrated that younger and older instar D. ergandros do discriminate siblings, but potential benefits were variable and not equally distributed. First, proportional survival was greater in large groups regardless of the within-group relatedness, so accepting immigrants increases probability of group survival (although relatedness was more important among smaller groups). Second, juvenile D. ergandros ate unrelated spiders instead of siblings when starved, so immigrants might represent a food reserve in times of food shortage. Third, subadult resident, sibling females cannibalised unrelated, immigrant females and their brothers instead of immigrant males when starved, suggesting that subadult female spiders may maximise outbreeding opportunities. These benefits provide selective pressure for groups to accept immigrants, but as benefits are realised differentially, conflict and cooperation will exist within spider groups similar to that shown in other group-living taxa.     

Kin recognition in an annual plant

Kin recognition is important in animal social systems. However, though plants often compete with kin, there has been as yet no direct evidence that plants recognize kin in competitive interactions. Here we show in the annual plant Cakile edentula, allocation to roots increased when groups of strangers shared a common pot, but not when groups of siblings shared a pot. Our results demonstrate that plants can discriminate kin in competitive interactions and indicate that the root interactions may provide the cue for kin recognition. Because greater root allocation is argued to increase below-ground competitive ability, the results are consistent with kin selection.     

Cog3p depletion blocks vesicle-mediated Golgi retrograde trafficking in HeLa cells

The conserved oligomeric Golgi (COG) complex is an evolutionarily conserved multi-subunit protein complex that regulates membrane trafficking in eukaryotic cells. In this work we used short interfering RNA strategy to achieve an efficient knockdown (KD) of Cog3p in HeLa cells. For the first time, we have demonstrated that Cog3p depletion is accompanied by reduction in Cog1, 2, and 4 protein levels and by accumulation of COG complex-dependent (CCD) vesicles carrying v-SNAREs GS15 and GS28 and cis-Golgi glycoprotein GPP130. Some of these CCD vesicles appeared to be vesicular coat complex I (COPI) coated. A prolonged block in CCD vesicles tethering is accompanied by extensive fragmentation of the Golgi ribbon. Fragmented Golgi membranes maintained their juxtanuclear localization, cisternal organization and are competent for the anterograde trafficking of vesicular stomatitis virus G protein to the plasma membrane. In a contrast, Cog3p KD resulted in inhibition of retrograde trafficking of the Shiga toxin. Furthermore, the mammalian COG complex physically interacts with GS28 and COPI and specifically binds to isolated CCD vesicles.     

Overlapping distribution of two glycosyltransferases in the Golgi apparatus of HeLa cells

Thin, frozen sections of a HeLa cell line were double labeled with specific antibodies to localize the trans-Golgi enzyme, beta 1,4 galactosyltransferase (GalT) and the medial enzyme, N- acetylglucosaminyltransferase I (NAGT I). The latter was detected by generating a HeLa cell line stably expressing a myc-tagged version of the endogenous protein. GalT was found in the trans-cisterna and trans- Golgi network but, contrary to expectation, NAGT I was found both in the medial- and trans-cisternae, overlapping the distribution of GalT. About one third of the NAGT I and half of the GalT were found in the shared, trans-cisterna. These data show that the differences between cisternae are determined not by different sets of enzymes but by different mixtures.     

Mitotic Golgi fragments in HeLa cells and their role in the reassembly pathway

Immunoelectron microscopy and stereology were used to identify and quantitate Golgi fragments in metaphase HeLa cells and to study Golgi reassembly during telophase. On ultrathin frozen sections of metaphase cells, labeling for the Golgi marker protein, galactosyltransferase, was found over multivesicular Golgi clusters and free vesicles that were found mainly in the mitotic spindle region. The density of Golgi cluster membrane varied from cell to cell and was inversely related to the density of free vesicles in the spindle. There were thousands of free Golgi vesicles and they comprised a significant proportion of the total Golgi membrane. During telophase, the distribution of galactosyltransferase labeling shifted from free Golgi vesicles towards Golgi clusters and the population of free vesicles was depleted. The number of clusters was no more than in metaphase cells so the observed fourfold increase in membrane surface meant that individual clusters had increased in size. More than half of these had cisterna(e) and were located next to "buds" on the endoplasmic reticulum. Early in G1 the number of clusters dropped as they congregated in the juxtanuclear region and fused. These results show that fragmentation of the Golgi apparatus yields Golgi clusters and free vesicles and reassembly from these fragments is at least a two-step process: (a) growth of a limited number of dispersed clusters by accretion and fusion of vesicles to form cisternal clusters next to membranous "buds" on the endoplasmic reticulum; (b) congregation and fusion to form the interphase Golgi stack in the juxtanuclear region.     

Kin recognition and cannibalism in polyphenic salamanders

We investigated kin discrimination among larvae of Arizona tigersalamanders (Ambystoma tigrinum nebulosum) which occur as "typical"morphs that feed mostly on invertebrate prey and occasionallyon conspecifics, and as "cannibal" morphs that feed primarilyon conspecifics. When housed with smaller larvae that differedin relatedness, both cannibals and typicals preferentially consumedless-related individuals. Cannibals ate typicals much quickerwhen the choice was between nonkin and siblings than when thechoice was between nonkin and cousins, indicating that cannibalscould distinguish different categories of relatives. Cannibalswere less likely to eat a larval sibling that was a cannibalmorph than a sibling that was a typical morph. Occluding animals'nares temporarily eliminated kin discrimination, implying thatolfaction is important in recognition. Larvae from differentsibships varied considerably in their ability to discriminatekin, and the greater the probability that a larva from a givensibship would develop into a cannibal morph, the more likelythe members of that sibship were to discriminate kin. Our resultsenable us to infer the functional significance of kin recognitionin this species and to develop an evolutionary model of themechanisms underlying the joint control of kin recognition andcannibalistic polyphenism.     

Evidence that the entire Golgi apparatus cycles in interphase HeLa cells: sensitivity of Golgi matrix proteins to an ER exit block.

We tested whether the entire Golgi apparatus is a dynamic structure in interphase mammalian cells by assessing the response of 12 different Golgi region proteins to an endoplasmic reticulum (ER) exit block. The proteins chosen spanned the Golgi apparatus and included both Golgi glycosyltransferases and putative matrix proteins. Protein exit from ER was blocked either by microinjection of a GTP-restricted Sar1p mutant protein in the presence of a protein synthesis inhibitor, or by plasmid-encoded expression of the same dominant negative Sar1p. All Golgi region proteins examined lost juxtanuclear Golgi apparatus-like distribution as scored by conventional and confocal fluorescence microscopy in response to an ER exit block, albeit with a differential dependence on Sar1p concentration. Redistribution of GalNAcT2 was more sensitive to low Sar1p(dn) concentrations than giantin or GM130. Redistribution was most rapid for p27, COPI, and p115. Giantin, GM130, and GalNAcT2 relocated with approximately equal kinetics. Distinct ER accumulation could be demonstrated for all integral membrane proteins. ER-accumulated Golgi region proteins were functional. Photobleaching experiments indicated that Golgi-to-ER protein cycling occurred in the absence of any ER exit block. We conclude that the entire Golgi apparatus is a dynamic structure and suggest that most, if not all, Golgi region-integral membrane proteins cycle through ER in interphase cells.     

Kin and sex recognition in a dioecious grass

Plants have evolved complex mechanisms to recognize and respond to the presence of neighboring plants, and the genetic identity of a neighbor has been shown to make a difference in this response. Studies have found that plants are able to differentiate among self- versus non-self and among sibling (kin) competitors. Here, we present data for the dioecious grass Distichlis spicata on seedling recognition of kin and sex. D. spicata exhibits extreme spatial segregation of the sexes (SSS) in the field, and previous work has shown that intra-sexual competition is less than inter-sexual competition in the field. In this experiment, we conducted experiments in the lab, exposing the seedlings to liquid media in which seedlings had been previously grown, rather than have the seedling physically contact one another. We found that inter-sexual interactions caused a decrease in the total dry weight and an increase in root/shoot ratio of the plants compared with intra-sexual interactions. These findings suggest that D. spicata plants can recognize and respond to plant sex and that inter-sexual competition contributes to SSS, even when additional interactions, such as mycorrhizal fungi are controlled, and physical interactions between plants are removed. In the kin recognition analysis, we found that plants paired with another plant from the same mother had significantly greater lateral root number and length than plants paired with non-kin, suggesting that in this highly clonal grass, kin recognition may be an important mechanism in competitive interactions.     

Heat-shock proteins associated with base excision repair enzymes in HeLa cells

Two enzymes of base excision repair (BER), uracil DNA glycosylase (UDG) and DNA polymerase beta (beta pol), from HeLa cells co-eluted from Superose 12 FPLC columns. The UDG was completely displaced from 150-180-kDa fractions to 30- 70-kDa fractions by brief treatment with 0.5 N NaCl, pH 3.0, as expected when protein-protein associations are disrupted, but beta pol was not displaced by this treatment. UDG was not essential to the presence of beta pol in the 150-180-kDa enzyme complex. beta pol and UDG apparently reside in separate but co-eluting structures. Immunoaffinity chromatography showed that the association of UDG and beta pol was accounted for by attachment in common to DNA and that the association was abolished by eliminating DNA. Evidence for base excision repairosomes containing UDG and beta pol in protein-protein assemblies was not found. However, UDG and human AP endonuclease (HAP1) were associated with HSP70 and HSP27, which are present in 150-180-kDa and 30-70-kDa proteins of cell sonicates. The association of HSPs with BER enzymes was confirmed by hydroxyl radical protein-protein footprinting and immunoaffinity tests. The association of HSPs and BER enzymes is a novel finding. HSP binding may account for the presence of BER enzymes in the two large size class fractions and HSPs may have functional roles in BER.     

Kin recognition and cannibalism in a subsocial spider

Evolution of cooperation and group living in spiders from subsocial family groups may be constrained by their cannibalistic nature. A tendency to avoid cannibalizing kin may facilitate tolerance among spiders and implies the ability to identify relatives. We investigated whether the subsocial spider Stegodyphus lineatus discriminates kin by recording cannibalism among juveniles in experiments during which amount of food and size difference among spiders in groups were varied. We hypothesized that family groups should be less cannibalistic than groups of mixed‐parental origin. Further, we tested whether food‐stress would influence cannibalism rates differently in kin and nonkin groups and the effect of relatedness on cannibalism within groups of spiders of variable size compared with those of homogenous size. In groups of six spiders, more spiders were cannibalized in nonsib groups than in sib groups under low food conditions. A tendency for nonkin biased cannibalism in starved spider pairs supported that kin recognition in S. lineatus is expressed when food is limited. Size variance of individuals within well‐fed groups of siblings and unrelated spiders had no influence on cannibalism rates. Apparently, both hunger and high density are important promoters of cannibalism. In addition to inclusive fitness benefits, we suggest that an ability to avoid cannibalizing kin will favour the evolution of cooperation and group living in phylogenetically pre‐adapted solitary species.     

Biosynthesis and modification of Golgi mannosidase II in HeLa and 3T3 cells

The biosynthesis and post-translational modification of mannosidase II, an enzyme required in the maturation of asparagine-linked oligosaccharides in the Golgi complex, has been investigated. Antibody raised against this enzyme purified from rat liver Golgi membranes was used to immunoprecipitate mannosidase II from rat liver, 3T3 cells, or HeLa cells. Mannosidase II immunoprecipitated from rat liver Golgi membranes, when analyzed by polyacrylamide gel electrophoresis, migrated with an apparent molecular weight of approximately 124,000. In contrast, the enzyme purified from rat liver Golgi membranes was shown to contain both the 124,000-dalton component and a 110,000-dalton polypeptide believed to result from degradation of intact mannosidase II during purification. Mannosidase II from 3T3 and HeLa cells migrated on polyacrylamide gels with apparent molecular weights of approximately 124,000 and 134,000-136,000, respectively. When immunoprecipitated from radiolabeled cultures, mannosidase II from both cell types was similar in the following respects: (a) the initial synthesis product had an apparent molecular weight of approximately 124,000; (b) in cultures treated with tunicamycin the initial synthesis product had an apparent molecular weight of approximately 117,000; (c) endoglycosidase H digestion of the initial synthesis product gave an apparent molecular weight similar to the tunicamycin-induced polypeptide; (d) the mature enzyme was mostly (HeLa) or entirely (3T3) resistant to digestion by endoglycosidase H. Loss of [35S]methionine from intracellular mannosidase II occurred with a half-life of approximately 20 h; there was no appreciable accumulation of labeled immuno-reactive material in the medium. HeLa mannosidase II, but not the 3T3 enzyme, was additionally modified 1-3 h after synthesis, the initial synthesis product being converted to a doublet with an apparent molecular weight of approximately 134,000-136,000. Evidence is presented that this mobility shift may result from O-glycosylation. Mannosidase II from both cell types could be labeled with [32P]phosphate or [35S]sulfate. The latter is apparently attached to oligosaccharide as indicated by inhibition of labeling by tunicamycin; the former was shown with the HeLa enzyme to be present as serine phosphate moieties. In addition, [3H]palmitate could be incorporated into the enzyme in 3T3 cells.(ABSTRACT TRUNCATED AT 400 WORDS)