Interactions between adaptor protein-1 of the clathrin coat and microtubules via type 1a microtubule-associated proteins.

The classical view suggests that adaptor proteins of the clathrin coat mediate the sorting of cargo protein passengers into clathrin-coated pits and the recruitment of clathrin into budding areas in the donor membrane. In the present study, we provide biochemical and morphological evidence that the adaptor protein 1 (AP-1) adaptor of the trans-Golgi network clathrin interacts with microtubules. AP-1 in cytosolic extracts interacted with in vitro assembled microtubules, and these interactions were inhibited by ATP depletion of the extracts or in the presence of 5'-adenylylimidodiphosphate. An overexpressed gamma-subunit of the AP-1 complex associated with microtubules, suggesting that this subunit may mediate the interaction of AP-1 with the cytoskeleton. Purified AP-1 did not interact with purified microtubules, but interaction occurred when an isolated microtubule-associated protein fraction was added to the reaction mix. The gamma-adaptin subunit of AP-1 specifically co-immunoprecipitated with a microtubule-associated protein of type 1a from rat brain cytosol. This suggests that type 1a microtubule-associated protein may mediate the association of AP-1 with microtubules in the cytoplasm. The microtubule binding activity of AP-1 was markedly inhibited in cytosol of mitotic cells. By means of its interaction with microtubule-associated proteins, we propose novel roles for AP-1 adaptors in modulating the dynamics of the cytoskeleton, the stability and shape of coated organelles, and the loading of nascent AP-1-coated vesicles onto appropriate microtubular tracks.

High proliferation rate characterizes the site of axis formation in the avian blastula-stage embryo.

Localized zones of high cell proliferation have been thought to be important in determining several phases of axis formation at early stages of chick development. It was suggested that a developmental center, a center of cellular activity such as proliferation and movement, is located in the posterior half of the area pellucida in the unincubated chick blastoderm. In the work reported here, we have used the bromodeoxyuridine (BrdU) incorporation procedure followed by immunoperoxidase detection to assess the rate of cell proliferation at particular sub-regions of pregastrulating chick blastoderms (stages X-XIII). Examination of whole-mount and histological sections of stages X through XII blastoderms, pulsed with BrdU, showed no distinguishable difference in labeled cells between particular regions of these blastoderms and also that there are no specific zones of high cellular proliferation in either the hypoblast or the epiblast layers of the area pellucida. However, our observations have shown a striking difference in the stage XIII blastoderm, in which a relatively high amount of labeled cells were detected all around the posterior region of the area opaca, the marginal zone, Koller's sickle and the epiblast. The relatively high proportion of cell divisions observed at the posterior end of a stage XIII blastoderm, the blastula stage of the avian embryo, may be associated with the major developmental ability of this region to initiate an embryonic axis. Directional axis formation, therefore, may be attributed to a region of proliferation in the posterior side of a stage XIII blastoderm.

The basolateral sorting signal of the polymeric immunoglobulin receptor contains two functional domains.

Basolateral sorting of the polymeric immunoglobulin receptor (pIgR) expressed in Madin-Darby canine kidney (MDCK) cells is mediated by a 17-residue sorting signal that resides in the cytoplasmic domain. We have recently analyzed the sequence requirements of the signal by alanine scanning mutagenesis. We found that basolateral sorting is mediated primarily by three amino acids: H656, R657 and V660. Individual mutations of each of these residues to Ala caused a substantial decrease in basolateral sorting and a corresponding increase in targeting to the apical surface. Structural analysis of 17-residue peptides corresponding to the signal revealed that V660 is in a beta-turn (probably type I) secondary structure, and its mutation to Ala destabilized the turn. H656 and R657 were not part of the turn and substitution of Arg657 to Ala had no effect on the turn stability. These results suggested that the signal is comprised of two structurally distinct domains: a critical V660 in the context of the beta-turn and an additional two residues (H656 and R657) that are not in the turn and probably are unimportant for its stability. Here we provide evidence suggesting that the two domains are distinguishable not only by their structure but also by their function. Basolateral targeting of pIgR mutants bearing Ala mutations at either 656 or 657 was not affected by treatment with brefeldin A (BFA), while basolateral targeting of pIgR containing an Ala substitution at position 660 was markedly and uniquely stimulated by BFA. Compared to single Ala substitutions, simultaneous mutations of H656 and R657 to Ala caused an additional minor effect on basolateral and apical sorting, whereas double mutations of V660 and either H656 or R657 resulted in a maximal decrease in basolateral targeting and corresponding increase in apical targeting. These results suggest the existence of two domains in the signal. When both domains are destroyed, basolateral targeting is maximally inhibited. The results also imply that V660 mediates basolateral sorting by a different mechanism from H656 and R657. We suggest that V660 and perhaps more generally the beta-turn may interact with BFA-sensitive adaptor complexes.

Side chain modified 5-deazafolate and 5-deazatetrahydrofolate analogues as mammalian folylpolyglutamate synthetase and glycinamide ribonucleotide formyltransferase inhibitors: synthesis and in vitro biological evaluation.

5-Deazafolate and 5-deazatetrahydrofolate (DATHF) analogues with the glutamic acid side chain replaced by homocysteic acid (HCysA), 2-amino-4-phosphonobutanoic acid (APBA), and ornithine (Orn) were synthesized as part of a larger program directed toward inhibitors of folylpolyglutamate synthetase (FPGS) as probes of the FPGS active site and as potential therapeutic agents. The tetrahydro compounds were also of interest as non-polyglutamatable inhibitors of the purine biosynthetic enzyme glycinamide ribonucleotide formyltransferase (GARFT). Reductive coupling of N2-acetamido-6-formylpyrido[2,3-d]pyrimidin-4(3H)-one with 4-aminobenzoic acid, followed by N10-formylation, mixed anhydride condensation of the resultant N2-acetyl-N10-formyl-5- deazapteroic acid with L-homocysteic acid, and removal of the N2-acetyl and N10-formyl groups with NaOH, afforded N-(5-deazapteroyl)-L-homocysteic acid (5-dPteHCysA). Mixed anhydride condensation of N2-acetyl-N10-formyl- 5-deazapteroic acid with methyl D,L-2-amino-4-(diethoxyphosphinyl)butanoic acid, followed by consecutive treatment with Me3SiBr and NaOH, yielded D,L-2-[(5-deazapteroyl)amino]-4-phosphonobutanoic acid (5-dPteAPBA). Treatment with NaOH alone led to retention of one ethyl ester group on the phosphonate moiety. Catalytic hydrogenation of N2-acetyl-N10-formyl-5-deazapteroic acid followed by mixed anhydride condensation with methyl L-homocysteate and deprotection with NaOH afforded N-(5,6,7,8-tetrahydro-5-deazapteroyl)-L-homocysteic acid (5-dH4PteHCysA). Similar chemistry starting from methyl D,L-2-amino-4-(diethoxyphosphinyl)butanoic acid and methyl N delta-(benzyloxycarbonyl)-L-ornithinate yielded D,L-2-[(5-deaza-5,6,7,8-tetrahydropteroyl)amino]-4-phosphonobut ano ic acid (5-dH4Pte-APBA) and N alpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-L-ornithine (5-dH4PteOrn), respectively. The 5-deazafolate analogues were inhibitors of mouse liver FPGS, and the DATHF analogues inhibited both mouse FPGS and mouse leukemic cell GARFT. Analogues with HCysA and monoethyl APBA side chains were less active as FPGS inhibitors than those containing an unesterified gamma-PO(OH)2 group, and their interaction with the enzyme was noncompetitive against variable folyl substrate. In contrast, Orn and APBA analogues obeyed competitive inhibition kinetics and were more potent, with Ki values as low as 30 nM. Comparison of the DATHF analogues as GARFT inhibitors indicated that the Orn side chain diminished activity relative to DATHF, but that the compounds with gamma-sulfonate or gamma-phosphonate substitution retained activity, with Ki values in the submicromolar range. The best GARFT inhibitor was the 5-dH4PteAPBA diastereomer mixture, with a Ki of 47 nM versus 65 nM for DATHF. None of the compounds showed activity against cultured WI-L2 or CEM human leukemic lymphoblasts at concentrations of up to 100 microM.(ABSTRACT TRUNCATED AT 400 WORDS)

Algal endosymbiosis in brown hydra: host/symbiont specificity.

Host/symbiont specificity has been investigated in non-symbiotic and aposymbiotic brown and green hydra infected with various free-living and symbiotic species and strains of Chlorella and Chlorococcum. Morphology and ultrastructure of the symbioses obtained have been compared. Aposymbiotic Swiss Hydra viridis and Japanese H. magnipapillata served as controls. In two strains of H. attenuata stable hereditary symbioses were obtained with Chlorococcum isolated from H. magnipapillata. In one strain of H. vulgaris, in H. oligactis and in aposymbiotic H. viridis chlorococci persisted for more than a week. Eight species of free-living Chlorococcum, 10 symbiotic and 10 free-living strains of Chlorella disappeared from the brown hydra within 1-2 days. In H. magnipapillata there was a graded distribution of chlorococci along the polyps. In hypostomal cells there were greater than 30 algae/cell while in endodermal cells of the mid-section or peduncle less than 10 algae/cell were found. In H. attenuata the algal distribution was irregular, there were up to five chlorocci/cell, and up to 20 cells/hydra hosted algae. In the dark most cells of Chlorococcum disappeared from H. magnipapillata and aposymbiotic hydra were obtained. Chlorococcum is thus an obligate phototroph, and host-dependent heterotrophy is not required for the preservation of a symbiosis. The few chlorococci that survived in the dark seem to belong to a less-demanding physiological strain. In variance with known Chlorella/H. viridis endosymbioses the chlorococci in H. magnipapillata and H. attenuata were tightly enveloped in the vacuolar membrane of the hosting cells with no visible perialgal space. Chlorococcum reproduced in these vacuoles and up to eight daughter cells were found within the same vacuole. We suggest that the graded or scant distribution of chlorococci in the various brown hydra, their inability to live in H. viridis and the inability of the various chlorellae to live in brown hydra are the result of differences in nutrients available to the algae in the respective hosting cells. We conclude that host/symbiont specificity and the various forms of interrelations we show in green and brown hydra with chlorococci and chlorellae are based on nutritional-ecological factors. These interrelations demonstrate successive stages in the evolution of stable obligatoric symbioses from chance encounters of preadapted potential cosymbionts.

Correlations between characteristics of some free-living Chlorella sp. and their ability to form stable symbioses with Hydra viridis.

Aposymbiotic polyps of Hydra viridis were infected with 17 strains of in vitro cultured Chlorella sp. Larvae of Artemia fed with the chlorellae were used as an infecting vector. Of the 17 strains, seven formed stable symbioses and one formed a transient infection that disappeared within several weeks. Chlorellae of the nine other strains were cleared out of the infected hydra within 2-3 days. There was a distinct correlation between the ability of the chlorellae to form stable symbioses and their ability to adapt and grow in media enriched with 0.5% proteose peptone. Only strains that grew in the latter medium formed symbioses with the hydra. The symbioses formed with the different strains of chlorellae differed from one another. Hydra infected with some strains greened completely while those infected with other strains greened only partially. The degree of infection varied also within each population, and there were differences in the distribution of the various chlorellae along the stalk and inside the digestive cells of the hydra. Growth rates of the infected hydra were all less that those of aposymbiotic hydra or of hydra hosting native zoochlorellae. We conclude that adaptability to a nutrient-rich environment inside the perialgal vacuole of the digestive cell and a sufficient growth rate therein are crucial to the ability of chlorellae to form stable symbioses with H. viridis. In time, co-adaptation of hydra and chlorellae would restore the normal growth rate of the former and bring about regularity to the form and extent of infection by the latter.

A rapid and convenient preparation of [4-3H]NADP and stereospecifically tritiated NADP3H.

The enzymatic preparation and chromatographic purification of [4-3H]NADP and NADPH stereospecifically labeled with 3H on either the A or B faces at position 4 have been simplified. Commercially available [1-3H]glucose was used as a starting material for the sequential synthesis of [4B-3H]NADPH, [4-3H]NADP, and [4A-3H]NADPH. These products were rapidly purified by step elution of DEAE-cellulose minicolumns so that [4B-3H]NADPH was produced and purified from [1-3H]glucose in 2 h, [4-3H]NADP in 5 h, and [4A-3H]NADPH in 8 h. Yields of these products were 65 to 88% starting with [1-3H]glucose. Analysis of the products by high-performance liquid chromatography indicated radiochemical purities of 82-95% for these compounds and specific activities equivalent to that of the starting material (10-15 Ci/mmol).

Intracellular infection of aposymbiotic Hydra viridis by a foreign free-living Chlorella sp.: initiation of a stable symbiosis.

An aposymbiotic strain of Hydra viridis became infected with free-living Chlorella sp. A stable symbiosis formed that differed in its characteristics from other known Chlorella/Hydra symbioses. The algae reproduced and formed clusters in host endodermal cells, inside large vacuoles filled with an electron-dense substance. A few algae were found to be digested by the hydra, but the apparently uncontrolled reproduction rate of the algae more than compensated for this loss. Surplus algae were expelled into the coelenteron and eventually into the surrounding medium. The expelled algae were repeatedly re-engulfed by the hydra during its feeding, forming a process of continuous reinfection. We suggest that such repeated reinfection of the hydra by the expelled algae provides the host with an endless number of Chlorella from which it might in time select a suitable adapted, controllable symbiont. The present newly formed symbiosis might serve as a model for the study of evolution of algal endosymbioses.