Actin is involved in the organization of the Golgi complex and Golgi-to-ER protein transport in mammalian cells. Little, however, is known about the regulation of the Golgi-associated actin cytoskeleton. We provide evidence that Cdc42, a small GTPase that regulates actin dynamics, controls Golgi-to-ER protein transport. We located GFP-Cdc42 in the lateral portions of Golgi cisternae and in COPI-coated and noncoated Golgi-associated transport intermediates. Overexpression of Cdc42 and its activated form Cdc42V12 inhibited the retrograde transport of Shiga toxin from the Golgi complex to the ER, the redistribution of the KDEL receptor, and the ER accumulation of Golgi-resident proteins induced by the active GTP-bound mutant of Sar1 (Sar1[H79G]). Coexpression of wild-type or activated Cdc42 and N-WASP also inhibited Golgi-to-ER transport, but this was not the case in cells expressing Cdc42V12 and N-WASP(ΔWA), a mutant form of N-WASP that lacks Arp2/3 binding. Furthermore, Cdc42V12 recruited GFP-N-WASP to the Golgi complex. We therefore conclude that Cdc42 regulates Golgi-to-ER protein transport in an N-WASP–dependent manner

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The distributions of three novel peptides, 7B2, neuromedin B, and neuromedin U, in rat, mouse, and human pituitaries, rat hypothalamus, and 30 human pituitary tumors were investigated with immunocytochemistry. Immunoreactivity for 7B2 was present in rat, mouse, and human gonadotropes, in intermediate lobe cells and posterior lobe nerve fibers in rats and mice, in rat hypothalamus (particularly in the median eminence), and in eight human pituitary gonadotropinomas. In gonadectomized rats, larger, more numerous LH beta- and 7B2-immunoreactive gonadotropes were seen than in controls. Extractable 7B2-like immunoreactivity was elevated but not significantly so in gonadectomized rat pituitaries [males: castrated, 37.4 +/- 4.3 (mean +/- SE); controls, 26.9 +/- 4.3; females: ovariectomized, 27.2 +/- 2.7; controls, 19.1 +/- 2.2 pmol/gland]. Neuromedin B immunoreactivity was found in normal rat and mouse thyrotropes and weakly in "thyroidectomy" cells in hypothyroid rats, in which extractable pituitary neuromedin B was significantly depleted (thyroidectomized, 87.0 +/- 14.0; methimazole-treated, 82.0 +/- 11.4; control, 230.7 +/- 25.6 fmol/gland). Hyperthyroid rat pituitaries showed increased TSH beta and neuromedin B immunoreactivities and neuromedin B content (TRH-treated, 385.2 +/- 30.2; T4-treated, 352.6 +/- 20.2; control, 230.7 +/- 25.6 fmol/gland). Neuromedin U immunoreactivity occurred in corticotropes of all species, in rat and mouse intermediate lobe, and throughout the rat hypothalamus, with immunoreactive cell bodies in the arcuate nucleus. Neuromedin U-immunoreactive cells were present in six of six human pituitary and five of six human extrapituitary corticotropinomas. In adrenalectomized rats, corticotropes were larger and more numerous than in controls, but extractable anterior pituitary neuromedin U-like immunoreactivity was not raised (adrenalectomized, 3.30 +/- 0.45; control, 3.32 +/- 0.27 pmol/gland). Our findings suggest that 7B2, neuromedin B, and neuromedin U may be involved in pituitary function.

Sperm binding activity has been detected in zona pellucida (ZP) glycoproteins and it is generally accepted that this activity resides in the carbohydrate moieties. In the present study we aim to identify some of the specific carbohydrate molecules involved in the bovine sperm-ZP interaction. We performed sperm binding competition assays, in vitro fecundation (IVF) in combination with different lectins, antibodies and neuraminidase digestion, and chemical and cytochemical analysis of the bovine ZP. Both MAA lectin recognising alpha-2,3-linked sialic acid and neuraminidase from Salmonella typhimurium with catalytic activity for alpha-2,3-linked sialic acid, demonstrated a high inhibitory effect on the sperm-ZP binding and oocyte penetration. These results suggest that bovine sperm-ZP binding is mediated by alpha-2,3-linked sialic acid. Experiments with trisaccharides (sialyllactose, 3'-sialyllactosamine and 6'-sialyllactosamine) and glycoproteins (fetuin and asialofetuin) corroborated this and suggest that at least the sequence Neu5Ac(alpha2-3)Gal(beta1-4)GlcNAc is involved in the sperm-ZP interaction. Moreover, these results indicate the presence of a sperm plasma membrane specific protein for the sialic acid. Chemical analysis revealed that bovine ZP glycoproteins contain mainly Neu5Ac (84.5%) and Neu5GC (15.5%). These two types of sialic acid residues are probably linked to Galbeta1,4GlcNAc and GalNAc by alpha-2,3- and alpha-2,6-linkages, respectively, as demonstrated by lectin cytochemical analysis. The use of a neuraminidase inhibitor resulted in an increased number of spermatozoa bound to the ZP and penetrating the oocyte. From this last result we hypothesize that a neuraminidase from cortical granules would probably participate in the block to polyspermy by removing sialic acid from the ZP.

Background: The zona pellucida (ZP), an extracellular matrix which surrounds mammalian oocytes, is formed by different glycoproteins. Several studies have revealed that carbohydrate residues present in glycoproteins of ZP play a key role in the sperm-egg recognition. However, the origin and the biochemical composition of ZP remain to be completely resolved.Methods: ZP glycoproteins from rat ovarian follicles were investigated at light and electron microscopic level by the application of lectins conjugated to peroxidase, digoxigenin, and colloidal gold in combination with enzyme and chemical treatment. A quantitative analysis was also performed.Results: ZP shows reactivity to WGA, DSA, LFA, AAA, RCA I, and MAA. SBA and PNA showed a variable reactivity ranging from negative to strongly positive. A uniform pattern of binding throughout ZP was observed with DSA, Con A, AAA, MAA, and LFA. However, labeling by RCA I and SBA was higher in the outer Z P while PNA and WGA showed a higher binding in the inner ZP. Lectin reactivity was detected in cortical granules, endoplasmic reticulum, Golgi apparatus, vesicles, and multivesicular bodies of oocytes.Conclusions: ZP contained the terminal disaccharides Galpl,4GlcNAc, Galpl,3GalNAc, and GalNAcpl,3Gal and the trisaccharides Neu5Aca2, 3Galpl,4GlcNAc, Neu5Ac-Galpl,3GalNAc, and Neu5Ac-GalNAcp1,3Gal sequences. The occurrence of Fucose residues (Y 1,6 linked to the inner core region of N-linked glycoproteins of Z P was demonstrated by the use of several fucose-specific lectins. Methylation-saponification treatment in combination with lectin cytochemistry reveals that Gal, GalNAc, and polyllactosamine residues of rat Z P glycoproteins contain sulphated groups. The reactivity observed in ooplasmic vesicles was similar to that of ZP, thus suggesting that the oocyte is the site of synthesis of ZP glycoproteins.