Arp2/3 activity is necessary for efficient formation of E-cadherin adhesive contacts

Classical cadherin adhesion molecules are fundamental determinants of cell-cell recognition that function in cooperation with the actin cytoskeleton. Productive cadherin-based cell recognition is characterized by a distinct morphological process of contact zone extension, where limited initial points of adhesion are progressively expanded into broad zones of contact. We recently demonstrated that E-cadherin ligation recruits the Arp2/3 actin nucleator complex to the plasma membrane in regions where cell contacts are undergoing protrusion and extension. This suggested that Arp2/3 might generate the protrusive forces necessary for cell surfaces to extend upon one another during contact assembly. We tested this hypothesis in mammalian cells by exogenously expressing the CA region of N-WASP. This fragment, which potently inhibits Arp2/3-mediated actin assembly in vitro, also effectively reduced actin assembly at cadherin adhesive contacts. Blocking Arp2/3 activity by this strategy profoundly reduced the ability of cells to extend cadherin adhesive contacts but did not affect cell adhesiveness. These findings demonstrate that Arp2/3 activity is necessary for cells to efficiently extend and assemble cadherin-based adhesive contacts.

Phosphatidylinositol 3-kinase C2 alpha is essential for ATP-dependent priming of neurosecretory granule exocytosis

Neurotransmitter release and hormonal secretion are highly regulated processes culminating in the calcium-dependent fusion of secretory vesicles with the plasma membrane. Here, we have identified a role for phosphatidylinositol 3-kinase C2 alpha (PI3K-C2 alpha) and its main catalytic product, PtdIns3P, in regulated exocytosis. In neuroendocrine cells, PI3K-C2 alpha is present on a subpopulation of mature secretory granules. Impairment of PI3K-C2 alpha function specifically inhibits the ATP-dependent priming phase of exocytosis. Overexpression of wild-type PI3K-C2 alpha enhanced secretion, whereas transfection of PC12 cells with a catalytically inactive PI3K-C2 alpha mutant or a 2xFYVE domain sequestering PtdIns3P abolished secretion. Based on these results, we propose that production of PtdIns3P by PI3K-C2 alpha is required for acquisition of fusion competence in neurosecretion.

The properties of CpG islands in the putative promoter regions of human immunoglobulin (Ig) genes

CpG island is a GC-rich motif occurred in gene promoter region, which can play important roles in gene silencing and imprinting. Here, we present a set of discriminant functions that can recognize the structural and compositional features of CpG islands in the putative promoter regions (PPRs) of human and mouse immunoglobulin (Ig) genes. We showed that the PPRs of both human and mouse Ig genes irrespective of gene chromosomal localization are apparently CpG island poor, with a low percentage of the CpG islands overlapped with the transcription start site (TSS). The human Ig genes that have CpG islands in the PPRs show a very narrow range of CpG densities. 47% of the Ig genes fall in the range of 3.5-4 CpGs/100 bp. In contrast, the non-Ig genes examined have a wide range of the density of CpG island, with 10.5% having the density of 8.1-15 CpGs/100 bp. Meantime, five patterns of the CpG distributions within the CpG islands have been classified: Pat A, B, C, D, and E. 21.6% and 10.8% of the Ig genes fall into the Pat B and Pat D groups, respectively, which were significantly higher than the non-Ig genes examined (8.2% and 3.8%). Moreover, the length of CpG islands is shorter in human Ig genes than in non-Ig genes but is much longer than in mouse orthologues. These findings provide a clear picture of non-neutral and nonrandom occurrence of the CpG islands in the PPRs of human and mouse Ig genes, which facilitate rational recommendations regarding their nomenclature. (C) 2005 Elsevier B.V. All rights reserved.

Lessons from tomographic studies of the mammalian Golgi

Basic structure studies of the biosynthetic machinery of the cell by electron microscopy (EM) have underpinned much of our fundamental knowledge in the areas of molecular cell biology and membrane traffic. Driven by our collective desire to understand how changes in the complex and dynamic structure of this enigmatic organelle relate to its pivotal roles in the cell, the comparatively high-resolution glimpses of the Golgi and other compartments of the secretory pathway offered to us through EM have helped to inspire the development and application of some of our most informative, complimentary (molecular, biochemical and genetic) approaches. Even so, no one has yet even come close to relating the basic molecular mechanisms of transport, through and from the Golgi, to its ultrastructure, to everybody's satisfaction. Over the past decade, EM tomography has afforded new insights into structure -function relationships of the Golgi and provoked a re-evaluation of older paradigms. By providing a set of tools for structurally dissecting cells at high-resolution in three-dimensions (3D), EM tomography has emerged as a method for studying molecular cell biology in situ. As we move rapidly toward the establishment of molecular atlases of organelles through advances in proteomics and genomics, tomographic studies of the Golgi offer the tantalizing possibility that one day, we will be able to map the spatio-temporal coordinates of Golgi-related proteins and lipids accurately in the context of 4D cellular space. (c) 2005 Elsevier B.V. All rights reserved.

Genomic organization of human arylamine N-acetyltransferase Type I reveals alternative promoters that generate different 5'-UTR splice variants with altered translational activities

In humans, a polymorphic gene encodes the drug-metabolizing enzyme NATI (arylamine N-acetyltransferase Type 1), which is widely expressed throughout the body. While the protein-coding region of NATI is contained within a single exon, examination of the human EST (expressed sequence tag) database at the NCBI revealed the presence of nine separate exons, eight of which were located in the 5'non-coding region of NATI. Differential splicing produced at least eight unique mRNA isoforms that could be grouped according to the location of the first exon, which suggested that NATI expression occurs from three alternative promoters. Using RT (reverse transcriptase)-PCR, we identified one major transcript in various epithelial cells derived from different tissues. In contrast, multiple transcripts were observed in blood-derived cell lines (CEM, THP-1 and Jurkat), with a novel variant, not identified in the EST database, found in CEM cells only. The major splice variant increased gene expression 9-11-fold in a luciferase reporter assay, while the other isoforrns were similar or slightly greater than the control. We examined the upstream region of the most active splice variant in a promoter-reporter assay, and isolated a 257 bp sequence that produced maximal promoter activity. This sequence lacked a TATA box, but contained a consensus Sp1 site and a CAAT box, as well as several other putative transcription-factor-binding sites. Cell-specific expression of the different NATI transcripts may contribute to the variation in NATI activity in vivo.

Mechanisms of acetohydroxyacid synthases

Acetohydroxyacid synthases are thiamin diphosphate- (ThDP-) dependent biosynthetic enzymes found in all autotrophic organisms. Over the past 4-5 years, their mechanisms have been clarified and illuminated by protein crystallography, engineered mutagenesis and detailed single-step kinetic analysis. Pairs of catalytic subunits form an intimate dimer containing two active sites, each of which lies across a dimer interface and involves both monomers. The ThDP adducts of pyruvate, acetaldehyde and the product acetohydroxyacids can be detected quantitatively after rapid quenching. Determination of the distribution of intermediates by NMR then makes it possible to calculate individual forward unimolecular rate constants. The enzyme is the target of several herbicides and structures of inhibitor-enzyme complexes explain the herbicide-enzyme interaction.

How an enzyme answers. multiple-choice questions

Acetohydroxyacid synthase (AHAS) is the first common enzyme in the pathway for the biosynthesis of branched-chain amino acids. Interest in the enzyme has escalated over the past 20 years since it was discovered that AHAS is the target of the sulfonylurea and imidazolinone herbicides. However, several questions regarding the reaction mechanism have remained unanswered, particularly the way in which AHAS I chooses' its second substrate. A new method for the detection of reaction intermediates enables calculation of the microscopic rate constants required to explain this phenomenon.

Suicide inhibition of acetohydroxyacid synthase by hydroxypyruvate

Acetohydroxyacid synthase (Ec 2.2.1.6) catalyses the thiamine diphosphate-dependent reaction between two molecules of pyruvate yielding 2-acetolactacte and CO2. The enzyme will also utilise hydroxypyruvate with a k(cat) value that is 12% of that observed with pyruvate. When hydroxypyruvate is the substrate, the enzyme undergoes progressive inactivation with kinetics that are characteristic of suicide inhibition. It is proposed that the dihydroxyethyl-thiamine diphosphate intermediate can expel a hydroxide ion forming an enol that rearranges to a bound acetyl group.

Mutations in the regulatory subunit of yeast acetohydroxyacid synthase affect its activation by MgATP

Isoleucine, leucine and valine are synthesized via a common pathway in which the first reaction is catalysed by AHAS (acetohydroxyacid synthase; EC 2.2.1.6). This heterotetrameric enzyme is composed of a larger subunit that contains the catalytic machinery and a smaller subunit that plays a regulatory role. The RSU (regulatory subunit) enhances the activity of the CSU (catalytic sub unit) and mediates end-product inhibition by one or more of the branched-chain amino acids, usually valine. Fungal AHAS differs front that in other organisms in that the inhibition by valine is reversed by MgATP. The fungal AHAS RSU also differs from that in other organisms in that it contains a sequence insert. We suggest that this insert may form the MgATP-binding site and we have tested this hypothesis by mutating ten highly conserved amino acid residues of the yeast AHAS RSU. The modified subunits were tested for their ability to activate the yeast AHAS CSU, to confer sensitivity to valine inhibition and to mediate reversal of the inhibition by MgATP. All but one of the mutations resulted in substantial changes in the properties of the RSU. Unexpectedly, four of them gave a protein that required mgATP in order for strong stimulation of the CSU and valine inhibition to be observed. A model to explain this result is proposed. Five of the mutations abolished MgATP activation and are suggested to constitute the binding site for this modulator.

Visualisation of macropinosome maturation by the recruitment of sorting nexins

We report that phosphoinositol-binding sorting nexin 5 ( SNX5) associates with newly formed macropinosomes induced by EGF stimulation. We used the recruitment of GFP-SNX5 to macropinosomes to track their maturation. Initially, GFP-SNX5 is sequestered to discrete subdomains of the macropinosome; these subdomains are subsequently incorporated into highly dynamic, often branched, tubular structures. Time-lapse videomicroscopy revealed the highly dynamic extension of SNX5-labelled tubules and their departure from the macropinosome body to follow predefined paths towards the perinuclear region of the cell, before fusing with early endosomal acceptor membranes. The extension and departure of these tubular structures occurs rapidly over 5-10 minutes and is dependent upon intact microtubules. As the tubular structures depart from the macropinosome there is a reduction in the surface area and an increase in tension of the limiting membrane of the macropinosome. In addition to the recruitment of SNX5 to the macropinosome, Rab5, SNX1 and EEA1 are also recruited by newly formed macropinosomes, followed by the accumulation of Rab7. SNX5 forms heterodimers with SNX1 and this interaction is required for endosome association of SNX5. We propose that the departure of SNX5-positive tubules represents a rapid mechanism of recycling components from macropinosomes thereby promoting their maturation into Rab7-positive structures. Collectively these findings provide a detailed real-time characterisation of the maturation process of the macropinocytic endosome.

GFAP analysis in aluminium chloride-treated rat brain regions

The neurotoxicity of aluminium chloride was assessed in maleWistar albino rats. Rats were treated with aluminium chloride dissolved in distilled water at a dose of 300 mg/kg body weight daily by oral gavage (1 ml) for 45 days. Controls were treated with distilled water only. Animals were sacrificed and different brain regions were dissected. GFAP analysis was carried out by Western blotting using mouse anti-GFAP monoclonal antibody (Pharmingen: 60311D) at 1:1000. Blots were developed with HRP-linked goat anti-mouse secondary antibody and quantified by densitometry.