Guy Keryer

Cloning and characterization of a cDNA encoding an A‐kinase anchoring protein located in the centrosome, AKAP450

A combination of protein kinase A type II (RII) overlay screening, database searches and PCR was used to identify a centrosomal A‐kinase anchoring protein. A cDNA with an 11.7 kb open reading frame was characterized and found to correspond to 50 exons of genomic sequence on human chromosome 7q21‐22. This cDNA clone encoded a 3908 amino acid protein of 453 kDa, that was designated AKAP450 (DDBJ/EMBL/GenBank accession No. AJ131693). Sequence comparison demonstrated that the open reading frame contained a previously characterized cDNA encoding Yotiao, as well as the human homologue of AKAP120. Numerous coiled‐coil structures were predicted from AKAP450, and weak homology to pericentrin, giantin and other structural proteins was observed. A putative RII‐binding site was identified involving amino acid 2556 of AKAP450 by mutation analysis combined with RII overlay and an amphipatic helix was predicted in this region. Immunoprecipitation of RII from RIPA‐buffer extracts of HeLa cells demonstrated co‐precipitation of AKAP450. By immunofluorecent labeling with specific antibodies it was demonstrated that AKAP450 localized to centrosomes. Furthermore, AKAP450 was shown to co‐purify in centrosomal preparations. The observation of two mRNAs and several splice products suggests additional functions for the AKAP450 gene.

Huntingtin Is Required for Mitotic Spindle Orientation and Mammalian Neurogenesis

Huntingtin is the protein mutated in Huntingtons disease, a devastating neurodegenerative disorder. We demonstrate here that huntingtin is essential to control mitosis. Huntingtin is localized at spindle poles during mitosis. RNAi-mediated silencing of huntingtin in cells disrupts spindle orientation by mislocalizing the p150(Glued) subunit of dynactin, dynein, and the large nuclear mitotic apparatus NuMA protein. This leads to increased apoptosis following mitosis of adherent cells in vitro. In vivo inactivation of huntingtin by RNAi or by ablation of the Hdh gene affects spindle orientation and cell fate of cortical progenitors of the ventricular zone in mouse embryos. This function is conserved in Drosophila, the specific disruption of Drosophila huntingtin in neuroblast precursors leading to spindle misorientation. Moreover, Drosophila huntingtin restores spindle misorientation in mammalian cells. These findings reveal an unexpected role for huntingtin in dividing cells, with potential important implications in health and disease.

The centrosome-nucleus complex and microtubule organization in the Drosophila oocyte

Molecular motors transport the axis-determining mRNAs oskar, bicoid and gurken along microtubules (MTs) in the Drosophila oocyte. However, it remains unclear how the underlying MT network is organized and how this transport takes place. We have identified a centriole-containing centrosome close to the oocyte nucleus. Remarkably, the centrosomal components, γ-tubulin and Drosophila pericentrin-like protein also strongly accumulate at the periphery of this nucleus. MT polymerization after cold-induced disassembly in wild type and in gurken mutants suggests that in the oocyte the centrosome-nucleus complex is an active center of MT polymerization. We further report that the MT network comprises two perpendicular MT subsets that undergo dynamic rearrangements during oogenesis. This MT reorganization parallels the successive steps in localization of gurken and oskar mRNAs. We propose that in addition to a highly polarized microtubule scaffold specified by the cortex oocyte, the repositioning of the nucleus and its tightly associated centrosome could control MT reorganization and, hence, oocyte polarization.

Identification of a high affinity binding protein for the regulatory subunit RII beta of cAMP-dependent protein kinase in Golgi enriched membranes of human lymphoblasts.

Immunocytochemical evidence of an association between the regulatory subunit RII of the cAMP‐dependent protein kinase (cAMP‐PK) and the Golgi apparatus in several cell types has been reported. In order to identify endogenous Golgi proteins binding RII, a fraction enriched in Golgi vesicles was isolated from human lymphoblasts. Only the RII beta isoform was detected in the Golgi‐rich fraction, although RII alpha has also been found to be present in these cells. A 85 kDa RII‐binding protein was identified in Golgi vesicles using a [32P]RII overlay of Western blots. The existence of an endogenous RII beta‐p85 complex in isolated Golgi vesicles was demonstrated by two independent means: (i) co‐immunoprecipitation of both proteins under non‐denaturing conditions with an antibody against RII beta and (ii) co‐purification of RII beta‐p85 complexes on a cAMP‐analogue affinity column. p85 was phosphorylated by both endogenous and purified catalytic subunits of cAMP‐pKII. Extraction experiments and protease protection experiments indicated that p85 is an integral membrane protein although it partitioned atypically during Triton X‐114 phase separation. We propose that p85 anchors RII beta to the Golgi apparatus of human lymphoblasts and thereby defines the Golgi substrate targets most accessible to phosphorylation by C subunit. This mechanism may be relevant to the regulation of processes involving the Golgi apparatus itself, such as membrane traffic and secretion, but also relevant to nearby nuclear events dependent on C subunit.

Involvement of phosphoinositide 3-kinase and Rac in membrane ruffling induced by IL-2 in T cells

IL‐2 is known to play a critical role in regulating T lymphocyte proliferation. We show here that IL‐2 also provokes an instantaneous and sustained membrane ruffling in cloned human or murine T cells as well as in lectin‐activated peripheral blood lymphocytes. In the IL‐2‐induced lamellipodia, tubulin is depolymerized whereas actin is strongly polymerized, forming caps. IL‐2‐induced membrane ruffling is protein kinase C (PKC) independent, as judged by the absence of effects of bisindolylmaleimide, an efficient inhibitor of all PKC isoforms. The formation of lamellipodia by IL‐2 is blocked by wortmannin and LY294002, two inhibitors of phosphoinositide 3‐kinase (PI3‐kinase). Moreover, expression in murine T cells of an inactive form of PI3‐kinase inhibits IL‐2‐induced membrane ruffling, whereas expression of a constitutively active p110 increases the basal membrane ruffling. Rac is also involved in IL‐2‐induced membrane ruffling since an inactive form of Rac (N17rac) blocks the IL‐2‐induced lamellipodia, whereas the constitutive form of Rac (Val12rac) can also lead to membrane ruffling. In the signaling cascade, Rac is downstream of PI3‐kinase since constitutive membrane ruffling in Val12rac cells is insensitive to wortmannin. Thus, through a signaling cascade involving PI3‐kinase and Rac, IL‐2 can induce profound alterations of the T cell cytoskeleton, a phenomenon which might be of importance for T cell physiology.

Centrioles resist forces applied on centrosomes during G2/M transition

Background information. Centrosome movements at the onset of mitosis result from a balance between the pulling and pushing forces mediated by microtubules. The structural stability of the centrosome core structure, the centriole pair, is correlated with a heavy polyglutamylation of centriole tubulin.

pARIS-htt: an optimised expression platform to study huntingtin reveals functional domains required for vesicular trafficking.

BackgroundHuntingtin (htt) is a multi-domain protein of 350 kDa that is mutated in Huntingtons disease (HD) but whose function is yet to be fully understood. This absence of information is due in part to the difficulty of manipulating large DNA fragments by using conventional molecular cloning techniques. Consequently, few studies have addressed the cellular function(s) of full-length htt and its dysfunction(s) associated with the disease.ResultsWe describe a flexible synthetic vector encoding full-length htt called pARIS-htt (A daptable, R NAi I nsensitive & S ynthetic). It includes synthetic cDNA coding for full-length human htt modified so that: 1) it is improved for codon usage, 2) it is insensitive to four different siRNAs allowing gene replacement studies, 3) it contains unique restriction sites (URSs) dispersed throughout the entire sequence without modifying the translated amino acid sequence, 4) it contains multiple cloning sites at the N and C-ter ends and 5) it is Gateway compatible. These modifications facilitate mutagenesis, tagging and cloning into diverse expression plasmids. Htt regulates dynein/dynactin-dependent trafficking of vesicles, such as brain-derived neurotrophic factor (BDNF)-containing vesicles, and of organelles, including reforming and maintenance of the Golgi near the cell centre. We used tests of these trafficking functions to validate various pARIS-htt constructs. We demonstrated, after silencing of endogenous htt, that full-length htt expressed from pARIS-htt rescues Golgi apparatus reformation following reversible microtubule disruption. A mutant form of htt that contains a 100Q expansion and a htt form devoid of either HAP1 or dynein interaction domains are both unable to rescue loss of endogenous htt. These mutants have also an impaired capacity to promote BDNF vesicular trafficking in neuronal cells.ConclusionWe report the validation of a synthetic gene encoding full-length htt protein that will facilitate analyses of its structure/function. This may help provide relevant information about the cellular dysfunctions operating during the disease. As proof of principle, we show that either polyQ expansion or deletion of key interacting domains within full-length htt protein impairs its function in transport indicating that HD mutation induces defects on intrinsic properties of the protein and further demonstrating the importance of studying htt in its full-length context.

Purification of the surface membrane-cytoskeleton complex (Cortex) of Paramecium and identification of several of its protein constituents.

In ciliates, the major morphogenetic events take place in the cortex, a complex of membranes and closely associated filamentous networks. To analyze the problems of assembly and morphogenesis at the molecular level in Paramecium, we have developed a method of purification of cortex fragments which retain their in situ organization and display a highly reproducible electrophoretic profile. The method used either a four-step sucrose gradient yielding a cortex + oral apparatus fraction or a six-step gradient which allowed the cortex fragments to be freed from the oral apparatuses (which were recovered separately). By comparative electrophoresis and immunological probing of these and other cell fractions or purified organelles, we could identify several of the major polypeptides resolved by SDS PAGE as components of specific cortical or oral structures. The purification method was successfully applied to morphological mutants, and the first case of a mutational modification of a cortical polypeptide was observed.

Endosome to Golgi transport is regulated by protein kinase A type IIα

Studies of RIIα-deficient B lymphoid cells and stable transfectants expressing the type IIα regulatory subunit (RIIα) of cAMP-dependent protein kinase (PKA), which is targeted to the Golgi-centrosomal area, reveal that the presence of a Golgi-associated pool of PKA type IIα mediates a change in intracellular transport of the plant toxin ricin. The transport of ricin from endosomes to the Golgi apparatus, measured as sulfation of a modified ricin (ricin sulf-1), increased in RIIα-expressing cells when PKA was activated. However, not only endosome-to-Golgi transport, but also retrograde ricin transport to the endoplasmic reticulum (ER), measured as sulfation and N-glycosylation of another modified ricin (ricin sulf-2), seemed to be increased in cells expressing RIIα in the presence of a cAMP analog, 8-(4-chlorophenylthio)-cAMP. Thus, PKA type IIα seems to be involved in both endosome-to-Golgi and Golgi-to-ER transport. Because ricin, after being retrogradely transported to the ER, is translocated to the cytosol, where it inhibits protein synthesis, we also investigated the influence of RIIα expression on ricin toxicity. In agreement with the other data obtained, 8-(4-chlorophenylthio)-cAMP and RIIα were found to sensitize cells to ricin, indicating an increased transport of ricin to the cytosol. In conclusion, our results demonstrate that transport of ricin from endosomes to the Golgi apparatus and further to the ER is regulated by PKA type IIα isozyme.

Role of cyclic AMP-dependent protein kinases in human villous cytotrophoblast differentiation

Summary Human trophoblast cells offer a unique in vitro model to study aspects of the dynamic processes occuring during cell fusion and syncytium formation. In the human placenta, mononuclear cytotrophoblasts aggregate and fuse to form a multinucleated syncytiotrophoblast. In vitro , the presence of 8-bromo-cAMP promotes syncytiotrophoblast formation, while an inhibitor of the cAMP dependent protein kinase (PKA) catalytic subunit, H-89, impairs cell fusion. This led us to review the pattern of expression and the subcellular localization of PKA subunits during syncytium formation as well as the role of specific A-kinase anchoring proteins in cytotrophoblast differentiation. Cytotrophoblasts expressed the RIα and RIIα regulatory subunits and Cα and Cβ catalytic subunits. RIα was down-regulated during syncytium formation. No change in RIIα mRNA or protein expression was observed, but there was a drastic subcellular redistribution. RIIα, located in the centrosomal Golgi area of cytotrophoblasts, was scattered throughout the cytoplasm in syncytiotrophoblast. Interestingly, an accumulation of RIIα was observed underneath the apical membrane of syncytiotrophoblast in vitro and in situ . As shown by overlay assay, RIIα appeared to bind to two major binding proteins, one of them being ezrin, an actin-binding protein. This suggests a key role of PKA type IIα in the secretory and transfer functions of the syncytiotrophoblast, but also in membrane reorganization associated with cell fusion and microvilli formation.