Abstract not available

A yeast two-hybrid assay suggested the possibility of an interaction between Cdc4p, a small EF-hand protein essential for cytokinesis, and Pik1p in S. pombe. This interaction was unexpected, as one function of Cdc4p is that of an essential light chain, bound to the first IQ-motif of type II myosins, whereas Pik1p is a phosphatidylinositol 4-kinase. The objective of this work was to analyze the effects of Pik1p lipid kinase activity on the cell cycle of S. pombe. Another goal of this study was to evaluate the functional significance of the interaction between Cdc4p and Pik1p. This was performed by generating two mutants of pik1: one that abolished lipid kinase activity (pik1-D709A) and one that abolished Pik1p Cdc4p-binding activity (pik1-R838A). Pik1p has a conserved IQ-motif in its C-terminal region. A mutation in this site (R838A), homologous to a residue which was mutated in myosin and abrogated the interaction with Cdc4p, prevented the interaction with Cdc4p in a yeast two-hybrid assay and ELISA. An increase in lipid kinase activity was observed in cell extracts upon ectopic expression of pik1-wt from an episome, which was abolished by a mutation in the lipid kinase domain of Pik1p (D709A), but not by the R838A mutation. However, little to no increase in lipid kinase activity was observed upon ectopic expression of pik1-wt and pik1-R838A in a strain carrying a conditionally lethal allele of cdc4 (cdc4-G107S). This mutation in Cdc4p was shown previously to prevent the interaction with Pik1p in yeast two-hybrid assays. Ectopic expression of pik1-wt suppressed cell proliferation, with disruption of actin cytoskeletal structures and contractile ring formation. These results were not observed with the ectopic expression of the pik1-R838A mutant or when pik1-wt was expressed in the cdc4-G107S strain. Ectopic expression of pik1-R838A resulted in cell shortening, likely through inhibition of growth, and many of the short cells showed an accumulation of the expressed Pik1p protein at the cell tips. Formation of the contractile ring appeared unaffected in cells with ectopic expression of the pik1-D709A mutant, but many of these cells had thick or more than one septum, characteristic of a septation defect. The ectopic expression phenotypes were dosage dependent since lower levels of expression greatly reduced the severity of the ectopic phenotypes. Pik1p lipid kinase activity is essential and, based on ectopic expression studies, is required for septation. There is a physical and functional interaction between Cdc4p and Pik1p which is not essential for cell viability, but suggests a role for Cdc4p in phosphoinositide metabolism.

A yeast two-hybrid assay suggested the possibility of an interaction between Cdc4p, a small EF-hand protein essential for cytokinesis, and Pik1p in S. pombe. This interaction was unexpected, as one function of Cdc4p is that of an essential light chain, bound to the first IQ-motif of type II myosins, whereas Pik1p is a phosphatidylinositol 4-kinase. The objective of this work was to analyze the effects of Pik1p lipid kinase activity on the cell cycle of S. pombe. Another goal of this study was to evaluate the functional significance of the interaction between Cdc4p and Pik1p. This was performed by generating two mutants of pik1: one that abolished lipid kinase activity (pik1-D709A) and one that abolished Pik1p Cdc4p-binding activity (pik1-R838A). Pik1p has a conserved IQ-motif in its C-terminal region. A mutation in this site (R838A), homologous to a residue which was mutated in myosin and abrogated the interaction with Cdc4p, prevented the interaction with Cdc4p in a yeast two-hybrid assay and ELISA. An increase in lipid kinase activity was observed in cell extracts upon ectopic expression of pik1-wt from an episome, which was abolished by a mutation in the lipid kinase domain of Pik1p (D709A), but not by the R838A mutation. However, little to no increase in lipid kinase activity was observed upon ectopic expression of pik1-wt and pik1-R838A in a strain carrying a conditionally lethal allele of cdc4 (cdc4-G107S). This mutation in Cdc4p was shown previously to prevent the interaction with Pik1p in yeast two-hybrid assays. Ectopic expression of pik1-wt suppressed cell proliferation, with disruption of actin cytoskeletal structures and contractile ring formation. These results were not observed with the ectopic expression of the pik1-R838A mutant or when pik1-wt was expressed in the cdc4-G107S strain. Ectopic expression of pik1-R838A resulted in cell shortening, likely through inhibition of growth, and many of the short cells showed an accumulation of the expressed Pik1p protein at the cell tips. Formation of the contractile ring appeared unaffected in cells with ectopic expression of the pik1-D709A mutant, but many of these cells had thick or more than one septum, characteristic of a septation defect. The ectopic expression phenotypes were dosage dependent since lower levels of expression greatly reduced the severity of the ectopic phenotypes. Pik1p lipid kinase activity is essential and, based on ectopic expression studies, is required for septation. There is a physical and functional interaction between Cdc4p and Pik1p which is not essential for cell viability, but suggests a role for Cdc4p in phosphoinositide metabolism.

α-actinin-4 is a non-muscle isoform of α-actinin that belongs to the spectrin superfamily. It comprises three functional regions: an N-terminal actin-binding region that consists of two calponin homology (CH) domains, a central region that consists of four copies of the spectrin-like repeat domain and a C-terminal calmodulin-like domain that is predicted to bind Ca²⁺. α-actinin-4 is organised as an antiparallel homodimer formed by the interaction of four spectrin-like repeats between the two monomers, giving a rod-like shape, with actin binding regions at both ends. α-actinin-4 is an abundant actin-bundling protein, which provides a direct link between actin filaments and integrins, and is believed to play an important role in stabilising cell shape and adhesion and regulating cell migration. It also acts as a tumor suppressor and influences the metastatic potential and invasiveness in human cancers. A cluster of three actin binding motifs have been identified in the CH domains (2X CH) from other members of the spectrin superfamily, utrophin and dystrophin. Two of them reside in the CH1 domain and the third resides in the first α-helix of the CH2 domain. In addition, a PIP2 binding site has been mapped on a region adjacent to actin-binding site-3. These observations imply the F-actin binding activity would be regulated by phosphoinositides. Five mutations of α-actinin-4, K122N, an alternative splice variant, K255E, T259I and S263P, have been reported to be involved in three human diseases, non-small lung cancer (NSCLC), small cell lung cancer (SCLC) and focal segmental glomerulosclerosis (FSGS). The mutation site within these mutants is located on the actin binding region. Therefore, the actin binding region is presumed to be associated with the progression of human disease. The aims of this thesis focused on the regulation of the F-actin binding activity of α-actinin-4 by phosphoinositides (PIP2 and PIP3), the calmodulin-like domain and Ca²⁺ , determination of the three-dimensional structure of the CH2 domain in solution and identification of the phosphoinositide binding site on the CH2 domain. In order to investigate the F-actin binding activity quantitatively, a novel in vitro F-actin binding assay (solid phase) was established to replace the semi-quantitative actin bundling assay. Using this novel solid phase F-actin binding assay, Ca²⁺ was shown to enhance the F-actin binding activity of α-actinin-4 in a concentration-dependent manner. The presence of 10 mM Ca²⁺ results in a two-fold increase in the F-actin binding activity. Both PIP2 and PIP3 inhibited the F-actin-binding activity of α-actinin-4 in a concentration-dependent manner with an approximate IC₅₀ of 75 and 45 μM, respectively. In order to characterise how phosphoinositides regulated the F-actin binding activity of α-actinin-4, the solution structure of α-actinin-4 CH2 domain was determined and the phosphoinositide binding residues within the CH2 domain were identified using NMR spectroscopy. The solution structure of α-actinin-4 CH2 domain contained six α-helices and was similar to that of other spectrin superfamily members. The strategy used in identification of the phosphoinositide binding site was an NMR-based 2D ¹H-¹⁵N HSQC ligand titration assay to replace the traditional semi-quantitative protein-lipid overlay assay. Using the NMR-based ligand titration assay, the recognition site for the inositol head group resides in residues Trp 172, Tyr 265 and His 266 and the binding region of acyl chains resides in the first α-helix structure which is one of the putative F-actin binding sites. In order to examine the interaction of phosphoinositides with this site, Y265A and H266E mutants of α-actinin-4 CH2 domain were generated using site-directed mutagenesis and verified the interaction with phosphoinositides and the inositol head group using an NMR-based ligand titration assay. These results confirmed the phosphoinositide binding site on the CH2 domain and residues, Tyr 265 and His 266, are critical for interacting with phosphoinositides. Wildtype and mutants (Y265A and H266E) of α-actinin-4 were expressed in mammalian cells as EGFP-fusion proteins. Wildtype α-actinin-4 was shown to be co-localised with focal adhesions and actin stress fibres. However, Y265A and H266E mutants of α-actinin-4 were co-localised with actin stress fibres but poorly co-localised with focal adhesions. Moreover, both Y265A and H266E mutants of α-actinin-4 were co-localised with actin in the cytoplasm rather than localised along the cell membrane after EGF stimulation for 30 minutes. These results suggested that PIP2 assists the co-localisation of α-actinin-4 with focal adhesions. Taken together, the results described in this thesis concluded that Ca²⁺ enhanced the F-actin binding activity of α-actinin-4 in vitro. However, phosphoinositides (PIP2 or PIP3) inhibited the F-actin binding activity in vitro. Moreover, the results described in this thesis provided a phosphoinositide binding site on α-actinin-4 CH2 domain. Binding to PIP2 is important to the localisation of α-actinin-4 in focal adhesions. / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Document formatted into pages; contains xv, 104 p.; also includes graphics (some col.). Includes bibliographical references (p. 93-104). Available online via OhioLINK's ETD Center

(ENGLISH) Phosphoinositides (PIs) are negatively charged glycerol-based phospholipids with inositol head (ring) which can be phosphorylated. Inositol ring phosphorylation yields in seven different PIs species which can be mono-, bis,- or tris-phosphorylated. Roles of cytoplasmic PIs have been extensively studied in for membrane and cytoskeletal dynamics, vesicular trafficking, ion channels and transporters and generating of second messengers. Nuclear PIs have been implicated in posttranscriptional processing of pre-mRNA, DNA transcription and chromatin remodelling. While cytoplasmic functions are very well described, the molecular mechanism of their nuclear functions are still poorly understood. In this study we focus on description of localization of nuclear PIs in particular functional nuclear compartments, which enable us to reveal PIs involvement in nuclear processes. We also focused on identification of nuclear PIs involved in the regulation of genes transcription and revealed detailed mechanism of PI(4,5)P2 a PHF8 interaction in the regulation of ribosomal genes transcription. By two independent approaches, we have described PIs localization to the nuclear membrane, nuclear speckles, small foci in the nucleoplasm, and the nucleolus. This spread nuclear localization suggests and confirms PI's...

Agonist-stimulated phosphoinositide (PPI) hydrolysis is a major signal transduction pathway in brain. These studies investigated neurotransmitter stimulated PPI hydrolysis in postmortem human brain. Preliminary studies using rat brain suggested that moderate postmortem delay has little effect on PPI hydrolysis and that human tissue might be reliably studied for differences in receptor-PLC coupling. Studies in human brain membranes (frontal cortex) indicated that the time course for GTPγS and carbachol/GTPγS-stimulated PPI hydrolysis was linear for at least 20 min. GTPγS-stimulated [3H] inositol phosphate (InsP) formation was enhanced by carbachol (232%) and 5-Hydroxytryptamine (5HT - 147%). SAX-HPLC seperation of [3H] inositol polyphosphates indicated that the major isomer of inositol trisphosphate (InsP3) was Ins(1,4,5)P3, the expected product of PtdIns(4,5)P2 hydrolysis. Ca2+ increased PPI hydrolysis progressively from 100 nM through 50 μM and synergistically enhanced carbachol/GTPγS stimulation. Comparisons of age-matched controls with Alzheimer's patients indicated that GTPγS, carbachol/GTPγS, and 5HT/GTPγS-stimulation of PPI hydrolysis is reduced approximately 50% in membranes prepared from Alzheimer's patients. Ca2+ stimulation of PPI hydrolysis was not different between controls and Alzheimer's patients suggesting that muscarinic cholinergic and serotonergic receptors are uncoupled from PLC in Alzheimer's disease. These studies indicate that there are changes in cholinergic and serotonergic signal transduction in Alzheimer's disease. Further, this method can be used to study signal transduction events in postmortem human brain.

La myopathie centronucléaire (CNM) est un groupe de maladies génétiques caractérisées au niveau histologique par des noyaux au centre des myofibres au lieu de la périphérie. Des mutations dans trois gènes (MTM1, DNM2 et BIN1) sont associées à cette pathologie. Récemment, l’implication d’un nouveau gène a été révélée dans une myopathie congénitale, le gène PYROXD1. Cependant, la base moléculaire responsable du déséquilibre à l'intérieur de la cellule reste incertaine et la relation entre le niveau histologique et les symptômes chez les patients n'est pas comprise. De plus, aucun traitement n'est disponible pour ces maladies. Au cours de ma thèse, j'ai centré mon travail sur l'utilisation du modèle de levure S. cerevisiae pour comprendre trois protéines associées au CNM : la myotubularine Mtm1, l'oxydoréductase Pyroxd1 et la dynamine Dnm2. Ces données révèlent qu’il est possible d’utiliser une simple cellule eucaryote afin d'élucider certains aspects moléculaires de ces protéines impliquées dans des maladies humaines. / Centronuclear myopathy (CNM) is a group of genetic disorders characterized at the histological level by nuclei at the center of the myofibers instead of the periphery. Mutations in three genes (MTM1, DNM2 and BIN1) are associated with this pathology. Recently the implication of a new gene has been revealed in a congenital myopathy, the PYROXD1 gene.However, the molecular basis responsible for the imbalance inside the cell remains unclear and the relation between the histological level and the symptoms in patients is not understood. Moreover, there is no treatment available for these diseases.During my thesis I have focused my work on using yeast S. cerevisiae model to understand three proteins associated to CNM: the myotubularin Mtm1, the oxidoreductase Pyroxd1 and the dynamin Dnm2. These data reveal that it is possible to use a single eukaryote cell to elucidate some molecular aspects of these proteins implicated in human disorders.

MTM1 et MTMR2 sont 2 phosphatases de phosphoinositides appartenant à la famille des myotubularines, conservée pendant l’évolution. Bien qu’étant très similaires, des mutations dans MTM1 entraînent la sévère myopathie XLCNM alors que les mutations dans MTMR2 entraînent la neuropathie CMT4B. On ne comprend pas encore les bases moléculaires de cette spécificité de tissu, et il n’existe aucun traitement spécifique pour ces maladies. J’ai tout d’abord caractérisé l’activité des 2 isoformes endogènes de MTMR2, nommés MTMR2-L et MTMR2-S. J’ai démontré que la différence fonctionnelle entre MTM1 et MTMR2 s’explique principalement par l’extension N-terminale de MTMR2, et que l’isoforme MTMR2-S dépourvu de cette extension entraîne les mêmes phénotypes que MTM1. Ensuite, grâce à l’injection d’AAV dans les souris Mtm1 KO, j’ai démontré que l’expression exogène des isoformes de MTMR2, et surtout de MTMR2-S, améliore grandement l’atrophie musculaire, la force musculaire et les marqueurs histologiques de ces souris myopathiques. Ces résultats révèlent une première base moléculaire expliquant les spécificités fonctionnelles de MTM1 et MTMR2, et montrent que MTMR2 est une cible thérapeutique potentielle pour la myopathie XLCNM. / MTM1 and MTMR2 are 2 phosphatases of phosphoinositides that belong to the myotubularin family conserved through evolution. Despite their high level of similarity, mutations in MTM1 lead to the severe XLCNM myopathy while mutations in MTMR2 lead to the CMT4B neuropathy. The molecular bases for the surprising tissue-specific functions of these ubiquitously expressed proteins was unclear. Moreover, there is no specific therapy for these diseases.I first characterized the activity of the two naturally occurring isoforms of MTMR2, that we named MTMR2-L (long) and MTMR2-S (short). I found that the functional differences between MTM1 and MTMR2 reside mostly in the N-terminal extension of MTMR2-L, and that the endogenous MTMR2-S isoform lacking this N-terminal extension behaves similarly as MTM1. Then, using the myopathic Mtm1 KO mouse and AAV-mediated expression, I showed that exogenous expression of MTMR2 isoforms, and specifically of MTMR2-S, strongly improved the muscle atrophy, muscle force and the histological hallmarks of the myopathic mice. These data reveal a first molecular basis for the functional specificities of MTM1 and MTMR2, and highlight MTMR2 as a therapeutic target for XLCNM myopathy.

Les septines sont une famille de protéines GTPases qui peuvent former des structures d'ordre supérieur, comme les filaments et les anneaux, et capables de se lier avec les membranes cellulaires par leur interaction avec les phosphoinositides (PIs) via un domaine polybasique en N-terminal de leur domaine de liaison au GTP. Nous avons montré par une analyse transcriptomique réalisée en utilisant les données GSE14323 que la septine 9 est significativement surexprimée dans la cirrhose induite par le virus de l'hepatite C (VHC). Nos résultats montrent, ainsi, que la septine 9 induit l’augmentation en taille des gouttelettes lipidiques (GLs) par un mécanisme dépendant le phosphatidylinositol-5-phosphate et des microtubules. Nous avons montré, également, que cette voie de régulation des GLs est exploité par le VHC. De plus, nous avons montré que la septine 9 est impliquée dans la régulation de la morphologie de l’appareil Golgi et la mise en place de la polarité cellulaire par son interaction avec les phosphoinositides via deux domaines polybasiques. Ces résultats apportent une nouvelle compréhension du mécanisme moléculaire de l’interaction des septines avec les phosphoinositides et montrent pour la première fois l’importance de cette interaction dans des fonctions cellulaires de la septine 9. / Septins are a GTPases proteins family that can form high order structures such as filaments and rings, and able to bind cell membranes by interacting with phosphoinositides via a polybasic domain located at the N-terminal of their GTP binding domain. Here, We show by the transcriptomic analysis performed using the GSE14323 dataset that septin 9 is significantly upregulated in hepatitis C virus induced cirrhosis. Our findings show that septin 9 induce the lipid droplet growth by a phosphatidylinositol-5-phosphate and microtubule-dependent mechanism hijacked by HCV. In addition, we have shown that the septin 9 is involved in Golgi apparatus morphology regulation and cell polarity installation by interacting with phosphoinositides via two polybasic domains. These results provide new understanding of the molecular mechanism of septins interaction with the phosphoinositides and show its importance in septin 9 cellular functions shown for the first time.