RIC8A roll hiire arengus ja funktsioon rakk-maatriks adhesioonis ning aktiini tsütoskeleti organiseerimises

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Hulkrakse organismi normaalse elutegevuse tagamiseks on oluline, et rakud saaksid vahetada otseste või kaudsete suhtlusteede kaudu olulist infot. Transmembraansed G-valk-seoselised retseptorid ja nendega interakteeruvad G valgud (guaniin-nukleotiidi siduvad valgud) on üks levinumaid väliskeskkonnast saadava info kanaleid, mis osalevad mitmete arenguliste, füsioloogiliste ja käitumuslike protsesside reguleerimisel. G-valk-seoselised retseptorid on sihtmärgiks paljudele signaalmolekulidele nagu näiteks hormoonid, neurotransmitterid ja kasvufaktorid aktiveerides seeläbi G valgud, mis omakorda käivitavad rakusisese signaalikaskaadi. Sellisel viisil kutsutakse rakkudes esile vastus, milleks võib olla näiteks migratsioon, jagunemine, või aktsioonipotentsiaali tekkimine. RIC8A on nende signaalikaskaadide asendamatu komponent reguleerides G valkude aktiivsust (nukleotiidivahetusfaktori roll) ning tagades rakus G valkude piisava koguse ja membraanse asetuse (chaperon’i roll). Kuna G-valk-seoselised retseptorid on sihtmärgiks rohkem kui poolele tänapäeva ravimitest, võib ka RIC8A uurimine omada meditsiinilist perspektiivi. RIC8A funktsiooni kohta on imetaja organismis vähe teada ning selle asjaolu parandamiseks analüüsiti antud doktoritöös valgu rolli hiire organismis, täpsemalt embrüogeneesis ja närvisüsteemis. Uurimusest tehti kaks peamist järeldust: RIC8A puudus närvisüsteemis viib tugeva neuro-motoorse häire tekkeni ning RIC8A võib olla vajalik rakkude ja ekstratsellulaarse maatriksi vahelise kontakti saavutamiseks. Viimase hüpoteesi kinnitamiseks uurisime rakk-maatriks interaktsioone kasutades mudelina RIC8A puudulikke primaarseid hiire rakke.Communication between cells is the key for the proper functioning of multicellular organisms. Sensing the environment for information about changes in the surrounding conditions and signals allows the cell to respond and adjust its properties in concert with the rest of the organism. G-protein coupled receptors (GPCRs), and G proteins (guanine nucleotide-binding proteins) that the former are coupled to, are critical for transducing the extracellular information to the inside of the cell, being involved in a multitude of developmental, physiological and behavioural processes. GPCRs communicate signals acquired through the binding of hormones, neurotransmitters, ions and even light particles to name a few. The subsequent activation of G proteins triggers a complex and a highly regulated intracellular signalling cascade, disturbance of which may result in many types of human diseases, such as cardiovascular, neurological and metabolic, as well as cancer. RIC8, one of the indispensable components of this signalling pathway, interacts directly with G protein α subunits, regulating their activity and abundance in cells. The function of RIC8A in mice is still poorly characterised. The main goal of this thesis was to analyse the phenotypes of three different Ric8a knockout mice: a total knockout (Ric8a-/-) and the specific depletions of Ric8a from the neural precursor cells and from differentiated neurons. Two major conclusions were drawn from this analysis. First, the ablation of RIC8A in the nervous system results in a neuromuscular phenotype and second, the lack of RIC8A results in cell migration defects during gastrulation and neurogenesis, probably due to defective adhesion of cells to the extracellular matrix. To elaborate on the latter finding, the adhesive properties of RIC8A-deficient mouse primary cells were evaluated

The role of integrin β1 in the heterogeneity of human embryonic stem cells culture

The maintenance of the pluripotency of human embryonic stem (hES) cells requires special conditions for culturing. These conditions include specific growth factors containing media and extracellular matrix (ECM) or an appropriate substrate for adhesion. Interactions between the cells and ECM are mediated by integrins, which interact with the components of ECM in active conformation. This study focused on the characterisation of the role of integrin β1 in the adhesion, migration and differentiation of hES cells. Blocking integrin β1 abolished the adhesion of hES cells, decreasing their survival and pluripotency. This effect was in part rescued by the inhibition of RhoA signalling with Y-27632. The presence of Y-27632 increased the migration of hES cells and supported their differentiation into embryoid bodies. The differences in integrin β1 recycling in the phosphorylation of the myosin light chain and in the localisation of TSC2 were observed between the hES cells growing as a single-cell culture and in a colony. The hES cells at the centre and borders of the colony were found to have differences in their morphology, migration and signalling network activity. We concluded that the availability of integrin β1 was essential for the contraction, migration and differentiation ability of hES cells

Expression Pattern and Localization Dynamics of Guanine Nucleotide Exchange Factor RIC8 during Mouse Oogenesis.

Targeting of G proteins to the cell cortex and their activation is one of the triggers of both asymmetric and symmetric cell division. Resistance to inhibitors of cholinesterase 8 (RIC8), a guanine nucleotide exchange factor, activates a certain subgroup of G protein α-subunits in a receptor independent manner. RIC8 controls the asymmetric cell division in Caenorhabditis elegans and Drosophila melanogaster, and symmetric cell division in cultured mammalian cells, where it regulates the mitotic spindle orientation. Although intensely studied in mitosis, the function of RIC8 in mammalian meiosis has remained unknown. Here we demonstrate that the expression and subcellular localization of RIC8 changes profoundly during mouse oogenesis. Immunofluorescence studies revealed that RIC8 expression is dependent on oocyte growth and cell cycle phase. During oocyte growth, RIC8 is abundantly present in cytoplasm of oocytes at primordial, primary and secondary preantral follicle stages. Later, upon oocyte maturation RIC8 also populates the germinal vesicle, its localization becomes cell cycle dependent, and it associates with chromatin and the meiotic spindle. After fertilization, RIC8 protein converges to the pronuclei and is also detectable at high levels in the nucleolus precursor bodies of both maternal and paternal pronucleus. During first cleavage of zygote RIC8 localizes in the mitotic spindle and cell cortex of forming blastomeres. In addition, we demonstrate that RIC8 co-localizes with its interaction partners Gαi1/2:GDP and LGN in meiotic/mitotic spindle, cell cortex and polar bodies of maturing oocytes and zygotes. Downregulation of Ric8 by siRNA leads to interferred translocation of Gαi1/2 to cortical region of maturing oocytes and reduction of its levels. RIC8 is also expressed at high level in female reproductive organs e.g. oviduct. Therefore we suggest a regulatory function for RIC8 in mammalian gametogenesis and fertility

Neurospecific deletion of <i>Ric8a</i> in mice.

<p>(<b>A</b>) Schematic representation of neuron-specific deletion of the <i>Ric8a</i> gene. First four exons of floxed <i>Ric8a</i> are removed by Cre-recombinase which expression is under the control of <i>Synapsin I</i> promoter (SynCre). Numbered boxes represent <i>Ric8a</i> exons and arrows <i>loxP</i> sites. (<b>B</b>) PCR-based genotyping of mice using DNA from tail samples to detect <i>SynCre</i> transgene, floxed <i>Ric8a</i> allele and <i>LacZ</i> allele respectively. <i>Ric8a</i>^<i>CKO</i> genotype is emphasized with dotted line. (<b>C</b>) Representative PCR showing the deletion of floxed <i>Ric8a</i> in <i>Ric8a</i>^<i>CKO</i> mouse nervous system and no deletion in non-neural organs. (<b>D</b> and <b>E</b>) Comparison of Cre-recombinase (in <i>SynCre</i>^<i>+/-</i><i>R26R</i>) and <i>Ric8a</i> (in <i>Ric8a</i>^{<i>lacZ/+</i>}) expression in E12.5 embryos by X-gal staining. (<b>F</b>) Down regulation of <i>Ric8a</i> mRNA expression in <i>Ric8a</i>^<i>CKO</i> mice relative to littermate control in hippocampus (HIP), spinal cord (SC), cardiac muscle (CM) and liver (LIV). (<b>G</b>) Deficiency of RIC8A protein in <i>Ric8a</i>^<i>CKO</i> (CKO) mice compared to littermate control (LM) in hippocampus (HIP), spinal cord (SC), spinal ganglia (SG) and cardiac muscle (CM). GAPDH was used as a reference. Abbreviations: del, PCR fragment from the deleted allele Drg, dorsal root ganglia; F, floxed allele; Hb, hindbrain; Mb, midbrain; Nt, neural tube; SC, spinal cord; SG, spinal ganglia; Syt, sympathetic trunk; Vno, vomeronasal organ; wt, PCR fragment from the wild-type allele; V, trigeminal ganglion; X, vagus ganglion; ** <i>P</i> < 0,01. Error bars represent mean ± SEM scores. Scale bars: (D and E) 1 mm.</p

Ablation of RIC8A Function in Mouse Neurons Leads to a Severe Neuromuscular Phenotype and Postnatal Death

<div><p>Resistance to inhibitors of cholinesterase 8 (RIC8) is a guanine nucleotide exchange factor required for the intracellular regulation of G protein signalling. RIC8 activates different Gα subunits via non-canonical pathway, thereby amplifying and prolonging the G protein mediated signal. In order to circumvent the embryonic lethality associated with the absence of RIC8A and to study its role in the nervous system, we constructed <i>Ric8a</i> conditional knockout mice using Cre/loxP technology. Introduction of a synapsin I promoter driven Cre transgenic mouse strain (<i>SynCre</i>) into the floxed <i>Ric8a</i> (<i>Ric8a</i>^<i>F/F</i>) background ablated RIC8A function in most differentiated neuron populations. Mutant <i>SynCre</i>^<i>+/-</i><i>Ric8</i>^{<i>lacZ/F</i>} mice were born at expected Mendelian ratio, but they died in early postnatal age (P4-P6). The mutants exhibited major developmental defects, like growth retardation and muscular weakness, impaired coordination and balance, muscular spasms and abnormal heart beat. Histological analysis revealed that the deficiency of RIC8A in neurons caused skeletal muscle atrophy and heart muscle hypoplasia, in addition, the sinoatrial node was misplaced and its size reduced. However, we did not observe gross morphological changes in brains of <i>SynCre</i>^<i>+/-</i><i>Ric8a</i>^{<i>lacZ/F</i>} mutants. Our results demonstrate that in mice the activity of RIC8A in neurons is essential for survival and its deficiency causes a severe neuromuscular phenotype.</p> </div

Localization pattern of RIC8, Gα_i1/2, LGN and NuMA at early cleavage stage of mouse embryo.

<p>(<b>A-C</b>) One-cell (1-cell) embryo at metaphase (Met) of first mitosis. (<b>D-L</b>) Two-cell (2-cell) mouse embryos. Embryos were double-labeled with RIC8 antibody (green) and Gα_i1/2, LGN or NuMA antibodies respectively (red). DNA was stained with DAPI (blue). (<b>d-i</b>) Higher magnification of overlapping regions (yellow to orange) of RIC8 and Gα_i1/2 or LGN in cortex area of blastomere (indicated with white arrowhead). Abbreviations: ms, mitotic spindle; pb, polar body. Scale bar: (<b>A-L</b>) 20 μm, (<b>d-i</b>) 10 μm.</p

GEF RIC8 co-localization with LGN during mouse oocyte maturation.

<p>(<b>A-F</b>) Mouse oocytes at meiosis I and (<b>G-I</b>) at metaphase block of meiosis II were double-labeled with RIC8 antibody (green) and LGN (red). DNA was stained with DAPI (blue). Localization of meiotic spindle is denoted with white arrows and yellow to orange colour in this area indicates the overlapping regions of RIC8 and LGN. Dotted white line indicates the borders of oocyte. Abbreviations: Ana/Tel I, anaphase/telophase of meiosis I; Met I or Met II, metaphase of meiosis I or meiosis II respectively; ms, meiotic spindle; pb, polar body. Scale bar: 10 μm.</p

RIC8 in folliculogenesis and in the reproductive tract of adult mouse.

<p>RIC8 was visualized with RIC8 antibody (red), and cell nuclei were visualized with DAPI (blue). (<b>A-F</b>) Transversal cryosections of ovary with oocytes in different follicular stages starting from primordial follicle to Graafian follicle and (<b>G-I</b>) different regions of oviduct are shown. (<b>H</b>) Higher magnification of the region of ampulla and (<b>i</b>, indicated by white box) isthmus. Abbreviations: A, antrum; Amp, ampulla region of oviduct; Cb, basal layer of cilia; Cc, ciliated cell; Ci, cilia; Co, cumulus oophorus; Cr, corona radiata; Cx, cell cortex, Ec, epithelial cells; Fc, follicular cell; Gc, granulosa cells; Gf, Graafian follicle; GV, germinal vesicle; Ist, isthmus region of oviduct; Lp, lamina propria; Lu, lumen; Pc, peg cell; Pf, primary follicle; Pmf, primordial follicle; Po, primary oocyte; Sf, secondary follicle. Scale bars: 50 μm.</p

Distribution of RIC8, Gα_i1/2, LGN and NuMA in the fertilized oocyte at pronuclear stages.

<p>(<b>A-I</b>) Mouse fertilized oocyte at pronuclear stages were double-labeled with RIC8 antibody (green) and NuMA, LGN or Gα_i1/2 antibodies respectively (red). DNA was stained with DAPI (blue). (<b>a-i</b>) Higher magnification of female or male (indicated by sex symbols) pronucleus. Yellow arrowheads point to small RIC8 foci localized in the nucleoplasm. White arrowheads point to the meiotic spindle. Dotted white line indicates the borders of oocyte. Abbreviations: ms, meiotic spindle; pb, polar body; PN, pronuclear stage. Scale bar: 10 μm.</p