The Golgi body is responsible for the modification and sorting of proteins and lipids in the secretory pathway. The Golgi must coordinate with other endomembrane compartments in order to target cargo to the correct destination. While our understanding of Golgi function is vast, we can extend our knowledge base by examining the functions of Golgi-associated proteins in developing animals. Lava lamp (Lva) is a Golgi-associated protein and a Drosophila golgin. Previously, Lva was shown to facilitate efficient membrane secretion required for cleavage furrow formation in early embryos. By acting as an adaptor molecule between Golgi and microtubule motility factors, Lva is thought to position Golgi bodies for targeted secretion during cellularization, the Drosophila cleavage stage of development. Here, I further characterize the role of Lva during animal development. I demonstrate that Lva is required for animal viability, and gamete production in females but not males. While Lva is expressed in many tissues, adult fat body cells are the most sensitive to decreased Lva activity, resulting in the disorganization of endomembrane compartments. Furthermore, this disruption in adult fat body cells correlates with a defect in neuroendocrine signaling, altering the activity of juvenile hormone. I propose that Lva activity in adult fat body cells is important for recognizing and/or processing juvenile hormone in order to support Drosophila oogenesis. Lva’s role in cellularization, which is a specialized form of cytokinesis in early embryos, provided insights into the combined processes of actomyosin-based contraction and membrane secretion. While some proteins have been implicated in cellularization, there are thought to be many more that have yet to be identified. In an effort to isolate additional genes involved in animal cell cytokinesis, we screened a unique collection of temperature sensitive (ts) mutations on the X-chromosome of Drosophila melanogaster. At the restrictive temperature, we identified five mutants that displayed a cellularization phenotype. For one of the mutants, fs(1)ts242, we narrowed the mutation to a region on the X chromosome consisting of 17 possible gene candidates. Identification of the gene should provide further elucidation of the mechanisms controlling actomyosin-based contraction and membrane secretion. / text

Abstract Skeletal muscle cells (myofibers) are huge multinucleated cells responsible for muscle contraction and hence for the everyday movements of the joints. The structure of these voluminous cells differs greatly from that of the mononucleated cells – the characteristic features of the myofibers include dozens of peripherally located nuclei, tightly packed contractile apparatus and a sophisticatedly organized endomembrane system. The basic physiology involving myofibers is quite well known, but scarce data exist on the membrane biology of the myofibers. The purpose of this study was to examine the localization of mRNA and the site of protein synthesis in the myofibers. The characterization of the membrane dynamics in muscle cells was also performed. In this study we utilized a primary cell culture model obtained from the rat flexor digitorum brevis (FDB) muscle. Also frozen sections from the rat extensor digitorum longus muscle were used. The precursor cells of the myofibers – myoblasts and myotubes – were also utilized in some experiments. Furthermore, methods of immunohistochemistry and molecular biology were applied extensively in this study. We found that in FDB myofibers the mRNA lies just under the plasma membrane. Protein synthesis seemed to be concentrated in the vicinity of nuclei locating beneath the plasma membrane but also in interfibrillar dot-like structures. Protein products moved hundreds of micrometers away from the nuclei of origin. Moreover, there were no barriers for protein movement into the core regions of the myofibers. Movement of proteins was found to be rapid in the cytosol and in the endomembrane system, too. Interestingly, when examining exocytic trafficking we observed that ER-to-Golgi trafficking significantly differed from that of mononucleated cells. Finally, myofibers were found to be able to generate lipid bodies under stress conditions. The dynamics of lipid bodies seemed to deviate from the dynamics found in other cells types. Nowadays not much muscle research with primary myofibers is done worldwide, and therefore dilemmas involving myofibers such as insulin resistance and myotoxicity of statins are mostly unresolved. The knowledge gained from this study may be used in the future to solve clinical problems related to the cell biology of the myofibers. / Tiivistelmä Luurankolihassolut eli myofiiberit ovat jättimäisiä monitumaisia soluja, jotka vastaavat lihassupistuksen aikaansaamisesta ja siten mahdollistavat jokapäiväisen liikkumisemme. Näiden suurten solujen rakenne poikkeaa selkeästi yksitumaisten solujen rakenteesta: myofiiberien tunnusomaisia piirteitä ovat kymmenet solun reunoille sijoittuneet tumat, tiiviisti pakkautunut supistumiskoneisto ja monimutkaisesti järjestynyt solukalvostojärjestelmä. Vaikka myofiiberien perusfysiologia tunnetaankin hyvin, niin tiedetään itse myofiiberien kalvostobiologiasta sangen vähän. Kokonaisuutena tämän tutkimuksen tarkoituksena oli tarkastella mRNA:n ja proteiinisynteesin sijaintia myofiibereissä. Lisäksi selvitimme lihassolujen kalvostodynamiikkaa. Tässä tutkimuksessa käytimme rotan flexor digitorum brevis (FDB) -lihaksesta saatua primääristä soluviljelymallia. Lisäksi hyödynsimme rotan extensor digitorum longus -lihaksesta hankittuja jääleikkeitä. Joissakin kokeissa käytimme myös myofiiberien esiastesoluja (myoblasteja ja myotuubeja). Immunohistokemian ja molekyylibiologian menetelmiä sovellettiin tutkimuksessa laajasti. Havaitsimme, että FDB –myofiibereissä mRNA sijaitsee aivan solukalvon alla. Proteiinisynteesi vaikutti olevan keskittynyt solukalvon alla sijaitsevien tumien ympärille, mutta myös solusisäisiin pistemäisiin rakenteisiin. Proteiinituotteet ylsivät satojen mikrometrien päähän alkuperäisestä tumastaan. Lisäksi proteiineille ei ilmennyt leviämisestettä myofiiberin sisäosiin. Leviämisen havaittiin olevan nopeaa sekä solulimassa että solulimakalvostoissa. Tutkiessamme solun eritystoimintaa huomasimme, että kuljetus ER:stä Golgin laitteeseen eroaa huomattavasti yksitumaisten solujen vastaavasta kuljetuksesta. Lopuksi havaitsimme myofiiberien pystyvän muodostamaan rasvapisaroita rasitusolosuhteissa. Rasvapisaroiden käyttäytyminen näytti myös poikkeavan siitä, mitä muissa soluissa on havaittu. Nykyään lihastutkimusta primäärisoluilla ei juuri tehdä maailmalla, minkä vuoksi myofiibereihin liittyvät lääketieteelliset pulmat kuten insuliiniresistenssi ja statiinien lihashaitat ovat suurelta osin ratkaisematta. Tästä tutkimuksesta saatuja tuloksia voitaneen jatkossa käyttää myofiiberien solubiologiaan liittyvien kliinisten ongelmien selvittämiseen.