In addition to classical adhesion structures like filopodia or focal adhesions, dendritic cells similar to macrophages and osteoclasts assemble highly dynamic F-actin structures called podosomes. They are involved in cellular processes such as extracellular matrix degradation, bone resorption by osteoclasts, and trans-cellular diapedesis of lymphocytes. Besides adhesion and migration, podosomes enable dendritic cells to degrade connective tissue by matrix metalloproteinases. SWAP-70 interacts with RhoGTPases and F-actin and regulates migration of dendritic cells. SWAP-70 deficient osteoclasts are impaired in F-actin-ring formation and bone resorption. In the present study, we demonstrate that SWAP-70 is not required for podosome formation and F-actin turnover in dendritic cells. Furthermore, we found that toll-like receptor 4 ligand induced podosome disassembly and podosome-mediated matrix degradation is not affected by SWAP-70 in dendritic cells. Thus, podosome formation and function in dendritic cells is independent of SWAP-70.

Abstract Pulmonary fibrosis, lung cancer and chronic obstructive pulmonary disease (COPD) are severe diseases and common death causes worldwide. Due to the lack of an effective therapy, the investigation of cell biological mechanisms behind these diseases is essential. An activation of stromal cells, including myofibroblasts, is a main feature found in the pathogenesis of lung diseases. Myofibroblasts express alpha-smooth muscle actin (α-SMA), have specific ultrastructure, produce extracellular matrix proteins and possess contractile capacity. Detailed structure and function of myofibroblasts and their roles in healthy and diseased lung are not yet wholly understood. The investigation of the myofibroblasts may further offer novel tools for the acquisition of proper diagnosis, prognosis and medical treatment. The study aimed to characterize the ultrastructural, functional and disease-specific features of stromal cells, particularly myofibroblasts, in interstitial and malignant lung diseases. The functional properties evaluated here were differentiation, invasive and contractile properties. The study material included in vitro stromal cells cultured from bronchoalveolar lavage (BAL) fluids. The appearance and location of myofibroblasts in different lung compartments of non-smokers and the COPD-patients were examined in vivo. The cells were investigated by light and electron microscopy. The α-SMA expression was analysed by gene or protein assays. The study demonstrated that stromal cells could be cultured from diagnostic BAL fluid samples and lung tissues. Cultured cells were a mixture of fibroblasts and myofibroblasts. A small proportion of cells exhibited progenitor-like features. Myofibroblasts revealed differential features in electron microscopy and invasive or contractile assays. When studying tissues from healthy and COPD lungs, myofibroblasts were located both in alveoli and airways. In alveoli myofibroblasts localized in widened alveolar tips which were newly described structures and locations of myofibroblasts in healthy and diseased lung. The amount of myofibroblasts in large airways, but not in peripheral lung, was increased in COPD. We concluded that myofibroblasts have several locations in normal and COPD lung, which suggests a function both in pulmonary regeneration and the pathogenesis of COPD. Smoking altered the phenotype of myofibroblasts regardless of its origin. / Tiivistelmä Keuhkofibroosi, keuhkosyöpä ja keuhkoahtaumatauti (COPD) ovat kansallisesti ja maailmanlaajuisesti yleisiä ja kuolemaan johtavia sairauksia. Taudinmääritys ja hoito ovat vaativia, eikä kaikille potilaille ole parantavaa hoitoa. Keuhkosairauksien kaikkia solubiologisia mekanismeja ei vielä tunneta, mikä on yksi syy lääkekehityksen ongelmiin. Interstitiaaleissa ja pahanlaatuisissa keuhkosairauksissa esiintyy paljon aktiivisia sidekudossoluja, kuten muuntuneita fibroblasteja eli myofibroblasteja. Ne tunnistetaan hienorakenteesta, jota voidaan tutkia elektronimikroskoopilla. Myofibroblastit ilmentävät myös solun sisäistä sileän lihaksen alfa-aktiinia (α-SMA), tuottavat sidekudoksen proteiineja ja kykenevät supistumaan. Myofibroblastien hienorakenteen ja toiminnan selvittäminen voi antaa lisätietoa keuhkosairauksien syntymekanismeista, jolloin diagnostiikkaa, ennustetta sekä hoitoja voidaan arvioida paremmin. Väitöskirjassa selvitettiin myofibroblastien hienorakennetta ja toimintaa eri keuhkosairauksissa. Tutkitut toiminnalliset ominaisuudet olivat erilaistumispotentiaali, invasiivisuus ja supistumiskyky. Sairauksien kliinistä käyttäytymistä ja potilaiden tupakointitottumuksia tarkasteltiin suhteessa solubiologiatason havaintoihin. Tutkimusmateriaali kerättiin taudinmäärityksen yhteydessä interstitiaalisia keuhkosairauksia, keuhkoahtaumatautia tai keuhkosyöpää sairastavilta potilailta. Tulosten mukaan bronkoalveolaarihuuhtelunesteestä (BAL) ja keuhkokudospaloista voidaan soluviljelymenetelmin kasvattaa ja ylläpitää solulinjoja. Viljellyt solut muodostivat sekasolupopulaatiota, joissa esiintyi pääosin fibroblasteja ja vaihteleva osuus myofibroblasteja. Pieni osa soluista ilmensi kantasoluille tyypillisiä piirteitä. Myofibroblastien tyyppipiirteet ja toiminnalliset ominaisuudet vaihtelivat taudeittain. Kudoksessa myofibroblasteja ilmentyi sekä keuhkorakkuloissa että ilmateissä. Keuhkorakkulatasolla myofibroblastit sijoittuivat irrallisten alveoliseinämien laajentuneisiin päihin, joita ei ole aiemmin tutkittu tieteellisessä kirjallisuudessa myofibroblastien yhteydessä. Keuhkoahtaumatauti ja tupakointi vähensivät näiden rakenteiden määrää perifeerisessä keuhkossa, kun taas suurissa ilmateissä keuhkoahtaumatauti lisäsi myofibroblasteja. Päättelimme, että myofibroblastit edistävät keuhkoahtaumataudin syntyä isoissa ilmateissä, mutta saattavat osallistua keuhkojen korjaukseen keuhkorakkuloissa ja pienissä ilmateissä.

In addition to classical adhesion structures like filopodia or focal adhesions, dendritic cells similar to macrophages and osteoclasts assemble highly dynamic F-actin structures called podosomes. They are involved in cellular processes such as extracellular matrix degradation, bone resorption by osteoclasts, and trans-cellular diapedesis of lymphocytes. Besides adhesion and migration, podosomes enable dendritic cells to degrade connective tissue by matrix metalloproteinases. SWAP-70 interacts with RhoGTPases and F-actin and regulates migration of dendritic cells. SWAP-70 deficient osteoclasts are impaired in F-actin-ring formation and bone resorption. In the present study, we demonstrate that SWAP-70 is not required for podosome formation and F-actin turnover in dendritic cells. Furthermore, we found that toll-like receptor 4 ligand induced podosome disassembly and podosome-mediated matrix degradation is not affected by SWAP-70 in dendritic cells. Thus, podosome formation and function in dendritic cells is independent of SWAP-70.

The importance of insulin delivery and action is best characterized in Type 2 Diabetes, a disease that is becoming a pandemic both nationally and globally. Obesity is a principal risk factor for Type 2 Diabetes, and adipocyte function abnormalities due to adipose hypertrophy and hyperplasia, have been linked to obesity. Numerous reports suggest that the intracellular and systemic consequences of adipocyte function abnormalities include adipocyte insulin resistance, enhanced production of free fatty acids, and production of inflammatory mediators. A hallmark of adipocyte insulin sensitivity is the stimulation of glucose transporter isoform 4 (GLUT4) trafficking events to promote glucose uptake. In the Type 2 diabetic and insulin resistant states the mechanism behind insulin-stimulated GLUT4 trafficking is compromised. Therefore, understanding the role of factors involved in glucose-uptake in adipose tissue is of great importance. Studies from our laboratory suggest an important role for the unconventional myosin, Myo1c, in promoting insulin-mediated glucose uptake in cultured adipocytes. Our observations suggest that depletion of Myo1c in cultured adipocytes results in a significant reduction in the ability of adipocytes to take up glucose following insulin treatment, suggesting Myo1c is required for insulin-mediated glucose uptake. A plausible mechanism by which Myo1c promotes glucose uptake in adipocytes has been suggested by further work from our laboratory in which expression of fluorescently-tagged Myo1c in cultured adipocytes induces significant membrane ruffling at the cell periphery, insulin-independent GLUT4 translocation to the cell periphery, and accumulation of GLUT4 in membrane ruffling regions. Taken together Myo1c seems to facilitate glucose uptake through remodeling of cortical actin. In the first part of this thesis I, in collaboration with others, uncovered a possible mechanism through which Myo1c regulates adipocyte membrane ruffling. Here we identified a novel protein complex in cultured adipocytes, comprising Myo1c and the mTOR binding partner, Rictor. Interestingly our studies in cultured adipocytes suggest that the Rictor-Myo1c complex is biochemically distinct from the Rictor-mTOR complex of mTORC2. Functionally, only depletion of Rictor but not Myo1c results in decreased Akt phosphorylation at serine 473, but depletion of either Rictor or Myo1c results in compromised cortical actin dynamic events. Furthermore we observed that whereas the overexpression of Myo1c in cultured adipocytes causes remarkable membrane ruffling, Rictor depletion in cells overexpressing Myo1c significantly reduces these ruffling events. Taken together our findings suggest that Myo1c, in conjunction with Rictor, modulates cortical actin remodeling events in cultured adipocytes. These findings have implications for GLUT4 trafficking as GLUT4 has been previously observed to accumulate in Myo1c-induced membrane ruffles prior to fusion with the plasma membrane. During our studies of adipocyte function we noticed that current siRNA electroporation methods present numerous limitations. To silence genes more effectively we employed a lentivirus-mediated shRNA delivery system, and to standardize this technology in cultured adipocytes we targeted Myo1c and MAP4K4. Using this technology we were able to achieve clear advantages over siRNA oligonucleotide electroporation techniques in stability and permanence of gene silencing. Furthermore we showed that the use of lentiviral vectors in cultured adipocytes did not affect insulin signaling or insulin-mediated glucose uptake events. Despite our inability to use lentiviral vectors to achieve gene silencing in mice we were able to achieve adipose tissue-specific gene silencing effects in mice following manipulation of the lentiviral conditional silencing vector, and then crossing resulting founders with aP2-Cre mice. Interestingly however, only founders from the MAP4K4 conditional shRNA vector, but not founders from the Myo1c conditional shRNA vector, showed gene knockdown, possibly due to position-effect variegation. Taken together, findings from these studies are important because they present an alternative means of achieving gene silencing in cultured adipocytes, with numerous advantages not offered by siRNA oligonucleotide electroporation methods. Furthermore, the in vivo, adipose tissue-specific RNAi studies offer a quick, inexpensive, and less technically challenging means of achieving adipose tissue-specific gene ablations relative to traditional gene knockout approaches.

Crucial to a cell’s ability to migrate is the organization of its plasma membrane and associated proteins in a polarized manner to interact with and respond to its surrounding environment. Cells interact with the extracellular matrix (ECM) through specialized contact sites, including podosomes and invadopodia. Tumor cells use F-actin-rich invadopodia to degrade ECM and invade tissues; related structures, termed podosomes, are sites of dynamic ECM interaction and degradation. We show here that supervillin (SV), a peripheral membrane protein that binds F-actin and myosin II,reorganizes the actin cytoskeleton and potentiates invadopodial function. Overexpressed SV increases the number of F-actin punctae, which are highly dynamic and co-localize with markers of podosomes and invadopodia. Endogenous SV localizes to the cores of Src-generated podosomes in COS-7 cells and with invadopodia in MDA-MB-231 cells. EGFP-SV overexpression increases the average amount of matrix degradation; RNAi-mediated downregulation of SV decreases degradation. Cortactin, an essential component of both podosomes and invadopodia, binds SV sequences in vitro and contributes to the formation of EGFP-SV induced punctae. Additionally, SV affects cortactin localization,which could provide a mechanism for SV action at invadopodia. The formation of cholesterol-rich membrane rafts is one method of plasma membrane organization. A property of membrane rafts is resistance to extraction with cold Triton X-100 and subsequent flotation to low buoyant densities. The actin cytoskeleton has been implicated in many signaling events localized to membrane rafts, but interactions between actin and raft components are not well characterized. Our laboratory isolated a heavy detergent resistant membrane fraction from neutrophils, called DRM-H, that contains at least 23 plasma membrane proteins. DRM-H is rich in cytoskeletal proteins, including fodrin, actin, myosin II, as well as supervillin. DRM-H also contains proteins implicated in both raft organization and membrane-mediated signaling. DRM-H complexes exhibit a higher buoyant density than do most DRMs (referred to as DRM-L), which are deficient in cytoskeletal proteins. By using similar purification methods, I find that COS-7 cells also contain cytoskeleton-associated DRMs. In addition, when transfected into COS-7 cells, estrogen receptor (ER)α associates with DRM-H, while ERβ is seen in both DRM-L and DRM-H populations, suggesting a role for DRM-H in nongenomic estrogen signaling. Thus, the cytoskeleton-associated DRM-H not limited to hematopoietic cells and could constitute a scaffold for membrane raftcytoskeleton signaling events in many cells. Taken together, our results show that SV is a component of cytoskeleton-associated membrane rafts as well as podosomes and invadopodia, and that SV plays a role in invadopodial function. SV, with its connections to both membrane rafts and the cytoskeleton, is well situated to mediate cortactin localization, activation state, and/or dynamics of matrix metalloproteases at the ventral cell surface for proper matrix degradation through invadopodia. The molecular dissection of invadopodia formation and function may contribute to a greater understanding of in vivo invasion, and thus, tumor cell metastasis.