Keratin 7 Is a Constituent of the Keratin Network in Mouse Pancreatic Islets and Is Upregulated in Experimental Diabetes

Keratin (K) 7 is an intermediate filament protein expressed in ducts and glands of simple epithelial organs and in urothelial tissues. In the pancreas, K7 is expressed in exocrine ducts, and apico-laterally in acinar cells. Here, we report K7 expression with K8 and K18 in the endocrine islets of Langerhans in mice. K7 filament formation in islet and MIN6 ?-cells is dependent on the presence and levels of K18. K18-knockout (K18?/?) mice have undetectable islet K7 and K8 proteins, while K7 and K18 are downregulated in K8?/? islets. K7, akin to F-actin, is concentrated at the apical vertex of ?-cells in wild-type mice and along the lateral membrane, in addition to forming a fine cytoplasmic network. In K8?/? ?-cells, apical K7 remains, but lateral keratin bundles are displaced and cytoplasmic filaments are scarce. Islet K7, rather than K8, is increased in K18 over-expressing mice and the K18-R90C mutation disrupts K7 filaments in mouse ?-cells and in MIN6 cells. Notably, islet K7 filament networks significantly increase and expand in the perinuclear regions when examined in the streptozotocin diabetes model. Hence, K7 represents a significant component of the murine islet keratin network and becomes markedly upregulated during experimental diabetes

Keratin 7 is a constituent of the keratin network in mouse pancreatic islets and is upregulated in experimental diabetes

Keratin (K) 7 is an intermediate filament protein expressed in ducts and glands of simple epithelial organs and in urothelial tissues. In the pancreas, K7 is expressed in exocrine ducts, and apico-laterally in acinar cells. Here, we report K7 expression with K8 and K18 in the endocrine islets of Langerhans in mice. K7 filament formation in islet and MIN6 β-cells is dependent on the presence and levels of K18. K18-knockout (K18‒/‒) mice have undetectable islet K7 and K8 proteins, while K7 and K18 are downregulated in K8‒/‒ islets. K7, akin to F-actin, is concentrated at the apical vertex of β-cells in wild-type mice and along the lateral membrane, in addition to forming a fine cytoplasmic network. In K8‒/‒ β-cells, apical K7 remains, but lateral keratin bundles are displaced and cytoplasmic filaments are scarce. Islet K7, rather than K8, is increased in K18 over-expressing mice and the K18-R90C mutation disrupts K7 filaments in mouse β-cells and in MIN6 cells. Notably, islet K7 filament networks significantly increase and expand in the perinuclear regions when examined in the streptozotocin diabetes model. Hence, K7 represents a significant component of the murine islet keratin network and becomes markedly upregulated during experimental diabetes.</p

TLNRD1 is a CCM complex component and regulates endothelial barrier integrity

We previously identified talin rod domain-containing protein 1 (TLNRD1) as a potent actin-bundling protein in vitro. Here, we report that TLNRD1 is primarily expressed in the vasculature in vivo and that its depletion leads to vascular abnormalities in vivo and loss of barrier integrity in cultured endothelial cells. We demonstrate that TLNRD1 is a component of the cerebral cavernous malformations (CCM) complex through its direct, high-affinity interaction with CCM2. Modeling and functional testing of TLNRD1 and CCM2 mutants reveal that their interaction is mediated by a hydrophobic C-terminal helix in CCM2 that attaches to a hydrophobic groove on the 4-helix domain of TLNRD1. Disruption of this binding interface leads to CCM2 and TLNRD1 accumulation in the nucleus and actin fibers. Notably, a CCM2 pathogenic mutation linked to vascular dementia in patients maps to the interface and disrupts the interaction. Our findings indicate that CCM2 controls TLNRD1 localization to the cytoplasm and inhibits its actin-bundling activity. Based on these results, we propose a new pathway by which the CCM complex modulates the actin cytoskeleton and vascular integrity by controlling TLNRD1 bundling activity

TLNRD1 is a CCM complex component and regulates endothelial barrier integrity

We previously identified talin rod domain-containing protein 1 (TLNRD1) as a potent actin-bundling protein in vitro. Here, we report that TLNRD1 is expressed in the vasculature in vivo. Its depletion leads to vascular abnormalities in vivo and modulation of endothelial cell monolayer integrity in vitro. We demonstrate that TLNRD1 is a component of the cerebral cavernous malformations (CCM) complex through its direct interaction with CCM2, which is mediated by a hydrophobic C-terminal helix in CCM2 that attaches to a hydrophobic groove on the four-helix domain of TLNRD1. Disruption of this binding interface leads to CCM2 and TLNRD1 accumulation in the nucleus and actin fibers. Our findings indicate that CCM2 controls TLNRD1 localization to the cytoplasm and inhibits its actin-bundling activity and that the CCM2-TLNRD1 interaction impacts endothelial actin stress fiber and focal adhesion formation. Based on these results, we propose a new pathway by which the CCM complex modulates the actin cytoskeleton and vascular integrity

Investigating the role of TLNRD1 in cerebral cavernous malformations and endothelial cells

Cerebral cavernous malformations (CCM) are cerebral lesions, which affect the vasculature of the central nervous system, and they are characterized by an enlarged and leaky endothelium. The disease has an estimated prevalence of 0.1-0,5 %, however, there are currently no effective drugs available against it, and common treatment methods rely on invasive brain surgery. The CCM disease is caused by loss-of-function mutations in the key proteins of the CCM complex, which regulates endothelial cell integrity. Recently, a novel cytoskeletal regulator, TLNRD1, has been shown to have high interaction potential with the CCM complex proteins and it has been suggested to function as a possible fourth CCM protein. Until now, the function of TLNRD1 in endothelial cells and its specific interactions with the CCM proteins have remained largely unexplored. The aim of this thesis was to elucidate the specific TLNRD1-CCM complex interactions and to confirm the presence of TLNRD1 in endothelial cells and to assess its role in regulating endothelial cell junction integrity. The interaction between TLNRD1 and the CCM complex was investigated by using a mitochondrial trapping system and siRNA-mediated silencing of key CCM proteins. Secondly, the presence and role of TLNRD1 in endothelial cells was explored using a primary endothelial cell line. Together, these experiments indicate a strong interaction between TLNRD1 and CCM2, with the 4-helix domain of TLNRD1 potentially functioning as the main interaction site. Additionally, the presence of endogenous TLNRD1 was confirmed in primary endothelial cells, and preliminary data suggest that TLNRD1 may be involved in maintaining cell junction integrity in endothelial cells

Yksi elokuva, kaksi käsikirjoitusta – alkuperäinen käsikirjoitus ja lopullinen leikkaus : case study: Jalasjärvi Nail Art

Opinnäytetyön tavoitteena oli tarkastella elokuvan kahta erillistä taiteellista lopputulosta – alkuperäistä käsikirjoitusta ja lopullista leikkausta sisällön ja tarinan kehittämisen näkökulmasta. Aihetta pohditaan suurimmaksi osaksi lopputyöelokuva Jalasjärvi Nail Artin kautta. Myös muita lähteitä, kuten ammattikirjallisuutta ja artikkeleita on käytetty opinnäytetyön tukena. Elokuva käydään läpi kohtauksittain, niin käsikirjoituksen kuin leikkauksenkin näkökulmasta. Tarkoitus on tarjota muille alan opiskelijoille työkaluja leikkaus- sekä käsikirjoitusprosessiin. Tutkimuksessa havaittiin, miten alkuperäinen käsikirjoitus muuttuu lopulliseen leikkaukseen ja minkä takia. Prosesseja tutkiessa huomattiin myös dramaturgisten oppien tärkeys ja paikkansa pitävyys. Opinnäytetyö olisi voinut olla havainnoiltaan tarkempi, jos aihe olis ollut tiedossa jo kirjoitus- tai kuvausvaiheessa. Aihe päätettiin vasta leikkauspöydän ongelmia puitaessa. Opinnäytetyössä on luottamuksellinen aineisto, johon lukijalla on mahdollisuus pyytää salasana. Lopputyöelokuva ei ole vielä julkinen opinnäytetyön julkaisupäivämäärään mennessä. Salasanan saa ottamalla yhteyttä Ada Pajariin [email protected] priority of this thesis was to examine two different art forms of a film – the original screenplay and the final cut from the aspect of developing story. The theme is reflected mostly by the graduation short film Jalasjärvi Nail Art. Other sources, such as professional literature and articles were also used in the thesis. The purpose of the thesis is to offer tools for editing and screenwriting processes for other students. Confidential material has been used in the making of this thesis. It has been deleted from this raport, since the film is not public when the thesis is being published