Janus kinase 2 activation mechanisms revealed by analysis of suppressing mutations

Background: Janus kinases (JAKs; JAK1 to JAK3 and tyrosine kinase 2) mediate cytokine signals in the regulation of hematopoiesis and immunity. JAK2 clinical mutations cause myeloproliferative neoplasms and leukemia, and the mutations strongly concentrate in the regulatory pseudokinase domain Janus kinase homology (JH) 2. Current clinical JAK inhibitors target the tyrosine kinase domain and lack mutation and pathway selectivity. Objective: We sought to characterize mechanisms and differences for pathogenic and cytokine-induced JAK2 activation to enable design of novel selective JAK inhibitors. Methods: We performed a systematic analysis of JAK2 activation requirements using structure-guided mutagenesis, cell-signaling assays, microscopy, and biochemical analysis. Results: Distinct structural requirements were identified for activation of different pathogenic mutations. Specifically, the predominant JAK2 mutation, V617F, is the most sensitive to structural perturbations in multiple JH2 elements (C helix [aC], Src homology 2-JH2 linker, and ATP binding site). In contrast, activation of K539L is resistant to most perturbations. Normal cytokine signaling shows distinct differences in activation requirements: JH2 ATP binding site mutations have only a minor effect on signaling, whereasJH2aCmutations reduce homomeric (JAK2-JAK2) erythropoietin signaling and almost completely abrogate heteromeric (JAK2-JAK1) IFN-gamma signaling, potentially by disrupting a dimerization interface on JH2. Conclusions: These results suggest that therapeutic approaches targeting the JH2 ATP binding site and aC could be effective in inhibiting most pathogenic mutations. JH2 ATP site targeting has the potential for reduced side effects by retaining erythropoietin and IFN-gamma functions. Simultaneously, however, we identified the JH2 aC interface as a potential target for pathway-selective JAK inhibitors in patients with diseases with unmutated JAK2, thus providing new insights into the development of novel pharmacologic interventions.Peer reviewe

Light-Induced Nanoscale Deformation in Azobenzene Thin Film Triggers Rapid Intracellular Ca2+ Increase via Mechanosensitive Cation Channels

Epithelial cells are in continuous dynamic biochemical and physical interaction with their extracellular environment. Ultimately, this interplay guides fundamental physiological processes. In these interactions, cells generate fast local and global transients of Ca2+ ions, which act as key intracellular messengers. However, the mechanical triggers initiating these responses have remained unclear. Light-responsive materials offer intriguing possibilities to dynamically modify the physical niche of the cells. Here, a light-sensitive azobenzene-based glassy material that can be micropatterned with visible light to undergo spatiotemporally controlled deformations is used. Real-time monitoring of consequential rapid intracellular Ca2+ signals reveals that the mechanosensitive cation channel Piezo1 has a major role in generating the Ca2+ transients after nanoscale mechanical deformation of the cell culture substrate. Furthermore, the studies indicate that Piezo1 preferably responds to shear deformation at the cell-material interphase rather than to absolute topographical change of the substrate. Finally, the experimentally verified computational model suggests that Na+ entering alongside Ca2+ through the mechanosensitive cation channels modulates the duration of Ca2+ transients, influencing differently the directly stimulated cells and their neighbors. This highlights the complexity of mechanical signaling in multicellular systems. These results give mechanistic understanding on how cells respond to rapid nanoscale material dynamics and deformations.Peer reviewe

Characterization of a stretching and compression device and its application on epithelial cells

Whether originated from the environment, surrounding cells or from the cell itself, all cells in a human being are subjected to mechanical signals. These signals, along with biochemical and electrical signals, control all cellular functions. Cells sense mechanical signals through a process called mechanotransduction. This allows cells to detect mechanical forces such as compression, stretching and shear stress, as well as the topography and toughness of the substrate. Mechanotransduction can affect cells directly at the protein level or through gene expression, and it affects, for example, differentiation, proliferation, viability and migration of cells. This Master of Science thesis focuses on a silicone based device designed to apply static or cyclic compression or stretching on cells. The development of this device has been ongo-ing for years, and as the latest advance a new version was designed. The new version is a step toward productization as it streamlines the manufacture of the device. The aim of this thesis was to characterize this new version of the device. The work can be divided into three parts. First, several commercial polydimethylsiloxane (PDMS) films were compared in order to choose the one that best suits the requirements of the device. This aimed to improve the efficiency of manufacture as well. Then, the stretching performance and repeatability of the device were characterized. Finally, the device was used in cell culture to apply compression to epithelial cells. Three commercial PDMS films in two thicknesses were compared for their autofluores-cence, optical resolution, biocompatibility and performance in the device. Differences between samples were small, but SILPURAN® (Wacker Chemie AG) in the thickness of 200 µm was chosen. In addition to showing good results in the tested parameters, the film is packed in a user-friendly way and showed no problems in adsorbing surface molecules. Also several modifications to the current device were tested. The actual characterization was carried out with a device with a slightly expanded vacuum chamber and a stabilator ring that decreased z-displacement. The maximum stretching was 8.6 ± 0.6 % and the undesired z-displacement less than 100 µm. Variation from device to device was 0.6 %-units and repeata-bility within a single device 0.2 %-units. Therefore, the variation originates from manufacture flaws, not the performance of the device itself Cell experiments were done with Madin-Darby Canine Kidney (MDCK) epithelial cells. One cell line expressed a genetically labeled occludin protein, and thus allowed the inspection of cell boarders in live cells. The other MDCK cell line expressed jRGECO1a, a live calcium indi-cator, and was used to study calcium signaling. After a six day culture period on a statically stretched device, strain was released thus creating a 15 % decrease in cell culture area. Cells were imaged before and after compression. The results show that epithelial cells became tightly packed due to compression. Average cross-sectional area of cells decreased 40 %, thus indicating active rearrangement of the cytoskeleton. Interestingly, calcium signaling de-creased after compression. This was probably a consequence of the tight packing. When cells had smaller space, they had less possibilities to change shape and migrate, which was seen as a decrease in calcium activity. All in all, the device was reliable and usable in cell culture conditions. However, further de-velopment is required to improve maximum stretching and linearity of stretching, and to make the setup more compact. Additionally, despite the advances in the manufacture of the device, production remains inefficient and calls for improvements

Characterization of a stretching and compression device and its application on epithelial cells

Whether originated from the environment, surrounding cells or from the cell itself, all cells in a human being are subjected to mechanical signals. These signals, along with biochemical and electrical signals, control all cellular functions. Cells sense mechanical signals through a process called mechanotransduction. This allows cells to detect mechanical forces such as compression, stretching and shear stress, as well as the topography and toughness of the substrate. Mechanotransduction can affect cells directly at the protein level or through gene expression, and it affects, for example, differentiation, proliferation, viability and migration of cells. This Master of Science thesis focuses on a silicone based device designed to apply static or cyclic compression or stretching on cells. The development of this device has been ongo-ing for years, and as the latest advance a new version was designed. The new version is a step toward productization as it streamlines the manufacture of the device. The aim of this thesis was to characterize this new version of the device. The work can be divided into three parts. First, several commercial polydimethylsiloxane (PDMS) films were compared in order to choose the one that best suits the requirements of the device. This aimed to improve the efficiency of manufacture as well. Then, the stretching performance and repeatability of the device were characterized. Finally, the device was used in cell culture to apply compression to epithelial cells. Three commercial PDMS films in two thicknesses were compared for their autofluores-cence, optical resolution, biocompatibility and performance in the device. Differences between samples were small, but SILPURAN® (Wacker Chemie AG) in the thickness of 200 µm was chosen. In addition to showing good results in the tested parameters, the film is packed in a user-friendly way and showed no problems in adsorbing surface molecules. Also several modifications to the current device were tested. The actual characterization was carried out with a device with a slightly expanded vacuum chamber and a stabilator ring that decreased z-displacement. The maximum stretching was 8.6 ± 0.6 % and the undesired z-displacement less than 100 µm. Variation from device to device was 0.6 %-units and repeata-bility within a single device 0.2 %-units. Therefore, the variation originates from manufacture flaws, not the performance of the device itself Cell experiments were done with Madin-Darby Canine Kidney (MDCK) epithelial cells. One cell line expressed a genetically labeled occludin protein, and thus allowed the inspection of cell boarders in live cells. The other MDCK cell line expressed jRGECO1a, a live calcium indi-cator, and was used to study calcium signaling. After a six day culture period on a statically stretched device, strain was released thus creating a 15 % decrease in cell culture area. Cells were imaged before and after compression. The results show that epithelial cells became tightly packed due to compression. Average cross-sectional area of cells decreased 40 %, thus indicating active rearrangement of the cytoskeleton. Interestingly, calcium signaling de-creased after compression. This was probably a consequence of the tight packing. When cells had smaller space, they had less possibilities to change shape and migrate, which was seen as a decrease in calcium activity. All in all, the device was reliable and usable in cell culture conditions. However, further de-velopment is required to improve maximum stretching and linearity of stretching, and to make the setup more compact. Additionally, despite the advances in the manufacture of the device, production remains inefficient and calls for improvements

Sandwich-erilaistusmenetelmän optimointi kahdelle ihmisen iPS-solulinjalle ja erilaistettujen sydänlihassolujen karakterisointi

Pluripotentteja kantasoluja voidaan erilaistaa sydänlihassoluiksi useilla eri menetelmillä. Erilaistettujen sydänlihassolujen avulla voidaan tutkia sydänsairauksia ja kehittää tautimalleja lääketestauksiin. Tulevaisuudessa potilaan omista soluista erilaistettuja sydänlihassoluja voidaan mahdollisesti käyttää kudosvaurioiden korjaamiseen. Tutkimuksen tavoitteena oli ottaa käyttöön sandwich-erilaistusmenetelmä opinnäytetyöpaikalla. Tarkoitus oli optimoida menetelmä kahdelle solulinjalle ja karakterisoida erilaistettuja sydänlihassoluja. Sandwich-erilaistusmenetelmällä kantasolut ohjataan erilaistumaan sydänlihassoluiksi mattomaisena kerroksena kasvutekijöiden avulla. Erilaistaminen tapahtuu matriksikerrosten välissä. Optimoitaville uudelleenohjelmoiduille kantasolulinjoille määritettiin menetelmän kannalta parhaiten toimiva solupitoisuus ja passage. Erilaistuneita soluja karakterisoitiin tutkimalla proteiinien ilmentymistä immunosytokemiallisten värjäysten avulla ja geenien ilmentymistä kvantitatiivisen reaaliaikaisen polymeraasiketjureaktion avulla. Toisella tutkituista linjoista saatiin erilaistettua yksisolukerroksena sykkiviä alueita, mutta toisella erilaistumista ei havaittu kertaakaan. Menetelmän toistettavuus oli heikko ja se oli hyvin herkkä passagen ja solupitoisuuden muutoksille. Jotta menetelmä voitaisiin ottaa käyttöön, sen optimoimista täytyy vielä jatkaa. Tulevaisuudessa menetelmää voitaisiin tutkia useammilla solulinjoilla, sillä tämän tutkimuksen otos oli hyvin pieni.Pluripotent stem cells can be differentiated into cardiomyocytes by several different methods. Differentiated cardiomyocytes can be used for studying cardiac diseases and for creating cell models for drug testing. In the future, cardiomyocytes differentiated from patient-derived cells may also be used in tissue repair. The objective of this study was to introduce the sandwich differentiation method in the Cardiology Research group. The purpose was to optimise the method for two cell lines and to characterise the differentiated cells. This method uses growth factors to direct stem cells to differentiate into a monolayer of cardiomyocytes. The differentiation is carried out between matrix layers. The two induced pluripotent stem cell lines were studied in order to find the most efficient cell density and passage. The differentiated cells were characterised by study-ing their protein expression using immunocytochemical staining and gene expression using quantitative real-time polymerase chain reaction. Areas of beating monolayer were achieved with one cell line, but with the other cell line no differentiation was observed. The repeatability of the method was weak and it was highly sensitive to alterations in passage and cell density. The method requires further optimisation in order for it to be taken into use. In the future, the method could be fur-ther studied with multiple cell lines, as in this study the sampling was very scarce

Pneumatic equiaxial compression device for mechanical manipulation of epithelial cell packing and physiology

It is well established that mechanical cues, e.g., tensile- compressive- or shear forces, are important co-regulators of cell and tissue physiology. To understand the mechanistic effects these cues have on cells, technologies allowing precise mechanical manipulation of the studied cells are required. As the significance of cell density i.e., packing on cellular behavior is beginning to unravel, we sought to design an equiaxial cell compression device based on our previously published cell stretching system. We focused on improving the suitability for microscopy and the user-friendliness of the system. By introducing a hinge structure to the substrate stretch generating vacuum chamber, we managed to decrease the z-displacement of the cell culture substrate, thus reducing the focal plane drift. The vacuum battery, the mini-incubator, as well as the custom-made vacuum pressure controller make the experimental setup more flexible and portable. Furthermore, we improved the efficiency and repeatability of manufacture of the device by designing a mold that can be used to cast the body of the device. We also compared several different silicone membranes, and chose SILPURAN® due to its best microscopy imaging properties. Here, we show that the device can produce a maximum 8.5% radial pre-strain which leads to a 15% equiaxial areal compression as the pre-strain is released. When tested with epithelial cells, upon compression, we saw a decrease in cell cross-sectional area and an increase in cell layer height. Additionally, before compression the cells had two distinct cell populations with different cross-sectional areas that merged into a more uniform population due to compression. In addition to these morphological changes, we detected an alteration in the nucleo-cytoplasmic distribution of YAP1, suggesting that the cellular packing is enough to induce mechanical signaling in the epithelium.publishedVersionPeer reviewe