Optimization of mechanosensitive cross-talk between matrix stiffness and protein density : independent matrix properties regulate spreading dynamics of myocytes

Cells actively sense differences in topology, matrix elasticity and protein composition of the extracellular microenvironment and adapt their function and morphology. In this study, we focus on the cross-talk between matrix stiffness and protein coating density that regulates morphology and proliferation dynamics of single myocytes. For this, C2C12 myocytes were monitored on L-DOPA functionalized hydrogels of 22 different elasticity and fibronectin density compositions. Static images were recorded and statistically analyzed to determine morphological differences and to identify the optimized extracellular matrix (ECM). Using that information, selected ECMs were used to study the dynamics before and after cell proliferation by statistical comparison of distinct cell states. We observed a fibronectin-density-independent increase of the projected cell area until 12 kPa. Additionally, changes in fibronectin density led to an area that was optimum at about 2.6 μg/cm2, which was confirmed by independent F-actin analysis, revealing a maximum actin-filament-to-cell-area ratio of 7.5%. Proliferation evaluation showed an opposite correlation between cell spreading duration and speed to matrix elasticity and protein density, which did not affect cell-cycle duration. In summary, we identified an optimized ECM composition and found that independent matrix properties regulate distinct cell characteristics

Low-level endothelial TRAIL-receptor expression obstructs the CNS-delivery of angiopep-2 functionalised TRAIL-receptor agonists for the treatment of glioblastoma

Glioblastoma (GBM) is the most malignant and aggressive form of glioma and is associated with a poor survival rate. Latest generation Tumour Necrosis Factor Related Apoptosis-Inducing Ligand (TRAIL)-based therapeutics potently induce apoptosis in cancer cells, including GBM cells, by binding to death receptors. However, the blood–brain barrier (BBB) is a major obstacle for these biologics to enter the central nervous system (CNS). We therefore investigated if antibody-based fusion proteins that combine hexavalent TRAIL and angiopep-2 (ANG2) moieties can be developed, with ANG2 promoting receptor-mediated transcytosis (RMT) across the BBB. We demonstrate that these fusion proteins retain the potent apoptosis induction of hexavalent TRAIL-receptor agonists. Importantly, blood–brain barrier cells instead remained highly resistant to this fusion protein. Binding studies indicated that ANG2 is active in these constructs but that TRAIL-ANG2 fusion proteins bind preferentially to BBB endothelial cells via the TRAIL moiety. Consequently, transport studies indicated that TRAIL-ANG2 fusion proteins can, in principle, be shuttled across BBB endothelial cells, but that low TRAIL receptor expression on BBB endothelial cells interferes with efficient transport. Our work therefore demonstrates that TRAIL-ANG2 fusion proteins remain highly potent in inducing apoptosis, but that therapeutic avenues will require combinatorial strategies, such as TRAIL-R masking, to achieve effective CNS transport

Golgi screen identifies the RhoGEF Solo as a novel regulator of RhoB and endocytic transport

The control of intracellular membrane trafficking by Rho GTPases is central to cellular homeostasis. How specific guanine nucleotide exchange factors and GTPase‐activating proteins locally balance GTPase activation in this process is nevertheless largely unclear. By performing a microscopy‐based RNAi screen, we here identify the RhoGEF protein Solo as a functional counterplayer of DLC3, a RhoGAP protein with established roles in membrane trafficking. Biochemical, imaging and optogenetics assays further uncover Solo as a novel regulator of endosomal RhoB. Remarkably, we find that Solo and DLC3 control not only the activity, but also total protein levels of RhoB in an antagonistic manner. Together, the results of our study uncover the first functionally connected RhoGAP‐RhoGEF pair at endomembranes, placing Solo and DLC3 at the core of endocytic trafficking.Deutsche ForschungsgemeinschaftProjekt DEA

Protein kinase D promotes activity‐dependent AMPA receptor endocytosis in hippocampal neurons

α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) type glutamate receptors (AMPARs) mediate the majority of fast excitatory neurotransmission in the brain. The continuous trafficking of AMPARs into and out of synapses is a core feature of synaptic plasticity, which is considered as the cellular basis of learning and memory. The molecular mechanisms underlying the postsynaptic AMPAR trafficking, however, are still not fully understood. In this work, we demonstrate that the protein kinase D (PKD) family promotes basal and activity‐induced AMPAR endocytosis in primary hippocampal neurons. Pharmacological inhibition of PKD increased synaptic levels of GluA1‐containing AMPARs, slowed down their endocytic trafficking and increased neuronal network activity. By contrast, ectopic expression of constitutive active PKD decreased the synaptic level of AMPARs, while increasing their colocalization with early endosomes. Our results thus establish an important role for PKD in the regulation of postsynaptic AMPAR trafficking during synaptic plasticity.Deutsche ForschungsgemeinschaftHungarian Brain Research ProgramNRDIO, HungaryProjekt DEA

Precision 3D‐printed cell scaffolds mimicking native tissue composition and mechanics

Cellular dynamics are modeled by the 3D architecture and mechanics of the extracellular matrix (ECM) and vice versa. These bidirectional cell‐ECM interactions are the basis for all vital tissues, many of which have been investigated in 2D environments over the last decades. Experimental approaches to mimic in vivo cell niches in 3D with the highest biological conformity and resolution can enable new insights into these cell‐ECM interactions including proliferation, differentiation, migration, and invasion assays. Here, two‐photon stereolithography is adopted to print up to mm‐sized high‐precision 3D cell scaffolds at micrometer resolution with defined mechanical properties from protein‐based resins, such as bovine serum albumin or gelatin methacryloyl. By modifying the manufacturing process including two‐pass printing or post‐print crosslinking, high precision scaffolds with varying Young's moduli ranging from 7‐300 kPa are printed and quantified through atomic force microscopy. The impact of varying scaffold topographies on the dynamics of colonizing cells is observed using mouse myoblast cells and a 3D‐lung microtissue replica colonized with primary human lung fibroblast. This approach will allow for a systematic investigation of single‐cell and tissue dynamics in response to defined mechanical and bio‐molecular cues and is ultimately scalable to full organs

Development of molecular tools to explore a function of protein kinase D in Golgi complex organization

Die Proteinkinase D (PKD) Familie der Serin/Threonin Kinasen umfasst in Säugerzellen drei Mitglieder: PKD1, PKD2 und PKD3. PKDs werden downstream von G-Protein gekoppelten Rezeptoren (GPCR) als Effektoren von Diacylglycerol (DAG) aktiviert. Sie sind an der Regulation zahlreicher zellulärer Prozesse, wie z.B. Zellmotilität, Überleben der Zelle bei oxidativem Stress und Aktivität von Transkriptionsfaktoren beteiligt. Am besten beschrieben ist die Funktion am Golgi Komplex. Hier kontrolliert PKD am Trans-Golgi Netzwerk (TGN) die Abschnürung von Transportvesikeln. Der Aktivierungsstatus von PKD ist stets streng reguliert und die jeweilige Funktion eng mit der subzellulären Lokalisierung verknüpft. Genetisch codierte, Fluoreszenz-basierte Kinaseaktivitätsreporter sind wichtige molekulare Werkzeuge, um die räumliche und zeitliche Veränderung von Kinaseaktivität auf subzellulärer Ebene zu beobachten und somit die Antwort der Kinase auf Veränderungen der physiologischen Umgebung zu erfassen. Im Rahmen dieser Arbeit wurden zwei dieser Reporter, die spezifisch für die Messung von PKD-Aktivität am Golgi Komplex eingesetzt werden können, entwickelt. Bei beiden Reportern wird die PKD-Aktivität über den Phosphorylierungsstatus der PKD-Substratsequenz von Phosphatidylinositol-4 Kinase IIIβ (PI4KIIIβ) visualisiert. Der erste Reporter, G-PKDrep wurde für Aktivitätsmessungen in fixierten Zellen entwickelt, wobei der Phosphorylierungsgrad der Substratsequenz mit einem phosphospezifischen Antikörper quantifiziert wird. Mit G-PKDrep-live wurde G-PKDrep zu einem FRET-basierten Reporter weiterentwickelt, mit dem PKD-Aktivität am Golgi Komplex in lebenden Zellen über die Zeit gemessen werden kann. Mit Hilfe von G-PKDrep konnte unter Verwendung der Mikrotubuli-zerstörenden Substanz Nocodazol eine Funktion von PKD in der Organisation des Golgi Komplex aufgezeigt werden. Hierbei wurde gezeigt, dass die durch Mikrotubuli-Verlust hervorgerufene Golgi-Fragmentierung abhängig von PKD-Aktivität ist. Zur Aufklärung der zugrunde liegenden Signalwege dieses Prozesses wurde eine globale SILAC-basierte Phosphoproteom-Studie durchgeführt. Durch Expression einer kinasetoten PKD wurden dabei 124 PKD-abhängige Phosphorylierungsstellen identifiziert, die unter Nocodazolbedingungen signifikant herabreguliert waren. Dabei wurden viele PKD-Zielsequenzen in Golgi-residenten Proteinen, z.B. Mitglieder des IGF2-Rezeptor Netzwerks, identifiziert. Zudem zeigte sich, dass PKD nach Nocodazolstimulation den MAP-Kinase Signalweg aktiviert, dessen downstream Effektoren zu Beginn der Mitose die Golgi-Fragmentierung initiieren. Im Zellzyklus fragmentiert der Golgi Apparat beginnend in der G2-Phase in einem sequenziellen Prozess, wobei die anfängliche Spaltung des Golgi-Bandes in einzelne Golgi-Stapel essenziell für den Eintritt der Zelle in die Mitose ist. Im Rahmen dieser Arbeit konnte gezeigt werden, dass die Trennung der Golgi-Stapel PKD-abhängig ist und dass PKD somit eine Funktion beim Eintritt der Zelle in die Mitose hat. Weiterhin konnte gezeigt werden, dass PKD bei diesem Prozess upstream vom MAP-Kinase Signalweg agiert. Zusammengefasst konnte mit Hilfe der in dieser Arbeit entwickelten und etablierten molekularen Werkzeuge eine bisher unbekannte Funktion von PKD in der Organisation des Golgi Komplex beschrieben werden.The protein kinase D (PKD) family of serine/threonine kinases comprises three members in mammalian cells, PKD1, PKD2 and PKD3. PKDs get activated downstream of GPCR activation, dependent on PKCs and DAG. They are involved in various cellular functions such as cell motility, oxidative stress response or regulation of transcription factor activity. At the TGN, PKD controls the fission of transport carriers, which are destined for the plasma membrane. Importantly, the cellular function of PKD is strictly associated with its subcellular localization and PKD´s activation state is tightly regulated. Genetically encoded, fluorescent kinase activity reporters are important molecular tools to track spatiotemporal changes on kinase activity on a subcellular level. Within this work, two different PKD-specific kinase activity reporters targeted to Golgi complex were developed. Both constructs report changes in PKD activity via the phosphorylation state of the PKD-specific substrate sequence of PI4KIIIβ. The first reporter, G-PKDrep was designed to measure PKD activity in fixed cells. Here, reporter phosphorylation was visualized and quantified using a phosphospecific antibody. In contrast, the newly developed G-PKDrep-live is based on FRET and thus allows tracking PKD activity at the TGN in living cells over the time. Using G-PKDrep and the microtubule-disrupting agent nocodazole, a function of PKD in Golgi maintenance was discovered. Specifically, it was shown that nocodazole-induced Golgi fragmentation was dependent on PKD activity. To elucidate the underlying signaling pathways in this process a global quantitative SILAC-based phosphoproteomic analysis was performed. In this screen, 124 PKD-dependent phosphorylation sites, which were significantly downregulated in nocodazole-treated cells expressing a kinase-dead PKD mutant, were detected. Among these PKD-dependent phosphorylation sites predominantly Golgi-resident proteins such as members of the IGF2 receptor network were present. Furthermore, it could be shown that members of the MAP kinase pathway were activated upon nocodazole stimulation in a PKD-dependent manner. Of note, MAPK downstream effectors cause Golgi dispersal in G2 phase of the cell cycle. Golgi ribbon cleavage into isolated Golgi stacks is essential for mitotic entry. In this work it could be shown that cleavage of the non-compact zones of the Golgi ribbon is dependent on PKD activity, which implicates a function for PKD in mitotic entry. Furthermore, it could be shown that PKD acts in this process upstream of the MAP kinase pathway. Taken together, the molecular tools developed and established in this work contributed to identify a critical role of PKD in Golgi complex function and maintenance

2-methoxyestradiol affects mitochondrial biogenesis pathway and succinate dehydrogenase complex flavoprotein subunit a in osteosarcoma cancer cells

Dysregulation of mitochondrial pathways is implicated in several diseases, including cancer. Notably, mitochondrial respiration and mitochondrial biogenesis are favored in some invasive cancer cells, such as osteosarcoma. Hence, the aim of the current work was to investigate the effects of 2-methoxyestradiol (2-ME), a potent anticancer agent, on the mitochondrial biogenesis of osteosarcoma cells. Materials and Methods: Highly metastatic osteosarcoma 143B cells were treated with 2-ME separately or in combination with L-lactate, or with the solvent (non-treated control cells). Protein levels of α-syntrophin and peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1α) were determined by western blotting. Impact of 2-ME on mitochondrial mass, regulation of cytochrome c oxidase I (COXI) expression, and succinate dehydrogenase complex flavoprotein subunit A (SDHA) was determined by immunofluorescence analyses. Inhibition of sirtuin 3 (SIRT3) activity by 2-ME was investigated by fluorescence assay and also, using molecular docking and molecular dynamics simulations. Results: Llactate induced mitochondrial biogenesis pathway via upregulation of COXI. 2-ME inhibited mitochondrial biogenesis via regulation of PGC-1�, COXI, and SIRT3 in a concentration-dependent manner as a consequence of nuclear recruitment of neuronal nitric oxide synthase and nitric oxide generation. It was also proved that 2-ME inhibited SIRT3 activity by binding to both the canonical and allosteric inhibitor binding sites. Moreover, regardless of the mitochondrial biogenesis pathway, 2-ME affected the expression of SDHA. Conclusion: Herein, mitochondrial biogenesis pathway regulation and SDHA were presented as novel targets of 2-ME, and moreover, 2-ME was demonstrated as a potent inhibitor of SIRT3. L-lactate was confirmed to exert pro-carcinogenic effects on osteosarcoma cells via the induction of the mitochondrial biogenesis pathway. Thus, L-lactate level may be considered as a prognostic biomarker for osteosarcom

Optimization of Mechanosensitive Cross-Talk between Matrix Stiffness and Protein Density: Independent Matrix Properties Regulate Spreading Dynamics of Myocytes

Cells actively sense differences in topology, matrix elasticity and protein composition of the extracellular microenvironment and adapt their function and morphology. In this study, we focus on the cross-talk between matrix stiffness and protein coating density that regulates morphology and proliferation dynamics of single myocytes. For this, C2C12 myocytes were monitored on L-DOPA functionalized hydrogels of 22 different elasticity and fibronectin density compositions. Static images were recorded and statistically analyzed to determine morphological differences and to identify the optimized extracellular matrix (ECM). Using that information, selected ECMs were used to study the dynamics before and after cell proliferation by statistical comparison of distinct cell states. We observed a fibronectin-density-independent increase of the projected cell area until 12 kPa. Additionally, changes in fibronectin density led to an area that was optimum at about 2.6 μg/cm2, which was confirmed by independent F-actin analysis, revealing a maximum actin-filament-to-cell-area ratio of 7.5%. Proliferation evaluation showed an opposite correlation between cell spreading duration and speed to matrix elasticity and protein density, which did not affect cell-cycle duration. In summary, we identified an optimized ECM composition and found that independent matrix properties regulate distinct cell characteristics