Endometrium On-a-Chip Reveals Insulin- and Glucose-induced Alterations in the Transcriptome and Proteomic Secretome

The molecular interactions between the maternal environment and the developing embryo are key for early pregnancy success and are influenced by factors such as maternal metabolic status. Our understanding of the mechanism(s) through which these individual nutritional stressors alter endometrial function and the in utero environment for early pregnancy success is, however, limited. Here we report, for the first time, the use of an endometrium-on-a-chip microfluidics approach to produce a multicellular endometrium in vitro. Isolated endometrial cells (epithelial and stromal) from the uteri of nonpregnant cows in the early luteal phase (Days 4-7) were seeded in the upper chamber of the device (epithelial cells; 4-6 × 104 cells/mL) and stromal cells seeded in the lower chamber (1.5-2 × 104 cells/mL). Exposure of cells to different concentrations of glucose (0.5, 5.0, or 50 mM) or insulin (Vehicle, 1 or 10 ng/mL) was performed at a flow rate of 1 µL/minute for 72 hours. Quantitative differences in the cellular transcriptome and the secreted proteome of in vitro–derived uterine luminal fluid were determined by RNA-sequencing and tandem mass tagging mass spectrometry, respectively. High glucose concentrations altered 21 and 191 protein-coding genes in epithelial and stromal cells, respectively (P < .05), with a dose-dependent quantitative change in the protein secretome (1 and 23 proteins). Altering insulin concentrations resulted in limited transcriptional changes including transcripts for insulin-like binding proteins that were cell specific but altered the quantitative secretion of 196 proteins. These findings highlight 1 potential mechanism by which changes to maternal glucose and insulin alter uterine function

The Lysosome Signaling Platform: Adapting With the Times

&lt;p&gt;Lysosomes are the terminal degradative compartment of autophagy, endocytosis and phagocytosis. What once was viewed as a simple acidic organelle in charge of macromolecular digestion has emerged as a dynamic organelle capable of integrating cellular signals and producing signal outputs. In this review, we focus on the concept that the lysosome surface serves as a platform to assemble major signaling hubs like mTORC1, AMPK, GSK3 and the inflammasome. These molecular assemblies integrate and facilitate cross-talk between signals such as amino acid and energy levels, membrane damage and infection, and ultimately enable responses such as autophagy, cell growth, membrane repair and microbe clearance. In particular, we review how molecular machinery like the vacuolar-ATPase proton pump, sestrins, the GATOR complexes, and the Ragulator, modulate mTORC1, AMPK, GSK3 and inflammation. We then elaborate how these signals control autophagy initiation and resolution, TFEB-mediated lysosome adaptation, lysosome remodeling, antigen presentation, inflammation, membrane damage repair and clearance. Overall, by being at the cross-roads for several membrane pathways, lysosomes have emerged as the ideal surveillance compartment to sense, integrate and elicit cellular behavior and adaptation in response to changing environmental and cellular conditions. &lt;/p&gt;</jats:p

The Lysosome Signaling Platform: Adapting With the Times

&lt;p&gt;Lysosomes are the terminal degradative compartment of autophagy, endocytosis and phagocytosis. What once was viewed as a simple acidic organelle in charge of macromolecular digestion has emerged as a dynamic organelle capable of integrating cellular signals and producing signal outputs. In this review, we focus on the concept that the lysosome surface serves as a platform to assemble major signaling hubs like mTORC1, AMPK, GSK3 and the inflammasome. These molecular assemblies integrate and facilitate cross-talk between signals such as amino acid and energy levels, membrane damage and infection, and ultimately enable responses such as autophagy, cell growth, membrane repair and microbe clearance. In particular, we review how molecular machinery like the vacuolar-ATPase proton pump, sestrins, the GATOR complexes, and the Ragulator, modulate mTORC1, AMPK, GSK3 and inflammation. We then elaborate how these signals control autophagy initiation and resolution, TFEB-mediated lysosome adaptation, lysosome remodeling, antigen presentation, inflammation, membrane damage repair and clearance. Overall, by being at the cross-roads for several membrane pathways, lysosomes have emerged as the ideal surveillance compartment to sense, integrate and elicit cellular behavior and adaptation in response to changing environmental and cellular conditions. &lt;/p&gt;</jats:p

The Lysosome Signaling Platform: Adapting With the Times

Lysosomes are the terminal degradative compartment of autophagy, endocytosis and phagocytosis. What once was viewed as a simple acidic organelle in charge of macromolecular digestion has emerged as a dynamic organelle capable of integrating cellular signals and producing signal outputs. In this review, we focus on the concept that the lysosome surface serves as a platform to assemble major signaling hubs like mTORC1, AMPK, GSK3 and the inflammasome. These molecular assemblies integrate and facilitate cross-talk between signals such as amino acid and energy levels, membrane damage and infection, and ultimately enable responses such as autophagy, cell growth, membrane repair and microbe clearance. In particular, we review how molecular machinery like the vacuolar-ATPase proton pump, sestrins, the GATOR complexes, and the Ragulator, modulate mTORC1, AMPK, GSK3 and inflammation. We then elaborate how these signals control autophagy initiation and resolution, TFEB-mediated lysosome adaptation, lysosome remodeling, antigen presentation, inflammation, membrane damage repair and clearance. Overall, by being at the cross-roads for several membrane pathways, lysosomes have emerged as the ideal surveillance compartment to sense, integrate and elicit cellular behavior and adaptation in response to changing environmental and cellular conditions. </p

Reactive oxygen species prevent lysosome coalescence during PIKfyve inhibition

Lysosomes are terminal, degradative organelles of the endosomal pathway that undergo repeated fusion-fission cycles with themselves, endosomes, phagosomes, and autophagosomes. Lysosome number and size depends on balanced fusion and fission rates. Thus, conditions that favour fusion over fission can reduce lysosome numbers while enlarging their size. Conversely, favouring fission over fusion may cause lysosome fragmentation and increase their numbers. PIKfyve is a phosphoinositide kinase that generates phosphatidylinositol-3,5-bisphosphate to modulate lysosomal functions. PIKfyve inhibition causes an increase in lysosome size and reduction in lysosome number, consistent with lysosome coalescence. This is thought to proceed through reduced lysosome reformation and/or fission after fusion with endosomes or other lysosomes. Previously, we observed that photo-damage during live-cell imaging prevented lysosome coalescence during PIKfyve inhibition. Thus, we postulated that lysosome fusion and/or fission dynamics are affected by reactive oxygen species (ROS). Here, we show that ROS generated by various independent mechanisms all impaired lysosome coalescence during PIKfyve inhibition and promoted lysosome fragmentation during PIKfyve re-activation. However, depending on the ROS species or mode of production, lysosome dynamics were affected distinctly. H2O2 impaired lysosome motility and reduced lysosome fusion with phagosomes, suggesting that H2O2 reduces lysosome fusogenecity. In comparison, inhibitors of oxidative phosphorylation, thiol groups, glutathione, or thioredoxin, did not impair lysosome motility but instead promoted clearance of actin puncta on lysosomes formed during PIKfyve inhibition. Additionally, actin depolymerizing agents prevented lysosome coalescence during PIKfyve inhibition. Thus, we discovered that ROS can generally prevent lysosome coalescence during PIKfyve inhibition using distinct mechanisms depending on the type of ROS.</jats:p

Reactive oxygen species prevent lysosome coalescence during PIKfyve inhibition

&lt;p&gt;Lysosomes are terminal, degradative organelles of the endosomal pathway that undergo repeated fusion-fission cycles with themselves, endosomes, phagosomes, and autophagosomes. Lysosome number and size depends on balanced fusion and fission rates. Thus, conditions that favour fusion over fission can reduce lysosome numbers while enlarging their size. Conversely, favouring fission over fusion may cause lysosome fragmentation and increase their numbers. PIKfyve is a phosphoinositide kinase that generates phosphatidylinositol-3,5-bisphosphate to modulate lysosomal functions. PIKfyve inhibition causes an increase in lysosome size and reduction in lysosome number, consistent with lysosome coalescence. This is thought to proceed through reduced lysosome reformation and/or fission after fusion with endosomes or other lysosomes. Previously, we observed that photo-damage during live-cell imaging prevented lysosome coalescence during PIKfyve inhibition. Thus, we postulated that lysosome fusion and/or fission dynamics are affected by reactive oxygen species (ROS). Here, we show that ROS generated by various independent mechanisms all impaired lysosome coalescence during PIKfyve inhibition and promoted lysosome fragmentation during PIKfyve re-activation. However, depending on the ROS species or mode of production, lysosome dynamics were affected distinctly. H2O2 impaired lysosome motility and reduced lysosome fusion with phagosomes, suggesting that H2O2 reduces lysosome fusogenecity. In comparison, inhibitors of oxidative phosphorylation, thiol groups, glutathione, or thioredoxin, did not impair lysosome motility but instead promoted clearance of actin puncta on lysosomes formed during PIKfyve inhibition. Additionally, actin depolymerizing agents prevented lysosome coalescence during PIKfyve inhibition. Thus, we discovered that ROS can generally prevent lysosome coalescence during PIKfyve inhibition using distinct mechanisms depending on the type of ROS.&lt;/p&gt; &lt;p&gt; &lt;/p&gt;</jats:p

Reactive oxygen species prevent lysosome coalescence during PIKfyve inhibition

AbstractLysosomes are terminal, degradative organelles of the endosomal pathway that undergo repeated fusion-fission cycles with themselves, endosomes, phagosomes, and autophagosomes. Lysosome number and size depends on balanced fusion and fission rates. Thus, conditions that favour fusion over fission can reduce lysosome numbers while enlarging their size. Conversely, favouring fission over fusion may cause lysosome fragmentation and increase their numbers. PIKfyve is a phosphoinositide kinase that generates phosphatidylinositol-3,5-bisphosphate to modulate lysosomal functions. PIKfyve inhibition causes an increase in lysosome size and reduction in lysosome number, consistent with lysosome coalescence. This is thought to proceed through reduced lysosome reformation and/or fission after fusion with endosomes or other lysosomes. Previously, we observed that photo-damage during live-cell imaging prevented lysosome coalescence during PIKfyve inhibition. Thus, we postulated that lysosome fusion and/or fission dynamics are affected by reactive oxygen species (ROS). Here, we show that ROS generated by various independent mechanisms all impaired lysosome coalescence during PIKfyve inhibition and accelerated lysosome fragmentation during re-activation. However, depending on the ROS species or mode of production, lysosome dynamics were affected distinctly. H2O2 impaired lysosome motility and reduced lysosome fusion with phagosomes, suggesting that H2O2 reduces lysosome fusogenecity. In comparison, inhibitors of oxidative phosphorylation, glutathione, and thioredoxin that produce superoxide, did not impair lysosome motility but instead promoted clearance of actin puncta on lysosomes formed during PIKfyve inhibition. Additionally, actin depolymerizing agents prevented lysosome coalescence during PIKfyve inhibition. Thus, we discovered that ROS can generally prevent lysosome coalescence during PIKfyve inhibition using distinct mechanisms depending on the type of ROS.</jats:p

Reactive oxygen species prevent lysosome coalescence during PIKfyve inhibition.

Lysosomes are terminal, degradative organelles of the endosomal pathway that undergo repeated fusion-fission cycles with themselves, endosomes, phagosomes, and autophagosomes. Lysosome number and size depends on balanced fusion and fission rates. Thus, conditions that favour fusion over fission can reduce lysosome numbers while enlarging their size. Conversely, favouring fission over fusion may cause lysosome fragmentation and increase their numbers. PIKfyve is a phosphoinositide kinase that generates phosphatidylinositol-3,5-bisphosphate to modulate lysosomal functions. PIKfyve inhibition causes an increase in lysosome size and reduction in lysosome number, consistent with lysosome coalescence. This is thought to proceed through reduced lysosome reformation and/or fission after fusion with endosomes or other lysosomes. Previously, we observed that photo-damage during live-cell imaging prevented lysosome coalescence during PIKfyve inhibition. Thus, we postulated that lysosome fusion and/or fission dynamics are affected by reactive oxygen species (ROS). Here, we show that ROS generated by various independent mechanisms all impaired lysosome coalescence during PIKfyve inhibition and promoted lysosome fragmentation during PIKfyve re-activation. However, depending on the ROS species or mode of production, lysosome dynamics were affected distinctly. H2O2 impaired lysosome motility and reduced lysosome fusion with phagosomes, suggesting that H2O2 reduces lysosome fusogenecity. In comparison, inhibitors of oxidative phosphorylation, thiol groups, glutathione, or thioredoxin, did not impair lysosome motility but instead promoted clearance of actin puncta on lysosomes formed during PIKfyve inhibition. Additionally, actin depolymerizing agents prevented lysosome coalescence during PIKfyve inhibition. Thus, we discovered that ROS can generally prevent lysosome coalescence during PIKfyve inhibition using distinct mechanisms depending on the type of ROS

Reactive oxygen species prevent lysosome coalescence during PIKfyve inhibition

&lt;p&gt;Lysosomes are terminal, degradative organelles of the endosomal pathway that undergo repeated fusion-fission cycles with themselves, endosomes, phagosomes, and autophagosomes. Lysosome number and size depends on balanced fusion and fission rates. Thus, conditions that favour fusion over fission can reduce lysosome numbers while enlarging their size. Conversely, favouring fission over fusion may cause lysosome fragmentation and increase their numbers. PIKfyve is a phosphoinositide kinase that generates phosphatidylinositol-3,5-bisphosphate to modulate lysosomal functions. PIKfyve inhibition causes an increase in lysosome size and reduction in lysosome number, consistent with lysosome coalescence. This is thought to proceed through reduced lysosome reformation and/or fission after fusion with endosomes or other lysosomes. Previously, we observed that photo-damage during live-cell imaging prevented lysosome coalescence during PIKfyve inhibition. Thus, we postulated that lysosome fusion and/or fission dynamics are affected by reactive oxygen species (ROS). Here, we show that ROS generated by various independent mechanisms all impaired lysosome coalescence during PIKfyve inhibition and promoted lysosome fragmentation during PIKfyve re-activation. However, depending on the ROS species or mode of production, lysosome dynamics were affected distinctly. H2O2 impaired lysosome motility and reduced lysosome fusion with phagosomes, suggesting that H2O2 reduces lysosome fusogenecity. In comparison, inhibitors of oxidative phosphorylation, thiol groups, glutathione, or thioredoxin, did not impair lysosome motility but instead promoted clearance of actin puncta on lysosomes formed during PIKfyve inhibition. Additionally, actin depolymerizing agents prevented lysosome coalescence during PIKfyve inhibition. Thus, we discovered that ROS can generally prevent lysosome coalescence during PIKfyve inhibition using distinct mechanisms depending on the type of ROS.&lt;/p&gt; &lt;p&gt; &lt;/p&gt;</jats:p