3-D retina organoids: Building platforms for therapies of the future.

Across scientific disciplines, 3-D organoid culture systems offer platforms to integrate basic research findings with clinical care. The National Eye Institute mounted a $1.1 million 3-D Retina Organoid Challenge. Organoids developed through the Challenge will be valuable resources for drug screening, disease modeling, and precision and regenerative medicine.

CTD2 Dashboard: a searchable web interface to connect validated results from the Cancer Target Discovery and Development Network.

Database URL: https://ctd2-dashboard.nci.nih.gov/.

Motor and tail homology 1 (Th1) domains antagonistically control myosin-1 dynamics.

Class 1 myosins are monomeric motor proteins that fulfill diverse functions at the membrane/cytoskeletal interface. All myosins-1 contain a motor domain, which binds actin, hydrolyzes ATP, and generates forces, and a TH1 domain, which interacts directly with membrane lipids. In most cases, TH1 is needed for proper subcellular localization and presumably function, although little is known about how this domain regulates the behavior of class 1 myosins in live cells. To address this, we used single molecule total internal reflection fluorescence microscopy to examine the dynamics of the well-characterized myosin-1a isoform during interactions with the cortex of living cells. Our studies revealed that full-length myosin-1a exhibits restricted mobility relative to TH1 alone. Motor domain mutations that disrupt actin binding increased the mobility of full-length myosin-1a, whereas mutations to the TH1 domain that are known to reduce steady-state targeting to the plasma membrane unexpectedly reduced mobility. Deletion of the calmodulin-binding lever arm in Myo1a mimicked the impact of actin-binding mutations. Finally, myosin-1b, which demonstrates exquisite sensitivity to mechanical load, exhibited dynamic behavior nearly identical to myosin-1a. These studies are the first, to our knowledge, to explore class 1 myosin dynamics at the single-molecule level in living cells; our results suggest a model where the motor domain restricts dynamics via a mechanism that requires the lever arm, whereas the TH1 domain allows persistent diffusion in close proximity to the plasma membrane.

Membrane-bound myo1c powers asymmetric motility of actin filaments.

Class I myosins are molecular motors that link cellular membranes to the actin cytoskeleton and play roles in membrane tension generation, membrane dynamics, and mechanosignal transduction. The widely expressed myosin-Ic (myo1c) isoform binds tightly to phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] via a pleckstrin homology domain located in the myo1c tail, which is important for its proper cellular localization. In this study, we found that myo1c can power actin motility on fluid membranes composed of physiological concentrations of PtdIns(4,5)P(2) and that this motility is inhibited by high concentrations of anionic phospholipids. Strikingly, this motility occurs along curved paths in a counterclockwise direction (i.e., the actin filaments turn in leftward circles). A biotinylated myo1c construct containing only the motor domain and the lever arm anchored via streptavidin on a membrane containing biotinylated lipid can also generate asymmetric motility, suggesting that the tail domain is not required for the counterclockwise turning. We found that the ability to produce counterclockwise motility is not a universal characteristic of myosin-I motors, as membrane-bound myosin-Ia (myo1a) and myosin-Ib (myo1b) are able to power actin gliding, but the actin gliding has no substantial turning bias. This work reveals a possible mechanism for establishing asymmetry in relationship to the plasma membrane.

Myosin-1A targets to microvilli using multiple membrane binding motifs in the tail homology 1 (TH1) domain.

One of the most abundant components of the enterocyte brush border is the actin-based monomeric motor, myosin-1a (Myo1a). Within brush border microvilli, Myo1a carries out a number of critical functions at the interface between membrane and actin cytoskeleton. Proper physiological function of Myo1a depends on its ability to bind to microvillar membrane, an interaction mediated by a C-terminal tail homology 1 (TH1) domain. However, little is known about the mechanistic details of the Myo1a-TH1/membrane interaction. Structure-function analysis of Myo1a-TH1 targeting in epithelial cells revealed that an N-terminal motif conserved among class I myosins and a C-terminal motif unique to Myo1a-TH1 are both required for steady state microvillar enrichment. Purified Myo1a bound to liposomes composed of phosphatidylserine and phosphoinositol 4,5-bisphosphate, with moderate affinity in a charge-dependent manner. Additionally, peptides of the N- and C-terminal regions required for targeting were able to compete with Myo1a for binding to highly charged liposomes in vitro. Single molecule total internal reflection fluorescence microscopy showed that these motifs are also necessary for slowing the membrane detachment rate in cells. Finally, Myo1a-TH1 co-localized with both lactadherin-C2 (a phosphatidylserine-binding protein) and PLCδ1-PH (a phosphoinositol 4,5-bisphosphate-binding protein) in microvilli, but only lactaderin-C2 expression reduced brush border targeting of Myo1a-TH1. Together, our results suggest that Myo1a targeting to microvilli is driven by membrane binding potential that is distributed throughout TH1 rather than localized to a single motif. These data highlight the diversity of mechanisms that enable different class I myosins to target membranes in distinct biological contexts.

Lipoxygenase-catalyzed phospholipid peroxidation: preparation, purification, and characterization of phosphatidylinositol peroxides.

The importance of understanding the mechanisms of modulation of cellular signaling cascades by the peroxidized membrane phospholipids (PLs) is well recognized. The enzyme-catalyzed peroxidation of PLs, as opposed to their oxidation by air and metal catalysis, is well controlled and rapid and yields well-defined PL peroxides which are highly desirable for biological studies. Therefore, here, we chose bovine liver phosphatidylinositol (PI), a crucial membrane PL which acts as the substrate for phospholipase C in cellular signal transduction, as a model membrane PL. We successfully generated the PI peroxides with soybean type-I lipoxygenase (LOX) in the presence of deoxycholate, which facilitates the LOX-mediated peroxidation of the polyunsaturated fatty acids esterified to the PL. The LOX-peroxidized PI, after enzymatic catalysis, was separated from the unoxidized PI in the reaction mixture by normal-phase, high-performance liquid chromatography (HPLC). The extent of LOX-mediated peroxidation of PI following HPLC purification was established by the analysis of lipid phosphorus, conjugated dienes by UV spectrophotometry, peroxides, and loss of fatty acids by gas chromatography. This study established the optimal conditions yielding approximately 46% of peroxidized PI from 300 microg of neat bovine liver PI that was peroxidized by soybean type-I LOX (50 microg) for 30 min in borate buffer (0.2 M, pH 9.0) containing 10 mM deoxycholate.

Calcium and calmodulin regulate mercury-induced phospholipase D activation in vascular endothelial cells.

Earlier, we reported that mercury, the environmental risk factor for cardiovascular diseases, activates vascular endothelial cell (EC) phospholipase D (PLD). Here, we report the novel and significant finding that calcium and calmodulin regulated mercury-induced PLD activation in bovine pulmonary artery ECs (BPAECs). Mercury (mercury chloride, 25 microM; thimerosal, 25 microM; methylmercury, 10 microM) significantly activated PLD in BPAECs. Calcium chelating agents and calcium depletion of the medium completely attenuated the mercury-induced PLD activation in ECs. Calmodulin inhibitors significantly attenuated mercury-induced PLD activation in BPAECs. Despite the absence of L-type calcium channels in ECs, nifedipine, nimodipine, and diltiazem significantly attenuated mercury-induced PLD activation and cytotoxicity in BPAECs. This study demonstrated the importance of calcium and calmodulin in the regulation of mercury-induced PLD activation and the protective action of L-type calcium channel blockers against mercury cytotoxicity in vascular ECs, suggesting mechanisms of mercury vasculotoxicity and mercury-induced cardiovascular diseases.

Redox-active antioxidant modulation of lipid signaling in vascular endothelial cells: vitamin C induces activation of phospholipase D through phospholipase A2, lipoxygenase, and cyclooxygenase.

We have earlier reported that the redox-active antioxidant, vitamin C (ascorbic acid), activates the lipid signaling enzyme, phospholipase D (PLD), at pharmacological doses (mM) in the bovine lung microvascular endothelial cells (BLMVECs). However, the activation of phospholipase A(2) (PLA(2)), another signaling phospholipase, and the modulation of PLD activation by PLA(2) in the ECs treated with vitamin C at pharmacological doses have not been reported to date. Therefore, this study aimed at the regulation of PLD activation by PLA(2) in the cultured BLMVECs exposed to vitamin C at pharmacological concentrations. The results revealed that vitamin C (3-10 mM) significantly activated PLA(2) starting at 30 min; however, the activation of PLD resulted only at 120 min of treatment of cells under identical conditions. Further studies were conducted utilizing specific pharmacological agents to understand the mechanism(s) of activation of PLA(2) and PLD in BLMVECs treated with vitamin C (5 mM) for 120 min. Antioxidants, calcium chelators, iron chelators, and PLA(2) inhibitors offered attenuation of the vitamin C-induced activation of both PLA(2) and PLD in the cells. Vitamin C was also observed to significantly induce the formation and release of the cyclooxygenase (COX)- and lipoxygenase (LOX)-catalyzed arachidonic acid (AA) metabolites and to activate the AA LOX in BLMVECs. The inhibitors of PLA(2), COX, and LOX were observed to effectively and significantly attenuate the vitamin C-induced PLD activation in BLMVECs. For the first time, the results of the present study revealed that the vitamin C-induced activation of PLD in vascular ECs was regulated by the upstream activation of PLA(2), COX, and LOX through the formation of AA metabolites involving oxidative stress, calcium, and iron.

Phospholipase A2 activation regulates cytotoxicity of methylmercury in vascular endothelial cells.

Mercury has been identified as a risk factor for cardiovascular disease among humans. Through diet, mainly fish consumption, humans are exposed to methylmercury, the biomethylated organic form of environmental mercury. As the endothelium is an important player in homeostasis of the cardiovascular system, here, the authors tested their hypothesis that methylmercury activates the lipid signaling enzyme phospholipase A(2) (PLA(2)) in vascular endothelial cells (ECs), causing upstream regulation of cytotoxicity. To test this hypothesis, the authors used bovine pulmonary artery ECs (BPAECs) cultured in monolayers, following labeling of their membrane phospholipids with [(3)H]arachidonic acid (AA). The cells were exposed to methylmercury chloride (MMC) and then the release of free AA (index of PLA(2) activity) and lactate dehydrogenase (LDH; index of cytotoxicity) were determined by liquid scintillation counting and spectrophotometry, respectively. MMC significantly activated PLA(2) in a dose-dependent (5 to 15 microM) and time-dependent (0 to 60 min) fashion. Sulfhydryl (thiol-protective) agents, calcium chelators, antioxidants, and PLA(2)-specific inhibitors attenuated the MMC-induced PLA(2) activation, suggesting the role of thiols, reactive oxygen species (ROS), and calcium in the activation of PLA(2) in BPAECs. MMC also induced the loss of thiols and increase of lipid peroxidation in BPAECs. MMC induced cytotoxicity in BPAECs as observed by the altered cell morphology and LDH leak, which was significantly attenuated by PLA(2) inhibitors. This study established that PLA(2) activation through thiols, calcium, and oxidative stress was associated with the cytotoxicity of MMC in BPAECs, drawing attention to the involvement of PLA(2) signaling in the methylmercury-induced vascular endothelial dysfunctions.

Mercury activates vascular endothelial cell phospholipase D through thiols and oxidative stress.

Currently, mercury has been identified as a risk factor of cardiovascular diseases among humans. Here, the authors tested the hypothesis that mercury modulates the activity of the endothelial lipid signaling enzyme, phospholipase D (PLD), which is an important player in the endothelial cell (EC) barrier functions. Monolayers of bovine pulmonary artery ECs (BPAECs) in culture, following labeling of membrane phospholipids with [32P]orthophosphate, were exposed to mercuric chloride (inorganic form), methylmercury chloride (environmental form), and thimerosal (pharmaceutical form), and the formation of phosphatidylbutanol as an index of PLD activity was determined by thin-layer chromatography and liquid scintillation counting. All three forms of mercury significantly activated PLD in BPAECs in a dose-dependent (0 to 50 microM) and time-dependent (0 to 60 min) fashion. Metal chelators significantly attenuated mercury-induced PLD activation, suggesting that cellular mercury-ligand interaction(s) is required for the enzyme activation and that chelators are suitable blockers for mercury-induced PLD activation. Sulfhydryl (thiol-protective) agents and antioxidants also significantly attenuated the mercury-induced PLD activation in BPAECs. Enhanced reactive oxygen species generation, as an index of oxidative stress, was observed in BPAECs treated with methylmercury that was attenuated by antioxidants. All the three different forms of mercury significantly induced the decrease of levels of total cellular thiols. For the first time, this study revealed that mercury induced the activation of PLD in the vascular ECs wherein cellular thiols and oxidative stress acted as signal mediators for the enzyme activation. The results underscore the importance of PLD signaling in mercury-induced endothelial dysfunctions ultimately leading to cardiovascular diseases.

Mercury activates phospholipase a(2) and induces formation of arachidonic Acid metabolites in vascular endothelial cells.

ABSTRACT Currently, mercury has been identified as a risk factor in cardiovascular diseases among humans. Here, we tested our hypothesis that mercury modulates the activity of the vascular endothelial cell (EC) lipid signaling enzyme phospholipase A(2) (PLA(2)), which is an important player in the EC barrier functions. Monolayers of bovine pulmonary artery ECs (BPAECs) in culture, following labeling of membrane phospholipids with [(3)H]arachidonic acid (AA), were exposed to the inorganic form of mercury, mercury chloride, and the release of free AA (index of PLA(2) activity) and formation of AA metabolites were determined by liquid scintillation counting and enzyme immunoassay, respectively. Mercury chloride significantly activated PLA(2) in BPAECs in a dose-dependent (0 to 50 muM) and time-dependent (0 to 120 min) fashion. Metal chelators significantly attenuated mercury-induced PLA(2) activation, suggesting that cellular mercury-ligand interaction is required for the enzyme activation and that chelators are suitable blockers for mercury-induced PLA(2) activation in ECs. Sulfhydryl (thiol-protective) agents, calcium chelating agents, and cPLA(2)-specific inhibitor also significantly attenuated the mercury-induced PLA(2), suggesting the role of thiol and calcium in the activation of cPLA(2) in BPAECs. Significant formation of AA metabolites, including the release of total prostaglandins, thromboxane B(2), and 8-isoprostane, were observed in BPAECs following their exposure to mercury chloride. Mercury chloride induced cytotoxicity as observed by the altered cell morphology and enhanced trypan blue uptake, which was attenuated by the cPLA(2) inhibitor AACOCF(3). The results of this study revealed that inorganic mercury-induced PLA(2) activation through the thiol and calcium signaling and the formation of bioactive AA metabolites further demonstrated the association of PLA(2) with the cytotoxicity of mercury in ECs. Overall, the results of the current study underscore the importance of PLA(2) signaling in mercury-induced endothelial dysfunctions.