The intracellular C-terminal domain (CTD) of AMPA (α-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid) receptor undergoes phosphorylation at specific locations during longterm potentiation (LTP). This modification enhances conductance through the AMPA receptor ion channel and thus potentially plays a crucial role in modulating receptor trafficking and signaling. However, because the CTD structure is largely unresolved, it is difficult to establish if phosphorylation induces conformational changes that might play a role in enhancing channel conductance. Herein, we utilize single molecule Fӧrster Resonance Energy Transfer (smFRET) spectroscopy to probe the conformational changes of a section of the AMPA receptor CTD, under the conditions of point-mutated phosphomimicry. Multiple analysis algorithms fail to identify stable conformational states within the smFRET distributions, consistent with a lack of welldefined secondary structure. Instead, our results show that phosphomimicry induces conformational rigidity to the CTD and such rigidity is electrostatically tunable.
Biocatalytic intracellular active nanoreactors (artificial organelles) aim to support their host cells. Here, we report the first successful micelle-based artificial organelles containing a salen−manganese complex (EUK) as catalase mimic with intracellular activity in HepG2 cells to act as reactive oxygen species (ROS) scavengers. Four different EUKs were synthesized and compared in their ability to convert hydrogen peroxide to water and oxygen as free compounds and when encapsulated into micelles assembled from the amphiphilic block copolymer poly(cholesteryl methacrylate)-block-poly(2-(dimethylamino)ethyl methacrylate). An EUK candidate with an asymmetric substitution of chemical groups at the ortho and the meta position (EUK-B) was identified as lead candidate. HepG2 cells continued proliferating when preincubated with low concentrations of EUK-B-containing micelles (M B ). Importantly, HepG2 cells equipped with M B showed improved viability compared to the controls when stressed with paraquat, a compound that induces ROS generation. The intracellular activity of M B was supported by lower amounts of intracellular detectable ROS. This first report on the combination of artificial enzymes and artificial organelles further extends the opportunities in therapeutic cell mimicry.
sues. Exploring the benefit of motors over their passively diffusing counterparts in cell culture or in animal models attracted substantial attention in the past years. [5,6] Early examples focused on externallydriven motors that made use of physical stimuli to propel in low-density media. Recent examples include the use of nearinfrared (NIR) light-activated motors to penetrate a 3D cell tumor culture, which selectively induced cell apoptosis upon NIR irradiation or resulted in depleted amyloid aggregation. [7] Similarly, magnetically driven motors made of CaCO 3 and Fe 3 O 4 showed an increased cellular uptake since their mobility allowed for more efficient approaching of the target area. [8] The decomposition of the core particle in the acidic environment of the lysosomes assisted cargo release. In a different approach, Shen et al. employed Zn-doped iron oxide rods that swarmed and eventually spun under a rotating magnetic field, inducing cell death. [9] This effect was also assessed in vivo, where the size of tumors generated in mice decreased 4× upon magnetic therapy. Further, ultrasound-powered motors used for antigen delivery showed a 5-7-fold increase in cell penetration compared to passive particles. [10] Alternatively, motors that harvest energy from their local environment are a powerful option to design truly autonomous motors. There are examples of motors that showed locomotion in simple 2D cell models as well as more complex 3D cultures and even in animal models. Mesoporous silica particles [11] or liposomes [12,13] were chosen to assess cellular uptake in 2D cultures, showing promising results. For instance, Llopis-Lorente et al. engineered a mesoporous silica motor that moved due to the conversion of urea into ammonia and CO 2 . [14] After entrapment in the lysosomes, a 4× lower motor concentration was needed to release the same amount of cargo as their passive particle counterparts, an aspect that was attributed to the enhanced diffusion of the motors. Further, Wilson and co-workers designed a drug-loaded liposome-based motor that gained locomotion in the acidic pH generated in the surroundings of HeLa cells. [15] These motors showed a chemo tactic behavior towards acidic environments where they reached speeds up to ≈9 µm s −1 at pH 4.6.Other motors were able to penetrate 3D cell environments or tissues. For example, we showed that collagenase-propelled motors posed velocities up to 22 µm s −1 in collagen fiber networks, being eventually able to enhance penetration into bone Nano/micromotors are self-propelled particles that use external stimuli to gain locomotion outperforming Brownian motion. Here, three different polymers are employed that are conjugated to silica particles through a pH-labile linker. At slightly acidic pH, the linkers hydrolyze and release the polymeric chains, resulting in enhanced locomotion. The motors show a maximum velocity of ≈3 µm s −1 in cell media when poly(ethylene glycol) methyl ether methacrylate is asymmetrically distributed on the surface of the particles. Furthe...
The behavior of two acrylate polymers, with carboxylic acid side groups, was investigated with regard to their pH-responsive interaction with phospholipid membranes.
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