Cellular Interior Research Articles

Evaluating the Expression of Efflux Pumps in Pseudomonas aeruginosa in Exposure to Sodium Dodecyl Sulfate, Didecyldimethylammonium Chloride, and Octenidine Dihydrochloride.

Emerging resistance of Gram-negative bacteria, including Pseudomonas aeruginosa, to commonly used detergents and disinfectant is encountering us with hazard. Inappropriate use of disinfectants has forced bacteria to gain resistance. The ability of bacteria to extrude substrates from the cellular interior to the external environment has enabled them to persist in exposure to toxic compounds, which is due to existence of transport proteins. Efflux pumps, in Gram-negative bacteria, are proteins responsible for exporting molecules outside of the cell, by crossing the two membranes. In this study, 40 P. aeruginosa strains from hospitals, clinics, and burn center laundries and 40 P. aeruginosa strains from urban laundries were collected. This study evaluated the minimum inhibitory concentration (MIC) level of sodium dodecyl sulfate (SDS), didecyldimethylammonium chloride (DDAC), and octenidine dihydrochloride (Od) in P. aeruginosa strains. The real-time PCR was carried out to evaluate the expression of MexAB-OprM, MexCD-OprJ, and MexXY-OprM efflux system. The obtained results indicated a higher MIC level for SDS, DDAC, and Od in medical laundries. The sub-MIC level of DDAC and Od increased the expression level of MexAB-OprM, MexCD-OprJ, and MexXY-OprM in P. aeruginosa strains, suggesting that efflux pumps contribute to disinfectant resistance in P. aeruginosa.

Barrier crossing in a viscoelastic medium under active noise: Predictions of Kramers’ flux-over-population method

The biochemical activity inside a cell has recently been suggested to act as a source of hydrodynamic fluctuations that can speed up or slow down enzyme catalysis [Tripathi et al., Commun. Phys. 5, 101 (2022).] The idea has been tested against and largely corroborated by simulations of activated barrier crossing in a simple fluid in the presence of thermal and athermal noise. The present paper attempts a wholly analytic solution to the same noise-driven barrier crossing problem but generalizes it to include viscoelastic memory effects of the kind likely to be present in cellular interiors. A calculation of the model’s barrier crossing rate, using Kramers’ flux-over-population formalism, reveals that in relation to the case where athermal noise is absent, athermal noise always accelerates barrier crossing, though the extent of enhancement depends on the duration τ0 over which the noise acts. More importantly, there exists a critical τ0—determined by the properties of the medium—at which Kramers’ theory breaks down and, on approach to which, the rate grows significantly. The possibility of such a giant enhancement is potentially open to experimental validation using optically trapped nanoparticles in viscoelastic media that are acted on by externally imposed colored noise.

Diffusive lensing as a mechanism of intracellular transport and compartmentalization.

While inhomogeneous diffusivity has been identified as a ubiquitous feature of the cellular interior, its implications for particle mobility and concentration at different length scales remain largely unexplored. In this work, we use agent-based simulations of diffusion to investigate how heterogeneous diffusivity affects the movement and concentration of diffusing particles. We propose that a nonequilibrium mode of membrane-less compartmentalization arising from the convergence of diffusive trajectories into low-diffusive sinks, which we call 'diffusive lensing,' is relevant for living systems. Our work highlights the phenomenon of diffusive lensing as a potentially key driver of mesoscale dynamics in the cytoplasm, with possible far-reaching implications for biochemical processes.

Diffusive lensing as a mechanism of intracellular transport and compartmentalization

While inhomogeneous diffusivity has been identified as a ubiquitous feature of the cellular interior, its implications for particle mobility and concentration at different length scales remain largely unexplored. In this work, we use agent-based simulations of diffusion to investigate how heterogeneous diffusivity affects the movement and concentration of diffusing particles. We propose that a nonequilibrium mode of membrane-less compartmentalization arising from the convergence of diffusive trajectories into low-diffusive sinks, which we call ‘diffusive lensing,’ is relevant for living systems. Our work highlights the phenomenon of diffusive lensing as a potentially key driver of mesoscale dynamics in the cytoplasm, with possible far-reaching implications for biochemical processes.

Deeper Insights into Mixed Crowding through Enzyme Activity, Dynamics, and Crowder Diffusion.

Given the fact that the cellular interior is crowded by many different kinds of macromolecules, it is important that in vitro studies be carried out in the presence of mixed crowder systems. In this regard, we have used binary crowders formed by the combination of some of the commonly used crowding agents, namely, Ficoll 70, Dextran 70, Dextran 40, and PEG 8000 (PEG 8), to study how these affect enzyme activity, dynamics, and crowder diffusion. The enzyme chosen is AK3L1, an isoform of adenylate kinase. To investigate its dynamics, we have carried out three single point mutations (A74C, A132C, and A209C) with the cysteine residues being labeled with a coumarin-based solvatochromic probe [CPM: (7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin)]. Both enzyme activity and dynamics decreased in the binary mixtures as compared with the sum of the individual crowders, suggesting a reduction in excluded volume (in the mixture). To gain deeper insights into the binary mixtures, fluorescence correlation spectroscopy studies were carried out using fluorescein isothiocyanate-labeled Dextran 70 and tetramethylrhodamine-labeled AK3L1 as the diffusion probes. Diffusion in binary mixtures was observed to be much more constrained (relative to the sum of the individual crowders) for the labeled enzyme as compared to the labeled crowder showing different environments being faced by the two species. This was further confirmed during imaging of the phase-separated droplets formed in the binary mixtures having PEG as one of the crowding agents. The interior of these droplets was found to be rich in crowders and densely packed, as shown by confocal and digital holographic microscopy images, with the enzymes predominantly residing outside these droplets, that is, in the relatively less crowded regions. Taken together, our data provide important insights into various aspects of the simplest form of mixed crowding, that is, composed of just two components, and also hint at the enhanced complexity that the cellular interior presents toward having a detailed and comprehensive understanding of the same.

Advancing the Boundaries of Immunotherapy in Lung Adenocarcinoma with Idiopathic Pulmonary Fibrosis by a Biomimetic Proteinoid Enabling Selective Endocytosis.

The coexistence of lung adenocarcinoma (LUAD) with idiopathic pulmonary fibrosis (IPF), which has been extensively documented as a prominent risk factor for checkpoint inhibitor-related pneumonitis (CIP) in patients undergoing immunotherapy, has long been considered a restricted domain for the use of immune checkpoint inhibitors (ICIs). To overcome it, an approach was employed herein to specifically target PD-L1 within the cellular interior, surpassing the conventional focus solely on the cytomembrane, thereby facilitating the development of ICIs capable of distinguishing between LUAD cells and noncancerous cells based on their distinctive endocytic propensities. By exploiting the aurophilicity-driven self-assembly of a PD-L1 binding peptide (PDBP) and subsequently encapsulating it within erythrocyte membranes (EM), the resulting biomimetic ICIs protein EMS-PDBP exhibited extraordinary selectivity in internalizing LUAD cells, effectively targeting PD-L1 within cancer cells while hindering its membrane translocation. The EMS-PDBP treatment not only reactivated the antitumor immune response in the LUAD orthotopic allograft mouse model but also demonstrated a favorable safety profile by effectively eliminating any immune-related adverse events (irAEs). Most significantly, EMS-PDBP successfully and safely restored the antitumor immune response in a mouse model of LUAD with coexistent IPF, thus shattering the confines of ICIs immunotherapy. The reported EMS-PDBP collectively offers a potential strategy for immune reactivation to overcome the limitations of immunotherapy in LUAD coexisting with IPF.

Soft X-ray Microscopy in Cell Biology: Current Status, Contributions and Prospects.

The recent advances achieved in microscopy technology have led to a significant breakthrough in biological research. Super-resolution fluorescent microscopy now allows us to visualize subcellular structures down to the pin-pointing of the single molecules in them, while modern electron microscopy has opened new possibilities in the study of protein complexes in their native, intracellular environment at near-atomic resolution. Nonetheless, both fluorescent and electron microscopy have remained beset by their principal shortcomings: the reliance on labeling procedures and severe sample volume limitations, respectively. Soft X-ray microscopy is a candidate method that can compensate for the shortcomings of both technologies by making possible observation of the entirety of the cellular interior without chemical fixation and labeling with an isotropic resolution of 40-70 nm. This will thus bridge the resolution gap between light and electron microscopy (although this gap is being narrowed, it still exists) and resolve the issue of compatibility with the former, and possibly in the near future, the latter methods. This review aims to assess the current state of soft X-ray microscopy and its impact on our understanding of the subcellular organization. It also attempts to look into the future of X-ray microscopy, particularly as relates to its seamless integration into the cell biology toolkit.

Controlling the size and adhesion of DNA droplets using surface- enriched DNA molecules.

Liquid droplets of biomolecules serve as organizers of the cellular interior and are of interest in biosensing and biomaterials applications. Here, we investigate means to tune the interfacial properties of a model biomolecular liquid consisting of multi-armed DNA 'nanostar' particles. We find that long DNA molecules that have binding affinity for the nanostars are preferentially enriched on the interface of nanostar droplets, thus acting as surfactants. Fluorescent measurements indicate that, in certain conditions, the interfacial density of the surfactant is around 20 per square micron, indicative of a sparse brush-like structure of the long, polymeric DNA. Increasing surfactant concentration leads to decreased droplet size, down to the sub-micron scale, consistent with droplet coalesence being impeded by the disjoining pressure created by the brush-like surfactant layer. Added DNA surfactant also keeps droplets from adhering to both hydrophobic and hydrophilic solid surfaces, apparently due to this same disjoining effect of the surfactant layer. We thus demonstrate control of the size and adhesive properties of droplets of a biomolecular liquid, with implications for basic biophysical understanding of such droplets, as well as for their applied use.

Molecular tracking of interactions between progenitor and endothelial cells via Raman and FTIR spectroscopy imaging: a proof of concept of a new analytical strategy for in vitro research

Circulating endothelial cell progenitors originating from the bone marrow are considered to be a powerful tool in the repair of endothelium damage. Due to their unique properties, endothelial progenitors are now broadly investigated to assess their clinical significance in diseases e.g., associated with brain endothelial dysfunction. However, their distinction in terms of the expression of specific markers remains ambiguous. Additionally, endothelial progenitor cells may change their repertoire of markers depending on the microenvironment of the tissue in which they are currently located. Here, we applied the label-free Raman and FTIR imaging to discriminate mice brain endothelium and endothelial progenitors. Cells cultured separately showed distinctly different spectral signatures extracted from the whole cellular interior as well as the detected intracellular compartments (nucleus, cytoplasm, perinuclear area, and lipid droplets). Then, we used these spectroscopic signals to examine the cells co-cultured for 24 h. Principal cluster analysis showed their grouping with the progenitor cells and segregation from brain endothelium at a level of the entire cell machinery (in FTIR images) which resulted from biochemical alternations in the cytoplasm and lipid droplets (in Raman images). The models included in partial least square regression indicated that lipid droplets are the key element for the classification of endothelial progenitor-brain endothelial cells interactions.

Nonmonotonic Modulation of the Protein-Ligand Recognition Event by Inert Crowders.

The ubiquitous event of a protein recognizing small molecules or ligands at its native binding site is crucial for initiating major biological processes. However, how a crowded environment, as is typically represented by a cellular interior, would modulate the protein-ligand search process is largely debated. Excluded volume-based theory suggests that the presence of an inert crowder would reinforce a steady stabilization and enhancement of the protein-ligand recognition process. Here, we counter this long-held perspective via the molecular dynamics simulation and Markov state model of the protein-ligand recognition event in the presence of inert crowders. Specifically, we demonstrate that, depending on concentration, even purely inert crowders can exert a nonmonotonic effect via either stabilizing or destabilizing the protein-ligand binding event. Analysis of the kinetic network of binding pathways reveals that the crowders would either modulate precedent non-native on-pathway intermediates or would devise additional ones in a multistate recognition event across a wide range of concentrations. As an important insight, crowders gradually shift the relative transitional preference of these intermediates toward a native-bound state, with ligand residence time at the binding pocket dictating the trend of nonmonotonic concentration dependence by simple inert crowders.

Selective Molecular Recognition and Indicator Displacement Sensing of Neurotransmitters in Cellular Environments.

Flexible, water-soluble hosts are capable of selective molecular recognition in cellular environments and can detect neurotransmitters such as choline in cells. Both cationic and anionic water-soluble self-folded deep cavitands can recognize suitable styrylpyridinium dyes in cellular interiors. The dyes selectively accumulate in nucleotide-rich regions of the cell nucleus and cytoplasm. The hosts bind the dyes and promote their relocation to the outer cell membrane: the lipophilic cavitands predominantly reside in membrane environments but are still capable of binding suitable targets in other cellular organelles. Incubating the cells with structurally similar biomarkers such as choline, cholamine, betaine, or butyrylcholine illustrates the selective recognition. Choline and butyrylcholine can be bound by the hosts, but minimal binding is seen with betaine or cholamine. Varying the dye allows control of the optical detection method, and both "turn-on" sensing and "turn-off" sensing are possible.

Effect of cellular structure on the mechanical properties of 316L stainless steel fabricated by EBF3

The cellular structure formed in additive manufactured 316L stainless steel (316L SS) plays a crucial role in achieving strength–ductility synergy. The cellular structure was also found in the 316L SS fabricated by electron beam freeform fabrication process (EBF3), and its microstructural characterization was analyzed. Interrupted tensile tests were carried out to demonstrate the effect of the cellular structure on the mechanical properties. The results showed that the microstructure of the as-deposited 316L SS consisted of the single austenite phase, and the enrichment of Cr and Mo elements was found in the cellular boundary. The orientation of the cellular structure and boundary was similar, and the obvious {100} preferred orientation was parallel to the building direction. The yield strength and fracture elongation of the as-deposited 316L SS parts were 316 MPa and 37.2%, respectively. Its ultimate tensile strength with a value of 632 MPa, was higher than that manufactured by conventional methods. The hardness and elastic modulus in the cellular interior were 2.88 ± 0.11 GPa and 227.45 ± 10.66 GPa, respectively. The cellular boundary had a higher hardness and a lower elastic modulus than the cellular interior. At the initial stage of tensile deformation, the cellular boundary deforms first due to its lower elastic modulus, causing the dislocation pileup and orientation change. The high dislocation density improved the slip resistance leading to the appearance of deformation twins after necking. The micron-dimples and nano-dimples formed in the fracture surface with the deformation of the cellular boundary and interior. Different from 316L SS fabricated by other additive manufacturing processes, the cellular structure improved the strain hardening capacity of the 316L SS fabricated by the EBF3 process.

Utilization of a cell-penetrating peptide-adaptor for delivery of human papillomavirus protein E2 into cervical cancer cells to arrest cell growth and promote cell death.

Human papillomavirus (HPV) is the causative agent of nearly all forms of cervical cancer, which can arise upon viral integration into the host genome and concurrent loss of viral regulatory gene E2. Gene-based delivery approaches show that E2 reintroduction reduces proliferative capacity and promotes apoptosis in vitro. This work explored if our calcium-dependent protein-based delivery system, TAT-CaM, could deliver functional E2 protein directly into cervical cancer cells to limit proliferative capacity and induce cell death. TAT-CaM and the HPV16 E2 protein containing a CaM-binding sequence (CBS-E2) were expressed and purified from Escherichia coli. Calcium-dependent binding kinetics were verified by biolayer interferometry. Equimolar TAT-CaM:CBS-E2 constructs were delivered into the HPV16+ SiHa cell line and uptake verified by confocal microscopy. Proliferative capacity was measured by MTS assay and cell death was measured by release of lactate dehydrogenase. As a control, human microvascular cells (HMECs) were used. As expected, TAT-CaM bound CBS-E2 with high affinity in the presence of calcium and rapidly disassociated upon its removal. After introduction by TAT-CaM, fluorescently labeled CBS-E2 was detected in cellular interiors by orthogonal projections taken at the depth of the nucleus. In dividing cells, E2 relocalized to regions associated with the mitotic spindle. Cells receiving a daily dose of CBS-E2 for 4 days showed a significant reduction in metabolic activity at low doses and increased cell death at high doses compared to controls. This phenotype was retained for 7 days with no further treatments. When subcultured on day 12, treated cells regained their proliferative capacity. Using the TAT-CaM platform, bioactive E2 protein was delivered into living cervical cancer cells, inducing senescence and cell death in a time- and dose-dependent manner. These results suggest that this nucleic acid and virus-free delivery method could be harnessed to develop novel, effective protein therapeutics.

Cryo-electron tomography on focused ion beam lamellae transforms structural cell biology.

Cryogenic electron microscopy and data processing enable the determination of structures of isolated macromolecules to near-atomic resolution. However, these data do not provide structural information in the cellular environment where macromolecules perform their native functions, and vital molecular interactions can be lost during the isolation process. Cryogenic focused ion beam (FIB) fabrication generates thin lamellae of cellular samples and tissues, enabling structural studies on the near-native cellular interior and its surroundings by cryogenic electron tomography (cryo-ET). Cellular cryo-ET benefits from the technological developments in electron microscopes, detectors and data processing, and more in situ structures are being obtained and at increasingly higher resolution. In this Review, we discuss recent studies employing cryo-ET on FIB-generated lamellae and the technological developments in ultrarapid sample freezing, FIB fabrication of lamellae, tomography, data processing and correlative light and electron microscopy that have enabled these studies. Finally, we explore the future of cryo-ET in terms of both methods development and biological application.

Combined online Bayesian and windowed estimation of background and signal localization facilitates active-feedback particle tracking in complex environments.

Despite successes in tracking single molecules in vitro, the extension of active-feedback single-particle methods to tracking rapidly diffusing and unconfined proteins in live cells has not been realized. Since the existing active-feedback localization methods localize particles in real time assuming zero background, they are ill-suited to track in the inhomogeneous background environment of a live cell. Here, we develop a windowed estimation of signal and background levels using recent data to estimate the current particle brightness and background intensity. These estimates facilitate recursive Bayesian position estimation, improving upon current Kalman-based localization methods. Combined, online Bayesian and windowed estimation of background and signal (COBWEBS) surpasses existing 2D localization methods. Simulations demonstrate improved localization accuracy and responsivity in a homogeneous background for selected particle and background intensity combinations. Improved or similar performance of COBWEBS tracking extends to the majority of signal and background combinations explored. Furthermore, improved tracking durations are demonstrated in the presence of heterogeneous backgrounds for multiple particle intensities, diffusive speeds, and background patterns. COBWEBS can accurately track particles in the presence of high and nonuniform backgrounds, including intensity changes of up to three times the particle's intensity, making it a prime candidate for advancing active-feedback single fluorophore tracking to the cellular interior.

Mitochondria-targeted and pH-triggered charge-convertible polymeric micelles for anticancer therapy

Mitochondria-targeted and pH-triggered charge-convertible polymeric micelles (GPTD) were developed for doxorubicin (DOX) delivery and targeted anticancer therapy. GPTD surface was negatively charged under physiological pH (7.4), while it converted to positive charge under tumor pH (6.8). Sustained release of DOX from the drug loaded polymeric micelles was observed and the drug release was accelerated by decreasing the medium pH from 7.4 to 6.8. Cellular uptake by U251 glioma cells showed that GPTD can enhance the accumulation of polymeric micelles inside the mitochondria after charge-mediated cellular internalization and release DOX into the cellular interior. In vivo anticancer activity performed on glioma Balb/c nude mice demonstrated that the DOX-containing GPTD could remarkably inhibit the tumor growth, and displayed a significant reduction of tumor tissue cells and fewer Ki67 positive cells compared to the PBS group. Hence, this mitochondrial targeted and pH-triggered charge-convertible polymeric micellar system showed substantial promise for anticancer therapy.

NLRP3 Inflammasome Simultaneously Involved in Autophagy and Phagocytosis of THP-1 Cells to Clear Aged Erythrocytes.

Autophagy and phagocytosis are two important processes that capture and digest materials found in cellular interiors and exteriors, respectively. Aged red blood cells (RBCs) are cleared by phagocytes in vivo. We focused on determining whether autophagy occurs after phagocytes swallow sunset erythrocytes, and whether the degree of autophagy is related to scavenging ability of phagocytes to erythrocytes. In addition, the ability of NLR family pyrin domain containing protein 3 (NLRP3) inflammasome to regulate erythrocyte clearance by phagocytes and its association with autophagy-related protein 16-like protein 1 (ATG16L1) are confirmed. We constructed a stable and low-NLRP3 expression THP-1 cell line using CRISPR/Cas9 technology. The analysis of erythrocyte clearance and autophagy of THP-1 cells with low NLRP3 expression showed that autophagy changes together when THP-1 engulfs aged RBCs. The occurrence of autophagy was dominated by microtubule-associated protein 1A/1B-light chain 3- (LC3-) associated phagocytosis accompanied by canonical autophagy. A negative correlation exists between the clearance of RBCs by THP-1 cells and the degree of autophagy. Downregulating the expression of NLRP3 in THP-1 cells can simultaneously inhibit the scavenging ability of THP-1 to erythrocytes and the degree of autophagy. In addition, the autophagy inhibitor bafilomycin A1 (BafA1) can enhance the phagocytosis ability of THP-1 to erythrocytes and promote the NLRP3 activation in THP-1 cells, while the autophagy inducer rapamycin inhibits the phagocytosis ability of THP-1 to RBCs and downregulates the NLRP3 activation. Results showed that autophagy and phagocytosis may be dynamic balance processes that can provide sufficient nutrition and energy to cells. Choosing NLRP3 as a target may regulate the phagocytic ability and the degree of autophagy in the meantime. These findings may be a potential strategy for regulating the clearance rate of phagocytes to aged RBCs and the secretion of proinflammatory cytokines to ensure transfusion safety.

Deformation faulting and dislocation-cell refinement in a selective laser melted 316L stainless steel

The selective laser melted (SLM) 316L stainless steel (316L SS) has shown superior tensile ductility and doubled yield strength compared to its wrought counterpart. The significantly improved yield strength has been attributed to the unique cellular substructures featured by Cr/Mo-segregation and trapped dislocations. The excellent ductility of SLMed 316L SS has been mainly understood from the pronounced deformation twinning, which, however, is just one of the dominant plastic deformation mechanisms in 316L SS. Here instead, we report two other fundamental deformation micro-mechanisms, i.e., deformation faulting and dislocation cell refinement, in the SLM 316L SS. Our results showed that deformation faulting and dislocation cell refinement characterize the whole tensile deformation significantly except for deformation twinning. These newly found mechanisms synergistically dominated the deformation behavior at low strain levels while deformation faulting and twinning play a crucial role at medium and high strain levels. The pre-existing stacking faults (SFs) in cellular substructure is mainly responsible for the significant deformation faulting. At the early deformation stage, these pre-existing SFs provide faulting nuclei for deformation faulting, leading to wide SFs. These wide SFs in different cellular interiors penetrate the cellular boundaries and overlap as the strain increases, resulting in long stacking fault ribbons (SFRs). We attributed the formation of DCs to the measured medium stacking fault energy (SFE) of ∼18 mJ/m2. Upon straining, equiaxed dislocation cells (DCs) were generated by new dislocation cell walls (DCWs) inside the cellular substructure, and refined by either the dissociation of fully developed DCWs or the continuously formed new DCWs. Together with deformation twinning, these two fundamental deformation mechanisms jointly led to a steady strain hardening rate during tension and thus a superior tensile ductility of the SLM 316L SS with high yield strength. These findings provide new insights into the excellent strength-ductility combination of SLMed 316L SS and the experimental basis for crystal plasticity modeling and simulation, as well.

Effect of annealing treatment on microstructure evolution and deformation behavior of 304 L stainless steel made by laser powder bed fusion

This paper focuses on the microstructural evolution of 304 L austenitic stainless steel (SS) manufactured by laser powder bed fusion (LPBF) after stress-relieving annealing (650 °C) and solution annealing (1050 °C). Multiple advanced characterizations were adopted to disclose the microstructural characteristics and investigate the annealing-driven dislocation migration process. At 650 °C, the dislocation density of cellular walls decreased slightly, associated with a slight decrease of strength. At 1050 °C, the dislocations of cellular walls migrated to more energetically favorable regions, forming subgrain boundaries with higher dislocation density and resulting in a strength-ductility trade-off. The temperature of 1050 °C could slightly increase the recrystallization volume fraction and induce the coalescence of multi-oriented fine-grained tribes into single-oriented grains. The nano-scale characterization indicated that the as-built samples and annealed samples at 650 °C contained the square lattice distortion networks composed of orthogonal strain ripples. However, after annealing at 1050 °C, only unidirectional strain ripples in square lattice distortion networks were retained due to unidirectional dislocation migration. Direct experimental results were provided that the local misorientation ranges of cellular interior, cellular walls, and newly formed subgrain boundaries were <0.2°, 0.2°-0.5°, and 0.5°-2°, respectively. After tensile deformation, interacting deformation twins occurred in <111> and 〈101〉 oriented grains of as-built and annealed specimens at 650 °C, while the twins occurred in all oriented grains of annealed specimens at 1050 °C due to the disappearance of cellular substructure and the increase of tensile elongation. This work yields new insights into misorientation across the cellular walls, dislocation migration process during annealing, strengthening mechanisms, and work hardening behaviors, which can be used to design and optimize future annealing routines for LPBF materials.

Decoding molecular plasticity of the dark proteome

The mechanisms by which intrinsically disordered proteins (IDPs) engage in rapid and highly selective binding is a subject of considerable interest and represents a central paradigm to nuclear pore complex (NPC) function. Nuclear transport receptors (NTRs) can move through the central channel of the NPC which is filled with hundreds of phenylalanine‐glycine‐rich nucleoporins (FG‐Nups) reaching millimolar concentrations with elusive conformational plasticity. Since site‐specific labeling of proteins with small but highly photostable fluorescent dyes inside cells remains the major bottleneck for directly studying protein dynamics in the cellular interior, we have now developed a semi‐synthetic strategy based on novel artificial amino acids that are easily and site‐specifically introduced into any protein by the natural machinery of the living cell via a newly developed thin‐film synthetic organelle that equips the living cell with up to three genetic codes. This allowed us to develop an experimental approach combining site‐specific fluorescent labeling of IDPs in non‐fixed cells with fluorescent lifetime imaging microscopy (FLIM) to directly decipher the plasticity of FG‐Nups via FRET. Our study enabled a conformational look on the condensated IDPs in the sub‐resolution (roughly (50 nm)3 small cavity) cavity of the NPC. By measuring the end‐to‐end distances of different segments of the labeled FG‐Nups using FLIM‐FRET, we can extract the scaling exponent, which directly describes the conformations of FG‐Nups at their functional status as well as the solvent quality in the cellular and even inner NPC environment.Reinkemeier CD, Lemke EA. Dual film‐like organelles enable spatial separation of orthogonal eukaryotic translation. Cell. 2021 Sep 16;184(19):4886‐4903.e21Celetti G, Paci G, Caria J, VanDelinder V, Bachand G, Lemke EA. The liquid state of FG‐nucleoporins mimics permeability barrier properties of nuclear pore complexes. J Cell Bio. (2020) Jan 6;219(1).Reinkemeier CD, Estrada Girona G, Lemke EA, 2019 Designer membraneless organelles enable codon reassignment of selected mRNAs in eukaryotes. Science, Mar 29;363(6434)Nikić I, Estrada Girona G, Kang JH, Paci G, rei S, Koehler C, Shymanska NV, Ventura Santos C, Spitz D, Lemke EA, Debugging Eukaryotic Genetic Code Expansion for Site‐Specific Click‐PAINT Super‐Resolution Microscopy. Angew Chem Int Ed Engl. (2016) Dec 23;55(52):16172‐16176