Rapid plasma membrane isolation via intracellular polymerization-mediated biomolecular confinement.

Plasma membrane isolation is a foundational process in membrane proteomic research, cellular vesicle studies, and biomimetic nanocarrier development, yet separation processes for this outermost layer are cumbersome and susceptible to impurities and low yield. Herein, we demonstrate that cellular cytosol can be chemically polymerized for decoupling and isolation of plasma membrane within minutes. A rapid, non-disruptive in situ polymerization technique is developed with cell membrane-permeable polyethyleneglycol-diacrylate (PEG-DA) and a blue-light-sensitive photoinitiator, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP). The photopolymerization chemistry allows for precise control of intracellular polymerization and tunable confinement of cytosolic molecules. Upon cytosol solidification, plasma membrane proteins and vesicles are rapidly derived and purified as nucleic acids and intracellular proteins as small as 15 kDa are stably entrapped for removal. The polymerization chemistry and membrane derivation technique are broadly applicable to primary and fragile cell types, enabling facile membrane vesicle extraction from shorted-lived neutrophils and human primary CD8 T cells. The study demonstrates tunable intracellular polymerization via optimized live cell chemistry, offers a robust membrane isolation methodology with broad biomedical utility, and reveals insights on molecular crowding and confinement in polymerized cells. STATEMENT OF SIGNIFICANCE: Isolating the minute fraction of plasma membrane proteins and vesicles requires extended density gradient ultracentrifugation processes, which are susceptible to low yield and impurities. The present work demonstrates that the membrane isolation process can be vastly accelerated via a rapid, non-disruptive intracellular polymerization approach that decouples cellular cytosols from the plasma membrane. Following intracellular polymerization, high-yield plasma membrane proteins and vesicles can be derived from lysis buffer and sonication treatment, respectively. And the intracellular content entrapped within the polymerized hydrogel is readily removed within minutes. The technique has broad utility in membrane proteomic research, cellular vesicle studies, and biomimetic materials development, and the work offers insights on intracellular hydrogel-mediated molecular confinement.

Engineering Cyborg Bacteria Through Intracellular Hydrogelation.

Natural and artificial cells are two common chassis in synthetic biology. Natural cells can perform complex tasks through synthetic genetic constructs, but their autonomous replication often causes safety concerns for biomedical applications. In contrast, artificial cells based on nonreplicating materials, albeit possessing reduced biochemical complexity, provide more defined and controllable functions. Here, for the first time, the authors create hybrid material-cell entities termed Cyborg Cells. To create Cyborg Cells, a synthetic polymer network is assembled inside each bacterium, rendering them incapable of dividing. Cyborg Cells preserve essential functions, including cellular metabolism, motility, protein synthesis, and compatibility with genetic circuits. Cyborg Cells also acquire new abilities to resist stressors that otherwise kill natural cells. Finally, the authors demonstrate the therapeutic potential by showing invasion into cancer cells. This work establishes a new paradigm in cellular bioengineering by exploiting a combination of intracellular man-made polymers and their interaction with the protein networks of living cells.

Synthetic Immunogenic Cell Death Mediated by Intracellular Delivery of STING Agonist Nanoshells Enhances Anticancer Chemo-immunotherapy.

Many favorable anticancer treatments owe their success to the induction immunogenic cell death (ICD) in cancer cells, which results in the release of endogenous danger signals along with tumor antigens for effective priming of anticancer immunity. We describe a strategy to artificially induce ICD by delivering the agonist of stimulator of interferon genes (STING) into tumor cells using hollow polymeric nanoshells. Following intracellular delivery of exogenous adjuvant, subsequent cytotoxic treatment creates immunogenic cellular debris that spatiotemporally coordinate tumor antigens and STING agonist in a process herein termed synthetic immunogenic cell death (sICD). sICD is indiscriminate to the type of chemotherapeutics and enables colocalization of exogenously administered immunologic adjuvants and tumor antigens for enhanced antigen presentation and anticancer adaptive response. In three mouse tumor models, sICD enhances therapeutic efficacy and restrains tumor progression. The study highlights the benefit of delivering STING agonists to cancer cells, paving ways to new chemo-immunotherapeutic designs.

Intracellular hydrogelation preserves fluid and functional cell membrane interfaces for biological interactions.

Cell membranes are an intricate yet fragile interface that requires substrate support for stabilization. Upon cell death, disassembly of the cytoskeletal network deprives plasma membranes of mechanical support and leads to membrane rupture and disintegration. By assembling a network of synthetic hydrogel polymers inside the intracellular compartment using photo-activated crosslinking chemistry, we show that the fluid cell membrane can be preserved, resulting in intracellularly gelated cells with robust stability. Upon assessing several types of adherent and suspension cells over a range of hydrogel crosslinking densities, we validate retention of surface properties, membrane lipid fluidity, lipid order, and protein mobility on the gelated cells. Preservation of cell surface functions is further demonstrated with gelated antigen presenting cells, which engage with antigen-specific T lymphocytes and effectively promote cell expansion ex vivo and in vivo. The intracellular hydrogelation technique presents a versatile cell fixation approach adaptable for biomembrane studies and biomedical device construction.

NMDAR signaling facilitates the IPO5-mediated nuclear import of CPEB3.

Cytoplasmic polyadenylation element-binding protein (CPEB)3 is a nucleocytoplasm-shuttling RNA-binding protein and predominantly resides in the cytoplasm where it represses target RNA translation. When translocated into the nucleus, CPEB3 binds to Stat5b and downregulates Stat5b-dependent transcription. In neurons, the activation of N-methyl-d-aspartate receptors (NMDARs) accumulates CPEB3 in the nucleus and redistributes CPEB3 in the nucleocytoplasmic compartments to control gene expression. Nonetheless, it is unclear which karyopherin drives the nuclear import of CPEB3 and which transport direction is most affected by NMDA stimulation to increase the nuclear pool of CPEB3. Here, we have identified that the karyopherins, IPO5 and CRM1, facilitate CPEB3 translocation by binding to RRM1 and a leucine-containing motif of CPEB3, respectively. NMDAR signaling increases RanBP1 expression and reduces the level of cytoplasmic GTP-bound Ran. These changes enhance CPEB3-IPO5 interaction, which consequently accelerates the nuclear import of CPEB3. This study uncovers a novel NMDA-regulated import pathway to facilitate the nuclear translocation of CPEB3.

Functional dissection of lysine deacetylases reveals that HDAC1 and p300 regulate AMPK.

First identified as histone-modifying proteins, lysine acetyltransferases (KATs) and deacetylases (KDACs) antagonize each other through modification of the side chains of lysine residues in histone proteins. Acetylation of many non-histone proteins involved in chromatin, metabolism or cytoskeleton regulation were further identified in eukaryotic organisms, but the corresponding enzymes and substrate-specific functions of the modifications are unclear. Moreover, mechanisms underlying functional specificity of individual KDACs remain enigmatic, and the substrate spectra of each KDAC lack comprehensive definition. Here we dissect the functional specificity of 12 critical human KDACs using a genome-wide synthetic lethality screen in cultured human cells. The genetic interaction profiles revealed enzyme-substrate relationships between individual KDACs and many important substrates governing a wide array of biological processes including metabolism, development and cell cycle progression. We further confirmed that acetylation and deacetylation of the catalytic subunit of the adenosine monophosphate-activated protein kinase (AMPK), a critical cellular energy-sensing protein kinase complex, is controlled by the opposing catalytic activities of HDAC1 and p300. Deacetylation of AMPK enhances physical interaction with the upstream kinase LKB1, leading to AMPK phosphorylation and activation, and resulting in lipid breakdown in human liver cells. These findings provide new insights into previously underappreciated metabolic regulatory roles of HDAC1 in coordinating nutrient availability and cellular responses upstream of AMPK, and demonstrate the importance of high-throughput genetic interaction profiling to elucidate functional specificity and critical substrates of individual human KDACs potentially valuable for therapeutic applications.

Vaccinia virus 4c (A26L) protein on intracellular mature virus binds to the extracellular cellular matrix laminin.

Vaccinia virus intracellular mature virus (IMV) binds to glycosaminoglycans (GAGs) on cells via three virion proteins, H3L, A27L, and D8L. In this study, we demonstrated that binding of IMV to BSC40 cells was competitively inhibited by soluble laminin but not by fibronectin or collagen V, suggesting that this cell surface extracellular matrix (ECM) protein may play a role in vaccinia virus entry. Moreover, IMV infection of GAG(-) sog9 cells was also inhibited by laminin, demonstrating that virion binding to laminin does not involve a prior interaction with GAGs. Furthermore, comparative envelope protein analyses of wild-type vaccinia virus strain Western Reserve, which binds to laminin, and of a mutant virus, IA27L, which does not, showed that the A26L open reading frame (ORF), encoding an envelope protein, was mutated in IA27L, resulting in A26L being absent from the IMV. Expression of the wild-type A26L ORF in IA27L resulted in laminin binding activity. Moreover, recombinant A26L protein bound to laminin in vitro with a high affinity, providing direct evidence that A26L is the laminin binding protein on IMV. In summary, these results reveal a novel role for the vaccinia viral envelope protein A26L in binding to the ECM protein laminin, an association that is proposed to facilitate IMV entry.