Functionalized chitosan for cancer nano drug delivery.

Chitosan is a biotechnological derivative of chitin receiving a widespread pharmaceutical and biomedical applications. It can be used to encapsulate and deliver cancer therapeutics with inherent pH-dependent solubility to confer drug targeting at tumour microenvironment and anti-cancer activity synergizing cancer cytotoxic drug actions. To further reduce the off-target and by-stander adverse effects of drugs, a high targeted drug delivery efficiency at the lowest possible drug doses is clinically required. The chitosan has been functionalized with covalent conjugates or complexes and processed into nanoparticles to encapsulate and control drug release, to avoid premature drug clearance, to deliver drugs passively and actively to cancer site at tissue, cell or subcellular levels, and to promote cancer cell uptake of nanoparticles through membrane permeabilization at higher specificity and scale. Nanomedicine developed using functionalized chitosan translates to significant preclinical improvements. Future challenges related to nanotoxicity, manufacturability, selection precision of conjugates and complexes as a function of cancer omics and their biological responses from administration site to cancer target need critical assessments.

α-Synuclein is required for sperm exocytosis at a post-fusion stage.

The sperm acrosome is a large dense-core granule whose contents are secreted by regulated exocytosis at fertilization through the opening of numerous fusion pores between the acrosomal and plasma membranes. In other cells, the nascent pore generated when the membrane surrounding a secretory vesicle fuses with the plasma membrane may have different fates. In sperm, pore dilation leads to the vesiculation and release of these membranes, together with the granule contents. α-Synuclein is a small cytosolic protein claimed to exhibit different roles in exocytic pathways in neurons and neuroendocrine cells. Here, we scrutinized its function in human sperm. Western blot revealed the presence of α-synuclein and indirect immunofluorescence its localization to the acrosomal domain of human sperm. Despite its small size, the protein was retained following permeabilization of the plasma membrane with streptolysin O. α-Synuclein was required for acrosomal release, as demonstrated by the inability of an inducer to elicit exocytosis when permeabilized human sperm were loaded with inhibitory antibodies to human α-synuclein. The antibodies halted calcium-induced secretion when introduced after the acrosome docked to the cell membrane. Two functional assays, fluorescence and transmission electron microscopies revealed that the stabilization of open fusion pores was responsible for the secretion blockage. Interestingly, synaptobrevin was insensitive to neurotoxin cleavage at this point, an indication of its engagement in cis SNARE complexes. The very existence of such complexes during AE reflects a new paradigm. Recombinant α-synuclein rescued the inhibitory effects of the anti-α-synuclein antibodies and of a chimeric Rab3A-22A protein that also inhibits AE after fusion pore opening. We applied restrained molecular dynamics simulations to compare the energy cost of expanding a nascent fusion pore between two model membranes and found it higher in the absence than in the presence of α-synuclein. Hence, our results suggest that α-synuclein is essential for expanding fusion pores.

Pulse Parameters and Thresholds for (ir)Reversible Electroporation on Hepatocellular Carcinoma Cells in Vitro.

Hepatocellular carcinoma is a leading cause of cancer-related death in many parts of the world. Traditional treatment options are not always effective. During the promising minimally invasive electroporation-based therapies, biological cell membranes are exposed to an external, sufficiently high, pulsed electric field which creates so-called nanopores into the lipid bilayer of the cell membrane. These pores can either be permanent (irreversible electroporation (IRE)), leading to apoptosis, or repairable (reversible electroporation (RE)), with continued cell function. In tumor therapy, RE is used to increase the diffusion of a chemotherapeutic drug during electrochemotherapy. For both IRE and RE, the success of the treatment is dependent on application of the appropriate electric field. Therefore, this study aims to define the pulse parameters and thresholds for IRE and RE on hepatocellular carcinoma (HepG2) cells in-vitro.In a custom-made in-vitro setup, HepG2 cell viability (0, 5, 10, and 15 min), and the peak temperature were measured after electroporation with the different IRE and RE pulsing protocols, to determine the most successful settings for IRE and RE. A CAM/PI flow cytometric assay was performed to confirm cell permeabilization for the RE pulsing protocols with the highest cell viability.The results indicated that an IRE pulsing protocol (70 pulses, 100 µs pulse length, and 100 ms interval) with an electric field strength of 4000 V/cm was needed as threshold for almost complete cell death of HepG2 cells. A RE pulsing protocol (8 pulses, 100 µs pulse length, and 1000 ms interval) with an electric field strength of 1000 V/cm was needed as threshold for viable and permeabilized HepG2 cells. The low peak temperatures (max 30.1°C for IRE, max 23.1°C for RE) within this study indicated that the reduction in HepG2 cell viability was caused by the applied electric field and was not a result of Joule heating.

In Vitro Methods to Investigate the Disassembly of the Golgi Ribbon During the G2-M Transition of the Cell Cycle.

The Golgi complex is the central hub of the secretory pathway. In mammalian cells, it is formed by stacks of flattened cisternae organized in a continuous membrane system, the Golgi ribbon, located near the centrosome. During G2, the Golgi ribbon is disassembled into isolated stacks that, at the onset of mitosis, are further fragmented into small tubular-vesicular clusters that disperse throughout the cytoplasm. Here, we describe a set of methods to study the Golgi complex in different phases of the cell cycle, drawing attention to reproducing the mitotic Golgi fragmentation to gain knowledge and acquire the skills to study the mechanisms that regulate mitotic Golgi reorganization as well as its biological significance. The investigations based on these assays have been instrumental in understanding that Golgi disassembly is not only a consequence of mitosis but is also required for mitotic entry and cell division.

Effect of Shock Waves on the Growth of Aspergillus niger Conidia: Evaluation of Germination and Preliminary Study on Gene Expression.

Shock waves, as used in medicine, can induce cell permeabilization, genetically transforming filamentous fungi; however, little is known on the interaction of shock waves with the cell wall. Because of this, the selection of parameters has been empirical. We studied the influence of shock waves on the germination of Aspergillus niger, to understand their effect on the modulation of four genes related to the growth of conidia. Parameters were varied in the range reported in protocols for genetic transformation. Vials containing conidia in suspension were exposed to either 50, 100 or 200 single-pulse or tandem shock waves, with different peak pressures (approximately 42, 66 and 83 MPa). In the tandem mode, three delays were tested. To equalize the total energy, the number of tandem "events" was halved compared to the number of single-pulse shock waves. Our results demonstrate that shock waves do not generate severe cellular effects on the viability and germination of A. niger conidia. Nevertheless, increase in the aggressiveness of the treatment induced a modification in four tested genes. Scanning electron microscopy revealed significant changes to the cell wall of the conidia. Under optimized conditions, shock waves could be used for several biotechnological applications, surpassing conventional techniques.

Efficient incorporation of p-azido-l-phenylalanine into the protein using organic solvents.

Azide, the most used photo-crosslinking group, facilitates the analysis of protein structure and function. This group is particularly useful when photochemically label antibodies and examine protein-protein interactions. The use of the expanded genetic code technique allows the special labeling of the functional azide group in proteins by adding the unnatural amino acid (UAA), p-azido-l-phenylalanine (AzF), in response to the amber codon during translation. However, a low UAA uptake rate due to mass transfer resistance in the cell membrane may lead to the early termination of the full-length protein. This study reports a general method for the efficient in vivo incorporation of AzF into the target protein by improving cell permeability using organic solvents. As expected, the yield of the full-length protein was significantly increased, which indicated that the AzF uptake was greatly improved due to the addition of organic solvents. Our method can serve as a good reference for improving the genetic incorporation of other kinds of UAAs into proteins.

Single-Cell Analysis of the Antimicrobial and Bactericidal Activities of the Antimicrobial Peptide Magainin 2.

Antimicrobial peptides (AMPs) inhibit the proliferation of or kill bacterial cells. To measure these activities, several methods have been used, which provide only the average value of many cells. Here, we report the development of a method to examine the antimicrobial and bactericidal activities of AMPs at the single-cell level (i.e., single-cell analysis) and apply this strategy to examine the interaction of an AMP, magainin 2 (Mag), with Escherichia coli cells. Using this method, we monitored the proliferation of single cells on agar in a microchamber and measured the distribution of the number of cells in each microcolony using optical microscopy. For method A, we incubated cells in the presence of various concentrations of AMPs for 3 h. The fraction of microcolonies containing only a single cell, Psingle, increased with the Mag concentration and reached 1 at a specific concentration, which corresponded to the MIC. For method B, after the interaction of a cell suspension with an AMP for a specific time, an aliquot was diluted to stop the interaction, and the proliferation of single cells then was monitored after a 3-h incubation; this method permits the definition of Psingle(t), the fraction of dead cells after the interaction. For the interaction of Mag with E. coli cells, Psingle(t) increased with the interaction time, reaching ~1 at 10 and 20 min for 25 and 13 µM Mag, respectively. Thus, these results indicate that a short interaction time between Mag and E. coli cells is sufficient to induce bacterial cell death. IMPORTANCE To elucidate the activity of antimicrobial peptides (AMPs) against bacterial cells, it is important to estimate the interaction time that is sufficient to induce cell death. We have developed a method to examine the antimicrobial and bactericidal activities of AMPs at the single-cell level (i.e., single-cell analysis). Using this method, we monitored the proliferation of single cells on agar in a microchamber and measured the distribution of the number of cells in each microcolony using optical microscopy. We found that during the interaction of magainin 2 (Mag) with E. coli cells, the fraction of dead cells, Psingle(t), increased with the interaction time, rapidly reaching 1 (e.g., 10 min for 25 µM Mag). This result indicates that Mag induces cell death after a short time of interaction.

AAV6 as an effective gene delivery vector for prolonged transgene expression in intervertebral disc cells in vivo.

Intervertebral disc degeneration is the main contributor to low back pain, now the leading cause of disability worldwide. Gene transfer, either in a therapeutic attempt or in basic research to understand the mechanisms of disc degeneration, is a fascinating and promising tool to manipulate the complex physiology of the disc. Viral vectors based on the adeno-associated virus (AAV) have emerged as powerful transgene delivery vehicles yet a systematic investigation into their respective tropism, transduction efficiency, and relative toxicity have not yet been performed in the disc in vivo. Herein, we used in vivo bioluminescence imaging to systematically compare multiple AAV serotypes, injection volumes, titers, promoters, and luciferase reporters to determine which result in high transduction efficiency of murine nucleus pulposus (NP) cells in vivo. We find that AAV6 using a CAG promoter to drive transgene expression, delivered into the NP of murine caudal discs at a titer of 1011 GC/mL, provides excellent transduction efficiency/kinetics and low toxicity in vivo. We also show, for the first time, that the transduction of NP cells can be significantly boosted in vivo by the use of small cell permeabilization peptides. Finally, to our knowledge, we are the first to demonstrate the use of optical tissue clearing and three-dimensional lightsheet microscopy in the disc, which was used to visualize fine details of tissue and cell architecture in whole intact discs following AAV6 delivery. Taken together, these data will contribute to the success of using AAV-mediated gene delivery for basic and translational studies of the IVD.

A Quantitative Flow Cytometry-Based Method for Autophagy Detection Across the Cell Cycle.

Autophagy is deregulated in cancer cells and often activated as a cellular stress response to anticancer therapies. Flow cytometry-based assays enable detection and quantification of various cellular markers in live or fixed cells. Here, a flow cytometry-based assay to characterize autophagy across the cell cycle is described. This method is based on selective plasma membrane permeabilization with digitonin and extraction of membrane-unbound LC3 protein followed by staining of the autophagosome-bound LC3 protein with antibody and labeling of DNA with propidium iodide. Staining with the LC3 antibody described here can be also combined with the staining of other cellular markers, allowing to quantitatively assess autophagy in relation to different cellular processes by flow cytometry.

Targeted protein degradation by Trim-Away using cell resealing coupled with microscopic image-based quantitative analysis.

"Trim-Away" technology enables rapid degradation of endogenous proteins without prior modification of protein-coding genes or mRNAs through delivery of antibodies that target proteins of interest. Although this approach can be readily applied to almost any cytosolic protein, strategies for cytosolic antibody delivery have been limited to microinjection or electroporation, which require skill-dependent operation or specialized equipment. Thus, the development of antibody delivery methods that are convenient, scalable, and preferably do not require detachment of adherent cells is required to extend the versatility of the Trim-Away method. Here, we developed a cell resealing technique optimized for Trim-Away degradation, which uses the pore-forming toxin streptolysin O (SLO) to permeabilize the cell membrane and delivered the antibodies of interest into HEK293T, HeLa, and HK-2 cell lines. We demonstrated the ability of Trim-Away protein degradation using IKKα and mTOR as targets, and we showed the availability of the developed system in antibody screening for the Trim-Away method. Furthermore, we effectively coupled Trim-Away with cyclic immunofluorescence and microscopic image-based analysis, which enables single-cell multiplexed imaging analysis. Taking advantage of this new analysis strategy, we were able to compensate for low signal-to-noise due to cell-to-cell variation, which occurs in the Trim-Away method because of the heterogenous contents of the introduced antibody, target protein, and TRIM21 in individual cells. Therefore, the reported cell resealing technique coupled with microscopic image analysis enables Trim-Away users to elucidate target protein function and the effects of target protein degradation on various cellular functions in a more quantitative and precise manner.

The ß-galactosidase assay in perspective: Critical thoughts for biosensor development.

Recently, the ß-galactosidase assay has become a key component in the development of assays and biosensors for the detection of enterobacteria and E. coli in water quality monitoring. The assay has often performed below its maximum potential, mainly due to a poor choice of conditions. In this study we establish a set of optimal conditions and provide a rough estimate of how departure from optimal values reduces the output of the assay potentially decreasing its sensitivity. We have established that maximum response for detecting low cell concentrations requires an induction of the samples using IPTG at a concentration of 0.2 mM during 180 min. Permeabilization of the samples is mandatory as lack of it results in an almost 60% reduction in assay output. The choice of enzyme substrate is critical as different substrates yield products with different extinction coefficients or fluorescence yields. The concentration of substrate used must be high enough (around 3 to 4 times Km) to ensure that the activity measured is not substrate limited. Finally, as the color/fluorescence of the reaction products is highly dependent on pH, care must be taken to ensure that pH at the time of reading is high enough to provide maximum signal.

Optimization of cell permeabilization in electron flow based mitochondrial function assays.

Permeable cell models have contributed much to the progress in mitochondrial research. Optimization of permeabilization is required to make the cell's plasma membrane permeable to small molecules while keeping the intracellular organelles and their membranes intact and fully functional. Here we report our assessment and optimization of commonly used permeabilizing agents including different saponin preparations, digitonin, and recombinant perfringolysin O employing a new electron flow based mitochondrial assay technology that utilizes a colorimetric redox dye. The results of this study provide guidance in optimizing the conditions for mitochondrial function assays with permeabilized cells using the novel redox dye-based format.

P2X7 Receptors and TMEM16 Channels Are Functionally Coupled with Implications for Macropore Formation and Current Facilitation.

P2X7 receptors (P2X7) are cationic channels involved in many diseases. Following their activation by extracellular ATP, distinct signaling pathways are triggered, which lead to various physiological responses such as the secretion of pro-inflammatory cytokines or the modulation of cell death. P2X7 also exhibit unique behaviors, such as "macropore" formation, which corresponds to enhanced large molecule cell membrane permeability and current facilitation, which is caused by prolonged activation. These two phenomena have often been confounded but, thus far, no clear mechanisms have been resolved. Here, by combining different approaches including whole-cell and single-channel recordings, pharmacological and biochemical assays, CRISPR/Cas9 technology and cell imaging, we provide evidence that current facilitation and macropore formation involve functional complexes comprised of P2X7 and TMEM16, a family of Ca2+-activated ion channel/scramblases. We found that current facilitation results in an increase of functional complex-embedded P2X7 open probability, a result that is recapitulated by plasma membrane cholesterol depletion. We further show that macropore formation entails two distinct large molecule permeation components, one of which requires functional complexes featuring TMEM16F subtype, the other likely being direct permeation through the P2X7 pore itself. Such functional complexes can be considered to represent a regulatory hub that may orchestrate distinct P2X7 functionalities.

Cell transmembrane potential in contactless permeabilization by time-varying magnetic fields.

BACKGROUND: Although experimental results proved the feasibility of using time-varying magnetic field as a contactless cells permeabilization method, the underlying mechanism is still poorly understood. In this study a numerical analysis of the time-dependent transmembrane potential (TMP) at cell membranes during permeabilization by time-varying magnetic fields was proposed, and a first quantification of mechanical stress induced by the magnetic and electric fields, hypothesized to play an important role in the permeabilization mechanism, was carried out. METHODS: Starting from the simulation of real in vitro experimental conditions, the analysis was widened quantifying the influence of pulse frequency, cell dimension and distance of the cell from the magnetic field source. The mechanical pressure on cell membrane due to the interaction between free charges and induced electric field and due to the gradient of the magnetic field was quantified in all those conditions in which the TMP values were not high enough to cause membrane permeabilization. RESULTS: TMP values induced by typical in-vitro experimental conditions were far below the values needed for membrane permeabilization, with a strong dependence on pulse frequency and distance of the cell from the coil. CONCLUSION: The preliminary assessment of the mechanical pressure on cell membrane showed that stress values evaluated in conditions in which TMP values were too low to cause membrane permeabilization were comparable to those known to influence the pores opening mechanisms. Results represent a significant step towards a better comprehension of the mechanism underlying cell membrane permeabilization by time-varying magnetic fields.

Conformational Plasticity of Cyclic Ras-Inhibitor Peptides Defines Cell Permeabilization Activity.

Cyclorasins 9A5 and 9A54 are 11-mer cyclic peptides that inhibit the Ras-Raf protein interaction. The peptides share a cell-penetrating peptide (CPP)-like motif; however, only cyclorasin 9A5 can permeabilize cells to exhibit strong cell-based activity. To unveil the structural origin underlying their distinct cellular permeabilization activities, we compared the three-dimensional structures of cyclorasins 9A5 and 9A54 in water and in the less polar solvent dimethyl sulfoxide (DMSO) by solution NMR. We found that cyclorasin 9A5 changes its extended conformation in water to a compact amphipathic structure with converged aromatic residues surrounded by Arg residues in DMSO, which might contribute to its cell permeabilization activity. However, cyclorasin 9A54 cannot adopt this amphipathic structure, due to the steric hindrance between two neighboring bulky amino-acid sidechains, Tle-2 and dVal-3. We also found that the bulkiness of the sidechains at positions 2 and 3 negatively affects the cell permeabilization activities, indicating that the conformational plasticity that allows the peptides to form the amphipathic structure is important for their cell permeabilization activities.

Complete Biotransformation of Protopanaxadiol-Type Ginsenosides into 20-O-ß-Glucopyranosyl-20(S)-protopanaxadiol by Permeabilized Recombinant Escherichia coli Cells Coexpressing ß-Glucosidase and Chaperone Genes.

The ginsenoside 20-O-ß-glucopyranosyl-20(S)-protopanaxadiol or compound K is an essential ingredient in functional food, cosmetics, and traditional medicines. However, no study has reported the complete conversion of all protopanaxadiol (PPD)-type ginsenosides from ginseng extract into compound K using whole-cell conversion. To increase the production of compound K from ginseng extract using whole recombinant cells, the ß-glucosidase enzyme from Caldicellulosiruptor bescii was coexpressed with a chaperone expression system (pGro7), and the cells expressing the coexpression system were permeabilized with ethylenediaminetetraacetic acid. The permeabilized cells carrying the chaperone coexpression system showed a 2.6-fold increase in productivity and yield as compared with nontreated cells, and completely converted all PPD-type ginsenosides from ginseng root extract into compound K with the highest productivity among the results reported so far. Our results will contribute to the industrial biological production of compound K.

Preparation of Plant Material for Analysis of Protein-Nucleic Acid Interactions by FRET-FLIM.

DNA-binding proteins are involved in the dynamic regulation of various cellular processes such as recombination, replication, and transcription. For investigating dynamic assembly and disassembly of molecular complexes in living cells, fluorescence microscopy represents a tremendous tool in biology. A fluorescence resonance energy transfer (FRET) approach coupled to fluorescence lifetime imaging microscopy (FLIM) has been used recently to monitor protein-DNA associations in plant cells. With this approach, the donor fluorophore is a GFP-tagged binding partner expressed in plant cells. A Sytox® Orange treatment converts nuclear nucleic acids to FRET acceptors. A decrease of GFP lifetime is due to FRET between donor and acceptor, indicating close association of the GFP binding partner and Sytox® Orange-stained DNA. In this chapter, we present a step-by-step protocol for the transient expression in N. benthamiana of GFP-tagged proteins and the fixation and permeabilization procedures used for the preparation of plant material aimed at detecting protein-nucleic acid interactions by FRET-FLIM measurements.

Structural and functional characterization of the membrane-permeabilizing activity of Nicotiana occidentalis defensin NoD173 and protein engineering to enhance oncolysis.

Defensins are an extensive family of host defense peptides found ubiquitously across plant and animal species. In addition to protecting against infection by pathogenic microorganisms, some defensins are selectively cytotoxic toward tumor cells. As such, defensins have attracted interest as potential antimicrobial and anticancer therapeutics. The mechanism of defensin action against microbes and tumor cells appears to be conserved and involves the targeting and disruption of cellular membranes. This has been best defined for plant defensins, which upon binding specific phospholipids, such as phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidic acid, form defensin-lipid oligomeric complexes that destabilize membranes, leading to cell lysis. In this study, to further define the anticancer and therapeutic properties of plant defensins, we have characterized a novel plant defensin, Nicotiana occidentalis defensin 173 (NoD173), from N. occidentalis. NoD173 at low micromolar concentrations selectively killed a panel of tumor cell lines over normal primary cells. To improve the anticancer activity of NoD173, we explored increasing cationicity by mutation, with NoD173 with the substitution of Q22 with lysine [NoD173(Q22K)], increasing the antitumor cell activity by 2-fold. NoD173 and the NoD173(Q22K) mutant exhibited only low levels of hemolytic activity, and both maintained activity against tumor cells in serum. The ability of NoD173 to inhibit solid tumor growth in vivo was tested in a mouse B16-F1 model, whereby injection of NoD173 into established subcutaneous tumors significantly inhibited tumor growth. Finally, we showed that NoD173 specifically targets PIP2 and determined by X-ray crystallography that a high-resolution structure of NoD173, which forms a conserved family-defining cysteine-stabilized-αß motif with a dimeric lipid-binding conformation, configured into an arch-shaped oligomer of 4 dimers. These data provide insights into the mechanism of how defensins target membranes to kill tumor cells and provide proof of concept that defensins are able to inhibit tumor growth in vivo.-Lay, F. T., Ryan, G. F., Caria, S., Phan, T. K., Veneer, P. K., White, J. A., Kvansakul, M., Hulett M. D. Structural and functional characterization of the membrane-permeabilizing activity of Nicotiana occidentalis defensin NoD173 and protein engineering to enhance oncolysis.

Simultaneous Recording of Subcellular Ca2+ Signals from the Cytosol and Sarco/Endoplasmic Reticulum: Compartmentalized Dye Loading, Imaging, and Analysis.

An increase in the cytosolic Ca2+ concentration triggers the contraction in cardiomyocytes. In these cells sarcoplasmic reticulum (SR) is the major source of Ca2+, and the release from this store is mediated by the ryanodine receptors (RyRs). These receptors are regulated by cytosolic and intra-SR [Ca2+]. The cytosolic Ca2+ regulation is well established, but there are some limitations to determine indirectly the intra-SR Ca2+ concentration and understand its role in the RyRs regulation. Therefore, the interest to directly measure the free intra-SR Ca2+ concentration ([Ca2+]SR) has led to the application of a low-affinity Ca2+ indicator (Fluo-5N AM) to follow changes of [Ca2+]SR in cardiomyocytes. However the loading of this AM-ester dye into the SR has remained a challenge in freshly isolated mouse cardiomyocytes. Here, we describe an optimized protocol to measure changes of [Ca2+]SR in mouse cardiomyocytes using fluorescent Ca2+ indicators and confocal microscopy. The application of this protocol allows to evaluate directly intra-SR Ca2+ in real time in various mouse models of cardiac disease, including transgenic animals harboring mutants of RyRs or other Ca2+ signaling proteins.

Liquid- and solid-like RNA granules form through specific scaffold proteins and combine into biphasic granules.

RNA granules consist of membrane-less RNA-protein assemblies and contain dynamic liquid-like shells and stable solid-like cores, which are thought to function in numerous processes in mRNA sorting and translational regulation. However, how these distinct substructures are formed, whether they are assembled by different scaffolds, and whether different RNA granule scaffolds induce these different substructures remains unknown. Here, using fluorescence microscopy-based morphological and molecular-dynamics analyses, we demonstrate that RNA granule scaffold proteins (scaffolds) can be largely classified into two groups, liquid and solid types, which induce the formation of liquid-like and solid-like granules, respectively, when expressed separately in cultured cells. We found that when co-expressed, the liquid-type and solid-type scaffolds combine and form liquid- and solid-like substructures in the same granules, respectively. The combination of the different types of scaffolds reduced the immobile fractions of the solid-type scaffolds and their dose-dependent ability to decrease nascent polypeptides in granules, but had little effect on the dynamics of the liquid-type scaffolds or their dose-dependent ability to increase nascent polypeptides in granules. These results suggest that solid- and liquid-type scaffolds form different substructures in RNA granules and differentially affect each other. Our findings provide detailed insight into the assembly mechanism and distinct dynamics and functions of core and shell substructures in RNA granules.