Intracellular Behavior Research Articles

Convergence of Artificial Protein Polymers and Intrinsically Disordered Proteins.

A flurry of research in recent years has revealed the molecular origins of many membraneless organelles to be the liquid phase separation of intrinsically disordered proteins (IDPs). Consequently, protein disorder has emerged as an important driver of intracellular compartmentalization by providing specialized microenvironments chemically distinct from the surrounding medium. Though the importance of protein disorder and its relationship to intracellular phase behavior are clear, a detailed understanding of how such phase behavior can be predicted and controlled remains elusive. While research in IDPs has largely focused on the implications of structural disorder on cellular function and disease, another field, that of artificial protein polymers, has focused on the de novo design of protein polymers with controllable material properties. A subset of these polymers, specifically those derived from structural proteins such as elastin and resilin, are also disordered sequences that undergo liquid-liquid phase separation. This phase separation has been used in a variety of biomedical applications, and researchers studying these polymers have developed methods to precisely characterize and tune their phase behavior. Despite their disparate origins, both fields are complementary as they study the phase behavior of intrinsically disordered polypeptides. This Perspective hopes to stimulate collaborative efforts by highlighting the similarities between these two fields and by providing examples of how such collaboration could be mutually beneficial.

Nonvectorial responses in photoreceptor cells stimulated by electrical fields

The eye possesses various different types of electrical properties, and ocular cells are exposed to endogenous electrical fields (EFs) under natural conditions. Cellular behaviors that are regulated by EFs include vectorial (migration, orientation, and elongation) and nonvectorial responses (apoptosis and proliferation). The aim of this study was to investigate the nonvectorial responses of photoreceptor cells stimulated by EFs and to decipher the underlying molecular mechanisms. To investigate the effects of endogenous EFs on the cell behavior, we designed a customized electrical instrument that was mimetic of endogenous electrical activity. We observed that 60 and 90 mV/mm voltage gradients dramatically enhanced cell proliferation. Gene expression profiling, together with signaling pathway and gene ontology analyses, collectively demonstrated that the Ca2+-dependent extracellular signal-regulated kinase pathway is involved in cellular responses to EFs. Moreover, the results of our study clearly demonstrated that EFs promote photoreceptor cell proliferation via increased Ca2+ influx, which subsequently activates the ERK pathway. EF stimulation affects ionic fluxes in photoreceptor cells and is an important regulator of some important aspects of photoreceptor cell behavior. In conclusion, the findings of our study offer new insights on how EFs affect intracellular Ca2+, which further influences intracellular signal transduction and cell behavior.

Characterization of the mechanical properties of cancer cells in 3D matrices in response to collagen concentration and cytoskeletal inhibitors.

Cellular processes, such as cell migration, adhesion, and proliferation depend on the interaction between the intracellular environment and the extracellular matrix (ECM). While many studies have explored the role of the microenvironment on cell behavior, the influence of 3D matrix mechanics on intracellular activity is not completely understood. To characterize the relationship between the mechanical components of the microenvironment and intracellular behavior, we use particle-tracking microrheology of metastatic breast cancer cells embedded in 3D collagen gels to quantify the intracellular activity from which the molecular motor activity and stiffness can be determined. Our results show that increasing collagen concentration of the 3D environments leads to an increase in intracellular stiffness and motor activity. Furthermore, our studies demonstrate that intracellular fluctuations depend on collagen concentration, even in the presence of a number of frontline chemotherapeutic and anti-MMP drugs, indicating that ECM concentration is an important and indispensable parameter to consider in drug screening.

Gold–carbon dots for the intracellular imaging of cancer-derived exosomes

As a novel fluorescent nanomaterial, gold–carbon quantum dots (GCDs) possess high biocompatibility and can be easily synthesized by a microwave-assisted method. Owing to their small sizes and unique optical properties, GCDs can be applied to imaging of biological targets, such as cells, exosomes and other organelles. In this study, GCDs were used for fluorescence imaging of exosomes. Tumor-specific antibodies are attached to the GCDs, forming exosome specific nanoprobes. The nanoprobes can label exosomes via immuno-reactions and thus facilitate fluorescent imaging of exosomes. When incubated with live cells, exosomes labeled with the nanoprobes can be taken up by the cells. The intracellular experiments confirmed that the majority of exosomes were endocytosed by cells and transported to lysosomes. The manner by which exosomes were taken up and the intracellular distribution of exosomes are unaffected by the GCDs. The experimental results successfully demonstrated that the presented nanoprobe can be used to study the intrinsic intracellular behavior of tumor derived exosomes. We believe that the GCDs based nanoprobe holds a great promise in the study of exosome related cellular events, such as cancer metastasis.

Studies on the preparation and controlled release of redox/pH-responsive zwitterionic nanoparticles based on poly-L-glutamic acid and cystamine

The enhancement of tumor intracellular drug uptake and resistance against nonspecific protein adsorption are essential for an injectable anticancer drug carrier. In the present study, a new type of redox/pH-responsive zwitterionic nanoparticles (NPs) was prepared using poly-L-glutamic acid and cystamine in aqueous solutions under mild conditions. The NPs showed surface charge convertible feature in response to pH change of the solutions. The NPs demonstrated excellent anti nonspecific protein adsorption. In vitro release profiles of the NPs, they showed redox/pH dual sensitivities in vitro release. The effective intracellular delivery behaviors were verified through investigation of cell viability, and confocal laser scanning microscopy observation of HeLa cells after incubation with the DOX-loaded NPs. The NPs were non-cytotoxic and would have potential applications as a drug delivery vehicle for enhancing intracellular uptake of anticancer drugs.

Subcellular storage and release mode of the novel 18F-labeled sympathetic nerve PET tracer LMI1195

Background18F-N-[3-bromo-4-(3-fluoro-propoxy)-benzyl]-guanidine (18F-LMI1195) is a new class of PET tracer designed for sympathetic nervous imaging of the heart. The favorable image quality with high and specific neural uptake has been previously demonstrated in animals and humans, but intracellular behavior is not yet fully understood. The aim of the present study is to verify whether it is taken up in storage vesicles and released in company with vesicle turnover.ResultsBoth vesicle-rich (PC12) and vesicle-poor (SK-N-SH) norepinephrine-expressing cell lines were used for in vitro tracer uptake studies. After 2 h of 18F-LMI1195 preloading into both cell lines, effects of stimulants for storage vesicle turnover (high concentration KCl (100 mM) or reserpine treatment) were measured at 10, 20, and 30 min. 131I-meta-iodobenzylguanidine (131I-MIBG) served as a reference. Both high concentration KCl and reserpine enhanced 18F-LMI1195 washout from PC12 cells, while tracer retention remained stable in the SK-N-SH cells. After 30 min of treatment, 18F-LMI1195 releasing index (percentage of tracer released from cells) from vesicle-rich PC12 cells achieved significant differences compared to cells without treatment condition. In contrast, such effect could not be observed using vesicle-poor SK-N-SH cell lines. Similar tracer kinetics after KCl or reserpine treatment were also observed using 131I-MIBG. In case of KCl exposure, Ca2+-free buffer with the calcium chelator, ethylenediaminetetracetic acid (EDTA), could suppress the tracer washout from PC12 cells. This finding is consistent with the tracer release being mediated by Ca2+ influx resulting from membrane depolarization.ConclusionsAnalogous to 131I-MIBG, the current in vitro tracer uptake study confirmed that 18F-LMI1195 is also stored in vesicles in PC12 cells and released along with vesicle turnover. Understanding the basic kinetics of 18F-LMI1195 at a subcellular level is important for the design of clinical imaging protocols and imaging interpretation.

Effect of the precooling rate on intracellular ice formation behavior

Effect of the precooling rate on intracellular ice formation behavior

Linking wound response and inflammation to regeneration in the zebrafish larval fin.

The study of regenerative biology aims to elucidate the innate ability of organisms to replace tissues or organs after they have been removed or damaged. The zebrafish is a powerful model for the analysis of intracellular signalling and cell behaviour and as such has made major contributions to our understanding of regenerative biology. The larval fin fold is an emerging model to understand how different signalling pathways interact to coordinate regeneration. Tissue damage causes the immediate release of signals that initiate wound closure and inflammation. Following this, regenerative cells proliferate and migrate to the damaged area. Each of these processes has been analysed using the larval fin fold model to provide a framework for how fin regeneration takes place. This review gives an overview of the current state of this field with particular emphasis on the different signalling networks that are required during fin fold regeneration.

Bioreducible nanocapsules for folic acid-assisted targeting and effective tumor-specific chemotherapy.

IntroductionIncreasing demands in precise control over delivery and functionalization of therapeutic agents for tumor-specific chemotherapy have led to a rapid development in nanocarriers. Herein, we report a nanocapsule (NC) system for tumor-oriented drug delivery and effective tumor therapy.Materials and methodsFunctionalized hyaluronan is utilized to build up the NC shells, in which bioreduction cleavable sites, targeting ligand folic acid (FA), and zwitterionic tentacles are integrated.ResultsThe hollow NCs obtained (~50 nm in diameter) showed well-defined spherical shell structures with a shell thickness of ~8 nm. These specially designed NCs (doxorubicin [DOX]/FA-Z-NCs) with high drug encapsulation content exhibited good biocompatibility in vitro and fast intracellular drug release behavior mediated by intracellular glutathione.ConclusionCellular uptake tests demonstrated rapid uptake of these functionalized NCs and effective escape from endosomes. Antitumor efficacy of the DOX/FA-Z-NCs was confirmed by the significant tumor growth inhibition effect as well as greatly reduced side effects, in contrast with those of the free drug DOX hydrochloride.

Investigating the Intracellular Behaviors of Liposomal Nanohybrids via SERS: Insights into the Influence of Metal Nanoparticles.

The recent proposition to combine liposomes with nanoparticles presents great opportunities to develop multifunctional drug delivery platforms. Although impressive progress has been made, attempts to elucidate the role nanoparticles play in the integral nanohybrids are still rather limited. Here, using surface-enhanced Raman scattering (SERS) technique, we investigate the influence of metal nanoparticles on the liposomal properties, ranging from drug release to intracellular movement. Specifically, we prepared SERS-active nanohybrids by attaching metal nanoparticles to liposomes and employed SERS signals to explore the intracellular behavior of the nanohybrids. Once deposited on the cell membrane, the nanohybrids entered tumor cells via clathrin-mediated endocytosis and then moved to lysosomes. In comparison with pure liposomes, metal nanoparticles in the nanohybrids had little influence on the properties of liposomes. This study fills the gap of the function of nanoparticles in the overall nanohybrids, which provides a significant prerequisite for efficient drug delivery in therapeutic applications.

Approaches for the successful isolation and cell culture of American Rickettsia species.

Rickettsia are intracellular vector-borne bacteria, which are the etiologic agent of severe infections that could inflict death to their host. The intracellular behaviour of Rickettsia makes the study of its genetics, proteomics and cellular processes very difficult. Hence, isolation remains an important experimental technique that permits the obtention of important yields of bacteria, useful for a broad range of experiments. Isolation of Rickettsia using passages in animals or embryonated eggs has been described for long time; however, it was until the 1990s that faster and more feasible approaches for cell culture were developed. Current isolation approaches are mainly based on shell vial culture, that varies according to the media, atmosphere or temperature conditions. These variations have allowed the establishment of isolates from different pathogenic and non-pathogenic Rickettsia species, using arthropod, animal or human samples. Purification method of bacteria has also witnessed changes alongside the quantification of its load in the resulting isolates, from the laborious and time consuming plaque assays, to the routinary use of real-time polymerase chain reaction (qPCR), which is faster and more accurate. This review discusses various approaches that have been used for the isolation and purification of different Rickettsia species along with the mention of some successful examples. It indicated that a successful strategy for the isolation of Rickettsia requires a careful selection of media, cell lines and culture conditions which now are not as time consuming as used to be.

Biocompatible protamine sulfate@silicon nanoparticle-based gene nanocarriers featuring strong and stable fluorescence.

The development of biocompatible and fluorescent gene carriers is of particular importance in the gene-delivery field. Taking advantage of the unique optical properties (e.g., strong and robust fluorescence) of silicon nanoparticles (SiNPs), as well as the excellent biocompatibility of silicon and protamine sulfate (PS, approved by the U.S. Food and Drug Administration (FDA) for clinical use), we herein present a type of PS-modified SiNP (PS@SiNP)-based gene carrier. Plasmid DNA (pDNA) with negative charges can be effectively bound onto the surface of the as-prepared fluorescent PS@SiNP-based gene carriers via electrostatic interactions. In particular, such resultant gene carriers possess stable and high fluorescence (photoluminescent quantum yield (PLQY): ∼25%). In addition, the PS@SiNP-based gene carriers show minimal toxic effects on normal mitochondrial metabolic activity (e.g., human retinal pigment epithelial (ARPE-19) cells preserve ∼90% of their cell viability after a 48 h incubation with the resultant carriers). Based on tracking the strong and stable fluorescence signals of SiNPs, the dynamic behavior of the PS@SiNP-based gene carriers in live cells (e.g., clathrin-mediated endocytosis, lysosomal escape, pDNA release, etc.) is investigated in a long-term manner, providing valuable information for understanding the intracellular behavior of gene vectors and designing high-efficacy gene carriers.

Physiological relevance of covalent protein modification by dietary isothiocyanates.

Isothiocyanates (ITCs), naturally occurring in abundance in cruciferous vegetables, are the most well-studied organosulfur compounds having an electrophilic reactivity. ITCs have been accepted as major ingredients of these vegetables that afford their health promoting potentials. ITCs are able to modulate protein functions related to drug-metabolizing enzymes, transporters, kinases and phosphatases, etc. One of the most important questions about the molecular basis for the health promoting effects of ITCs is how they modulate cellular target proteins. Although the molecular targets of ITCs remains to be validated, dietary modulation of the target proteins via covalent modification by ITCs should be one of the promising strategies for the protection of cells against oxidative and inflammatory damage. This review discusses the plausible target proteins of dietary ITCs with an emphasis on possible involvement of protein modification in their health promoting effects. The fundamental knowledge of ITCs is also included with consideration of the chemistry, intracellular behavior, and metabolism.

An Updated View on the Rck Invasin of Salmonella: Still Much to Discover.

Salmonella is a facultative intracellular Gram-negative bacterium, responsible for a wide range of food- and water-borne diseases ranging from gastroenteritis to typhoid fever depending on hosts and serotypes. Salmonella thus represents a major threat to public health. A key step in Salmonella pathogenesis is the invasion of phagocytic and non-phagocytic host cells. To trigger its own internalization into non-phagocytic cells, Salmonella has developed different mechanisms, involving several invasion factors. For decades, it was accepted that Salmonella could only enter cells through a type three secretion system, called T3SS-1. Recent research has shown that this bacterium expresses outer membrane proteins, such as the Rck protein, which is able to induce Salmonella entry mechanism. Rck mimics natural host cell ligands and triggers engulfment of the bacterium by interacting with the epidermal growth factor receptor. Salmonella is thus able to use multiple entry pathways during the Salmonella infection process. However, it is unclear how and when Salmonella exploits the T3SS-1 and Rck entry mechanisms. As a series of reviews have focused on the T3SS-1, this review aims to describe the current knowledge and the limitations of our understanding of the Rck outer membrane protein. The regulatory cascade which controls Rck expression and the molecular mechanisms underlying Rck-mediated invasion into cells are summarized. The potential role of Rck-mediated invasion in Salmonella pathogenesis and the intracellular behavior of the bacteria following a Salmonella Rck-dependent entry are discussed.

Intracellular metal binding and redox behavior of human DJ-1.

DJ-1 is a conserved, ubiquitous protein associated to a large number of intracellular processes. Human DJ-1 has been linked to several pathologies, including hereditary forms of Parkinson's disease, cancer, and amyotrophic lateral sclerosis. Several cytoprotective functions of DJ-1 have been reported, however, its actual mechanisms of action remain elusive. In vitro, DJ-1 has been shown to bind zinc and copper(II) at its active site, which contains a conserved cysteine (C106), and copper(I) at a different binding site. C106 is essential to DJ-1 function, and is easily oxidized upon oxidative stress. Here, we investigated the metal-binding- and redox properties of DJ-1 in living human cells by in-cell NMR. Intracellular DJ-1 is surprisingly free from interactions with any other cellular components and as such is clearly detectable by NMR. Metal-bound forms of DJ-1 were not observed upon treating the cells with excess zinc or copper. No copper binding was observed when co-expressing DJ-1 with the copper chaperone for superoxide dismutase 1 (SOD1). Co-expression of DJ-1 with SOD1 itself did not promote copper binding to SOD1, excluding a previously suggested function of DJ-1 as a copper chaperone. Overall, our data do not support the role of DJ-1 as a metalloprotein. Conversely, oxidative treatment to the cells caused the complete and selective oxidation of C106 to sulfinic acid, consistent with the reported role of DJ-1 as a redox sensor.

Multifunctional nanoparticles co-delivering EZH2 siRNA and etoposide for synergistic therapy of orthotopic non-small-cell lung tumor

Malignant proliferation and metastasis in non-small cell lung carcinoma (NSCLC) are great challenges for effective clinical treatment through conventional chemotherapy. The combinational therapy strategy of RNA interfering (RNAi) technology and chemotherapeutic agents have been reported to be promising for effective cancer therapy. In this study, based on multifunctional nanoparticles (NPs), the simultaneous delivery of etoposide (ETP) and anti-Enhancer of Zeste Homologue 2 (EZH2) siRNA for the effective treatment of orthotopic lung tumor was achieved. The NPs exhibited pH/redox dual sensitivity verified by particle size changes, morphological changes, and in vitro release of drugs. Confocal microscopy analysis confirmed that the NPs exhibited endosomal escape property and on-demand intracellular drug release behavior, which can protect siRNA from degradation and facilitate the chemotherapeutic effect respectively. In vitro tumor cell motility study demonstrated that EZH2 siRNA loaded in NPs can decrease the migration and invasion capabilities of tumor cells by downregulating the expression of EZH2 mRNA and protein. In particular, an antiproliferation study revealed that the co-delivery of siRNA and ETP in the multifunctional NPs can induce a synergistic therapeutic effect on NSCLC. In vivo targeting evaluation showed that cRGDyC-PEG modification on NPs exhibited a low distribution in normal organs and an obvious accumulation in orthotopic lung tumor. Furthermore, targeted NPs co-delivering siRNA and ETP showed superior inhibition on tumor growth and metastasis and produced minimal systemic toxicity. These findings indicated that multifunctional NPs can be utilized as a co-delivery system, and that the combination of EZH2 siRNA and ETP can effectively treat NSCLC.

ERP arrangement: a strategy for assembled genomic contig rearrangement based on replication profiling in bacteria

BackgroundThe reduced cost of sequencing has made de novo sequencing and the assembly of draft microbial genomes feasible in any ordinary biology lab. However, the process of finishing and completing the genome remains labor-intensive and computationally challenging in some cases, such as in the study of complete genome sequences, genomic rearrangements, long-range syntenic relationships, and structural variations.MethodsHere, we show a contig reordering strategy based on experimental replication profiling (eRP) to recapitulate the bacterial genome structure within draft genomes. During the exponential growth phase, the majority of bacteria show a global genomic copy number gradient that is enriched near the replication origin and gradually declines toward the terminus. Therefore, if genome sequencing is performed with appropriate timing, the short-read coverage reflects this copy number gradient, providing information about the contig positions relative to the replication origin and terminus.ResultsWe therefore investigated the appropriate timing for genomic DNA sampling and developed an algorithm for the reordering of the contigs based on eRP. As a result, this strategy successfully recapitulates the genomic structure of various structural mutants with draft genome sequencing.ConclusionsOur strategy was successful for contig rearrangement with intracellular DNA replication behavior mechanisms and can be applied to almost all bacteria because the DNA replication system is highly conserved. Therefore, eRP makes it possible to understand genomic structural information and long-range syntenic relationships using a draft genome that is based on short reads.

Disulfide‐bond‐containing agamatine‐cystaminebisacrylamide polymer demonstrates better transfection efficiency and lower cytotoxicity than polyethylenimine in NIH/3T3 cells

Previously, we synthesized a non-viral vector containing disulfide bond by polymerization of agamatine (AGM) and N,N'-cystaminebisacrylamide (CBA). In this study, we investigated the transfection efficiency of disulfide bond (SS) containing AGM-CBA polymer in gene delivery into NIH/3T3 cells, and examined the factors affecting its transfection efficiency by comparing with polyethylenimine (PEI). In addition, experiments were carried out to determine the mechanisms of cell entry pathways and intracellular behavior of AGM-CBA/pDNA polyplexes. The transfection efficiency of AGM-CBA/pDNA with different weight ratios and different amounts of pDNA was measured and the pathways mediated transfection processes were studied by using various endocytosis inhibitors. To determine the intracellular behavior of AGM-CBA/pDNA polyplexes, the transfection efficiencies of AGM-CBA/pDNA and PEI/pDNA polyplexes with different combination structures were determined by using reporter gene and fake plasmid DNA. The transfection efficiency of AGM-CBA/pDNA polyplexes was correlated with its weight ratio of AGM-CBA and pDNA, and the amount of pDNA. Both AGM-CBA/pDNA and PEI/pDNA polyplexes enter into cell by clathrin- and caveolae-mediated endocytic pathways. However, AGM-CBA/pDNA showed different intracellular behavior in NIH/3T3 cells compared to PEI/pDNA polyplexes. It was hypothesized that disulfide bond in AGM-CBA could be an important factor contributing to its intracellular behavior and better transfection efficiency. Overall, AGM-CBA demonstrated better transfection efficiency and lower cytotoxicity than PEI in NIH/3T3 cells as a gene delivery vector.

Complex Consisting of β-Glucan and Antigenic Peptides with Cleavage Site for Glutathione and Aminopeptidases Induces Potent Cytotoxic T Lymphocytes.

The efficient induction of antigen-specific immune responses requires not only promotion of the uptake of antigens and adjuvant molecules into antigen-presenting cells but also control of their intracellular behavior. We previously demonstrated that the β-glucan schizophyllan (SPG) can form complexes with CpG oligonucleotides with attached dA40 (CpG-dA/SPG), which can accumulate in macrophages in the draining inguinal lymph nodes and induce strong immune responses. In this study, we prepared various conjugates composed of antigenic peptide (OVA257-264) and dA40 and made complexes with SPG. The conjugates with a disulfide bond between OVA257-264 and dA40 were easily cleaved by glutathione. The resultant peptides with a hydrophobic amino acid at the C-terminal end was recognized by puromycin-insensitive leucine aminopeptidase (PILS-AP), which trims antigenic peptide precursors and prepares peptides of eight or nine amino acids in length, which is the optimal length for binding to major histocompatibility complex (MHC)-I. The conjugate exposed to such enzymes induced a high antigen presentation level. The antigen presentation level was almost the same before and after the complexation with SPG. Immunization with a mixture of dA-OVA257-264/SPG and CpG-dA/SPG induced high antigen-specific cytotoxic T-lymphocyte activity at a much lower peptide dose than in previous studies. These results can be strongly ascribed to not only the cell-specific delivery by SPG but also the control of the intracellular behavior by the introduction of cleavage sites. Therefore, peptide-dA/SPG complexes could be used as potent vaccine antigens for the treatment of cancers and infectious diseases.

Intracellular Survival of Staphylococcus aureus in Endothelial Cells: A Matter of Growth or Persistence.

The Gram-positive human pathogen Staphylococcus aureus is a leading cause of severe bacterial infections. Recent studies have shown that various cell types could readily internalize S. aureus and infected cells have been proposed to serve as vehicle for the systemic dissemination of the pathogen. Here we focused on the intracellular behavior of the Community-Associated Methicillin-Resistant S. aureus strain USA300. Supporting earlier observations, we found that wild-type S. aureus strain USA300 persisted for longer period within endothelial cells than within macrophages and that a mutant displaying the small colony variant phenotype (ΔhemDBL) had increased intracellular persistence. Time-lapse microscopy revealed that initial persistence of wild-type bacteria in endothelial cells corresponded to distinct single cell events, ranging from active intracellular bacterial proliferation, leading to cell lysis, to non-replicating bacterial persistence even 1 week after infection. In sharp contrast, ΔhemDBL mutant bacteria were essentially non-replicating up to 10 days after infection. These findings suggest that internalization of S. aureus in endothelial cells triggers its persistence and support the notion that endothelial cells might constitute an intracellular persistence niche responsible for reported relapse of infection after antibiotic therapy.