Internalization of novel non-viral vector TAT-streptavidin into human cells

BACKGROUND: The cell-penetrating peptide derived from the Human immunodeficiency virus-1 transactivator protein Tat possesses the capacity to promote the effective uptake of various cargo molecules across the plasma membrane in vitro and in vivo. The objective of this study was to characterize the uptake and delivery mechanisms of a novel streptavidin fusion construct, TAT(47–57)-streptavidin (TAT-SA, 60 kD). SA represents a potentially useful TAT-fusion partner due to its ability to perform as a versatile intracellular delivery vector for a wide array of biotinylated molecules or cargoes. RESULTS: By confocal and immunoelectron microscopy the majority of internalized TAT-SA was shown to accumulate in perinuclear vesicles in both cancer and non-cancer cell lines. The uptake studies in living cells with various fluorescent endocytic markers and inhibiting agents suggested that TAT-SA is internalized into cells efficiently, using both clathrin-mediated endocytosis and lipid-raft-mediated macropinocytosis. When endosomal release of TAT-SA was enhanced through the incorporation of a biotinylated, pH-responsive polymer poly(propylacrylic acid) (PPAA), nuclear localization of TAT-SA and TAT-SA bound to biotin was markedly improved. Additionally, no significant cytotoxicity was detected in the TAT-SA constructs. CONCLUSION: This study demonstrates that TAT-SA-PPAA is a potential non-viral vector to be utilized in protein therapeutics to deliver biotinylated molecules both into cytoplasm and nucleus of human cells

Parvovirus Induced Alterations in Nuclear Architecture and Dynamics

The nucleus of interphase eukaryotic cell is a highly compartmentalized structure containing the three-dimensional network of chromatin and numerous proteinaceous subcompartments. DNA viruses induce profound changes in the intranuclear structures of their host cells. We are applying a combination of confocal imaging including photobleaching microscopy and computational methods to analyze the modifications of nuclear architecture and dynamics in parvovirus infected cells. Upon canine parvovirus infection, expansion of the viral replication compartment is accompanied by chromatin marginalization to the vicinity of the nuclear membrane. Dextran microinjection and fluorescence recovery after photobleaching (FRAP) studies revealed the homogeneity of this compartment. Markedly, in spite of increase in viral DNA content of the nucleus, a significant increase in the protein mobility was observed in infected compared to non-infected cells. Moreover, analyzis of the dynamics of photoactivable capsid protein demonstrated rapid intranuclear dynamics of viral capsids. Finally, quantitative FRAP and cellular modelling were used to determine the duration of viral genome replication. Altogether, our findings indicate that parvoviruses modify the nuclear structure and dynamics extensively. Intranuclear crowding of viral components leads to enlargement of the interchromosomal domain and to chromatin marginalization via depletion attraction. In conclusion, parvoviruses provide a useful model system for understanding the mechanisms of virus-induced intranuclear modifications

G2/M checkpoint regulation and apoptosis facilitate the nuclear egress of parvoviral capsids

The nuclear export factor CRM1-mediated pathway is known to be important for the nuclear egress of progeny parvovirus capsids in the host cells with virus-mediated cell cycle arrest at G2/M. However, it is still unclear whether this is the only pathway by which capsids exit the nucleus. Our studies show that the nuclear egress of DNA-containing full canine parvovirus. capsids was reduced but not fully inhibited when CRM1-mediated nuclear export was prevented by leptomycin B. This suggests that canine parvovirus capsids might use additional routes for nuclear escape. This hypothesis was further supported by our findings that nuclear envelope (NE) permeability was increased at the late stages of infection. Inhibitors of cell cycle regulatory protein cyclin-dependent kinase 1 (Cdk1) and pro-apoptotic caspase 3 prevented the NE leakage. The change in NE permeability could be explained by the regulation of the G2/M checkpoint which is accompanied by early mitotic and apoptotic events. The model of G2/M checkpoint activation was supported by infection-induced nuclear accumulation of cyclin B1 and Cdk1. Both NE permeability and nuclear egress of capsids were reduced by the inhibition of Cdk1. Additional proof of checkpoint function regulation and promotion of apoptotic events was the nucleocytoplasmic redistribution of nuclear transport factors, importins, and Ran, in late infection. Consistent with our findings, post-translational histone acetylation that promotes the regulation of several genes related to cell cycle transition and arrest was detected. In conclusion, the model we propose implies that parvoviral capsid egress partially depends on infection-induced G2/M checkpoint regulation involving early mitotic and apoptotic events.Peer reviewe

Light-Induced Nanoscale Deformation in Azobenzene Thin Film Triggers Rapid Intracellular Ca2+ Increase via Mechanosensitive Cation Channels

Epithelial cells are in continuous dynamic biochemical and physical interaction with their extracellular environment. Ultimately, this interplay guides fundamental physiological processes. In these interactions, cells generate fast local and global transients of Ca2+ ions, which act as key intracellular messengers. However, the mechanical triggers initiating these responses have remained unclear. Light-responsive materials offer intriguing possibilities to dynamically modify the physical niche of the cells. Here, a light-sensitive azobenzene-based glassy material that can be micropatterned with visible light to undergo spatiotemporally controlled deformations is used. Real-time monitoring of consequential rapid intracellular Ca2+ signals reveals that the mechanosensitive cation channel Piezo1 has a major role in generating the Ca2+ transients after nanoscale mechanical deformation of the cell culture substrate. Furthermore, the studies indicate that Piezo1 preferably responds to shear deformation at the cell-material interphase rather than to absolute topographical change of the substrate. Finally, the experimentally verified computational model suggests that Na+ entering alongside Ca2+ through the mechanosensitive cation channels modulates the duration of Ca2+ transients, influencing differently the directly stimulated cells and their neighbors. This highlights the complexity of mechanical signaling in multicellular systems. These results give mechanistic understanding on how cells respond to rapid nanoscale material dynamics and deformations.Peer reviewe

Toward Xeno-Free Differentiation of Human Induced Pluripotent Stem Cell-Derived Small Intestinal Epithelial Cells

The small intestinal epithelium has an important role in nutrition, but also in drug absorption and metabolism. There are a few two-dimensional (2D) patient-derived induced pluripotent stem cell (iPSC)-based intestinal models enabling easy evaluation of transcellular transport. It is known that animal-derived components induce variation in the experimental outcomes. Therefore, we aimed to refine the differentiation protocol by using animal-free components. More specifically, we compared maturation of 2D-cultured iPCSs toward small intestinal epithelial cells when cultured either with or without serum, and either on Geltrex or on animal-free, recombinant laminin-based substrata. Differentiation status was characterized by qPCR, immunofluorescence imaging, and functionality assays. Our data suggest that differentiation toward definitive endoderm is more efficient without serum. Both collagen-and recombinant laminin-based coating supported differentiation of definitive endoderm, posterior definitive endoderm, and small intestinal epithelial cells from iPS-cells equally well. Small intestinal epithelial cells differentiated on recombinant laminin exhibited slightly more enterocyte specific cellular functionality than cells differentiated on Geltrex. Our data suggest that functional small intestinal epithelial cells can be generated from iPSCs in serum-free method on xeno-free substrata. This method is easily converted to an entirely xeno-free method.publishedVersionPeer reviewe

Parvovirus nonstructural protein 2 interacts with chromatin-regulating cellular proteins

Autonomous parvoviruses encode at least two nonstructural proteins, NS1 and NS2. While NS1 is linked to important nuclear processes required for viral replication, much less is known about the role of NS2. Specifically, the function of canine parvovirus (CPV) NS2 has remained undefined. Here we have used proximity-dependent biotin identification (BioID) to screen for nuclear proteins that associate with CPV NS2. Many of these associations were seen both in noninfected and infected cells, however, the major type of interacting proteins shifted from nuclear envelope proteins to chromatin-associated proteins in infected cells. BioID interactions revealed a potential role for NS2 in DNA remodeling and damage response. Studies of mutant viral genomes with truncated forms of the NS2 protein suggested a change in host chromatin accessibility. Moreover, further studies with NS2 mutants indicated that NS2 performs functions that affect the quantity and distribution of proteins linked to DNA damage response. Notably, mutation in the splice donor site of the NS2 led to a preferred formation of small viral replication center foci instead of the large coalescent centers seen in wild-type infection. Collectively, our results provide insights into potential roles of CPV NS2 in controlling chromatin remodeling and DNA damage response during parvoviral replication.Peer reviewe

Protein Diffusion in Mammalian Cell Cytoplasm

We introduce a new method for mesoscopic modeling of protein diffusion in an entire cell. This method is based on the construction of a three-dimensional digital model cell from confocal microscopy data. The model cell is segmented into the cytoplasm, nucleus, plasma membrane, and nuclear envelope, in which environment protein motion is modeled by fully numerical mesoscopic methods. Finer cellular structures that cannot be resolved with the imaging technique, which significantly affect protein motion, are accounted for in this method by assigning an effective, position-dependent porosity to the cell. This porosity can also be determined by confocal microscopy using the equilibrium distribution of a non-binding fluorescent protein. Distinction can now be made within this method between diffusion in the liquid phase of the cell (cytosol/nucleosol) and the cytoplasm/nucleoplasm. Here we applied the method to analyze fluorescence recovery after photobleach (FRAP) experiments in which the diffusion coefficient of a freely-diffusing model protein was determined for two different cell lines, and to explain the clear difference typically observed between conventional FRAP results and those of fluorescence correlation spectroscopy (FCS). A large difference was found in the FRAP experiments between diffusion in the cytoplasm/nucleoplasm and in the cytosol/nucleosol, for all of which the diffusion coefficients were determined. The cytosol results were found to be in very good agreement with those by FCS

Structural dynamics of tight junctions modulate the properties of the epithelial barrier

<div><p>Tight junctions are dynamic structures that are crucial in establishing the diffusion and electrical barrier of epithelial monolayers. Dysfunctions in the tight junctions can impede this barrier function and lead to many pathological conditions. Unfortunately, detailed understanding of the non-specific permeation pathway through the tight junctions, the so-called leak pathway, is lacking. We created computational models of the leak pathway to describe the two main barrier measures, molecular permeability and transepithelial electric resistance while using common structural dynamics. Our results showed that the proposed alternatives for the leak pathway, the bicellular strand opening dynamics and the tricellular pores, contribute together with distinct degrees, depending on the epithelium. The models can also capture changes in the tight junction barrier caused by changes in tight junction protein composition. In addition, we observed that the molecular permeability was markedly more sensitive to changes in the tight junction structure and strand dynamics compared with transepithelial electric resistance. The results highlight that our model creates a good methodological framework to integrate knowledge on the tight junction structure as well as to provide insights and tools to advance tight junction research.</p></div

Mechanical impact stimulation platform tailored for high-resolution light microscopy

High frequency (HF) mechanical vibration has been used in vitro to study the cellular response to mechanical stimulation and induce stem cell differentiation. However, detailed understanding of the effect of the mechanical cues on cellular physiology is lacking. To meet this limitation, we have designed a system, which enables monitoring of living cells by high-resolution light microscopy during mechanical stimulation by HF vibration or mechanical impacts. The system consists of a commercial speaker, and a 3D printed sample vehicle and frame. The speaker moves the sample in the horizontal plane, allowing simultaneous microscopy. The HF vibration (30–200 Hz) performances of two vehicles made of polymer and aluminum were characterized with accelerometer. The mechanical impacts were characterized by measuring the acceleration of the aluminum vehicle and by time lapse imaging. The lighter polymer vehicle produced higher HF vibration magnitudes at 30–50 Hz frequencies than the aluminum vehicle. However, the aluminum vehicle performed better at higher frequencies (60–70 Hz, 90–100 Hz, 150 Hz). Compatibility of the system in live cell experiments was investigated with epithelial cells (MDCKII, expressing Emerald-Occludin) and HF (0.56 Gpeak, 30 Hz and 60 Hz) vibration. Our findings indicated that our system is compatible with high-resolution live cell microscopy. Furthermore, the epithelial cells were remarkable stable under mechanical vibration stimulation. To conclude, we have designed an inexpensive tool for the studies of cellular biophysics, which combines versatile in vivo like mechanical stimuli with live cell imaging, showing a great potential for several cellular applications.publishedVersionPeer reviewe

Correction to: Depletion of nuclear import protein karyopherin alpha 7 (KPNA7) induces mitotic defects and deformation of nuclei in cancer cells

Following publication of the original article [1], the authors notified us that the Additional File 1 contains reviewer comments instead of the Supplementary tables