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.

BACKGROUND: 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. AIMS: 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. MATERIALS AND RESULTS: 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. CONCLUSIONS: 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.

Taking flight: an educational primer for use with "A novel mechanism for activation of myosin regulatory light chain by protein kinase C-delta in Drosophila".

Muscles are required for animal movement, feeding, heartbeat, and reproduction. Disruption of muscle function can lead to mobility impairments and diseases like muscular dystrophy and cardiac myopathy; therefore, research in this area has significant implications for public health. Recent work by Vaziri and colleagues has taken genetic, cell biological, and biochemical approaches to identify Protein kinase C-d (Pkcδ) as a novel regulator of the essential myosin light chain 2 (MLC2) by phosphorylation. The authors determine which residues of MLC2 are modified by Pkcδ and show that phosphorylation by Pkcδ is required for proper sarcomere assembly and function. This study underscores the importance of Drosophila melanogaster as a model system for muscle function and highlights how protein phosphorylation is a vital part of post-translational gene regulation.

A real-time assay for cell-penetrating peptide-mediated delivery of molecular cargos.

Cell-penetrating peptides (CPPs) are capable of transporting molecules to which they are tethered across cellular membranes. Unsurprisingly, CPPs have attracted attention for their potential drug delivery applications, but several technical hurdles remain to be overcome. Chief among them is the so-called 'endosomal escape problem,' i.e. the propensity of CPP-cargo molecules to be endocytosed but remain entrapped in endosomes rather than reaching the cytosol. Previously, a CPP fused to calmodulin that bound calmodulin binding site-containing cargos was shown to efficiently deliver cargos to the cytoplasm, effectively overcoming the endosomal escape problem. The CPP-adaptor, "TAT-CaM," evinces delivery at nM concentrations and more rapidly than we had previously been able to measure. To better understand the kinetics and mechanism of CPP-adaptor-mediated cargo delivery, a real-time cell penetrating assay was developed in which a flow chamber containing cultured cells was installed on the stage of a confocal microscope to allow for observation ab initio. Also examined in this study was an improved CPP-adaptor that utilizes naked mole rat (Heterocephalus glaber) calmodulin in place of human and results in superior internalization, likely due to its lesser net negative charge. Adaptor-cargo complexes were delivered into the flow chamber and fluorescence intensity in the midpoint of baby hamster kidney cells was measured as a function of time. Delivery of 400 nM cargo was observed within seven minutes and fluorescence continued to increase linearly as a function of time. Cargo-only control experiments showed that the minimal uptake which occurred independently of the CPP-adaptor resulted in punctate localization consistent with endosomal entrapment. A distance analysis was performed for cell-penetration experiments in which CPP-adaptor-delivered cargo showing wider dispersions throughout cells as compared to an analogous covalently-bound CPP-cargo. Small molecule endocytosis inhibitors did not have significant effects upon delivery. The real-time assay is an improvement upon static endpoint assays and should be informative in a broad array of applications.

Akirin is critical for early tinman induction and subsequent formation of the heart in Drosophila melanogaster.

The regulation of formation of the Drosophila heart by the Nkx 2.5 homologue Tinman is a key event during embryonic development. In this study, we identify the highly conserved transcription cofactor Akirin as a key factor in the earliest induction of tinman by the Twist transcription cofactor. akirin mutant embryos display a variety of morphological defects in the heart, including abnormal spacing between rows of aortic cells and abnormal patterning of the aortic outflow tract. akirin mutant embryos have a greatly reduced level of tinman transcripts, together with a reduction of Tinman protein in the earliest stages of cardiac patterning. Further, akirin mutants have reduced numbers of Tinman-positive cardiomyoblasts, concomitant with disrupted patterning and organization of the heart. Finally, despite the apparent formation of the heart in akirin mutants, these mutant hearts exhibit fewer coordinated contractions in akirin mutants compared with wild-type hearts. These results indicate that Akirin is crucial for the first induction of tinman by the Twist transcription factor, and that the success of the cardiac patterning program is highly dependent upon establishing the proper level of tinman at the earliest steps of the cardiac developmental pathway.

Improved cardiac contraction imaging in live Drosophila embryos.

Drosophila melanogaster is a powerful model organism in which to address the genetics of cardiac patterning and heart development. This system allows the pairing of live imaging with the myriad available genetic and transgenic techniques to not only identify the genes that are critical for heart development, but to assess their impact on heart function in living organisms. There are several described methods to assess cardiac function in Drosophila. However, these approaches are restricted to imaging of mid- to late-instar larval and adult hearts. This technical hurdle therefore does not allow for the recording and analysis of cardiac function in embryos bearing strong mutations that do not hatch into larvae. Our technical innovation lies in transgenically labeling the cells of the Drosophila heart and using line scan-based confocal imaging to repeatedly image the walls of the heart. By plotting this line scan as a kymograph, heart contractions can be visualized and assayed, thereby allowing for quantification of physiological defects. This method can be used to obtain physiological data from known mutations that affect cardiac development yet are incapable of hatching into larvae for conventional analysis.•Use transgenic methods to label heart proper walls•Use high-speed line scanning to capture position of heart proper walls•Create X vs. time plot to visualize and quantify contractions over imaging period.

Breaking in and busting out: cell-penetrating peptides and the endosomal escape problem.

Cell-penetrating peptides (CPPs) have long held great promise for the manipulation of living cells for therapeutic and research purposes. They allow a wide array of biomolecules from large, oligomeric proteins to nucleic acids and small molecules to rapidly and efficiently traverse cytoplasmic membranes. With few exceptions, if a molecule can be associated with a CPP, it can be delivered into a cell. However, a growing realization in the field is that CPP-cargo fusions largely remain trapped in endosomes and are eventually targeted for degradation or recycling rather than released into the cytoplasm or trafficked to a desired subcellular destination. This 'endosomal escape problem' has confounded efforts to develop CPP-based delivery methods for drugs, enzymes, plasmids, etc. This review provides a brief history of CPP research and discusses current issues in the field with a primary focus on the endosomal escape problem, for which several promising potential solutions have been developed. Are we on the verge of developing technologies to deliver therapeutics such as siRNA, CRISPR/Cas complexes and others that are currently failing because of an inability to get into cells, or are we just chasing after another promising but unworkable technology? We make the case for optimism.

A versatile cell-penetrating peptide-adaptor system for efficient delivery of molecular cargos to subcellular destinations.

Cell penetrating peptides have long held great potential for delivery of biomolecular cargos for research, therapeutic and diagnostic purposes. They allow rapid, relatively nontoxic passage of a wide variety of biomolecules through the plasma membranes of living cells. However, CPP-based research tools and therapeutics have been stymied by poor efficiency in release from endosomes and a great deal of effort has been made to solve this 'endosomal escape problem.' Previously, we showed that use of a reversible, noncovalent coupling between CPP and cargo using calmodulin and a calmodulin binding motif allowed efficient delivery of cargo proteins to the cytoplasm in baby hamster kidney and other mammalian cell lines. The present report demonstrates the efficacy of our CPP-adaptor scheme for efficient delivery of model cargos to the cytoplasm using a variety of CPPs and adaptors. Effective overcoming of the endosomal escape problem is further demonstrated by the delivery of cargo to the nucleus, endoplasmic reticulum and peroxisomes by addition of appropriate subcellular localization signals to the cargos. CPP-adaptors were also used to deliver cargo to myotubes, demonstrating the feasibility of the system as an alternative to transfection for the manipulation of hard-to-transfect cells.

Identification of a vacuolar proton channel that triggers the bioluminescent flash in dinoflagellates.

In 1972, J. Woodland Hastings and colleagues predicted the existence of a proton selective channel (HV1) that opens in response to depolarizing voltage across the vacuole membrane of bioluminescent dinoflagellates and conducts protons into specialized luminescence compartments (scintillons), thereby causing a pH drop that triggers light emission. HV1 channels were subsequently identified and demonstrated to have important functions in a multitude of eukaryotic cells. Here we report a predicted protein from Lingulodinium polyedrum that displays hallmark properties of bona fide HV1, including time-dependent opening with depolarization, perfect proton selectivity, and characteristic ΔpH dependent gating. Western blotting and fluorescence confocal microscopy of isolated L. polyedrum scintillons immunostained with antibody to LpHV1 confirm LpHV1's predicted organellar location. Proteomics analysis demonstrates that isolated scintillon preparations contain peptides that map to LpHV1. Finally, Zn2+ inhibits both LpHV1 proton current and the acid-induced flash in isolated scintillons. These results implicate LpHV1 as the voltage gated proton channel that triggers bioluminescence in L. polyedrum, confirming Hastings' hypothesis. The same channel likely mediates the action potential that communicates the signal along the tonoplast to the scintillon.

Novel cell-penetrating peptide-adaptors effect intracellular delivery and endosomal escape of protein cargos.

The use of cell-penetrating peptides (CPPs) as biomolecular delivery vehicles holds great promise for therapeutic and other applications, but development has been stymied by poor delivery and lack of endosomal escape. We have developed a CPP-adaptor system capable of efficient intracellular delivery and endosomal escape of user-defined protein cargos. The cell-penetrating sequence of HIV transactivator of transcription was fused to calmodulin, which binds with subnanomolar affinity to proteins containing a calmodulin binding site. Our strategy has tremendous advantage over prior CPP technologies because it utilizes high-affinity non-covalent, but reversible coupling between CPP and cargo. Three different cargo proteins fused to a calmodulin binding sequence were delivered to the cytoplasm of eukaryotic cells and released, demonstrating the feasibility of numerous applications in living cells including alteration of signaling pathways and gene expression.

Akirin: a context-dependent link between transcription and chromatin remodeling.

Embryonic patterning relies upon an exquisitely timed program of gene regulation. While the regulation of this process via the action of transcription factor networks is well understood, new lines of study have highlighted the importance of a concurrently regulated program of chromatin remodeling during development. Chromatin remodeling refers to the manipulation of the chromatin architecture through rearrangement, repositioning, or restructuring of nucleosomes to either favor or hinder the expression of associated genes. While the role of chromatin remodeling pathways during tumor development and cancer progression are beginning to be clarified, the roles of these pathways in the course of tissue specification, morphogenesis and patterning remains relatively unknown. Further, relatively little is understood as to the mechanism whereby developmentally critical transcription factors coordinate with chromatin remodeling factors to optimize target gene loci for gene expression. Such a mechanism might involve direct transcription factor/chromatin remodeling factor interactions, or could likely be mediated via an unknown intermediary. Our group has identified the relatively unknown protein Akirin as a putative member of this latter group: a secondary cofactor that serves as an interface between a developmentally critical transcription factor and the chromatin remodeling machinery. This role for the Akirin protein suggests a novel regulatory mode for regulating gene expression during development.

Akirin links twist-regulated transcription with the Brahma chromatin remodeling complex during embryogenesis.

The activities of developmentally critical transcription factors are regulated via interactions with cofactors. Such interactions influence transcription factor activity either directly through protein-protein interactions or indirectly by altering the local chromatin environment. Using a yeast double-interaction screen, we identified a highly conserved nuclear protein, Akirin, as a novel cofactor of the key Drosophila melanogaster mesoderm and muscle transcription factor Twist. We find that Akirin interacts genetically and physically with Twist to facilitate expression of some, but not all, Twist-regulated genes during embryonic myogenesis. akirin mutant embryos have muscle defects consistent with altered regulation of a subset of Twist-regulated genes. To regulate transcription, Akirin colocalizes and genetically interacts with subunits of the Brahma SWI/SNF-class chromatin remodeling complex. Our results suggest that, mechanistically, Akirin mediates a novel connection between Twist and a chromatin remodeling complex to facilitate changes in the chromatin environment, leading to the optimal expression of some Twist-regulated genes during Drosophila myogenesis. We propose that this Akirin-mediated link between transcription factors and the Brahma complex represents a novel paradigm for providing tissue and target specificity for transcription factor interactions with the chromatin remodeling machinery.

Nap1-mediated actin remodeling is essential for mammalian myoblast fusion.

Myoblast fusion is crucial for the formation, growth, maintenance and regeneration of healthy skeletal muscle. Unfortunately, the molecular machinery, cell behaviors, and membrane and cytoskeletal remodeling events that govern fusion and myofiber formation remain poorly understood. Using time-lapse imaging approaches on mouse C2C12 myoblasts, we identify discrete and specific molecular events at myoblast membranes during fusion and myotube formation. These events include rearrangement of cell shape from fibroblast to spindle-like morphologies, changes in lamellipodial and filopodial extensions during different periods of differentiation, and changes in membrane alignment and organization during fusion. We find that actin-cytoskeleton remodeling is crucial for these events: pharmacological inhibition of F-actin polymerization leads to decreased lamellipodial and filopodial extensions and to reduced myoblast fusion. Additionally, shRNA-mediated inhibition of Nap1, a member of the WAVE actin-remodeling complex, results in accumulations of F-actin structures at the plasma membrane that are concomitant with a decrease in myoblast fusion. Our data highlight distinct and essential roles for actin cytoskeleton remodeling during mammalian myoblast fusion, provide a platform for cellular and molecular dissection of the fusion process, and suggest a functional conservation of Nap1-regulated actin-cytoskeleton remodeling during myoblast fusion between mammals and Drosophila.

Myoblast fusion in fly and vertebrates: new genes, new processes and new perspectives.

Muscle formation and repair depends critically on the fusion of myoblasts. Despite the importance of this process, little is known about the cellular and molecular mechanisms regulating fusion. Forward genetic screens in Drosophila melanogaster have uncovered genes that, when mutated, prevent myoblast fusion. Analyses of these gene products have indicated that the actin cytoskeleton and its regulation play a central role in the fusion process. In this review, we discuss recent advances in the field, including new imaging approaches to analyze fusion as well as a description of novel genes required for fusion. In particular, we highlight what has been learned about the requirement of a specific actin structure at the site of fusion. We also place these findings from Drosophila within the context of myoblast fusion in vertebrates.

SCAR/WAVE and Arp2/3 are crucial for cytoskeletal remodeling at the site of myoblast fusion.

Myoblast fusion is crucial for formation and repair of skeletal muscle. Here we show that active remodeling of the actin cytoskeleton is essential for fusion in Drosophila. Using live imaging, we have identified a dynamic F-actin accumulation (actin focus) at the site of fusion. Dissolution of the actin focus directly precedes a fusion event. Whereas several known fusion components regulate these actin foci, others target additional behaviors required for fusion. Mutations in kette/Nap1, an actin polymerization regulator, lead to enlarged foci that do not dissolve, consistent with the observed block in fusion. Kette is required to positively regulate SCAR/WAVE, which in turn activates the Arp2/3 complex. Mutants in SCAR and Arp2/3 have a fusion block and foci phenotype, suggesting that Kette-SCAR-Arp2/3 participate in an actin polymerization event required for focus dissolution. Our data identify a new paradigm for understanding the mechanisms underlying fusion in myoblasts and other tissues.

Runx1 prevents wasting, myofibrillar disorganization, and autophagy of skeletal muscle.

Disruptions in the use of skeletal muscle lead to muscle atrophy. After short periods of disuse, muscle atrophy is reversible, and even after prolonged periods of inactivity, myofiber degeneration is uncommon. The pathways that regulate atrophy, initiated either by peripheral nerve damage, immobilization, aging, catabolic steroids, or cancer cachexia, however, are poorly understood. Previously, we found that Runx1 (AML1), a DNA-binding protein that is homologous to Drosophila Runt and has critical roles in hematopoiesis and leukemogenesis, is poorly expressed in innervated muscle, but strongly induced in muscle shortly after denervation. To determine the function of Runx1 in skeletal muscle, we generated mice in which Runx1 was selectively inactivated in muscle. Here, we show that Runx1 is required to sustain muscle by preventing denervated myofibers from undergoing myofibrillar disorganization and autophagy, structural defects found in a variety of congenital myopathies. We find that only 29 genes, encoding ion channels, signaling molecules, and muscle structural proteins, depend upon Runx1 expression, suggesting that their misregulation causes the dramatic muscle wasting. These findings demonstrate an unexpected role for electrical activity in regulating muscle wasting, and indicate that muscle disuse induces compensatory mechanisms that limit myofiber atrophy. Moreover, these results suggest that reduced muscle activity could cause or contribute to congenital myopathies if Runx1 or its target genes were compromised.

Identification of the omega4406 regulatory region, a developmental promoter of Myxococcus xanthus, and a DNA segment responsible for chromosomal position-dependent inhibition of gene expression.

When starved, Myxococcus xanthus cells send signals to each other that coordinate their movements, gene expression, and differentiation. C-signaling requires cell-cell contact, and increasing contact brought about by cell alignment in aggregates is thought to increase C-signaling, which induces expression of many genes, causing rod-shaped cells to differentiate into spherical spores. C-signaling involves the product of the csgA gene. A csgA mutant fails to express many genes that are normally induced after about 6 h into the developmental process. One such gene was identified by insertion of Tn5 lac at site omega4406 in the M. xanthus chromosome. Tn5 lac fused transcription of lacZ to the upstream omega4406 promoter. In this study, the omega4406 promoter region was identified by analyzing mRNA and by testing different upstream DNA segments for the ability to drive developmental lacZ expression in M. xanthus. The 5' end of omega4406 mRNA mapped to approximately 1.3 kb upstream of the Tn5 lac insertion. A 1.0-kb DNA segment from 0.8 to 1.8 kb upstream of the Tn5 lac insertion, when fused to lacZ and integrated at a phage attachment site in the M. xanthus chromosome, showed a similar pattern of developmental expression as Tn5 lac Omega4406. The DNA sequence upstream of the putative transcriptional start site was strikingly similar to promoter regions of other C-signal-dependent genes. Developmental lacZ expression from the 1.0-kb segment was abolished in a csgA mutant but was restored upon codevelopment of the csgA mutant with wild-type cells, which supply C-signal, demonstrating that the omega4406 promoter responds to extracellular C-signaling. Interestingly, the 0.8-kb DNA segment immediately upstream of Tn5 lac omega4406 inhibited expression of a downstream lacZ reporter in transcriptional fusions integrated at a phage attachment site in the chromosome but not at the normal omega4406 location. To our knowledge, this is the first example in M. xanthus of a chromosomal position-dependent effect on gene expression attributable to a DNA segment outside the promoter region.

Phosphorylation of histone H3: a balancing act between chromosome condensation and transcriptional activation.

In recent years, the covalent modification of histone tails has emerged as a crucial step in controlling the transcription of eukaryotic genes. Phosphorylation of the serine 10 residue of the N-terminal tail of histone H3 is crucial for chromosome condensation and cell-cycle progression during mitosis and meiosis. In addition, this modification is important during interphase because it enables the transcription of an increasing number of genes that are activated as a consequence of a variety of cell-signaling events. The location of the serine 10 residue in close proximity to other modifiable amino acids in the histone H3 tail enables the possibility of an interaction between phosphorylation of serine 10 and methylation and/or acetylation of lysine 9 and lysine 14. Finally, the finding that the histone H3.3 variant, which has a conserved N-terminal tail, can replace histone H3 at sites of active transcription, adds a new layer of complexity and possibilities to the regulation of transcription through changes in chromatin structure.

Protein phosphatase 2A activity affects histone H3 phosphorylation and transcription in Drosophila melanogaster.

Transcriptional activation of the heat shock genes during the heat shock response in Drosophila has been intimately linked to phosphorylation of histone H3 at serine 10, whereas repression of non-heat-shock genes correlates with dephosphorylation of histone H3. It is then possible that specific kinase and/or phosphatase activities may regulate histone phosphorylation and therefore transcription activation and repression, respectively. We find that treatment of cells with strong phosphatase inhibitors interferes with the genome-wide dephosphorylation of histone H3 normally observed at non-heat-shock genes during heat shock. Mutants in protein phosphatase type 2A (PP2A) also display reduced genome-wide H3 dephosphorylation, and sites of H3 phosphorylation that do not contain heat shock genes remain transcriptionally active during heat shock in PP2A mutants. Finally, the SET protein, a potent and highly selective inhibitor of PP2A activity that inhibits PP2A-mediated dephosphorylation of Ser10-phosphorylated H3, is detected at transcriptionally active regions of polytene chromosomes. These results suggest that activation and repression of gene expression during heat shock might be regulated by changes in PP2A activity controlled by the SET protein.