Recombinant Streptolysin O (Functional) (A121187)

In striated muscles, approximately 300 myosin molecules form a single thick filament in myofibrils. Each myosin is continuously displaced by another myosin to maintain the thick filament structure. Our previous study using a fluorescence recovery after photobleaching (FRAP) technique showed that the myosin replacement rate is decreased by inhibition of protein synthesis, but myosin is still exchangeable. This result prompted us to examine whether myosin in the cytoplasm is involved in myosin replacement in myofibrils. To address this, FRAP was measured in green fluorescent protein (GFP)-tagged myosin heavy chain 3 (Myh3) expressing myotubes that were treated with streptolysin-O (SLO), which forms pores specifically in the plasma membrane to induce leakage of cytoplasmic proteins. Our biochemical data demonstrated that the cytoplasmic myosin content was reduced in SLO-permeabilized semi-intact myotubes. Furthermore, FRAP experiments showed a sluggish substitution rate of GFP-Myh3 in SLO-permeabilized myotubes. Taken together, these results demonstrate that the myosin substitution rate is significantly reduced by a decreased amount of myosin in the cytoplasm and that cytoplasmic myosin contributes to myosin replacement in myofibrils.

© 2017 Japanese Society of Animal Science.

Logical operations using biological molecules, such as DNA computing or programmable diagnosis using DNA, have recently received attention. Challenges remain with respect to the development of such systems, including label-free output detection and the rapidity of operation. Here, we propose integration of biological nanopores with DNA molecules for development of a logical operating system. We configured outputs "1" and "0" as single-stranded DNA (ssDNA) that is or is not translocated through a nanopore; unlabeled DNA was detected electrically. A negative-AND (NAND) operation was successfully conducted within approximately 10 min, which is rapid compared with previous studies using unlabeled DNA. In addition, this operation was executed in a four-droplet network. DNA molecules and associated information were transferred among droplets via biological nanopores. This system would facilitate linking of molecules and electronic interfaces. Thus, it could be applied to molecular robotics, genetic engineering, and even medical diagnosis and treatment.

Rab is a small GTP-binding protein family that regulates various pathways of vesicular transport. Although more than 60 Rab proteins are targeted to specific organelles in mammalian cells, the mechanisms underlying the specificity of Rab proteins for the respective organelles remain unknown. In this study, we reconstituted the Golgi targeting of Rab6A in streptolysin O (SLO)-permeabilized HeLa cells in a cytosol-dependent manner and investigated the biochemical requirements of targeting. Golgi-targeting assays identified Bicaudal-D (BICD)2, which is reportedly involved in the dynein-mediated transport of mRNAs during oogenesis and embryogenesis in Drosophila, as a cytosolic factor for the Golgi targeting of Rab6A in SLO-permeabilized HeLa cells. Subsequent immunofluorescence analyses indicated decreased amounts of the GTP-bound active form of Rab6 in BICD2-knockdown cells. In addition, fluorescence recovery after photobleaching (FRAP) analyses revealed that overexpression of the C-terminal region of BICD2 decreased the exchange rate of GFP-Rab6A between the Golgi membrane and the cytosol. Collectively, these results indicated that BICD2 facilitates the binding of Rab6A to the Golgi by stabilizing its GTP-bound form. Moreover, several analyses of vesicular transport demonstrated that Rab6A and BICD2 play crucial roles in Golgi tubule fusion with the endoplasmic reticulum (ER) in brefeldin A (BFA)-treated cells, indicating that BICD2 is involved in coat protein I (COPI)-independent Golgi-to-ER retrograde vesicular transport.

Copyright © 2015 Elsevier B.V. All rights reserved.

We previously reported that the small GTPase Rab27 and its effectors regulate isoproterenol (IPR)-stimulated amylase release from rat parotid acinar cells. Although activation of Rab27 by a specific guanine nucleotide exchange factor (GEF) is thought to be required for amylase release, its activation mechanism is poorly understood, because GEF for Rab27 has not been reported in parotid acinar cells. In the present study, we investigated the possible involvement of MADD/DENN/Rab3GEP, which was recently described as a Rab27-GEF in melanocytes, in amylase release from rat parotid acinar cells. Reverse transcription-PCR analyses indicated that mRNA of DENND family members, including MADD, was expressed in parotid acinar cells. MADD protein was also expressed in the cytosolic fraction of parotid acinar cells. Incubation of an antibody against the C-terminal 150 amino acids of MADD (anti-MADD-C antibody) with streptolysin O-permeabilized parotid acinar cells caused not only inhibition of IPR-induced amylase release but also reduction in the amount of GTP-Rab27. Our findings indicated that MADD functions as a GEF for Rab27 in parotid acinar cells and that its GEF activity for Rab27, i.e., GDP/GTP cycling, is required for IPR-induced amylase release.

Copyright © 2013 Elsevier Inc. All rights reserved.

Cell-based assay systems that can serve as cellular models of aberrant function in pathogenic organs would be novel and useful tools for screening drugs and clarifying the molecular mechanisms of various diseases. We constructed model cells that replicated the conditions in diabetic hepatocytes by using the cell resealing technique, which enables the exchange of cytosol. The plasma membrane of HeLa cells was permeabilized with the streptococcal toxin streptolysin O, and cytosol that had been prepared from wild-type or db/db diabetic mice was introduced into the resulting semi-intact cells. By resealing the plasma membrane by exposure to Ca(2+), we created WT or Db model cells, in which the cytosolic conditions replicated those of healthy or diabetic liver. Interestingly, phosphorylation of p38 MAPK was promoted, whereas the level of endosomal phosphatidylinositol-3-phosphate was decreased, in Db cells. We investigated several endocytic pathways in WT and Db cells, and found that retrograde endosome-to-Golgi transport was delayed in a p38 MAPK-dependent manner in Db cells. Furthermore, the degradation pathway of the EGF receptor from endosomes to lysosomes was enhanced in Db cells, and this did not depend on the activation of p38 MAPK. The disease model cell system should become a powerful tool for the detection of aberrant processes in cells under pathogenic conditions and for therapeutic applications.

The annexins, a family of Ca(2+)- and lipid-binding proteins, are involved in a range of intracellular processes. Recent findings have implicated annexin A1 in the resealing of plasmalemmal injuries. Here, we demonstrate that another member of the annexin protein family, annexin A6, is also involved in the repair of plasmalemmal lesions induced by a bacterial pore-forming toxin, streptolysin O. An injury-induced elevation in the intracellular concentration of Ca(2+) ([Ca(2+)](i)) triggers plasmalemmal repair. The highly Ca(2+)-sensitive annexin A6 responds faster than annexin A1 to [Ca(2+)](i) elevation. Correspondingly, a limited plasmalemmal injury can be promptly countered by annexin A6 even without the participation of annexin A1. However, its high Ca(2+) sensitivity makes annexin A6 highly amenable to an unproductive binding to the uninjured plasmalemma; during an extensive injury accompanied by a massive elevation in [Ca(2+)](i), its active pool is severely depleted. In contrast, annexin A1 with a much lower Ca(2+) sensitivity is ineffective at the early stages of injury; however, it remains available for the repair even at high [Ca(2+)](i). Our findings highlight the role of the annexins in the process of plasmalemmal repair; a number of annexins with different Ca(2+)-sensitivities provide a cell with the means to react promptly to a limited injury in its early stages and, at the same time, to withstand a sustained injury accompanied by the continuous formation of plasmalemmal lesions.

© 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Phosphatidylinositol-3-phosphate (PI3P) is a lipid that is enriched specifically in early endosomes. Given that early endosomes containing PI3P act as a microdomain to recruit proteins that contain a PI3P-binding domain (FYVE domain), the equilibrium between the production and degradation of PI3P influences a variety of processes, including endocytosis and signal transduction via endosomes. In the study reported herein, we have developed a novel analytical method to quantify the amount of PI3P in endosomes by introducing a GST-2xFYVE protein probe into semi-intact cells. The GST-2xFYVE probe was targeted specifically to intracellular PI3P-containing endosomes, which retained their small punctate structure, and allowed the semi-quantitative measurement of intracellular PI3P. Using the method, we found that treatment of HeLa cells with H(2)O(2) decreased the amount of PI3P in endosomes in a p38 MAPK-dependent manner. In addition, H(2)O(2) treatment delayed transport through various endocytic pathways, especially post-early endosome transport; the retrograde transport of cholera toxin was especially dependent on the amount of PI3P in endosomes. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

2011 Elsevier B.V. All rights reserved.

Cytotoxic T lymphocytes and natural killer cells destroy target cells via the polarized exocytosis of lytic effector proteins, perforin and granzymes, into the immunologic synapse. How these molecules enter target cells is not fully understood. It is debated whether granzymes enter via perforin pores formed at the plasma membrane or whether perforin and granzymes are first endocytosed and granzymes are then released from endosomes into the cytoplasm. We previously showed that perforin disruption of the plasma membrane induces a transient Ca(2+) flux into the target cell that triggers a wounded membrane repair response in which lysosomes and endosomes donate their membranes to reseal the damaged membrane. Here we show that perforin activates clathrin- and dynamin-dependent endocytosis, which removes perforin and granzymes from the plasma membrane to early endosomes, preserving outer membrane integrity. Inhibiting clathrin- or dynamin-dependent endocytosis shifts death by perforin and granzyme B from apoptosis to necrosis. Thus by activating endocytosis to preserve membrane integrity, perforin facilitates granzyme uptake and avoids the proinflammatory necrotic death of a membrane-damaged cell.

Oligonucleotide-templated reactions are attracting attention as a method for RNA detection in living cells. Previously, a reduction-triggered fluorescence probe has been reported that is based on azide reduction to switch fluorescence on. In this article, we report a more advanced probe, a reduction-triggered fluorescent amplification probe that is capable of amplifying a target signal. Azidomethyl fluorescein was newly synthesized and introduced into a probe. Azido-masked fluorescein on the probe showed a strong turn-on fluorescence signal upon oligonucleotide-templated Staudinger reduction. The catalytic reaction of the probe offered a turnover number of 50 as fluorescence readout within 4 h. Finally, probes were introduced into human leukemia HL-60 cells by use of streptolysin O pore-forming peptide. We successfully detected and quantitated the 28S rRNA and beta-Actin mRNA signal above the background by flow cytometry. In addition, the same RNA targets were imaged by fluorescence microscopy. The data suggest that a reduction-triggered amplification probe may be a powerful tool in analyzing the localization, transcription, or processing of RNA species in living eukaryotic cells.

The organelles within secretory and endocytotic pathways in mammalian cells have acidified lumens, and regulation of their acidic pH is critical for the trafficking, processing and glycosylation of cargo proteins and lipids, as well as the morphological integrity of the organelles. How organelle lumen acidification is regulated, and how luminal pH elevation disturbs these fundamental cellular processes, is largely unknown. Here, we describe a novel molecule involved in Golgi acidification. First, mutant cells defective in Golgi acidification were established that exhibited delayed protein transport, impaired glycosylation and Golgi disorganization. Using expression cloning, a novel Golgi-resident multi-transmembrane protein, named Golgi pH regulator (GPHR), was identified as being responsible for the mutant cells. After reconstitution in planar lipid bilayers, GPHR exhibited a voltage-dependent anion-channel activity that may function in counterion conductance. Thus, GPHR modulates Golgi functions through regulation of acidification.