Tag Archives: confocal microscopy

Enhancement of internalization of diphtheria toxin recombinant fragments in sensitive cells mediated by toxin’s T-domain

K. Yu. Manoilov, A. J. Labyntsev, N. V. Korotkevych, D. V. Kolybo

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: manoilovmail@gmail.com

Subunit B of diphtheria toxin (DT) and its R-domain differ by the presence of T-domain. The aim of the present work was to analyze the interaction of these toxin fragments with mammalian cells in order to evaluate the T-domain’s influence on endocytosis in resistant cells. Internalization of recombinant fluorescent subunit B and R-domain was characterized in toxin-resistant L929 cells derived from mouse connective tissue and toxin-sensitive Vero cells from African green monkey kidney. It was found that during incubation of cells in the presence of both subunit B and R-domain in the culture medium, Vero cells internalize more molecules of subunit B than of R-domain. Under the same conditions, L929 cells internalize more molecules of R-domain than of subunit B. Colocalization of fluorescent subunit B and R-domain in L929 was rapid and proceeded almost completely at the early period of incubation compared to Vero cells in which it was slow and occurred gradually. The obtained data suggest that T-domain affects internalization and endosomal transport of DT in cells indirectly correlated with their toxin sensitivity. It was concluded that T-domain participates in intracellular endosomal transport and sorting of DT only in toxin-sensitive cells by enhancing the internalization of toxin molecules.

Calix[4]arene C-99 inhibits myosin ATPase activity and changes the organization of contractile filaments of myometrium

R. D. Labyntseva1, A. A. Bevza1, A. О. Lul’ko1, S. О. Cherenok2,
V. I. Kalchenko2, S. О. Kosterin1

1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: labyntseva@biochem.kiev.ua;
2Institute of Organic Chemistry, National Academy of Sciences of Ukraine, Kiev

Calix[4]arenes are cup-like macrocyclic (polyphenolic) compounds, they are regarded as promising molecular “platforms” for the design of new physiologically active compounds. We have earlier found that сalix[4]arenе C-99 inhibits the ATPase activity of actomyosin and myosin subfragment-1 of pig uterus іn vitro. The aim of this study was to investigate the interaction of calix[4]arene C-99 with myosin from rat uterine myocytes. It was found that the ATPase activity of myosin prepared from pre-incubated with 100 mM of calix[4]arene C-99 myocytes was almost 50% lower than in control. Additionally, we have revealed the effect of calix[4]arene C-99 on the subcellular distribution of actin and myosin in uterus myocytes by the method of confocal microscopy. This effect can be caused by reorganization of the structure of the contractile smooth muscle cell proteins due to their interaction with calix[4]arene. The obtained results demonstrate the ability of calix[4]arene C-99 to penetrate into the uterus muscle cells and affect not only the myosin ATPase activity, but also the structure of the actin and myosin filaments in the myometrial cells. Demonstrated ability of calix[4]arene C-99 can be used for development of new pharmacological agents for efficient normalization of myometrial contractile hyperfunction.

Seeing is believing! Live confocal imaging of microvascular networks in situ: morphology, Ca(2+) signalling and tone

T. Burdyga, L. Borysova

Department of Cellular and Molecular Physiology,
Institute of Translational Medicine,
University of Liverpool, Crown Street, Liverpool, L69 3BX, UK

2- and 3-dimensional confocal imaging of Fluo-4 loaded ureteric microvesells in situ allowed us to demonstrate distinct morphology, Ca2+ signalling and contractility in myocytes of arcade arterioles and pericytes of arcade venules. In myocytes and pericytes, Ca2+ signals arise exclusively from Ca2+ release from the sarcoplasmic reticulum through inositol 1,4,5-trisphosphate receptors. Са2+ transients in pericytes are less oscillatory, slower and longer-lasting than those in myocytes. The data obtained suggest differences in the mechanisms controlling local blood flow in precapillary arterioles and postcapillary venules.

Effect of diphtheria toxin T-domain on endosomal pH

A. J. Labyntsev, N. V. Korotkevych, D. V. Kolybo, S. V. Komisarenko

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: lab.andrey@gmail.com

A key step in the mode of cytotoxic action of diphtheria toxin (DT) is the transfer of its catalytic domain (Cd) from endosomes into the cytosol. The main activity in this process is performed by the transport domain (Td), but the molecular mechanism of its action remains unknown. We have previously shown that Td can have some influence on the endosomal transport of DT. The aim of this work was to study the effect of diphtheria toxin on the toxin compartmentalization in the intracellular transporting pathway and endosomal pH. We used recombinant fragments of DT, which differed only by the presence of Td in their structure, fused with fluorescent proteins. It was shown that the toxin fragment with Td moved slower by the pathway early-late endosomes-lysosomes, and had a slightly different pattern of colocalization with endosomal markers than DT fragment without Td. In addition, endosomes containing DT fragments with Td had a constant pH of about 6.5 from the 10th to 50th minute of observation, for the same time endosomes containing DT fragments without Td demons­trated a decrease in pH from 6.3 to 5.5. These results indicate that Td inhibits acidification of endosomal medium. One of possible explanations for this may be the effect of the ion channel formed by the T-domain on the process of the endosomal acidification. This property of Td may not only inhibit maturation of endosomes but also inhibit activation of endosomal pH-dependent proteases, and this promotes successful transport of Cd into the cell cytosol.

Signal function of cytokinin 6-benzylaminopurine in the reaction of Triticum aestivum L. mesophyll cells to hyperthermia

M. M. Musienko, V. V. Zhuk, L. M. Batsmanova

ESC Institute of Biology, Taras Shevchenko National University of Kyiv, Ukraine;
e-mail: zhuk_bas@voliacable.com

The signaling effect of 6-benzylaminopurine (BAP) on leaf mesophyll cells of Triticum aestivum L. under hyperthermic conditions was studied­. It was found that BAP regulated photosynthetic pigment, hydrogen peroxide content and activity of antioxidant enzymes, namely superoxide dismutase, ascorbate peroxidase and catalase under high-temperature conditions. The additive effect of BAP and high temperature on the activation of cell antioxidant systems was demonstrated. BAP regulated reducing processes in mesophyll leaf cells under high-temperature conditions.

Effect of the T-domain on intracellular transport of diphtheria toxin

А. J. Labyntsev, D. V. Kolybo, E. S. Yurchenko,
A. A. Kaberniuk, N. V. Korotkevych, S. V. Komisarenko

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: lab.andrey@gmail.com

Subunit B of diphtheria toxin (DT), which consists of two domains: R (receptor-binding) and T (transmembrane), plays an important role in toxin-receptor binding on the cell-targets and in transportation of catalytic subunit A to the cell cytosol. Recombinant analogues of the subunit B are promising representatives in the unique class of transporting proteins, able to deliver different types of biologically active molecules to cell cytosol. In the development of these protein constructs understanding of the role of each of the DT fragments in determination of transporting pathways of endocytosed complex toxin-receptor is urgently required.
We have studied in this work the T-domain effect on intracellular transport of recombinant fragments of DT. We have compared intracellular transport of the R-domain and the subunit B, the last one consisted of both R-domain and T-domain. Recombinant fragments of DT used in this work were labeled with fluorescent proteins, which allowed applying colocalization technique for our study. Application of confocal microscopy technique revealed differences in transportation of recombinant derivates of DT in Vero cells: R domain moved faster than subunit B to tubular compartments. Analysis of R-domain and subunit B transportation confirmed almost linear increase of their colocalization with the time regarding to Pearsons correlation coefficient (PCC). However, amount of colocalized with R-domain subunit B were not linearly increased with time according to Manders coefficient (M1), this could indicate the ability of subunit B to transport to such compartments that R-domain do not reach. Possible role of the T-domain in intracellular transportation and compartmentalization of the toxin may be associated with the ability of the T-domain to form a proton channels and its ability to interact with COPI complex.