Two-photon imaging of cancer cell extravasation in live mice

Abstract MDA-MB-231 breast cancer cells were engineered to express cytoplasmic paxillin-GFP and nuclear H2B-mCherry. In order to image extravasation, the cancer cells were injected in the blood stream of nude mice. Using 2-photon excitation microscopy we can simultaneously excite the two probes and also visualize the autofluorescence of tissues. A skin flap was opened to visualize blood vessels and recognize the position of the cancer cells. Two-photon imaging showed that after an initial phase in which the cells are non-adherent, some cells spread on the internal surface of the capillaries. Days later some cells started to appear on the external side of the capillary. The extravasated cells extend very long protrusions into the tissue. The goal was to determine if at the end of the long protrusion, if it is possible to observe the formation of focal adhesions by imaging paxillin-GFP. Preliminary results show that when cells start to adhere to the blood vessel wall they form focal adhesions as determined by the characteristic elongated features observed in the paxillin-GFP channel. New approaches will allow the tracking of the tip of the protrusion to determine if focal adhesions are forming there as the cells extravasate. This is important in establishing the mechanism of cell extravasation and migration in tissues. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1412. doi:10.1158/1538-7445.AM2011-141

Real-time imaging of 3-dimensional cancer cell movement in tissues

Abstract Our knowledge of how cells move in 3D in tissues is limited due to the lack of imaging methods that can produce 3D images fast enough and with sufficient resolution. Cancer cells migrate in 3D by forming adhesion points at the end of very long cellular protrusions. These protrusions are very thin and it is difficult to visualize adhesions along the protrusion surface. Conventional 3D stack reconstruction has relatively low resolution unless it is done using many frames. This results in a very slow acquisition in 3D confocal microscopy. Faster methods of 3D data acquisition (spinning disk microscopy) cannot be easily implemented since there is significant amount of scatter in tissues. A major obstacle in imaging adhesions is to find and track them so that they will not go out of focus. We are developing a new method which is based on orbiting imaging around cellular protrusions to visualize protein dynamics during extravasation. A feedback mechanism controls the center of the orbit to be at the center of the fluorescence distribution. A program reconstructs the shape of the protrusions in 3D. The fluorescence intensity in one or more channels is also simultaneously measured. The fluorescence intensity of one channel is used to paint the protrusion shape, which results in the 3D reconstruction of the protrusion. During the orbit, the second channel of the microscope measures the second harmonic generation (SHG) signal. We then correlated the appearance of bright fluorescence spots on the protrusion surface with the points of contact of the protrusion. This method will enable imaging of cancer cell invasion in 3-dimentions in live mice in real time. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4750. doi:10.1158/1538-7445.AM2011-475

Optimization of WAVE2 complex–induced actin polymerization by membrane-bound IRSp53, PIP3, and Rac

WAVE2 activates the actin-related protein (Arp) 2/3 complex for Rac-induced actin polymerization during lamellipodium formation and exists as a large WAVE2 protein complex with Sra1/PIR121, Nap1, Abi1, and HSPC300. IRSp53 binds to both Rac and Cdc42 and is proposed to link Rac to WAVE2. We found that the knockdown of IRSp53 by RNA interference decreased lamellipodium formation without a decrease in the amount of WAVE2 complex. Localization of WAVE2 at the cell periphery was retained in IRSp53 knockdown cells. Moreover, activated Cdc42 but not Rac weakened the association between WAVE2 and IRSp53. When we measured Arp2/3 activation in vitro, the WAVE2 complex isolated from the membrane fraction of cells was fully active in an IRSp53-dependent manner but WAVE2 isolated from the cytosol was not. Purified WAVE2 and purified WAVE2 complex were activated by IRSp53 in a Rac-dependent manner with PIP3-containing liposomes. Therefore, IRSp53 optimizes the activity of the WAVE2 complex in the presence of activated Rac and PIP3

Non-Equilibrium Thermodynamic Theory of 4-Component Lead-Free Solder

We investigated non-equilibrium thermodynamic theories for 4-component lead-free solders based on classical thermodynamics using one-dimensional oscillator model to obtain their specific heat and the coefficient of linear thermal expansion. We analyzed In and Sn reactions in SnAgBiIn solder as a representative lead-free material and experimentally obtained an N c factor for expressing the state of non-equilibrium from the formation of metallic compounds. It was considered that the state of equilibrium did not occur experimentally, and that metallic compounds InSn 4 formed in some parts of the 2-component eutectic solder. We then built a theory as onedimensional oscillator model by approximating this N c factor, as a representation of the non-equilibrium behavior of high temperature lead-free solder. Using this model representing the non-equilibrium state, the correlation between specific heat and coefficient of linear thermal expansion was derived theoretically, which was found to be linear in mathematical studies. In fact, it was learned that experimental results of this correlation also tended to be linear. This suggested that the derived non-equilibrium theory was practically useful, moreover other thermodynamic characteristics could be analyzed by this non-equilibrium thermodynamic theory using one-dimensional oscillator model

Raster-image-correlation spectroscopy of paxillin-GFP-expressing breast cancer cell in vitro and in vivo

Abstract Raster-image-correlation spectroscopy (RICS) is a noninvasive technique to detect and quantify events in the living cell, including concentrations of molecules and their diffusion coefficients. Any cell containing a fluorophore that can be imaged with a laser scanning microscope can be analyzed with RICS. We obtained RICS images with an Olympus FluoView FV1000 confocal microscope using Olympus FluoView software to acquire data and SimFCS software to perform RICS analysis. Paxillin is involved in the assembly of focal adhesions, which was linked to green fluorescent protein (GFP) for the current study. In this study, we describe RICS of paxillin-GFP expression in breast cancer cells (MDA-MB-231) in vitro and in vivo. Slow-moving membrane-bound paxillin proteins were measured in live breast cancer cells in vitro. Paxillin-GFP-expressing breast cancer cells (1×106) were injected in the epigastric cranials vein of the nude mouse. Paxillin-GFP-expressing breast cancer cells became attached to the inner vessel wall within 3 hours after injection. Rapidly-moving cytosolic paxillin-GFP molecules were imaged with RICS. With the ability to measure the molecular dynamics of paxillin in cancer cells in vitro and in vivo by RICS, we are now capable of studying the role of both slow-moving paxillin in the cell membrane and rapidly-moving cytosolic paxillin in cancer-cell behavior. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5183. doi:1538-7445.AM2012-518

High-efficacy targeting of colon-cancer liver metastasis with Salmonella typhimurium A1-R via intra-portal-vein injection in orthotopic nude-mouse models.

Liver metastasis is the main cause of colon cancer-related death and is a recalcitrant disease. We report here the efficacy and safety of intra-portal-vein (iPV) targeting of Salmonella typhimurium A1-R on colon cancer liver metastasis in a nude-mouse orthotopic model. Nude mice with HT29 human colon cancer cells, expressing red fluorescent protein (RFP) (HT29-RFP), growing in the liver were administered S. typhimurium A1-R by either iPV (1×104 colony forming units (CFU)/100 μl) or, for comparison, intra-venous injection (iv; 5×107 CFU/100 μl). Similar amounts of bacteria were delivered to the liver with the two doses, indicating that iPV delivery is 5×103 times more efficient than iv delivery. Treatment efficacy was evaluated by tumor fluorescent area (mm2) and total fluorescence intensity. Tumor fluorescent area and fluorescence intensity highly correlated (p<0.0001). iPV treatment was more effective compared to both untreated control and iv treatment (p<0.01 and p<0.05, respectively with iPV treatment with S. typhimurium arresting metastatic growth). There were no significant differences in body weight between all groups. The results of this study suggest that S. typhimurium A1-R administered iPV has potential for peri-operative adjuvant treatment of colon cancer liver metastasis

RPT2/NCH1 subfamily of NPH3-like proteins is essential for the chloroplast accumulation response in land plants

葉緑体が光に集まる反応を制御する新たな因子の発見. 京都大学プレスリリース. 2016-08-30.In green plants, the blue light receptor kinase phototropin mediates various photomovements and developmental responses, such as phototropism, chloroplast photorelocation movements (accumulation and avoidance), stomatal opening, and leaf flattening, which facilitate photosynthesis. In Arabidopsis, two phototropins (phot1 and phot2) redundantly mediate these responses. Two phototropin-interacting proteins, NONPHOTOTROPIC HYPOCOTYL 3 (NPH3) and ROOT PHOTOTROPISM 2 (RPT2), which belong to the NPH3/RPT2-like (NRL) family of BTB (broad complex, tramtrack, and bric à brac) domain proteins, mediate phototropism and leaf flattening. However, the roles of NRL proteins in chloroplast photorelocation movement remain to be determined. Here, we show that another phototropin-interacting NRL protein, NRL PROTEIN FOR CHLOROPLAST MOVEMENT 1 (NCH1), and RPT2 redundantly mediate the chloroplast accumulation response but not the avoidance response. NPH3, RPT2, and NCH1 are not involved in the chloroplast avoidance response or stomatal opening. In the liverwort Marchantia polymorpha, the NCH1 ortholog, MpNCH1, is essential for the chloroplast accumulation response but not the avoidance response, indicating that the regulation of the phototropin-mediated chloroplast accumulation response by RPT2/NCH1 is conserved in land plants. Thus, the NRL protein combination could determine the specificity of diverse phototropin-mediated responses

Avian seed dispersal in a mycoheterotrophic orchid Cyrtosia septentrionalis

光合成をやめたラン科植物ツチアケビにおける鳥による種子散布 -動物に種子散布を託す初めてのラン科植物の発見-. 京都大学プレスリリース. 2015-05-12.Orchids produce remarkably small seeds lacking endosperm, which are considered to be predominantly wind-dispersed. Here, we report avian seed dispersal in a mycoheterotrophic orchid Cyrtosia septentrionalis, which occurs under closed canopies where wind is less dependable. Because some orchids occupy similar habitats and have fleshy fruits, shifts in seed dispersal strategy may be a previously unnoticed mechanism promoting the orchid's evolutionary success