Journal: The Journal of Neuroscience

Article Title: Mitochondrial Dynamics and Bioenergetic Dysfunction Is Associated with Synaptic Alterations in Mutant SOD1 Motor Neurons

doi: 10.1523/JNEUROSCI.1233-11.2012

Figure Lengend Snippet: Impaired mitochondrial fusion in the soma and motor axons of G93A SOD1 motor neurons. A, Cell bodies from non-transgenic control and G93A SOD1 motor neurons containing mitoDendra-labeled mitochondria before (−1 min) and after (0 min) photo-activation. Subsets of mitochondria (green and red fluorescence) were followed over time (10, 40, 70, and 100 min) by live imaging microscopy. Note that the appearance of yellow fluorescence, as a result of the mixing between green and red mitochondria (i.e. fusion, indicated by arrows), was delayed in mutant SOD1 motor neurons. Scale bar, 10 μm. B, Fusion rates were obtained in single optical z-sections by measuring the colocalization (in %) of red over green fluorescent mitochondria at the indicated time points. The correlation coefficient (r) for each group is indicated. n (somas) = 8 non-transgenic, and 7 G93A. *P<0.05 by ANOVA with repeated measurements. C, In the inset, example of a time-lapse recording of axonal mitochondria (numbers indicate time in min) showing a fusion event (arrow). Scale bar, 5 μm. The graph shows the analysis of fusion (% of fusion events of total moving mitochondria) in control and G93A SOD1 motor axons. n (axons) = 24 non-transgenic, 28 WT and 16 G93A axonal segments. *P<0.05 versus non-transgenic. D, Time-lapse microscopy of mitochondrial transport in the soma of non-transgenic and G93A SOD1 motor neurons. All mitochondria in a ROI of the soma were photo-converted, while only non-photo-converted (green fluorescent) mitochondria were followed over time. Note a decrease of mobile mitochondria towards the ROI in mutant SOD1 motor neurons compared to controls. Scale bar, 10 μm. E, Analysis of the transport of green mitochondria over the photo-activated area (no green mitochondria present). The correlation coefficient (r) for each group is indicated. n (somas) = 8 non-transgenic, and 7 G93A. *P<0.05 by ANOVA with repeated measurements. F, Analysis of the transport of red mitochondria over the non-photo-activated area (no red mitochondria present). The correlation coefficient (r) for each group is indicated. n (somas) = 8 non-transgenic, and 7 G93A. *P<0.05 by ANOVA with repeated measurements. All data obtained from 3–5 independent experiments. The error bars represent ± SE.

Article Snippet: Colocalization and Integrated Morphometric Analysis applications were from Metamorph software (Universal Imaging Co.).

Techniques: Transgenic Assay, Control, Labeling, Activation Assay, Fluorescence, Imaging, Microscopy, Mutagenesis, Time-lapse Microscopy

Journal: Pharmaceutical research

Article Title: The Extracellular Microenvironment Explains Variations in Passive Drug Transport across Different Airway Epithelial Cell Types

doi: 10.1007/s11095-013-1069-5

Figure Lengend Snippet: Confocal 3D image analyses of the mixed cell cultures confirmed TEER values of the monolayer or multilayer architecture of airway epithelial cells. Images were acquired on day 8 under ALI conditions. (a) Mixed cells on the inserts were incubated for 30 min (37°C, 5% CO2) with dye mixtures containing Hoe, MTR, and LTG staining cell nuclei, mitochondria, and lysosomes. Zeiss LSM confocal microscopy was used for the examination with z-axis scanning. Two dimensional images in xy planes show cell nuclei (blue), mitochondria (red), and lysosomes (green) and cell architecture on the insert are shown in yz planes with the arrows indicating “apical medium”, “cell”, and “membrane”. (b) Development of tight junctions in cell-to-cell contacts was examined for the mixed cell cultures on day 8 in ALI condition. Actin filaments in the cells grown on the inserts were stained with Alexa Fluor® 488 phalloidin (green) and cell nuclei stained with Hoe (blue). For the mixed cells (Calu-3/NHBE = 99/1), the vertical arrows represent “apical medium”, “cell layers”, and “porous membrane”. As shown in the yz planes, the mixed cells with more NHBE cells (Calu-3/NHBE = 1/9 or 1/99) formed multilayers with varied thickness of layers. The arrows point to the top cell layer, bottom cell layer and porous membrane support.

Article Snippet: Cell volume (μm 3 ) was calculated by Metamorph in the confocal 3D images and was used as a representative cytometric parameter to evaluate the distribution profiles in the pure and mixed cell populations.

Techniques: Incubation, Staining, Confocal Microscopy, Membrane

Journal: Pharmaceutical research

Article Title: The Extracellular Microenvironment Explains Variations in Passive Drug Transport across Different Airway Epithelial Cell Types

doi: 10.1007/s11095-013-1069-5

Figure Lengend Snippet: The fraction of Calu-3 and NHBE cells in cell populations consisting of pure (a) Calu-3 and NHBE cultures and mixed cell cultures (b, c) were estimated by fitting the distribution of cell volumes in the mixed cell population using a normal mixture statistical model. Cell volume (μm3) was calculated by Metamorph in the confocal 3D images and was used as a representative cytometric parameter to evaluate the distribution profiles in the pure and mixed cell populations. All the mixed cell cultures (Calu-3/NHBE = (b) 99/1, 9/1, 1/1, (c) 1/9, and 1/99) showed bimodal distributions with two distinct cell populations as indicated with the blue arrows (group 1 and 2). For the multilayers in the mixed cell cultures with more NHBE cells (Calu-3/NHBE= 1/9 or 1/99), the bottom cell layers and top cells were separately analyzed. As shown in (c), bottom cell layers consisted of two cell populations while top cells showed unimodal distribution, reflecting one cell-type in the top layer.

Article Snippet: Cell volume (μm 3 ) was calculated by Metamorph in the confocal 3D images and was used as a representative cytometric parameter to evaluate the distribution profiles in the pure and mixed cell populations.

Techniques: