Movement of Bax from the Cytosol to Mitochondria during Apoptosis

Bax, a member of the Bcl-2 protein family, accelerates apoptosis by an unknown mechanism. Bax has been recently reported to be an integral membrane protein associated with organelles or bound to organelles by Bcl-2 or a soluble protein found in the cytosol. To explore Bcl-2 family member localization in living cells, the green fluorescent protein (GFP) was fused to the NH2 termini of Bax, Bcl-2, and Bcl-XL. Confocal microscopy performed on living Cos-7 kidney epithelial cells and L929 fibroblasts revealed that GFP–Bcl-2 and GFP–Bcl-XL had a punctate distribution and colocalized with a mitochondrial marker, whereas GFP–Bax was found diffusely throughout the cytosol. Photobleaching analysis confirmed that GFP–Bax is a soluble protein, in contrast to organelle-bound GFP–Bcl-2. The diffuse localization of GFP–Bax did not change with coexpression of high levels of Bcl-2 or Bcl-XL. However, upon induction of apoptosis, GFP–Bax moved intracellularly to a punctate distribution that partially colocalized with mitochondria. Once initiated, this Bax movement was complete within 30 min, before cellular shrinkage or nuclear condensation. Removal of a COOH-terminal hydrophobic domain from GFP–Bax inhibited redistribution during apoptosis and inhibited the death-promoting activity of both Bax and GFP– Bax. These results demonstrate that in cells undergoing apoptosis, an early, dramatic change occurs in the intracellular localization of Bax, and this redistribution of soluble Bax to organelles appears important for Bax to promote cell death

Scanning electron microscopy of cells and tissues under fully hydrated conditions

A capability for scanning electron microscopy of wet biological specimens is presented. A membrane that is transparent to electrons protects the fully hydrated sample from the vacuum. The result is a hybrid technique combining the ease of use and ability to see into cells of optical microscopy with the higher resolution of electron microscopy. The resolution of low-contrast materials is ≈100 nm, whereas in high-contrast materials the resolution can reach 10 nm. Standard immunogold techniques and heavy-metal stains can be applied and viewed in the fluid to improve the contrast. Images present a striking combination of whole-cell morphology with a wealth of internal details. A possibility for direct inspection of tissue slices transpires, imaging only the external layer of cells. Simultaneous imaging with photons excited by the electrons incorporates data on material distribution, indicating a potential for multilabeling and specific scintillating markers