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In correlative light and electron microscopy (CLEM) workflows, identifying the same sub-cellular features in tissue by both light (LM) and electron microscopy (EM) remains a challenge. Furthermore, use of cryo-fixation for EM is desirable to capture rapid biological phenomena. Here, we describe a workflow that incorporates cryo-confocal laser scanning microscopy into the CLEM process, mapping cells in brain slices to re-image them with serial section scanning electron microscopy (ssSEM) array tomography. The addition of Airyscan detection increased the signal-to-noise ratio (SNR), allowing individual spines in thick frozen tissue to be visualized at a sufficient spatial resolution, providing a new tool for a CLEM approach to capture biological dynamics. [ABSTRACT FROM AUTHOR]

The seismonastic movement of Mimosa pudica is triggered by a sudden loss of turgor pressure. In the present study, we compared the cell cytoskeleton by immunofluorescence analysis before and after movement, and the effects of actin- and microtubule-targeted drugs were examined by injecting them into the cut pulvinus. We found that fragmentation of actin filaments and microtubules occurs during bending, although the actin cytoskeleton, but not the microtubules, was involved in regulation of the movement. Transmission electron microscopy revealed that actin cables became loose after the bending. We injected phosphatase inhibitors into the severed pulvinus to examine the effects of such inhibitors on the actin cytoskeleton. We found that changes in actin isoforms, fragmentation of actin filaments and the bending movement were all inhibited after injection of a tyrosine phosphatase inhibitor. We thus propose that the phosphorylation status of actin at tyrosine residues affects the dynamic reorganization of actin filaments and causes seismonastic movement. [ABSTRACT FROM AUTHOR]

3-Amino-1,2,4-triazole (3-AT) is known as an inhibitor of catalase to whose active center it specifically and covalently binds. Subcellular fractionation and immunoelectronmicroscopic observation of the yeast Candida tropicalis revealed that, in 3-AT-treated cells in which the 3-AT was added to the n-alkane medium from the beginning of cultivation, catalase transported into peroxisomes was inactivated and was present as insoluble aggregated forms in the organelle. The aggregation of catalase in peroxisomes occurred only in these 3-AT-treated cells and not in cells in which 3-AT was added at the late exponential growth phase. Furthermore, 3-AT did not affect the transportation of catalase into peroxisomes. The appearance of aggregation only in cells to which 3-AT was added from the beginning of cultivation suggests that, in the process of catalase transportation into yeast peroxisomes, some conformational change may take place and that correct folding may be inhibited by the binding of 3-AT to the active center of catalase. Accordingly, 3-AT will be an interesting compound for investigation of the transport machinery of the peroxisomal tetrameric catalase. [Copyright &y& Elsevier]

In an n-alkane-assimilating yeast, Candida tropicalis, carnitine acetyltransferase (CAT; EC 2.3.1.7) was localized in both peroxisomes and mitochondria. Both CATs were encoded by one gene, CT-CAT, although the initiation sites of translation were suggested to be different. In the present study, the genes corresponding to the supposed C. tropicalis peroxisomal and mitochondrial CATs, which were truncated from the CT-CAT gene, were individually expressed in Saccharomyces cerevisiae, using the C. tropicalis isocitrate lyase promoter (UPR-ICL), which is inducible by oleic acid in concert with proliferation of peroxisomes in S. cerevisiae [Umemura, K., Atomi, H., Kanai, T., Teranishi, Y., Ueda, M., and Tanaka, A. (1995) Appl. Microbiol. Biotechnol. 43, 489–492]. The 71 kDa precursor of mitochondrial CAT, initiating at the first Met, was found to be processed to the mature size (66 kDa) in S. cerevisiae and immunoelectronmicroscopical observation revealed that this enzyme was localized in mitochondria. On the other hand, 68 kDa CAT, initiating at the second Met (residue No. 19), had no cleavable signal and was translocated into peroxisomes and cytosol, but not into mitochondria. The aniino-terniinal amino acid sequences of individually expressed CATs were identical to those of CATs isolated from alkane-grown C. tropicalls cells, respectively. These results demonstrated that only the 71 kDa protein yielded the 66 kDa protein and that peroxisomal and mitochondrial CATs arose from the difference in the initiation sites of translation. [ABSTRACT FROM AUTHOR]

The peroxisomal isocitrate lyase (ICL) of an n-alkane-utilizable yeast, Candida tropicalis, could be transported into the peroxisomes of Saccharomyces cerevisiae MT8-1 cells grown in an oleic acid medium, even if strains were different. To identify the important regions to direct C. tropicalis ICL (CT-ICL) to its proper subcellular location in S. cerevisiae, we observed the subcellular localization of the expressed products of mutant CT-ICL genes in S. cerevisiae by immunoelectron microscopy. Under the control of the UPR-ICL promoter, which enhanced gene expression by proliferation of the peroxisomes, the following mutant CT-ICL genes were constructed and expressed: (i) truncated or mutant genes in the C-terminal region which apparently contained peroxisomal targeting signals, Δ550, Δ549–550, Δ548–550, Δ548–550 + SKL and Δ340–550; and (ii) four internal deletion mutant genes, Δ41–115, Δ41–239, Δ41–325 and Δ237–339. The results showed that three C-terminal amino acid residues were not essential for targeting the peroxisomes in this enzyme. In cells harboring the deleted internal regions of mutant ICL genes, the expressed polypeptides did not target the peroxisomes and the polypeptides, except for Δ41–115, were mistargeted to mitochondria. We observed an unknown structure which we called a “protein aggregate body (PAB),” in which various mutant CT-ICL polypeptides aggregated. [ABSTRACT FROM PUBLISHER]

The time course of BMY-28864-dependent morphological changes in A9540 was studied by electron microscopy. Scanning electron microscopy revealed that BMY-28864 often induced cell surface deformations such as abnormal swelling and bulging around budding sites and bud scars. The cell membrane damage was visualized by freeze-fracturing technique as deep pit-like invaginations. Transmission electron microscopy with thin-sectioned specimens also demonstrated that BMY-28864 induced cell membrane invaginations together with cell membrane detachment from the cell wall, nuclear membrane fragmentation and mitochondrial aberration. Statistical sequence analysis of the prominent BMY-28864-dependent morphological changes of the yeast cells led to the conclusion that BMY-28864 first attacked the cell membrane and then caused disintegration of other intracytoplasmic organelles, resulting in the lethal effect. [ABSTRACT FROM PUBLISHER]