Although peroxisomes oxidize lipids, the metabolism of lipid bodies and peroxisomes is thought to be largely uncoupled from one another. In this study, using oleic acid–cultured Saccharomyces cerevisiae as a model system, we provide evidence that lipid bodies and peroxisomes have a close physiological relationship. Peroxisomes adhere stably to lipid bodies, and they can even extend processes into lipid body cores. Biochemical experiments and proteomic analysis of the purified lipid bodies suggest that these processes are limited to enzymes of fatty acid β oxidation. Peroxisomes that are unable to oxidize fatty acids promote novel structures within lipid bodies (“gnarls”), which may be organized arrays of accumulated free fatty acids. However, gnarls are suppressed, and fatty acids are not accumulated in the absence of peroxisomal membranes. Our results suggest that the extensive physical contact between peroxisomes and lipid bodies promotes the coupling of lipolysis within lipid bodies with peroxisomal fatty acid oxidation.

Investigation of Caenorhabditis elegans act-5 gene function revealed that intestinal microvillus formation requires a specific actin isoform. ACT-5 is the most diverged of the five C. elegans actins, sharing only 93% identity with the other four. Green fluorescent protein reporter and immunofluorescence analysis indicated that act-5 gene expression is limited to microvillus-containing cells within the intestine and excretory systems and that ACT-5 is apically localized within intestinal cells. Animals heterozygous for a dominant act-5 mutation looked clear and thin and grew slowly. Animals homozygous for either the dominant act-5 mutation, or a recessive loss of function mutant, exhibited normal morphology and intestinal cell polarity, but died during the first larval stage. Ultrastructural analysis revealed a complete loss of intestinal microvilli in homozygous act-5 mutants. Forced expression of ACT-1 under the control of the act-5 promoter did not rescue the lethality of the act-5 mutant. Together with immuno-electron microscopy experiments that indicated ACT-5 is enriched within microvilli themselves, these results suggest a microvillus-specific function for act-5, and further, they raise the possibility that specific actins may be specialized for building microvilli and related structures.

Nerve cells communicate with each other at specialized intercellular junctions named synapses. Although synaptic structure has been studied for decades, many important questions addressing function in terms of synaptic architecture have yet to be answered. Traditional chemical fixation based electron microscopy has proved to be too slow to capture the constantly changing structure, such as the fusion of the synaptic vesicle with the plasma membrane [1]. Using rapid freezing techniques, mainly slam freezing, to study synaptic structure yields only about 10-20 µm of good ultrastructural detail. Any cellular structure beyond this depth range will be subject to extensive distortion caused by ice crystal damage [2]. Therefore, an alternative method has being developed. The intact tissue is frozen under high hydrostatic pressure resulting in an increased layer of vitrification. High pressure freezing (HPF) followed by freeze substitution (FS) has produced excellent preservation of the subcellular structure of various biological samples. However, the brain tissue is among the most difficult tissue to cryofix due to its delicate structure and high percentage of water content. One solution to this problem would be to "mild" aldehyde fix and apply cryoprotection prior to high pressure freezing. However, this approach not only suffers the disadvantage of chemical fixation, but also introduces a new potential osmotic problem. It is unlikely that the morphology achieved by this method represents the near-nature state in vivo.In order to overcome the above problems, we have developed a method of rapidly freezing the tissue from a vibratomed living brain slice (200 µm). The slice was constantly incubated in artificial cerebral spinal fluid (ACSF) equilibrated with 95%O2/5%CO2 at 37 ºC. The cerebral cortex was quickly punched out and immediately frozen in the Leica EM PACT high-pressure freezing machine followed by freeze substitution in a Leica AFS apparatus using a medium containing acetone and osmium tetroxide, then infiltrated and embedded in EMbed 812 resin [3]. The 60 nm thin sections were cut and stained with uranyl acetate and lead citrate prior to EM observation. To compare the effect of sample preparation, we also examined brain tissue that had been pre-fixed and cryoprotected prior to freezing.Here, we present two sets of experimental results. Group I is the directly frozen living brain tissue (Figs.1A, 2A); Group II is pre-fixed and sucrose cryoprotected tissue that underwent the same HPF and FS process as Group I (Figs.1B, 2B). In general, Group II yields better morphology characterized by the smoothness of plasma membrane, and minimal ice damage to the cellular structure. However, the contrast for synaptic vesicles was poor and the number of the vesicles at the presynaptic terminal is less than that in Group I, suggesting the loss the vesicles during the process. Conversely, Group I clearly demonstrates more structural detail, such as synaptic vesicles and their associated microfilaments. This result is comparable ...