Nanoscale three-dimensional imaging of the human myocyte.

The ventricular human myocyte is spatially organized for optimal ATP and Ca(2+) delivery to sarcomeric myosin and ionic pumps during every excitation-contraction cycle. Comprehension of three-dimensional geometry of the tightly packed ultrastructure has been derived from discontinuous two-dimensional images, but has never been precisely reconstructed or analyzed in human myocardium. Using a focused ion beam scanning electron microscope, we created nanoscale resolution serial images to quantify the three-dimensional ultrastructure of a human left ventricular myocyte. Transverse tubules (t-tubule), lipid droplets, A-bands, and mitochondria occupy 1.8, 1.9, 10.8, and 27.9% of the myocyte volume, respectively. The complex t-tubule system has a small tortuosity (1.04±0.01), and is composed of long transverse segments with diameters of 317±24nm and short branches. Our data indicates that lipid droplets located well beneath the sarcolemma are proximal to t-tubules, where 59% (13 of 22) of lipid droplet centroids are within 0.50µm of a t-tubule. This spatial association could have an important implication in the development and treatment of heart failure because it connects two independently known pathophysiological alterations, a substrate switch from fatty acids to glucose and t-tubular derangement.

Cryptococcus neoformans Yop1 , an endoplasmic reticulum curvature-stabilizing protein, participates with Sey1 in influencing fluconazole-induced disomy formation.

Cryptococcus neoformans, an opportunistic fungal pathogen, manifests an intrinsic adaptive mechanism of resistance toward fluconazole (FLC) termed heteroresistance. Heteroresistance is characterized by the emergence of minor resistant subpopulations at levels of FLC that are higher than the strain's minimum inhibitory concentration. The heteroresistant clones that tolerate high concentrations of FLC often contain disomic chromosome 4 (Chr4). SEY1 , GLO3 , and GCS2 on Chr4 are responsible for endoplasmic reticulum (ER) integrity and important for Chr4 disomy formation under FLC stress. We sought an evidence of a direct relationship between ER morphology and Chr4 disomy formation. Deletion of the YOP1 gene on Chr7, which encodes an ER curvature-stabilizing protein that interacts with Sey1 , perturbed ER morphology without affecting FLC susceptibility or the frequency of FLC-induced disomies. However, deletion of both YOP1 and SEY1 , not only perturbed ER morphology more severely than in sey1∆ or yop1∆ strains, but also abrogated the FLC-induced disomy. Although the heteroresistance phenotype was retained in the sey1∆yop1∆ strains, tolerance to FLC appeared to have resulted not from chromosome duplication but from gene amplification restricted to the region surrounding ERG11 on Chr1. These data support the importance of ER integrity in C. neoformans for the formation of disomy under FLC stress.

Characterization of the chromosome 4 genes that affect fluconazole-induced disomy formation in Cryptococcus neoformans.

Heteroresistance in Cryptococcus neoformans is an intrinsic adaptive resistance to azoles and the heteroresistant phenotype is associated with disomic chromosomes. Two chromosome 1 (Chr1) genes, ERG11, the fluconazole target, and AFR1, a drug transporter, were reported as major factors in the emergence of Chr1 disomy. In the present study, we show Chr4 to be the second most frequently formed disomy at high concentrations of fluconazole (FLC) and characterize the importance of resident genes contributing to disomy formation. We deleted nine Chr4 genes presumed to have functions in ergosterol biosynthesis, membrane composition/integrity or drug transportation that could influence Chr4 disomy under FLC stress. Of these nine, disruption of three genes homologous to Sey1 (a GTPase), Glo3 and Gcs2 (the ADP-ribosylation factor GTPase activating proteins) significantly reduced the frequency of Chr4 disomy in heteroresistant clones. Furthermore, FLC resistant clones derived from sey1Δglo3Δ did not show disomy of either Chr4 or Chr1 but instead had increased the copy number of the genes proximal to ERG11 locus on Chr1. Since the three genes are critical for the integrity of endoplasmic reticulum (ER) in Saccharomyces cerevisiae, we used Sec61ß-GFP fusion as a marker to study the ER in the mutants. The cytoplasmic ER was found to be elongated in sey1Δ but without any discernable alteration in gcs2Δ and glo3Δ under fluorescence microscopy. The aberrant ER morphology of all three mutant strains, however, was discernable by transmission electron microscopy. A 3D reconstruction using Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) revealed considerably reduced reticulation in the ER of glo3Δ and gcs2Δ strains. In sey1Δ, ER reticulation was barely detectable and cisternae were expanded extensively compared to the wild type strains. These data suggest that the genes required for maintenance of ER integrity are important for the formation of disomic chromosomes in C. neoformans under azole stress.