Contractile and energetic properties of human skeletal muscle have been studied for many years in vivo in the body. It has been, however, difficult to identify the specific role of muscle fibres in modulating muscle performance. Recently it has become possible to dissect short segments of single human muscle fibres from biopsy samples and make them work in nearly physiologic conditions in vitro. At the same time, the development of molecular biology has provided a wealth of information on muscle proteins and their genes and new techniques have allowed analysis of the protein isoform composition of the same fibre segments used for functional studies. In this way the histological identification of three main human muscle fibre types (I, IIA and IIX, previously called IIB) has been followed by a precise description of molecular composition and functional and biochemical properties. It has become apparent that the expression of different protein isoforms and therefore the existence of distinct muscle fibre phenotypes is one of the main determinants of the muscle performance in vivo. The present review will first describe the mechanisms through which molecular diversity is generated and how fibre types can be identified on the basis of structural and functional characteristics. Then the molecular and functional diversity will be examined with regard to (1) the myofibrillar apparatus; (2) the sarcolemma and the sarcoplasmic reticulum; and (3) the metabolic systems devoted to producing ATP. The last section of the review will discuss the advantage that fibre diversity can offer in optimizing muscle contractile performance.

Human skeletal muscle fibres: molecular and functional diversity

The ultrastructure of yeast: Cell wall structure and formation

One stop mycology

The fission yeast, Schizosaccharomyces pombe, has been used as a model eukaryote to study processes such as the cell cycle and cell morphology In this single-celled organism, growing in a straight line and maintaining the nucleus in the centre of the cell depend on intracellular positional information Microtubules and microtubular transport are important for generating positional information within the fission yeast cell, and these molecular mechanisms are also probably relevant for generating positional information in other eukaryotic cells

A journey into space.

Cryptococcal meningitis is a fungal infection, caused by Cryptococcus neoformans, which is prevalent in immunocompromised patient populations. Treatment failures of this disease are emerging in the clinic, usually associated with long-term treatment with existing antifungal agents. The fungal cell wall is an attractive target for drug therapy because the syntheses of cell wall glucan and chitin are processes that are absent in mammalian cells. Echinocandins comprise a class of lipopeptide compounds known to inhibit 1,3-beta-glucan synthesis, and at least two compounds belonging to this class are currently in clinical trials as therapy for life-threatening fungal infections. Studies of Saccharomyces cerevisiae and Candida albicans mutants identify the membrane-spanning subunit of glucan synthase, encoded by the FKS genes, as the molecular target of echinocandins. In vitro, the echinocandins show potent antifungal activity against Candida and Aspergillus species but are much less potent against C. neoformans. In order to examine why C. neoformans cells are less susceptible to echinocandin treatment, we have cloned a homolog of S. cerevisiae FKS1 from C. neoformans. We have developed a generalized method to evaluate the essentiality of genes in Cryptococcus and applied it to the FKS1 gene. The method relies on homologous integrative transformation with a plasmid that can integrate in two orientations, only one of which will disrupt the target gene function. The results of this analysis suggest that the C. neoformans FKS1 gene is essential for viability. The C. neoformans FKS1 sequence is closely related to the FKS1 sequences from other fungal species and appears to be single copy in C. neoformans. Furthermore, amino acid residues known to be critical for echinocandin susceptibility in Saccharomyces are conserved in the C. neoformans FKS1 sequence.

A glucan synthase FKS1 homolog in cryptococcus neoformans is single copy and encodes an essential function.

The ultrastructure of regenerating cell wall in Schizosaccharomyces pombe protoplasts was studied with a high resolution, low voltage scanning electron microscope (LVSEM). In contrast to the transmission electron microscopy, the LVSEM images give three-dimensional information on the cell wall regeneration in yeast protoplasts. We found that, after only a few minutes of incubation, the protoplasts began to show protuberances in a unipolar manner, and a fibrilar network was formed asymmetrically which covered the whole surface of the protoplasts after 5 hr. The network consisted of microfibrils about 8 to 10 nm wide, forming flat and wavy bundles of various widths and lengths, up to about 200 nm wide and 1 micron long, mainly made of yeast glucan. Free ends of microfibrils were seldom found. Interfibrillar spaces were progressively filled with granular particles and finally the complete cell wall was formed after 12 hr. The fibrillar network was destroyed by the digestion with beta (1----3)-glucanase. When protoplasts were regenerating in the presence of aculeacin A, the fibrillar networks were not formed, resulting in incomplete cell wall formation. These observations suggest that beta-glucan is the main component of the microfibrils and that it plays an important role in the formation of the cell wall in S. pombe.

Cell Wall Formation in Regenerating Protoplasts of Schizosaccharomyces pombe: Study by High Resolution, Low Voltage Scanning Electron Microscopy

High Resolution, Low Voltage Scanning Electron Microscopy of Uncoated Yeast Cells Fixed by the Freeze-Substitution Method

Certain digital image-processing methods, which are useful for nonperiodic structural images, have been applied to high-resolution SEM images for the improvement of resolution. Samples utilized in the present study consisted of magnetic tape coated with gold, T4 phage coated with gold-palladium, and uncoated specimens of Prolamellar body (PLB) in Cucurbita moschata. These images were blurred and otherwise disturbed by electronic noise, though the images were taken at the limit of efficiency of intrinsic instrument. The major image-processing tool was the Laplacian filter, which subtracts the Laplacian from the original image. Noise, which is a serious problem in digital processing of high-resolution SEM images, was suppressed by the nonlinear type smoothing method. Also, the noise was evaluated by an autocorrelation function and a power spectrum of the image. By using these methods of “deblurring” and noise removal, we achieved better resolution, and structural details of our biological specimens were revealed.

Application of the Laplacian filter to high‐resolution enhancement of SEM images

The refined field emission SEM, S-900 LV which gives better resolution especially at low voltages below 5 kV was developed for ultrahigh resolution scanning electron microscopy. A visualization test at x 300,000 was made using a gold-evaporated magnetic tape, and the resolution was found to be about 1 nm at 2.5 kV. The ultrastructure of the cell wall, especially the reverting glucan network, from the protoplast of Schizosaccharomyces pombe was studied using this improved ultrahigh-resolution low-voltage SEM (UHR-LVSEM). The results with uncoated reverting protoplasts observed with this microscope revealed that the network was originally formed as secreted particles scattered on the protoplast surface and these were subsequently stretched to microfibrils about 2 nm thick. The microfibrils were twisted around each other and joined together so that they developed into 8-nm-thick fibrils, forming a ribbon-shaped network of glucans about 16-nm-thick which covered the entire protoplast surface. The UHR-LVSEM images of reverting protoplasts treated with glucanase confirmed that the particles scattered on the protoplast surface in the initial stage of regeneration were glucan in nature.

Ultrahigh-resolution low-voltage SEM reveals ultrastructure of the glucan network formation from fission yeast protoplast.

An ultra high resolution scanning microscope (UHRSEM) which is composed of a cold cathode field emission electron gun and an in-lens system for specimens, has been introduced. Probe size is estimated approximately 0.5∼0.7 nm at an accelerating voltage of 30 kV by calculation, and it was confirmed experimentally using high atomic number fine particles. Fine metal oxide particles and semiconductor materials were observed directly without any metal coating. A necessity of the UHRSEM has been discussed especially for low voltage SEM (LVSEM) to obtain fine topographical details of the samples, which are very sensitive to the scanning electron beams.

Takashi Nagatani

Papers

Cell Wall Formation in Regenerating Protoplasts of Schizosaccharomyces pombe: Study by High Resolution, Low Voltage Scanning Electron Microscopy

High Resolution, Low Voltage Scanning Electron Microscopy of Uncoated Yeast Cells Fixed by the Freeze-Substitution Method

Application of the Laplacian filter to high‐resolution enhancement of SEM images

Ultrahigh-resolution low-voltage SEM reveals ultrastructure of the glucan network formation from fission yeast protoplast.

Ultra High Resolution Scanning Electron Microscopy