Obese and Slim Yeast Cells

Microscopic Insights into Cellular Lipid Metabolism

August 22, 2008

Lipids are on everyone’s lips nowadays, whether ω-3/6 fatty acids, good and bad cholesterol or just plain fat that has the annoying habit of accumulating on our hips. Serious diseases such as obesity, arteriosclerosis and type 2 diabetes mellitus are directly connected with lipid metabolism disorders. The model system yeast ("baker’s yeast") provides excellent possibilities for exploring lipid-associated diseases, including the use of high-resolution microscopy.

Fat as a risk factor

In accordance with its central importance for the organism, lipid metabolism is controlled by diverse regulation mechanisms. However, these mechanisms are overtaxed by our modern lifestyle of too much food and a lack of exercise. The dramatic development of lipid-associated diseases in industrialised countries has tended to propagate a negative image of lipid substances. Yet fat in a wide variety of forms is an indispensable ingredient of all cells. Even the much-maligned triglycerides play a vital role as a buffer for excess and potentially dangerous fatty acids in our circulation or as an energy store.

Only recently was a main fat degrading enzyme (ATGL, Adipose Tri­glyceride Lipase) found in fat tissue [1], demonstrating that research into fats is, now as ever, a "goldmine" for discovering new biomedically relevant key factors (see also: GOLD – Genomics of lipid-associated disorders, a project conducted within the framework of the Austrian Genome Research program GEN-AU, and the special research project LIPOTOX, sponsored by FWF [Austrian Science Fund]).

A big look at small cells

Besides molecular-biological, genomic and proteomic techniques, high-resolution light microscopy is important for gaining innovative insights into cellular lipid synthesis, lipid and membrane dynamics and morphological changes in connection with lipid metabolism disorders.

With its diameter of 5–8 μm, the yeast cell used to be considered an unsuitable specimen for light microscopic examination. Actually, the technological progress made in microscopy over the last few years through improved imaging techniques and excellent objectives can now resolve sub-cellular structures of yeast cells (Figure 1). Confocal laser scanning microscopy offers special advantages for three-dimensional analysis of cells observed under physiological conditions over several generations [2]. Diffraction problems and stray light are minimal due to the thin cell thickness. The typical lateral and axial resolution is approx. 150 nm and 350 nm, respectively. The three-dimensional reconstruction achieved by recording a large number of "optical sections" and the simultaneous detection of several fluorophores provide completely new insights into the spatial and dynamic protein and lipid interactions in live cells.

The availability of fluorescing protein variants (e.g. GFP – green fluorescent protein) in connection with extremely simple cloning techniques has created the basis for localisation and expression studies of all proteins of the yeast proteome (of which there are roughly 6,000) [3]. Robust preparation protocols for live cell microscopy and vital staining allow sim­ultaneous observation of large cell populations, an excellent basis for collecting quantitative micro­scopy data.

Fig. 1: Visualisation of sub-cellular structures of yeast by GFP tagging of proteins and confocal laser scanning microscopy (Leica TCS SP2 Multi-Photon System; 488 nm excitation; 500–550 nm detection; 1.4 N.A. 100x oil). From top left to bottom right: Cell nucleus, endoplasmic reticulum, plasma membrane, cytosol. Scale = 5 μm.

Through thick and thin – yeast in lipid metabolism research

Storage fats are found in the cell in the form of fat droplets. These, however, are not passive fat depots, but dynamic organelles with numerous proteins and specific biochemical functions. The biogenesis of lipid droplets is closely connected to the synthesis of the storage fats: if synthesis is switched off due to mutation, no lipid droplets are formed, and "slim" yeast cells are produced. These mutants react particularly sensitively to excess fatty acids which can no longer be incorporated into storage fat. This lipotoxicity of fatty acids is also observed in similar form in animal cells. We may therefore deduce that the synthesis of storage fat is an essential valve for rendering excess fatty acids metabolically harmless. The biogenesis and dynamics of these lipid droplets can be imaged under a high-resolution microscope by staining with vital dyes or GFP-labelled proteins (Figure 2).

If the fat-splitting enzymes are switched off through mutation, this leads to an accumulation of triglycerides in the cell and "obese" yeast cells are produced. This defect leads to stunted growth, too, suggesting that fat degradation provides important metabolic products for cell growth [4]. The enzymes Tgl3 and Tgl4 that are involved in the fat splitting process in yeast are structurally related to ATGL and their function can be partially replaced with mouse ATGL. This finding confirms the high degree of functional and structural conservation of lipid metabolism enzymes between yeast and animal cells.

Fig. 2: A) Yeast mutants with defective triglyceride breakdown accumulate Nile Red-positive lipid droplets (LD). Scale = 5 μm.
Fig. 2: B) Visualisation of LD with the aid of GFP-labelled proteins. Left, fluorescence; Right, Differential Interference contrast. Upper row: LD in live cells, lower row: LD released from squeezed cells.

The spatial organisation of lipid metabolism

Lipid metabolism is spread out over different areas of the cell and is subjected to a complex control process. To characterise the spatial organisation, about 600 proteins of lipid metabolism were localised as GFP fusions at high resolution [5]. Among other observations, this study has led to the identification of previously unknown proteins of the lipid droplets. Currently, all approx. 6,000 chromosomally expressed GFP fusions are being examined with the aid of confocal laser scanning microscopy and the localisation data are being made available to the scientific community in the specially designed Yeast Protein Localisation database, YPL.db [6] (Figure 3). It can be clearly shown that protein localisation is not static, but is decisively influenced by the state of development of the cell and by disorders in lipid metabolism.

Fig. 3: Yeast protein localisation database

Microscopy-based high-content screens

The availability of extensive collections of yeast mutants (approx. 4,500 viable deletion mutants) and suitable fluorescence dyes for lipid stores prompts the use of microscopy-based screens for lipid metabolism mutants. A recently conducted microscopy-based screen actually identified a yeast homolog of seipin that is defective in patients with Bernardinelli-Seip congenital lipodystrophy 2. The absence of this protein in yeast leads to a disturbed lipid droplet morphology [7, 8]. This implies that the identification and characterisation of preserved factors of lipid storage in yeast has tremendous potential for the understanding of the molecular causes of lipid metabolism disorders in humans.

Future developments

The technological progress being made in micro­scopy plus the extensive repertoire of yeast technologies provide fascinating opportunities for understanding lipid metabolism disorders. The recently developed technique of CARS microscopy (Coherent Anti-Stokes Raman Scattering) is particularly useful for the selective detection of lipid molecules and therefore has enormous potential for imaging lipid species without the use of special fluorescence dyes. In combination with genomic and proteomic techniques, fascinating new ways of exploring lipid synthesis and dynamics in live (yeast) cells are emerging with a view to understanding lipid metabolism-associated diseases in humans.

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