Biomedical Engineering Reference
In-Depth Information
Chapter 21
Microscopic and Spectroscopic Techniques to Investigate
Lipid Droplet Formation and Turnover in Yeast
Heimo Wolinski and Sepp D. Kohlwein
Abstract
In spite of some progress in understanding the molecular basis of lipid-associated disorders, major questions
about the regulation of synthesis and degradation of lipids and the interaction of these processes with other
aspects of cellular physiology are still unresolved. Studies in reference organisms such as various yeast spe-
cies, the fruit fl y
Drosophila melanogaster
, or the nematode
Caenorhabditis elegans
complement efforts in
mouse models as well as clinical studies in humans to address these questions. Imaging techniques play a
pivotal role in understanding lipid droplet biology, and the implementation of imaging-based high-content
screens of mutant collections has led to the identifi cation of novel molecular players. This study focuses on
novel fl uorescent probes as well as spectroscopic imaging techniques to investigate lipid droplet formation
and turnover in yeast. The application and limitations of such techniques in understanding lipid storage
and turnover are discussed.
Key words
Yeast,
Saccharomyces cerevisiae
, Neutral lipid storage, Lipolysis, Fluorescence microscopy,
Green fl uorescent protein, BODIPY 493/504, LD540, Nile Red, CARS microscopy
1
Introduction
Storage of neutral lipids such as triglycerides (TAG) in cytosolic
lipid droplets (LDs) is an important cellular process and essential
under conditions of fatty acid overload. Research on LD physiology
has attracted great interest in cell biology and biomedicine in
recent years [
1
-
4
]. The molecular function of a key regulator of
TAG synthesis, the phosphatidate phosphatase Pah1, and its mam-
malian ortholog lipin was fi rst described in yeast, underscoring the
important role of yeast as a reference organism [
5
-
7
]. The discov-
ery of novel enzymes and their regulators executing lipid degrada-
tion in mammalian cells [
8
-
13
] has led to a paradigm shift in our
understanding of how cells execute fat degradation. These studies
have unveiled molecular links between lipid degradation, signaling,
and cancer development and progression. Similarly, yeast expresses
the Tgl4 lipase, an ortholog of the mammalian adipose triglyceride
