Chapter Twenty‐Five Kinetoplastida: Model Organisms for Simple Autophagic Pathways?
Introduction
The invention of the lysosome was certainly a major breakthrough in the development of eukaryotic cells, as it enabled them to get rid of any kind of cellular material without losing the basic chemical components, such as amino acids, bases, sugars, or lipids. In the most primitive way, a bunch of acid hydrolases, sequestered in a membrane‐surrounded vesicle, are sufficient to do the job. Here, the vesicle meanders through the cell, thereby engulfing and grabbing small parts of the cytosol or cytoplasm, respectively, which are degraded to building blocks of macromolecules, finally reused by the cell (Marzella et al., 1981). This process is highly unspecific and only marginally controlled but sufficient for a steady turnover of biomacromolecules and individual damaged organelles. From here more sophisticated functions for lysosomal digestion and nutrient supply have evolved, which include fusion of lysosomes with phagosomes on phagocytosis or endosomes on receptor‐mediated endocytosis (Morgan et al., 2002a, Morgan et al., 2002b). In addition, lysosomes serve as an intracellular defense machinery to destroy bacteria or viruses on infection of the cell (Kirkegaard et al., 2004). Most specific and controlled, however, are the various forms of autophagy. In its simplest form, autophagy is a limited form of self‐digestion for the benefit of cell survival, such as during starvation. In this case, bulk cytosol or disposable organelles are surrounded by specifically formed membranes of unknown origin called phagophores. These are thought to originate at phagophore assembly sites or preautophagosomal structures (PAS). Upon completion, the phagophore forms a double‐membrane vesicle, termed an autophagosome, that fuses with lysosomes forming so‐called autophagolysosomes (or autolysosomes). If essential nutrients reappear in time, the cell will recover and replace missing organelles and molecules. Otherwise, affected cells may enter a controlled cell death pathway and dissolve, altruistically leaving nutrients for surrounding cells. This form of autophagy has been called programmed cell death (PCD) type II (Levine and Yuan, 2005, Maiuri et al., 2007).
Even more interesting is autophagy as a mechanism to maintain the functional integrity of a cell during changing environmental conditions. For example, at a certain time hepatocytes may have to produce bulk quantities of serum proteins and thus contain abundant ER and Golgi membranes. If, under changing conditions, these organelles become obsolete and should be removed, they will be specifically labeled for digestion, engulfed by phagophores and delivered to lysosomes for degradation. Thus, removal of defined organelles during the life span of a cell is a common and regular process and needs a precise and controlled targeting. There is even a specific denotation such as pexophagy for the removal of peroxisomes or mitophagy for the removal of mitochondria (Dunn et al., 2005, Mijaljica et al., 2007). Likewise, during differentiation a cell adjusts to the organism's needs and has to remodel or replace some organelles to fulfill its duties. Autophagy is thus for an average cell a continuous and indispensable mechanism to cope with cellular needs (e.g. turnover of housekeeping enzymes) and changing life cycle (e.g. differentiation) or environmental (e.g. starvation or stress response) conditions. These different functional elements make it also a process of considerable complexity with a plethora of different factors and the involvement of a variety of protein‐protein, protein‐organelle, and membrane‐membrane interactions (Codogno and Meijer, 2005, Yorimitsu and Klionsky, 2005). As described throughout this volume and elsewhere, some of the interaction partners and molecular events have already been dissected, while others are still missing or of mysterious function.
Using bioinformatic approaches, only a limited number of genes seem to be involved in autophagy in trypanosomes as compared with yeast and higher eukaryotes (Herman et al., 2006, Rigden et al., 2005). This may reflect the specific situation for the order kinetoplastida, which branched off the evolutionary development at a very early time point (Baldauf et al., 2000) and offers the hope of finding a less complex network of molecular partners in these model organisms (Klionsky, 2006). On the other hand, several members of this order are very important pathogens (Table 25.1) such as Trypanosoma brucei (sleeping sickness), Trypanosoma cruzi (Chagas' disease) and Leishmania donovani (kala‐azar). A precise understanding of this elementary cellular process in these cells may provide new targets for the development of effective and safe drugs to treat the respective parasitic diseases. Here we present a comprehensive and largely complete picture of what is currently known about autophagy in the trypanosomatids.
Section snippets
Experimental Procedures to Handle the Different Species of the Order Kinetoplastida
The order kinetoplastida was named for the presence of a Feulgen‐positive structure in a distinct region of the single mitochondrion in these organisms (Feulgen stain is used to detect chromosomal, or in this case mitochondrial, DNA). This compartment contains the mtDNA, which comprises up to several thousand minicircles and some dozen maxicircles intercalated to a cuboidlike structure easily detected in electron micrographs (Shapiro, 1993). Whereas only maxicircles contain genetic information,
Microautophagy
Microautophagy seems to be a continuous process in any eukaryotic cell to have a constant turnover of cytosol/cytoplasm. The most obvious morphological difference between macro‐ and microautophagy is the form of sequestration. While macroautophagy involves formation of a cytosolic double‐membrane vesicle that picks up cellular materials and eventually fuses with the lysosome, microautophagy describes the direct invagination or embracing protrusions of the lysosome to take up parts of the
Fluorescence microscopy
Rapamycin treatment of various cells such as yeast (Chung et al., 1992), myoblasts (Jayaraman and Marks, 1993), and T‐lymphocytes (Morice et al., 1993) leads to a cell cycle arrest especially in G1/S‐phase. Because of the cell cycle progression by binary fission, in trypanosomatids the number of nuclei and kinetoplasts within one cell are indicative of the respective cell cycle phase (Woodward and Gull, 1990); therefore, inhibition of proliferation can be easily visualized.
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Cells are subjected
Concluding Remarks
As judged from an in silico survey, members of the order kinetoplastida seem to perform autophagy in a rather primitive way, as only half of the respective proteins expressed in yeast have homologs in trypanosomes and Leishmania (Rigden et al., 2005). Moreover, they represent an early evolutionary branch point. This led to the idea that trypanosomes may serve as model organisms to investigate autophagy in a simple form. However, it should be kept in mind that the presence or absence of gene
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