Elsevier

Methods in Enzymology

Volume 451, 2008, Pages 373-408
Methods in Enzymology

Chapter Twenty‐Five Kinetoplastida: Model Organisms for Simple Autophagic Pathways?

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Abstract

Phylogenetic analyses based on defined proteins or different RNA species have revealed that the order kinetoplastida belongs to the early‐branching eukaryotes and may thus contain organisms in which complex cellular events are easier to analyze. This view was further supported by results from a bioinformatic survey that suggested that nearly half of the autophagy‐related proteins existent in yeast are missing in trypanosomatids. On the other hand, these organisms have evolved a highly sophisticated machinery to escape from the different host immune‐response strategies and have learned to cope with extremely variable environmental conditions by morphological and functional reorganization of the cell. For both the stress response and the differentiation processes, autophagy seems to be an indispensable prerequisite. So far autophagy has not been systematically investigated in trypanosomatids. Here we present technical information on how to handle the different parasites belonging to this order and give an overview of the current status of autophagy research in these organisms.

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.

  • 1

    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

References (72)

  • F. Hesse et al.

    A novel cultivation technique for long‐term maintenance of bloodstream form trypanosomes in vitro

    Mol. Biochem. Parasitol.

    (1995)
  • T. Jayaraman et al.

    Rapamycin‐FKBP12 blocks proliferation, induces differentiation, and inhibits cdc2 kinase activity in a myogenic cell line

    J. Biol. Chem.

    (1993)
  • H. Jungwirth et al.

    Diazaborine treatment of Baker's yeast results in stabilization of aberrant mRNAs

    J. Biol. Chem.

    (2001)
  • S.M. Landfear

    Molecular genetics of nucleoside transporters in Leishmania and African trypanosomes

    Biochem. Pharmacol.

    (2001)
  • S.N. Moreno et al.

    Calcium homeostasis in Trypanosoma cruzi amastigotes: presence of inositol phosphates and lack of an inositol 1,4,5‐trisphosphate‐sensitive calcium pool

    Mol. Biochem. Parasitol.

    (1992)
  • G.W. Morgan et al.

    The kinetoplastida endocytic apparatus

  • G.W. Morgan et al.

    The endocytic apparatus of the kinetoplastida

  • W.G. Morice et al.

    Rapamycin‐induced inhibition of p34cdc2 kinase activation is associated with G1/S‐phase growth arrest in T lymphocytes

    J. Biol. Chem.

    (1993)
  • M. Parsons et al.

    Biogenesis and function of peroxisomes and glycosomes

    Mol. Biochem. Parasitol.

    (2001)
  • S. Redmond et al.

    RNAit: An automated web‐based tool for the selection of RNAi targets in Trypanosoma brucei

    Mol. Biochem. Parasitol.

    (2003)
  • B. Reuner et al.

    Cell density triggers slender to stumpy differentiation of Trypanosoma brucei bloodstream forms in culture

    Mol. Biochem. Parasitol.

    (1997)
  • N.L. Uzcategui et al.

    Cloning, heterologous expression, and characterization of three aquaglyceroporins from Trypanosoma brucei

    J. Biol. Chem.

    (2004)
  • D. Vertommen et al.

    Differential expression of glycosomal and mitochondrial proteins in the two major life‐cycle stages of Trypanosoma brucei

    Mol. Biochem. Parasitol.

    (2008)
  • S.L. Baldauf et al.

    A kingdom‐level phylogeny of eukaryotes based on combined protein data

    Science

    (2000)
  • S. Bannai et al.

    Transport of cystine and cysteine and cell growth in cultured human diploid fibroblasts: Effect of glutamate and homocysteate

    J. Cell Physiol.

    (1982)
  • C. Berberich et al.

    The metacyclic stage‐expressed meta‐1 gene is conserved between Old and New World Leishmania species

    Mem. Inst. Oswaldo Cruz

    (1998)
  • A. Biederbick et al.

    Monodansylcadaverine (MDC) is a specific in vivo marker for autophagic vacuoles

    Eur. J. Cell Biol.

    (1995)
  • M. Clamp et al.

    The Jalview Java alignment editor

    Bioinformatics

    (2004)
  • C.E. Clayton

    Life without transcriptional control? From fly to man and back again

    EMBO J.

    (2002)
  • P. Codogno et al.

    Autophagy and signaling: Their role in cell survival and cell death

    Cell Death Differ.

    (2005)
  • W. de Souza et al.

    The paraxial structure of the flagellum of trypanosomatidae

    J. Parasitol.

    (1980)
  • A. Djikeng et al.

    RNA interference in Trypanosoma brucei: Cloning of small interfering RNAs provides evidence for retroposon‐derived 24‐26‐nucleotide RNAs

    RNA

    (2001)
  • W.A.J. Dunn et al.

    Pexophagy: The selective autophagy of peroxisomes

    Autophagy

    (2005)
  • M. Duszenko et al.

    Cysteine eliminates the feeder cell requirement for cultivation of Trypanosoma brucei bloodstream forms in vitro

    J. Exp. Med.

    (1985)
  • R.C. Edgar

    MUSCLE: A multiple sequence alignment method with reduced time and space complexity

    BMC Bioinformatics

    (2004)
  • K. Figarella et al.

    Prostaglandin D2 induces programmed cell death in Trypanosoma brucei bloodstream form

    Cell Death Differ.

    (2005)
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