Giardia, a protist, is providing new insights into the evolution
of the eukaryotic cell and of endoplasmic reticulum,
microtubules and mitochondria
by Dr. Bohdan J. Soltys
Here you will learn some of the new and exciting things about the biology of protists, the most primitive eukaryotic, or non-bacterial, organisms. Recent discoveries concerning Giardia lamblia are emphasized and microscopic imaging of this organism and its subcellular organelles using both fluorescence and electron microscopy is a major highlight. There is also a tribute page in honor of Keith Porter, pioneer of electron microscopy, which features comparative microscopic imaging of mammalian cells, including human cells, which in contrast to Giardia represent the most highly evolved eukaryotic organisms.
Visitors since March 2006
(website launched in 1999)
- Giardia Homepage
- I. Introduction
- II. Identification and characterization of Giardia’s endomembranes
- III. Identity and organization of Giardia’s cytoskeleton
- IV. Did Giardia lose mitochondria in evolution?
- V. Giardia references
- VI. Links and recommended books
- Microscopy of Mammalian Cells – A Keith Porter Tribute Page
- Giardia Homepage
Giardia lamblia is considered one of the deepest branching or most primitive eukaryotes in existence (see reference 1 and 2 listed below) and some scientists have called Giardia a ‘missing link’ in the evolution of eukaryotic cells from prokaryotic cells. Eukaryotic cells, by definition, are those containing a nucleus and include all organisms from protists up to human, while prokaryotic cells are bacteria. Giardia lamblia was the first eukaryotic cell to ever be seen using one of the first good quality microscopes developed by Antonie van Leeuwenhoek, a Dutchman, back in the late 1600’s. Van Leeuwenhoek was an amateur scientist with no higher education who nevertheless made extraordinary contributions to biology. Although Giardia is a single cell organism, van Leeuwenhoek called it an ‘animacule’ because he thought it had an amazing similarity with the general appearance of animals. What does Giardia look like to you in the fluorescence micrograph below?
As a primitive eukaryote, understanding how Giardia performs basic cellular functions will be helpful in elucidating the mechanisms present in all higher eukaryotic cells (3, 4). In comparison, a model organism such as yeast, which many scientists study, is actually a higher eukaryote and provides information about only relatively recent evolutionary developments. Also, the giardial genome is not much more complex than that of yeast, making its complete sequencing a feasible and deserving objective (4). There are also applied reasons for studying Giardia. Giardia is of significant environmental and medical importance worldwide, being a waterborne pathogen and an important intestinal parasite in humans (5, 6) .
A few basic facts about Giardia should be mentioned. The life cycle of Giardia alternates between trophozoite and cyst. Giardia lacks mitochondria and peroxisomes and until recently (see below) was reported to also lack a Golgi apparatus and endoplasmic reticulum. The dormant water-resistant cyst causes infection while the rapidly dividing trophozoite causes the symptoms of giardiasis. The processes of encystation and excystation are both problems in cell differentiation. Giardia is relatively easy to culture and work with in the laboratory. Encystation can be artificially induced in culture. De novo assembly of endomembranes has been suggested to occur during encystation, correlated with regulated secretion of cyst wall proteins. (3, 4).
All the fluorescence and electron microscopic imaging shown on this website comes from my own laboratory research while I was a postdoctoral researcher in the Department of Biochemistry at McMaster University (Hamilton, Ontario, Canada). I have published fundamental studies that deal with different aspects of Giardia cell biology (7-9). These studies identify three important areas of current basic research on this organism.
Despite there having already been 30 years of research on Giardia’s subcellular structure, I and Rad Gupta have provided the first definitive evidence for the presence of endoplasmic reticulum (ER) in this organism (Reference 7). You may click on electron microscopic (EM) pictures or you can read the actual publication. Permission to make these items available on the Internet was obtained from the Journal of Cell Science and the Company of Biologists Ltd.
1.Low magnification EM of Giardia.
2.Higher magnification EM showing ER membranes. The ER is labelled with antibody against the ER protein called Bip. The antibody is bound to colloidal gold markers, which in EM appear as black dots.
3.Read the actual publication.You will need Adobe Acrobat Reader.
For a review of this study, see the ‘Headlines’ article which appeared in Trends in Cell Biology (7). This work depended on (i) the use of cryotechniques to preserve endomembranes and (ii) raising an antibody against recombinant giardial Bip, the hsp70 homolog resident in ER in higher eukaryotes, to serve as a definitive molecular label. This work confirmed a central dogma in cell biology, namely that the endomembrane system and nucleus co-evolved in the same evolutionary event and that all eukaryotic cells would possess both. In addition to characterizing the structural organization of the ER, this study also identified membrane systems in trophozoites which appeared to represent a Golgi apparatus. Although a Golgi apparatus was previously reported by others to form uniquely during encystation (4), our studies led us to conclude that a Golgi apparatus is present throughout the life cycle but in functionally different forms. Further confirmation of this will require molecular labels which specifically identify the Golgi. Giardia should prove to be an excellent model system for studies into developmental changes in Golgi structure and function, vesicular transport and regulated versus constitutive secretion, with the changes that occur during encystation and excystation serving as models of cell differentiation.
The processes of cytokinesis and cell differentiation are driven by dramatic restructuring of cytoskeletal structures. I have published a detailed study of the organization of the microtubule-based cytoskeleton in trophozoites (8) which also included a partial investigation of postranslational tubulin modifications (postranslational modifications are chemical reactions that change the biological activity and/or localization of a protein). In the case of tubulin, postranslational modifications may alter the assembly/disassembly dynamics of microtubules. You can click on some fluorescence or electon microscopic pictures:
1.Immunofluorescence micrograph of Giardia microtubules. Cells were labeled with a fluorescent antibody against acetylated tubulin.
2. Electron micrographs of flagellar axonemes and the median body. Microtubules in these structures, which look like railroad tracks when viewed longitudinally (A and B) or as circles when viewed in cross section (C),are labeled with antibody against acetylated tubulin bound to colloidal gold markers, which appear as black dots.
3. Flagella microtubules in cross section Giardia flagella have the typical 9(2)+2 axonemal structure found in higher eukaryotes (nine sets of two microtubules arranged in a circle with two microtubules in the center) and not a more primitive version.
4. Longitudinal sections of cytoplasmic axonemes Kinetosomes from which microtubules are nucleated are seen at the top of part B.
5. Electron micrographs showing labeling of the adhesive disk with antibody to acetylated tubulin. The adhesive disk is a microtubule-based structure only found in Giardia.
Since postranslational modifications of tubulin are known to significantly alter microtubule assembly-disassembly dynamics, further investigation of these throughout the life cycle are warranted. Thus far, the cytoskeleton of trophozoites appears to be a rather stable structure, with all microtubules being acetylated . Future work should include examination of cytoskeleton dynamics during encystation and excystation, when dramatic structural rearrangements occur. Moreover, mitosis in Giardia has not yet been adequately described. Since it is no doubt the simplest mitosis in all eukaryotes, Giardia may be an excellent model system for understanding mitosis in general. The mitotic spindle is also a potential target for therapeutic intervention in cases of giardiasis, and tests of anti-mitotic drugs would benefit from an understanding of Giardia’s mitotic spindle physiology.
A large number of protists, including Giardia, lack mitochondria. In the past this has been taken as evidence that these organisms existed before the endosymbiosis event which led to mitochondria, and hence were more primitive than other protists. In endosymbiosis, a theory made popular in its modern version by Dr. Lynn Margulis 30 years ago, oxygen respiring bacteria invaded a host cell and formed a permanent relationship living within it, evolving into mitochondria. This endosymbiotic event is thought to have occured more than 1000 million years ago [our planetary system formed 4600 million years ago; the first bacterial cell appeared 3900 million years ago; the first protists appeared 2000 milllion years ago; man’s ancestors appeared 4 million years ago]. Mitochondria in cells actually still look like bacteria and grow and divide at their own pace. They even have their own DNA, although most genes over time have been transferred to the nucleus.
Despite the fact that Giardia lacks mitochondria, I and Rad Gupta published work showing the presence of a protein related to mitochondrial hsp60 in Giardia (9). The evidence included biochemical immunoblot detection of a protein of the correct molecular weight and both immunoflourescence and electron microscopic localization of reactivity at discrete sites in the cytoplasm.
1.Immunofluorescence micrograph of Giardia.Cells were labeled with antibody against mammalian hsp60. Hsp60 is considered a mitochondrial protein in higher eukaryotes. The fluorescent dots throughout the cytoplasm are suggestive of organelle labeling.
2. Double label immunofluorescence.Hsp60 antibody labeling in A is compared with anti-tubulin labeling of the same cells in B. Some microtubule structures are identified in B: MB=median body, AF=anterior flagella, AD=adhesive disk.
Electron microscopic localization of hsp60 showed that hsp60 labeling was in the cytoplasm and was not associated with any type of membranous structure (not shown). To explain the findings we suggested that Giardia originally had mitochondria but lost them in evolution. More recent studies in the higher protist Trichomonas vaginalis, which contain hydrogenosomes but no mitochondria, showed molecular evidence for the presence of mitochondrial heat shock proteins within hydogenosomes (the hydrogenosome is a double membraned redox organelle found in certain anaerobic protists). Palmer et al (10) have reviewed this work. The results led to the suggestion that hydogenosomes evolved (or de-evolved, depending on how you look at it) from mitochondria by a process of reductive, as opposed to acquisitive, evolution. Since Giardia has been regarded as the most primitive eukaryote in existence, Palmer et al (10) also cite our work as evidence from diplomonads to support the idea that the earliest eukaryotic cell contained mitochondria which were subsequently lost. Thus, the timing for the endosymbiotic event that gave rise to mitochondria is currently being pushed backwards. We are faced with the possibility that no representative of the premitochondrial stage of eukaryotic evolution may be alive today. The endosymbiotic event that gave rise to mitochondria in fact may have occurred as far back as the very origin of the first eukaryotic cell. The key to resolving this issue would be to obtain further molecular data in Giardia. The cloning of a variety of mitochondrial proteins will be necessary. It may be very difficult, however, to exclude lateral gene transfer of proteins from another species, particularly bacterial. The proteins would have to contain mitochondrial targeting sequences to definitively distinquish them from prokaryotic homologs.
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1. Sogin, ML 1991. Early evolution and the origin of eukaryotes. Current Opinion in Genetics and Development 1:457-463.
2. Nasmuth, K 1996. A homage to Giardia. Current Biology 6:1042.
3. Gillin, F et al.1996. Cell biology of the primitive eukaryote Giardia lamblia. Annual Review of Microbiology 50, 679-705.
4. Lujan, HD, Mowatt, MR and Nash, TE 1997. Mechanisms of Giardia lamblia differentiation into cysts. Microbiology and Molecular Biology Review. 61:294-304.
5. Marshall, MM, et al. 1997. Waterborne protozoan pathogens. Clinical Microbiology Review 10:67-85.
6. Finch, GR 1996. Water industry challenge waterborne parasites Part II. Environmental Science and Engineering 9:35,36,38.
7.Soltys, BJ, Falah, M and RS Gupta. 1996. Identification of endoplasmic reticulum in the primitive eukaryote Giardia lamblia using cryoelectron microscopy and antibody to Bip. Journal of Cell Science 109:1909-1917 .*See also the ‘Headlines’ article entitled ‘The Primitive ER’ which reviews this work in Trends in Cell Biology 1996. 6:378.
8. Soltys, BJ and RS Gupta. 1994. Immunoelectron microscopy of Giardia lamblia cytoskeleton using antibody to acetylated alpha tubulin. Journal of Eukaryotic Microbiology 41: 625-632.
9. Soltys, BJ and RS Gupta. 1994. Presence and cellular distribution of a 60-kDa protein related to mitochondrial hsp60 in Giardia lamblia. Journal of Parasitology 80: 580-590.
10. Palmer, JD 1997. Organelle genomes: going, going, gone! Science 275:790-791.
11. Roger AJ, Svard SG, Tovar J, Clark CG, Smith MW, Gillin FD, Sogin ML 1998 A mitochondrial-like chaperonin 60 gene in Giardia lamblia: evidence that diplomonads once harbored an endosymbiont related to the progenitor of mitochondria. Proc Natl Acad Sci USA 95: 229-34.
This site is a featured website at YAHOO! where you can find links to other sites on model systems and microorganisms:
B. BOOKS FOR STUDENTS, HOBBYISTS AND AMATEUR SCIENTISTS
1. Explore the World Using Protozoa by R.O. Anderson and M. Druger
Paperback (1997) Natl Science Teachers Assn; ISBN: 0873551591; highly recommended.
2. Guide to Microlife by K.G. Rainis and B.J. Russell
Paperback (1997) Franklin Watts, Inc.; ISBN: 0531112667
3. How to Know the Protozoa by Theodore L. Jahn
Paperback 2nd edition (1979) WCB/McGraw-Hill; ISBN: 0697047598
4. A World in a Drop of Water : Exploring With a Microscope by A. Silverstein, V.B. Silverstein
Paperback (1998) Dover Pubns; ISBN: 0486403815; Under $4!
5. Antoni Van Leeuwenhoek : First to See Microscopic Life (Great Minds of Science series) by L. Yount
Library Binding (1996) Enslow Publishers, Inc.; ISBN: 0894906801
6. Five Kingdoms : An Illustrated Guide to the Phyla of Life on Earth by L. Margulis, K.V. Schwartz, S.J. Gould
Paperback 3rd edition (1998) W H Freeman & Co; ISBN: 0716730278
C. BOOKS FOR GRADUATE STUDENTS AND PROFESSIONAL SCIENTISTS
1. Protozoa and Other Protists by Michael A. Sleigh
Hardcover 3rd edition (1999) Edward Arnold; ISBN: 0521573629
2. Protoctista Glossary by L. Margulis, H.I. McKhann, L. Olendzenski
Hardcover (1999) Jones & Bartlett Pub; ISBN: 0867200812