Giardia lamblia: cell biology and microscopy of one of the most ...
Giardia lamblia: cell biology and microscopy of one of the most ...
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<strong>Giardia</strong> <strong>lamblia</strong>: <strong>cell</strong> <strong>biology</strong> <strong>and</strong> <strong>microscopy</strong> <strong>of</strong> <strong>one</strong> <strong>of</strong> <strong>the</strong> <strong>most</strong> primitive eukaryotes<strong>Giardia</strong> <strong>lamblia</strong>:<strong>cell</strong> <strong>biology</strong> <strong>and</strong> <strong>microscopy</strong><strong>of</strong> <strong>one</strong> <strong>of</strong> <strong>the</strong> <strong>most</strong> primitive eukaryotes.SUMMARY<strong>Giardia</strong>, a protist, is providing new insights into <strong>the</strong>evolution<strong>of</strong> <strong>the</strong> eukaryotic <strong>cell</strong> <strong>and</strong> <strong>of</strong> endoplasmic reticulum,microtubules <strong>and</strong> mitochondriaby Dr. Bohdan J. SoltysHere you will learn some <strong>of</strong> <strong>the</strong> new <strong>and</strong> exciting things about <strong>the</strong><strong>biology</strong> <strong>of</strong> protists, <strong>the</strong> <strong>most</strong> primitive eukaryotic, or non-bacterial,organisms. Recent discoveries concerning <strong>Giardia</strong> <strong>lamblia</strong> are emphasized<strong>and</strong> microscopic imaging <strong>of</strong> this organism <strong>and</strong> its sub<strong>cell</strong>ular organellesusing both fluorescence <strong>and</strong> electron <strong>microscopy</strong> is a major highlight.There is also a tribute page in honor <strong>of</strong> Keith Porter, pi<strong>one</strong>er <strong>of</strong> electron<strong>microscopy</strong>, which features comparative microscopic imaging <strong>of</strong>mammalian <strong>cell</strong>s, including human <strong>cell</strong>s, which in contrast to <strong>Giardia</strong>represent <strong>the</strong> <strong>most</strong> highly evolved eukaryotic organisms.SINCE FEB 2001CONTENTS❍<strong>Giardia</strong> HomepageI. IntroductionII. Identification <strong>and</strong> characterization <strong>of</strong> <strong>Giardia</strong>'shttp://www.geocities.com/CollegePark/Lab/4551/ (1 <strong>of</strong> 10) [2003/10/21 21:37:47]
<strong>Giardia</strong> <strong>lamblia</strong>: <strong>cell</strong> <strong>biology</strong> <strong>and</strong> <strong>microscopy</strong> <strong>of</strong> <strong>one</strong> <strong>of</strong> <strong>the</strong> <strong>most</strong> primitive eukaryotes❍❍endomembranesIII. Identity <strong>and</strong> organization <strong>of</strong> <strong>Giardia</strong>'s cytoskeletonIV. Did <strong>Giardia</strong> lose mitochondria in evolution?V. <strong>Giardia</strong> referencesVI. Links <strong>and</strong> recommended booksMicroscopy <strong>of</strong> Mammalian Cells - A Keith Porter Tribute PageAbout <strong>the</strong> AuthorI. INTRODUCTION<strong>Giardia</strong> <strong>lamblia</strong> is considered <strong>one</strong> <strong>of</strong> <strong>the</strong> deepest branching or <strong>most</strong>primitive eukaryotes in existence (see reference 1 <strong>and</strong> 2 listed below) <strong>and</strong>some scientists have called <strong>Giardia</strong> a 'missing link' in <strong>the</strong> evolution <strong>of</strong>eukaryotic <strong>cell</strong>s from prokaryotic <strong>cell</strong>s. Eukaryotic <strong>cell</strong>s, by definition, arethose containing a nucleus <strong>and</strong> include all organisms from protists up tohuman, while prokaryotic <strong>cell</strong>s are bacteria. <strong>Giardia</strong> <strong>lamblia</strong> was <strong>the</strong> firsteukaryotic <strong>cell</strong> to ever be seen using <strong>one</strong> <strong>of</strong> <strong>the</strong> first good qualitymicroscopes developed by Antonie van Leeuwenhoek, a Dutchman, backin <strong>the</strong> late 1600's. Van Leeuwenhoek was an amateur scientist with nohigher education who never<strong>the</strong>less made extraordinary contributions to<strong>biology</strong>. Although <strong>Giardia</strong> is a single <strong>cell</strong> organism, van Leeuwenhoekcalled it an 'animacule' because he thought it had an amazing similaritywith <strong>the</strong> general appearance <strong>of</strong> animals. What does <strong>Giardia</strong> look like toyou in <strong>the</strong> fluorescence micrograph below?http://www.geocities.com/CollegePark/Lab/4551/ (2 <strong>of</strong> 10) [2003/10/21 21:37:47]
<strong>Giardia</strong> <strong>lamblia</strong>: <strong>cell</strong> <strong>biology</strong> <strong>and</strong> <strong>microscopy</strong> <strong>of</strong> <strong>one</strong> <strong>of</strong> <strong>the</strong> <strong>most</strong> primitive eukaryotesAs a primitive eukaryote, underst<strong>and</strong>ing how <strong>Giardia</strong> performs basic<strong>cell</strong>ular functions will be helpful in elucidating <strong>the</strong> mechanisms present inall higher eukaryotic <strong>cell</strong>s (3, 4). In comparison, a model organism such asyeast, which many scientists study, is actually a higher eukaryote <strong>and</strong>provides information about only relatively recent evolutionarydevelopments. Also, <strong>the</strong> giardial genome is not much more complex thanthat <strong>of</strong> yeast, making its complete sequencing a feasible <strong>and</strong> deservingobjective (4). There are also applied reasons for studying <strong>Giardia</strong>. <strong>Giardia</strong>is <strong>of</strong> significant environmental <strong>and</strong> medical importance worldwide, being awaterborne pathogen <strong>and</strong> an important intestinal parasite in humans (5, 6).A few basic facts about <strong>Giardia</strong> should be menti<strong>one</strong>d. The life cycle <strong>of</strong><strong>Giardia</strong> alternates between trophozoite <strong>and</strong> cyst. <strong>Giardia</strong> lacksmitochondria <strong>and</strong> peroxisomes <strong>and</strong> until recently (see below) was reportedto also lack a Golgi apparatus <strong>and</strong> endoplasmic reticulum. The dormantwater-resistant cyst causes infection while <strong>the</strong> rapidly dividing trophozoitecauses <strong>the</strong> symptoms <strong>of</strong> giardiasis. The processes <strong>of</strong> encystation <strong>and</strong>excystation are both problems in <strong>cell</strong> differentiation. <strong>Giardia</strong> is relativelyeasy to culture <strong>and</strong> work with in <strong>the</strong> laboratory. Encystation can behttp://www.geocities.com/CollegePark/Lab/4551/ (3 <strong>of</strong> 10) [2003/10/21 21:37:47]
<strong>Giardia</strong> <strong>lamblia</strong>: <strong>cell</strong> <strong>biology</strong> <strong>and</strong> <strong>microscopy</strong> <strong>of</strong> <strong>one</strong> <strong>of</strong> <strong>the</strong> <strong>most</strong> primitive eukaryotes4. Longitudinal sections <strong>of</strong> cytoplasmic ax<strong>one</strong>mes Kinetosomes from whichmicrotubules are nucleated are seen at <strong>the</strong> top <strong>of</strong> part B.5. Electron micrographs showing labeling <strong>of</strong> <strong>the</strong> adhesive disk withantibody to acetylated tubulin. The adhesive disk is a microtubule-basedstructure only found in <strong>Giardia</strong>.Since postranslational modifications <strong>of</strong> tubulin are known tosignificantly alter microtubule assembly-disassembly dynamics, fur<strong>the</strong>rinvestigation <strong>of</strong> <strong>the</strong>se throughout <strong>the</strong> life cycle are warranted. Thus far,<strong>the</strong> cytoskeleton <strong>of</strong> trophozoites appears to be a ra<strong>the</strong>r stable structure,with all microtubules being acetylated . Future work should includeexamination <strong>of</strong> cytoskeleton dynamics during encystation <strong>and</strong> excystation,when dramatic structural rearrangements occur. Moreover, mitosis in<strong>Giardia</strong> has not yet been adequately described. Since it is no doubt <strong>the</strong>simplest mitosis in all eukaryotes, <strong>Giardia</strong> may be an ex<strong>cell</strong>ent modelsystem for underst<strong>and</strong>ing mitosis in general. The mitotic spindle is also apotential target for <strong>the</strong>rapeutic intervention in cases <strong>of</strong> giardiasis, <strong>and</strong> tests<strong>of</strong> anti-mitotic drugs would benefit from an underst<strong>and</strong>ing <strong>of</strong> <strong>Giardia</strong>'smitotic spindle physiology.IV. DID GIARDIA LOSE MITOCHONDRIA?A large number <strong>of</strong> protists, including <strong>Giardia</strong>, lack mitochondria. In <strong>the</strong>past this has been taken as evidence that <strong>the</strong>se organisms existed before<strong>the</strong> endosymbiosis event which led to mitochondria, <strong>and</strong> hence were moreprimitive than o<strong>the</strong>r protists. In endosymbiosis, a <strong>the</strong>ory made popular inits modern version by Dr. Lynn Margulis 30 years ago, oxygen respiringbacteria invaded a host <strong>cell</strong> <strong>and</strong> formed a permanent relationship livingwithin it, evolving into mitochondria. This endosymbiotic event is thoughtto have occured more than 1000 million years ago [our planetary systemformed 4600 million years ago; <strong>the</strong> first bacterial <strong>cell</strong> appeared 3900million years ago; <strong>the</strong> first protists appeared 2000 milllion years ago;man's ancestors appeared 4 million years ago]. Mitochondria in <strong>cell</strong>sactually still look like bacteria <strong>and</strong> grow <strong>and</strong> divide at <strong>the</strong>ir own pace.They even have <strong>the</strong>ir own DNA, although <strong>most</strong> genes over time have beentransferred to <strong>the</strong> nucleus.http://www.geocities.com/CollegePark/Lab/4551/ (6 <strong>of</strong> 10) [2003/10/21 21:37:47]
<strong>Giardia</strong> <strong>lamblia</strong>: <strong>cell</strong> <strong>biology</strong> <strong>and</strong> <strong>microscopy</strong> <strong>of</strong> <strong>one</strong> <strong>of</strong> <strong>the</strong> <strong>most</strong> primitive eukaryotesDespite <strong>the</strong> fact that <strong>Giardia</strong> lacks mitochondria, I <strong>and</strong> Rad Guptapublished work showing <strong>the</strong> presence <strong>of</strong> a protein related to mitochondrialhsp60 in <strong>Giardia</strong> (9). The evidence included biochemical immunoblotdetection <strong>of</strong> a protein <strong>of</strong> <strong>the</strong> correct molecular weight <strong>and</strong> bothimmun<strong>of</strong>lourescence <strong>and</strong> electron microscopic localization <strong>of</strong> reactivity atdiscrete sites in <strong>the</strong> cytoplasm.1.Immun<strong>of</strong>luorescence micrograph <strong>of</strong> <strong>Giardia</strong>.Cells were labeled withantibody against mammalian hsp60. Hsp60 is considered a mitochondrialprotein in higher eukaryotes. The fluorescent dots throughout <strong>the</strong>cytoplasm are suggestive <strong>of</strong> organelle labeling.2. Double label immun<strong>of</strong>luorescence.Hsp60 antibody labeling in A iscompared with anti-tubulin labeling <strong>of</strong> <strong>the</strong> same <strong>cell</strong>s in B. Somemicrotubule structures are identified in B: MB=median body, AF=anteriorflagella, AD=adhesive disk.Electron microscopic localization <strong>of</strong> hsp60 showed that hsp60 labelingwas in <strong>the</strong> cytoplasm <strong>and</strong> was not associated with any type <strong>of</strong> membranousstructure (not shown). To explain <strong>the</strong> findings we suggested that <strong>Giardia</strong>originally had mitochondria but lost <strong>the</strong>m in evolution. More recentstudies in <strong>the</strong> higher protist Trichomonas vaginalis, which containhydrogenosomes but no mitochondria, showed molecular evidence for <strong>the</strong>presence <strong>of</strong> mitochondrial heat shock proteins within hydogenosomes (<strong>the</strong>hydrogenosome is a double membraned redox organelle found in certainanaerobic protists). Palmer et al (10) have reviewed this work. The resultsled to <strong>the</strong> suggestion that hydogenosomes evolved (or de-evolved,depending on how you look at it) from mitochondria by a process <strong>of</strong>reductive, as opposed to acquisitive, evolution. Since <strong>Giardia</strong> has beenregarded as <strong>the</strong> <strong>most</strong> primitive eukaryote in existence, Palmer et al (10)also cite our work as evidence from diplomonads to support <strong>the</strong> idea that<strong>the</strong> earliest eukaryotic <strong>cell</strong> contained mitochondria which weresubsequently lost. Thus, <strong>the</strong> timing for <strong>the</strong> endosymbiotic event that gaverise to mitochondria is currently being pushed backwards. We are facedwith <strong>the</strong> possibility that no representative <strong>of</strong> <strong>the</strong> premitochondrial stage <strong>of</strong>eukaryotic evolution may be alive today. The endosymbiotic event thathttp://www.geocities.com/CollegePark/Lab/4551/ (7 <strong>of</strong> 10) [2003/10/21 21:37:47]
<strong>Giardia</strong> <strong>lamblia</strong>: <strong>cell</strong> <strong>biology</strong> <strong>and</strong> <strong>microscopy</strong> <strong>of</strong> <strong>one</strong> <strong>of</strong> <strong>the</strong> <strong>most</strong> primitive eukaryotesgave rise to mitochondria in fact may have occurred as far back as <strong>the</strong>very origin <strong>of</strong> <strong>the</strong> first eukaryotic <strong>cell</strong>. The key to resolving this issuewould be to obtain fur<strong>the</strong>r molecular data in <strong>Giardia</strong>. The cloning <strong>of</strong> avariety <strong>of</strong> mitochondrial proteins will be necessary. It may be verydifficult, however, to exclude lateral gene transfer <strong>of</strong> proteins fromano<strong>the</strong>r species, particularly bacterial. The proteins would have to containmitochondrial targeting sequences to definitively distinquish <strong>the</strong>m fromprokaryotic homologs.SINCE FEB 2001Material presented here is protected by copyright.V. GIARDIA REFERENCES1. Sogin, ML 1991. Early evolution <strong>and</strong> <strong>the</strong> origin <strong>of</strong> eukaryotes. CurrentOpinion in Genetics <strong>and</strong> Development 1:457-463.2. Nasmuth, K 1996. A homage to <strong>Giardia</strong>. Current Biology 6:1042.3. Gillin, F et al.1996. Cell <strong>biology</strong> <strong>of</strong> <strong>the</strong> primitive eukaryote <strong>Giardia</strong><strong>lamblia</strong>. Annual Review <strong>of</strong> Micro<strong>biology</strong> 50, 679-705.4. Lujan, HD, Mowatt, MR <strong>and</strong> Nash, TE 1997. Mechanisms <strong>of</strong> <strong>Giardia</strong><strong>lamblia</strong> differentiation into cysts. Micro<strong>biology</strong> <strong>and</strong> Molecular BiologyReview. 61:294-304.5. Marshall, MM, et al. 1997. Waterborne protozoan pathogens. ClinicalMicro<strong>biology</strong> Review 10:67-85.6. Finch, GR 1996. Water industry challenge waterborne parasites PartII. Environmental Science <strong>and</strong> Engineering 9:35,36,38.7.Soltys, BJ, Falah, M <strong>and</strong> RS Gupta. 1996. Identification <strong>of</strong> endoplasmicreticulum in <strong>the</strong> primitive eukaryote <strong>Giardia</strong> <strong>lamblia</strong> using cryoelectron<strong>microscopy</strong> <strong>and</strong> antibody to Bip. Journal <strong>of</strong> Cell Science 109:1909-1917.*See also <strong>the</strong> ‘Headlines' article entitled ‘The Primitive ER' which reviewsthis work in Trends in Cell Biology 1996. 6:378.8. Soltys, BJ <strong>and</strong> RS Gupta. 1994. Immunoelectron <strong>microscopy</strong> <strong>of</strong> <strong>Giardia</strong><strong>lamblia</strong> cytoskeleton using antibody to acetylated alpha tubulin. Journal <strong>of</strong>http://www.geocities.com/CollegePark/Lab/4551/ (8 <strong>of</strong> 10) [2003/10/21 21:37:47]
<strong>Giardia</strong> <strong>lamblia</strong>: <strong>cell</strong> <strong>biology</strong> <strong>and</strong> <strong>microscopy</strong> <strong>of</strong> <strong>one</strong> <strong>of</strong> <strong>the</strong> <strong>most</strong> primitive eukaryotesEukaryotic Micro<strong>biology</strong> 41: 625-632.9. Soltys, BJ <strong>and</strong> RS Gupta. 1994. Presence <strong>and</strong> <strong>cell</strong>ular distribution <strong>of</strong> a60-kDa protein related to mitochondrial hsp60 in <strong>Giardia</strong> <strong>lamblia</strong>. Journal<strong>of</strong> Parasitology 80: 580-590.10. Palmer, JD 1997. Organelle genomes: going, going, g<strong>one</strong>! Science275:790-791.11. Roger AJ, Svard SG, Tovar J, Clark CG, Smith MW, Gillin FD, SoginML 1998 A mitochondrial-like chaperonin 60 gene in <strong>Giardia</strong> <strong>lamblia</strong>:evidence that diplomonads once harbored an endosymbiont related to <strong>the</strong>progenitor <strong>of</strong> mitochondria. Proc Natl Acad Sci USA 95: 229-34.VI. RECOMMENDED BOOKS AND LINKSA. LINKSFor links to websites on o<strong>the</strong>r protists, its a must to check out ProtistImage DataThis site is a featured website at YAHOO! where you can find links too<strong>the</strong>r sites on model systems <strong>and</strong> microorganisms:1. http://dir.yahoo.com/Science/Research/Model_Systems/2. http://dir.yahoo.com/Science/Biology/Micro<strong>biology</strong>/Microorganisms/B. BOOKS FOR STUDENTS, HOBBYISTS AND AMATEURSCIENTISTS1. Explore <strong>the</strong> World Using Protozoa by R.O. Anderson <strong>and</strong> M. DrugerPaperback (1997) Natl Science Teachers Assn; ISBN: 0873551591; highlyrecommended.2. Guide to Microlife by K.G. Rainis <strong>and</strong> B.J. RussellPaperback (1997) Franklin Watts, Inc.; ISBN: 05311126673. How to Know <strong>the</strong> Protozoa by Theodore L. JahnPaperback 2nd edition (1979) WCB/McGraw-Hill; ISBN: 06970475984. A World in a Drop <strong>of</strong> Water : Exploring With a Microscope by A.Silverstein, V.B. Silversteinhttp://www.geocities.com/CollegePark/Lab/4551/ (9 <strong>of</strong> 10) [2003/10/21 21:37:47]
<strong>Giardia</strong> <strong>lamblia</strong>: <strong>cell</strong> <strong>biology</strong> <strong>and</strong> <strong>microscopy</strong> <strong>of</strong> <strong>one</strong> <strong>of</strong> <strong>the</strong> <strong>most</strong> primitive eukaryotesPaperback (1998) Dover Pubns; ISBN: 0486403815; Under $4!5. Antoni Van Leeuwenhoek : First to See Microscopic Life (Great Minds <strong>of</strong>Science series) by L. YountLibrary Binding (1996) Enslow Publishers, Inc.; ISBN: 08949068016. Five Kingdoms : An Illustrated Guide to <strong>the</strong> Phyla <strong>of</strong> Life on Earth by L.Margulis, K.V. Schwartz, S.J. GouldPaperback 3rd edition (1998) W H Freeman & Co; ISBN: 0716730278C. BOOKS FOR GRADUATE STUDENTS AND PROFESSIONALSCIENTISTS1. Protozoa <strong>and</strong> O<strong>the</strong>r Protists by Michael A. SleighHardcover 3rd edition (1999) Edward Arnold; ISBN: 05215736292. Protoctista Glossary by L. Margulis, H.I. McKhann, L. OlendzenskiHardcover (1999) J<strong>one</strong>s & Bartlett Pub; ISBN: 0867200812Where to next on this website?1. BACK TO THE TOP2. MICROSCOPY OF MAMMALIAN CELLS3. ABOUT THE AUTHORThis page hosted byGet your own Free Home Pagehttp://www.geocities.com/CollegePark/Lab/4551/ (10 <strong>of</strong> 10) [2003/10/21 21:37:47]
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