Genomics and Cell Biology of the Apicomplexa
Transcript of Part 1: Biology of Apicomplexan Parasites
00:00:03.03 Hello, my name is David Roos and I'm a Professor of Biology 00:00:08.11 at the University of Pennsylvania located in Philadelphia. 00:00:12.12 Today I am here to talk about my favorite group of organisms, 00:00:15.15 the Phylum Apicomplexa, a large group of protozoan organisms 00:00:21.21 which are parasites and make their living inside their host cells. 00:00:26.16 Just by way of example, here are four parasites in the genus toxoplasma 00:00:31.15 and you can see their nuclei stained in blue and we will see some of the other 00:00:35.04 organelles associated with them later on in the talk 00:00:39.08 and by way of comparison, the length of these organisms 00:00:43.12 from one end to the other is about 10 microns long. 00:00:46.09 One one-hundredth-thousandth of a meter, 00:00:48.18 and in contrast, a mammalian host cell, 00:00:51.23 a human host cell in fact in this case is about 10 times that size. 00:00:56.19 In this seminar series, we have three talks, 00:00:59.08 and in the first I will be introducing the parasites, the organisms in question, 00:01:03.03 and talking a little bit about how they grow, how they live, how they replicate, 00:01:08.08 a little bit about their clinical relevance, and also a great deal about 00:01:15.07 how they serve as fascinating windows into the biology of eukaryotic cells, 00:01:21.21 enabling us to understand a little bit more about what are common aspects of cell biology, 00:01:28.28 and what are novel aspects in cell biology that highlight the diversity of life on earth 00:01:34.03 and also particular features which we might be able to 00:01:37.05 as potential targets for therapeutic intervention in trying to target these organisms. 00:01:45.00 So letâ€™s look a little bit deeper at the phylum Apicomplexa. 00:01:49.08 This is a schematic view of the tree of life divided into three great domains 00:01:56.03 defined as the eukaryotes, the eubacteria, and the archaebacteria. 00:02:00.21 Eubacteria and archaebacteria are as different from each other as either is from the eukaryotes. 00:02:08.15 Both of these groups lack nuclei although 00:02:12.05 they do of course have their own genetic material, 00:02:14.25 and the vast majority of life on earth is in fact bacterial or archaebacterial life, 00:02:20.28 and we wonâ€™t' be talking about that today although there are other 00:02:24.00 discussions in the iBioseminar series which describe these organisms. 00:02:28.11 We'll be focusing on the eukaryotes, nucleated cells that include 00:02:32.24 the animals and plants, fungi, that are a part of our everyday existence. 00:02:37.20 But you'll note that the two great Linnaean kingdoms 00:02:40.24 of animals and plants are just branches off on the edge of this tree, 00:02:44.12 and in fact even among the eukaryotes, the vast majority of life is microbial diversity. 00:02:51.26 Protozoans, unicellular or in some cases colonial unicellular organisms 00:02:58.06 including many species that we care quite a lot about. 00:03:01.11 All of those which are underlined here are human pathogens 00:03:06.16 that are of concern. Some of you may have encountered giardia, for example, 00:03:12.17 in drinking water from a mountain stream that perhaps you shouldn't have. 00:03:16.16 Some of these others cause more severe disease, and we will be talking 00:03:20.01 today about the phylum Apicomplexa, indicated in red. 00:03:23.25 A group that includes more than five thousand known species, 00:03:29.02 although to be honest, I couldn't tell you very much about most of them. 00:03:33.10 This group does include many dozens, scores, perhaps hundreds of species 00:03:39.00 in the phylum Plasmodium that is responsible for malaria. 00:03:42.14 Five species of which cause malaria in humans 00:03:46.23 and I urge those of you interested in further information about this to take a look at the 00:03:51.10 iBioseminar by Joseph DeRisi that described some aspects of malaria biology. 00:03:57.12 We'll be talking a little bit more about malaria in the course of these talks. 00:04:02.00 Plasmodium parasites infect red blood cells and only red blood cells in this stage of their life 00:04:10.10 and only those in humans and a few species of relatively closely related monkeys 00:04:17.24 exquisite tissue and species specificity and cause devastating disease, 00:04:23.25 as in the case of this child with a coma, although this particular child 00:04:28.15 survived his infection with no serious adverse effects. Many individuals do not. 00:04:35.22 There are thought to be hundreds of millions of cases of malaria every year word wide, 00:04:41.28 chiefly in Sub-Saharan Africa, South Asia, and in South America. 00:04:48.00 And something on the order of two million people die every year 00:04:53.24 from this disease, particularly from Plasmodium falciparum 00:04:57.08 and particularly in Sub-Saharan Africa. 00:04:59.16 Among the other five thousand species of organisms include many 00:05:04.20 that are of importance, particularly in immunosuppressed individuals. 00:05:10.10 Cryptosporidium for example, with something of the transmission cycle 00:05:14.09 indicated here, something that you can pick up from contaminated water 00:05:18.04 causes a devastating diarrhea in immuno-compromised patients, 00:05:22.23 and for which we have no effective treatment. A serious problem in patients 00:05:28.15 with severe HIV AIDS or with other immuno-suppressive disorders 00:05:33.11 perhaps a treatment for cancer chemotherapy or for transplantation. 00:05:38.12 Toxoplasma is also an opportunistic pathogen that causes problems 00:05:44.08 in HIV patients, in this case the lesions shown in the CT scan 00:05:50.04 of a patient with Toxoplasmic encephalitis. 00:05:52.25 But this parasite is classically known as the leading source of 00:05:56.14 congenital neurological birth defects throughout the world. 00:06:00.08 Chronic infections are on the order of one third of the population 00:06:05.28 are thought to be chronically infected in the US, in South America, in Europe, in Asia, 00:06:12.10 in Africa, globally, a ubiquitous pathogen normally innocuous 00:06:17.29 but under certain circumstances, for example, during pregnancy 00:06:21.22 a serious problem. Now, despite the very different diseases 00:06:29.05 that these organisms cause, they all share 00:06:32.11 a common ancestry and exhibit many similar features. 00:06:36.01 For example, all of them, as unicellular organisms infect a cell 00:06:40.28 in the case of Plasmodium that may be a red blood cell, 00:06:43.25 in the case of Toxoplasma, it may be a nucleated cell in the muscle 00:06:48.05 or in the brain. In the case of Cryptosporidium it may be 00:06:50.14 a cell in the gut. They then differentiate to form a different kind of cell, 00:06:56.02 a cell that you wouldn't even think to look at it 00:06:58.27 was the same cell at all, and are transmitted often from one organism to another. 00:07:03.05 Plasmodium, malaria parasites, are transmitted by mosquitos. 00:07:08.12 Theileria, an important veterinary pathogen of cattle are transmitted by ticks. 00:07:14.08 Toxoplasma are transmitted by cats and this is the reason why pregnant women 00:07:18.24 are often told not to empty the kitty litter box. 00:07:23.00 And so this allows us to overlay their lifecycles one on top of the other 00:07:28.27 and to take advantage of experimental opportunities for example 00:07:33.04 in things that we can study in Cryptosporidium to gain insights 00:07:35.27 into what's happening in Plasmodium. 00:07:37.27 To study Plasmodium, to understand what happens in Toxoplasma, 00:07:41.08 this concept of model organisms, biological concept of the guinea pig, 00:07:45.22 allowing us to use a guinea pig or a fruit fly or a parasite to understand 00:07:51.19 aspects of the biology of organisms we care more about 00:07:56.15 is a fundamental principle of biology, and one that we will explore further. 00:08:03.13 Most of the research in my laboratory focuses on Toxoplasma, 00:08:08.28 and for the specific reason that this organism has turned out 00:08:13.08 to be the most experimentally tractable of all of the Apicomplexan parasites. 00:08:19.00 It's easily cultivated in the laboratory. We have excellent models 00:08:23.04 for human disease, a serious problem for some of these parasites, 00:08:26.15 where, for example, malaria parasites that cause disease in birds 00:08:31.06 or in mice or in lizards may provide at best an incomplete model for human disease. 00:08:40.07 We can carry out genetic crosses, much as Mendel did with his pea plants 00:08:44.29 and in the case of Toxoplasma those crosses need to be done in cats, 00:08:48.22 doesn't bother the cat, but it certainly not the most convenient way 00:08:53.03 to do experiments, but at least it's something that we can do 00:08:55.29 if we are interested in re-assorting genes, putting together genes 00:09:00.11 from one mutant with another. 00:09:02.01 And fortunately we are not restricted to doing our genetics in cats 00:09:05.24 because this parasite is readily amenable to molecular genetic analysis. 00:09:14.06 Toxoplasma exhibits extraordinary ultrastructural resolution 00:09:19.26 as you can see from these transmission electron micrographs 00:09:24.16 and we'll talk more about that in just a moment. 00:09:27.03 The complete genome sequence is known for actually several of the isolates 00:09:31.12 of Toxoplasma, and we have a wide range of functional genomic and 00:09:36.00 bioinformatic tools, which we will talk about in the third of the sessions in this seminar series. 00:09:44.22 All of these parasites are obligate intracellular parasites, 00:09:50.07 they live inside host cells, and within those host cells, 00:09:55.05 they establish a unique compartment, the parasitophorous vacuole, 00:09:59.01 which you can see surrounding these two parasites living inside in this case a human fibroblast. 00:10:06.15 And that vacuole, which we know relatively little about, 00:10:09.20 is the key factor in mediating communication between the parasite and its host cell. 00:10:15.13 Despite being so greatly divergent from animals, plants, fungi, 00:10:23.08 more familiar eukaryotic cells, Toxoplasma and all of these parasites, 00:10:28.01 harbor a virtually complete set of canonical eukaryotic organelles, 00:10:33.10 that we have come to know and love from introductory cell biology. 00:10:37.02 They have a nucleus, they have a golgi apparatus and other components 00:10:41.14 of the secretory pathway, and many other organelles including two endosymbiotic organelles. 00:10:48.13 But interestingly, they have only one of each of these organelles, 00:10:52.28 so we can think of Toxoplasma as a minimalist eukaryote, 00:10:57.19 stripped down to its barebones minimum, an organism which has 00:11:01.21 all of the organelles that we might be interested in 00:11:03.29 studying in a way that we can study genetically, 00:11:07.11 that we can study cell biologically, and yet without such a wide range of diversity 00:11:17.08 that we can hope to try to make sense of what's going on where. 00:11:21.18 So for example, imagine we were to consider the host cell side cytoplasm here, 00:11:27.25 the host cell mitochondrion, a little bit of ER, here is a ribosome. 00:11:32.07 This ribosome here is presumably making protein, 00:11:36.06 but I have no idea what protein that ribosome is engaged in manufacturing. 00:11:42.26 Whereas in contrast if we take a look at the ribosome on a parasite, let's say this ribosome down here, 00:11:48.24 we have good reason to believe that this ribosome is likely to be making 00:11:53.03 a secretory protein that will enter into the single interconnected 00:11:57.04 endoplasmic reticulum network, pass up via the nuclear envelope 00:12:03.00 to the single golgi apparatus up at the apical end of the cell, 00:12:06.15 and from there to the apical secretory organelles that define the phylum Apicomplexa. 00:12:15.05 So let's take a closer look at some of those organelles. 00:12:22.02 So all of these organelles that we've described, 00:12:27.17 the nucleus, the golgi apparatus, the endoplasmic reticulum, mitochondria, 00:12:32.21 even plastids are generic organelles that we see throughout the eukaryotic domain. 00:12:40.20 But these parasites also harbor a variety of unique organelles, most notably 00:12:46.24 the apical complex that gives the phylum its name, 00:12:50.08 up here at the apical end of the parasite where invasion 00:12:53.17 occurs includes a variety of specialized organelles known as rhoptries, 00:13:01.11 here are smaller organelles, the micronemes, that play a key role in invasion. 00:13:06.29 So let's take a closer look at these organelles, these apical complex organelles, 00:13:13.19 that are responsible for secreting proteins essential for parasite invasion. 00:13:19.03 We'll take a look at those, and we'll take a look at the involvement in invasion 00:13:24.07 in a beautiful time-lapse video sequence taken in real time by Gary Ward 00:13:31.13 at the University of Vermont. Here you can see a single parasite 00:13:35.08 as it moves along gliding over the surface of cells, and now watch 00:13:39.14 it stops and at this point it would be secreting proteins out of the rhoptries, 00:13:44.08 as it penetrates into the host cell through this narrow constriction 00:13:49.23 of a moving junction, establishing that intracellular parasitophorous vacuole, 00:13:54.28 within which the parasite will live and replicate. 00:13:58.07 Here are two more parasites living in the progeny of one parasite 00:14:03.20 that had invaded, living within this cell. Now this raises a number of interesting points. 00:14:12.00 These parasites are obviously dividing more rapidly than the host cell itself. 00:14:19.04 One parasite has invaded giving rise to two, and while they are still within 00:14:23.11 a single host cell, and this process of proliferation is of course key 00:14:27.16 to the pathogenesis of the parasite. And we'll take a look at structures 00:14:41.13 that are involved in that pathogenesis or that are involved in the replication of parasites 00:14:47.29 as a potential means of understanding the diversity of eukaryotic replication, 00:14:53.20 but also as a potential target for interfering with the replication and survival of these cells. 00:15:01.17 So let's look back at the morphology of these parasites. 00:15:06.02 We've discussed the nucleus and the golgi apparatus and generic secretory structures. 00:15:11.16 We've discussed the micronemes and rhoptries, parts of the 00:15:16.02 secretory pathway that are critical for invasion. 00:15:22.05 The apical complex also includes a variety of cytoskeletal structures. 00:15:26.29 The conoid here, a fascinating spiral organelle whose function 00:15:31.09 is quite 00:15:39.02 And underlying the entire parasite, the inner membrane complex, 00:15:43.20 a series of flattened vesicles which are sutured together in a patchwork associated 00:15:49.21 with cytoskeletal structures, such that the surface membrane of the parasite 00:15:54.11 consists of a plasma membrane, but also these inner two membranes 00:15:58.28 and those cytoskeletal components, which are essential for parasite survival 00:16:04.07 and replication as we shall see. All of these organelles can be labeled 00:16:12.25 in living parasites if need be, with fluorescent protein reporters in any color of the rainbow. 00:16:19.12 We can study the secretory of the organelles, both parasite specific and generic. 00:16:25.10 The cytoskeletal structures including generic structures such as microtubules 00:16:30.27 and parasite specific organelles such as the inner membrane complex, 00:16:34.26 the endosymbiotic organelles, and so on. And, being able to study these 00:16:41.12 in living parasites, being able to manipulate them, allows us to study both 00:16:46.03 pathogen specific processes, which we might use to interfere with parasite survival, 00:16:51.15 as well as the evolution of eukaryotic organelles in general, 00:16:55.25 studying the biogenesis of the golgi apparatus for example 00:16:59.27 or the structure of microtubules in addition to the beautiful structure 00:17:05.14 of the coronoid organelle or parasite replicative processes 00:17:09.04 as we will be discussing here, and indicated as daughter parasites 00:17:13.17 that are assembling within the mother. For the next few minutes, 00:17:17.23 I'd like to concentrate on the process of parasite replication, 00:17:21.18 a process that is normally quite familiar, one cell goes to two goes to four, 00:17:28.08 but which we will discuss because it is critical to the pathogenesis of these parasites. 00:17:35.28 It is after all the frank tissue destruction which is responsible 00:17:40.14 for neurological birth defects as parasites in the fetus 00:17:45.04 destroy tissue before they come under control. 00:17:49.03 It is the tissue destruction that is responsible for lesions 00:17:53.03 like that encephalitic lesion we saw in the brain of a HIV patient. 00:17:58.04 And this is a common feature of many pathogenic microorganisms 00:18:01.23 although there are certainly organisms that cause disease by interfering 00:18:06.00 with say normal cellular signaling pathways. 00:18:09.03 It's actually the replication of the organisms themselves 00:18:12.11 which is responsible for disease in many other organisms 00:18:15.21 and in this way we can think of the problem as very much analogous to that 00:18:23.08 of cancer cells where it is not the mutation in an individual cell which is 00:18:27.10 responsible for disease, but the uncontrolled proliferation of cells 00:18:31.29 and therefore cancer chemotherapy is typically targeted 00:18:35.20 at blocking that proliferation in much the same way, much antimicrobial therapy 00:18:42.00 is targeted specifically at blocking the replication of parasites. 00:18:47.06 So if we understand more about that replication process 00:18:50.16 and particularly novel features that we might be about to specifically target, 00:18:55.20 we may be able to interfere with them in useful ways. 00:19:00.14 Here we see a micrograph of host cells which have been infected with a 00:19:08.10 single parasite, and that parasite is divided once, twice, 00:19:12.20 giving rise to four parasites living within that parasitophorous vacuole. 00:19:17.02 Here's another parasitophorous vacuole another parasite infected 00:19:20.21 maybe a little bit earlier, replicated three times, giving rise to eight parasites, 00:19:25.19 and yet another with sixteen parasites. As we follow over time, 00:19:31.03 over the next 24 to 48 hours, those parasites will replicate much more rapidly 00:19:37.12 than the host cell, swell up like a fat sausage, and a few hours later burst out 00:19:42.17 so that there is no residue, no evident cellular material here at all. 00:19:50.22 And if we were to look inside an encephalitic lesion, 00:19:53.23 this is precisely the kind of thing we would see. Destruction of tissue 00:19:58.13 and perhaps inflammation that is a result of that tissue destruction. 00:20:05.01 Now this process is quite different from the process of replication described 00:20:12.09 in the text books for Plasmodium, or at least superficially, 00:20:14.26 so after all Toxoplasma divides from one to two to four to eight 00:20:19.12 the way mammalian cells, plant cells, bacterial cells do, in contrast Plasmodium parasites, 00:20:26.22 as illustrated in these beautiful images drawn by Laurie Bannister of the UK 00:20:32.08 a single Plasmodium parasite, showing all of the features 00:20:36.12 that we looked at in Toxoplasma, infects the cell, in this case a red blood cell, 00:20:41.02 and undergoes a process of de-differentiation, 00:20:43.22 turning into what is known as a ring stage parasite responsible for setting up 00:20:47.25 that intracellular home within which the parasite will live 00:20:51.19 for the remainder of its tenure inside the red cell. Within the red cell, 00:20:57.27 it specializes to a trophozoite form parasite, which engulfs hemoglobin, 00:21:03.20 degrading the protein and detoxifying the heme as it is polymerized 00:21:07.28 into a para-crystalline structure, and finally, multiple parasites are assembled, 00:21:13.23 bursting out in the lysis of the red blood cell. Superficially, a very different process, 00:21:23.16 but in fact a process that is more similar than one might originally think. 00:21:27.26 Here's the process again in Plasmodium, this time in actual images of parasites 00:21:33.18 labeled with a fluorescent protein reporter. Parasites invading, 00:21:39.00 developing ring stage parasites, maturing into trophozoites, 00:21:43.14 we can see the beginning of that crystal of heme shown in a shadow 00:21:47.16 starting to segment to produce the schizont that will then rupture out 00:21:52.11 completing the cycle and going on to infect a new series of red blood cells. 00:21:56.27 This process is difficult to study in malaria parasites for a variety of reasons, 00:22:02.02 including the fact that red blood cells are inhospitable environments 00:22:06.22 for a variety of cell biological studies, and the fact that this complicated process 00:22:12.11 is very difficult to follow particularly in real time. In contrast, we can look at 00:22:19.12 Toxoplasma parasites, in this case labeled with a fluorescent protein reporter 00:22:24.06 linked to a histone protein, providing, incidentally, a quantitative marker 00:22:29.10 for DNA content in these parasites, and what you can see is eight parasites 00:22:34.01 living inside this vacuole, with the eight nuclei labeled in green. 00:22:40.26 And as we start the movie, we can follow the replication of the parasites 00:22:45.13 as the nuclei grow and divide and now we see sixteen nuclei but only eight parasites. 00:22:55.26 If we continue to watch though, for a few minutes longer, what we will see 00:23:00.22 is the emergence of the daughter parasites from the mother, 00:23:04.18 leaving off this vestigial material, which will not be incorporated into the daughters. 00:23:11.10 Waste material that is left behind as the parasites go on to mature. 00:23:16.23 So this uncoupling of nuclear replication from cell division 00:23:23.01 is a little bit unusual, and if we look in closer detail, we can see that in fact that process 00:23:28.17 is more akin to the process of schizogony that we know from malaria parasites 00:23:34.05 and the key to doing this has been the labeling of the inner membrane 00:23:38.05 complex, and this particular set of experiments carried out 00:23:41.20 by graduate student Ke Hu, we can label the inner membrane complex 00:23:47.12 in such a way that it is most brightly visualized as it's starting to assemble daughters 00:23:52.20 and so we can define as time zero, these parasites that are beginning to divide. 00:23:58.13 There are two parasites and two bright dots in each, 00:24:01.23 as the new inner membrane complex starts to assemble. 00:24:05.13 Over the next few minutes, you see those grow further until they expand 00:24:11.11 and bud out of the mother, picking up the plasma membrane 00:24:14.24 as they go and sloughing off residual material. 00:24:17.24 This process takes about two hours, and at the end of that 00:24:22.25 we will see no more changes in the inner membrane complex for an entire cell cycle. 00:24:27.15 Eight hours later, we see the process repeated in the same cells, 00:24:32.00 now four cells each of which develops two bright dots 00:24:35.05 which grow and elongate and expand and so in contrast 00:24:40.01 to the binary fission that we see dividing cells in half in mammalian cells, 00:24:47.23 virtually all animal cells, in most fungal cells, in plant cells, in bacterial cells, 00:24:55.26 this process is a little bit different. Conceptually more akin to the assembly 00:25:01.08 of viruses within an infected cell. Two daughters are assembled 00:25:06.02 within the mother and then they emerge and we know that this is the case 00:25:10.28 quite clearly because we can even see rare cases 00:25:14.24 of what one might imagine as schizont. Here's a case 00:25:18.07 of four Toxoplasma parasites, three of which are making two daughters, 00:25:23.00 but one indicated in the red arrows, is actually making four daughters. 00:25:27.18 Here's another case of vacuole consisting of not sixteen, but seventeen 00:25:34.20 parasites, so somehow in the last replicative cycle, 00:25:38.19 one of the mother parasites gave rise to not two, 00:25:41.26 but three daughters for a total of seventeen and in this case 00:25:46.09 we can see five daughter cells that are giving rise to three daughters each 00:25:54.24 for that next round. So we can say in conclusion that 00:26:00.24 the Toxoplasma replicates like Plasmodium, using the process of 00:26:05.20 schizogeny, known in Toxoplasma as endodyogeny, but endodyogeny and schizogeny 00:26:13.03 are really the same sort of thing, assembling daughters within the mother 00:26:16.17 here we can see the daughter inner membrane complex 00:26:19.04 schematically shown in yellow in contrast to that of the mother 00:26:23.02 and as the daughter scaffolding develops, it will then emerge from the mother, 00:26:29.26 picking up its plasma membrane and maintaining 00:26:32.13 that key apical polarity that is essential for parasite invasion. 00:26:37.22 Similarly in Plasmodium, we can see the assembly of the inner membrane 00:26:42.09 complex, but in this case producing not two, but typically sixteen daughter 00:26:48.11 parasites as they grow. So these provide landmarks 00:26:52.22 for us to assemble a picture of the cell cycle of Toxoplasma 00:26:57.14 and by analogy Plasmodium as well. And in work carried out 00:27:01.14 by graduate student Manami Nishi, we now know a great deal about this process. 00:27:06.19 We know that the key first step is in fact not the assembly 00:27:11.22 of the inner membrane complex, but another cytoskeletal structure 00:27:16.13 the centrioles of these proteins, which begin apically oriented, 00:27:22.01 migrate to the basal end of the cell, where they then divide, 00:27:27.22 migrate back up to the apical end, and associate with other organelles 00:27:32.09 starting to put together in a concerted fashion, all the components 00:27:37.01 that are essential for a daughter cell, associating with the golgi apparatus, 00:27:41.12 or plastid organelle, and the nucleus and other structures as well. 00:27:47.04 Last on this list is in fact the mitochondria. Watch this remarkable process. 00:27:53.03 Here we see the assembly of the inner membrane complex, 00:27:56.19 two daughters that are developing as bright green dots that then grow, grow further, 00:28:02.17 and start to emerge from the mother so now we have four daughters 00:28:08.06 emerging from the two mothers, ready to go, but wait, no mitochondrion. 00:28:13.19 All the mitochondrion is left in the residual part of the mother 00:28:18.07 and now in the space of ten minutes, that mitochondrion attaches probably 00:28:23.02 to microtubules associated with the inner membrane complex and zips up to the top 00:28:28.10 of the parasites which then proceed to pick up 00:28:31.25 the plasma membrane and bud out of the mother. 00:28:35.02 So in some studies like these have allowed us to put together a complete 00:28:40.04 timetable that is rigorously adhered to for organellar replication 00:28:45.15 in these parasites, beginning with the replication of the centrioles 00:28:49.00 and successive packaging of the golgi apparatus, the plastid, the nucleus, 00:28:54.25 assembly of this daughter scaffolding which then picks up the endoplasmic reticulum 00:28:59.19 and the mitochondrion and finally the specialized secretorial organelles, 00:29:03.24 the rhoptries and micronemes are assembled de novo in each parasite. 00:29:07.25 So in answer to the question that we started with 00:29:12.08 of how do we build a parasite, we do so with a process that's significantly different 00:29:19.04 than the familiar cell cycle processes that have been defined in yeast cells 00:29:24.22 and in mammalian cells, where the hallmark marker of S-phase, 00:29:30.12 DNA replication, is completely subsumed within the process 00:29:35.03 that we normally think of as associated with M-phase, organellar replication, 00:29:40.22 mitosis, cytokinesis, this large region indicated here in pink 00:29:46.27 and encompassing approximately 80% of the parasite cell cycle, 00:29:50.20 including the process of DNA replication. This argues certainly 00:29:56.23 that there are likely to be significant modifications 00:29:59.27 of the familiar checkpoints associated with cell cycle control, and changes 00:30:06.08 that will be interesting to explore as we characterize the biology 00:30:10.02 of these parasites further. So in answer to the question 00:30:13.24 of how we build a parasite cell? The answer appears to be 00:30:17.26 that we hang everything onto the cytoskeleton, 00:30:22.04 building the parasite from the top down in a process that ensures 00:30:27.26 the maintenance of polarity that is so essential to parasite survival 00:30:32.06 and has a number of other interesting implications as well, because 00:30:36.03 everything in the daughter parasite is put there by choice 00:30:40.15 Residual material, waste products for example, are sloughed off behind, 00:30:45.27 allowing the parasites to survive without classical lysosomes. 00:30:52.10 So I hope that this tour through the biology of Toxoplasma parasites 00:30:57.28 and the ways in which we use this as a model organism to study 00:31:02.28 the biology of Apicomplexa parasites in general, has given you some 00:31:07.01 insight into the fascinating cell biological processes that are involved 00:31:12.10 both parasite specific features and features that are more general to eukaryotes, 00:31:19.06 and I hope that will encourage you to read more about the biology 00:31:23.13 of these organisms, perhaps to work on these organisms yourself. 00:31:26.22 And in the next lecture I'd like to take you through the biology 00:31:31.00 of one particularly interesting organelle, this organelle, the plastid, 00:31:35.05 or apicoplast, an organelle that reveals some 00:31:39.28 remarkable aspects of organellar evolution in eukaryotic cells. 00:31:45.14 Here in a malaria parasites, the nucleus, golgi apparatus, 00:31:51.22 secretory structures, inner membrane complex that we described 00:31:55.17 mitochondrion and finally the apicoplast 00:31:58.18 which will be the subject of the next iBioLecture.