Studies of Dynamin 2 in Megakaryocytes

! University of Copenhagen Bachelor Thesis Studies of Dynamin 2 in Megakaryocytes Amalie Dahl Haue Niels Borregaard Supervisor University of Copenhagen Herv´e Falet Supervisor Harvard Medical School December 3, 2013 ! i! ! ! Preamble ! The present thesis is based on experiments conducted at Division of Translational Medicine, Brigham & Women's Hospital, Harvard Medical School in Boston. The experiments were designed by associate biologist Hervé Falet and conducted under supervision of postdoc Markus Bender. The thesis was written under supervision of professor Niels Borregaard, University of Copenhagen. The thesis has the following structure. The introduction includes an account of relevant background knowledge, including an experimental motivation, a brief description of molecular structures and mechanisms central to the experiments and a presentation of the hypothesis. Materials and methods presents a description of the experiments conducted - a methodological triad comprising mutagenesis, retroviral production and viral transfection. The result section presents sequencing results following mutagenesis and two sets of pictures, one of retroviral production and one of viral transfection. The discussion includes a critical evaluation of the data and the experimental setup. ! ! ! ! ! ! ! ! ! ! ! ii! ! ! Resumé ! Formålet med nærværende opgave var at undersøge dynamin 2’s betydning for dannelse og funktion af blodplader. Konditioneret knockout af dynamin 2 i megakaryocytter hos mus er årsag til thrombocytopeni med macro-thrombocytter, splenomegali og megakaryocyt hyperplasi. I mennesker er mutationer i genet der koder for dynamin 2 årsag til Charcot-Marie Tooth sygdom. Med henblik på at undersøge funktionen af dynamin 2 i megakaryocytter, blev en række forsøg udført. Disse talte tre, nemlig mutagenese, retroviral produktion og retroviral transfektion. Det eksperimentelle mål var (i) at lokalisere dynamin 2 i megakaryocytter og (ii) at revertere blodpladedefekten i knockout mus ved at transfektere dymanin 2 ind megakaryocytter fra disse. Da der ikke er antistoffer tilgængelige mod dynamin 2 blev lokalisationen forsøgt gennemført ved at undersøge lokalisationen af et fusionsprotein bestående af dynamin 2 og et fluorescerende protein. Dog lykkedes det hverken at lokalisere dynamin 2, eller at opnå tilstrækkelig transfektionseffektivitet af dynamin 2 i megakaryocytter, hvorfor spørgsmålet fortsat er ubesvaret. I forlængelse af de eksperimentelle resultater følger en evaluering af eksperimenterne. Der konkluderes, at resultaterne var inkonklusive grundet begrænset erfaring og tid. Dermed er projektets egentlige konklusion ikke af videnskabelig karakter, men består i et erhvervet kompetencesæt. Herved forstås, at jeg i forbindelse med udarbejdelsen af opgaven har oparbejdet en beskeden laboratorieerfaring. Ydermere er jeg blevet bekendt med anvendelsen af den bibliografiske database Pubmed i forbindelse med at indhente og selektere videnskabelig information. ! ! ! ! ! ! ! ! ! iii! ! ! ! ! Abstract ! The aim of the present thesis was to obtain an in-depth description of the role of dynamin 2 in platelet formation and activation of platelets. In mice, dynamin 2 knockout in the megakaryocyte lineage causes thrombocytopenia with macro-thrombocytes, splenomegaly and megakaryocyte hyperplasia. In humans, Charcot-Marie-Tooth disease is caused by dynamin 2 mutations. On that account, a series of experiments were designed to study megakaryocytes from dynamin 2 knockout mice. The experiments included mutagenesis, retroviral production and viral infection. The aim was to (i) locate dynamin 2 in megakaryocytes and (ii) rescue platelet deficiency in knockout megakaryocytes by transfecting dynamin 2 into the megakaryocytes. Since there is no antibody against dynamin 2, a fusion protein comprising of dynamin 2 and a fluorescent protein was constructed. Theoretically, fluorescence should then be used to locate dynamin 2. However, locating dynamin 2 failed. Consequently, the aim remains open to examination. Subsequent to a presentation of the experimental results, the thesis comments on the experimental results and evaluates the experimental setup. It concludes that the results were inconclusive due to limited expertise and time. Hence, the main conclusion is not strictly scientific. Rather, it consists in the fact, that I gained modest laboratory experience in collecting data. Further, I became familiar with the bibliographic database Pubmed and learned how to use the tool in means of finding and selecting information. ! ! ! ! ! ! ! ! ! 1 ! ! Table of Contents INTRODUCTION 2! EXPERIMENTAL MOTIVATION! 2! THEORETICAL COMPONENTS! 3! DYNAMIN! 3! THROMBOPOIESIS! 5! CHARCOT-MARIE-TOOTH DISEASE! 6! EXPERIMENTAL APPROACH! 7! EXPERIMENTAL MODEL! 7! HYPOTHESIS! 8! EXPERIMENTAL OBJECT! 9! MATERIALS AND METHODS 9! PLASMID CONSTRUCT! 9! MUTAGENESIS! 9! DNA PURIFICATION!10! RETROVIRAL PRODUCTION!11! MEGAKARYOCYTE TREATMENT!12! ISOLATION!12! TRANSFECTION!12! METHODOLOGICAL ACHIEVEMENTS!12! RESULTS 13! MUTAGENESIS!13! RETROVIRAL PRODUCTION!14! MEGAKARYOCYTE TRANSFECTION!15! DISCUSSION 16! DESCRIPTIVE SUMMARY!16! EXPERIMENTAL EVALUATION!17! EXPERIMENTAL LIMITS!17! CONCLUSION!18! REFERENCES 19! ! ! ! Front-page illustration from: http://www.abovetopsecret.com/forum/thread682667/pg2 ! 2 Introduction Experimental Motivation Platelets, the smallest member of the blood corpuscle, are unique in two aspects. Structurally, they are anucleate. Functionally, they are the cells regulating hemostasis. These structural and functional characteristics make them a prevalent object of scientific investigations(1). In 1882 Giulio Bizzozero, the man who came to be known as the discoverer of platelets, described the existence of ‘constant blood particles, differing from red and white blood cells (…)’(2). Ever since, platelets have been studied intensely resulting in a deeper cellular understanding, which has formed the basis for various treatment regimes. In spite of many pharmacological contributions already, the field is still evolving(3). Despite subject of scientific investigation for centuries, much remains to be revealed about the mechanisms underlying normal platelet function. In particular the description of platelet formation and activation continually generates more questions than answers(1). With that in mind, a series of experiments were designed to study functional aspects of thrombopoiesis, the formation of platelets. The study included patients suffering from Charcot-Marie Tooth disease and a conditional dynamin 2 knockout mice. Charcot-Marie Tooth disease is an autosomal dominant neuromuscular disorder, which is caused by mutations in DNM2, the gene coding for dynamin 2. Specific DMN2 mutations lead to thrombocytopenia in Charcot-Marie Tooth disease patients(4) and the conditional megakaryocyte lineage specific dynamin 2 knockout mice suffer from thrombocytopenia (unpublished data, Falet, H.). Hence, Charcot-Marie Tooth patients and conditional dynamin 2 knockout mice share affection site and their phenotypes overlap. Combined, these two features are the rational behind studying them jointly.!! ! Figure 1 Experimental origin In summary, an in-depth description of thrombopoiesis is the incentive behind the experiments that the present thesis presents. The hypothesis is that dynamin 2 is responsible for the fragmentation of platelets from their precursors and thereby the platelet synthesis. The hypothesis is grounded in two features. One is a clinical observation and one is theoretical hook derived from well-studied functions of dynamin isoforms elsewhere in the organism. The clinical observation is that Charcot-Marie Tooth disease is caused by DNM2!mutations and that they suffer from thrombocytopenia(4). The theoretical hook is that the prime functions of ! 3 dynamin are obtained through interaction with the plasma membrane(5). Three methods constitute the experimental approach. One is mutagenesis, one is retroviral production and one is retroviral transfection as presented in figure 2. The experimental subjects comprise a plasmid construct and megakaryocytes isolated from mouse embryos. The plasmid was constructed in such a way that dynamin 2 is fused with Dendra2. Dendra2 is a fluorescent protein, which has been modified from Dendra, a fluorescent protein derived from octocoral Dendronephthya sp(6, 7). The mutagenesis was performed to acquire the correct DNA sequence in the plasmid used for transfection. Retroviral production was performed to allow transfection of the plasmid into the megakaryocytes. Finally, retroviral transfection was performed to induce the expression of Dendra2-dynamin 2 in megakaryocytes, and thus locate dynamin 2. ! Figure 2 Experimental flowchart Theoretical Components Dynamin Dynamins are large GTPases involved in a wide range of cellular functions, including membrane remodeling, endocytosis, intracellular trafficking and interaction with the actin and microtubule cytoskeletal networks. The mammalian genome encodes three dynamin isoforms. They share characteristics, in that they have GTPase activity, have affinity for various intracellular proteins, ability to oligomerize and are capable of binding lipid. Together, these characteristics enable dynamins to induce conformational changes, interact with a variety of other proteins and induce structural changes in the cellular membranes, resulting in membrane scission(5). ! 4 Initially, dynamin was identified as a protein associated with microtubules (8). Subsequently, various dynamin isoforms have been identified. Dynamin 1 is expressed mainly in neurons(9), dynamin 2 is ubiquitously expressed(10, 11) and dynamin 3 was originally purified from testis(12). ! Figure 3 Three-dimensional structure of dynamin, PH: pleckstrin homology, GED: GTPase effector domain, PRD: proline rich domain(13) Five domains make up the primary structure of a dynamin monomer, as illustrated in figure 3. The G domain, located at the very amino-terminus, is the site of GTPase activity. The middle domain and GTPase effector domain facilitate dimerization of dynamin(14). The pleckstrin homology domain is capable of binding to negatively charged membrane bound phospholipids, preferentially phosphatidylinositol-4,5-bisphophate. The binding of the pleckstrin homology domain enhances GTPase activity(15). The carboxy-terminus part of the stalk, also termed GTPase effector domain, is functionally associated with the G domain as well and regulates the GTPase activity. Hence, it drives the dynamics of dynamin(16). The proline rich domain is capable of binding to various intracellular proteins. The interaction between dynamin and other intracellular proteins is involved in guiding dynamin molecules to the plasma membrane (17, 18). Further, the proline rich domain has various phosphorylation sites. Thus, it serves as a covalent modification site and facilitates the interaction between dynamin and a variety of other intracellular proteins(18). ! 5 A three-dimensional reconstruction of dynamin implies a ‘T’ shape characterized by three densities. They have been termed head, stalk and foot. Figure 3 presents the relationship between the primary structure and the three-dimensional structure of dynamin. The GTPase domain forms the head, the middle domain and GED form the stalk and the PH domain forms the foot(13). The function of dynamin has been described in terms of a corkscrew model. According to the corkscrew model, dynamin function results from a conformational change in the head and stalk that collectively generates a twisting motion extending from the dynamin assembly. It has been assumed that the twisting motion is the sum of two forces. One results from a force parallel with the plasma membrane and one causes a force orthogonal to the plasma membrane (19, 20). ! ! Figure 4 Structural and functional overview of DNM(21) Thrombopoiesis Thrombopoiesis, the formation of blood platelets, takes place in the bone marrow. It is a highly regulated process and is essential for hemostasis. In humans, the daily platelet production amounts to approximately 1 x 1011, and the circulatory life span is roughly 10 days(22). Megakaryocytes are the myeloid cells that give rise to platelets(23) following a maturation process that temporally ranges from 4 to 7 days, originating at the megakaryoblast and ultimately leading to the formation of megakaryocytes(24). Megakaryocytes send long protrusions from the sinusoids of the bone marrow to the circulation, where these protrusions also called pro-platelets undergo fragmentation to form platelets. It has been estimated that one megakaryocyte gives rise to 1000 to 3000 platelets(22). Pro-platelets contain a characteristic distribution of microtubulin suggesting that it is involved in the fragmentation of platelets(25). ! 12 Megakaryocyte Treatment Isolation At day 0, fetal liver cells from mouse embryos aged 13.5-14.5 days were collected in DMEM with 10% fetal-bovine-serum and 1% Penicillin/Streptococcus. Tissue was saved for genotyping. Cells were passed with 1 mL pipet, 18G, 22G and 25G needles. Then, cells were transferred to a BD 40µm nylon moisten cell strainer (BD Ref. 352340). Cells were spun at 200g for 5 minutes at room temperature and the pellets were resuspended in medium with 50ng/mL thrombopoietin and incubated at 37°C. Transfection At day 2, fetal liver cells were transfected. They were resuspended carefully, transferred to a 15 mL Falcon tube and spun at 200g for 5 minutes at room temperature. The pellets were resuspended in 1.5mL fresh DMEM. Then, 1 mL virus and 2.5µL polybrene were added to each well, transferred to a 6-well plate and spun (Sorvall, RT6000B) at 800g for 90 minutes at room temperature. Thereafter, the transfected cells were incubated for 90 minutes at 37°C. Then, cells were transferred to 15 mL Falcon tubes, spun at 200g for 5 minutes and resuspended in 2 mL DMEM with thrombopoietin and incubated overnight in a 6-well plate at 37°C. At day 3, megakaryocytes were resuspended in 1.5 mL fresh medium and isolated over a bovine-serum-albumin gradient at 1.5%/3.0% and cells were resuspended in fresh medium with thrombopoietin. At day 4, cells were visualized in a fluorescent-light-microscope and pictures were taken. Methodological Achievements I gained experience in molecular biological procedures from the experiments conducted. The molecular biological procedures count mutagenesis comprised of polymerase chain reaction and bacterial transformation, retroviral production and infection. Hence, I became familiar with two ways in which DNA multiplication may come about, one being polymerase chain reaction and one being bacterial growth. Finally, I became aware of the fact that retrovirus functions as a critical tool in manipulating cellular protein synthesis. !! ! 13 Results Mutagenesis Test digestions were performed to confirm successful transformation into the XL-10 ultracompetent cells. ! Figure 11 Test digestion with NcoI, left: 6 samples and 2 negative controls on 0.8% agarose gel at 120V for 30 minutes, right: expected band pattern Figure 11 demonstrates that digestion of the purified plasmid yields a band corresponding to the expected band size. Furthermore, digestion with the restriction enzyme BglII that has three cleavage sides on the plasmid yielded the expected band pattern. A picture of the gel is shown in figure 12. ! Figure 12 Test digestion with BglII, left: 6 samples and 2 negative controls on 0.8% agarose gel at 120V for 30 minutes, right: expected band pattern To confirm successful mutation, two samples were sent for sequencing. Results are presented in figure 13.! !!! Figure 13 Sequencing results: From left: sequence prior to cysteine insert, 2 samples after successful cysteine insert. The arrows mark site of interest.! ! ! ! 14 Retroviral Production Pictures were taken 48 hours after transfection to confirm successful transfection of the HEK 293T cells. The pictures provide a qualitative estimate of the transfection efficiency and they demonstrate that the distribution of dynamin 2 is confined to the cytoplasm as expected. ! Figure 14 HEK 293T cells, 4X bright field and fluorescence, 48 hours after transfection The estimate of the transfection efficiency does not transfer to information on the retroviral production, explicitly. Hence, neither the quality nor the quantity of the virus was determined. ! 15 Megakaryocyte Transfection Pictures of the transfected megakaryocytes are shown in figure 15, 16 and 17. The size of the cells is used to identify the megakaryocytes. The megakaryocyte diameter in mice ranges from 20-30µm(34). ! Figure 15 Transfected wild type megakaryocyte, bright field and fluorescence, 48 hours after transfection ! Figure 16 Transfected knockout megakaryocytes, bright field and fluorescence, 48 hours after transfection Figure 17 Transfection trend in knockout megakaryocytes, bright field fluorescence and fluorescence, 48 hours after transfection ! 16 Discussion Descriptive Summary Figure 15 and 16 reveal that the transfection is successful in wild type and knockout cells. However, the transfection efficiency is low, reducing the informative potential of the pictures. Furthermore, no pro-platelet formation occurs. Based on the experimental model that shows decreased platelet count in knockout mice, sparse formation of pro-platelets is to be expected. The distribution of dynamin 2 is more scattered in megakaryocytes than in HEK 293T cells. Yet, a clear pattern in the location of dynamin 2 cannot be deduced from the pictures. The combination of relatively low transfection efficiency and the apparent lack of specific location of dynamin 2 prohibits any conclusions to be drawn whether rescuing of the knockout megakaryocyte has been obtained. A comparison between the wild type and knockout megakaryocytes (figure 15 and 16 bright field pictures), shows that the wild type cells are more numerous and bigger, suggesting a difference between the behavior of megakaryocytes in vivo and in vitro, since the experimental model is characterized by megakaryocyte hyperplasia. Either, the pictures are not representative, or the megakaryocyte progenitors respond differently to thrombopoietin in vivo and in vitro. The response to thrombopoietin in vivo may be integrated with other stimuli, allowing the megakaryocytes to proliferate in the absence of dynamin 2. Alternatively, it can be hypothesized that megakaryocyte proliferation in dynamin 2 knockout mice accelerates postpartum or later in the embryonic development. Accordingly, megakaryocyte hyperplasia does not present in cultures from embryos. Figure 17 illustrates the trend of the transfected cells, which can be categorized in to three, namely: untransfected cells, resting transfected cells and active transfected cells. The terms resting and active are coupled to the appearance of the cell membrane active cells meaning cells where the cell membrane appears disrupted. As opposed to figure 15 and 16, figure 17 indirectly pictures the multi lobular nucleus of the megakaryocytes, making the identification of the cell more reliable. Antibody staining against lineage specific surface molecules in the megakaryocyte lineage is required to confirm the nature of the cells. It seems that there is a difference between the location of dynamin 2 in active and resting cells, in that the location of dynamin 2 in active cells is limited to specific areas in close proximity to the cell membrane. Additionally, the fluorescence is stronger in the resting cells than in the active cells, suggesting that the expression of dynamin 2 varies depending on the functional identity of the cells. ! 17 In summary, the quality of the pictures is not good enough to allow conclusions to be drawn. First, the combination of low transfection efficiency and no pro-platelet formation hinders visualization of transfected pro-platelets. Second, the unspecific localization of dynamin 2 in transfected cells does not point to any clear relation between thrombopoiesis and function of dynamin 2. Experimental Evaluation Other than the low transfection efficiency, it is a major problem that no pro-platelets are seen in the pictures. Since there are no substantial differences between pro-platelets formation of wild type and knockout megakaryocytes, absence of pro-platelets is most likely a consequence of the experimental circumstances. The lack of pro-platelet formation may result from (i) the viral transfection or (ii) the Dendra2-intervention. In case of (i) the viral transfection, including centrifugation may be too harsh on the cells. As a result, the cells may be more susceptible to apoptosis. Further investigation of this possibility requires identification of apoptotic bodies. In line with that, it should be noted, that the concentration of the viral supernatant used for transfection is not adjusted. The element gives rise to two problems. First, the cell cultures are not necessarily treated alike. Second, the amount of viral supernatant used for transfection may be either too little or too much to obtain sufficient expression levels. In case of the subtler (ii), the experimental setup assumes that Dendra2 does not disrupt the functional integrity of dynamin 2. To date, successful labeling of other polymerizing proteins have been reported, among them another nucleotide hydrolyzing protein, namely tubulin (35). Assuming that dynamin 2 orchestrates platelet fragmentation, sparse pro-platelet formation may result in a drop in the functional integrity of dynamin 2 in transfected cells, caused by Dendra2. Based on that, the properties of dynamin 2 and Dendra2-dynamin 2 ought to be studied separately to ensure reliable data. Experimental Limits From the experimental model and the Charcot-Marie Tooth disease patients, it seems that dynamin 2 is a protein of great importance in mice and humans. Nevertheless the patients and the experimental model differ, and the differences are critical to keep in mind in the attempt to map thrombocytopenia in Charcot-Marie Tooth disease patients and the mouse model. For example, the Charcot-Marie Tooth disease patients do express dynamin 2, although the function is altered due to mutations. In contrast, the knockout of dynamin 2 in the experimental model is lineage specific. Hence, the patients and the model differ fundamentally, aside from differences resulting from the fact that they are different animals. Possibly, genetic redundancy may preserve some functions of dynamin 2 in the Charcot-Marie Tooth disease patients. That possibility may explain why thrombocytopenia in Charcot-Marie Tooth disease ! 18 patients is limited to one mutation site. In addition, the presence of dynamin 2 in the patients, albeit mutated, may sustain some dynamin 2 functions. Conclusion Experimentally, the site-directed mutagenesis yielded positive results, whereas the attempt to locate dynamin 2 with Dendra2 was inconclusive because of low transfection efficiency combined with sparse pro-platelet formation. Hence, the experiments gave no basis to confirm or reject the hypothesis, namely that dynamin 2 facilitates the fragmentation of platelets from pro-platelets. While acknowledging the fact that the experiments were conducted with limited expertise and time, the thesis evaluates the experimental setup and draws attention to certain features of the experiments. Specifically, the evaluation raises two main questions regarding the experimental procedures, (i) that they may be too harsh on the megakaryocytes and (ii) that Dendra2 may interrupt the functional integrity of dynamin 2. Personally, the work gave me insight in to central molecular biological experimental procedures that have a broad application in research and clinically. Additionally, I studied platelet development and function and dynamin structure and function. Thereby I learned to acquire knowledge from scientific articles and communicate these insights in writing. !! ! 19 References 1. Thon JN, Italiano JE. Platelets: production, morphology and ultrastructure. Handbook of experimental pharmacology. 2012(210):3-22. 2. Ribatti D, Crivellato E. 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