Mechanisms of asymmetric cell division

MECHANISMS OF ASYMMETRIC CELL DIVISION GOH LIHUI (WU LIHUI) B.SC (HONS) NANYANG TECHNOLOGICAL UNIVERSITY, SINGAPORE A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NUS GRADUATE SCHOOL FOR INTEGRATIVE SCIENCES AND ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2011 Acknowledgements I would like to express my gratitude to my supervisor Prof. Yang Xiaohang for his kind guidance and support and patience for me during my one year as research officer and four years of Ph.D studies. He not only gave me a lot of encouragement and feedback, but also a large degree of freedom for me to express and carry out my own ideas. All these allowed me to overcome the many obstacles in my course of study. I would also like to thank Prof. Wang Yue whom took me under his group and for the last eight months of my studies. I would also like to thank Dr. Wang Huashan, Dr. Lin Shuping, Dr. Lee Meichin, Ms. Sung Ying Ying, Mr. Huang Zhenxing and Ms. Lee Chai Ling for all the selfless advice, support, help and encouragement. Their willingness to share their knowledge and to render their helping hands whenever possible will always be remembered by me. I would also like to thank all the past and present members of both Yang Xiaohang group and Wang Yue group for their friendship and kind help. I would also like to thank Prof. Tang Bor Luen and Prof. Thomas Leung of my Thesis Advisory Committee for their generous advice and relentless effort in helping me throughout my course of study. Last but not least, I am very thankful and grateful to my family members and my friends whom unconditionally supported me, whom always stood by me, constantly giving me comfort and encouragement whenever I needed it. Their love sustained me for my entire period of candidature and enabled me to overcome all obstacles in my path. i Table of contents Acknowledgements . i Table of contents . ii Summary . vi List of Tables . viii List of Figures ix List of Abbreviations xii Chapter 1: Introduction . Drosophila melanogaster as a model organism for study . Asymmetric cell division Drosophila neuroblasts in neurogenesis . Asymmetric cell division in neuroblast 10 Setting up cell polarity . 11 Mitotic spindle orientation 13 Asymmetric segregation of cell fate determinants . 14 Asymmetric localization of cell fate determinants 17 Localizing the cell fate determinants by apical exclusion 17 aPKC substrate specificity is linked with cell cycle 19 Lgl as a molecular buffer 20 ii Other regulators of asymmetric cell division . 21 Cancer and stem cell asymmetric cell division 23 Chapter 2: Materials and methods 25 Molecular biology 25 DNA application . 25 RNA application 34 Protein application . 36 Drosophila melanogaster manipulation 37 General maintenance of Drosophila melanogaster . 37 Fly stocks used in the study . 37 RNAi knockdown in Drosophila melanogaster 38 Generation of revertants . 38 Isogenize and removal of background mutation . 39 Recombination . 39 Climbing assay 39 Larval brain dissection . 40 Pupal nota dissection . 40 Cell line manipulation 41 General maintenance of cell line . 41 RNAi treatment of cells 41 iii Transfection of cells . 42 Biochemistry 42 Antibodies 42 SDS-PAGE . 43 Western blot 44 Co-immunoprecipitation 44 Immunohistochemistry 45 List of antibodies used . 45 Immunofluorescence staining of larval brains 46 Immunofluorescence staining of pupal notum . 46 Chapter 3: clueless . 48 Introduction . 48 clueless was identified in an RNAi screen to be involved in asymmetric cell division of the adult sensory organ precursor. . 51 clueless . 54 Generation of clueless mutant . 58 clu169 mutant is semi lethal 61 clu169 mutants display a weak phenotype of mislocalized Miranda 65 Mutations in clu rescue the tumor formation phenotype and mislocalization of cell fate determinants in lgl mutants. 67 Deletion of clu rescues lgl mutant phenotype by down-regulation of aPKC. . 71 iv Down-regulation of aPKC in lgl clu double mutant does not rescue the mislocalization of aPKC. 76 Clu forms a complex with aPKC . 79 Deletion of clu does not cause mitochondria clustering in the larval brain neuroblasts. 80 Down-regulation of aPKC in lgl clu double mutant is independent of transcription. . 83 Deletion of clu also rescues phenotype caused by overexpression of aPKCCAAX in larval brain neuroblasts. 86 Clu is associated with the active Par6-aPKC-Baz complex . 89 Summary of results 91 Future work and direction . 94 Chapter 4: Discussion . 102 The equilibrium of Par6-aPKC-Baz and Par6-aPKC-Lgl complex 102 clu mutant is protected by Lgl . 104 parkin and clu . 104 The lack of mitochondria clustering 105 Clueless about clueless 106 Neurodegeneration versus cancer 107 Potential novel target for cancer therapy . 109 References . 111 v Summary Asymmetric cell division is an important process by which a cell divides to give two different daughter cells. This process is important in stem cells whereby a single division produces two cells, one of which is the stem cell itself and the other, committed or differentiated daughter cell. This process is highly controlled, as failure of which would result in depletion of stem cells or uncontrolled cell proliferation, leading to diseases like Parkinson’s disease or cancer. Asymmetric cell division is well studied in the model organism Drosophila neuroblast and sensory organ precursor. It is known that proteins in the apical complex such as atypical Protein Kinase C (aPKC), Bazooka (Baz), Partitioning Defective (Par6), Inscuteable, Partner of Inscuteable and G-protein alpha-I subunit direct cell fate determinants such as Numb, Partner of Numb, Miranda, Prospero, and Brain Tumor to localize as a basal crescent in the cell such that they would be inherited by only one of the daughter cells. However, detailed mechanism of this directing of cell fate determinants is largely unknown. Using a UAS-GAL4 system to drive expression of RNAi constructs in the central and peripheral nervous system, I identified clueless (clu) as one of the candidate genes that are involved in asymmetric cell division of the larval brain neuroblasts or sensory organ precursor cells. Clueless is expressed during the larval stages and enriched in the neuroblast. clu mutants display a weak but obvious phenotype of mislocalized Miranda and Numb crescent in a small percentage of dividing neuroblast. Deletion of clu in lethal giant larvae (lgl) mutants largely rescues the phenotype of mislocalized Miranda. The brain vi size of clu, lgl double mutant is also considerably smaller than that of lgl mutants. Biochemistry data suggests that this rescue is due to down-regulation of aPKC. This down-regulation of aPKC is independent of transcription and translation and did not rescue the mislocalization of the apical aPKC crescent. Par6-aPKC can form a complex with either Baz or Lgl to exist in either an active or inactive complex. Co-immunoprecipitation of Clu showed that aPKC and Bazooka form a complex with Clu but not Lgl. This result suggests that Clu forms a complex with Par6aPKC-Baz. Clu may be responsible for the stability of the active Par6-aPKC-Baz complex. vii List of Tables Table 1: List of primers used for sequencing . 31 Table 2: List of primers used for production of double stranded oligo DNA 32 Table 3: List of primers used for PCR . 32 Table 4: List of primers used for dsRNA DNA template generation 35 Table 5: List of primers used for real time PCR . 36 Table 6: Fly stocks used in this study . 38 viii List of Figures Figure 1: Image adapted from FlyMove (www. flymove.uni-muenster.de). Life cycle of Drosophila melanogaster. Figure 2: Image adapted from (Neumüller and Knoblich, 2009). Three different modes of asymmetric cell division: niche derived, external polarity cues and intrinsic asymmetry. . Figure 3: Schematic diagram showing the division of embryonic neuroblasts. Figure 4: Schematic diagram showing Type I and Type II neuroblasts. . Figure 5: Schematic diagram to summarize key players involved in asymmetric cell division of the neuroblast. . 11 Figure 6: Schematic diagram showing the model for phosphorylation of Numb and Mira by the Par complex. . 21 Figure 7: Image source from (Caussinus and Gonzalez, 2005). Demonstration of ability of mutant larval brain to form tumors when injected into adult hosts. . 24 Figure 8: Schematic diagram showing localization of Par complex proteins, Pins and Gαi, cell fate determinants Pon and Numb in the neuroblast and sensory organ precursor . 50 Figure 9: Schematic diagram showing division of SOP to give rise to socket, hair, glial, sheath and neuron cells. 51 Figure 10: RNAi knockdown of clu (CG8443) in adult notum (A, B) and in larval brain neuroblasts (C). 53 Figure 11: Western blot analysis of whole embryonic lysate, whole larval lysate, whole larval brain lysate of yw and clu169. . 55 Figure 12: Anti-Clu (FITC) and anti-Deadpan (Cy3) staining of third instar larval brain of yw (A) and clu169(B). . 56 ix Discussion Potential novel target for cancer therapy The notion of cancer stem cells was coined when various groups of researchers discover that tumors generally consist of two populations of cells, one which is non-tumorigenic and the other which is tumorigenic and is capable of forming tumors in vivo (Al-Hajj et al., 2003; Singh et al., 2004). This indicates that there are two separate population of cells in a tumor, one population which closely resembles differentiated cells and not possess the ability to form tumors when cultured in vivo. The other population, however, closely resembles stem cells and could generate both populations of tumorigenic and non-tumorigenic cells again when cultured in vivo. These cells, termed as tumor initiating cells or cancer stem cells, are suspected to be resistant to many forms of cancer treatment, and may be the root cause of cancer relapse and metastasis in many cancer patients (Florian et al., 2011). While it is unclear if mis-regulation of asymmetric cell division is involved in these tumor initiating cells and tumorigenesis, parallel comparisons drawn between tumors form in both Drosophila and mice due to loss of function in genes involved in asymmetric cell division like Numb, Lgl and Pros have lead us to suspect that these tumor initiating cells have defective asymmetric cell division (Agrawal et al., 1995; Klezovitch et al., 2004; Bello et al., 2006; Wang et al., 2006). Most cancer treatment available now mainly targets the differentiated, non-tumorigenic cells that are non-proliferative while tumor initiating cells are resistant to them (Harrison and Lerner, 1991), and we are in need of designing drugs or therapies that are more directed at these tumorigenic cells. Clu may be a potential target for drugs and therapeutics design to target tumor initiating cells and suppress their tumorigenesis process. 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(2005) 'Locomotion defects, together with Pins, regulates heterotrimeric G-protein signaling during Drosophila neuroblast asymmetric divisions.', Genes Dev 19(11): 1341-53. 124 | P a g e [...]... particularly important in stem cells where asymmetric cell division of the stem cell would produce two distinct daughter cells: one committed cell or differentiated cell and another which remained as a stem cell This stem cell can undergo the same process to produce more committed or differentiated cells while it itself remained as a pluripotent cell Asymmetric cell division can arise from three different... 30-60 days 3|Page Introduction Asymmetric cell division Asymmetric cell division is an important cellular process whereby a single cell divides via mitosis to two different daughter cells While symmetric divisions give rise to two daughter cells that are exactly identical, asymmetric cell division generates two daughter cells that can be different in size, cellular or protein contents, and/ or differentiation... by the lack of nuclear Asense and cytoplasmic Prospero, also divides asymmetrically to regenerate itself and to produce a smaller cell This smaller cell, unlike the GMC formed in asymmetric division of the Type I neuroblasts, have the capability to divide numerous times Termed as intermediate progenitors or transit amplifying cell, the smaller cell produced by asymmetric cell division of the Type II... amplifying cell can further divide asymmetrically to regenerate itself as well as to produce a GMC 9|Page Introduction Asymmetric cell division in neuroblast Asymmetric cell division in the Drosophila neuroblasts enables a single cell to divide and give rise to two cells with very distinct fate Many molecular switches and machineries are required to facilitate this process Asymmetric cell division in... components of the machineries had been identified Many of the key players of asymmetric cell division identified in embryonic neuroblasts were also found to be conserved in the larval brain neuroblasts (Figure 5) These key players are mainly involved in one or more of the following processes: setting up cell polarity, orientation of the mitotic spindle, and asymmetric segregation of the cell fate determinants... two daughter cells which arise from a single neuroblast involves many cellular processes, interaction and cross-talk between the molecules which are highly regulated Asymmetric localization of cell fate determinants As discussed earlier, the ultimate aim of asymmetric cell division is to segregate the cell fate determinants to only the basal GMC For this segregation to be complete, the cell fate determinants... attract one of the spindle poles, allowing the spindle to align to the apical-basal axis set up by the Par complex Asymmetric segregation of cell fate determinants The ultimate aim of setting up cell polarity and aligning mitotic spindle to the apicalbasal axis such that the cleavage plane of the cell is orthogonal to the apical-basal axis is to ensure that there is basal localization of cell fate determinants... key steps of asymmetric cell division in neuroblasts are to set up the apical-basal polarity of the cell and to align the mitotic spindle along the apical-basal axis This set-up then directs cell fate determinants to localize to the basal cortex and is asymmetrically segregated during mitosis so that only the basal GMC would receive the 16 | P a g e Introduction cell fate determinants The cell fate... to inhibit cell growth and renewal of the GMC, promoting its exit of the cell cycle and differentiation Hence, the larger cell at the apical side which does not receive these cell fate determinants remains as a neuroblast while the GMC at the basal side which receives these cell fate determinants would exit the cell cycle and commits itself to differentiate The generation of these distinct cell fates... mitosis, both cells are similar However, due to differences in their environment, only one cell would receive the signal to remain as a stem cell (Figure 2A) For the external polarity cues mechanism, on the other hand, the cell relies on the polarity of the external environment to induce asymmetric segregation of cell fate determinants within the cell (Figure 2B) Last but not least, cells which divide . in stem cells where asymmetric cell division of the stem cell would produce two distinct daughter cells: one committed cell or differentiated cell and another which remained as a stem cell. This. Asymmetric cell division in neuroblast 10 Setting up cell polarity 11 Mitotic spindle orientation 13 Asymmetric segregation of cell fate determinants 14 Asymmetric localization of cell fate determinants. dissection 40 Pupal nota dissection 40 Cell line manipulation 41 General maintenance of cell line 41 RNAi treatment of cells 41 iv Transfection of cells 42 Biochemistry 42 Antibodies