Characterization of Bazooka and apkc in asymmetric Drosophila cyst stem cell division

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1 Characterization of Bazooka and apkc in asymmetric Drosophila cyst stem cell division BY WEI SHEN B.S., Jiangnan University, 2010 THESIS Submitted as partial fulfillment of the requirements for the degree of Master of Science in Bioengineering in the Graduate College of the University of Illinois at Chicago, 2012 Chicago, Illinois Defense Committee: Jun Cheng, PhD, Chair and Advisor Michael Cho, PhD Jason Yuan, MD/PhD, Medicine

2 This thesis is dedicated to my parents, and to my one and only, Claire, whose support and trust inspired me to follow the path of my choosing. WS ii

3 ACKNOWLEDGMENTS I would like to thank my thesis committee Dr. Cheng, Dr. Cho and Dr. Yuan for their unwavering support and assistance. I would like to especially thank my advisor, Dr. Jun Cheng, for all his guidance, support and encouragement during the two years. Dr. Cheng is a wonderful mentor and instructs me in the way of learning, the way of thinking and the way of being a scientist. I have yet not to meet anyone with more passion and enthusiasm for research. I have been very fortunate to have worked with an exceptional group of colleagues in LLIULM. I must thank all my fellow lab mates for their assistance, encouragement and friendship for the past two years. They have made this a truly memorable experience. iii

4 TABLE OF CONTENTS CHAPTER PAGE 1. INTRODUCTION Stem cell Asymmetric stem cell division Drosophila male cyst stem cell as a model Par proteins: Interpretation of cell polarity Signaling through Par proteins METHODS AND MATERIALS Fly husbandry and stains Immunoflurescent staining Time-lapse live-cell imaging SPECIFIC AIM 1: LOCALIZATION OF BAZOOKA AND APKC IN DROSOPHILA CYST STEM CELLS Dynamic Bazooka expression throughout the CySCs cycle Expression of apkc throughout the CySCs cycle Interaction between Bazooka and apkc Discussion SPECIFIC AIM 2: FUNCTIONS OF BAZOOKA AND APKC IN ASYMMETRIC DROSOPHILA CYST STEM CELLS DIVISION RNAi-mediated knockdown of Bazooka or apkc lead to testis morphology change RNAi-mediated knockdown of Bazooka or apkc increasd stem cell number Bazooka and apkc are required for spindle repositioning in Drosophila CySCs asymmetric division Spindle dynamics analysis revealed Bazooka and apkc regulate the mitotic spindle movement Proximal and distal spindle poles move independently Bazooka or apkc knockdown altered spindle pole dynamics throughout mitosis Bazooka or apkc knockdown shorten the mitosis duration Expression of E-cadherin was disrupted by RNAi-mediated knockdown of bazooka or apkc Discussion iv

5 TABLE OF CONTENTS (continued) CHAPTER PAGE 5. DISCUSSION AND CONCLUSIONS Discussion Study limitation Future work Conclusions CITED LITERACTURE APPENDIX VITA v

6 LIST OF TABLES TABLE PAGE I. SUMMARY OF INDIVIDUAL SPINDLE POLE VELOCITY II. SUMMARY OF MITOTIC SPINDLE DYNAMICS III. MITOSIS DURATION vi

7 LIST OF FIGURES FIGURE PAGE 1. Mechanisms of asymmetric stem cell division The Drosophila testis stem cell niche and asymmetric stem cell division Dynamic CySCs spindle movement and repositioning Distribution of cell fate determinants in the C. elegans one cell embryo and the Drosophila neuroblast and SOP cell An overview of Par complex signaling Pixel intensity analysis Bazooka localization in Drosophila CySCs changed at different cell cycles Pixel intensity analysis of Bazooka expression throughout the Drosophila CySCs cortex apkc localization in Drosophila CySCs changed at different cell cycles Pixel intensity analysis of apkc expression throughout the Drosophila CySCs cortex Bazooka localization in Drosophila apkc knockdown CySCs changed at different cell cycles Pixel intensity analysis of Bazooka expression throughout the apkc knockdown CySC cortex Expression of apkc in Drosophila Bazooka knockdown CySCs Pixel intensity analysis of apkc expression throughout Bazooka knockdown CySCs cortex Bazooka or apkc knockdown in CySCs severely affected the testes morphology and robustness Bazooka or apkc knockdown resulted in symmetric CySCs division and stem cells number increase vii

8 LIST OF FIGURES (continued) FIGURE PAGE 17. Bazooka and apkc are both required for CySCs anaphase spindle repositioning Histogram of proximal and distal spindle pole velocity throughout mitosis in control group Histogram of proximal and distal spindle pole velocity throughout mitosis in Bazooka and apkc knockdown groups Histogram of spindle movement (distance) throughout mitosis Histogram of spindle movement (velocity) throughout mitosis Bazooka and apkc affected mitosis duration in CySCs division RNAi-mediated knockdown of Bazooka or apkc disrupted the aggregation of E-cadherin to the hub-cysc interface in CySCs viii

9 LIST OF ABBREVIATIONS AB anterior daughter blastomere AJ adherens junction Apc2 Adenomatous polyposis coli 2 apkc atypical protein kinase C AurA Aurora A Bam bag-of-marbles Baz Bazooka Brat Brain tumor CNS central nervous system CySC cyst stem cell Dig Discs large E-cad E-cadherin Fas III Fasciclin III GMC ganglion mother cell GSC germline stem cells HA hemagglutinin Insc Inscuteable JAK-STAT Janus kinase and signal transducer and activator of transcription Lgl Lethal giant larva P1 posterior blastomere Par Partitioning defective ix

10 LIST OF ABBREVIATIONS (continued) Pins Partner of Inscuteable PDZ postsynaptic density/discs large/zonula occludens PH3 Thr 3-phosphorylated histone H3 Pon Partner of Numb PNS peripheral nervous system Pros Prospero SOP sensory organ precursors Upd Unpaired x

11 SUMMARY Stem cells reside in specific microenvironments, known as niches, which provide the stem cells with essential signals required to maintain stem cell identity. Asymmetric stem cell division is important for maintaining tissue homeostasis since it determines the fate of the two dividing daughter cells -- a daughter cell that is displaced outside of the niche is committed for differentiation while the other daughter cell remaining within the niche is committed for self-renewal. The intrinsic cellular machinery of asymmetric stem cell division remains, however, poorly understood. In this study, combined with developmental biology, live-cell imaging, and imaging processing, the importance of polarity proteins (i.e. Bazooka and apkc) in regulating the asymmetric stem cell division is discussed. More specifically, this study was conducted to examine the function of intrinsic cellular polarization in Drosophila cyst stem cells (CySCs) by directly comparing with RNAi-mediated knockdown Bazooka and apkc during cell division. The goal of this study is to meet the following specific aims: 1) To elucidate the expression of Bazooka and apkc in Drosophila CySCs, especially their dynamic localization throughout the cell cycle; 2) To investigate the function of Bazooka and apkc in regulating the anaphase spindle repositioning, which maintains CySCs asymmetric divisions. In order to examine the localization of Bazooka and apkc, confocal microscopy was used to visualize the expression of Bazooka and apkc within the CySCs. Then a pixel intensity analysis program was used to quantitatively xi

12 reveal their preferential localization throughout the cell cycle. Results showed bazooka was evenly distributed from interphase to metaphase, but illustrated a higher expression level in the basal cortex during anaphase. Additionally, apkc also displayed different localizations at different cell cycles. These results demonstrated dynamic localization of Bazooka and apkc during asymmetric CySCs division. Time-lapse live-cell imaging provides a means to directly investigate dynamic cellular process, demonstrating the function of Bazooka and apkc in regulating the CySCs asymmetric divisions. Compared with control, RNAimediated knockdown of either Bazooka or apkc severely disrupted the anaphase spindle repositioning, leading to symmetric stem cell division. Spindle pole dynamics and mitosis duration analysis illustrated that both proximal and distal spindle pole migrate drastically slower in Bazooka and apkc knockdown groups than the control group. Moreover, the duration from Prophase to Metaphase became shorter. Meanwhile, the E-cadherin, which is highly localized to the hub-cysc interface in widetype, was disrupted and distributed throughout the cell cortex in Bazooka and apkc knockdown. These results suggested both Bazooka and apkc play essential role in cellular polarization during anaphase spindle repositioning to ensure the asymmetric CySCs division. To sum up, the results obtained from time-lapse live-cell imaging provided diverse aspects of cellular dynamics, including the dynamic CySCs shape throughout the cell cycle, anaphase spindle repositioning, and mitosis xii

13 duration. The findings of this study demonstrated the significance of intrinsic cellular machinery in asymmetric stem cell division, revealing the role of Bazooka and apkc proteins in establishing cell polarity in the CySCs. Moreover, this study may also lead to further investigation of mechanisms of the stem cell niche and cellular machinery ensuring asymmetric cell divisions. xiii

14 1. INTRODUCTION 1.1 Stem cell Stem cells are defined to have remarkable potential to develop into many different cell types in the body during early life and growth. When a stem cell divides, each daughter cell has the potential either to remain stem cell identity or become another type of daughter cells that differentiate into diverse specialized cell types. Stem cells are distinguished from other cell types by two important characteristics. First, they are capable of dividing and renewing themselves for long periods. Stem cells are unspecialized cells that do not have any tissuespecific structures that allow it to perform specialized functions. However, stem cells can renew themselves through cell division, sometimes after long periods. Second, under certain physiologic or experimental conditions, they can be induced to become tissue- or organ-specific cells with specialized functions, known as differentiation. The process of differentiation is triggered by signals inside and outside the cells. The internal signals are controlled by a cell's genes, and carry coded instructions for all cellular structures and functions. The external signals for cell differentiation include molecules secreted by other cells, physical contact with neighboring cells, and certain molecules in the microenvironment. 1

15 2 1.2 Asymmetric stem cell division During the development of a multicellular organism, various types of cells have to arise from just a few progenitor cells, the stem cells. Stem cells are capable to divide symmetrically or asymmetrically, providing a simple method to maintain tissue homeostasis and eventually generate an entire organism. Symmetric cell divisions generate daughter cells that are identical and destined to acquire the same fate, whereas asymmetric cell divisions generate daughter cells that are destined for both self-renewal and differentiation. During the process of wound healing and regeneration, the stem cells divide symmetrically to propagate the number of stem cells to replenish the lost cells. Asymmetric stem cell division allows stem cells to self renew and differentiate for the purpose of preserving stem cell identity and producing cells that undergo differentiation. There is a tight balance between self-renewal and differentiation, which is crucial for tissue homeostasis (Morrison and Kimble 2006). Defects in this balance may result in tumorigenesis by overpopulation of stem cell daughters, or lead to aging/tissue degeneration due to failure in preserving stem cell functions. In order to maintain this vital balance, many stem cells control both the symmetric and asymmetric division outcome (Neumuller and Knoblich 2009). In brief, there are two main types of mechanism sustain the asymmetric cell division outcome. The first relies on the asymmetric placement of daughter cells relative to external cues. We refer this mechanism as extrinsic regulation

16 3 (Fig 1a), in which the only difference between the daughter cells is their position relative to the stem cell niche, an environment that instructs stem cell identity. Two daughter cells may initially have equivalent developmental potential, but they may acquire different fates owing to exposure to different external signals (Yamashita, Fuller et al. 2005). The second involves the asymmetric partitioning of cell components that determine the cell fate. We refer to this mechanism as intrinsic regulation (Fig 1b), and this includes regulated assembly of cell polarity factors and regulated segregation of cell fate determinants (Doe, Chu- LaGraff et al. 1991; Betschinger and Knoblich 2004). Drosophila germline stem cell (GSC) is one of the deeply studied and well characterized model systems, whose asymmetric stem cell divisions are regulated by extrinsic mechanism. GSCs divide asymmetrically to generate one daughter that remains in the stem cell niche and the other daughter that is placed away the niche. Cells that make up the stem cell niche include cap cells in the Drosophila ovary (Xie and Spradling 2000) and hub cells in the Drosophila testis (Kiger, Jones et al. 2001; Tulina and Matunis 2001). In the ovary, the cap cells secrete ligands called Decapentaplegic (DPP) and Glass bottom boat (GBB) that activate the signaling pathway in GSCs and prevent the differentiation of GSCs by repressing the transcription of bag-of-marbles (Bam), a key differentiation-promoting factor (Chen and McKearin 2003; Chen and McKearin 2003; Song, Wong et al. 2004). In the testis, the hub cells secrete ligand called Unpaired (Upd) that activates the Janus kinase and signal transducer and activator of transcription (JAK-STAT) signaling pathway in GSCs

17 4 and cyst stem cells (CySCs) and prevent them from differentiating (Leatherman and Dinardo 2008). Zfh-1, a transcription factor that is specifically expressed in cyst cells, is identified as a downstream target of the JAK-STAT pathway (Kiger, Jones et al. 2001; Tulina and Matunis 2001; Yamashita, Fuller et al. 2005). Caenorhabditis elegans (C. elegans) zygote provides as a classic example of an asymmetric division that is controlled by an intrinsic mechanism. C. elegan divides asymmetrically to produce one larger blastomere fated to make ectoderm, and one smaller blastomere that produces mesoderm, endoderm and finally germ line in a series of asymmetric division (Doe and Bowerman 2001). The asymmetric division of C. elegans zygote requires asymmetric localization of the PAR-3, PAR-6 and atypical protein kinase C (apkc) complex at the cortex, which in turn govern both mitotic spindle orientation and asymmetric segregation of cytoplasmic cell fate determinants, including riboprotein particles known as P granules and PIE-1, a transcriptional repressor required for germline fate (Strome and Wood 1983; Mello, Draper et al. 1992; Mello, Schubert et al. 1996; Reese, Dunn et al. 2000).

18 Figure 1. Mechanisms of asymmetric stem cell division. Two primary mechanisms are shown. (a) Asymmetric stem cell division by extrinsic cues (i.e. the stem cell niche). The two daughters of stem cell division are exposed to distinct cellular environments either inside or outside the stem cell niche, leading to asymmetric fate choice. (b) Asymmetric localization of cell polarity regulators or fate determinants initiates the asymmetric division; they are differentially distributed in the dividing cells, making the division asymmetrically. (Yamashita, Yuan et al. 2010) 5

19 6 1.3 Drosophila male cyst stem cells as model Asymmetric cell division is a mechanism highly conserved from bacteria to fungi, plants, and mammals (Hawkins and Garriga 1998), demanding stem cell populations that interact with external microenvironment or other stem cell types. To unveil the regulatory mechanisms of asymmetric division, a few animal models are specifically studied due to their ability to closely resemble the cell/tissue type of interest. The Drosophila melanogaster testis is a classic model system for exploring the stem cell division behavior in a specialized microenvironment (Fig. 2a). This specialized microenvironment is known as the stem cell niche. At the apical tips of the testis, the germline stem cells (GSCs) anchor to the hub cells via E-cadherin based cell-cell junctions. Hub cells are 8 16 somatically derived cells that are considered as a major component of the niche (Morrison and Kimble 2006). The GSCs asymmetric division produces one GSC and one gonialblast (GB), and then GB undergoes transit-amplifying divisions to become a 16-cell spermatogonium. The niches are also composed of cyst stem cells (CySCs), which maintain contact with the hub via cell processes (Leatherman and Dinardo 2008). Similar to GSCs, the CySCs also divide asymmetrically, producing one CySC and one cyst cell. A pair of CySCs encapsulates a GSC; however, the cyst cells that envelop the GB do not divide, but they continue to grow and encase the GB and its progeny throughout spermatogenesis (Yamashita, Jones et al. 2003; Cheng, Tiyaboonchai et al. 2011).

20 7 The interaction of stem cell with the niche is important for their normal function (Fig. 2b). The signaling ligand Unpaired (Upd) secreted from the hub cells activates the Janus kinase signal transducer and activator of transcription (JAK-STAT) pathway in both GSCs and CySCs, instructing the stem cell selfrenew (Kiger, Jones et al. 2001; Tulina and Matunis 2001; Leatherman and Dinardo 2008). Zfh-1 is a transcription factor that is restrictively expressed in CySCs and quickly down-regulated in cyst cells, and it is identified as a downstream target of the JAK-STAT pathway. Moreover, overexpression of Zfh- 1 in cyst stem cells leads to CySCs hyperproliferation, which is followed by hyperproliferation of GSCs (Leatherman and Dinardo 2008). These studies elucidate the complex interaction between two stem cells population and reveal that CySCs play an instructive role in GSC fate determination.

21 Figure 2. The Drosophila testis stem cell niche and asymmetric stem cell division. (a). Hub cells (yellow) adhere to the apical tip of the testis. Surrounding the hub are germline stem cells (GSCs, purple) and somatic cyst stem cells (CySCs, green). GSCs and CySCs divide and produce daughter cells that remain in the niche (self-renewal) or leave the niche and differentiate. GSCs give rise to spermatogonia (light blue); CySCs give rise to cyst cells (light green), which encase the developing spermatogonia. (b) GSCs and CySCs are attached to the hub cells via adherens junctions. In the testis, the hub cells synthesize a ligand called Unpaired (Upd) that activates the Janus kinase signal transducer and activator of transcription (JAK-STAT) pathway in the GSCs and CySCs to specify their stem cell identity. The Zfh-1 transcription factor controls CySCs identity. Together with hub cells, CySCs dictate GSCs identity. The epidermal growth factors receptor (EGFR) signaling ensures the encapsulation of germ cells by the cyst cells. (Fuller and Spradling 2007) 8

22 9 It is important to note that asymmetric divisions can be governed by both intrinsic partitioning of fate regulators and asymmetric exposure to extrinsic cues. Asymmetric division of Drosophila germline stem cells do not seem to rely on partitioning of cell fate determinants. Although each germline stem cell is marked by a cytoplasmic organelle called spectrosome, the function of this asymmetrically distributed organelle remains uncertain (Deng and Lin 1997). Additionally, the mechanism controlling the centrosome orientation in GSCs is intracellular, and depends on polarity cues from the GSC-hub interface. During mitosis, the mother centrosomes localize near adherens junctions at the hub- GSC interface and associate with a robust array of microtubules (Yamashita, Mahowald et al. 2007). Moreover, centrosomin, a centrosomal protein, along with periocentriolar matrix tethers centrosomes to astral microtubules, the Adenomatous polyposis coli 2 (Apc2) links astral microtubules to adherens junctions. GSCs lacking any of the forementioned proteins have the centrosomes misoriented. The polarity cue that positions the mother centrosome to the hub-gsc interface is likely to be the adhesion protein E-cadherin, which is located exclusively at the hub-gsc interface, as is Apc2 (Yamashita, Jones et al. 2003; Inaba, Yuan et al. 2010). When E-cadherin is expressed ectopically at the hub-gsc interface, Apc2 is also mislocalized, resulting in centrosome misorientation. E-cadherin therefore is required for the polarizing proteins toward the hub cells, and this polarizing proteins maybe a way which the GSCs achieve asymmetric stem cell division.

23 10 Like GSCs, CySCs undergo asymmetric division in stem cell niche, however, using a cellular mechanism strikingly distinct from GSC or any other known stem cells. It is reported that the mitotic centrosomes keep a consistent position respect to the hub-gsc interface when they are oriented in the GSCs, which allows the spindle to be oriented perpendicular to the hub-gsc interface and the mother centrosome to be retained in the daughter cell that remains at the hub (Yamashita, Jones et al. 2003; Yamashita, Mahowald et al. 2007). Additionally, in Drosophila embryonic neuroblast, the mitotic spindles assemble orthogonally to the polarity axis and later rotate to align with the polarity axis during the first division, and subsequently align with the polarity axis in the later divisions (Kaltschmidt, Davidson et al. 2000; Rebollo, Roldan et al. 2009). In contrast to Drosophila male GSCs or neuroblast, which divides in asymmetrically in a fixed cell polarity, CySCs employ a different spindle position pattern during mitosis (Cheng, Tiyaboonchai et al. 2011). The mitotic spindles in CySCs exhibit no consistent orientation until the onset of anaphase, when one spindle pole quickly retracts to the hub-cysc interface and the mitotic spindle is almost always juxtaposed with the hub-cysc interface (Fig 3). Besides that, compared with the reports that male GSCs and neuroblasts maintain their round shape throughout the cycle, CySCs undergo dynamic cell shape during cell cycle (Cheng, Tiyaboonchai et al. 2011). The shape of the CySCs is assumed flat with a thin projection attaching the hub during interphase, then the CySCs subsequently round up slightly during mitosis, probably giving enough room for anaphase spindle repositioning.

24 11 Recent study illuminated that the CySC division requires some different molecular mechanisms from GSCs (Cheng, Tiyaboonchai et al. 2011). For example, while GSC requires Apc2 but not Moesin, CySC requires Moesin but not Apc2 for spindle repositioning. In addition, the requirement of orientated centrosome, Dynein, and Moesin is necessary for the spindle repositioning during anaphase, which in turn ensures the consistent CySCs asymmetric division (Cheng, Tiyaboonchai et al. 2011). The basic cellular machinery for spindle repositioning awaits further investigation, but it provides a new but excellent model for studying the asymmetric cell division. It would also be significant to find out the detailed cellular machinery and biomechanical regulation that may be causing the CySCs to slightly round-up and activate the anaphase spindle repositioning process.

25 Figure 3. Dynamic CySCs spindle movement and repositioning. A CySC rounds up during mitosis. Unlike the GSC s mitotic spindle that is consistently oriented, CySC spindles forms away the bub and are dynamic during metaphase. Around the onset of anaphase, one of the spindle poles retracts to the hub-cysc interface, pulling the entire spindle near the hub. (Cheng, Tiyaboonchai et al. 2011) 12

26 Par proteins: Interpretation of cell polarity Spindle orientation in the asymmetrically dividing stem cells is required for the cellular polarity, and this can be either regulated through E-cadherin based cell-cell junctions (den Elzen, Buttery et al. 2009; Inaba, Yuan et al. 2010), or through the proteins involved in polarization of the cell, known as polarity proteins. The polarity genes were originally discovered in C. elegans zygotes in a screen for partitioning defect mutants (Kemphues, Priess et al. 1988). Six mutants were identified that share a similar phenotype: a switch from asymmetric division into abnormal symmetric division that results in equal sizes of anterior daughter blastomere (AB) and posterior blastomere (P1) cells (Fig 4a). The genes were named after their mutation: par-1 to par-6. Except for par-2, these genes are conserved in Drosophila. In 1998, the fly cell polarization gene bazooka was cloned and was found to encode a protein closely resembling Par- 3 (Kuchinke, Grawe et al. 1998). A mammalian PAR-3 homolog that can bind an atypical protein kinase C (apkc) was found to adopt an apical-basal asymmetrical localization in mammalian epithelial cells (Izumi, Hirose et al. 1998), and this lead to the identification of C. elegans apkc as a new protein with a Par loss-of-function phenotype and an asymmetric localization in anterior cortex, like Par-3 and Par-6 (Tabuse, Izumi et al. 1998). Drosophila neural stem cells are one of stem cells that rely on intrinsic asymmetric division (Wu, Egger et al. 2008). The asymmetric division of these cells was shown to depend on the segregation of cell fate determinants, which is

27 14 used to generate cell-fate diversity in both the central nervous system (CNS) and the peripheral nervous system (PNS). The neural precursors in the CNS are called neuroblasts (Fig 4b). Each neuroblast divides asymmetrically to produce an apical daughter, which remains a neuroblast, and a smaller basal daughter called a ganglion mother cell (GMC) (Jan and Jan 1998; Doe and Bowerman 2001). Another neural precursors in the PNS are called sensory organ precursors (SOPs). Each SOP is made of the progeny of a single SOP (Fig 4c), generated through several rounds of asymmetric cell divisions (Bardin, Le Borgne et al. 2004). Par (Partition defective) proteins are widely investigated due to their important role in integrating diverse signals that regulate cell polarity (Munro and Bowerman 2009; Nelson 2009; Prehoda 2009). Drosophila uses the homologues of these polarity proteins to set-up an apical-basal polarity in neuroblasts and an anterior-posterior polarity in SOPs. Par-3 homolog Bazooka and Par-6 both localize to the apical cortex in neuroblasts, but to the posterior cortex in SOP (Neumuller and Knoblich 2009). The atypical protein kinase C (apkc) gene encodes a serine/threonine kinase that was later added to the par family. The apkc protein is shown to form a complex with Bazooka and Par-6 (Kemphues, Priess et al. 1988; Tabuse, Izumi et al. 1998). The basal/anterior cortex however is marked by a different polarity protein. Drosophila neuroblasts and SOP cells use the lethal giant larvae (Lgl) protein to mark their basal/anterior cortexes (Atwood and Prehoda 2009). All of the Par proteins are enriched to some degree at or near the cell cortex, and par mutants are

28 15 defective in cell polarization that may disrupt the motor regulation and necessary establishment condition in the cell cortex for asymmetric movement of cellular components (Kemphues 2000).

29 Figure 4. Distribution of cell fate determinants in the C. elegans one cell embryo and the Drosophila neuroblast and SOP cell. (a) The C. elegans embryo has an anterior-posterior polarity defined by an anterior Par-3/Par-6/PKC3 complex and a posterior Par1/Par2 complex. (b) The Drosophila neuroblast has an apical-basal polarity, defined by the apical Baz/Par-6/aPKC complex and the basal Lgl marker. The differentiation determinants Numb, Brat and Pros are asymmetrically distributed into the basal GMC daughter cell via their cargo proteins Pon and Mira. (c) The Drosophila SOP cell has an anterior-posterior polarity defined by anterior Lgl and a posterior Par-3/Par-6/aPKC complex. The cell fate determinants Numb, Brat, Pros and Pon are asymmetrically segregated in the anterior piib daughter. (Gonczy 2008) 16

30 Signaling through Par proteins Polarized distribution of cytoplasmic components and the proper alignment of the mitotic apparatus are prerequisite for asymmetric divisions (Whittaker 1980; Strome and Wood 1982). Polarity proteins, which play major roles in integrating diverse signals that regulate cell polarity (Fig 5), are involved in the set-up of fate determinant that guarantee the different differentiation potential of daughter cells in asymmetric cell division. Polarity cue is initially manifested as an apical complex comprising Bazooka, Par-6 and atypical protein kinase C (apkc), which is also called Par complex proteins (Joberty, Petersen et al. 2000; Lin, Edwards et al. 2000; Wodarz, Ramrath et al. 2000). Par-6 and apkc associate through their aminoterminal PB1 domain (Hirano, Yoshinaga et al. 2005), and Par-6 inhibits the basal activity for apkc, but also can act as a targeting subunit for the kinase domain, recruiting the substrates for phosphorylation (Yamanaka, Horikoshi et al. 2001). One of Par-6 substrates is Bazooka. Bazooka and Par-6 are PDZ (PSD95/Dig/ZO1) domain-containing proteins with scaffolding or adaptor functions (Etemad-Moghadam, Guo et al. 1995; Hung and Kemphues 1999). Par-6 binds to Bazooka through a PDZ-PDZ interaction, but apkc can also bind through its kinase domain directly to the carboxyl-terminal half of Bazooka, and phosphorylate a Ser residue (Nagai-Tamai, Mizuno et al. 2002). Importantly, Bazooka, Par-6 and apkc do not form a constitutive complex. Their interactions are regulated by multiple protein kinases, by small GTPases, and by competition

31 18 for other binding partners. These regulations can alter the subcellular distribution of the polarity proteins, and their function. Perhaps the most significant gap in our understanding has been information linking the Par proteins to downstream localization events. That is, how do the Par proteins mediate asymmetric distribution of other molecules? -- Kemphues, 2000 Although recent cell biological and biochemical works have begun to solve it, the gap has not yet closed. Studies of mechanisms in diverse systems have revealed a core signaling pathway that interacts with numerous other pathways in diverse ways, indicating that the Par complex proteins polarize cells by a number of different mechanisms. In Drosophila neuroblast, for instance, the function of Par complex proteins in asymmetric cell division is deeply studied. The neuroblast polarization is a dynamic process that result in the formation of two mutually exclusive cortical domains-- a basal domain containing fate determinants that specify differentiation and an apical domain containing polarity regulatory factors. During interphase, apkc forms a complex with Par-6 and Lgl, which is uniformly distributed along the cell cortex (Betschinger, Eisenhaber et al. 2005). At the early stage of mitosis, Aurora A (AurA) is activated by Bora in a Cdc-2 dependent manner (Hutterer, Berdnik et al. 2006). Activated AurA

32 19 phosphorylates Par-6 in the Lgl/aPKC complex. Unphosphorylated Par-6 suppresses apkc activity, indicating that upon Par-6 phosphorylation apkc is active. apkc subsequently phosphorylates Lgl, which can no longer bind to the complex and will translocate to the basal cortex. Dissociated Lgl enables Bazooka to bind apkc. Besides AurA, there is another apkc activator, the Rho GTPase Cdc42, which can also activate apkc via binding to Par-6 (Lin, Edwards et al. 2000). Once activated, the apical and basal polarity complexes can restrict each other s localization. The major components segregated to the basal part of cell contain Miranda, Prospero (Pros), Numb, Partner of Numb (Pon) and Brain tumor (Brat) (Kraut, Chia et al. 1996; Lu, Jan et al. 2000; Bellaiche, Radovic et al. 2001). During mitosis, apical apkc activity is required to restrict basal fate determinants from occupying the apical cortex. Recent work has discovered a direct mechanism for polarization of basal domain proteins by apkc in which phosphorylation of Numb or Miranda leads to release apkc from the basal cortex (Lee, Robinson et al. 2006). Proper segregation of fate determinants during the asymmetric cell division is required for the mitotic spindle aligning with the polarity axis. Spindle alignment is important that two cortical cleavage furrow forms between the two cortical domains, properly segregating their contents into the two daughter cells. The cellular machinery that controls spindle orientation must interface with cortical polarity regulatory factors (Prehoda 2009). The primary connection between the spindle and cortical polarity is the protein Inscuteable (Insc), which links them by binding Bazooka and the adapter protein Partner of Inscuteable

33 20 (Pins) (Kraut, Chia et al. 1996; Schober, Schaefer et al. 1999; Yu, Morin et al. 2000). Additionally, it has been found that astral microtubules can induce polarity through the kinesin Khc72 and the tumor suppressor Discs large (Dig) (Siegrist and Doe 2007). Dlg binds to Pins, providing the connection to Insc and cortical polarization. Disruption of some Par complex proteins results in random migration of the apical spindle (Cai, Yu et al. 2003; Lee 2009) or lead to misoriented spindles by affecting the overall apical-basal polarity (Bowman, Neumuller et al. 2006), indicating the importance of cell polarity in spindle orientation. Although many of the proteins have been identified, the exact nature of the mechanical linkage between the spindle apparatus and the cortical organization still requires further investigation.

34 Figure 5. An overview of Par complex signaling. Par complex proteins (apkc/par-3(bazooka)/par-6) play major roles integrating diverse signals that regulate cell polarity. Inputs (bottom) and outputs (top) with cellular functions are targeted by these pathways. (McCaffrey and Macara 2009) 21

35 22 Here I used the Drosophila cyst stem cell as model to study the regulatory mechanism of Bazooka and apkc in mediating the asymmetric stem cell division. I first analyzed the expression of Bazooka and apkc throughout the cell cycle in Drosophila cyst stem cells. The aggregation of Bazooka to basal cortex during anaphase is independent of apkc. Meanwhile, by means of timelapse live-cell imaging, I investigated the role of Bazooka and apkc in regulating the asymmetric Drosophila cyst stem cell division outcome by performing RNAi-mediated knockdown of Bazooka and apkc, which strikingly abolishing the expression of Bazooka and apkc in cyst stem cells. Compared with the control group, RNAi-mediated knockdown of either Bazooka or apkc severely disrupted the anaphase spindle repositioning, leading to symmetric stem cell division. Spindle pole dynamics analysis illustrated that both proximal and distal spindle poles migrate drastically slower in Bazooka and apkc knockdown groups than the control group. Moreover, the duration from Prophase/Prometaphase to Metaphase became shorter than the control groups, resulting in overall shorter mitosis duration. These results suggested that Bazooka and apkc are required to regulate the anaphase spindle repositioning in Drosophila cyst stem cell. Meanwhile, the aggregation of E-cadherin to the hub-cysc interface was disrupted and showed ectopic distribution throughout the cell cortex in the Bazooka and apkc knockdown groups, indicating that Bazooka and apkc might also be involved in the maintenance of extrinsic regulatory mechinary. These results demonstrated that both Bazooka and apkc

36 23 play essential roles in cellular polarization during anaphase spindle repositioning to ensure the asymmetric CySCs divisions.

37 2. METHODS AND MATERIALS 2.1 Fly husbandry and stains All fly stocks were raised on standard Bloomington medium at 25 C, and young flies (0 to 1-day-old adults) were used for all experiments. The following fly stocks were used: c587-gal4 (Manseau, Baradaran et al. 1997; Kai and Spradling 2003) from S. Hou (National Cancer Institute, NIH); UAS-α-tubulin- GFP (Grieder, de Cuevas et al. 2000) from A. Spradling (Carnegie Institution, Baltimore, MD, USA); UAS-DEFL from H. Oda (JT Biohistory Research Hall, Japan); UAS-Bazooka-RNAi (V2914), UAS-DaPKC-RNAi (V2907) from Vienna Drosophila RNAi Center; UAS-Bazooka-EYFP, UAS-aPKC (caxx-1)-ha (Lee, Robinson et al. 2006) from Lee (University of Oregon, Or, USA). 2.2 Immunoflurescent staining Samples were fixed for minutes with 4% formaldehyde in PBS, permeabilized for 30 minutes in PBST (0.1% Triton X-100 in PBS), incubated overnight at 4 C with primary antibodies, washed with PBST (20 minutes, three times), incubated overnight at 4 C with AlexaFluor-conjugated secondary antibodies (1:100, Molecular Probes) and washed again with PBST (20 min, three times). Samples were then mounted in VECTASHIELD (H-1200, Vector Laboratory) and imaged using a Zeiss Z1 confocal microscope. The primary antibodies included mouse anti-γ-tubulin (1:100; GTU-88, Sigma), mouse anti- 24

38 25 Fasciclin III [1:80, developed by C. Goodman (University of California, Berkeley) and obtained from the Developmental Studies Hybridoma Bank (DSHB)], rabbit anti-thr 3-phosphorylated histone H3 (1:80, Millipore), goat anti-vasa (1:70; dc- 13, Santa Cruz), Mouse anti-adducin [1:100, developed by H. Lipshitz (The Hospital for Sick Children, Toronto) and obtained from the Developmental Studies Hybridoma Bank (DSHB)], rabbit anti-hemagglutinin (1:80, Cell Signaling). Images were processed using Image J. Anaphase was determined by the morphology of mitotic chromatin (stained by Thr 3-phosphorylated histone H3), as well as cell shape and spindle (pole-to-pole) length: anaphase was determined by having two segregating chromatin masses and rounded (oval) cell shape and the onset of spindle elongation, while telophase was identified by two segregating chromatin masses and a peanut cell shape constricted by the contractile ring. In order to further quantitatively analyze the target protein localization at the CySCs cortex, a program running inside Matlab software was applied to trace the pixel intensity of target protein throughout the cell cortex. Starting from the hub-cysc interface, the region of interest can be outlined with the multiline tool set at a thickness of five pixels and returns a data log of the pixel intensity values. Then I used Excel to make line graphs using pixel intensity data from program, so that I can compare the intensity of target protein expression near the hub with expression around the rest of the cell cortex (Fig 6). The average intensity for each pixel point was obtained to make a line graph to compare the distribution in early stage of mitosis (Pro/Metaphase) and that in anaphase.

39 Figure 6. Pixel intensity analysis. (a) Schematic of tracing protein pixel intensity along the circumference of CySC, starting from the hub-cysc interface. (b) An example of freehand outlining the circumference of CySC, then (c) program would show the outline in an orthogonal coordinate. (d) A data log of pixel intensity would be plotted based on the tracing and take the average of five pixel thickness. 26

40 Time-lapse live-cell imaging Newly enclosed control (α-tub-gfp), Baz-RNAi (α-tub-gfp/ Baz-RNAi) and apkc-rnai (α-tub-gfp/ apkc-rnai) flies were dissected inside Drosophila culture medium containing Schneider s Drosophila medium and 10% fetal bovine serum and 1% Penicillin Streptomycin. The testis tips were placed inside a sterile glass=bottom chamber, and was mounted on a three-axis computer-controlled piezoelectric stage and imaged using an inverted microscope equipped with an electron multiplier cooled CCD camera. Fourdimensional image sequences (x, y, z, and time) were acquired every 60 s. A semi-automatic tracking program was used to trace location of proximal spindle pole, distal spindle pole and hub-cysc interface. The pattern matching was completed with National Instrument Labview software, and then their coordinates were imported to the distance-velocity calculation program inside Matlab software to calculate the following the factors: Mitotic spindle separation distance Proximal Spindle Pole to hub-cysc interface distance Distal Spindle Pole to hub-cysc interface distance Mitotic Spindle angle Mitotic Spindle angle (Absolute Value) Proximal Spindle Pole movement velocity (one frame = 1min) Distal Spindle Pole movement velocity (one frame = 1min) Mitotic spindle separation velocity (one frame = 1min) Mitotic Spindle angular velocity (one frame = 1min)

41 28 All the calculation results would be saved in Excel file and imported to the Histogram Output program inside the Matlab Software, allowing to plot the histograms from the calculated excel files and compute the p-values between two groups (control and Baz-RNAi/ apkc-rnai) of study.

42 3. SPECIFIC AIM 1: LOCALIZATION OF BAZOOKA AND APKC IN DROSOPHILA CYST STEM CELLS Currently, little is known about where Bazooka and apkc localize and how they regulate the asymmetric cell division in Drosophila cyst stem cells (CySCs). First of all, to better understand the function of Bazooka and apkc in the CySC asymmetric division, their localizations were investigated in CySCs. Bazooka and apkc have been reported to localize to the apical cortex in Drosophila larval neuroblast (Lu, Ackerman et al. 1999; Schober, Schaefer et al. 1999), however to basal cortex in sensory organ precursors (SOPs) (Gho and Schweisguth 1998; Lu, Usui et al. 1999), which depends on the polarity pattern and core groups of polarity genes in distinct cell division (Jan and Jan 2001). 3.1 Dynamic Bazooka expression throughout the cell cycle I investigated the Bazooka and apkc localization by using EYFP-bound Bazooka (UAS-Baz-EYFP) and hemagglutinin (HA)- bound apkc (UAS-aPKC- HA). Both expressions were activated by C587-Gal4, which specifically drives UAS expression in cyst stem cells and cyst cells, allowing me to visualize the distribution of Bazooka and apkc in CySCs throughout the cell cycle. Besides that, each group of testes was immunostained for the hub cells marker Fas III, the germline cell marker Vasa, centrosome marker ϒ -tub and mitotic chromatin marker Thr 3-phosphorylated histone H3 (PH3). Images obtained from confocal microscopy showed that the Bazooka was localized evenly throughout the CySC 29

43 30 cortex during interphase until metaphase, and aggregated to the basal cortex during anaphase (Fig 7). The categorization criteria is based on the morphology of PH3 signal, which can be observed by condensed chromosomes as cells enter prophase and separated to the opposite ends of the cell as two masses when it proceeds to anaphase. However, it is hard to certify the preferential distribution of Bazooka because of the low expression level and high background noise distraction. In this case, I used a pixel intensity analysis program to track the expression level throughout the cell cortex.

44 31 Figure 7. Bazooka localization in Drosophila CySCs changed at different cell cycles. (a) Bazooka was evenly expressed through the cell cortex during interphase. (b) Bazooka remained evenly localized throughout the cell cortex during the early stage of mitosis, indicated by the Thr 3- phosphorylated histone H3 (PH3) amassed in the mid-plate. (c) Bazooka aggregated to the basal cortex during anaphase, indicated by two separated masses of PH3 signals. Blue: Fas III (hub cells, indicated as asterisk), γ-tubulin (centrosome) and PH3 (mitotic chromatin); green: Bazooka-EYFP; red: Vasa (germ cells). Dotted lines outline CySC cell shape. Mitotic chromatin separation is indicated by a double-headed arrow. Scale bar: 5 μm.

45 32 Totally twenty-one CySCs without PH3 signal and thirteen CySCs with PH3 signal were analyzed via pixel intensity analysis program, among which eleven CySCs were in early stage of mitosis (from prophase to metaphase) and two CySCs were in anaphase. Averaging data of pixel intensity level illustrated a general map of Bazooka localization with respect to the cell cortex. The analysis clearly showed that Bazooka was almost equally distributed through the cell cortex during the interphase and early stage of mitosis, illustrated by the similar Bazooka expression level throughout the cell cortex (Fig 8a, b, d). Strikingly, Bazooka aggregated to the basal cortex during anaphase, showing higher Bazooka level in the middle section of the curve, indicated as the basal cell cortex (Fig 8c, d). The peak value in the basal cortex (pixel intensity=300) was almost two times larger than that in other part of cell cortex (average pixel intensity=160). The result demonstrated the specificity of the dynamic Bazooka localization change throughout cell cycle. However, the exact timing for such aggregation is unknown yet. One scenario is that the recruitment of Bazooka to the basal cortex occurs before the onset of anaphase, which may ensure the proper basal anchor between distal spindle pole and basal cell cortex. Another possibility is that during the anaphase, the expression of Bazooka is repressed by the apical fate determinants, which is elucidated in SOPs that Bazooka is phosphorylated by Numb and forms basal crescent (Bellaiche, Radovic et al. 2001).

46 Figure 8. Pixel intensity analysis of Bazooka expression throughout the Drosophila CySCs cortex. Circumference of CySC was traced and the pixel intensity was analyzed (see Methods for detail). (a) 21 CySCs in interphase, (b) 11 CySCs in the early stage of mitosis, and (c) 2 CySCs in anaphase were analyzed. (d) Average pixel intensity for Bazooka expression during interphase, early stage mitosis, and anaphase was shown. 33

47 Expression of apkc throughout the cell cycle Similarly, the pixel intensity analysis of apkc revealed the cortical localization throughout the cell cycle. Different from EFYP-bound Bazooka, which is an endogenous enhanced yellow fluorescent protein, apkc is hemagglutinin (HA)-bound and need to bind to anti-ha antibody in order to be visualized via fluorescent microscopy. Meanwhile, since both anti-ha and anti-ph3 (mitotic chromatin marker) are from the same donor--rabbit, both mitotic chromatin and apkc might emerge in the same channel. In order to obtain clear apkc expression in CySCs, I only used anti-γ-tubulin (centrosome) instead of anti-ph3 to eliminate the distraction from PH3 signal. Using same GAL4-UAS system to overexpress the apkc in CySCs, apkc can be observed as cortical distribution throughout the CySC cortex. Strikingly, in some CySCs displayed dual centrosome, apkc seemed to aggregate to the basal cortex. However, it would be hasty to use dual centrosome as mitotic marker. For example, replicated centrosomes are already split during early interphase in Drosophila neuroblast (Januschke. 2011), however, the centrosome migration pattern in Drosophila CySCs is unknown. Thus, lacking mitotic marker made it hard to distinguish the specific stage during the mitosis. Similar with Bazooka, the expression of apkc in CySC with single centrosome can be observed evenly distributed throughout the cell cortex (Fig 9a). As for those CySC with dual centrosomes, it became hard to tell their accurate preferential localization because of the relative dim image quality (Fig 9b).

48 35 Figure 9. apkc localization in Drosophila CySCs changed at different cell cycles. (a) apkc was evenly expressed throughout the cell cortex within CySCs with a single centrosome. (b) apkc seemed to aggregate to the basal cortex within CySCs with dual centrosomes, indicated by two separated γ-tubulin signal. Green: Fas III (hub cells, indicated as asterisk) and γ-tubulin (centrosome); blue: apkc-ha; red: Vasa (germ cells). Dotted lines outlined CySC cell shape. Scale bar: 5 μm.

49 36 In order to unearth the preferential expression of apkc during the Drosophila cyst stem cell cycle, the same pixel intensity analysis also applied to the apkc localization. However, different from Bazooka preferential localization, CySCs with apkc-ha were divided into two groups: one was made up of CySCs with single centrosome; the other was made up of CySCs with dual centrosomes. Totally seventeen CySCs in first group and five CySCs in second group were analyzed by pixel intensity analysis program. Averaging data of pixel intensity level illustrated a general map of apkc localization with respect to the CySC cortex. Consistent with the confocal images showed above, apkc was almost equally distributed throughout the cell cortex within CySCs with single centrosome, which was also similar to the Bazooka expression during interphase. Strikingly, pixel intensity in second group showed relatively higher apkc expression in the middle section of the curve, indicated as the basal cell cortex. The averaging peak value in the basal cortex (average pixel intensity= 230) was almost two times larger than that in other part of cell cortex (average pixel intensity= 110). Specifically, among all the CySCs with dual centrosomes, most of them preformed higher expression at the basal cortex, while the other two CySCs displayed relatively similar apkc expression level throughout the cell cortex (Fig 10b. Red and green curves). This suggested that apkc experienced dynamic distribution throughout the CySCs cycle; however, further investigation with mitotic marker is needed to reveal the intact apkc expression throughout Drosophila CySC cycle.

50 Figure 10. Pixel intensity analysis of apkc throughout the Drosophila CySC cortex. Circumference of CySCs was traced and the pixel intensity was analyzed (see Methods for detail). (a) seventeen CySCs with a single centrosome, and (b) five CySCs with dual centrosomes were analyzed. (c) Average pixel intensity was shown. 37

51 Interaction between Bazooka and apkc During asymmetric division of Drosophila neuroblasts, proper recruitment of apkc to the apical domain requires the activity of the so-called Par complex, of which apkc is a component but also includes Bazooka and Par-6 (McCaffrey and Macara 2009). Both Bazooka and Par-6 interact directly with apkc that Bazooka interacts with the apkc kinase domain and is an enzymatic substrate (Suzuki and Ohno 2006; Goldstein and Macara 2007). To address the potential functional relationship between Bazooka and apkc, I examined Bazooka localization in apkc knockdown CySCs and apkc localization in Bazooka knockdown CySCs. Similar method as investigation of Bazooka and apkc localization in CySCs was performed; the hub cells were identified by Fas III, along with the germline cell marker Vasa, centrosome marker ϒ -tub and mitotic chromatin marker phosphorylation of histone H3 (PH3) (Fig 20). When Bazooka was expressed in apkc knockdown CySCs (C687-Gal4 > UAS-Baz-EYFP/ UASaPKC-RNAi), it remained localized evenly in the cell cortex during interphase and early mitosis stage (Fig 11 a, b, 12). Moreover, Bazooka preferentially aggregated to the basal cell cortex during anaphase, which was similar to the expression in control group (C687-Gal4 > UAS-Baz-EYFP, Fig 11c, 12). The result indicated that Bazooka localization is independent of apkc; however, Bazooka may directly bind to lateral membrane and rely on basal cytoskeletal cues, anchoring the minus end of microtubule (Harris and Peifer 2005).

52 39 Figure 11. Bazooka localization in Drosophila apkc knockdown CySCs changed at different cell cycles. (a) Bazooka was evenly expressed throughout the cell cortex during interphase. (b) Bazooka remained evenly localized throughout the cell cortex during the early stage of mitosis, indicated by the Thr 3- phosphorylated histone H3 (PH3) mass in the mid-plate. (c) Bazooka aggregated to the basal cortex during anaphase, indicated by two separated masses of PH3 signals. Blue: Fas III (hub cells, indicated as asterisk), γ-tubulin (centrosome) and PH3 (mitotic chromatin); green: Bazooka-EYFP; red: Vasa (germ cells). Dotted lines outlined CySC cell shape. Mitotic chromatin separation was indicated by a double-headed arrow. Scale bar: 5 μm.

53 Figure 12. Pixel intensity analysis of Bazooka expression throughout the apkc knockdown CySCs cycle. Circumference of CySCs was traced and the pixel intensity was analyzed (see Methods for detail). (a) 11 CySCs in interphase, (b) 14 CySCs in the early stage of mitosis, and (c) 3 CySCs in anaphase were analyzed. (d) Average pixel intensity of Bazooka expression during interphase, early stage mitosis, and anaphase was shown. 40

54 41 I also examined the apkc localization in Bazooka knockdown CySCs to further investigate the interaction of Bazooka and apkc. Similar method as investigation of apkc localization in CySCs was performed: the hub cells were identified by Fas III, along with the germline cell marker Vasa, centrosome marker ϒ -tub (Fig 13). When apkc was expressed in Bazooka knockdown CySCs (C687-Gal4 > UAS-aPKC-HA/ UAS-Bazooka-RNAi), it remained evenly localized in the cell cortex within CySCs with a single centrosome (Fig 13, 14). Moreover, the expression level was similar to that in control group (C687-Gal4 > UAS-aPKC-HA). However, within CySCs with dual centrosomes, apkc remained equally distributed throughout the cell cortex. Specifically, totally ten CySCs with a single centrosome and three CySCs with dual centrosomes were analyzed by the pixel intensity analysis program. And averaging value of pixel intensity level illustrated a general map of apkc localization with respect to the Bazooka knockdown CySC cortex. Both averaging data illustrated similar expression level throughout cell cortex. These results indicated that the aggregation of apkc to basal cortex was disrupted by Bazooka knockdown. Lacking mitotic marker or specific cell cycle stage marker, however, the detailed interaction mechanism remained unkown. It is possible that Bazooka is a positive regulator of apkc than functions as upstream factor mediating the apkc aggregating to the basal cell cortex during mitosis. Together, these results suggested that the dynamic apkc localization is Bazooka dependent.

55 Figure 13. Expression of apkc in Drosophila Bazooka knockdown CySCs. (a) apkc was evenly expressed throughout the cell cortex within the CySCs with a single centrosome. (b) apkc seemed to remain evenly distributed throughout the cell cortex within the CySCs with dual centrosomes, indicated by two separated γ-tubulin masses. Green: Fas III (hub cells, indicated as asterisk), γ- tubulin (centrosome); blue: apkc-ha; red: Vasa (germ cells). Dotted lines outlined CySC shape. Scale bar: 5 μm. 42

56 Figure 14. Pixel intensity analysis of apkc expression throughout Bazooka knockdown CySCs cycle. Circumference of CySCs was traced and the pixel intensity was analyzed (see Methods for detail). (a) ten CySCs with a single centrosome, and (b) three CySCs with dual centrosomes were analyzed. (c) Average pixel intensity was shown. 43

57 Discussion This study elucidated the dynamic localization of Bazooka and apkc throughout Drosophila CySC cycle. Assisted with the mitotic chromatin specific staining, known as phosphorylated of histone H3 (PH3), mitotic cells can be clearly identified. The early stage of mitosis (from prometaphase to metaphase) and anaphase can be distinguished by the mitotic chromatin separation and elongated cell shape. Strikingly, both Bazooka and apkc evenly localized throughout the cell cortex, and then aggregated to the basal CySC cortex throughout cell cycle. However, the present data do not provide to the exact time of such recruitment to the basal cortex. It is possible that the progression of Bazooka to basal cortex during anaphase might activate the anchoring cue distribution, which in turn represses the basal fate determinant to the opposite side of cells. In the one-cell Caenorhabditis elegans (C. elegans) embryo, the cortical dynein motors maintain a basal level of activity that propels microtubules along the cortex (Gusnowski and Srayko 2011), and the pulling forces are active in late metaphase/anaphase. The anaphase-promoting complex (APC) activation is required for asymmetric positioning of mitotic spindle, simultaneously triggering spindle displacement and anaphase onset (McCarthy Campbell, Werts et al. 2009). In other words, in light of the function of Bazooka and apkc involving in the formation of a submembraneous protein scaffold to anchor daughter centrosome to cortical region (Izumi, Hirose et al. 1998; Wodarz, Ramrath et al. 2000), it is likely that Bazooka and apkc are already recruited to basal cell

58 45 cortex, providing enough time for spindle pole linking properly to the right cortical site. It is likely that the Bazooka and apkc provide critical spatial information during cell division. An important question lies in how the localization of Bazooka and apkc are regulated at different cell cycles. Recent findings suggest that the polarization mechanism regulated by Bazooka and apkc may be partly conserved in different cell types and the expression is dependent on context. For example, in the C. elegans zygotes, Par-3 (homolog of Bazooka) aggregates to anterior cortex that requires the expression of Par-6, apkc and Par-2 (Etemad- Moghadam, Guo et al. 1995; Hung and Kemphues 1999); however, in Drosophila neuroblasts and embryonic epithelial cells, apical Bazooka localization is independent of apkc (Harris and Peifer 2005). And in mammalian epithelial cells, Bazooka localizes at the basal cortex but is associated with tight junctions (Izumi, Hirose et al. 1998). Nevertheless, in Drosophila CySCs, I found out that Bazooka and apkc localization is dynamic with respect to cell cycle. Bazooka and apkc are distributed evenly throughout the cell cortex then aggregate to the basal cortex, displaying different localizations at different cell cycles. Although the detailed molecular mechanism and signal pathway by which the Bazooka and apkc recruited to the basal cortex await further investigation, other well-studied stem cell types shed light on the puzzle. Inscuteable (Insc) constructs the primary connection between the spindle and cortical polarity. Insc binds to the cortical polarity comprising Bazooka and the adapter protein Partner of Inscuteable (Pins) (Kraut, Chia et al. 1996; Schober, Schaefer et al. 1999; Yu,

59 46 Morin et al. 2000), showing the essential role of Insc in Drosophila neuroblast asymmetric division that recruits Bazooka to the apical cortex, and directs Numb to the opposite pole (Doe, Chu-LaGraff et al. 1991; Zipursky and Rubin 1994; Knoblich, Jan et al. 1995). Strikingly, because of lacking Insc in Drosophila SOPs, Bazooka forms crescent on the posterior cortex, also at the opposite pole of the Numb crescent, which in parts due to the alternative binding partners without the Insc recruiting Bazooka (Bellaiche, Radovic et al. 2001; Roegiers, Younger- Shepherd et al. 2001). Typically the apical/anterior Par proteins are required to prevent basal/posterior Par proteins form localizing apically/anteriorly, vice versa. Furthermore, most of the interactions among Par complex proteins via PDZ (postsynaptic density/discs large/zonula occludens) domains of Bazooka are required for the individual enrichment in a specific region of the cell cortex (Etemad-Moghadam, Guo et al. 1995; Tabuse, Izumi et al. 1998; Hung and Kemphues 1999; Wu, Feng et al. 2007). That also accounts for another possibility that, in Drosophila CySCs, Bazooka and apkc form distinctive complex with other polarity proteins. Interestingly, the atypical PKCs are named as such because, unlike canonical PKCs, they are not activated by Ca 2+ and diacylglycerol, but contain a similar serine/threonine kinase domain (Newton 2001). Given this functional difference, it is not surprising that apkcs contain distinct regulatory domain structure compared to their canonical relatives. At its amino terminus, apkc contains a PB1 domain that binds to the polarity protein Par-6, which is a potent repressor of apkc kinase activity (Yamanaka, Horikoshi et al. 2001; Atwood,

60 47 Chabu et al. 2007). This repression is partially relieved when Cdc42 binds to Par- 6, providing a possible mechanism for coupling localization to activation. Par-6 repression of apkc can also be relieved by phosphorylation of the Par-6 PB1 domain by the mitotic kinase Aurora A, which bridge Bazooka with apkc and Par-6 (Wirtz-Peitz, Nishimura et al. 2008). This intriguing model provides a compelling link between a central regulator of polarity and the cell cycle. In light of the reports sparkling the versatility of the deployment of these molecular components in different types of asymmetric division, I hypothesized that Bazooka and apkc might be repressed towards basal cortex, and anchored in a basal-dependent manner to basal target site, which may totally opposite to that in Drosophila neuroblast (Harris and Peifer 2005). Several feedback mechanisms are possible involving Bazooka and apkc. As mentioned above, Bazooka is a positive regulator of apkc localization and activity, presumably through Cdc42 (Chen and Macara 2006). Bazooka is also an apkc substrate, and although Bazooka is found to remain basal localization in apkc knockdown, these crescents are weaker than in wildtype group, suggesting that robust Bazooka localization and activity requires apkc activity. Bazooka/aPKC and other regulators may form another feedback loop (for example, Lgl in Drosophila neuroblast), but in this case, the interaction may be mutually antagonistic. The antagonistic regulators inhibit Bazooka/aPKC activity and perhaps localization (Betschinger, Eisenhaber et al. 2005; Lee, Robinson et al. 2006). And Bazooka/aPKC are also their substrate, which leads to their own inactivation and possibly cortical release. What role feedback plays in Drosophila

61 48 stem cell polarity will require further investigation, but it would not be surprising if robust symmetry breaking were the result of the combined action of positive and negative feedback loops.

62 4. SPECIFIC AIM 2: FUNCTIONS OF BAZOOKA AND APKC IN ASYMMETRIC DROSOPHILA CYST STEM CELLS DIVISION Time-lapse live-cell imaging provides a novel means to directly investigate dynamic cellular processes. With the aid of time-lapse live-cell imaging, Bazooka and apkc s dynamic processes in regulating asymmetric Drosophila cyst stem cells (CySCs) divisions were widely investigated in this study. 4.1 RNAi-mediated knockdown of Bazooka or apkc lead to testis morphology change In order to investigate the function of Bazooka and apkc in mediating the asymmetric CySCs division, I first generated samples with expression of α- tubulin-gfp, which is a major component of microtubule, using cyst lineagespecific driver (C587-gal4> UAS- α-tub-gfp). The live-cell imaging provided me a visualized CySC cell shape throughout the cell cycle. Similar to previous report (Cheng, Tiyaboonchai et al. 2011), CySCs attached to hub cells via a thin projection and formed a flat shape encapsulated round GSCs. Strikingly, the CySCs rounded up but maintained attachment to the hub via a thin projection. Thus, the ability to consistently identify CySCs allowed me to study the function of Bazooka and apkc in asymmetric CySC cell division. RNAi-mediated knockdown of Bazooka and apkc were expressed, along with α-tubulin-gfp, by using cyst lineage-specific driver (C587-gal4> UAS- α-tub- 49

63 50 GFP/UAS-Baz-RNAi and UAS- α-tub-gfp/uas-apkc-rnai). Strikingly, it was showed that the testis morphology was severely affected in both Bazooka and apkc knockdown groups (Fig 15a). The testis size was almost two times smaller than control (C587-gal4> UAS- α-tub-gfp), and became fragile and vulnerable. To verify whether the intriguing phenomenon resulted from the specificity of effect of Bazooka and apkc knockdown in CySCs, I further examined the parents that contained no driver (UAS-Baz-RNAi and UAS-aPKC-RNAi). The samples showed similar testis size and morphology as control (Fig 15b), which suggested that the overall testis morphology was affected by the knockdown of Bazooka or apkc in CySCs, resulting in abnormal morphology and fragileness.

64 Figure 15. Bazooka or apkc knockdown in CySCs severely affected the testes morphology and robustness. (a) Testes affected by the Bazooka or apkc knockdown in CySCs (left and middle panel) showed smaller size and fragility compared to control shown in the right panel. (b) UAS-Baz-RNAi and UASaPKC-RNAi testes without any driver showed normal morphology and robustness as control. Scale bar: 100 μm. 51

65 RNAi-mediated knockdown of Bazooka or apkc increased stem cell number Meanwhile, I also hypothesized that the expression of Bazooka or apkc knockdown in CySCs may also affect the proper stem cell number. Fixed samples were examined with α-tub-gfp expression in cyst cells using C587- GAL4 driver, along with the identification of hub cell by Fas III and germline cells by Vasa. This allowed me to quantify the germline stem cells (GSCs), CySCs and hub cells number within the niche. In control (n=14), the average number of hub cells was 8.6 and that of GSCs was 9.3, which were both consistent with typical wildtype hub cells and GSCs number (Hardy, Tokuyasu et al. 1979). However, the number of GSCs increased significantly in both Bazooka knockdown group (n=11) and apkc knockdown group (n=5), of which the GSCs in Bazooka knockdown group raised to 12 and that in apkc knockdown group was 15 (Fig 16a, student t-test: p<0.003 and p<0.002, respectively). The number of CySCs was quantified based on the α-tub-gfp expression that either Bazooka or apkc knockdown group occupied significantly increased CySCs number, compared to control group (Fig 16b, student t-test: p<0.003 and p<0.003, respectively). Nevertheless, the number of hub cells in knockdown group was comparable with control group (Fig 16c, student t-test: p>0.1 and p>0.2, respectively). In control group, 100% of mitotic cells divided asymmetrically: one daughter cell maintained attachment to the hub, while the other was displaced

66 53 away the hub (Fig 16d). However, the asymmetric division outcome in Bazooka and apkc knockdown groups reduced to 74% and 60% (Fig 16d, student t-test: p<0.003 and p<0.009, respectively), remaining two daughter cells attached to the hub. These results might also account for the increase of CySCs and GSCs number. This might be possibly because the role of Bazooka and apkc in regulating the cellular polarity and ensuring the asymmetric cell division, and the disruption of asymmetric fate determinant governed by the polarity proteins leads to symmetric cell divisions. The increase in GSCs number might be secondary to the increase in CySCs, as CySCs play an instructive role in establishing the GSCs identity (Leatherman and Dinardo 2008; Leatherman and Dinardo 2010). The finding demonstrated the knockdown of either Bazooka or apkc in CySCs disrupted the asymmetric cell division and resulted in symmetric cell division. Together, these results suggested that the importance of Bazooka and apkc in mediating asymmetric CySCs divisions and maintaining the proper stem cell number.

67 Figure 16. Bazooka or apkc knockdown resulted in symmetric CySCs division and stem cells number increase. (a) RNAi-mediated knockdown of Bazooka or apkc in CySCs significantly increased the number of GSCs (Mean + S.D.) and (b) CySCs (Mean + S.D.). P-values are calculated using Student s t- test. (c) The number of hub cells was unaffected in Bazooka or apkc knockdown groups (Mean + S.D.). (d) Frequency of CySCs asymmetric and symmetric divisions. 54

68 Bazooka and apkc are required for spindle repositioning in asymmetric Drosophila CySCs division Bazooka and apkc control cellular polarity in many systems, including Caenorhabditis. elegans (C. elegans) early embryos (Kemphues, Priess et al. 1988; Cheeks, Canman et al. 2004), Drosophila neuroblast (Lu, Jan et al. 2000; Bellaiche, Radovic et al. 2001) and sensory organ precursors (SOPs) (Jan and Jan 2001; Roegiers, Younger-Shepherd et al. 2001). In order to further investigate the function of Bazooka and apkc in asymmetric Drosophila CySCs division, especially seeking for effect of basal recruitment during anaphase CySCs spindle repositioning, I performed time-lapse live-cell imaging by epifluorescence microscope to unveil the characteristics of Bazooka and apkc in regulating the spindle repositioning. I used UAS- α-tub-gfp, driven by C587- Gal4, to facilitate the analysis of mitotic spindle in CySCs. First of all, observation from live-cell imaging of control group (C587-gal4; UAS- α-tub-gfp, n=12) revealed the dynamic CySCs shape throughout the cell cycle: during interphase, CySCs formed a water drop - like shape, with a thin projection reaching the hub. Then they rounded up during mitosis, but maintained attachment to hub via a thin projection, while some CySCs appeared to have full contract with the hub. Besides that, mitotic spindles showed stereotypical movement, which could be identified by dynamic spindle rocking back and forth during metaphase; then, one of the spindle poles was almost adjacent to the hub-cysc interface within 2 μm during anaphase (Fig 17a), known as spindle association (Cheng, Tiyaboonchai et al. 2011). In most cases, non-associated

69 56 mitotic spindles rotated within the cell for a long time (on average, 12.6 min for metaphase duration, Table I). Subsequently, right before the onset of anaphase, one spindle pole always retracted to hub-cysc interface, and the other located to the opposite end of cell, known as anaphase spindle repositioning, then CySCs quickly underwent division (on average, 3.2 min for anaphase duration, Table I). The spindle pole retracting to hub-cysc interface along with anaphase spindle repositioning can be seen in almost every mitotic CySCs (Fig 17e, 92%, n=12) and eventually resulted in asymmetric cell division (Fig 16c, 100%). And the angle of anaphase spindle was preferable to be perpendicular with respect to hub-cysc interface (Fig 17d, 67%, n=12), with one spindle pole positioned within the crescent where the CySC contacted the hub. These results were in accordance with the previous conception that asymmetric cell divisions of CySCs are ensured by mitotic spindle repositioning during anaphase (Cheng, Tiyaboonchai et al. 2011). The abnormal testis morphology resulted from RNAi-mediated knockdown of Bazooka (C587-gal4; UAS- α-tub-gfp/uas-baz-rnai) or apkc (C587-gal4; UAS- α-tub-gfp/uas-apkc-rnai) in CySCs arouse my interest to investigate their effect on asymmetric division in CySCs. As described above, both Bazooka and apkc displayed dynamic localization during asymmetric CySCs division. When Bazooka or apkc was knockdown in CySCs, anaphase spindle repositioning, identified by spindle association, was significantly reduced to 26% (n=19) and 27% (n=16), compared with 92% in control (Fig 17e, student t-test: p< and p<0.02, respectively). Specifically, in either Bazooka or apkc

70 57 knockdown groups, most mitotic cells underwent anaphase, identified by separating microtubule clusters and elongated cell shape, without either spindle pole adjacent to the hub-cysc interface (Fig 17. b, c). Additionally, the angle of the CySCs anaphase spindle with respect to the hub-cysc interface became random in either Bazooka or apkc knockdown groups (Fig 17d).

71 Figure 17. Bazooka and apkc are both required for CySCs anaphase spindle repositioning. (a) An example of control CySC anaphase spindle (two spindle poles indicated by arrowheads) with one pole (blue arrowhead) closely associated with the hub-cysc interface (hub cell is indicated by asterisk). Scale bar: 10 μm. (b) An example of Bazooka knockdown group and (c) apkc knockdown group during anaphase, neither spindle poles was adjacent to the hub-cysc interface. (d) The anaphase spindle orientation was random in Bazooka and apkc knockdown groups. (e) Frequency of spindle association and non-associated with hub-cysc interface during anaphase in control, Bazooka and apkc knockdown groups. 58

72 Spindle dynamics analysis revealed Bazooka and apkc regulate the mitotic spindle movement In order to fully investigate the function of Bazooka and apkc in regulating the mitotic spindle repositioning, it is necessary to understand the mechanism of spindle repositioning and how the spindle poles interact with apical, basal, or both cortex Proximal and distal spindle poles move independently Studies in yeast and C. elegans have provided strong evidence that spindle-cell cortex forces are generated at the astral microtubule-cortex interface by the action of cortically attached microtubule-based motors, proteins affecting microtubule length, and/or by proteins controlling the dynamics of astral microtubule-cortex interactions. Thus, spindle positioning requires dynamic attachment of astral microtubules to polarized cortical domains and generation of forces exerted on these cortically-attached astral microtubules aligns the mitotic spindle (Cowan and Hyman 2004; Pearson and Bloom 2004). However, several important questions remain to be answered for Drosophila CySCs anaphase spindle repositioning: Do both proximal and distal spindle pole interact with the cortex during spindle repositioning? What microtubule-binding proteins are required for spindle-cortex interaction?

73 60 First of all, to fully understand the spindle repositioning mechanism, I need to trace the movement of individual spindle pole throughout distinct mitosis stage to determine their dynamics, and most importantly whether they move independently. Control group images were analyzed by the spindle pole tracing program (see Method 2.3). Result showed that during prophase/prometaphase and metaphase both spindle poles showed vigorous movement, and interestingly, the distal spindle pole moved much more vigorously than the proximal spindle pole. Specifically, the average velocity of distal spindle pole is 3.4 μm/min during prophase/prometaphase and 3.9 μm/min during metaphase (Fig 18a, b, Table I), which is 54% and 63% faster than that of proximal spindle pole during prophase/prometaphase and metaphase (2.2 μm/min and 2.4 μm/min, student t-test: p< and p<0.02, respectively), while the peak velocity of distal spindle pole reached 6.6 μm/min and 7.1 μm/min during prophase/prometaphase and metaphase, which is 50% and 61% faster than that of proximal spindle pole (4.4 μm/min and 4.4 μm/min). However, there was no significant difference during anaphase or telophase (Fig 18c, d, Table I). It is possible that proximal and distal spindle pole were interacted with apical/basal cell cortex associated with force-generated protein. These results suggested that the individual spindle pole moved independently from prophase to metaphase, however, the detailed machinery that responsible for the spindlecortex interaction is still unknown.

74 Figure 18. Histogram of proximal and distal spindle pole velocity throughout mitosis in control group. (a) Proximal and distal spindle pole velocity during prophase/prometaphase (student t-test: p < ). (b) Proximal and distal spindle pole velocity during metaphase (p < 2E-8). (c) Proximal and distal spindle pole velocity during anaphase (p > 0.9). (b) Proximal and distal spindle pole velocity during telophase (p > 0.8). 61

75 62 I further investigated the proximal/distal dynamics in either Bazooka or apkc knockdown groups. As described above, anaphase spindle repositioning was disrupted and the anaphase spindle association decreased drastically, I hypothesized that the cortical polarity proteins involved Bazooka and apkc should communicate in some way with mitotic spindle to position it properly. Strikingly, compared to control group, both proximal and distal spindle pole in Bazooka and apkc knockdown groups showed vigorous movement in both prophase/prometaphase and metaphase. However, the proximal and distal spindle poles showed similar dynamic pattern (Fig 19, Table I). And the average and peak velocity was comparable in each mitosis stage. It is possibly because Bazooka and apkc mediated the spindle-cortex interaction that the knockdown of Bazooka and apkc disrupted the proper spindle pole dynamics.

76 63 Figure 19. Histogram of proximal and distal spindle pole velocity throughout mitosis in Bazooka or apkc knockdown groups. (a) Proximal and distal spindle pole velocity in Bazooka knockdown group during prophase/prometaphase (student t-test: p < 0.001), metaphase (p > 0.3), anaphase (p > 0.6) and telophase (p > 0.6). (b) Proximal and distal spindle pole velocity in apkc knockdown group during prophase/prometaphase (student t-test: p > 0.05), metaphase (p > 0.6), anaphase (p > 0.07) and telophase (p > 0.5).

77 TABLE I. SUMMARY OF INDIVIDUAL SPINDLE POLE VELOCITY (μm/min). 64

78 Bazooka or apkc knockdown altered spindle pole dynamics throughout mitosis Spindle movement could be due to the direct interaction between astral microtubules and the CySCs cortex via the regulation of Bazooka and apkc. I further investigated how the knockdown of Bazooka or apkc affected the spindle dynamics throughout mitosis. Similarly, the mitotic spindle pole movement tracing program was used to quantitatively illustrate the mitotic spindle dynamic in CySCs division, revealing the role of Bazooka and apkc in regulating the anaphase spindle repositioning. Consistent with the non-associated anaphase spindle in Bazooka or apkc knockdown groups described above, the distribution of the mitotic spindle angle and the distance between each spindle pole to the hub-cysc interface showed significant difference in Bazooka and apkc knockdown groups compared to control group. Specifically, the proximal spindle pole showed much more distant to hub-cysc interface (Fig 20a, Table II, mean value: 3.3 μm and 3.7 μm, respectively, compared to 2.6 μm in control group), and the distal spindle pole, on the other hand, showed much closer to hub-cysc interface (Fig 20b, Table II, mean value: 7.2 μm and 6.2 μm, respectively, compared to 7.7 μm in control group). Additionally, most of the mitotic spindle angles were in large degree (Fig 20d, Table II, mean value: 40 degree and 53 degree, respectively, compared to 22 degree in control). However, the mitotic spindle separation in Bazooka knockdown group displayed similar to control group (Fig 20c, Table II, mean value: 6.2 μm compared to 6.1 μm in control), while that in apkc knockdown

79 66 group showed significant decrease (Fig 20c, Table II, mean value: 5.6 μm). That might be partially because the size of CySCs in apkc knockdown was 1.4 times smaller than the Bazooka knockdown and control group, so the mitotic spindle was separated in the relatively limited room. These results illustrated that both the mitotic spindles in Bazooka and apkc knockdown groups throughout mitosis resided much more preferentially parallel with respect to the hub-cysc interface. It was also demonstrated the spindle association was disrupted by RNAimediated knockdown of Bazooka or apkc. Thus, Bazooka and apkc are both required for anaphase spindle repositioning that ensure the asymmetric CySCs division.

80 Figure 20. Histogram of spindle movement (distance) throughout mitosis. (a) Distribution of proximal spindle pole to hub-cysc interface distance in control, Bazooka and apkc knockdown group (student t-test p< and p<0.0001, respectively, compared with control). (b) Distribution of distal spindle pole to hub- CySC interface distance in control, Bazooka and apkc knockdown group (student t-test p<0.04 and p<0.0001, respectively, compared with control group). (c) Distribution of spindle pole separation distance in control, Bazooka and apkc knockdown group (student t-test p>0.8 and p<0.02, respectively, compared with control group). (d) Distribution of spindle angle in control, Bazooka and apkc knockdown group (student t-test p< and p<0.0001, respectively, compared with control group). 67

81 68 In order to get deeper insight of the function of Bazooka and apkc in asymmetric CySCs division regulation, the spindle pole tracing program was also utilized to quantify the distribution of distinct spindle pole body velocity as well as spindle angular velocity throughout the mitosis, which revealed the overall mitotic spindle dynamics in Bazooka or apkc knockdown CySCs. The distribution of distal spindle pole velocity revealed that the movement of distal spindle pole with respect to hub-cysc interface was drastically slower in Bazooka or apkc knockdown group than that in control group (Fig 21b, Table II, mean value: 3.0 μm/min and 2.9 μm/min, respectively, compared to 3.7 μm/min in control). Additionally, proximal spindle pole velocity in Bazooka knockdown group (Fig 21a, Table II, mean value: 2.2 μm/min) was significantly slower compared to apkc knockdown group (mean value: 2.6 μm/min) or control group (mean value: 2.5 μm/min). However, both mitotic spindle separation velocity and mitotic spindle velocity showed similar distribution in control, Bazooka and apkc knockdown groups (Fig 21c, d, Table II). Based on the observation described above that each spindle pole may move independently basically depend on distinct force-generating proteins, it is possible that Bazooka and apkc both directly or indirectly regulate the interaction of basal astral microtubules with the basal cell cortex, and Bazooka, to some extent, might be also involved in regulating the apical interaction. Thus, the results suggested that RNAi-mediated knockdown of Bazooka or apkc disrupted the mitotic spindle-cortex interaction, which in turn disrupted anaphase spindle repositioning. However, the relationship between Bazooka/aPKC and

82 69 force-generating proteins in CySCs is still unknown, and the intact molecular interaction between Bazooka and apkc and mitotic spindle remains further investigation.

83 Figure 21. Histogram of spindle movement (velocity) throughout mitosis. (a) Distribution of proximal spindle pole velocity in control, Bazooka and apkc knockdown group (student t-test p<0.005 and p>0.8, respectively, compared with control). (b) Distribution of distal spindle pole velocity in control, Bazooka and apkc knockdown group (student t-test p<0.001 and p<0.0001, respectively, compared with control group). (c) Distribution of spindle pole separation velocity in control, Bazooka and apkc knockdown group (student t-test p>0.8 and p>0.3, respectively, compared with control group). (d) Distribution of spindle angular velocity in control, Bazooka and apkc knockdown group (student t-test p>0.07 and p>0.2, respectively, compared with control group). 70

84 TABLE II. SUMMARY OF MITOTIC SPINDLE DYNAMICS 71

85 Bazooka or apkc knockdown shorten the mitosis duration Based on the live-cell imaging, I also noticed that the durations in mitosis were different in each groups. Two spindle poles migrating around the nuclei can be visualized during prophase. During metaphase, two spindle poles faced each other on opposite cell ends. Then one spindle pole would retract close to the hub-cysc interface on the onset of anaphase then the cell would elongate and a contractile ring developed between two daughters, indicated as telophase (Fig 22a, b). Typically during the mitosis in control group (Fig 22c, Table III), the entire mitosis duration last average 31.8 min (Table III), with a relatively long duration of prophase/prometaphase (mean value: 12.6 min, Table III) and metaphase duration (mean value: 12.4 min), and relatively short duration of anaphase (mean value: 3.2 min, Table III) and telophase duration (mean value: 3.6 min, Table III). Strikingly, RNAi-mediated knockdown of Bazooka or apkc resulted in decreased mitosis duration. Specifically, the durations of prophase/prometaphase (mean value: 8.7 min and 9.2 min, respectively, Table III) and metaphase (mean value: 8.8 min and 8.9 min, respectively, Table III) were significant decreased in both Bazooka and apkc knockdown groups (Fig 22c, Table III). However, the duration of anaphase (mean value: 3.4 min and 3.3 min, respectively, Table III) and telophase (mean value: 3.9 min and 3.3 min, respectively, Table III) remained similar as control group. Thus, the knockdown of Bazooka or apkc significantly reduced the mitosis duration by decreasing the duration from Prometaphase to Metaphase. It is possibly because the RNAi-

86 73 mediated knockdown of Bazooka or apkc leads to abolishing the downstream protein kinases regulation. A region in the carboxyl terminus of Bazooka binds to the LIMK2 protein kinase, and attenuates LIMK2 kinase activity (Chen and Macara 2006). The principal function of LIMK2 is to phosphorylate and inhibit cofilin, an actin-severing protein that is essential for actin remodeling during junction formation (Ivanov, McCall et al. 2004), so Bazooka may locally alter actin dynamics through this mechanism. Yet, it is not clear how universal this mechanism is and how Bazooka and apkc work exactly in regulating the forcegenerating protein kinase.

87 Figure 22. Bazooka and apkc affected CySCs mitosis duration. (a) Schematic diagram of cell division. Time was given in min relative to onset of anaphase (0min), which was evident by extension of mitotic spindle. (b) An example of CySC mitosis duration with respect to time from prophase (-25 min) to anaphase (0 min), then to telophase (5 min). Hub cells were indicated by asterisk. Yellow dotted lines outlined CySC cell shape. Spindle poles were indicated by the arrowheads. (c) Mitosis duration among control, Bazooka knockdown and apkc knockdown group. Mean values, standard deviation and student t-test p value were listed in Table III. 74

88 TABLE III. MITOSIS DURATION (min) 75

89 Expression of E-cadherin was disrupted by RNAi-mediated knockdown of bazooka or apkc Adhesion molecules are required to keep the stem cells attached to the hub. In Drosophila testis, GSCs and CySCs attach to hub cells via adherens junctions (Raymond, Deugnier et al. 2009; Marthiens, Kazanis et al. 2010). Recently, E-cadherin was investigated to play an important role in the polarization of Drosophila male GSCs for its requirement in GSC maintenance (Inaba, Yuan et al. 2010). E-cadherin is also required for CySC maintenance (Voog, D'Alterio et al. 2008), GSCs lacking E-cadherin rapidly lost from the niche, suggesting that adherens junctions mediate GSCs adhesion to the hub in addition to their role in orienting centrosomes and spindles. The hub-cysc interface is also enriched with E-cadherin, which is thought to provide a polarity cue for dynein to pull one of the spindle poles (Cheng, Tiyaboonchai et al. 2011). When wildtype E-cadherin-GFP (E-cad DEFL ) was expressed using cyst-specific driver (C587-gal4>UAS- E-cad DEFL ), it localized exclusively to the hub-cysc interface (Fig 23a, a ). Because of the various cell shape, E-cadherin was concentrated at the CySC projection attached to the hub, or appeared at broader interface between hub and CySC. Then I expressed RNAi-mediated knockdown of Bazooka or apkc to address their function in CySC adherens junctions (UAS- E-cad DEFL /UAS-Baz-RNAi and UAS- E-cad DEFL /UAS-aPKC-RNAi), using cyst cell lineage driver. The expression of E-cadherin in Bazooka and apkc knockdown groups both predominantly localized to the hub-cysc interface,

90 77 however, in almost 50% Bazooka knockdown group and 40% in apkc knockdown group, the expression of E-cadherin also ectopically appeared to the CySC cortex outside the hub-cysc interface (Fig 23b, c, b and c ). Even within the same testis, the expression of E-cadherin shows heterogeneous distribution among CySCs in Bazooka and apkc knockdown groups. The result suggested that both Bazooka and apkc are required for E-cadherin aggregation at the hub- CySC interface. However, the cellular machinery that responsible for E-cadherin interacting with Bazooka or apkc and regulating the cell polarity during cell cycle remained uncertain.

91 Figure 113. RNAi-mediated knockdown of Bazooka or apkc disrupted the aggregation of E-cadherin to the hub-cysc interface in CySCs. (a) E-cadherin was highly localized at the hub-cysc interface in control group. Red indicates Vasa (germ cells); greed indicates E-cadherin; blue indicates Fas III (hub cells, asterisk indicates the hub cells center). (b, c) Examples of disrupted E-cadherin distribution in Bazooka knockdown groups (b) and apkc knockdown group (c). (a, b, c ) green channel only displayed the E-cadherin expression in control, Bazooka and apkc knockdown groups, respectively. 78