Asymmetric Cell Division Protein Complex Structure And Function Studies

The main contributions of this dissertation were:For the first time, we solved the crystal structures of two protein complexes mPins (partner of Inscuteable/mInsc (inscuteable), mPins/NuMA (nuclear mitotic apparatus), which are involved in asymmetric cell division. With combined efforts of biochemical and molecular tools, we carefully analyzed each structure and discussed the relationship between both complexes. The revealed structural information provides strong structural bases underlying the molecular mechanism of asymmetric cell division.Stem cells are defined by their abilities of self-renewal and differentiation through asymmetric cell division, whereby each stem cell divides to generate one daughter with a stem-like fate and one daughter that differentiates. Asymmetric division is a particularly attractive strategy because it manages both tasks with a single division. However, a disadvantage of asymmetric cell division is that it leaves stem cell unable to expand in number. To achieve this, Stem cells rely on symmetric divisions that are defined as the generation of daughter cells that are destined to acquire the same fate. It is very important for stem cell to balance two kinds of division modes. Defects in regulation of the switch between symmetric and asymmetric divisions can be deleterious. A defect favouring symmetric divisions results in tumorigenesis whereas a defect favouring asymmetric divisions results in decreased capacity for tissue repair.The role of asymmetric cell division in stem-cell control, coupled with the mechanisms that regulate this process, have been extensively studied. In brief, two main type of mechanism govern asymmetric cell divisions. The first relies on the asymmetric portioning of cell components that determine cell fate, which called intrinsic mechanism. The second involves he asymmetric placement of daughter cells relative to external cues, which called extrinsic mechanism. Intrinsic mechanisms include regulated assembly of cell polarity factors and regulated segregation of cell fate determinants. A classic example of an asymmetric division that is controlled by an intrinsic mechanism is provided by Drosophila neural stem cells (Neuroblast, NB). NBs delaminate from the vental neuroectoderm and undergo stem cell-like, asymmetric division, to self-renew and to generate smaller daughter cells, called ganglion mother cells (GMCs). Each GMC divides only once to generate two neurons and/or glial cells. NBs delaminate from the neuroectoderm and rotate their mitotic spindle 90 degree perpendicular to the epithelial plane. Subsequent NB divisions are thus oriented along the apical-basal axis. The polarity of NBs is established by the Par complex, Insc, and Pins which localized at the apical cortex. The cell fate determinants such as Numb, Pros, and Brat are localized at the basal cortex. To ensure asymmetric segregation of cell fate determinants, the orientation of mitotic spindle needs to be coordinated with their asymmetric localization. In embryonic NB, this coordination is achieved by Insc, Pins, and Gai. Although these process and signal network have been studied for many years, detailed mechanism of asymmetric division is still not clear. How is NB polarity and spindle orientation coordinated during NB division? What then maintains the apical localization of Insc/Par complex and orientation of apical-basal spindle axis through subsequent mitotic cell cycles? What is the relationship between different complexes involved in asymmetric cell division? We believe that the detailed structures of these complexes and elucidation of the interaction between these complexes can provide strong supports to understand molecular mechanism of asymmetric cell division.This dissertation consists of 6 chapters and contents are summarized as follows.In the first chapter, the recent achievements of stem cell research and division modes of stem cell will be introduced. Different asymmetric division modes in several model organisms will be compared. Also differences of molecular mechanism between these asymmetric division modes will be discussed. Protein complexes which play a very important role in asymmetric division will be summarized. Importantly, the aims and significance of the work will be presented.In the second chapter materials and methods which used in the experiments are presented.In the third chapter, the content focuses on the interaction of mPins and mlnsc. Pins and Insc play important roles in the establishment and maintenance of cell polarity. We confirmed that mPins can interact with mInsc in vitro with a binding affinity about 0.67μM. The minimal binding sites in mPins and mInsc were mapped at TPR47 of mPins and the N-terminal domain (NTD) of mInsc. To gain detailed structural information of Pins/Insc interaction, we firstly turned to NMR-based techniques. However, the homogeneity of mInsc NTD is poor although it folds well, and the behavior was not improved in the presence of Pins TPR region. On the other hand, the TPR4-7 heavily aggregates with concentration increase and thus it is not amendable for NMR-based structural analysis. Then we tried our best to get crystals of Pins/Insc complexes. After extensive screening of protein boundaries and buffer conditions, we finally got high quality crystal structure of Pins/Insc complex with diffraction of 1.1 A. The mPins TPR repeats adopts canonical TPR folds, each containing two antiparallelα-helices. Tandem arrays of TPR motif generate a right-handed helical structure with an amphipathic channel for mInsc peptide binding. The complex structure was further confirmed by point mutagenesis.In the forth chapter, the main content focuses on the interaction of mPins and NuMA. mPins/NuMA plays an important role in the spindle orientation. We confirmed that mPins can interact with NuMA in vitro with a binding affinity of 0.50μM. The minimal binding sites in mPins and NuMA were mapped at TPR17 of mPins and the C-terminal domain (CTD) of NuMA. To gain detailed structural information of Pins/NuMA interaction, we firstly turned to NMR-based techniques. However, the homogeneity of NuMA CTD is poor although it folds well, and the behavior was not improved in the presence of Pins TPR region. So it is not amendable for NMR-based structural analysis. Then we tried our best to get crystals of Pins/NuMA complexes. After extensive screening of protein boundaries and buffer conditions, we finally got high quality crystal structure of Pins/NuMA complex with diffraction of 2.3 A. The mPins TPR repeats adopts canonical TPR folds, each containing two antiparallelα-helices. Tandem arrays of TPR motif generate a right-handed helical structure with an amphipathic channel for NuMA peptide binding. The complex structure was further confirmed by point mutagenesis.In the fifth chapter, we discuss the relationship between two complexes (mPins/mInsc, mPins/NuMA). The two complexes both localize at the apical cortex, and are bridged by mPins. It is known that mInsc is recruited to the apical cortex by binding to Par complex, and the apical localization of Pins depends on Insc. Apical localized Pins further recruits NuMA which associates with microtubules through dynein/dynactin complex to the apical region. To test the hypothesis that Pins, Insc and NuMA may form a ternary complex, we incubated the three proteins together and found that NuMA and Insc cannot simultaneously bind to mPins, and the presence of mInsc blocks the binding between NuMA and mPins.In the last chapter, a summary was made and a prospect was presented.