Regulation of cell traction force by DAP-kinase

中文摘要 收縮力在許多重要的生理功能上，都扮演著不可或缺的角色；像是胚胎發育、血管新生和傷口癒合都依賴適當的組織收縮而成。就細胞層級而言，收縮力的大小對細胞移動、分裂和存活也是十分重要的。當正常細胞面對新環境時，它必須要先貼附然後重組骨架否則就會慢慢凋亡。細胞收縮力的產生與細胞骨架重組及細胞表面受器的訊息傳遞息息相關，這些表面受器包括了integrin、cadherins、selectins和其它附著性分子。 本實驗採用顯微鏡追蹤法，偵測並相對性的比較細胞收縮力的大小，而且更進一步用該方法來探討細胞調控本身收縮力之相關分子機制。我們以死亡訊息相關分子( DAP-kinase)為主要對象，研究其與細胞收縮力之間的關係，進一步証實該分子能夠透過調節integrin的活性來改變細胞的收縮力。 死亡訊息相關分子( DAP-kinase/DAPK)是一種促進細胞凋亡的絲胺酸/酥胺酸蛋白激酶，分子量約有160 kDa，其活性受到鈣調節分子(calmodulin)的調控。當細胞移動時，部份DAPK會聚在細胞的前緣，調控細胞的收縮及運動。過量表現DAP-kinase會使細胞產生典型的凋亡特微，像是貼附變差、外表變圓、細胞膜變成不規則的泡泡狀，以及自噬小體的產生。DAPK包括數個不同功能的蛋白單元，像是激酶單位、鈣調節分子作用單位、骨架連結單元以及蛋白未端的凋亡訊息單元。將DAPK的鈣調節分子作用單元移除可以產生具持續催化能力的變種(ΔCaM)，如果將它的激酶單位第42個胺基酸由K置換為A時(K42A)，則反而會導致其失去催化的能力。先前的研究顯示出過量表現ΔCaM時會抑制integrin的活性，而我們的研究結果則顯示在細胞大量表現ΔCaM會明顯的降低其收縮力，相反的，大量表現K42A時則會升高。我們更進一步研究，當添加活化integrin的抗體的或是大量表現它下游的活化性分子時，都能夠逆轉ΔCaM使收縮力變弱的情形。總結以上，我們的研究結果清楚的証明了DAPK可以透過integrin來調節細胞的收縮力表現。Abstract Traction force is important in various fundamental processes, for instance, embryonic morphogenesis, angiogenesis and wound healing. At cellular level, traction force also has a great influence on cell migration, proliferation and survival. When normal cells are placed a new environment, either they adhere well and reassemble cytoskeleton, or they move toward cell death. Generation of traction forces is involved cytoskeleton rearrangement and cell surface receptors like integrin, cadherins, selectins and other cell adhesion molecules with their signaling in all these cellular events. In our research we use traction force microscopy to detect relative amount of forces exerted by cells, and study how the signaling event can influence the contraction. Here we demonstrated that death-associated protein kinase (DAP-kinase/DAPK) actually alters the cell traction force via regulating integrin activity. DAPK is a 160 kDa calcium/calmodulin-dependant Ser/Thr protein kinase that promotes cell death. It appears to be located within the cell interior and regulate cell shape and movement. Overpression of DAP-kinase leads to cell death including membrane blabbing, cell rounding and the formation of autophagic vesicles. DAP-kinase contains multiple functional domains, such as kinase domain, calmodulin regulatory domain, eight ankyrin repeats, cytoskeleton binding domain and a C-terminal death domain. The deletion of calmodulin regulatory domain (ΔCaM) offers DAP-kinase a constitutively active ability. When replacing lysine with alanine in kinase domain (K42A), this technique makes a kinase inactive mutant. Previous studies of these DAP-kinase mutants showed that the cells expressing ΔCaM mutant downregulate integrin activity. In our results, overexpression of ΔCaM mutant significantly reduced the cell traction forces. On the contrary, overexpression of kinase dead mutant (K42A) had also led the increase on traction force. Moreover, to enforce activation of integrin both with activating antibody or with downstream elements, i.e., overexpression of FAK or talin F3 domain, associated downstream signals activated by integrin, both can reverse the effect. Therefore, our results clearly demonstrated that DAPK can regulate cellular traction forces from suppressing integrin activityContents CONTENTS 1 FIGURE INDEX 2 ABSTRACT IN CHINESE 3 ABSTRACT IN ENGLISH 5 Chapter I. INTRODUCTION 1.1 Introduce fo traction forces generation and detection methods 7 Traction forces generation and transmission 7 Forces measurement studies 8 1.2 Role of integrin in adhesion and signaling mechanism 16 Bidirectional signaling mechanism 17 Focal adhesions and FAK 19 Talin 21 1.3 DAP-kinase 26 Functions of each domain 27 Chapter II. MATERIALS AND METHODS 2.1 Cells and cell culture 30 2.2 Tration force microscopy 31 2.3 Transient transfection into NIH 3T3 cells 37 Chapter III. RESULTS 3.1 DAP-kinase induces reduction of traction forces in NIH 3T3 cells 38 3.2 Activation of integrins can reverse the DAP-kinase-induced effect on cell traction forces 40 3.3 Overexpressing CD2-FAK partially rescue the decrease of cell traction forces induced by DAP-kinase 41 3.4 The talin FERM 3 (F3) domain is able to restore the traction forces reduced by DAP- kinase 43 3.5 Preliminary study on the DAP kinase regulation site on integrins by using GD25 beta1- null cells 45 Chapter IV. DISCUSSION 60 Chapter V. REFERENCES 7