**5.2.1 Cell polarization and lamellipodium extension at the leading edge**

An asymmetrical organization of intracellular activities is required for a cell to move, that means the molecular processes at the leading and trailing edges of a moving cell must be different. Establishing and maintaining cell polarity in response to extracellular stimuli appear to be mediated by Rho family GTPases.

Cdc42 is well accepted as a master regulator of cell polarity in eukaryotic organisms ranging from yeast to human.Cdc42 was first studied in a budding yeast model for its involvement in cell polarity. During the cell cycle, yeast cells adopt alternative states of growth to nonfocused isotropic growth. In the absence of Cdc42, *Saccharomyces cerevisiae* fail to establish focused apical growth and, cells expand isotropically (Pruyne and Bretscher 2000). Cdc42 regulates cell polarity by deciding the location of lamellipodia formation (Srinivasan, Wang et al. 2003). In addition, Cdc42 directs the localization of the microtubule-organizing center

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ROCKs and MLCK have been suggested to act in concert to regulate different aspects of cell contractility, since ROCK appears to be required for MLC phosphorylation which are associated with actin filaments in the cell body, and MLCK is required at the cell periphery

Tail detachment occurs when cell-substrate linkages are preferentially disrupted at the rear of a migrating cell, while the leading edge remains attached to the ECM and continues to elongate (Palecek, Huttenlocher et al. 1998). Mechanisms underlying the focal complex disassembly and tail detachment depend on the type of cell and strength of adhesion to the extracellular matrix at the trailing edge (Wear, Schafer et al. 2000). In slow moving cells, tail detachment depend on the action of a calcium-dependent, non-lysosomal cysteine protease calpain that cleaves focal complex components like talin and cytoplasmic tail of β1 and β3 integrins along the trailing edge (Potter, Tirnauer et al. 1998). Strong tension forces exerted across the cells at the rear adhesions is required to break the physical link between integrin and the actin cytoskeleton. Rho and myosin II are involved in this event. Furthermore, Rho plays important roles in reducing adhesion and promoting tail detachment in fibroblasts,

which have relatively large focal adhesion complexes (Cox and Huttenlocher 1998).

A number of studies have suggested that Rho family GTPases are involved in the regulation of nuclear signaling. Rac and Cdc42, but not Rho, have been demonstrated to regulate the activation of JNK and reactivate kinase p38RK in certain cell types (Seger and Krebs 1995). Expression of constitutively active forms of Rac and Cdc42 in HeLa, NIH-3T3, and Cos cells stimulates JNK and p38 activity (Coso, Chiariello et al. 1995). Furthermore, these same effects were observed with oncogenic GEFs for these Rho proteins (Minden, Lin et al. 1995). However in human kidney 293 T cells, Cdc42 and the Rho protein, but not Rac, induces the activation of JNK (Teramoto, Crespo et al. 1996). Upon activation, JNKs and p38 translocate to the nucleus where they phosphorylate transcription factors, including c-Jun, ATF2, and Elk (Derijard, Hibi et al. 1994; Gille, Strahl et al. 1995). Further, Rac has been shown to activate PEA3, a member of the Ets family, in a JNK-dependent manner (O'Hagan, Tozer et al. 1996). Activated p38 phosphorylates ATF2, Elk, Max, and the cAMP response element

PAKs are the farthest known upstream kinases that connect Rho GTPases to JNK and p38 through GTP-dependent bindings to Rac and Cdc42 *in vitro* and are activated after binding to activated Rac and Cdc42. (Manser, Chong et al. 1995). In addition, certain constitutively active forms of PAK can activate JNK and p38 (Zhang, Han et al. 1995). Further, a mutant effector of Rac that cannot bind to PAK remains a potent JNK activator (Westwick, Lambert et al. 1997). These observations suggest that other kinases, in addition to PAK, participate in the signalling from Rho GTPases to JNK. Supporting this, MLK3 and MEKK4 are found to be regulated by Cdc42 and Rac, and selectively activate the JNK pathway (Gerwins, Blank et al. 1997). It has also been reported that Cdc42/Rac can bind to MLK3 both *in vitro* and *in vivo* and that the coexpression of activated Cdc42/Rac mutants elevates the enzymatic activity of MLK3 in Cos-7 cells (Teramoto, Coso et al. 1996; Gerwins, Blank et al. 1997). In addition, Rho, Rac and Cdc42 stimulate the activation of the serum responsive factor (SRF) (Hill, Wynne et al. 1995). SRF forms a complex with TCF/Elk proteins to stimulate transcription

(Totsukawa, Yamakita et al. 2000).

**5.2.5 Adhesion disassembly and tail detachment** 

**5.3 Rho GTPases and transcriptional activation** 

binding protein.

(MTOC) and Golgi apparatus to the front of the nucleus, oriented toward the direction of movement. MTOC orientation at the leading edge then facilitates the delivery of Golgi derived vesicles to the leading edge and microtubule growth into the lamellipodium (Rodriguez, Schaefer et al. 2003). It has been further studied that Cdc42 exerts its effect on MTOC through its downstream effector, PAK1 (Li, Hannigan et al. 2003).
