The roles of cyclin-dependent kinase 5 in dendrite and synapse development

Since the isolation of cyclin-dependent kinase 5 (Cdk5), this proline-directed serine/threonine kinase has been demonstrated as an important regulator of neuronal migration, neuronal survival and synaptic functions. Recently, a number of players implicated in dendrite and synapse development have been identified as Cdk5 substrates. Neurite extension, synapse and spine maturation are all modulated by a myriad of extracellular guidance cues or trophic factors. Cdk5 was recently demonstrated to regulate signaling downstream of some of these extracellular factors, in addition to modulating Rho GTPase activity, which regulates cytoskeletal dynamics. In this communication, we summarize our existing knowledge on the pathways and mechanisms through which Cdk5 affects dendrite, synapse and spine development.     

Role of the Wnt and GTPase pathways in breast cancer tumorigenesis and treatment

Breast cancer (BC) is the most frequently diagnosed cancer among women in all the populations of the world. Although the BC mortality rate has declined, resistance to treatment is still a significant challenge for patient survival. Various cellular signaling pathways, such as Wnt and Rho/GTPase have been linked to the development, migration, and metastasis of BC, and also in treatment resistance mechanisms. Some studies have shown an association between two important cellular pathways, Wnt and Rho/GTPase, in cytoskeleton activation and cancer invasion. However, their involvement in BC has received little attention. This review summarizes the Wnt and Rho/GTPases signaling pathway functions, and also the crosstalk between these pathways in the progression, metastasis, and drug resistance mechanisms in BC. Considering the signaling pathways involved in BC tumorigenesis, future studies will need to investigate possible molecular interventions and new opportunities for the development of personalized strategies for BC treatment in order to improve overall outcomes.     

RhoGDIβ affects HeLa cell spindle orientation following UVC irradiation

The molecular signals that regulate mitotic spindle orientation to determine the proper division axis play a critical role in the development and maintenance of tissue homeostasis. However, deregulation of signaling events can result in spindle misorientation, which in turn can trigger developmental defects and cancer progression. Little is known about the cellular signaling pathway involved in the misorientation of proliferating cells that evade apoptosis after DNA damage. In this study, we found that perturbations to spindle orientation were induced in ultraviolet C (UVC)-irradiated surviving cells. N-terminal truncated Rho GDP-dissociation inhibitor β (RhoGDIβ), which is produced by UVC irradiation, distorted the spindle orientation of HeLa cells cultured on Matrigel. The short hairpin RNA-mediated knockdown of RhoGDIβ significantly attenuated UVC-induced misorientation. Subsequent expression of wild-type RhoGDIβ, but not a noncleavable mutant, RhoGDIβ (D19A), again led to a relative increase in spindle misorientation in response to UVC. Our findings revealed that RhoGDIβ impacts spindle orientation in response to DNA damage.     

RhoJ Regulates α5β1 Integrin Trafficking to Control Fibronectin Remodeling during Angiogenesis

1. Download : Download high-res image (177KB)
2. Download : Download full-size image

     

Nucleotide binding to the G12V-mutant of Cdc42 investigated by X-ray diffraction and fluorescence spectroscopy: two different nucleotide states in one crystal

The 2.5 A crystal structure of the full length human placental isoform of the Gly12 to Val mutant Cdc42 protein (Cdc42(G12V)) bound to both GDP/Mg2+ and GDPNH2 (guanosine-5'-diphospho-beta-amidate) is reported. The crystal contains two molecules in the asymmetric unit, of which one has bound GDP/Mg2+, while the other has bound GDPNH2 without a Mg2+ ion. Crystallization of the protein was induced via hydrolysis of the Cdc42 x GppNHp complex by the presence of contaminating alkaline phosphatase activity in combination with the crystallization conditions. This prompted us to compare the binding characteristics of GDPNH2 vs. GDP. The amino group of GDPNH2 drastically reduces the affinity to Cdc42 in comparison with that of GDP, causes the loss of the Mg2+ ion, and apparently also increases the conformational flexibility of the protein as seen in the crystal. Both the switch I and switch II regions are visible in the electron density of the GDP-bound molecule, but not in the molecule bound to GDPNH2. The C-terminus containing the CaaX-motif is partly ordered in both molecules due to an intramolecular disulfide bond formed between Cys105/Cys188 and Cys305/Cys388, respectively.     

Evolutionarily conserved modules in actin nucleation: lessons from Dictyostelium discoideum and plants

Summary. The actin cytoskeleton plays a central part in the dynamic organization of eukaryotic cell structure. Nucleation of actin filaments is a crucial step in the establishment of new cytoskeletal structures or modification of existing ones, providing abundant targets for regulatory processes. A substantial part of our understanding of actin nucleation derives from studies on yeast and metazoan cells. However, recent advances in structural and functional genome analysis in less traditional models, such as plants or Dictyostelium discoideum, provide an emerging picture of an evolutionarily conserved core of at least two actin nucleation mechanisms, one mediated by the Arp2/3 complex and the other one by the formin-based module. A considerable degree of conservation is found also in the systems controlling the balance between filamentous and globular actin (profilin, actin-depolymerizing factor/cofilin) and even in certain regulatory aspects, such as the involvement of Rho-related small GTPases. Identification of such conserved elements provides a prerequisite for the characterization of evolutionarily variable aspects of actin regulation which may be responsible for the rich morphological diversity of eukaryotic cells.Correspondence and reprints: Department of Plant Physiology, Faculty of Sciences, Charles University, Vininá 5, 128 44 Praha 2, Czech Republic.     

Hsp42 is the general small heat shock protein in the cytosol of Saccharomyces cerevisiae

Small heat shock proteins (sHsps) are ubiquitous molecular chaperones that prevent the unspecific aggregation of proteins. So far, Hsp26 was the only unambiguously identified member of the sHsp family in Saccharomyces cerevisiae. We show here that the sHsp system in the cytosol of S. cerevisiae consists of two proteins, Hsp26 and Hsp42. Hsp42 forms large dynamic oligomers with a barrel-like structure. In contrast to Hsp26, which functions predominantly at heat shock temperatures, Hsp42 is active as a chaperone under all conditions tested in vivo and in vitro. Under heat shock conditions, both Hsp42 and Hsp26 suppress the aggregation of one-third of the cytosolic proteins. This subset is about 90% overlapping for Hsp42 and Hsp26. The sHsp substrates belong to different biochemical pathways. This indicates a general protective function of sHsps for proteome stability in S. cerevisiae. Consistent with this observation, sHsp knockout strains show phenotypical defects. Taken together, our results define Hsp42 as an important player for protein homeostasis at physiological and under stress conditions.     

Small GTPases in vesicle trafficking

Plant small GTPases belonging to the Rop, Arf, and Rab families are regulators of vesicle trafficking. Rop GTPases regulate actin dynamics and modulate H(2)O(2) production in polar cell growth and pathogen defence. A candidate Rop GDP to Rop GTP exchange factor (RopGEF) SPIKE1 is involved in the morphogenesis of leaf epidermal cells. The ArfGEF GNOM regulates the endosomal recycling of the PIN proteins, which are involved in polar auxin transport. Intracellular localisation of small GTPases and functional studies using dominant mutant versions of Arf and Rab GTPases are defining novel plant-specific membrane compartments, especially those that participate in endosomal vesicle trafficking.     

RhoA/ROCK regulation of neuritogenesis via profilin IIa-mediated control of actin stability

Neuritogenesis, the first step of neuronal differentiation, takes place as nascent neurites bud from the immediate postmitotic neuronal soma. Little is known about the mechanisms underlying the dramatic morphological changes that characterize this event. Here, we show that RhoA activity plays a decisive role during neuritogenesis of cultured hippocampal neurons by recruiting and activating its specific kinase ROCK, which, in turn, complexes with profilin IIa. We establish that this previously uncharacterized brain-specific actin-binding protein controls neurite sprouting by modifying actin stability, a function regulated by ROCK-mediated phosphorylation. Furthermore, we determine that this novel cascade is switched on or off by physiological stimuli. We propose that RhoA/ROCK/PIIa-mediated regulation of actin stability, shown to be essential for neuritogenesis, may constitute a central mechanism throughout neuronal differentiation.     

ROCK-generated contractility regulates breast epithelial cell differentiation in response to the physical properties of a three-dimensional collagen matrix

Breast epithelial cells differentiate into tubules when cultured in floating three-dimensional (3D) collagen gels, but not when the cells are cultured in the same collagen matrix that is attached to the culture dish. These observations suggest that the biophysical properties of collagenous matrices regulate epithelial differentiation, but the mechanism by which this occurs is unknown. Tubulogenesis required the contraction of floating collagen gels through Rho and ROCK-mediated contractility. ROCK-mediated contractility diminished Rho activity in a floating 3D collagen gel, and corresponded to a loss of FAK phosphorylated at Y397 localized to 3D matrix adhesions. Increasing the density of floating 3D collagen gels also disrupted tubulogenesis, promoted FAK phosphorylation, and sustained high Rho activity. These data demonstrate the novel finding that breast epithelial cells sense the rigidity or density of their environment via ROCK-mediated contractility and a subsequent down-regulation of Rho and FAK function, which is necessary for breast epithelial tubulogenesis to occur.