Hepatocyte polarity establishment and apical lumen formation are organized by Par3, Cdc42, and aPKC in conjunction with Lgl.

Hepatocytes differ from columnar epithelial cells by their multipolar organization, which follows the initial formation of central lumen-sharing clusters of polarized cells as observed during liver development and regeneration. The molecular mechanism for hepatocyte polarity establishment, however, has been comparatively less studied than those for other epithelial cell types. Here, we show that the tight junction protein Par3 organizes hepatocyte polarization via cooperating with the small GTPase Cdc42 to target atypical protein kinase C (aPKC) to a cortical site near the center of cell-cell contacts. In 3D Matrigel culture of human hepatocytic HepG2 cells, which mimics a process of liver development and regeneration, depletion of Par3, Cdc42, or aPKC results in an impaired establishment of apicobasolateral polarity and a loss of subsequent apical lumen formation. The aPKC activity is also required for bile canalicular (apical) elongation in mouse primary hepatocytes. The lateral membrane-associated proteins Lgl1 and Lgl2, major substrates of aPKC, seem to be dispensable for hepatocyte polarity establishment because Lgl-depleted HepG2 cells are able to form a single apical lumen in 3D culture. On the other hand, Lgl depletion leads to lateral invasion of aPKC, and overexpression of Lgl1 or Lgl2 prevents apical lumen formation, indicating that they maintain proper lateral integrity. Thus, hepatocyte polarity establishment and apical lumen formation are organized by Par3, Cdc42, and aPKC; Par3 cooperates with Cdc42 to recruit aPKC, which plays a crucial role in apical membrane development and regulation of the lateral maintainer Lgl.

SKA1 regulates actin cytoskeleton remodelling via activating Cdc42 and influences the migration of pancreatic ductal adenocarcinoma cells.

OBJECTIVES: Spindle and kinetochore-associated protein 1(SKA1), originally identified as a protein essential for proper chromosome segregation, has been recently linked to multiple malignancies. This study aimed to explore the biological, clinical role and molecular mechanism of SKA1 in pancreatic carcinogenesis. MATERIALS AND METHODS: SKA1 expression was detected in 145 pancreatic ductal adenocarcinoma (PDAC) specimens by immunohistochemistry. Biological behaviour assays were used to determine the role of SKA1 in PDAC progression in vitro and in vivo. Using isobaric tags for relative and absolute quantitation (iTRAQ), SKA1's downstream proteins were examined. Moreover, cytochalasin B and ZCL278 were used to explore the changes of SKA1-induced signalling and cell morphology, with further confirmation by immunoblotting and immunofluorescence assays. RESULTS: Increased SKA1 expression was significantly correlated with tumour size and cellular differentiation degree in PDAC tissues. Furthermore, elevated levels of SKA1 reflected shorter overall survival (P = .019). As for biological behaviour, SKA1 acted as a tumour promotor in PDAC, overexpression of SKA1 facilitates cell proliferation, migration and invasion in vitro and in vivo. Mechanistically, we demonstrated that SKA1 enhanced pancreatic cancer aggressiveness by inhibiting G2/M arrest and regulating actin cytoskeleton organization via activating Cdc42. CONCLUSIONS: This study revealed novel roles for SKA1 as an important regulator of actin cytoskeleton organization and an oncogene in PDAC cells, which may provide insights into developing novel therapeutics.

miR-29a/b/c function as invasion suppressors for gliomas by targeting CDC42 and predict the prognosis of patients.

BACKGROUND: The lethality and poor outcome of high-grade gliomas result from the tumour relentless invasion. miR-29a/b/c downexpressions contribute to several human tumourigenesis. However, their relevance to prognosis and invasion in gliomas remains unclear. METHODS: Relationships of miR-29a/b/c and CDC42 expressions to grade and survival-time in 147 human gliomas were analysed by in situ hybridisation and immunohistochemistry. Dual-luciferase reporter assay was used to identify CDC42 as a target of miR-29a/b/c. Underlining mechanisms by which miR-29a/b/c inhibited glioma cell migration and invasion were studied by in vitro and in vivo assays. RESULTS: miR-29a/b/c expressions were inversely correlated with glioma grades, but positively correlated with patients' survival. Two distinct subgroups of grade I-IV glioma patients with different prognoses were identified according to miR-29a/b/c expressions. miR-29a/b/c overexpressions suppressed glioma cell migration and invasion through targeting CDC42 and subsequently decreasing phosphorylated PAK1/2/3, LIMK1/2 and cofilin, the pivotal downstream effectors of CDC42. Moreover, CDC42 expression was positively correlated with glioma grades, but inversely correlated with miR-29a/b/c expressions and patients' survival. In glioblastoma cell lines, CDC42-knockdown could mimic the anti-tumour effects of miR-29a/b/c. CONCLUSIONS: miR-29a/b/c are important tumour suppressors and novel prognostic biomarkers of gliomas, and miR-29a/b/c and CDC42 are potential therapeutic candidates for malignant gliomas.

Probing Cdc42 Polarization Dynamics in Budding Yeast Using a Biosensor.

Cdc42 is a small guanosine triphosphatase (GTPase) that plays a central role in polarity development in diverse cell types. Since the activity of Cdc42 is dynamically controlled in time and space, it is required to develop a biosensor to monitor its activation in vivo. In this chapter, we describe the construction and usage of a simple and robust biosensor for monitoring active Cdc42 in budding yeast. This affinity-based biosensor uses a red fluorescent protein fused to a Cdc42- and Rac-interactive binding motif from one of the Cdc42 effector proteins. Because it binds specifically to the GTP-bound Cdc42, this biosensor can be used to monitor Cdc42 activation in vivo. This or similar biosensors can be widely used for studying GTPase signaling in other cell types because of the conserved CRIB motif present among GTPase targets.

Aerobic exercise regulates Rho/cofilin pathways to rescue synaptic loss in aged rats.

PURPOSE: The role of exercise to prevent or reverse aging-induced cognitive decline has been widely reported. This neuroprotection is associated with changes in the synaptic structure plasticity. However, the mechanisms of exercise-induced synaptic plasticity in the aging brain are still unclear. Thus, the aim of the present study is to investigate the aging-related alterations of Rho-GTPase and the modulatory influences of exercise training. METHODS: Young and old rats were used in this study. Old rats were subjected to different schedules of aerobic exercise (12 m/min, 60 min/d, 3d/w or 5d/w) or kept sedentary for 12 w. After 12 w of aerobic exercise, the synapse density in the cortex and hippocampus was detected with immunofluorescent staining using synaptophysin as a marker. The total protein levels of RhoA, Rac1, Cdc42 and cofilin in the cortex and hippocampus were detected with Western Blot. The activities of RhoA, Rac1 and Cdc42 were determined using a pull down assay. RESULTS: We found that synapse loss occurred in aging rats. However, the change of expression and activity of RhoA, Rac1 and Cdc42 was different in the cortex and hippocampus. In the cortex, the expression and activity of Rac1 and Cdc42 was greatly increased with aging, whereas there were no changes in the expression and activity of RhoA. In the hippocampus, the expression and activity of Rac1 and Cdc42 was greatly decreased and there were no changes in the expression and activity of RhoA. As a major downstream substrate of the Rho GTPase family, the increased expression of cofilin was only observed in the cortex. High frequency exercise ameliorated all aging-related changes in the cortex and hippocampus. CONCLUSIONS: These data suggest that aerobic exercise reverses synapse loss in the cortex and hippocampus in aging rats, which might be related to the regulation of Rho GTPases.

FLIM FRET Visualization of Cdc42 Activation by Netrin-1 in Embryonic Spinal Commissural Neuron Growth Cones.

Netrin-1 is an essential extracellular chemoattractant that signals through its receptor DCC to guide commissural axon extension in the embryonic spinal cord. DCC directs the organization of F-actin in growth cones by activating an intracellular protein complex that includes the Rho GTPase Cdc42, a critical regulator of cell polarity and directional migration. To address the spatial distribution of signaling events downstream of netrin-1, we expressed the FRET biosensor Raichu-Cdc42 in cultured embryonic rat spinal commissural neurons. Using FLIM-FRET imaging we detected rapid activation of Cdc42 in neuronal growth cones following application of netrin-1. Investigating the signaling mechanisms that control Cdc42 activation by netrin-1, we demonstrate that netrin-1 rapidly enriches DCC at the leading edge of commissural neuron growth cones and that netrin-1 induced activation of Cdc42 in the growth cone is blocked by inhibiting src family kinase signaling. These findings reveal the activation of Cdc42 in embryonic spinal commissural axon growth cones and support the conclusion that src family kinase activation downstream of DCC is required for Cdc42 activation by netrin-1.

Ratiometric Imaging Using a Single Dye Enables Simultaneous Visualization of Rac1 and Cdc42 Activation.

Biosensors that report endogenous protein activity in vivo can be based on environment-sensing fluorescent dyes. The dyes can be attached to reagents that bind selectively to a specific conformation of the targeted protein, such that binding leads to a fluorescence change. Dyes that are sufficiently bright for use at low, nonperturbing intracellular concentrations typically undergo changes in intensity rather than the shifts in excitation or emission maxima that would enable precise quantitation through ratiometric imaging. We report here mero199, an environment-sensing dye that undergoes a 33 nm solvent-dependent shift in excitation. The dye was used to generate a ratiometric biosensor of Cdc42 (CRIB199) without the need for additional fluorophores. CRIB199 was used in the same cell with a FRET sensor of Rac1 activation to simultaneously observe Cdc42 and Rac1 activity in cellular protrusions, indicating that Rac1 but not Cdc42 activity was reduced during tail retraction, and specific protrusions had reduced Cdc42 activity. A novel program (EdgeProps) used to correlate localized activation with cell edge dynamics indicated that Rac1 was specifically reduced during retraction.

GTPases Rho distribution in intraepithelial and invasive neoplasias of the uterine cervix.

PURPOSE OF INVESTIGATION: To evaluate the distribution of GTPases RhoA, RhoB, and Cdc42 in cervical intraepithelial neoplasias (CIN) and invasive neoplasias of the uterine cervix. MATERIALS AND METHODS: samples of neoplastic lesions of the uterine cervix of 44 patients were classified in: CIN I (n = 10), CIN II (n = 10), CIN III (n = 09), and invasive carcinoma (n = 15). Antibodies anti-RhoA, anti-RhoB, and anti-Cdc42 were used and staining was classified as: negative, mild, moderate, and intense positive. RESULTS: When compared with dysplastic cells, superficial cells showed: higher expression of RhoB in CIN I (p = 0.0018), and lower expression of Cdc42 in CIN I (p = 0.0225). The authors observed higher expression of RhoA (p = 0.0002) and RhoB (p = 0.0046) in CIN dysplastic cells when compared with invasive carcinoma cells. CONCLUSIONS: GTPases Rho may be involved with the regulation of biological processes, important to the progression of cervical neoplasias. Probably, RhoA is important for maintenance of cell differentiation and RhoB protects cells from malignant cervical neoplasia.

A new genetically encoded single-chain biosensor for Cdc42 based on FRET, useful for live-cell imaging.

Cdc42 is critical in a myriad of cellular morphogenic processes, requiring precisely regulated activation dynamics to affect specific cellular events. To facilitate direct observations of Cdc42 activation in live cells, we developed and validated a new biosensor of Cdc42 activation. The biosensor is genetically encoded, of single-chain design and capable of correctly localizing to membrane compartments as well as interacting with its upstream regulators including the guanine nucleotide dissociation inhibitor. We characterized this new biosensor in motile mouse embryonic fibroblasts and observed robust activation dynamics at leading edge protrusions, similar to those previously observed for endogenous Cdc42 using the organic dye-based biosensor system. We then extended our validations and observations of Cdc42 activity to macrophages, and show that this new biosensor is able to detect differential activation patterns during phagocytosis and cytokine stimulation. Furthermore, we observe for the first time, a highly transient and localized activation of Cdc42 during podosome formation in macrophages, which was previously hypothesized but never directly visualized.