Final Report Summary - CDC42 AND GLIOMA (Specific functions of individual Cdc42 and polarity protein variants in cellular processes and glioblastoma progression)
The cellular cytoskeleton is involved in numerous basic functions of cells such as the uptake of nutrition and signaling molecules via endocytosis or the polarization and locomotion of cells, which is for instance crucial for neural precursor cells to reach their proper destination in the developing brain. The protein Cdc42, a member of the so called RhoGTPase family of proteins, is a key regulator of the cytoskeleton and thereby of the aforementioned processes. Cdc42 exists in two variants; one is ubiquitously expressed and also called placental isoform, while the other variant, the so called brain isoform is restricted to brain tissue cells. Most studies have focused on the former and specific functions of the brain-restricted variant remain largely obscure. Our study therefore aimed at characterizing the functional differences between the two Cdc42 variants and especially at describing new functions of the poorly studied brain isoform in order to understand its role in brain tissue cells. In addition, recent observations from Etienne-Manneville’s lab uncovered altered expression levels of Cdc42 isoforms in brain tumors. Our study therefore also aimed at understanding the potential impact of these alterations on the behavior of cells that may turn into tumor cells.
In our project we studied two brain tissue cell types, astrocytes and neural precursor cells, which can give raise to brain tumors when mutating into cancerogenous cells. In order to characterize the specific functions of each Cdc42 isoform we studied the behavior of these cells upon depletion of either Cdc42 variant using the RNAi technique. We thereby found that only the ubiquitously expressed “placental” Cdc42 variant controls the directed movement of astrocytes to their final destination in response to wounding or to the presence of chemoattractants. Chemoattractants are molecules that guide the pathway of cells through the tissue. In this process the placental isoform seems to be required to maintain a polarized structure of the cells towards their direction of locomotion. In neural precursor cells however, the ubiquitously expressed, but also the brain restricted isoform was required for the directed migration towards a gradient of chemoattractants. In further experiments, we unraveled that the brain isoform is the major regulator of endocytosis. We therefore hypothesize that in neural precursor cells the brain restricted Cdc42 variant might support the migration of these cells towards a chemoattractant gradient by contributing its role in endocytosis. In this process endocytosis may support the processing of chemoattractants. In further experiments we demonstrated that the attachment of specific lipid tails is necessary for the two Cdc42 variants to fulfil there functions in cells, thereby identifying a molecular basis for the functional differences of the two variants. In addition, we identified the protein RhoGDI3 as a new regulator for the brain restricted variant. We were able to demonstrate that RhoGDI3 negatively regulates the activity of the brain isoform as RNAi-mediated depletion of RhoGDI3 was found to enhance processes controlled by the brain variant including endocytosis and neural precursor migration.
In conclusion, we were able to contribute specific functions to each of the two Cdc42 isoforms and especially characterized novel functions for the brain restricted variant, whose role in the brain had been poorly studied in the past. With RhoGDI3 we additionally identified a novel regulator controlling the specific functions of the brain isoform. Our study thus has a strong impact on the understanding of molecular mechanisms contributing to basic functions in brain tissue cells such as cellular locomotion. Cellular locomotion plays a role during fundamental processes as for instance during brain development, when neural precursor cells move through the tissue to get to their final destination where they differentiate into mature neurons. Furthermore, defects in directed cellular movement can contribute to the development and invasion of tumors, as cells that do not follow their normal pathways during migration may infiltrate new tissues. Our characterization of the role of the ubiquitously expressed “placental” Cdc42 isoform in astrocyte and neural precursor locomotion and the additional role of the brain isoform in the latter will thus impact on future studies aiming at the understanding of the molecular causes of brain tumor development and invasiveness.
In our project we studied two brain tissue cell types, astrocytes and neural precursor cells, which can give raise to brain tumors when mutating into cancerogenous cells. In order to characterize the specific functions of each Cdc42 isoform we studied the behavior of these cells upon depletion of either Cdc42 variant using the RNAi technique. We thereby found that only the ubiquitously expressed “placental” Cdc42 variant controls the directed movement of astrocytes to their final destination in response to wounding or to the presence of chemoattractants. Chemoattractants are molecules that guide the pathway of cells through the tissue. In this process the placental isoform seems to be required to maintain a polarized structure of the cells towards their direction of locomotion. In neural precursor cells however, the ubiquitously expressed, but also the brain restricted isoform was required for the directed migration towards a gradient of chemoattractants. In further experiments, we unraveled that the brain isoform is the major regulator of endocytosis. We therefore hypothesize that in neural precursor cells the brain restricted Cdc42 variant might support the migration of these cells towards a chemoattractant gradient by contributing its role in endocytosis. In this process endocytosis may support the processing of chemoattractants. In further experiments we demonstrated that the attachment of specific lipid tails is necessary for the two Cdc42 variants to fulfil there functions in cells, thereby identifying a molecular basis for the functional differences of the two variants. In addition, we identified the protein RhoGDI3 as a new regulator for the brain restricted variant. We were able to demonstrate that RhoGDI3 negatively regulates the activity of the brain isoform as RNAi-mediated depletion of RhoGDI3 was found to enhance processes controlled by the brain variant including endocytosis and neural precursor migration.
In conclusion, we were able to contribute specific functions to each of the two Cdc42 isoforms and especially characterized novel functions for the brain restricted variant, whose role in the brain had been poorly studied in the past. With RhoGDI3 we additionally identified a novel regulator controlling the specific functions of the brain isoform. Our study thus has a strong impact on the understanding of molecular mechanisms contributing to basic functions in brain tissue cells such as cellular locomotion. Cellular locomotion plays a role during fundamental processes as for instance during brain development, when neural precursor cells move through the tissue to get to their final destination where they differentiate into mature neurons. Furthermore, defects in directed cellular movement can contribute to the development and invasion of tumors, as cells that do not follow their normal pathways during migration may infiltrate new tissues. Our characterization of the role of the ubiquitously expressed “placental” Cdc42 isoform in astrocyte and neural precursor locomotion and the additional role of the brain isoform in the latter will thus impact on future studies aiming at the understanding of the molecular causes of brain tumor development and invasiveness.