Biology

Introduction and significance
The article of Yang et al (2008) described the results gathered from their investigation on the possible role of the mammalian Cdc42 protein in the development of filopodia, migration of cells and the cell cycle.  The investigation was triggered by the accumulation of reports claiming that Cdc42 plays a major role in the normal physiology of a cell.  It should be understood that Cdc42 was initially examined in lower eukaryotes such as Saccharomyces cerevisiae, as well as Caenorhabditis elegans, using dominant negative expression.  These earlier reports showed results that suggested that Cdc42 induced the progression of the cell cycle, as well as other cell signaling pathways.  On the other hand, recent studies on Cdc42 in mammalian cells showed the opposite, wherein the capacity of cells for proliferation remained the same after direct knockout of the gene coding for the Cdc42 protein.  Another recent study also described that Cdc42 was responsible for the development of filopodia in embryonic stem cell-derived fibroblastoid cells.  Given the discrepancies in cellular function of Cdc42, Yang et al (2008) thus opted to clarify the issue of the function of mammalian Cdc42 protein.  The results of their investigation would provide a better understanding of the nature of the Cdc42 protein, as it is highly likely that earlier studies using lower eukaryotes were limited in terms of experimental conditions.  The information that would be generated in their investigation would serve as foundation for future functional studies on the Cdc42 protein.  More importantly, the identification of the function of the mammalian Cdc42 protein would determine whether this macromolecule would be a good target protein for future therapeutic approaches in controlling cell proliferation and cell migration, both of which are hallmarks of cancer.

Methods and results
Conditional knockout mice of Cdc42-- and Cdc42GAP-- were generated from Cdc42floxflow homozygous mice.  The loxPCre technique was employed to detect the Cdc42 gene in embryonic fibroblasts of the conditional knockout mice.  Briefly, Cre recombinase was employed to remove the first 28 amino acids of the Cdc42 polypeptide, of which were encoded by the second exon.  PCR was performed for each loxPCre experiment to verify that exon 2 was indeed absent in the PCR product.  The intact DNA segment was expected to be 762 base pairs in length while the exon 2-excised DNA segment would only be 176 base pairs in length.

In order to produce Cdc42, as well as CDC42-- genotypes, the mouse embryonic fibroblasts were introduced to retrovirus-expressing Cre recombinaseEGFP.  Using the various genotypes of Cdc42, functional assays were then performed to determine the actual effect of endogenous or exogenous Cdc42 during cell culture.  This experiment showed that both Cdc42 and Cdc42 floxflox cultures were capable of expressing the endogenous protein, similar to that of Cdc42GAP and Cdc42GAP--.  On the other hand, Cdc42--, as well as Cdc42--Cdc42 cultures did not show any endogenous Cdc42.  In addition, the Cdc42--Cdc42 cultured cells were capable of producing endogenous Cdc42, similar to that of wild-type cells.

In order to determine the interaction of Cdc42 with Rac1 and RhoA proteins, the effector-domain binding assay was employed.  This experiment showed that even in the absence of Cdc42 in knockout embryonic fibroblast cells, the level of RhoA expression remained the same.  On the other hand, the absence of Cdc42 in cells resulted in a two-fold decrease in the level of expression of Rac1 protein.  Such observation suggested that Rac1 may be directly interacting with Cdc42 and that the RhoA protein is working independent of Cdc42.

In order to determine the effect of Cdc42 in the morphology of mouse embryonic fibroblasts, the phalloidin protein was localized in cells using immunostaining techniques.  The deletion of the Cdc42 gene in these cells resulted in irregular cellular shapes, mainly characterized by protrusions.  On the hand, Cdc42GAP-- cells were observed to have extended arms within the cells, also known as filopodia.  Despite the absence of the Cdc42 gene, the embryonic fibroblasts maintained their capacity to form stress fibers.

In order to determine the association of Cdc42 and cell migration, cells from the knockout mice were plated and monitored at different time points.  The experiment showed that cell spreading occurred by the first hour of plating and the cell extensions appeared by the fifth hour of plating.  In addition, cell adhesion was also observed in the knockout cells at this time point.  In order to determine the role of Cdc42 on cell growth, each genotype was assayed for cell proliferation at different time points.  The knockout cells showed poor or defective growth, as compared to that of wild type cells.  The role of Cdc42 in cell signaling was examined using phosphorylation assays of ERK and JNK.  The experiment showed that the phosphorylation of JNK was highly dependent on the presence of Cdc42.

The results of all experiments described in the research article are convincing, as the details presented in the paper were straightforward and comprehensive.  The appropriate controls were also included in each experimental setup, which further strengthens the study.  The images presented in the article are also very clear and convincing.  Since the report was highly experimental, there was no need for any statistical tests to check for robustness of data.

Discussion
The investigation on the function of Cdc42 in mammalian embryonic fibroblasts indicates that the Cdc42 protein is an essential protein for migration, filopodia production, cell signaling and proliferation.  Such report is timely because it provides a direct examination of an issue that could have been left uncovered despite the conflicting reports of earlier studies.  In addition, the report is important because it provides additional information on the Cdc42 protein.  One important fact that has prompted the investigators to perform the study was that most of the publications on Cdc42 focused on lower vertebrates.  Their report would thus be an essential addition to the short list of research articles that describe the results of functional analyses of the Cdc42 protein in mammalian cells.  The information that has been generated in this study will pave the way for future research efforts on Cdc42.  This protein may serve as an interesting target protein that may be used for molecular therapeutics of specific medical conditions associated with highly proliferating and migrating cells, both of which are hallmarks of cancer.  It is also very interesting to find that the investigators also attempted to replenish each particular cellular function by reintroducing the Cdc42 protein to Cdc42-deficient cells.  Such attempts would also facilitate in the design of future therapeutic treatments for specific medical conditions that remain untreatable to this date.