Cell–Cell Adhesion Prevents Mutant Cells Lacking Myosin II from Penetrating Aggregation Streams ofDictyostelium

AbstractWhen a small number of fluorescently labeled myosin II mutant cells (mhcA−) are mixed with wild-type cells and development of the chimeras is observed by confocal microscopy, the mutant cells are localized to the edges of aggregation streams and mounds. Moreover, the mutant cells stick to wild-type cells and become distorted (Shelden and Knecht, 1995). Two independent adhesion mechanisms, Contact Sites A and Contact Sites B, function during the aggregation stage and either one or both might be responsible for excluding the myosin II null cells. We have mixedmhcA−cells with cells in which the appearance of Contact Sites B is delayed (strain TL72) as well as cells which lack Contact Sites A (strain GT10) and double mutants in which both adhesion mechanisms are affected (strain TL73). In all chimeras, themhcA−cells were distorted by interactions with the adhesion mutant cells, indicating that it does not require significant adhesive interaction to distort the flaccid cortex ofmhcA−cells.mhcA−cells were excluded from streams composed of cells lacking either Contact Sites A or Contact Sites B but mixed randomly with cells lacking both adhesion systems. By 10 hr of development, cells of strain TL73 acquire Contact Sites B adhesion. If cells of this strain were mixed with labeledmhcA−cells, allowed to develop for 9 hr, and then dissociated before replating, the myosin II null cells were seen to be distorted and excluded from the reaggregates. Thus the exclusion ofmhcA−cells from streams can be accomplished by either Contact Sites A or B. When chimeras of labeled TL73 and wild-type cells were made, the TL73 cells were found to be randomly mixed into aggregation streams. This result indicates that adhesive sorting does not function during aggregation and so cannot account for the exclusion ofmhcA−cells from streams. We hypothesize that the flaccid cortex ofmhcA−cells cannot generate sufficient protrusive force to break the contacts between adhered cells in aggregation streams but can enter streams where the cells are weakly adherent

The role that myosin II plays in altering cell shape and behavior during development in {\it Dictyostelium discoideum\/}

Dictyostelium axenic cell lines have been tested for vegetative motility and cell behavior. The data shows that different cell lines display significant differences in vegetative motility and cell behavior in a substrate-dependent manner. NC4A2 cells behave more like wild type NC4 than do other axenic cell lines. For developmental cells, the interval of cAMP pulsing decreases as development proceeds for wild-type and mhcA\sp- cells. The cAMP pulsing of mhcA\sp- cells is slower than wild type cells. When 2% of fluorescently labeled wild type cells are mixed with mhcA\sp- cells, they have a lower mean rate than when alone, which suggests that the mhcA\sp- cells interfere with wild type cells in cell movement. When 2% of labeled mhcA\sp- are mixed with cells in which one of the two adhesion mechanisms is affected, or double mutants in which both adhesion mechanisms are affected, the mhcA\sp- cells are distorted by interactions with the adhesion mutant cells. mhcA\sp- cells are excluded from streams composed of cells lacking either one of the two adhesion systems but mix randomly with cells lacking both adhesion systems, suggesting that the flaccid cortex of mhcA\sp- cells cannot generate sufficient protrusive force to break the contacts between adhered cells in streams but can enter streams where the cells are weakly adherent. Cell lines lacking either the essential (mlcE\sp-) or regulatory (mlcR\sp-) myosin light chains have been used to distinguish the two functions of myosin. Neither cells show significant actin activated ATPase activity measured by in vivo or in vitro assays. The mlcR\sp- cells are distorted and localized to the edges of wild type streams indistinguishable from mhcA\sp- cells; the mlcE\sp- cells are randomly mixed in the streams and are not distorted. The similarity of mlcR\sp- and mhcA\sp- cell behavior is consistent with the reduced amount of myosin in the cortex of mlcR\sp- cells. The behavior of the mlcE\sp- cells suggests that actin binding activity and/or normally localized filaments of myosin II in the absence of motor function is sufficient to stiffen the actin cortex in a manner similar to actin crosslinking proteins.

Defining the function of xeroderma pigmentosum group F protein in psoralen interstrand cross-link-mediated DNA repair and mutagenesis.

Many commonly used drugs, such as psoralen and cisplatin, can generate a very unique type of DNA damage, namely ICL (interstrand cross-link). An ICL can severely block DNA replication and transcription and cause programmed cell death. The molecular mechanism of repairing the ICL damage has not been well established. We have studied the role of XPF (xeroderma pigmentosum group F) protein in psoralen-induced ICL-mediated DNA repair and mutagenesis. The results obtained from our mutagenesis studies revealed a very similar mutation frequency in both human normal fibroblast cells and XPF cells. The mutation spectra generated in both cells, however, were very different: most of the mutations generated in the normal fibroblast cells were T167-->A transversions, whereas most of the mutations generated in the XPF cells were T167-->G transversions. When a wild-type XPF gene cDNA was stably transfected into the XPF cells, the T167-->A mutations were increased and the T167-->G mutations were decreased. We also determined the DNA repair capability of the XPF cells using both the host-cell reactivation and the in vitro DNA repair assays. The results obtained from the host-cell reactivation experiments revealed an effective reactivation of a luciferase reporter gene from the psoralen-damaged plasmid in the XPF cells. The results obtained from the in vitro DNA repair experiments demonstrated that the XPF nuclear extract is normal in introducing dual incisions during the nucleotide excision repair process. These results suggest that the XPF protein has important roles in the psoralen ICL-mediated DNA repair and mutagenesis

The initiative role of XPC protein in cisplatin DNA damaging treatment-mediated cell cycle regulation

XPC is an important DNA damage recognition protein involved in DNA nucleotide excision repair. We have studied the role of the XPC protein in cisplatin treatment-mediated cell cycle regulation. Through the comparison of microarray data obtained from human normal fibroblasts and two individual XPC-defective cell lines, 486 genes were identified as XPC-responsive genes in the cisplatin treatment (with a minimal 1.5-fold change) and 297 of these genes were further mapped to biological pathways and gene ontologies. The cell cycle and cell proliferation-related genes were the most affected genes by the XPC defect in the cisplatin treatment. Many other cellular function genes were also affected by the XPC defect in the treatment. Western blot hybridization results revealed that the XPC defect reduced the p53 responses to the cisplatin treatment. The ability to activate caspase-3 was also attenuated in the XPC cells with the treatment. These results suggest that the XPC protein plays a critical role in initiating the cisplatin DNA damaging treatment-mediated signal transduction process, resulting in activation of the p53 pathway and cell cycle arrest that allow DNA repair and apoptosis to take place. These results reveal an important role of the XPC protein in the cancer prevention