Growth arrest of PPP2R5C and PPP2R5D double knockout mice indicates a genetic interaction and conserved function for these PP2A B subunits.

Protein phosphatase 2A (PP2A) is a heterotrimeric phosphatase that controls a wide range of cellular functions. The catalytic activity and intracellular location of PP2A are modulated by its association with regulatory B subunits, including B56 proteins, which are encoded by five separate genes in humans and mice. The specific effects of each B56 protein on PP2A activity and function are largely unknown. As part of an effort to identify specific PP2A-B56 functions, we created knockout strains of B56ß, B56δ, and B56ε using CRISPR/Cas9n. We found that none of the individual B56 genes are essential for mouse survival. However, mice that have both B56δ and B56γ inactivated (B56δγ-), arrest fetal development around Day E12. The hearts of B56δγ- mice have a single outflow vessel rather than having both an aorta and a pulmonary artery. Thus, there appears to be strong genetic interaction between B56δ and B56γ, and together they are necessary for heart development. Of note, both these proteins have been shown to localize to the nucleus and have the most related peptide sequences of the B56 family members. Our results suggest there are B56 subfamilies, which work in conjunction to regulate specific PP2A functions.

Evaluation of the differentiation status of neural stem cells based on cell morphology and the expression of Notch and Sox2.

Neural stem cells (NSCs) isolated from a variety of sources are being developed as cellular therapies aimed at treating neurodegenerative diseases. During NSC culture and expansion it is important the cells do not differentiate prematurely because this may have an unfavorable effect on product quality and yield. In our study, we evaluated the use of Notch and Sox2 as markers for undifferentiated human and mouse NSCs. The expression of Notch2 and Sox2 during extensive-passage, low-oxygen culture and differentiation conditions were analyzed to confirm that the presence of these signature proteins directly correlates with the ability of NSCs to form new neurospheres and differentiate into multiple cell types. Using expression of Notch1, Notch2 and Sox2 as a reference, we then used flow cytometry to identify a specific morphological profile for undifferentiated murine and human NSCs. Our studies show that Notch and Sox2 expression, along with flow cytometry analysis, can be used to monitor the differentiation status of NSCs grown in culture for use in cellular therapies.

Live Cell Imaging of Chromosome Segregation During Mitosis.

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56γ regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

PP2A-B56γ is required for an efficient spindle assembly checkpoint.

The Spindle Assembly Checkpoint (SAC) is part of a complex feedback system designed to ensure that cells do not proceed through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. The formation of the kinetochore complex and the implementation of the SAC are regulated by multiple kinases and phosphatases. BubR1 is a phosphoprotein that is part of the Cdc20 containing mitotic checkpoint complex that inhibits the APC/C so that Cyclin B1 and Securin are not degraded, thus preventing cells going into anaphase. In this study, we found that PP2A in association with its B56γ regulatory subunit, are needed for the stability of BubR1 during nocodazole induced cell cycle arrest. In primary cells that lack B56γ, BubR1 is prematurely degraded and the cells proceed through mitosis. The reduced SAC efficiency results in cells with abnormal chromosomal segregation, a hallmark of transformed cells. Previous studies on PP2A's role in the SAC and kinetochore formation were done using siRNAs to all 5 of the B56 family members. In our study we show that inactivation of only the PP2A-B56γ subunit can affect the efficiency of the SAC. We also provide data that show the intracellular locations of the B56 subunits varies between family members, which is consistent with the hypothesis that they are not completely functionally redundant.

The protein phosphatase 2A B56γ regulatory subunit is required for heart development.

BACKGROUND: Protein Phosphatase 2A (PP2A) function is controlled by regulatory subunits that modulate the activity of the catalytic subunit and direct the PP2A complex to specific intracellular locations. To study PP2A's role in signal transduction pathways that control growth and differentiation in vivo, a transgenic mouse lacking the B56γ regulatory subunit of PP2A was made. RESULTS: Lack of PP2A activity specific to the PP2A-B56γ holoenzyme, resulted in the formation of an incomplete ventricular septum and a decrease in the number of ventricular cardiomyocytes. During cardiac development, B56γ is expressed in the nucleus of α-actinin-positive cardiomyocytes that contain Z-bands. The pattern of B56γ expression correlated with the cardiomyocyte apoptosis we observed in B56γ-deficient mice during mid to late gestation. In addition to the cardiac phenotypes, mice lacking B56γ have a decrease in locomotive coordination and gripping strength, indicating that B56γ has a role in controlling PP2A activity required for efficient neuromuscular function. CONCLUSIONS: PP2A-B56γ activity is required for efficient cardiomyocyte maturation and survival. The PP2A B56γ regulatory subunit controls PP2A substrate specificity in vivo in a manner that cannot be fully compensated for by other B56 subunits.

Generation of mice that conditionally express the activation domain of Notch2.

The Notch pathway is an intercellular signaling mechanism frequently used for controlling cell fate during organogenesis. There are four structurally related Notch receptors in mice and humans, and Notch1 and Notch2 are essential genes. In this report we describe the construction of a transgenic mouse strain that expresses the Notch2 intracellular domain in response to cell lineage specific expression of Cre recombinase. This approach bypasses the requirement for ligand- receptor interaction and allows the direct determination of the consequences of Notch2 activation in vivo. Exogenous expression of the Notch2 intracellular domain resulted in the developmental arrest of secondary heart field derived cardiomyocytes during the transition from immature alpha-Smooth Muscle Actin expressing cells to mature alpha-Actinin positive cardiomyocytes. In contrast, a cell nonautonomous mesenchymal expansion was observed in semilunar valves. This new conditionally expressed allele of Notch2 can be used in studies by investigators interested in the effects of Notch2 activation in vivo.

Notch2 is required for the proliferation of cardiac neural crest-derived smooth muscle cells.

Mutations in Notch receptors and their ligands have been identified as the cause of human congenital heart diseases, indicating the importance of the Notch signaling pathway during heart development. In our study, we use Cre-Lox technology to inactivate Notch2 in several cardiac cell lineages to determine the functional requirements for Notch2 during mammalian heart development. Inactivation of Notch2 in cardiac neural crest cells resulted in abnormally narrow aortas and pulmonary arteries due to a decrease in smooth muscle tissue. The reduction in smooth muscle tissue was not due to cell migration defects but instead was found to be caused by less proliferation in smooth muscle cells during mid to late gestation. Our findings demonstrate that Notch2 is required cell autonomously for proper formation of the heart outflow tract and provides insights into the role of Notch2 in vascular smooth muscle development and the cardiovascular defects associated with Alagille syndrome.

Differential activation of kinases in ex vivo and in vivo irradiated mice lymphocytes.

Various kinases, such as tyrosine, protein kinase C (PKC) and MAP kinase, play important role in the cellular response to radiation, but little is known about the specific response in the whole animal. Most studies, except a few, are based on single cells. There is a paucity of data where signaling following whole body irradiation is concerned. In this study a comparison has been made between the activities of these kinases following ex vivo and in vivo irradiation. Tyrosine kinase activity showed no difference in the lymphocytes irradiated ex vivo or in vivo. A significant differential dose-dependent response could be observed in PKC activity. PKC was seen to be activated at the higher dose, i.e., 1 Gy in, in vivo irradiated lymphocytes, whereas in ex vivo irradiated lymphocytes, PKC was seen to be activated at the lower dose, i.e., 0.1 Gy. MAP kinase activity was seen to decrease with an increasing dose in ex vivo irradiated lymphocytes. In vivo MAP kinase activity was seen to increase as the dose increased, with maximum activation at 3 Gy. These kinases are being used to manipulate the tumor response to radiotherapy. Thus it is essential to study the behavior of the above kinases in the whole animal because the difference in response of a single cell to the whole animal may be different.

Dose-dependent differential expression of protein kinase C isozymes in mouse lymphocytes after gamma irradiation in vivo and ex vivo.

Protein kinase C (PKC, now known as Prkc) plays an important role in the response of cells to radiation, but little is known about the specific response of each isozyme in the radiation-induced response of cells in whole animals. However, most studies are based on single cells. There is a paucity of data on signaling after whole-body irradiation. In this study, a comparison has been made between the expression of Prkc isozymes after in vivo and ex vivo irradiation. There was a significant difference in the dose response of the isozymes. In animals in which lymphocytes were irradiated ex vivo, the expression of the Prkca isozyme was found to be maximum at 3 Gy, while in vivo irradiation did not increase the expression beyond that of 1 Gy. Prkcd was marginally activated after 0.1 Gy ex vivo irradiation, whereas there was significant activation of expression after in vivo irradiation with 3 Gy. The response of Prkcz was found to be similar to that of Prkcd. Prkc is a crucial enzyme that is being used to manipulate the response of tumors to radiotherapy. Conventional radiotherapy is delivered at low doses, and hence only those isozymes that are activated at these doses should be taken into consideration. Moreover, the differences between the response of a single cell and that of the whole animal must be considered.