A Role for Protein Phosphatase 2A in the Proliferation-Quiescence Decision

Metazoans precisely control the number of cell divisions during organ or tissue development throughout their lifetime. In adult metazoans, most differentiated cells no longer proliferate and lie in a quiescent state, also termed cell cycle exit. The decision to proliferate or to lie in quiescence is essential for development and its dysregulation may lead to defects in organogenesis, wound healing and regeneration as well as tumor formation. However, at what stage of the cell cycle the proliferation-quiescence decision occurs and what molecular mechanisms control this decision remain controversial. Here my thesis work revealed a novel role for PP2A in promoting the transition to quiescence upon terminal differentiation during tissue development. Using Drosophila eyes and wings as a model, I found that compromising PP2A activity during the final cell cycle prior to a developmentally controlled cell cycle exit leads to extra cell divisions and delayed normal exit. By systematically testing the regulatory subunits of Drosophila PP2A, I discovered that the B56 family member widerborst (wdb) is required for the role of PP2A in promoting the transition to quiescence. In particular, the PP2A/B56 complex targets cyclin-dependent kinase 2 several hours after mitosis to promote entry into quiescence, indicating when the decision occurs and how PP2A impacts the decision. I also investigated the dynamic features of the proliferation-quiescence transition using time-lapse, live imaging in mammalian cell culture. By monitoring the proliferation-quiescence transition without cell synchronization, I discovered that the quiescent state is heterogeneous. Mammalian cells can enter into either a transient or a prolonged quiescent state after mitosis, prior to the next round of cell cycle even under conditions of abundant nutrients. Notably, I showed that two sister cells born of the same mitosis can make different cell cycle decisions, with one cell entering long-term quiescence while the other re-entering the cell cycle. Consistent with my work in the Drosophila model, PP2A in mammals also plays a conserved role in promoting the entry into quiescence. The novel role of PP2A in modulation of the proliferation-quiescence decision may contribute to its tumor suppressor role and impact the emerging problem of tumor dormancy.PhDMolecular, Cellular and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/133389/1/sunda_1.pd

Time-Lapse Microscopy

Time-lapse microscopy is a powerful, versatile and constantly developing tool for real-time imaging of living cells. This review outlines the advances of time-lapse microscopy and refers to the most interesting reports, thus pointing at the fact that the modern biology and medicine are entering the thrilling and promising age of molecular cinematography

Quantification of the morphological characteristics of hESC colonies

The maintenance of the undifferentiated state in human embryonic stem cells (hESCs) is critical for further application in regenerative medicine, drug testing and studies of fundamental biology. Currently, the selection of the best quality cells and colonies for propagation is typically performed by eye, in terms of the displayed morphological features, such as prominent/abundant nucleoli and a colony with a tightly packed appearance and a well-defined edge. Using image analysis and computational tools, we precisely quantify these properties using phase-contrast images of hESC colonies of different sizes (0.1–1.1 mm2) during days 2, 3 and 4 after plating. Our analyses reveal noticeable differences in their structure influenced directly by the colony area A. Large colonies (A > 0.6 mm2) have cells with smaller nuclei and a short intercellular distance when compared with small colonies (A  0.6 mm2) due to the proliferation of the cells in the bulk. This increases the colony density and the number of nearest neighbours. We also detect the self-organisation of cells in the colonies where newly divided (smallest) cells cluster together in patches, separated from larger cells at the final stages of the cell cycle. This might influence directly cell-to-cell interactions and the community effects within the colonies since the segregation induced by size differences allows the interchange of neighbours as the cells proliferate and the colony grows. Our findings are relevant to efforts to determine the quality of hESC colonies and establish colony characteristics database

Automated processing of zebrafish imaging data: a survey

Due to the relative transparency of its embryos and larvae, the zebrafish is an ideal model organism for bioimaging approaches in vertebrates. Novel microscope technologies allow the imaging of developmental processes in unprecedented detail, and they enable the use of complex image-based read-outs for high-throughput/high-content screening. Such applications can easily generate Terabytes of image data, the handling and analysis of which becomes a major bottleneck in extracting the targeted information. Here, we describe the current state of the art in computational image analysis in the zebrafish system. We discuss the challenges encountered when handling high-content image data, especially with regard to data quality, annotation, and storage. We survey methods for preprocessing image data for further analysis, and describe selected examples of automated image analysis, including the tracking of cells during embryogenesis, heartbeat detection, identification of dead embryos, recognition of tissues and anatomical landmarks, and quantification of behavioral patterns of adult fish. We review recent examples for applications using such methods, such as the comprehensive analysis of cell lineages during early development, the generation of a three-dimensional brain atlas of zebrafish larvae, and high-throughput drug screens based on movement patterns. Finally, we identify future challenges for the zebrafish image analysis community, notably those concerning the compatibility of algorithms and data formats for the assembly of modular analysis pipelines

Development and validation of computational models of cellular interaction

In this paper we take the view that computational models of biological systems should satisfy two conditions – they should be able to predict function at a systems biology level, and robust techniques of validation against biological models must be available. A modelling paradigm for developing a predictive computational model of cellular interaction is described, and methods of providing robust validation against biological models are explored, followed by a consideration of software issues

Consequences of altered eicosanoid patterns for nociceptive processing in mPGES-1-deficient mice

Cyclooxygenase-2 (COX-2)-dependent prostaglandin (PG) E2 synthesis in the spinal cord plays a major role in the development of inflammatory hyperalgesia and allodynia. Microsomal PGE2 synthase-1 (mPGES-1) isomerizes COX-2-derived PGH2 to PGE2. Here, we evaluated the effect of mPGES-1-deficiency on the noci-ceptive behavior in various models of nociception that depend on PGE2 synthesis. Surprisingly, in the COX-2-dependent zymosan-evoked hyperalgesia model, the nociceptive behavior was not reduced in mPGES-1-deficient mice despite a marked decrease of the spinal PGE2 synthesis. Similarly, the nociceptive behavior was unaltered in mPGES-1-deficient mice in the formalin test. Importantly, spinal cords and primary spinal cord cells derived from mPGES-1-deficient mice showed a redirection of the PGE2 synthesis to PGD2, PGF2α and 6-keto-PGF1α (stable metabolite of PGI2). Since the latter prostaglandins serve also as mediators of noci-ception they may compensate the loss of PGE2 synthesis in mPGES-1-deficient mice

RGS14 is a mitotic spindle protein essential from the first division of the mammalian zygote.

Heterotrimeric G protein alpha subunits, RGS proteins, and GoLoco motif proteins have been recently implicated in the control of mitotic spindle dynamics in C. elegans and D. melanogaster. Here we show that regulator of G protein signaling-14 (RGS14) is expressed by the mouse embryonic genome immediately prior to the first mitosis, where it colocalizes with the anastral mitotic apparatus of the mouse zygote. Loss of Rgs14 expression in the mouse zygote results in cytofragmentation and failure to progress to the 2-cell stage. RGS14 is found in all tissues and segregates to the nucleus in interphase and to the mitotic spindle and centrioles during mitosis. Alteration of RGS14 levels in exponentially proliferating cells leads to cell growth arrest. Our results indicate that RGS14 is one of the earliest essential product of the mammalian embryonic genome yet described and has a general role in mitosis

Role of STIL overexpression in supernumerary centriole formation, chromosomal instability and cancer development in mice

Centrosome is the major microtubule-organizing center in mammalian cell and consists of a pair of centrioles embedded in pericentriolar material. Centrosomes are important for bipolar spindle formation and correct chromosome segregation during mitosis. Disruption of normal centrosome function leads to aneuploidy and chromosome missegregation. Many cancer cells harbor supernumerary centrosomes, which were shown to correlate to chromosomal instability, clinical aggressiveness, and poor prognosis. Nevertheless, the question, whether amplified centrosomes can drive tumorigenesis in vivo remains unresolved. The process of centrosome duplication is tightly controlled by a small set of proteins including the kinase protein PLK4 and the structural centriole proteins as STIL and SAS6. Depletion of any one of these proteins’ blocks centrosome duplication and, conversely, overexpression causes centrosome amplification. In different PLK4 overexpression in vivo models, it remains arguable, whether extra centrosomes could derive tumorigenesis and whether the generated tumors might be induced by the additional serine/threonine kinase functions of PLK4 besides its role in regulating centriole duplication. On the other hand, the structural centrosome protein STIL is involved only in centriole replication without any other known functions up to now and its overexpression leads to the formation of supernumerary centrioles in tissue culture. Therefore, investigating the role of centrosome aberrations in chromosomal instability and tumor formation in vivo using STIL overexpression (STILOE) mouse model will add important information to the conflicting data generated by PLK4 overexpressing mice. Accordingly, we generated a new transgenic Cre-LoxP mouse model, B6-STIL, that overexpresses STIL when bred with a Cre-deleter line leading to STOP cassette excision. These mice were used for (i) generation and characterization of mice with ubiquitous STIL overexpression (STILOE) for the assessment of spontaneous centrosome amplification-driven tumor development; (ii) generation and characterization of mouse embryonic fibroblasts (MEFs) derived from these STILOE mice to determine STIL overexpression levels and the development of centrosome amplification, mitotic aberrations; (iii) generation and characterization of mice with tamoxifen-inducible epithelium-specific STIL overexpression (K14(CreERT2);STILOE) with and without active TP53, which were used in skin carcinogenesis assays, to determine the relative contribution of supernumerary centrosomes and chromosomal instability to chemical tumor induction and progression. Our results showed a graded overexpression of STIL mRNA and protein in early MEFs passages and tissues from heterozygous STILOE and homozygous STILOE mice, leading to significant centrosome amplification and chromosomal aberrations via aberrant mitoses. Importantly, MEFs with high levels of STIL-induced centrosome amplification showed a proliferative disadvantage with a strong selective pressure to eliminate cells overexpressing STIL by apoptosis and senescence. In line, rates of both, spontaneous tumor formation in STILOE mice and chemically induced skin tumors in K14(CreERT2);STILOE animals are largely reduced as compared to controls. Thus, centrosome amplification induced by STIL overexpression seems to inhibit tumor formation rather than enhancing it in vivo in mammals