MAPK uncouples cell cycle progression from cell spreading and cytoskeletal organization in cycling cells.

Integrin-mediated cytoskeletal tension supports growth-factor-induced proliferation, and disruption of the actin cytoskeleton in growth factor-stimulated cells prevents the re-expression of cyclin D and cell cycle re-entry from quiescence. In contrast to cells that enter the cell cycle from G0, cycling cells continuously express cyclin D, and are subject to major cell shape changes during the cell cycle. Here, we investigated the cell cycle requirements for cytoskeletal tension and cell spreading in cycling mammalian cells that enter G1-phase from mitosis. Disruption of the actin cytoskeleton at progressive time-points in G1-phase induced cell rounding, FA disassembly, and attenuated both integrin signaling and growth factor-induced p44/p42 mitogen-activated protein kinase activation. Although cyclin D expression was reduced, the expression of cyclin A and entry into S-phase were not affected. Moreover, expression of cyclin B1, progression through G2- and M-phase, and commitment to a new cell cycle occurred normally. In contrast, cell cycle progression was strongly prevented by inhibition of MAPK activity in G1-phase, whereas cell spreading, cytoskeletal organization, and integrin signaling were not impaired. MAPK inhibition also prevented cytoskeleton-independent cell cycle progression. Thus, these results uncouple the requirements for cell spreading and cytoskeletal organization from MAPK signaling, and show that cycling mammalian cells can proliferate independently of actin stress fibers, focal adhesions, or cell spreading, as long as a threshold level of MAPK activity is sustained.

Inhibition of protein kinase B activity induces cell cycle arrest and apoptosis during early G1 phase in CHO cells.

Inhibition of PKB (protein kinase B) activity using a highly selective PKB inhibitor resulted in inhibition of cell cycle progression only if cells were in early G1 phase at the time of addition of the inhibitor, as demonstrated by time-lapse cinematography. Addition of the inhibitor during mitosis up to 2 h after mitosis resulted in arrest of the cells in early G1 phase, as deduced from the expression of cyclins D and A and incorporation of thymidine. After 24 h of cell cycle arrest, cells expressed the cleaved caspase-3, a central mediator of apoptosis. These results demonstrate that PKB activity in early G1 phase is required to prevent the induction of apoptosis. Using antibodies, it was demonstrated that active PKB translocates to the nucleus during early G1 phase, while an even distribution of PKB was observed through cytoplasm and nucleus during the end of G1 phase.

Attachment of HeLa cells during early G1 phase.

Both growth factor directed and integrin dependent signal transduction were shown to take place directly after completion of mitosis. The local activation of these signal transduction cascades was investigated in early G1 cells. Interestingly, various key signal transduction proteins were found in blebs at the cell membrane within 30 min after mitosis. These membrane blebs appeared in round, mitotic-like cells and disappeared rapidly during spreading of the cells in G1 phase. In addition to tyrosine-phosphorylated proteins, the blebs contained also phosphorylated FAK and phosphorylated MAP kinase. The formation of membrane blebs in round, mitotic cells before cell spreading is not specific for mitotic cells, because similar features were observed in trypsinized cells. Just before cell spreading also these cells exhibited membrane blebs containing active signal transduction proteins. Inhibition of signal transduction did not affect membrane bleb formation, suggesting that the membrane blebs were formed independent of signal transduction.

Novel role of cPLA(2)alpha in membrane and actin dynamics.

Actin-directed processes such as membrane ruffling and cell migration are regulated by specific signal transduction pathways that become activated by growth factor receptors. The same signaling pathways that lead to modifications in actin dynamics also activate cPLA(2)alpha. Moreover, arachidonic acid, the product of cPLA(2)alpha activity, is involved in regulation of actin dynamics. Therefore, it was investigated whether cPLA(2)alpha plays a role in actin dynamics, more specifically during growth factor-induced membrane ruffling and cell migration. Upon stimulation of ruffling and cell migration by growth factors, endogenous cPLA(2)alpha and its active phosphorylated form were shown to relocate at protrusions of the cell membrane involved in actin and membrane dynamics. Inhibition of cPLA(2)alpha activity with specific inhibitors blocked growth factor-induced membrane and actin dynamics, suggesting an important role for cPLA(2)alpha in these processes.

The ROS-NOX connection in cancer and angiogenesis.

Initially viewed as dangerous byproducts of aerobic life, reactive oxygen species (ROS) nowadays appear to be essential secondary messengers of many signaling cascades and cellular functions. The establishment of ROS as important signaling molecules has been confirmed by the existence of specialized ROS producing complexes expressed in nonphagocytic cells, the NADPH oxidase complex (NOX). Because of the diversity of their proteic targets (besides lipids and DNA), ROS have multiple and sometimes contradictory functions. In the present review, we focus on several different signaling pathways influenced by ROS and NOX in tumorigenesis, focusing on proliferation and angiogenesis. We review the ROS targets regulating proliferation, including cellular signaling (phosphatases, AP1, and nuclear factor-kappa B [NF-kappaB]) and cell cycle targets (CDC25, cyclin D, and forkhead proteins), and the role of NOX during proliferation. Finally, we review the direct and indirect involvement of ROS and NOX in (tumor) angiogenesis through the regulation of different biologic systems such as vascular endothelial growth factor, angiotensin II, hypoxia-inducible factor, AP1, and inflammation.

Collaborative Learning in Higher Education: Evoking Positive Interdependence.

Collaborative learning is a widely used instructional method, but the learning potential of this instructional method is often underused in practice. Therefore, the importance of various factors underlying effective collaborative learning should be determined. In the current study, five different life sciences undergraduate courses with successful collaborative-learning results were selected. This study focuses on factors that increased the effectiveness of collaboration in these courses, according to the students. Nine focus group interviews were conducted and analyzed. Results show that factors evoking effective collaboration were student autonomy and self-regulatory behavior, combined with a challenging, open, and complex group task that required the students to create something new and original. The design factors of these courses fostered a sense of responsibility and of shared ownership of both the collaborative process and the end product of the group assignment. In addition, students reported the absence of any free riders in these group assignments. Interestingly, it was observed that students seemed to value their sense of achievement, their learning processes, and the products they were working on more than their grades. It is concluded that collaborative learning in higher education should be designed using challenging and relevant tasks that build shared ownership with students.

Activation of cytosolic phospholipase A2 in Her14 fibroblasts by hydrogen peroxide: a p42/44(MAPK)-dependent and phosphorylation-independent mechanism.

Reactive oxygen species (ROS) have been implicated in the pathogenesis of diseases as well as various normal cellular processes. It has been suggested that ROS function as mediators of signal transduction, given that they can mimic growth factor-induced signaling. The ROS H2O2 has been reported to activate phospholipase A2 (PLA2) and, therefore, we investigated if and through which pathway ROS activate cytosolic PLA2 (cPLA2) in Her14 fibroblasts. cPLA2 was activated concentration-dependently by H2O2 in a transient manner. In addition, the lipophilic cumene hydroperoxide was shown to induce cPLA2 activity in the same manner. H2O2-induced cPLA2 activity in Her14 cells was partially phosphorylation-dependent, which was mediated through the Raf-MEK-p42/44(MAPK) pathway and occurred partially through a phosphorylation-independent mechanism. ROS can lead to changes in the (micro) viscosity of membranes due to the presence oxidized lipids, thereby increasing the substrate availability for cPLA2. In support of this, treatment of Her14 cells with H2O2 induced lipid peroxidation time-dependently as determined from degradation of lipid arachidonate and linoleate and the formation of aldehydic degradation products. Furthermore, H2O2 induced translocation of cPLA2 to the membrane fraction in a calcium-independent fashion, with a concomitant increase in cPLA2 activity. Collectively, the results suggest that oxidative stress-induced cPLA2 activity is partially phosphorylation-dependent and is further increased due to increased substrate availability by the action of ROS on membranes.

Signal transduction and actin in the regulation of G1-phase progression.

Regulation of cell proliferation is dependent on the integration of signal transduction systems that are activated by external signal molecules, such as growth factors and extracellular matrix components. Dependent on these signal transduction networks, the cells decide in the G1 phase to continue proliferation or, alternatively, to stop cell-cycle progression and undergo apoptosis, differentiation, or quiescence. The MAP kinase and PI-3 kinase pathways have been demonstrated to play an essential role in these G1-phase decisions. Interestingly, actin has been demonstrated to mutually interfere with signal transduction. In addition, it has been indicated that the FOXO transcription factors are involved in these decisions, as well. Actin has been demonstrated to play an important role in the regulation of G1-phase progression. Because of its properties as a structural protein, actin is essential in cytokinesis and in cell spreading and, thus, is involved in G1-phase progression. As an intermediate factor in signal transduction, actin is likely to be involved in cell-cycle regulation induced by external signal molecules. And, finally, actin has been demonstrated to play a direct role in transcription. These observations indicate a prominent role of actin in the regulation of G1-phase progression.

Role of signal transduction and actin in G1 phase progression.

Progression through the cell cycle of mammalian cells is dependent upon external factors such as growth- and ECM factors. These factors exert their effect predominantly during the G1 phase of the cell cycle. When cells are cultured in suspension or when growth factors are withdrawn from the medium, cells will stop cell cycle progression and enter a quiescent state. Cells will remain in this quiescent state until extracellular conditions change and cells are stimulated to re-enter the cell cycle. This stimulation is mediated by various signal transduction cascades such as the mitogen-activated protein kinase (MAPK) pathway and the phosphatidylinositol 3-kinase (PI3-kinase) pathway. In Chinese hamster ovary cells at least two serum-dependent points exist during G1 phase that lead to diffent cellular responses. The first point is located immediately after mitosis and is suggested to link with apoptosis. The second point is located in late G1 phase and probably corresponds with cellular differentiation. Signal transduction is mutually related to the cytoskeleton, especially the actin microfilament system. The actin microfilament system influences signal transduction and several signal transduction pathways influence the actin structure. Here we describe the role of the MAPK and PI3-kinase activities and of actin microfilaments in progression through the cell cycle and their role in the two G1 checkpoints.

Specific production rate of VHH antibody fragments by Saccharomyces cerevisiae is correlated with growth rate, independent of nutrient limitation.

Saccharomyces cerevisiae carrying a multicopy integrated expression vector containing the gene encoding a Llama antibody fragment, has been cultivated in continuous cultures both under carbon and nitrogen limiting conditions with galactose as the sole carbon source. VHH-R2 expression was under control of the inducible GAL7 promoter. Induction however, was independent of the galactose consumption rate and maximal at all growth rates. VHH-R2 was secreted with 70% efficiency at all growth rates and under both limitations. The specific production rate increased linear with increasing growth rate in a growth-associated manner. However, when grown under nitrogen limitation at growth rates above 0.09 h(-1), the extracellular VHH-R2 was less active or part of the VHH-R2 was in an inactive form. From our results we conclude that to obtain a maximal amount of VHH per kilogram biomass per hour, VHH production should be done in carbon limited continuous cultures at high specific growth rates.

Transient Intervals of Hyper-Gravity Enhance Endothelial Barrier Integrity: Impact of Mechanical and Gravitational Forces Measured Electrically.

BACKGROUND: Endothelial cells (EC) guard vascular functions by forming a dynamic barrier throughout the vascular system that sensitively adapts to 'classical' biomechanical forces, such as fluid shear stress and hydrostatic pressure. Alterations in gravitational forces might similarly affect EC integrity, but remain insufficiently studied. METHODS: In an unique approach, we utilized Electric Cell-substrate Impedance Sensing (ECIS) in the gravity-simulators at the European Space Agency (ESA) to study dynamic responses of human EC to simulated micro- and hyper-gravity as well as to classical forces. RESULTS: Short intervals of micro- or hyper-gravity evoked distinct endothelial responses. Stimulated micro-gravity led to decreased endothelial barrier integrity, whereas hyper-gravity caused sustained barrier enhancement by rapid improvement of cell-cell integrity, evidenced by a significant junctional accumulation of VE-cadherin (p = 0.011), significant enforcement of peripheral F-actin (p = 0.008) and accompanied by a slower enhancement of cell-matrix interactions. The hyper-gravity triggered EC responses were force dependent and nitric-oxide (NO) mediated showing a maximal resistance increase of 29.2±4.8 ohms at 2g and 60.9±6.2 ohms at 4g vs. baseline values that was significantly suppressed by NO blockage (p = 0.011). CONCLUSION: In conclusion, short-term application of hyper-gravity caused a sustained improvement of endothelial barrier integrity, whereas simulated micro-gravity weakened the endothelium. In clear contrast, classical forces of shear stress and hydrostatic pressure induced either short-lived or no changes to the EC barrier. Here, ECIS has proven a powerful tool to characterize subtle and distinct EC gravity-responses due to its high temporal resolution, wherefore ECIS has a great potential for the study of gravity-responses such as in real space flights providing quantitative assessment of a variety of cell biological characteristics of any adherent growing cell type in an automated and continuous fashion.

Molecular events associated with reactive oxygen species and cell cycle progression in mammalian cells.

Cell cycle progression is regulated by a wide variety of external factors, amongst them are growth factors and extracellular matrix factors. During the last decades evidence has been obtained that reactive oxygen species (ROS) may also play an important role in cell cycle progression. ROS may be generated by external and internal factors. In this overview we describe briefly the generation of ROS and their effects on processes that have been demonstrated to play an essential role in cell cycle progression, including such systems as signal transduction cascades, protein ubiquitination and degradation, and the cytoskeleton. These different effects of ROS influence cell cycle progression dependent upon the amount and duration of ROS exposure. Activation of growth factor stimulated signaling cascades by low levels of ROS result in increased cell cycle progression, or, in case of prolonged exposure, to a differentiation like growth arrest. From many studies it seems clear that the cyclin kinase inhibitor protein p21 plays a prominent role, leading to cell cycle arrest at higher but not directly lethal levels of ROS. Dependent upon the nature of p21 induction, the cell cycle arrest may be transient, coupled to repair processes, or permanent. At high concentrations of ROS all of the above processes are activated, in combination with enhanced damage to the building blocks of the cell, leading to apoptosis or even necrosis.

Causes and Consequences of Age-Related Changes in DNA Methylation: A Role for ROS?

Recent genome-wide analysis of C-phosphate-G (CpG) sites has shown that the DNA methylome changes with increasing age, giving rise to genome-wide hypomethylation with site­specific incidences of hypermethylation. This notion has received a lot of attention, as it potentially explains why aged organisms generally have a higher risk of age-related diseases. However, very little is known about the mechanisms that could cause the occurrence of these changes. Moreover, there does not appear to be a clear link between popular theories of aging and alterations in the methylome. Some of the most fruitful of these theories attribute an important role to reactive oxygen species, which seem to be responsible for an increase in oxidative damage to macromolecules, such as DNA, during the lifetime of an organism. In this review, the connection between changes in DNA methylation and these reactive oxygen species is discussed, as well as the effect of these changes on health. Deeper insights into the nature, causes and consequences of the aging methylome might provide a deeper understanding of the molecular mechanisms of aging and eventually contribute to the development of new diagnostic and therapeutic tools.

Ground-based facilities for simulation of microgravity: organism-specific recommendations for their use, and recommended terminology.

Research in microgravity is indispensable to disclose the impact of gravity on biological processes and organisms. However, research in the near-Earth orbit is severely constrained by the limited number of flight opportunities. Ground-based simulators of microgravity are valuable tools for preparing spaceflight experiments, but they also facilitate stand-alone studies and thus provide additional and cost-efficient platforms for gravitational research. The various microgravity simulators that are frequently used by gravitational biologists are based on different physical principles. This comparative study gives an overview of the most frequently used microgravity simulators and demonstrates their individual capacities and limitations. The range of applicability of the various ground-based microgravity simulators for biological specimens was carefully evaluated by using organisms that have been studied extensively under the conditions of real microgravity in space. In addition, current heterogeneous terminology is discussed critically, and recommendations are given for appropriate selection of adequate simulators and consistent use of nomenclature.

The influence of reactive oxygen species on cell cycle progression in mammalian cells.

Cell cycle regulation is performed by cyclins and cyclin dependent kinases (CDKs). Recently, it has become clear that reactive oxygen species (ROS) influence the presence and activity of these enzymes and thereby control cell cycle progression. In this review, we first describe the discovery of enzymes specialized in ROS production: the NADPH oxidase (NOX) complexes. This discovery led to the recognition of ROS as essential players in many cellular processes, including cell cycle progression. ROS influence cell cycle progression in a context-dependent manner via phosphorylation and ubiquitination of CDKs and cell cycle regulatory molecules. We show that ROS often regulate ubiquitination via intermediate phosphorylation and that phosphorylation is thus the major regulatory mechanism influenced by ROS. In addition, ROS have recently been shown to be able to activate growth factor receptors. We will illustrate the diverse roles of ROS as mediators in cell cycle regulation by incorporating phosphorylation, ubiquitination and receptor activation in a model of cell cycle regulation involving EGF-receptor activation. We conclude that ROS can no longer be ignored when studying cell cycle progression.

Kinetics of PKCε activating and inhibiting llama single chain antibodies and their effect on PKCε translocation in HeLa cells.

Dysregulation of PKCε is involved in several serious diseases such as cancer, type II diabetes and Alzheimer's disease. Therefore, specific activators and inhibitors of PKCε hold promise as future therapeutics, in addition to being useful in research into PKCε regulated pathways. We have previously described llama single chain antibodies (VHHs) that specifically activate (A10, C1 and D1) or inhibit (E6 and G8) human recombinant PKCε. Here we report a thorough kinetic analysis of these VHHs. The inhibiting VHHs act as non-competitive inhibitors of PKCε activity, whereas the activating VHHs have several different modes of action, either increasing V(max) and/or decreasing K(m) values. We also show that the binding of the VHHs to PKCε is conformation-dependent, rendering the determination of affinities difficult. Apparent affinities are in the micromolar range based on surface plasmon resonance studies. Furthermore, the VHHs have no effect on the activity of rat PKCε nor can they bind the rat form of the protein in immunoprecipitation studies despite the 98% identity between the human and rat PKCε proteins. Finally, we show for the first time that the VHHs can influence PKCε function also in cells, since an activating VHH increases the rate of PKCε translocation in response to PMA in HeLa cells, whereas an inhibiting VHH slows down the translocation. These results give insight into the mechanisms of PKCε activity modulation and highlight the importance of protein conformation on VHH binding.

An undergraduate course to bridge the gap between textbooks and scientific research.

This article reports on a one-semester Advanced Cell Biology course that endeavors to bridge the gap between gaining basic textbook knowledge about cell biology and learning to think and work as a researcher. The key elements of this course are 1) learning to work with primary articles in order to get acquainted with the field of choice, to learn scientific reasoning, and to identify gaps in our current knowledge that represent opportunities for further research; 2) formulating a research project with fellow students; 3) gaining thorough knowledge of relevant methodology and technologies used within the field of cell biology; 4) developing cooperation and leadership skills; and 5) presenting and defending research projects before a jury of experts. The course activities were student centered and focused on designing a genuine research program. Our 5-yr experience with this course demonstrates that 1) undergraduate students are capable of delivering high-quality research designs that meet professional standards, and 2) the authenticity of the learning environment in this course strongly engages students to become self-directed and critical thinkers. We hope to provide colleagues with an example of a course that encourages and stimulates students to develop essential research thinking skills.

The development of activating and inhibiting camelid VHH domains against human protein kinase C epsilon.

The 10 isozymes of the protein kinase C (PKC) family can have different roles on the same biological process, making isozyme specific analysis of function crucial. Currently, only few pharmacological compounds with moderate isozyme specific effects exist thus hampering research into individual PKC isozymes. The antigen binding regions of camelid single chain antibodies (VHHs) could provide a solution for obtaining PKC isozyme specific modulators. In the present study, we have successfully selected and characterized PKCɛ specific VHH antibodies from two immune VHH libraries using phage display. The VHHs were shown to exclusively bind to PKCɛ in ELISA and immunoprecipitation studies. Strikingly, five of the VHHs had an effect on PKCɛ kinase activity in vitro. VHHs A10, C1 and D1 increased PKCɛ kinase activity in a concentration-dependent manner (EC(50) values: 212-310nM), whereas E6 and G8 inhibited PKCɛ activity (IC(50) values: 103-233nM). None of these VHHs had an effect on the activity of the other novel PKC isozymes PKCδ and PKCθ. To our knowledge, these antibodies are the first described VHH activators and inhibitors for a protein kinase. Furthermore, the development of PKCɛ specific modulators is an important contribution to PKC research.