Illuminating Cells: Mechanistic Heuristics in the Age of Fluorescence Microscopy

Many cellular processes are carried out by large macromolecular assemblies. We systematically analyzed large macromolecular assemblies in the cytoplasm of mouse macrophages (RAW264.7 cell line), cells with crucial roles in immunity and inflammation. Fractionation of the cytoplasmic fraction was performed using sucrose density gradient centrifugation, and individual fractions were subjected in parallel to (i) identification of constituent proteins by mass spectrometry and (ii) structural visualization by electron microscopy. Macromolecular assemblies present in the fractions were analyzed by integrating available data using bioinformatic approaches. We identified 368 unique proteins in our sample. Among these are components of some well-characterized assemblies involved in diverse cellular processes and structures including translation, proteolysis, protein folding, metabolism, and the cytoskeleton, as well as less characterized proteins that may correspond to additional components of known assemblies or other homo- or hetero-oligomeric structures. Single-particle analysis of electron micrographs of negatively stained samples allowed the identification of clearly distinguishable two-dimensional projections of discrete protein assemblies. Among these, we can identify small ribosomal subunits and preribosomal particles, the 26S proteasome complex and small ringlike structures resembling the molecular chaperone complexes. In addition, a broad range of discrete and different complexes were seen at size ranges between 11 to 38 nm in diameter. Our procedure selects the assemblies on the basis of abundance and ease of isolation, and therefore provides an immediately useful starting point for further study of structure and function of large assemblies. Our results will also contribute toward building a molecular cell atlas.

Structural Analysis of Supramolecular Assemblies by Cryo-Electron Tomography

The paper presents some evidential and epistemic strategies that appear frequently in old and present-day Romanian parliamentary debates. By evidential and epistemic strategy we mean the manipulation of evidential and epistemic markers, either grammaticalized or pragmatic, in order to achieve various interactional and rhetorical aims. We have in view not only encoded evidential and epistemic meanings, but also (interactional) inferentially conveyed evidential and epistemic meanings. Modality can be manipulated in various ways: the usual ranking of evidentials can be reinterpreted for argumentative means: i.e., hearsay can be sometimes rejected, while, in other cases, it can be presented as more reliable than perceptual evidence.

In recent years, theories of social understanding have moved away from arguing that just one epistemic strategy, such as theory-based inference or simulation constitutes our ability of social understanding. Empirical observations speak against any monistic view and have given rise to pluralistic accounts arguing that humans rely on a large variety of epistemic strategies in social understanding. We agree with this promising pluralist approach, but highlight two open questions: what is the residual role of mindreading, i.e. the indirect attribution of mental states to others within this framework, and how do different strategies of social understanding relate to each other? In a first step, we aim to clarify the arguments that might be considered in evaluating the role that epistemic strategies play in a pluralistic framework. On this basis, we argue that mindreading constitutes a core epiststrategy in human social life that opens new central spheres of social understanding. In a second step, we provide an account of the relation between different epistemic strategies which integrates and demarks the important role of mindreading for social understanding.

Optimizing immuno-labeling for correlative fluorescence and electron microscopy on a single specimen

Engineering of LOV-domains for their use as protein tags

Cell biological research in the wheat pathogen Zymoseptoria tritici (formerly Mycosphaerella graminicola) has led to a good understanding of the histology of the infection process. Expression profiling and bioinformatic approaches, combined with molecular studies on signaling pathways, effectors and potential necrosis factors provides first insight into the complex interplay between the host and the pathogen. Cell biological studies will help to further our understanding of the infection strategy of the fungus. The cellular organization and intracellular dynamics of the fungus itself is largely unexplored. Insight into essential cellular processes within the pathogen will expand our knowledge of the basic biology of Z. tritici, thereby providing putative new anti-fungal targets.

The third dimension: cell biology comes alive

Sulphur (S) is required in considerable amounts by all organisms, while selenium (Se) is beneficial for plants and essential for animals albeit in only small amounts. Due to their chemical similarity that is in parts also shared with molyb- denum and chromium, inorganic sulphate and selenate are taken up by plants and assimilated in reduced form into organic compounds, most notably cysteine and selenocysteine, respectively. Uptake, reduction, and storage of S and Se compounds underlie complex cellular processes that need to be understood before successful translation into improved plants for human and animal diets can be achieved. Genetic engineering, breeding, and plant production approaches use insights from cell biology and basic research to introduce tailor-made desirable traits related to S and Se metabolism. Several examples for this approach will be discussed. In terms of enhanced crop quality, the so-called push and pull approaches to improve seed S amino acid compositions draw heavily from cell biology research. In pull or sink approaches, the expression of S-rich seed storage proteins is put under the control of seed-specific promoters. Such proteins possibly carry a targeting signal for the endoplasmic reticulum to achieve deposition in protein bodies. The regulation of some health-promoting compounds of Se together with S compounds such as glucoraphanin in Brassicaceae has received considerable attention in the recent past. However, to achieve high contents of Se metabolites simultaneously with the health-promoting S-containing compounds is challenging due to the crosstalk between the two pathways. The concept of S-enhanced defence linking S nutrition of plants with enhanced synthesis of S-containing defence compounds has been supported by considerable experimental data from basic research. Some of the

Molecular Biology of the Cell (MBC) begins its 16th year, and I am proud to have been asked to serve as its new Editor-in-Chief. I follow in the footsteps of two visionaries in scientific publication, David Botstein and Keith Yamamoto. Under their leadership, MBC set the tone as a journal whose exclusive motivation was to serve the scientific community and to facilitate the communication of important findings. The following philosophy of Molecular Biology of the Cell, first articulated in 1992, remains our guide: Molecular Biology of the Cell is published by the nonprofit American Society for Cell Biology (ASCB) and is free from commercial oversight and influence. We believe that the reporting of science is an integral part of research itself and that scientific journals should be instruments in which scientists are at the controls. Hence, MBC serves as an instrument of the ASCB membership and as such advocates the interests of contributors and readers through fair, prompt, and thorough review, coupled with responsible editorial adjudication and thoughtful suggestions for revision and clarification. Our most essential review criterion is that the work significantly advances our knowledge, and/or provides new concepts or approaches that extend our understanding. At MBC, every editorial decision is rendered solely by active working scientists—true peers of the contributors. The ASCB and MBC are committed to promoting the concept of open access to the scientific literature. MBC seeks to facilitate communication among scientists by publishing original papers that include full documentation of Methods and Results, with Introductions and Discussions that frame questions and interpret findings clearly (even for those outside an immediate circle of experts); exploiting technical advances to enable rapid dissemination of articles prior to print publication, and transmitting and archiving videos, large datasets, and other materials that enhance understanding; making accepted manuscripts freely accessible within days of acceptance via MBC in Press and presenting final online format only 2 months after acceptance. Key to our philosophy is the statement that “the reporting of science is an integral part of research itself, and scientific journals should be instruments in which scientists are at the controls. Hence, MBC serves as an instrument of the ASCB membership and as such advocates the interests of contributors and readers.” In other words, MBC is our journal. We are active participants—as editors, reviewers, authors, and readers—in determining how MBC affects research and researchers in cell biology. What are our responsibilities, and how do we exercise them?

Echinoderm eggs as a model for discoveries in cell biology.

We propose a general approach for encoding epistemic strategies for playing incomplete information games. A game strategy involves selecting actions in order to maximise an outcome (e.g., winning the game). In an epistemic strategy the selection of actions is based on reasoning about the knowledge of other players. We show how epistemic strategies can be encoded by supplementing a GDL-II game description with a set of epistemic rules to produce a GDL-III game that an appropriate reasoner can use to play the original GDL-II game. We prove the formal correctness of this approach and provide a practical evaluation to show its efficacy for playing the co-operative multi-player game of Hanabi. It was found that the encoded epistemic rules were able to provide players with a strategy that allowed them to play Hanabi near optimally.

This chapter maps out four influential positions in the sociology of morality taken by Weber, Simmel, Durkheim, and Marx. These authors’ differing substantive claims about morality are understood in terms of their differing epistemic strategies, fundamental conceptual assumptions that frame sociological inquiry. Epistemic strategies, most often divided simply into holism and methodological individualism, are here classified according to Kontopoulos’s five-part scheme based on the extent to which the theory in question invokes emergent phenomena, features of social life irreducible to individual action. Weber’s methodological individualism frames a subjectivist account of personal values. Simmel’s compositionist epistemology, which proposes institutions and relations irreducible to individual action, grants morality a limited objectivity that develops historically through the growth in scale of social groups. Durkheim’s holist epistemology frames a moralizing sociology in which moral questions have scientific answers, derivable from knowledge of the objective mechanisms of social integration and solidarity. The hierarchical epistemology of Marx’s thought is centered on a view of society as a strongly emergent but self-contradictory system of relations; complete moral integration is seen as impossible within a class society, so that the answers to normative questions vary by one’s location in class relations. Each of these four classical theories relativizes morality, but in different ways and with differing effects. In the fifth epistemic strategy, heterarchy or tangled-systems theory, sociology is reflexively implicated in the social relations it studies. The implications of heterarchical reflexivity for the sociology of morality are sketched briefly at the conclusion of this chapter.

Stiffness control of cell culture platforms provides researchers in cell biology with the ability to study different experimental models in conditions of mimicking physiological or pathological microenvironments. Nevertheless, the signal transduction pathways and drug sensibility of cancer cells have been poorly characterized widely using biomimetic platforms because the limited experience of cancer cell biology groups about handling substrates with specific mechanical properties. The protein cross-linking and stiffening control are crucial checkpoints that could strongly affect cell adhesion and spreading, misrepresenting the data acquired, and also generating inaccurate cellular models. Here, we introduce a simple method to adhere to polyacrylamide (PAA) hydrogels on glass coverslips without any special treatment for mechanics studies in cancer cell biology. By using a commercial photosensitive glue, Loctite 3525, it is possible to polymerize PAA hydrogels directly on glass surfaces. Furthermore, we describe a cross-linking reaction method to attach proteins to PAA as an alternative method to Sulfo-SANPAH cross-linking, which is sometimes difficult to implement and reproduce. In this chapter, we describe a reliable procedure to fabricate ECM protein-cross-linked PAA hydrogels for mechanotransduction studies on cancer cells.

INTRODUCTION: Cell biology is a subject of difficult comprehension among the topics discussed in Biology in High School. Usually, whether the teacher lacks teaching alternative methods, these classes can become purely theoretical and abstract to students. Moreover, understanding cell biology is essential for learning other subjects, such as Biochemistry. OBJECTIVES: A theoretical-practical course was offered to 20 students from a high school of Curitibanos-SC in order to provide a better understanding of cell biology, as well as to verify whether the students' concept about this topic changed after the course. MATERIALS AND METHODS: Prior to the course, the students answered a questionnaire, composed by questions regarding which topics are studied in cell biology, the student previous experience observing some sample under a light microscope, and other questions for the selection of students according to their interest in the biological area. At the end of the course, the participants answered another questionnaire, including the same question about what cell biology studies, whether they had previously handled a microscope, among other questions that aimed the course evaluation by the participants. DISCUSSION AND RESULTS: The first questionnaire was answered by 37 students, and 64.9% had the perception of what Cell Biology studies. Moreover, 59.5% stated that they had already visualized cells under the light microscope, 32.4% did not observe them and 8.1% did not remember. The second questionnaire was answered by 13 students present on the last day of the course. All students agreed the course will assist them in their studies and were able to explain how, and 84.6% satisfactorily answered what cell biology studies. Also, 92.3% of the students affirmed they had not handled a microscope before attending the course. CONCLUSION: It is concluded that the course improved the students' perception about cell biology, contributing to their knowledge about this subject.