Quantitative cell biology with the Virtual Cell

Cell biological processes are controlled by an interacting set of biochemical and electrophysiological events that are distributed within complex cellular structures. Computational models, comprising quantitative data on the interacting molecular participants in these events, provide a means for applying the scientific method to these complex systems. The Virtual Cell is a computational environment designed for cell biologists, to facilitate the construction of models and the generation of predictive simulations from them. This review summarizes how a Virtual Cell model is assembled and describes the physical principles underlying the calculations that are performed. Applications to problems in nucleocytoplasmic transport and intracellular calcium dynamics will illustrate the power of this paradigm for elucidating cell biology.     

Quantitative cell biology of tip growth in moss

Plant Molecular Biology - Here we review, from a quantitative point of view, the cell biology of protonemal tip growth in the model moss Physcomitrium patens. We focus on the role of the...     

The cell biology of cross-presentation and the role of dendritic cell subsets

The cell biology of cross-presentation is reviewed regarding exogenous antigen uptake, antigen degradation and entry into the major histocompatibility complex class I pathway. Whereas cross-presentation is not associated with enhanced phagocytic ability, certain receptors may favour uptake for cross-presentation for example mannose receptor for soluble glycoproteins. Perhaps, the defining property of the cross-presenting cell is some specialization in host machinery for handling and transport of antigen across organelles. Both cytosolic and vacuolar pathways are discussed. Which dendritic cell (DC) subset is the cross-presenting cell is explored. Cross-presentation is found within the CD8(+) subset resident in lymphoid organs. The role of other DC subsets (especially the migratory CD8(-) DC) and the route of antigen delivery are also discussed. Further consideration is given to antigen transfer between DC subsets and differential presentation to naive vs memory T cells.     

Quantitative biology: where modern biology meets physical sciences

Quantitative methods and approaches have been playing an increasingly important role in cell biology in recent years. They involve making accurate measurements to test a predefined hypothesis in order to compare experimental data with predictions generated by theoretical models, an approach that has benefited physicists for decades. Building quantitative models in experimental biology not only has led to discoveries of counterintuitive phenomena but has also opened up novel research directions. To make the biological sciences more quantitative, we believe a two-pronged approach needs to be taken. First, graduate training needs to be revamped to ensure biology students are adequately trained in physical and mathematical sciences and vice versa. Second, students of both the biological and the physical sciences need to be provided adequate opportunities for hands-on engagement with the methods and approaches necessary to be able to work at the intersection of the biological and physical sciences. We present the annual Physiology Course organized at the Marine Biological Laboratory (Woods Hole, MA) as a case study for a hands-on training program that gives young scientists the opportunity not only to acquire the tools of quantitative biology but also to develop the necessary thought processes that will enable them to bridge the gap between these disciplines.What does a mathematician looking at bacterial division under a microscope have in common with a biologist programming a stochastic simulation of microtubule growth? For one, both can be found at the Physiology Course at the Marine Biological Laboratory (MBL) in Woods Hole, MA, which brings together graduate students and young postdocs who have a passion for quantitative biology. Students enter the course with a wide array of scientific backgrounds, including chemistry, molecular biology, mathematics, and theoretical physics. Although at first hesitant to step outside their comfort zones, students leave the course confident and courageous in their abilities to work across traditional academic boundaries. Having experienced this transformation ourselves as participants in the 2014 Physiology Course, we wanted to share some of our insights and how they have influenced our perspectives on the present challenges and exciting future of quantitative cell biology.“When you cannot express [what you are speaking about] in numbers, your knowledge is of a meager and unsatisfactory kind; it may be the beginning of knowledge, but you have scarcely, in your thoughts, advanced to the stage of science.” The need for quantification in the life sciences could not have been better worded than it is in this quote from Lord Kelvin. One of the key take-home messages from the course has been the crucial need for advancement of quantitative cell biology, which uses accurate measurements to refine a hypothesis, with the aim of comparing experimental data with predictions generated by theoretical models. We strongly believe that quantitative approaches not only aid in better addressing existing biological questions but also enable the formulation of new ones.The present time is particularly ripe for implementing quantitative approaches in cell biology, due to the wealth of data available and the depth of control we now have over many experimental systems. In the past 20 years, we have sequenced the human genome, broken the diffraction limit in microscopy, and begun to explore the possibilities of the micron-scaled experiments with microfluidics. With these tools in hand, the means to obtain quantitative data are not limited to a select few model systems; this level of experimental detail allows us to craft theoretical models that not only fit the data but have real predictive power. We can then return to our respective experimental systems with new hypotheses and interrogate them anew, reaping the benefits of an approach that has benefited physicists for decades.Building quantitative models in biology has been a powerful approach that has often revealed counterintuitive phenomena and insights while at the same time leading to novel research directions. This is of particular importance today, as experiments are becoming increasingly expensive and are rapidly accumulating vast amounts of data. It is now possible to perform “virtual” preliminary experiments in silico using quantitative models and pre-existing data and only then move to “real” laboratory experiments to test the developed hypotheses. Researchers trained this way can perform more focused experiments instead of adopting the traditional exploratory mode in the lab, saving both time and resources. However, we recognize that a majority of biology graduates have not been rigorously trained in the mathematical and physical sciences. Similarly, many physics graduates often remember their introductory biology classes simply for the rote memorization of protein names and signaling pathways, leading to the wrong assumption that biology is all about remembering three-letter abbreviations such as WNT, MYC, and so on. This can often create a misleading picture of biology.These challenges could be overcome by finding a common language between biologists, physicists, and mathematicians. A simple example of this is the word “model.” The same word can mean very different things to scientists depending upon their training: to a physicist it refers to quantitative visualization of a process via certain well-defined mathematical parameters; a biologist, on the other hand, might use the word to refer to a schematic depiction (also called a cartoon) of a biochemical reaction. We aim to reduce this gap between biological and physical sciences and bring these two communities together.One way to train young scientists in such an approach is to provide opportunities for hands-on engagement with the methods and thought processes necessary to partake in both fields. The MBL Physiology Course is an excellent case study for a training program that gives young scientists the building blocks and community necessary for success in bridging quantitative/physical sciences and biology. The course starts with a weeklong boot camp designed to bring students of different backgrounds up to speed on basic tools in quantitative biology. Students purify proteins, program in MATLAB, and build microscopes. The most important skill that biologists acquire is not simply learning how to write lines of MATLAB code, but rather phrasing biological phenomena in mathematical terms through equations and simulations. Building confocal microscopes and optical tweezers on a bare optical table creates trust in the tools we depend on to acquire quantitative data. Physicists, on the other hand, learn to purify motor proteins like kinesins and dyneins from native sources (squid), in the process coming face-to-face with the natural context of the biological questions that they are addressing.This interdisciplinary approach helps students from diverse backgrounds develop a common language. After the boot camp, students work together on three 2-week-long research projects under the guidance of leading scientists. Projects range from studying the spatial organization of the human oral microbiome and observing the development of the Caenorhabditis elegans embryo all the way to performing computational simulations of cytoskeletal polymers. By working together in a highly informal and stimulating environment, physicists learn to appreciate biological problems and biologists begin to see biological phenomena in a new light as a result of the novel physical tools and methodologies they learn from their peers. As an example, course participants Rikki Garner and Daniel Feliciano successfully collaborated to study how competition between two highly processive microtubule motors that work in opposition controls microtubule length. While Rikki (mentored by Jané Kondev) tackled the question theoretically using a random walk model, Daniel, under the mentorship of Joe Howard, carried out the experimental measurements via an in vitro assay to test Rikki''s predictions. Other examples of quantitative and biological expertise coming together to address biological questions include studying the displacement and transport of proteins at the interface between cells and synthetic supported lipid bilayers (Figure 1), observing and quantifying the cytoplasmic streaming as well as the filter-feeding flow vortices in the giant single-celled organism Stentor coeruleus (Figure 2), and imaging the spatial organization of complex oral microbial communities (Figure 3).Open in a separate windowFIGURE 1:Proteins are organized based on size at the membrane interface. The membrane interface between a cell and a supported lipid bilayer (SLB) was formed by the interaction of synthetic adhesion molecules, one protein (bound to the membrane via a His-tag) and another protein that interacts and binds with the membrane-bound protein (expressed in the S2 cells). Note that the long noninteracting protein (21 nm, magenta colored) but not the shorter noninteracting protein (8 nm, magenta colored), which is bigger than the synthetic interacting dimer (16 nm, red–green colored) is excluded from the cell–SLB interface. Both of these noninteracting proteins are bound to the SLB and do not interact with the cell. Scale bar: 10 μm. (Prepared by L.Z. and Nan Hyung Hong under the guidance of Matt Bakalar, Eva Schmid, and Dan Fletcher.)Open in a separate windowFIGURE 2:Stentor coeruleus is a giant single-celled organism that feeds by creating flow vortices in water and directing prey into its oral opening using this flow. (A) Maximum intensity projection of time-lapse images showing flow fields in the feeding flow generated by S. coeruleus. The flow is generated by the coordinated ciliary beating of the mouth cilia. (B) Flow velocity and flow directions were quantified by the particle image velocimetry method. The circularity of flow has also been indicated—the blue cloud around the oral cilia indicates clockwise flow, and red indicates anticlockwise flow. Scale bar: 100 μm. (Prepared by S.S. under the guidance of Mark Slabodnick, Tatyana Makushok, and Wallace Marshall in collaboration with Jack Costello, Providence College.)Open in a separate windowFIGURE 3:Spatial organization of complex microbial communities in an oral plaque sample taken from a volunteer as seen by combinatorial labeling and spectral imaging–fluorescent in situ hybridization (CLASI-FISH). Microbes seen here are Corynebacterium (pink), Neisseriaceae (blue), Fusobacterium (green), Pasteurellaceae (yellow), Streptococcus (cyan), and Actinomyces (red). (Prepared by Bryan Weinstein, Lishibanya Mohapatra, and Matti Gralka under the guidance of Blair Rossetti, Jessica Mark Welch, and Gary Borisy.)An invaluable aspect of the course is the informal nature of the interaction. There are a wide variety of morning seminar speakers, and in the question-and-answer sessions following the talks, the speakers discuss not only science but also the successes and failures they experienced while moving across the boundaries of biological and physical sciences. The interactive and collaborative nature of the course encourages students to not just learn from one another but to actually teach one another. “Chalk talks” and interactions happen spontaneously and are the strongest indication of the richness of the intellectual exchange among members of the community. Prime examples in the 2014 course are the chalk talks on Python (Bryan Weinstein), Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) cloning (Dan Dickinson), and the basics of microfluidics (Sindy Tang).Although we have benefited immensely from this interdisciplinary course, we understand that it might not be feasible for all graduate students to participate in such courses. Nevertheless, we believe that the scientific community should work together to replicate elsewhere, at least partially, the strengths of this course to allow students to benefit from this approach. Students should be encouraged to host and attend seminars from speakers with diverse backgrounds, which will expose them to research areas different from their own. Biology students should also be exposed to mathematical and statistical instruction early in their research careers, preferably at the undergraduate level, to enable them to build strong foundations. Students should also be encouraged to participate in short-term, low-pressure interdisciplinary collaborations to broaden their understanding and initiate interactions with other fields. Short summer/winter schools—for example, the physical biology of the cell courses at Cold Spring Harbor and the International Centre for Theoretical Physics (Italy)–International Centre for Theoretical Sciences (India) Winter School on Quantitative Systems Biology, 2013, in Bangalore, India—can serve as the perfect stage for this.In conclusion, we expect that quantitative approaches will be indispensable for better addressing biological questions in the future. Our experience is that combining traditional experimental cell biology with quantitative thinking leads to hitherto unknown scientifically rich domains, and we ourselves have found this exploratory journey to be both achievable and rewarding. Although bridging the gap may appear to be difficult at times, it is extremely satisfying when accomplished, and doing it within a highly motivated and supportive community is what makes the connection possible and extremely useful.     

The role of endothelial cell biology in endocarditis

The treatment of endocarditis remains a challenge for physicians, even in times of modern antibiotic treatment. Depending on its cause, endocarditis can either be of infectious or non-infectious origin. Infective endocarditis is caused by bacterial (or fungal) pathogens, and the clinical course is critically dependent on the virulence factors of the specific microorganisms involved. Therefore, the clinical type of endocarditis can be divided into an acute and more aggressive form and a subacute form (endocarditis lenta). Much of our knowledge regarding the pathogenesis of infective endocarditis is based on studies of the virulence of Staphylococcus aureus, which has become the most frequent cause of infective endocarditis nowadays. However, independently of the underlying cause of endocarditis (infectious or noninfectious), the pathogenesis involves the damage and disturbance of endothelial function and the formation of associated “vegetation”. Surprisingly little is known about the specific role of the endothelium in the pathogenesis of endocarditis. This review will thus give insights into current knowledge of the pathogenesis of endocarditis with a focus on the role of the endothelium.     

Cancer cell metabolism: the essential role of the nonessential amino acid,glutamine

Biochemistry textbooks and cell culture experiments seem to be telling us two different things about the significance of external glutamine supply for mammalian cell growth and proliferation. Despite the fact that glutamine is a nonessential amino acid that can be synthesized by cells from glucose‐derived carbons and amino acid‐derived ammonia, most mammalian cells in tissue culture cannot proliferate or even survive in an environment that does not contain millimolar levels of glutamine. Not only are the levels of glutamine in standard tissue culture media at least ten‐fold higher than other amino acids, but glutamine is also the most abundant amino acid in the human bloodstream, where it is assiduously maintained at approximately 0.5 mM through a combination of dietary uptake, de novo synthesis, and muscle protein catabolism. The complex metabolic logic of the proliferating cancer cells' appetite for glutamine—which goes far beyond satisfying their protein synthesis requirements—has only recently come into focus. In this review, we examine the diversity of biosynthetic and regulatory uses of glutamine and their role in proliferation, stress resistance, and cellular identity, as well as discuss the mechanisms that cells utilize in order to adapt to glutamine limitation.     

The role of theory in an emerging new plant reproductive biology

Recent empirical studies hint at an end to the historical solitude between pollination and mating system approaches to plant reproductive character evolution. Now is an opportune time to distill theoretical results into comprehensible insight, and to integrate these findings into the emerging new plant reproductive biology. We outline four theoretical insights for understanding the evolution of reproductive characters, and show how these allow researchers to dissect complex ecological scenarios into clear and evolutionarily relevant components.     

A role for chemistry in stem cell biology

Although stem cells hold considerable promise for the treatment of numerous diseases including cardiovascular disease, neurodegenerative disease, musculoskeletal disease, diabetes and cancer, obstacles such as the control of stem cell fate, allogenic rejection and limited cell availability must be overcome before their therapeutic potential can be realized. This requires an improved understanding of the signaling pathways that affect stem cell fate. Cell-based phenotypic and pathway-specific screens of natural products and synthetic compounds have recently provided a number of small molecules that can be used to selectively control stem cell proliferation and differentiation. Examples include the selective induction of neurogenesis and cardiomyogenesis in murine embryonic stem cells, osteogenesis in mesenchymal stem cells and dedifferentiation in skeletal muscle cells. Such molecules will likely provide new insights into stem cell biology, and may ultimately contribute to effective medicines for tissue repair and regeneration.     

Pluripotent stem cell heterogeneity and the evolving role of proteomic technologies in stem cell biology

Stem cells represent obvious choices for regenerative medicine and are invaluable for studies of human development and drug testing. The proteomic landscape of pluripotent stem cells (PSCs), in particular, is not yet clearly defined; consequently, this field of research would greatly benefit from concerted efforts designed to better characterize these cells. In this concise review, we provide an overview of stem cell potency, highlight the types and practical implications of heterogeneity in PSCs and provide a detailed analysis of the current view of the pluripotent proteome in a unique resource for this rapidly evolving field. Our goal in this review is to provide specific insights into the current status of the known proteome of both mouse and human PSCs. This has been accomplished by integrating published data into a unified PSC proteome to facilitate the identification of proteins, which may be informative for the stem cell state as well as to reveal areas where our current view is limited. These analyses provide insight into the challenges faced in the proteomic analysis of PSCs and reveal one area--the cell surface subproteome--that would especially benefit from enhanced research efforts.     

Quantitative microscopy and systems biology: seeing the whole picture

Understanding cellular function requires studying the spatially resolved dynamics of protein networks. From the isolated proteins we can only learn about their individual properties, but by investigating their behavior in their natural environment, the cell, we obtain information about the overall response properties of the network module in which they operate. Fluorescence microscopy methods provide currently the only tools to study the dynamics of molecular processes in living cells with high temporal and spatial resolution. Combined with computational approaches they allow us to obtain insights in the reaction-diffusion processes that determine biological function on the scale of cells.