Protoplasts as tools in cell biology

Studies of plant protoplasts using both fluorescent dyes and electron dense probes have demonstrated endocytosis in plants. Ultrastructural work with soybean protoplasts using cationized ferritin (CF) revealed an endocytotic pathway from coated pits at the plasma membrane to coated vesicles, the partially coated reticulum, Golgi bodies, multivesicular bodies and finally the vacuole. Endocytosis may be responsible for membrane retrieval from the cell surface or degradation of elicitors or toxins during host-pathogen interactions. Immunofluorescence studies of dividing plant protoplasts have provided new information about the preprophase band (PPB) of microtubules and the shape of spindles. Studies of PPBs in soybean protoplast cultures permitted detailed examination of PPB development and an assessment of the usefulness of the PPB index for identifying morphogenic cultures. In multinucleate protoplasts the size and number of PPBs were apparently not controlled by nuclear number. Research with conifer protoplasts resulted in the discovery of new features of gymnosperm spindles.     

Fluorescent sterols as tools in membrane biophysics and cell biology

Cholesterol is an important constituent of cellular membranes playing a fundamental role in many biological processes. This sterol affects membrane permeability, lateral lipid organization, signal transduction and membrane trafficking. Intracellular sterol transport modes and pathways as well as the regulation of sterol metabolism and disposition in various tissues are areas of intense research. Progress is intimately linked to development and use of appropriate analogs, which closely mimic the properties of cholesterol while allowing to be detected by spectroscopic or microscopic methods. This review provides an overview of various fluorescent sterols used in membrane biophysics and cell biology including analogs of cholesterol and cholesteryl esters. Attention is paid to the natural fluorescent sterol dehydroergosterol (DHE). A survey of the many applications of DHE in biological research is presented. Special emphasis is on recent developments in fluorescence microscopy instrumentation to visualize DHE as an intrinsically fluorescent analog of cholesterol in living cells.     

Proteomic tools for cell biology

Acquisition of large bodies of genomic sequence is facilitating the use of global techniques to assay cellular function. DNA microarrays have enabled the measurement of global mRNA levels and are able to detect changes in gene expression between different cellular states. Since much of the regulation of physiolgical processes happens post-translationally, measuring only the mRNA levels gives an incomplete picture. Strategies to assay global expression, localization, or interaction of proteins fall into the emerging field of proteomics, with various combinations of techniques being utilized to separate and identify proteins. In this review, we will present a general overview of the currently available proteomic tools and then give examples of how these tools are being utilized to answer questions in cell biology.     

Engineered riboswitches as novel tools in molecular biology

During the last years the great importance of RNA for regulating gene expression in all organisms has become obvious. Consequently, several recent approaches aim to utilize the outstanding chemical properties of RNA to develop artificial RNA regulators for conditional gene expression systems. A combination of rational design, in vitro selection and in vivo screening systems has been used to create a versatile set of RNA based molecular switches. These tools rely on diverse mechanisms and exhibit activity in several organisms. In this review, we summarize recent developments in the application of engineered riboswitches for gene regulation in vivo.     

Chemical genetics: tailoring tools for cell biology

Chemical genetics is a research approach that uses small molecules as probes to study protein functions in cells or whole organisms. Here, I review the parallels between classical genetic and chemical-genetic approaches and discuss the merits of small molecules to dissect dynamic cellular processes. I then consider the pros and cons of different screening approaches and specify strategies aimed at identifying and validating cellular target proteins. Finally, I highlight the impact of chemical genetics on our current understanding of cell biology and its potential for the future.     

2'-Functionalized nucleic acids as structural tools in molecular biology

Modified oligonucleotides bearing 2'-reactive groups or 2'-conjugated molecules have found wide application as structural tools in molecular biology. Of principal interest has been the use of 2'-reactive oligonucleotides for cross-linking with biomolecules and of 2'-conjugated oligonucleotides in hybridization assays. In this review we compare a range of electrophilic, nucleophilic and photoreactive groups for cross-linking and conjugation studies.     

Lipophilic triphenylphosphonium cations as tools in mitochondrial bioenergetics and free radical biology

Lipophilic phosphonium cations were first used to investigate mitochondrial biology by Vladimir Skulachev and colleagues in the late 1960s. Since then, these molecules have become important tools for exploring mitochondrial bioenergetics and free radical biology. Here we review why these molecules are useful in mitochondrial research and outline some of the ways in which they are now being utilized.Translated from Biokhimiya, Vol. 70, No. 2, 2005, pp. 273–283.Original Russian Text Copyright ¢ 2005 by Ross, Kelso, Blaikie, James, Cochemé, Filipovska, Da Ros, Hurd, Smith, Murphy.This revised version was published online in April 2005 with corrections to the post codes.     

A program system for interactive measurements on digitized cell images.

Using a high precision image scanner and a PDP-8/F minicomputer, we have developed a program system for interactive measurements on microscopic images. By giving simple keyboard commands, the operator can run the image scanner and manipulate the digitized images. The interface between the operator and the microscope-computer system is a Tektronix 4010 graphic terminal. The system allows objects to be isolated and parameters to be calculated from each object, e.g., parameters characterizing shape of the object, irregularity in light transmission over the object, area, integrated light transmission, etc. Objects are isolated and parameters are calculated under complete operator control using interactive computer graphics technique. Calculated parameters may be stored in dedicated data records, which are stored in files for later statistical analysis. The system also includes a statistical evaluation part. Technically, the system consists of a command scanner, which translates commands into internal representation, a parser, which checks the syntax of the commands, and an interpreter, which executes the commands. The system is designed so that new commands can be added easily.     

Avanti lipid tools: Connecting lipids,technology, and cell biology

Lipid research is challenging owing to the complexity and diversity of the lipidome. Here we review a set of experimental tools developed for the seasoned lipid researcher, as well as, those who are new to the field of lipid research. Novel tools for probing protein–lipid interactions, applications for lipid binding antibodies, enhanced systems for the cellular delivery of lipids, improved visualization of lipid membranes using gold-labeled lipids, and advances in mass spectrometric analysis techniques will be discussed. Because lipid mediators are known to participate in a host of signal transduction and trafficking pathways within the cell, a comprehensive lipid toolbox that aids the science of lipidomics research is essential to better understand the molecular mechanisms of interactions between cellular components. This article is part of a Special Issue entitled Tools to study lipid functions.     

Mechanistic modeling confronts the complexity of molecular cell biology

Mechanistic modeling has the potential to transform how cell biologists contend with the inescapable complexity of modern biology. I am a physiologist–electrical engineer–systems biologist who has been working at the level of cell biology for the past 24 years. This perspective aims 1) to convey why we build models, 2) to enumerate the major approaches to modeling and their philosophical differences, 3) to address some recurrent concerns raised by experimentalists, and then 4) to imagine a future in which teams of experimentalists and modelers build—and subject to exhaustive experimental tests—models covering the entire spectrum from molecular cell biology to human pathophysiology. There is, in my view, no technical obstacle to this future, but it will require some plasticity in the biological research mind-set.     

Information literacy in biology education: an example from an advanced cell biology course

Information literacy skills are critically important for the undergraduate biology student. The ability to find, understand, evaluate, and use information, whether from the scientific literature or from Web resources, is essential for a good understanding of a topic and for the conduct of research. A project in which students receive information literacy instruction and then proceed to select, update, and write about a current research topic in an upper-level cell biology course is described. Students research the chosen topic using paper and electronic resources, generate a list of relevant articles, prepare abstracts based on papers read, and, finally, prepare a "state-of-the-art" paper on the topic. This approach, which extends over most of one semester, has resulted in a number of well-researched and well-written papers that incorporate some of the latest research in cell biology. The steps in this project have also led to students who are prepared to address future projects on new and complex topics. The project is part of an undergraduate course in cell biology, but parts of the assignments can be modified to fit a variety of subject areas and levels.     

Receptor-selective coactivators as tools to define the biology of specific receptor-coactivator pairs

In the absence of specific high-affinity agonists and antagonists, it has been difficult to define the target genes and biological responses attributable to many of the orphan nuclear receptors (ONRs). Indeed, it appears that many members of this receptor superfamily are not regulated by classical small molecules but rather their activity is controlled by interacting cofactors. Motivated by this finding, we have developed an approach to genetically isolate specific receptor-cofactor pairs in cells, allowing us to define the biological responses attributable to each complex. This is accomplished by using combinatorial peptide phage display to engineer the receptor interacting domain of each cofactor such that it interacts selectively with one nuclear receptor. In this study, we describe the customization of PGC-1alpha and its use to study the biology of the estrogen-related receptor alpha (ERRalpha) in cultured liver cells.     

Firefly luciferase as a tool in molecular and cell biology

The unique properties of firefly luciferase and the cloning of the gene for this enzyme have spawned a number of novel applications of this protein. We summarize a few of these applications including its use as a reporter gene, as a model for the study of protein import into peroxisomes, and as a component of a heterologous gene expression system.     

Learning cell biology as a team: a project-based approach to upper-division cell biology

To help students develop successful strategies for learning how to learn and communicate complex information in cell biology, we developed a quarter-long cell biology class based on team projects. Each team researches a particular human disease and presents information about the cellular structure or process affected by the disease, the cellular and molecular biology of the disease, and recent research focused on understanding the cellular mechanisms of the disease process. To support effective teamwork and to help students develop collaboration skills useful for their future careers, we provide training in working in small groups. A final poster presentation, held in a public forum, summarizes what students have learned throughout the quarter. Although student satisfaction with the course is similar to that of standard lecture-based classes, a project-based class offers unique benefits to both the student and the instructor.     

Use of high throughput genomic tools for the study of endothelial cell biology

The endothelium is an active, dynamic and heterogeneous organ. It lines the vessels in every organ system and regulates diverse and important biological functions. Over the past several years researchers have gained enormous insights into endothelial cell function in physiological processes such as coagulation and vascular reactivity, and pathophysiological disease states such as inflammation and atherosclerosis. Despite our expanding knowledge of endothelial cell biology, the molecular mechanisms underlying these functions remain largely unknown. The newly developed high throughput genomic tools and accompanying analytical methods provide powerful approaches for identifying new endothelial cell genes and characterizing their role in health and disease. Here, we review some of the recent genomics and proteomic advances that are providing new methodologies for endothelial cell and vascular biology research.     

Volumetric modeling and interactive cutting of deformable bodies

A new approach for the interactive simulation of viscoelastic object cutting is presented. Two synchronized geometrical models at different resolutions are used, both derived from medical images. In contrast with most previous approaches, the blade deforms the object, and cutting occurs once a contact pressure threshold is exceeded. Moreover, we achieve interactive simulation rates by embedding a high-resolution geometry within a regular grid with arbitrary resolution. This allows to trade off accuracy for speed in the computation of deformations. The input data is a high-resolution volumetric model of the objects. The surface model of the object, used for rendering as well as collision detection and response, is a polygonal level set of the volumetric data. It is embedded in the volume model using barycentric coordinates.Cutting is performed by removing voxels at the fine level, and updating the surface and volume models accordingly. We introduce a new data structure, which we call a Dynamic Branched Grid, in order to preserve the fine-level topology at the coarse level. When an element of the coarse volumetric model is cut, it is replaced by a number of superimposed elements with the same size and at the same rest position as the original one. Each new element is assigned a part of material contained in the original one, and the mass and stiffness are recomputed accordingly. The well-known problem of creating small, ill-shaped finite elements while remeshing is thus completely avoided.