Molecular Cell Biology Research Research Articles

The technological landscape and applications of single-cell multi-omics.

Single-cell multi-omics technologies and methods characterize cell states and activities by simultaneously integrating various single-modality omics methods that profile the transcriptome, genome, epigenome, epitranscriptome, proteome, metabolome and other (emerging) omics. Collectively, these methods are revolutionizing molecular cell biology research. In this comprehensive Review, we discuss established multi-omics technologies as well as cutting-edge and state-of-the-art methods in the field. We discuss how multi-omics technologies have been adapted and improved over the past decade using a framework characterized by optimization of throughput and resolution, modality integration, uniqueness and accuracy, and we also discuss multi-omics limitations. We highlight the impact that single-cell multi-omics technologies have had in cell lineage tracing, tissue-specific and cell-specific atlas production, tumour immunology and cancer genetics, and in mapping of cellular spatial information in fundamental and translational research. Finally, we discuss bioinformatics tools that have been developed to link different omics modalities and elucidate functionality through the use of better mathematical modelling and computational methods.

High-performance gene expression and knockout tools using sleeping beauty transposon system

BackgroundSimilar to retro−/lenti- virus system, DNA transposons are useful tools for stable expression of exogenous genes in mammalian cells. Sleeping Beauty (SB) transposon has adopted for integrating genes into host genomes in recent studies. However, SB-derived vector system for proteins purifying/tracking and gene knockout are still not available.ResultsIn this study, we generated a series of vectors (termed as pSB vectors) containing Sleeping Beauty IRDR-L/R that can be transposed by SB transposase. Gateway cassette was combined to the pSB vectors to facilitate the cloning. Vectors with various tags, Flag, Myc, HA, V5 and SFB, were generated for multiple options. Moreover, we incorporated the CRISPR-Cas9 cassette into the pSB plasmids for gene knockout. Indeed, using one of these vectors (pSB-SFB-GFP), we performed Tandem Affinity Purification and identified that NFATc1 is a novel binding partner of FBW7. We also knocked out RCC2 and BRD7 using pSB-CRISPR vector respectively, and revealed the novel roles of these two proteins in mitosis.ConclusionOur study demonstrated that the pSB series vectors are convenient and powerful tools for gene overexpression and knockout in mammalian cells, providing a new alternative approach for molecular cell biology research.

Real-Time Single Molecule Visualization of SH2 Domain Membrane Recruitment in Growth Factor Stimulated Cells.

In the last decade, single molecule tracking (SMT) techniques have emerged as a versatile tool for molecular cell biology research. This approach allows researchers to monitor the real-time behavior of individual molecules in living cells with nanometer and millisecond resolution. As a result, it is possible to visualize biological processes as they occur at a molecular level in real time. Here we describe a method for the real-time visualization of SH2 domain membrane recruitment from the cytoplasm to epidermal growth factor (EGF) induced phosphotyrosine sites on the EGF receptor. Further, we describe methods that utilize SMT data to define SH2 domain membrane dynamics parameters such as binding (τ), dissociation (k d), and diffusion (D) rates. Together these methods may allow us to gain greater understanding of signal transduction dynamics and the molecular basis of disease-related aberrant pathways.

A decade of molecular cell biology: achievements and challenges

Nature Reviews Molecular Cell Biology celebrated its 10-year anniversary during this past year with a series of specially commissioned articles. To complement this, here we have asked researchers from across the field for their insights into how molecular cell biology research has evolved during this past decade, the key concepts that have emerged and the most promising interfaces that have developed. Their comments highlight the broad impact that particular advances have had, some of the basic understanding that we still require, and the collaborative approaches that will be essential for driving the field forward.

Cost-effective gene transfection by DNA compaction at pH 4.0 using acidified, long shelf-life polyethylenimine

Introduction of genetic material into cells is an essential prerequisite for current research in molecular cell biology. Although transfection with commercially available reagents results in excellent gene expression, their high costs are obstacles to experimentation with a large number or large scales of transfection. The cationic polymer linear-polyethylenimine (MW 25,000) (PEI), one of the most cost-effective vehicles, facilitates DNA compaction by polyplex formation, which leads to efficient delivery of DNA into cells by endocytosis. However, the use of PEI is still limited because of substantial cytotoxicity and intolerable deterioration in transfection efficiency by its low stability. Here, we show that acidification of PEI is important for its transfection activity. Dissolving PEI powder in 0.2N HCl confers a long shelf-life for PEI storage at 4 and -80 degrees C, and the polyplex formation of plasmid DNA with PEI is optimized in lactate-buffered saline at pH 4.0. Furthermore, changing the culture medium at 8-12 h posttransfection can minimize the cytotoxicity of PEI without sacrificing the high transfection efficiency comparable to that of commercial reagents. The cost per test using acidified PEI is drastically reduced to approximately 1:10,000, compared with commercial reagents. Thus, we conclude that acidification of PEI satisfactorily accomplishes cost-effective, high-efficiency transfection.

Molecular Motors: Design, Mechanism, and Control

Theoretical modeling and computer simulations of molecular motors provide insight that engineers can exploit to design and control artificial nanomotors.For obvious reasons, the study of molecular motors has been a traditional area of research in molecular cell biology and biochemistry, but in recent years, it has attracted physicists' as well as engineers. Exploring the design and mechanisms of these motors from an engineering perspective requires investigating their structure and dynamics using the fundamental principles of physics at the subcellular level. The insights gained from such fundamental research could also find practical applications in designing and manufacturing artificial nanomotors - motors whose typical size is usually in the rage of a few nanometers to a few tens of nanometers. In contrast to man-made macroscopically large motors, natural nanomotors have evolved over billions of years. While discussing the design, mechanism, and control of molecular motors, this article also compares their macroscopic counterparts to emphasize common features as well as differences.

Modeling protein synthesis from a physicist’s perspective: A toy model

Proteins are polymers of amino acids. These macromolecules are synthesized by intracellular machines called ribosomes. Although the experimental investigation of protein synthesis has been a traditional area of research in molecular cell biology, important quantitative models of protein synthesis have been reported in research journals devoted to statistical physics and related interdisciplinary topics. From the perspective of a physicist, protein synthesis is the classical transport of interacting ribosomes on a messenger RNA (mRNA) template that dictates the sequence of the amino acids on the protein. We discuss appropriate simplification of the models and methods. In particular, we develop and analyze a simple toy model using some elementary techniques of nonequilibrium statistical mechanics and predict the average rate of protein synthesis and the spatial organization of the ribosomes in the steady state.

Generation of Live‐Cell Microarrays by Means of DNA‐Directed Immobilization of Specific Cell‐Surface Ligands

Everything in its place: The DNA-directed immobilization of specific cell-surface ligands on a DNA microarray enables the generation of positionally encoded arrays of live cells. Such arrays can be used for fundamental research in molecular cell biology or as a screening platform in the area of drug development

The big catch

The availability of short interfering RNAs (siRNAs) to silence gene expression has revolutionized research in molecular cell biology. But these synthetic siRNAs rely on cellular enzymes for their activity.

Cytokinetic analysis of cell cycle and sub-phases in MOLT-4 cells by cyclin E + A/DNA multiparameter flow cytometry

Cell cycle analysis has become increasingly important in verifying the effect of anti-tumor drugs and cytokinetic research. In the early methods of cell cycle analysis, the flow cytometry relied on DNA content, and therefore, the cell cycle could be only broken into three stages: G(0)/G(1), S, and G(2)/M phase. It could not distinguish the G(0), G(1), G(2), and M phase cells, let alone the sub-phases in G(1) phase. In cell cycle, expression of cyclin E living up to the maximal level in the cells undergoing transition from G(1) to S phase, and G(2) + M cells are cyclin E negative. Expression of cyclin A is progressively increasing during S phase and is maximal in G(2) phase cells. Therefore, in the current study we established a cyclin E + A/DNA multiparameter flow cytometric technique by using a mixture of cyclin E and cyclin A antibodies, which can identify six stages in the whole cell cycle: G(0), early G(1), late G(1), S, G(2), and M phase. Furthermore, we found that cyclin E + A/DNA multiparameter flow cytometry could also be used for stathmokinetic analysis of lymphocyte leukemia MOLT-4 cells after addition of the stathmokinetic agent vinblastine to cultures of exponentially growing MOLT-4 cells. We believe that this new technique will provide a much better tool for molecular cell biology research and especially for cell proliferation kinetics investigations.

Rapid two-dimensional analysis of proteins by ultra-thin layer gel electrophoresis.

Identification of qualitative and/or quantitative protein expression differences as well as characterization of specific cell proteomes would further advance molecular cell biology research. Today, one of the most commonly used tools for proteome analysis is two-dimensional gel electrophoresis. Although this technology is informative, it is extremely cumbersome, time-consuming and lacks automation and proper reproducibility. In this paper, we propose an automated separation/detection system capable of rapid two-dimensional analysis of proteins by ultra-thin layer gel electrophoresis with real time imaging of the separated components, using fiber optics based laser induced fluorescence technology. The approach is based on electric field mediated separation in capillary dimensions, along with noncovalent, "in migratio" fluorescent staining methodology. The advantage of the technology discussed over existing techniques is its simplicity, speed and good detection sensitivity.

Can we identify the forces that drive the folding of integral membrane proteins?

Protein folding has been at the forefront of molecular cell biology research for several years. However, integral membrane proteins have eluded detailed molecular level study until recently. One reason is the often apparently insurmountable problem of mimicking the natural membrane bilayer with lipid or detergent mixtures. There is nevertheless a large body of information on lipid properties and in particular on phosphatidylcholine and phosphatidylethanolamine lipids, which are common to many biological membranes. We have exploited this knowledge to design efficient in vitro, lipid-bilayer folding systems for membrane proteins. Bacteriorhodopsin has been used as a model system for our initial studies: we have shown that a rate-limiting apoprotein folding step and the overall folding efficiency seem to be controlled by particular properties of the lipid bilayer. The properties of interest are the stored curvature elastic energy within the bilayer and the lateral pressure that the lipid chains exert on their neighbouring folding protein. These are generic properties of the bilayer that can be achieved with simple mixtures of many types of biological lipid and seem to be important in vivo.

An Integrated System for Transmission Electron Microscopy.

Macromolecular microscopy is becoming an increasingly important tool for structural biology. The development of improved capabilities for three-dimensional electron microscopy is critical for optimal progress in emerging integrative research in molecular cell biology. These techniques currently suffer from several severe disadvantages related to the tremendous time and effort required to acquire and analyze the data.For several years we have been developing software for automated and intelligent acquisition of transmission electron micrographs [1,2,3]. Our overall goal is to develop a system for rapid and routine structure determination of macromolecular assemblies from specimens preserved in vitreous ice. Ultimately we plan to develop an integrated system that can produce an electron density map of a structure within a few hours of inserting a specimen into the electron microscope. With this goal in mind it is essential that the images be acquired using a digital camera rather than film.