Current Cell Biology Research Articles

A Comprehensive Review of Organ-on-a-Chip Technology and Its Applications.

Organ-on-a-chip (OOC) is an emerging technology that simulates an artificial organ within a microfluidic cell culture chip. Current cell biology research focuses on in vitro cell cultures due to various limitations of in vivo testing. Unfortunately, in-vitro cell culturing fails to provide an accurate microenvironment, and in vivo cell culturing is expensive and has historically been a source of ethical controversy. OOC aims to overcome these shortcomings and provide the best of both in vivo and in vitro cell culture research. The critical component of the OOC design is utilizing microfluidics to ensure a stable concentration gradient, dynamic mechanical stress modeling, and accurate reconstruction of a cellular microenvironment. OOC also has the advantage of complete observation and control of the system, which is impossible to recreate in in-vivo research. Multiple throughputs, channels, membranes, and chambers are constructed in a polydimethylsiloxane (PDMS) array to simulate various organs on a chip. Various experiments can be performed utilizing OOC technology, including drug delivery research and toxicology. Current technological expansions involve multiple organ microenvironments on a single chip, allowing for studying inter-tissue interactions. Other developments in the OOC technology include finding a more suitable material as a replacement for PDMS and minimizing artefactual error and non-translatable differences.

Epigallocatechin gallate is a potent inhibitor of cystathionine beta-synthase: Structure-activity relationship and mechanism of action

Epigallocatechin gallate (EGCG) is the main bioactive component of green tea. Through screening of a small library of natural compounds, we discovered that EGCG inhibits cystathionine β-synthase (CBS), a major H2S-generating enzyme. Here we characterize EGCG's mechanism of action in the context of CBS-derived H2S production. In the current project, biochemical, pharmacological and cell biology approaches were used to characterize the effect of EGCG on CBS in cellular models of cancer and Down syndrome (DS). The results show that EGCG binds to CBS and inhibits H2S-producing CBS activity almost 30-times more efficiently than the canonical cystathionine formation (IC50 0.12 versus 3.3 μM). Through screening structural analogs and building blocks, we identified that gallate moiety of EGCG represents the pharmacophore responsible for CBS inhibition. EGCG is a mixed-mode, CBS-specific inhibitor with no effect on the other two major enzymatic sources of H2S, CSE and 3-MST. Unlike the prototypical CBS inhibitor aminooxyacetate, EGCG does not bind the catalytic cofactor of CBS pyridoxal-5′-phosphate. Molecular modeling suggests that EGCG blocks a substrate access channel to pyridoxal-5′-phosphate. EGCG inhibits cellular H2S production in HCT-116 colon cancer cells and in DS fibroblasts. It also exerts effects that are consistent with the functional role of CBS in these cells: in HCT-116 cells it decreases, while in DS cells it improves viability and proliferation. In conclusion, EGCG is a potent inhibitor of CBS-derived H2S production. This effect may contribute to its pharmacological effects in various pathophysiological conditions.

Unparalleled Approaches to Directly Visualize DNA‐Binding Proteins and Biomolecular Condensates

The structure, regulation, and maintenance of DNA are critical to cellular function. Detailed molecular insights into these processes are essential for understanding their associated pathological conditions, establishing and validating biological models, and developing novel therapeutics. As a result, direct, real‐time observations of individual proteins and biomolecular condensates interacting with DNA are needed to obtain a thorough understanding of their mechanisms and dynamics, and to validate and complete the current biological models. Technologies, such as the C‐Trap, offer an unique opportunity to meet these challenges by enabling researchers to observe DNA processes in real‐time at the single‐molecule level. Here, we present our efforts for further enabling discoveries in the field of DNA‐protein interactions using the combination of optical tweezers with correlative confocal fluorescence microscopy. We present several examples in which our technologies enhance the understanding of DNA repair mechanisms, chromatin structure and stability, DNA replication, DNA editing tools (e.g. CRISPR/Cas), and biomolecular condensates. Importantly, we show that advances in single‐molecule technologies can be turned into easy‐to‐use instruments that enable discoveries to all biologists previously reserved for the world’s most specialized biophysics labs. This approach complements current structural or cell biology methods and provides key insights to complete the molecular mechanisms and models describing essential DNA‐binding processes.

Not Going with the Flow: How Cells Adapt Internal Physics

Defining the principles underlying the organization of biomolecules within cells is a key challenge of current cell biology research. Persson etal. now identify a powerful layer of regulation that allows cells to decouple diffusion from temperature by modulating their intracellular viscosity. This so-called viscoadaptation is mediated through trehalose and glycogen activities, which alter diffusion dynamics and self-assembly propensity inside the cell globally.

Cell Type Purification by Single-Cell Transcriptome-Trained Sorting

SummaryMuch of current molecular and cell biology research relies on the ability to purify cell types by fluorescence-activated cell sorting (FACS). FACS typically relies on the ability to label cell types of interest with antibodies or fluorescent transgenic constructs. However, antibody availability is often limited, and genetic manipulation is labor intensive or impossible in the case of primary human tissue. To date, no systematic method exists to enrich for cell types without a priori knowledge of cell-type markers. Here, we propose GateID, a computational method that combines single-cell transcriptomics with FACS index sorting to purify cell types of choice using only native cellular properties such as cell size, granularity, and mitochondrial content. We validate GateID by purifying various cell types from zebrafish kidney marrow and the human pancreas to high purity without resorting to specific antibodies or transgenes.

Solvent-Assisted Micromolding of Biohybrid Hydrogels to Maintain Human Hematopoietic Stem and Progenitor Cells Ex Vivo.

Array-format cell-culture carriers providing tunable matrix cues are instrumental in current cell biology and bioengineering. A new solvent-assisted demolding approach for the fabrication of microcavity arrays with very small feature sizes down to single-cell level (3 µm) of very soft biohybrid glycosaminoglycan-poly(ethylene glycol) hydrogels (down to a shear modulus of 1 kPa) is reported. It is further shown that independent additional options of localized conjugation of adhesion ligand peptides, presentation of growth factors through complexation to gel-based glycosaminoglycans, and secondary gel deposition for 3D cell embedding enable a versatile customization of the hydrogel microcavity arrays for cell culture studies. As a proof of concept, cell-instructive hydrogel compartment arrays are used to analyze the response of human hematopoietic stem and progenitor cells to defined biomolecular and spatial cues.

Intensification and redistribution of protrusive activity is a feature of tumor transformation and is associated with an increase of the invasive potential of cells

The abilities of tumor cells to invade and metastasize are frequent causes of death of cancer patients. Studying the mechanisms of cell motility alterations and acquisition of enhanced metastatic potential as the result of transformation is an important aspect in current cell biology. The initial and determinant step of cell motility is the formation of active cell edge with protrusions based on the Arp2/3-dependent actin polymerization. We used three different cell systems as examples of different models of tumor transformation to study the alteration and redistribution of protrusive activity caused by transformation in fibroblasts. We analyzed relationships between detected alterations and the acquisition of increased invasive potential by cells. Active edge of untransformed fibroblasts occupies about 50% of the cell perimeter and is concentrated at the cell front. There are well pronounced stable regions at the lateral cell edges. Tumor transformation causes redistribution of protrusive activity of fibroblasts irrespective of their origin and the nature of transforming agents. The length of active edges significantly increases, up to 92% of the total perimeter in fibrosarcoma cells of tumor origin. These cells have practically no stable edges. The intensity of protrusive activity of transformed cells is also increased. Single transformed cells show a decrease in the directionality and rate of migration on 2D substrate without special stimulation. Instead, they gain the capacity to migrate in 3D and to invade matrigel. These abilities increase in parallel with the intensification of edge activity. We showed that invasive abilities are not associated with the activation of matrix metalloproteinases in the studied cell systems. Our data demonstrate that the increase of length of active edge could be considered as an additional feature of cell transformation together with the reduction of stress fiber and focal adhesions and that the excessive protrusive activity results in the development of explorative migration of tumor cells.

Three-dimensional (3D) printing of mouse primary hepatocytes to generate 3D hepatic structure.

PurposeThe major problem in producing artificial livers is that primary hepatocytes cannot be cultured for many days. Recently, 3-dimensional (3D) printing technology draws attention and this technology regarded as a useful tool for current cell biology. By using the 3D bio-printing, these problems can be resolved.MethodsTo generate 3D bio-printed structures (25 mm × 25 mm), cells-alginate constructs were fabricated by 3D bio-printing system. Mouse primary hepatocytes were isolated from the livers of 6–8 weeks old mice by a 2-step collagenase method. Samples of 4 × 107 hepatocytes with 80%–90% viability were printed with 3% alginate solution, and cultured with well-defined culture medium for primary hepatocytes. To confirm functional ability of hepatocytes cultured on 3D alginate scaffold, we conducted quantitative real-time polymerase chain reaction and immunofluorescence with hepatic marker genes.ResultsIsolated primary hepatocytes were printed with alginate. The 3D printed hepatocytes remained alive for 14 days. Gene expression levels of Albumin, HNF-4α and Foxa3 were gradually increased in the 3D structures. Immunofluorescence analysis showed that the primary hepatocytes produced hepatic-specific proteins over the same period of time.ConclusionOur research indicates that 3D bio-printing technique can be used for long-term culture of primary hepatocytes. It can therefore be used for drug screening and as a potential method of producing artificial livers.

Reconstitutions of mitochondrial inner membrane remodeling.

Biological membranes exhibit function-related shapes, leading to a plethora of complex and beautiful cell and cell organellar morphologies. Most if not all of these structures have evolved for a particular physiological reason. The shapes of these structures are formed by physical forces that operate on membranes. To create particular shaped cells and cell organelles, membranes must undergo deformations which are determined by the structure and elasticity of the membrane and this process is most probable driven by proteins, lipids and/or interplay of both Zimmerberg and Kozlov (2006). Therefore, an important question of current cell biology in conjunction with physics and mathematics is to elucidate the functional cause for these different membrane morphologies as well as how they are formed. One of the most peculiar membrane shapes is observed in mitochondria. These organelles are surrounded by two membranes and especially the convoluted inner membrane displays a complex ultra-structure. A molecular understanding of how this membrane is shaped is missing to a large extent. Unlike membrane remodeling in classical curvature-dependent processes like clathrin-mediated endocytosis, mitochondria are most likely shaped by integral membrane proteins. Following, we will review the current knowledge of inner mitochondrial membrane architecture and discuss recent findings and advances in understanding the factors that shape this membrane. We will address pending questions especially with regard to the experimentally challenging nature of investigating membrane bending by hydrophobic integral membrane proteins.

Shedding microvesicles released by a human microglial cell line (CHME-5): A good platform for studying the biology of the system

Cell-to-cell communication is a field of growing interest in the current cell biology since the recent discovery of multiple and unexpected mechanisms by which cells can propagate information among them through active biomolecules. One of these mechanisms is based on the release of membrane particles of different size, generally called microvesicles (MVs), filled by either proteins or nucleic acids whose composition seems to precisely reflect that of the cell of origin.

Reflection on teaching of extracellular matrix in current medical cell biology

Extracellular matrix （ECM） is not only one of current hot-points in medical cell biology, but also one tricky part for undergraduates to learn. This article compared chapters of ECM in medical cell biology courses and spotlighted that ECM chapter was often neglected in some domes- tic universities. Then it analyzed the possible causes, such as variable arrangement of ECM section in current textbooks. Lastly, the article recommended several suggestions, including giving a timely re- vision of the old ECM knowledge, designing an appropriate strategy for teaching, enumerating certain representative diseases to improve the ECM education. It appealed our teachers to pay more attention to how to make the module of ECM master well in near future. Key words: Medical cell biology; Extracellular matrix; Educational reform; Medical un- dergraduate

From fat cell biology to public health preventive strategies – pinpointing the critical period for obesity prevention

The prevalence of childhood and adolescence obesity is increasing to alarming proportions worldwide and poses a major public health problem by significantly elevating the risks of chronic diseases. There is strong evidence that childhood overweight and obesity are risk factors for severe obesity over the whole life course. In fact, longitudinal studies have found that most overweight/obese children would become overweight and obese adults. There is a lack of coupling in the scientific literature between adipose tissue development and biology to obesity prevention and treatment strategies. This is of utmost importance, especially regarding childhood and adolescence, as the major scientific paradigm in studies of adiposity is that the major number of adipocytes is set for life at this early age. This review discusses the current adipose cell biology paradigms to pinpoint the critical factors and periods in childhood overweight and obesity and, consecutively, to develop relevant prevention strategies.

Spot Variation Fluorescence Correlation Spectroscopy Allows for Superresolution Chronoscopy of Confinement Times in Membranes

Resolving the dynamical interplay of proteins and lipids in the live-cell plasma membrane represents a central goal in current cell biology. Superresolution concepts have introduced a means of capturing spatial heterogeneity at a nanoscopic length scale. Similar concepts for detecting dynamical transitions (superresolution chronoscopy) are still lacking. Here, we show that recently introduced spot-variation fluorescence correlation spectroscopy allows for sensing transient confinement times of membrane constituents at dramatically improved resolution. Using standard diffraction-limited optics, spot-variation fluorescence correlation spectroscopy captures signatures of single retardation events far below the transit time of the tracer through the focal spot. We provide an analytical description of special cases of transient binding of a tracer to pointlike traps, or association of a tracer with nanodomains. The influence of trap mobility and the underlying binding kinetics are quantified. Experimental approaches are suggested that allow for gaining quantitative mechanistic insights into the interaction processes of membrane constituents.

Regulation of phosphorylase kinase by low concentrations of Ca ions upon muscle contraction: the connection between metabolism and muscle contraction and the connection between muscle physiology and Ca-dependent signal transduction

It had long been one of the crucial questions in muscle physiology how glycogenolysis is regulated in connection with muscle contraction, when we found the answer to this question in the last half of the 1960s. By that time, the two principal currents of muscle physiology, namely, the metabolic flow starting from glycogen and the mechanisms of muscle contraction, had already been clarified at the molecular level thanks to our senior researchers. Thus, the final question we had to answer was how to connect these two currents. We found that low concentrations of Ca ions (10−7–10−4 M) released from the sarcoplasmic reticulum for the regulation of muscle contraction simultaneously reversibly activate phosphorylase kinase, the enzyme regulating glycogenolysis. Moreover, we found that adenosine 3′,5′-monophosphate (cyclic AMP), which is already known to activate muscle phosphorylase kinase, is not effective in the absence of such concentrations of Ca ions. Thus, cyclic AMP is not effective by itself alone and only modifies the activation process in the presence of Ca ions (at that time, cyclic AMP-dependent protein kinase had not yet been identified). After a while, it turned out that our works have not only provided the solution to the above problem on muscle physiology, but have also been considered as the first report of Ca-dependent protein phosphorylation, which is one of the central problems in current cell biology. Phosphorylase kinase is the first protein kinase to phosphorylate a protein resulting in the change in the function of the phosphorylated protein, as shown by Krebs and Fischer. Our works further showed that this protein kinase is regulated in a Ca-dependent manner. Accordingly, our works introduced the concept of low concentrations of Ca ions, which were first identified as the regulatory substance of muscle contraction, to the vast field of Ca biology including signal transduction.

MicroRNAs in Human Diseases: From Cancer to Cardiovascular Disease

The great discovery of microRNAs (miRNAs) has revolutionized current cell biology and medical science. miRNAs are small conserved non-coding RNA molecules that post-transcriptionally regulate gene expression by targeting the 3' untranslated region of specific messenger RNAs for degradation or translational repression. New members of the miRNA family are being discovered on a daily basis and emerging evidence has demonstrated that miRNAs play a major role in a wide range of developmental process including cell proliferation, cell cycle, cell differentiation, metabolism, apoptosis, developmental timing, neuronal cell fate, neuronal gene expression, brain morphogenesis, muscle differentiation and stem cell division. Moreover, a large number of studies have reported links between alterations of miRNA homeostasis and pathological conditions such as cancer, psychiatric and neurological diseases, cardiovascular disease, and autoimmune disease. Interestingly, in addition, miRNA deficiencies or excesses have been correlated with a number of clinically important diseases ranging from cancer to myocardial infarction. miRNAs can repress the gene translation of hundreds of their targets and are therefore well-positioned to target a multitude of cellular mechanisms. As a consequence of extensive participation in normal functions, it is quite logical to ask the question if abnormalities in miRNAs should have importance in human diseases. Great discoveries and rapid progress in the past few years on miRNAs provide the hope that miRNAs will in the near future have a great potential in the diagnosis and treatment of many diseases. Currently, an explosive literature has focussed on the role of miRNA in human cancer and cardiovascular disease. In this review, I briefly summarize the explosive current studies about involvement of miRNA in various human cancers and cardiovascular disease.

The Role of MicroRNAs in Regulatory T Cells and in the Immune Response

The discovery of microRNA (miRNA) is one of the major scientific breakthroughs in recent years and has revolutionized current cell biology and medical science. miRNAs are small (19~25nt) noncoding RNA molecules that post-transcriptionally regulate gene expression by targeting the 3' untranslated region (3'UTR) of specific messenger RNAs (mRNAs) for degradation of translation repression. Genetic ablation of the miRNA machinery, as well as loss or degradation of certain individual miRNAs, severely compromises immune development and response, and can lead to immune disorders. Several sophisticated regulatory mechanisms are used to maintain immune homeostasis. Regulatory T (Treg) cells are essential for maintaining peripheral tolerance, preventing autoimmune diseases and limiting chronic inflammatory diseases. Recent publications have provided compelling evidence that miRNAs are highly expressed in Treg cells, that the expression of Foxp3 is controlled by miRNAs and that a range of miRNAs are involved in the regulation of immunity. A large number of studies have reported links between alterations of miRNA homeostasis and pathological conditions such as cancer, cardiovascular disease and diabetes, as well as psychiatric and neurological diseases. Although it is still unclear how miRNA controls Treg cell development and function, recent studies certainly indicate that this topic will be the subject of further research. The specific circulating miRNA species may also be useful for the diagnosis, classification, prognosis of diseases and prediction of the therapeutic response. An explosive literature has focussed on the role of miRNA. In this review, I briefly summarize the current studies about the role of miRNAs in Treg cells and in the regulation of the innate and adaptive immune response. I also review the explosive current studies about clinical application of miRNA.

The cytologic criteria of malignancy

Cytology and cell biology are two separate fields that share a focus on cancer. Cancer is still diagnosed based on morphology, and surprisingly little is known about the molecular basis of the defining structural features. Cytology uses the smallest possible biopsy for diagnosis by reducing morphologic "criteria of malignancy" to the smallest scale. To begin to develop common ground, members of the American Society of Cytopathology Cell Biology Liaison Working Group classify some of the "criteria of malignancy" and review their relation to current cell biology concepts. The criteria of malignancy are extremely varied, apparently reflecting many different pathophysiologies in specific microenvironments. Criteria in Group 1 comprise tissue-level alterations that appear to relate to resistance to anoikis, alterations in cell adhesion molecules, and loss of apical-basal polarity. Criteria in Group 2 reflect genetic instability, including chromosomal and possibly epigenetic instability. Criteria in Groups 3 are subcellular structural changes involving cytoplasmic components, nuclear lamina, chromatin and nucleoli that cannot be accounted for by genetic instability. Some distinct criteria in Group 3 are known to be induced by cancer genes, but their precise structural basis remains obscure. The criteria of malignancy are not closely related to the histogenetic classification of cancers, and they appear to provide an alternative, biologically relevant framework for establishing common ground between cytologists and cell biologists. To understand the criteria of malignancy at a molecular level would improve diagnosis, and likely point to novel cell physiologies that are not encompassed by current cell biology concepts.

Desmosomal molecules in and out of adhering junctions: normal and diseased States of epidermal, cardiac and mesenchymally derived cells.

Current cell biology textbooks mention only two kinds of cell-to-cell adhering junctions coated with the cytoplasmic plaques: the desmosomes (maculae adhaerentes), anchoring intermediate-sized filaments (IFs), and the actin microfilament-anchoring adherens junctions (AJs), including both punctate (puncta adhaerentia) and elongate (fasciae adhaerentes) structures. In addition, however, a series of other junction types has been identified and characterized which contain desmosomal molecules but do not fit the definition of desmosomes. Of these special cell-cell junctions containing desmosomal glycoproteins or proteins we review the composite junctions (areae compositae) connecting the cardiomyocytes of mature mammalian hearts and their importance in relation to human arrhythmogenic cardiomyopathies. We also emphasize the various plakophilin-2-positive plaques in AJs (coniunctiones adhaerentes) connecting proliferatively active mesenchymally-derived cells, including interstitial cells of the heart and several soft tissue tumor cell types. Moreover, desmoplakin has also been recognized as a constituent of the plaques of the complexus adhaerentes connecting certain lymphatic endothelial cells. Finally, we emphasize the occurrence of the desmosomal transmembrane glycoprotein, desmoglein Dsg2, out of the context of any junction as dispersed cell surface molecules in certain types of melanoma cells and melanocytes. This broadening of our knowledge on the diversity of AJ structures indicates that it may still be too premature to close the textbook chapters on cell-cell junctions.

Focal Adhesion: A Focal Point in Current Cell Biology and Molecular Medicine

Cell-extracellular matrix (ECM) is an important property of virtually all cells in multi-cellular organisms. Cell-ECM adhesion research, therefore, has broad impact on biology and medicine. Studies over the past three decades have resulted in tremendous advance in our understanding of the molecular basis and functions of cell-ECM adhesion. Here, I focus on some of the general lessons that we have learned from recent studies on cell-ECM adhesion. In addition, I highlight several topics in this rapidly advancing research area. These topics, which include assembly, disassembly and regulation of cell-ECM adhesion structures, the molecular mechanisms of bi-directional signaling through cell-ECM adhesions, and the tissue and organ pathobiology of cell-ECM adhesion, are pertinent to our understanding of cell-ECM adhesion and signaling.

Systematic Analysis of the Epidermal Growth Factor Receptor by Mass Spectrometry Reveals Stimulation-dependent Multisite Phosphorylation

Multisite phosphorylation of proteins is a general mechanism for modulation of protein function and molecular interactions. Definition of phosphorylation sites and elucidation of the functional interplay between multiple phosphorylated residues in proteins are, however, a major analytical challenge in current molecular cell biology and proteomic research. In the present study, we used mass spectrometry to determine the major phosphorylated residues of the human epidermal growth factor (EGF) receptor at various well defined cellular conditions. Activation of EGF receptor was achieved by several types of stimulation, i.e. by sodium pervanadate, EGF, and integrin-dependent adhesion. The contribution of cell-matrix adhesion was also determined by activating the EGF receptor by EGF in cells kept in suspension. We developed an analytical strategy that combined miniaturized sample preparation techniques and MALDI tandem mass spectrometry and determined a total of nine phosphorylation sites in the EGF receptor. We discovered one novel phosphorylation site (Ser967) and revealed constitutive phosphorylation of Thr669, Ser967, Ser1002, and Tyr1045 and stimulation-dependent differential phosphorylation of Tyr1068, Tyr1086, Ser1142, Tyr1148, and Tyr1173. The EGF receptor was purified from HeLa cells or ECV304 cells by immunoprecipitation and SDS-PAGE and then digested with trypsin. Phosphopeptides in the range of 0.8-3.7 kDa were recovered by combinations of IMAC, perfusion chromatography, and graphite powder chromatography and subsequently detected and sequenced by MALDI quadrupole time-of-flight tandem mass spectrometry. Two phosphorylation sites were detected in the peptide 1137GSHQISLDNPDYQQDFFPK1155; however, only Tyr1148 was phosphorylated upon EGF treatment; in contrast Ser1142 was only phosphorylated by integrin-dependent adhesion in the absence of EGF treatment, suggesting differential phosphorylation of this region by distinct stimuli. This MALDI MS/MS-based analytical approach demonstrates the feasibility of systematic analysis of signaling molecules by mass spectrometry and provides new insights into the dynamics of receptor signaling processes.