Characterization of heterogeneous primary human cartilage-derived cell population using non-invasive live-cell phase-contrast time-lapse imaging

Reliable and reproducible cell therapy strategies to treat osteoarthritis demand an improved characterization of the cell and heterogeneous cell population resident in native cartilage tissue. Using live-cell phase-contrast time-lapse imaging (PC-TLI), this study investigates the morphological attributes and biological performance of the three primary biological objects enzymatically isolated from primary human cartilage: connective tissue progenitors (CTPs), non-progenitors (NPs) and multi-cellular structures (MCSs). The authors’ results demonstrated that CTPs were smaller in size in comparison to NPs (P < 0.001). NPs remained part of the adhered cell population throughout the cell culture period. Both NPs and CTP progeny on day 8 increased in size and decreased in circularity in comparison to their counterparts on day 1, although the percent change was considerably less in CTP progeny (P < 0.001). PC-TLI analyses indicated three colony types: single-CTP-derived (29%), multiple-CTP-derived (26%) and MCS-derived (45%), with large heterogeneity with respect to cell morphology, proliferation rate and cell density. On average, clonal (CL) (P = 0.009) and MCS (P = 0.001) colonies exhibited higher cell density (cells per colony area) than multi-clonal (MC) colonies; however, it is interesting to note that the behavior of CL (less cells per colony and less colony area) and MCS (high cells per colony and high colony area) colonies was quite different. Overall effective proliferation rate (EPR) of the CTPs that formed CL colonies was higher than the EPR of CTPs that formed MC colonies (P = 0.02), most likely due to CTPs with varying EPR that formed the MC colonies. Finally, the authors demonstrated that lag time before first cell division of a CTP (early attribute) could potentially help predict its proliferation rate long-term. Quantitative morphological characterization using non-invasive PC-TLI serves as a reliable and reproducible technique to understand cell heterogeneity. Size and circularity parameters can be used to distinguish CTP from NP populations. Morphological cell and colony features can also be used to reliably and reproducibly identify CTP subpopulations with preferred proliferation and differentiation potentials in an effort to improve cell manufacturing and therapeutic outcomes.     

Cellular and molecular mechanisms of cerebellar granule cell migration

The real-time observation of cell movement in brain slice preparations reveals that in the developing brain, postmitotic neurons alter their shape concomitantly with changes in the mode, direction, tempo, and rate of migration as they traverse different cortical layers. Although it has been hypothesized that orchestrated activities of multiple external cues and cell-cell contact are essential for controlling the cortical-layer-specific changes in cell migration, signaling mechanisms and external guidance cues related to the alteration of neuronal cell migration remain to be determined. In this article, we will first review recent studies on position-specific changes in granule cell behavior through different migratory terrains of the developing cerebellar cortex. We will then present possible roles for the coordinated activity of Ca2+ channels, NMDA type of glutamate receptors, and intracellular Ca2+ fluctuations in controlling cerebellar granule cell movement. Furthermore, we will discuss the crucial roles of brain-derived neurotrophic factor (BDNF), neuregulin (NRG), stromal cell-derived factor 1alpha (SDF-1alpha), ephrin-B2, and EphB2 receptor in providing directional cues promoting granule cell migration from the external granular layer (EGL) to the internal granular layer (IGL). Finally, we will demonstrate that endogenous somatostatin controls the migration of granule cells in a cortical layer-specific manner: Endogenous somatostatin accelerates granule cell movement near the birthplace within the EGL, but significantly slows down the movement near their final destination within the IGL.     

Cellular and molecular mechanisms of cerebellar granule cell migration

The real-time observation of cell movement in brain slice preparations reveals that in the developing brain, postmitotic neurons alter their shape concomitantly with changes in the mode, direction, tempo, and rate of migration as they traverse different cortical layers. Although it has been hypothesized that orchestrated activities of multiple external cues and cell-cell contact are essential for controlling the cortical-layer-specific changes in cell migration, signaling mechanisms and external guidance cues related to the alteration of neuronal cell migration remain to be determined. In this article, we will first review recent studies on position-specific changes in granule cell behavior through different migratory terrains of the developing cerebellar cortex. We will then present possible roles for the coordinated activity of Ca²⁺ channels, NMDA type of glutamate receptors, and intracellular Ca²⁺ fluctuations in controlling cerebellar granule cell movement. Furthermore, we will discuss the crucial roles of brain-derived neurotrophic factor (BDNF), neuregulin (NRG), stromal cell-derived factor 1α (SDF-1α), ephrin-B2, and EphB2 receptor in providing directional cues promoting granule cell migration from the external granular layer (EGL) to the internal granular layer (IGL). Finally, we will demonstrate that endogenous somatostatin controls the migration of granule cells in a cortical layer-specific manner: Endogenous somatostatin accelerates granule cell movement near the birthplace within the EGL, but significantly slows down the movement near their final destination within the IGL.     

Cellular and molecular mechanisms of neuronal migration in neocortical development

The complicated mammalian brain structure arises from accurate movements of neurons from their birthplace to their final locations. Detailed observation of this migration process by various methods revealed that neuronal migration is highly motile and that there are different modes of migration. Moreover, mouse mutants or human disorders that disrupt normal migration have provided significant insights into molecular pathways that control the neuronal migration. Although our knowledge is still fragmentary, it is becoming clear that various molecules are participating in this process. In this review, we outline about the cellular and molecular mechanisms of neuronal migration in the cerebral cortex.     

Cellular and molecular mechanisms controlling the migration of melanocytes and melanoma cells

During embryonic development in vertebrates, the neural crest‐derived melanoblasts migrate along the dorsolateral axis and cross the basal membrane separating the dermis from the epidermis to reach their final location in the interfollicular epidermis and epidermal hair follicles. Neoplastic transformation converts melanocytes into highly invasive and metastatic melanoma cells. In vitro, these cells extend various types of protrusions and adopt two interconvertible modes of migration, mesenchymal and amoeboid, driven by different signalling molecules. In this review, we describe the major contributions of natural mouse mutants, mouse models generated by genetic engineering and in vitro culture systems, to identification of the genes, signalling pathways and mechanisms regulating the migration of normal and pathological cells of the melanocyte lineage, at both the cellular and molecular levels.     

Quantitative measurement of cell migration using time-lapse videomicroscopy and non-linear system analysis

Epithelial cells of the mammary gland possess the inherent capacity to form epithelial monolayers in vitro. This requires coordination of cell migration, cell-cell contact formation, and cell proliferation. Using time-lapse phase contrast videomicroscopy we have observed mammary gland epithelial cells over different time scales. We show the generation of a complete polarized epithelial monolayer in real-time, starting from a few cells. We subsequently concentrated on the early stages of this process by tracking epithelial cells during phases of polarized migration. We performed migration analysis using fractal measures. With this technology the structure of seemingly random processes not accessible to the usual methods of linear analysis can be measured. As a control and proof of principle approach we applied infection of cells with an adenoviral vector, which is used as a gene targeting vector for many applications. Infection markedly influenced the patterns of migratory behavior. We, therefore, believe that time-lapse videomicroscopy in combination with fractal analysis can contribute to differential characterization of distinct cellular migration patterns. This will be useful in situations of long-term alterations in cell culture systems.     

Cellular mechanisms of nuclear migration

Subcellular mobility, positioning, and directional movement of the nucleus in a certain site of the cell or cenocyte and, less frequently, intercellular translocation of the nucleus accompany the cell and tissue differentiation, change of their functions, and the organism growth and development and its response to stress, plant–microbial interactions, symbiosis, and many other processes in plants and animals. The nucleus movement is performed and directed through the interaction between dynamic cytoskeleton components and nucleus by means of signal-binding proteins, including motor and linker. The cell responds to the external signal by mobilization and polar reconstruction of the cytoskeleton components, as a result of which the nucleus displacement by means of actomyosin or microtubule mechanisms in cooperation with dynein and kinesin occurs. In plants, the actomyosin mechanism is involved in the nucleus migration; it allows the nucleus to move rapidly and over significant distances in response to environmental stimuli. An important role in the nucleus translocation belongs to the linker complexes of the proteins that are inserted in the nuclear envelope, that connect and transmit signals from the plasmalemma to the cytoplasm and nucleoplasm, and that provide the skeletal basis for many subcellular compartments. Changes in the protein composition, conformational modifications of the proteins, and displacement of linkers from the nuclear envelope result in the nucleus detachment from the cytoskeleton, and change in the form, mechanical rigidity, and positioning of the nucleus.     

Automated time-lapse microscopy and high-resolution tracking of cell migration

We describe a novel fully automated high-throughput time-lapse microscopy system and evaluate its performance for precisely tracking the motility of several glioma and osteoblastic cell lines. Use of this system revealed cell motility behavior not discernable with conventional techniques by collecting data (1) from closely spaced time points (minutes), (2) over long periods (hours to days), (3) from multiple areas of interest, (4) in parallel under several different experimental conditions. Quantitation of true individual and average cell velocity and path length was obtained with high spatial and temporal resolution in “scratch” or “wound healing” assays. This revealed unique motility dynamics of drug-treated and adhesion molecule-transfected cells and, thus, this is a considerable improvement over current methods of measurement and analysis. Several fluorescent vital labeling methods commonly used for end-point analyses (GFP expression, DiO lipophilic dye, and Qtracker nanocrystals) were found to be useful for time-lapse studies under specific conditions that are described. To illustrate one application, fluorescently labeled tumor cells were seeded onto cell monolayers expressing ectopic adhesion molecules, and this resulted in consistently reduced tumor cell migration velocities. These highly quantitative time-lapse analysis methods will promote the creation of new cell motility assays and increase the resolution and accuracy of existing assays.Joseph S. Fotos and Vivek P. Patel contributed equally to this work     

Cellular and molecular mechanisms of memory

There has been nearly a century of interest in the idea that information is encoded in the brain as specific spatio-temporal patterns of activity in distributed networks and stored as changes in the efficacy of synaptic connections on neurons that are activated during learning. The discovery and detailed report of the phenomenon generally known as long-term potentiation opened a new chapter in the study of synaptic plasticity in the vertebrate brain, and this form of synaptic plasticity has now become the dominant model in the search for the cellular bases of learning and memory. To date, the key events in the cellular and molecular mechanisms underlying synaptic plasticity are starting to be identified. They require the activation of specific receptors and of several molecular cascades to convert extracellular signals into persistent functional changes in neuronal connectivity. Accumulating evidence suggests that the rapid activation of the genetic machinery is a key mechanism underlying the enduring modification of neural networks required for the laying down of memory. The recent developments in the search for the cellular and molecular mechanisms of memory storage are reviewed.     

神经元迁移的细胞和分子机制

在脑的发育过程中,神经元的正确迁移是正常脑组织发生的一个必不可少的环节。在过去的几十年中,通过不同的学科方法,对于神经元迁移的机制有了较好的理解。在细胞水平上,神经元迁移需要3个重复事件的精确调控;在分子水平,与神经元迁移相关的胞外信号分子已经被鉴定,而且大量的胞内信号通路也已经被阐明。     

Cellular bases of olfactory circuit assembly revealed by systematic time-lapse imaging

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飞蝗嗅觉的细胞与分子机制研究进展

生命的进化依赖于其周边的化学环境,通过对这些化学物质的感受,适应环境,生命得以繁衍。直到现在,各种有机体仍然保留着这种古老而有效的感知方式。飞蝗是世界性的农业大害虫,其很多行为如远距离迁飞、聚集、取食、产卵等是其造成灾害的重要生物学因素,而这些行为都与其感受化学信息相关。深入研究飞蝗感受化学信息的机制对于揭示生物感受化学信息的分子和细胞机制的多样性,设计出可以激发或钝化这些蛋白质的引诱剂或忌避剂,进而防治害虫等具有重要意义。该文主要介绍了该课题组在东亚飞蝗(Locusta migratoria manilesis)感受化学信息机制方面的一些进展。通过超微结构研究发现在飞蝗触角上至少有毛形、锥形、腔锥形和刺形4种类型的化学感受器,明确了各种感受器的超微结构特征,其中毛形和锥形是重要的嗅觉感受器。以此为基础,单感受器电位记录试验结果表明飞蝗触角上的毛形感受器至少有7种功能亚型,其中5种亚型每个感受器含有2个神经原,2种亚型每个感受器含有3种神经原。初步明确了飞蝗毛形感受器神经原对一些化学信息的编码特征。在飞蝗的触角中鉴定出了飞蝗气味分子结合蛋白(LmigOBP1),通过免疫细胞化学定位实验证明该蛋白特异表达在飞蝗毛形和锥形感受器的淋巴液中,而且在胚胎即将孵化前就开始表达,此后在各个胚后发育时期都表达,说明该蛋白可能参与飞蝗胚后发育的所有阶段的嗅觉活动。采用荧光竞争结合实验方法,明确了LmigOBP1对有15～17个碳原子的直链的脂肪族醇、酯或醛有很强的亲和力,说明该蛋白有结合特异性。采用生物信息学技术模拟出了更为合理的LmigOBP1的三维结构,通过对接实验,提出了飞蝗气味分子结合蛋白结合腔中可能参与结合十五醇的氨基酸残基。之后通过定点氨基酸突变将59位的丝氨酸、74位的天冬酰氨和87位的缬氨酸分别用丙氨酸替代获得三个突变体蛋白(S59A、N74A、V87A),通过与野生型蛋白荧光竞争结合实验结果的比较,发现突变体S59A的结合模式与野生型相同,N74A几乎丧失了全部结合能力,而V87A则对有些气味分子的结合能力有较大改变。因此,位于结合腔开口处的74位天冬酰氨是该蛋白的重要结合位点,而位于结合腔底部的87位缬氨酸也是结合位点。结合前人的结果,我们首次提出了昆虫气味分子结合蛋白依赖位于结合腔开口处的亲水性氨基酸实现对气味分子的初始识别的假说。文章最后对今后研究的一些重点进行了讨论。     

Cellular and molecular mechanisms of muscle atrophy

Skeletal muscle is a plastic organ that is maintained by multiple pathways regulating cell and protein turnover. During muscle atrophy, proteolytic systems are activated, and contractile proteins and organelles are removed, resulting in the shrinkage of muscle fibers. Excessive loss of muscle mass is associated with poor prognosis in several diseases, including myopathies and muscular dystrophies, as well as in systemic disorders such as cancer, diabetes, sepsis and heart failure. Muscle loss also occurs during aging. In this paper, we review the key mechanisms that regulate the turnover of contractile proteins and organelles in muscle tissue, and discuss how impairments in these mechanisms can contribute to muscle atrophy. We also discuss how protein synthesis and degradation are coordinately regulated by signaling pathways that are influenced by mechanical stress, physical activity, and the availability of nutrients and growth factors. Understanding how these pathways regulate muscle mass will provide new therapeutic targets for the prevention and treatment of muscle atrophy in metabolic and neuromuscular diseases.     

肝纤维化的细胞和分子机制

肝脏纤维化(hepatic fibrosis)是多种慢性肝病的共同病理基础,是进一步向肝硬化发展的中心环节。肝脏内一些免疫细胞如枯否细胞(Kupffer cell,KC)、树突状细胞(dendritic cell,DC)、T淋巴细胞、NK细胞(nature killer cell,NK cell)、B细胞等在多种致病因素刺激下激活,释放多种细胞因子和趋化因子,引起一系列病理变化,共同参与肝纤维化的发生和发展过程。本文主要从肝脏内各类免疫细胞以及分泌的细胞因子方面,对肝纤维化形成机制的最新研究进展进行综述。     

A fast and robust quantitative time-lapse assay for cell migration

We describe a simple and widely applicable method to measure cell migration in time-lapse sequences of fluorescently labeled cells in culture. Briefly, binarized cell images obtained after thresholding were cumulatively projected, and the covered areas were measured. This procedure determines the time course of the track area successively covered by the cell population. Under conditions where cell growth is negligible, a robust index of cell motility is derived from normalized plots for the displacement of cells over time. We applied this method to quantitatively examine the migration of B35 neuroblastoma cells transiently expressing GFP and to C6 glioma cells after staining with Hoechst 33258. This sensitive assay detected the influence of agents which inhibit actin polymerization (cytochalasin B) or interfere with the maintenance of cell polarity (methyl-beta-cyclodextrin) on cell migration. Thus, this assay is a versatile tool to measure quickly the migration of different cell types using different labeling strategies.     

Autophagy pathway: Cellular and molecular mechanisms

Macroautophagy/autophagy is an essential, conserved self-eating process that cells perform to allow degradation of intracellular components, including soluble proteins, aggregated proteins, organelles, macromolecular complexes, and foreign bodies. The process requires formation of a double-membrane structure containing the sequestered cytoplasmic material, the autophagosome, that ultimately fuses with the lysosome. This review will define this process and the cellular pathways required, from the formation of the double membrane to the fusion with lysosomes in molecular terms, and in particular highlight the recent progress in our understanding of this complex process.     

Cellular and molecular mechanisms in focal epileptogenesis

In the review, it is presented an analysis of experimental data about cellular and molecular mechanisms of focal epileptogenesis. Basic principals of synchronized burst activity development in epileptogenic focus are considered. The roles of synaptic activities and extrasynaptic membrane excitability for epileptiform activity development are discussed. The various pathways of Ca2+ entry into neurones as well as an involvement of Ca2+/calmodulin-dependent protein phosphorylation in mechanisms of epileptogenesis are analyzed. In vitro and in vivo experimental models of epileptogenesis (especially, kindling and audiogenic seizures) allowing to study the predisposition of neuronal circuit to epileptiform activity development are discussed.