Long-distance electrical coupling via tunneling nanotubes

Tunneling nanotubes (TNTs) are nanoscaled, F-actin containing membrane tubes that connect cells over several cell diameters. They facilitate the intercellular exchange of diverse components ranging from small molecules to organelles and pathogens. In conjunction with recent findings that TNT-like structures exist in tissue, they are expected to have important implications in cell-to-cell communication. In this review we will focus on a new function of TNTs, namely the transfer of electrical signals between remote cells. This electrical coupling is not only determined by the biophysical properties of the TNT, but depends on the presence of connexons interposed at the membrane interface between TNT and the connected cell. Specific features of this coupling are compared to conventional gap junction communication. Finally, we will discuss possible down-stream signaling pathways of this electrical coupling in the recipient cells and their putative effects on different physiological activities. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

Tunneling nanotubes: emerging view of their molecular components and formation mechanisms

Cell-to-cell communication is essential for the development and maintenance of multicellular organisms. The tunneling nanotube (TNT) is a recently recognized distinct type of intercellular communication device. TNTs are thin protrusions of the plasma membrane and allow direct physical connections of the plasma membranes between remote cells. The proposed functions for TNTs include the cell-to-cell transfer of large cellular structures such as membrane vesicles and organelles, as well as signal transduction molecules in a wide variety of cell types. Moreover TNT and TNT-related structures are thought to facilitate the intercellular spreading of virus and/or pathogenic proteins. Despite their contribution to normal cellular functions and importance in pathological conditions, virtually nothing is known about the molecular basis for their formation. We have recently shown that M-Sec (also called TNFaip2) is a key molecule for TNT formation. In cooperation with the RalA small GTPase and the exocyst complex, M-Sec can induce the formation of functional TNTs, indicating that the remodeling of the actin cytoskeleton and vesicle trafficking are involved in M-Sec-mediated TNT formation. Discovery of the role of M-Sec will accelerate our understanding of TNTs, both at the molecular and physiological levels.     

Impact of image segmentation on high-content screening data quality for SK-BR-3 cells

Background  

High content screening (HCS) is a powerful method for the exploration of cellular signalling and morphology that is rapidly being adopted in cancer research. HCS uses automated microscopy to collect images of cultured cells. The images are subjected to segmentation algorithms to identify cellular structures and quantitate their morphology, for hundreds to millions of individual cells. However, image analysis may be imperfect, especially for "HCS-unfriendly" cell lines whose morphology is not well handled by current image segmentation algorithms. We asked if segmentation errors were common for a clinically relevant cell line, if such errors had measurable effects on the data, and if HCS data could be improved by automated identification of well-segmented cells.     

Tunneling-nanotube development in astrocytes depends on p53 activation

Tunneling nanotubes (TNTs) can be induced in rat hippocampal astrocytes and neurons with H(2)O(2) or serum depletion. Major cytoskeletal component of TNTs is F-actin. TNTs transfer endoplasmic reticulum, mitochondria, Golgi, endosome and intracellular as well as extracellular amyloid β. TNT development is a property of cells under stress. When two populations of cells are co-cultured, it is the stressed cells that always develop TNTs toward the unstressed cells. p53 is crucial for TNT development. When p53 function is deleted by either dominant negative construct or siRNAs, TNT development is inhibited. In addition, we find that among the genes activated by p53, epidermal growth factor receptor is also important to TNT development. Akt, phosphoinositide 3-kinase and mTOR are involved in TNT induction. Our data suggest that TNTs might be a mechanism for cells to respond to harmful signals and transfer cellular substances or energy to another cell under stress.     

Image analysis for understanding embryo development: a bridge from microscopy to biological insights

The digital reconstruction of the embryogenesis of model organisms from 3D+time data is revolutionizing practices in quantitative and integrative Developmental Biology. A manual and fully supervised image analysis of the massive complex data acquired with new microscopy technologies is no longer an option and automated image processing methods are required to fully exploit the potential of imaging data for biological insights. Current developments and challenges in biological image processing include algorithms for microscopy multiview fusion, cell nucleus tracking for quasi-perfect lineage reconstruction, segmentation, and validation methodologies for cell membrane shape identification, single cell gene expression quantification from in situ hybridization data, and multidimensional image registration algorithms for the construction of prototypic models. These tools will be essential to ultimately produce the multilevel in toto reconstruction that combines the cell lineage tree, cells, and tissues structural information and quantitative gene expression data in its spatio-temporal context throughout development.     

Selective block of tunneling nanotube (TNT) formation inhibits intercellular organelle transfer between PC12 cells

Organelle exchange between cells via tunneling nanotubes (TNTs) is a recently described form of intercellular communication. Here, we show that the selective elimination of filopodia from PC12 cells by 350 nM cytochalasin B (CytoB) blocks TNT formation but has only a weak effect on the stability of existing TNTs. Under these conditions the intercellular organelle transfer was strongly reduced, whereas endocytosis and phagocytosis were not affected. Furthermore, the transfer of organelles significantly correlated with the presence of a TNT-bridge. Thus, our data support that in PC12 cells filopodia-like protrusions are the principal precursors of TNTs and CytoB provides a valuable tool to selectively interfere with TNT-mediated cell-to-cell communication.     

Computer-aided 2D and 3D quantification of human stem cell fate from in vitro samples using Volocity high performance image analysis software

Accurate automated cell fate analysis of immunostained human stem cells from 2- and 3-dimensional (2D-3D) images would improve efficiency in the field of stem cell research. Development of an accurate and precise tool that reduces variability and the time needed for human stem cell fate analysis will improve productivity and interpretability of the data across research groups. In this study, we have created protocols for high performance image analysis software Volocity? to classify and quantify cytoplasmic and nuclear cell fate markers from 2D-3D images of human neural stem cells after in vitro differentiation. To enhance 3D image capture efficiency, we optimized the image acquisition settings of an Olympus FV10i? confocal laser scanning microscope to match our quantification protocols and improve cell fate classification. The methods developed in this study will allow for a more time efficient and accurate software based, operator validated, stem cell fate classification and quantification from 2D and 3D images, and yield the highest ≥94.4% correspondence with human recognized objects.     

Rhes Tunnels: A Radical New Way of Communication in the Brain's Striatum?

Ras homolog enriched in the striatum (Rhes) is a striatal enriched protein that promotes the formation of thin membranous tubes resembling tunneling nanotubes (TNT)—“Rhes tunnels”—that connect neighboring cell and transport cargoes: vesicles and proteins between the neuronal cells. Here the literature on TNT-like structures is reviewed, and the implications of Rhes-mediated TNT, the mechanisms of its formation, and its potential in novel cell-to-cell communication in regulating striatal biology and disease are emphasized. Thought-provoking ideas regarding how Rhes-mediated TNT, if it exists, in vivo, would radically change the way neurons communicate in the brain are discussed.     

Automated Quantification and Integrative Analysis of 2D and 3D Mitochondrial Shape and Network Properties

Mitochondrial morphology and function are coupled in healthy cells, during pathological conditions and (adaptation to) endogenous and exogenous stress. In this sense mitochondrial shape can range from small globular compartments to complex filamentous networks, even within the same cell. Understanding how mitochondrial morphological changes (i.e. “mitochondrial dynamics”) are linked to cellular (patho) physiology is currently the subject of intense study and requires detailed quantitative information. During the last decade, various computational approaches have been developed for automated 2-dimensional (2D) analysis of mitochondrial morphology and number in microscopy images. Although these strategies are well suited for analysis of adhering cells with a flat morphology they are not applicable for thicker cells, which require a three-dimensional (3D) image acquisition and analysis procedure. Here we developed and validated an automated image analysis algorithm allowing simultaneous 3D quantification of mitochondrial morphology and network properties in human endothelial cells (HUVECs). Cells expressing a mitochondria-targeted green fluorescence protein (mitoGFP) were visualized by 3D confocal microscopy and mitochondrial morphology was quantified using both the established 2D method and the new 3D strategy. We demonstrate that both analyses can be used to characterize and discriminate between various mitochondrial morphologies and network properties. However, the results from 2D and 3D analysis were not equivalent when filamentous mitochondria in normal HUVECs were compared with circular/spherical mitochondria in metabolically stressed HUVECs treated with rotenone (ROT). 2D quantification suggested that metabolic stress induced mitochondrial fragmentation and loss of biomass. In contrast, 3D analysis revealed that the mitochondrial network structure was dissolved without affecting the amount and size of the organelles. Thus, our results demonstrate that 3D imaging and quantification are crucial for proper understanding of mitochondrial shape and topology in non-flat cells. In summary, we here present an integrative method for unbiased 3D quantification of mitochondrial shape and network properties in mammalian cells.     

FogBank: a single cell segmentation across multiple cell lines and image modalities

Background

Many cell lines currently used in medical research, such as cancer cells or stem cells, grow in confluent sheets or colonies. The biology of individual cells provide valuable information, thus the separation of touching cells in these microscopy images is critical for counting, identification and measurement of individual cells. Over-segmentation of single cells continues to be a major problem for methods based on morphological watershed due to the high level of noise in microscopy cell images. There is a need for a new segmentation method that is robust over a wide variety of biological images and can accurately separate individual cells even in challenging datasets such as confluent sheets or colonies.

Results

We present a new automated segmentation method called FogBank that accurately separates cells when confluent and touching each other. This technique is successfully applied to phase contrast, bright field, fluorescence microscopy and binary images. The method is based on morphological watershed principles with two new features to improve accuracy and minimize over-segmentation.First, FogBank uses histogram binning to quantize pixel intensities which minimizes the image noise that causes over-segmentation. Second, FogBank uses a geodesic distance mask derived from raw images to detect the shapes of individual cells, in contrast to the more linear cell edges that other watershed-like algorithms produce.We evaluated the segmentation accuracy against manually segmented datasets using two metrics. FogBank achieved segmentation accuracy on the order of 0.75 (1 being a perfect match). We compared our method with other available segmentation techniques in term of achieved performance over the reference data sets. FogBank outperformed all related algorithms. The accuracy has also been visually verified on data sets with 14 cell lines across 3 imaging modalities leading to 876 segmentation evaluation images.

Conclusions

FogBank produces single cell segmentation from confluent cell sheets with high accuracy. It can be applied to microscopy images of multiple cell lines and a variety of imaging modalities. The code for the segmentation method is available as open-source and includes a Graphical User Interface for user friendly execution.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0431-x) contains supplementary material, which is available to authorized users.     

Vertebrate neural stem cell segmentation, tracking and lineaging with validation and editing

This protocol and the accompanying software program called LEVER (lineage editing and validation) enable quantitative automated analysis of phase-contrast time-lapse images of cultured neural stem cells. Images are captured at 5-min intervals over a period of 5-15 d as the cells proliferate and differentiate. LEVER automatically segments, tracks and generates lineage trees of the stem cells from the image sequence. In addition to generating lineage trees capturing the population dynamics of clonal development, LEVER extracts quantitative phenotypic measurements of cell location, shape, movement and size. When available, the system can include biomolecular markers imaged using fluorescence. It then displays the results to the user for highly efficient inspection and editing to correct any errors in the segmentation, tracking or lineaging. To enable high-throughput inspection, LEVER incorporates features for rapid identification of errors and for learning from user-supplied corrections to automatically identify and correct related errors.     

Segmentation of the Clustered Cells with Optimized Boundary Detection in Negative Phase Contrast Images

Cell image segmentation plays a central role in numerous biology studies and clinical applications. As a result, the development of cell image segmentation algorithms with high robustness and accuracy is attracting more and more attention. In this study, an automated cell image segmentation algorithm is developed to get improved cell image segmentation with respect to cell boundary detection and segmentation of the clustered cells for all cells in the field of view in negative phase contrast images. A new method which combines the thresholding method and edge based active contour method was proposed to optimize cell boundary detection. In order to segment clustered cells, the geographic peaks of cell light intensity were utilized to detect numbers and locations of the clustered cells. In this paper, the working principles of the algorithms are described. The influence of parameters in cell boundary detection and the selection of the threshold value on the final segmentation results are investigated. At last, the proposed algorithm is applied to the negative phase contrast images from different experiments. The performance of the proposed method is evaluated. Results show that the proposed method can achieve optimized cell boundary detection and highly accurate segmentation for clustered cells.     

3D cell nuclei segmentation based on gradient flow tracking

Background  

Reliable segmentation of cell nuclei from three dimensional (3D) microscopic images is an important task in many biological studies. We present a novel, fully automated method for the segmentation of cell nuclei from 3D microscopic images. It was designed specifically to segment nuclei in images where the nuclei are closely juxtaposed or touching each other. The segmentation approach has three stages: 1) a gradient diffusion procedure, 2) gradient flow tracking and grouping, and 3) local adaptive thresholding.     

Automated classification and quantification of F-actin-containing ruffles in confocal micrographs

Membrane ruffles are actin-rich protrusions of the plasma membrane that can be observed on the surface of many cell types. Phase contrast and fluorescent microscopy are widely used in the analysis of ruffles, which are commonly identified in cells stained with fluorescently labeled phalloidin. Currently, comparison of cellular ruffle formation under different experimental conditions is generally qualitative or semiquantitative. Ruffle structures are often defined using manual tracing and thresholding methods. Here, we report the rapid and accurate segmentation of ruffles from two-dimensional confocal projections of cells using automated method based on well-established image processing and analysis methods. Line-shaped ruffles were detected using line detectors and were then separated from the filtered images. Automated categorizing of the segmented line structures enabled accurate quantification of the ruffles. This automated approach is efficient and reliable and hence can serve as a powerful tool in studies of the mechanism of ruffle formation.     

Transfer of mitochondria via tunneling nanotubes rescues apoptotic PC12 cells

Tunneling nanotubes (TNTs) are F-actin-based membrane tubes that form between cells in culture and in tissues. They mediate intercellular communication ranging from electrical signalling to the transfer of organelles. Here, we studied the role of TNTs in the interaction between apoptotic and healthy cells. We found that pheochromocytoma (PC) 12 cells treated with ultraviolet light (UV) were rescued when cocultured with untreated PC12 cells. UV-treated cells formed a different type of TNT with untreated PC12 cells, which was characterized by continuous microtubule localized inside these TNTs. The dynamic behaviour of mCherry-tagged end-binding protein 3 and the accumulation of detyrosinated tubulin in these TNTs indicate that they are regulated structures. In addition, these TNTs show different biophysical properties, for example, increased diameter allowing dye entry, prolonged lifetime and decreased membrane fluidity. Further studies demonstrated that microtubule-containing TNTs were formed by stressed cells, which had lost cytochrome c but did not enter into the execution phase of apoptosis characterized by caspase-3 activation. Moreover, mitochondria colocalized with microtubules in TNTs and transited along these structures from healthy to stressed cells. Importantly, impaired formation of TNTs and untreated cells carrying defective mitochondria were unable to rescue UV-treated cells in the coculture. We conclude that TNT-mediated transfer of functional mitochondria reverse stressed cells in the early stages of apoptosis. This provides new insights into the survival mechanisms of damaged cells in a multicellular context.Apoptosis is an important regulatory mechanism of tissue homeostasis. It is triggered by the extrinsic pathway through the activation of proapoptotic receptors or by the intrinsic pathway through the destabilization of mitochondria in response to various forms of cell injury or stress.¹ Notably, stressed cells are also strongly influenced by intercellular communicative networks. This includes diffusible growth factors, cytokines and other small molecules secreted from neighbouring cells, which can modulate the fate of distressed cells. For example, stem cells release growth factors to protect dysfunctional neurons in the brain.² In tumour stroma, activated fibroblasts are thought to promote tumour progression by secreting growth factors that act in a paracrine manner.³ Moreover, contact-dependent signalling, for example, via adhesion molecules, can trigger contact inhibition or protection of endothelial cells.⁴ In addition, gap junctions have been shown to be involved in the transfer of death or survival molecules in different cell types.⁵ Therefore, the signals transferred from neighbouring cells influence the viability of target cells through different pathways.In 2004, our group described a previously unrecognized form of cell-to-cell interaction based on nanoscaled, F-actin-containing membrane tubes.^{6, 7} These tubes, referred to as membrane or tunneling nanotubes (TNTs), were subsequently found in numerous cell types in culture and in tissues.^{8, 9, 10, 11} Importantly, TNTs facilitate the intercellular exchange of diverse cellular signals and components ranging from electrical signalling to organelles.^{12, 13, 14, 15} Moreover, pathogens such as human immunodeficiency virus (HIV) and prions can spread between cells along TNTs.^{16, 17} Consistent with the model that TNTs are involved in cell-to-cell communication, apoptosis regulators may be transferred via TNTs between apoptotic and healthy cells to alter the fate of recipient cells. Indeed, it has been shown that TNTs can propagate the death signal Fas ligand between T lymphocytes to induce cell death.^{18, 19} TNTs have been also proposed to participate in the rescue of injured cardiomyoblasts or endothelial cells by mesenchymal stem cells (MSCs) through transferred mitochondria.^{20 ,21} However, the rescue mechanism by how and when this event was accomplished remains elusive.In this study, we found that PC12 cells stressed by ultraviolet (UV) radiation were rescued from apoptosis when cocultured with untreated, healthy PC12 cells. Single-cell analysis showed that stressed cells in the early stages of apoptosis form a new type of TNT to interact with untreated cells. These TNTs have a distinct cytoskeletal composition and biophysical properties when compared with TNTs interconnecting normal PC12 cells. We also observed the presence and transport of mitochondria in the TNTs formed by stressed cells. Notably, the rescue effect was inhibited when the formation of TNTs were impaired by incubating with an F-actin-depolymerizing drug, or when the mitochondria of rescuer cells were damaged. Our results suggest that the delivery of functional mitochondria via TNTs mediates the recovery of PC12 cells in the early stages of apoptosis.     

Cell communication by tunneling nanotubes: Implications in disease and therapeutic applications

Intercellular communication is essential for the development and maintenance of multicellular organisms. Tunneling nanotubes (TNTs) are a recently recognized means of long and short distance communication between a wide variety of cell types. TNTs are transient filamentous membrane protrusions that connect cytoplasm of neighboring or distant cells. Cytoskeleton fiber-mediated transport of various cargoes occurs through these tubules. These cargoes range from small ions to whole organelles. TNTs have been shown to contribute not only to embryonic development and maintenance of homeostasis, but also to the spread of infectious particles and resistance to therapies. These functions in the development and progression of cancer and infectious disease have sparked increasing scrutiny of TNTs, as their contribution to disease progression lends them a promising therapeutic target. Herein, we summarize the current knowledge of TNT structure and formation as well as the role of TNTs in pathology, focusing on viral, prion, and malignant disease. We then discuss the therapeutic possibilities of TNTs in light of their varied functions. Despite recent progress in the growing field of TNT research, more studies are needed to precisely understand the role of TNTs in pathological conditions and to develop novel therapeutic strategies.     

Automated fluorescence image cytometry. DNA quantification and detection of chlamydial infections

Digitized fluorescence microscopy in conjunction with automated image segmentation is a promising approach for screening clinical specimens quickly and reliably. This paper describes the hardware and software of a prototype image-based cytometer that can identify fluorescent objects, discriminate true objects from artifacts and divide overlapping pairs of objects. The use of this image cytometer is discussed for: (1) the measurement of the DNA ploidy distribution of isolated mature rat liver nuclei labeled with 4',6-diamidine-2-phenylindole; (2) the comparison of the DNA ploidy distributions of the same samples measured by image cytometry (ICM) and flow cytometry (FCM); and (3) the quantification of chlamydial infection by double labeling cells with antichlamydiae antibody and Hoechst 33258 for nuclear DNA analysis. Ploidy distributions measured by the automated image cytometer compared favorably to those obtained by FCM. All pairs of overlapping nuclei were automatically detected by an additional computer algorithm, and those pairs that were clearly more than one nucleus by visual inspection were correctly divided. The irregular morphology of the chlamydiae-infected cells meant that 26% of them were not correctly identified in the fluorescein-stained images (as judged by manual inspection), but all cells were nevertheless detected correctly from the images of the Hoechst-stained samples. Automated fluorescence ICM yielded results similar to those obtained with FCM and had the additional benefit of maintaining cell and tissue architecture while preserving the opportunity for subsequent manual inspection of the specimen.     

New Automated Single-Cell Technique for Segmentation and Quantitation of Lipid Droplets

Lipid droplets are the major organelle for intracellular storage of triglycerides and cholesterol esters. Various methods have been attempted for automated quantitation of fluorescently stained lipid droplets using either thresholding or watershed methods. We find that thresholding methods deal poorly with clusters of lipid droplets, whereas watershed methods require a smoothing step that must be optimized to remove image noise. We describe here a novel three-stage hybrid method for automated segmentation and quantitation of lipid droplets. In this method, objects are initially identified by thresholding. They are then tested for circularity to distinguish single lipid droplets from clusters. Clusters are subjected to a secondary watershed segmentation. We provide a characterization of this method in simulated images. Additionally, we apply this method to images of fixed cells containing stained lipid droplets and GFP-tagged proteins to provide a proof-of-principle that this method can be used for colocalization studies. The circularity measure can additionally prove useful for the identification of inappropriate segmentation in an automated way; for example, of non-cellular material. We will make the programs and source code available to the community under the Gnu Public License. We believe this technique will be of interest to cell biologists for light microscopic studies of lipid droplet biology.