Photomodulatable fluorescent proteins for imaging cell dynamics and cell fate

An organism arises from the coordinate generation of different cell types and the stereotypical organization of these cells into tissues and organs. Even so, the dynamic behaviors, as well as the ultimate fates, of cells driving the morphogenesis of an organism, or even an individual organ, remain largely unknown. Continued innovations in optical imaging modalities, along with the discovery and evolution of improved genetically-encoded fluorescent protein reporters in combination with model organism, stem cell and tissue engineering paradigms are providing the means to investigate these unresolved questions. The emergence of fluorescent proteins whose spectral properties can be photomodulated is one of the most significant new developments in the field of cell biology where they are primarily used for studying protein dynamics in cells. Likewise, the use of photomodulatable fluorescent proteins holds great promise for use in developmental biology. Photomodulatable fluorescent proteins also represent attractive and emergent tools for studying cell dynamics in complex populations by facilitating the labeling and tracking of individual or defined groups of cells. Here, we review the currently available photomodulatable fluorescent proteins and their application in model organisms. We also discuss prospects for their use in mice, and by extension in embryonic stem cell and tissue engineering paradigms.     

Single-Molecule Microscopy Reveals Membrane Microdomain Organization of Cells in a Living Vertebrate

It has been possible for several years to study the dynamics of fluorescently labeled proteins by single-molecule microscopy, but until now this technology has been applied only to individual cells in culture. In this study, it was extended to stem cells and living vertebrate organisms. As a molecule of interest we used yellow fluorescent protein fused to the human H-Ras membrane anchor, which has been shown to serve as a model for proteins anchored in the plasma membrane. We used a wide-field fluorescence microscopy setup to visualize individual molecules in a zebrafish cell line (ZF4) and in primary embryonic stem cells. A total-internal-reflection microscopy setup was used for imaging in living organisms, in particular in epidermal cells in the skin of 2-day-old zebrafish embryos. Our results demonstrate the occurrence of membrane microdomains in which the diffusion of membrane proteins in a living organism is confined. This membrane organization differed significantly from that observed in cultured cells, illustrating the relevance of performing single-molecule microscopy in living organisms.     

Expression of green or red fluorescent protein (GFP or DsRed) linked proteins in nonmuscle and muscle cells

The introduction of the green fluorescent protein (GFP) plasmids that allow proteins and peptides to be expressed with a fluorescent tag has had a major impact on the field of cell biology. It has enabled the dynamics of a wide variety of proteins to be analyzed that could not otherwise be detected in live cells. Transient transfections of muscle and nonmuscle cells with plasmids encoding various cytoskeletal proteins ligated to green fluorescent protein or Ds red protein allow changes in the cytoskeletal network to be studied in the same cell for time periods up to several days. With this approach, proteins that could not be purified and directly labeled with fluorescent dyes and microinjected into cells can now be expressed and visualized in a wide variety of cells. Procedures are presented for transfection of the nonmuscle cell, PtK2, and primary cultures of embryonic chick myocytes, and for studying the live transfected cells.     

A Cre‐inducible fluorescent reporter for observing apical membrane dynamics

The ability to image living tissues with fluorescent proteins has revolutionized the fields of cell and developmental biology. Fusions between fluorescent proteins and various polypeptides are allowing scientists to image tissues with sub‐cellular resolution. Here, we describe the generation and activity of a genetically engineered mouse line expressing a fusion between the green fluorescent protein (GFP) and the apically localized protein Crumbs3 (Crb3). This reporter drives Cre‐inducible expression of Crb3–GFP under control of the EF1a regulatory domains. The fusion protein is broadly expressed in embryonic and adult tissues and shows apical restriction in the majority of epithelial cell types. It displays a variably penetrant gain of function activity in the neural tube. However, in several cell types, over‐expression of Crb3 does not appear to have any effect on normal development or maintenance. Detailed analysis of kidneys expressing this reporter indicates normal morphology and function highlighting the utility for live imaging. Thus, the EF1a^Crb3–GFP mouse line will be of broad use for studying membrane and/or tissue dynamics in living tissues. genesis 53:285–293, 2015. © 2015 Wiley Periodicals, Inc.     

Live cell imaging: approaches for studying protein dynamics in living cells

In the last decade, the long-standing biologist's dream of seeing the molecular events within the living cell came true. This technological achievement is largely due to the development of fluorescence microscopy technologies and the advent of green fluorescent protein as a fluorescent probe. Such imaging technologies allowed us to determine the subcellular localization, mobility and transport pathways of specific proteins and even visualize protein-protein interactions of single molecules in living cells. Direct observation of such molecular dynamics can provide important information about cellular events that cannot be obtained by other methods. Thus, imaging of protein dynamics in living cells becomes an important tool for cell biology to study molecular and cellular functions. In this special issue of review articles, we review various imaging technologies of microscope hardware and fluorescent probes useful for cell biologists, with a focus on recent development of live cell imaging.     

Application of real-time confocal microscopy for observation of living cells in tissue specimens.

Measurement of intracellular Ca2+ concentration ([Ca2+]i) has been a fundamental technique in cell biology. However, most investigations have used cultured or isolated cells as an experimental model, and consequently can provide only limited insight into the mechanisms that operate in tissue in situ. Useful information may be obtained by studying intact tissue specimens. High-speed confocal microscopes that can acquire digital images at video rate have recently been developed. These confocal microscopes which can acquire data in real-time enable [Ca2+]i dynamics of individual cells in intact tissue specimens to be observed. The present paper examines the use of fluorescent microscopy and confocal microscopy for [Ca2+]i imaging of living tissue. We analyzed the dynamics of the duodenal gland, lacrimal gland, intestinal smooth muscles, arterioles, myenteric plexus, and dorsal root ganglion. In these specimens, individual cells exhibited different [Ca2+]i dynamics, and the responses to transmitters/modulators were heterogeneous. In conclusion, real-time imaging provides a useful tool for observing dynamic changes in cells in situ, and it may lead to improve understanding tissue physiology.     

Molecular imaging and stem cell research

During the last decade, there has been enormous progress in understanding both multipotent stem cells such as hematopoietic stem cells and pluripotent stem cells such as embryonic stem cells and induced pluripotent stem cells. However, it has been challenging to study developmental potentials of these stem cells because they reside in complex cellular environments and aspects of their distribution, migration, engraftment, survival, proliferation, and differentiation often could not be sufficiently elucidated based on limited snapshot images of location or environment or molecular markers. Therefore, reliable imaging methods to monitor or track the fate of the stem cells are highly desirable. Both short-term and more permanent monitoring of stem cells in cultures and in live organisms have benefited from recently developed imaging approaches that are designed to investigate cell behavior and function. Confocal and multiphoton microscopy, time-lapse imaging technology, and series of noninvasive imaging technologies enable us to investigate cell behavior in the context of a live organism. In turn, the knowledge gained has brought our understanding of stem cell biology to a new level. In this review, we discuss the application of current imaging modalities for research of hematopoietic stem cells and pluripotent stem cells and the challenges ahead.     

Dissecting cell adhesion architecture using advanced imaging techniques

Cell adhesion to extracellular matrix proteins or to other cells is essential for the control of embryonic development, tissue integrity, immune function and wound healing. Adhesions are tightly spatially regulated structures containing over one hundred different proteins that coordinate both dynamics and signaling events at these sites. Extensive biochemical and morphological analysis of adhesion types over the past three decades has greatly improved understanding of individual protein contributions to adhesion signaling and, in some cases, dynamics. However, it is becoming increasingly clear that these diverse macromolecular complexes contain a variety of protein sub-networks, as well as distinct sub-domains that likely play important roles in regulating adhesion behavior. Until recently, resolving these structures, which are often less than a micron in size, was hampered by the limitations of conventional light microscopy. However, recent advances in optical techniques and imaging methods have revealed exciting insight into the intricate control of adhesion structure and assembly. Here we provide an overview of the recent data arising from such studies of cell:matrix and cell:cell contact and an overview of the imaging strategies that have been applied to study the intricacies and hierarchy of proteins within adhesions.Key words: adhesion, migration, microscopy, dynamics, cytoskeleton, photobleaching, super-resolution imaging, fluorescence     

Variable-angle epifluorescence microscopy: a new way to look at protein dynamics in the plant cell cortex

Live-cell microscopy imaging of fluorescent-tagged fusion proteins is an essential tool for cell biologists. Total internal reflection fluorescence microscopy (TIRFM) has joined confocal microscopy as a complementary system for the imaging of cell surface protein dynamics in mammalian and yeast systems because of its high temporal and spatial resolution. Here we present an alternative to TIRFM, termed variable-angle epifluorescence microscopy (VAEM), for the visualization of protein dynamics at or near the plasma membrane of plant epidermal cells and root hairs in whole, intact seedlings that provides high-signal, low-background and near real-time imaging. VAEM uses highly oblique subcritical incident angles to decrease background fluorophore excitation. We discuss the utilities and advantages of VAEM for imaging of fluorescent fusion-tagged marker proteins in studying cortical cytoskeletal and membrane proteins. We believe that the application of VAEM will be an invaluable imaging tool for plant cell biologists.     

A femto-injection technique for dynamic analysis of protein function in living embryonic stem cells

The potential of a femto-injection technique for use in analyzing protein dynamics in embryonic stem (ES) cells was investigated. First, we showed that fluorescent proteins could be injected in a quantitative fashion into individual mouse ES cells. Second, we demonstrated that the technique could identify functional differences between proteins by analyzing the effect of a nuclear localization signal on the behavior of glutathione S-transferase conjugated to green fluorescent protein. The analysis showed a clear difference in the distribution of the protein when the nuclear localization signal was present. Our results confirm that the non-destructive, quantitative and time controllable aspects of the technique provide considerable advantages for the analysis of protein behavior in living ES cells. To the best of our knowledge, this is the first report of the successful introduction of proteins into living ES cells by an injection technique.     

In situ stem cell therapy: novel targets, familiar challenges

Tissue engineering approaches for expanding, differentiating and engrafting embryonic or adult stem cells have significant potential for tissue repair but harnessing endogenous stem cell populations offers numerous advantages over these approaches. There has been rapid basic biological progress in the identification of stem cell niches throughout the body and the molecular factors that regulate their function. These niches represent novel therapeutic targets and efforts to use them involve the familiar challenges of delivering molecular medicines in vivo. Here we review recent progress in the use of genes, proteins and small molecules for in situ stem cell control and manipulation, with a focus on using stem cells of the central nervous system for neuroregeneration.     

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 µm of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.     

Identification of candidate regulators of embryonic stem cell differentiation by comparative phosphoprotein affinity profiling

Embryonic stem cells are a unique cell population capable both of self-renewal and of differentiation into all tissues in the adult organism. Despite the central importance of these cells, little information is available regarding the intracellular signaling pathways that govern self-renewal or early steps in the differentiation program. Embryonic stem cell growth and differentiation correlates with kinase activities, but with the exception of the JAK/STAT3 pathway, the relevant substrates are unknown. To identify candidate phosphoproteins with potential relevance to embryonic stem cell differentiation, a systems biology approach was used. Proteins were purified using phosphoprotein affinity columns, then separated by two-dimensional gel electrophoresis, and detected by silver stain before being identified by tandem mass spectrometry. By comparing preparations from undifferentiated and differentiating mouse embryonic stem cells, a set of proteins was identified that exhibited altered post-translational modifications that correlated with differentiation state. Evidence for altered post-translational modification included altered gel mobility, altered recovery after affinity purification, and direct mass spectra evidence. Affymetrix microarray analysis indicated that gene expression levels of these same proteins had minimal variability over the same differentiation period. Bioinformatic annotations indicated that this set of proteins is enriched with chromatin remodeling, catabolic, and chaperone functions. This set of candidate phosphoprotein regulators of stem cell differentiation includes products of genes previously noted to be enriched in embryonic stem cells at the mRNA expression level as well as proteins not associated previously with stem cell differentiation status.     

A quick guide to light microscopy in cell biology

Light microscopy is a key tool in modern cell biology. Light microscopy has several features that make it ideally suited for imaging biology in living cells: the resolution is well-matched to the sizes of subcellular structures, a diverse range of available fluorescent probes makes it possible to mark proteins, organelles, and other structures for imaging, and the relatively nonperturbing nature of light means that living cells can be imaged for long periods of time to follow their dynamics. Here I provide a brief introduction to using light microscopy in cell biology, with particular emphasis on factors to be considered when starting microscopy experiments.     

Global expression of cell surface proteins in embryonic stem cells

Background

Recent studies have shown that embryonic stem (ES) cells globally express most genes in the genome at the mRNA level; however, it is unclear whether this global expression is propagated to the protein level. Cell surface proteins could perform critical functions in ES cells, so determining whether ES cells globally express cell surface proteins would have significant implications for ES cell biology.

Methods and Principal Findings

The surface proteins of mouse ES cells were purified by biotin labeling and subjected to proteomics analysis. About 1000 transmembrane or secreted cell surface proteins were identified. These proteins covered a large variety if functional categories including signal transduction, adhesion and transporting. More over, mES cells promiscuously expressed a wide variety of tissue specific surface proteins. And many surface proteins were expressed heterogeneously on mES cells. We also find that human ES cells express a wide variety of tissue specific surface proteins.

Conclusions/Significance

Our results indicate that global gene expression is not simply a result of leaky gene expression, which could be attributed to the loose chromatin structure of ES cells; it is also propagated to the functional level. ES cells may use diverse surface proteins to receive signals from the diverse extracellular stimuli that initiate differentiation. Moreover, the promiscuous expression of tissue specific surface proteins illuminate new insights into the strategies of cell surface marker screening.     

Proteomic analyses reveal common promiscuous patterns of cell surface proteins on human embryonic stem cells and sperms

Background

It has long been proposed that early embryos and reproductive organs exhibit similar gene expression profiles. However, whether this similarity is propagated to the protein level remains largely unknown. We have previously characterised the promiscuous expression pattern of cell surface proteins on mouse embryonic stem (mES) cells. As cell surface proteins also play critical functions in human embryonic stem (hES) cells and germ cells, it is important to reveal whether a promiscuous pattern of cell surface proteins also exists for these cells.

Methods and Principal Findings

Surface proteins of hES cells and human mature sperms (hSperms) were purified by biotin labelling and subjected to proteomic analyses. More than 1000 transmembrane or secreted cell surface proteins were identified on the two cell types, respectively. Proteins from both cell types covered a large variety of functional categories including signal transduction, adhesion and transporting. Moreover, both cell types promiscuously expressed a wide variety of tissue specific surface proteins, and some surface proteins were heterogeneously expressed.

Conclusions/Significance

Our findings indicate that the promiscuous expression of functional and tissue specific cell surface proteins may be a common pattern in embryonic stem cells and germ cells. The conservation of gene expression patterns between early embryonic cells and reproductive cells is propagated to the protein level. These results have deep implications for the cell surface signature characterisation of pluripotent stem cells and germ cells and may lead the way to a new area of study, i.e., the functional significance of promiscuous gene expression in pluripotent and germ cells.     

Detection of Calcium Transients in Embryonic Stem Cells and Their Differentiated Progeny

A central issue in stem cell biology is the determination of function and activity of differentiated stem cells, features that define the true phenotype of mature cell types. Commonly, physiological mechanisms are used to determine the functionality of mature cell types, including those of the nervous system. Calcium imaging provides an indirect method of determining the physiological activities of a mature cell. Camgaroos are variants of yellow fluorescent protein that act as intracellular calcium sensors in transfected cells. We expressed one version of the camgaroos, Camgaroo-2, in mouse embryonic stem (ES) cells under the control of the CAG promoter system. Under the control of this promoter, Camgaroo-2 fluorescence was ubiquitously expressed in all cell types derived from the ES cells that were tested. In response to pharmacological stimulation, the fluorescence levels in transfected cells correlated with cellular depolarization and hyperpolarization. These changes were observed in both undifferentiated ES cells as well as ES cells that had been neurally induced, including putative neurons that were differentiated from transfected ES cells. The results presented here indicate that Camgaroo-2 may be used like traditional fluorescent proteins to track cells as well as to study the functionality of stem cells and their progeny.