Live-cell Studies Research Articles

Comparison study of live cells by atomic force microscopy, confocal microscopy, and scanning electrochemical microscopy

In this report, three kinds of scanning probe microscopy techniques, atomic force microscopy (AFM), confocal microscopy (CM), and scanning electrochemical microscopy (SECM), were used to study live cells in the physiological environment. Two model cell lines, CV-1 and COS-7, were studied. Time-lapse images were obtained with both contact and tapping mode AFM techniques. Cells were more easily scratched or moved by contact mode AFM than by tapping mode AFM. Detailed surface structures such as filamentous structures on the cell membrane can be obtained and easily discerned with tapping mode AFM. The toxicity of ferrocenemethanol (Fc) on live cells was studied by CM in reflection mode by recording the time-lapse images of controlled live cells and live cells with different Fc concentrations. No significant change in the morphology of cells was caused by Fc. Cells were imaged by SECM with Fc as the mediator at a biased potential of 0.35 V (vs. Ag/AgCl with a saturated KCl solution). Cells did not change visibly within 1 h, which indicated that SECM was a noninvasive technique and thus has a unique advantage for the study of soft cells, since the electrode scanned above the cells instead of in contact with them. Reactive oxygen species (ROS) generated by the cells were detected and images based on these chemical species were obtained. It is demonstrated that SECM can provide not only the topographical images but also the images related to the chemical or biochemical species released by the live cells.Key words: live cells, atomic force microscopy, confocal microscopy, scanning electrochemical microscopy.

Getting across the plasma membrane and beyond: intracellular uses of colloidal semiconductor nanocrystals.

Semiconductor nanocrystals (NCs) are increasingly being used as photoluminescen markers in biological imaging. Their brightness, large Stokes shift, and high photostability compared to organic fluorophores permit the exploration of biological phenomena at the single-molecule scale with superior temporal resolution and spatial precision. NCs have predominantly been used as extracellular markers for tagging and tracking membrane proteins. Successful internalization and intracellular labelling with NCs have been demonstrated for both fixed immunolabelled and live cells. However, the precise localization and subcellular compartment labelled are less clear. Generally, live cell studies are limited by the requirement of fairly invasive protocols for loading NCs and the relatively large size of NCs compared to the cellular machinery, along with the subsequent sequestration of NCs in endosomal/lysosomal compartments. For long-period observation the potential cytotoxicity of cytoplasmically loaded NCs must be evaluated. This review focuses on the challenges of intracellular uses of NCs.

Experimentally manipulating fungi with optical tweezers

A short review of the use of optical tweezers in fungal cell biological research is provided. First, we describe how optical tweezers work. Second, we review how they have been used in various experimental live-cell studies to manipulate intracellular organelles, hyphal growth and branching, and whole cells. Third, we indicate how optically trapped microbeads can be used for the localized delivery of chemicals or mechanical stimulation to cells, as well as permitting measurements of the growth forces generated by germ tubes. Finally, the effects of optical trapping on fungal cell viability and growth are assessed.

Setup of a fluorescence lifetime and spectral correlated acquisition system for two-photon microscopy

In this article we present a complete laser scanning microscope designed for simultaneous spectral and lifetime measurements from every point of the specimen located within the field of view. The pulsed laser source used for two-photon excitation provides good spatial resolution with minimal invasivity. In addition, the detection module was optimized for minimal photon loss, allowing laser power minimization and further reduction of cells photodamage. Analysis of biological samples illustrates the performances of this configuration, particularly when applied to fluorescent resonance energy transfer (FRET) measurements. Indeed, multiparametric acquisition is particularly useful to discriminate between FRET and artifactual response due to acquisition invasivity or cell heterogeneity. Combined with adapted homemade driving software, this system is stable, portable, and optimized for living cell studies.

Live-cell studies with the Axio Observer

The Axio Observer inverted research microscope from Carl Zeiss, available with three different stand formats and a variety of other optional features, gives users a flexible and powerful tool for performing fluorescence microscopy.

Optimizing Laser Source Operation for Confocal and Multiphoton Laser Scanning Microscopy

Confocal laser scanning microscopy (CLSM) and multiphoton laser scanning microscopy (MPLSM) are methods both widely used by life-sciences researchers for imaging fluorescently labeled live cells and fixed tissue specimens. Key to the success of both CLSM and MPLSM is the application of a suitable laser source, namely one that provides sufficient average or peak power at the correct wavelength to excite fluorescence. High stability of the laser source output is required for three-dimensional imaging, time-lapse studies of live cells, and quantitative studies and inter-experiment comparisons. The laser technology associated with the design of such lasers is mature, yet is unfortunately rather complex. This complexity can be off-putting for the life-sciences researcher who needs to optimize the system for the best possible images, but this apprehension can be overcome by understanding the function of the system components. This unit summarizes the optimization of the most commonly used laser sources for CLSM and MPLSM, including power and wavelength tuning and methods for cleaning optical components.

MICROTUBULE DYNAMICS AND ORGANIZATION IN THE PLANT CORTICAL ARRAY

Live-cell studies have brought fresh insight into the organizational activities of the plant cortical array. Plant interphase arrays organize in the absence of a discrete microtubule organizing center, having plus and minus ends distributed throughout the cell cortex. Microtubule nucleation occurs at the cell cortex, frequently followed by minus-end detachment from origin sites. Microtubules associate tightly with the cell cortex, resisting lateral and axial translocation. Slow, intermitant loss of dimers from minus ends, coupled with growth-biased dynamic instability at the plus ends, results in the migration of cortically attached microtubules across the cell via polymer treadmilling. Microtubule-microtubule interactions, a direct consequence of treadmilling, result in polymer reorientation and creation of polymer bundles. The combined properties of microtubule dynamics and interactions among polymers constitute a system with predicted properties of self-organization.

Motion as a phenotype: the use of live-cell imaging and machine visual screening to characterize transcription-dependent chromosome dynamics.

BackgroundGene transcriptional activity is well correlated with intra-nuclear position, especially relative to the nuclear periphery, which is a region classically associated with gene silencing. Recently however, actively transcribed genes have also been found localized to the nuclear periphery in the yeast Saccharomyces cerevisiae. When genes are activated, they become associated with the nuclear pore complex (NPC) at the nuclear envelope. Furthermore, chromosomes are not static structures, but exhibit constrained diffusion in real-time, live-cell studies of particular loci. The relationship of chromosome motion with transcriptional activation and active-gene recruitment to the nuclear periphery has not yet been investigated.ResultsWe have generated a yeast strain that enables us to observe the motion of the galactose-inducible GAL gene locus relative to the nuclear periphery in real-time under transcriptionally active and repressed conditions. Using segmented geometric particle tracking, we show that the repressed GAL locus undergoes constrained diffusive movement, and that transcriptional induction with galactose is associated with an enrichment in cells with GAL loci that are both associated with the nuclear periphery and much more constrained in their movement. Furthermore, we report that the mRNA export factor Sac3 is involved in this galactose-induced enrichment of GAL loci at the nuclear periphery. In parallel, using a novel machine visual screening technique, we find that the motion of constrained GAL loci correlates with the motion of the cognate nuclei in galactose-induced cells.ConclusionTranscriptional activation of the GAL genes is associated with their tethering and motion constraint at the nuclear periphery. We describe a model of gene recruitment to the nuclear periphery involving gene diffusion and the mRNA export factor Sac3 that can be used as a framework for further experimentation. In addition, we applied to the analysis of chromosome motion a machine visual screening approach that used unbiased visual data rather than segmented geometric data. This novel analytical approach will allow for high-throughput study of processes that can be monitored via alterations in chromosome motion and connectivity with the nuclear periphery.

The cell biology of wood formation: from cambial divisions to mature secondary xylemThis review is one of a selection of papers published in the Special Issue on Plant Cell Biology.

The development of secondary xylem has been studied historically from an anatomical point of view, but recent developments in cell and molecular biology have revitalized this field. An integrated view of cell structure with physiology is emerging for each stage of the developing xylem cells’ lives, from birth in the cambium to programmed cell death. High-quality structural information is essential in building this type of integrated view, but conventional electron microscopy of developing wood cells has been problematic. The importance of adequately preserving cells of the secondary vascular system is illustrated with examples from dormant cambium, cell division, and secondary cell wall deposition. In many cases, contemporary gene expression studies can be viewed in the context of both new structural information and pioneering live cell studies done in the early 1900s to increase our understanding of secondary xylem development.

GFP-based FRET analysis in live cells

Fluorescence resonance energy transfer (FRET) is a widely utilized optical technique for measuring small distances of 1–10 nm in live cells. In recent years, its application has been greatly popularized by the discovery of green fluorescent protein (GFP) and many improved variants which make good donor–acceptor fluorophore pairs. GFP-based proteins are structurally stable, relatively inert, and can be reliably attached to points of interest. The combination of easy access to the GFP-based FRET technique and its obvious usefulness in many applications can lead to complacency. Potential problems such as light contaminants, e.g., bleed-through and cross-talk, and inconsistent donor and acceptor concentrations are easily overlooked and can lead to errors in FRET calculation and data interpretation. In this article, we outline possible pitfalls of GFP-based FRET and approaches that address these issues, including a “Spectra FRET” technique that can be easily applied to live cell studies.

FRET Study of CAD‐Rad9 Protein‐Protein Interaction

The highly regulated rate limiting protein in the de novo synthesis of pyrimidines is the CPSase portion of the multi-enzyme CAD. Both full length CAD and the CPSase segment alone have previously been found to co-immunoprecipitate with the DNA damage checkpoint protein Rad9 in vitro. The interaction increases the enzymatic activity of CPSase in vitro twofold. Live cell studies of the interaction are being performed using fluorescence resonance energy transfer (FRET) imaging in order to visualize the interaction in vivo. YFP-CAD and CFP-Rad9 fusion proteins have been constructed for FRET protein-protein interaction analysis. Changes in localization and interaction are being studied in response to DNA damage as well as cell synchronization in an attempt to determine the dynamics of the interactions under different conditions. Preliminary data suggests that CAD is absent from the nucleus, contrary to a previously published study. Thus, the Rad9-CAD interaction may occur in the cytoplasm. The project is funded by the NIH.

Enhanced Green Fluorescent Protein (GFP) fluorescence after polyelectrolyte caging

Discovery of Green Fluorescent Protein (GFP) constituted an important improvement for living cell studies on submicron resolution allowing in vivo fluorescence labeling. We studied the photo-physical properties of single GFP molecules incorporated in a charged polyelectrolyte environment by means of single molecule spectroscopy. The fluorescence characteristics change dramatically in terms of photo-stability,lifetime and blinking behavior so that the proteins scale up to quantum dots. The reported results highlight interesting applications in the design of fluorescent markers and in the development of optical data storage architectures.

How actin crosslinking and bundling proteins cooperate to generate an enhanced cell mechanical response

Actin-crosslinking proteins organize actin filaments into dynamic and complex subcellular scaffolds that orchestrate important mechanical functions, including cell motility and adhesion. Recent mutation studies have shown that individual crosslinking proteins often play seemingly non-essential roles, leading to the hypothesis that they have considerable redundancy in function. We report live-cell, in vitro, and theoretical studies testing the mechanical role of the two ubiquitous actin-crosslinking proteins, α-actinin and fascin, which co-localize to stress fibers and the basis of filopodia. Using live-cell particle tracking microrheology, we show that the addition of α-actinin and fascin elicits a cell mechanical response that is significantly greater than that originated by α-actinin or fascin alone. These live-cell measurements are supported by quantitative rheological measurements with reconstituted actin filament networks containing pure proteins that show that α-actinin and fascin can work in concert to generate enhanced cell stiffness. Computational simulations using finite element modeling qualitatively reproduce and explain the functional synergy of α-actinin and fascin. These findings highlight the cooperative activity of fascin and α-actinin and provide a strong rationale that an evolutionary advantage might be conferred by the cooperative action of multiple actin-crosslinking proteins with overlapping but non-identical biochemical properties. Thus the combination of structural proteins with similar function can provide the cell with unique properties that are required for biologically optimal responses.

Quantum Dot-Based Cell Motility Assay

Because of their favorable physical and photochemical properties, colloidal CdSe/ZnS-semiconductor nanocrystals (commonly known as quantum dots) have enormous potential for use in biological imaging. In this report, we present an assay that uses quantum dots as markers to quantify cell motility. Cells that are seeded onto a homogeneous layer of quantum dots engulf and absorb the nanocrystals and, as a consequence, leave behind a fluorescence-free trail. By subsequently determining the ratio of cell area to fluorescence-free track area, we show that it is possible to differentiate between invasive and noninvasive cancer cells. Because this assay uses simple fluorescence detection, requires no significant data processing, and can be used in live-cell studies, it has the potential to be a powerful new tool for discriminating between invasive and noninvasive cancer cell lines or for studying cell signaling events involved in migration.

Antiandrogens prevent stable DNA-binding of the androgen receptor

The androgen receptor (AR) is essential for development of the male gender and in the growth of the majority of prostate cancers. Agonists as well as most antagonists induce translocation of the receptor to the nucleus, whereas only agonists can activate AR function. Antagonists are therefore used in the therapy of metastasized prostate cancer. To obtain insight into the mechanism by which antagonists block AR function in living cells, we studied nuclear mobility and localization of green fluorescent protein (GFP)-tagged AR in the presence of either the agonist R1881 or the antagonists bicalutamide and hydroxyflutamide. As controls we investigated a non-DNA-binding AR mutant (A573D) and two mutants (W741C and T877A) with broadened ligand specificity. We demonstrate that in the presence of R1881, AR localizes in numerous intranuclear foci and, using complementary fluorescence recovery after photobleaching (FRAP) approaches and computer modelling, that a fraction of AR ( approximately 10-15%) is transiently immobilized in a DNA-binding-dependent manner (individual ARs being immobile for approximately 45 seconds). By contrast, antagonist-bound GFP-AR showed no detectable immobile fraction and the mobility was similar to that of the R1881-liganded non-DNA-binding mutant (A573D), indicating that antagonists do not induce the relatively stable DNA-binding-dependent immobilization observed with agonist-bound AR. Moreover, in the presence of bicalutamide and hydroxyflutamide GFP-AR was homogeneously distributed in the nucleus. Binding of bicalutamide and hydroxyflutamide to GFP-AR(W741C) and GFP-AR(T877A), respectively, resulted in similar mobility and heterogeneous nuclear distribution as observed for R1881-liganded GFP-AR. The live cell studies indicate that the investigated antagonists interfere with events early in the transactivation function of the AR.

RNA association and nucleocytoplasmic shuttling by ataxin-1

Spinocerebellar ataxia type 1 (SCA1) is a dominant neurodegenerative disease caused by the expression of mutant ataxin-1 containing an expanded polyglutamine tract. Ataxin-1 is a nuclear protein that localizes to punctate inclusions similar to neuronal nuclear inclusions seen in many polyglutamine expansion disease proteins. We demonstrate that ataxin-1 localization to inclusions and inclusion dynamics within the nucleus are RNA and transcription dependent, but not dependent on the polyglutamine tract. Ataxin-1 nuclear inclusions are distinct from other described nuclear bodies but recruit the mRNA export factor, TAP/NXF1, in a manner that is enhanced by cell heat shock. By FRAP protein dynamic studies in live cells, we found that wild-type, but not mutant, ataxin-1 was capable of nuclear export. These results suggest that the normal role of ataxin-1 may be in RNA processing, perhaps nuclear RNA export. Thus, nuclear retention of mutant ataxin-1 may be an important toxic gain of function in SCA1 disease.

Membrane destabilizing properties of cell-penetrating peptides

Although cell-penetrating peptides (CPPs), also denoted protein transduction domains (PTDs), have been widely used for intracellular delivery of large and hydrophilic molecules, the mechanism of uptake is still poorly understood. In a recent live cell study of the uptake of penetratin and tryptophan-containing analogues of Tat(48–60) and oligoarginine, denoted TatP59W, TatLysP59W and R 7W, respectively, it was found that both endocytotic and non-endocytotic uptake pathways are involved [Thorén et al., Biochem. Biophys. Res. Commun. 307 (2003) 100–107]. Non-endocytotic uptake was only observed for the arginine-rich peptides TatP59W and R 7W. In this paper, the interactions of penetratin, R 7W, TatP59W and TatLysP59W with phospholipid vesicles are compared in the search for an understanding of the mechanisms for cellular uptake. While R 7W, TatP59W and TatLysP59W are found to promote vesicle fusion, indicated by mixing of membrane components, penetratin merely induces vesicle aggregation. Studies of the leakage from dye-loaded vesicles indicate that none of the peptides forms membrane pores and that vesicle fusion is not accompanied by leakage of the aqueous contents of the vesicles. These observations are important for a proper interpretation of future experiments on the interactions of these peptides with model membranes. We suggest that the discovered variations in propensity to destabilize phospholipid bilayers between the peptides investigated, in some cases sufficient to induce fusion, may be related to their different cellular uptake properties.

Infrared microspectroscopy of individual human cervical cancer (HeLa) cells suspended in growth medium.

We report for the first time the infrared spectra of individual human cervical cancer (HeLa) cells suspended in buffer or cell culture medium. Although we did not establish whether these cells were viable at the time of spectral data acquisition, we believe that the methodology used is applicable to the study of live cells. Data were collected either from entire cells, using 25- to 40-microm apertures, or via an imaging approach, where pixels measuring 6.25 x 6.25 microm were assembled to form a map of a cell in suspension. Measurements were carried out both in reflection/absorption and in transmission modes. The results reported here might have far-reaching implications for the use of infrared microspectroscopy to monitor cell proliferation, drug response, and other cell biological parameters in live cells.

Annexin 2 Binding to Phosphatidylinositol 4,5-Bisphosphate on Endocytic Vesicles Is Regulated by the Stress Response Pathway

Annexin 2 is a Ca(2+)-binding protein that has an essential role in actin-dependent macropinosome motility. We show here that macropinosome rocketing can be induced by hyperosmotic shock, either alone or synergistically when combined with phorbol ester or pervanadate. Rocketing was blocked by inhibitors of phosphatidylinositol-3-kinase(s), p38 mitogen-activated protein (MAP) kinase, and calcium, suggesting the involvement of phosphoinositide signaling. Since various phosphoinositides are enriched on inwardly mobile vesicles, we examined whether or not annexin 2 binds to any of this class of phospholipid. In liposome sedimentation assays, we show that recombinant annexin 2 binds to phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5P(2)) but not to other poly- and mono-phosphoinositides. The affinity of annexin 2 for PtdIns-4,5P(2) (K(D) approximately 5 microm) is comparable with those reported for a variety of PtdIns-4,5P(2)-binding proteins and is enhanced in the presence of Ca(2+). Although annexin 1 also bound to PtdIns-4,5P(2), annexin 5 did not, indicating that this is not a generic annexin property. To test whether annexin 2 binds to PtdIns-4,5P(2) in vivo, we microinjected rat basophilic leukemia cells stably expressing annexin 2-green fluorescent protein (GFP) with fluorescently tagged antibodies to PtdIns-4,5P(2). Annexin 2-GFP and anti-PtdIns-4,5P(2) IgG co-localize at sites of pinosome formation, and annexin 2-GFP relocalizes to intracellular membranes in Ptk cells microinjected with Arf6Q67L, which has been shown to stimulate PtdIns-4,5P(2) synthesis on pinosomes through activation of phosphatidylinositol 5 kinase. These results establish a novel phospholipid-binding specificity for annexin 2 consistent with a role in mediating the interaction between the macropinosome surface and the polymerized actin tail.

The androgen receptor ligand-binding domain stabilizes DNA binding in living cells

The androgen receptor (AR) is a member of the steroid receptor family, a group of transcription factors that activate steroid-regulated genes. Live cell studies of several steroid receptors have shown that the mobility of the liganded receptor is strongly reduced compared to the unliganded receptor. To investigate the nature of this reduced mobility, we generated Hep3B cells stably expressing green fluorescent protein (GFP)-AR at physiological levels. Computer-aided analysis of photobleaching experiments showed that in the presence of ligand on average one out of five ARs is immobilized, each individual AR being immobile for 1–2 min. This immobilization depended on DNA binding since GFP-ARs mutated in the DNA-binding domain were not immobilized. Interestingly, a truncated AR lacking the ligand-binding domain (LBD) displayed substantially shorter immobilizations, in the order of seconds, although its transcriptional activation function was stronger. Our data suggest the LBD has a role in maintaining the stability of AR–DNA complexes.