Laura Furia

Live-cell 3D super-resolution imaging in thick biological samples

We demonstrate three-dimensional (3D) super-resolution live-cell imaging through thick specimens (50–150 μm), by coupling far-field individual molecule localization with selective plane illumination microscopy (SPIM). The improved signal-to-noise ratio of selective plane illumination allows nanometric localization of single molecules in thick scattering specimens without activating or exciting molecules outside the focal plane. We report 3D super-resolution imaging of cellular spheroids.

DNA damage in stem cells activates p21, inhibits p53, and induces symmetric self-renewing divisions

DNA damage leads to a halt in proliferation owing to apoptosis or senescence, which prevents transmission of DNA alterations. This cellular response depends on the tumor suppressor p53 and functions as a powerful barrier to tumor development. Adult stem cells are resistant to DNA damage-induced apoptosis or senescence, however, and how they execute this response and suppress tumorigenesis is unknown. We show that irradiation of hematopoietic and mammary stem cells up-regulates the cell cycle inhibitor p21, a known target of p53, which prevents p53 activation and inhibits p53 basal activity, impeding apoptosis and leading to cell cycle entry and symmetric self-renewing divisions. p21 also activates DNA repair, limiting DNA damage accumulation and self-renewal exhaustion. Stem cells with moderate DNA damage and diminished self-renewal persist after irradiation, however. These findings suggest that stem cells have evolved a unique, p21-dependent response to DNA damage that leads to their immediate expansion and limits their long-term survival.

A computational platform for robotized fluorescence microscopy (I): High-content image-based cell-cycle analysis

Hardware automation and software development have allowed a dramatic increase of throughput in both acquisition and analysis of images by associating an optimized statistical significance with fluorescence microscopy. Despite the numerous common points between fluorescence microscopy and flow cytometry (FCM), the enormous amount of applications developed for the latter have found relatively low space among the modern high‐resolution imaging techniques. With the aim to fulfill this gap, we developed a novel computational platform named A.M.I.CO. (Automated Microscopy for Image‐Cytometry) for the quantitative analysis of images from widefield and confocal robotized microscopes. Thanks to the setting up of both staining protocols and analysis procedures, we were able to recapitulate many FCM assays. In particular, we focused on the measurement of DNA content and the reconstruction of cell‐cycle profiles with optimal parameters. Standard automated microscopes were employed at the highest optical resolution (200 nm), and white‐light sources made it possible to perform an efficient multiparameter analysis. DNA‐ and protein‐content measurements were complemented with image‐derived information on their intracellular spatial distribution. Notably, the developed tools create a direct link between image‐analysis and acquisition. It is therefore possible to isolate target populations according to a definite quantitative profile, and to relocate physically them for diffraction‐limited data acquisition. Thanks to its flexibility and analysis‐driven acquisition, A.M.I.CO. can integrate flow, image‐stream and laser‐scanning cytometry analysis, providing high‐resolution intracellular analysis with a previously unreached statistical relevance.

A computational platform for robotized fluorescence microscopy (II): DNA damage, replication, checkpoint activation, and cell cycle progression by high‐content high‐resolution multiparameter image‐cytometry

Dissection of complex molecular‐networks in rare cell populations is limited by current technologies that do not allow simultaneous quantification, high‐resolution localization, and statistically robust analysis of multiple parameters. We have developed a novel computational platform (Automated Microscopy for Image CytOmetry, A.M.I.CO) for quantitative image‐analysis of data from confocal or widefield robotized microscopes. We have applied this image‐cytometry technology to the study of checkpoint activation in response to spontaneous DNA damage in nontransformed mammary cells. Cell‐cycle profile and active DNA‐replication were correlated to (i) Ki67, to monitor proliferation; (ii) phosphorylated histone H2AX (γH2AX) and 53BP1, as markers of DNA‐damage response (DDR); and (iii) p53 and p21, as checkpoint‐activation markers. Our data suggest the existence of cell‐cycle modulated mechanisms involving different functions of γH2AX and 53BP1 in DDR, and of p53 and p21 in checkpoint activation and quiescence regulation during the cell‐cycle. Quantitative analysis, event selection, and physical relocalization have been then employed to correlate protein expression at the population level with interactions between molecules, measured with Proximity Ligation Analysis, with unprecedented statistical relevance.

Cellular Heterogeneity During Embryonic Stem Cell Differentiation to Epiblast Stem Cells is Revealed by the ShcD/RaLP Adaptor Protein

The Shc family of adaptor proteins are crucial mediators of a plethora of receptors such as the tyrosine kinase receptors, cytokine receptors, and integrins that drive signaling pathways governing proliferation, differentiation, and migration. Here, we report the role of the newly identified family member, ShcD/RaLP, whose expression in vitro and in vivo suggests a function in embryonic stem cell (ESC) to epiblast stem cells (EpiSCs) transition. The transition from the naïve (ESC) to the primed (EpiSC) pluripotent state is the initial important step for ESCs to commit to differentiation and the mechanisms underlying this process are still largely unknown. Using a novel approach to simultaneously assess pluripotency, apoptosis, and proliferation by multiparameter flow cytometry, we show that ESC to EpiSC transition is a process involving a tight coordination between the modulation of the Oct4 expression, cell cycle progression, and cell death. We also describe, by high‐content immunofluorescence analysis and time‐lapse microscopy, the emergence of cells expressing caudal‐related homeobox 2 (Cdx2) transcription factor during ESC to EpiSC transition. The use of the ShcD knockout ESCs allowed the unmasking of this process as they presented deregulated Oct4 modulation and an enrichment in Oct4‐negative Cdx2‐positive cells with increased MAPK/extracellular‐regulated kinases 1/2 activation, within the differentiating population. Collectively, our data reveal ShcD as an important modulator in the switch of key pathway(s) involved in determining EpiSC identity. STEM CELLS2012;30:2423–2436

PML is required for telomere stability in non-neoplastic human cells.

Telomeres interact with numerous proteins, including components of the shelterin complex, whose alteration, similarly to proliferation-induced telomere shortening, initiates cellular senescence. In tumors, telomere length is maintained by Telomerase activity or by the Alternative Lengthening of Telomeres mechanism, whose hallmark is the telomeric localization of the promyelocytic leukemia (PML) protein. Whether PML contributes to telomeres maintenance in normal cells is unknown. We show that in normal human fibroblasts the PML protein associates with few telomeres, preferentially when they are damaged. Proliferation-induced telomere attrition or their damage due to alteration of the shelterin complex enhances the telomeric localization of PML, which is increased in human T-lymphocytes derived from patients genetically deficient in telomerase. In normal fibroblasts, PML depletion induces telomere damage, nuclear and chromosomal abnormalities, and senescence. Expression of the leukemia protein PML/RARα in hematopoietic progenitors displaces PML from telomeres and induces telomere shortening in the bone marrow of pre-leukemic mice. Our work provides a novel view of the physiologic function of PML, which participates in telomeres surveillance in normal cells. Our data further imply that a diminished PML function may contribute to cell senescence, genomic instability, and tumorigenesis.

RNAi screens identify CHD4 as an essential gene in breast cancer growth

Epigenetic regulation plays an essential role in tumor development and epigenetic modifiers are considered optimal potential druggable candidates. In order to identify new breast cancer vulnerabilities and improve therapeutic chances for patients, we performed in vivo and in vitro shRNA screens in a human breast cancer cell model (MCF10DCIS.com cell line) using epigenetic libraries. Among the genes identified in our screening, we deeply investigated the role of Chromodomain Helicase DNA binding Protein 4 (CHD4) in breast cancer tumorigenesis. CHD4 silencing significantly reduced tumor growth in vivo and proliferation in vitro of MCF10DCIS.com cells. Similarly, in vivo breast cancer growth was decreased in a spontaneous mouse model of breast carcinoma (MMTV-NeuT system) and in metastatic patient-derived xenograft models. Conversely, no reduction in proliferative ability of non-transformed mammary epithelial cells (MCF10A) was detected. Moreover, we showed that CHD4 depletion arrests proliferation by inducing a G0/G1 block of cell cycle associated with up-regulation of CDKN1A (p21). These results highlight the relevance of genetic screens in the identification of tumor frailties and the role of CHD4 as a potential pharmacological target to inhibit breast cancer growth.

Confocal Microscopy for High‐Resolution and High‐Content Analysis of the Cell Cycle

Optical fluorescence microscopy offers a wide range of technological solutions to address many questions in biomedical research. Spatial resolution has been greatly improved by the use of confocal microscopes, providing a 3‐D analysis of the intracellular space. Automation has contributed to make confocal analysis available for high‐content image cytometry studies. However, the storage, browsing, and analysis of the amount of data generated can challenge the feasibility of such studies. Presented in this chapter is a multistep acquisition and analysis protocol that can bypass such difficulties by an analysis‐driven data collection. Cell‐cycle analysis of low‐resolution data can be employed to select cell populations of interest that can then be imaged at extremely high resolution and subjected to high‐content analysis. Curr. Protoc. Cytom. 70:7.42.1‐7.42.14.

High‐Resolution Cytometry for High‐Content Cell Cycle Analysis

One of the major limitations of flow cytometry (FCM) is the absence of an intracellular view. Automated microscopy and image analysis, together with technological developments, led to new approaches in cytometry that bypass the above limitation, introducing high resolution, high content, and large statistical sampling. However, few attempts have been made, until now, to translate the wide repertoire of FCM assays into high‐content image screening. This unit describes the implementation of an acquisition and analysis protocol for evaluation of the cell cycle by automated microscopy. The approach grants the possibility to perform simultaneous analysis of a high number of different parameters. A large part of this unit is devoted to the description of hardware features that can optimize the recorded information together with the acquisition and analysis procedures employed to produce good‐quality data. Curr. Protoc. Cytom. 70:7.41.1‐7.41.15.

Exploiting the tunability of stimulated emission depletion microscopy for super-resolution imaging of nuclear structures

Imaging of nuclear structures within intact eukaryotic nuclei is imperative to understand the effect of chromatin folding on genome function. Recent developments of super-resolution fluorescence microscopy techniques combine high specificity, sensitivity, and less-invasive sample preparation procedures with the sub-diffraction spatial resolution required to image chromatin at the nanoscale. Here, we present a method to enhance the spatial resolution of a stimulated-emission depletion (STED) microscope based only on the modulation of the STED intensity during the acquisition of a STED image. This modulation induces spatially encoded variations of the fluorescence emission that can be visualized in the phasor plot and used to improve and quantify the effective spatial resolution of the STED image. We show that the method can be used to remove direct excitation by the STED beam and perform dual color imaging. We apply this method to the visualization of transcription and replication foci within intact nuclei of eukaryotic cells.A known limitation of super-resolution STED microscopy is the need of high laser power which can cause photobleaching and phototoxicity. Here the authors further optimize this method and show that modulating STED intensity during acquisition results in an enhanced resolution and reduced background.