Nanoscale Distribution of Nuclear Sites by Super-Resolved Image Cross-Correlation Spectroscopy

Deciphering the spatiotemporal coordination between nuclear functions is important to understand its role in the maintenance of human genome. In this context, super-resolution microscopy has gained considerable interest because it can be used to probe the spatial organization of functional sites in intact single-cell nuclei in the 20\u2013250 nm range. Among the methods that quantify colocalization from multicolor images, image cross-correlation spectroscopy (ICCS) offers several advantages, namely it does not require a presegmentation of the image into objects and can be used to detect dynamic interactions. However, the combination of ICCS with super-resolution microscopy has not been explored yet. Here, we combine dual-color stimulated emission depletion (STED) nanoscopy with ICCS (STED-ICCS) to quantify the nanoscale distribution of functional nuclear sites. We show that super-resolved ICCS provides not only a value of the colocalized fraction but also the characteristic distances associated to correlated nuclear sites. As a validation, we quantify the nanoscale spatial distribution of three different pairs of functional nuclear sites in MCF10A cells. As expected, transcription foci and a transcriptionally repressive histone marker (H3K9me3) are not correlated. Conversely, nascent DNA replication foci and the proliferating cell nuclear antigen(PCNA) protein have a high level of proximity and are correlated at a nanometer distance scale that is close to the limit of our experimental approach. Finally, transcription foci are found at a distance of 130 nm from replication foci, indicating a spatial segregation at the nanoscale. Overall, our data demonstrate that STED-ICCS can be a powerful tool for the analysis of the nanoscale distribution of functional sites in the nucleus

Investigating nanoscale chromatin alterations involved in neuroblastoma transformation by optical nanoscopy

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood and is characterized by remarkable heterogeneity. This work aims to characterize changes in chromatin nanoscale architecture being associated with NB transformation. We employed an NB cell line overexpressing the non-coding RNA NDM29, which promotes cell differentiation toward a neuronal phenotype. The nuclear shape, volume and architecture were assessed in both malignant and neuronlike cells by confocal microscopy. Moreover, heterochromatin and euchromatin organization was investigated by stimulated emission depletion (STED) microscopy. The results showed that the nuclei of neuron-like cells have a reduced volume and a more elongated shape compared to those of malignant cells and a different spatial arrangement of euchromatin and heterochromatin. Altogether these data point to an alteration of nuclear organization associated to NB, paving the way towards a better comprehension of the disease

New insights in Progerin-induced modifications of chromatin landscapes

Genome structure, expression, and regulation are crucial in maintaining the physiological state of any cell. However, even a single variation in one of these processes can induce genomic instability, leading to different pathologies, including aging and cancer. Our work focuses on a particular model for of laminopathies disease, Hutchinson Gilford Progeria Syndrome, HGPS. HGPS is a genetic disorder in which patients show aging-related symptoms in the first years of their lives. HPGS is caused by a mutation in the Lamin A gene, LMNA, that causes the permanent anchoring of the mutated protein, named Progerin, to the nuclear membrane. This permanent bond causes abnormal tractions leading to a crumpled nuclear morphology that affects chromatin organization, inducing alteration in replication, transcription, and DNA repair. Indeed, one single point mutation in the LMNA gene results in massive damage to many cellular processes. Here, we exploit Structured Illumination microscopy, SIM, to investigate altered heterochromatin distribution in HGPS-model and compare the results with the control cell line. In particular, we explore how Progerin affects facultative heterochromatin organization in the proximity of the Nuclear Pore Complex, NUP, at the nuclear lamina level. To this aim, we perform multicolor SIM of DNA, Progerin or lamin (respectively HGPS or control), facultative heterochromatin (tagging histone H3K9me2) and NUP. Then, we combine SIM with Image Cross-Correlation Spectroscopy analysis, ICCS, to evaluate the differences in distances between NUP and H3K9me2. Our approach enables the visualization of Progerin-induced alterations of chromatin nanoscale organization at the single-cell level and will hopefully lead to a deeper understanding of the molecular mechanisms associated with HGPS development

Measuring nanoscale distances by structured illumination microscopy and image cross-correlation spectroscopy (Sim-iccs)

Since the introduction of super-resolution microscopy, there has been growing interest in quantifying the nanoscale spatial distributions of fluorescent probes to better understand cellular processes and their interactions. One way to check if distributions are correlated or not is to perform colocalization analysis of multi-color acquisitions. Among all the possible methods available to study and quantify the colocalization between multicolor images, there is image cross-correlation spectroscopy (ICCS). The main advantage of ICCS, in comparison with other co-localization tech-niques, is that it does not require pre-segmentation of the sample into single objects. Here we show that the combination of structured illumination microscopy (SIM) with ICCS (SIM-ICCS) is a simple approach to quantify colocalization and measure nanoscale distances from multi-color SIM images. We validate the SIM-ICCS analysis on SIM images of optical nanorulers, DNA-origami-based model samples containing fluorophores of different colors at a distance of 80 nm. The SIM-ICCS analysis is compared with an object-based analysis performed on the same samples. Finally, we show that SIM-ICCS can be used to quantify the nanoscale spatial distribution of functional nuclear sites in fixed cells

Decrypting the spatial relationship between peripheral chromatin and nuclear lamina in Hutchinson-Gilford Progeria Syndrome using super-resolution microscopy techniques

The genetic condition known as Hutchinson-Gilford Progeria Syndrome (HGPS) causes the early onset of aging symptoms and premature death. HGPS is caused by a single nucleotide mutation in the LMNA gene, resulting in a truncated form of the protein Lamin A known as ΔLA50 or Progerin. This protein modification causes aberrant lamina network organization inducing nuclear and DNA structure abnormalities, leading to the loss of peripheral heterochromatin. In this study, we examine variations in chromatin compaction in a cellular model of HPGS using Expansion Microscopy (ExM). This super-resolution microscopy technique is based on embedding biological samples into a water-absorbant polymer network that, following mechanical homogenization, enables a four-fold linear expansion in water, allowing nanoscale biophysical studies using conventional microscopes. The image analysis of the HGPS cell model shows a significant variation in chromatin compaction that seems to disrupt its physical association with the nuclear lamina. Furthermore, this loss of compaction leads to a detectable spatial separation between the lamina network, mainly composed of the mutated form Progerin, and the peripheral heterochromatin. Therefore, to quantify this distance variation caused by Progerin-induced chromatin disorganization, we analyzed a batch of control cells expressing wild-typer lamin A and HGPS cells, finding a one-order of magnitude difference between the two sets. In order to validate these results, we perform super-resolution Stimulated Emission Depletion (STED) imaging. The next step will be performing Correlative Light Electron Microscopy (CLEM) on non-expanded cells to push the resolution of our analysis even forward and to investigate chromatin compaction on a nanoscale level

Combining Expansion Microscopy and STED Nanoscopy for the Study of Cellular Organization

Expansion microscopy (ExM) is a novel method that allows super-resolutionimaging with conventional microscopes(1, 2). It consists in soaking the cellswith a polymer, inducing the polymerization to form a dense meshworkthroughout the cell, cross-linking the fluorophores to the polymer and, afterdigestion of cellular protein, rehydrating of the sample. The swelling ofthe polymer gel led to a fourfold isotropic stretching of the sample. Therefore,it increases the distance between two objects that otherwise couldnot be seen as two different things with an ordinary microscope. One of the drawback of sucha technique is the long preparation made of several stages, i.e. immunostaining,gelation, digestion and expansion. They are really crucial steps for a good im-aging post-expansion.In our work we present a comparison between ExM and stimulated emissiondepletion (STED) nanoscopy(3). Our aim is to study the e possible combinationof STED and ExM as a method to further enhance the final resolution achiev-able. We will in particularly take advantage of the use of separation of photonsby lifetime tuning (SPLIT) STED (4).We show application of these methods from single fixed cells to slices of fixedmouse retina tissue.We are also interested in applying the approach to high-density compartmentslike the cell nucleus to decipher the high-order structure organization of chro-matin-DN