Novel roles for A-type lamins in telomere biology and the DNA damage response pathway.

A-type lamins are intermediate filament proteins that provide a scaffold for protein complexes regulating nuclear structure and function. Mutations in the LMNA gene are linked to a variety of degenerative disorders termed laminopathies, whereas changes in the expression of lamins are associated with tumourigenesis. The molecular pathways affected by alterations of A-type lamins and how they contribute to disease are poorly understood. Here, we show that A-type lamins have a key role in the maintenance of telomere structure, length and function, and in the stabilization of 53BP1, a component of the DNA damage response (DDR) pathway. Loss of A-type lamins alters the nuclear distribution of telomeres and results in telomere shortening, defects in telomeric heterochromatin, and increased genomic instability. In addition, A-type lamins are necessary for the processing of dysfunctional telomeres by non-homologous end joining, putatively through stabilization of 53BP1. This study shows new functions for A-type lamins in the maintenance of genomic integrity, and suggests that alterations of telomere biology and defects in DDR contribute to the pathogenesis of lamin-related diseases.

Translocation frequencies and chromosomal proximities for selected mouse chromosomes in primary B lymphocytes.

Chromosome positions within the nucleus of mammalian cells are nonrandom and it is assumed that chromosomal neighborhoods affect the probability of translocations. Four chromosomes can be involved in c-myc-activating chromosomal translocations in mouse plasmacytoma (PCT): the c-myc gene on mouse chromosome 15 can be juxtaposed to either one of the immunoglobulin (Ig) loci on chromosomes 12 (IgH), 16 (Igλ), or 6 (Igκ). In the BALB/c mouse, the translocation between chromosomes 12 and 15, T(12;15), is most common (90%) while the other two possible translocations, T(6;15) and T(16;15), are much less common (<10%). In contrast, in the BALB/cRb6.15 mouse, T(6;15) is found with the same frequency as T(12;15). We, therefore, examined the distance between chromosomes 15 and 12, 6, and 16 in primary mouse B lymphocytes in order to examine the effect of the chromosome proximity on the translocation frequency. We performed three-dimensional fluorescent in situ hybridization (3D-FISH) with chromosome paints. We acquired three-dimensional image stacks with 90 slices per stack and used constrained iterative deconvolution. The nucleus and chromosomes were segmented from this image stack and the interchromosomal distances were measured. Chromosomes 6 and 15 were found in close proximity in BALB/cRb6.15 mice (82%), whereas they did not share this neighborhood relationship in BALB/c mice. No other chromosome combinations showed such a high percentage of close proximities in either mouse strain. Chromosome positions contribute to translocation frequencies in mouse PCTs. The BALB/cRb6.15 mouse data argue for a proximity relationship of chromosomes that engage in illegitimate recombination. These positions are not, however, the only contributing factor as the T(12;15) translocation preference in BALB/c mice could not be supported by significantly elevated proximity of chromosomes 12 and 15 versus 12 and 16 or 12 and 6. Moreover, while there is a significant increase in T(6;15) in BALB/cRb6.15 mice, T(12;15) still occurs in this mouse strain.

Segmentation and analysis of the three-dimensional redistribution of nuclear components in human mesenchymal stem cells.

To better understand the impact of changes in nuclear architecture on nuclear functions, it is essential to quantitatively elucidate the three-dimensional organization of nuclear components using image processing tools. We have developed a novel image segmentation method, which involves a contrast enhancement and a subsequent thresholding step. In addition, we have developed a new segmentation method of the nuclear volume using the fluorescent background signal of a probe. After segmentation of the nucleus, a first-order normalization is performed on the signal positions of the component of interest to correct for the shape of the nucleus. This method allowed us to compare various signal positions within a single nucleus, and also on pooled data obtained from multiple nuclei, which may vary in size and shape. The algorithms have been tested by analyzing the spatial localization of nuclear bodies in relation to the nuclear center. Next, we used this new tool to study the change in the spatial distribution of nuclear components in cells before and after caspase-8 activation, which leads to cell death. Compared to the morphological TopHat method, this method gives similar but significantly faster results. A clear shift in the radial distribution of centromeres has been found, while the radial distribution of telomeres was changed much less. In addition, we have used this new tool to follow changes in the spatial distribution of two nuclear components in the same nucleus during activation of apoptosis. We show that after caspase-8 activation, when centromeres shift to a peripheral localization, the spatial distribution of PML-NBs does not change while that of centromeres did. We propose that the use of this new image segmentation method will contribute to a better understanding of the 3D spatial organization of the cell nucleus.

From micro to nano: recent advances in high-resolution microscopy.

Improving the spatial resolution of optical microscopes is important for a vast number of applications in the life sciences. Optical microscopy allows intact samples and living cells to be studied in their natural environment, tasks that are not possible with other microscopy methods (e.g. electron microscopy). Major advances in the past two decades have significantly improved microscope resolution. By using interference and structured light methods microscope resolution has been improved to approximately 100 nm, and with non-linear methods a ten times improvement has been demonstrated to a current resolution limit of approximately 30 nm. These methods bring together old theoretical concepts such as interference with novel non-linear methods that improve spatial resolution beyond the limits that were previously assumed to be unreachable.

The three-dimensional organization of telomeres in the nucleus of mammalian cells.

BACKGROUND: The observation of multiple genetic markers in situ by optical microscopy and their relevance to the study of three-dimensional (3D) chromosomal organization in the nucleus have been greatly developed in the last decade. These methods are important in cancer research because cancer is characterized by multiple alterations that affect the modulation of gene expression and the stability of the genome. It is, therefore, essential to analyze the 3D genome organization of the interphase nucleus in both normal and cancer cells. RESULTS: We describe a novel approach to study the distribution of all telomeres inside the nucleus of mammalian cells throughout the cell cycle. It is based on 3D telomere fluorescence in situ hybridization followed by quantitative analysis that determines the telomeres' distribution in the nucleus throughout the cell cycle. This method enables us to determine, for the first time, that telomere organization is cell-cycle dependent, with assembly of telomeres into a telomeric disk in the G2 phase. In tumor cells, the 3D telomere organization is distorted and aggregates are formed. CONCLUSIONS: The results emphasize a non-random and dynamic 3D nuclear telomeric organization and its importance to genomic stability. Based on our findings, it appears possible to examine telomeric aggregates suggestive of genomic instability in individual interphase nuclei and tissues without the need to examine metaphases. Such new avenues of monitoring genomic instability could potentially impact on cancer biology, genetics, diagnostic innovations and surveillance of treatment response in medicine.

Quantitative comparison of PET and Bremsstrahlung SPECT for imaging the in vivo yttrium-90 microsphere distribution after liver radioembolization.

BACKGROUND: After yttrium-90 ((90)Y) microsphere radioembolization (RE), evaluation of extrahepatic activity and liver dosimetry is typically performed on (90)Y Bremsstrahlung SPECT images. Since these images demonstrate a low quantitative accuracy, (90)Y PET has been suggested as an alternative. The aim of this study is to quantitatively compare SPECT and state-of-the-art PET on the ability to detect small accumulations of (90)Y and on the accuracy of liver dosimetry. METHODOLOGY/PRINCIPAL FINDINGS: SPECT/CT and PET/CT phantom data were acquired using several acquisition and reconstruction protocols, including resolution recovery and Time-Of-Flight (TOF) PET. Image contrast and noise were compared using a torso-shaped phantom containing six hot spheres of various sizes. The ability to detect extra- and intrahepatic accumulations of activity was tested by quantitative evaluation of the visibility and unique detectability of the phantom hot spheres. Image-based dose estimates of the phantom were compared to the true dose. For clinical illustration, the SPECT and PET-based estimated liver dose distributions of five RE patients were compared. At equal noise level, PET showed higher contrast recovery coefficients than SPECT. The highest contrast recovery coefficients were obtained with TOF PET reconstruction including resolution recovery. All six spheres were consistently visible on SPECT and PET images, but PET was able to uniquely detect smaller spheres than SPECT. TOF PET-based estimates of the dose in the phantom spheres were more accurate than SPECT-based dose estimates, with underestimations ranging from 45% (10-mm sphere) to 11% (37-mm sphere) for PET, and 75% to 58% for SPECT, respectively. The differences between TOF PET and SPECT dose-estimates were supported by the patient data. CONCLUSIONS/SIGNIFICANCE: In this study we quantitatively demonstrated that the image quality of state-of-the-art PET is superior over Bremsstrahlung SPECT for the assessment of the (90)Y microsphere distribution after radioembolization.

Molecular image analysis: quantitative description and classification of the nuclear lamina in human mesenchymal stem cells.

The nuclear lamina is an intermediate filament network that provides a structural framework for the cell nucleus. Changes in lamina structure are found during changes in cell fate such as cell division or cell death and are associated with human diseases. An unbiased method that quantifies changes in lamina shape can provide information on cells undergoing changes in cellular functions. We have developed an image processing methodology that finds and quantifies the 3D structure of the nuclear lamina. We show that measurements on such images can be used for cell classification and provide information concerning protein spatial localization in this structure. To demonstrate the efficacy of this method, we compared the lamina of unmanipulated human mesenchymal stem cells (hMSCs) at passage 4 to cells activated for apoptosis. A statistically significant classification was found between the two populations.

Robust nuclear lamina-based cell classification of aging and senescent cells.

Changes in the shape of the nuclear lamina are exhibited in senescent cells, as well as in cells expressing mutations in lamina genes. To identify cells with defects in the nuclear lamina we developed an imaging method that quantifies the intensity and curvature of the nuclear lamina. We show that this method accurately describes changes in the nuclear lamina. Spatial changes in nuclear lamina coincide with redistribution of lamin A proteins and local reduction in protein mobility in senescent cell. We suggest that local accumulation of lamin A in the nuclear envelope leads to bending of the structure. A quantitative distinction of the nuclear lamina shape in cell populations was found between fresh and senescent cells, and between primary myoblasts from young and old donors. Moreover, with this method mutations in lamina genes were significantly distinct from cells with wild-type genes. We suggest that this method can be applied to identify abnormal cells during aging, in in vitro propagation, and in lamina disorders.

The nuclear lamina promotes telomere aggregation and centromere peripheral localization during senescence of human mesenchymal stem cells.

Ex vivo, human mesenchymal stem cells (hMSCs) undergo spontaneous cellular senescence after a limited number of cell divisions. Intranuclear structures of the nuclear lamina were formed in senescent hMSCs, which are identified by the presence of Hayflick-senescence-associated factors. Notably, spatial changes in lamina shape were observed before the Hayflick senescence-associated factors, suggesting that the lamina morphology can be used as an early marker to identify senescent cells. Here, we applied quantitative image-processing tools to study the changes in nuclear architecture during cell senescence. We found that centromeres and telomeres colocalised with lamina intranuclear structures, which resulted in a preferred peripheral distribution in senescent cells. In addition, telomere aggregates were progressively formed during cell senescence. Once formed, telomere aggregates showed colocalization with gamma-H2AX but not with TERT, suggesting that telomere aggregates are sites of DNA damage. We also show that telomere aggregation is associated with lamina intranuclear structures, and increased telomere binding to lamina proteins is found in cells expressing lamina mutants that lead to increases in lamina intranuclear structures. Moreover, three-dimensional image processing revealed spatial overlap between telomere aggregates and lamina intranuclear structures. Altogether, our data suggest a mechanical link between changes in lamina spatial organization and the formation of telomere aggregates during senescence of hMSCs, which can possibly contribute to changes in nuclear activity during cell senescence.

Alterations of centromere positions in nuclei of immortalized and malignant mouse lymphocytes.

BACKGROUND: The three-dimensional (3D) positions of centromeres have been studied in several cell systems. However, data on centromere positions during cellular transformation remain elusive. This study has focused on mouse lymphocytes and investigated the centromere positions in primary, immortalized, and tumor cells. METHODS: Eighty-to-ninety z-slices of each mouse lymphocyte nucleus were acquired using a sampling distance of 107 nm in the xy plane and 200 nm along z for each z-stack, using an Axioplan 2 microscope, an AxioCam HR CCD, a 63x/1.4 oil objective, and the Axiovision 3.1 software (Carl Zeiss, Canada). A constrained iterative algorithm (Schaefer et al., J Microsc 2001;204:99-107) was used for deconvolution. Centromere positions in 3D images were analyzed using CentroView, a program we developed to measure nuclear centromere positions. RESULTS: Using CentroView we determined the positions of centromeres in primary lymphocytes, immortalized and malignant mouse B cells. We show that centromeres exhibit altered nuclear positions in immortalized and malignant B cells. These changes are independent of previously described cell cycle-dependent centromere dynamics. CONCLUSIONS: The 3D positions of centromeres are altered during cellular transformation. In lymphocytes, centromeres are found in more central nuclear positions following immortalization and transformation. These nuclear changes reflect structural remodeling of mammalian nuclei during oncogenesis and may impact on the structural organization of chromosomes. How centromeric changes are linked to nuclear remodeling can now be quantitatively examined using the tools of this study.

Changes in lamina structure are followed by spatial reorganization of heterochromatic regions in caspase-8-activated human mesenchymal stem cells.

Apoptosis is fundamental to the regulation of homeostasis of stem cells in vivo. Whereas the pathways underlying the molecular and biochemical details of nuclear breakdown that accompanies apoptosis have been elucidated, the precise nature of nuclear reorganization that precedes the demolition phase is not fully understood. Here, we expressed an inducible caspase-8 in human mesenchymal stem cells, and quantitatively followed the early changes in nuclear organization during apoptosis. We found that caspase-8 induces alteration of the nuclear lamina and a subsequent spatial reorganization of both centromeres, which are shifted towards a peripheral localization, and telomeres, which form aggregates. This nuclear reorganization correlates with caspase-3 sensitivity of lamina proteins, because the expression of lamin mutant constructs with caspase-3 hypersensitivity resulted in a caspase-8-independent appearance of lamina intranuclear structures and telomere aggregates, whereas application of a caspase inhibitor restrains these changes in nuclear reorganization. Notably, upon activation of apoptosis, we observed no initial changes in the spatial organization of the promyelocytic leukemia nuclear bodies (PML-NBs). We suggest that during activation of the caspase-8 pathway changes in the lamina structure precede changes in heterochromatin spatial organization, and the subsequent breakdown of lamina and PML-NB.

c-Myc induces chromosomal rearrangements through telomere and chromosome remodeling in the interphase nucleus.

In previous work, we showed that telomeres of normal cells are organized within the 3D space of the interphase nucleus in a nonoverlapping and cell cycle-dependent manner. This order is distorted in tumor cell nuclei where telomeres are found in close association forming aggregates of various numbers and sizes. Here we show that c-Myc overexpression induces telomeric aggregations in the interphase nucleus. Directly proportional to the duration of c-Myc deregulation, we observe three or five cycles of telomeric aggregate formation in interphase nuclei. These cycles reflect the onset and propagation of breakage-bridge-fusion cycles that are initiated by end-to-end telomeric fusions of chromosomes. Subsequent to initial chromosomal breakages, new fusions follow and the breakage-bridge-fusion cycles continue. During this time, nonreciprocal translocations are generated. c-Myc-dependent remodeling of the organization of telomeres thus precedes the onset of genomic instability and subsequently leads to chromosomal rearrangements. Our findings reveal that c-Myc possesses the ability to structurally modify chromosomes through telomeric fusions, thereby reorganizing the genetic information.