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Super-resolution fluorescence microscopy has revolutionized multicolor imaging of nuclear structures due to the combination of high labeling specificity and high resolution. Here we expanded the recently developed fBALM (DNA structure fluctuation-assisted binding activated localization microscopy) method by developing a stable methodological sequence that enables dual-color imaging of high-resolution genomic DNA together with an immunofluorescently labeled intranuclear protein. Our measurements of the nuclear periphery, imaging DNA and LaminB1 in biologically relevant samples, show that this novel dual-color imaging method is feasible for further quantitative evaluations. We were able to study the relative spatial signal organization between DNA and LaminB1 by means of highly specific colocalization measurements at nanometer resolution. Measurements were performed with and without the antifade embedding medium ProLong Gold, which proved to be essential for imaging of LaminB1, but not for imaging of SytoxOrange labeled DNA. The localization precision was used to differentiate between localizations with higher and lower amounts of emitting photons. We interpret high intensity localizations to be renatured DNA sections in which a high amount of Sytox Orange molecules were bound. This could give insight into the denaturation kinetics of DNA during fBALM. These results were further complemented by measurements of γH2AX and H3K9me3 signal organization to demonstrate differences within the chromatin landscape, which were quantified with image processing methods such as Voronoi segmentation.

Wilhelm Bernhard\'s revolutionary microscopy techniques helped him put forward the hypothesis of specialized compartmentalization of the nucleus. He also described for the first time the nuclear bodies and peri-chromatin fibrils, and demonstrated that these granules contain an RNA component. The tradition of biennial workshops, named after this great scientist, continues, and this year it took place in the heart of Burgundy, in Dijon, France (May 20-24, 2019, organized by INSERM UMR1231, UBFC), where well-fed participants emphasized the importance of viewing the cell nucleus as a hub of specialized colloidal compartments that orchestrate replication, transcription and nuclear transport.

Although many protein labeling probes have been developed to elucidate the trafficking and turnover processes of cell surface proteins, real-time tracking of intracellular proteins remains a challenging task. Herein, we describe a new design to construct a cell-permeable, photostable, and far-red fluorescent turn-on probe to enable no-wash, organelle-specific, and long-term visualization of intracellular SNAP-tagged proteins in living cells. When the probe was used in dual-color pulse chase labeling experiments to differentiate between preLamin and mature Lamin, our results reveal that the shape of mature Lamin can be altered by the newly synthesized preLamin and that this alteration is progressive, cumulative, and due to a concentration-dependent dominant-negative effect of preLamin. We believe that this probe can also be applied to other intracellular proteins whose cellular localization and synthesis changes dynamically in response to external stimuli.

In the last decades, atomic force microscopy (AFM) underwent a rapid and stunning development, especially for studying mechanical properties of biological samples. The numerous discoveries relying to this approach, have increased the credit of AFM as a versatile tool, and potentially eligible as a diagnostic equipment. Meanwhile, it has become strikingly evident that lamins are involved on the onset and development of certain diseases, including cancer, Hutchinson-Gilford progeria syndrome, cardiovascular pathologies, and muscular dystrophy. A new category of pathologies has been defined, the laminopathies, which are caused by mutations in the gene encoding for A-type lamins. As the majority of medical issues, lamins, and all their related aspects can be considered as a quite complex problem. Indeed, there are many facets to explore, and this definitely requires a multidisciplinary approach. One of the most intriguing aspects concerning lamins is their remarkable contribute to cells mechanics. Over the years, this has led to the speculation of the so-called \"structural hypothesis\", which attempts to elucidate the etiology and some features of the laminopathies. Among the various techniques tried to figure out the role of lamins in the cells mechanics, the AFM has been already successfully applied, proving its versatility. Therefore, the present work aims both to highlight the qualities of AFM and to review the most relevant knowledge about lamins, in order to promote the study of the latter, taking advantage from the former. Microsc. Res. Tech. 80:97-108, 2017. © 2016 Wiley Periodicals, Inc.

To evaluate the intracellular force transmission between the nucleus and cytoskeleton, we optimized a single cell-based assay that involves the manipulation of living, adherent cells with a fine glass microneedle and a microscope-mounted micromanipulator. The user inserts the microneedle into the cytoplasm and then, using a custom-programmable computer script, pulls the needle laterally toward the cell periphery. Normalized cross-correlation is applied to recorded time-lapse image sequences to determine average displacements within predefined regions of the nucleus and the cytoskeleton. These regional displacements, together with calculations of nuclear elongation, nuclear centroid translocation, and nuclear shape changes, enable quantitative assessments of nucleo-cytoskeletal coupling in both normal and disease conditions and provide an improved understanding of the role of specific nuclear envelope proteins in intracellular force propagation.

The dynamic organisation of the cell nucleus is profoundly modified during growth, development and senescence as a result of changes in chromatin arrangement and gene transcription. A plethora of data suggests that the nuclear lamina is a key player in chromatin dynamics and argues in favour of a major involvement of prelamin A in fundamental mechanisms regulating cellular senescence and organism ageing. As the best model to analyse the role of prelamin A in normal ageing, we used cells from centenarian subjects. We show that prelamin A is accumulated in fibroblasts from centenarians owing to downregulation of its specific endoprotease ZMPSTE24, whereas other nuclear envelope constituents are mostly unaffected and cells do not enter senescence. Accumulation of prelamin A in nuclei of cells from centenarians elicits loss of heterochromatin, as well as recruitment of the inactive form of 53BP1, associated with rapid response to oxidative stress. These effects, including the prelamin-A-mediated increase of nuclear 53BP1, can be reproduced by rapamycin treatment of cells from younger individuals. These data identify prelamin A and 53BP1 as new targets of rapamycin that are associated with human longevity. We propose that the reported mechanisms safeguard healthy ageing in humans through adaptation of the nuclear environment to stress stimuli.

Laminopathies are rare monogenic diseases, some of them exhibiting features of the metabolic syndrome. These diseases are mainly due to mutations in LMNA, encoding A-type lamins. One LMNA polymorphism, rs4641, has been associated with the metabolic syndrome, but results have been controversial. We therefore investigated the effect of single nucleotide polymorphisms (SNPs) in the LMNA gene in combination with four other genes encoding enzymes influencing lamin post-translational maturation on risk of metabolic syndrome (MS). Twenty-three tagging SNPs characterising the haplotypic variability of five genes (LMNA, ICMT, ZMPSTE24, FNTA and FNTB) were genotyped in 3,916 French men and women who took part in the prospective DESIR study. Single locus and haplotype analyses were performed but did not detect any significant association with the risk of MS. No robust interaction between SNPs located in different genes on the risk of MS was identified. In conclusion, we did not observe any convincing evidence that common polymorphisms of the lamina pathway could modulate the risk of MS.

BACKGROUND: Current research in depression aims to delineate genes involved in neuronal plasticity that are altered in the disease or its treatment. We have shown antidepressant induced increases in three interrelated genes, cell adhesion molecule L1 (CAM-L1), laminin, and cAMP response element binding protein (CREB), and a reciprocal decrease in these genes consequent to stress. Presently we hypothesized that CAM-L1, CREB, and laminin may be altered in post mortem brains of depressed subjects.
METHODS: Studies were performed in the prefrontal and in the ventral parieto-occipital cortices, of 59 brains from depressed, bipolar, and schizophrenic subjects, and normal controls, obtained from the Stanley Foundation Brain Collection. mRNA and protein levels were determined by RT-PCR and Western blot analysis, respectively.
RESULTS: Levels of CAM-L1 and of phosphorylated CREB (pCREB) were increased in the prefrontal cortex of the depressed group, while CAM-L1, laminin and pCREB were decreased in the parieto-occipital cortex. Depressed subjects receiving antidepressants differed from subjects not receiving antidepressants in the expression of CAM-L1 and laminin in the parieto-occipital cortex, and in the expression of pCREB in the prefrontal cortex.
CONCLUSIONS: The present findings of specific alterations in depression and antidepressant treatment particularly in CAM-L1 suggest that this gene may play an important role in the pathophysiology and treatment of depression.

Farnesyl protein transferase (FT), an enzyme that catalyzes the first step in the posttranslational modification of ras and a number of other polypeptides, has emerged as an important target for the development of anticancer agents. SCH66336 is one of the first FT inhibitors to undergo clinical testing. We report a Phase I trial to assess the maximum tolerated dose, toxicities, and biological effectiveness of SCH66336 in inhibiting FT in vivo. Twenty patients with solid tumors received 92 courses of escalating SCH66336 doses given orally twice a day (b.i.d.) for 7 days out of every 3 weeks. Gastrointestinal toxicity (nausea, vomiting, and diarrhea) and fatigue were dose-limiting at 400 mg of SCH66336 b.i.d. Moderate reversible renal insufficiency, secondary to dehydration from gastrointestinal toxicity, was also seen. Inhibition of prelamin A farnesylation in buccal mucosa cells of patients treated with SCH66336 was demonstrated, confirming that SCH66336 inhibits protein farnesylation in vivo. One partial response was observed in a patient with previously treated metastatic non-small cell lung cancer, who remained on study for 14 months. This study not only establishes the dose for future testing on this schedule (350 mg b.i.d.) but also provides the first evidence of successful inhibition of FT in the clinical setting and the first hint of clinical activity for this class of agents.