Advanced Fluorescence Microscopy Techniques-FRAP, FLIP, FLAP, FRET and FLIM

Fluorescence microscopy provides an efficient and unique approach to study fixed and living cells because of its versatility, specificity, and high sensitivity. Fluorescence microscopes can both detect the fluorescence emitted from labeled molecules in biological samples as images or photometric data from which intensities and emission spectra can be deduced. By exploiting the characteristics of fluorescence, various techniques have been developed that enable the visualization and analysis of complex dynamic events in cells, organelles, and sub-organelle components within the biological specimen. The techniques described here are fluorescence recovery after photobleaching (FRAP), the related fluorescence loss in photobleaching (FLIP), fluorescence localization after photobleaching (FLAP), Forster or fluorescence resonance energy transfer (FRET) and the different ways how to measure FRET, such as acceptor bleaching, sensitized emission, polarization anisotropy, and fluorescence lifetime imaging microscopy (FLIM). First, a brief introduction into the mechanisms underlying fluorescence as a physical phenomenon and fluorescence, confocal, and multiphoton microscopy is given. Subsequently, these advanced microscopy techniques are introduced in more detail, with a description of how these techniques are performed, what needs to be considered, and what practical advantages they can bring to cell biological research

Importin-β modulates the permeability of the nuclear pore complex in a Ran-dependent manner

Soluble karyopherins of the importin-β (impβ) family use RanGTP to transport cargos directionally through the nuclear pore complex (NPC). Whether impβ or RanGTP regulate the permeability of the NPC itself has been unknown. Here, we identify a stable pool of impβ at the NPC. A subpopulation of this pool is rapidly turned-over by RanGTP, likely at Nup153. Impβ, but not transportin-1 (TRN1), alters the pore's permeability in a Ran-dependent manner, suggesting that impβ is a functional component of the NPC. Upon reduction of Nup153 levels, inert cargos more readily equilibrate across the NPC yet active transport is impaired. When purified impβ or TRN1 are mixed with Nup153 in vitro, higher-order, multivalent complexes form. RanGTP dissolves the impβ•Nup153 complexes but not those of TRN1•Nup153. We propose that impβ and Nup153 interact at the NPC's nuclear face to form a Ran-regulated mesh that modulates NPC permeability

The methylated N-terminal tail of RCC1 is required for stabilisation of its interaction with chromatin by Ran in live cells

<p>Abstract</p> <p>Background</p> <p>Regulator of chromosome condensation 1 (RCC1) is the guanine nucleotide exchange factor for Ran GTPase. Localised generation of Ran-GTP by RCC1 on chromatin is critical for nucleocytoplasmic transport, mitotic spindle assembly and nuclear envelope formation. Both the N-terminal tail of RCC1 and its association with Ran are important for its interaction with chromatin in cells. In vitro, the association of Ran with RCC1 induces a conformational change in the N-terminal tail that promotes its interaction with DNA.</p> <p>Results</p> <p>We have investigated the mechanism of the dynamic interaction of the α isoform of human RCC1 (RCC1α) with chromatin in live cells using fluorescence recovery after photobleaching (FRAP) of green fluorescent protein (GFP) fusions. We show that the N-terminal tail stabilises the interaction of RCC1α with chromatin and this function can be partially replaced by another lysine-rich nuclear localisation signal. Removal of the tail prevents the interaction of RCC1α with chromatin from being stabilised by Ran^T24N, a mutant that binds stably to RCC1α. The interaction of RCC1α with chromatin is destabilised by mutation of lysine 4 (K4Q), which abolishes α-N-terminal methylation, and this interaction is no longer stabilised by Ran^T24N. However, α-N-terminal methylation of RCC1α is not regulated by the binding of Ran^T24N. Conversely, the association of Ran with precipitated RCC1α does not require the N-terminal tail of RCC1α or its methylation. The mobility of RCC1α on chromatin is increased by mutation of aspartate 182 (D182A), which inhibits guanine-nucleotide exchange activity, but RCC1α^D182Acan still bind nucleotide-free Ran and its interaction with chromatin is stabilised by Ran^T24N.</p> <p>Conclusions</p> <p>These results show that the stabilisation of the dynamic interaction of RCC1α with chromatin by Ran in live cells requires the N-terminal tail of RCC1α. α-N-methylation is not regulated by formation of the binary complex with Ran, but it promotes chromatin binding through the tail. This work supports a model in which the association of RCC1α with chromatin is promoted by a conformational change in the α-N-terminal methylated tail that is induced allosterically in the binary complex with Ran.</p

Genomic Alteration in Head and Neck Squamous Cell Carcinoma (HNSCC) Cell Lines Inferred from Karyotyping, Molecular Cytogenetics, and Array Comparative Genomic Hybridization

<div><p>Genomic alteration in head and neck squamous cell carcinoma (HNSCC) was studied in two cell line pairs (HN30-HN31 and HN4-HN12) using conventional C-banding, multiplex fluorescence <i>in situ</i> hybridization (M-FISH), and array comparative genomic hybridization (array CGH). HN30 and HN4 were derived from primary lesions in the pharynx and base of tongue, respectively, and HN31 and HN12 were derived from lymph-node metastatic lesions belonging to the same patients. Gain of chromosome 1, 7, and 11 were shared in almost all cell lines. Hierarchical clustering revealed that HN31 was closely related to HN4, which shared eight chromosome alteration cases. Large C-positive heterochromatins were found in the centromeric region of chromosome 9 in HN31 and HN4, which suggests complex structural amplification of the repetitive sequence. Array CGH revealed amplification of 7p22.3p11.2, 8q11.23q12.1, and 14q32.33 in all cell lines involved with tumorigenesis and inflammation genes. The amplification of 2p21 (<i>SIX3</i>), 11p15.5 (<i>H19</i>), and 11q21q22.3 (<i>MAML2</i>, <i>PGR</i>, <i>TRPC6</i>, and <i>MMP</i> family) regions, and deletion of 9p23 (<i>PTPRD</i>) and 16q23.1 (<i>WWOX</i>) regions were identified in HN31 and HN12. Interestingly, partial loss of <i>PTPRD</i> (9p23) and <i>WWOX</i> (16q23.1) genes was identified in HN31 and HN12, and the level of gene expression tended to be the down-regulation of <i>PTPRD</i>, with no detectable expression of the <i>WWOX</i> gene. This suggests that the scarcity of <i>PTPRD</i> and <i>WWOX</i> genes might have played an important role in progression of HNSCC, and could be considered as a target for cancer therapy or a biomarker in molecular pathology.</p></div

C-banded metaphase spread of four head and neck squamous cell carcinoma cell lines.

<p>C-banded metaphase spread of HN30, HN31, HN4, and HN12 are shown in (a, b, c, and d). Arrows indicate chromosome 9, and arrowheads indicate chromosomes 1. Scale bars represent 10 μm.</p

mRNA expression of candidate genes in four head and neck squamous cell carcinoma cell lines and a human mucoepidermoid pulmonary carcinoma NCI as compared with a non-tumorigenic human skin keratinocyte cell line HaCaT by real time quantitative reverse transcription PCR.

<p>The X-axis indicates relative expression level to HaCaT, and the Y-axis indicates expression level of each cell line. The level of mRNA expression of <i>SIX3</i>, <i>H19</i>, <i>PTPRD</i>, and <i>WWOX</i> are shown in (a, b, c, and d). Data were expressed as mean ± standard deviation. The levels of statistical significance were represented as * for <i>P</i> ≤ 0.05, ** for <i>P</i> ≤ 0.01, and *** for <i>P</i> ≤ 0.001. N/A indicates expression not detectable.</p

Graphical representation of two-dimensional unsupervised hierarchical clustering of chromosomal aberrations in four head and neck squamous cell carcinoma cell lines (10 metaphases per cell line).

<p>Each column refers to a metaphase in each cell line, and each row to type of chromosomal abnormality. Red indicates the presence of each abnormality. Black indicates the absence of each abnormality.</p

DAPI stained- and M-FISH (multiplex fluorescence <i>in situ</i> hybridization) karyotypes of four head and neck squamous cell carcinoma cell lines.

<p>DAPI stained and M-FISH karyotype of a representative metaphase indicates for HN30 (a and b), HN31(c and d), HN4 (e and f), and HN12 (g and h). Scale bars represent 10 μm.</p