Cristina Flors

A super-resolution map of the vertebrate kinetochore

A longstanding question in centromere biology has been the organization of CENP-A–containing chromatin and its implications for kinetochore assembly. Here, we have combined genetic manipulations with deconvolution and super-resolution fluorescence microscopy for a detailed structural analysis of chicken kinetochores. Using fluorescence microscopy with subdiffraction spatial resolution and single molecule sensitivity to map protein localization in kinetochore chromatin unfolded by exposure to a low salt buffer, we observed robust amounts of H3K9me3, but only low levels of H3K4me2, between CENP-A subdomains in unfolded interphase prekinetochores. Constitutive centromere-associated network proteins CENP-C and CENP-H localize within CENP-A–rich subdomains (presumably on H3-containing nucleosomes) whereas CENP-T localizes in interspersed H3-rich blocks. Although interphase prekinetochores are relatively more resistant to unfolding than sur-rounding pericentromeric heterochromatin, mitotic kinetochores are significantly more stable, reflecting mitotic kinetochore maturation. Loss of CENP-H, CENP-N, or CENP-W had little or no effect on the unfolding of mitotic kinetochores. However, loss of CENP-C caused mitotic kinetochores to unfold to the same extent as their interphase counterparts. Based on our results we propose a new model for inner centromeric chromatin architecture in which chromatin is folded as a layered boustrophedon, with planar sinusoids containing interspersed CENP-A–rich and H3-rich subdomains oriented toward the outer kinetochore. In mitosis, a CENP-C–dependent mechanism crosslinks CENP-A blocks of different layers together, conferring extra stability to the kinetochore.

Singlet Oxygen Generation by the Genetically Encoded Tag miniSOG

The genetically encodable fluorescent tag miniSOG is expected to revolutionize correlative light- and electron microscopy due to its ability to produce singlet oxygen upon light irradiation. The quantum yield of this process was reported as ΦΔ = 0.47 ± 0.05, as derived from miniSOGs ability to photooxidize the fluorescent probe anthracene dipropionic acid (ADPA). In this report, a significantly smaller value of ΦΔ = 0.03 ± 0.01 is obtained by two methods: direct measurement of its phosphorescence at 1275 nm and chemical trapping using uric acid as an alternative probe. We present insight into the photochemistry of miniSOG and ascertain the reasons for the discrepancy in ΦΔ values. We find that miniSOG oxidizes ADPA by both singlet oxygen-dependent and -independent processes. We also find that cumulative irradiation of miniSOG increases its ΦΔ value ~10-fold due to a photoinduced transformation of the protein. This may be the reason why miniSOG outperforms other fluorescent proteins reported to date as singlet oxygen generators.

Second-Harmonic Generation in GFP-like Proteins

The second-order nonlinear optical properties of green fluorescent proteins (GFPs), such as the photoswitchable Dronpa and enhanced GFP (EGFP), have been studied at both the theoretical and experimental levels. In the case of Dronpa, both approaches are consistent in showing the rather counterintuitive result of a larger second-order nonlinear polarizability (or first hyperpolarizability, beta) for the protonated state, which has a higher transition energy, than for the deprotonated, fluorescent state with its absorption at lower energy. Moreover, the value of beta for the protonated form of Dronpa is among the highest reported for proteins. In addition to the pH dependence, we have found a wavelength dependence in the beta values. These properties are essential for the practical use of Dronpa or other GFP-like fluorescent proteins as second-order nonlinear fluorophores for symmetry-sensitive nonlinear microscopy imaging and as nonlinear optical sensors for electrophysiological processes. An accurate value of the first hyperpolarizability is also essential for any qualitative analysis of the nonlinear images.

DNA and chromatin imaging with super-resolution fluorescence microscopy based on single-molecule localization.

With the expansion of super‐resolution fluorescence microscopy methods, it is now possible to access the organization of cells and materials at the nanoscale by optical means. This review discusses recent progress in super‐resolution imaging of isolated and cell DNA using single‐molecule localization methods. A high labeling density of photoswitchable fluorophores is crucial for these techniques, which can be provided by sequence independent DNA stains in which photoblinking reactions can be induced. In particular, unsymmetrical cyanine intercalating dyes in combination with special buffers can be used to image isolated DNA with a spatial resolution of 30–40 nm. For super‐resolution imaging of chromatin, cell permeant cyanine dyes that bind the minor groove of DNA have the potential to become a useful alternative to the labeling of histones and other DNA‐associated proteins. Other recent developments that are interesting in this context such as high density labeling methods or new DNA probes with photoswitching functionalities are also surveyed. Progress in labeling, optics, and single‐molecule localization algorithms is being rapid, and it is likely to provide real insight into DNA structuring in cells and materials.

Quantification of Photosensitized Singlet Oxygen Production by a Fluorescent Protein

Fluorescent proteins are increasingly becoming actuators in a range of cell biology techniques. One of those techniques is chromophore-assisted laser inactivation (CALI), which is employed to specifically inactivate the function of target proteins or organelles by producing photochemical damage. CALI is achieved by the irradiation of dyes that are able to produce reactive oxygen species (ROS). The combination of CALI and the labelling specificity that fluorescent proteins provide is useful to avoid uncontrolled photodamage, although the inactivation mechanisms by ROS are dependent on the fluorescent protein and are not fully understood. Herein, we present a quantitative study of the ability of the red fluorescent protein TagRFP to produce ROS, in particular singlet oxygen ((1)O(2)). TagRFP is able to photosensitize (1)O(2) with an estimated quantum yield of 0.004. This is the first estimation of a quantum yield of (1)O(2) production value for a GFP-like protein. We also find that TagRFP has a short triplet lifetime compared to EGFP, which reflects relatively high oxygen accessibility to the chromophore. The insight into the structural and photophysical properties of TagRFP has implications in improving fluorescent proteins for fluorescence microscopy and CALI.

Super-resolution fluorescence microscopy as a tool to study the nanoscale organization of chromosomes.

Chromatin organization spans a wide range of structural complexity. Substructures at the 10-200nm scale are poorly characterized, especially in living cells, due to the limitations of electron microscopy and standard optical microscopy. Recently developed super-resolution fluorescence microscopy methods represent an exciting opportunity to access those substructures, and recent progress with these techniques has yielded insights into chromatin organization at different condensation stages. Recent studies have focused on confronting the challenges that are specific to chromatin super-resolution imaging, such as the high packing density of mitotic chromosomes and difficulties in interpreting interphase chromatin images. Building on these first results and with ongoing rapid technical advances in super-resolution fluorescence imaging there is great potential to uncover new features with unprecedented detail.

Photoswitching of monomeric and dimeric DNA-intercalating cyanine dyes for super-resolution microscopy applications

A growing trend in far-field super resolution fluorescence microscopy based on single molecule photoswitching involves the replacement of photoactivatable fluorophores by common organic dyes in which photoswitching reactions or blinking can be induced. This alternative strategy can greatly simplify the sample preparation and imaging scheme in some cases, and enables its application to a wider range of biological systems. This methodology has been applied successfully to unveil the nanoscale organisation of proteins, but little progress has been seen to date in DNA super-resolution imaging. Previous results have shown that blinking can be induced in the DNA-intercalating dimeric dye YOYO-1 in combination with a reducing buffer, and in turn super-resolution images of DNA can be reconstructed. However, monomeric intercalating dyes like YO-PRO-1 are more advantageous for biological applications. This paper shows that both YO-PRO-1 and YOYO-1 can be used in super-resolution imaging, and different sample preparation strategies are compared in terms of spatial resolution and homogeneity of the reconstructed super-resolution images. Moreover, ensemble and single-molecule experiments provide insight into the switching mechanism. The dyes YOYO-1 and YO-PRO-1 hold great potential for their use in nanoscale imaging of DNA topology in biology and nanoscience.

Light- and singlet oxygen-mediated antifungal activity of phenylphenalenone phytoalexins.

The light-induced singlet oxygen production and antifungal activity of phenylphenalenone phytoalexins isolated from infected banana plants (Musa acuminata) are reported. Upon absorption of light energy all studied phenylphenalenones sensitise the production of singlet oxygen in polar and non-polar media. Antifungal activity of these compounds towards Fusarium oxysporum is enhanced in the presence of light. These results, together with the correlation of IC50 values under illumination with the quantum yield of singlet oxygen production and the enhancing effect of D2O on the antifungal activity, suggest the intermediacy of singlet oxygen produced by electronic excitation of the phenylphenalenone phytoalexins.

Constitutively active RhoA inhibits proliferation by retarding G1 to S phase cell cycle progression and impairing cytokinesis

The actions of RhoA in cytoskeletal regulation have been extensively studied. RhoA also contributes to proliferation and oncogenic transformation by less well-characterized means. Elevated RhoA signalling has been associated with human cancer; through increased RhoA expression, mutation or elevated expression of activating Rho guanine-nucleotide exchange factors (GEFs), or from deletion or decreased expression of inhibitory Rho GTPase-activating proteins (GAPs). Unlike the Ras oncogene, constitutively-activated GTPase-deficient RhoA mutants have not been identified in tumours. To investigate the effects of active RhoA on proliferation, we generated Swiss3T3 cells that inducibly express wild-type RhoA or GTPase-deficient active V14RhoA. We found that V14RhoA inhibited cell proliferation by retarding entry into the DNA synthetic cell cycle phase and blocking successful completion of cytokinesis, resulting in an increased incidence of binucleate cells. These effects were associated with inhibition of mitogen-induced activation of the MAPK pathway, and suppression of several proteins involved in mitosis, including anillin, ECT2 and cyclin B1 which would be expected to result in reduced activation of endogenous RhoA at the cell equator. Accumulation of active RhoA protein in the midbody of cells in telophase was inhibited in V14RhoA-expressing cells, suggesting that RhoA inactivation must occur prior to re-activation. Defective cytokinesis was also associated with prominent actin structures in V14RhoA-expressing cells, which might be incompatible with equatorial furrowing. Using super-resolution imaging based on single-molecule switching, we have significantly improved the resolution of active RhoA in midbodies. These results indicate that constitutively-active RhoA antagonizes several cellular activities that contribute to proliferation, highlighting the importance for cycling between GTP/GDP-bound states.

Correlative Atomic Force Microscopy and Localization‐Based Super‐Resolution Microscopy: Revealing Labelling and Image Reconstruction Artefacts

Hybrid microscopy: A correlative microscopy tool that combines in situ super-resolution fluorescence microscopy based on single-molecule localization and atomic force microscopy is presented. Direct comparison with high- resolution topography allows the authors to improve fluorescence labeling and image analysis in super-resolution imaging.