Biophysical ordering transitions underlie genome 3D re-organization during cricket spermiogenesis.

Spermiogenesis is a radical process of differentiation whereby sperm cells acquire a compact and specialized morphology to cope with the constraints of sexual reproduction while preserving their main cargo, an intact copy of the paternal genome. In animals, this often involves the replacement of most histones by sperm-specific nuclear basic proteins (SNBPs). Yet, how the SNBP-structured genome achieves compaction and accommodates shaping remain largely unknown. Here, we exploit confocal, electron and super-resolution microscopy, coupled with polymer modeling to identify the higher-order architecture of sperm chromatin in the needle-shaped nucleus of the emerging model cricket Gryllus bimaculatus. Accompanying spermatid differentiation, the SNBP-based genome is strikingly reorganized as ~25nm-thick fibers orderly coiled along the elongated nucleus axis. This chromatin spool is further found to achieve large-scale helical twisting in the final stages of spermiogenesis, favoring its ultracompaction. We reveal that these dramatic transitions may be recapitulated by a surprisingly simple biophysical principle based on a nucleated rigidification of chromatin linked to the histone-to-SNBP transition within a confined nuclear space. Our work highlights a unique, liquid crystal-like mode of higher-order genome organization in ultracompact cricket sperm, and establishes a multidisciplinary methodological framework to explore the diversity of non-canonical modes of DNA organization.

Sequence-specific DNA labelling for fluorescence microscopy.

The preservation of nucleus structure during microscopy imaging is a top priority for understanding chromatin organization, genome dynamics, and gene expression regulation. In this review, we summarize the sequence-specific DNA labelling methods that can be used for imaging in fixed and/or living cells without harsh treatment and DNA denaturation: (i) hairpin polyamides, (ii) triplex-forming oligonucleotides, (iii) dCas9 proteins, (iv) transcription activator-like effectors (TALEs) and (v) DNA methyltransferases (MTases). All these techniques are capable of identifying repetitive DNA loci and robust probes are available for telomeres and centromeres, but visualizing single-copy sequences is still challenging. In our futuristic vision, we see gradual replacement of the historically important fluorescence in situ hybridization (FISH) by less invasive and non-destructive methods compatible with live cell imaging. Combined with super-resolution fluorescence microscopy, these methods will open the possibility to look into unperturbed structure and dynamics of chromatin in living cells, tissues and whole organisms.

A general highly efficient synthesis of biocompatible rhodamine dyes and probes for live-cell multicolor nanoscopy.

The development of live-cell fluorescence nanoscopy is powered by the availability of suitable fluorescent probes. Rhodamines are among the best fluorophores for labeling intracellular structures. Isomeric tuning is a powerful method for optimizing the biocompatibility of rhodamine-containing probes without affecting their spectral properties. An efficient synthesis pathway for 4-carboxyrhodamines is still lacking. We present a facile protecting-group-free 4-carboxyrhodamines' synthesis based on the nucleophilic addition of lithium dicarboxybenzenide to the corresponding xanthone. This approach drastically reduces the number of synthesis steps, expands the achievable structural diversity, increases overall yields and permits gram-scale synthesis of the dyes. We synthesize a wide range of symmetrical and unsymmetrical 4-carboxyrhodamines covering the whole visible spectrum and target them to multiple structures in living cells - microtubules, DNA, actin, mitochondria, lysosomes, Halo-tagged and SNAP-tagged proteins. The enhanced permeability fluorescent probes operate at submicromolar concentrations, allowing high-contrast STED and confocal microscopy of living cells and tissues.

Mechanism of spindle pole organization and instability in human oocytes.

Human oocytes are prone to assembling meiotic spindles with unstable poles, which can favor aneuploidy in human eggs. The underlying causes of spindle instability are unknown. We found that NUMA (nuclear mitotic apparatus protein)-mediated clustering of microtubule minus ends focused the spindle poles in human, bovine, and porcine oocytes and in mouse oocytes depleted of acentriolar microtubule-organizing centers (aMTOCs). However, unlike human oocytes, bovine, porcine, and aMTOC-free mouse oocytes have stable spindles. We identified the molecular motor KIFC1 (kinesin superfamily protein C1) as a spindle-stabilizing protein that is deficient in human oocytes. Depletion of KIFC1 recapitulated spindle instability in bovine and aMTOC-free mouse oocytes, and the introduction of exogenous KIFC1 rescued spindle instability in human oocytes. Thus, the deficiency of KIFC1 contributes to spindle instability in human oocytes.

Blinking Fluorescent Probes for Tubulin Nanoscopy in Living and Fixed Cells.

Here we report a small molecule tubulin probe for single-molecule localization microscopy (SMLM), stimulated emission depletion (STED) microscopy and MINFLUX nanoscopy, which can be used in living and fixed cells. We explored a series of taxane derivatives containing spontaneously blinking far-red dye hydroxymethyl silicon-rhodamine (HMSiR) and found that the linker length profoundly affects the probe permeability and off-targeting in living cells. The best performing probe, HMSiR-tubulin, is composed of cabazitaxel and the 6'-regioisomer of HMSiR bridged by a C6 linker. Microtubule diameter of ≤50 nm was routinely measured in SMLM experiments on living and fixed cells. HMSiR-tubulin allows a complementary use of different nanoscopy techniques for investigating microtubule functions and developing imaging methods. For the first time, we resolved the inner microtubule diameter of 16 ± 5 nm by optical nanoscopy and thereby demonstrated the utility of a self-blinking dye for MINFLUX imaging.

Parental genome unification is highly error-prone in mammalian embryos.

Most human embryos are aneuploid. Aneuploidy frequently arises during the early mitotic divisions of the embryo, but its origin remains elusive. Human zygotes that cluster their nucleoli at the pronuclear interface are thought to be more likely to develop into healthy euploid embryos. Here, we show that the parental genomes cluster with nucleoli in each pronucleus within human and bovine zygotes, and clustering is required for the reliable unification of the parental genomes after fertilization. During migration of intact pronuclei, the parental genomes polarize toward each other in a process driven by centrosomes, dynein, microtubules, and nuclear pore complexes. The maternal and paternal chromosomes eventually cluster at the pronuclear interface, in direct proximity to each other, yet separated. Parental genome clustering ensures the rapid unification of the parental genomes on nuclear envelope breakdown. However, clustering often fails, leading to chromosome segregation errors and micronuclei, incompatible with healthy embryo development.

Far-red switching DNA probes for live cell nanoscopy.

Herein we present DNA probes composed of Hoechst 33258 and spontaneously blinking far-red hydroxymethyl silicon-rhodamine (HMSiR). The best performing probe, 5-HMSiR-Hoechst, contains the 5'-regioisomer, shows ∼400-fold fluorescence increase upon DNA binding and is compatible with wash-free single molecule localization and 3D stimulated emission depletion microscopy of chromatin nanostructures in living cells.

Enhancing the biocompatibility of rhodamine fluorescent probes by a neighbouring group effect.

Fluorescence microscopy is an essential tool for understanding dynamic processes in living cells and organisms. However, many fluorescent probes for labelling cellular structures suffer from unspecific interactions and low cell permeability. Herein, we demonstrate that the neighbouring group effect which results from positioning an amide group next to a carboxyl group in the benzene ring of rhodamines dramatically increases cell permeability of the rhodamine-based probes through stabilizing a fluorophore in a hydrophobic spirolactone state. Based on this principle, we create probes targeting tubulin, actin and DNA. Their superb staining intensity, tuned toxicity and specificity allows long-term 3D confocal and STED nanoscopy with sub-30 nm resolution. Due to their unrestricted cell permeability and efficient accumulation on the target, the new probes produce high contrast images at low nanomolar concentrations. Superior performance is exemplified by resolving the real microtubule diameter of 23 nm and selective staining of the centrosome inside living cells for the first time.

Rhodamine-Hoechst positional isomers for highly efficient staining of heterochromatin.

Hoechst conjugates to fluorescent dyes are popular DNA stains for live-cell imaging, but the relationship between their structure and performance remains elusive. This study of carboxyrhodamine-Hoechst 33258 conjugates reveals that a minimal change in the attachment point of the dye has dramatic effects on the properties of the final probe. All tested 6'-carboxyl dye-containing probes exhibited dual-mode binding to DNA and formed a dimmer complex at high DNA concentrations. The 5'-carboxyl dye-containing probes exhibited single-mode binding to DNA which translated into increased brightness and lower cytotoxicity. Up to 10-fold brighter nuclear staining by the newly developed probes allowed acquisition of stimulated emission depletion (STED) nanoscopy images of outstanding quality in living and fixed cells. Therefore we were able to estimate a diameter of ∼155 nm of the heterochromatin exclusion zones in the nuclear pore region in living cells and intact chicken erythrocytes and to localize telomeres relative to heterochromatin in living U-2 OS cells. Employing the highly efficient probes for two-color STED allowed visualization of DNA and tubulin structures in intact nucleated erythrocytes - a system where imaging is greatly hampered by high haemoglobin absorbance.

Synthesis and fluorescent properties of N(9)-alkylated 2-amino-6-triazolylpurines and 7-deazapurines.

The synthesis of novel fluorescent N(9)-alkylated 2-amino-6-triazolylpurine and 7-deazapurine derivatives is described. A new C(2)-regioselectivity in the nucleophilic aromatic substitution reactions of 9-alkylated-2,6-diazidopurines and 7-deazapurines with secondary amines has been disclosed. The obtained intermediates, 9-alkylated-2-amino-6-azido-(7-deaza)purines, were transformed into the title compounds by CuAAC reaction. The designed compounds belong to the push-pull systems and possess promising fluorescence properties with quantum yields in the range from 28% to 60% in acetonitrile solution. Due to electron-withdrawing properties of purine and 7-deazapurine heterocycles, which were additionally extended by triazole moieties, the compounds with electron-donating groups showed intramolecular charge transfer character (ICT/TICT) of the excited states which was proved by solvatochromic dynamics and supported by DFT calculations. In the 7-deazapurine series this led to increased fluorescence quantum yield (74%) in THF solution. The compounds exhibit low cytotoxicity and as such are useful for the cell labelling studies in the future.

Emission wavelength dependence on the rISC rate in TADF compounds with large conformational disorder.

Large vibronic coupling between the local and charge-transfer triplet states is required for efficient reverse intersystem crossing in TADF compounds. This is ensured by low steric hindrance between donor and acceptor molecular units. However, flexible molecular cores show large conformational disorder and emission wavelength instability in solid films.

Concentration effects on spontaneous and amplified emission in benzo[c]fluorenes.

Deep-blue-emitting benzo[c]fluorene-cored compounds featuring twisted peripheral moieties for suppressed concentration quenching of emission were synthesized and investigated as potential materials for light amplification. This detailed study of the effect of concentration on the spontaneous and stimulated emission, excited-state lifetime and susceptibility to form aggregates obtained for different benzofluorenes, has enabled the understanding of the concentration dependence of the amplified spontaneous emission (ASE) threshold and revealed the optimal concentration for the lowest threshold. The weak concentration quenching accompanied by high fluorescence quantum yield (>40%) and radiative decay rate (>5 × 10(8) s(-1)) have enabled the attainment of the lowest ASE threshold in the neat amorphous film of benzofluorene bearing dihexylfluorenyl peripheral moieties. Aggregate formation was found to negligibly affect the emission efficiency of the benzofluorene films; however, it drastically increased ASE threshold via the enhanced scattering of directional stimulated emission, and thereby implied the necessity to utilize homogeneous glassy films as the lasing medium. Although the bulky dihexylfluorenyl groups at the periphery ensured the formation of glassy benzofluorene films with the ASE threshold as low as 900 W cm(-2) (under nanosecond excitation), they adversely affected carrier drift mobility, which implied a tradeoff between ASE and charge transport properties for the lasing materials utilized in the neat form. Such a low ASE threshold attained in air is among the lowest reported for solution-processed neat films. The low threshold and enhanced photostability of benzofluorenes against fluorene compounds in air show great potential for benzofluorene-cored molecular glasses as active media for lasing applications.