Divinylanthracene-Containing Tetracationic Organic Cyclophane with Near-Infrared Photoluminescence.

Near-infrared (NIR) light is known to have outstanding optical penetration in biological tissues and to be non-invasive to cells compared with visible light. These characteristics make NIR-specific light optimal for numerous biological applications, such as the sensing of biomolecules or in theranostics. Over the years, significant progress has been achieved in the synthesis of fluorescent cyclophanes for sensing, bioimaging, and making optoelectronic materials. The preparation of NIR-emissive porphyrin-free cyclophanes is, however, still challenging. In an attempt for fluorescence emissions to reach into the NIR spectral region, employing organic tetracationic cyclophanes, we have inserted two 9,10-divinylanthracene units between two of the pyridinium units in cyclobis(paraquat-p-phenylene). Steady-state absorption, fluorescence, and transient-absorption spectroscopies reveal the deep-red and NIR photoluminescence of this cyclophane. This tetracationic cyclophane is highly soluble in water and has been employed successfully as a probe for live-cell imaging in a breast cancer cell line (MCF-7).

Polysilicon Microchips Functionalized with Bipyridinium-Based Cyclophanes for a Highly Efficient Cytotoxicity in Cancerous Cells.

The use of micrometric-sized vehicles could greatly improve selectivity of cytotoxic compounds as their lack of self-diffusion could maximize their retention in tissues. We have used polysilicon microparticles (SiµP) to conjugate bipyridinium-based compounds, able to induce cytotoxicity under regular intracellular conditions. Homogeneous functionalization in suspension was achieved, where the open-chain structure exhibits a more dense packing than cyclic analogues. The microparticles internalized induce high cytotoxicity per particle in cancerous HeLa cells, and the less densely packed functionalization using cyclophanes promotes higher cytotoxicity per bipy than with open-chain analogues. The self-renewing ability of the particles and their proximity to cell membranes may account for increased lipid peroxidation, achieving toxicity at much lower concentrations than that in solution and in less time, inducing highly efficient cytotoxicity in cancerous cells.

Mechanical-Bond-Induced Exciplex Fluorescence in an Anthracene-Based Homo[2]catenane.

Collisional intermolecular interactions between excited states form short-lived dimers and complexes that lead to the emergence of excimer/exciplex emission of lower energy, a phenomenon which must be differentiated from the photoluminescence (PL) arising from the monomeric molecules. Although the utilization of noncovalent bonding interactions, leading to the generation of excimer/exciplex PL, has been investigated extensively, precise control of the aggregates and their persistence at very low concentrations remains a rare phenomenon. In the search for a fresh approach, we sought to obtain exciplex PL from permanent structures by incorporating anthracene moieties into pyridinium-containing mechanically interlocked molecules. Beyond the optical properties of the anthracene moieties, their π-extended nature enforces [π···π] stacking that can overcome the Coulombic repulsion between the pyridinium units, affording an efficient synthesis of an octacationic homo[2]catenane. Notably, upon increasing the ionic strength by adding tetrabutylammonium hexafluorophosphate, the catenane yield increases significantly as a result of the decrease in Coulombic repulsions between the pyridinium units. Although the ground-state photophysical properties of the free cyclophane and the catenane are similar and show a charge-transfer band at ∼455 nm, their PL characters are distinct, denoting different excited states. The cyclophane emits at ∼562 nm (quantum yield ϕF = 3.6%, emission lifetime τs = 3 ns in MeCN), which is characteristic of a disubstituted anthracene-pyridinium linker. By contrast, the catenane displays an exciplex PL at low concentration (10-8 M) with an emission band centered on 650 nm (ϕF = 0.5%, τs = 14 ns) in MeCN and at 675 nm in aqueous solution. Live-cell imaging performed in MIAPaCa-2 prostate cancer cells confirmed that the catenane exciplex emission can be detected at micromolar concentrations.

Age-associated alterations in the micromechanical properties of chromosomes in the mammalian egg.

PURPOSE: The incidence of aneuploidy in eggs from women of advanced reproductive age can exceed 60%, making the mammalian egg a unique model system to study the mechanisms of chromosome segregation errors. METHODS: Here we applied a novel biophysical chromosome stretching approach to quantify mechanical stiffness of meiotic chromosomes in the mammalian egg and then documented how these properties changed in a mouse model of physiologic reproductive aging. RESULTS: We found significant differences in chromosome micromechanics, and thus in higher order chromosome structure, coincident with advanced reproductive age, a time that is also unequivocally associated with an increase in egg aneuploidy. CONCLUSIONS: These findings have important implications for both reproductive and cancer biology where aneuploidy plays a central role in aging related disease states.

Amphiastral mitotic spindle assembly in vertebrate cells lacking centrosomes.

The role of centrosomes and centrioles during mitotic spindle assembly in vertebrates remains controversial. In cell-free extracts and experimentally derived acentrosomal cells, randomly oriented microtubules (MTs) self-organize around mitotic chromosomes and assemble anastral spindles. However, vertebrate somatic cells normally assemble a connected pair of polarized, astral MT arrays--termed an amphiaster ("a star on both sides")--that is formed by the splitting and separation of the microtubule-organizing center (MTOC) well before nuclear envelope breakdown (NEB). Whether amphiaster formation requires splitting of duplicated centrosomes is not known. We found that when centrosomes were removed from living vertebrate cells early in their cell cycle, an acentriolar MTOC reassembled, and, prior to NEB, a functional amphiastral spindle formed. Cytoplasmic dynein, dynactin, and pericentrin are all recruited to the interphase aMTOC, and the activity of kinesin-5 is needed for amphiaster formation. Mitosis proceeded on time and these karyoplasts divided in two. However, ~35% of aMTOCs failed to split and separate before NEB, and these entered mitosis with persistent monastral spindles. Chromatin-associated RAN-GTP--the small GTPase Ran in its GTP bound state--could not restore bipolarity to monastral spindles, and these cells exited mitosis as single daughters. Our data reveal the novel finding that MTOC separation and amphiaster formation does not absolutely require the centrosome, but, in its absence, the fidelity of bipolar spindle assembly is highly compromised.

Kinesins to the core: The role of microtubule-based motor proteins in building the mitotic spindle midzone.

In mammalian cultured cells the initiation of cytokinesis is regulated - both temporally and spatially - by the overlapping, anti-parallel microtubules of the spindle midzone. This region recruits several key central spindle components: PRC-1, polo-like kinase 1 (Plk-1), the centralspindlin complex, and the chromosome passenger complex (CPC), which together serve to stabilize the microtubule overlap, and also to coordinate the assembly of the cortical actin/myosin cytoskeleton necessary to physically cleave the cell in two. The localization of these crucial elements to the spindle midzone requires members of the kinesin superfamily of microtubule-based motor proteins. Here we focus on reviewing the role played by a variety of kinesins in both building and operating the spindle midzone machinery during cytokinesis.

Tektin 2 is required for central spindle microtubule organization and the completion of cytokinesis.

During anaphase, the nonkinetochore microtubules in the spindle midzone become compacted into the central spindle, a structure which is required to both initiate and complete cytokinesis. We show that Tektin 2 (Tek2) associates with the spindle poles throughout mitosis, organizes the spindle midzone microtubules during anaphase, and assembles into the midbody matrix surrounding the compacted midzone microtubules during cytokinesis. Tek2 small interfering RNA (siRNA) disrupts central spindle organization and proper localization of MKLP1, PRC1, and Aurora B to the midzone and prevents the formation of a midbody matrix. Video microscopy revealed that loss of Tek2 results in binucleate cell formation by aberrant fusion of daughter cells after cytokinesis. Although a myosin II inhibitor, blebbistatin, prevents actin-myosin contractility, the microtubules of the central spindle are compacted. Strikingly, Tek2 siRNA abolishes this actin-myosin-independent midzone microtubule compaction. Thus, Tek2-dependent organization of the central spindle during anaphase is essential for proper midbody formation and the segregation of daughter cells after cytokinesis.

Live-cell analysis of mitotic spindle formation in taxol-treated cells.

Taxol functions to suppress the dynamic behavior of individual microtubules, and induces multipolar mitotic spindles. However, little is known about the mechanisms by which taxol disrupts normal bipolar spindle assembly in vivo. Using live imaging of GFP-alpha tubulin expressing cells, we examined spindle assembly after taxol treatment. We find that as taxol-treated cells enter mitosis, there is a dramatic re-distribution of the microtubule network from the centrosomes to the cell cortex. As they align there, the cortical microtubules recruit NuMA to their embedded ends, followed by the kinesin motor HSET. These cortical microtubules then bud off to form cytasters, which fuse into multipolar spindles. Cytoplasmic dynein and dynactin do not re-localize to cortical microtubules, and disruption of dynein/dynactin interactions by over-expression of p50 "dynamitin" does not prevent cytaster formation. Taxol added well before spindle poles begin to form induces multipolarity, but taxol added after nascent spindle poles are visible-but before NEB is complete-results in bipolar spindles. Our results suggest that taxol prevents rapid transport of key components, such as NuMA, to the nascent spindle poles. The net result is loss of mitotic spindle pole cohesion, microtubule re-distribution, and cytaster formation.