Animal cell cytokinesis requires a contractile ring of crosslinked actin filaments and myosin motors. How contractile rings form and are stabilized in dividing cells remains unclear. We address this problem by focusing on septins, highly conserved proteins in eukaryotes whose precise contribution to cytokinesis remains elusive. We use the cleavage of the Drosophila melanogaster embryo as a model system, where contractile actin rings drive constriction of invaginating membranes to produce an epithelium in a manner akin to cell division. In vivo functional studies show that septins are required for generating curved and tightly packed actin filament networks. In vitro reconstitution assays show that septins alone bundle actin filaments into rings, accounting for the defects in actin ring formation in septin mutants. The bundling and bending activities are conserved for human septins, and highlight unique functions of septins in the organization of contractile actomyosin rings.

Single-molecule epifluorescence microscopy was used to observe the translational motion of GPI-linked and native I-E(k) class II MHC membrane proteins in the plasma membrane of CHO cells. The purpose of the study was to look for deviations from Brownian diffusion that might arise from barriers to this motion. Detergent extraction had suggested that these proteins may be confined to lipid microdomains in the plasma membrane. The individual I-E(k) proteins were visualized with a Cy5-labeled peptide that binds to a specific extracytoplasmic site common to both proteins. Single-molecule trajectories were used to compute a radial distribution of displacements, yielding average diffusion coefficients equal to 0.22 (GPI-linked I-E(k)) and 0.18 microm(2)/s (native I-E(k)). The relative diffusion of pairs of proteins was also studied for intermolecular separations in the range 0.3-1.0 microm, to distinguish between free diffusion of a protein molecule and diffusion of proteins restricted to a rapidly diffusing small domain. Both analyses show that motion is predominantly Brownian. This study finds no strong evidence for significant confinement of either GPI-linked or native I-E(k) in the plasma membrane of CHO cells.

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An interesting property of several yellow-emitting mutants of the green fluorescent protein (GFP) is that they switch between a fluorescent and a nonfluorescent state on a time scale of seconds. This peculiar blinking behavior was observed in single-molecule fluorescence studies of GFP mutants in poly(acrylamide) gels et al. Nature 1997, 388, 355.). Utilizing primarily the yellow-emitting phenolate anion mutant EGFP, we report new single-molecule experiments studying the effect of several parameters on the blinking process: pH, host matrix, and pumping intensity. The primary measurement in these studies is the observed distribution of on-times and off-times. The on-time dynamics of EGFP are independent of pH over the range of 6-10, thus making protonation/deprotonation of the chromophore unlikely as the source of the blinking. The excitation intensity, however, has a considerable effect on the blinking: the on-times are shorter at high intensity. We compare these results to ensemble bleaching measurements which find the bleaching quantum yield of EGFP in agarose gel at pH 8 to be (8 ( 2) × 10 -6 . The probability of termination of single-molecule emission per photon absorbed is in agreement with the bulk bleaching quantum yield, thus suggesting that the two processes are related.

We elucidate the crystalline nature and the three-dimensional orientation of isolated organic nanocrystals embedded in a sol-gel matrix, using a polarized nonlinear microscopy technique that combines two-photon fluorescence and second harmonic generation. This technique allows the distinction between mono-crystalline structures and nano-scale poly-crystalline aggregates responsible for incoherent second harmonic signals.PACS numbers: 78.67. Bf, 61.82.Rx The optical properties of nanoparticles have recently attracted much attention. In addition to metallic and semiconductor nanoparticles which are now used as biomarkers and as the building blocks of nanostructured materials [1], their organic counterparts constitute an interesting alternative. Advances in molecular engineering have enabled the design of molecular structures of various resonances and symmetries with optimized one-and two-photon absorption cross sections [2], or combining different optical properties such as luminescence and second harmonic generation (SHG) [3]. In addition, macroscopic molecular arrangements have been optimized using the tensorial oriented gas model [4], which predicts that an enhancement of the SHG efficiency is expected from the non-centrosymmetric crystalline arrangement of efficient nonlinear molecules. Molecular nanocrystals can be therefore envisioned as a new class of multi-functional nano-scale materials. In the case of organic nanocrystals however, the traditional crystalline characterization techniques have raised many practical barriers due to their low concentration and fragility. Consequently, the elucidation of their crystalline nature has been so far indirect or averaged over a large number of nanocrystals [5].In this letter, we show that two-photon nonlinear microscopy permits in-situ characterization of isolated organic nanocrystals grown in an amorphous sol-gel matrix. The diagnostic is based on polarization resolved two-photon excited fluorescence (TPF) and SHG. TPF is an incoherent process allowed in centrosymmetric media, which exhibits a specific anisotropy depending on the medium symmetry. On the other hand, SHG is the signature of a crystalline non-centrosymmetric phase in the sample, with a sensitivity down to the nanometric scale [6,7]. We show that the polarization analysis of both TPF and SHG from nanocrystals allows the unambiguous discrimination between isolated mono-crystalline and poly-crystalline systems. Moreover, once a nanocrystal has been identified as mono-crystalline, a detailed model for both TPF and SHG polarization responses accounting for the unit-cell symmetry allows the determination of its three-dimensional orientation within the host matrix.The organic nanocrystals that we investigate are based upon the α-((4'-methoxyphenyl)methylene)-4-nitro-benzene-acetonitrile) molecule (CMONS), which exhibits efficient luminescence and quadratic nonlinearity under two-photon excitation [8,9,10]. The bulk crystalline phases of such crystals have three possible polymorphic forms, two being noncen...

Essential cellular functions as diverse as genome maintenance and tissue morphogenesis rely on the dynamic organization of filamentous assemblies. For example, the precise structural organization of DNA filaments has profound consequences on all DNA-mediated processes including gene expression, whereas control over the precise spatial arrangement of cytoskeletal protein filaments is key for mechanical force generation driving animal tissue morphogenesis. Polarized fluorescence is currently used to extract structural organization of fluorescently labeled biological filaments by determining the orientation of fluorescent labels, however with a strong drawback: polarized fluorescence imaging is indeed spatially limited by optical diffraction, and is thus unable to discriminate between the intrinsic orientational mobility of the fluorophore labels and the real structural disorder of the labeled biomolecules. Here, we demonstrate that quantitative single-molecule polarized detection in biological filament assemblies allows not only to correct for the rotational flexibility of the label but also to image orientational order of filaments at the nanoscale using superresolution capabilities. The method is based on polarized direct stochastic optical reconstruction microscopy, using dedicated optical scheme and image analysis to determine both molecular localization and orientation with high precision. We apply this method to double-stranded DNA in vitro and microtubules and actin stress fibers in whole cells.iological processes are inherently driven by the molecularscale organization of biomolecular assemblies, which arrange in precise structures that are essential for biological functions in cells and tissues. The extent to which the biological function depends on the underlying molecular-scale organization is particularly evident in filamentous assemblies, such as DNA filaments and cytoskeletal protein filaments. Changes in the local higher-order organization of DNA filaments is tightly linked to essential DNA-mediated processes including control of gene expression, DNA replication, and DNA repair. However, how specific DNA-binding proteins affect DNA filament architecture and thus DNA-mediated functions is poorly understood (1). Similarly, the spatial organization of cytoskeletal filaments in cells and tissues is also weakly explored, despite their central role in generating forces and driving cell motility, cell division, and tissue morphogenesis (2). Electron microscopy has been widely used to provide molecular-scale images of the structure of such filament assemblies; however, it typically involves several daylong sample preparation and ultrathin sectioning of the biological material, thus limiting investigations in whole cells and tissues.Polarized fluorescence imaging is a powerful approach for elucidating the structural organization of filament assemblies because it is compatible with a wide variety of microscopy techniques, thus enabling studies across multiple spatial and temporal scales. Polarized fluorescenc...

The principles of crystal engineering have been used to design a family of structures with potential as octupolar nonlinear optical (NLO) materials. The major aim in such an exercise, a carry-over of molecular symmetry into the crystal, is possible with a retrosynthetic approach. An appropriate choice of precursor trigonal molecules leads from the concept of the dimeric Piedfort unit. The crystal structures and NLO properties of a series of 2,4,6-triaryloxy-1,3,5-triazines, 1−6, are reported. These compounds consistently form quasi-trigonal or trigonal networks that are two-dimensionally noncentrosymmetric. Substitutional variations on the phenyl moieties that were expected to maintain or to perturb this trigonal network have been explored. Molecular nonlinearities have been measured by Harmonic Light Scattering (HLS) experiments. Among the compounds studied, 2,4,6-triphenoxy-1,3,5-triazine, 1 adopts a noncentrosymmetric crystal structure with a measurable SHG powder signal. All these crystal structures are stabilized by weak intermolecular interactions such as herringbone, π···π, C−H···O, and C−H···N hydrogen bonding. These octupolar molecules are more isotropic than the classical p-nitroaniline based dipolar NLO molecules, and this is advantageous from the viewpoint of potential electrooptic applications.