Simultaneous hermaphrodites experience unique conflicts of interest during reproduction, some of which are reflected in their complex mating behaviours. We here provide the first detailed description of the mating behaviour of a marine flatworm of the genus Macrostomum, a cosmopolitan group of microturbellaria. Mating in this species is usually initiated by the precopulatory behaviours circling and reeling, then leads to reciprocal copulation where worms mutually insert their copulatory stylet, and often ends in an intriguing postcopulatory sucking behaviour. We provide detailed data on the frequencies and durations of the different behaviours, and examine some biotic and abiotic factors that could influence the mating rate. We further speculate on the function of sucking and suggest that it could be an adaptation for the digestion of sperm and/or the removal of seminal components, which may function as allohormones. Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx

Root hairs emerge from epidermal root cells (trichoblasts) and differentiate by highly localized tip growth. Microtubules (MTs) are essential for establishing and maintaining the growth polarity of root hairs. The current knowledge about the configuration of the MT cytoskeleton during root hair development is largely based on experiments on fixed material, and reorganization and in vivo dynamics of MTs during root hair development is at present unclear. This in vivo study provides new insights into the mechanisms of MT (re)organization during root hair development in Arabidopsis (Arabidopsis thaliana). Expression of a binding site of the MT-associated protein-4 tagged with green fluorescent protein enabled imaging of MT nucleation, growth, and shortening and revealed distinct MT configurations. Depending on the dynamics of the different MT populations during root hair development, either repeated two-dimensional (x, y, t) or repeated three-dimensional (x, y, z, t) scanning was performed. Furthermore, a new image evaluation tool was developed to reveal important data on MT instability. The data show how MTs reorient after apparent contact with other MTs and support a model for MT alignment based on repeated reorientation of dynamic MT growth.

Telomeres are complex end structures that confer functional integrity and positional stability to human chromosomes. Despite their critical importance, there is no clear view on telomere organization in cycling human cells and their dynamic behavior throughout the cell cycle. We investigated spatiotemporal organization of telomeres in living human ECV-304 cells stably expressing telomere binding proteins TRF1 and TRF2 fused to mCitrine using four dimensional microscopy. We thereby made use of controlled light exposure microscopy (CLEM), a novel technology that strongly reduces photodamage by limiting excitation in parts of the image where full exposure is not needed. We found that telomeres share small territories where they dynamically associate. These territories are preferentially positioned at the interface of chromatin domains. TRF1 and TRF2 are abundantly present in these territories but not firmly bound. At the onset of mitosis, the bulk of TRF protein dissociates from telomere regions, territories disintegrate and individual telomeres become faintly visible. The combination of stable cell lines, CLEM and cytometry proved essential in providing novel insights in compartment-based nuclear organization and may serve as a model approach for investigating telomere-driven genome-instability and studying long-term nuclear dynamics. ' 2008 International Society for Advancement of Cytometry Key termstelomere; TRF1; TRF2; nuclear organization; chromatin dynamics; CLEM; live cell imaging TELOMERES are the natural ends of linear chromosomes. A mammalian telomere consists of a double stranded array of simple TTAGGG repeats ending in a single stranded overhang that folds back to form a T-loop structure (1). In combination with sufficient telomere repeats, a complex of indirect and direct telomere binding proteins, dubbed shelterin, assures proper telomere structure and function. The specificity of shelterin for telomeric DNA is due to the recognition of TTAGGG repeats by three of its components: the homodimers telomeric repeat binding factor 1 and 2 (TRF1 and TRF2) that bind the duplex part of telomeres, and protection of telomeres 1 that binds to the single stranded TTAGGG repeats present at the three-overhang and in the D loop of the T-loop configuration (2-5).The nucleus has a meticulous organization with functional relevance to the cell assuring proper gene expression, replication and genome stability (6-8). Telomeres are considered to play an important role in spatial genome organization. Whereas yeast telomeres are known to form few large silencing clusters at the heterochromatic nuclear periphery (9,10), there is no consensus on how telomeres are organized in the mammalian nucleus. From an architectural point of view, human telomeres have been proposed to be anchored to a nuclear scaffold, thereby possibly providing struc-

The organization of proteins in space and time is essential to their function. To accurately quantify subcellular protein characteristics in a population of cells with regard for the stochasticity of events in a natural context, there is a fast-growing need for image-based cytometry. Simultaneously, the massive amount of data that is generated by image-cytometric analyses, calls for tools that enable pattern recognition and automated classification. In this article, we present a general approach for multivariate phenotypic profiling of individual cell nuclei and quantification of subnuclear spots using automated fluorescence mosaic microscopy, optimized image processing tools, and supervised classification. We demonstrate the efficiency of our analysis by determination of differential DNA damage repair patterns in response to genotoxic stress and radiation, and we show the potential of data mining in pinpointing specific phenotypes after transient transfection. The presented approach allowed for systematic analysis of subnuclear features in large image data sets and accurate classification of phenotypes at the level of the single cell. Consequently, this type of nuclear fingerprinting shows potential for high-throughput applications, such as functional protein assays or drug compound screening. ' 2009 International Society for Advancement of Cytometry

SummaryNuclear migration is a fundamental mechanism necessary for the proper growth and development of many eukaryotic organisms. In this study root hairs of Arabidopsis thaliana were used as a research model to gain insight into the dynamics of nuclear migration. Root hairs are long tubular outgrowths of epidermal cells and are responsible for the uptake of water and nutrients. During the development of root hairs, the nucleus migrates into the hair after the bulge is formed. The position of the nucleus relative to the tip plays an essential role in the growth process. However, what is happening to the nucleus in full-grown root hairs is still unclear. To study nuclear dynamics in living root hair cells, stably transformed plants with the fusion proteins Histone2B-YFP and NLS-GFP-GUS were used. Four-dimensional confocal laser scanning microscopy made it possible to monitor the exact position of the nucleus in different root hairs. To analyse the sequential positions of the nuclei in the root hairs, a new computer-assisted method was developed. After track analysis a number of parameters could be extracted from the movies, such as the average speed, the amplitude, direction factor and the range of movement in the root hairs. Our results show that nuclei do not reach a final position in full-grown root hairs and this sustained movement seems to be more similar in root hairs lying close to each other. Moreover, with this methodology it could be quantitatively demonstrated that the integrity of actin is necessary for nuclear movement.

SummaryTelomeres are the complex end structures that confer functional integrity and positional stability to human chromosomes. Telomere research has long been dominated by length measurements and biochemical analyses. Recently, interest has shifted towards the role of their threedimensional organization and dynamics within the nuclear volume. In the mammalian interphase nucleus, there is increasing evidence for a telomeric configuration that is non-random and is cell cycle and cell type dependent. This has functional implications for genome stability. Objective and reproducible representation of the spatiotemporal organization of telomeres, under different experimental conditions, requires quantification by reliable automated image processing techniques. In this paper, we describe methods for quantitative telomere analysis in cell nuclei of living human cells expressing telomere-binding fusion proteins. We present a toolbox for determining telomere positions within the nucleus with subresolution accuracy and tracking telomeres in 4D controlled light exposure microscopy (CLEM) recordings. The use of CLEM allowed for durable imaging and thereby improved segmentation performance considerably. With minor modifications, the underlying algorithms can be expanded to the analysis of other intranuclear features, such as nuclear bodies or DNA double stranded break foci.

A macro function was developed to run in conjunction with the popular image analysis package NIH Image, to allow simultaneous determination of mapping positions of one or two separate probes with respect to cytogenetic bands by dual color fluorescence in situ hybridization (FISH) and DAPI banding, and by determination of their fractional distance from pter (FLpter). In order to allow maximal flexibility, a user‐defined line along the chromosome is used for measurements. Algorithms were developed to detect the ends of the chromosome and the cytogenetic bands. Results of the analysis are presented in graphical form, comprising a display of the DAPI intensity along the chromosome, the positions of the probe(s), the locations of bands as determined by analysis of the second derivative of the DAPI intensity profile, and a standard ideogram of the chromosome for comparison. The approach was validated and compared to visual assignment of probes to DAPI bands using the cosmid clone PYGM, which has been previously mapped to chromosome 11q13, and has been used as a landmark for mapping of other probes (3). © 1996 Wiley‐Liss, Inc.

The contractile cycle of the cardiac myocyte is essentially controlled by the concentration of intracellular calcium ([Ca2+]i). Measurement of [Ca2+]i using Ca2+-dependent fluorescence and simultaneous monitoring of cell dynamics enable characterization of a variety of substances interacting with ion channels and contractile proteins. In this report we describe a novel method featuring up to 480 frames/s for monitoring rapid changes in cellular calcium and cell length, in which every individual cycle allows effective evaluation of major cell parameters. Computers aid in determination of time to peak (in ms), time to 50% decrease (ms), diastolic Ca2+ (relative fluorescence units, rfu), systolic Ca2+ (rfu), Ca2+ transients (rfu), DeltaCa2+/Delta(t) rise (rfu/s), and DeltaCa2+/Delta(t) fall (rfu/s). Contractile parameters are as follows: maximum cell length (microm), minimum cell length (microm), absolute cell shortening (microm), peak DeltaL/Delta(t) shortening (microm/s), and peak DeltaL/Delta(t) relaxation (microm/s). In summary, we succeeded in demonstrating that this system is a unique and valuable tool that allows simultaneous and accurate assessment of contractile parameters and of calcium movements of isolated adult cardiac myocytes.