Summary Telomeres are essential for nuclear organization in yeast and during meiosis in mouse. Exploring telomere dynamics in living human cells by advanced time-lapse confocal microscopy allowed us to evaluate the spatial distribution of telomeres within the nuclear volume. We discovered an unambiguous enrichment of telomeres at the nuclear periphery during post-mitotic nuclear assembly, while telomeres were localized more internally during the rest of the cell cycle. Telomere enrichment at the nuclear rim was mediated by physical tethering of telomeres to the nuclear envelope, likely via specific interactions between the shelterin subunit RAP1 with the nuclear envelope protein Sun1. Genetic interference revealed a critical role in cell cycle progression for Sun1, but no effect on telomere positioning for RAP1. Our results shed light on the dynamic relocalization of human telomeres during the cell cycle and suggest redundant pathways for tethering telomeres to the nuclear envelope.

Scanning Electron Microscopy (SEM) has long been the standard in imaging the sub-micrometer surface ultrastructure of both hard and soft materials. In the case of biological samples, it has provided great insights into their physical architecture. However, three of the fundamental challenges in the SEM imaging of soft materials are that of limited imaging resolution at high magnification, charging caused by the insulating properties of most biological samples and the loss of subtle surface features by heavy metal coating. These challenges have recently been overcome with the development of the Helium Ion Microscope (HIM), which boasts advances in charge reduction, minimized sample damage, high surface contrast without the need for metal coating, increased depth of field, and 5 angstrom imaging resolution. We demonstrate the advantages of HIM for imaging biological surfaces as well as compare and contrast the effects of sample preparation techniques and their consequences on sub-nanometer ultrastructure.

This study identifies zwint-1 as a novel substrate for AurB during mitosis. Phosphorylation is required for outer kinetochore assembly during prometaphase. However, zwint-1 dephosphorylation is required at metaphase for checkpoint silencing.

Kinetochore dynein has been implicated in microtubule capture, correcting inappropriate microtubule attachments, chromosome movement, and checkpoint silencing. It remains unclear how dynein coordinates this diverse set of functions. Phosphorylation is responsible for some dynein heterogeneity (Whyte, J., Bader, J. R., Tauhata, S. B., Raycroft, M., Hornick, J., Pfister, K. K., Lane, W. S., Chan, G. K., Hinchcliffe, E. H., Vaughan, P. S., and Vaughan, K. T. (2008) J. Cell Biol. 183, 819 -834), and phosphorylated and dephosphorylated forms of dynein coexist at prometaphase kinetochores. In this study, we measured the impact of inhibiting polo-like kinase 1 (Plk1) on both dynein populations. Phosphorylated dynein was ablated at kinetochores after inhibiting Plk1 with a small molecule inhibitor (5-Cyano-7-nitro-2-(benzothiazolo-Noxide)-carboxamide) or chemical genetic approaches. The total complement of kinetochore dynein was also reduced but not eliminated, reflecting the presence of some dephosphorylated dynein after Plk1 inhibition. Although Plk1 inhibition had a profound effect on dynein, kinetochore populations of dynactin, spindly, and zw10 were not reduced. Plk1-independent dynein was reduced after p150Glued depletion, consistent with the binding of dephosphorylated dynein to dynactin. Plk1 phosphorylated dynein intermediate chains at Thr-89 in vitro and generated the phospho-Thr-89 phosphoepitope on recombinant dynein intermediate chains. Finally, inhibition of Plk1 induced defects in microtubule capture and persistent microtubule attachment, suggesting a role for phosphorylated dynein in these functions during prometaphase. These findings suggest that Plk1 is a dynein kinase required for recruitment of phosphorylated dynein to kinetochores.Cytoplasmic dynein plays important roles at spindle poles, the cell cortex, and kinetochores during mitosis. The mechanisms that allow dynein to associate specifically with each site remain unknown. One possibility is suggested by the identification of a novel mitotic phosphorylation site in the dynein intermediate chains (ICs) 2 (1). This phosphorylated form of dynein (phospho-Thr-89, pT89) is specific for kinetochores from nuclear envelope breakdown (NEB) to metaphase. Previous work suggests that pT89 dynein is recruited to kinetochores through a direct interaction with zw10 but undergoes dephosphorylation in response to bioriented microtubule attachment and chromosome alignment. This dephosphorylation of dynein shifts binding to dynactin and stimulates poleward transport of checkpoint proteins; a transition required for the silencing of the spindle assembly checkpoint at metaphase. Defects in this process induce metaphase arrest/delay.One prediction of this model is that dynein could interact with multiple proteins at kinetochores, depending on the degree of microtubule attachment, microtubule occupancy, and chromosome alignment. Consistent with this scenario, several mitotic proteins have been implicated in interacting with or anchoring kinetochore dynein, including dy...

One of the fundamental limitations of optical microscopy is that of diffraction, or in essence, how small a beam of light can be focused by using an optical lens system. This constraint, or barrier if you will, was theoretically described by Ernst Abbe in 1873 and is roughly equal to half the wavelength of light used to probe the system. Many structures, particularly those within cells, are much smaller than this limit and thus are difficult to visualize. Over the last two decades, a new field of super-resolution imaging has been created and been developed into a broad range of techniques that allow routine imaging beyond the far-field diffraction limit of light. In this unit we outline the basic principles of the various super-resolution imaging modalities, paying particular attention to the technical considerations for biological imaging. Furthermore, we discuss their various applications in the imaging of both fixed and live biological samples.