Richard W. Cole

The small molecule Hesperadin reveals a role for Aurora B in correcting kinetochore–microtubule attachment and in maintaining the spindle assembly checkpoint

The proper segregation of sister chromatids in mitosis depends on bipolar attachment of all chromosomes to the mitotic spindle. We have identified the small molecule Hesperadin as an inhibitor of chromosome alignment and segregation. Our data imply that Hesperadin causes this phenotype by inhibiting the function of the mitotic kinase Aurora B. Mammalian cells treated with Hesperadin enter anaphase in the presence of numerous monooriented chromosomes, many of which may have both sister kinetochores attached to one spindle pole (syntelic attachment). Hesperadin also causes cells arrested by taxol or monastrol to enter anaphase within <1 h, whereas cells in nocodazole stay arrested for 3–5 h. Together, our data suggest that Aurora B is required to generate unattached kinetochores on monooriented chromosomes, which in turn could promote bipolar attachment as well as maintain checkpoint signaling.

A synergy of technologies: combining laser microsurgery with green fluorescent protein tagging.

When focused through an objective lens with a high numerical aperture, nanosecond pulses of high-intensity green (532-nm) laser light can be used to selectively destroy any cellular component whose boundaries can be defined by light microscopy. These components include, for example, chromosomes, spindle fibers, bundles of keratin, or actin filaments, mitochondria, vacuoles, and so forth. In addition, the definition of poorly resolved components can be enhanced for selective destruction by tagging one or more of their constituent proteins with green fluorescence protein (GFP). As a example we show that the centrosome in living PtK1 cells can be clearly defined, and then destroyed by green laser light, after transforming the cells with gamma-tubulin/GFP fusion protein. In some transformed cells it is even possible to target and selectively destroy just one of the centrioles.

Live-cell imaging

It would be hard to argue that live-cell imaging has not changed our view of biology. The past 10 years have seen an explosion of interest in imaging cellular processes, down to the molecular level. There are now many advanced techniques being applied to live cell imaging. However, cellular health is often under appreciated. For many researchers, if the cell at the end of the experiment has not gone into apoptosis or is blebbed beyond recognition, than all is well. This is simply incorrect. There are many factors that need to be considered when performing live-cell imaging in order to maintain cellular health such as: imaging modality, media, temperature, humidity, PH, osmolality, and photon dose. The wavelength of illuminating light, and the total photon dose that the cells are exposed to, comprise two of the most important and controllable parameters of live-cell imaging. The lowest photon dose that achieves a measureable metric for the experimental question should be used, not the dose that produces cover photo quality images. This is paramount to ensure that the cellular processes being investigated are in their in vitro state and not shifted to an alternate pathway due to environmental stress. The timing of the mitosis is an ideal canary in the gold mine, in that any stress induced from the imaging will result in the increased length of mitosis, thus providing a control model for the current imagining conditions.

A Novel Method to Quantify Angiogenesis in vivo Using Multi-photon Imaging

w3-1 n nProgress towards understanding the basic mechanisms of angiogenesis, the formation of new blood vessels from existing vasculature, and therapies to control it have been limited by the lack of tools to measure angiogenesis in appropriate living tissues. While in vivo angiogenesis assays provide the best environment to follow angiogenesis, most assays are tempered by the inherent difficulty in obtaining reproducible data. Surgical methods induce inflammatory response and local variations in the site of implants can impact the outcome of the assay. The key elements of better assays should focus on approaches that can measure growth over time and minimize artifacts induced by experimental manipulation. Using multi-photon intra-vital imaging, it is possible to repeatedly image (Z-series) the same area over a period of days or even weeks.The multi-photon excitation produces the lowest photon dose in the sample of any imaging modality.These abilities, coupled with high spatial resolution achievable deep (0.5 mm) into living tissue allows non-invasivemonitoring of vessel growth over time.We are developing the technology to visualize the behavior and growth of blood vessels of mice after treatment with Vascular Endothelial Growth Factor delivered though direct injection of protein and by expression of endogenous growth factor in keratinocytes. Transgenic mice expressing GFP (Green Fluorescent Protein) driven by the Tie II promoter or direct injection of dextran-conjugated fluorophores enable the visualization of blood vessel structure are imaged and mapped over time.

Intermediate Magnification Imaging System for Whole Organs/Organisms.

There are many techniques for 3D imaging of biological specimens such as confocal, two-photon and, wide-field fluorescence microscopy, CAT scan, MRI, and optical coherence tomography. There are also many derivatives of these techniques, each having its strengths and weaknesses. Due to the differences in resolution, depth-of-field, and field-of-view, it is often difficult to compare images from the relatively high-resolution microscopy methods to the later lower-resolution high-volume imaging methods. Effectively making this comparison could be very powerful in relating organ or organism level information to cellular level processes. Several microscope systems are being developed that bridge this gap [1-2]. The system reported here based on [1] has several advantages for 3-D imaging of large objects such as whole organs, organisms or embryos utilizing reflection, transmission and fluorescent imaging modes. It has higher resolution than MNR, and low cost. Additionally, the ability to use commercially available antibody/ fluorescent markers including green fluorescent proteins, allows a large number of molecules to be labeled and their distributions imaged. The juxtaposition of proteins within objects, such as a mouse embryo can be easily determined by using multiple labels and/or multiple imaging modes. With image algorithms, some borrowed from CAT and MRI imaging, as well as digital microscopic imaging, this technology has the potential to evolve and improve in terms of resolution, detection of a number of labels at lower levels, and the variety of specimen types and sizes that can be imaged. Instruments of this type will be especially important in bridging the gap between medical imaging and microscopy.