Jennifer C. Waters

Association of spindle assembly checkpoint component XMAD2 with unattached kinetochores

The spindle assembly checkpoint delays anaphase until all chromosomes are attached to a mitotic spindle. The mad (mitotic arrest-deficient) and bub (budding uninhibited by benzimidazole) mutants of budding yeast lack this checkpoint and fail to arrest the cell cycle when microtubules are depolymerized. A frog homolog of MAD2 (XMAD2) was isolated and found to play an essential role in the spindle assembly checkpoint in frog egg extracts. XMAD2 protein associated with unattached kinetochores in prometaphase and in nocodazole-treated cells and disappeared from kinetochores at metaphase in untreated cells, suggesting that XMAD2 plays a role in the activation of the checkpoint by unattached kinetochores. This study furthers understanding of the mechanism of cell cycle checkpoints in metazoa and provides a marker for studying the role of the spindle assembly checkpoint in the genetic instability of tumors.

Accuracy and precision in quantitative fluorescence microscopy

The light microscope has long been used to document the localization of fluorescent molecules in cell biology research. With advances in digital cameras and the discovery and development of genetically encoded fluorophores, there has been a huge increase in the use of fluorescence microscopy to quantify spatial and temporal measurements of fluorescent molecules in biological specimens. Whether simply comparing the relative intensities of two fluorescent specimens, or using advanced techniques like Förster resonance energy transfer (FRET) or fluorescence recovery after photobleaching (FRAP), quantitation of fluorescence requires a thorough understanding of the limitations of and proper use of the different components of the imaging system. Here, I focus on the parameters of digital image acquisition that affect the accuracy and precision of quantitative fluorescence microscopy measurements.

Vertebrate Shugoshin Links Sister Centromere Cohesion and Kinetochore Microtubule Stability in Mitosis

Drosophila MEI-S332 and fungal Sgo1 genes are essential for sister centromere cohesion in meiosis I. We demonstrate that the related vertebrate Sgo localizes to kinetochores and is required to prevent premature sister centromere separation in mitosis, thus providing an explanation for the differential cohesion observed between the arms and the centromeres of mitotic sister chromatids. Sgo is degraded by the anaphase-promoting complex, allowing the separation of sister centromeres in anaphase. Intriguingly, we show that Sgo interacts strongly with microtubules in vitro and that it regulates kinetochore microtubule stability in vivo, consistent with a direct microtubule interaction. Sgo is thus critical for mitotic progression and chromosome segregation and provides an unexpected link between sister centromere cohesion and microtubule interactions at kinetochores.

Pathways of spindle assembly

Recent studies have revealed that, in some systems, chromatin has the ability to stabilize microtubules and organize them into bipolar spindles independently of kinetochores and centrosomes. In addition, several molecules have been identified recently that are necessary for spindle assembly; these include proteins that regulate microtubule dynamics, proteins that organize microtubule minus ends into spindle poles, and members of the kinesin superfamily that reside on the chromosome arms.

Evaluating performance in three-dimensional fluorescence microscopy

In biological fluorescence microscopy, image contrast is often degraded by a high background arising from out of focus regions of the specimen. This background can be greatly reduced or eliminated by several modes of thick specimen microscopy, including techniques such as 3‐D deconvolution and confocal. There has been a great deal of interest and some confusion about which of these methods is ‘better’, in principle or in practice. The motivation for the experiments reported here is to establish some rough guidelines for choosing the most appropriate method of microscopy for a given biological specimen. The approach is to compare the efficiency of photon collection, the image contrast and the signal‐to‐noise ratio achieved by the different methods at equivalent illumination, using a specimen in which the amount of out of focus background is adjustable over the range encountered with biological samples. We compared spot scanning confocal, spinning disk confocal and wide‐field/deconvolution (WFD) microscopes and find that the ratio of out of focus background to in‐focus signal can be used to predict which method of microscopy will provide the most useful image. We also find that the precision of measurements of net fluorescence yield is very much lower than expected for all modes of microscopy. Our analysis enabled a clear, quantitative delineation of the appropriate use of different imaging modes relative to the ratio of out‐of‐focus background to in‐focus signal, and defines an upper limit to the useful range of the three most common modes of imaging.

A High-Resolution Multimode Digital Microscope System

In this chapter we describe the development of a high-resolution, multimode digital imaging system based on a wide-field epifluorescent and transmitted light microscope and a cooled charge-coupled device (CCD) camera. Taylor and colleagues (Farkas et al., 1993; Taylor et al., 1992) have reviewed the advantages of using multiple optical modes to obtain quantitative information about cellular processes and described instrumentation they have developed for multimode digital imaging. The instrument described here is somewhat specialized for our microtubule and mitosis studies, but it is also applicable to a variety of problems in cellular imaging including tracking proteins fused to the green fluorescent protein (GFP) in live cells (Cubitt et al., 1995; Heim and Tsien, 1996; Olson et al., 1995). For example, the instrument has been valuable for correlating the assembly dynamics of individual cytoplasmic microtubules (labeled by conjugating X-rhodamine to tubulin) with the dynamics of membranes of the endoplasmic reticulum (ER, labeled with DiOC6) and the dynamics of the cell cortex [by differential interference contrast (DIC)] in migrating vertebrate epithelial cells (Waterman-Storer and Salmon, 1997). The instrument has also been important in the analysis of mitotic mutants in the powerful yeast genetic system Saccharo-myces cerevisiae. Yeast cells are a major challenge for high-resolution imaging of nuclear or microtubule dynamics because the preanaphase nucleus is only about 2 μm wide in a cell about 6 μm wide. We have developed methods for visualizing nuclear and spindle dynamics during the cell cycle using high-resolution digitally enhanced DIC (DE-DIC) imaging (Yang et al., 1997; Yeh et al., 1995). Using genetic and molecular techniques. Bloom and coworkers (Shaw et al., 1997a,b) have been able to label the cytoplasmic astral microtubules in dividing yeast cells by expression of cytoplasmic dynein fused to GFP. Overlays of GFP and DIC images of dividing cells have provided the opportunity to see for the first time the dynamics of cytoplasmic microtubules in live yeast cells and how these dynamics and microtubule interactions with the cell cortex change with mitotic cell cycle events in wild-type and in mutant strains (Shaw et al., 1997a,b). Our high-resolution multimode digital imaging system is shown in Fig. 1 and diagrammed in Fig. 2. The legend to Fig. 2 provides model numbers and sources of the key components of the instrument. There are three main parts to the system: a Nikon FXA microscope, a Hamamatsu C4880 cooled CCD camera, and a MetaMorph digital imaging system. First we will consider our design criteria for the instrument, then consider separately the major features of the microscope components, the cooled CCD camera, and the MetaMorph digital imaging system. The reader is referred to other sources for general aspects of microscope optics (Inoue and Oldenbourg, 1993; Keller, 1995; Spencer, 1982); DIC microscopy (Salmon, 1998); fluorescence microscopy (Taylor and Salmon, 1989); video cameras, slow-scan cameras, and video microscopy (Aikens et al., 1989; CLMIB staff, 1995; Inoue, 1986; Inoue and Spring, 1997; Shotten, 1995) and 3D imaging methods (Carrington et al., 1995; Hiroka et al., 1991; Pawley, 1995). Fig. 1 Photograph of the multimode digital imaging system including the Nikon FXA upright microscope sitting on an air table (Newport Corp., Irvine, CA, VW-3660 with 4-inch-high tabletop). Images are captured with a Hamamatsu C4880 cooled CCD camera. Image acquisition, ... Fig. 2 Component parts are L1, 100-watt quartz halogen lamp; S1, Uniblitz shutter (No. 225L2A1Z523398, Vincent Associates, Rochester, NY); G, ground glass diffuser: FBI, manual filter changers including KG4 heat cut and green interference filters; I1, field ... II. Design Criteria A. Fluorescence Considerations When we began building our instrument 4 years ago, our primary objective was to obtain quantitative time-lapse records of spindle microtubule dynamics and chromosome movements for mitosis in live tissue culture cells and in in vitro assembled spindles in Xenopus egg extracts. Fluorescence optical components were chosen, in part, based on the fluorophores that were available for labeling microtubules, chromosomes, and other microtubule- or spindle-associated components. Microtubules could be fluorescently labeled along their lengths by incorporating X-rhodamine-labeled tubulin into the cytoplasmic tubulin pool (Fig. 3; Murray et al., 1996; Salmon et al., 1994). In addition, we needed to use fluorescence photoactivation methods to produce local marks on spindle microtubules to study the dynamics of their assembly (Mitchison and Salmon, 1993; Waters et al., 1996). This is accomplished by the addition of a cagedfluorescein-labeled tubulin to the cytoplasmic pool and fluorescence photoactivation with a 360-nm microbeam as described by Mitchison and coworkers (Mitchison, 1989; Sawin and Mitchison, 1991; Sawin et al., 1992). In extracts and some living cells, chromosomes can be vitally stained with the DNA intercalating dyes DAPI or Hoescht 33342 (Murray et al., 1996; Sawin and Mitchison, 1991). Thus, in fluorescence modes, we needed to be able to obtain images in a “red channel” for X-rhodamine microtubules, a “green channel” for photoactivated fluorescein marks, and a “blue channel” for chromosomes. Fig. 3 Views of a living, dividing yeast, Saccharomyces cerevisiae, by (A) fluorescence of GFP protein bound to nuclear histones and (B) DIC.* (C) Images in DIC of the 0.24-μm spacing between rows of surface pores of the diatom Amphipleura illuminated ... B. Live Cell Considerations Photobleaching and photodamage are a major concern during time-lapse imaging of fluorescently tagged molecules in live cells or in in vitro extracts. Minimizing these problems requires that the specimen be illuminated by light shuttered between camera exposures, that the imaging optics have high transmission efficiencies, that the camera detectors have high quantum efficiency at the imaging wavelengths, and that light can be integrated on the detector to reduce illumination intensity and allow longer imaging sessions without photo-damage. This later point is true not only for epifluorescence but also for transmitted light (phase-contrast or DIC) imaging. In our studies, the detector also needed a high dynamic range (12 bits = 4096 gray levels), as we wanted to be able to quantitate the fluorescence of a single microtubule or the whole mitotic spindle, which could have 1000 or more microtubules. In addition, the red, green, and blue images needed to be in focus at the same position on the detector so that the fluorophores within the specimen could be accurately correlated.

Mad2 binding by phosphorylated kinetochores links error detection and checkpoint action in mitosis

The spindle checkpoint must detect the presence of unattached or improperly attached kinetochores and must then inhibit progression through the cell cycle until the offending condition is resolved. Detection probably involves attachment-sensitive kinetochore phosphorylation (reviewed in [1,2]). A key player in the checkpoints response is the Mad2 protein, which prevents activation of the anaphase-promoting complex (APC) by the Cdc20 protein [3-8]. Microinjection of Mad2 antibodies results in premature anaphase onset [9,10], and excess Mad2 protein causes arrest in mitosis [5,11]. We have previously shown that Mad2 localizes to unattached kinetochores in vertebrate cells, and that this localization ceases as kinetochores accumulate microtubules [10,12,13]. But how is Mad2 binding limited to unattached kinetochores? Here, we used lysed PtK1 cells to study kinetochore phosphorylation and Mad2 binding. We found that Mad2 binds to phosphorylated kinetochores, but not to unphosphorylated ones. Our data suggest that it is kinetochore protein phosphorylation that promotes Mad2 binding to unattached kinetochores. Thus, we have identified a probable molecular link between attachment-sensitive kinetochore phosphorylation and the inhibition of anaphase. The complete pathway for error control in mitosis can now be outlined.

Live-Cell Fluorescence Imaging

This chapter examines the ways to optimize the signal-to-noise ratio while keeping the specimen healthy. Live cells expressing fluorescent protein fusions are usually dim compared to fixed specimens, both because the fluorescent proteins are not very bright and because there is, in most cases, only one fluorophores per protein. It is also favorable to choose cells that are expressing low levels of fluorescent protein fusions to minimize the difference from the levels of the endogenous protein in vivo. Long camera exposure times, which allow accumulation of weak signals, must be often avoided to reduce photobleaching and phototoxicity and to acquire images quickly enough to capture cell dynamics. Choices, such as objective lens and camera, determine the signal-to-noise ratio of an imaging system. Optimizing the imaging system to maximize signal and minimize noise is critical for live-cell fluorescence imaging. Imaging with high signal-to-noise ratio will allow detection of low concentrations of fluorescent fusion proteins with illumination conditions that are less likely to damage cells. Automation of an imaging system allows collection of multidimensional data while helping to maintain focus and minimize specimen exposure to light. Under all imaging conditions, maintaining and verifying cell health is essential to the validity of the experimental results.

Chapter 10 A High-Resolution Multimode Digital Microscope System

This chapter describes the development of a high-resolution, multimode digital imaging system based on a wide-field epifluorescent and transmitted light microscope, and a cooled charge-coupled device (CCD) camera. The three main parts of this imaging system are Nikon FXA microscope, Hamamatsu C4880 cooled CCD camera, and MetaMorph digital imaging system. This chapter presents various design criteria for the instrument and describes the major features of the microscope components—the cooled CCD camera and the MetaMorph digital imaging system. The Nikon FXA upright microscope can produce high resolution images for both epifluorescent and transmitted light illumination without switching the objective or moving the specimen. The functional aspects of the microscope set-up can be considered in terms of the imaging optics, the epi-illumination optics, the transillumination optics, the focus control, and the vibration isolation table. This instrument is somewhat specialized for microtubule and mitosis studies, and it is also applicable to a variety of problems in cellular imaging, including tracking proteins fused to the green fluorescent protein in live cells. The instrument is also valuable for correlating the assembly dynamics of individual cytoplasmic microtubules (labeled by conjugating X-rhodamine to tubulin) with the dynamics of membranes of the endoplasmic reticulum (labeled with DiOC6) and the dynamics of the cell cortex (by differential interference contrast) in migrating vertebrate epithelial cells. This imaging system also plays an important role in the analysis of mitotic mutants in the powerful yeast genetic system Saccharomyces cerevisiae.

Navigating challenges in the application of superresolution microscopy

In this review, Lambert and Waters focus on the current practical limitations of superresolution microscopy (SRM) and provide information and resources to help biologists navigate through common pitfalls when designing an SRM experiment.