Electron microscopy (EM) can play an important role in modern gen- etic analysis because of its ability to identify subtle structural fea- tures of complex macromolecular assemblies; its high resolution can therefore provide important evi- dence about mutant phenotypes. A classic example of the contribu- tion of EM to the characterization of mutants can be seen in the mol- ecular dissection of the pathway for T4 phage assembly (reviewed in Ref. 1) and, more recently, in the molecular analysis of flagellar beat2n3. In both these examples, the logic is the same - wild-type structures are characterized by EM; a catalogue of protein components is established by biochemistry; and mutants showing abnormal function are selected. Mutant and wild-type strains are then com- pared for differences in structure and chemical composition. In some cases, missing proteins can be cor- related with missing structures and aberrant function, so a stmcture- function link is established. As a final test, missing proteins can be restored genetically or by add-back experiments in vitro, and the loss of function rescued.

Our research is directed towards understanding the structure and function of the machinery for moving chromosomes, that is, the mitotic spindle. Although mitotic spindles are fragile structures, and they are easily altered by methods used to study their structure and chemistry, we believe that the same strategy that has been used so suc- cessfully with phage and flagella can be applied to understanding the molecular mechanisms for moving chromosomes. But, because mitotic spindles lack the structural symmetry of a phage or an axon- eme, the EM methods by which different samples can be compared require a more extensive, three- dimensional (3D) analysis. In this article, we summarize our efforts to develop such methods.

Two organisms that offer particu- lar promise for an analysis of mitosis by the coordinated use of genetics, biochemistry and EM are the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae. The power of yeast genetics is well known and a relatively large num- ber of mitotic mutants have already

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Mapping the three-dimensional

organization of microtubules in mitotic spindles of yeast

Kent McDonald, Eileen T. O’Toole, David N. Mastronarde, Mark Winey and J. Richard McIntosh

been isolated from each of these organisms (see Refs 4 and 5 for reviews). As with all spindles, con- ventional biochemical analysis will be a problem, but immuno- cytochemical methods such as those used by Rout and Kilmarti& to identify components of the spin- dle pole body (SPB) in S. cerevisiae could provide the necessary bio- chemical information. The yeasts are also favourable material for EM studies because of their small size and simple, yet representa- tive, spindle structure. As shown by the pioneering work of Byers and Goetsch7 and, especially, Peterson and Riss, the S. cerevisiae spindle is small (with a maximum length of -10 pm) and has only one microtubule (MT) for each of the kinetochores on the 16 chromo- somes of a haploid cell. Likewise, the spindle of S. pombe is small (-11 km long at maximum length) and contains ~50 MTs, of which 2-4 are connected with each kinetochore on the three meta- phase chromosomes9,10. By com- parison, the average mammalian spindle has between 1000 and 3000 MTs, more than 15 MTs at- tached to each kinetochore, and it may be up to 20 km longll.

light and electron microscopy of yeast spindles

Despite improvements in light microscopy (LM) and image pro- cessing for LM (reviewed in Refs 12 and 13), the small size of yeast spindles makes them diffi- cult to characterize by this tech- nique (Fig. 1). LM is useful for determining the percentage of a population that expresses a given phenotype and for showing the general features of mutant spindle structure (Ref. 14; Figs la-c), but other features, such as the relative

position of individual spindle MTs, are impossible to see without the better spatial resolution of EM (Figs Id-f). To make that point more explicitly, the scale bars for Figures Id-g have been made to represent 0.2 pm, which is the limit of resolution for light microscopy. Structures closer together than this distance will be seen in the light microscope as one object.

For a detailed understanding of the distribution of spindle MTs, it is necessary to make 3D models from serial EM cross sections. Serial longitudinal sections of spindles can be used for some 3D reconstructions15, but not in yeast, where the spindle MTs are packed closely. Following MTs from one section to the next is difficult when their axes are oblique to the plane of the section (Fig. Id). In transverse section, however (Figs le-f), MTs are clearly distinguish- able, and identifying successive images of the same MT is usually straightforward. Data from many such sections can be assembled and displayed as a model (Fig. lg).

Although there are obvious advantages to EM imaging, there are also disadvantages. The work is comparatively time-consuming, and high-quality work requires a keen attention to many techni- cal details (Table 1). Fortunately, since the pioneering work on 3D EM of mitotic spindles (reviewed in Ref. 16), many improvements have been made in both the preparative procedures and the tools available for ultrastructural analysis. There are now better ways to fix cells, improved micro- tomes that make serial sectioning easier, and better and less costly computer equipment for making 3D reconstructions.

Kent McDonald is at the Electron Microscope Laboratory, 26 Ciannini Hall, University of California, Berkeley, CA 94720-3330, USA; and Eileen O’Toole, David Mastronarde, Mark Winey and J. Richard McIntosh are at the Dept of Molecular, Cellular and Developmental

Biology, University of Colorado, Boulder, CO 80309-0347, USA.

trends in CELL BIOLOGY (Vol. 6) June 1996 0 1996 Elsevier Science Ltd 235 PII:SO962-8924(96)40003-4

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spindle structure in fungi goes back to the pioneering work in 1982 of Heath and Rethoret17, and to the studies of S. pombe by Tanaka and KanbelO and S. cere- visiae by Baba and Osumi18.

We routinely use high-pressure freezing19 to cryofix yeast cells9J0 because the yield of well-frozen cells is high, but other methods of cryofixation will work also10~18. An added benefit of the high- pressure-freezing method is that it is not necessary to ‘prefix’ the cells chemically or digest their walls enzymatically prior to EM processing, a method that is used routinely for conventional EM fix- ation of yeast. Once frozen, the cells are freeze-substituted21,22, embed- ded and serially sectioned by routine methods23. Spindles are photographed at magnifications of 125 000 on the microscope, using a rotating, tilting specimen- holder to get images of MTs in cross section (Figs le-f).

Data entry and alignment To track MTs accurately through

serial sections, the images on the negatives must be converted to digital form and aligned adequately with each other. Currently, we record images on electron micro- scope film, then digitize the serial images with a video camera and a motorized, computer-driven light table24. This approach has several advantages. First, it allows a gen- erously large area to be recorded during microscopy to guarantee that the region of interest can be tracked through the series, yet the final digitized images can be re- stricted to just the size needed for study of the spindle. Second, areas larger than -1000 X 1000 pixels can be digitized easily from film, but may be difficult to record digitally direct from the electron micro- scope. Finally, during digitization, images can be translated and ro- tated into good initial alignment. Nevertheless, we anticipate that direct recording of digital images using charge coupled device (CCD) technology on the microscope will save significant effort in 3D spindle-reconstruction studies in the future.

Typically, section-to-section magnification changes and distor- tions of up to 10% arise during

trends in CELL BIOLOGY (Vol. 6) June 1996

FIGURE 1

The mitotic spindle of Schizosaccharomyces pombe visualized by different imaging methods. (a,b) Light microscopy of a metaphase (a) and an anaphase (b) spindle stained with DAPI (blue) to

reveal the DNA, and fluorescently-labelled antibody against tubulin (red) to show the spindle. (c) A similar image of 5. pombe mutant in the cut7 1+locus. Spindle pole body (SPB) function has been impaired, and a single SPB initiates multiple shafts of MTs. Light microscopy reveals the overall design

of the aberrant spindle, but the details of spindle disorganization cannot be seen. (d) Electron microscopy provides better resolution for visualizing the details of spindle design. This section was cut

approximately parallel to the longitudinal axis of an early anaphase spindle, and, while some of the spindle MTs are clearly visible, one section is insufficient for an understanding of spindle design. Bar,

200 nm. (e,f) Electron micrographs of spindle cross sections show the spindle MTs clearly. The relative positions of neighbouring MTs allow the tracking of individual MTs from section to section, so MTs can

be characterized by whether they end on kinetochores (light blue) or whether they run pole to pole (dark blue) or start at one SPB and end before reaching the other (red and green). Bar, 200 nm. (9) A model of the spindle generated from sections such as those shown in Figs 1 e-f. The magenta objects

near the centre of the model represent the kinetochores. Bar, 200 nm. Materials for Figs 1 c, e, f and g were kindly provided by R. Ding, Fred Hutchison Cancer Research Center, Seattle, USA.

The importance of cryofixation Of all the processing steps be-

tween the live cell and the 3D image (Table l), none is more im- portant than the initial fixation. Artifacts introduced at this stage cannot be corrected later and will show up as errors in the final

236

models. Fortunately, cryofixation vastly improves the quality of yeast ultrastructure, giving the user confidence that the images produced of spindle MTs are an accurate reflection of the MTs in the living cell. The history of cryofixation for preservation of

MISCELLANEA

sectioning and microscopy. In re- constructions of large volumes from mammalian spindlesl’,“, we have found it essential to align im- ages using linear transformations that correct for these distortions. For the simpler and smaller yeast spindles (Figs lg and Za), rota- tional and translational align- ments alone are sufficient; how- ever, for some mutant spindles containing greater numbers of MTs spread out over a large area (Fig. Zb), full alignment, including distortion correction, is necessary.

Modelling After alignment, the objects of

interest can be modelled by using programs developed specifically for tracking spindle MTsg,Z0~24. Although it is possible to have the computer make a model automati- cally by using MT-recognition programs, with yeast spindles it is usually more efficient to model the entire spindle manually. A single MT is modelled using a computer-controlled cursor to place a model point at the centre of the MT on each successive sec- tion. Initially, it may be difficult to recognize MTs that are not in perfect cross section, but this skill. develops quickly. The ability to riffle quickly through a series of spindle images greatly facilitates the identification of all MTs. Structures other than MTs, such as SPBs or kinetochores, can be out- lined as separate contours. Because we know the position of poles and kinetochores (in the case of S. pombe, at leastg), the MTs can be identified and distinguished by different user-defined colours (Fig. lg). The model can be rotated in any direction to give the optimal viewing angle or it can be imaged in stereo. Perhaps most impor- tantly, the relationship between different spindle components can be studied quantitatively.

Analysis Detailed 3D analysis is necess-

ary for a clear understanding of mitosis, even with wild-type cells. In S. cerevisiae, it is not even poss- ible to identify the stages of mito- sis, except for spindle elongation (anaphase B), by LM alone. From a 3D EM analysis of budding yeast spindleszO, we found that there

TABLE 1 - STEPS REQUIRED TO GO FROM LIVING YEAST CELLS THROUGH ELECTRON MICROSCOPY TO THREE-DIMENSIONAL MODELS’

Processing step Operational conriderationb

Healthy live cells

Ctyofixation

Freeze-substitution

Serial sectioning

Microscopy

Data entry and alignment

Model building

Analysis

If possible, have growth facility, e.g. shaker bath, next

to freezing apparatus. High-pressure freezing is the method of choice; other

methods will give lower yields of well-frozen cells. Substitute the cells in 1% osmium tetroxide plus

0.05% uranyl acetate in acetone at -90°C for

2-3 days. Trim the block face -200 km wide by 50 km high.

Sections should be 40-50 nm thick and a total of 300 serial sections or more will ensure that even the

longest spindles will be included. Use a rotating, tilting specimen-holder for optimal

view of MTs in cross section. Record tilt angles so

correct spindle lengths can be calculated. Use video digitization to create computer-image files

from selected regions of EM negatives. Align MT images manually, followed by computer correction

of sectioning distortions. For spindles with small numbers of MTs (e.g. wild-

type), model individual MTs manually. Use computer to display 3D models and to calculate

spacings between different MT classes, total MT lengths and other structural parameters.

+or each processing step in the left-hand column, a corresponding notation in the right-hand column indicates one or more important technical considerations for that stage in the processing.

bEM, electron microscope; MT, microtubule.

FIGURE 2

A comparison of models from a wild-type 2.7 km Saccharomyces cerevisiae spindle (a) and a comparable spindle (2.5 km) from a strain that is mutant at the C/X20

locus, grown for 4 h at the restrictive temperature (b). The models reveal clearly the major difference between mutant and wild-type spindles in terms of the

number, type and length-distribution of MTs that they contain. Bar, 100 nm. We thank D. Burke, Univ. Virginia, Charlottesville, USA for providing the cdc20 strain.

Acknowledgements

Support for this work was provided by grants to 1. R. M. (NIH award RR00592) and M. W. (National Science Foundation award MCB-9357033, and the Pew Scholars Program in the Biological Sciences award POO2OSC). We thank D. Burke, Univ. of Virginia, Charlottesville, USA for providing the cdc20 strains of 5. cerevisbe,

R. Ding, Fred Hutchison Cancer Research Center, Seattle, USA for providing materials for Fig. 1, Mary Morphew, Univ. Colorado, Boulder, USA for the images in Fig. 3, and Berl Oakley, Ohio State Univ., Columbus, USA for antibodies to y-tubulin.

trends in CELL BIOLOGY (Vol. 6) June 1996 237

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FIGURE 3

Sections of Schizosaccharomyces pombe embedded in Lowicryl K4M (a) or LR White (b). The sections have been labelled by reaction with antibodies to Aspergillus nidulans

y-tubulin (a generous gift of Berl Oakley, Ohio State Univ., Columbus, USA), followed by incubation with secondary

antibodies conjugated to IO-nm colloidal gold. Most of the label is associated with the inner surface of the nuclear

envelope at the spindle pole body (SPB). This level of detail could not be resolved with the light microscope.

Bar, 100 nm. We thank Mary Morphew, Univ. Colorado, Boulder, USA for providing these images.

was no apparent metaphase con- figuration of kinetochore MTs and that chromosome-to-pole move- ment (anaphase A) is likely to be asynchronous. The value of 3D analysis is also evident in S. pombe, where reconstructions allow the determination of whether a given MT is associated with a kineto- chore, is attached on one SPB or the other, or is continuous be- tween the poles9 (Fig. 1); this al- lows identification of the MT type and assessment of its probable function.

3D analysis is also valuable in the characterization of mutant pheno- types - for example, in cdc20 mu- tants of S. cerevisiae. The CDCZO gene product appears to be involved in a number of MT-dependent processes in the yeast life cycle, including mitosisz6. Temperature- sensitive alleles of CDC20 have been characterized by LM (Ref. 26), but such images could not de- termine whether the apparently brighter staining of mutant spin- dles by antibodies against tubulin was due to an increase in MTs or to a change in the character of the spindle staining. Serial-section

EM demonstrates unambiguously that the spindles in c&?O~ cells grown at the restrictive tem- perature contain more and longer MTs than wild-type cells of com- parable length (Fig. 2) and possess additional interesting structural features (E. T. O’Toole et al., un- published).

Perspectives Having demonstrated the power

of 3D descriptions of wild-type S. pombe and S. cerevisiae spindles91z0, what are some of the future appli- cations of this technique? One aim is to use 3D reconstruction methods to analyse mitotic mu- tant phenotypes in both yeasts, comparing them with what we already know about the detailed structure of wild-type spindles. Of the two, S. cerevisiae is the more fully-developed genetic system, but S. pombe may be the more attrac- tive model for studying some as- pects of mitosis. S. pombe has sim- ple, but discrete, kinetochores that have 2-4 MTs attached dur- ing metaphase and anaphase A (Ref. 9), and its three chromo- somes are condensed during mi- tosis, making it possible to visual- ize them by LM. By contrast, S. cerevisiae has 16 chromosomes, which do not condense appreci- ably during mitosis; it does not have a conventional metaphase20; and it does not have structured kinetochores that can be identi- fied by EM. Despite these differ- ences, the principal method of chromosome segregation in both S. pombe and S. cerevisiae is spindle elongation, or anaphase B. We ex- pect that mutations affecting this process can be studied effectively in both types of yeast.

Another goal is to model 3D distributions of specific spindle proteins by using antibody-label- ling in combination with serial- section reconstruction techniques. Our descriptions of spindles could then include the 3D distributions of important, non-MT proteins relative to important spindle landmarks. This work is already under way and Figure 3 (J. R. McIntosh, unpublished results) shows the localization of y-tubu- lin in S. pombe relative to the in- terphase SPB. This reveals that y-tubulin, an isoform thought to

be important for MT initiation, is localized on the inner surface of the nuclear envelope as well as on the SPB itself, even at a time when the cell contains no intra- nuclear MTs. This suggests that the MT-initiating activity of y- tubulin can be regulated as a function of time in the cell cycle. Having a 3D model, instead of random, 2D slices, will be impor- tant because it will allow a better visualization of the antibody dis- tribution relative to other spindle structures. For example, in the case of y-tubulin, 3D modelling may reveal an asymmetric distri- bution of labelling during SPB duplication. The modelling of motor enzymes, MT-associated proteins and other structural pro- teins identified in screens for mi- totic mutants should be helpful in understanding the role of each molecule in moving chromosomes.

Finally, we expect that con- tinued improvements in compu- ter hardware and software, and especially in direct digital imaging from transmission electron micro- scopes, will make 3D modelling more accessible to all researchers. At the moment, anyone with ac- cess to EM facilities can make use of the methods described here if they are willing to make a modest investment in computer hardware. We are currently running the mod- elling and analysis programs on an SGI Iris Indigo XZ4000; such programs can be run on a variety of platforms, although not yet on personal computers (PCs). The ex- ecutable version of our modelling programz7 and our analysis pro- grams are available at no cost from the Boulder Laboratory for 3D Fine Structure by contacting Jim Kremer at the following e-mail ad- dress:kremer@beagle.colorado. edu, or by telephoning: (303) 492- 7980. Another valuable resource for 3D EM methods is the 3D EM Home Page on the Internet (URLhttp://rcr-www.med.nyu. edu/3dem/HomePage.html.Inthis article, we have focused on meth- ods for reconstructing mitotic spindle distributions, but we hope it is evident that these basic methods can be readily adapted to provide 3D reconstructions for genetic analysis of other cellular organelles.

238 trends in CELL BIOLOGY (Vol. 6) June 1996

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In the past few years major advances have occurred in our understanding of prohormone processing’, intracellular vesicular trafficking2 and secretory vesicle fusion with the plasma mem- brane3. The goal of this, the Third International Annaberg Conference*, was to bring together investigators in the converging areas of protein sort- ing and processing in the secretory pathway. This conference first took place in 1991 as an informal gathering of several laboratories and has now evolved into a major international meeting that combines scientific inten- sity with an equally demanding social program (Fig. 1) held on the ski slopes and in the discotheques of Annaberg, a picturesque village near Salzburg.

Polarized sorting Given the functional conservation of

components of secretory vesicles in yeast and mammalian cells, it is poss- ible that transport vesicles targeted to specialized membrane domains would possess specific components

in addition to a generic set of SNARE- like factors. Kai Simons (Heidelberg, Germany), in his opening lecture, pro- posed that, at least for polarized epi- thelial cells, an alternative pathway might exist. He demonstrated that vesicle transport to the basolateral

Fusion and confusion in the secretory pathway

Gabriele Seethaler, Sharon Tooze and Dennis Shields

surface utilizes ‘classical’ coatomer- SNARE components, whereas protein sorting to the apical surface uses a dif- ferent set of components including an annexin isoform, 13a, VIP21 -caveolin, a cholesterol-binding protein and poss- ibly lectin-type proteins such as VIP-36

(Ref. 4). He proposed that sphingo- lipids, glycolipids, cholesterol, VIP21, VIP-36 and endogenous lectins associ- ate preferentially in the TCN to sort apically targeted proteins. Whether these ‘apical factors’ facilitate differ- ential vesicle targeting in other polar- ized cells, neurons or hepatocytes will no doubt be forthcoming in the near future.

Protein targeting in the Golgi apparatus

How membrane proteins are tar- geted to, retained in and recycled to the trans Colgi network (TCN) is under- stood for only a few examples. George Banting (Bristol, UK) presented evi- dence suggesting that TGN38, a type-l transmembrane protein localized to

the TGN that recycles to and from the plasma membrane, may be involved in the organization of the TGN itself. Overexpression of TGN38 affects its localization and also that of furin and y-adaptin, and results in fragmen- tation of the TGN. Using TGN38 tagged with green fluorescent protein and mutagenesis of the conserved cytoplasmic tail motif SDYQRL, he showed that the presence of the ser- ine residue enhanced appearance of TGN38 on the plasma membrane and in late endosomes, suggesting this residue plays a role in return to the TGN. The importance of serine resi- dues in TGN retention was underscored by the work of Gary Thomas (Portland, Oregon, USA) who has been investi- gating the cytoplasmic tail of another TCN membrane protein, the furin- processing enzyme. He demonstrated that the cytoplasmic tail of furin is phosphorylated on serine residues by casein kinase II (Ref. 5). Mutagenesis of these residues greatly reduced the endocytosis of furin from endosomes

Cabriele Seethaler is at SchieRstand- strasse 12, 5061 Elsbethen, Austria; Sharon Tooze is at the Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London, UK WCZA 3PX; and Dennis Shields is at the Dept of Developmental and Molecular Biology, Albert Einstein College of Medicine of Yeshiva University, 1300 Morris Park Avenue, Bronx, NY 10461, USA.

trends in CELL BIOLOGY (Vol. 6) June 1996 0 1996 Elsevier Science Ltd PII: SO962-8924(96)30020-Z

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