Mitotic Spindle: Laser Microsurgery inYeast Cells

Dispatch

Pedro Carvalho1,2 and David Pellman1

Laser microsurgery has led to remarkable discoveriesin a number of cell types. Two recent studies haveshown that this classical technology can now beemployed with small yeast cells. This advance willenable regional ablation to be combined with facilegenetic manipulation in a eukaryotic cell.

Some cell biologists just like to watch, while othersprefer to poke, prod and manipulate. The pokers andprodders have a distinguished record of uncoveringfundamental biological principals from simple,elegant experiments. This began with developmentalbiologists, who used transplantation experiments toidentify regions of embryos that specified differentcell fates, and has extended to classic experimentsthat continue to define some of the central issues inmodern cell biology. Microsurgery experiments byRay Rappaport [1] revealed the role of overlappingmicrotubule arrays in determining the cell cleavageplane during cytokinesis. Nicklas and Koch [2]demonstrated the importance of tension on kineto-chores to signal the completion of chromosomealignment on the mitotic spindle. Laser cutting exper-iments [3,4] uncovered the existence of pulling forcesgenerated by astral microtubules on the anaphasespindle in certain eukaryotic cell types.

Because most cell biologists now study relativelysmall tissue culture cells, rather than the largerembryonic cells used in the pioneering studies, lasermicrosurgery has become a weapon of choice for themanipulators [5,6]. More recently, the use of GFP-tagged proteins has enabled lasers to be targeted morespecifically to even smaller structures, such as the cen-trosome [6,7]. The manipulators continue to push theenvelop of smallness, and two recent reports in CurrentBiology [8,9] have now demonstrated the utility of lasermicrosurgery in yeast, the smallest and most geneticallytractable eukaryotic system for studying cell division.

“Why bother with laser microsurgery on yeast?” theold-fashioned geneticist might ask. “Yeasts are moreeasily ‘dissected’ by mutations. And anyway, isn’t thisusing a cannon to kill a gnat?” Although we like to thinkof genetic mutations as ‘smart bombs’ to study genefunction, disrupting a multifunctional gene product notuncommonly has more of a ‘scud missile’ effect, pro-ducing hard to interpret collateral damage. Thisproblem can sometimes be addressed by studying

specific alleles, but often such informative alleles arenot available. Furthermore, mutations tend to inactivategene functions globally, while cell biologists oftenreally want information about local activity. Cell biolo-gists also need functional information with rapid timeresolution, and functional inactivation through condi-tional mutations can be slow.

All of this makes an appealing case to try lasermicrosurgery on yeast. This appeal is only enhancedwhen one considers the fact that the engines drivingintracellular movements are often ‘over-built’,consisting of several overlapping mechanisms. Thus,genetics can be used to strip down over-builtprocesses, and laser microsurgery can then be usedto characterize individual mechanisms. The power ofthis combined genetic/microsurgery approach hasbeen elegantly demonstrated by experiments in thenematode Caenorhabditis elegans [4].

The first step to apply laser microsurgery to yeastwas to see if these small cells could survive the insult.Working with the fission yeast Schizosaccharomycespombe, Khodjakov et al. [8] performed a carefulseries of experiments to show that irradiation with agreen laser beam did not cause a general disruptionin cell integrity but rather produced small, approxi-mately 500 nm2 ‘scars’, similar to those previouslyobserved in animal cells. Most importantly, manyaspects of normal cell division continued after lasermicrosurgery. Tolic-Norrelykke et al. [9] observed thatS. pombe cells could similarly survive the even longerwavelength irradiation from the excitation beam of atwo-photon microscope. After a recent keynote talkat this year’s American Society for Cell Biologymeeting on cytokinesis, Ray Rappaport fielded aquestion concerning potential artifacts resulting fromthe surgical manipulation of cells. His response wassimple: “I always designed an experiment where theendpoint was the division of the cell.” Rappaport’stime-tested design remains the gold standard.

As a proof of principle for the use of laser surgery infission yeast, Tolic-Norrelykke et al. [9] and Khodjakovet al. [8] both targeted their heavy artillery into a well-characterized process: the forces that controlelongation of the mitotic spindle. Like other eukary-otes, fission yeast cells assemble a microtubule-based bipolar spindle which elongates at anaphase,distributing sister chromosomes to the two daughtercells [10] (Figure 1A). The two half-spindles are linkedat the spindle midzone, where antiparallel ‘polar’microtubules adopt a characteristic square-packeddistribution [11,12]. The midzone houses a wealth ofmicrotubule-associated proteins (MAPs), motors andsignaling molecules [12]. During spindle elongation,the midzone motors slide the half-spindles apart, andthe polar microtubules polymerize from their plus-ends; the MAPs and motors maintain the interactionbetween the half-spindles in the face of all this activ-ity [12–15] (Figure 1B).

Current Biology, Vol. 14, R748–R750, September 21, 2004, ©2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cub.2004.09.010

1Department of Pediatric Oncology, Dana-Farber CancerInstitute and Division of Hematology/Oncology, Children’sHospital Boston and Harvard Medical School, Boston,Massachusetts 02115, USA. 2Institute for BiomedicalSciences Abel Salazar, Porto, Portugal.E-mail: david_pellman@dfci.harvard.edu

The first question both groups [8,9] wanted toaddress was the origin of the forces for spindleelongation. Although there was abundant evidencethat this would come from ‘pushing’ forces on the polefrom the central spindle [13,14], some recent studieshad raised the possibility that astral microtubulescould contribute to the rate of spindle elongation [16].After blowing away the spindle midzone in anaphasecells, however, both groups [8,9] observed that thespindle poles promptly collapsed inward, presumablyas a result of the elastic recoil of the nuclear envelope[8,9] (Figure 1C). This inward movement strongly sug-gests that, in fission yeast, astral microtubules exertlittle, if any, pulling forces on the spindle poles; themain driver for spindle elongation is indeed pushing bythe central spindle.

By following the severed spindles by timelapsemicroscopy, both groups [8,9] made an additionaland remarkable observation: in most spindles thathad been severed during mid-anaphase, the twohalves eventually came together and the resulting‘healed’ spindles resumed elongation at a normalrate. This was only observed in mid-anaphase cells,and spindle healing therefore correlated with exten-sive overlap of antiparallel microtubules. Moreover,when Khodjakov et al. [8] severed spindles close totheir insertion into one spindle pole body (SPB), the

larger, midzone-containing, spindle fragment stillelongated. This elongation had the hallmarks of anormal anaphase B: antiparallel microtubules slidpast each other and microtubules polymerized fromtheir plus ends, as evidenced by the use of photo-bleaching to place a fiduciary mark in the midzone ofthe long spindle fragment (Figure 1D). In fact, if thespindle is severed from both SPBs, it still elongates,albeit at a slower rate [8]. Thus, the SPBs are notabsolutely required for spindle elongation, althoughthey clearly promote many aspects of cell division, forexample the channeling spindle forces onto specificdomains of the nuclear envelope.

Tolic-Norrelykke et al. [9] applied the lasermicrosurgery to another interesting process in fissionyeast mitosis: the mechanism by which astralmicrotubules position the pre-anaphase spindle priorto spindle elongation. Geometrical constraints canposition elongating spindles within rod-shapedfission yeast; however, astral microtubule–corticalinteractions appear to make this process more effi-cient. Indeed, a cell-cycle checkpoint has been pro-posed to delay anaphase if spindle alignment isimpaired [16–18].

A key open question is whether the astral micro-tubules push or pull the spindle into alignment. Tolic-Norrelykke et al. [9] filmed astral microtubule–cortical

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Figure 1. Microtubule-dependent forces on the spindle pole bodies during the cell cycle in fission yeast.

(A) Microtubule interactions with the cell ends generate the pushing force (arrows) to center the nucleus in interphase. As cellsprogress into mitosis, a short spindle assembles in the nucleus. Interactions of astral microtubules with cortical regions around thenucleus appear to orient along the longer cell axis (arrows). A number of microtubule-associated proteins, microtubule motors andregulatory proteins promote anaphase spindle elongation by sliding apart overlapping antiparallel microtubules at the spindlemidzone — boxed area detailed in (B). (C) Severing the spindle by laser microsurgery resulted in the inward movement of the spindlepoles (red arrows). Strikingly, most of the mid-anaphase cut spindles re-annealed and elongated at a normal rate. (D) Laser dissec-tion of spindle poles did not abolish spindle elongation. Bleaching marks (gray boxes) made in off-center severed spindles movesymmetrically away from the midzone. This experiment demonstrates that the spindle elongates by force generated by the sliding ofantiparallel microtubules coupled to polymerization of microtubules from their plus-end.

B C

A Interphase Pre-anaphase Anaphase

D

Laser beam

Newly polymerized microtubules

Photobleaching-induced mark

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interactions during spindle orientation and measuredthe rate at which the angle between the spindle andthe cell axis changed. Centering movements occurredwhen polymerizing astral microtubules seemed topush the spindle pole away from the cortex (Figure 1A‘Pre-anaphase’). Moreover the rate of these move-ments increased by about 40% if the spindle wassevered, strongly suggesting that the poles arepushed by polymerizing astral microtubules againstthe resistance of the intact spindle.

A mathematical model of the process was formu-lated, from which the authors estimate the magnitudeof the pushing force to be between 1–10 pN. Thus, aswith the centering of the fission yeast nucleus duringinterphase [19], pushing forces by astral microtubulesare an important and possibly the only mechanism forpositioning preanaphase spindles in fission yeast[9] (Figure 1A).

Is laser microsurgery in yeast a one-off shot, or isthis the beginning of a sustained campaign? Only timewill tell, but a number of potential applications imme-diately suggest themselves. In both budding andfission yeast, there is an asymmetric distribution of anumber of molecules between the mother and daugh-ter SPBs [20]. Ablation of one or the other pole shouldbe useful to test the functional consequences of theseasymmetries. In fact, Khodjakov et al. [8] have prelimi-nary evidence that ablation of both poles, but noteither pole individually, abolishes pole-derived signalsfor cytokinesis. Therefore, we suspect that laser micro-surgery in yeast is here to stay. It may now be addedto the growing list of biophysical and computationaltools that, coupled to the ease of genetic manipulation,make yeast certainly the most fun, and possibly themost fruitful, system to study cell division.

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