Author Correction

Wilson, P. G., Simmons, R. and Shigali, S. (2004). Novel nuclear defects in KLP61F-deficient mutants in Drosophila are partiallysuppressed by loss of Ncd function. J. Cell Sci. 117, 4921-4933.

The name of the last author is S. Saighal not S. Shigali.

The authors apologise for this error.

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IntroductionSpindle bipolarity is essential for accurate segregation ofchromosomes to daughter cells at cell division. Members of theBimC family of kinesin related motor proteins are essential forspindle bipolarity across species; loss of BimC function resultsin monopolar rather than bipolar spindles (Hildebrandt andHoyt, 2000; Kashina et al., 1997). BimC kinesins are bipolar(Kashina et al., 1996) homotetramers (Cole et al., 1994) andspindle-associated during mitosis (Sharp et al., 1999a). Despitethe implications of spindle localization and spindle defects, theBimC kinesin Eg5 in Xenopusremains statically positioned inspindles as microtubules flux toward spindle poles (Kapoorand Mitchison, 2001) as if Eg5 is tethered to an immobilenon-microtubule spindle matrix rather than to dynamicmicrotubules fluxing toward spindle poles. It is not yet clearwhether BimC kinesins apply motive force to spindlemicrotubules to establish or to maintain spindle bipolarity orwhether these kinesins prevent collapse of a non-microtubulespindle matrix that is connected in some way to spindlemicrotubules.

Non-microtubule components of spindles includeconstituents of the nuclear matrix. First defined as non-chromatin material in the interphase nucleus (Fawcett, 1966),the nuclear matrix is in part composed of a nuclear lamina anda ribonucleoprotein network involved in gene expression

(Nickerson, 2001). The nuclear lamina is a supportivemeshwork of nuclear-specific intermediate filaments calledlamins and lamin-binding proteins (Holaska et al., 2002). Thenuclear lamina is attached to the inner nuclear envelope and toheterochromatin at the nuclear periphery (Holaska et al., 2002).A number of nuclear matrix proteins have been implicated inspindle assembly, including NuMa in vertebrates (Becker et al.,2003; Compton and Cleveland, 1994; Dionne et al., 1999; Duet al., 2002; Khodjakov et al., 2003; Levesque et al., 2003;Merdes et al., 1996; Saredi et al., 1996; Tulu et al., 2003),TPX2 in Xenopusand humans (Bayliss et al., 2003; Eyers andMaller, 2004; Garrett et al., 2002; Gruss et al., 2002; Wittmannet al., 2000), the chromatin bound Skeletor protein inDrosophila(Silverman-Gavrila and Forer, 2003; Walker et al.,2000) and the recently identified nucleolar protein NuSAP invertebrates (Raemaekers et al., 2003). Although inactivationgenerates defects in spindle and/or centrosome organization,the precise role of these nuclear proteins in spindle assemblyis not clear. Although still controversial (Bloom, 2002; Scholeyet al., 2001; Wells, 2001), some components of the nuclearmatrix have been proposed to form a spindle matrix that assistsin spindle assembly. However, spindle defects need not reflectan active role in spindle assembly, but rather collapse of acompressible non-microtubule matrix that is connected tospindle microtubules.

4921

KLP61F in Drosophila and other BimC kinesins areessential for spindle bipolarity across species; loss of BimCfunction generates high frequencies of monopolar spindles.Concomitant loss of Kar3 kinesin function increases thefrequency of bipolar spindles although the underlyingmechanism is not known. Recent studies raise the questionof whether BimC kinesins interact with a non-microtubulespindle matrix rather than spindle microtubules. Here wepresent cytological evidence that loss of KLP61F functiongenerates novel defects during M-phase in the organizationand integrity of the nuclear lamina, an integral componentof the nuclear matrix. Larval neuroblasts andspermatocytes of klp61F mutants showed deep involutionsin the nuclear lamina extending toward the centrallylocated centrosomes. Repositioning of centrosomes to formmonopolar spindles probably does not cause invaginationsas similar invaginations formed in spermatocytes lacking

centrosomes entirely. Immunofluorescence microscopyindicated that non-claret disjunctional (Ncd) is acomponent of the nuclear matrix in somatic cells andspermatocytes. Loss of Ncd function increases thefrequency of bipolar spindles in klp61F mutants. Nucleardefects were incompletely suppressed; micronuclei formednear telophase at the poles of bipolar spindle in klp61F ncdspermatocytes. Our results are consistent with a model inwhich KLP61F prevents Ncd-mediated collapse of a non-microtubule matrix derived from the interphase nucleus.

Supplementary material available online at http://jcs.biologists.org/cgi/content/full/117/21/4921/DC1

Key words: Spindle, Centrosome, BimC, Kar3, Mitosis, Meiosis,Nuclear matrix, Kinesin, Spindle matrix

Summary

Novel nuclear defects in KLP61F-deficient mutants inDrosophila are partially suppressed by loss of NcdfunctionPatricia G. Wilson*, Robert Simmons and Sheena ShigaliGeorgia State University, Department of Biology, 24 Peachtree Center, Atlanta, GA 30303, USA*Author for correspondence (e-mail: biopgw@gsu.edu)

Accepted 27 May 2004Journal of Cell Science 117, 4921-4933 Published by The Company of Biologists 2004doi:10.1242/jcs.01334

Research Article

4922

Members of the Kar3 family of kinesins provide thestrongest link between BimC kinesins and the nucleus. TheKar3 family of kinesins was first identified in Saccharomycescerevisiaeas a karyogamy (Kar) mutant that was defective innuclear fusion (Meluh and Rose, 1990). BimC function inspindle assembly is antagonized by Kar3 kinesins; concomitantloss of BimC and Kar3 function increases the frequency ofbipolar spindles in comparison with the frequency ofmonopolar spindles in bimC mutants (O’Connell et al., 1993;Saunders and Hoyt, 1992). The motor domains of BimC(Barton et al., 1995; Sawin et al., 1992) and Kar3 kinesins(Endow and Komma, 1998; Vale and Milligan, 2000)translocate toward the plus and minus ends of microtubules,respectively. Collectively, these results suggested that geneticinteractions between BimC and Kar3 kinesins reflect theapplication of opposing motive forces to spindle microtubulesto establish or maintain spindle bipolarity (Cottingham andHoyt, 1997; Gaglio et al., 1996; Mountain et al., 1999;Saunders and Hoyt, 1992; Sharp et al., 1999b). Paradoxically,Kar3 kinesins are not essential for assembly of mitotic spindlesor for mitosis. Kar3 kinesins are nuclear during interphase invertebrate cells (Kuriyama et al., 1995; Mountain andCompton, 2000; Walczak et al., 1997). Nuclear localization ofKar3 kinesins in vertebrates and genetic interactions withBimC kinesins across species raise the question of whetherKar3 kinesins are associated with a non-microtubule spindlematrix derived from the interphase nucleus.

Non-claret disjunctional (Ncd) in Drosophilais probably themost extensively studied member of the Kar3 family ofkinesins. Ncd was the first of this family to be identifiedbecause of a mutant phenotype; Ncd-deficient females arenearly sterile (Sturtevant, 1929) and attributed to chromosomenon-disjunction during female meiosis (Kimble and Church,1983). Female meiotic spindles are unstable, showingmicrotubule minus ends splayed into broad rather than focusedspindle poles (Endow and Komma, 1997; Matthies et al.,1996). In contrast to vertebrate homologs, Ncd does not shownuclear localization in the brief interphase between mitoses inearly embryos, but is spindle and microtubule associatedthroughout the cell cycle (Endow and Komma, 1996; Hatsumiand Endow, 1992; Sharp et al., 2000a; Sharp et al., 1999b). Yet,loss of Ncd function prevents collapse of bipolar spindles whenKLP61F is immunoinactivated in embryos (Sharp et al.,1999b) or knocked down by RNAi in cultured cells (Goshimaand Vale, 2003). Ncd protein is highly expressed in the femalegerm line (Hatsumi and Endow, 1992), but expression insomatic cells or in the male germ line has not been reported.Thus, the functional relationship between Ncd and other Kar3homologs has not been fully established.

Here we apply genetic, immunological, and cytologicalmethods to determine whether genetic interactions betweenKLP61F and Ncd exist in somatic cells and the male germ lineand if so, whether genetic interactions impact disposition ofnuclear material during M-phase.

Materials and MethodsFly husbandry and geneticsDrosophilaflies were maintained on standard medium in humidifiedchambers at 25°C. The klp61F1, klp61F3 (Heck et al., 1993), klp61F4

(Wilson et al., 1997) and ncd1 mutants (Komma et al., 1991) have

been described. The ncd point mutant described herein as ncd9 wasisolated in a screen to uncouple ncd and ca (O’Tousa and Szauter,1980) and obtained from Scott Hawley. Df(3L)bab PGwas obtainedfrom James Posakony (Godt et al., 1993). Chromosomes bearingmutations in KLP61F and Ncd were constructed by meioticrecombination and standard genetic methods. The genotypes ofklp61F ncd mutant stocks were confirmed by cytological andimmunoblot analyses. Mutant animals were maintained asheterozygotes with TM6B or TM6C balancer chromosomes bearingdominant mutations in Tubby, Humoraland/or Stubble (Lindsley andZimm, 1992).

DNA sequence analysisDNA was extracted from larval brains of klp61F4 ncd1 and ncd9

mutants with DNeasy Tissue Kit (Quiagen). Mutant genes wereamplified with Fail Safe PCR System (Epicentre) and selectedoligonucleotides (Integrated Device Technology). Oligonucleotidesequences and reaction conditions are available upon request.Amplification products were purified from agarose gels withQiaQuick Gel Extraction Kit (Quiagen), sequenced (DNA Corefacility, Georgia State University) and analyzed with MacVectorsoftware (Accelrys). Sequence analysis of independent amplificationproducts confirmed mutational changes. The molecular lesion inklp61F4 is probably the result of aberrant excision of the mutatingtransposon (Wilson et al., 1997). The ncd9 mutant contains a deletionof a single nucleotide in the motor domain. Sequences for klp61F4

and ncd9 have been deposited in GenBank as AY729989 andAY729990, respectively.

AntibodiesAntibodies against Ncd were directed against bacterially expressedfusion protein. The Ncd stalk domain was expressed in bacteria bycloning the 0.6 kbp EcoRI/HindIII fragment of pBSncd (McDonaldand Goldstein, 1990) into pQE32 (Qiagen). Fusion protein expressionwas induced with IPTG as directed by the manufacturer. Two NewEngland white rabbits were immunized and boosted (ResearchAnimal Care Facility, University of Wisconsin-Madison) withimmunogen in slices of 10% SDS-PAGE gels. To affinity purifyantibodies, fusion protein was fractionated on preparative 10% SDS-PAGE gels and transferred to nitrocellulose membranes with standardmethods. Strips of membrane-bound bacterial fusion protein, revealedby Ponceau S (Sigma-Aldrich) staining, were incubated for 30minutes in 10 ml PBS-TT, PBS with 0.2% Triton-X-100 (v/v) and0.2% Tween 20 (v/v), and then with 1:10 dilution of crude antiserumin 10 ml PBS-TT for 2 hours. Antibodies were eluted with 100 mMGlycine HCl (pH 2.3) and combined with an equal volume of 3 MTris-HCl (pH 8.0). Antibodies were stabilized by addition of BSA toapproximately 100 µg/ml, repeatedly dialyzed in a more than 1000×volume of PBS, brought to 50% glycerol (v/v), and frozen in aliquotsat –86°C.

Immunochemical methodsTo perform immunoblot analysis, larval brains and imaginal discswere dissected out of third instar larvae in PBS and macerated in 1%SDS. Protein concentrations were established with BCA ProteinAssay system (Pierce) and 50 µg of each extract was fractionated ina wide lane of a 10% SDS-PAGE gel and transferred to Protran 0.2µm nitrocellulose (Fisher Scientific) by standard methods. Antibodiesagainst Ncd and KLP61F were diluted 1:1000 in TBS-TT (TBS with2% Triton-X 100, 2% Tween 20, pH 7.5) supplemented with 5% drymilk. Membranes were incubated in antibody solution with for 1-2hours and washed three times for 15 minutes with 50 ml TBS-TT.Membranes were then incubated in a solution of anti-rabbit antibodiesconjugated to horseradish peroxidase (Jackson ImmunoResearch

Journal of Cell Science 117 (21)

4923KLP61F Ncd, and the nuclear matrix

Laboratories) diluted 1:2000 in TBS-TT supplemented with 5% drymilk for 1 hour and washed three times for 15 minutes with 50 mlTBS-TT. Signal was detected with SuperSignal West DuraChemiluminescent System (Pierce) as directed by the manufacturer.

To perform immunofluorescence, tissues were spread under acoverslip in PEMS (PIPES 80 mM, EGTA 10 mM, MgCl 10 mM, 100mM sucrose, pH 6.8), placed in a ~50 µl PEMS with 1% (w/v)glycerol. Tissue was gently spread under a silanized coverslip bywicking media from beneath the cover slip with a tissue. Slides werefrozen in liquid nitrogen, the coverslip was flicked off with a razorblade and the slide was placed in 100% methanol cooled in dry icefor 6 minutes. Slides were moved to a postfixative of 4%paraformaldehyde in PBS for 6-12 minutes and washed in severalchanges of PBS-TT at room temperature. Following application ofPBS-TT supplemented with 3% BSA, primary antibodies diluted inPBS-TT with 3% BSA were applied for 1-2 hours. Following severalwashes in PBS-TT, tissues were incubated with conjugated secondaryantibodies for 1-8 hours. Samples were washed in PBS-TT andmounted in Vectashield (Vector) or Prolong (Pierce). Antibodiesagainst Ncd, KLP61F, and γ-tubulin were diluted 1:200 and mousemonoclonal antibodies against lamin (Harold Saumweber), E7antibodies against tubulin (Developmental Studies Hybridoma Bank)and mAb1A1 antibodies against Skeletor (Kristen Johansen) wereused at 1:5-10. When necessary, secondary antibodies were directedagainst the FCγ fragment of mouse IgG or the µ chain of IgM (JacksonImmunoResearch Laboratories). Chromatin was routinely stainedwith a combination of DAPI and SYTO 43 (Molecular Probes).Phenotypic analysis of antibody localization was based on inspectionof more than 100 M-phase cells and a greater number of interphasecells in at least ten larval brains and 20 testes.

Immunofluorescence microscopy and TEMExcept when noted, immunofluorescence images are singleprojections of 3-9 µm stacks of deconvolved sections obtained withDelta Vision Imaging system with a 60× 1.4 oil emersion objective atroom temperature. Wide-field images were obtained with a QuantixCCD and 40× 1.2 dry objective mounted on an IX70 Olympusmicroscope. Digitized images were imported into Photoshop andprocessed for presentation. To perform ultrastructural analysis, larvalbrains of wild-type and mutant animals were dissected in trialdehydefixative, 2% paraformaldehyde, 2% glutaraldehyde, 2% acrolein, 0.13M NaCacodylate, 1 mM CaCl2, 1.5% DMSO, pH 7.2 (Kalt andTandler, 1971) and transferred to fresh trialdehyde fixative containing0.2% tannic acid on ice for 1 hour. Brains were washed three timesfor 30 minutes in 0.1 M Na Cacodylate (pH 7.4) and held in 1% OsO4in 0.1 M sucrose, 0.1 M Na Cacodylate (pH 7.4) for 2 hours at roomtemperature. Following four washes in H2O for 20 minutes, brainswere stained in 2% uranyl acetate for 2 hours, held four times in H2Ofor at least 20 minutes and then dehydrated in a series of 30%, 50%,70% and 95% ethanol. Following two washes in pure ethanol and twowashes in propylene oxide, brains were passed through a graded seriesof propylene oxide and Embed 812/Araldite resin. Brains were finallypassed through two exchanges of pure resin and polymerized at 60°C

for 24 hours in individual silicone molds. EM-grade fixatives, stainsand resin were obtained from Electron Microscopy Sciences. Blockswere mounted, trimmed, and ~70 nm sections were obtained with adiamond knife in an RMC MTX ultramicrotome. Specimens wereexamined with a LEO 906 transmission electron microscope at kV80.Images were captured with a Soft Imaging Systems CCD camera andimported into Photoshop for processing.

ResultsThis study was initiated with a genetic test to determinewhether loss of Ncd suppressed the lethal effects of mutationsin KLP61F (Heck et al., 1993; Wilson et al., 1997). Animalsbearing mutant alleles of both KLP61F and Ncd wereconstructed by meiotic recombination. Alleles of KLP61Fincluded klp61F1, klp61F3 and klp61F4. The hypomorphicklp61F1 and klp61F3 mutants (Wilson et al., 1997) containtransposon insertions in the upstream non-coding region ofKLP61F (Heck et al., 1993) and express a low level of wild-type KLP61F protein (Wilson, 1999). The klp61F4 mutant is asevere loss-of-function allele (Wilson et al., 1997). Alleles ofNcd included the protein null ncd1 allele (Komma et al., 1991)and ncd9 that contains a nucleotide deletion in the motordomain (Materials and Methods). General references here todouble mutants are designated as klp61F ncd mutants forsimplicity.

Mutant klp61F ncd adults were recovered in almost allcombinations tested (Table 1). In most combinations, fullyformed adults often died within pupal cases and viable adultsdied within a few days of hatching, possibly because ofresidual klp61Fmutant effects and/or additive effects of klp61Fand ncd on animal viability. Although viable adults were notrecovered, klp61F4ncd1 mutant animals survived longer asthird instar larvae and showed increased cell proliferation incomparison to klp61F4 mutants, as indicated by the larger sizeof larval brains and imaginal discs. These results indicated thatNcd function limits cell proliferation and contributes tolethality of klp61Fmutants.

Immunoblot analysis of Ncd and KLP61F expressionImproved viability and increased cell proliferation in klp61Fncd mutants suggested that Ncd is expressed in the soma ofwild-type animals. To examine Ncd expression directly,antibodies were directed against the stalk domain of Ncd andused to probe crude extracts of larval brains of wild-typeanimals by immunoblot analysis. Affinity purified antibodiesagainst Ncd recognized 90 kDa and 150 kDa proteins that werenot recognized by pre-immune antibodies (Fig. 1A). The 90kDa protein was designated Ncd since it was close to the size

Table 1. Viability of klp61F ncd double mutantsExpected no. Observed no.

Genotype Heterozygotes* of homozygotes† of homozygotes‡ Observed/expected

klp61F1 ncd9/klp61F4 ncd1 170 85 24 0.28klp61F1 ncd9 201 101 46 0.46klp61F3 ncd1/klp61F1 ncd9 309 155 192 1.24klp61F4 ncd1/Df(3) bab PG ncd1 200 100 0 NA

*Heterozygous progeny bearing a mutant and wild-type copy of KLP61Fandncd.†The expected number of fully viable homozygous mutant adults is ù0.5 of the number of viable heterozygous adults.‡Scored within 48 hours of eclosion from pupal case.

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expected and was not detected in ncd1 or klp61F4 ncd1 mutants(Fig. 1B). These results show that Ncd is expressed in somatictissue as well as in the female germ line.

At the onset of this study, neither the level of proteinexpression nor the mutational change in klp61F4 was known.Membranes here were probed with antibodies against the stalkdomain of KLP61F (Wilson, 1999). The ~130 kDa KLP61Fprotein was detected inncd1 mutant brains, but not in klp61F4

ncd1 mutants (Fig. 1B). DNA sequence analysis of the klp61F4

gene revealed an insertion of four nucleotides near the startcodon that shifts the translational reading frame. Conceptualtranslation of the mutant gene predicted expression of atruncated 6.3 kDa polypeptide of 66 amino acids that includesthe amino terminal 40 amino acids of KLP61F. These dataindicate that klp61F4 is effectively a protein null allele and thatklp61F4 ncd1 mutants lack KLP61F as well as Ncd.

Spindle organization in klp61F ncd mutantsWe next compared spindle organization in wild type, klp61Fand klp61F ncdmutants. Spindles in wild-type animals arebipolar and spindle poles are organized by centrosomes,designated here as biastral spindles (Fig. 2A). Spindles inklp61F mutants are predominantly monopolar (Fig. 2B) ormonastral bipolar structures (Wilson et al., 1997) in which onepole lacks detectable immunostaining of γ-tubulin or shows avery tiny mass (Fig. 2C). The frequency of biastral spindleswas increased in klp61F ncdmutants in comparison with thecorresponding klp61Fmutant. Biastral spindles comprised lessthan 30% (Wilson et al., 1997) and 15% of the spindles inklp61F1 and klp61F3 mutant brains, respectively, but more than96% of >100 spindles scored in klp61F1 ncd9 (n>3) andklp61F3 ncd1 (n>3) mutant brains. Biastral spindles were notobserved in klp61F4 mutants (Wilson et al., 1997), but

comprised more than 66% of >150 spindles klp61F4 ncd1

mutants (n>3). Biastral spindles in klp61F ncd mutantssometimes showed multiple masses of γ-tubulin at spindlepoles and an apparently polyploid complement of chromatin,suggesting that mitosis and/or cytokinesis failed in a previouscell cycle. The frequency of monastral bipolar spindles wasalso decreased in all mutant combinations, but mostdramatically in klp61F1 mutants in which monastral bipolarspindles comprised ~65% of the spindles scored (Wilson et al.,1997). Some spindles in klp61F ncdmutants could not beunambiguously assigned as monopolar or monastral bipolar,showing multiple centrosomal asters and condensedchromosomes, but without recognizable spindle organization.Tabulation of spindle defects in klp61F and klp61F ncdmutants is available online (Table S1, see supplementarymaterial). Collectively, these results indicate that neitherKLP61F nor Ncd are essential for crosslinking and/or bundlingmicrotubules into bipolar spindles. Furthermore, Ncdcontributes to assembly of monastral bipolar spindles as wellas monopolar spindles in klp61Fmutants.

Nuclear defects in the soma of KLP61F-deficientmutantsSpindle pole bodies in yeast move through the nuclearenvelope to side-by-side positions when temperature sensitivebimC mutants are shifted to non-permissive temperatures(Saunders and Hoyt, 1992). To examine the relationshipbetween centrosomes and the nuclear envelope here, larvalbrains of wild-type and mutant animals were stained withantibodies against lamins and γ-tubulin as well as a chromatindye and examined by deconvolution immunofluorescencemicroscopy (Fig. 3). The nuclear envelope in Drosophilabecomes fenestrated at spindle poles (Hiraoka et al., 1990),appearing in larval brains of wild-type animals as a fusiform

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Fig. 1. Immunoblot analysis of Ncd and KLP61F expression.(A) Crude extracts of ovaries of wild-type females probed withantibodies from immune (I) and preimmune (P) antibodies againstNcd. (B) Crude extracts of larval brains of wild type (wt), ncd1 andklp61F4 ncd1 mutants fractionated in three wide lanes (underscores)of a 10% SDS-PAGE gel. Parallel channels of an Immuneticsmanifold were probed with antibodies against Ncd (Ab Ncd) andKLP61F (Ab KLP61F). Positions of Ncd (*) and KLP61F (**)proteins are indicated.

Fig. 2. Spindle organization in klp61Fand klp61F ncd mutants.Larval brains of wild type (A), klp61F3 (B,C) andklp61F3 ncd1 (D)animals were stained with antibodies against α-tubulin (green), γ-tubulin (red) and a fluorescent chromatin dye (blue). (C) Arrowindicates a tiny dot of γ-tubulin at a nearly anastral spindle pole andasterisk indicates chromosomes at opposing pole. Bar, 2 µm.

4925KLP61F Ncd, and the nuclear matrix

structure with diffuse lamin staining extending toward theplasma membrane (Fig. 3A). The nuclear lamina in klp61F3

and klp61F4 mutant cells with monopolar spindles (n>50),inferred by monopolar organization of chromosomes and

centrosomes, showed involutions that extended towardcentrally located centrosomes as well as blebbing and diffusestaining throughout cells (Fig. 3B,C). Similar staining wasdetected in cells (n>20) of klp61F4 ncd1, klp61F3 ncd1 andklp61F3 ncd1/klp61F4 ncd1 mutants with monopolar structures.Invaginations in the nuclear lamina extending toward centrallylocated centrosomes suggested that the nuclear lamina is,directly or indirectly, attached to centrosomal material and/orcentrosomal asters.

Ultrastructural analysis of the nuclear lamina in thesoma of klp61F ncd mutantsUnexpectedly, cells in klp61F ncd mutants with bipolarspindles, inferred by metaphase alignment of chromosomesand bipolar positioning of centrosomes, showed extensiveblebbing and regions of disorganization (Fig. 3D). The nuclearlamina in ncd1 mutants was similar to the nuclear lamina inwild-type animals (data not shown), indicating that the defectscould not be attributed to loss of Ncd. However, diffusestaining of lamins in klp61F ncd mutant animals madeevaluation of nuclear lamina integrity difficult to evaluate byimmunofluorescence. To examine the nuclear lamina at ahigher resolution, larval brains in the wild type and klp61F4

ncd1/klp61F3 ncd1 mutants were examined by transmissionelectron microscopy (Fig. 4). Transheterozygotes klp61F4

ncd1/klp61F3 ncd1 mutant animals were chosen for theseexperiments because mutant effects were representative. Thenuclear lamina in interphase cells of klp61F ncdmutants wasindistinguishable from the nuclear lamina in wild-type animals.Mitotic cells (n=8) in wild-type brains showed a nuclear laminalying between condensed chromatin and mitochondria in thecytoplasm (Fig. 4A). Although 1 of 20 mitotic cells in mutantbrains showed a nuclear lamina similar to the nuclear lamina

in wild-type brains, a nuclear lamina could not bedetected or appeared to be very disorganized (Fig. 4B)in 16 of the remaining mitotic cells inklp61F ncdmutant brains (n=5). The positioning of condensed

Fig. 3.Nuclear lamina organization in klp61F and klp61F ncdmutants. Larval brains of wild type (A) and klp61F3 (B), klp61F3

ncd1/Df(3L)bab PG (C) and klp61F3 ncd1/klp61F4 (D) mutantsstained with antibodies against lamins (grayscale), γ-tubulin (red)and a chromatin dye (blue). Arrows in (B, C) indicate involutions ofthe nuclear lamina that extend toward centrosomes. (D) Cell withbipolar positioning of centrosomes and metaphase alignment ofchromosomes, showing regions of (arrow) disorganization andblebbing of nuclear lamina. Images of γ-tubulin and chromatin aresingle projected images of a stack of deconvolved sections. Imagesof lamin staining are single deconvolved sections chosen to showinvolutions in nuclear lamina. Bar, 5 µm.

Fig. 4.TEM of nuclear lamina defects in klp61F ncdmutants. Somatic cells in larval brains of wild-type (A) andklp61F4 ncd1/klp61F3 ncd1 mutant (B-D) animals withnuclear lamina indicated with red arrows, condensedchromatin indicated by blue arrows and mitochondriaindicated by green arrows. (A) Nuclear lamina in a wild-type cell lies between condensed chromatin andcytoplasmic mitochondria, shown in the insert at 2×magnification. (B) Somatic cell in klp61F4 ncd1/klp61F3

ncd1 larval brain lacks detectable nuclear lamina lyingbetween mitochondrion and condensed chromatin. A regionbetween a mitochondrion and chromatin is shown in theinset at 2× magnification. (C) Nuclear lamina surroundingseparate masses of condensed chromatin. (D) Region lyingbetween marked lamina is shown at 3× magnification.Fenestration of the nuclear lamina in prometaphase ormetaphase cells was inferred from uniform stainingthroughout mitotic cells whereas interphase cells showedlighter staining of nucleoplasm in comparison to darkerstaining of the cytoplasm and by position of condensedchromatin near the cell center rather than the nuclearperiphery. Interphase cells were more than tenfold morefrequent than cells in mitosis. Bars, 2 µm.

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chromatin in these 17 cells suggested the presence of a bipolarspindle. The three remaining mitotic cells showed a nuclearlamina lying between three masses of chromatin (Fig. 4C,D)that probably corresponds to the deep involutions in the nuclearlamina detected by immunofluorescence (Fig. 3B,C). Oneexplanation for the disorganized state of the nuclear lamina inklp61F and klp61F ncdmutants is that cells are delayed inmitosis, allowing continued depolymerization of nuclearlamins. This view is consistent with the disorganizedappearance of the nuclear lamina in clone 8 cells that weredelayed in mitosis by treatment with the APC inhibitor MG132(Fig. S1, see supplementary material). Another explanation,that is not mutually exclusive, is that KLP61F function or thecombined functions of KLP61F and Ncd are required, directlyor indirectly, to maintain normal organization of the nuclearlamina during mitosis.

KLP61F localization and function in male meiosisThe results thus far could not determine whether involutions inthe nuclear lamina in mutant cells with monopolar spindleswere secondary to microtubule dependent repositioning ofcentrosomes or whether involutions reflected collapse of a non-microtubule spindle matrix that was connected in some way tocentrosomes. Since spermatocytes in klp61F mutant testes canhave too many centrosomes or none at all (Wilson et al., 1997),we were interested in whether nuclear matrix defects wereevident in klp61Fmutant spermatocytes lacking centrosomes.

We first examined KLP61F localization in wild-type testeswith confocal microscopy. Immunostaining of KLP61F inwild-type primary spermatocytes in G2 showed diffusecytoplasmic distribution that was similar to immunostaining ofsomatic cells in interphase (Wilson, 1999). Although KLP61Flocalizes to centrosomal asters and spindles in somatic cells ofwild-type animals (Wilson, 1999) and cultured clone 8 cells(Fig. S1, see supplementary material), KLP61F remaineddiffusely distributed and did not show detectable localizationto centrosomal asters at the G2/M transition (Fig. 5A) or tomeiotic spindles at metaphase (Fig. 5B). KLP61F showedseptin-like localization in midbodies in late telophase ofmeiosis (Fig. 5C,D) that was similar to its localization duringtelophase of mitosis in germ cells (Wilson, 1999). Male germcells within a cyst proceed through spermatogenesis in nearsynchrony (Fuller, 1993) and an exceptional cyst caught in thetransition from metaphase to anaphase (Fig. 5E) revealed thetransition in KLP61F localization; KLP61F showed slightenrichment in the polar regions of spermatocytes at metaphase(Fig. 5F). KLP61F repositioned to the interpolar region ofspindles near anaphase (Fig. 5G) whereas spermatocytes in lateanaphase and early telophase showed KLP61F nearly removedfrom the cellular pool and localized in a plane that bisected theentire spermatocyte (Fig. 5H). We found similar localization(data not shown) with antibodies against the phosphorylatedBimC Box of Eg5 (Sharp et al., 1999a) and with antibodiesagainst KLP61F tagged with the myc epitope (Barton et al.,1995). Taken together, these results show the germ-cell-

specific localization of KLP61F to cleavagefurrows and forming ring canals neartelophase. However, in contrast to mitosis insomatic cells and germ cells, meioticspindles assemble in wild-typespermatocytes without detectable spindleenrichment of KLP61F.

Failure to detect enriched centrosomaland spindle localization of KLP61Fsuggested either that KLP61F function isnot required for male meiosis or thatenriched spindle localization is not requiredfor KLP61F function. To address thisquestion, we examined male meiosis inlarval testes of klp61F3 homozygotes,klp61F3/klp61F1 transheterozygotes andklp61F3/Df(3L) bab PG animals thatexpress one copy of klp61F3. Testes are notgenerated in klp61F4 mutants, precludinganalysis here of spermatocytes lackingKLP61F entirely. Immunofluorescenceanalysis indicated that centrosomesseparated the G2/M transition in thoseklp61F mutant spermatocytes with twoor more centrosomes (Fig. 6A).Acentrosomal meiotic spermatocytesshowed microtubules, but did not showmicrotubule asters or detectable spindlestructures (Fig. 6B). Althoughspermatocytes containing centrosomesshowed a range of spindle defects, classicmonopolar spindles (Fig. 6C) comprisedless than 10% of spindle structures,

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Fig. 5. Atypical localization of KLP61F in spermatocytes. Wild-type testes stained withantibodies against KLP61F (red), a fluorescent chromatin dye (blue) and α-tubulin (green,A-C) and γ-tubulin (lavender, D). (A-D) Single projection of a stack of optical sections.Spermatocytes at the G2/M transition failed to show KLP61F enrichment at centrosomes(A) or spindle localization at metaphase (B). Spermatocytes are shown at telophase ofmeiosis II (C) and meiosis I (D) with septin-like localization of KLP61F. (E) Lowmagnification wide-field image of a cyst. Asterisks indicate spermatocytes at highermagnification in metaphase (F), anaphase (G) and late anaphase/telophase (H). Bar (A-D),2 µm; (E), 10 µm; (F-H), 5 µm.

4927KLP61F Ncd, and the nuclear matrix

substantially lower than the 50-75% frequencyfound in somatic cells of these mutants (Wilsonet al., 1997). The lower frequency of monopolarspindles in mutant spermatocytes most likelyreflects progression through meiosis inspermatocytes with spindle defects (Kemphues etal., 1980; Rebollo and Gonzalez, 2000; White-Cooper et al., 1993) whereas somatic cells withmonopolar spindles are severely delayed inmitosis (Gatti and Baker, 1989). Most meioticspindles had characteristics of both monopolarand monastral bipolar spindles, showingmicrotubules splayed into broad spindle polesthat lacked detectable γ-tubulin or showingsplintered spindle poles with foci of γ-tubulin atthe ends of microtubule bundles (Fig. 6C,D).Bivalents were closely apposed to spindle poles,positioned symmetrically or asymmetricallywithin microtubule bundles. Despite theabnormal organization of microtubules,spermatocytes in telophase showed a cytokineticfurrow that divided the spermatocyte unequally(Fig. 6E), with one daughter receiving most or allof the bivalents and centrosomes. Telophase wasalso abnormal in that bivalents typically remainedpaired, as indicated by the presence of ~3-4chromatin masses in spermatocytes at telophasethat were similar in size to the chromatin massesnear metaphase. Nonetheless, these data indicatethat, despite its dispersed distribution, loss ofKLP61F function in spermatocytes produces the same range ofspindle defects that are found in somatic cells.

Nuclear matrix defects in spermatocytes with andwithout centrosomesConfocal microscopy was used to evaluate the relationshipbetween centrosomes and the nuclear lamina in klp61Fmutantspermatocytes. Similar to wild-type spermatocytes (Fig. 7A),centrosomes separated at the G2/M transition in klp61F3 and

klp61F3/klp61F4 mutant spermatocytes with an intact nuclearenvelope (Fig. 7D). Fusiform structures, indicative of a bipolarspindle in wild-type spermatocytes (Fig. 7B), were not detectedin klp61F mutant spermatocytes. Rather, the nuclear laminashowed deep involutions that were occupied by closelyapposed centrosomes (Fig. 7E). Remarkably, acentrosomalspermatocytes in the same cyst showed similar involutions(Fig. 7E). The nuclear lamina appeared to collapse aroundbivalents in spermatocytes and form micronuclei, irrespectiveof the presence or absence of centrosomes (Fig. 7E). Several

Fig. 6. Spindle organization in klp61Fmutant spermatocytes. Testes of klp61F3

mutants stained with antibodies against α-tubulin (green), γ-tubulin (red) and afluorescent chromatin dye (blue). Arrows in (B) indicate an acentrosomalspermatocyte, (E) bivalent at a metaphase position in microtubule bundle, and (F) acleavage furrow forming an asymmetric telophase spindle. Images arerepresentative spindle organization in klp61F3/klp61F3, klp61F3/klp61F1

transheterozygotes and klp61F3/Df(3L)bab PGmutants. Bar, 5 µm.

Fig. 7. Nuclear lamina organizationin klp61F mutant spermatocytes.Testes from wild type (A-C) andklp61F3 mutants (D-F) stained withantibodies against γ-tubulin (red),lamin (green) and a fluorescentchromatin dye (blue). Images aresingle projections of a stack ofoptical sections. (A) Antibodiesagainst γ-tubulin crossreact with amitochondrial antigen(s) in testes,providing a useful marker formitochondrial derivatives and theprogression of spermatocytedevelopment. (E,F) Arrows indicateacentrosomal spermatocytes. (E) Toshow the apposition of centrosomesand chromatin relative to invaginations of nuclear lamina, the lasso tool of Photoshop was used to replace the projected stack of lamin stainingwith lamin staining in a single optical section in the spermatocyte outlined in dots. Bar, 5 µm.

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inferences can be drawn from these observations. Oneinference is that formation of involutions and collapse of thenuclear lamina in klp61F mutant spermatocytes are probablynot caused by microtubule-dependent repositioning ofcentrosomes. Another inference is that failure of most homologpairs to disjoin in klp61F mutant spermatocytes probablyreflects collapse of the nuclear lamina around bivalents. A finalinference is that KLP61F function is required, directly orindirectly, to prevent collapse of the nuclear matrix during M-phase.

Nuclear lamina defects at poles of bipolar spindles inklp61F ncd spermatocytesWe next asked whether loss of Ncd function both restoredspindle bipolarity and prevented formation of micronuclei inklp61F ncdmutant spermatocytes. For these experiments, weexamined homozygous klp61F3 ncd1 mutants and mutantshomozygous for ncd1, but transheterozygous forklp61F3,klp61F4 or the deficiencyDf(3L)bab PGin which the wild-typeKLP61F gene has been deleted. Immunostaining of α- and γ-

tubulin in mutant spermatocytes indicated that spindles atmetaphase of both meiotic divisions were typically bipolarwith a single centrosome at each pole (Fig. 8A). Homolog pairsand sister chromatids disjoined and segregated to opposingspindle poles at anaphase of meiosis I and meiosis II,respectively (Fig. 8B). However, many spermatocytes (~50%)showed several small nuclei at the poles of telophase spindles(n>50) and some post-meiotic spermatids also containedmicronuclei (Fig. 8B,C). These observations suggested thepresence of residual nuclear matrix defects in klp61F ncdmutants, despite assembly of bipolar spindles.

Immunostaining of lamins in klp61F ncd mutant testesshowed that virtually all primary spermatocytes contained asingle nucleus, indicating that formation of micronuclei did notreflect progression through meiosis with multiple nuclei.Although a single nuclear lamina was present in spermatocytesat metaphase and anaphase of meiosis I and meiosis II (Fig.8D), 50-75% of spermatocytes at telophase of meiosis I andmeiosis II (n>50) showed a nuclear lamina about individualhomolog pairs or chromosomes clustered at centrosomes (Fig.8E). Cysts of post-meiotic spermatids showed multiple nuclei

(Fig. 8F), but the frequency was typically less than10%, suggesting that fusion of individual nuclearenvelopes in mutant spermatocytes is delayed orblocked. The nuclear laminas in the wild type(Fig. 8G) and ncd1 mutant spermatocytes wereindistinguishable (Fig. 8H,I), indicating that thenuclear lamina defects in klp61F ncdmutants areprobably not a result of loss of Ncd alone. Giventhe apparent collapse of the nuclear laminaaround bivalents in klp61F mutants, the simplestexplanation for these results is that micronuclei inklp61F ncdmutant spermatocytes reflects residualdefects caused by loss of KLP61F function.

Ncd localization in wild-type and KLP61F-deficient spermatocytesNcd-mediated suppression of klp61F mutantsraised the question of Ncd function in spindle andnuclear matrix organization in spermatocytes. Incontrast to embryos (Hatsumi and Endow, 1992;Sharp et al., 2000a; Sharp et al., 1999b), Ncd wasdistributed throughout the interphase nucleus andenriched near heterochromatin attached to thenuclear lamina in wild-type spermatocytes (Fig.9A). Similar localization was detected in somaticcells of larval brains and in cultured clone 8 cells(Fig. S2, see supplementary material). Near theG2/M transition, Ncd showed diffuse distributionthroughout the nucleus (Fig. 9B), as if Ncd isreleased from chromatin-associated material asbivalents become condensed. Spermatocytes atmetaphase of both meiotic divisions showedimmunostaining of Ncd near centrosomes, butimmunostaining in spindles was weak (Fig. 9C) orundetectable. Ncd localized to midbodies neartelophase as it does in somatic cells (Fig. 9D).Immunostaining of nuclei, centrosomes andspindle fibers are caused by Ncd and not the 150kDa protein detected by immunoblot analysis

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Fig. 8.Micronuclei in klp61F ncd spermatocytes at telophase. Testes in klp61F ncdmutants (A-F), wild-type animals (G) and ncd1 mutants (H,I) were stained withantibodies against γ-tubulin (green) and a fluorescent chromatin dye (blue) and α-tubulin (green, A-C) or lamins (green, D-I). (A,D) Multiple nuclei were notdetected in metaphase of meiosis, but were detected at the poles of telophasespindles (B, arrows) and in spermatids (C,F arrows). Multiple nuclei were notdetected in wild-type (G) or in ncd1 (H) spermatocytes in telophase of meiosis or inpost-meiotic spermatocytes (I). Bar, 2 µm.

4929KLP61F Ncd, and the nuclear matrix

(Fig. 1) as staining was not detected in spermatocytes (Fig. 9E)or in somatic cells (Fig. S2, see supplementary material) ofncd1 mutants. In contrast to spermatocytes, immunostaining ofNcd in larval brains and cultured clone 8 cells showedimmunostaining of pole-to-pole fibers in mitotic cells (Fig. S2,see supplementary material) that were similar to the pole-to-pole fibers observed in embryos (Endow and Komma, 1996b).These observations show that Ncd localization in somatic cellsand spermatocytes is similar during interphase and telophase,but the precise pattern in prometaphase and metaphasevaries. Ncd localization in primary spermatocytes and inspermatocytes near the G2/M transition in klp61F3 and otherklp61F mutants was similar to localization in wild-typespermatocytes. However, Ncd was diffusely distributed inmeiotic spermatocytes (Fig. 9F) and immunostaining ofspindles, centrosomes or micronuclei was not detected. Takentogether, these observations indicate that Ncd is a componentof the nuclear matrix and is mislocalized in klp61F mutantspermatocytes. One implication of these findings is thatKLP61F is required for disposition of nuclear matrixconstituents during M-phase.

Skeletor localization in klp61F mutant spermatocytesImmunostaining of Skeletor provided another marker forthe nuclear matrix in klp61F and klp61F ncd mutantspermatocytes. In contrast to the rapid mitotic cycles in earlyembryos (Walker et al., 2000), Skeletor was below the levelof detection in primary spermatocytes until chromatincondensation was detectable near the G2/M transition (Fig.10A). Skeletor showed similar mitosis-specific expression inproliferating germ cells and in cultured clone 8 cells (data not

shown), suggesting that Skeletor expression is upregulated nearthe G2/M transition in somatic cells and spermatocytes, as is

Fig. 9.Ncd localization in klp61Fand ncdmutantspermatocytes. Spermatocytes in wild type (A-D),ncd1 (E) and klp61F3/klp61F1 (F) mutants stainedwith a blue fluorescent chromatin dye and antibodiesagainst Ncd (red), γ-tubulin (green, A,C,E), α-tubulin(green, B,D) and lamin (green, F). Arrows in (A)indicate enriched Ncd near heterochromatin inprimary spermatocyte. (C) Arrow indicatesoverexposure of nuclear staining of Ncd in primaryspermatocyte for comparison with spindle stainingmarked by asterisks. (F) Arrow indicates laminaaround bivalent in spermatocyte near prometaphase.Bar, 5 µm.

Fig. 10.Skeletor localization in ncd, klp61F and klp61F ncdmutant.Testes of the wild type (A-C), ncd1 (E), klp61F3 (F), klp61F3

ncd1/Df(3L)bab PG ncd1 (klp61F3/Df ncd1) (G) and klp61F3

ncd1/klp61F4 ncd1 (klp61F3/4 ncd1) (D) mutants stained withantibodies against Skeletor (green), γ-tubulin (red) and a chromatindye (blue). (A) Arrows indicate spermatocytes near the G2/Mtransition with low levels of Skeletor. (C) Arrow show enrichedSkeletor localization near chromatin and (F) aggregates of Skeletorin klp61F3 spermatocytes near prometaphase. Bar, 5 µm.

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the nucleolar protein NuSAP (Raemaekers et al., 2003).Skeletor was diffusely distributed throughout the nucleus priorto prometaphase, but fusiform structures were not observeduntil metaphase (Fig. 10B,D). Skeletor usually appeared tobe enriched near segregating chromatin during anaphaseand chromatin-associated during telophase (Fig. 10C).Immunostaining of Skeletor in klp61F3 mutant spermatocytesresembled immunostaining in wild-type spermatocytes nearthe G2/M transition (Fig. 10G). However, meioticspermatocytes showed immunostaining of aggregates (Fig.10F) that were typically associated with chromatin withinmicronuclei (Fig. S3, see supplementary material). In contrastto klp61F mutant spermatocytes, Skeletor localization inklp61F3 ncd1 and ncd1 mutant spermatocytes was similar tothat in wild-type spermatocytes (Fig. 10E,G,H). The simplestinterpretation of these results is that neither KLP61F nor Ncdare essential for Skeletor localization and that Skeletorcollapses with the nuclear matrix in KLP61F-deficient mutants.

DiscussionHere we present cytological evidence that loss of KLP61Ffunction generates spindle defects as well as novel defects inorganization of the nuclear matrix during M-phase in somaticcells and spermatocytes. Our results also show that Ncd isnuclear during interphase and spindle-associated in M-phase inthe soma and male germ line. Loss of Ncd function increasesthe frequency of biastral spindles in klp61F mutants, but failsor incompletely restores nuclear matrix defects. These findingsraise new questions about the molecular basis of geneticinteractions between KLP61F and Ncd.

Novel nuclear defects in klp61F and klp61F ncd mutantsSomatic cells in klp61F and klp61F ncd mutants withmonopolar spindles showed deep invaginations in the nuclearlamina that extended toward centrally located centrosomes(Fig. 3B,C). Similar involutions were found in klp61F mutantspermatocytes judged to be near prometaphase (Fig. 7E),irrespective of the presence or absence of centrosomes (Fig.7E). These observations suggest that the driving force informing invaginations in the nuclear lamina is associatedwith nuclear and/or cytoplasmic material rather than withcentrosomes or centrosome organized microtubules. Acontribution of nuclear forces to repositioning of centrosomeshas precedence in yeast; spindle-pole bodies in preassembledspindles move through the nuclear envelope to side-by-sidepositions when temperature-dependent BimC function isinactivated at non-permissive temperatures (Saunders andHoyt, 1992). Because spindle pole bodies assume face-to-face positions when microtubules are depolymerized (Jacobset al., 1988), side-by-side positions suggests that nuclearforces contribute to spindle defects in BimC-deficient yeastas well.

Nuclear defects in somatic cells differed from those inspermatocytes, raising the question of whether KLP61Ffunction in somatic cells and spermatocytes is mediated by acommon mechanism or two different mechanisms. Argumentscan be made for and against a common mechanism. Thestrongest argument for a common mechanism is the strikingsimilarity of spindle defects in somatic cells (Wilson et al.,

1997) and spermatocytes (Fig. 6). Another argument is theability of ncdmutants to suppress the klp61Fmutant phenotypein both cell types (Fig. 2, Fig. 8). At first glance, other aspectsof the mutant phenotype are not consistent with a commonfunction. Somatic cells in KLP61F-deficient animals showedextensive disorganization of the nuclear lamina (Fig. 3),including cells showing bipolar positioning of centrosomes andmetaphase alignment of chromosomes. In contrast to somaticcells, the nuclear lamina appeared to collapse around bivalentsnear prometaphase and form micronuclei in klp61F mutantspermatocytes (Fig. 7). These differences could reflectdifferent functions in somatic cells and spermatocytes.Alternatively, the difference may reflect cell cycle regulation;the spindle assembly checkpoint is active in somatic cells(Gatti and Baker, 1989), but inactive (Kemphues et al., 1982)or severely abrogated in spermatocytes (Rebollo and Gonzalez,2000). This view is consistent with the disorganized state ofthe nuclear lamina in cultured clone 8 cells that were delayedin mitosis with an inhibitor of APC (Fig. S2, see supplementarymaterial). Thus, disorganization of the nuclear lamina insomatic cells and formation of micronuclei in spermatocytes inKLP61F-deficient mutants could reflect a common underlyingdefect in different cell types.

KLP61F shows overlapping, but differential localizationduring mitosis and male meiosis. In somatic cells, KLP61F ishighly enriched near centrosomal asters during prophase,spindle-associated in metaphase and located in midbodies intelophase (Wilson, 1999). In meiotic spermatocytes, KLP61Ffails to show centrosomal enrichment or spindle association,but in late anaphase/early telophase KLP61F localizes to asphere that bisects the entire spermatocyte and then follows theingressing cleavage furrow (Fig. 5). Similar localization inproliferating germ cells in telophase was found to reflectinteractions, directly or indirectly, with components of fusomes(Wilson, 1999). We cannot draw firm conclusions from thefailure to detect KLP61F localization to centrosomal asters orto spindles as a small pool could escape our detection methods.However, given static positioning of Eg5 in spindles assembledin Xenopusegg extracts (Kapoor and Mitchison, 2001), thefailure to detect KLP61F localization to male meiotic spindlesmay indicate that KLP61F is not associated with spindlemicrotubules, but with non-microtubule binding partners. Inmost cell types, BimC kinesins are diffusely distributedthroughout the cytoplasm during interphase. Localization ofBimC kinesins to spindles in vertebrate cells has been linkedto phosphorylation of a Cdk1 target site in the conserved BimCBox near the carboxyl tail region of these kinesins (Blangy etal., 1995; Sawin and Mitchison, 1995; Sharp et al., 1999a),postulated to elicit or strengthen intrinsic microtubule bindingactivity and spindle localization. However, phosphorylation ofthe BimC Box of Cut7 in Saccharomyces pombeis not requiredfor spindle association or for Cut7 function in assembly of abipolar spindle (Drummond and Hagan, 1998). It is possiblethat KLP61F and other BimC kinesins crosslink microtubulesand non-microtubule binding partners during interphase. BimCBox phosphorylation may downregulate microtubule bindingactivity and allow interactions with non-microtubule bindingpartners to direct KLP61F localization during M-phase.Identification of non-microtubule binding partners and geneticanalysis of BimC Box function in KLP61F localization couldtest these possibilities.

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4931KLP61F Ncd, and the nuclear matrix

Ncd and the nuclear matrixSimilar to Kar3 kinesins in vertebrates (Kuriyama et al., 1995;Mountain et al., 1999; Walczak et al., 1996), Ncd is nuclearduring interphase in spermatocytes (Fig. 9A) and in somaticcells (Fig. S1A, see supplementary material) as well as insomatically derived clone 8 cells (Fig. S1C, see supplementarymaterial). It is not clear why Ncd fails to show nuclearlocalization in early embryos, but the rapidity of the cell cyclein early embryos may preclude complete reorganization of thenuclear envelope and nuclear entry of Ncd through nuclearpores. Two lines of cytological evidence suggest that Ncd maybe associated with the nuclear matrix during interphase insomatic and male germ line cells. First, Ncd shows subnuclearenrichment near heterochromatin attached to the nuclearenvelope (Fig. 9 and Fig. S1, see supplementary material).Given that fibrillar components of the nuclear matrix connectchromatin to the inner nuclear membrane (Nickerson, 2001),subnuclear enrichment could reflect localization of Ncd tofibrillar components of the nuclear matrix and/or localizationto chromatin-associated material at these sites. Second, Ncdlocalizes to fibers extending between the poles of metaphasespindles in somatic cells and cultured clone 8 cells (Fig. S2,see supplementary material), reminiscent of Ncd localizationin embryos (Endow and Komma, 1997). The functionalsignificance of these fibers is not clear since pole to pole fibershave not been reported in female meiotic spindles (Endow andKomma, 1997; Endow and Komma, 1998; Hatsumi andEndow, 1992) and we did not detect strong immunostaining ofsimilar fibers in meiotic spermatocytes (Fig. 9). However, thereis a precedence for localization of other nuclear matrix proteinsto spindle fibers. The nuclear matrix protein NuSAP islocalized to spindle-associated fibers in cultured vertebratecells and loss of its function generates defects in spindleorganization and chromosome segregation (Raemaekers et al.,2003). The nuclear matrix protein Skeletor also localizes tofibers in embryonic spindles although the fibers do not extendthe full distance between spindle poles (Walker et al., 2000).Loss of Ncd function did not appreciably alter Skeletordistribution in spermatocytes (Fig. 10), indicating that Ncd isnot necessary or plays only a very limited role in Skeletorlocalization. Nonetheless, our findings are consistent with theview that Ncd is a component of the nuclear matrix in somaticcells and spermatocytes.

Cooperation between KLP61F and Ncd in assembly ofbipolar spindlesAt this point, we can only speculate on the relationshipbetween spindle and nuclear defects in klp61Fmutants and thefunctional significance of Ncd-mediated suppression. With fewexceptions, cooperation between BimC and Kar3 kinesins inspindle assembly has been ascribed to application ofantagonistic motive forces to spindle microtubules to establishor maintain centrosome separation (Kashina et al., 1997; Sharpet al., 2000b). According to this view, nuclear defects inKLP61F-deficient animals could be secondary to primarydefects in spindle organization; increasing the frequency ofbipolar spindles in klp61F ncdmutants results in a decreasedfrequency of nuclear defects. However, this explanation doesnot easily explain formation of micronuclei at the poles ofbipolar spindles in klp61F ncd spermatocytes (Fig. 8).

Moreover, collapse of the nuclear lamina about bivalentscannot be ascribed to spindle defects since similar defects arenot found in meiotic spermatocytes of β2tn mutants (P.G.W.,unpublished) that lack microtubules and spindle structuresowing to loss of an essential testis specific β-tubulin(Kemphues et al., 1982; Kemphues et al., 1983; Kemphues etal., 1980). An alternative interpretation of our findings is thatspindle defects are secondary; spindle defects reflect collapseof a nonmicrotubule spindle matrix that is derived from thenuclear matrix and attached to centrosomes and/or spindlemicrotubules. According to this view, interactions betweenKLP61F and nonmicrotubule binding partners prevent collapseof a compressible spindle matrix when nuclear and cytoplasmiccontents mix at prometaphase, whether or not KLP61F isspindle associated.

Our results are in part unexpected because they question theassumed relationship between localization and function of amicrotubule-dependent motor protein. KLP61F is required forspindle bipolarity, but its function in male meiosis does notrequire spindle association. Conversely, KLP61F localizes tocleavage furrows, but it is not required for cytokinesis. Withthese contradictions in mind, further work must address themechanism of KLP61F function in spindle organization andthe functional significance of nuclear localization of Ncd.

We are grateful for discussion and support from Gary Borisy andtechnical assistance from John Peloquin during early stages of thiswork. This work was supported by Scientist Development Grant(9930257N PGW) from the American Heart Association-National andFaculty Support Program Award (FY04-RS-3 PGW) from GeorgiaState University.

ReferencesBarton, N. R., Pereira, A. J. and Goldstein, L. S. (1995). Motor activity and

mitotic spindle localization of the Drosophila kinesin-like protein KLP61F.Mol. Biol. Cell 6, 1563-1574.

Bayliss, R., Sardon, T., Vernos, I. and Conti, E. (2003). Structural basisof Aurora-A activation by TPX2 at the mitotic spindle. Mol. Cell 12, 851-862.

Becker, B. E., Romney, S. J. and Gard, D. L. (2003). XMAP215, XKCM1,NuMA, and cytoplasmic dynein are required for the assembly andorganization of the transient microtubule array during the maturation ofXenopus oocytes. Dev. Biol.261, 488-505.

Blangy, A., Lane, H. A., d’Herin, P., Harper, M., Kress, M. and Nigg, E.A. (1995). Phosphorylation by p34cdc2 regulates spindle association ofhuman Eg5, a kinesin-related motor essential for bipolar spindle formationin vivo. Cell 83, 1159-1169.

Bloom, K. (2002). Yeast weighs in on the elusive spindle matrix: new filamentsin the nucleus. Dev. Biol.99, 4757-4759.

Cole, D. G., Saxton, W. M., Sheehan, K. B. and Scholey, J. M. (1994). A“slow” homotetrameric kinesin-related motor protein purified fromDrosophila embryos. J. Biol. Chem. 269, 22913-22916.

Compton, D. A. and Cleveland, D. W. (1994). NuMA, a nuclear proteininvolved in mitosis and nuclear reformation. Curr. Opin. Cell Biol.6, 343-346.

Cottingham, F. R. and Hoyt, M. A. (1997). Mitotic spindle positioning inSaccharomyces cerevisiae is accomplished by antagonistically actingmicrotubule motor proteins. J. Cell Biol.138, 1041-1053.

Dionne, M. A., Howard, L. and Compton, D. A. (1999). NuMA is acomponent of an insoluble matrix at mitotic spindle poles. Cell Motil.Cytoskeleton42, 189-203.

Drummond, D. R. and Hagan, I. M. (1998). Mutations in the bimC box ofCut7 indicate divergence of regulation within the bimC family of kinesinrelated proteins. J. Cell Sci. 111, 853-865.

Du, Q., Taylor, L., Compton, D. A. and Macara, I. G. (2002). LGN blocksthe ability of NuMA to bind and stabilize microtubules. A mechanism formitotic spindle assembly regulation. Curr. Biol. 12, 1928-1933.

4932

Endow, S. A. and Komma, D. J. (1996). Centrosome and spindle function ofthe Drosophila Ncd microtubule motor visualized in live embryos usingNcd-GFP fusion proteins. J. Cell Sci.109, 2429-2442.

Endow, S. A. and Komma, D. J.(1997). Spindle dynamics during meiosis inDrosophila oocytes.J. Cell Biol.137, 1321-1336.

Endow, S. A. and Komma, D. J. (1998). Assembly and dynamics of ananastral:astral spindle: the meiosis II spindle of Drosophila oocytes. J. CellSci. 111, 2487-2495.

Eyers, P. A. and Maller, J. L. (2004). Regulation of Xenopus Aurora Aactivation by TPX2. J. Biol. Chem. 279, 9008-9015.

Fawcett, D. W. (1966). An Atlas of Fine Structure: The Cell, Its Organelles,and Inclusions. 2nd edn. Philadelphia, PA: W. B. Saunders.

Fuller, M. T. (1993). Spermatogenesis In The Development of Drosophilamelanogaster(ed. M. Bate and A. M. Arias), pp. 71-147. Cold SpringHarbor, NY: Cold Spring Harbor Laboratory Press.

Gaglio, T., Saredi, A., Bingham, J. B., Hasbani, M. J., Gill, S. R., Schroer,T. A. and Compton, D. A. (1996). Opposing motor activities are requiredfor the organization of the mammalian mitotic spindle pole. J. Cell Biol.135, 399-414.

Garrett, S., Auer, K., Compton, D. A. and Kapoor, T. M. (2002). hTPX2 isrequired for normal spindle morphology and centrosome integrity duringvertebrate cell division. Curr. Biol. 12, 2055-2059.

Gatti, M. and Baker, B. S. (1989). Genes controlling essential cell-cyclefunctions in Drosophila melanogaster. Genes Dev.3, 438-453.

Godt, D., Couderc, J. L., Cramton, S. E. and Laski, F. A. (1993). Patternformation in the limbs of Drosophila: bric a brac is expressed in both agradient and a wave-like pattern and is required for specification and propersegmentation of the tarsus. Development119, 799-812.

Goshima, G. and Vale, R. D. (2003). The roles of microtubule-based motorproteins in mitosis: comprehensive RNAi analysis in the Drosophila S2 cellline. J. Cell Biol.162, 1003-1016.

Gruss, O. J., Wittmann, M., Yokoyama, H., Pepperkok, R., Kufer, T.,Sillje, H., Karsenti, E., Mattaj, I. W. and Vernos, I. (2002). Chromosome-induced microtubule assembly mediated by TPX2 is required for spindleformation in HeLa cells. Nat. Cell Biol.4, 871-879.

Hatsumi, M. and Endow, S. A. (1992). The Drosophila ncd microtubulemotor protein is spindle-associated in meiotic and mitotic cells. J. Cell Sci.103, 1013-1020.

Heck, M. M., Pereira, A., Pesavento, P., Yannoni, Y., Spradling, A. C. andGoldstein, L. S. (1993). The kinesin-like protein KLP61F is essential formitosis in Drosophila. J. Cell Biol.123, 665-679.

Hildebrandt, E. R. and Hoyt, M. A. (2000). Mitotic motors inSaccharomyces cerevisiae. Biochim. Biophys. Acta1496, 99-116.

Hiraoka, Y., Agard, D. A. and Sedat, J. W. (1990). Temporal and spatialcoordination of chromosome movement, spindle formation, and nuclearenvelope breakdown during prometaphase in Drosophila melanogasterembryos. J. Cell Biol.111, 2815-2828.

Holaska, J. M., Wilson, K. L. and Mansharamani, M. (2002). The nuclearenvelope, lamins and nuclear assembly. Curr. Opin. Cell Biol.14, 357-364.

Jacobs, C. W., Adams, A. E., Szaniszlo, P. J. and Pringle, J. R. (1988).Functions of microtubules in the Saccharomyces cerevisiae cell cycle. J. CellBiol. 107, 1409-1426.

Kalt, M. R. and Tandler, B. (1971). A study of fixation of earlyamphibian embryos for electron microscopy. J. Ultrastruct. Res.36, 633-645.

Kapoor, T. M. and Mitchison, T. J. (2001). Eg5 is static in bipolar spindlesrelative to tubulin: evidence for a static spindle matrix. J. Cell Biol. 154,1125-1133.

Kashina, A. S., Baskin, R. J., Cole, D. G., Wedaman, K. P., Saxton, W. M.and Scholey, J. M. (1996). A bipolar kinesin. Nature379, 270-272.

Kashina, A. S., Rogers, G. C. and Scholey, J. M. (1997). The bimC familyof kinesins: essential bipolar mitotic motors driving centrosome separation.Biochim. Biophys. Acta1357, 257-271.

Kemphues, K. J., Raff, E. C., Raff, R. A. and Kaufman, T. C. (1980).Mutation in a testis-specific beta-tubulin in Drosophila: analysis of its effectson meiosis and map location of the gene. Cell 21, 445-451.

Kemphues, K. J., Kaufman, T. C., Raff, R. A. and Raff, E. C. (1982). Thetestis-specific beta-tubulin subunit in Drosophila melanogaster has multiplefunctions in spermatogenesis. Cell 31, 655-670.

Kemphues, K. J., Raff, E. C. and Kaufman, T. C. (1983). Genetic analysisof B2t, the structural gene for a testis-specific beta- tubulin subunit inDrosophila melanogaster. Genetics105, 345-356.

Khodjakov, A., Copenagle, L., Gordon, M. B., Compton, D. A. and

Kapoor, T. M. (2003). Minus-end capture of preformed kinetochore fiberscontributes to spindle morphogenesis. J. Cell Biol.160, 671-683.

Kimble, M. and Church, K. (1983). Meiosis and early cleavage in Drosophilamelanogaster eggs: effects of the claret-non-disjunctional mutation. J. CellSci. 62, 301-318.

Komma, D. J., Horne, A. S. and Endow, S. A. (1991). Separation of meioticand mitotic effects of claret non-disjunctional on chromosome segregationin Drosophila. EMBO J.10, 419-424.

Kuriyama, R., Kofron, M., Essner, R., Kato, T., Dragas-Granoic, S.,Omoto, C. K. and Khodjakov, A. (1995). Characterization of a minus end-directed kinesin-like motor protein from cultured mammalian cells. J. CellBiol. 129, 1049-1059.

Levesque, A. A., Howard, L., Gordon, M. B. and Compton, D. A. (2003).A functional relationship between NuMA and kid is involved in both spindleorganization and chromosome alignment in vertebrate cells. Mol. Biol. Cell14, 3541-3552.

Lindsley, D. L. and Zimm, R. (1992). The Genome of Drosophilamelanogaster. New York, NY: Academic Press.

Matthies, H. J., McDonald, H. B., Goldstein, L. S. and Theurkauf, W. E.(1996). Anastral meiotic spindle morphogenesis: role of the non-claretdisjunctional kinesin-like protein. J. Cell Biol.134, 455-464.

McDonald, H. B. and Goldstein, L. S. (1990). Identification andcharacterization of a gene encoding a kinesin-like protein in Drosophila.Cell 61, 991-1000.

Meluh, P. B. and Rose, M. D. (1990). KAR3, a kinesin-related gene requiredfor yeast nuclear fusion. Cell 60, 1029-1041.

Merdes, A., Ramyar, K., Vechio, J. D. and Cleveland, D. W. (1996). Acomplex of NuMA and cytoplasmic dynein is essential for mitotic spindleassembly. Cell 87, 447-458.

Mountain, V. and Compton, D. A. (2000). Dissecting the role of molecularmotors in the mitotic spindle. Anat. Rec.261, 14-24.

Mountain, V., Simerly, C., Howard, L., Ando, A., Schatten, G. andCompton, D. A. (1999). The kinesin-related protein, HSET, opposes theactivity of Eg5 and cross-links microtubules in the mammalian mitoticspindle. J. Cell Biol.147, 351-366.

Nickerson, J. (2001). Experimental observations of a nuclear matrix. J. CellSci.141, 463-474.

O’Connell, M. J., Meluh, P. B., Rose, M. D. and Morris, N. R. (1993).Suppression of the bimC4 mitotic spindle defect by deletion of klpA, a geneencoding a KAR3-related kinesin-like protein in Aspergillus nidulans. J.Cell Biol. 120, 153-162.

O’Tousa, J. and Szauter, P. (1980). The initial characterization of non-claretdisjunctional (ncd): evidence that cand is the double mutant, ca ncd. D. I. S.55, 119.

Peel, D. J. and Milner, M. J. (1992). The expression of PS integrins inDrosophila melanogaster imaginal disc cell lines. Rouxs Arch. Dev. Biol.201, 120-123.

Raemaekers, T., Ribbeck, K., Beaudouin, J., Annaert, W., van Camp, M.,Stockmans, I., Smets, N., Bouillon, R., Ellenberg, J. and Carmeliet, G.(2003). NuSAP, a novel microtubule-associated protein involved in mitoticspindle organization. J. Cell Biol.162, 1017-1029.

Rebollo, E. and Gonzalez, C. (2000). Visualizing the spindle checkpoint inDrosophila spermatocytes. EMBO Rep.1, 65-70.

Saredi, A., Howard, L. and Compton, D. A. (1996). NuMA assembles intoan extensive filamentous structure when expressed in the cell cytoplasm. J.Cell Sci. 109, 619-630.

Saunders, W. S. and Hoyt, M. A. (1992). Kinesin-related proteins requiredfor structural integrity of the mitotic spindle. Cell 70, 451-458.

Sawin, K. E. and Mitchison, T. J. (1995). Mutations in the kinesin-likeprotein Eg5 disrupting localization to the mitotic spindle. Proc. Natl. Acad.Sci. USA92, 4289-4293.

Sawin, K. E., LeGuellec, K., Philippe, M. and Mitchison, T. J. (1992).Mitotic spindle organization by a plus-end-directed microtubule motor.Nature359, 540-543.

Scholey, J. M., Rogers, G. C. and Sharp, D. J. (2001). Mitosis, microtubules,and the matrix. J. Cell Biol.154, 261-266.

Sharp, D. J., McDonald, K. L., Brown, H. M., Matthies, H. J., Walczak,C., Vale, R. D., Mitchison, T. J. and Scholey, J. M. (1999a). The bipolarKinesin, KLP61F, cross-links microtubules within interpolar microtubulebundles of Drosophila, embryonic mitotic spindles. J. Cell Biol.144, 125-138.

Sharp, D. J., Yu, K. R., Sisson, J. C., Sullivan, W. and Scholey, J. M.(1999b). Antagonistic microtubule-sliding motors position mitoticcentrosomes in Drosophila early embryos. Nat. Cell Biol. 1, 51-54.

Journal of Cell Science 117 (21)

4933KLP61F Ncd, and the nuclear matrix

Sharp, D. J., Brown, H. M., Kwon, M., Rogers, G. C., Holland, G. andScholey, J. M. (2000a). Functional coordination of three mitotic motors inDrosophila embryos. Mol. Biol. Cell 11, 241-253.

Sharp, D. J., Rogers, G. C. and Scholey, J. M. (2000b). Roles of motorproteins in building microtubule-based structures: a basic principle ofcellular design. Biochim. Biophys. Acta1496, 128-141.

Silverman-Gavrila, R. V. and Forer, A. (2003). Myosin localization duringmeiosis I of crane-fly spermatocytes gives indications about its role indivision. Cell Motil. Cytoskeleton55, 97-113.

Sturtevant, A. H. (1929). The claret mutant phenotpe of Drosophila simulans:a study of chromosome elimination and cell lineage. Z. Wiss Zool.135, 326-356.

Tulu, U. S., Rusan, N. M. and Wadsworth, P. (2003). Peripheral, non-centrosome-associated microtubules contribute to spindle formation incentrosome-containing cells. Curr. Biol. 13, 1894-1899.

Vale, R. D. and Milligan, R. A. (2000). The way things move: looking underthe hood of molecular motor proteins. Science288, 88-95.

Walczak, C. E., Mitchison, T. J. and Desai, A. (1996). XKCM1: a Xenopuskinesin-related protein that regulates microtubule dynamics during mitoticspindle assembly. Cell 84, 37-47.

Walczak, C. E., Verma, S. and Mitchison, T. J. (1997). XCTK2: a kinesin-related protein that promotes mitotic spindle assembly in Xenopus laevisegg extracts. J. Cell Biol.136, 859-870.

Walker, D. L., Wang, D., Jin, Y., Rath, U., Wang, Y., Johansen, J. andJohansen, K. M. (2000). Skeletor, a novel chromosomal protein thatredistributes during mitosis provides evidence for the formation of a spindlematrix. J. Cell Biol.151, 1401-1412.

Wells, W. A. (2001). Searching for a spindle matrix. J. Cell Biol.154, 1102-1104.

White-Cooper, H., Alphey, L. and Glover, D. M. (1993). The cdc25homologue twine is required for only some aspects of the entry into meiosisin Drosophila. J. Cell Sci. 106, 1035-1044.

Wilson, P. G. (1999). BimC motor protein KLP61F cycles between mitoticspindles and fusomes in Drosophila germ cells. Curr. Biol. 9, 923-926.

Wilson, P. G., Fuller, M. T. and Borisy, G. G. (1997). Monastral bipolarspindles: implications for dynamic centrosome organization. J. Cell Sci.110, 451-464.

Wittmann, T., Wilm, M., Karsenti, E. and Vernos, I. (2000). TPX2, a novelxenopus MAP involved in spindle pole organization. J. Cell Biol.149, 1405-1418.