2012 - A Nuclear -Derived Proteinaceous Matrix Embeds the Microtubule Spindle Apparatus During Mitosis

7/28/2019 2012 - A Nuclear -Derived Proteinaceous Matrix Embeds the Microtubule Spindle Apparatus During Mitosis

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3532 | C. Yao et al . Molecular Biology o the Cell

MBoC | ARTICLE

A nuclear-derived proteinaceous matrix embedsthe microtubule spindle apparatus during mitosisChang u Yao a, Uttama Rath b , Helder Maiato c, David Sharp b , Jack Girton a, Kristen M. Johansen a,and Jrgen Johansen aaDepartment o Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011; bDepartmento Physiology and Biophysics, Albert Einstein College o Medicine, Bronx, NY 10461; cInstituto de Biologia Molecular e Celular, Universidade do Porto, 4150-180 Porto, Portugal

ABSTRACT The concept o a spindle matrix has long been proposed. Whether such a struc-ture exists, however, and what its molecular and structural composition are have remainedcontroversial. In this study, using a live-imaging approach in Drosophila syncytial embryos, wedemonstrate that nuclear proteins reorganize during mitosis to orm a highly dynamic, vis-cous spindle matrix that embeds the microtubule spindle apparatus, stretching rom pole topole. We show that this internal matrix is a distinct structure rom the microtubule spindleand rom a lamin Bcontaining spindle envelope. By injection o 2000-kDa dextran, we showthat the disassembling nuclear envelope does not present a di usion barrier. Furthermore,when microtubules are depolymerized with colchicine just be ore metaphase the spindlematrix contracts and coalesces around the chromosomes, suggesting that microtubules act asstruts stretching the spindle matrix. In addition, we demonstrate that the spindle matrixprotein Megator requires its coiled-coil amino-terminal domain or spindle matrix localiza-tion, suggesting that speci c interactions between spindle matrix molecules are necessary orthem to orm a complex con ned to the spindle region. The demonstration o an embedding

spindle matrix lays the groundwork or a more complete understanding o microtubule dy-namics and o the viscoelastic properties o the spindle during cell division.

INTRODUCTIONDuring cell division the entire nucleus undergoes a dramatic reor-ganization as the cell prepares to segregate its duplicated chro-mosomes. For many years the prevailing view on organisms pos-sessing an open mitosis has held that the nucleus completelydisassembled during early mitotic stages, thus enabling cytoplas-mic microtubules emanating rom the separated centrosomes to

orm a mitotic spindle. This cytocentric view largely discounted

any nuclear contributions to the ormation and/or unction o themitotic spindle (Johansen and Johansen, 2009; Simon and Wilson,

2011; Sandquist et al ., 2011). However, in Drosophila we recentlyidenti ed two nuclear proteins, Chromator (Rath et al ., 2004;Ding et al ., 2009; Yao et al ., 2012) and Megator (Qi et al ., 2004;Lince-Faria et al ., 2009), rom two di erent nuclear compartmentsthat interact with each other and redistribute during prophase to

orm a molecular complex that persists in the absence o polymer-ized tubulin (Johansen et al ., 2011). Chromator is localized to

polytene chromosome interbands during interphase (Rath et al .,2004, 2006; Yao et al ., 2012), whereas Megator occupies the nu-clear rim and the intranuclear space surrounding the chromo-somes (Zimowska et al ., 1997; Qi et al ., 2004). Chromator has noknown orthologues in other species; however, Megator is the ho-mologue o mammalian Tpr (Zimowska et al ., 1997). The Megator/Tpr amily o proteins is highly conserved through evolution, andstructural homologues are present rom yeast to humans (DeSouza and Osmani, 2009). Moreover, in addition to Megator, theAspergillus Mlp1 and human Tpr spindle matrix proteins have ashared unction as spatial regulators o spindle assembly check-point proteins during metaphase (Lee et al ., 2008; De Souza et al .,2009; Lince-Faria et al ., 2009). Both Chromator and Megator are essential proteins required or normal mitosis to occur in

Monitoring Editor Yixian Zheng

Carnegie Institution

Received: Jun 6, 2012Revised: Jul 12, 2012Accepted: Jul 26, 2012

This article was published online ahead o print in MBoC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E12-06-0429) on August 1, 2012.Address correspondence to: Jrgen Johansen ([email protected]), Kristen M.Johansen ([email protected]).

2012 Yao et al. This article is distributed by The American Society or Cell Biol-ogy under license rom the author(s). Two months a ter publication it is availableto the public under an AttributionNoncommercialShare Alike 3.0 UnportedCreative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).ASCB, The American Society or Cell Biology , and Molecular Biology o the Cell are registered trademarks o The American Society o Cell Biology.

Abbreviations used: DMSO, dimethyl sul oxide; GFP, green fuorescent protein;MAP, microtubule-associated protein; NE, nuclear envelope; NLS, nuclear local-ization signal; ROI, region o interest; YFP, yellow fuorescent protein.


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Volume 23 September 15, 2012 A nuclear-derived spindle matrix | 3533

apparatus rom pole to pole. The ndings urther suggest that thespindle matrix may directly contribute to the viscoelastic microme-chanical properties (Shimamoto et al ., 2011) o the spindle.

RESULTSThe spindle matrix embeds the microtubule spindleapparatusFigure 1 shows time-lapse imaging o Chromatorgreen fuores-cent protein (GFP) and tubulin-mCherry during mitosis in syncytial

Drosophila (Qi et al ., 2004; Lince-Faria et al ., 2009; Ding et al .,2009). These ndings suggest that these proteins are molecular components o the hitherto-elusive spindle matrix that, based ontheoretical considerations o the requirements or orce produc-tion, has been proposed to help constrain and stabilize the micro-tubule-based spindle apparatus (Pickett-Heaps et al ., 1982;Pickett-Heaps and Forer, 2009). Here we demonstrate that thisnuclear-derived internal spindle matrix is a highly dynamic,sel -contained structure that embeds the microtubule spindle

FIGURE 1: Con ocal time-lapse analysis o Chromator-GFP during mitosis in syncytial Drosophila embryos. (A) Relativedynamics o Chromator-GFP (green) and tubulin-mCherry (red) during a complete mitotic cycle. Scale bar, 10 m.(B) Chromator-GFP at metaphase. Arrowheads indicate the gap between Chromator-GFPs spindle matrix and centrosomallocalization. Scale bar, 10 m. (C) Relative localization o Jupiter-GFP (green) and tubulin-mCherry (red) at metaphase.Scale bar, 5 m. (D) Relative localization o Chromator-GFP (green) and tubulin-mCherry (red) at metaphase. Scale bar,5 m. (E , G) Line-scan plots o pixel intensity across the spindle along the white lines in C and D or Jupiter-GFP/tubulin-mCherry and Chromator-GFP/tubulin-mCherry, respectively. The images in C and D are both rom a single con ocal

optical plane. The asterisks indicate the likely position o microtubule K- bers. (F, H) Plots o the correlation between pixelintensity between Jupiter-GFP/tubulin-mCherry and Chromator-GFP/tubulin-mCherry across the spindle along the whitelines in C and D, respectively. The regression line and the value o Pearsons coe cient are indicated or each plot.


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strongly correlated ( r = 0.73 0.10, n = 17; Figure 1F), whereaspixel intensities in line scans o Chromator-GFP and tubulin-mCherry showed little correlation ( r = 0.32 0.07, n = 17; Figure1H). Taken together, these observations are consistent with the hy-pothesis that the Chromator-de ned spindle matrix is part o a vis-cous, gel-like structure that embeds the microtubule-based spin-dle apparatus. Furthermore, the ndings suggest that althoughthis matrix orms independently o microtubules, its morphologyand dynamic behavior during mitosis are governed by microtubule

spindle dynamics.To urther test this hypothesis, we depolymerized tubulin by in- jecting colchicine into embryos expressing GFP-Chromator and tu-bulin-mCherry or histone H2Av-RFP be ore prophase (Figure 2;Supplemental Movies S4 and S5). Under these conditions Chroma-tor still relocates rom the chromosomes to the matrix (Figure 2, Aand B); however, in the absence o microtubule spindle ormationthe Chromator-de ned matrix did not undergo any dynamic changesbut instead statically embedded the condensed chromosomes or extended periods ( >20 min). The movement observed within thematrix is caused by Brownian motion o the chromosomes. O inter-est, Chromator under these conditions still relocated to the cen-trosomes, suggesting that this is a microtubule-independent pro-cess. Control embryos injected with vehicle only underwent normal

Drosophila embryos. The results show that Chromator has reorga-nized away rom the chromosomes as they begin to condense and

lls the entire nuclear space be ore microtubule invasion (Figure1A and Supplemental Movie S1; see also Supplemental Movie S5

or a clearer view o this transition). As spindle microtubules orm,Chromator distribution attains a spindle-like morphology whilealso translocating to the centrosomes (Figure 1A). At anaphaseand telophase Chromator dynamics closely mirror that o the mi-crotubules be ore relocating back to the chromosomes in the

orming daughter nuclei. This dynamic behavior o Chromator dur-ing mitosis is very di erent rom microtubule-associated proteins(MAPs) such as Jupiter (Karpova et al ., 2006; Supplemental MovieS2). Although Chromator is present throughout the spindle, itspoleward boundary does not extend all the way to the centrosome(Figure 1B and Supplemental Movie S3), as also observed or theputative spindle pole matrix protein NuMA (Radulescu and Cleve-land, 2010). O interest, in line scans o pixel intensity across thespindle we ound that peak intensities o the MAP Jupiter coincidewith that o microtubules, indicating colocalization (Figure 1, C andE), whereas peak intensities o Chromator are notably distinct romthose o microtubules and in many cases show an alternating pat-tern (Figure 1, D and G). Moreover, pixel intensities in line scansacross the spindle or Jupiter-GFP and tubulin-mCherry were

FIGURE 2: Spindle matrix dynamics a ter colchicine injection be ore nuclear envelope breakdown. (A) Two image panelsrom the beginning and end o a time-lapse sequence o Chromator-GFP (green) and tubulin-mCherry (red) a ter

colchicine injection. (B) Two image panels rom the beginning and end o a time-lapse sequence o Chromator-GFP(green) and histone H2Av-RFP (red). (C) Plot o the average pixel intensity in regions o interest (ROIs) outside thenucleus (red) and inside the nucleus (blue) as a unction o time in a colchicine-injected embryo. The two image insertscorrespond to the area outlined by a white boxes in A be ore and a ter NE breakdown, respectively. The ROIs areindicated by white squares. The di erence in expression levels o Chromator-GFP in A and B is due to use o high- andlow-expression driver lines, respectively.


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centrosomes, and NE breakdown and dispersal o nuclear laminssuch as lamin B (lamin Dm0 in Drosophila ) is not completed until justbe ore the end o metaphase (Sta strom and Staehelin, 1984; Paddyet al ., 1996; Civelekoglu-Scholey et al ., 2010). This raises the ques-tion o whether the NE or the nuclear lamina presents a di usionbarrier during the early stages o mitosis and thus may contribute tothe con nement o spindle matrix proteins. To test whether this isthe case, we injected fuorescein-labeled dextrans o molecular mass 70, 500, or 2000 kDa, which are up to 10 times the molecular mass o the spindle matrix proteins Chromator and Megator, intotubulin-mCherryexpressing embryos treated with colchicine. Theresults showed that all three molecular-mass dextrans enteredthe nuclear space a ter NE breakdown on approximately the sametimescale as tubulin-mCherry (Figures 3 and 4), indicating theabsence o any signi cant di usion barriers to spindle matrix pro-teins. Furthermore, in colchicine-injected embryos lamin B disperseswithin 2 min, on a timescale similar to that o uninjected embryos(Figure 5), and does not accumulate in the nuclear space. In con-trast, the Chromator-de ned matrix persists around the chromo-somes or at least 10 times longer. Taken together, these ndingssuggest that the Chromator-de ned internal spindle matrix is adistinct and independent structure rom both the microtubule-basedspindle apparatus and rom the lamin Bcontaining spindle enve-lope previously described in Xenopus egg extracts (Zheng, 2010)and that the spindle matrix is held together by cohesive molecular interactions within the matrix.

The 70- and 500-kDa dextrans incorporateinto the spindle matrixO interest, we noted that 70- and 500-kDa dextrans accumulatedwithin the nuclear space in a way similar to tubulin in colchicine-in-

jected embryos, as illustrated in Figure 3 or 500-kDa dextran. Thissuggested that branched macromolecular polysaccharides can beincorporated into the spindle matrix. To urther explore this possibil-ity, we injected fuorescein-conjugated 70-, 500-, and 2000-kDa

dextrans into tubulin-mCherryexpressing embryos without colchi-cine treatment. As exempli ed in Figure 4A or 70-kDa dextran,both 70- and 500-kDa dextrans accumulate in the nuclear spacebe ore microtubule spindle ormation, and its dynamics during mi-tosis until the end o telophase, when it gets excluded rom the

orming daughter nuclei (Supplemental Movie S7), closely resem-bles that o the spindle matrix proteins Chromator and Megator (Supplemental Movies S1 and S8). In contrast, although the 2000-kDa dextran did enter and equilibrate within the nuclear space atthe time o NE breakdown, it did not show any enrichment withinthe spindle region (Figure 4B). We speculate that this di erence be-tween 70- and 2000-kDa dextrans is due to potential size exclusion-ary properties o the spindle matrix. These data provide additional

support or the concept o a viscous matrix made up o macromol-ecules enriched in the spindle region by cohesive interactions.

The amino-terminal region o Megator is required or itsspindle matrix localizationMegator is a large, 260-kDa protein (Mtor-FL) with an extendedamino-terminal coiled-coil domain (Mtor-NTD) and an unstructuredcarboxy-terminal domain (Mtor-CTD). Coiled-coil domains areknown protein interaction domains, as previously demonstrated or the spindle pole matrix protein NuMA (Radulescu and Cleveland,2010). There ore, to explore whether Megators coiled-coil domainis required or Megators spindle matrix localization, we conductedtime-lapse imaging o ull-length, yellow fuorescent protein (YFP)tagged Megator (Mtor-FL), green fuorescent protein (GFP)tagged

mitosis indistinguishable rom wild-type preparations (Supplemen-tal Movie S6). Moreover, as illustrated in Figure 2C, unpolymerizedtubulin accumulates within the nuclear space, as measured by rela-tive average pixel intensity, to 1.6 0.2 (n = 12, rom ve di erentpreparations) times the levels outside the nuclear space in thecolchicine-injected embryos (see also Figure 2, A and C, and Sup-plemental Movie S4). This nding suggests the presence o one or more tubulin-binding proteins within the spindle matrix.

The nuclear envelope and lamin B do not contribute to theinternal spindle matrixDrosophila embryos have semiopen mitosis in which the nuclear envelope (NE) initially breaks down only in the region o the

FIGURE 3: The 500-kDa dextran enters and accumulates in thenuclear space on the same timescale as tubulin in colchicine-injectedembryos. (A) Image panels rom a time-lapse sequence rom atubulin-mCherry (red)expressing embryo coinjected with fuorescein-labeled dextran o molecular mass 500 kDa (green) and colchicine.Time is in seconds. Scale bar, 10 m. (B) Plot o the normalizedaverage pixel intensity in ROIs outside the nucleus and inside thenucleus o tubulin (red) and 500-kDa dextran (green) as a unction otime in a colchicine-injected embryo. The solid and stippled linescorrespond to areas inside and outside a nucleus, respectively, asoutlined by the white boxes in A. The approximate time o NEbreakdown is indicated by an arrow.


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mosomal localization during interphase. Furthermore, i microtu-bules are prevented rom orming by colchicine injection be ore

prophase, both Mtor-FL and Mtor-NTD still relocate to the spindlematrix and, as with the Chromator-de ned matrix, do not undergoany dynamic changes but statically embed the condensed chromo-somes (Figure 6E and Supplemental Movie S10). In contrast, under these conditions Mtor-CTD disperses on a rapid timescale in


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matrix physically be linked to microtubulesand that changes to the shape and orm o the matrix in turn are governed by microtu-bule dynamics. One possible mechanism toaccomplish this is exempli ed by NuMA,which, together with dynein, unctions as aspindle pole matrix that tethers and ocusesthe majority o spindle microtubules to thepoles largely independently o centrosomes(Dumont and Mitchison, 2009; Radulescuand Cleveland, 2010). Thus we propose thata spindle pole matrix may be a constituento a larger pole-to-pole matrix that couplesthis matrix to microtubule dynamics.

In Xenopus egg extracts it was sug-gested that a membranous lamin Bcon-taining envelope derived rom the nuclear membrane could be part o the spindle ma-trix (Tsaiet al ., 2006; Zheng, 2010). However,our ndings clearly demonstrate that theinternal matrix as de ned by the Chroma-tor and Megator proteins is physically dis-tinct rom such a structure and that the inter-nal matrix persists a ter dispersal o lamin Bin nuclei arrested at metaphase. Nonethe-less, the interplay between microtubules,the spindle matrix, and NE dynamics duringmitosis is likely to be nely tuned and mutu-ally dependent (Zheng, 2010). For example,evidence has been provided that the NE

and lamin B in systems with semiopen mitosis may contribute to therobustness o spindle unction and assembly during prometaphaseand that the gradual disassembly o the lamin B envelope is coupledto proper spindle maturation during metaphase (Civelekoglu-Scholey et al ., 2010).

In this study we present evidence by injection o highmolecular weight dextrans that the disassembling NE and nuclear lamina a ter their initial breakdown are not likely to present a di usion barrier tomost known proteins. O interest, even in the absence o such a di -

usion barrier we show that ree tubulin (possibly as /-tubulin dim-ers) accumulates coextensively with the spindle matrix protein Chro-mator in colchicine-treated embryos independently o tubulinpolymerization. We propose that this enrichment is dependent onone or more proteins within the spindle matrix with tubulin-bindingactivity. A similar enrichment within the nuclear region o ree tubulina ter NE breakdown has recently been reported in Caenorhabditis elegans embryos (Hayashi et al ., 2012). The enhanced accumulationo ree tubulin within the nascent spindle region may serve as a gen-

eral mechanism to promote the e cient assembly o the microtu-bule-based spindle apparatus (Hayashi et al ., 2012) and be medi-ated by spindle matrix constituents. The accumulation o tubulin inthe nucleus under microtubule depolymerization conditions is not ageneral property o cytoplasmic proteins, as exempli ed by the dy-nactin complex component DNC-1 in the nematode (Hayashi et al .,2012).

A surprising nding o the present study is that nonproteina-ceous polysaccharide macromolecules such as dextrans have theability to be incorporated into the spindle matrix. However, the re-sults o previous studies showed that the spindle pole protein NuMAis highly poly(ADP-ribosyl)ated (Radulescu and Cleveland, 2010)and that poly(ADP-ribose) is required or spindle assembly and unc-tion in Xenopus (Chang et al ., 2004). Thus it is possible that the size,

tubulin-mCherryexpressing embryos during metaphase. As shownin the image sequence o Figure 7 and in Supplemental Movie S12,as the microtubules undergo depolymerization, the Chromator-de-

ned matrix contracts and coalesces around the chromosomes. Thereduction in the length o the spindle matrix was almost 60% rom

when the rst image was obtained a ter colchicine injection to whenmicrotubules were depolymerized (Figure 7B). This suggests thatthe spindle matrix is stretched by the microtubules. A similar resultwas obtained in S2 cells expressing the spindle matrix protein Me-gator (Lince-Faria et al ., 2009), suggesting that the properties o thespindle matrix described here are a general eature o mitosis andnot con ned to only syncytial nuclei. Furthermore, the expectationwould be that i microtubules were stabilized at metaphase insteado depolymerized, then the shape and orm o the spindle matrixwould not change. To test this prediction, we injected the microtu-bule-stabilizing agent Taxol into Mtor-FL and tubulin-mCherryex-pressing embryos during metaphase. As shown in SupplementalMovie S13, under these conditions both the spindle matrix and the

microtubules do not undergo any dynamic changes but maintaintheir metaphase usi orm spindle morphology or extended timeperiods o >14 min.

DISCUSSIONIn this study we showed that at least two proteins rom di erentnuclear compartments reorganize during mitosis to orm a spindlematrix that embeds the microtubule spindle apparatus and that islikely to be part o a molecular complex stretching rom pole topole. As also indicated by previous experiments in S2 cells (Lince-Faria et al ., 2009), the present observations are not compatible witha rigid matrix structure but instead with a highly dynamic viscousmatrix made up o protein polymers orming a gel-like meshwork.For such a matrix to be stretched implies that components o the

FIGURE 5: Lamin B in colchicine-injected embryos disperses on a timescale similar to that ouninjected embryos during mitosis. (A) Image panels rom a time-lapse sequence rom a histoneH2Av-RFP (red) and lamin B-GFP (green)expressing embryo. (B) Image panels rom atime-lapse sequence rom a histone H2Av-RFP (red) and lamin B-GFP (in green)expressingembryo injected with colchicine be ore nuclear envelope breakdown. Time is in minutes andseconds. Scale bars, 10 m.


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FIGURE 6: Time-lapse analysis o the spindle matrix protein Megator in syncytial embryos. (A) Relative dynamics oull-length Megator-YFP (Mtor-FL) and histone H2Av-RFP (H2Av) during a complete mitotic cycle. The images show their

distribution at interphase 1, metaphase, and interphase 2, respectively. The diagram beneath the images shows thedomain structure o Megator with the coiled-coil region in black, the CTD in white, and the endogenous NLS in red.Scale bar, 20 m. (B) Relative dynamics o a truncated, GFP-tagged, carboxy-terminal construct o Megator (Mtor-CTD)and histone H2Av-RFP (H2Av) during a complete mitotic cycle. The images show their distribution at interphase 1,metaphase, and interphase 2, respectively. Mtor-CTD is diagrammed below the images. Scale bar, 20 m. (C) Relativedynamics o a truncated, GFP-tagged, amino-terminal construct o Megator (Mtor-NTD) and histone H2Av-RFP (H2Av)during interphase and metaphase. Mtor-NTD is diagrammed below the images. Scale bar, 10 m. (D) The localizationpatterns o Mtor-FL, Mtor-NTD, and Mtor-CTD at interphase. Mtor-FL localizes to the nuclear interior, as well as to thenuclear rim, Mtor-NTD is present at the nuclear rim with no or very little interior nuclear localization, and Mtor-CTD isdi usively present in the nucleoplasm without detectable nuclear rim localization. (E) Top, three images rom a time-lapse sequence o Mtor-FL-YFP (green) and histone H2Av-RFP (red) a ter colchicine injection at interphase. Middle,three images rom a time-lapse sequence o Mtor-CTD-GFP (green) and histone H2Av-RFP (red) a ter colchicine injectionat interphase. Bottom, three images rom a time-lapse sequence o Mtor-NTD-GFP (green) and histone H2Av-RFP (red)a ter colchicine injection at interphase. Time is in minutes and seconds. Scale bars, 10 m.


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polymer meshwork with hydrogel-like prop-erties within the nuclear pore (Frey et al .,2006). I , as suggested here, the spindle ma-trix is a similar gel-like assembly o weaklyassociated protein polymers, its exact stoi-chiometry and composition may not be criti-cal and it likely would be able to accommo-date the inclusion o a wide array o proteins.However, it is important to note that not allnuclear proteins relocate to the spindle ma-trix during mitosis. For example, both laminB and C (Paddy et al ., 1996; Katsani et al .,2008) disperse, as does the nucleoporinNup58 (Katsani et al ., 2008). Furthermore, inthis study we demonstrate that the amino-terminal coiled-coil region o Megator is re-quired or its spindle matrix localization dur-ing mitosis, whereas the carboxy-terminalregion disperses. In uture experiments itwill be o interest to determine the nature o the speci c molecular interactions that gov-ern which proteins are incorporated into thematrix.

Regardless o the exact compositionand structure o the spindle matrix, thedemonstration here o a sel -containedmacromolecular structure embedding thespindle apparatus during mitosis will haveimportant implications or our understand-ing o microtubule dynamics (Dumont andMitchison, 2009). Furthermore, in a recentstudy o the micromechanical properties o the metaphase spindle, the e ective viscos-ity o the spindle region was measured tobe 100 times higher than in the surround-

ing cytoplasm (Shimamoto et al ., 2011).This di erence was attributed largely to the actions o motor andnonmotor proteins cross-linking microtubules, with the assumptiono negligible contributions rom the spindle medium. However, theresults o this study suggest that a gel-like spindle matrix is likely todirectly contribute to the viscoelastic mechanical properties o thespindle.

MATERIALS AND METHODSDrosophila melanogaster stocks and transgenic fiesFly stocks were maintained according to standard protocols(Roberts, 1998), and Canton S was used or wild-type preparations.Full-length, GFP-tagged Chromator constructs under native or GAL-4

promoter control have been previously characterized (Ding et al .,2009). Tubulin-mCherry, Jupiter-GFP, and lamin-GFP fy stocks (stocks25774, 6836, and 7378, respectively) and a tubulin-GAL-4 driver line(stock 7062) were obtained rom the Bloomington Drosophila StockCenter, Indiana University (Bloomington, IN). The Megator YFP-trapfy line (w [1118 ]; PBac{602.P.SVS-1}Mtor [CPTI001044]) was obtained

rom the Drosophila Genetic Resource Center, Kyoto Institute o Technology (Kyoto, Japan; stock 115129). The H2AvDmRFP1 trans-genic line was the gi t o S. Heidmann and has been previously de-scribed (Deng et al ., 2005). For the Megator-CTD construct under native promoter control a genomic region o 949 nucleotides up-stream and 9 nucleotides downstream o the ATG start codon wasPCR ampli ed and used with an in- rame GFP tag, as well aswith Megator carboxy-terminal coding sequence corresponding to

branching, and charge distribution o such polymeric carbohydratemodi cations o spindle matrix proteins might play a role in regulat-ing its assembly and unction. Furthermore, these modi cationsmight contribute directly to the viscoelastic properties o the spindleand contribute to the modulation o microtubule dynamics andspindle stabilization.

An issue or the spindle matrix hypothesis has been to accountor its molecular composition and structure, especially as the num-

ber and diversity o its possible constituents has grown (reviewed inJohansen et al ., 2011). In Drosophila , in addition to Megator andChromator, the nuclear proteins Skeletor, EAST, and Mad2 havebeen demonstrated to be associated with the spindle matrix (Walker

et al ., 2000; Qi et al ., 2005; Katsani et al ., 2008; Lince-Faria et al .,2009; Ding et al ., 2009). Another candidate nuclear spindle matrixprotein that relocates to the spindle region during mitosis in a mi-crotubule-independent manner is the nucleoporin Nup107 (Katsaniet al ., 2008). Thus it is becoming clear that during mitosis manydisassembled components o interphase nuclear structure do notsimply disperse but rather reorganize, making important contribu-tions to mitotic progression (De Souza and Osmani, 2009; Johansenand Johansen, 2007, 2009; Simon and Wilson, 2011). For example,many nuclear pore complex constituents in addition to Megator/Tpr and Nup107 have been demonstrated to relocate to the spindleregion in both invertebrates and vertebrates (reviewed in De Souzaand Osmani, 2009; Johansen et al ., 2011). O interest, certain nu-clear pore proteins have been shown to orm a three-dimensional

FIGURE 7: Depolymerization o microtubules at metaphase leads to contraction o the spindlematrix. (A) Two image panels rom the beginning and end o a time-lapse sequence oChromator-GFP (green) and tubulin-mCherry (red) a ter colchicine injection. The image sequencebegins 30 s a ter colchicine injection. Scale bar, 10 m. (B) Image sequence o Chromator-GFPa ter colchicine injection in the spindle outlined by white rectangles in A. Time is in minutes andseconds. Scale bar, 5 m.


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residues 17582347, and inserted into the pUAST vector using stan-dard techniques (Sambrook and Russell, 2001). For the Megator-NTDconstruct under native promoter control the same upstream regionas or the Mtor-CTD construct was used with an in- rame GFP tag,with Megator amino-terminal coding sequence corresponding toresidues 11757, and with the NLS rom the NLS-pECFP vector (Clontech, Mountain View, CA) and inserted into the pPFHW vector (Murphy, 2003) using standard techniques (Sambrook and Russell,2001). Transgenic Mtor-CTD and Mtor-NTD fy lines were generatedby P-element trans ormation by BestGene (Chino Hills, CA). Fly linesexpressing combinations o transgenes were generated by standardgenetic crosses.

Time-lapse con ocal microscopy and injectionsTime-lapse imaging o the fuorescently tagged constructs in livesyncytial embryos were per ormed using a TCS SP5 tandem scan-ning microscope (Leica, Wetzlar, Germany) or an UltraView spinning-disk con ocal system (PerkinElmer, Waltham, MA) as previously de-scribed (Ding et al ., 2009). In brie , 0- to 1.5-h embryos werecollected rom apple juice plates and aged 1 h. The embryos weremanually dechorinated, trans erred onto a coverslip coated with athin layer o heptane glue, and covered with a drop o halocarbonoil 700. Time-lapse image sequences o a single z -plane or o z -stacks covering the depth o the mitotic apparatus were obtainedusing a Plan-Apochromat 63/1.4 numerical aperture objective.For colchicine injections, colchicine (Sigma-Aldrich, St. Louis, MO)was dissolved in dimethyl sul oxide (DMSO) to a concentration o 100 mg/ml as a stock solution. The nal concentration o colchicine

or injection was 1 mg/ml by diluting the stock solution with PEMbu er (80 mM Na 1,4-piperazinediethanesul onic acid, pH 6.9,1 mM MgCl2, 1 mM ethylene glycol tetraacetic acid, 5% glycerol).Injections o 100200 pl o 1 mg/ml colchicine into each embryowere per ormed with an IM-300 programmable microinjector sys-tem (Narishige, Tokyo, Japan) connected to the Leica con ocal TCSSP5 microscope system, as previously described (Brust-Mascher and

Scholey, 2009). For Taxol injections, 100200 pl o 20 mg/ml Taxol(Sigma-Aldrich) in DMSO was injected into each embryo. Controlinjections were per ormed with DMSO alone or with PEM bu er with 1% DMSO. Fluorescein-labeled dextrans o molecular mass 70,500, or 2000 kDa (Invitrogen, Carlsbad, CA) were injected into syn-cytial embryos using standard methods (Brust-Mascher and Scholey,2009).

Image quanti cation and analysisImage processing and quanti cation were carried out with the Im-ageJ 1.45 so tware (National Institutes o Health, Bethesda, MD) or with Photoshop (Adobe, San Jose, CA). QuickTime movies weregenerated with QuickTime Pro 7.6.6 (Apple, Cupertino, CA). Scatter

plots, average pixel intensities o regions o interest, and determina-tion o Pearsons correlation coe cient o the measured fuores-cence intensity o line scans generated in ImageJ were per ormedand calculated using Excel (Microso t, Redmond, CA).

ACKNOWLEDGMENTSWe thank members o our laboratory or discussion, advice, andcritical reading o the manuscript. We also acknowledge Atrez Nor-wood or technical assistance. We especially thank S. Heidmann, H.White-Cooper, and J. Roote or providing fy stocks. Work in thelaboratory o J.J. and K.M.J. is supported by National Science Foun-dation Grant MCB0817107, and work in the laboratory o H.M. issupported by the Human Frontier Research Program.


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2012 - A Nuclear -Derived Proteinaceous Matrix Embeds the Microtubule Spindle Apparatus During Mitosis