a capillary electrophoretic method for monitoring the presence of α-tubulin in nuclear preparations

9
ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 330 (2004) 1–9 www.elsevier.com/locate/yabio 0003-2697/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.03.059 A capillary electrophoretic method for monitoring the presence of -tubulin in nuclear preparations Nilhan Gunasekera, 1 Guohua Xiong, Karin Musier-Forsyth, and Edgar Arriaga ¤ Department of Chemistry, University of Minnesota, 207 Pleasant St. SE, Minneapolis, MN 55455, USA Received 30 July 2003 Available online 10 May 2004 Abstract A sensitive capillary electrophoretic method was developed to detect the presence of -tubulin, a microtubular cytoskeletal component, in isolated nuclear preparations. These preparations are treated with anti--tubulin primary mouse antibodies and then stained with a Xuorescently labeled anti-mouse IgG antibody. The stained preparation is then analyzed by capillary electrophoresis with laser-induced Xuorescence detection, a technique that allows for sensitive detection of Xuorescently labeled species. Using this method, it is feasible to count individual subcellular aggregates containing -tubulin (SATs), estimate the number of -tubulin mole- cules per SAT, determine the cumulative intensity of all SATs as an estimate of the relative level of -tubulin in a preparation, and obtain their apparent electrophoretic mobility distribution. The method was validated by comparing SATs from untreated cells with those from colchicine-treated cells. Since colchicine is a microtubule-disrupting agent, treatment reduced the number of SATs per cell as well as the cumulative intensity of all SATs in a preparation. In contrast, the apparent electrophoretic mobility distribution was not inXuenced by colchicine treatment, suggesting that this parameter is not strongly dependent on the -tubulin content. Given the zeptomolar sensitivity of laser-induced Xuorescence detection and the widespread availability of antibodies, the approach used here represents an improvement in the detection of cytoskeletal impurities in subcellular fractions. 2004 Elsevier Inc. All rights reserved. Keywords: Capillary electrophoresis; Colchicine; Cytoskeleton; -Tubulin; Nuclei The cytoskeleton helps cells maintain their shape. It forms an extensive network around the nucleus of eukaryotic cells and allows organelles (such as mito- chondria and lysosomes) to move within the cytosol [1]. Imaging shows that microtubules, which constitute a major portion of the cytoskeletal network, are tightly associated with the nucleus and radiate outward toward the plasma membrane from the perimeter of the nucleus [2–7]. The close association of the cytoskeleton to the nuclear perimeter is also evidenced by the ability to isolate a cytoskeleton-nuclear matrix fraction from homogenized cells [8]. When studying nuclear function based on isolated nuclear preparations, the presence of cytoskeletal com- ponents is not desired. It can lead to biases in data inter- pretation since nonnuclear organelles, such as mitochondria, microsomes, and lysosomes, as well as ribosomes can also be associated with the cytoskeleton [9,10]. A previous study detected mitochondrial, endo- plasmic reticular and plasma membrane proteins in nuclear preparations, and suggested that these were associated with cytoskeletal remnants found in isolated nuclear fractions [9]. The presence of cytosolic ribo- somes in a nuclear preparation was proposed to explain an earlier report suggesting that nuclei are capable of protein translation [11,12]. Mitochondrial components have also been detected in nuclear preparations through succinate dehydrogenase or acyl-CoA synthetase assays [13]. The presence of actin, a cytoskeletal component, in preparations isolated nuclei from Amoeba proteus also indicated that the cytoskeleton plays a role in determin- ing the size of the nucleus [14]. Although microscopy facilitates observation of the abundance and morphological features of the cytoskele- ton in nuclear preparations [14], it has low throughput ¤ Corresponding author. Fax: 1-6126267541. E-mail address: [email protected] (E. Arriaga). 1 Present address: University of Wisconsin–Marathon County, 518 S. 7th Avenue, Wasau, WI 54401, USA.

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Page 1: A capillary electrophoretic method for monitoring the presence of α-tubulin in nuclear preparations

ANALYTICALBIOCHEMISTRY

Analytical Biochemistry 330 (2004) 1–9

www.elsevier.com/locate/yabio

A capillary electrophoretic method for monitoring the presenceof �-tubulin in nuclear preparations

Nilhan Gunasekera,1 Guohua Xiong, Karin Musier-Forsyth, and Edgar Arriaga¤

Department of Chemistry, University of Minnesota, 207 Pleasant St. SE, Minneapolis, MN 55455, USA

Received 30 July 2003Available online 10 May 2004

Abstract

A sensitive capillary electrophoretic method was developed to detect the presence of �-tubulin, a microtubular cytoskeletalcomponent, in isolated nuclear preparations. These preparations are treated with anti-�-tubulin primary mouse antibodies and thenstained with a Xuorescently labeled anti-mouse IgG antibody. The stained preparation is then analyzed by capillary electrophoresiswith laser-induced Xuorescence detection, a technique that allows for sensitive detection of Xuorescently labeled species. Using thismethod, it is feasible to count individual subcellular aggregates containing �-tubulin (SATs), estimate the number of �-tubulin mole-cules per SAT, determine the cumulative intensity of all SATs as an estimate of the relative level of �-tubulin in a preparation, andobtain their apparent electrophoretic mobility distribution. The method was validated by comparing SATs from untreated cells withthose from colchicine-treated cells. Since colchicine is a microtubule-disrupting agent, treatment reduced the number of SATs per cellas well as the cumulative intensity of all SATs in a preparation. In contrast, the apparent electrophoretic mobility distribution wasnot inXuenced by colchicine treatment, suggesting that this parameter is not strongly dependent on the �-tubulin content. Given thezeptomolar sensitivity of laser-induced Xuorescence detection and the widespread availability of antibodies, the approach used hererepresents an improvement in the detection of cytoskeletal impurities in subcellular fractions. 2004 Elsevier Inc. All rights reserved.

Keywords: Capillary electrophoresis; Colchicine; Cytoskeleton; �-Tubulin; Nuclei

The cytoskeleton helps cells maintain their shape. It pretation since nonnuclear organelles, such as

forms an extensive network around the nucleus ofeukaryotic cells and allows organelles (such as mito-chondria and lysosomes) to move within the cytosol [1].Imaging shows that microtubules, which constitute amajor portion of the cytoskeletal network, are tightlyassociated with the nucleus and radiate outward towardthe plasma membrane from the perimeter of the nucleus[2–7]. The close association of the cytoskeleton to thenuclear perimeter is also evidenced by the ability toisolate a cytoskeleton-nuclear matrix fraction fromhomogenized cells [8].

When studying nuclear function based on isolatednuclear preparations, the presence of cytoskeletal com-ponents is not desired. It can lead to biases in data inter-

¤ Corresponding author. Fax: 1-6126267541.E-mail address: [email protected] (E. Arriaga).1 Present address: University of Wisconsin–Marathon County,

518 S. 7th Avenue, Wasau, WI 54401, USA.

0003-2697/$ - see front matter 2004 Elsevier Inc. All rights reserved.doi:10.1016/j.ab.2004.03.059

mitochondria, microsomes, and lysosomes, as well asribosomes can also be associated with the cytoskeleton[9,10]. A previous study detected mitochondrial, endo-plasmic reticular and plasma membrane proteins innuclear preparations, and suggested that these wereassociated with cytoskeletal remnants found in isolatednuclear fractions [9]. The presence of cytosolic ribo-somes in a nuclear preparation was proposed to explainan earlier report suggesting that nuclei are capable ofprotein translation [11,12]. Mitochondrial componentshave also been detected in nuclear preparations throughsuccinate dehydrogenase or acyl-CoA synthetase assays[13]. The presence of actin, a cytoskeletal component, inpreparations isolated nuclei from Amoeba proteus alsoindicated that the cytoskeleton plays a role in determin-ing the size of the nucleus [14].

Although microscopy facilitates observation of theabundance and morphological features of the cytoskele-ton in nuclear preparations [14], it has low throughput

Page 2: A capillary electrophoretic method for monitoring the presence of α-tubulin in nuclear preparations

2 N. Gunasekera et al. / Analytical Biochemistry 330 (2004) 1–9

and is unlikely to detect small components of cytoskele-ton either bound to nuclei or cosuspended in the nuclearpreparation. Since the cytoskeleton is made of microtu-bules, intermediate Wlaments, and actin [2], detection ofany of these components in a nuclear preparation wouldindicate the presence of cytoskeleton remnants. In par-ticular, detection of �-tubulin, which polymerizes toform microtubules, would facilitate detection of cyto-skeletal impurities in nuclear preparations.

Capillary electrophoresis with laser-induced Xuores-cence detection (CE-LIF)1 is an analytical method thathas been used to detect organelles and other subcellularsized particles that have an intrinsic electrophoreticmobility [15–23]. A net surface charge density on theseparticles, a consequence of the charged phospholipidsand exposed proteins on their surface, causes particles toundergo electrophoresis in the presence of an electricWeld. Thus, when CE-LIF is performed in a biologicallycompatible medium (e.g., 250 mM sucrose, 10 mMHepes, pH 7.4), organelles and other subcellular sizedparticles will move toward the LIF detector at a speedthat is a function of their apparent electrophoreticmobility. Due to the high sensitivity of LIF, Xuorescentlylabeled particles are detected as they pass individuallythrough the detector.

In a previous report, we demonstrated the usefulnessof CE-LIF for the detection and characterization ofnucleic acid-containing particles (NAPs) in nuclearpreparations from mammalian cells [15]. In these prepa-rations, NAPs include not only intact nuclei, but alsounwanted components such as nuclear fragments, largeDNA-protein complexes, and possibly other subcellularaggregates rich in nucleic acids. Mitochondrial contami-nation in nuclear preparations was also reported [15].Due to the close interaction between the cytoskeletonand the nuclei, cytoskeleton remnants are another sourceof impurity in nuclear preparations [8–11,14].

In this work, we demonstrate the use of CE-LIF todetect the presence of subcellular aggregates containing�-tubulin (SATs) in nuclear preparations. For this pur-pose, we Xuorescently immunolabeled SATs in thesesamples with an anti-�-tubulin primary antibody fol-lowed by a Xuorescently labeled secondary antibody.This CE-LIF method was successfully used to detect andcount the number of SATs and to determine changes in�-tubulin levels in nuclear preparations isolated fromcell cultures treated with colchicine, a microtubule-dis-rupting agent [24–26]. In addition, the CE-LIF methodallows the determination of the apparent electrophoreticmobility distribution of SATs, providing an added

1 Abbreviations used: CE-LIF, capillary electrophoresis with laser-induced Xuorescence detection; NAPs, nucleic acid-containingparticles; SATs, subcellar aggregates containing �-tubulin; PBS,phosphate-buVered saline; colchicine, N-(5,6,7,9-tetrahydro-1,2,3,10-tetramethoxy-9-oxobenzo[a] heptelen-7-yl)-,(S)-acetamide.

dimension in the characterization of cytoskeletal impuri-ties in nuclear preparations.

Materials and methods

Chemicals

Tris[hydroxymethyl]aminomethane (Tris), (N-[2-hydro-xyethyl]piperazine-N-ethanesulphonic acid) (Hepes),phosphate-buVered saline (PBS), Dulbecco’s modiWedEagle’s medium, calf serum, and N-(5,6,7,9-tetrahydro-1,2,3,10-tetramethoxy-9-oxobenzo[a]heptelen-7-yl)-,(S)-acetamide (colchicine) were purchased from Sigma (St.Louis, MO). Trypan blue stain was purchased from Bio-Whitaker (Walkersville, MD). Magnesium chloride andsucrose were purchased from Fisher (Fair Lawn, NJ).Fluorescein, hexidium iodide, and antibodies were pur-chased from Molecular Probes (Eugene, OR). The pri-mary antibody used in immunolabeling was the mousemonoclonal antibovine �-tubulin. This antibody speciW-cally binds to �-tubulin and has been shown to bind tomouse microtubules [27]. The Xuorescently labeledsecondary antibody was an anti-mouse Alexa Fluor 488-labeled goat IgG antibody (6.2 mol of Alexa Fluor 488per mole of antibody) from Molecular Probes. This sec-ondary antibody binds to the primary antibody and thusmakes it possible to visualize microtubules via Xuores-cence microscopy.

Cell culture

NS-1 (mouse hybridoma) cells were cultured at 37 °Cand 5% CO2 by splitting cells 1:4 every 2 days in Dul-becco’s modiWed Eagle’s medium supplemented with10% calf serum.

Colchicine treatment of cell cultures

The cell cultures were treated with 1.4, 14, or 140�Mcolchicine for 15 h at 37°C and 5% CO2 [28]. A control cellculture was grown under similar conditions, in the absenceof colchicine. At the end of the incubation period the cellcultures were washed by centrifugation (600g, 10 min)three times with PBS to remove the cell medium.

Cell viability determination

The percentage of live cells in cultures was deter-mined using the Trypan blue exclusion test, by incubat-ing a 100-�L aliquot of cells with 100�L of 0.4% Trypanblue. The cells with compromised plasma membranes arestained dark by this dye, while live cells remain light. Thecell viability (percentage of live cells) can be determinedby counting the number of dark vs light cells in a givenvolume using a hemocytometer (Sigma).

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N. Gunasekera et al. / Analytical Biochemistry 330 (2004) 1–9 3

Nuclear isolation

Bulk nuclear preparations were isolated from NS-1cells as described previously [15]. At the end of theisolation procedure the Wnal nuclear pellet was resus-pended in buVer I (2.2 M sucrose, 1 mM magnesiumchloride, 10 mM Tris, pH 7.4). Each nuclear preparationwas split into three portions. The Wrst portion wasstained with the antibody system, the second portionwas stained with hexidium iodide, and the third was notstained (negative control).

Immunolabeling of nuclear preparations

The washed nuclear aliquots were incubated 1:1 (v/v)with the mouse monoclonal antibody, anti-�-tubulin(2 �g/mL) for 1.5 h at 37 °C. They were then washed threetimes with PBS to remove excess antibody. The aliquotswere next incubated 1:1 (v/v) with Alexa Fluor 488-labeled goat anti-mouse IgG antibody (20 �g/mL) for 1 hin the dark at room temperature. After this incubation,the aliquots were washed three times with PBS toremove unbound antibody and suspended in the CE runbuVer A (250 mM sucrose, 10 mM Hepes, pH 7.4). Theexcitation maximum for the Xuorescent label of the sec-ondary antibody, Alexa Fluor, is at 495 nm while theemission maximum is at 519 nm.

Hexidium iodide labeling of nuclear preparations

Aliquots from colchicine-treated and untreated sam-ples were mixed with an equal volume of 1.0 �M hexi-dium iodide and kept at room temperature for at least15 min. Samples were then washed by centrifugation(600g, 10 min) and resuspended in buVer A prior to CEanalysis. Hexidium iodide has an excitation maximum at518 nm and an emission maximum at 600 nm.

Imaging of immunolabeled nuclear preparations

The nuclear preparations treated with colchicine wereimaged using a Nikon TE300 Xuorescence microscope(Fryer, Huntley, IL) using a green Wlter setting (excita-tion, 465–495 nm; emission, 515–550 nm; dichroic,505 nm). These images were analyzed using Image J soft-ware to determine the ratio of mean intensities betweennuclear images from colchicine-treated and untreatedcell cultures. BrieXy, the mean pixel intensity was deter-mined for the Xuorescent area in each image. The meanintensity of the background was subtracted from theabove Xuorescent mean intensity.

Capillary electrophoresis with laser-induced Xuorescence

Uncoated, 50 �m i.d., 150 �m o.d. fused silica capillar-ies (Polymicro Technologies, Phoenix, AZ) were used for

the separation of NAPs or SATs in nuclear preparations.The CE-LIF instrument used to analyze these prepara-tions has been previously described [18]. The opticaldetection system was aligned using a 10¡9 M solution ofXuorescein (Molecular Probes). BrieXy, during a conti-nuous Xow of Xuorescein through the capillary, the posi-tion of the sheath-Xow cuvette detector housing thecapillary is adjusted until the signal from Xuorescein ismaximized.

CE analysis of nuclear preparationsAn aliquot of the stained sample (11 § 3 nL) was

injected into the capillary at 400 V/cm. After the injec-tion, a vial containing buVer A replaced the sample vial,and electromigration proceeded at 400 V/cm for at least20 min. The Xuorescence of each individual SAT or NAPwas detected as it migrated out of the capillary. At theend of each separation, the capillary was reconditionedby pressure Xushing using buVer A contained within asyringe Wtted to the capillary through an adapter (ValcoInstruments, Houston, TX).

Detection of Xuorescent NAPs and SATsThe optical system contribution to peak intensity

error was assessed with standard Xuorescent micro-spheres (0.748 �m o.d.) (Polysciences, Warrington, PA).The relative standard deviation for the individual micro-sphere Xuorescence intensity was 22%. This value repre-sents the error expected in a determination based on aXuorescence measurement. NAPs labeled with the DNA-intercalating dye hexidium iodide or SATs labeled withthe Alexa Fluor conjugated anti-�-tubulin antibody sys-tem were detected as they migrated out of the capillaryby excitation with a 488 nm Ar-ion laser line (7.1 mW;Model 532-BS-A04, Melles Griot, Carlsbad, CA). Scat-tering was reduced by using a long-pass Wlter (505AELP, Omega Optical, Brattleboro, VT). Hexidiumiodide Xuorescence was monitored by selecting the Xuo-rescence in the 608–662 nm range with a bandpass Wlter(635DF55, Omega Optical). The Alexa Fluor signal wassimilarly detected using a bandpass Wlter (535DF35,Omega Optical) that selects the Xuorescence in the 518–552 nm range. The Xuorescence intensity of eachdetected event was measured using a R1471 (Hamama-tsu, Bridgewater, NJ) photomultiplier tube. The outputof the photomultiplier tube was digitized at 100 Hz usinga NiDaq I/O board (PCI-MIO-16XE-50, NationalInstruments, Austin, TX) and the data were saved asbinary Wle [18].

Data analysis and calculationsThe procedures for data analysis have been described

previously [18]. BrieXy, two electropherograms wereobtained from the raw data using an Igor-Pro (Wave-metrics Lake Oswego, OR) algorithm: one for broadevents and another for narrow events (i.e., those events

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4 N. Gunasekera et al. / Analytical Biochemistry 330 (2004) 1–9

attributed to NAPs or SATs). In the Wrst case, narrowevents were eliminated using an 18-point median Wlter. Asecond routine (PickPeaks) was used to process thenarrow event electropherogram in order to select andtabulate those events that had a signal-to-noise ratiolarger than Wve times the standard deviation of the back-ground [18]. For each event, PickPeaks determines themigration time and the peak intensity. A similar routinewas used to determine the peak width at 10% of the max-imum peak intensity for each event.

The number of �-tubulin molecules per event (n�) wascalculated from the peak height (h) using the expression

n� D 3300 £ h. (1)

The constant in Eq. (1) takes into account the detec-tor response, (3.8 § 0.4) £ 1018 peak area/mol of second-ary antibody, (n D 4), and the ratio of peak area to peakheight (48 § 8), (n D 9). It was also assumed that theinteractions between (i) �-tubulin and the primary anti-body and (ii) the primary and secondary antibodies have1:1 stoichiometry.

The distribution of peak intensities for each SATevent was determined using the PickPeaks program. Thecumulative SAT intensity, deWned as an estimate of therelative SAT content per 100 cells, was determined byintegrating this distribution. The cumulative intensity ofthe unlabeled sample was subtracted from that of thelabeled SAT sample, divided by the number of cells con-tributing to the signal and multiplied by 100.

The total electrophoretic mobility (�T) of a detectedevent can be calculated using the separation voltage (V),capillary length (L) and migration time (tM) as follows[29]:

�T D L2/(V · tM). (2)

In a bare fused-silica capillary the total electropho-retic mobility has three contributors: the intrinsic elec-trophoretic mobility (�e), the electro-osmotic Xow(�EOF), and a correction factor due to interactionbetween the migrating species (e.g., SATs or NAPs) andthe capillary wall (C).

�T D�e C �EOF C C. (3)

The electro-osmotic Xow contribution was estimatedat 6.9 £ 10¡4 cm2 V¡1 s¡1 using the previously establishedcurrent-monitoring method [30]. From the �T and the�EOF, the apparent electrophoretic mobility (�app) can becalculated as

�app D�T ¡ �EOF. (4)

Based on the broad peaks (i.e., those corresponding tospecies with appreciable diVusion during the separation),run-to-run variation of apparent electrophoretic mobili-ties was determined to be 2%. For the narrow events (i.e.,SATs or NAPs), the individual migration time values,

extracted from the data by the PickPeaks program, andEqs. (2) and (4) were used to calculate the apparent elec-trophoretic mobility. The collections of individual �appvalues were plotted as histograms.

Results and discussion

Microscopy reveals the presence of �-tubulin in nuclei

The microtubular-cytoskeleton network is typicallyimaged using immunolabeling and Xuorescence micros-copy. In this approach, Xuorescently labeled secondaryantibodies are bound to anti-�-tubulin antibodies, whichare speciWcally bound to �-tubulin, the main componentof microtubules [3–7]. In the present work, we used thisdual antibody labeling strategy to detect the presence ofmicrotubules in isolated nuclear preparations.

Fig. 1 shows a representative set of nuclei labeled withanti-�-tubulin antibodies isolated from cell culturestreated with or without colchicine. Colchicine disruptsthe cytoskeleton by binding to the tubulin monomersand thus preventing them from polymerizing to form thebackbone of the cytoskeleton [24–26]. The nuclei of cellsnot treated with colchicine are Xuorescent (109 intensityunits/pixel), indicating the presence of microtubules(Fig. 1A, top panel). Similar Xuorescence intensity wasnoted for the 1.4 �M colchicine treatment (Fig. 1B, toppanel; 109 intensity units/pixel), suggesting that this col-chicine concentration is not suYcient to disrupt the cyto-skeleton. In contrast, the Xuorescence intensities for the14 and 140 �M colchicine treatment (13 and 14 intensityunits/pixel, top panels of Figs. 1C and D, respectively)indicate colchicine has disrupted the microtubular-cyto-skeletal network. The morphology of the nuclei alsoappears to change with the colchicine concentration,with higher concentrations resulting in a less sphericalstructure (Fig. 1, bottom panels).

Fig. 1. Fluorescence images of isolated nuclei showing the presence of�-tubulin. NS1 cells were incubated with 0 �M (A), 1.4 �M (B), 14 �M(C), and 140 �M (D) colchicine in tissue culture medium for 15 h at 5%CO2 and 37 °C. The top and bottom panels in each case show the Xuo-rescence and phase-contrast image, respectively. Nuclei were isolatedfrom these cell cultures and stained with a Xuorescent antibody sys-tem, as described under Materials and methods. The magniWcationused was 60£; the bar in panel A denotes 5 �m.

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N. Gunasekera et al. / Analytical Biochemistry 330 (2004) 1–9 5

The change in nuclear morphology observed whenthe cells were treated with 14 and 140 �M colchicine mayalso indicate the loss of cell viability. Therefore, we usedthe Trypan blue test to determine the viability of cell cul-tures treated with colchicine. The percentage viabilitiesof the colchicine-treated cell cultures were 62 § 4%(140 �M), 84 § 9% (14 �M), and 95 § 5% (1.4 �M). More-over, the cell density of the 14 �M colchicine-treatedculture (2.0 § 0.7 £ 105 cells/mL) was not signiWcantlydiVerent from that of the untreated culture (2.3 § 0.5 £105 cells/mL), whereas the 140 �M colchicine-treatedsample had a lower cell density (1.4 § 0.2 £ 105 cells/mL).Taking into consideration cell density, viability, and theextent of cytoskeletal disruption, 14 �M colchicine wasselected for the treatment of cell cultures used in the CEanalyses described below.

Capillary electrophoresis of subcellular aggregatescontaining �-tubulin

Prior to CE analysis, the Wnal isolated nuclear prepa-ration was treated with the anti �-tubulin Xuorescentantibody system described under Materials and meth-ods, followed by centrifugation to pellet out SATs. Anyunbound antibodies, free �-tubulin, and small microtu-bule fragments not bound to nuclear species areexpected to stay in the supernatant at this step. Thus, therecovered pellet contains SATs consisting of nuclei withattached cytoskeleton remnants and large cytoskeletalaggregates.

CE-LIF of this pellet conWrmed the presence of SATsin the Wnal nuclear preparation (Fig. 2). Trace A is anelectropherogram that shows events corresponding toSATs found in a typical immunolabeled nuclear pellet.The arrow points to an expansion of the region from»232.5 to 240.2 s, which shows a representative collec-tion of the narrow individually separated events. Theentire electropherogram showed a total of 743 eventswith a peak width of 23 § 6 ms. As a control, the pellet ofan unlabeled preparation was analyzed by CE-LIF. Asshown in Fig. 2 (trace C), the number of narrow eventsin this case was low, indicating that most of the detectedevents are indeed SATs. Replicate analyses determinedthat the number of narrow events was always higher forthe immunolabeled (794 § 165, n D 3) than for the unla-beled (20 § 2, n D 2) nuclear pellets. Based on these val-ues, the fraction of false positives to the total number ofdetected events is less than 2.5%. Thus, about 97.5% ofthe narrow events correspond to SATs.

The CE-LIF analysis of the supernatant remainingafter isolation of the immunolabeled nuclear pellet(Fig. 2, trace B) produces electropherograms that show atypical broad peak (peak width D 37.2 s) that saturatesthe detector (110 V). This peak most likely results fromspecies that diVuse appreciably during the separation.These species include the free Xuorescent antibody,

complexes of the primary and secondary antibodies, andlabeled microtubule remnants that failed to pellet outduring the centrifugation step. Thus, based on peakwidth, CE-LIF analysis distinguishes between SATs(narrow events) and species with signiWcant diVusion(broad events).

Estimation of �-tubulin molecules per SAT

The amount of �-tubulin bound to an SAT was calcu-lated using Eq. (1). This calculation is based on theassumptions that (i) the stoichiometry between antibod-ies and between the primary antibody and �-tubulin is1:1, and (ii) the detector response (peak area/mole) wasthe same for the Xuorescently labeled secondary anti-body free in solution or attached to an SAT via a pri-mary antibody. The numbers of �-tubulin molecules/SAT for all the SATs detected in the electropherogramshown in Fig. 2A are represented as a histogram distri-bution in Fig. 3. The number of �-tubulin molecules perSAT, (10.5 § 0.3) £ 103, corresponds to a level of impu-rity that cannot be detected easily by any other bioana-lytical technique. In fact, as few as 170 �-tubulin

Fig. 2. Electropherograms of a nuclear preparation stained with a Xuo-rescent anti-�-tubulin antibody system. The top electropherogram (A)corresponds to a sample stained with the anti-�-tubulin antibody sys-tem, as described under Materials and methods. The arrow points toan expansion of the 232.5–240.2 s region of this trace. The middle elec-tropherogram (B) corresponds to the supernatant collected after thestained nuclear preparation is washed to remove unbound antibodyand other material not bound to nuclei. The bottom electropherogram(C) results from analysis of an unstained nuclear preparation. Forclarity, traces B and C are oVset by 15 and 4 V, respectively. The sam-ple was injected electrokinetically (400 V/s, 5 s) into a bare fused-silicacapillary (39.7 cm) and electrophoresed at 400 V/cm in buVer A.

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6 N. Gunasekera et al. / Analytical Biochemistry 330 (2004) 1–9

molecules per SAT can be detected using the CE-LIFmethod described here. Of course, due to the assump-tions made above and the variation in the detectorresponse (i.e., 22% relative standard deviation), the levelof �-tubulin molecules per SAT reported here should beconsidered approximate.

Colchicine treatment lowers number of SATs

Table 1 summarizes the total number of SATsdetected in immunolabeled nuclear pellets from cell cul-tures untreated or treated with 14�M colchicine. Asindicated, without colchicine treatment, 794 § 165(n D 3) SATs were detected, while this number wasreduced to 258 § 94 (n D 3) when the cell culture wastreated with 14�M colchicine. The diVerence betweenthese averages is signiWcant (90% conWdence level).

The comparison between the treated and untreatednuclear preparations is more meaningful if the SATs arereported on a per cell basis. As reported in Table 1, SATs

Fig. 3. Histogram of number of �-tubulin molecules per SAT. Thenumber of �-tubulin molecules per SATs for the events detected inFig. 2A was calculated using Eq. (1). This calculation assumes 1:1 stoi-chiometry between antibodies and between the primary antibody andthe �-tubulin. It is also assumed an equal detector response for theAlexa Fluor 488-labeled antibody in solution and bound to SATs.

decreased signiWcantly (90% conWdence level) from8.8 § 3.6 to 2.5 § 0.7 SATs per cell upon treatment with14 �M colchicine. When corrected for the number offalse positives (0.5 § 0.2 SATs per cell), these valuesshow that there is a 4-fold decrease in SATs as a result ofthe colchicine treatment.

Ideally, if all detected SATs were caused by cytoskele-ton remnants bound to intact nuclei, there should havebeen only one SAT per cell. Fragmented nuclei presentin nuclear preparations [15] could increase the numberof SATs per cell, because each of the fragments may con-tain microtubular remnants. Furthermore, it is possiblethat large nonnuclear bound cytoskeleton aggregates orantibody aggregates may be present in the Wnal nuclearpellet contributing to the observed 2.5 § 0.7 SATs percell (Table 1).

The eVect of colchicine treatment on reducing thepresence of �-tubulin in a nuclear preparation can alsobe appreciated if the cumulative intensity, resulting fromadding the individual intensities for all detected SATs inan electropherogram, is compared between the colchi-cine-treated and the untreated cases (Table 1). Thecumulative intensity ratio of untreated to colchicine-treated nuclear preparations is 3.6 § 1.8.

Electrophoretic mobility distributions of SATs and NAPs

Fig. 4 shows a histogram of the apparent electropho-retic mobility values for each SAT, calculated with Eqs.(2) and (4). As described above, SATs are likely a hetero-geneous mixture of nuclei, nuclear fragments, and non-nuclear bound cytoskeleton aggregates. Therefore, it isnot surprising to Wnd such a wide electrophoretic mobil-ity range. As reported in various studies, the intrinsicelectrophoretic mobility (�e) of biological species is afunction of the surface charge and size [19,21]. Therefore,the observed mobility values for SATs are aVected bythese parameters. For those SATs that are due to nucleiwith bound cytoskeleton, the presence of the cytoskele-ton contributes to the overall electrical charge of the

Table 1Number and intensity of SATs in colchicine-treated and untreated culturesa

a Reported values correspond to the average of three consecutive runs, except for the negative control, which corresponds to two runs. Errors rep-resent one standard deviation.

b Values were determined using the data analysis procedures described under Materials and methods.c Values were calculated by dividing the total number of SATs by the total number of cells expected in each CE-LIF analysis when considering

the analyzed volume, the cell count in the original culture, and the various dilutions.d Values were determined by adding the peak intensity of each SAT in a given CE-LIF analysis. This value was corrected for contributions from

false positives by subtracting the cumulative peak intensity of those events detected in the CE-LIF analysis of nuclear pellets from cells not treatedwith colchicine or antibodies. The value is normalized to 100 cells.

Cell treatment Total number of SATsb SATs per cellc Cumulative SAT intensityd

Colchicine Antibodies

¡ + 794 § 165 8.8 § 3.6 7.2 § 2.1+ + 258 § 94 2.5 § 0.7 2.0 § 0.8¡ ¡ 20 § 2 0.5 § 0.2 —

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N. Gunasekera et al. / Analytical Biochemistry 330 (2004) 1–9 7

nucleus. Also, as reported previously, the cytoskeletonmay also be a determinant of nuclear size [14]. Since theresidual cytoskeleton bound to a nucleus is expected tovary, diVerent nuclei may display diVerent electropho-retic mobilities dependent on their cytoskeletal con-tent. It is also expected that antibodies used inimmunolabeling of SATs will have an eVect on chargeand size of SATs, thereby aVecting SATs electropho-retic mobility. Thus, the presence of cytoskeletal com-ponents would be expected to directly and indirectly(through antibody electrical charge contributions)aVect the apparent electrophoretic mobility of SATs.However, when cells were treated with colchicine,changes in the electrophoretic mobility distributions ofSATs were not observed (data not shown). Therefore,the cytoskeleton may not be the major contributor tothe apparent electrophoretic mobilities of SATs. Giventhat capillary wall interactions with proteins have beenshown to aVect overall migration times [31], it is pres-ently hypothesized that the correction factor in Eq. (3)may be a key factor in deWning the apparent electro-phoretic mobility of SATs. Decreasing the contributionfrom this correction factor may provide the means tobetter observe the eVect that the cytoskeleton has onthe mobility of SATs. Unfortunately, polymer-coatedcapillaries (e.g., polyacryloylaminopropanol coated),that are eVective in decreasing interactions betweenother organelle types (e.g., mitochondria) and capillarywalls, were not eVective when used with SATs as SATsdid not migrate out from the capillary during electro-phoresis. Thus, while the apparent electrophoreticmobility distribution serves as a new descriptor ofcytoskeletal contamination in nuclear preparations, the

Fig. 4. Apparent electrophoretic mobility distribution of SATs. Thehistogram is the average of the apparent mobility distributions ofSATs in three consecutive injections of the same nuclear pellet. Themobilities were determined as described under Materials and methods.The number of events in each bin (N) was normalized by the totalnumber of events in the corresponding distribution (NT D 743, 660, or979) and multiplying by 100. Bin size is 6.0 £ 10¡6 cm2/V s. CE-LIFconditions are the same as in Fig. 2.

key factors in the electrophoretic mobility of SATsrequire further investigation.

To determine the eVect of colchicine treatment on theapparent electrophoretic mobility of nuclear related par-ticles, we carried out CE-LIF analysis of hexidiumiodide-stained nuclear fractions from untreated and14�M colchicine-treated cells. By comparing apparentmobility distributions of NAPs from untreated (Fig. 5B)and colchicine-treated (Fig. 5A) cells, we Wnd thatcolchicine treatment did not have a detectable eVect onthese distributions. As for SATs, cytoskeletal remnantsbound to NAPs do not seem to be the main contributorsto the apparent electrophoretic mobility values of NAPs.As deWned in the introduction, NAPs are a heterogenoussample, consisting of intact nuclei, nuclear fragments,large DNA–protein complexes, etc. We cannot rule outthe possibility that if the sole species in NAPs werenuclei, then the eVect of colchicine on the mobility ofindividual nuclei may have been detectable.

Fig. 5. Apparent electrophoretic mobilities of NAPs. Mobility histo-grams are shown for NAPs resulting from cells treated with 14 �M col-chicine (A) and from an untreated cell culture (B). N is the number ofevents in each bin. Bin size is 1.2 £ 10¡5 cm2/V s. These histograms cor-respond to a combination of events detected in three replicate electro-pherograms. The treated sample was incubated with colchicine asdescribed under Materials and methods. Both samples were stainedwith 0.5 �M hexidium iodide for 30 min at room temperature prior toanalysis. CE-LIF conditions are the same as in Fig. 2. The mobilitieswere determined as described under Materials and methods.

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8 N. Gunasekera et al. / Analytical Biochemistry 330 (2004) 1–9

Conclusions

In this work, the presence of SATs in nuclear prepara-tions was detected by CE-LIF for the Wrst time. Thisunique method provides three parameters describingcytoskeletal impurities in nuclear preparations: thenumber of SATs per cell, the amount of �-tubulin in apreparation, and the apparent electrophoretic mobilitydistribution of the detected SATs. The Wrst two parame-ters clearly indicate that treatment of cells with colchi-cine prior to nuclear isolation leads to a decrease incytoskeletal impurities. The third parameter, the appar-ent electrophoretic mobility of SATs, was not aVected bytreatment with colchicine indicating that cytoskeletalremnants may not be the main factor aVecting theobserved mobility. This CE-LIF analysis is complemen-tary to the microscopic methods used to monitor thepresence of cytoskeletal components in biological sam-ples. However, it forgoes some of the time-consumingsteps in microscopic analyses including cell Wxing, imag-ing, and complicated image analysis procedures. Mostimportantly, the sensitivity of the CE-LIF approachdescribed here should allow for the detection of subze-ptomole levels (e.g., 170 �-tubulin molecules per SAT) ofimpurities in subcellular fractions. Future applicationsmay include the determination of other cytoskeletalproteins or the endoplasmic reticulum in nuclear prepa-rations.

Acknowledgments

This work was supported by NIH Grants GM61969(EA) and GM49928 (KMF). E.A. also acknowledgessupport from NIH K02-AG21453.

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