Copyright C Munksgaard 2001Traffic 2001 2: 385–394

Munksgaard International Publishers ISSN 1398-9219

Nuclear Relocation of Normal Huntingtin

Tao Tao and Alan M. Tartakoff*

Pathology Department and Cell Biology Program, Case

Western Reserve University, School of Medicine,

Cleveland, OH, USA 44106

*Corresponding author: Alan M. Tartakoff,

amt10@po.cwru.edu

In Huntington’s Disease (HD), the huntingtin protein(Htt) includes an expanded polyglutamine domain.Since mutant Htt concentrates in the nucleus ofaffected neurons, we have inquired whether normalHtt (Q16ª23) is also able to access the nucleus. We ob-serve that a major pool of normal full-length Htt ofHeLa cells is anchored to endosomes and also detectRNase-sensitive nuclear foci which include a 70-kDaN-terminal Htt fragment. Agents which damage DNAtrigger caspase-3-dependent cleavage of Htt and dra-matically relocate the 70kDa fragment to the nucleo-plasm. Considering that polyglutamine tracts stimu-late caspase activation, mutant Htt is therefore poisedto enter the nucleus. These considerations help ration-alize the nuclear accumulation of Htt which is charac-teristic of HD and provide a first example of involve-ment of caspase cleavage in release of membrane-bound proteins which subsequently enter the nucleus.

Key words: apoptosis, caspase 3, endosome, hunt-ingtin, nuclear import

Received 15 February 2001, accepted for publication26 March 2001

The trinucleotide repeat disorder, Huntington’s disease (HD),is due to an abnormal form of huntingtin (Htt) (.360kDa (Qn-Htt)) which includes a polyglutamine tract of 37–130 residuesencoded by CAG repeats. Htt lacks putative transmembranedomains or signal sequences (Figure 1A). Cytological and sub-cellular fractionation studies document its presence in thecytoplasm, but its detailed subcellular localization is not clear.For example, different publications report association of Httwith ‘particulate fractions’, the plasma membrane, endosom-es, clathrin, Golgi membranes, synaptic vesicles and microtu-bules (1–6). A secure characterization of its localization isnecessary in order to understand its biology.

In HD, Qn-Htt is also found in the cytoplasm of most cells; how-ever, Qn-Htt (or its fragments – see below) forms striking nu-clear inclusions in affected neurons, especially in the corpusstriatum, cortex and neocortex. Nuclear inclusions of proteinswith polyglutamine tracts are also characteristic of distinct re-

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gions of the brain in other trinucleotide repeat diseases. Sinceubiquitin, Hsp70 and proteasomal subunits localize to these in-clusions, they may be sites of degradation (7–9).

Considering that Htt is not normally concentrated in the nu-cleus, it has not been clear why Qn-Htt is in the nucleus ofaffected cells. One unexplored possibility is that Htt has theintrinsic ability to relocate upon stress, regardless of whetherit includes a polyglutamine tract, and that affected cells havebeen exposed to – and cannot combat – such stress. Manyother proteins do relocate to the nucleus upon stress (10, 11).Proteolytic fragmentation of Qn-Htt – as is found in affectedregions of the brain – might facilitate relocation (12, 13).Moreover, since polyglutamine-containing domains can as-sociate with each other (14, 15), encounters with polyglutam-ine-containing proteins could result in progressive accumu-lation or aggregation of Qn-Htt, possibly stabilized by trans-glutaminase-catalyzed cross-linking. Interestingly, recentstudies indicate that exposure of cells to appropriate hor-mones can regulate such aggregation (16, 17).

To model aspects of HD and related diseases, transgenic miceand Drosophila have been investigated. Most of these studiessupport the hypothesis that overexpression of polyglutamine-containing proteins in the nucleus is toxic; however, aggre-gation itself is not required (18–26). In fact, some nuclear in-clusions are also found in non-neuronal tissues (27).

Evidence against a central role of the nuclear inclusions per

se has also been obtained in studies of transfected intact ortruncated Htt in neuroblastomas, kidney epithelial cells,striatal cells, the PC12 pheochromocytoma, etc. (17, 28–36).Other authors report that aggregates of fragments of Qn-Httwhich form in the nucleus can be replaced by cytoplasmicaggregates by adding a nuclear export signal and vice versa,without affecting toxicity (29, 37).

The observations described in the preceding two paragraphsmake use of distinct models. For example, in the transfectionstudies, different fragments of Htt with different lengths ofpolyglutamine are overexpressed using different promoters indifferent cell types which are examined after a few days. Acomparable variety of experimental conditions is used for thetransgenics. It is therefore not clear which observations arethe most relevant to HD.

Given these disparate observations and hypotheses, we haveinvestigated the distribution of normal Htt expressed at nor-mal levels from its own promoter. We document a nuclearpool of an N-terminal 70kDa fragment of Htt and observe

Tao and Tartakoff

Figure1: Structure of huntingtin and localization of wild-type Htt in HeLa cells. As shown in panel A, wild-type Htt in-cludes a modest polyglutamine tract near its amino terminus(Q6ª34), a putative caspase-3 cleavage site and two putative basicnuclear localization signal domains (NLS A: RPKKELSATKKDRV;NLS B: RRKGKEKEAGEQASVPLSPKKG). Htt lacks transmembranedomains or signal sequences. The regions of Htt which are recog-nized by the antibodies used are designated (AP194, MAb-N,HDB4E10, MAb-C, HDC8A4). In panel B, HeLa cells were stainedwith MAb-N. As shown, most cytoplasmic Htt localizes adjacent toone face of the nucleus. Tiny foci of fluorescence (arrow) are alsoseen in more peripheral areas of the cytoplasm. Scale barΩ5 mm.

that agents which cause DNA damage greatly increase theamount of the nuclear fragment. Equivalent cell-specific per-turbations may initiate nuclear entry of Qn-Htt in neurons inHD.

Results

Polyglutamine tract lengths of Htt in HeLa cells

The lengths of the polyglutamine tracts of the Huntingtonalleles of HeLa cells were determined to lie well within thenormal range, having 16 and 23 CAG repeats (Methods).

Localization of Htt in the cytoplasm

We have localized Htt in HeLa cells using a monoclonal anti-body (MAb) which recognizes an epitope near the N-ter-minus of Htt (MAb-N:MAb-2166). As shown in Figure 1(B),Htt is largely cytoplasmic and is usually concentrated at oneface of the nucleus. Its nonuniform distribution suggests that itis not soluble. A similar distribution is obtained with other anti-bodies which recognize the extreme N-terminus of Htt(AP194) or carboxy-terminal epitopes (MAb-2168, HDC8A4).Additionally, tiny foci of fluorescence are seen in more periph-eral areas of the cytoplasm. Similar distributions are seen in

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NT2 cells and in rat striatal neurons, where the signal also ex-tended into processes.

As shown in Figure 2(A), the endoplasmic reticulum marker,protein disulfide isomerase, has a more symmetric peri-nuclear distribution than Htt. Moreover, when Triton X-100lysates of HeLa cells are fractionated on sucrose gradientsand analyzed by immunoblotting, Htt is recovered at the top,not in association with polysomes (not shown).

Htt is more broadly distributed than Golgi markers (a-manno-sidase (Figure 2), b-COP (not shown)). Its distribution re-sembles that of early endosomes detected with an antibodyto Rho B (38) to a greater extent than that of late endosomes(detected with an antibody to Rab7 (39)) or the lysosomalmembrane marker, LAMP-1 (not shown (40)). To further in-vestigate the relation of Htt to endosomes, we have followedthe fluid-phase endocytic tracer, fluoresceinated-BSA. Al-though the distribution of this tracer after a 20-min pulsedoes not resemble the distribution of Htt, the two distri-butions are quite similar after a 1-h chase.

The Golgi perturbants, brefeldin A (Figure 2A) and monensin(not shown), do not alter the distribution of Htt over severalhours, although the distribution of Golgi markers is affected.An association with microtubules is implied by experimentsin which cells are treated with nocodazole before fixation: asshown, this treatment causes Htt to become broadly distrib-uted throughout the cytoplasm.

Density perturbation of Htt-containing vesicles

To learn whether Htt associates with endosomes, we havefractionated HeLa cells which have endocytosed 15nM col-loidal gold–BSA for 90min. As shown in Figure 2(B), muchof the full-length Htt acquires distinct sedimentation charac-teristics in Percoll gradients, by comparison to controls lack-ing gold. The position of the Golgi marker, a-mannosidase, isunchanged in such experiments. The density-shifted peak ofHtt is distinct from the localization of the lysosomal marker,b-hexosaminidase (not shown).

Some Htt does not show a density shift in these experiments,presumably because some is truly soluble (and remains atthe top of the gradient) and perhaps because some endo-somes rupture and lose their gold.

Differential extraction of cytoplasmic Htt

When the plasma membrane is permeabilized with minimaldoses of digitonin, followed by fixation, Htt (as well as ER,Golgi and endosomal markers (not shown)) can still be de-tected by indirect immunofluorescence. Thus, Htt is an-chored to cytoplasmic structures and does not primarily as-sociate with cholesterol-rich membranes. This associationpersists in cells pretreated with nocodazole (not shown).When digitonin treatment is followed by various extractions(0.5 M KCl, 25mM EDTA, 200mg/mL RNase A) before fix-ation, the distribution of most cytoplasmic Htt is notaffected. Parallel Western blotting studies show that the

Relocation of Normal Huntingtin

Figure2: Htt associates with endosomes and depends on the integrity of microtubules. As shown in panel A, the distribution ofcytoplasmic Htt (a, b) is not altered by the Golgi perturbant, brefeldin A (BFA, 5 mg/mL, 1h), although the Golgi marker is dispersed underthe same conditions (c, d). Htt was extensively dispersed throughout the cytoplasm when cells were pretreated with 10 mM nocodazole for2h to depolymerize microtubules before fixation (e). By comparison to several subcellular organelles, the distribution of Htt is similar to thatof endosomes labeled by endocytosed fluorescent-BSA (Cells were allowed to endocytose fluorescent BSA (1.0mg/mL, Sigma .9771) for20min, washed and reincubated for 60 min) (f) and early endosomes (Rho B, g), but different from that of the ER (PDI, h). Panel B showsPercoll gradient analysis of homogenates of control cells and cells that have endocytosed colloidal gold–BSA for 90min. Note that a newHtt peak appears in high-density fractions after endocytosis. The distribution of lysosomes and the Golgi marker, a-mannosidase, is notchanged (not shown). Scale barΩ5 mm.

limited soluble pool of Htt increases upon exposure to 0.5M KCl.

In agreement with earlier biochemical experiments with cru-de membrane fractions (2), cytosolic Htt is extensively ex-tracted by treatment with the nonspecific detergent, Triton X-100, as are ER and Golgi markers (not shown).

Localization of Htt in the nucleus by indirect

immunofluorescence (Figure 3A)

Little Htt is normally detected in the nucleus with MAb-N;however, examination in the confocal microscope or pretreat-ment with Triton X-100 considerably increases the visibility ofintranuclear Htt-positive inclusions, presumably by removingcytoplasmic and nucleoplasmic Htt. These foci can also bedetected with the antibody raised against the extreme N-terminus of Htt (AP194), but not with MAb-C. These in-clusions bear no obvious relation to the nuclear envelope, asdetected with a MAb which recognizes Nup153. Moreover,

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they do not associate with the nucleoli, Cajal bodies, or PMLbodies (not shown).

In parallel experiments using Triton X-100, the Htt-positivenuclear inclusions are extracted by RNase A (Figure 3A), butnot by KCl, EDTA or DNase I (not shown).

We conclude that a large fraction of cytoplasmic full-lengthHtt is anchored to endosomes and depends on microtubulesfor its localization. As explained in the next section, the nu-clear pool – by contrast – is an N-terminal 70kDa fragmentwhich associates with RNA.

Size of cytosolic and nuclear Htt

To evaluate the size of Htt in the nucleus, both completecell lysates and nuclear and cytosolic fractions isolatedfrom them were analyzed by Western blotting (Figure 3B).As shown in Figure 4(B), the predominant form of Htt inthe cell lysate is full-length (350kDa); however, a minor

Tao and Tartakoff

Figure3: Localization and size of nuclear Htt. In panel A, although most cytoplasmic Htt resists extraction by digitonin (which permeabil-izes the plasma membrane), Triton X-100 removes almost all cytoplasmic Htt signal (c, d). Strikingly, after Triton X-100, foci of nuclear Httare detected by MAb-N (c), but not by MAb-C (d). Htt-positive foci are sensitive to RNase A (e), but resist DNase I, KCl or EDTA extraction(not shown). a and b are control HeLa cells stained with MAb-N or MAb-C, respectively. In panel B, the Western blots show that at steadystate, there are two forms of Htt: a major form (350kDa) which accounts for.90% of the total; and a 70-kDa form which is recovered innuclear fractions. The 70-kDa form is detected only by MAb-N, suggesting that it corresponds to an N-terminal fragment and that cleavageprecedes the nuclear entry of Htt. W, whole HeLa lysate; S, low-speed supernatant; N, nuclear fraction. Since there are two basic putativenuclear localization signals in Htt (see Figure 1), fluorescent BSA conjugates of corresponding peptides were microinjected into the cytoplasmof HeLa cells. These NLS conjugates did not migrate into the nucleus over 4 h at 37 æC. A positive control conjugate (NLSLTAg–rhodamine–BSA) did enter the nucleus over this period, suggesting that the import of Htt is not by the classical importin a/b pathway. Scale barΩ5 mm.

70kDa fragment is enriched in nuclei. The fragment lacksthe epitope recognized by MAb-C, but can be recognizedby an antibody (AP194) raised against a synthetic Htt pep-tide (residues 1–17). MAb-C and HDB4E10 react only withthe full-length form. The abundance of the 70kDa fragmentis markedly reduced by culture in the presence of the pan-caspase inhibitor, Z-VAD-FMK (not shown).

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Import and export of Htt

The surprising presence of a nuclear pool of Htt might reflectcyclic entry of Htt into the nucleus. Since Htt includes severalputative leucine-rich nuclear export signals, we have treatedcells with leptomycin B for 0–8h. This drug inhibits Crm1,which exports proteins with such nuclear export signals (41).Although a shuttling Rev-GFP fusion protein can be trapped

Relocation of Normal Huntingtin

Figure4: Agents which damage DNAcause nuclear accumulation of Htt. Inpanel A, 5mg/mL actinomycin D (AD)causes striking relocation of Htt to the nu-cleus in HeLa cells (b). Neither 0.05 mg/mLactinomycin D (which inhibits only pol I) (c)or 50mg/mL a-amanitin (which inhibits onlypol II) (d) nor the combination of 0.05 mg/mL actinomycin D and 50mg/mL a-amani-tin (e) has the same effect. The well-char-acterized DNA damage agent, cisplatin,also causes conspicuous nuclear accumu-lation of Htt (f). In panel B, HeLa cells wereincubated with either the pan-caspase in-hibitor (Pan-Cs-In), Z-VAD-FMK, the cas-pase-1 inhibitor (Cs-1-In), Z-WEHD-FMK,or the caspase-3 inhibitor (Cs-3-In), Ac-DEVD-FMK for 1,2h before adding eitheractinomycin D or cisplatin (Cin). Both Z-VAD-FMK and Ac-DEVD-FMK stop thecleavage of Htt, but Z-WEHD-FMK doesnot. (g) and (h) are confocal images takenat a slightly lower magnification than theother images. Scale barΩ5 mm.

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Tao and Tartakoff

in the nucleus by this treatment, the titer of nuclear Htt doesnot increase (not shown).

Since the size of the nuclear fragment of Htt exceeds thatwhich can diffuse through nuclear pores, some specific im-port mechanism must be involved. We have synthesizedpeptides corresponding to two putative basic nuclear localiz-ation signals (NLS) of Htt and coupled them to fluorescentBSA. Upon microinjection into the cytoplasm of HeLa cells,they do not enter the nucleus over 4h (not shown). We there-fore conclude that importin a (which binds many importcargos with basic NLSs) is unlikely to be involved.

Nuclear relocation of Htt

Strikingly, following on our observation of RNase-sensitivityof nuclear Htt, we identified a condition which causes majorrelocation of Htt to the nucleus of HeLa cells. We first ob-served that incubation with 5mg/mL actinomycin D (AD) forseveral hours, which inhibits RNA polymerases I and II,caused a major increase in the nuclear signal (Figure 4). Thispool of Htt is not extracted by Triton X-100 (not shown).

We were surprised to see that low doses of AD which inhibitonly polymerase I or doses of a-amanitin (10ª8 M) whichselectively inhibit RNA polymerase II, do not have a compar-able effect (Figure 4A). We therefore tested the effects of a-amanitin and low doses of AD (for polymerase I), only tofind no more than a modest effect. We conclude that someconsequence of high doses of AD other than inhibition ofRNA polymerases underlies the relocation of Htt to the nu-cleus. Since high doses of AD damage DNA and triggerapoptosis (e.g. 42–44), we then tested the effects of otheragents which damage DNA (45–47) and observed that cis-platin (Figure 4A) and etoposide (not shown) also cause nu-clear relocation of Htt.

Western analysis of cells before and after AD or cisplatintreatment shows that these agents cause a major reductionof full-length Htt and corresponding increase of the 70-kDafragment. The remaining full-length Htt and a 280-kDa frag-ment are enriched in cytoplasmic extracts of cells and the70-kDa fragment is enriched in isolated nuclei. Smaller frag-ments are not seen (Figure 4 and not shown).

The pan-caspase inhibitor, Z-VAD-FMK, blocks cleavage dueto AD, as does the caspase-3 inhibitor, Ac-DEVD-FMK. Thecaspase-1 inhibitor, Z-WEHD-FMK, does not block cleavage.Thus, caspase-3 is likely to account for the cleavage of Htt.

Discussion

Each of the trinucleotide repeat polyglutamine diseases (HD,the spinocerebellar ataxias, DRPLA, SBMA) shows distinctregional specificity within the brain. This specificity presum-ably reflects features of the prevailing environment of eachregion that impact primarily on one or another of the corre-sponding proteins. Apart from their polyglutamine tracts,

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these proteins do not resemble each other. It therefore isimportant to understand the fundamental biology of theseproteins in the absence of polyglutamine expansion.

No function has been ascribed to Htt. Its structure is notstrongly indicative of any specific role or subcellular localiz-ation, although there are indications that it binds several pro-teins (48, 49). Htt is not required at the level of individualneurons for survival or differentiation in culture (50); however,it is required in extraembryonic tissues for normal embryo-genesis (51).

Most cytological studies conclude that endogenous Htt islargely cytoplasmic; however, there is no agreement as towhere it concentrates. We and others also have detectedboth a membrane-bound and a soluble pool, which may bein equilibrium with each other. Possible nuclear pools of mi-nor amounts of endogenous Htt have frequently been de-scribed; however, there is no agreement as to whether theintact protein is present (1, 52).

These controversies have been compounded by investigationof transfected cells and transgenic organisms, both sinceoverexpression is generally involved (which may saturate anynormal binding sites and may activate caspases), and sincein many of these studies severely truncated forms of Htt areexpressed. Especially in the context of investigations of thegenesis and distribution of nuclear and cytoplasmic ‘in-clusions’, such expression systems generate observationswhich are difficult to relate to normal or pathogenic events.

Given these uncertainties, we have investigated normal Htt,without forcing overexpression. We observe that full-lengthHtt is cytoplasmic, and that an N-terminal fragment accumu-lates at nuclear foci, which may or may not be aggregated.In the cytoplasm, a major pool of full-length Htt is anchoredto endosomes, thereby suggesting that any malfunction ofthe endocytic path (or other aspects of membrane traffic)may influence the fate of wt or polyglutamine-expanded Htt.An association with endocytic structures may also be relatedto the deficit in neurotransmission which is characteristic ofHD (e.g. (18)).

DNA damage, like other stresses, causes a number of pro-teins to relocate to the nucleus, where at least some of themsuch as the AP endonuclease, NF-kB and the tumor sup-pressor, p53, play a protective role (53–55). Since the mag-nitude of such relocation is striking for Htt, it is reasonableto propose that prevailing stresses normally cause modestamounts of Htt to enter the nucleus. There has been no pre-vious evidence that caspases mediate the nuclear relocationof proteins from binding sites on cytoplasmic membranes.Proteolytic release and nuclear relocation are known in othercontexts, however (56–59).

Although normal Htt may protect cells against apoptosis(60), nuclear entry of Qn-Htt may have detrimental effects,e.g. due to interaction with transcription factors which in-

Relocation of Normal Huntingtin

clude polyglutamine tracts (61, 62). Indeed, the expressionof polyglutamine-containing fragments of Htt does alter thetranscriptional profile of neurons (63). Since polyglutaminetracts can activate caspases (64–66) and since Qn-Htt ap-pears especially prone to in vitro caspase cleavage (67) –judging from the present evidence that Htt enters the nu-cleus – one can think of Qn-Htt as being poised to relocate.

Different caspases have been implicated in Htt cleavage andin HD (32, 68, 69); however, our observations are clear-cutin implicating caspase-3 in Htt cleavage upon DNA damage.Possibly, since caspase-1 cleavage sites exist near the casp-ase-3 site, they may be attacked under other circumstanceswhich also relocate the 70-kDa fragment. In agreement withsome (37) but not all (70) previous observations, we observethat the basic putative NLSs of Htt do not have NLS activity.The covalent NLS and path of its import therefore remain tobe identified.

Our concluding model for the transport of Htt thus involvesits release from a binding site on endosomes (Figure 5). It willbe of interest to identify the anchoring sites, the physiologicalcircumstances which cause its cleavage, the signals andtransporters which mediate its entry into the nucleus, and thepossible transcriptional consequences of its entry. The pres-ent observations show that these events are of interest in thecontext of normal Htt as well as for Qn-Htt and thereforesuggest that nuclear Htt is of biological importance (Table 1).

Materials and Methods

Cell growth and treatment

Adherent HeLa cells were grown in DMEM (GIBCO BRL) plus 10% de-complemented FBS at 37 æC in a 10% CO2/air incubator. For microscopic

Figure5: Model of Htt relocation. At steady state, most Htt isin the cytoplasm, associated with the surface of endosomes. Wepostulate the existence of an adaptor protein which mediates mem-brane binding, since Htt itself does not have a hydrophobic domain.A lesser soluble pool is also present. Limited amounts of cyto-plasmic Htt normally are cleaved by caspase 3, enter the nucleusand concentrate in RNase-sensitive foci. This process is dramati-cally exaggerated upon caspase activation by agents which causeDNA damage.

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examination, cultures were on glass coverslips. The polyglutamine repeatsfor the Huntington genes in HeLa cells were determined by Dianon Sys-tems (Stratford, CT, USA).

Cells were incubated with either 10mg/mL of actinomycin D (Sigma A1410(St Louis, MO, USA)) for 5h at 37 æC or 20mg/mL of cisplatin (SigmaP4394) (freshly prepared in PBS) for 16h. Cells were washed in PBS, lysedin prewarmed 1¿SDS-PAGE loading buffer and immediately boiled.

Antibodies

Anti-Htt antibodies: MAb-N (raised against Htt181ª810; Chemicon, MAb2166 (Temecula, Ca, USA)); MAb-C (raised against Htt2146–2254; Chem-icon, MAb 2168); rabbit AP194 (raised against a synthetic peptide includ-ing residues .1–17, from Dr A. Sharp, CWRU); Monoclonals: HDB4E10(Htt1844–2131), HDC8A4 (Htt2703–2911) (both from Dr G. Morris, NEWales Institute, UK (71)).

Other antibodies: Rabbit anti-a-mannosidase (from Dr M. Farquhar,UCSD); rabbit anti-Rho B (Santa Cruz, sc-180, from Dr C. Carlin, CWRU);goat anti-Rab 7 (Santa Cruz, sc-6563, from Dr C. Carlin, CWRU); mouseanti-protein disulfide isomerase (from Dr S. Pimplikar, CWRU); rabbit anti-Gar 1 (from Dr G. Matera, CWRU); rabbit anti-p80 coilin (serum R288,from Dr E. Chan, The Scripps Research Institute).

Indirect immunofluorescence

Coverslips were fixed with 3.7% formaldehyde for 30min on ice and thenquenched and permeabilized with 0.1 M glycine (pH7.0), 0.1% Triton X-100 in PBS. Coverslips were stained with primary antibodies for 1h atroom temperature and subsequently labeled with FITC – or Rhodamine-conjugated secondary/tertiary antibodies from Jackson ImmunoResearchLaboratories, Inc. (West Grove, PA, USA).

Detergent extractions

After washing with PBS, HeLa cells received either 40mg/mL of digitonin(Calbiochem, La Jolla, CA, USA) or 0.5% Triton X-100 (Fisher, Pittsburgh,PA, USA) in cold PBS and were kept on ice for 5min. Cells were washedwith PBS and then incubated in PBS with 5mM MgCl2 with or withouteither 200mg/mL of RNase A, 50 U/mL of DNase I, 0.5 M KCl or 25mM

EDTA on ice for 30min. The supernatants were removed and the cellswere either fixed in formaldehyde or dissolved in SDS-containing bufferfor Western blotting. The supernatants were clarified by centrifugation for30min at 18500¿g before precipitation with 10% TCA and Western blot-ting.

Preparation of Htt-NLS peptide conjugates

High-purity BSA (Sigma 7638) was labeled with FITC (Molecular Probes,F-1906 (Eugene, OR, USA)) or RITC (Pierce 46112 (Rockford, IL, USA)).Two putative Htt NLS peptides (NLS A: CRPKKELSATKKDRV; NLS B:CRRKGKEKEAGEQASVPLSPKKG) or an NLS peptide from SV40 large Tantigen (CGTGPKKKRKVGG) were cross-linked to the fluorescent BSA bysulfo-SMCC (Pierce, Rockford, IL, USA) (72). The conjugates were de-salted by gel filtration. SDS-PAGE was used to estimate coupling ratios.

Microinjection

Microinjections of 2.5mg/mL of FITC-BSA-NLSHtt conjugates and3.6mg/mL of RITC-BSA-NLSLTAg conjugate were made into the HeLacell cytoplasm. Microinjected HeLa cells were either fixed immediatelyor fixed after a 4-h reincubation at 37 æC.

Isolation of nuclear fractions

Ninety per cent confluent HeLa cells were scraped into isotonic buffer(0.15 M NaCl, 1.5 M MgCl2, 10mM Tris-HCl, pH7.4, 1mM PMSF, 1mg/mLaprotinin, 1mg/mL leupeptin, 1mg/mL pepstatin). Triton X-100 was addedto 0.5%. After 5min on ice, cells were broken with a Dounce homogenizer.

Tao and Tartakoff

Table1: Comparison of properties of wildtype and mutant Htt

Normal Htt Normal Htt Mutant Htt Mutant Httafter AD in unaffected cells in affected cells

Cytoplasmic Endosome/soluble Largely absent Unchanged Largely absentlocalizationCleavage Modest Extensive ? ExtensiveNuclear Foci Nucleoplasm ? Accumulation/localization aggregationApoptosis Anti-apoptotic Ongoing Sensitized Post-apoptosis

Lysates were spun at 200¿g for 10min at 4 æC to sediment nuclei. Thesupernatant was frozen immediately. The nuclear pellet was frozen aftertwo washes by resuspension in isotonic buffer.

Density perturbation

Preparation of 15nM colloidal gold–BSA: Four milliliters of 1% HAuCl4 (Al-drich Chemical Co., Milwaukee, WI, USA) was mixed with 200mL H2Oand boiled. Ten milliliters of 1% sodium citrate was added and the boilingcontinued for 10min after the color turned burgundy. The resulting solutionwas adjusted to pH7.0 and stabilized by addition of BSA (55mg/mL, final).Fourteen milliliters of 1% PEG 20000 was added and the colloidal gold–BSA was pelleted by centrifugation at 45000¿g for 80min. The pelletwas resuspended in PBS and frozen at ª20 æC in a total volume of 2mL.

Endocytosis of colloidal gold–BSA: One half a mililiter of the gold–BSAsolution was pelleted and resuspended in 10mL of complete DMEM andadded to a 150-mm plate of 90% confluent HeLa cells for 90min at 37 æC.Control HeLa cells were not exposed to gold–BSA.

Subcellular fractionation: After endocytosis, the control and experimentalcells were washed with cold PBS and recovered by scraping into PBS.Cells were sedimented, resuspended in 2mL of homogenization buffer(0.25 M sucrose, 10mM HEPES, pH7.2) and spun at 300¿g for 5min.The pellet was resuspended in 1mL of homogenization buffer with 1mM

PMSF, homogenized with 45 strokes of a Dounce homogenizer and load-ed on top of 9mL of 23% Percoll (Amersham 17–0891–01 (Princeton, NJ,USA)) in homogenization buffer and centrifuged at 23000rpm in a Ti50fixed-angle rotor (Beckman Instruments, Palo Alto, CA, USA) for 60min.Htt, a-mannosidase and b-hexosaminidase in each fraction and in the pel-let were detected either by Western blotting or enzyme activity assay.

Western blotting analysis: After boiling in SDS loading buffer, equal vol-umes of fractions were electrophoresed on 6% SDS-polyacrylamide gelsand transferred to Immobilon-P membranes (Millipore Cat. IPVH00010).Membranes were blocked in TBS-T (20mM Tris-HCl, pH7.6, 137mM NaCl,0.1% Tween-20) containing 5% skim milk and incubated with primaryantibodies (MAb-N (1 :1000), MAb-C (1 :1000) and a-mannosidase(1 :2000)) in the same solution. Membranes were developed followingthe protocol of ECL (Amersham).

B-hexosaminidase activity assay: Forty-five microliters of each Percollgradient fraction was mixed with 150mL of assay buffer (0.1 M 2-(N-mor-pholino) ethane sulfonic acid (Sigma M-3885), pH6.5, 0.2% Triton X-100)and 50mL of enzyme substrate (4mM p-nitrophenyl-acetyl-b-D-glucosami-nide (Sigma N-9376)) was added. After incubation at 37 æC for 90min,100mL of reaction mixture was mixed with 100mL of stop solution (0.5 Mglycine, pH10.0) and the absorbance was measured at 405nm.

Inhibition of cleavage of Htt by caspase inhibitors

Ninety per cent confluent HeLa cells were incubated with either 20mM ofthe pan-caspase inhibitor, Z-VAD-FMK (R & D Systems, Inc., or 50mM of

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the caspase-3 inhibitor, Ac-DEVD-FMK (Bio-Rad) or the caspase-1 in-hibitor, Z-WEHD-FMK (R & D Systems, Inc., Minneapolis, MN, USA) for1–2h at 37 æC before adding 10mg/mL of actinomycin D or 20mg/mL ofcisplatin. The cells were incubated at 37 æC for either 6h (for actinomycinD) or 16h (for cisplatin) before lysis.

Acknowledgments

This research was supported by a generous grant from the Wills Founda-tion.

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