Molecular and Biophysical Characterization of Assembly-Starter

Molecular and Biophysical Characterization ofAssembly-Starter Units of Human Vimentin

Norbert Mucke1†, Tatjana Wedig2†, Andrea Burer3, Lyuben N. Marekov4

Peter M. Steinert4‡, Jorg Langowski1, Ueli Aebi3 andHarald Herrmann2*

1Division of Biophysics ofMacromolecules, GermanCancer Research Center, ImNeuenheimer Feld 280D-69120 Heidelberg, Germany

2Division of Cell BiologyGerman Cancer ResearchCenter, Im Neuenheimer Feld280, D-69120 HeidelbergGermany

3Maurice E. Muller Institutefor Structural BiologyBiozentrum, University ofBasel, Klingelbergstrasse 70CH-4056 Basel, Switzerland

4Laboratory of Skin BiologyNIAMS, National Institutes ofHealth, Bethesda, MD20892-2752, USA

We have developed an assembly protocol for the intermediate filament(IF) protein vimentin based on a phosphate buffer system, which enablesthe dynamic formation of authentic IFs. The advantage of this physiologi-cal buffer is that analysis of the subunit interactions by chemical cross-linking of internal lysine residues becomes feasible. By this system, wehave analyzed the potential interactions of the coiled-coil rod domainswith one another, which are assumed to make a crucial contribution to IFformation and stability. We show that headless vimentin, which dimerizesunder low salt conditions, associates into tetramers of the A22-typeconfiguration under assembly conditions, indicating that one of the effectsof increasing the ionic strength is to favor coil 2–coil 2 interactions.Furthermore, in order to obtain insight into the molecular interactionsthat occur during the first phase of assembly of full-length vimentin, weemployed a temperature-sensitive variant of human vimentin, which isarrested at the “unit-length filament” (ULF) state at room temperature,but starts to elongate upon raising the temperature to 37 8C. Mostimportantly, we demonstrate by cross-linking analysis that ULF formationpredominantly involves A11-type dimer–dimer interactions. The presenceof A22 and A12 cross-linking products in mature IFs, however, indicatesthat major rearrangements do occur during the longitudinal annealingand radial compaction steps of IF assembly.

q 2004 Elsevier Ltd. All rights reserved.

Keywords: assembly; chemical cross-linking; intermediate filament;analytical ultracentrifugation; vimentin*Corresponding author

Introduction

Intermediate filament (IF) proteins are principalstructural components of all metazoan cells.1–5

Remarkably, they form two entirely different typesof fiber systems: (i) a meshwork-like assemblywithin the nucleus; and (ii) desmosome-anchoredor microtubule-associated individual IFs in thecytoplasm.6,7 Both systems segregate completely

even when assembled in the same compartmentafter forced expression, for instance, of cytoplasmicIF proteins in the nucleus.5,8 In contrast to nuclearIF proteins, the lamins, which in man are encodedby only three genes, cytoplasmic IF proteins consti-tute a highly heterogeneous superfamily encodedby at least 62 genes in the human genome.9 Duringembryonic development they are expressed incomplex patterns tightly coupled to programs ofcellular differentiation.10 This suggests that individ-ual IF proteins such as epidermal keratins, musculardesmin or the neurofilament triplet proteins, mayexhibit quite different molecular properties withregard to intrafilamentous associations as well asinteraction with other constituents of the differen-tially expressed cytoskeletal proteins.11

Vimentin is a marker protein for mesenchymalcells in mammalian species.12 However, it has alsobeen found in all other vertebrates looked at, from

0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.

†N.M. and T.W. contributed equally to this work.‡Deceased.

E-mail address of the corresponding author:[email protected]

Abbreviations used: B, colligative second virialcoefficient; DST, disulfosuccinimidyl tartrate; IF,intermediate filament; STEM, scanning transmissionelectron microscopy; TEB, transient electricbirefringence; ULF, unit-length filament; wt, wild-type.

doi:10.1016/j.jmb.2004.04.039 J. Mol. Biol. (2004) 340, 97–114

birds to fish.13–15 In contrast to keratins, whichform heteropolymers assembled from obligateheterodimers of two subgroups of sequence-relatedproteins, vimentin is able to form homopolymericfilaments. However, in the living cell it mayrepresent, as a kind of “multi-purpose” molecule,the obligate partner for IF proteins unable to self-assemble such as nestin and, to some degree, theglial acidic fibrillary protein GFAP.5,7,16

Together with microtubules and microfilaments,IFs constitute, dynamically cross-bridged byvarious associated proteins, the cytoskeletonproper. The former two filament systems are madefrom globular proteins, tubulin and actin, bothexhibiting nucleotidase activity, which is instru-mental in the generation of polar polymers. Incontrast, IFs are formed from extended fibrousproteins that are organized as parallel, in-registercoiled-coil dimers that do not exhibit any knownenzymatic activity. Moreover, IFs are much moreflexible than microtubules and microfilaments.17

At their first level of organization dimers associatelaterally into tetrameric complexes. This has beenshown by various methods, and the preferredtype of overlap has been determined to be, underlow ionic strength conditions, one in which twodimers interact in an approximately half-staggered,anti-parallel configuration via their coil 1 (forconcepts and overview, see published work2,3,18).

For tubulin and actin alike their recruitment intothe cytoskeleton has been shown to be dependenton chaperones and associated proteins.19 Muchless is known for IF proteins. However, tetramers,which are supposed to be the smallest stable unitsin vitro for cytoplasmic IF proteins, have indeedbeen demonstrated to exist, albeit in small amountsonly, in the living cell.20 The first IF-like assembliesfound in vitro are produced by laterally interactingtetramers to yield fibers of about 60 nm lengthand around 17 nm width, so-called “unit-lengthfilaments” (ULFs). Such ULFs are able to anneallongitudinally into filaments.21 ULFs or shortfilaments might be candidate complexes to betransported throughout the cells. Indeed, fusion ofsmall “dots” into particles, i.e. small aggregates ofULFs, and “squiggles”, i.e. short IFs, has beenobserved in living cells using GFP-taggedvimentin.22

We and others have long relied on a regimen tokeep IF proteins in the form of soluble oligomers:It involves denaturation in 8 M urea followed bydialysis into a low salt/high pH buffer such as5 mM Tris–HCl (pH 8.4), containing reducing andchelating agents to prevent oxidative cross-linkingby cysteine residues and to control divalentions.2,3,21 To induce assembly, a buffer is addedthat yields a neutral pH and a monovalent saltconcentration of up to 170 mM.23–25 In order toinvestigate whether vimentin could also be keptsoluble at physiological pH, we experimentedwith the bona fide physiological buffering ion, i.e.phosphate. A phosphate buffer of very low ionicstrength (0.7 mM, pH 7.5) was originally used in a

series of experiments by Kooijman andcolleagues.26 Employing transient electric bi-refringence (TEB) measurements, these authorsclaimed to have traced vimentin to dimers,tetramers (and/or octamers) and hexamers. More-over, by this technique they also attempted toinvestigate the assembly mechanism of IFs. How-ever, since TEB prevented the use of higher ionicstrength, they induced assembly by addition of 0.3to 2.5 mM magnesium chloride. Although somekind of filament assembly occurred under theseconditions,26 the structures obtained appearedvery different from bona fide IFs by conventionaland scanning transmission electron microscopy(TEM and STEM).27 The fibers formed were ratherstiff and very thick, with multiples of the numberof molecules per IF cross-section usually found.Another matter of concern was the occurrence ofhexameric species of vimentin,28 an oligomericspecies not found in our previous analytical ultra-centrifugation studies in any significant amount.21

For rigorous determination of the distinct oligo-meric species obtained from IF proteins undervarious assembly and centrifugation conditions,we combined data from sedimentation equilibriumruns employing a global fit algorithm with thoseobtained from sedimentation velocity runs usingthe time derivative method. In particular, weestablished a method that reliably discriminatestetrameric from dimeric, hexameric and octamericspecies under the “non-ideal” conditions neededto keep IF proteins soluble.29,30 We thereby definedconditions for recombinant human vimentin thatkeep the protein almost exclusively in a tetramericcomplex at 20 8C. By addition of various amountsof potassium chloride, we were able to induce theformation of bona fide IFs at different rates.Moreover, by analytical ultracentrifugation andprotein-chemical cross-linking, we determinedthat vimentin missing the non-a-helical amino-terminal end domain (“headless” vimentin),associated under physiological conditions intotetramers and higher aggregates. Taken together,we obtained more insight into the in vitro assemblymechanism of IFs that may also be relevant for IFassembly in the living cell. Last but not least, partlyby serendipity we generated a point-mutatedhuman vimentin that is arrested mainly at theULF state when assembled at room temperaturebut which immediately assembles into bona fide IFswhen the temperature is raised to 37 8C.

Results

Following complex formation of vimentin byanalytical ultracentrifugation

We have analyzed the association state ofrecombinant human vimentin by sedimentationequilibrium and sedimentation velocity ultracentri-fugation under low ionic strength and high pHconditions, i.e. 5 mM Tris–HCl (pH 8.4), with and

98 Assembly-Starter Units of Vimentin

without 1 mM EDTA, 0.1 mM EGTA and 1 mMDTT (5 mM Tris buffer (þ ) and (2 ), respectively),versus low ionic strength and physiological pHconditions, i.e. 2 mM sodium phosphate (Na-phos-phate), pH 7.5. Since vimentin remains solubleafter dialysis from buffers containing high concen-trations of urea into low ionic strength/high pHbuffer, conditions similar to those obtained withour Tris buffer system are usually chosen to recon-stitute soluble complexes that can be used as astarting material for assembly experiments.21 Thephosphate buffer system has been employed,among other low ionic strength buffers, forassembly studies as followed by transient electricalbirefringence (TEB).26 Here, we have attempted toinvestigate the homogeneity of the vimentin com-plexes formed in the two buffer systems. Inparticular, we investigated if and to what degreetetramer formation was accompanied by dimeric,hexameric or octameric complexes. Moreover, wewanted to quantitatively determine the effect oftruncation of the non-a-helical head and taildomains, respectively, on complex formation.

Recombinant wild-type human vimentin, calledhere wt vimentin, was analyzed in 5 mM Trisbuffer (þ ) without salt and at protein concen-trations ranging from 0.4 to 1.4 mg/ml bysedimentation equilibrium centrifugation. Weobserved a systematic deviation of the residualsfrom a single-exponential fit at a protein concen-tration of 1.4 mg/ml but not at 0.4 mg/ml

(Figure 1a, top panel, open circles versus filledsquares). This type of concentration-dependentdeviation is characteristic for a non-ideal system.29

The same type of behavior is exhibited by headlessvimentin (Figure 1b), indicating that it is mainlythe highly acidic charges accumulated within therod domain that determine this non-ideal sedimen-tation behavior rather than the highly basic headdomain.These data can be expressed as molecular mass

ðMrÞ values and extrapolated to zero concentration(0.0 mg/ml), thereby generating concentration-independent values for the molecular mass ofthe smallest soluble complexes.30 For wt vimentin,the value obtained was 2.10(^0.01) £ 105 (Figure 2,filled circles). This value corresponds very closelyto a tetrameric complex with a theoretical Mr of215,000. It is well within the expected error due tothe highly asymmetric shape of the molecule. Incontrast, calculations for headless vimentin yieldedvalues of 1.02(^0.06) £ 105, indicating that thehead-truncated vimentin is essentially a dimerunder these conditions with a theoretical value of93,000 (Figure 2, filled triangles). In order toinvestigate whether changing the ionic strengthwould lead to the formation of higher oligomers,we performed additional analytical ultracentrifuga-tion experiments under more physiological con-ditions. Accordingly, in 25 mM Tris–HCl (pH 8.4),1 mM EDTA, 1 mM DTT, 50 mM NaCl, a concen-tration-dependent increase of the molecular mass

Figure 1. Sedimentation equilibrium centrifugation analysis of recombinant human (a) wt vimentin and (b) headlessvimentin. The top panels present the residuals to the fit expressed as the difference between experimental and fittedvalues. The bottom panels reveal the concentration distribution of the probes within the cells. Filled squares,0.38 mg/ml for wt vimentin and 0.49 mg/ml for headless vimentin; open circles, 1.44 mg/ml for wt vimentin and1.26 mg/ml for headless vimentin. Centrifugation was carried out at (a) 7000 rpm and (b) 10,000 rpm, respectively, at20 8C until equilibrium was reached (20 hours). Note that the systematic error obtained with the higher protein concen-tration is typical for a non-ideal system. Extrapolation to zero concentration yields the molecular mass of the smallestsoluble subunit (see Figure 2).

Assembly-Starter Units of Vimentin 99

was observed with headless vimentin (Figure 2,open squares). This effect was even more pro-nounced when headless vimentin was analyzed in5 mM Tris–HCl (pH 7.5), with 100 mM KCl (Figure2, open diamonds). From these data we concludethat tetramerization of vimentin at low ionicstrength is indeed mediated by the very basic,non-a-helical head domain. However, headlessvimentin forms, both at high and physiologicalpH, exclusively dimers, and only with increasingionic strength do these dimers progressivelyassociate to tetramers.

Application of a global fit program

In order to investigate whether and to whatextent higher oligomeric complexes were presentthat went undetected by standard data analysisprograms that extrapolate to zero protein concen-tration, we applied the program Winnonlin (seeMaterials and Methods) that allows us to performa global, non-linear least-squares fit of the dataobtained at different speeds (7000 and 10,000 rpm)and protein concentrations (0.2 to 1.1 mg/ml). Inthe single-exponential fit mode, the plot of thedeviation of individual data points from the fittedfunction exhibits a systematic divergence(Figure 3a, top panel). In contrast, the fittingroutine including the second virial coefficientyielded now a precise fitting of the data points.

The residuals no longer deviated systematically butonly in a stochastic manner (Figure 3b, top panel).

Thus, Winnonlin provides a reliable algorithm fordescribing the association state and sedimentationbehavior of the extended, highly charged vimentinoligomers in the analytical ultracentrifuge. Inaddition, Winnonlin enabled us to quantitativelydetermine the molecular mass and hence the oligo-merization state of various mutant proteins com-pared to wt vimentin (Table 1). In particular, theassociation characteristics of these molecules indifferent buffers, i.e. Tris–HCl buffer, pH 7.5 and8.4, and Na-phosphate buffer at pH 7.5, with andwithout salt could now be investigated with highprecision. As documented in Table 1, wt vimentinwas determined to associate exclusively into tetra-meric complexes in 5 mM Tris–HCl at pH 8.4. Themolecular mass was only slightly influenced bythe presence (þ ), or absence (2 ), of EDTA, EGTAand DTT, thereby yielding values of 197,000 and192,000, respectively. Nevertheless, a significantinfluence of the charged compounds is clearly indi-cated by the lower value for the colligative secondvirial coefficient B (Table 1) as determined by theglobal fit program Winnonlin. However, at pH 7.5in the presence of these chelators (þ ), the for-mation of higher aggregates is already apparentby the relatively higher molecular mass, i.e.226,000, compared to that obtained without (2 ),i.e. 207,000 (see Table 1). Hence, both sets of valuesare in excellent agreement with the calculated Mr

of 215,000. Moreover, the slightly higher valueobtained at pH 7.5 (þ ) indicates that a reversibleinteraction of tetramers with one another occursunder these conditions of slightly enhanced ionicstrength that is mediated by the chelators (seebelow). This is further documented by the negativeB value and the high value for the curve fittingerror (Table 1, see Stdev).

With this reliable global fit program at hand toinvestigate the association state of elongated,highly charged molecules, we analyzed the contri-bution of the non-a-helical head and tail domainsto tetramerization. Accordingly, headless vimentinwas shown to be a dimer with a mass of 90,000compared to the calculated mass of 93,000. Thesame applied to the “rod”, whose calculated Mr

was almost indistinguishable from that obtainedby centrifugation (78,000 versus 82,000; see Table 1).In contrast, tailless vimentin sedimented as a tetra-mer both under (þ ) conditions at pH 8.4 and (2 )conditions at pH 7.5 (188,000 and 186,000; seeTable 1), in close agreement with the calculated Mr

of 190,000. Here too, the negative value for B indi-cates the occurrence of interactions leading to theformation of larger oligomers (Table 1).

Vimentin oligomers are present inphosphate buffer

The association properties of wt vimentin in anear-physiological buffer were investigated by per-forming sedimentation equilibrium runs in 2 mM

Figure 2. Determination of the apparent molecularmass values from recombinant wt vimentin at 5 mMTris–HCl (pH 8.4) (filled circles) and headless vimentinunder the following three conditions: (1) 5 mM Tris–HCl (pH 8.4) (filled triangles); (2) 25 mM Tris–HCl(pH 8.4), 50 mM NaCl (open squares); (3) 5 mM Tris(pH 7.5), 100 mM KCl (open diamonds). The wt vimentinwas not run under conditions (2) and (3), since saltinduces its assembly into higher-order structures. Thesedimentation equilibrium centrifugation runs of bothproteins were performed as described in the legend toFigure 1. The individual data points represent valuesobtained from one centrifugation run performed at agiven loading concentration.


Na-phosphate buffer (pH 7.5). Under these con-ditions, wt vimentin migrated as a homogenoustetramer species with an Mr of 202,000, which is ingood agreement with the calculated Mr of 215,000.In contrast, under these conditions headlessvimentin migrated as a dimer with Mr of 92,000,very close to the calculated Mr of 93,000 (Table 1).

Determination of association constants

The ionic strength-dependent association of bothwt and headless vimentin was evaluated with theprogram Winnonlin by keeping the correspondingvalue for the molecular mass fixed (see Table 1)and solving the sedimentation equation for the

Figure 3. Global non-linear, least-squares fit of equilibrium sedimentation data from wt vimentin. (a) A global singleexponential fit; (b) inclusion of the second virial coefficient into the calculation. The lines in the bottom panels of a andb represent data generated by a global fit for experiments employing four different concentrations (0.2 mg/ml to1.1 mg/ml) at two rotor speeds (program Winnonlin). The top panels display the residuals to the fit expressed as thedifference between experimental and fitted values. The inclusion of the second virial coefficient into the fit yieldedmuch more regular values; compare right side of both panels in particular.

Table 1. Determination of the molecular mass and the association state of wt vimentin and mutated vimentins bysedimentation equilibrium ultracentrifugation

Proteina pHb Mnonlinc £ 103 Bd (l/g) Stdeve £ 1023 Association statef Mcalc

g £ 103

5 mM Tris–HClwt 8.4 (þ) 197 (189, 206) 0.13 5 T 215wt 8.4 (2) 192 (181, 205) 0.27 4 T 215wt 7.5 (þ) 226 (199, 255) 20.02 19 T/O 215/430wt 7.5 (2) 207 (195, 219) 0.03 7 T 215DH 8.4 (þ) 90 (83, 98) 0.31 5 D 93DT 8.4 (þ) 188 (178, 199) 0.08 9 T 190DT 7.5 (2) 186 (177, 196) 20.05 4 T 190Rod 8.4 (þ) 82 (70, 94) 0.20 9 D 78

2 mM NaPi

wt 7.5 (2) 202 (191, 223) 0.08 4 T 215DH 7.5 (2) 92 (83, 101) 0.31 5 D 93

a wt, wt vimentin; DH, headless vimentin; DT, tailless vimentin; rod, head- and tailless vimentin.b (þ), with and (2), without 1 mM EDTA, 0.1 mM EGTA, 1 mM DTT.c Molecular mass determined by global fitting (program Winnonlin) of scans with different loading concentrations. For more details

see Figure 3.d B represents the colligative second virial coefficient as reported by Winnonlin. Charge effects and excluded volume effects are

indicated by a positive value of B. In contrast an ideal self-association will lead to a negative value for B.e Square-root of variance as calculated by Winnonlin.f D, dimer; T, tetramer; O, octamer.g Theoretical molecular mass of the respective complexes as calculated from the primary sequence of vimentin.


equilibrium constant of the smallest soluble andhigher-order oligomers. Accordingly, for wtvimentin in 5 mM Tris–HCl (pH 7.5) (þ ), we deter-mined an association constant of 0.3 £ 106 l/molfor a tetramer to octamer association (Table 2).Evidently, slight changes of the ionic strength, forexample, as introduced by the inclusion of 1 mMEDTA, are sufficient to shift the equilibriumtowards higher-order oligomers. Correspondingly,the addition of small amounts of salt leads to thegeneration of even more complex oligomermixtures and, with further increase of the ionicstrength, to filament formation (data not shown).

Unlike wt vimentin, headless vimentin does notpolymerize into IFs upon salt addition.21 Neverthe-less, it is not known what effect the increase of theionic strength has on its association behavior.Therefore, we investigated the dependence of com-plex formation of headless vimentin at various saltand pH conditions. In 5 mM Tris–HCl (pH 7.5),with 100 mM KCl, headless vimentin revealed anassociation constant of 1.5 £ 106 l/mol, indicativeof a substantial association of dimers to tetramers(Table 2). A similar association was also observedunder other ionic strength conditions such asthose of the two IF assembly buffers, i.e. 22.5 mMTris–HCl (pH 7.5), 50 mM NaCl, and 2 mM Na-phosphate (pH 7.5), 100 mM KCl, as evidenced byassociation constants of 0.7 £ 106 and 0.4 £ 106 l/mol, respectively (see Table 2). At higher pH, i.e.in the above Tris assembly buffer, but now at pH8.4, association of dimers to tetramers wasobserved too, albeit with a distinctly lower associ-ation constant of 0.2 £ 106 l/mol (see Table 2).

Determination of s-values

In order to investigate the homogeneity of thesoluble oligomers with respect to their size andshape, we performed sedimentation velocity cen-trifugation runs with wt, headless and taillessvimentin, as well as the vimentin rod. The samples

were analyzed in 5 mM Tris–HCl (2 ), at pH 7.5and pH 8.4, and at relatively low protein concen-tration (,0.1 mg/ml) to avoid non-idealityphenomena. Interestingly, at pH 7.5 wt vimentinsedimented with an s-value of 5.5 S whereas atpH 8.4 it sedimented with 4.7 S (Figure 4a, openversus filled squares; and Table 3). Since the Mr

values determined by sedimentation equilibriumcentrifugation were the same at these two pHvalues (see Table 1), the different s-values indicatedthat the tetramers differed significantly in size,shape or stiffness (Table 3). Moreover, the Mr deter-mined from the fitted sedimentation profilesyielded values in good agreement with the sedi-mentation equilibrium data, i.e. 209,000 at pH 8.4and 200,000 at pH 7.5 (Table 3) compared to thevalues obtained by sedimentation equilibrium, i.e.192,000 and 207,000 (Table 1). From this agreementand the fact that the sedimentation velocity curvescould be fitted with a single component(Figure 4a), it may be concluded that the samplewas of high homogeneity in terms of its oligomericstate. Notably, the tailless vimentin behaved identi-cally with wt vimentin except that evidently asecond species with a higher s-value was presentin this sample (Figure 4b, open versus filledsquares). This species was calculated to representless than 10% of the entire sample. In contrast,both headless vimentin and the vimentin rod didnot exhibit a pronounced pH-dependent shift oftheir s-values (Figure 4a and b, open versus filledcircles; Table 3). Moreover, both the headlessvimentin and the vimentin rod sedimented ashomogenous species. The difference of s-valuesobtained for wt and tailless vimentin at the twopH values was of the order of 0.76 S and 0.65 S,respectively, whereas for headless vimentin andthe vimentin rod the difference was only 0.20 Sand 0.17 S, respectively (see Table 3).

To more completely investigate the influence ofpH and ionic strength on the apparent sedimen-tation behavior, we also measured wt vimentin

Table 2. Determination of the binding constants of wt vimentin and headless vimentin by sedimentation equilibriumultracentrifugation for a tetramer/octamer and dimer/tetramer interaction, respectively

Protein pHa Salt Kb £ 106 (l/mol) Bc (l/g) Stdevd £ 1023

5 mM Tris–HClwt 7.5 (þ) – 0.3 (0.2, 0.4)e 0.07 7DH 7.5 (2) 100 mM KCl 1.5 (1.0, 2.4) 0.03 4

25 mM Tris–HClDH 7.5 (þ) 50 mM NaCl 0.7 (0.1, 1.0) 0.01 6DH 8.4 (þ) 50 mM NaCl 0.2 (0.1, 0.2) 0.02 5

2 mM NaPi

DH 7.5 (2) 100 mM KCl 0.4 (0.3, 0.6) 0.00 5

Scans were taken at 7000 rpm and 10,000 rpm. For more details see Figure 3 and Material and Methods.a (þ ), with and (2), without 1 mM EDTA, 0.1 mM EGTA, 1 mM DTT.b Binding constants were derived by global fitting (program Winnonlin) of scans from runs with different loading concentrations.

Molecular masses of the complexes were fixed at 207,000 for wt vimentin and 92,000 for headless vimentin.c Colligative second virial coefficient as reported by Winnonlin (see Table 1).d Square-root of variance as calculated with Winnonlin.e Only scans of runs performed at 7000 rpm were taken.


reconstituted in 5 mM Tris–HCl (pH 9.5) (Figure 4c,filled circles) and obtained an even further shifteds-value of 4.1 S compared to 4.7 S at pH 8.4 and5.5 S at pH 7.5 (Figure 4a and Table 3). This indi-cated that an increase in negative charge, becauseof the deprotonation of the arginine and the lysineresidues at higher pH, enhances the structuralreorganization of the individual vimentin mol-ecules within the tetrameric complex, therebyyielding more rigid complexes. Therefore, in orderto manipulate the charge repulsion phenomenaencountered at the low ionic strength used inthese pH experiments, we raised the ionic strengthby adding 5 mM NaCl to the buffer. Notably, thisimmediately increased the s-value by more thanone unit, close to that measured at pH 7.5(Figure 4c, open circles; and Table 3).

Whereas lowering the pH from 8.4 to 7.5 onlyslightly increased the s-value of headless vimentinand the vimentin rod (see Figure 4a and c; andTable 3), changes in ionic strength had a muchstronger effect during sedimentation equilibriumruns (see Figure 2). Therefore, we performed sedi-

mentation velocity experiments in filamentassembly buffer with higher ionic strength, i.e.5 mM Tris–HCl (pH 7.5), 1 mM EDTA, 0.1 mMEGTA, 1 mM DTT, 200 mM NaCl. Accordingly, theheadless vimentin sedimented with an apparents-value of 4.4 S, indicating it was present mainlyin the form of tetrameric complexes (Figure 4d,open circles). Similarly, the vimentin rod sedimen-ted with an s-value of 4.1 S. However, here weobserved the generation of large oligomers as aresult of raising the ionic strength (Figure 4d,open squares).

Salt-dependent association ofheadless vimentin

Analysis of the sedimentation behavior of head-less vimentin had shown that under low salt con-ditions it is in a dimeric and in filament assemblybuffer predominantly in a tetrameric state (seeTables 1 and 3). Therefore, the mode of associationof headless vimentin dimers upon salt additionwas investigated by glycerol spraying, rotary

Figure 4. Sedimentation velocity analysis of vimentin and various domain-deleted forms of vimentin, i.e. headlessand tailless vimentin as well as the vimentin rod. The curves displayed are representative g(s*) plots. a, wt vimentin(squares) and headless vimentin (circles) were analyzed in 5 mM Tris–HCl buffer at pH 8.4 (filled symbols) andpH 7.5 (open symbols). b, Analysis of tailless vimentin (squares) and vimentin rod (circles) in 5 mM Tris–HCl bufferat pH 8.4 (filled symbols) and pH 7.5 (open symbols). c, wt vimentin analyzed in 5 mM Tris–HCl buffer (pH 9.5), inthe absence (filled circles) and presence (open circles) of 5 mM NaCl. d, Analysis of headless vimentin (circles) andvimentin rod (squares) in 5 mM Tris–HCl (pH 7.5), 1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 200 mM NaCl. Bothvimentin fragments exhibit a peak at 4.1 S. The vimentin rod exhibits, in addition, a shoulder that indicates thepresence of higher-order complexes. Protein concentrations: a–c, 0.1 mg/ml; d, 1.0 mg/ml. Scans were recorded at230 nm (a–c) and 280 nm (d). Note in a and b that the head-deleted mutants, i.e. headless vimentin and rod, sedimentslower than the head-containing forms, i.e. wt and tailless vimentin.


metal shadowing electron microscopy. In theseexperiments, both at pH 8.4 and 7.5, headlessvimentin yielded ,45 nm long rods (Figure 5, leftpanels). After addition of 150 mM NaCl most par-ticles observed were rods of a symmetric tripartitenature. The center third was slightly thicker thanthe two flanking parts, indicating that two dimerrods had associated in an overlapping, half-stag-gered mode (Figure 5, right panels). In addition,both of the free end segments appeared to be ableto bend around the stiff center, indicating thatlinker L12 was outside of the overlap. Whereas atpH 8.4 tetramers were 65 nm long, at pH 7.5 theirlength became slightly smaller, i.e. 62 nm. Bymeasuring larger numbers of particles the majorityof the tetramers were indeed in an extended,,65 nm long form (histograms in Figure 5, rightpanels). The 40 nm to 50 nm long rods occasionallyobserved were most likely normal half-staggeredtetramers, the end segments of which werepartially masked during the adhesion and solventevaporation process.

Modeling the shape of soluble vimentin

After having determined the s-values and oligo-meric states of wt vimentin and its three domain-deleted variants, headless, tailless and rod, wenow could use these data to model the theoreticalshape of the complexes employing the programSednterp (see Materials and Methods). Since elec-

tron microscopy of soluble vimentin complexeshas demonstrated their elongated, rod-like shape(Herrmann et al.;21 see also Figure 5), we modeledthe complexes formed by vimentin and its deriva-tives as prolate ellipsoids. Correspondingly, wtvimentin at pH 7.5 can be described as an ellipsoidof 73 nm length and 3.3 nm width at pH 7.5(Table 3), whereas tailless vimentin at the samepH is only 53 nm long. Hence, the distal 10 nm ateither end of the tetramer represent vimentin’s taildomains. The maximum length of the dimericvimentin rod is calculated as 49 nm. This correlateswell with data obtained by glycerol spraying/rotary metal shadowing of the desmin rod exhibit-ing a peak value of 50 nm.31 The value for theheadless vimentin dimer is 10 nm larger (59 nm)due to the tail domains residing at one end of theparticle. At higher pH, i.e. pH 8.4, the lengths areincreasing, which correlates well with the lowers-values (see Table 3). The rather small value fortailless vimentin in comparison to the rod may beexplained by the approximation of the actual struc-ture by a prolate ellipsoid, which represents, ofcourse, an oversimplification.

Assembly of vimentin in phosphate buffer

Human recombinant vimentin, when assembledunder standard conditions, forms full-width,60 nm long unit-length filaments after one secondof assembly (Figure 6a, inset) that elongate into

Table 3. Sedimentaion velocity data of wt vimentin and mutated vimentins by analytical ultracentrifugation

Probe s20,wa (S) Msed

b £ 103 Number of runs Concentration (g/l) Prolatec 2a (nm)/2b (nm)

5 mM Tris–HCl (pH 8.4)wt vimentin 4.7 (0.30) 209 (24) 5 0.1 92/3.0DH 2.7 (0.11) 104 (11) 3 0.1 67/2.3Rod 2.7 (0.13) 78 (5) 3 0.1 54/2.4DT 5.3 (0.17) 186 (19) 3 0.1 64/3.4

5 mM Tris–HCl (pH 7.5)wt vimentin 5.5 (0.24) 200 (29) 8 0.1 73/3.3DH 2.9 (0.13) 107 (8) 3 0.1 59/2.5Rod 2.8 (0.20) 74 (2) 3 0.1 49/2.5DT 5.9 0.06) 176 (20) 7 0.1 53/3.7

2 mM NaPi (pH 7.5)wt vimentin 5.6 (0.05) 217 (6) 3 0.1 n.c.DH 2.9 (0.01) 92.3 (3.5) 3 0.1 n.c.

2 mM NaPi (pH 7.5) þ 100 mM KClDH 4.1 (0.00) 105 (2) 3 0.1 n.c.DH 4.2 132 1 0.5 n.c.

5 mM Tris–HCl (pH 7.5) þ 200 mM NaClDH 4.4 159 1 0.5 84/2.9

All runs were performed in the absence of EDTA, EGTA, and DTT except for the one performed in 5 mM Tris–HCl (pH 7.5),200 mM NaCl. Here we included 1 mM EDTA, 0.1 mM EGTA and 1 mM DTT. The values in parentheses are the standard deviationsof the mean values obtained from various runs.

a Sedimentation coefficients were corrected to standard conditions.b Molecular mass M was determined by the dependency of the peak (s-value) and the peak broadening (diffusion coefficient)

within the program DCDT þ ; an ideal behavior of one component will give the true molecular mass; non-ideality will lead to a shar-pened boundary and therefore to a higher molecular mass and sample inhomogeneity will give a higher diffusion coefficient valueand this will decrease the determined M value.

c Prolates were calculated with the program SEDNTERP using the calculated translational frictional coefficient ratio;50 2a is theoverall length of the major axis and 2b is the overall length of the minor axis; n.c., not calculated.


long IFs within one hour (Figure 6a) with an aver-age diameter of 9.7(^2.3) nm (values obtainedfrom Gauss fit of the data shown in Figure 6b,black bars). In this regime, soluble tetramers arefirst produced in 5 mM Tris–buffer (pH 8.4), andare then mixed with an equal volume of “assemblyinitiation buffer” consisting of 40 mM Tris–HCl(pH 7.0), and 100 mM NaCl, thus yielding a “fila-ment assembly buffer” consisting of 22.5 mMTris–HCl (pH 7.5), and 50 mM NaCl. In the phos-phate buffer regime, soluble tetramers are formedin 2 mM Na-phosphate (pH 7.5), and are thenmixed with an equal volume of a buffer consisting

of 2 mM Na-phosphate (pH 7.5), plus 200 mM pot-assium chloride. This yields ULFs and IFs thatappear indistinguishable from “Tris-filaments”,with an average diameter of 9.4(^2.7) nm (Figure6b and c). Also, kinetic measurements performedin the Ostwald viscometer demonstrated that theassembly process in the phosphate buffer systemis comparable to that in the Tris system (Figure 7).The increase in viscosity is critically dependent onthe protein concentration employed (Figure 7a) aswell as the ionic strength (Figure 7b). For example,with 0.3 mg/ml of protein only approximately60% of the specific viscosity is obtained comparedto the 0.5 mg/ml profiles, and at 0.1 mg/ml lessthan 10% of the specific viscosity is achieved.Lowering the salt concentration from 100 mM to50 mM leads to the reduction of the specificviscosity from 0.21 to 0.10 after one hour ofassembly. This indicates that the plateau valuemay not have been reached yet at this lower ionicstrength (Figure 7b).

Interference of headless vimentin and vimentincoil 1 with the assembly of wt vimentin

Since headless vimentin has the ability to formtetrameric complexes upon increase of the ionicstrength (see Figure 5 and Table 3), we wanted toinvestigate whether headless vimentin dimers ortetramers could also interact with wt vimentinoligomers. More specifically, we wanted to knowif they would form complexes with wt vimentin atthe tetrameric, the ULF and the IF state, respect-ively. Moreover, since based on the cross-linkingdata (see below) we assumed that most of thetetramer formation of headless vimentin dimersinvolves the A22 dimer–dimer interaction mode,we wondered if and how coil 1 would bind to wtvimentin in comparison to headless vimentin.Therefore, we added to wt vimentin in 2 mMNa-phosphate buffer (pH 7.5), recombinant coil 1,coil 2 or headless vimentin before IF assembly wasstarted by addition of assembly initiation buffer.In a second set of experiments, we added thevimentin fragments five seconds and one hour,respectively, after initiation of assembly of wtvimentin. After one hour of incubation, IFs werepelleted and the supernatants analyzed by SDS-polyacrylamide gel electrophoresis (Figure 8a).Both coil 1 (lanes 3–5) and headless vimentin(lanes 6–8) were predominantly found in thesupernatant together with varying amounts of wtvimentin. For control, lane 1 shows the amount ofwt vimentin in solution before centrifugation,whereas lane 2 displays what is left in the super-natant when wt vimentin is assembled for onehour in the absence of a vimentin fragment. There-fore, coil 1 is indeed able to bind to wt vimentintetramers as evidenced by their presence in thesupernatant (lane 3). Similarly, some wt vimentinis retained in the supernatant when coil 1 is addedto ULFs (lane 4). In contrast, when coil 1 is addedto preformed IFs no significant amount of wt

Figure 5. Length determination of rodlets formed fromrecombinant headless vimentin upon dialysis from 8 Murea into (a) 5 mM Tris–HCl (pH 8.4), or (b) 2 mMNa-phosphate (pH 7.5). The protein was subjected toglycerol spraying/rotary metal shadowing eitherdirectly, left panels (2 ), or after incubation with100 mM KCl for one hour, right panels ( þ ). The lengthof approximately 500 particles each was measured andhistogrammed. Note that in the presence of salt the rodsare 50% longer and that the middle segment is thickerthan the protruding end segments.


vimentin is retained in the supernatant (comparelanes 5 and 2). Similar results were obtained withheadless vimentin, except that the amounts of wtvimentin present in the supernatant were slightlyhigher when headless vimentin was added to pre-formed ULFs or IFs, respectively (lanes 7 and 8).In absolute contrast, under no condition tested didcoil 2 retain any wt vimentin in the supernatant(data not shown).

In order to investigate whether under assemblyconditions coil 1 or coil 2 have an affinity forvimentin tetramers, we performed analytical ultra-centrifugation of both recombinant coil 1 and coil2 with and without wt vimentin. In filamentassembly buffer, coil 1 sediments as a singlespecies with an s20,w-value of 2.5 S (Figure 8b, con-

tinuous line). In the presence of wt vimentin, how-ever, a second peak of 6.0 S is observed (opencircles). In contrast, coil 2 in filament buffer yieldsonly one peak of 2.5 S (broken line) both in theabsence and the presence of wt vimentin. Takentogether, under filament forming conditions bothcoil 1 and coil 2 form dimers, but only the coil 1dimer is able to associate with vimentin tetramers.At this stage, the number of coil 1 dimers boundper tetramer has not been determined, but fromsymmetry considerations one may expect at leasttwo to be incorporated. We suggest that coil 1dimers, in contrast to coil 2 dimers, interact to asignificant extent with the head domains of wtvimentin tetramers. Thereby “unproductive”complexes are formed that neither take part in the

Figure 6. Electron microscopic analysis of negatively stained IFs assembled from human recombinant vimentin at37 8C for one hour in (a) 22.5 mM Tris–HCl (pH 7.5), 50 mM NaCl and (c) 2 mM Na-phosphate (pH 7.5), 100 mMKCl. b, In both cases, the diameter of IFs was between 8 nm and 11 nm for both types of IFs with peak values between9 nm and 10 nm. Filled bars, assembly in Tris–HCl buffer; hatched bars, assembly in phosphate buffer. The insets in aand c depict ULFs found in abundance ten seconds after initiation of assembly. The shortest filaments in these insetsexhibit an average length of approximately 60 nm. The scale bar represents 100 nm.

Figure 7. Viscometric analysis of IF assembly of human recombinant vimentin in the phosphate buffer system.a, Variation of the protein concentration: vimentin was assembled at 0.5 mg/ml (filled circles), 0.3 mg/ml (filleddiamonds) and 0.1 mg/ml (filled squares) in 2 mM Na-phosphate (pH 7.5), and 100 mM KCl at 37 8C. For comparison,0.5 mg/ml vimentin was assembled in 22.5 mM Tris–HCl (pH 7.5), and 50 mM NaCl (filled triangles). b, Variation ofthe salt concentration: at a protein concentration of 0.5 mg/ml wt vimentin was assembled in 2 mM Na-phosphate(pH 7.5), in the presence of 100 mM KCl (open circles), 75 mM KCl (open triangles) and 50 mM KCl (open squares).


formation of ULFs nor in the elongation reaction ofULFs to IFs.

Cross-linking of headless vimentin

Samples of wt vimentin and headless vimentinwere cross-linked with DST essentially as

described32 except for the use of Na-phosphate buf-fer. As expected, in 2 mM Na-phosphate buffer wtvimentin forms tetramers, and in the presence of150 mM NaCl or 100 mM KCl it forms large poly-mers, consistent with IF formation (Figure 9, leftpanel). These data are in agreement with the ana-lytical ultracentrifugation analyses shown above.However, using the commercial precast 4%–12%gels, two partially resolved tetramer bands wereroutinely observed (data not shown). The two tet-ramer bands were excised from the gels, digestedwith CNBr and trypsin, and the resulting peptidesanalyzed by HPLC. Accordingly, the upper minorband consisted of tetramers in the A22 alignmentmode whereas the lower major band representedtetramers in the A11 alignment mode. In contrast,headless vimentin in 2 mM Na-phosphate bufferyielded dimers in the absence of salt, and tetramerswith no higher oligomers in the presence of 0.15 MNaCl (Figure 9, right panel). HPLC analyses ofCNBr/trypsin peptides of these tetramer bandsrevealed the three cross-linked peptides character-istic for each of the A11 and A22 alignment modesof wt vimentin tetramers (Figure 10, blue andgreen arrows, respectively), although in somewhatreduced yields (Table 4). In addition, five novelcross-linked species (Figure 10, red arrows) wererecovered with headless vimentin, documenting a9–20 residue shift with respect to the typical align-ment of molecules within wt vimentin (Table 4).Taken together, these data indicate that some speci-ficity in terms of preferred molecular alignment forwt vimentin has been lost in headless vimentin.Moreover, these data indicate that vimentin headdomain sequences are required for precise align-ment of the wt molecules within tetramers, and byextension, within intact IFs.

Figure 8. Interference of recombinant human vimentinfragments with the assembly of wt vimentin. a, Gelelectrophoretic analysis of material obtained in the high-speed supernatant after mixing equimolar amounts ofwt vimentin with coil 1 (lanes 3–5) or headless vimentin(lanes 6–8) in 2 mM Na-phosphate (pH 7.5),immediately before (lanes 3 and 6), five minutes after(lanes 4 and 7) and one hour after addition of assemblyinitiation buffer (lanes 5 and 8). Lane 1 represents theamount of wt vimentin employed in the assemblyexperiments, lane 2 the corresponding materialrecovered in the supernatant after initiation of assemblyin the absence of any vimentin fragment. Note that bothcoil 1 and headless vimentin retain significantly morevimentin in the non-pelletable fraction when addedearly after initiation of assembly compared to whenadded to mature filaments. b, Analytical ultracentrifu-gation analysis of the complexes formed after additionof filament buffer to coil 1 (continuous line), coil 1 pluswt vimentin (open circles), and coil 2 plus wt vimentin(broken line). Note that in the presence of coil 2 onlyone peak at around 2.5 S is obtained. This indicates thatit did not form a complex with wt vimentin, whichinstead polymerized completely into IFs and was there-fore pelleted. In contrast, coil 1 formed a complex withwt vimentin and retained part of it in solution. This com-plex sedimented at around 6 S, slightly faster than thevimentin tetramer alone (5.6 S in parallel experiments).

Figure 9. Gel electrophoretic analysis of cross-linkingproducts obtained from wt vimentin (left panel) andheadless vimentin (D80). Recombinant protein was dia-lyzed from 8 M urea into 2 mM Na-phosphate (pH 7.5),and either left untreated (left lanes) or cross-linkedbefore (middle lane) or after initiation of assembly byaddition of 150 mM NaCl (right lanes). The probes weredenatured by heating with SDS-sample buffer andloaded onto 4%–15% gradient polyacrylamide gels. M,monomer; D, dimer; T, tetramer; L, loading slot.


Figure 10. HPLC fractionation of DST cross-linked CNBr/tryptic peptides derived from wt vimentin (upper panels)and headless vimentin (lower panels). Left panels, unreacted protein. Middle panels, dimers formed in 2 mMNa-phosphate buffer (pH 7.5). Right panels, tetrameric species formed in 2 mM Na-phosphate buffer (pH 7.5), plus150 mM NaCl (for the proteins analyzed see marked bands in Figure 9). Red dots indicate cross-linked peptides inthe one-molecule state (dimers). The arrows indicate cross-linked products characteristic for tetramers in the A11

(blue) and A22 (green) orientation, respectively. Note that the A11 cross-link fragments are significantly reduced in thedigest from the headless vimentin. The red arrows point to new fragments (see Table 4).

Table 4. Yields of cross-links within wt and headless vimentin

Cross-linkwt vimentin Headless vimentin

1 mola (dimer) 2 mola (tetramer) 1 molb (dimer) 2 molb (tetramer)

Intramolecular1B-42/1B-42 0.27 0.30 0.25 0.231A-01/1A-01 0.20 0.27 0.22 0.301A-17/1A-17 0.07 0.06 0.05 0.06L1-05/L1-05 0.24 0.21 0.20 0.212A-19/2A-19 0.33 0.37 0.31 0.29L12-15/L12-15 0.17 0.21 0.16 0.18

Intermolecular A11

L1-05/1B-89 0.18 0.051A-01/2A-19 0.22 0.031A-17/L12-15 0.08 0.02(new) 1A-26/L12-15 0.05

Intermolecular A22

2B-83/2B-23 0.17c 0.08c

(new) 2B-83/2B-02 0.13(new) 2B-83/2B-04 0.152B-100/2B-02 0.19 0.09(new) 2A-19/2B-100 0.182A-19/2B-112 0.25 0.12(new) 2B-43/2B-43 0.10

a Cross-linking with DST was performed in 2 mM NaPi (pH 7.5).b Cross-linking was performed one hour after the addition of 150 mM NaCl.c Note that the value for the 2B-83/2B-23 fragment is relatively low in headless vimentin, probably due to the new cross-links (new)

formed between 2B-83 and 2B-02 as well as 2B-04; the same holds true for the 2B-100/2B-02 and the 2A-19/2B-112 cross-links.


Assembly of a temperature-sensitivehuman vimentin

By serendipity we found that mutation of lysine139, the first amino acid residue of linker L1 inhuman vimentin, to cysteine yielded a proteinwhich at 37 8C assembled completely normallyinto ULFs within ten seconds and normal IFs afterone hour (Figure 11a and c), but that was impededin elongation when assembly was carried out atroom temperature (Figure 11b and d). Whereas

ULFs formed also at the lower temperature withinseconds (Figure 11b), fusion of ULFs into long IFsproceeded very slowly even after one hour of incu-bation (Figure 11d). This temperature dependencewas also reflected in the behavior exhibited by theviscometric analyses (Figure 11e). Whereas theincrease in viscosity at 21 8C was similar to that at37 8C one minute after initiation of assembly, thevalue for the 21 8C experiment plateaued veryquickly, unlike the increase observed at 37 8C(Figure 11e). When the assembly experiments

Table 5. Yields of cross-links within the temperature-sensitive vimentin mutant K139C

Cross-link 1 mola (dimer) 2 mola (tetramer) ULF at 22 8Cb IF at 37 8Cb

Intramolecular1B-42/1B-42 0.34 0.33 0.28 0.331A-01/1A-01 0.22 0.27 0.31 0.331A-17/1A-17 0.09 0.09 0.06 0.01L1-05/L1-05 0.21 0.29 0.23 0.182A-19/2A-19 0.41 0.57 0.31 0.45L12-15/L12-15 0.12 0.11 0.07 0.01

Intermolecular A11

L1-05/1B-89 0.18 0.19 0.161A-01/2A-19 0.23 0.25 0.201A-17/L12-15 0.07 0.09 0.17

Intermolecular A22

2B-23/2B-83 0.17c 0.00 0.212B-02/2B-100 0.10c 0.00 0.192A-19/2B-112 0.12c 0.00 0.30

Intermolecular A12

L1-05/2B-83 0.00 0.00 0.221B-90/2A-19 0.00 0.00 0.10

a Cross-linking was carried out in 2 mM NaPi (pH 7.5).b Cross-linking was carried out one hour after addition of 150 mM NaCl.c Note that these cross-link fragments are absent in the “ULF at 22 8C”-fraction, indicating that they are formed only during cross-

linking as a result of the change of the ionic conditions by addition of the cross-linking reagent (see effect of EDTA, Table 1).

Figure 11. Assembly of the recombinant human temperature-sensitive vimentin mutant, Vim K139C. a–d, Nega-tively stained preparations of structures formed at 37 8C (a and c) and 21 8C (b and d) for ten seconds (a and b) andone hour (c and d), respectively. The scale bar represents 100 nm. e, Temperature-dependent viscometric analysis ofVim K139C assembly. Abscissa, time (minutes); ordinate, specific viscosity. Assembly was initiated at t ¼ ten minutesand the change in viscosity of the protein solution was assessed in an Ostwald viscometer at the temperature indicatedfor each curve.


were performed at increasingly higher tempera-tures, the viscosity values increased correspond-ingly so that the viscosity profiles progressivelyapproached that exhibited at 37 8C. Most impor-tantly, the short filaments obtained after one hourof assembly at 21 8C (see Figure 11d) were no“dead end” structures, since upon raising the tem-perature to 37 8C they immediately started toelongate to yield eventually long bona fide IFs. Thiswas corroborated by viscometry, demonstratingthat after a short lag time the viscosity rapidlyincreased to normal values (data not shown).Taken together, these data clearly documentthat the ULF state is indeed a distinct assemblyintermediate that can be separated from theother principal states, i.e. elongation andcompaction.

Analysis of the cross-linking products of ULFsobtained from the temperature-sensitive mutant at23 8C revealed a very interesting result when com-pared to the cross-linking products obtained fromthe IFs formed at 37 8C (Table 5). Whereas thethree cross-linked peptides indicative of the align-ment mode A11 were found identically at bothtemperatures, corresponding A22 as well as A12

fragments were present only at 37 8C, as routinelyseen with wt IFs,33 but were not found in thedigests of ULFs formed at 23 8C (Table 5). Thisfinding is consistent with a model that ULFs aremade exclusively from A11-type tetramers, andthat A22 and A12-type dimer–dimer interactionsare occurring only at the later stages of assembly,most likely as a consequence of a complex set ofsubunit rearrangements. Evidently, the A22 cross-link products obtained predominantly for the tetra-mers made from headless vimentin are absent fromULFs assembled at 23 8C. This strongly indicatesthat the majority of tetramers present in ULFs areindeed in the A11 mode and that, once ULFs areformed, A22-type configurations cannot beadopted. Moreover, the small amounts of A22-typecross-links found under non-polymerization con-ditions are most likely due to coil 2–coil 2 contactsformed during the cross-linking procedure bylongitudinally annealing A11-type tetramers.

Discussion

The molecular mechanisms underlying the for-mation of cytoplasmic IF systems are, to a largeextent, still elusive.5 However, recent in vivostudies with GFP-tagged vimentin, keratin andneurofilament-triplet proteins have indicated thatshort filaments may be transported by specificmotor protein complexes to their sites ofintegration into the IF cytoskeleton.34–36 Because ofthe limitations set by the resolution of the lightmicroscope, short filaments of around 0.2 mmwould appear as “dots” rather than filaments.However, since these dots evidently elongateduring transport to become “squiggles”,22 whicheventually integrate into the vimentin IF network,

it is not too far-fetched to assume that these dotsdo actually represent ULFs or multiples thereof.The integration of such squiggles into IFs mostlikely poceeds by an end-on annealing mechanism.Hence, the three distinct structural states of IFproteins observed by fluorescent light microscopyin vivo (i.e. dots, squiggles and filaments) wouldrepresent the three distinct phases described by usfor in vitro IF assembly.37

A phosphate buffer system for IF assembly

Here, we have investigated the in vitro assemblyof IFs in a near-physiological buffer, i.e. 2 mMNa-phosphate (pH 7.5), plus 100 mM potassiumchloride. We document that the molecular inter-actions occurring in the Na-phosphate buffer arevery much the same as those in the Tris system. Inparticular, we have determined the oligomericstate of the soluble vimentin complexes in the twobuffer systems by analytical ultracentrifugationand chemical cross-linking. Most importantly, byanalytical ultracentrifugation, vimentin indeedforms a homogenous population of tetramers inphosphate buffer at pH 7.5. Moreover, by cross-linking the A11-type tetramer predominates,whereas the A22-type tetramer occurs as a minorspecies only. Both types of tetramers werepreviously identified by cross-linking of IFs intriethanolamine.33 However, since in Na-phosphatebuffer vimentin can both be assembled into bonafide IFs and cross-linked, this system is far superiorover other cross-linking regimes.

A concern was, however, whether the phosphateion would interfere with assembly, since it had pre-viously been shown that in the presence of 10 mMNa-phosphate buffer keratin filaments unravelextensively.38 Interestingly, Kooijman et al.28 havedemonstrated by transient electrical birefringencethat vimentin can assemble into fibers in 0.7 mMphosphate buffer when magnesium ions areadded. Assessed by conventional transmissionelectron microscopy and STEM,27 however, thesefibers did by no means represent IFs and thereforewe investigated further, if and under what con-ditions IFs would actually form. We have nowdocumented that in 2 mM Na-phosphate buffer,ULFs are formed, which elongate further to regular10 nm filaments, when assembly is induced byaddition of 100 mM monovalent ions. Thesefilaments are indistinguishable from thoseobtained in the standard Tris buffer system of22.5 mM Tris–HCl (pH 7.5), with 50 mM NaCl, asdetermined both by electron microscopy andviscometry, and hence represent bona fide IFs.

The assembly-starter unit

One inherent problem with IF assembly is theconsiderable polymorphism encountered duringin vitro reconstitution experiments.3,39 Since theassembly process actually starts already duringdialysis when the sample is freed of urea and


soluble oligomers are formed, we wondered aboutthe homogeneity of these samples. Recentexperiments had shown that vimentin dimerizesalready at around 6 M urea and that tetramers areprominently formed at 4.5 M urea.21,40–42 However,when urea was completely removed and theprotein equilibrated in 5 mM Tris–HCl (pH 8.4),in the presence of chelating agents, analytical ultra-centrifugation experiments indicated thatoligomers as large as octamers were alreadypresent.21 It was therefore not clear whether suchoctameric complexes would serve as regular inter-mediates of IF assembly, or if they would give riseto the structural polymorphism detected by STEMwhen the filament mass-per-length of in vitroassembled IF populations was investigated.21 Evenmore disturbing, Kooijman and colleagues hadreported that vimentin, in addition to tetramers,yielded dimers and hexamers after reconstitutionin low ionic strength Tris–HCl, bis-Tris–HCl orNa-phosphate buffers as determined by TEBmeasurements.28,43

The data reported here clearly document thatvimentin under low salt conditions forms nearlyexclusively tetramers, both in Tris–HCl buffer(pH 7.5 or 8.4), and in 2 mM Na-phosphate buffer(pH 7.5). Unlike Kooijman and colleagues, we didnot observe any significant dimer formation butonly tetramers at 0.7 mM Na-phosphate.28,43 In ourhands, in the absence of urea, dimers wereobtained only at very high pH values, i.e. abovepH 11.25 (data not shown). In summary, the globalfit method used by us to analyze the sedimentationequilibrium centrifugation data in combinationwith the time derivative method used to analyzethe sedimentation velocity data, enabled us toaccurately and reproducibly determine the associ-ation state of vimentin. More specifically, weshowed that in both the Tris–HCl and theNa-phosphate buffer system wt vimentin formeda homogenous species of tetrameric complexeswith less than 5% faster sedimenting species.

Interestingly, it was shown previously thatlateral association of tetramers into ULF-like struc-tures occurred even at pH 8.5 when monovalentions were present at 50 mM.44 Under these con-ditions uniform full-width IFs of 66 nm length butno elongated filaments were observed. Neverthe-less, the ULFs generated under these conditionsare productive as bona fide IFs formed upondialysis against 170 mM NaCl (pH 7.0). These datastrongly indicate that ULF formation is muchmore robust than elongation, since it takes place athigh pH provided the ionic strength is raisedappropriately. In line with this, the linker L1mutant VimK139C at room temperature formsULFs indistinguishable from wt vimentin but doesnot elongate significantly unless the temperatureis raised to 37 8C. This indicates, since wt vimentindoes assemble into IFs at room temperature, thatcomplex rearrangements take place duringelongation that depend on the correct linkergeometry.

Analysis of early assembly intermediates

Whereas lateral association of tetramers intoULFs is extremely fast, it can effectively bearrested by the addition of glutaraldehyde.21,27

More specific cross-linkers such as disulfosuccini-midyltartrate (DST) that cross-link the free aminogroups of lysine side-chains need much longerincubation times (30 minutes) to yield significantamounts of cross-link products. Hence, they can-not be used to effectively arrest IF assembly. Inthis context, our finding that a point-mutatedvimentin (K139C) does not associate significantlyabove ULFs at room temperature, enabled us toanalyze the relative orientation and alignment ofdimers with respect to one another by cross-link-ing within ULFs. The only major cross-linkedfragments found indicate that within ULFsformed by the temperature-sensitive vimentinvariant at 23 8C, only A11-type dimer–dimerinteractions occur. This, in turn, means thatULFs are exclusively formed by in-register lateralassociation of ,60 nm long A11-type tetramers.Alternatively, “tetramer-switching type” reactionsmay occur upon salt addition, thus giving rise tothe A22-type interactions, which may, however, besterically hindered for cross-linking by the DST,becoming accessible only after further rearrange-ments in mature IFs. A schematic representationis depicted in Figure 12. This latter model isdeduced from the fact that ULFs of vimentin(K139C), “frozen” at room temperature, immedi-ately start to longitudinally anneal into longer,radially loosely packed IFs upon temperatureshift to 37 8C. Analysis of the correspondingcross-linking products revealed that these fila-ments gave rise to the typical pattern of A11, A22

and A12 fragments that is obtained from maturewt vimentin IFs. An alternative explanationwould infer that ULFs indeed are built exclu-sively from A11-type tetramers (Figure 12, rightmodel) and reorganize only upon longitudinalannealing and/or radial compaction. The lattermodel is strongly supported by the fact that, incontrast to the lateral association reaction oftetramers, the elongation and compaction reac-tions take much longer as expected for complexrearrangements.

Materials and Methods

Protein chemical methods

The cloning and expression of wt vimentin and itsdomain-deleted fragments has been described.21,45

Note that both coil 1 and coil 2 form dimers undervarious salt conditions.45 The stepwise dialysis of pro-teins into low salt buffers and subsequent assemblyexperiments including viscometry were done asbefore.21 The phosphate buffer used for IF assemblyconsisted of 2 mM sodium phosphate (pH 7.5),100 mM KCl.


Cross-linking with disulfosuccinimidyl tartrate (DST)

wt and headless vimentin were equilibrated in 2 mMNa-phosphate (pH 8.0) at a concentration of 0.1 mg/ml.DST (Pierce Chemical Company, Rockford, IL) wasadded to 0.5 mM (from a 50 mM stock in DMSO) andreacted for 30 minutes at 23 8C or 37 8C.32,33 In somecases, KCl was added to 0.15 M (final concentration)30 minutes prior to addition of DST. Reactions wereterminated with 0.1 M NH4HCO3 and then resolved onpre-cast 4%–12% polyacrylamide gradient Tris–glycineSDS gels (Novex Invitrogen Life Technologies, Carlsbad,CA). Desired monomer, dimer (one molecule) and tetra-mer (two molecules) species were recovered in larger-scale preparative experiments by excision from 4%–15%slab gels, essentially as before.33 Excised proteins werefreed of SDS, cleaved with CNBr followed by trypsin,and peptides were resolved by HPLC.33 Comparisons ofprofiles between control and cross-linked digestsallowed us to identify cross-linked species, most ofwhich were identical to known intramolecular linkages,or intermolecular linkages of the A11 or A22 types.

32,33 Insome cases, a few novel peaks were recovered, cleavedwith periodate and the separate pieces sequenced asbefore to identify the cross-linked partners. Yields ofcross-linked peptides were calculated based on the peakheight and molar extinction coefficient of the peptidesof known composition.

Analytical ultracentrifugation

Analytical ultracentrifugation experiments werecarried out using a Beckman analytical ultracentrifuge(model Optima XLA) equipped with an ultravioletabsorption optical system.

Sedimentation velocity experiments were carried outin double-sector charcoal-Epon cells at 20 8C at40,000 rpm for headless vimentin and the rod. wt vimen-tin and tailless vimentin were run at both 30,000 rpmand 40,000 rpm. Scans were recorded at 230 nm or280 nm using a spacing of 0.1 cm with four averages in

a continuous scan mode. Data were analyzed with theprogram DCDT þ [Version 1.13]46 that implementsthe algorithms described by Walter Stafford47,48 to obtainthe sedimentation and diffusion coefficients and there-fore the molecular mass values of the soluble subunits.The further development of the algorithm to calculateg(s*) was taken to decrease the errors of the calculatedvalues. The scans to be analyzed were selected from theend of the run; the time window for the analysis wasestimated as suggested in the DCDT þ manual.

Sedimentation equilibrium runs were carried out insix-channel charcoal-Epon cells at 20 8C by first pre-sedi-menting the sample for two hours at double rotor speed.Scans were recorded at 7000 rpm and at 10,000 rpmevery four hours until no change could be detectedbetween successive runs; this usually took 20 hours. Thebaseline of the centrifugation run was determined byspinning the sample down at 48,000 rpm at the end ofthe experiment and averaging the absorbance in the firstone-third of the scan. The data were analyzed with theprogram Winnonlin V1.035 (J. Lary, M. L. Johnson andD. A. Yphantis; freely downloadable†) based on thealgorithm used in the program NONLIN.49 A global,single-exponential fit over four or five different loadingconcentrations (0.3–1.2 mg/ml) and two rotor speeds(7000 rpm and 10,000 rpm) was calculated. Baselineswere fixed and only the concentration, the molecularmass, and the colligative second virial coefficient werefitted.

The molecular mass according to the amino acidsequence, the absorbance per mg/ml at 280 nm, thehydration, the netcharge, and the partial specific volumewere calculated with the program SEDNTERP V1.05(J. Philo, D. Hayes, T. Laue; freely downloadable‡).

The viscosity of the various buffers was measuredusing a Schott KPG Ubbelohde capillary viscosimeterwith automatic sampler (Schott, Hofheim, Germany).

Figure 12. Hypothetical scheme of the ULF formation from tetrameric subunits of the two principal approximatelyhalf-staggered, antiparallel dimer-dimer associations, A11 and A22.

† ftp://alpha.bbri.org/rasmb/spinms_dos/uconn_uaf‡ ftp://alpha.bbri.org/rasmb/spin/ms_dos/


ftp://alpha.bbri.org/rasmb/spinms_dos/uconn_uaf

ftp://alpha.bbri.org/rasmb/spin/ms_dos/

For buffers with and without KCl, it was determined tobe h20,buf ¼ 1.00 mPa s. For 22.5 mM Tris buffer plus50 mM NaCl we obtained h20,buf ¼ 1.02 mPa s, and for5 mM Tris buffer plus 200 mM NaCl we measuredh20,buf ¼ 1.03 mPa s. The density of all buffers wasmeasured to be 1.00 g/ml employing a DMA 5000density meter (Anton Paar, Graz, Austria).Calculationsfor modelling prolate ellipsoids from the s20,w valueswere done in SEDNTERP V1.05. Parameters such asmolecular mass, partial specific volume and hydrationwere calculated from the amino acid sequence, and theassociation state of the protein. The friction coefficientf=fp (Teller method) was used to calculate the size of theprolate ellipsoid.

Electron microscopy

Negatively stained specimens were recorded with aZeiss model 900 electron microscope (Zeiss, Oberkochen,Germany) as described.21 Glycerol spraying/rotary metalshadowing of samples has been reported elsewhere.21,27

Acknowledgements

We thank Monika Brettel for excellent technicalassistance in an early phase of the work and EvaGundel for expert secretarial assistance throughoutthe writing process. This work has been supportedby grants from the Deutsche Forschungs-gemeinschaft (to H.H., DFG HE 1853/4-1) and theSwiss National Science Foundation, The M.E.Muller Foundation of Switzerland, and the CantonBasel Stadt (to U.A.). Last but not least, we thankWerner W. Franke for continuous interest andsupport.

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Edited by M. Moody

(Received 10 December 2003; received in revised form 6 April 2004; accepted 20 April 2004)


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Molecular and Biophysical Characterization of Assembly-Starter