Binding of nucleic acids to intermediate filaments of the vimentin type and their effects on filament formation and stability.

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Binding of Nucleic Acids to Intermediate Filaments of the Vimentin Type and Their Effects on Filament Formation and Stability a

a

Peter Traub , Elfriede Mothes , Robert L. Shoeman a

b

, Rasmus Schröder & Annemarie Scherbarth

a

a

Max-Planck-Institut für Zellbiologie , Rosenhof, W-6802 , Ladenburg bei Heidelberg b

Max-Planck-Institut für medizinische Forschung , W-6900 , Heidelberg , Federal Republic of Germany Published online: 21 May 2012.

To cite this article: Peter Traub , Elfriede Mothes , Robert L. Shoeman , Rasmus Schröder & Annemarie Scherbarth (1992) Binding of Nucleic Acids to Intermediate Filaments of the Vimentin Type and Their Effects on Filament Formation and Stability, Journal of Biomolecular Structure and Dynamics, 10:3, 505-531, DOI: 10.1080/07391102.1992.10508665 To link to this article: http://dx.doi.org/10.1080/07391102.1992.10508665

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Journal of Biomolecular Structure & Dynamics, /SSN 0739-1102 Volume 10, Issue Number 3 (1992), €>Adenine Press (1992).

Binding of Nucleic Acids to Intermediate Filaments of the Vim en tin TYpe and Their Effects on Filament Formation and Stability Peter Traub 1, Elfriede Mothes 1, Robert L. Shoeman 1, Rasmus Schroder2 and Annemarie Scherbarth 1 Downloaded by [Rutgers University] at 12:13 09 April 2015

1

Max-Planck-lnstitut filr Zellbiologie, Rosenhof W-6802 Ladenburg bei Heidelberg

2

Max-Planck-Institut filr medizinische Forschung W-6900 Heidelberg Federal Republic of Germany

Abstract Guanine-rich polynucleotides such as poly(dG), oligo(dG) 12• 18 or poly(rG) were shown to exert a strong inhibitory effect on vimentin filament assembly and also to cause disintegration of preformed filaments in vitro. Gold-labeled oligo(dGhs was preferentially localized at the physical ends ofthe aggregation and disaggregation products and at sites along filaments with a basic periodicity of 22.7 nm. Similar effects were observed with heat-denatured eukaryotic nuclear DNA or total rRNA. although these nucleic acids could affect filament formation and structure only at ionic strengths lower than physiological. However, whenever filaments were formed or stayed intact, they appeared associated with the nucleic acids. These electron microscopic observations were corroborated by sucrose gradient analysis of complexes obtained from preformed vimentin filaments and radioactively labeled heteroduplexes. Among the duplexes of the DNA type, particularly poly(dG) · poly(dC), and, of those of the RNA type, preferentially poly(rA) · poly(rU), were carried by the filaments with high efficiency into the pellet fraction. Single-stranded ISS and 28S rRNA interacted only weakly with vimentin filaments. Nevertheless, in a mechanically undisturbed environment, vimentin filaments could be densely decorated with intact 40S and 60S ribosomal subunits as revealed by electron microscopy. These results indicate that, in contrast to single-stranded nucleic acids with their compact random coil configuration, double-stranded nucleic acids with their elongated and flexible shape have the capability to stably interact with the helically arranged, surface-exposed amino-terminal polypeptide chains of vimentin filaments. Such interactions might be of physiological relevance in regard to the transport and positioning of nucleic acids and nucleoprotein particles in the various compartments of eukaryotic cells. Conversely, nucleic acids might be capable of affecting the cytoplasmic organization of vimentin filament networks through their filament-destabilizing potentials.

Introduction Intermediate filaments (IFs) (I) are thought to fulfil cytoskeletal functions in eukaryotic cells due to their characteristic three-dimensional distribution in the cytoplasm,

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their rigidity of structure, their relatively low dynamics in comparison with those of microtubules and microfilaments and their association with many subcellular structures. As "mechanical integrators of cellular space" (2), they are supposed to play important roles in the structural and functional organization of such cells in that they maintain distinct spatial relationships between various cellular constituents permitting their optimal interaction with each other during the life cycle of the cell. Since many of these substructures are membrane-bounded, the capability of IFs and their subunit proteins to interact with membrane-associated proteins (reviewed in 3,4) and lipid bilayers (5-8) appears to be of particular physiological relevance. However, in addition to these binding capacities, IF subunit proteins exhibit strong binding affinities for nucleic acids, especially for single-stranded DNAs and RNAs of high guanine content (9-12). The affinity, for instance, of vimentin for oligonucleotide models of telomere DNA with its guanine-rich repeat sequences extending into a short single-strand overhang (13), together with its capacity to interact with lamin B (14-16), might provide an explanation for the association ofvimentin filaments with the nucleus. Since IF proteins also interact in vitro with a distinct stoichiometry with core his tones ( 17), it was postulated that they also fulfil nuclear functions in addition to the role they play as cytoskeletal elements (18). The properties and, to a limited extent, the cellular function(s) ofiF(protein)s can be understood on the basis oftheirstructural organization (reviewed in 3, 19-24). While the backbone of IFs consists largely of coiled-coil rope structures made up of the well-conserved a-helical rod domains of the different constituent subunit proteins, their periphery is occupied by the non-a-helical terminal regions of the subunit proteins protruding from the surface of the filament core (25). Since these end regions vary in size and amino acid sequence and thus in their chemical properties, they mainly determine the reactivities of IFs and their interactions with other cellular components. However, while the formation and stability ofiFs are thought to rest on hydrophobic and, to a lesser extent, ionic interactions between the a-helical rod domains of the subunit proteins, there is a considerable body of evidence that the arginine-rich, non-a-helical head domains of the subunits are also involved in these aspects. This was shown by the inhibition of filament formation, for instance from protofilamentous vimentin, in the presence of free arginine but not free lysine (26) or in the presence of the isolated amino-terminal polypeptide of vimentin (27). Employing site-directed mutagenesis of vimentin eDNA, a nonapeptide motif in the head domain was shown to be essential for filament assembly (28). Moreover, in addition to proteolytic truncation ofiF proteins at their amino termini (26, 29-32) and deimination of amino-terminal arginine residues (33), phosphorylation of serine and threonine residues in close neighborhood to these arginine residues (3442) causes inhibition of filament formation and destabilization of filament structure. Similar effects are exerted by negatively charged phospholipids (5)which show high affinities for the arginine-rich amino termini ofiF proteins (43). Although the amino-terminal head domain thus appears to be occupied as a stabilizing element in the formation and maintenance of filament structure, it might, nevertheless, be able to mediate the interaction of IFs with other cellular substructures and components such as proteins of the plasma membrane-associated cytoskeleton or the lipid bilayer of membranes. This seems plausible since a limited number of amino-

Nucleic Acid Binding to Filaments

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terminally truncated subunit proteins can be tolerated in IFs without causing their severe destabilization or can be incorporated into IFs in filament reconstitution assays (30). In contrast to the involvement of the amino-terminal head domain in filament formation and stabilization, the carboxy-terminal tail region seems to be of considerably less importance in this respect, although there is some experimental evidence against this supposition (44-46).1t might therefore be readily available for interactions ofiFs with other cellular substructures or components such as lamin B of the nuclear envelope (14,15) or skeletal elements and vesicular bodies in the axoplasm of neurons (47,48). On the basis of the foregoing considerations, it is conceivable that polyanionic substances with high affinities for the arginine-rich amino-terminal head regions ofiF proteins bind to IFs and, when offered in large excess, exert negative effects oh filament assembly and stability. Good candidates along this line would be nucleic acids or nucleoprotein particles. As mentioned above, polynucleotides of the RNA and DNA type, particularly those of high guanine content, have been demonstrated to bind with high efficiency to the amino termini of protofilamentous IF proteins. The present investigation was conducted to show whether IFs can bind nucleic acids through their surface-exposed amino-terminal head domains and whether these compounds are able to destabilize preexisting IFs or to inhibittheir formation when present in large amounts. The detection of such relationships is of cell biological relevance in regard to the possibility that IFs, as a rnajor part of the cytoskeleton, might serve as a scaffold for the intracellular distribution and functioning of nucleic acids and the structural organization of their complexes with proteins. It would also provide information on the potential influence of nucleic acids on the distribution and dynamics of IF networks in the cytoplasm.

Materials and Methods Materials

Poly(dG) · poly(dC), poly(dA) · poly(d1), poly(rl) · poly(IC), poly(rA) · poly(rU), poly(dG), oligo(dG) 12. 18 and poly(rG) were purchased from Pharmacia-LKB (Freiburg, FRG), calf thymus DNA and gold-conjugated streptavidin from Sigma (Deisenhofen, FRG). Lysine-specific endoproteinase Lys-C was obtained from Boehringer Mannheim (Mannheim, FRG), pancreatic RNase A from Serva (Heidelberg, FRG). Calf thymus DNA dissolved in 10 mM Tris-HCl, pH 7.6, 6 mM 2-mercaptoethanol (buffer I) was sonicated to reduce the viscosity and, for denaturation, heated at IOOC for lO min and quickly cooled in ice water. All reactions were normalized to contain equal masses of the various nucleic acids employed due to differences and heterogeneity in their lengths, as well as to the uncertainties concerning the length and molecular mass ofvimentin IFs. To provide a point of reference, a mass ratio ofvimentin:oligo(dG) 12_18 of 40:1, such as employed in experiments described in Figure lC, corresponds to a molar ratio of vimentin monomers:nucleic acid of about 3:1. Vimentin was prepared from cultured Ehrlich ascites tumor (EAT) cells as described previously (49). The Oxytricha oligonucleotide telomere model was prepared as described (13). For the preparation of ribosomes, EAT cells grown in minimal essential Eagle's


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medium (Flow Laboratories, Meckenheim, FRG) supplemented with 5% fetal calf serum (Boehringer Mannheim) and harvested at a cell density ofS.5 X 105 cells/ml were homogenized in a buffer containing 50 mM Tris-HCl, pH 7.6, 25 mM KCl, 5 mM MgC12, 250 mM sucrose, 1% Triton X-100 by 20 strokes in a tightly-fitting Dounce homogenizer (unless otherwise stated, all operations were carried out at 2C). Mter removal of the nuclei by centrifugation at 14,000 gav for 5 min, the supernatant was layered on top of a cushion of2 M sucrose in 50 mM Tris-HCl, pH 7.6, 25 mM KCl, 5 mM MgC12 and centrifuged for 20 h at 255,000 gav· The ribosome pellet was carefully rinsed with 50 mM Tris-HCl, pH 7.6, 500 mM KCl, 1.5 mM MgC1 2 and dissolved in the same buffer using a tightly-fitting Dounce homogenizer. To dissociate the ribosomes into their subunits, the solution was adjusted to 1 mM puromycin (Sigma) and incubated at 37C for 15 min. The ribosomal subunits were separated by centrifugation at 104,000 gav for 9.5 h on 10 to 30 % (w/w) sucrose gradients in 50 mM Tris-HCl, pH 7.6, 500 mM KCl, 5 mM MgC12, pelleted by centrifugation at 255,000 gav for 11 h, redissolved in buffer I in a tightly-fitting Dounce homogenizer, frozen in liquid N 2 and stored at -SOC. Total rRNA was isolated from EAT cell ribosomes with phenol-chloroform (4:1) in the presence of 1% Na-dodecylsulfate (SDS) according to conventional methods and finally dialysed against buffer I. 3H-labeled total rRNA was likewise obtained from ribosomes of EAT cells grown in the presence of [5,6-3H] uridine (l.S5 TBq/ mmol, Amersham-Buchler, Braunschweig, FRG). eH] lSS and eH] 2SS rRNA were separated by centrifugation on 5 to 20% (w/w) sucrose gradients in 10 mM TrisHCl, pH 7.6, 3 mM EDTA at 104,000 gav for 22.5 h. Mter precipitation with 2 vol. ethanol at- 20C, the RNAs were dissolved in and dialysed against buffer I, frozen in liquid N 2 and stored at -SOC.

Radiolabeling of Polynucleotides Nucleic acids (see Results section) were 5'-end labeled with 32P in a phosphate exchange reaction employing T4 polynucleotide kinase (Pharmacia-LKB) and [A._3 2P)-ATP (Amersham-Buchler), according to standard protocols (50). Alternatively, poly(dG) · poly(dC) and poly(dA) · poly( dT) were labeled with 3H in a twostep exonuclease-polymerase replacement synthesis reaction employing T4 DNA polymerase and either eH)-dGTP (414 GBq/mmol; Amersham-Buch1er) or eH]dATP (1.1 TBq/mmol; Amersham-Buch1er), essentially as described (50). In brief, 2.51Jg ofthe po1ynucleotides were incubated for 20 min at 37C in 100 jJl buffer which contained 1.6 units of T4 DNA polymerase (Pharmacia-LKB), 0.1 mM dNTPs (Pharmacia-LKB) minus either dGTP (for labeling of poly(dG).poly( dC)) or dATP (forlabeling of poly(dA).poly( dT)), 33 mM Tris-acetate, pH 7.9, 66 mM K-acetate, 10 mM Mg-acetate and 0.5 mM dithiothreitol. This solution was then transferred to another tube containing either 900 pmol eH)-dGTP or 330 pmol eH]-dATP dried down from the original ethanol solution. The reaction mixtures were then incubated 15 min at 37C, followed by the addition of either dGTP or dATP to a final concentration ofO.l mM and incubated for a further40 min at 37C. All 32P- and 3H-labeled nucleic acids were separated from unincorporated label and the buffer was exchanged to 10 mM Tris-HCl, pH 7.4, 0.1 mM EDTA by gel filtration on NICK columns


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(Pharmacia-LKB), according to the manufacturer's instructions. The specific activity ranged from 1-3 X 106 cpmfl.tg for 32P-labeled and 5-8 X 105 cpmfl.tg for 3H-labeled nucleic acids. Oligo(dGb was labeled with biotin by the addition of dUTP-11biotin (Sigma) in a polymerase reaction catalyzed by terminal deoxynucleotide transferase (Pharmacia-LKB), essentially as described (50). Control reactions supplemented with [a- 32P]-dCTP demonstrated that the extent of labeling was low, as desired; on the average, 0.8 biotinylated nucleotide was added per oligo(dG)25 molecule (i.e., 8 of 10 oligo(dG )25 molecules were labeled). Alternatively, a 3' -aminomodified oligo(dGh5 oligonucleotide was synthesized on 3'-Amino-ON CPG and subsequently biotinylated with Biotin-:XX-NHS using the Cruachem "Easy Label" kit (Beckman Instruments, Munchen, FRG). Biotinylated oligo(dG) 25 was purified by gel filtration on NICK columns, as described above. Reaction of Vimentin and its Filaments with Nucleic Acids and Ribosomes

For electron microscopy, vim en tin filaments were formed by incubating a solution of vimentin at 100 f..lg/ml in 10 mM Tris-HCI, pH 7.6, 150 mM KCI, 6 mM 2mercaptoethanol (buffer II) in the absence (control) and presence of increasing amounts of various nucleic acids (see Results section) for 1.5 to 2 hat 37C. In some cases, the KCl concentration was reduced to 50 mM or 100 mM and the 50 mM KCI buffer supplemented with 2 mM Mg2+. To study the stability ofvimentin filaments in the presence of nucleic acids, solutions of control filaments assembled as specified were incubated together with different quantities of the various nucleic acids (see Results section) for another 1 to 1.5 hat 37C. Similarly, 20 f..lg of preformed vimentin filaments were allowed to react with 0.32 OD 260 units of 40S and 0.65 OD 260 units of 60S ribosomal subunits, respectively, in 200 111 of buffer II for 30 min at 37C. Duplicates of the reaction mixtures were treated with RNaseA, using 10 ng enzyme/ OD260 unit of ribosomal subunit, for 1 h at room temperature. The binding of nucleic acids to preformed vimentin filaments was also studied by sucrose gradient centrifugation. Filaments were assembled from 500 11g ofvimentin in 2 ml of buffer II or buffer II containing 2 mM Mg2+ at 37C for 2 h and then mixed with 250 111 of a solution of radioactively labeled nucleic acid (see Results section) in filament assembly buffer; in some cases, the final reaction mixtures contained an additional 2 mM Mg2+ and 2 mM EDTA The reaction mixtures were layered on top of 10.4 ml2.5 to 10% (w/w) sucrose gradients in the respective filament assembly buffer and allowed to stand at room temperature for 0.5 to 1 h. Centrifugation was at 40,000 rpm (200,000g8 v) and 4C for 3 to 7 h (depending on the molecular size of the nucleic acid and the ionic composition ofthe sucrose gradient) in the SW 40 Ti rotor of the Beckman L8-70 M centrifuge. For gradient analysis, 10 1.26 ml fractions were collected. The pellets were dissolved in 500111 ofSDS-sample buffer. I 00 fll of each pellet solution and 250 fll of each gradient fraction were subjected to radioactivity measurement. The remainder of each gradient fraction was mixed with 100 111 of 100% trichloroacetic acid and incubated at 50C for 30 min. Mtercentrifugation, the precipitates were dissolved in 100111 ofSDS-sample buffer. 10 fll of each fraction sample was analyzed by SDS-polyacrylamide gradient slab gel electrophoresis (SDS-PAGE) (51). Since the vimentin concentration ofthe pellet

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solutions was usually very high, these were appropriately diluted. The Coomassie Brilliant Blue-stained vimentin bands were scanned at 590 nm.


Mixtures containing 150 flg ofvimentin filaments and either 1.25 OD260 units of 40S or 2.7 OD 260 units of 60S ribosomal subunits, respectively, in l ml of buffer II were incubated at 37C for 30 min, layered on top of discontinuous sucrose gradients consisting of l ml of 5%, 1 ml of3.5% and 1.2 ml of2.5% (w/w) sucrose in buffer II and centrifuged at 21,000 rpm (45,000 gav) and 4C for 3.5 h (40S) or 2.5 h (60S), respectively, in the SW 60 Ti rotor of the Beckman L8-70 M centrifuge. The gradients were fractionated by withdrawing l ml portions from the top by means of a syringe. Protein was precipitated and analyzed by SDS-PAGE as described above. Digestion of Vimentin Filament-Nucleic Acid Mixtures with Lysine-Specific Endoproteinase Lys-C

500 flgportionsofvimentin were incubated with l mgofheat-denaturedcalfthymus DNA and lmg of total rRNA, respectively, in l ml of buffer I containing an additional 50, 100 or 150 mM KCl for 1 hat 37C. The mixtures were then supplemented with 0.15 units of proteinase Lys-C and incubated further at 37C. At the time points 0, 5, 10, 20, 30, 45, 60, 120, 180 and 240 min after addition of the proteinase, 100 fll portions were withdrawn and mixed with 25 fll of 5 X SDS-sample buffer. 20 fll of each sample was subjected to SDS-PAGE. Decoration of Vimentin Filaments with Biotinylated Oligo (dG)25 and Gold-Conjugated Streptavidin and Electron Microscopy

Preformed vimentin filaments (100 flg ofvimentin/ml buffer II) were incubated with various amounts of oligo (dGb (5-15 Jlg/ml) for 10 min at 37C. The reaction products were applied to carbon films (prepared on freshly cleaved mica) for 30 sec, briefly washed with distilled H 20, stained with uranyl acetate and transferred to copper grids. For visualization ofthe complexes, 0.1 flg ofbiotinylated oligo (dG b was allowed to react with 0.12 flg of 10 nm (or 0.5 flg of 5 nm) gold-conjugated streptavidin in 30 fll of 10 mM Tris-HCl, pH 7.4, 0.1 mM EDTA for 1.5 min at 37C. Following the addition of KCl to a final concentration of 150 mM, a carbon film with vimentin filaments adsorbed to it was placed on the surface of the oligo (dG)25 -streptavidin solution for 3 min at room temperature. The film was washed in buffer I for 10 sec, stained with uranyl acetate and transferred to a copper grid. The reaction products obtained with biotinylated oligo(dGb were viewed in a Zeiss EM 902 electron microscope, while all other negatively stained specimens of this study were observed in a Philips Model 400T electron microscope. The axial distances of gold particles along the filaments were measured and their distribution analyzed mathematically. First, a Fourier power spectrum of the distribution was calculated to identify a underlying periodicity. With this result, Gaussian curves were fit to clusters of measurement values around multiples of the basic periodicity, giving mean value and standard deviation of the individual clusters.

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Figure 1: Assembly of vimentin filaments in the presence of increasing amounts of poly(dG), oligo(dG) 12_18 or poly(rG). Filaments were reconstituted at 150 mM KC1 in the absence (A) and presence ofpoly(dG) at a vimentin: poly(dG) mass ratio of 10:1 (B). Oligo(dG) 12_18 was used at vimentin: oligonucleotide mass ratios of 40:1 (C) and 10:1 (D), poly(rG) also at vimentin: polynucleotide ratios of 40:1 (E) and 10:1 (F)_ The reaction products were negatively stained and viewed in a Philips Model400T electron microscope. Magnification: X 80,000. Bar: 100 nm.


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Figure 2: Disassembly ofvimentin filaments by poly(dG). Vimentin filaments were reconstituted at 150 mM KCl and then incubated in the absence (A) or presence of poly (dG) at the following mass ratios of vimentin:poly(dG)- 40: l (B); 20: l (C) and 5: l (D). The preparations were negatively stained and viewed in a Philips Model400T electron microscope. Magnification: X 80,000. Bar: 100 nm.

Results and Discussion

Electron Microscopy of IF:G-Homopolymer Complexes Assuming that the binding of the amino-terminal head domain ofvimentin to its corresponding a-helical rod domain during filament formation or in intact filaments (27) is reversible, it should be possible to prevent it from interacting with this site by the addition of polynucleotides to which it alternatively may bind with a particularly high affinity. Indeed, when the assembly of IFs from protofilamentous vimentin was carried out in the presence of poly(dG ), a dramatic inhibition of this process by the polynucleotide was observed (Figure lA,B). Already at the low mass ratio of vimentin: poly(dG) = 80: 1, the filaments produced were considerably shorter than the control filaments and their ends often appeared splayed. Irregularly formed filaments were also detected on the electron microscopy grids. Whenever such irregularities were observed, the respective filament regions were more densely and also diffusely stained, indicating their association with the polynucleotide (not

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Figure 3: Negative staining and electron microscopy of vimentin filaments and their reaction products with oligo(dG )25• Note that in comparison with the control filaments (A) the oligonucleotide-treated filaments are fragmented and that the fragments either have splayed ends (B) or terminate in globular structures which might have arisen by bursting of swellings occurring in internal filament regions (C and D). In panel (D), the progressive formation of such swellings can be seen in three neighboring filaments. In contrast to the smooth surface of the control filaments, the surface of oligonucleotide-treated filaments is generally rough due to local displacement of the amino-terminal polypeptide chains of the filament subunit proteins. The specimens were viewed in a Zeiss EM 902 electron microscope. Magnification: X 200,000. Bar. 100 nm.

shown). The impairment of the assembly process was enhanced with increasing amounts of poly(dG) in the reaction mixture until at ratios of vimentin: poly(dG) = 10: l (Figure lB) or 5:1 (not shown) only very short filament fragments and electron-dense aggregates ofvimentin-poly(dG) adducts were formed. Even oligo-deoxyriboguanylic acids of considerably smaller molecular size (oligo(dG)12_18) interfered severely with vimentin filament assembly. As depicted in Figure lC, at a vimentin: oligo(dG) 12_18

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ratio of 40:1, the filaments formed were rather short and often associated with irregular protein-nucleic acid complexes, whereas at a ratio of 10:1 no filament fragments but only small and fuzzy vimentin-oligo(dG) 12_18 aggregates could be detected (Figure 1D). Figure 1E,F illustrates that poly( rG) was in no way inferior to poly(dG) or oligo(dG) 12_18 in inhibiting vimentin filament formation, although RNAs generally show a weaker affinity for IF proteins than DNAs. However, if the RNAs are rich in guanine, their affinities for the amino-terminal head domains of IF proteins are also high. These same G-homopolymers were also able to disassemble preexisting filaments (Figure 2 for poly(dG), Figure 3 for oligo(dGb and data not shown for oligo(dG) 12_18 or poly(rG)). The resulting structures were similar with all of the G-homopolymers; however, in general, higher concentrations of polynucleotides were needed to produce the same end products from preexisting filaments as those obtained in filament reconstitution assays. This is illustrated with poly(dG) in Figure 2. At a mass ratio ofvimentin:poly( dG) of 40:1, a partial unraveling of some ofthe filaments into a braided or rope-like structure was observed (Figure 2B). Fragmentation of the filaments and small aggregates of partially disrupted filaments and nucleic acids were observed at a ratio of 20:1 (Figure 2C). When the amount of poly(dG) was increased to yield a ratio ofvimentin:nucleic acid of 5:1, extensive fragmentation and large clumps of filament fragment-nucleic acid complexes were seen (Figure 2D). Thus, in comparison to the data presented in Figure 1, disassembly of preformed filaments requires a concentration ofpoly(dG) about 4 times higher than that required to affect assembly ofvimentin filaments (i.e., compare Figure 1B with the components at a ratio of 80:1 with Figure 2C at a ratio of 20:1 ). In order to demonstrate that the changes in filament structure seen in the previous experiments were indeed brought about by the physical interaction of the various nucleic acids with the filaments, preassembled vimentin filaments were incubated with oligo(dGb (Figure 3) and with preformed complexes ofbiotinylated oligo(dGb and gold-conjugated streptavidin (Figure 4). While Figure 3 illustrates the effect of the oligonucleotide alone on the filaments at higher magnification, Figure 4 shows a clear relationship between filament breakage or distortion and the extent of oligonucleotide binding indirectly visualized by the deposition of gold particles. In comparison with the control filaments, which were characterized by great length and a distinct, smooth surface (Figure 3A), the filaments treated with oligo(dGb were severely fragmented (Figure 3B,C), splayed at their physical ends (Figure 3B), terminated in voluminous thickenings (Figure 3C) and exhibited hooks, fringes and fraying regions along their entire longitudinal extension (Figure 3D). In general, the contour of the oligonucleotide-treated filaments was rather fuzzy due to these local perturbations. It appeared as if the filaments were progressively disrupted at their physical ends by the binding of the oligonucleotide molecules with the production of large, globular, filament-associated aggregations (Figure 3C). These could have been formed at natural filament ends or arisen from swellings in the interior of the filaments, as can be seen in the lower right hand comer of Figures 2C and D. The generation of such swellings eventually leading to filament breakage can be followed in the upper right hand comer of Figure 3D, where in neighboring filaments three stages of severity of perturbation in the filament structure can be seen.

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Figure 4: Negative staining and electron microscopy of vimentin filaments treated with a mixture of biotinylated oligo(dG) 25 and 10 nm (or 5 nm) gold-conjugated streptavidin. This composite picture shows various examples of filaments at different stages of oligonucleotide-induced disassembly, ranging from occupation of individual, surface-exposed amino-terminal polypeptide chains by the oligonucleotide molecules, swellings in internal filament regions to bending and fragmentation of individual filaments. All these oligonucleotide-induced distortions are visualized by the deposition of gold-conjugated streptavidin at splayed filament ends, at globular, terminal structures, at swellings in internal filament regions, at kinks produced by the local reaction of the filament with multiple oligonucleotide molecules and at ill-defined vimentin-oligonucleotide complexes. In the upper, central micrograph, gold particles remote from filaments indicate the reaction of sub filament strands with biotinylated oligo(dG}zs. Note in the lower left hand field the seemingly periodic distribution of gold particles along the filaments. The specimens were viewed in a Zeiss EM 902 electron microscope. Magnification: X 150,000. Bar: 100 nm.

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Figure 5: Quantitative analysis of the axial separation of gold particles along vimentin filaments treated as in Figure 3. Panel A shows the Fourier power spectrum of the 128 measured distances seen in the insert. It reveals a peak at the lowest spatial frequency of0.044 nm -•, corresponding to a basic periodicity of 22.73 nm underlying the distribution of measured values. The total distribution of gold particle distances can be understood as an overlay of several Gaussian distributions around multiples of this basic periodicity. The mean values and standard deviations of such Gaussian fits are shown in panel B, where the linear fit together with very small standard deviations should be noted.

All of these types of distorted filament structures could be labeled with goldconjugated streptavidin, indicating the presence ofbiotinylated oligo(dGhs molecules. As depicted in the composite picture of Figure 4, gold particles were preferentially deposited at the fraying and thickening ends of individual vimentin filaments. Particles attached to internal regions of filaments decorated swellings and very often kinks. In the latter cases, massive accumulations of electron-dense material together with the deposition of multiple gold particles were noticed. Gold particles were also detected remote from filaments, but in such instances the respective areas of the electron microscopy grids were covered with subfilamentous protein material which was obviously derived from disrupted vim en tin filaments. Clean, vimentin-free grid areas were also normally free of gold particles. Gold particles were also found along the length of otherwise seemingly intact filaments, and it was the general impression as if at those sites the filament structure became disturbed. These defects could have been present on the original, untreated filaments and caused, for instance, by false alignment of proto filaments or they may have been induced by the binding of the oligonucleotide molecules. Interestingly, individual gold particles on seemingly intact filaments were sometimes bound with an apparently periodic distribution, as seen, for instance, in the lower left hand field of Figure 4.1t should be noted that the decoration of filaments with streptavidin-gold was limited by both the fragility of the filaments in the presence of an excess of oligonucleotide and the employment of non-saturating amounts of the gold-label. The quantitative evaluation of the distribution of gold particles along the longitudinal extension of the filaments showed that among a total of 128 measurements the minimal distance was 18 nm and the maximal222 nm (Figure 5A, insert). Calculation of the Fourier power spectrum of the distribution revealed a periodicity of 22 to 23 nm (Figure SA) which is in good agreement with the longitudinal periodicity generally observed in the surface structure ofiFs (25,54). The total distribution of axial distances could thus be interpreted


517


as an overlay of several Gaussian distributions around multiples of the basic periodicity. Gaussian fits calculated accordingly were in excellent agreement with this model (Figure 5B). Thus it seems as if theN-terminal head domains of the constituent protein subunits are exposed on the filament surface in a helical arrangement with a periodicity of approximately 22 nm and that for steric reasons the oligo(dGb molecules can bind only to those N-termini which are exposed on the upper side of the substratum-attached filaments. These results indicate that oligo- and polynucleotides with high affinities for the amino-terminal head domain of vimentin may initiate filament disassembly by binding either to the intact ftlament surface or, preferentially, to ends of or defective sites along the filaments, thereby weakening the filament structure. This initiallabilization makes additional amino-terminal head domains available to polynucleotide molecules and eventually leads to fragmentation of the filaments. However, displacement of a limited number of amino termini from the filament surface must not necessarily lead to filament disruption since IFs are able to tolerate the incorporation of a certain number of amino-terminally truncated subunit proteins without being grossly affected in their overall structure (30). While this conclusion is based on the static model ofiF structure, the dissociation effects ofG-rich polynucleotides on IFs can also be reconciled with the concept that IFs are dynamic structures which constantly assemble and disassemble. This view has recently been developed on the basis of results obtained from transfection (52) and microinjection (53) experiments, in which it has been shown that newly-synthesized vimentin subunits or labelled protofilaments can incorporate or exchange all along the length of preformed filaments, rapidly and without apparent polarity. Furthermore, Vikstrom et a/. (53) propose that the IF structure more closely resembles a partially disordered braid of highly flexible, loosely packed polymers, rather than a rigid crystalline lattice. This concept would not only account for their observations of rapid exchange ofiF subunits, but would also be easy to reconcile with the present observations on the interactions ofvimentin IFs with nucleic acids. Since it is known that the binding site ofvimentin for nucleic acids is the amino-terminal head domain, which is also highly essential for filament formation and stability, the ability of a nucleic acid to perturb IF assembly or stability would be a function of its affinity for the head domain and its ability to drive the IF:protoftlament equilibrium to the right

Effects of Naturally Occurring Nucleic Acids on IF Assembly and Stability An additional series of experiments addressed the question of whether naturally occurring DNAs and RNAs are endowed with similar capacities as polyguanylic acids to interfere with vimentin filament formation or to distort the structure of intact filaments. A biochemical approach to answer this question is based on the fact that vimentin filaments survive treatment with lysine-specific endoproteinase despite carboxy-terminal truncation of their subunit proteins by 27 amino acid residues (producing a molecule termed L-vimentin), whereas protofilamentous vimentin is totally degraded by this enzyme (30,31). If naturally occurring nucleic acids block vimentin filament assembly or disrupt intact filaments, that fraction of vimentin which is not incorporated into or released from filaments should be

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Figure 6: Assembly ofvimentin filaments at different KCl concentrations in the absence and presence of heat-denatured calf thymus DNA or total EAT cell rRNA, respectively, and digestion ofvimentin with lysine-specific endoproteinase Lys-C. Vimentin filaments were reconstituted at 50 mM (A-C), 100 mM (D-F) and 150 mM (G-1) KCl in the absence of nucleic acid (A,D,G) and the presence ofheat-denatured calf thymus DNA (B,E,H) and total EAT cell rRNA (C,F,I). The mass ratio ofvimentin: nucleic acid was 1:2. The assembly mixtures were incubated with lysine-specific endoproteinase Lys-C and at the time points 0, 5, 10, 20, 30, 45, 60, 120, 180 and 240 min (lanes 1 to 10) equal portions of the digestion mixtures were subjected to SDS-PAGE analysis.

degradable by the proteinase. Since it was known that naturally occurring RNAs and DNAs with their more or less random base compositions bind vimentin less efficiently than polyguanylic acids, their effect on filament formation was also studied at subphysiological ionic strength. The purpose of this strategy was to reduce somewhat the potential of vimentin to assemble into IFs and to partially diminish the stability of the filaments.



519

Figure 6 shows the kinetics of the degradation by lysine-specific endoproteinase of vimentin assembled into IFs at 50 mM (Figure 6A-C), 100 mM (Figure 6D-F) and 150 mM (Figure 6G-I) KCl in the absence (Figure 6A,D,G) and presence of either heat-denatured calf thymus DNA (Figure 6B,E,H) or EAT cell total rRNA (Figure 6C,F,I). When filament formation had been performed at 50 mM KCl in the absence of nucleic acids and the filaments were subsequently digested, a substantial amount ofL-vimentin was detectable on the SDS-polyacrylamide gel, even after a digestion period of 4h, indicating the incorporation of an at least equivalent quantity of vimentin into filaments (Figure 6A). The fact that the amount of L-vimentin decreased somewhat with increasing digestion time suggests that the filaments formed were less compact than the filaments produced at higher ionic strength and therefore tended to disintegrate during digestion (see, e.g. Figure 6G). llowever, when filament formation at the same 50 mM KCl had been allowed to proceed in the presence of single-stranded DNA or rRNA, subsequent treatment of the reaction mixtures with proteinase caused rapid and complete degradation ofvimentin to small polypeptides (Figure 6B,C). This demonstrated that the nucleic acids had totally prevented the formation ofiFs. This differential response ofthe filament assembly assays performed in the absence and presence of nucleic acids was still observable when the filaments had been reconstituted at 100 mM KCl (Figure 6D-F), but it was totally abolished when filament assembly had been conducted at 150 mM KCl (Figure 6GI). The somewhat slower degradation of vim en tin in the reconstitution mixtures containing 100 mM KCl and in the presence of nucleic acids points to the formation of a significant amount ofiFs which, however, were disassembled during the course of proteolytic digestion. The results of this biochemical assay were confirmed by electron microscopy. Figure 7 shows negatively stained vimentin filaments reconstituted at 50, 100 and 150 mM KCl (Figure 7A,E,I) as well as reaction products obtained at the same KCl concentrations but in the presence of heat-denatured calf thymus DNA (Figure 7B,F,J), EAT cell rRNA (Figure 7C,G,K) and the duplex DNA poly(dG) ·poly(dC) (Figure 7D,H,L). As expected, all of these nucleic acids prevented filament formation at 50 mM KCl. Surprisingly, single-stranded DNA caused the formation oflong ribbons of varying diameter which were decidedly different from normal 10 nm filaments (Figure 7B), whereas at 100 mM KCl the first signs of normal IF formation in the presence of this nucleic acid were detectable (Figure 7F). At the same KCl concentration, rRNA blocked filament assembly (Figure 7G). However, filament formation could take place in the presence of poly(dG) · poly(dC) (Figure 7H), although this synthetic DNA duplex was found to efficiently interact with vimentin at low ionic strength. The affinity of the polynucleotide for the amino-terminal head region ofvimentin was obviously not sufficient to successfully out compete the ahelical rod domains of the vim en tin tetramers for binding of this protein domain. Finally, at 150 mM KCl, all three of these nucleic acids tested failed to suppress the formation ofvimentin filaments (Figure 7J-L). The inspection of the filaments which had formed despite the presence of nucleic acids revealed that they were heavily decorated with densely staining material (Figure 7H,J-L). Similar complexes were observed when a synthetic, double-stranded oligonucleotide telomere model ( 13) was used (data not shown). Since only nucleic

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520

Figure 7: Negative staining and electron microscopy ofvimentin filaments assembled at different KCI concentrations and in the absence and presence of heat-denatured calf thymus DNA, EAT cell rRNA and poly(dG) · poly(dC), respectively. Vimentin filaments were reconstituted at 50 mM (A-D), 100 mM (E-H) and !50 mM (1-L) KCI in the absence of nucleic acid (A,E,I) and the presence of heat-denatured calf thymus DNA(B,F,J), rRNA(C,G,K) and poly(dG).poly(dC) (D,H,L). The mass ratios were: vimentin: calf thymus DNA = 1:2, vimentin: rRNA = 1:2 and vimentin: poly(dG).poly(dC) = 1:1. The negatively stained specimens were viewed in a Philips Model400T electron microscope. Magnification: X 80,000. Bar: 100 nm.



521

acids were present as additional components in the filament reconstitution mixtures, this material could be either only free nucleic acid or consist of complexes of nonfilamentous vimen tin with these nucleic acids. The latter case, however, could be precluded because probing of the reconstitution mixtures with lysine-specific endoproteinase had revealed the total absence of vimentin not incorporated into IFs (Figure 6H,I). However, there was still the possibility that the association products observed on the electron microscopy grids had formed, in a secondary reaction, during washing of the loaded carbon films with distilled water. It was known from previous studies that the affinity of polynucleotides for vimentin increases with diminishing ionic strength. Moreover, reduction of the salt concentration could have made the surface-exposed amino-terminal polypeptide chains more accessible to the nucleic acid molecules through a peripheral decondensation of the filaments. In order to prevent such a secondary association, the loaded carbon films were washed several times with filament reconstitution buffer, to remove as much free nucleic acid material as possible, and only then with distilled water before staining with uranyl acetate. However, this additional treatment did not bring about any change in the electron microscopic appearance of the reaction products (data not shown) which, therefore, has to be ascribed to the direct association of the nucleic acids with the reconstituted vimentin filaments. To find out whether single-stranded DNA rRNA and synthetic duplex DNA had the potential to disassemble preexisting filaments assembled at 50, 100 and 150 mM KCl, these were treated at the different salt concentrations with the various nucleic acids, negatively stained and examined by electron microscopy. While filaments formed at 50 mM KCl were disintegrated by all three nucleic acids, those assembled at higher KCl concentrations appeared to be resistant to such treatment. However, in all cases in which intact IFs could be detected, these were again densely decorated with nucleic acid material (data not shown). Interaction of IFs with Heteroduplex DNA, Heteroduplex RNA and rRNA: Influence of

Mi+. Filament Stability and Strandedness ofNucleic Acid

It was also known from previous investigations that Mg2+ has a negative influence on the interaction of polynucleotides with vimentin (9) and a positive effect on filament assembly and stability. On the basis ofthese observations, it was assumed that Mg2+ might suspend the inhibition of filament formation by nucleic acids. In a series of experiments identical to that described in Figure 7, with the exception that all reconstitution mixtures additionally contained 2 mM MgC12, vimentin was allowed to react with the various nucleic acids. Already at 50 mM KCl, the polynucleotides did not interfere anymore with the incorporation ofvimentin into IFs. Only poly(dG) · poly(dC) seemed to have retained some inhibitory activity under these ionic conditions. At the higher concentrations of 100 and 150 mM KCl, normal filament formation took place despite the presence of nucleic acids. Intriguingly, Mg2+ did not prevent the binding of nucleic acids to the filaments( data not shown and Table I, discussed below).

The latter observation induced a more detailed biochemical investigation of the stability of some of the vim en tin filament-nucleic acid adducts detected by electron

522

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Table I Interaction ofvimentin filaments with radiolabeled nucleic acids and sucrose gradient analysis of the association products. The ionic conditions under which the reactions were performed were: T 10 K 150, 10 mM Tris-HCI, pH 7.6,150 mM KCI, 6 mM 2-mercaptoethanol (buffer II); T 10Mg 2K 150o the same as buffer II plus 2 mM MgCI2; T 10M&EDTA.tK150, the same as buffer II plus 4 mM MgCI2 and 2 mM EDTA Whenever the reaction mixtures contained MgCI 2, the ionic composition of the sucrose gradients was I 0 mM Tris-HCI, pH 7.6, 2 mM MgCI2, 150 mM KCI, 6 mM 2-mercaptoethanol; otherwise the sucrose solutions were prepared in buffer II. The amount in percent of nucleic acid stably bound to the vimentin filaments (.~) was obtained by subtraction of the quantity of nucleic acid pelleted in the absence of filaments(- vim fil.) from that pelleted in the presence of filaments(+ vim fil.). Percentage of nucleic acid in the pellet -vim fil. +vim fil. A

Nucleic acid

Ionic conditions

poly(dG) · poly(dC)

TIO Kl50 TIO Mg2Kl50 T 10 Mg4EDTA2K 150

75.3 79.3 88.4

1.8 1.8 5.2

73.5 77.5 83.2

poly(dA) · poly(dT)

TIO Kl50 Tw Mg2K1so TIO M&EDTA2Kl50

13.9 15.1 60.1

0.4 0.3 3.4

13.5 14.8 56.7

poly(rl) · poly(rC)

Tw K1so TIO Mg2Kl50 TIO Mg4EDTA2Kl50

20.3 19.7 43.8

2.5 16.8 26.0

17.8 2.9 17.8

poly(rA) · poly(rU)

TIO Kl50 TIO Mg2Kl50 TIO M&EDTA2Kl50

75.1 44.4 64.2

15.5 19.2 25.5

59.6 25.2 38.7

ISs rRNA 28s rRNA

T 10 Mg4EDTA2K 150 TIO M&EDTA2Kl50

6.4 15.1

2.0 9.1

4.4 6.0

microscopy. In a first set of ex~eriments, vimentin filaments reconstituted at 150 mM KC1 were allowed to react with H-1abe1ed poly(dG) · poly(dC) and poly(d.A) · poly(dl), respectively, and subjected to sucrose gradient centrifugation under the same ionic conditions. These two heteroduplexes were selected because they represent doublestranded DNAs with considerably different affinities for vimentin at low ionic strength (11 ). Figure 8A-D shows the distribution ofvimentin and the two polynucleotides in the sucrose gradients. Whereas in the absence ofvimentin filaments virtually all polynucleotide material stayed in the upper parts of the sucrose gradients (Figure 8A,C), in the presence of vimentin filaments close to 75% of poly(dG) · poly(dC) (Figure 8B) and 15% of poly(dA) · poly(dT) (Figure 80) (see also Table I) were pelleted together with approximately 20% of the vim en tin filaments. The fact that poly( dG) ·poly( dC) bound with a much higher efficiency to the vim entin filaments than poly(d.A) · poly(dT) shows that the complexes formed were not just unspecific aggregation products but that the filaments made specific use of their surfaceexposed amino-terminal polypeptide chains in binding the polynucleotides, mirroring the binding characteristics of proto filamentous vimentin. It is striking that only a relatively small fraction ofthe vimentin filaments reached the bottom ofthe sucrose gradients; the remainder apparently disintegrated with the formation of more

523


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Coalignment of vimentin intermediate filaments with microtubules depends on kinesin.

Microtubule-dependent transport and dynamics of vimentin intermediate filaments.

Methods for Determining the Cellular Functions of Vimentin Intermediate Filaments.

Intermediate filament assembly and stability in vitro: effect and implications of the removal of head and tail domains of vimentin by the human immunodeficiency virus type 1 protease.

Direct observation of subunit exchange along mature vimentin intermediate filaments.

Vimentin Intermediate Filaments Template Microtubule Networks to Enhance Persistence in Cell Polarity and Directed Migration.

Fluorescence study of the nucleic acid binding site of vimentin.

stromal cells.

Human immunodeficiency virus type 1 protease cleaves the intermediate filament proteins vimentin, desmin, and glial fibrillary acidic protein.

Epithelial cell migration requires the interaction between the vimentin and keratin intermediate filaments.

Trinitrophenylation of nucleic acids and their constituents.

Immunogold staining of intermediate-sized filaments of the vimentin type in human skin: a postembedding immunoelectron microscopic study.

Lateral association and elongation of vimentin intermediate filament proteins: A time-resolved light-scattering study.

Vimentin filament organization and stress sensing depend on its single cysteine residue and zinc binding.

Self-assembly enhances the strength of fibers made from vimentin intermediate filament proteins.

Structure of the Intermediate Filament-Binding Region of Desmoplakin.

Liprin-α1 is a regulator of vimentin intermediate filament network in the cancer cell adhesion machinery.

Bidirectional Interplay between Vimentin Intermediate Filaments and Contractile Actin Stress Fibers.

HeLa cells contain intermediate-sized filaments of the prekeratin type.

Tiny masking locked nucleic acids effectively bind to mRNA and inhibit binding of microRNAs in relation to thermodynamic stability.

Binding and cleavage of nucleic acids by the "hairpin" ribozyme.

Aldolase binding to actin-containing filaments. Formation of paracrystals.

Intermediate filament reorganization during mitosis is mediated by p34cdc2 phosphorylation of vimentin.

Intermediate filaments in normal thyrocytes: modulation of vimentin expression in primary cultures.