Quantitative analysis of the self-assembly strategies of intermediate filaments from tetrameric vimentin

In vitro assembly of intermediate filaments from tetrameric vimentin consists of a very rapid phase of tetramers laterally associating into unit-length filaments and a slow phase of filament elongation. We focus in this paper on a systematic quantitative investigation of two molecular models for filament assembly, recently proposed in (Kirmse et al. J. Biol. Chem. 282, 52 (2007), 18563-18572), through mathematical modeling, model fitting, and model validation. We analyze the quantitative contribution of each filament elongation strategy: with tetramers, with unit-length filaments, with longer filaments, or combinations thereof. In each case, we discuss the numerical fitting of the model with respect to one set of data, and its separate validation with respect to a second, different set of data. We introduce a high-resolution model for vimentin filament self-assembly, able to capture the detailed dynamics of filaments of arbitrary length. This provides much more predictive power for the model, in comparison to previous models where only the mean length of all filaments in the solution could be analyzed. We show how kinetic observations on low-resolution models can be extrapolated to the high-resolution model and used for lowering its complexity.     

Assembly of vimentin in vitro and its implications concerning the structure of intermediate filaments

After dialysis against 10 mM-Tris-acetate (pH 8.5), vimentin that has been purified in the presence of urea is present in the form of tetrameric 2 to 3 nm X 48 nm rods known as protofilaments. These building blocks in turn polymerize into intermediate filaments (10 to 12 nm diameter) when they are dialyzed against a solution of physiological ionic strength and pH. By varying the ionic conditions under which polymerization takes place, we have identified two classes of assembly intermediates whose structures provide clues as to how an intermediate filament may be constructed. The structure of the first class, seen when assembly takes place at 10 to 20 mM-salt at pH 8.5, strongly suggests that one of the initial steps of filament assembly is the association of protofilaments into pairs with a half-unit axial stagger. Increasing the ionic strength of the assembly buffer leads to the emergence of short, full-width intermediate filaments at approximately 50 mM-salt at pH 8.5. In the presence of additional protofilaments, these short filaments elongate to many micrometers when the ionic strength and pH are further adjusted to physiological levels. The electron microscope images of the assembly intermediates suggest that vimentin-containing intermediate filaments are made up of eight protofilaments, assembled such that there is an approximately 22 nm axial stagger between neighboring protofilaments. We propose that this half-unit staggering of protofilaments is a fundamental feature of intermediate filament structure and assembly, and that it could account for the 20 to 22 nm axial repeat seen in all intermediate filaments examined so far.     

Engagement of vimentin intermediate filaments in hypotonic stress

Intermediate filaments (IFs) play a key role in the control of cell structure and morphology, cell mechano-responses, migration, proliferation, and apoptosis. However, the mechanisms regulating IFs organization in motile adhesive cells under certain physical/pathological conditions remain to be fully understood. In this study, we found hypo-osmotic–induced stress results in a dramatic but reversible rearrangement of the IF network. Vimentin and nestin IFs are partially depolymerized as they are redistributed throughout the cell cytoplasm after hypo-osmotic shock. This spreading of the IFs requires an intact microtubule network and the motor protein associated transportation. Both nocodazole treatment and depletion of kinesin-1 (KIF5B) block the hypo-osmotic shock–induced rearrangement of IFs showing that the dynamic behavior of IFs largely depends on microtubules and kinesin-dependent transport. Moreover, we show that cell survival rates are dramatically decreased in response to hypo-osmotic shock, which was more severe by vimentin IFs depletion, indicating its contribution to osmotic endurance. Collectively, these results reveal a critical role of vimentin IFs under hypotonic stress and provide evidence that IFs are important for the defense mechanisms during the osmotic challenge.     

Analysis of the mechanism of assembly of mouse keratin 1/keratin 10 intermediate filaments in vitro suggests that intermediate filaments are built from multiple oligomeric units rather than a unique tetrameric building block.

The question as to whether keratin intermediate filaments (KIF) are built from a unique "building block" consisting of a pair of coiled-coil molecules has been studied by examining the earliest stages of reassembly of mouse K1/K10 KIF in vitro. Particles formed in protein solutions of about 45 micrograms/ml (near or below the critical concentration for assembly) or 0.5-1.65 mg/ml were monitored by turbidity, visualized by electron microscopy, and their structures resolved biochemically using crosslinking, limited proteolysis, and amino acid sequencing. The rate of KIF reassembly in vitro is limited by an initial slow step involving the formation of a three- or four-molecule oligomer. At 2 min, the particles in solution are about 65 nm long and consist of two molecules aligned antiparallel and staggered. A few minutes later, a three- and/or four-molecule species appears that may be the rate-limiting particle(s). It is also 65 nm long, but contains one or two additional molecules aligned in register but antiparallel with respect to one of the molecules on the two-molecule particle. The present data cannot establish whether the rate-limiting particle contains three or four molecules, or in fact consists of a mixture of both. Below the critical concentration for KIF assembly, it exists in solution in rapid exchange with particles containing one and two molecules. In solutions above the critical concentration for assembly, once this oligomer has formed in sufficient quantity, further assembly into KIF occurs rapidly; 90, 110, and 130-nm particles soon appear by apparent addition of a single molecule or oligomers containing two, three, four, or even several molecules. Within about 20 min short KIF about 200-500 nm long appear which later elongate to long (greater than 1 micron) KIF. These data suggest that KIF assembly requires the initial correct alignment of three or four molecules which, once formed, provides a template for further rapid addition of molecules leading to KIF assembly. Furthermore, the data establish that KIF are built from alternating rows of in-register and staggered antiparallel molecules. The present data confirm independently the observations of the previous paper and do not support earlier notions that IF are built from a tetrameric building block consisting of a pair of in-register molecules. Finally, the data suggest that the mechanism of assembly in vitro and the dynamic in vivo assembly-disassembly characteristics of KIF in particular and IF in general are mediated through a variety of small oligomeric species ranging in size from one to several molecules.     

Microtubule-dependent transport and dynamics of vimentin intermediate filaments

We studied two aspects of vimentin intermediate filament dynamics—transport of filaments and subunit exchange. We observed transport of long filaments in the periphery of cells using live-cell structured illumination microscopy. We studied filament transport elsewhere in cells using a photoconvertible-vimentin probe and total internal reflection microscopy. We found that filaments were rapidly transported along linear tracks in both anterograde and retrograde directions. Filament transport was microtubule dependent but independent of microtubule polymerization and/or an interaction with the plus end–binding protein APC. We also studied subunit exchange in filaments by long-term imaging after photoconversion. We found that converted vimentin remained in small clusters along the length of filaments rather than redistributing uniformly throughout the network, even in cells that divided after photoconversion. These data show that vimentin filaments do not depolymerize into individual subunits; they recompose by severing and reannealing. Together these results show that vimentin filaments are very dynamic and that their transport is required for network maintenance.     

Interaction of surfactant protein A with the intermediate filaments desmin and vimentin

Surfactant protein A (SP-A), a member of the collectin family that modulates innate immunity, has recently been involved in the physiology of reproduction. Consistent with the activation of ERK-1/2 and COX-2 induced by SP-A in myometrial cells, we reported previously the presence of two major proteins recognized by SP-A in these cells. Here we identify by mass spectrometry one of these SP-A targets as the intermediate filament (IF) desmin. In myometrial preparations derived from desmin-deficient mice, the absence of binding of SP-A to any 50 kDa protein confirmed the identity of this SP-A-binding site as desmin. Our data based on partial chymotrypsin digestion of pure desmin suggested that SP-A recognizes especially its rod domain, which is known to play an important role during the assembly of desmin into filaments. In line with that, electron microscopy experiments showed that SP-A inhibits in vitro the polymerization of desmin filaments. SP-A also recognized in vitro polymerized filaments in a calcium-dependent manner at a physiological ionic strength but not the C1q receptor gC1qR. Furthermore, Texas Red-labeled SP-A colocalized with desmin filaments in myometrial cells. Interestingly, vimentin, the IF characteristic of leukocytes, is one of the major proteins recognized by SP-A in protein extracts of U937 cells after PMA-induced differentiation of this monocytic cell line. Interaction of SP-A with vimentin was further confirmed using recombinant vimentin in solid-phase binding assays. The ability of SP-A to interact with desmin and vimentin, and to prevent polymerization of desmin monomers, shed light on unexpected and wider biological roles of this collectin.     

Nestin promotes the phosphorylation-dependent disassembly of vimentin intermediate filaments during mitosis

The expression of the intermediate filament (IF) protein nestin is closely associated with rapidly proliferating progenitor cells during neurogenesis and myogenesis, but little is known about its function. In this study, we examine the effects of nestin expression on the assembly state of vimentin IFs in nestin-free cells. Nestin is introduced by transient transfection and is positively correlated with the disassembly of vimentin IFs into nonfilamentous aggregates or particles in mitotic but not interphase cells. This nestin-mediated disassembly of IFs is dependent on the phosphorylation of vimentin by the maturation/M-phase-promoting factor at ser-55 in the amino-terminal head domain. In addition, the disassembly of vimentin IFs during mitosis appears to be a unique feature of nestin-expressing cell types. Furthermore, when the expression of nestin is downregulated by the nestin-specific small interfering RNA in nestin-expressing cells, vimentin IFs remain assembled throughout all stages of mitosis. Previous studies suggest that nonfilamentous vimentin particles are IF precursors and can be transported rapidly between different cytoplasmic compartments along microtubule tracks. On the basis of these observations, we speculate that nestin may play a role in the trafficking and distribution of IF proteins and potentially other cellular factors to daughter cells during progenitor cell division.     

Desmin/vimentin intermediate filaments are dispensable for many aspects of myogenesis

An expression vector was prepared containing a cDNA coding for a truncated version of the intermediate filament (IF) protein desmin. The encoded truncated desmin protein lacks a portion of the highly conserved alpha-helical rod region as well as the entire nonhelical carboxy-terminal domain. When transiently expressed in primary fibroblasts, or in differentiating postmitotic myoblasts and multinucleated myotubes, the truncated protein induces the complete dismantling of the preexisting vimentin or desmin/vimentin IF networks, respectively. Instead, in both cell types vimentin and desmin are packaged into hybrid spheroid bodies scattered throughout the cytoplasm. Despite the complete lack of intact IFs, myoblasts and myotubes expressing truncated desmin assemble and laterally align normal striated myofibrils and contract spontaneously in a manner indistinguishable from that of control myogenic cells. In older cultures the spheroid bodies shift from a longitudinal to a predominantly transverse orientation and loosely align along the I-Z-I-regions of striated myofibrils (Bennett, G.S., S. Fellini, Y. Toyama, and H. Holtzer. 1979. J. Cell Biol. 82:577-584), analogous to the translocation of intact desmin/vimentin IFs in control muscle. These results suggest the need for a critical reexamination of currently held concepts regarding the functions of desmin IFs during myogenesis.     

Synemin and vimentin are components of intermediate filaments in avian erythrocytes

Synemin, a high-molecular-weight protein associated with intermediate filaments in muscle, and vimentin, an intermediate-filament subunit found in many different cell types, have been identified by immunologic and electrophoretic criteria as components of intermediate filaments in mature avian erythrocytes. Desmin, the predominant subunit of intermediate filaments in muscle, has not been detected in these cells. Two dimensional immunoautoradiography of proteolytic fragments of synemin and vimentin demonstates that the erythrocyte proteins are highly homologous, if not identical, to their muscle counterparts. Double immunoflurorescence reaveals that erythrocyte synemin and vimentin co-localize in a cytoplasmic network of sinuous filaments that extends from the nucleus to the plasma membrane and resists aggregation by colcemid. Erythrocytes that are attached to glass cover slips can be sonicated to remove nuclei and nonadherent regions of the plasma membrane; this leaves elliptical patches of adherent membrane that retain mats of vimentin- and synemin-containing intermediate filaments, as seen by immunofluorescence and rotary shadowing. Similarly, mechanical enucleation of erythrocyte ghosts in suspension allows isolation of plasma membranes that retain a significant fraction of the synemin and vimentin, as assayed by electrophoresis, and intermediate filaments, as seen in thin sections. Both synemin and vimentin remain insoluble along with spectrin and actin, in solutions containing nonionic detergent and high salt. However, brief exposure of isolated membrane to distilled water releases the synemin and vimentin together in nearly pure form, before the release of significant amounts of spectrin and actin. These data suggest that avian erythrocyte intermeditate filaments are somehow anchored to the plasma membrane; erythrocytes may thus provide a simple system for the study of intermediate filaments and their mode of interaction with membranes. In addition, these data, in conjunction with previous data from muscle, indicate that synemin is capable of associating with either desmin or vimentin and may thus perform a special role in the structure or function of intermediate filaments in erythrocytes as well as muscle.     

A kinetic model of intermediate formation during assembly of cholera toxin B-subunit pentamers

Cholera toxin is the most important virulence factor produced by Vibrio cholerae. The pentameric B-subunit of the toxin can bind to GM1-ganglioside receptors, leading to toxin entry into mammalian cells. Here, the in vitro disassembly and reassembly of CtxB(5) (the B subunit pentamer of cholera toxin) is investigated. When CtxB(5) was acidified at pH 1.0 and then neutralized, the B-subunits disassembled and could no longer migrate as SDS-stable pentamers on polyacrylamide gels or be captured by GM1. However, continued incubation at neutral pH resulted in the B-subunits regaining the capacity to be detected by GM1 enzyme-linked immunosorbent assay (t(12) approximately 8 min) and to migrate as SDS-stable pentamers (t(12) approximately 15 min). Time-dependent changes in Trp fluorescence intensity during B-subunit reassembly occurred with a half-time of approximately 8 min, similar to that detected by GM1 enzyme-linked immunosorbent assay, suggesting that both methods monitor earlier events than B-pentamer formation alone. Based on the Trp fluorescence intensity measurements, a kinetic model of the pathway of CtxB(5) reassembly was generated that depended on trans to cis isomerization of Pro-93 to give an interface capable of subunit-subunit interaction. The model suggests formation of intermediates in the reaction, and these were successfully detected by glutaraldehyde cross-linking.     

Role of the aromatic residues in the near-amino terminal motif of vimentin in intermediate filament assembly in vitro

Type III and IV intermediate filament (IF) proteins share a conserved sequence motif of -Tyr-Arg-Arg-X-Phe- at the near-amino termini. To characterize significance of the aromatic residues in the motif, we prepared vimentin mutants in which Tyr-10 and Phe-14 are substituted with Asn and Ser (Vim[Y10N], Vim[F14S] and Vim[Y10N, F14S]), and examined assembly properties in vitro by electron microscopy and viscosity measurements. At 2 s after initiation of assembly reaction at pH 7.2 and 150 mM NaCl, all the vimentin mutants formed so-called unit-length filaments (ULFs) that were slightly larger than ULFs of wild-type vimentin. In following filament elongation, Vim[Y10N, F14S] and Vim[Y10N] performed longitudinal annealing of ULFs very rapidly and formed IFs within only 2.5 and 5 min, respectively, while Vim[F14S] and wild-type vimentin gave IFs by 40-60 min. The IFs of Vim[Y10N, F14S] and Vim[Y10N], however, tended to intertwine each other and formed bundles in parts of the specimens. The intertwinements decreased as the salt concentration decreased, and optimal salt concentration for the two mutants to form normal IFs was 50 mM. These results suggest that the aromatic residues, especially Tyr-10, in the motif have a role in controlling intermolecular interactions involved in IF assembly in vitro and suppress undesirable filament intertwinements at physiological ionic strength.     

Efficient interaction of nonpolar lipids with intermediate filaments of the vimentin type

Based on the finding that vimentin isolated and purified from cultured mammalian cells is heavily contaminated by neutral lipids, the binding of a series of radioactively labeled nonpolar lipids to pure, delipidated vimentin was investigated. Employing gel permeation chromatography of the complexes on Sephacryl S-300, cholesterol, cholesteryl fatty acid esters and mono-, di- and triglycerides were found to efficiently associate with vimentin. These compounds also showed a strong tendency to bind to vimentin filaments. While the non-alpha-helical head piece of vimentin did not interact with neutral lipids under the above assay conditions, the alpha-helical rod domain was highly active. When cholesterol or 1,2-dioleoyl-glycerol was incorporated into phospholipid vesicles, the affinity of the liposomes for vimentin filaments was considerably increased. However, in sucrose density gradient equilibrium centrifugation the filament-vesicle adducts were only stable when the liposomes contained negatively charged phospholipids. These results suggest that the association of intermediate filaments with lipid vesicles is initiated by interaction of the arginine-rich N-termini of their subunit proteins with the negatively charged vesicle surface and stabilized by partial insertion of the protein molecules into the lipid bilayer, particularly at those sites where immiscible, nonpolar lipids create defects in phospholipid packing. Very likely, nonpolar lipids play a significant role in the interaction of intermediate filaments with natural membrane systems.     

Dissecting the 3-D structure of vimentin intermediate filaments by cryo-electron tomography

Vimentin polymerizes via complex lateral interactions of coiled-coil dimers into long, flexible filaments referred to as intermediate filaments (IFs). Intermediate in diameter between microtubules and microfilaments, IFs constitute the third cytoskeletal filament system of metazoan cells. Here we investigated the molecular basis of the 3-D architecture of vimentin IFs by cryo-electron microscopy (cryo-EM) as well as cryo-electron tomography (Cryo-ET) 3-D reconstruction. We demonstrate that vimentin filaments in cross-section exhibit predominantly a four-stranded protofibrilar organization with a right-handed supertwist with a helical pitch of about 96 nm. Compact filaments imaged by cryo-EM appear surprisingly straight and hence appear very stiff. In addition, IFs exhibited an increased flexibility at sites of partial unraveling. This is in strong contrast to chemically fixed, negatively stained preparations of vimentin filaments that generally exhibit smooth bending without untwisting. At some point along the filament unraveling may be triggered and propagates in a cooperative manner so that long stretches of filaments appear to have unraveled rapidly in a coordinated fashion.     

An electron microscopic study of the interaction in vitro of vimentin intermediate filaments with vesicles prepared from Ehrlich ascites tumor cell lipids

The interaction of intermediate filaments prepared from pure, delipidated vimentin with vesicles obtained from Ehrlich ascites tumor (EAT) cell lipids was studied employing sucrose density gradient centrifugation in combination with electron microscopy. In negative stain electron microscopy, preformed vimentin filaments were seen in lateral association with lipid vesicles; end-on contacts of filaments with liposomes were rarely detected. When the reaction of filaments with vesicles was carried out at 0 degree C, sucrose density gradient equilibrium centrifugation of the reaction products led to the banding of relatively light filament-vesicle meshworks in clear separation from free filaments and free vesicles. With certain vimentin and lipid preparations, occasionally partial breakdown of the filaments during centrifugation and banding of vesicle-free fragments in denser regions of the sucrose gradients was observed. However, when the reaction mixtures were incubated at 37 degrees C prior to sucrose gradient analysis, all filaments were released from vesicles and totally fragmented during centrifugation. Electron microscopy showed unraveling of the filament fragments into subfilament strands. Employing lipid vesicles labeled with [3H]cholesterol, a low but significant amount of radioactivity was found to be associated with the fragments in a non-vesicular form. Filament reconstitution experiments performed in the presence of EAT cell lipids revealed an inhibitory effect of vesicles on filament assembly, particularly at lower temperatures. The mechanical labilization of the filament structure by lipid vesicles might play a role in the redistribution of intermediate filaments in the course of certain cellular processes involving turnover and fragmentation of intracellular membrane systems.     

Expression of glial and vimentin type intermediate filaments in cultures derived from human glial material

Several cultures established from biopsies of apparently normal adult human glial material showed no cells positive for glial fibrillary acidic protein (GFA) when examined after seven or more cumulative population doublings (CPD), although the established glioma line U251 MG showed approximately 3% GFA-positive cells, and U333 CG/343 MG clone 3 showed greater than 98% GFA-positive cells. Both the human glia delivered cultures and the glioma lines were positive when assayed with sera specific for vimentin. We therefore investigated the expression of GFA as a function of cumulative population doublings after the establishment of primary cultures. Under our experimental conditions, although GFA-positive cells were clearly present in the primary cultures accounting for some 3%-14% of the cells present, the GFA marker was subsequently lost, and the proliferating cultures expressed only the vimentin type of intermediate filament. Those cells that were GFA-positive also appeared to be vimentin-positive. GFA expression was not reinduced in cultures that had lost the GFA marker by treatment with dibutyryl cyclic AMP. We discuss two alternate hypotheses for the origin of the GFA-negative cells: (1) the cultures area of astrocyte origin but lost the ability to express GFA on culturing; (2) the cultures originate from cells of nonastrocyte origin present in the primary material.     

Rigidity of circulating lymphocytes is primarily conferred by vimentin intermediate filaments

Lymphocytes need rigidity while in circulation, but must abruptly become deformable to undergo transmigration into tissue. Previously, the control of leukocyte deformability has been attributed to microfilaments or microtubules, but the present studies demonstrate the greater importance of vimentin intermediate filaments (IFs). In circulating T lymphocytes, IFs form a distinctive spherical cage that undergoes a rapid condensation into a juxtanuclear aggregate during chemokine-induced polarization. Measurements of the resistance of peripheral blood T lymphocytes to global deformation demonstrate that their rigidity is primarily dependent on intact vimentin filaments. Microtubules, in contrast, are not sufficient to maintain rigidity. Thus, vimentin IFs are a primary source of structural support in circulating human lymphocytes, and their regulated collapse is likely to be an essential element in chemokine-induced transendothelial migration.     

Structural analysis of vimentin and keratin intermediate filaments by cryo-electron tomography

Intermediate filaments are a large and structurally diverse group of cellular filaments that are classified into five different groups. They are referred to as intermediate filaments (IFs) because they are intermediate in diameter between the two other cytoskeletal filament systems that is filamentous actin and microtubules. The basic building block of IFs is a predominantly alpha-helical rod with variable length globular N- and C-terminal domains. On the ultra-structural level there are two major differences between IFs and microtubules or actin filaments: IFs are non-polar, and they do not exhibit large globular domains. IF molecules associate via a coiled-coil interaction into dimers and higher oligomers. Structural investigations into the molecular building plan of IFs have been performed with a variety of biophysical and imaging methods such as negative staining and metal-shadowing electron microscopy (EM), mass determination by scanning transmission EM, X-ray crystallography on fragments of the IF stalk and low-angle X-ray scattering. The actual packing of IF dimers into a long filament varies between the different families. Typically the dimers form so called protofibrils that further assemble into a filament. Here we introduce new cryo-imaging methods for structural investigations of IFs in vitro and in vivo, i.e., cryo-electron microscopy and cryo-electron tomography, as well as associated techniques such as the preparation and handling of vitrified sections of cellular specimens.     

A Highlights from MBoC Selection: Endoplasmic spreading requires coalescence of vimentin intermediate filaments at force-bearing adhesions

For cells to develop long-range forces and carry materials to the periphery, the microtubule and organelle-rich region at the center of the cell—the endoplasm—needs to extend to near the cell edge. Depletion of the actin cross-linking protein filamin A (FlnA) causes a collapse of the endoplasm into a sphere around the nucleus of fibroblasts and disruption of matrix adhesions, indicating that FlnA is involved in endoplasmic spreading and adhesion growth. Here, we report that treatment with the calpain inhibitor N-[N-(N-acetyl-l-leucyl)-l-leucyl]-l-norleucine (ALLN) restores endoplasmic spreading as well as focal adhesion (FA) growth on fibronectin-coated surfaces in a Fln-depleted background. Addback of calpain-uncleavable talin, not full-length talin, achieves a similar effect in Fln-depleted cells and indicates a crucial role for talin in endoplasmic spreading. Because FA maturation involves the vimentin intermediate filament (vIF) network, we also examined the role of vIFs in endoplasmic spreading. Wild-type cells expressing a vimentin variant incapable of polymerization exhibit deficient endoplasmic spreading as well as defects in FA growth. ALLN treatment restores FA growth despite the lack of vIFs but does not restore endoplasmic spreading, implying that vIFs are essential for endoplasm spreading. Consistent with that hypothesis, vIFs are always displaced from adhesions when the endoplasm does not spread. In Fln-depleted cells, vIFs extend beyond adhesions, nearly to the cell edge. Finally, inhibiting myosin II–mediated contraction blocks endoplasmic spreading and adhesion growth. Thus we propose a model in which myosin II–mediated forces and coalescence of vIFs at mature FAs are required for endoplasmic spreading.