For example, in preparation for the crystallization of the amino-acid-long coil 1A of human vimentin, analytical ultracentrifugation experiments revealed that this segment did not form a stable dimer Strelkov et al. Hence, a coiled-coil structure was observed for coil 1A only in the context of the entire coil 1 Chernyatina et al. In contrast, coil 1B forms a well-organized left-handed coiled coil, as determined by X-ray crystallography and by site-directed spin labeling electron paramagnetic resonance SDSL-EPR analysis Aziz et al.
However, there is a possibility that at the very end of coil 1B, a hendecad residue repeat segment can form and thereby allow for the elongation of the helical domain by four amino acids into linker L12 see Fig. In lamins, another glutamic acid followed the consensus motif such that four glutamic acids, apposed to each other on the two chains, would terminate the coiled coil.
This high charge density at the end of a coiled coil seemed to be unfavorable, and, therefore, the first atomic structure to be determined was from the last 28 amino acids of coil 2 of vimentin.
Also, the atomic structure disclosed that both helices harbor an intrahelical salt bridge between Lys and Asp In addition, the structure revealed one interhelical salt bridge between Glu of the one chain and Arg of the other chain, which is assumed to stabilize the coiled coil Burkhard et al. Notably, these four amino acids are evolutionarily highly conserved in IF proteins as diverse as human hair keratins and Hydra lamin, indicating they mediate a principal molecular interaction in filament formation Herrmann et al.
Assembly mechanism of intermediate filament IF proteins. A Sequence comparison of coil 1A containing the IF consensus motif 1 and of the carboxy-terminal end of coil 2 IF consensus harboring motif 2 see Fig. Amino acids are shown in the one-letter code.
Blue represents identical amino acids, yellow represents three or at least two identical amino acids, and the remaining residues are of very similar character; heptad indicates the position of each amino acid in the coiled-coil heptad pattern abcdefg.
Left Head-to-tail polymerization of lamin A dimers. The two arrowheads indicate the direction from the carboxy-terminal to the amino-terminal end of the dimer, referring to the two globular domains, which represent the carboxy-terminal tail domains reprinted from Heitlinger et al.
Right The same type of head-to-tail polymers formed by a human lamin A variant that is missing most of its tail amino acids — Here, the helices are oriented such that they either form a four-stranded coiled coil or two two-stranded heterocoiled coils, each formed by one coil 1A and one coil 2 chain. Note that all four helices have the same orientation.
N, amino terminus; C, carboxyl terminus. D Schematic representation of the three-phase assembly model for cytoplasmic IFs—involving, first, lateral association and then longitudinal annealing, followed by a maturation phase that involves radial compaction—as originally proposed by Herrmann and Aebi The degree of radial compaction—from 17 to 11 nm—is to some extent buffer-dependent see Herrmann et al. Originally, coil 2 was also subdivided into two helical segments—coil 2A and coil 2B—connected by linker L2.
However, as all three subsegments showed the same number of amino acids in all IF proteins investigated i. As the structural appearance of such a stutter in an IF protein coiled-coil dimer was unknown, the atomic structure of a segment covering most of coil 2B of vimentin amino acids — was determined by X-ray crystallography. Corresponding structures for coil 2B were subsequently obtained for lamin A Strelkov et al.
In both cases, the coiled-coil structural organization followed the one originally elucidated for vimentin Strelkov et al. Eventually, the atomic structure of the entire coil 2 of human vimentin was solved, showing that this originally defined coil 2A and L2 represent hendecad segments Nicolet et al. Hence, coil 2 is predicted to be amino acids long, with the first 36 amino acids forming hendecad repeats instead of heptad repeats, and another hendecad repeat residing within the second half of coil 2.
As mentioned above, vimentin dimerizes in 6 m urea, and tetramers are formed in 5 m urea. Further dialysis into low-ionic-strength buffer such as 2 m m Tris-HCl pH 9 preserves tetramers, as shown by analytical ultracentrifugation. This biophysical behavior is consistently shown by all cytoplasmic IF proteins Lichtenstern et al.
Chemical cross-linking of entire filaments followed by sequencing of cross-linked peptides revealed the existence of four types of tetrameric orientations Parry and Steinert The globular structures observed by rotary metal shadowing techniques represent the tail domain and, thus, serve as a marker for the directionality of the assembled fiber made from dimeric complexes by head-to-tail association Fig.
Moreover, note that also the tailless lamin A still assembles and that now the head-to-tail overlap region can be seen directly Fig. From a large series of such EM images, the length of the overlap has been determined to be in the range of 2—4 nm Heitlinger et al.
For cytoplasmic IFs, this overlap distance was determined to be 1—2 nm by chemical cross-linking experiments Parry and Steinert Accordingly, EM showed a highly stringent periodicity in vertebrate cytoplasmic IFs of 43 nm Henderson et al. From these data, we proposed that this overlap could involve the formation of two heterodimeric coiled-coil segments between helices from the first three heptads of coil 1A and those from the last three heptads of coil 2 Fig. The exact mode of the A 11 organization has been determined jointly by SDSL-EPR, using spin-labeled vimentin filaments and X-ray diffraction analysis of an extended coil 1B fragment of vimentin: The interaction of the two coiled coils is centered around the glutamic acids in position Fig.
In contrast, the globular subunits of an actin filament are arranged in a single helix, with 13 subunits packed in six left-handed turns, showing an axial increase of 2. Accordingly, the structure can also be described as a right-handed long-pitch two-start helix Dominguez and Holmes Because of this distinct subunit arrangement, the actin filament is polar, and this polarity has a consequence for assembly: Actin filaments show a fast-growing and a slow-growing end for review, see Pollard In contrast, because of the antiparallel association of the two dimers in a tetramer as a building block for assembly, IFs are nonpolar.
Different from actin and tubulin, which are globular proteins with nucleotidase activity that is intimately connected to the assembly process, IF proteins are fibrous and lack enzymatic activity.
ULFs are productive precursors for the formation of long filaments Fig. In phase 2, elongation occurs by longitudinal annealing of ULFs via coil 2, such that filaments containing two, three, and more ULFs are formed.
These filaments show a high flexibility, with an apparent persistence length l p of 0. These desmin aggregates have indeed been discussed as a cause for disease development in the wider context of protein aggregate myopathies Clemen et al. This property dictates the way that they can be isolated, purified, and crystallized; in addition, it may influence investigations of their assembly and their interaction with other cellular constituents.
However, these were, in most cases, met with caution and skepticism, as they were obtained together with various heterogeneous, in many cases somewhat unraveled, filamentous structures. These were of different lengths, depending on the disassembly or reassembly conditions used, and occurred in the presence of phosphate buffer or even 4 m urea Franke et al.
Notably, epidermal keratin IFs showed a distinct subfilamentous organization that could best be described as a partially unraveled, multistranded helix of two to four protofibrils when reconstituted filaments were deposited onto an EM grid and washed with phosphate buffer before negative staining Aebi et al.
However, it was not clear in what order tetramers would associate laterally and longitudinally to form an IF. Hence, the molecular mechanism of assembly was not immediately evident. As it turned out, the mode of assembly very strongly impacts on the homogeneity of the filaments formed. In contrast, assembly of tetramers by dialysis into assembly buffer results in homogeneous filaments in terms of their MPL Fig.
Determination of intermediate filament IF architecture by complementary experimental methods. Left Dark-field electron micrographs of unstained freeze-dried single IFs top together with the corresponding MPL tracings bottom.
Right MPL histograms obtained from measuring several hundred segments along many filaments. B Red and green fluorescently labeled vimentin were each assembled separately for 1 h before being mixed to trace how assembly continued over the next two days.
Inspection of the assembly products in sample chambers confined in height, so that long filaments could swiftly equilibrate on the surface, was performed by total internal reflection fluorescence TIRF microscopy. Reprinted from Winheim et al. C IFs obtained from cultured human rhabdomyosarcoma-derived RD cells in the course of their extraction with 0.
D Single vimentin IFs were double-labeled with a mouse monoclonal antibody detecting an epitope at the beginning of coil 2 of the central rod red and a rabbit serum detecting the last nine amino acids of the carboxy-terminal tail domain green.
The inset clearly depicts the peripheral space occupied by the tips of the carboxy-terminal vimentin tail domains around the filament core. One way to follow the assembly of IFs over time is by turbidometric methods, in which changes of light scattering are followed over time at nm Steinert and Gullino ; Steinert et al. This revealed nm-long filaments that produced mature IFs when further dialyzed into a buffer of physiological ionic strength and pH m m NaCl, 10 m m imidazole, pH 7.
To follow quantitatively the kinetics of assembly of vimentin, viscometry was performed by a kick-start assembly schedule Hofmann et al. Visual inspection of the mature filament assemblies by EM revealed them to be homogeneous and aggregate-free.
With a standard Ostwald capillary viscometer, the time resolution was in the minute range: Typically, measurements were performed at 1 min after initiation of assembly, and then every 5 min.
Depending on the ionic conditions and temperature, the increase in relative viscosity was very fast and leveled off by 10 min of assembly, indicating that, on average, the filaments had reached a length larger than the mesh size and thereby had established the properties of a network Herrmann et al. Accordingly, the mesh size of vimentin IFs was determined to be nm at 0.
The mechanism underlying this network formation is based on the rapid lateral association of A 11 tetramers Fig.
Accordingly, the assembly reaction was quenched after initiation of assembly from one second onward by addition of assembly buffer containing the strong fixative glutaraldehyde at various time points Herrmann et al. After one minute, this background had disappeared, indicating the consumption of the ULF-assembly precursors. As measured in a quantitative study, the mean filament length grew from 55 nm at 5 sec to nm by 1 min, indicating that these filaments on average consist of three ULFs Kirmse et al.
Note that, during longitudinal annealing of two ULFs, the length grows only by 43 nm because of the overlap of the two coil 2 segments in the annealing zone. Every further addition of an ULF yields a nm increase in length. To describe the kinetics of assembly, a suite of different types of complexes that can theoretically assemble from tetramers, such as 8-mers, mers, mers, and further multiples of 4, was investigated.
The resulting association constants were applied to the experimentally derived mean length values and compared with a scenario in which tetramers predominantly formed eight-mers and multiples thereof, which eventually yielded the best approximation to the measured assembly data Kirmse et al.
In a next step, the assembly process was modeled according to the experimentally determined filament length histograms at different time points and by considering geometrical constraints, as well as the diffusion behavior of rod-like aggregates Portet et al.
This way, it became clear how the measured IF assembly differs from the monomer-addition-type polymerization shown, for example, by actin and tubulin.
With respect to the late stage of IF assembly, it was shown by TIRF microscopy of preassembled IFs that filaments of micrometer length and longer do longitudinally anneal, although it takes days for a significant number of elongation events to occur Winheim et al. The segmental organization of IFs has also been visualized in IFs of cultured cells. In the case of human rhabdomyosarcoma-derived RD cells, which contain both vimentin and the muscle-specific desmin, individual IFs show alternating segments, being either rich in vimentin green or in desmin red indicating that vimentin and desmin segregate from each other up to the level of ULFs Fig.
Referring to this different assembly mechanism of IFs, it has been mooted by Douglas that the complex evolution in the assembly dynamics of the IFs, as proposed by Portet and colleagues Portet et al. The reason for this behavior might reside in the fact that IF proteins are polyelectrolytes with a negatively charged rod domain and a very basic head domain.
As a consequence, the IF is built as a complex network of ionic interactions formed by the acidic rod and the basic head domain, complemented by additional hydrophobic interactions of the head and the rod with neighboring subunits within the filament.
In cells, additional mechanisms using various posttranslational modifications exist to remodel IF networks dynamically Snider and Omary An important organizational feature of IFs is the positioning of the protein tail domain within the filament.
Also, a direct demonstration of the tails protruding from the filament axis was shown by EM after glycerol spraying coupled with rotary metal shadowing of filaments assembled from the low-molecular-weight and high-molecular-weight forms of the neurofilament triplet proteins NF-L, NF-H formed in vitro.
As documented in Figure 2 F, the long NF-H tails project radially, thereby giving the neurofilaments a millipede-like appearance see also Hisanaga and Hirokawa ; Heins and Aebi The tail domains are probably of general significance for the unusual resistance of IFs to mechanical stress displayed, for example, when filaments are stretched in bulk on an EM grid Fig.
Mechanical properties of intermediate filaments IFs. A A desmin IF network stretched on a glow-discharged carbon-coated copper grid followed by glutaraldehyde fixation and negative staining for electron microscopy.
Reprinted from Kreplak et al. The upper left panel shows the filament before being pulled; the lower left image documents the breakage of the filament through the action of the AFM cantilever after being operated under high force; the white boxed area is shown enlarged on the right : arrows indicate the length of the stretched filament segments after pulling and breaking, as well as the length of the original filament segment, now appearing as a gap of nm.
C Rheological investigation of the effect of disease-causing point mutations in the human desmin tail domain symbols in left box on IF strain stiffening when compared with wild-type and tailless desmin symbols in right box.
Note that tailless desmin shows no strain stiffening at all. D Inhibition of vimentin filament assembly in the presence of a peptide representing the last 58 amino acids of coil 2, as described in Strelkov et al. Note that the presence of this peptide effectively prevents longitudinal annealing of unit-length filaments ULFs , but not their formation by lateral association of tetramers. In E , a selected x — y slice through a tomographic reconstruction stack is shown.
In this view, filaments are either compact or on occasion partially unraveled, thereby exposing individual octameric protofibrils that show a twisted arrangement red arrows.
In F , an x — z slice of the same tomographic reconstruction stack is shown, yielding a prominent packing pattern of cross-sectioned IFs, with four protofibrils per filament white arrows. Scale bars, nm. Arrows point to partially unraveled IFs. E , F , Adapted from Goldie et al.
Because of their exposure to the filament surface, the IF tails have important roles in the gelation—or network formation—of long filaments Beck et al. Instead of becoming stiffer, like wild-type desmin networks, when being mechanically stressed, tailless desmin filament networks actually become softer Fig. As an important medical consequence, desmin tail mutations causing both cardiomyopathy and skeletal muscle disease show altered strain-stiffening behavior Fig.
MPL measurements by STEM of tail-truncated vimentin variants made it clear that the first 30 amino acids of the tail domain are directly engaged in the packing of the coiled-coil domains within the filament core.
Hence, tailless vimentin contains more tetramers per filament cross section than wild-type vimentin i. Despite their high resistance to mechanical stress, IFs are highly dynamic polymers both in vivo and in vitro. Hence, by simply challenging them with peptides e. When added during assembly, these peptides allow the formation of ULF-like structures but completely inhibit their longitudinal annealing Fig.
A molecular basis for such a drastic effect might lie in the fact that IFs are built from four protofibrillar strands, with appropriate space around and within the individual protofibrils.
Hence, the dimeric head-to-tail overlaps within a protofibril can be rather freely accessed by the peptides.
The impact of these peptides on cellular organization has also been shown directly in vivo, when, after its microinjection into serum-starved cells, the entire vimentin IF network retracted into the nuclear periphery and the cells showed extensive membrane ruffling Helfand et al. The subfilamentous structure of vimentin IFs is documented in fine detail by cryo-electron tomography of unfixed, unstained specimens. Accordingly, in selected x — y slices through tomographic reconstruction stacks, some filament segments appear flexible and partially unraveled, whereas others look stiffer and compact Fig.
Cytoskeletal filaments provide the basis for cell movement. For instance, cilia and eukaryotic flagella move as a result of microtubules sliding along each other. In fact, cross sections of these tail-like cellular extensions show organized arrays of microtubules. Other cell movements, such as the pinching off of the cell membrane in the final step of cell division also known as cytokinesis are produced by the contractile capacity of actin filament networks.
Actin filaments are extremely dynamic and can rapidly form and disassemble. In fact, this dynamic action underlies the crawling behavior of cells such as amoebae. At the leading edge of a moving cell, actin filaments are rapidly polymerizing; at its rear edge, they are quickly depolymerizing Figure 5. A large number of other proteins participate in actin assembly and disassembly as well. Figure 5: Cell migration is dependent on different actin filament structures.
These protrusive structures contain actin filaments, with elongating barbed ends orientated toward the plasma membrane. B During cellular arm extension, the plasma membrane sticks to the surface at the leading edge. C Next, the nucleus and the cell body are pushed forward through intracellular contraction forces mediated by stress fibers.
D Then, retraction fibers pull the rear of the cell forward. Filopodia: molecular architecture and cellular functions. Nature Reviews Molecular Cell Biology 9, All rights reserved. Figure Detail. This page appears in the following eBook. Aa Aa Aa. Microtubules and Filaments. What Is the Cytoskeleton Made Of? The cytoskeleton of eukaryotic cells is made of filamentous proteins, and it provides mechanical support to the cell and its cytoplasmic constituents.
All cytoskeletons consist of three major classes of elements that differ in size and in protein composition. Microtubules are the largest type of filament, with a diameter of about 25 nanometers nm , and they are composed of a protein called tubulin.
Actin filaments are the smallest type, with a diameter of only about 6 nm, and they are made of a protein called actin. Intermediate filaments, as their name suggests, are mid-sized, with a diameter of about 10 nm.
Figure 5. Interactive network plot of intermediate filament proteins with multiple localizations. The numbers in the connecting nodes show the proteins that are localized to intermediate filaments and to one or more additional locations. Only connecting nodes containing more than one protein and at least 0.
The circle sizes are related to the number of proteins. Note that this calculation is only done for proteins with dual localizations.
Each node is clickable and results in a list of all proteins that are found in the connected organelles. Transcriptome analysis and classification of genes into tissue distribution categories Figure 6 shows that a larger portion of genes encoding proteins that localize to intermediate filaments are detected in some tissues or in many tissues, while a smaller portion are detected in all tissues, compared to all genes presented in the Cell Atlas.
This is well in-line with the known tissue type-dependent expression patterns of intermediate filament proteins Herrmann H et al. Figure 6. Bar plot showing the percentage of genes in different tissue distribution categories for intermediate filament-associated protein-coding genes compared to all genes in the Cell Atlas.
Parikh K et al. Cell Res. Nat Methods. J Proteomics. J Proteome Res. PLoS One. E Semple JW et al. We use cookies to enhance the usability of our website. If you continue, we'll assume that you are happy to receive all cookies. More information. Intermediate filaments have no role in cell movement. Their function is purely structural. They bear tension, thus maintaining the shape of the cell, and anchor the nucleus and other organelles in place.
Figure 2 shows how intermediate filaments create a supportive scaffolding inside the cell. The intermediate filaments are the most diverse group of cytoskeletal elements. Several types of fibrous proteins are found in the intermediate filaments.
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