The structure of Get4 reveals an α-solenoid fold adapted for multiple interactions in tail-anchored protein biogenesis

Tail-anchored proteins play important roles in protein translocation, membrane fusion and apoptosis. They are targeted to the endoplasmic reticulum membrane via the guided-entry of tail-anchored proteins (Get) pathway. We present the 2 Å crystal structure of Get4 which participates in early steps of the Get pathway. The structure shows an α-solenoid fold with particular deviations from the regular pairwise arrangement of α-helices. A conserved hydrophobic groove accommodates the flexible C-terminal region in trans. The structural organization of the Get4 helical hairpin motifs provides a scaffold for protein-protein interactions in the Get pathway.     

Molecular dynamics simulations of discoidal bilayers assembled from truncated human lipoproteins

Human apolipoprotein A-1 (apo A-1) is the major protein component of high-density lipoproteins. The apo A-1 lipid-binding domain was used as a template for the synthesis of amphipathic helical proteins termed membrane scaffold proteins, employed to self-assemble soluble monodisperse discoidal particles called Nanodiscs. In these particles, membrane scaffold proteins surround a lipid bilayer in a belt-like fashion forming bilayer disks of discrete size and composition. Here we investigate the structure of Nanodiscs through molecular dynamics simulations in which Nanodiscs were built from scaffold proteins of various lengths. The simulations showed planar or deformed Nanodiscs depending on optimal length and alignment of the scaffold proteins. Based on mean surface area per lipid calculations, comparison of small-angle x-ray scattering curves, and the relatively planar shape of Nanodiscs made from truncated scaffold proteins, one can conclude that the first 17 to 18 residues of the 200-residue apo A-1 lipid-binding domain are not involved in formation of the protein "belts" surrounding the lipid bilayer. To determine whether the addition of an integral membrane protein has an effect on the overall structure of a Nanodisc, bacteriorhodopsin was embedded into a Nanodisc and simulated using molecular dynamics, revealing a planar disk with a slightly rectangular shape.     

The vertebrate skeletal muscle thick filaments are not three-stranded. Reinterpretation of some experimental data

Computer simulation of mass distribution within the model and Fourier transforms of images depicting mass distribution are explored for verification of two alternative modes of the myosin molecule arrangement within the vertebrate skeletal muscle thick filaments. The model well depicting the complete bipolar structure of the thick filament and revealing a true threefold-rotational symmetry is a tube covered by two helices with a pitch of 2 x 43 nm due to arrangement of the myosin tails along a helical path and grouping of all myosin heads in the crowns rotated by 240 degrees and each containing three cross-bridges separated by 0 degrees, 120 degrees, and 180 degrees. The cross-bridge crown parameters are verified by EM images as well as by optical and low-angle X-ray diffraction patterns found in the literature. The myosin tail arrangement, at which the C-terminus of about 43-nm length is near-parallel to the filament axis and the rest of the tail is quite strongly twisted around, is verified by the high-angle X-ray diffraction patterns. A consequence of the new packing is a new way of movement of the myosin cross-bridges, namely, not by bending in the hinge domains, but by unwrapping from the thick filament surface towards the thin filaments along a helical path.     

Conformational transitions in the membrane scaffold protein of phospholipid bilayer nanodiscs

Phospholipid bilayer nanodiscs are model membrane systems that provide an environment where membrane proteins are highly stable and monodisperse without the use of detergents or liposomes. Nanodiscs consist of a discoidal phospholipid bilayer encircled by two copies of an amphipathic alpha helical membrane scaffold protein, which is modeled from apolipoprotein A-1. Hydrogen exchange mass spectrometry was used to probe the structure and dynamics of the scaffold protein in the presence and absence of lipid. On nanodisc self-assembly, the entire scaffold protein gained significant protection from exchange, consistent with a large, protein-wide, structural rearrangement. This protection was short-lived and the scaffold protein was highly deuterated within 2 h. Several regions of the scaffold protein, in both the lipid-free and lipid-associated states, displayed EX1 unfolding kinetics. The rapid deuteration of the scaffold protein and the presence of correlated unfolding events both indicate that nanodiscs are dynamic rather than rigid bodies in solution. This work provides a catalog of the expected scaffold protein peptic peptides in a nanodisc-hydrogen exchange mass spectrometry experiment and their deuterium uptake signatures, data that can be used as a benchmark to verify correct assembly and nanodisc structure. Such reference data will be useful control data for all hydrogen exchange mass spectrometry experiments involving nanodiscs in which transmembrane or lipid-associated proteins are the primary molecule(s) of interest.     

Arrangement of myosin heads in relaxed thick filaments from frog skeletal muscle

The distribution of myosin heads on the surface of frog skeletal muscle thick filaments has been determined by computer processing of electron micrographs of isolated filaments stained with tannic acid and uranyl acetate. The heads are arranged in three strands but not in a strictly helical manner and so the structure has cylindrical symmetry. This accounts for the "forbidden" meridional reflections seen in diffraction patterns. Each layer-line therefore represents the sum of terms of Bessel orders 0, +/- 3, +/- 6, +/- 9 and so on. These terms interact so that, unlike a helical object without terms from overlapping Bessel orders, as the azimuth is changed, the amplitude on a layer-line at a particular radius varies substantially and its phase does not alter linearly. Consequently, a three-dimensional reconstruction cannot be produced from a single view. We have therefore used tilt series of three individual filaments to decompose the data on layer-lines 0 to 6 into terms of Bessel orders up to +/- 9 using a least-squares procedure. These data had a least-squares residual of 0.32 and enabled a three-dimensional reconstruction to be obtained at a nominal resolution of 6 nm. This showed, at a radius of about 10 nm, three strands of projecting morphological units with three units spaced along each strand every 42.9 nm axially. We have identified these units with pairs of myosin heads. Successive units along a strand are perturbed axially, azimuthally and radially from the positions expected if the structure was perfectly helical. This may simply be a consequence of steric restrictions in packing the heads on the thick filament surface, but could also reflect an underlying non-helical arrangement of myosin tails, which would be consistent with the thick filament shaft being constructed from three subfilaments in which the tails were arranged regularly. There was also material at a radius of about 6 nm spaced 42.9 nm axially, which we tentatively identified with accessory proteins. The filament shaft had a pronounced pattern of axial staining.     

Predicting Transmembrane Helix Packing Arrangements using Residue Contacts and a Force-Directed Algorithm

Alpha-helical transmembrane proteins constitute roughly 30% of a typical genome and are involved in a wide variety of important biological processes including cell signalling, transport of membrane-impermeable molecules and cell recognition. Despite significant efforts to predict transmembrane protein topology, comparatively little attention has been directed toward developing a method to pack the helices together. Here, we present a novel approach to predict lipid exposure, residue contacts, helix-helix interactions and finally the optimal helical packing arrangement of transmembrane proteins. Using molecular dynamics data, we have trained and cross-validated a support vector machine (SVM) classifier to predict per residue lipid exposure with 69% accuracy. This information is combined with additional features to train a second SVM to predict residue contacts which are then used to determine helix-helix interaction with up to 65% accuracy under stringent cross-validation on a non-redundant test set. Our method is also able to discriminate native from decoy helical packing arrangements with up to 70% accuracy. Finally, we employ a force-directed algorithm to construct the optimal helical packing arrangement which demonstrates success for proteins containing up to 13 transmembrane helices. This software is freely available as source code from http://bioinf.cs.ucl.ac.uk/memsat/mempack/.     

AN ultrastructural study of cross-bridge arrangement in the frog thigh muscle thick filament.

We have developed thick filament isolation methods that preserve the relaxed cross-bridge order of frog thick filaments such that the filaments can be analyzed by the convergent techniques of electron microscopy, optical diffraction, and computer image analysis. Images of the filaments shadowed by using either unidirectional shadowing or rotary shadowing show a series of subunits arranged along a series of right-handed near-helical strands that occur every 43 nm axially along the filament arms. Optical filtrations of images of these shadowed filaments show 4-5 subunits per half-turn of the strands, consistent with a three-stranded arrangement of the cross-bridges, thus supporting our earlier results from negative staining and computer-image analysis. The optical diffraction patterns of the shadowed filaments show a departure from the pattern expected for helical symmetry consistent with the presence of cylindrical symmetry and a departure of the cross-bridges from helical symmetry. We also describe a modified negative staining procedure that gives improved delineation of the cross-bridge arrangement. From analysis of micrographs of these negatively stained filament tilted about their long axes, we have computed a preliminary three-dimensional reconstruction of the filament that clearly confirms the three-stranded arrangement of the myosin heads.     

Cryo-electron tomography of Marburg virus particles and their morphogenesis within infected cells

Several major human pathogens, including the filoviruses, paramyxoviruses, and rhabdoviruses, package their single-stranded RNA genomes within helical nucleocapsids, which bud through the plasma membrane of the infected cell to release enveloped virions. The virions are often heterogeneous in shape, which makes it difficult to study their structure and assembly mechanisms. We have applied cryo-electron tomography and sub-tomogram averaging methods to derive structures of Marburg virus, a highly pathogenic filovirus, both after release and during assembly within infected cells. The data demonstrate the potential of cryo-electron tomography methods to derive detailed structural information for intermediate steps in biological pathways within intact cells. We describe the location and arrangement of the viral proteins within the virion. We show that the N-terminal domain of the nucleoprotein contains the minimal assembly determinants for a helical nucleocapsid with variable number of proteins per turn. Lobes protruding from alternate interfaces between each nucleoprotein are formed by the C-terminal domain of the nucleoprotein, together with viral proteins VP24 and VP35. Each nucleoprotein packages six RNA bases. The nucleocapsid interacts in an unusual, flexible "Velcro-like" manner with the viral matrix protein VP40. Determination of the structures of assembly intermediates showed that the nucleocapsid has a defined orientation during transport and budding. Together the data show striking architectural homology between the nucleocapsid helix of rhabdoviruses and filoviruses, but unexpected, fundamental differences in the mechanisms by which the nucleocapsids are then assembled together with matrix proteins and initiate membrane envelopment to release infectious virions, suggesting that the viruses have evolved different solutions to these conserved assembly steps.     

Interpretation of the X-ray scattering profiles of chromatin at various NaCl concentrations by a simple chain model.

In order to interpret the change in the X-ray scattering profiles from rat thymus chromatin, extensive model calculation was carried out. Chromatin is modelled as a string of subunits (nucleosomes) in which disorder is introduced into the positions of adjacent subunits. Disposition parameters characterizing the arrangement of subunits were estimated for various states of chromatin, so that the main feature of the scattering profiles is described. The result indicated that the structure of chromatin changes, as the NaCl concentration increases, from the extended "beads-on-a string" structure to the condensed helical structure. The latter has an outer diameter of about 26 nm with 3-4 nucleosomes per turn. In the intermediate state, it has a loose helical structure. The estimation of disorder suggested that the arrangement of subunits is appreciably disordered even in the condensed helical filament at 50 mM NaCl. Our model for chromatin condensation seems to support models of the "crossed linker" type.     

A novel, multilayer structure of a helical peptide.

X-ray diffraction analysis at 1.5 A resolution has confirmed the helical conformation of a de novo designed 18-residue peptide. However, the crystal structure reveals the formation of continuous molecular layers of parallel-packed amphiphilic helices as a result of much more extensive helix-helix interactions than predicted. The crystal packing arrangement, by virtue of distinct antiparallel packing interactions, segregates the polar and apolar surfaces of the helices into discrete and well-defined interfacial regions. An extensive "ridges-into-grooves" interdigitation characterizes the hydrophobic interface, whereas an extensive network of salt bridges and hydrogen bonds dominates the corresponding hydrophilic interface.     

Fourier transform infrared spectroscopic studies of Ca(2+)-binding proteins

The secondary structures of calmodulin and parvalbumin are well established from X-ray diffraction and nuclear magnetic resonance spectroscopic studies, which indicate that these proteins are predominantly alpha-helical in character. Recent infrared studies have nevertheless suggested that the helical structures present in these proteins in solution are not the standard alpha-helix but rather some kind of distorted helices [Trewhella, J., et al. (1989) Biochemistry 28, 1294]. The evidence for this was the unusually low amide I frequency for calmodulin and troponin C in 2H2O solution. The studies presented here, however, suggest that the helical structures in these proteins are not significantly distorted, for two reasons. First, distorted helical structures have weaker hydrogen bonds than the standard alpha-helix and would therefore be expected to absorb at a higher rather than a lower frequency. Second, distorted helical structures would absorb at an unusual frequency in H2O solutions which is not the case for the proteins studied here. The band frequency of these proteins is observed to occur at a frequency observed with other proteins known to contain predominantly alpha-helical structures. Quantitative analysis of the FT-IR spectra of calmodulin (67% alpha-helix) and parvalbumin (68% alpha-helix) in H2O in the presence of Ca2+ gives helical contents similar to those reported by X-ray studies. This raises the question as to why these proteins in H2O show a normal frequency for the presence of alpha-helical structures and an abnormal frequency in 2H2O. Addition of deuterated glycerol to the proteins in 2H2O solutions results in a significant shift of absorbance to higher frequency.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stereochemical theory of the 3-dimensional structure of globular proteins. I. Highly helical intermediate structures

The authors analyze the physical prerequisites on which the proposed stereochemical theory of the three-dimensional structure of globular proteins is based. The theory represents a stereochemical modelling of the mechanism of protein self-organization suggested earlier by one of the authors. According to this mechanism, a highly helical intermediate structure(s) is formed at first and then it passes into the native one. In the highly-helical intermediate structure the arrangement of the polypeptide chain in space is the same as in the native structure. These two structures differ mainly by the secondary structure of the chain. The transition into the native structure proceeds under the effect of long-range interactions which transform the excess alpha-helices into beta-structural and irregular conformations. The so-called s-helices are considered (the alpha-helix, whose hydrophobic groups form a separate cluster on its surface). s-Helices can be obtained on the greater part of the polypeptide chain of any globular protein. In the unfolded protein chain they are the most stable and rapidly formed structures. It has been shown that namely s-helices are the initial blocks for the formation of the highly-helical intermediate structure. Stereochemical principles of the s-helix packing that permit to predict the three-dimensional structure of highly helical proteins have been found. According to these principles the highly helical structure represents the packing of hydrophobic surfaces and s-helices. In their turn, hydrophobic surfaces are formed as a result of complementary interaction of borders of hydrophobic clusters of two s-helices according to the "knob-hole" principle.     

Intermediate filament-like cytoskeleton of Caulobacter crescentus

Eukaryotic cytoskeleton consists of three main types of filaments: actin microfilaments, microtubules and intermediate filaments (IFs). Actin and tubulin-like proteins are also found in bacteria where they perform diverse cytoskeletal functions. IFs, however, are considered to be a characteristic constituent of metazoan cells only, where they (among other functions) are involved in determination and maintenance of cell shape and cellular integrity. Surprisingly, a coiled coil-rich protein called crescentin was recently shown to play a key role in determining the complex curved and helical cell shapes of the bacterium Caulobacter crescentus, and to exhibit several characteristic properties of animal IF proteins. First, the arrangement of the coiled coil domains of crescentin closely resembles the tripartite molecular architecture of IF proteins. Second, crescentin also possesses the defining biochemical property of IF proteins to assemble into 10-nm-wide filaments in vitro without cofactors. Furthermore, crescentin forms a higher-order helical structure in vivo, which is localized asymmetrically along the concave side of the cell. In close association with the cell membrane, the crescentin structure promotes the helical growth of the cell and thereby determines a curved or a helical shape, depending on the length of the cell. The unexpected finding of an IF-like element in a bacterium raises several interesting questions concerning, for example, the molecular mechanisms whereby complex and asymmetric cell shapes are generated by different bacteria, or the functional and evolutionary relatedness of crescentin to animal IF proteins.     

Influence of the arrangement and secondary structure of melittin peptides on the formation and stability of toroidal pores

Melittin interactions with lipid bilayers and melittin formed pores are extensively studied to understand the mechanism of the toroidal pore formation. Early experimental studies suggested that melittin peptide molecules are anchored by their positively charged residues located next to the C-terminus to only one leaflet of the lipid bilayer (asymmetric arrangement). However, the recent non-linear spectroscopic experiment suggests a symmetric arrangement of the peptides with the C-terminus of the peptides anchored to both bilayers. Therefore, we present here a computational study that compares the effect of symmetric and asymmetric arrangements of melittin peptides in the toroidal pore formation. We also investigate the role of the peptide secondary structure during the pore formation. Two sets of the symmetric and asymmetric pores are prepared, one with a helical peptide from the crystal structure and the other set with a less helical peptide. We observe a stable toroidal pore being formed only in the system with a symmetric arrangement of the less helical peptides. Based on the simulation results we propose that the symmetric arrangement of the peptides might be more favorable than the asymmetric arrangement, and that the helical secondary structure is not a prerequisite for the formation of the toroidal pore.     

Signal Transduction Cross Talk Mediated by Jun N-Terminal Kinase-Interacting Protein and Insulin Receptor Substrate Scaffold Protein Complexes

Scaffold proteins have been established as important mediators of signal transduction specificity. The insulin receptor substrate (IRS) proteins represent a critical group of scaffold proteins that are required for signal transduction by the insulin receptor, including the activation of phosphatidylinositol 3 kinase. The c-Jun NH₂-terminal kinase (JNK)-interacting proteins (JIPs) represent a different group of scaffold molecules that are implicated in the regulation of the JNK. These two signaling pathways are functionally linked because JNK can phosphorylate IRS1 on the negative regulatory site Ser-307. Here we demonstrate the physical association of these signaling pathways using a proteomic approach that identified insulin-regulated complexes of JIPs together with IRS scaffold proteins. Studies using mice with JIP scaffold protein defects confirm that the JIP1 and JIP2 proteins are required for normal glucose homeostasis. Together, these observations demonstrate that JIP proteins can influence insulin-stimulated signal transduction mediated by IRS proteins.The c-Jun NH₂-terminal kinase (JNK)-interacting proteins (JIPs) are implicated in the regulation of the JNK signal transduction pathway (8, 28). The JIP1 and JIP2 proteins are structurally related with similar modular domains (SH3 and PTB) and binding sites for the mixed-lineage protein kinase (MLK) group of mitogen-activated protein kinase (MAPK) kinase kinases, the MAPK kinase MKK7, and JNK (19). These JIP proteins also interact with the microtubule motor protein kinesin, several guanine nucleotide exchange factors, the phosphatase MKP7, Src-related protein kinases, and AKT to form multifunctional protein complexes (19).One potential physiological role of JIP scaffold proteins is the response to metabolic stress, insulin resistance, and diabetes. Several lines of evidence support this hypothesis. First, JIP1 is required for metabolic stress-induced activation of JNK in white adipose tissue (12). Second, MLKs that interact with JIP proteins are implicated as essential components of a signaling pathway that mediates the effects of metabolic stress on JNK activation (13). Third, studies have demonstrated that the human Jip1 gene may contribute to the development of type 2 diabetes, because a Jip1 missense mutation was found to segregate with type 2 diabetes (26). Collectively, these data suggest that JIP proteins play a role in the cellular response to metabolic stress and the regulation of insulin resistance.It is established that the insulin receptor substrate (IRS) group of scaffold proteins plays a central role in insulin signaling (27). Treatment of cells with insulin causes tyrosine phosphorylation of the insulin receptor, the recruitment of IRS proteins to the insulin receptor, and the subsequent tyrosine phosphorylation of IRS proteins on multiple residues that act as docking sites for insulin-regulated signaling molecules, including phosphatidylinositol 3 kinase (27). Negative regulation of IRS proteins is implicated as a mechanism of insulin resistance and can be mediated by multiple pathways, including IRS protein phosphorylation and degradation. Thus, the mTOR/p70^S6K (21, 22, 24) and the SOCS-1/3 (20) signaling pathways can regulate IRS protein degradation. Multisite phosphorylation on Ser/Thr residues can also regulate IRS protein function, including JNK phosphorylation of IRS1 on the inhibitory site Ser-307 that prevents recruitment of IRS1 to the activated insulin receptor (2).The IRS and JIP groups of scaffold proteins may function independently to regulate JNK-dependent and insulin-dependent signal transduction. However, functional connections between these scaffold proteins have been identified. Thus, studies using Jip1^−/− mice demonstrate that JIP1 is required for high-fat-diet-induced JNK activation in white adipose tissue, IRS1 phosphorylation on the inhibitory site Ser-307, and insulin resistance (12). These data suggest that JIP scaffold proteins function cooperatively with IRS proteins to regulate signal transduction by the insulin receptor. The purpose of this study was to examine cross talk between the JIP and IRS scaffold complexes. We demonstrate that the JIP and IRS scaffold complexes physically interact in an insulin-dependent manner and confirm that JIP proteins influence normal glucose homeostasis.     

A miniprotein scaffold used to assemble the polyproline II binding epitope recognized by SH3 domains

SH3 domains are molecular-recognition modules that function by interacting with proteins containing sequences in polyproline II (PPII) conformation. The main limitation in designing short-ligand peptides to interact with these domains is the preservation of this helical arrangement, for which a high content of proline is needed. We have overcome this limitation by using a protein scaffold provided by the avian pancreatic polypeptide (APP), a natural hormone of 36 amino acid residues. The APP protein contains a PPII stretch packed against an alpha-helix. We have designed a structure in which some residues of the APP PPII helix are replaced by a sequence motif, named RP1, which interacts with the SH3 domain of the Abelson tyrosine kinase (Abl-SH3). This design, which we call APP-RP1, is folded and, as shown by circular dichroism, has a structural content similar to that of natural APP (APP-WT). The stability of both miniproteins has been compared by unfolding experiments; the designed APP-RP1 is almost 20 deg. C more stable than the wild-type and has a higher Gibbs energy function. This increase in stability has an entropic origin. Isothermal titration calorimetry and fluorescence spectroscopy show that the thermodynamics of the binding of the APP-RP1 molecule to Abl-SH3 is comparable to that of the shorter RP1 peptide. Furthermore, the mutation by Tyr of two proline residues in APP-RP1, which are essential for the binding of some linear peptides to Abl-SH3, demonstrates the effectiveness of the scaffold in enhancing the variability in the design of high-affinity and high-specificity ligands for any SH3 domain. The application of this strategy may help in the design of ligands for other polyproline-recognition domains such as WW, PX or EVH1, and even for the in vivo application of these miniproteins.     

Helix propagation in trifluoroethanol solutions.

Helix propagation of the S-peptide sequence (residues 1-19 of ribonuclease A) in 2,2,2-trifluoroethanol (TFE) solutions has been investigated with CD and nmr Overhauser effect spectroscopies. In this study, the S-peptide helix is covalently initiated at the N-terminus through disulfide bonds to a helix scaffold derived from the N-terminal sequence of the bee venom peptide apamin. The entire S-peptide sequence of this hybrid sequence peptide becomes helical at high proportions of TFE. Residues 14-19 of the S-peptide are not helical in the free peptide in TFE, nor are they helical in ribonuclease A. The "helix stop" signal encoded by the S-peptide sequence near residue 13 does not persist at high TFE with this hybrid sequence peptide. The helix-stabilizing effects of TFE are due at least in part to facilitated propagation of an extant helix. This stabilizing effect appears to be a general solvation effect and not due to specific interaction of the helical peptide with TFE. Specifically these data support the idea that TFE destabilizes the coil state by less effective hydrogen bonding of the peptide amide to the solvent.     

Appearance of “β-Like” Circular Dichroism Spectra on Protein Aggregation That Is not Accompanied by Transition to β-Structure

CD spectra in the 200 to 250 nm spectral region for small ordered aggregates (trimers-pentamers) of tobacco mosaic virus (TMV) coat protein (CP) and for long virus-like helical aggregates of TMV CP were compared. It was found that small (4S) TMV CP aggregates have a CD spectrum typical of a protein with high -helix content, which agrees well with results of X-ray diffraction studies. But in the long helical aggregates (and in the TMV virions) TMV CP gives -like CD spectra similar to those of many other aggregated proteins. From X-ray diffraction data, it is well known that TMV CP subunits do not change their secondary or tertiary structure on assembly into virions or the helical repolymerized protein. Thus, the change in the shape of 200 to 250 nm CD spectra cannot be employed as the sole criterion of the conversion of a protein to -structure in the course of aggregation.     

A new look at the cellular scaffold by embedment-free electron microscopy method

The basic scaffold of most cells is afforded by the cytoskeleton (comprising microfilaments, intermediate filaments and the microtubules). The conventional methods of electron microscopy fail to visualize filamentous cell structure. They can show only these filaments lying at the section surface. Heavy metal staining (I), and the optical properties of the resins used for embedding are similar to those of proteins hence most proteinaceous structures remain unresolved and the cytoplasm seems to be quite homogenous (II). Aldehyde fixation could cross-link proteins and lead to the emergence of artificial structures (III). These limitations may be overcome by the use of the embedment-free electron microscopy (EF-EM). This technique present cellular scaffold as a purified, isolated, three-dimensional network with various thickness of filaments. Our study on the dynamic aspect of cellular scaffold indicate that the thickness and arrangement of filaments depend on cell type and both physiological or pathological environments. Thank also to the adaptation of immunocytochemistry to EF-EM it was possible to understand the nuclear matrix and cytomatrix structure in relation to function. Thus, combination these methods revealed findings suggesting the nuclear homing of proapoptotic proteins and their association with intermediate filaments.