Protein synthesis in resting and stimulated human lymphocytes

Summary The ribosomal profiles in lysates from resting and phytohemagglutinin stimulated human lymphocytes have been analyzed by sucrose gradient centrifugation. The percentage of polyribosomes increased during lymphocyte transformation reaching a maximal value of 60 to 70% of the total ribosomes after 72 hours of mitogen addition. This time period coincides with maximalin vivo protein synthesis. On the other hand, in nonstimulated lymphocytes, about 25% of the ribosomal particles appeared as aggregates, independently of the incubation period.Experiments performed with homologous cell free systems containing ribosomes and supernatant fluids prepared from unstimulated or activated lymphocytes demonstrate that the mixtures containing both components from stimulated lymphocytes are several fold more active in polypeptide synthesis than the systems which contain ribosomal particles and cell sap from resting cells. Assays carried out with mixtures combining the components from both sources indicate that the increased activity depends on ribosomes as well as on the supernatant fractions.Dedicated to Professor LUIS F. LELOIR on the occasion of his 70^th birthday.     

Cytospectrophotometry of hepatocyte RNA in regenerating and neoplastic liver]

The present work consists in a quantitative cytospectrophotometric investigation of the cytoplasmic hyperbasophilia that characterizes the foci of neoplastic transformation and the tumor cells in rats fed hepatocarcinogens. It reveals that the increase in the dye-binding capacity shown by the cytoplasmic RNA of these cell populations results primarily form a qualitative alteration which raises the affinity for basic dyes by a factor of nearly 2, and also to a change in concentration due to volumetric changes which may again double the staining intensity of these hepatocytes. This phenomenon of hyperbasophilia differs radically from the weak variations in basophilia observed in normal regenerating liver and in hyperplastic liver parenchyma of rats fed the carcinogenic diet in which cases the changes appear to be related mainly to de nova RNA synthesis. Biochemical assays on cellular fractions indicate that the ribosomes are the organelles responsible for the hyperbasophilic properties that hepatocytes acquire in areas of neoplastic transformation.     

Effects of hyperthermia on spectrin expression patterns of murine lymphocytes

In this study the influence of whole-body hyperthermia on the distribution of spectrin in murine lymphocytes isolated from various lymphoid tissues is examined. Lymphocytes normally vary in terms of the pattern of spectrin distribution within the cell. In certain populations of lymphocytes, spectrin is distributed into a dense submembranous aggregate that can be easily identified by immunofluorescence microscopy. In these lymphocytes, little or no spectrin is seen at the plasma membrane region in the rest of the cell. Other lymphocytes have no such cytoplasmic aggregates, and the protein is seen at the region of the plasma membrane. Following whole-body hyperthermia (40.5 degrees C for 90 min) there is a 100% increase in cells exhibiting polar spectrin aggregates in the spleen, while lymphocytes from the thymus show no alteration in the number of cells showing such aggregates. The increase in the percentage of splenic cells that express aggregated spectrin is a result of increases occurring in both T- and B-cell subsets. This increase gradually returns to control levels by 48 h post-heating. During recovery to control levels this phenomenon is resistant to additional changes when a second heat treatment is applied. The effects described above are not observed when the experiments are performed in vitro; therefore, it is likely that the in vivo heat-induced alteration in the splenic lymphocyte population reflects the physiological response of lymphocytes to stimuli during a natural fever. The role that spectrin may play in the modulation of lymphocyte membrane properties is discussed.     

Cytodifferentiation of the chick pancreas. II. Ultrastructure of the acinar cells

The ultrastructural changes occurring during the differentiation of the pancreatic acinar cell were studied in White Leghorn chick embryos from the onset of pancreatic morphogenesis on day 3 of incubation (day 3) to hatching. Generally, the changes included a loss of some structures, the addition of others and modification of existing structures. Numerous cytoplasmic filaments which were present in the early migrating cells of the pancreatic bud were no longer present on day 5. The nucleoli enlarged temporarily on days 5–6 and then resumed a reduced size. The Golgi apparatus enlarged by day 6 and remained this way throughout the embryonic period. Associated with these changes was the initial appearance of the zymogen granules on day 5. The endoplasmic reticulum was present initially in both the smooth and the rough forms. The rough form and the outer nuclear membrane were both initially studded intermittently with aggregates of ribosomes. Subsequently, there was an increase in the number of attached ribosomes, an increase in the amount of rough reticulum and a decrease in smooth membranes. The ribosomes attached to the membranes appeared to augment the large free ribosome population characteristic of the early cells. Mitochondria did not appear to increase in number but there was an increase in size. The granules varied in kind, number and size with developmental age. The first granules formed (days 3–5) appeared to be miniatures of the mature type. Subsequently, a heterogeneity of granule morphologies was present.     

Quantitative studies on RNA accumulation in human PHA-stimulated lymphocytes during blast transformation

The net RNA accumulation in PHA-stimulated individual lymphocytes during blast transformation was assayed. The RNA content of the cells was measured by UV microspectrophotometry and the concentration of ribosomes in the cytoplasm of non-stimulated and transforming lymphocytes was determined by counting RNP particles in electronmicrographs.The mean RNA content of non-stimulated lymphocytes was 1.9 ± 0.8 × 10⁻¹²g/cell. During transformation the RNA content increased considerably, the maximal increase being about ten-fold. This increase was proportional to the increase in cellular dry mass. In fact, the RNA concentration was constant during transformation and approximately the same in non-stimulated and transforming lymphocytes. Furthermore the ribosome concentration was approximately the same in the cytoplasm of non-stimulated and transforming lymphocytes. However, a pronounced transition of single ribosomes to ribosomal clusters occurred during transformation and was one of the earliest structural signs of stimulation observed.     

THE FINE STRUCTURE OF MITOSIS IN RAT THYMIC LYMPHOCYTES

The fine structure of rat thymic lymphocytes from early prophase to late telophase of mitosis is described, using material fixed at pH 7.3 either in 1 per cent OsO4 or in glutaraldehyde followed by 2 per cent OsO4. The structure of the centriolar complex of interphase thymocytes is analyzed and compared with that of centrioles during division. The appearance of daughter centrioles is the earliest clearly recognizable sign of prophase. Daughter centrioles probably retain a secondary relation to the primary centriole, while the latter appears to be related, both genetically and spatially, to the spindle apparatus. The nuclear envelope persists in recognizable form to help reconstitute the envelopes of the daughter nuclei. Ribosome bodies (dense aggregates of ribosomes) accumulate, beginning at late prophase, and are retained by the daughter cells. Cytokinesis proceeds by formation of a ribosome-free plate at the equator with a central plate of vesicles which may coalesce to form the new plasma membrane of the daughter cells. Stages in the formation of the midbody are illustrated.     

Dimethyl sulfoxide induces microtubule formation in cultured arterial smooth muscle cells

Prolonged exposure of cultured arterial smooth muscle cells to 1% dimethyl sulfoxide (DMSO) resulted in a remarkable increase in cytoplasmic microtubules and an appearance of bundle-like aggregates of microtubules associated with ribosomes (microtubule-ribosome-complex). In this complex, fine filaments of 8 to 16 nm diameter were interspersed, some of which displayed continuity with the microtubules and were studded with a single array of ribosomes. The microtubule-ribosome-complex may represent an unusual aggregate of microtubules containing incompletely polymerized forms of newly synthesized tubulin induced by prolonged effect of DMSO.     

Isolation of a cDNA clone encoding S-adenosylmethionine decarboxylase. Expression of the gene in mitogen-activated lymphocytes

S-Adenosylmethionine decarboxylase was purified from bovine liver and digested with endopeptidase Lys-C; the resulting peptides were chromatographically separated. Peptides containing either methionine or tryptophan were subjected to sequence analysis. An oligonucleotide mixture of 48 sequences, which was 17 nucleotides in length, was synthesized based on one of these peptide sequences. This synthetic oligonucleotide mixture was labeled and used to screen a bovine cDNA library in phage lambda gt11. A clone was identified which contained a 1350-nucleotide insert. This insert contained nucleotide sequences coding for amino acid sequences of two of the peptides that were analyzed, thus proving that this cDNA clone codes for S-adenosylmethionine decarboxylase. A subcloned fragment from the coding region of the cDNA was used as a probe to analyze the expression of this gene in mitogen-activated lymphocytes. Northern blots revealed two message species of 2.4 and 3.6 kilobases in length. Both mRNAs were coordinately expressed and were present in polysomes. The levels of these mRNAs increased approximately 4-fold by 9 h after activation of the cells. The magnitude of the increase in these messages is to be compared with an 8- to 10-fold increase in the rate of synthesis of the protein. The apparent increase in translational efficiency of this message upon lymphocyte activation was confirmed by analyzing polysomes from these cells. In resting lymphocytes, the average size of polysomes containing mRNA coding for S-adenosylmethionine decarboxylase was 1.4 ribosomes per mRNA, and this value increased to 2.7 in stimulated cells. Thus, it appears that the increase in translational efficiency of this mRNA arises from an elevated rate of translational initiation, leading to more ribosomes per polysome encoding this particular message. This is not a general effect on the expression of all proteins, since there is no change in the translational efficiency of cytoplasmic actin upon activation of lymphocytes.     

Mitochondrial and cytoplasmic ribosomes and their activity in blood and culture form Trypanosoma brucei

Ribosomes of Trypanosoma brucei, a parasitic, flagellated protozoan (order Kinetoplastida), were identified on sucrose density gradients by their radioactively labeled nascent peptides. Ultraviolet absorption revealed only cytoplasmic ribosomes which served as internal sedimentation markers. Synthesis on cytoplasmic ribosomes was completely inhibited by cycloheximide. In the presence of this antibiotic, nascent peptides were associated with ribosomes of lower sedimentation coefficient than the cytoplasmic ribosomes. Chloramphenicol blocked synthesis on these ribosomes which are probably the mitochondrial ribosomes. These ribosomes differed from the cytoplasmic ribosomes in several ways. Their sedimentation coefficient was about 72S rather than 84S. The stability of the 72S ribosomes was less sensitive to pancreatic ribonuclease and low Mg-++ concentrations, dissociating below 0.1 mM Mg++. The 72S ribosomes were more sensitive to elevated KCl concentrations, dissociation above 0.25 M. Protein synthetic activity associated with the 72S class of ribosomes was found in trypanosomes grown in rats. Under these conditions no cytochromes or fully active Krebs cycle is present in these cells and respiration is insensitive to cyanide.     

Mobility of ribosomes bound to microsomal membranes. A freeze-etch and thin-section electron microscope study of the structure and fluidity of the rough endoplasmic reticulum

The lateral mobility of ribosomes bound to rough endoplasmic reticulum (RER) membranes was demonstrated under experimental conditions. High- salt-washed rough microsomes were treated with pancreatic ribonuclease (RNase) to cleave the mRNA of bound polyribosomes and allow the movement of individual bound ribosomesmfreeze-etch and thin-section electron microscopy demonstrated that, when rough microsomes were treated with RNase at 4 degrees C and then maintained at this temperature until fixation, the bound ribosomes retained their homogeneous distribution on the microsomal surface. However, when RNase- treated rough microsomes were brought to 24 degrees C, a temperature above the thermotropic phase transition of the microsomal phospholipids, bound ribosomes were no longer distributed homogeneously but, instead, formed large, tightly packed aggregates on the microsomal surface. Bound polyribosomes could also be aggregated by treating rough microsomes with antibodies raised against large ribosomal subunit proteins. In these experiments, extensive cross-linking of ribosomes from adjacent microsomes also occurred, and large ribosome-free membrane areas were produced. Sedimentation analysis in sucrose density gradients demonstrated that the RNase treatment did not release bound ribosomes from the membranes; however, the aggregated ribosomes remain capable of peptide bond synthesis and were released by puromycin. It is proposed that the formation of ribosomal aggregates on the microsomal surface results from the lateral displacement of ribosomes along with their attached binding sites, nascent polypeptide chains, and other associated membrane proteins; The inhibition of ribosome mobility after maintaining rough microsomes at 4 degrees C after RNase, or antibody, treatment suggests that the ribosome binding sites are integral membrane proteins and that their mobility is controlled by the fluidity of the RER membrane. Examination of the hydrophobic interior of microsomal membranes by the freeze-fracture technique revealed the presence of homogeneously distributed 105-A intramembrane particles in control rough microsomes. However, aggregation of ribosomes by RNase, or their removal by treatment with puromycin, led to a redistribution of the particles into large aggregates on the cytoplasmic fracture face, leaving large particle-free regions.     

Proteins of mammalian mitochondrial ribosomes.

The bovine mitochondrial system is being developed as a model system for studies on mammalian mitochondrial ribosomes. Information is emerging on the structural organization and RNA binding properties of proteins in these mitochondrial ribosomes. Unexpectedly, these ribosomes appear to interact directly with GTP, via a high affinity binding site on the small subunit. Despite major differences in their RNA content and physical properties, mammalian mitochondrial and cytoplasmic ribosomes contain about the same number of proteins. The proteins in each kind of ribosome have a similar size distribution, and both sets are entirely coded by nuclear genes, raising the possibility that these different ribosomes may contain the same set of proteins. Comparison of bovine mitochondrial and cytoplasmic r-proteins by co-electrophoresis in two-dimensional gels reveals that most of the cytoplasmic ribosomal proteins are more basic than the mitochondrial ribosomal proteins, and that none are co-migratory with mitochondrial ribosomal proteins, suggesting that the proteins in the two ribosomes are different. To exclude the possibility that the electrophoretic differences result only from post-translational modification of otherwise identical proteins, antibodies against several proteins from the large subunit of bovine mitochondrial ribosomes were tested against cytoplasmic ribosomes by solid phase radioimmunoassay and against cytoplasmic ribosomal proteins on Western blots. The lack of cross-reaction of these antibodies with cytoplasmic r-proteins suggests that mitochondrial ribosomal proteins have different primary structures and thus are most likely encoded by a separate set of nuclear genes.     

Changes in intracellular cAMP reported by a Redistribution assay using a cAMP-dependent protein kinase-green fluorescent protein chimera

We report on a novel method to monitor changes in intracellular cAMP concentration ([cAMP]i) within intact living cells using a chimeric fusion of the catalytic subunit of cAMP-dependent protein kinase to green fluorescent protein (PKAcat-GFP). In stably transfected unstimulated fibroblasts, fusion protein fluorescence is highly concentrated in aggregates throughout the cytoplasm and absent in the nucleus. Elevation of [cAMP]i disperses GFP fluorescence from the cytoplasmic aggregates within minutes. Spot-photobleach measurements show that the rate of exchange of GFP-labeled catalytic subunits at these aggregates increases in proportion to [cAMP]i. For any given stimulus, the response curve for dispersal of GFP fluorescence from aggregates agrees closely with the increase in total [cAMP]i as measured by standard in vitro methods (SPA). The redistribution of fluorescence is completely reversible: reduction of [cAMP]i results in return of fluorescence to the cytoplasmic aggregates. Consistent behaviour of PKAcat-GFP is seen in different cell backgrounds. We demonstrate that PKA Redistribution assays are suitable for measurement of changes in [cAMP]i brought about by both Gs- and Gi-protein-coupled receptor stimulation as well as by inhibition of cAMP phosphodiesterases.     

Protein Electrophoretic Analysis of Amyloplast and Cytoplasm Ribosomes from Lotus Cotyledon

Amyloplasts and cytoplasmic ribosomes in cotyledon cells of lotus (Nelvmbo nucifeva Gaertn. ) have been observed on the basis of morphology. Isolation of these ribosomes by centrifugation through 30% to 55% (W/V) sucrose density gradient resulted in three bands of amyloplasts ribosomes and four bands of cytoplasmic ribosomes. The authors used these ribosomes bands for SDS-PAGE electrophoresis to analyse ribosomes of proteins. The patterns of SDS-PAGE between cytoplasmic ribosomes of proteins and amyloplasts ribosomes of proteins were different. The amyloplasts ribosomes of proteins showed 26 kD and 23 kD bands, and the cytoplasmic ribosomes of proteins showed 65 kD band. The analysis of electrophoretic patterns of the cytoplasmic ribosomes of proteins showed that there was a newly synthesized ribosomes protein with 19 kD molecular weight in 18 to 20 days after fertilization.     

Comparative studies on mitochondrial development in yeasts

Summary High molecular weight mitochondrial RNA from Saccharomyces cerevisiae can be isolated rapidly and in relatively high yield from mitochondria prepared from cells prefixed with glutaraldehyde and disrupted mechanically. The RNA has lower electrophoretic mobilities than corresponding species from cytoplasmic ribosomes, and can also be distinguished from cytoplasmic RNA on the basis of the sensitivity of the mobility to temperature. RNA from cytoplasmic ribosomes and mitochondria of Candida parapsilosis shows a similar differential response to temperature.Mitochondrial ribosomes in Saccharomyces cerevisiae do not appear to be distinguishable from the cytoplasmic particles on the basis of sedimentation velocity. They can be identified, however, by pulse-labelling cells in the presence of cycloheximide. Cytoplasmic ribosomes under these conditions do not label. The labelling of mitochondrial ribosomes is sensitive to chloramphenicol, and is dispersed over the polysomal or ribosomal aggregate region of density gradients.     

Light-induced Activation of Cytoplasmic Protein Synthesis in Verticillium agaricinum

The level of protein synthetic activity in dark-grown cultures of Verticillium agaricinum was significantly enhanced by light. As expected the enhancement of protein synthetic activity was accompanied by a transformation of cytoplasmic monoribosomes to polyribosomes. Amino acid incorporation studies utilizing the synthetic mRNA, poly (U), suggest that the transformation was preceded by an activation of pre-existing ribosomes. The change in ribosome activity related, at least in part, to an increase in the level of peptidyl-tRNA associated with the ribosomes. In this regard the response of V. agaricinum ribosomes was similar to ribosome activation in several higher plant systems. The initial response at the level of the ribosome remains to be elucidated.     

ELECTRON MICROSCOPE STUDY OF MITOCHONDRIAL 60S AND CYTOPLASMIC 80S RIBOSOMES FROM LOCUSTA MIGRATORIA

Purified mitochondrial ribosomes (60S) have been isolated from locust flight muscle. Purification could be achieved after lysis of mitochondria in 0.055 M MgCl₂. Mitochondrial 60S and cytoplasmic 80S ribosomes were investigated by electron microscopy in tissue sections, in sections of pellets of isolated ribosomes, and by negative staining of ribosomal suspensions. In negatively stained preparations, mitochondrial ribosomes show dimensions of ~270 x 210 x 215 Å; cytoplasmic ribosomes measure ~295 x 245 x 255 Å. From these values a volume ratio of mitochondrial to cytoplasmic ribosomes of 1: 1.5 was estimated. Despite their different sedimentation constants, mitochondrial ribosomes after negative staining show a morphology similar to that of cytoplasmic ribosomes. Both types of particles show bipartite profiles which are interpreted as "frontal views" and "lateral views." In contrast to measurements on negatively stained particles, the diameter of mitochondrial ribosomes in tissue sections is ~130 Å, while the diameter of cytoplasmic ribosomes is ~ 180–200 Å. These data suggest a volume ratio of mitochondrial to cytoplasmic ribosomes of 1:3. Subunits of mitochondrial ribosomes (40S and 25S) were obtained by incubation under dissociating conditions before fixation in glutaraldehyde. After negative staining, mitochondrial large (40S) subunits show rounded profiles with a shallow groove on a flattened side of the profile. Mitochondrial small subunits (25S) display elongated, triangular profiles.     

Intraepithelial lymphocytes in the male reproductive tract of rats and rhesus monkeys.

Although lymphocytes are never present in 'normal ' seminiferous epithelium, they are found in the terminal portions of the seminiferous tubles near their junctions with the tubuli recti. Intraepithelia lymphocytes are also found in the tubuli recti testis, ductuli efferentes, epididymis and ductus deferens. The ultrastructural morphology of these cells closely resembles that of the intraepithelial lymphocytes in the intestinal mucosa and those obtained from the lymph nodes, spleen blood and thoracic duct. The mucleus is spherical and is characterized by clumps of chromatin near the nuclear membrane. A thin rim of cytoplasm is usually found, and is remarkably free of most cell organelles except for free ribosomes. Frequently, a blunt cytoplasmic process can be seen extending from one end of the cell. Membrane-bounded granules and other dense bodies are occasionally encountered in the cytoplasm. The possible functional significance of intraepithelial lymphocytes in the male reproductive tract is discussed.     

Protein synthesis in resting and growth-stimulated human peripheral lymphocytes : Evidence for regulation by a non-messenger RNA

Stimulation of lymphocyte growth is accompanied by an early increase in the rate of protein synthesis. This increase is dependent upon the flow of inactive free ribosomes into polysomes, which is limited by a rate-controlling step at initiation [2]. Addition of actinomycin D (actD) to lymphocytes caused a gradual reduction in protein synthesis in resting cells, but rapidly inhibited both the elevation of protein synthesis and the activation of free ribosomes which normally follow exposure to mitogens. Since actD does not affect protein synthesis in enucleated lymphocytes [4], the effect in intact cells must be mediated by a nuclear event, which available data indicate is RNA synthesis. ActD prevented the accumulation of 80S initiation complexes which normally occurs in resting lymphocytes treated with pactamycin and cycloheximide, showing that its locus of action was at some point in initiation. The decline in rate of protein synthesis began without detectable lag when resting lymphocytes were treated with actD. However, after growth stimulation, a delay of ca 50 min occurred before the protein synthetic rate declined in response to actD. These observations agree with the hypothesis that the concentration of some moderately short-lived RNA is rate-limiting for protein synthesis in resting lymphocytes, and that an early event in growth stimulation is a rise in the amount of this component to levels which are no longer rate-limiting. This permits an increased flow of ribosomes into polysomes and a consequent rise in protein synthesis. Available evidence indicates that the regulatory RNA is neither mRNA nor rRNA, but may either be one of the small cytoplasmic RNAs whose function is unknown, or tRNA_i^met.     

Early Development and Ultrastructure of the Major Veins and Their Sheath Tissues in the Maize Leaves

The report described the ultrastructural changes that occurred in the major veins and their associated bundle sheaths (BS) of the maize ( Zea mays L. ) leaf blade in the process of their differentiation from three adjacent cells in the middle layer of the ground meristem, the minimal number of cells involved with the initiation of a procambial strand and the associated BS. The inner cell underwent two successive unequal periclinal divisions: a smaller cell that later differentiated into the adaxial BS cell precursor, and a larger one that divided once again periclinally yielding an abaxial BS cell precursor and a centrally located procambial initial cell. One of the two lateral cells immediately adjacent to either side of the inner cell also divided periclinally; these derivatives, along with another lateral cell of the original three-celled unit formed the precursor cells of the lateral BS. Prior to the initiation of protophlcem differentiation, all of the procambial cells showed ultrastructural characteristics basically similar to the procambial initial. They possessed a prominent nucleus with electron-dense aggregates of heterochromatin, a dense cytoplasm rich in ribosomes, proplastids and mitochondria; also a thin wall containing numerous plasmodesmata. In many cases, only short pieces of rough endoplasmic reticulum cistemae and a few small sized vacuoles were present. In adclifton, evidence of cytoplasmic disintegration leading to new vacuole formation was noted in the process of proeambium development. It was observed that certain endoplasmic reticulum was engaged in the sequestration and lysis of cytoplasm. No apparent uhrastmctuml difference was found between the BS cell precursors and the procambial initials, that was, the distinction between the procambium and the surrounding BS cells occurred gradually after vein initiation, The major ultrastmctural changes which occurred during the differentiation of the meristematic BS cells into the vacuolated cells were (1) a proplastid to chloroplast transformation going through a prolamellar body stage, and (2) the appearance of the multi-concentric membrane complex which might play a role in the degradation of some ribosomes and other cytoplasmic components during the differentiation of BS cells.     

FINE STRUCTURAL ALTERATIONS OF INTERPHASE NUCLEI OF LYMPHOCYTES STIMULATED TO GROWTH ACTIVITY IN VITRO

This report describes fine structural changes of interphase nuclei of human peripheral blood lymphocytes stimulated to growth by short-term culture with phytohemagglutinin. Chromatin is found highly labile, its changes accompanying the sequential increases of RNA and DNA synthesis which are known to occur in lymphocyte cultures. In "resting" lymphocytes, abundant condensed chromatin appears as a network of large and small aggregates. Early in the response to phytohemagglutinin, small aggregates disappear during increase of diffuse chromatin regions. Small aggregates soon reappear, probably resulting from disaggregation of large masses of condensed chromatin. Loosened and highly dispersed forms then appear prior to the formation of prophase chromosomes. The loosened state is found by radioautography to be most active in DNA synthesis. Small nucleoli of resting lymphocytes have concentric agranular, fibrillar, and granular zones with small amounts of intranucleolar chromatin. Enlarging interphase nucleoli change chiefly (1) by increase in amount of intranucleolar chromatin and alteration of its state of aggregation and (2) by increase in granular components in close association with fibrillar components.