Cellulose Microfibril Formation Research Articles

Extraction of lignocellulosic fiber and cellulose microfibrils from agro waste-palmyra fruit peduncle: Water retting, chlorine-free chemical treatments, physio-chemical, morphological, and thermal characterization

In this paper, lignocellulosic fibers and cellulose microfibrils (CMFs) were extracted from palmyra fruit peduncle waste and investigated as naturally derived cellulosic materials for their potential use as reinforcement materials in composite applications. The physicochemical, mechanical, and thermal properties of the extracted fiber were studied. Physical and morphological analysis results revealed an extracted fiber diameter of 82.5 μm with a very rough surface, providing excellent interfacial bonding performance with the polymer matrix. Chemical, mechanical, and thermal results showed that the fibers consist mainly of cellulose as their crystallized phase, with a cellulose content of 56.5 wt% and a tensile strength of 693.3 MPa, along with thermal stability up to 252 °C. The chemically extracted CMFs exhibit a short, rough-surfaced, cylindrical cellulose structure with a diameter range of 10–15 μm. These CMFs demonstrate excellent thermal stability, withstanding temperatures up to 330 °C. Furthermore, the formation of CMFs is evident from a substantial increase in the crystallinity index, which increased from 58.2 % in the raw fibers to 78.2 % in the CMFs. FT-IR analysis further confirms the successful removal of non-cellulosic materials through chlorine-free chemical treatments. These findings strongly support the potential use of extracted fibers and CMFs as reinforcement materials in polymers.

Plant cell wall construction crew

Structural Biology Plants produce a complex cell wall in which cellulose, a glucose polymer, is a major component. Cellulose fibers are formed from close-packed single chains of cellulose that have been proposed to be formed by multimeric complexes (18 or more subunits) of the enzyme cellulose synthase, which exists in several isoforms. Purushotham et al. determined a cryo–electron microscopy structure of a trimer of a single isoform of cellulose synthase. A large channel forms a path for cellulose chains through the membrane-embedded complex. The structure also reveals oligomeric interfaces and provides a framework for modeling the larger complexes seen in plant membranes. The close arrangement of exit sites for nascent glycan chains is consistent with the enzyme complex playing a role in directing cellulose microfibril formation. Science , this issue p. [1089][1] [1]: /lookup/doi/10.1126/science.abb2978

Analysis of a cellulose synthase catalytic subunit from the oomycete pathogen of crops Phytophthora capsici

Phytophthora capsici Leonian is an important oomycete pathogen of crop vegetables, causing significant economic losses each year. Its cell wall, rich in cellulose, is vital for cellular integrity and for interactions with the host organisms. Predicted cellulose synthase (CesA) proteins are expected to catalyze the polymerization of cellulose, but this has not been biochemically demonstrated in an oomycete. Here, we present the properties of the four newly identified CesA proteins from P. capsici and compare their domain organization with that of CesAs from other lineages. Using a newly constructed glucosyltransferase-deficient variant of Saccharomyces cerevisiae with low residual background activity, we have achieved successful heterologous expression and biochemical characterization of a CesA protein from P. capsici (PcCesA1). Our results demonstrate that the individual PcCesA1 enzyme produces cellobiose as the major reaction product. Co-immunoprecipitation studies and activity assays revealed that several PcCesA proteins interact together to form a complex whose multiproteic nature is most likely required for cellulose microfibril formation. In addition to providing important insights into cellulose synthesis in the oomycetes, our data may assist the longer term identification of cell wall biosynthesis inhibitors to control infection by pathogenic oomycetes.

Architecture of a catalytically active homotrimeric plant cellulose synthase complex.

Cellulose is an essential plant cell wall component and represents the most abundant biopolymer on Earth. Supramolecular plant cellulose synthase complexes organize multiple linear glucose polymers into microfibrils as load-bearing wall components. We determined the structure of a poplar cellulose synthase CesA homotrimer that suggests a molecular basis for cellulose microfibril formation. This complex, stabilized by cytosolic plant-conserved regions and helical exchange within the transmembrane segments, forms three channels occupied by nascent cellulose polymers. Secretion steers the polymers toward a common exit point, which could facilitate protofibril formation. CesA's N-terminal domains assemble into a cytosolic stalk that interacts with a microtubule-tethering protein and may thus be involved in CesA localization. Our data suggest how cellulose synthase complexes assemble and provide the molecular basis for plant cell wall engineering.

Cellulose synthase 'class specific regions' are intrinsically disordered and functionally undifferentiated.

Cellulose synthases (CESAs) are glycosyltransferases that catalyze formation of cellulose microfibrils in plant cell walls. Seed plant CESA isoforms cluster in six phylogenetic clades, whose non-interchangeable members play distinct roles within cellulose synthesis complexes (CSCs). A 'class specific region' (CSR), with higher sequence similarity within versus between functional CESA classes, has been suggested to contribute to specific activities or interactions of different isoforms. We investigated CESA isoform specificity in the moss, Physcomitrella patens (Hedw.) B. S. G. to gain evolutionary insights into CESA structure/function relationships. Like seed plants, P. patens has oligomeric rosette-type CSCs, but the PpCESAs diverged independently and form a separate CESA clade. We showed that P. patens has two functionally distinct CESAs classes, based on the ability to complement the gametophore-negative phenotype of a ppcesa5 knockout line. Thus, non-interchangeable CESA classes evolved separately in mosses and seed plants. However, testing of chimeric moss CESA genes for complementation demonstrated that functional class-specificity is not determined by the CSR. Sequence analysis and computational modeling showed that the CSR is intrinsically disordered and contains predicted molecular recognition features, consistent with a possible role in CESA oligomerization and explaining the evolution of class-specific sequences without selection for class-specific function.

Synthesis and Self-Assembly of Cellulose Microfibrils from Reconstituted Cellulose Synthase.

Cellulose, the major component of plant cell walls, can be converted to bioethanol and is thus highly studied. In plants, cellulose is produced by cellulose synthase, a processive family-2 glycosyltransferase. In plant cell walls, individual β-1,4-glucan chains polymerized by CesA are assembled into microfibrils that are frequently bundled into macrofibrils. An in vitro system in which cellulose is synthesized and assembled into fibrils would facilitate detailed study of this process. Here, we report the heterologous expression and partial purification of His-tagged CesA5 from Physcomitrella patens Immunoblot analysis and mass spectrometry confirmed enrichment of PpCesA5. The recombinant protein was functional when reconstituted into liposomes made from yeast total lipid extract. The functional studies included incorporation of radiolabeled Glc, linkage analysis, and imaging of cellulose microfibril formation using transmission electron microscopy. Several microfibrils were observed either inside or on the outer surface of proteoliposomes, and strikingly, several thinner fibrils formed ordered bundles that either covered the surfaces of proteoliposomes or were spawned from liposome surfaces. We also report this arrangement of fibrils made by proteoliposomes bearing CesA8 from hybrid aspen. These observations describe minimal systems of membrane-reconstituted CesAs that polymerize β-1,4-glucan chains that coalesce to form microfibrils and higher-ordered macrofibrils. How these micro- and macrofibrils relate to those found in primary and secondary plant cell walls is uncertain, but their presence enables further study of the mechanisms that govern the formation and assembly of fibrillar cellulosic structures and cell wall composites during or after the polymerization process controlled by CesA proteins.

A single heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro

Plant cell walls are a composite material of polysaccharides, proteins, and other noncarbohydrate polymers. In the majority of plant tissues, the most abundant polysaccharide is cellulose, a linear polymer of glucose molecules. As the load-bearing component of the cell wall, individual cellulose chains are frequently bundled into micro and macrofibrils and are wrapped around the cell. Cellulose is synthesized by membrane-integrated and processive glycosyltransferases that polymerize UDP-activated glucose and secrete the nascent polymer through a channel formed by their own transmembrane regions. Plants express several different cellulose synthase isoforms during primary and secondary cell wall formation; however, so far, none has been functionally reconstituted in vitro for detailed biochemical analyses. Here we report the heterologous expression, purification, and functional reconstitution of Populus tremula x tremuloides CesA8 (PttCesA8), implicated in secondary cell wall formation. The recombinant enzyme polymerizes UDP-activated glucose to cellulose, as determined by enzyme degradation, permethylation glycosyl linkage analysis, electron microscopy, and mutagenesis studies. Catalytic activity is dependent on the presence of a lipid bilayer environment and divalent manganese cations. Further, electron microscopy analyses reveal that PttCesA8 produces cellulose fibers several micrometers long that occasionally are capped by globular particles, likely representing PttCesA8 complexes. Deletion of the enzyme's N-terminal RING-finger domain almost completely abolishes fiber formation but not cellulose biosynthetic activity. Our results demonstrate that reconstituted PttCesA8 is not only sufficient for cellulose biosynthesis in vitro but also suffices to bundle individual glucan chains into cellulose microfibrils.

Comparative Structural and Computational Analysis Supports Eighteen Cellulose Synthases in the Plant Cellulose Synthesis Complex.

A six-lobed membrane spanning cellulose synthesis complex (CSC) containing multiple cellulose synthase (CESA) glycosyltransferases mediates cellulose microfibril formation. The number of CESAs in the CSC has been debated for decades in light of changing estimates of the diameter of the smallest microfibril formed from the β-1,4 glucan chains synthesized by one CSC. We obtained more direct evidence through generating improved transmission electron microscopy (TEM) images and image averages of the rosette-type CSC, revealing the frequent triangularity and average cross-sectional area in the plasma membrane of its individual lobes. Trimeric oligomers of two alternative CESA computational models corresponded well with individual lobe geometry. A six-fold assembly of the trimeric computational oligomer had the lowest potential energy per monomer and was consistent with rosette CSC morphology. Negative stain TEM and image averaging showed the triangularity of a recombinant CESA cytosolic domain, consistent with previous modeling of its trimeric nature from small angle scattering (SAXS) data. Six trimeric SAXS models nearly filled the space below an average FF-TEM image of the rosette CSC. In summary, the multifaceted data support a rosette CSC with 18 CESAs that mediates the synthesis of a fundamental microfibril composed of 18 glucan chains.

Cellulose Microfibril Formation by Surface-Tethered Cellulose Synthase Enzymes.

Cellulose microfibrils are pseudocrystalline arrays of cellulose chains that are synthesized by cellulose synthases. The enzymes are organized into large membrane-embedded complexes in which each enzyme likely synthesizes and secretes a β-(1→4) glucan. The relationship between the organization of the enzymes in these complexes and cellulose crystallization has not been explored. To better understand this relationship, we used atomic force microscopy to visualize cellulose microfibril formation from nickel-film-immobilized bacterial cellulose synthase enzymes (BcsA-Bs), which in standard solution only form amorphous cellulose from monomeric BcsA-B complexes. Fourier transform infrared spectroscopy and X-ray diffraction techniques show that surface-tethered BcsA-Bs synthesize highly crystalline cellulose II in the presence of UDP-Glc, the allosteric activator cyclic-di-GMP, as well as magnesium. The cellulose II cross section/diameter and the crystal size and crystallinity depend on the surface density of tethered enzymes as well as the overall concentration of substrates. Our results provide the correlation between cellulose microfibril formation and the spatial organization of cellulose synthases.

Synthesis and water state characterization of polysodium acrylate/cellulose microfibril hydrogels

The structure of polysodium acrylate (PSA) hydrogel was modified using N, N-methylenebisacrylamide and cellulose microfibrils (CMFs) as a cross-linker and filler, respectively. CMF was obtained from banana fiber by acid hydrolysis process. The resulting structure and morphology of CMF and PSA/CMF hydrogels were characterized by Fourier transform infrared spectroscopy (FTIR), x-ray diffraction (XRD) analysis and scanning electron microscopy. From the FTIR and XRD spectra, the formation of CMF from banana fiber is confirmed. Differential scanning calorimetric study was carried out on the swollen PSA/CMF hydrogels to determine the state and quantification of water present inside the hydrogels. The mechanical characterization of PSA/CMF hydrogels was studied in brief by dynamic mechanical analysis technique.

Apo- and Cellopentaose-bound Structures of the Bacterial Cellulose Synthase Subunit BcsZ

Cellulose, a very abundant extracellular polysaccharide, is synthesized in a finely tuned process that involves the activity of glycosyl-transferases and hydrolases. The cellulose microfibril consists of bundles of linear β-1,4-glucan chains that are synthesized inside the cell; however, the mechanism by which these polymers traverse the cell membrane is currently unknown. In Gram-negative bacteria, the cellulose synthase complex forms a trans-envelope complex consisting of at least four subunits. Although three of these subunits account for the synthesis and translocation of the polysaccharide, the fourth subunit, BcsZ, is a periplasmic protein with endo-β-1,4-glucanase activity. BcsZ belongs to family eight of glycosyl-hydrolases, and its activity is required for optimal synthesis and membrane translocation of cellulose. In this study we report two crystal structures of BcsZ from Escherichia coli. One structure shows the wild-type enzyme in its apo form, and the second structure is for a catalytically inactive mutant of BcsZ in complex with the substrate cellopentaose. The structures demonstrate that BcsZ adopts an (α/α)(6)-barrel fold and that it binds four glucan moieties of cellopentaose via highly conserved residues exclusively on the nonreducing side of its catalytic center. Thus, the BcsZ-cellopentaose structure most likely represents a posthydrolysis state in which the newly formed nonreducing end has already left the substrate binding pocket while the enzyme remains attached to the truncated polysaccharide chain. We further show that BcsZ efficiently degrades β-1,4-glucans in in vitro cellulase assays with carboxymethyl-cellulose as substrate.

Formation of plant cell wall supramolecular structure

Plant cell wall is an example of a widespread natural supramolecular structure: its components are considered to be the most abundant organic compounds renewable by living organisms. Plant cell wall includes numerous components, mainly polysaccharidic; its formation is largely based on carbohydrate-carbohydrate interactions. In contrast to the extracellular matrix of most other organisms, the plant cell compartment located outside the plasma membrane is so structured that has been named "wall". The present review summarizes data on the mechanisms of formation of this supramolecular structure and considers major difficulties and results of research. Existing approaches to the study of interactions between polysaccharides during plant cell wall formation have been analyzed, including: (i) characterization of the structure of natural polysaccharide complexes obtained during cell wall fractionation; (ii) analysis of the interactions between polysaccharides "at mixing in a tube"; (iii) study of the interactions between isolated individual plant cell wall matrix polysaccharides and microfibrils formed by cellulose-synthesizing microorganisms; and (iv) investigation of cell wall formation and modification directly in plant objects. The key stages in formation of plant cell wall supramolecular structure are defined and characterized as follows: (i) formation of cellulose microfibrils; (ii) interactions between matrix polysaccharides within Golgi apparatus substructures; (iii) interaction between matrix polysaccharides, newly secreted outside the plasma membrane, and cellulose microfibrils during formation of the latter; (iv) packaging of the formed complexes and individual polysaccharides in cell wall layers; and (v) modification of deposited cell wall layers.

Cellulose Biosynthesis: Current Views and Evolving Concepts

To outline the current state of knowledge and discuss the evolution of various viewpoints put forth to explain the mechanism of cellulose biosynthesis. * Understanding the mechanism of cellulose biosynthesis is one of the major challenges in plant biology. The simplicity in the chemical structure of cellulose belies the complexities that are associated with the synthesis and assembly of this polysaccharide. Assembly of cellulose microfibrils in most organisms is visualized as a multi-step process involving a number of proteins with the key protein being the cellulose synthase catalytic sub-unit. Although genes encoding this protein have been identified in almost all cellulose synthesizing organisms, it has been a challenge in general, and more specifically in vascular plants, to demonstrate cellulose synthase activity in vitro. The assembly of glucan chains into cellulose microfibrils of specific dimensions, viewed as a spontaneous process, necessitates the assembly of synthesizing sites unique to most groups of organisms. The steps of polymerization (requiring the specific arrangement and activity of the cellulose synthase catalytic sub-units) and crystallization (directed self-assembly of glucan chains) are certainly interlinked in the formation of cellulose microfibrils. Mutants affected in cellulose biosynthesis have been identified in vascular plants. Studies on these mutants and herbicide-treated plants suggest an interesting link between the steps of polymerization and crystallization during cellulose biosynthesis. * With the identification of a large number of genes encoding cellulose synthases and cellulose synthase-like proteins in vascular plants and the supposed role of a number of other proteins in cellulose biosynthesis, a complete understanding of this process will necessitate a wider variety of research tools and approaches than was thought to be required a few years back.

Callose deposition in the primary wall of suspension cells and regenerating protoplasts, and its relationship to patterned cellulose synthesis

Monitoring cell-wall formation in vivo with Fluorescent Brightener 28, by fluorescence microscopy, revealed that tobacco protoplasts regeneration started within 30 min indicated by cellulose microfibril formation at distinct sites on the protoplast surface. Oriented cellulose microfibril deposition was apparent before elongation and indicated the early polarization of protoplasts. The sequence of cellulose microfibril deposition correlates with an helicoidal-like texture. Within 6 h, a texture was completed. Tobacco suspension cells, stained by decolourized aniline blue, showed radiant granular callose fluorescence in cell plates and transverse walls. During the culture cycle of suspension cells, transverse fibrillar deposits of callose gradually appeared in the lateral walls during the log-phase, and subsequently disappeared in the early stationary phase of the cell culture. Similar callose transitions were observed in regenerated elongating protoplasts. Culture cells of Morinda citrifolia L. only showed transient granular depositions in the lateral walls. The callose formations did not result from artificial wounding. The transient appearance of callose might be related to cellulose microfibril deposition. Keywords: aniline blue, Calcofluor White ST, callose, cellulose, Fluorescent Brightener 28, Morinda citrifolia, Nicotiana tabacum.

Incorporation of [14C]Glucose into Crystalline Cellulose in Aberrant Fibers of a Cotton Mutant

Ligon lintless‐1 is a dominant simply inherited mutant of cotton (Gossypium hirsutum L.) containing markedly short cotton fibers with extensively thickened secondary walls. The incorporation of [14C]glucose into crystalline cellulose in the primary and secondary walls was studied to determine the relation between the extent of crystalline cellulose microfibril formation and the pattern of microfibril deposition to the aberrant growth and developmental pattern in the mutant cotton. The results show that the rate of crystalline cellulose formation in the primary walls of the mutant fibers correlates with the reduced rate of fiber elongation and primary wall formation. There is a five‐fold increase in the rate of crystalline cellulose formed per millimeter of fiber length during secondary wall formation in the mutant fibers compared to the rate in the wild‐type fibers. The Ligon lintless−1 gene mutation affects the growth and development of the cotton fibers with accompanying changes in the rate of formation of crystalline cellulose microfibrils in the primary and secondary walls. This increase in crystalline cellulose microfibrils in secondary walls is most likely due either to an increase in synthetic activity of the individual cellulose synthase complexes or to an increase in number of synthetic complex sites per unit of fiber length in the mutant.

Tunnel structures in Acetobacter xylinum

The architecture of cellulose pellicles produced by the bacterium Acetobacter xylinum was observed using a scanning electron microscope. The existence of tunnels with regular orientation argues for some kind of coordination during the pellicle formation and against a random formation of cellulose microfibrils.

Plasma-membrane rosettes involved in localized wall thickening during xylem vessel formation of Lepidium sativum L.

Developing xylem vessel elements in roots of cress, Lepidium sativum L., were freeze-fractured after rapid freezing in nitrogen slush (without cryoprotection). With the double-replica technique, both the plasmatic fracture face (PF) and the extraplasmatic fracture face (EF) of the plasma membrane were exposed. The EF revealed abundant, but rather indistinct "terminal globules"; whereas the PF showed numerous "rosettes". Terminal globules and rosettes were localized, restricted to regions of secondary wall thickening only, and showed comparale frequencies per μm(2), supporting the assumption that they are part of the same synthase complex. The abundance of rosettes in regions of high cellulose production supports their postulated involvement in cellulose microfibril formation. With up to 191 rosettes per μm(2), the rosettes appear to be too densely arranged to be directly aligned on individual microtubules. This favors the channelling hypothesis of synthase movement in the plasma membrane.

Oriented ?rosette? alignment during cellulose formation in mung bean hypocotyl

Cell-wall microfibrils formed on the surface of plant cells may be either deposited without obvious order, as in primary wall formation of higher plants, or in parallel orientations, as during secondary wall formation of higher plants and in the crossed microfibrillar layers in various algae. The mechanism of orientation is an actual cytological theme with controversial hypotheses trying to correlate components of the cytoskeleton with the microfibril orientation [1, 2]. Mainly based on inhibitor experiments, there is broad agreement that in most cases microtubules play a key role in the orientation mechanism. With the recent freeze-fracture findings of "roset te" particle complexes in the plasma membrane of a variety of cellulose forming algae, moss, fern and some higher plant systems (reviews in [3, 5]), it has become rather probable that these rosette structures, and perhaps associated "terminal globules" in the other half of the membrane, are involved in cellulose microfibril formation, or even are the synthases themselves ([3-8], for discussion see [3, 4]). We now have freeze-fractured the plasma membrane of higher plant cells involved in secondary wall formation to check for the possible existence of oriented rosette arrangements during the deposition of parallel wall microfibrils. We used mung beans (Vigna radiata (L.) Wilczek) cultivated for 6 days with twice daily watering. The hypocotyl was cut into thin slices, and immediately after sectioning was frozen between double replica holders in nitrogen slush. The further procedures were as described [3]. The plasmatic fracture face (PF) of the plasma membrane of (not further defined) cells with parallel microfibril arrangement (as judged from the microfibril imprints) was relatively rich in rosettes, and showed unequal particle density depending on the region of the cell surface. Some rosettes did not show a correlated position with respect to other rosettes, but quite frequently the rosettes occurred in tracks (Fig. 1 a). There was no regular distance between the individual rosettes in such a track, and the alignment was somewhat zigzag, with up to ca. 50 nm lateral deviation from the extrapolated center line of such a track following a microfibril imprint (arrows). This arrangement would allow cooperation of adjacent rosettes in formation of microfibril bands, if each rosette would form a 3.5 nm elementary microfibril (Fig. 1 b) as postulated earlier [3]. It furthermore would allow formation of many parallel microfibrils. The adjacent tracks of rosettes were often, but not always, in parallel, and not with regular spacing to the neighbour track. These findings of an oriented arrangement of rosettes in mung bean plasma membranes are the first such case for a higher plant system, and lend further support to the postulated role of rosettes in cellulose microfibril formation [3-8]. There are some indications that individual rosettes arranged in lines also occur in the basal parts of Funaria caulonema cells [8]. The arrangement of rosettes in tracks somewhat resembles the rows of rosettes involved in formation of the parallel microfibrils in Closterium, but there the groups of rosettes show equal center-to-center spacing [5]. From a variety of experimental data, mainly involving serial section analyses of cortical microtubule arrangement with respect to microfibril orientation after inhibition of the crystallization process, it has become rather

Cell wall development in Oocystis solitaria in the presence of polysaccharide binding dyes

Cell wall and thus cellulose microfibril formation in the presence of Congo red or Calcofluor white by Oocystis solitaria autospores was investigated ultrastructurally and chemically. The prevention of microfibril formation by both substances is accompanied by drastic changes of microfibril synthesis and orientation as well as the morphology of plasma-membrane-associated E-face terminal complexes. Removal of Congo red and Calcofluor white from the culture medium results in the recovery of microfibril formation, of a normal patterned microfibril arrangement, and of terminal complexes with extending microfibril imprints.

Congo red and calcofluor white inhibition ofAcetobacter xylinum cell growth and of bacterial cellulose microfibril formation: Isolation and properties of a transient, extracellular glucan related to cellulose

Chemical analysis of pellicles ofAcetobacter xylinum grown in cultures containing increasing amounts of Calcofluor White or Congo Red shows that the inhibition of cellulose formation by both direct dyes has two aspects, internal and external. Internally, each dye seems to act as a general cell poison, although not a strong one. Externally, each dye interrupts cellulose microfibril assembly by forming a complex with previously synthesized polysaccharide(s). Both effects contribute to diminution of cellulose production. The polysaccharide(s) which forms a soluble complex with Congo Red external to the cell may be isolated by dilution. It is a homoglucan that yields cellobiose, cellotriose and possibly cellotetrose after partial acid hydrolysis. The glucan is hydrolyzed at the same rate as bacterial cellulose by glusulase to yield glucose. It contains little or no ninhydrin-positive material. Observations by electron microscopy show that the glucan is fibrillar. Heating the precipitate in 1 M NaOH for 10 minutes at 100 °C dissolves more than three quarters of the substance but does not alter its ultrastructure or composition. No discrete reflections are observed by powder X-ray diffraction. The foregoing results are consistent with the conclusion that Congo Red (and presumably Calcofluor White) interrupts the normal, extracellular association of previously synthesized 1 → 4-β-glucans by forming a soluble complex with a fraction of these glucans. The interruption of the association prevents the formation of the microfibril.