Mechanobiology: Shaping the future of cellular form and function.

Mechanobiology-the field studying how cells produce, sense, and respond to mechanical forces-is pivotal in the analysis of how cells and tissues take shape in development and disease. As we venture into the future of this field, pioneers share their insights, shaping the trajectory of future research and applications.

Plant Cell Wall Loosening by Expansins.

Expansins comprise an ancient group of cell wall proteins ubiquitous in land plants and their algal ancestors. During cell growth, they facilitate passive yielding of the wall's cellulose networks to turgor-generated tensile stresses, without evidence of enzymatic activity. Expansins are also implicated in fruit softening and other developmental processes and in adaptive responses to environmental stresses and pathogens. The major expansin families in plants include α-expansins (EXPAs), which act on cellulose-cellulose junctions, and ß-expansins, which can act on xylans. EXPAs mediate acid growth, which contributes to wall enlargement by auxin and other growth agents. The genomes of diverse microbes, including many plant pathogens, also encode expansins designated expansin-like X. Expansins are proposed to disrupt noncovalent bonding between laterally aligned polysaccharides (notably cellulose), facilitating wall loosening for a variety of biological roles.

Structure and growth of plant cell walls.

Plant cells build nanofibrillar walls that are central to plant growth, morphogenesis and mechanics. Starting from simple sugars, three groups of polysaccharides, namely, cellulose, hemicelluloses and pectins, with very different physical properties are assembled by the cell to make a strong yet extensible wall. This Review describes the physics of wall growth and its regulation by cellular processes such as cellulose production by cellulose synthase, modulation of wall pH by plasma membrane H+-ATPase, wall loosening by expansin and signalling by plant hormones such as auxin and brassinosteroid. In addition, this Review discusses the nuanced roles, properties and interactions of cellulose, matrix polysaccharides and cell wall proteins and describes how wall stress and wall loosening cooperatively result in cell wall growth.

The nonlinear mechanics of highly extensible plant epidermal cell walls.

Plant epidermal cell walls maintain the mechanical integrity of plants and restrict organ growth. Mechanical analyses can give insights into wall structure and are inputs for mechanobiology models of plant growth. To better understand the intrinsic mechanics of epidermal cell walls and how they may accommodate large deformations during growth, we analyzed a geometrically simple material, onion epidermal strips consisting of only the outer (periclinal) cell wall, ~7 µm thick. With uniaxial stretching by >40%, the wall showed complex three-phase stress-strain responses while cyclic stretching revealed reversible and irreversible deformations and elastic hysteresis. Stretching at varying strain rates and temperatures indicated the wall behaved more like a network of flexible cellulose fibers capable of sliding than a viscoelastic composite with pectin viscosity. We developed an analytic framework to quantify nonlinear wall mechanics in terms of stiffness, deformation, and energy dissipation, finding that the wall stretches by combined elastic and plastic deformation without compromising its stiffness. We also analyzed mechanical changes in slightly dehydrated walls. Their extension became stiffer and more irreversible, highlighting the influence of water on cellulose stiffness and sliding. This study offers insights into the structure and deformation modes of primary cell walls and presents a framework that is also applicable to tissues and whole organs.

Single-molecule tracking reveals dual front door/back door inhibition of Cel7A cellulase by its product cellobiose.

Degrading cellulose is a key step in the processing of lignocellulosic biomass into bioethanol. Cellobiose, the disaccharide product of cellulose degradation, has been shown to inhibit cellulase activity, but the mechanisms underlying product inhibition are not clear. We combined single-molecule imaging and biochemical investigations with the goal of revealing the mechanism by which cellobiose inhibits the activity of Trichoderma reesei Cel7A, a well-characterized exo-cellulase. We find that cellobiose slows the processive velocity of Cel7A and shortens the distance moved per encounter; effects that can be explained by cellobiose binding to the product release site of the enzyme. Cellobiose also strongly inhibits the binding of Cel7A to immobilized cellulose, with a Ki of 2.1 mM. The isolated catalytic domain (CD) of Cel7A was also inhibited to a similar degree by cellobiose, and binding of an isolated carbohydrate-binding module to cellulose was not inhibited by cellobiose, suggesting that cellobiose acts on the CD alone. Finally, cellopentaose inhibited Cel7A binding at micromolar concentrations without affecting the enzyme's velocity of movement along cellulose. Together, these results suggest that cellobiose inhibits Cel7A activity both by binding to the "back door" product release site to slow activity and to the "front door" substrate-binding tunnel to inhibit interaction with cellulose. These findings point to strategies for engineering cellulases to reduce product inhibition and enhance cellulose degradation, supporting the growth of a sustainable bioeconomy.

A rich and bountiful harvest: Key discoveries in plant cell biology.

The field of plant cell biology has a rich history of discovery, going back to Robert Hooke's discovery of cells themselves. The development of microscopes and preparation techniques has allowed for the visualization of subcellular structures, and the use of protein biochemistry, genetics, and molecular biology has enabled the identification of proteins and mechanisms that regulate key cellular processes. In this review, seven senior plant cell biologists reflect on the development of this research field in the past decades, including the foundational contributions that their teams have made to our rich, current insights into cell biology. Topics covered include signaling and cell morphogenesis, membrane trafficking, cytokinesis, cytoskeletal regulation, and cell wall biology. In addition, these scientists illustrate the pathways to discovery in this exciting research field.

Dynamic Structural Change of Plant Epidermal Cell Walls under Strain.

The molecular foundations of epidermal cell wall mechanics are critical for understanding structure-function relationships of primary cell walls in plants and facilitating the design of bioinspired materials. To uncover the molecular mechanisms regulating the high extensibility and strength of the cell wall, the onion epidermal wall is stretched uniaxially to various strains and cell wall structures from mesoscale to atomic scale are characterized. Upon longitudinal stretching to high strain, epidermal walls contract in the transverse direction, resulting in a reduced area. Atomic force microscopy shows that cellulose microfibrils exhibit orientation-dependent rearrangements at high strains: longitudinal microfibrils are straightened out and become highly ordered, while transverse microfibrils curve and kink. Small-angle X-ray scattering detects a 7.4 nm spacing aligned along the stretch direction at high strain, which is attributed to distances between individual cellulose microfibrils. Furthermore, wide-angle X-ray scattering reveals a widening of (004) lattice spacing and contraction of (200) lattice spacing in longitudinally aligned cellulose microfibrils at high strain, which implies longitudinal stretching of the cellulose crystal. These findings provide molecular insights into the ability of the wall to bear additional load after yielding: the aggregation of longitudinal microfibrils impedes sliding and enables further stretching of the cellulose to bear increased loads.

Loosenin-Like Proteins from Phanerochaete carnosa Impact Both Cellulose and Chitin Fiber Networks.

Microbial expansin-related proteins are ubiquitous across bacterial and fungal organisms and reportedly play a role in the modification and deconstruction of cell wall polysaccharides, including lignocellulose. So far, very few microbial expansin-related proteins, including loosenins and loosenin-like (LOOL) proteins, have been functionally characterized. Herein, four LOOLs encoded by Phanerochaete carnosa and belonging to different subfamilies (i.e., PcaLOOL7 and PcaLOOL9 from subfamily A and PcaLOOL2 and PcaLOOL12 from subfamily B) were recombinantly produced and the purified proteins were characterized using diverse cellulose and chitin substrates. The purified PcaLOOLs weakened cellulose filter paper and cellulose nanofibril networks (CNF); however, none significantly boosted cellulase activity on the selected cellulose substrates (Avicel and Whatman paper). Although fusing the family 63 carbohydrate-binding module (CBM63) of BsEXLX1 encoded by Bacillus subtilis to PcaLOOLs increased their binding to cellulose, the CBM63 fusion appeared to reduce the cellulose filter paper weakening observed using wild-type proteins. Binding of PcaLOOLs to alpha-chitin was considerably higher than that to cellulose (Avicel) and was pH dependent, with the highest binding at pH 5.0. Amendment of certain PcaLOOLs in fungal liquid cultivations also impacted the density of the cultivated mycelia. The present study reveals the potential of fungal expansin-related proteins to impact both cellulose and chitin networks and points to a possible biological role in fungal cell wall processing. IMPORTANCE The present study deepens investigations of microbial expansin-related proteins and their applied significance by (i) reporting a detailed comparison of diverse loosenins encoded by the same organism, (ii) considering both cellulosic and chitin-containing materials as targeted substrates, and (iii) investigating the impact of the C-terminal carbohydrate binding module (CBM) present in other expansin-related proteins on loosenin function. By revealing the potential of fungal loosenins to impact both cellulose and chitin-containing networks, our study reveals a possible biological and applied role of loosenins in fungal cell wall processing.

Building an extensible cell wall.

This article recounts, from my perspective of four decades in this field, evolving paradigms of primary cell wall structure and the mechanism of surface enlargement of growing cell walls. Updates of the structures, physical interactions, and roles of cellulose, xyloglucan, and pectins are presented. This leads to an example of how a conceptual depiction of wall structure can be translated into an explicit quantitative model based on molecular dynamics methods. Comparison of the model's mechanical behavior with experimental results provides insights into the molecular basis of complex mechanical behaviors of primary cell wall and uncovers the dominant role of cellulose-cellulose interactions in forming a strong yet extensible network.

Mutations in the Pectin Methyltransferase QUASIMODO2 Influence Cellulose Biosynthesis and Wall Integrity in Arabidopsis.

Pectins are abundant in the cell walls of dicotyledonous plants, but how they interact with other wall polymers and influence wall integrity and cell growth has remained mysterious. Here, we verified that QUASIMODO2 (QUA2) is a pectin methyltransferase and determined that QUA2 is required for normal pectin biosynthesis. To gain further insight into how pectin affects wall assembly and integrity maintenance, we investigated cellulose biosynthesis, cellulose organization, cortical microtubules, and wall integrity signaling in two mutant alleles of Arabidopsis (Arabidopsis thaliana) QUA2, qua2 and tsd2 In both mutants, crystalline cellulose content is reduced, cellulose synthase particles move more slowly, and cellulose organization is aberrant. NMR analysis shows higher mobility of cellulose and matrix polysaccharides in the mutants. Microtubules in mutant hypocotyls have aberrant organization and depolymerize more readily upon treatment with oryzalin or external force. The expression of genes related to wall integrity, wall biosynthesis, and microtubule stability is dysregulated in both mutants. These data provide insights into how homogalacturonan is methylesterified upon its synthesis, the mechanisms by which pectin functionally interacts with cellulose, and how these interactions are translated into intracellular regulation to maintain the structural integrity of the cell wall during plant growth and development.

Regiospecific Cellulose Orientation and Anisotropic Mechanical Property in Plant Cell Walls.

Cellulose microfibrils (CMFs) are a major load-bearing component in plant cell walls. Thus, their structures have been studied extensively with spectroscopic and microscopic characterization methods, but the findings from these two approaches were inconsistent, which hampers the mechanistic understanding of cell wall mechanics. Here, we report the regiospecific assembly of CMFs in the periclinal wall of plant epidermal cells. Using sum frequency generation spectroscopic imaging, we found that CMFs are highly aligned in the cell edge region where two cells form a junction, whereas they are mostly isotropic on average throughout the wall thickness in the flat face region of the epidermal cell. This subcellular-level heterogeneity in the CMF alignment provided a new perspective on tissue-level anisotropy in the tensile modulus of cell wall materials. This finding also has resolved a previous contradiction between the spectroscopic and microscopic imaging studies, which paves a foundation for better understanding of the cell wall architecture, especially structure-geometry relationships.

Microbial Expansins.

Expansins are small proteins that loosen plant cell walls and cellulosic materials without lytic activity. First discovered in plants, expansin genes are found in the genomes of numerous bacteria and fungi that interact with plants in pathogenic and mutualistic patterns, as well as in microbes that feed on plant debris. Horizontal gene transfer from plants to microbes and between microbes accounts for expansins' irregular taxonomic distribution. Expansins facilitate plant colonization by Bacillus, Clavibacter, and Trichoderma species, a list likely to grow as knowledge of microbial expansin function deepens. Studies have documented a synergistic action of expansins for cellulose digestion by cellulases, but only rarely to an extent that is commercially relevant. Expansins' biophysical actions remain enigmatic because of limited understanding of cell wall structure. Deeper understanding of microbial expansins may lead to novel approaches for biomass deconstruction and biocontrol of plant diseases.

Conservation of endo-glucanase 16 (EG16) activity across highly divergent plant lineages.

Plant cell walls are highly dynamic structures that are composed predominately of polysaccharides. As such, endogenous carbohydrate active enzymes (CAZymes) are central to the synthesis and subsequent modification of plant cells during morphogenesis. The endo-glucanase 16 (EG16) members constitute a distinct group of plant CAZymes, angiosperm orthologs of which were recently shown to have dual ß-glucan/xyloglucan hydrolase activity. Molecular phylogeny indicates that EG16 members comprise a sister clade with a deep evolutionary relationship to the widely studied apoplastic xyloglucan endo-transglycosylases/hydrolases (XTH). A cross-genome survey indicated that EG16 members occur as a single ortholog across species and are widespread in early diverging plants, including the non-vascular bryophytes, for which functional data were previously lacking. Remarkably, enzymological characterization of an EG16 ortholog from the model moss Physcomitrella patens (PpEG16) revealed that EG16 activity and sequence/structure are highly conserved across 500 million years of plant evolution, vis-à-vis orthologs from grapevine and poplar. Ex vivo biomechanical assays demonstrated that the application of EG16 gene products caused abrupt breakage of etiolated hypocotyls rather than slow extension, thereby indicating a mode-of-action distinct from endogenous expansins and microbial endo-glucanases. The biochemical data presented here will inform future genomic, genetic, and physiological studies of EG16 enzymes.

Expansin gene loss is a common occurrence during adaptation to an aquatic environment.

Expansins comprise a superfamily of plant cell wall loosening proteins that can be divided into four individual families (EXPA, EXPB, EXLA and EXLB). Aside from inferred roles in a variety of plant growth and developmental traits, little is known regarding the function of specific expansin clades, for which there are at least 16 in flowering plants (angiosperms); however, there is evidence to suggest that some expansins have cell-specific functions, in root hair and pollen tube development, for example. Recently, two duckweed genomes have been sequenced (Spirodela polyrhiza strains 7498 and 9509), revealing significantly reduced superfamily sizes. We hypothesized that there would be a correlation between expansin loss and morphological reductions seen among highly adapted aquatic species. In order to provide an answer to this question, we characterized the expansin superfamilies of the greater duckweed Spirodela, the marine eelgrass Zostera marina and the bladderwort Utricularia gibba. We discovered rampant expansin gene and clade loss among the three, including a complete absence of the EXLB family and EXPA-VII. The most convincing correlation between morphological reduction and expansin loss was seen for Utricularia and Spirodela, which both lack root hairs and the root hair expansin clade EXPA-X. Contrary to the pattern observed in other species, four Utricularia expansins failed to branch within any clade, suggesting that they may be the result of neofunctionalization. Last, an expansin clade previously discovered only in eudicots was identified in Spirodela, allowing us to conclude that the last common ancestor of monocots and eudicots contained a minimum of 17 expansins.

Disentangling loosening from softening: insights into primary cell wall structure.

How cell wall elasticity, plasticity, and time-dependent extension (creep) relate to one another, to plant cell wall structure and to cell growth remain unsettled topics. To examine these issues without the complexities of living tissues, we treated cell-free strips of onion epidermal walls with various enzymes and other agents to assess which polysaccharides bear mechanical forces in-plane and out-of-plane of the cell wall. This information is critical for integrating concepts of wall structure, wall material properties, tissue mechanics and mechanisms of cell growth. With atomic force microscopy we also monitored real-time changes in the wall surface during treatments. Driselase, a potent cocktail of wall-degrading enzymes, removed cellulose microfibrils in superficial lamellae sequentially, layer-by-layer, and softened the wall (reduced its mechanical stiffness), yet did not induce wall loosening (creep). In contrast Cel12A, a bifunctional xyloglucanase/cellulase, induced creep with only subtle changes in wall appearance. Both Driselase and Cel12A increased the tensile compliance, but differently for elastic and plastic components. Homogalacturonan solubilization by pectate lyase and calcium chelation greatly increased the indentation compliance without changing tensile compliances. Acidic buffer induced rapid cell wall creep via endogenous α-expansins, with negligible effects on wall compliances. We conclude that these various wall properties are not tightly coupled and therefore reflect distinctive aspects of wall structure. Cross-lamellate networks of cellulose microfibrils influenced creep and tensile stiffness whereas homogalacturonan influenced indentation mechanics. This information is crucial for constructing realistic molecular models that define how wall mechanics and growth depend on primary cell wall structure.

Global cellulose biomass, horizontal gene transfers and domain fusions drive microbial expansin evolution.

Plants must rearrange the network of complex carbohydrates in their cell walls during normal growth and development. To accomplish this, all plants depend on proteins called expansins that nonenzymatically loosen noncovalent bonding between cellulose microfibrils. Surprisingly, expansin genes have more recently been found in some bacteria and microbial eukaryotes, where their biological functions are largely unknown. Here, we reconstruct a comprehensive phylogeny of microbial expansin genes. We find these genes in all eukaryotic microorganisms that have structural cell wall cellulose, suggesting expansins evolved in ancient marine microorganisms long before the evolution of land plants. We also find expansins in an unexpectedly high diversity of bacteria and fungi that do not have cellulosic cell walls. These bacteria and fungi inhabit varied ecological contexts, mirroring the diversity of terrestrial and aquatic niches where plant and/or algal cellulosic cell walls are present. The microbial expansin phylogeny shows evidence of multiple horizontal gene transfer events within and between bacterial and eukaryotic microbial lineages, which may in part underlie their unusually broad phylogenetic distribution. Overall, expansins are unexpectedly widespread in bacteria and eukaryotes, and the contribution of these genes to microbial ecological interactions with plants and algae has probbaly been underappreciated.

Pectin methylesterase selectively softens the onion epidermal wall yet reduces acid-induced creep.

De-esterification of homogalacturonan (HG) is thought to stiffen pectin gels and primary cell walls by increasing calcium cross-linking between HG chains. Contrary to this idea, recent studies found that HG de-esterification correlated with reduced stiffness of living tissues, measured by surface indentation. The physical basis of such apparent wall softening is unclear, but possibly involves complex biological responses to HG modification. To assess the direct physical consequences of HG de-esterification on wall mechanics without such complications, we treated isolated onion (Allium cepa) epidermal walls with pectin methylesterase (PME) and assessed wall biomechanics with indentation and tensile tests. In nanoindentation assays, PME action softened the wall (reduced the indentation modulus). In tensile force/extension assays, PME increased plasticity, but not elasticity. These softening effects are attributed, at least in part, to increased electrostatic repulsion and swelling of the wall after PME treatment. Despite softening and swelling upon HG de-esterification, PME treatment alone failed to induce cell wall creep. Instead, acid-induced creep, mediated by endogenous α-expansin, was reduced. We conclude that HG de-esterification physically softens the onion wall, yet reduces expansin-mediated wall extensibility.

Cellulose synthase interactive1- and microtubule-dependent cell wall architecture is required for acid growth in Arabidopsis hypocotyls.

Auxin-induced cell elongation relies in part on the acidification of the cell wall, a process known as acid growth that presumably triggers expansin-mediated wall loosening via altered interactions between cellulose microfibrils. Cellulose microfibrils are a major determinant for anisotropic growth and they provide the scaffold for cell wall assembly. Little is known about how acid growth depends on cell wall architecture. To explore the relationship between acid growth-mediated cell elongation and plant cell wall architecture, two mutants (jia1-1 and csi1-3) that are defective in cellulose biosynthesis and cellulose microfibril organization were analyzed. The study revealed that cell elongation is dependent on CSI1-mediated cell wall architecture but not on the overall crystalline cellulose content. We observed a correlation between loss of crossed-polylamellate walls and loss of auxin- and fusicoccin-induced cell growth in csi1-3. Furthermore, induced loss of crossed-polylamellate walls via disruption of cortical microtubules mimics the effect of csi1 in acid growth. We hypothesize that CSI1- and microtubule-dependent crossed-polylamellate walls are required for acid growth in Arabidopsis hypocotyls.

Xyloglucan in the primary cell wall: assessment by FESEM, selective enzyme digestions and nanogold affinity tags.

Xyloglucan has been hypothesized to bind extensively to cellulose microfibril surfaces and to tether microfibrils into a load-bearing network, thereby playing a central role in wall mechanics and growth, but this view is challenged by newer results. Here we combined high-resolution imaging by field emission scanning electron microscopy (FESEM) with nanogold affinity tags and selective endoglucanase treatments to assess the spatial location and conformation of xyloglucan in onion cell walls. FESEM imaging of xyloglucanase-digested cell walls revealed an altered microfibril organization but did not yield clear evidence of xyloglucan conformations. Backscattered electron detection provided excellent detection of nanogold affinity tags in the context of wall fibrillar organization. Labelling with xyloglucan-specific CBM76 conjugated with nanogold showed that xyloglucans were associated with fibril surfaces in both extended and coiled conformations, but tethered configurations were not observed. Labelling with nanogold-conjugated CBM3, which binds the hydrophobic surface of crystalline cellulose, was infrequent until the wall was predigested with xyloglucanase, whereupon microfibril labelling was extensive. When tamarind xyloglucan was allowed to bind to xyloglucan-depleted onion walls, CBM76 labelling gave positive evidence for xyloglucans in both extended and coiled conformations, yet xyloglucan chains were not directly visible by FESEM. These results indicate that an appreciable, but still small, surface of cellulose microfibrils in the onion wall is tightly bound with extended xyloglucan chains and that some of the xyloglucan has a coiled conformation.

Cellulose synthase complexes act in a concerted fashion to synthesize highly aggregated cellulose in secondary cell walls of plants.

Cellulose, often touted as the most abundant biopolymer on Earth, is a critical component of the plant cell wall and is synthesized by plasma membrane-spanning cellulose synthase (CESA) enzymes, which in plants are organized into rosette-like CESA complexes (CSCs). Plants construct two types of cell walls, primary cell walls (PCWs) and secondary cell walls (SCWs), which differ in composition, structure, and purpose. Cellulose in PCWs and SCWs is chemically identical but has different physical characteristics. During PCW synthesis, multiple dispersed CSCs move along a shared linear track in opposing directions while synthesizing cellulose microfibrils with low aggregation. In contrast, during SCW synthesis, we observed swaths of densely arranged CSCs that moved in the same direction along tracks while synthesizing cellulose microfibrils that became highly aggregated. Our data support a model in which distinct spatiotemporal features of active CSCs during PCW and SCW synthesis contribute to the formation of cellulose with distinct structure and organization in PCWs and SCWs of Arabidopsis thaliana This study provides a foundation for understanding differences in the formation, structure, and organization of cellulose in PCWs and SCWs.