RESUMEN

Microscale deformation processes, such as reorientation, buckling, and sliding of collagen fibrils, determine the mechanical behavior and function of collagenous tissue. While changes in the structure and composition of tendon have been extensively studied, the deformation mechanisms that modulate the interaction of extracellular matrix (ECM) constituents are not well understood, partly due to the lack of appropriate techniques to probe the behavior. In particular, the role of glycosaminoglycans (GAGs) in modulating collagen fibril interactions has remained controversial. Some studies suggest that GAGs act as crosslinkers between the collagen fibrils, while others have not found such evidence and postulate that GAGs have other functions. Here, we introduce a new framework, relying on orientation-dependent indentation behavior of tissue and computational modeling, to evaluate the shear-mediated function of GAGs in modulating the collagen fibril interactions at a length scale more relevant to fibrils compared to bulk tests. Specifically, we use chondroitinase ABC to enzymatically deplete the GAGs in tendon; measure the orientation-dependent indentation response in transverse and longitudinal orientations; and infer the microscale deformation mechanisms and function of GAGs from a microstructural computational model and a modified shear-lag model. We validate the modeling approach experimentally and show that GAGs facilitate collagen fibril sliding with minimal crosslinking function. We suggest that the molecular reconfiguration of GAGs is a potential mechanism for their microscale, strain-dependent viscoelastic behavior. This study reveals the mechanisms that control the orientation-dependent indentation response by affecting the shear deformation and provides new insights into the mechanical function of GAGs and collagen crosslinkers in collagenous tissue.

Asunto(s)

Matriz Extracelular , Glicosaminoglicanos , Glicosaminoglicanos/química , Tendones/fisiología , Colágeno/química , Simulación por Computador , Fenómenos Biomecánicos

RESUMEN

The spatial arrangement and interactions of the extracellular matrix (ECM) components control the mechanical behavior of tissue at multiple length scales. Changes in microscale deformation mechanisms affect tissue function and are often hallmarks of remodeling and disease. Despite their importance, the deformation mechanisms that modulate the mechanical behavior of collagenous tissue, particularly in indentation and compression modes of deformation, remain poorly understood. Here, we develop an integrated computational and experimental approach to investigate the deformation mechanisms of collagenous tissue at the microscale. While the complex deformation arising from indentation with a spherical probe is often considered a pitfall rather than an opportunity, we leverage this orientation-dependent deformation to examine the shear-regulated interactions of collagen fibrils and the role of crosslinks in modulating these interactions. We specifically examine tendon and cervix, two tissues rich in collagen with quite different microstructures and mechanical functions. We find that interacting, crosslinked collagen fibrils resist microscale longitudinal compressive forces, while widely used constitutive models fail to capture this behavior. The reorientation of collagen fibrils tunes the compressive stiffness of complex tissues like cervix. This study offers new insights into the mechanical behavior of collagen fibrils during indentation, and more generally, under longitudinal compressive forces, and illustrates the mechanisms that contribute to the experimentally observed orientation-dependent mechanical behavior. STATEMENT OF SIGNIFICANCE: Remodeling and disease can affect the deformation and interaction of tissue constituents, and thus mechanical function of tissue. Yet, the microscale deformation mechanisms are not well characterized in many tissues. Here, we develop a combined experimental-computational approach to infer the microscale deformation mechanisms of collagenous tissues with very different functions: tendon and cervix. Results show that collagen fibrils resist microscale forces along their length, though widely-used constitutive models do not account for this mechanism. This deformation process partially modulates the compressive stiffness of complex tissues such as cervix. Computational modeling shows that crosslink-mediated shear deformations are central to this unexpected behavior. This study offers new insights into the deformation mechanisms of collagenous tissue and the function of collagen crosslinkers.

Asunto(s)

Colágeno , Matriz Extracelular , Tendones , Simulación por Computador , Estrés Mecánico , Fenómenos Biomecánicos

RESUMEN

The cervix acts as a dynamic barrier between the uterus and vagina, retaining the fetus during pregnancy and allowing birth at term. Critical to this function, the physical properties of the cervix change, or remodel, but abnormal remodeling can lead to preterm birth (PTB). Although cervical remodeling has been studied, the complex 3D cervical microstructure has not been well-characterized. In this complex, dynamic, and heterogeneous tissue microenvironment, the microstructural changes are likely also heterogeneous. Using quantitative, 3D, second-harmonic generation microscopy, we demonstrate that rat cervical remodeling during pregnancy is not uniform across the cervix; the collagen fibers orient progressively more perpendicular to the cervical canals in the inner cervical zone, but do not reorient in other regions. Furthermore, regions that are microstructurally distinct early in pregnancy become more similar as pregnancy progresses. We use a finite element simulation to show that heterogeneous regional changes influence cervical funneling, an important marker of increased risk for PTB; the internal cervical os shows ∼6.5x larger radial displacement when fibers in the inner cervical zone are parallel to the cervical canals compared to when fibers are perpendicular to the canals. Our results provide new insights into the microstructural and tissue-level cervical changes that have been correlated with PTB and motivate further clinical studies exploring the origins of cervical funneling. STATEMENT OF SIGNIFICANCE: Cervical funneling, or dilation of the internal cervical os, is highly associated with increased risk of preterm birth. This study explores the 3D microstructural changes of the rat cervix during pregnancy and illustrates how these changes influence cervical funneling, assuming similar evolution in rats and humans. Quantitative imaging showed that microstructural remodeling during pregnancy is nonuniform across cervical regions and that initially distinct regions become more similar. We report, for the first time, that remodeling of the inner cervical zone can influence the dilation of the internal cervical os and allow the cervix to stay closed despite increased intrauterine pressure. Our results suggest a possible relationship between the microstructural changes of this zone and cervical funneling, motivating further clinical investigations.

Asunto(s)

Cuello del Útero , Nacimiento Prematuro , Animales , Cuello del Útero/diagnóstico por imagen , Simulación por Computador , Femenino , Embarazo , Nacimiento Prematuro/etiología , Ratas , Útero

RESUMEN

Although aluminum (AL) toxicity has been widely studied in monocotyledonous crop plants, the mechanism of Al impact on economically important dicotyledonous plants is poorly understood. Here, we report the spatial pattern of Al-induced root growth inhibition, which is closely associated with inhibition of H(+)-ATPase activity coupled with decreased surface negativity of plasma membrane (PM) vesicles isolated from apical 5-mm root segments of squash (Cucurbita pepo L. cv Tetsukabuto) plants. High-sensitivity growth measurements indicated that the central elongation zone, located 2 to 4 mm from the tip, was preferentially inhibited where high Al accumulation was found. The highest positive shifts (depolarization) in zeta potential of the isolated PM vesicles from 0- to 5-mm regions of Al-treated roots were corresponded to pronounced inhibition of H(+)-ATPase activity. The depolarization of PM vesicles isolated from Al-treated roots in response to added Al in vitro was less than that of control roots, suggesting, particularly in the first 5-mm root apex, a tight Al binding to PM target sites or irreversible alteration of PM properties upon Al treatment to intact plants. In line with these data, immunolocalization of H(+)-ATPase revealed decreases in tissue-specific H(+)-ATPase in the epidermal and cortex cells (2--3 mm from tip) following Al treatments. Our report provides the first circumstantial evidence for a zone-specific depolarization of PM surface potential coupled with inhibition of H(+)-ATPase activity. These effects may indicate a direct Al interaction with H(+)-ATPase from the cytoplasmic side of the PM.

Asunto(s)

Aluminio/toxicidad , Cucurbitaceae/efectos de los fármacos , Raíces de Plantas/efectos de los fármacos , ATPasas de Translocación de Protón/metabolismo , Adaptación Fisiológica , Aluminio/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Cucurbitaceae/metabolismo , Inhibidores Enzimáticos , Inmunohistoquímica , Técnicas In Vitro , Potenciales de la Membrana/efectos de los fármacos , Modelos Biológicos , Epidermis de la Planta/citología , Epidermis de la Planta/efectos de los fármacos , Raíces de Plantas/fisiología , ATPasas de Translocación de Protón/antagonistas & inhibidores

RESUMEN

Symplastic intercellular transport in plants is achieved by plasmodesmata (PD). These cytoplasmic channels are well known to interconnect plant cells to facilitate intercellular movement of water, nutrients, and signaling molecules including hormones. However, it is not known whether Al may affect this cell-to-cell transport process, which is a critical feature for roots as organs of nutrient/water uptake. We have microinjected the dye lucifer yellow carbohydrazide into peripheral root cells of an Al-sensitive wheat (Triticum aestivum cv Scout 66) either before or after Al treatment and followed the cell-to-cell dye-coupling through PD. Here we show that the Al-induced root growth inhibition is closely associated with the Al-induced blockage of cell-to-cell dye coupling. Immunofluorescence combined with immuno-electron microscopic techniques using monoclonal antibodies against 1-->3-beta-D-glucan (callose) revealed circumstantial evidence that Al-induced callose deposition at PD may responsible for this blockage of symplastic transport. Use of 2-deoxy-D-glucose, a callose synthesis inhibitor, allowed us to demonstrate that a reduction in callose particles correlated well with the improved dye-coupling and reduced root growth inhibition. While assessing the tissue specificity of this Al effect, comparable responses were obtained from the dye-coupling pattern in tobacco (Nicotiana tabacum) mesophyll cells. Analyses of the Al-induced expression of PD-associated proteins, such as calreticulin and unconventional myosin VIII, showed enhanced fluorescence and co-localizations with callose deposits. These results suggest that Al-signal mediated localized alterations to calcium homeostasis may drive callose formation and PD closure. Our data demonstrate that extracellular Al-induced callose deposition at PD could effectively block symplastic transport and communication in higher plants.

Asunto(s)

Aluminio/toxicidad , Comunicación Celular , Glucanos/metabolismo , Nicotiana/metabolismo , Plantas Tóxicas , Triticum/metabolismo , Aluminio/farmacología , Transporte Biológico , Señalización del Calcio , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Calreticulina , Técnica del Anticuerpo Fluorescente , Glucanos/biosíntesis , Líquido Intracelular/metabolismo , Microscopía Inmunoelectrónica , Miosinas/química , Miosinas/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Raíces de Plantas/ultraestructura , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Fracciones Subcelulares/metabolismo , Nicotiana/fisiología , Nicotiana/ultraestructura , Triticum/fisiología , Triticum/ultraestructura

RESUMEN

Eleven aluminum stress-induced genes derived from plants (wheat, Arabidopsis and tobacco) were introduced into Saccharomyces cerevisiae to test if expression of these genes confers Al tolerance. Al sensitivity tests showed that expression of two genes, either an Arabidopsis gene for blue copper binding protein (BCB), or a tobacco gene for the GDP dissociation inhibitor (NtGDI1), conferred Al tolerance. Determinations of total content and localization of Al ions in these transformants suggested that the BCB gene product functions in restricting Al uptake, while expression of the NtGDI1 gene promotes release of Al ions after uptake.

Asunto(s)

Aluminio/toxicidad , Proteínas de Arabidopsis , Proteínas Portadoras/genética , Proteínas de Unión al GTP/genética , Inhibidores de Disociación de Guanina Nucleótido , Proteínas de Plantas/genética , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Aluminio/farmacocinética , Transporte Biológico/efectos de los fármacos , Transporte Biológico/genética , Proteínas Portadoras/metabolismo , Gránulos Citoplasmáticos/metabolismo , Proteínas de Unión al GTP/metabolismo , Galactosa/farmacología , Regulación Fúngica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Glucosa/farmacología , Proteínas de Plantas/metabolismo , Saccharomyces cerevisiae/metabolismo , Transformación Genética

RESUMEN

Using monoclonal tubulin and actin antibodies, Al-mediated alterations to microtubules (MTs) and actin microfilaments (MFs) were shown to be most prominent in cells of the distal part of the transition zone (DTZ) of an Al-sensitive maize (Zea mays L.) cultivar. An early response to Al (1 h, 90 µM) was the depletion of MTs in cells of the DTZ, specifically in the outermost cortical cell file. However, no prominent changes to the MT cytoskeleton were found in elongating cells treated with Al for 1 h in spite of severe inhibition of root elongation. Al-induced early alterations to actin MFs were less dramatic and consisted of increased actin fluorescence of partially disintegrated MF arrays in cells of the DTZ. These tissue- and development-specific alterations to the cytoskeleton were preceded by and/or coincided with Al-induced depolarization of the plasma membrane and with callose formation, particularly in the outer cortex cells of the DTZ. Longer Al supplies (>6 h) led to progressive enhancements of lesions to the MT cytoskeleton in the epidermis and two to three outer cortex cell files. Our data show that the cytoskeleton in the cells of the DTZ is especially sensitive to Al, consistent with the recently proposed specific Al sensitivity of this unique, apical maize root zone.

Asunto(s)

Allium/fisiología , Aluminio/toxicidad , 2,4-Dinitrofenol , Allium/metabolismo , Cicloheximida/farmacología , Dinitrofenoles/farmacología , Nitratos/metabolismo , Proteínas de Plantas/metabolismo