Structure of Cellulose Microfibrils in Primary Cell Walls from Collenchyma

In the primary walls of growing plant cells, the glucose polymer cellulose is assembled into long microfibrils a few nanometers in diameter. The rigidity and orientation of these microfibrils control cell expansion; therefore, cellulose synthesis is a key factor in the growth and morphogenesis of plants. Celery (Apium graveolens) collenchyma is a useful model system for the study of primary wall microfibril structure because its microfibrils are oriented with unusual uniformity, facilitating spectroscopic and diffraction experiments. Using a combination of x-ray and neutron scattering methods with vibrational and nuclear magnetic resonance spectroscopy, we show that celery collenchyma microfibrils were 2.9 to 3.0 nm in mean diameter, with a most probable structure containing 24 chains in cross section, arranged in eight hydrogen-bonded sheets of three chains, with extensive disorder in lateral packing, conformation, and hydrogen bonding. A similar 18-chain structure, and 24-chain structures of different shape, fitted the data less well. Conformational disorder was largely restricted to the surface chains, but disorder in chain packing was not. That is, in position and orientation, the surface chains conformed to the disordered lattice constituting the core of each microfibril. There was evidence that adjacent microfibrils were noncovalently aggregated together over part of their length, suggesting that the need to disrupt these aggregates might be a constraining factor in growth and in the hydrolysis of cellulose for biofuel production.Growth and form in plants are controlled by the precisely oriented expansion of the walls of individual cells. The driving force for cell expansion is osmotic, but the rate and direction of expansion are controlled by the mechanical properties of the cell wall (Szymanski and Cosgrove, 2009). Expanding, primary cell walls are nanocomposite materials in which long microfibrils of cellulose, a few nanometers in diameter, run through a hydrated matrix of xyloglucans, pectins, and other polymers (Knox, 2008; Mohnen, 2008; Szymanski and Cosgrove, 2009; Scheller and Ulvskov, 2010). Native cellulose microfibrils are partially crystalline (Nishiyama, 2009; Fernandes et al., 2011). Formerly, primary wall cellulose was thought to have a unique crystal structure called cellulose IV₁ (Dinand et al., 1996), but NMR evidence suggests the presence of forms similar to the better characterized cellulose Iα and Iβ crystalline forms together with large quantities of less ordered cellulose (Wickholm et al., 1998; Sturcová et al., 2004; Wada et al., 2004). Nevertheless, cellulose is much more ordered than any other component of the primary cell wall (Bootten et al., 2004), in keeping with its key role of providing strength and controlling growth.The stiffness of the cell wall is greatest in the direction of the cellulose microfibrils, where growth is directional and the predominant microfibril orientation is usually transverse to the growth direction (Green, 1999; MacKinnon et al., 2006; Szymanski and Cosgrove, 2009). Expansion of the cell wall then requires either widening of the spacing between microfibrils (Marga et al., 2005) or slippage between them (Cosgrove, 2005), or both, and the microfibrils reorient toward the direction of growth (Anderson et al., 2010). Polymer cross bridges between microfibrils (McCann et al., 1990) are thought to resist these deformations of the cell wall nanostructure and, thus, to control the rate of growth. Until recently, most attention was focused on bridging xyloglucans, hydrogen bonded to microfibril surfaces (Scheller and Ulvskov, 2010). However, there is evidence that not all xyloglucans are appropriately positioned (Fujino et al., 2000; Park and Cosgrove, 2012a) and that other bridging polymers may be involved (Zykwinska et al., 2007). It has also been suggested that bundles of aggregated microfibrils, not single microfibrils, might be the key structural units in primary cell walls (Anderson et al., 2010), as in wood (Fahlén and Salmén, 2005; Fernandes et al., 2011). If so, single microfibrils could bridge between microfibril bundles. In summary, the growth of plant cells is not well understood, and we need more information on how cellulose orientation is controlled and on the nature of the bridging polymers, the cellulose surfaces to which these polymers bind, and the cohesion between microfibril surfaces that might mediate aggregation.Cellulose microfibrils are synthesized at the cell surface by large enzyme complexes having hexagonal symmetry, sometimes called “rosettes” (Somerville, 2006). Each complex contains multiple cellulose synthases that differ between primary cell walls and wood, although the appearance of the complexes is similar (Somerville, 2006; Atanassov et al., 2009). The simultaneous synthesis, from the same end, of all the chains in a native cellulose microfibril is why they are parallel (Nishiyama et al., 2002, 2003), in contrast to the entropically favored antiparallel structure found in man-made celluloses like rayon (Langan et al., 2001). The number of chains in a microfibril and the number of cellulose synthases in the synthetic complex are evidently related. It is commonly assumed that the number of chains is divisible by six, matching the hexagonal rosette symmetry, and 36-chain models (Himmel et al., 2007) bounded by the hydrophilic [110] and [1-10] crystal faces, as in algal celluloses (Bergenstråhle et al., 2008), have been widely adopted. The assembly and orientation of cellulose are connected, as several cellulose synthase mutants have phenotypes defective in cellulose orientation and plant form as well as depleted in cellulose content (Paredez et al., 2008). In certain other mutant lines, the crystallinity of the microfibrils appears to be affected (Fujita et al., 2011; Harris et al., 2012; Sánchez-Rodríguez et al., 2012).Therefore, a detailed understanding of the structure of primary wall cellulose microfibrils would help us to understand cellulose synthesis as well as the growth and structural mechanics of living plants (Burgert and Fratzl, 2009). Primary cell walls and their cellulose skeletons also affect food quality characteristics like the crispness of salad vegetables and apples (Malus domestica; Jarvis, 2011). When biofuels are produced from lignocellulosic biomass, lignification leads to recalcitrance (Himmel et al., 2007), but some of the cell types in Miscanthus spp., switchgrass (Panicum virgatum), and arable crop residues have only primary walls with no lignin, and recalcitrance then depends on the nature of the cellulose microfibrils (Beckham et al., 2011).A relatively detailed structure has recently been proposed for the microfibrils of spruce (Picea spp.) wood (Fernandes et al., 2011), which are 3.0 nm in diameter, allowing space for only about 24 cellulose chains. Evidence from x-ray diffraction supported a “rectangular” shape (Matthews et al., 2006) bounded by the [010] and [200] faces. There was considerable disorder increasing toward the surface, and the microfibrils were aggregated into bundles about 15 to 20 nm across, with some, but not all, of the lateral interfaces being resistant to water (Fernandes et al., 2011). Disordered domains are a feature of other strong biological materials such as spider silk (van Beek et al., 2002).Therefore, it is of interest whether any of these features of wood cellulose might also be found in the cellulose microfibrils of primary (growing) cell walls. It would be particularly useful to characterize the disorder known to be present in primary wall microfibrils, that is, to define how cellulose that is not measured as “crystalline” differs from crystalline cellulose. Many of the experiments leading toward a structure for wood cellulose were dependent on exceptionally uniform orientation of the cellulose microfibrils (Sturcová et al., 2004; Fernandes et al., 2011). However, in growing cell walls, the microfibrils are not uniformly oriented. When microfibrils are first laid down at the inner face of the primary cell wall, their orientation is normally transverse to the direction of growth, but as the cell wall expands, the microfibrils reorient so that the orientation distribution, integrated across the thickness of the expanded cell wall, becomes progressively closer to random (Cosgrove, 2005; MacKinnon et al., 2006).This technical problem does not apply to the cell walls of celery (Apium graveolens) collenchyma, which are similar in composition to other primary cell walls but have their microfibrils oriented relatively uniformly along the cell axis (Sturcová et al., 2004; Kennedy et al., 2007a, 2007b). Some structural information on celery collenchyma cellulose has already been derived from spectroscopic and scattering experiments (Sturcová et al., 2004; Kennedy et al., 2007a, 2007b), confirming the disorder expected in a primary wall cellulose. Some of these experiments were analogous to what has been done on spruce cellulose (Fernandes et al., 2011), but insufficient data are available to specify the number of chains in each primary wall microfibril, the nature and location of the disorder, and the presence or absence of direct contact between microfibrils. Here, we report x-ray and neutron scattering and spectroscopic experiments addressing these questions and leading to a proposed structure for primary wall cellulose microfibrils. Characterizing a structure containing so much disorder presented unusual challenges, but disorder appears to be central to the enigmatic capacity of primary wall cellulose to provide high strength and yet to permit and control growth.     

The in situ supermolecular structure of type I collagen.

BACKGROUND: The proteins belonging to the collagen family are ubiquitous throughout the animal kingdom. The most abundant collagen, type I, readily forms fibrils that convey the principal mechanical support and structural organization in the extracellular matrix of connective tissues such as bone, skin, tendon, and vasculature. An understanding of the molecular arrangement of collagen in fibrils is essential since it relates molecular interactions to the mechanical strength of fibrous tissues and may reveal the underlying molecular pathology of numerous connective tissue diseases. RESULTS: Using synchrotron radiation, we have conducted a study of the native fibril structure at anisotropic resolution (5.4 A axial and 10 A lateral). The intensities of the tendon X-ray diffraction pattern that arise from the lateral packing (three-dimensional arrangement) of collagen molecules were measured by using a method analogous to Rietveld methods in powder crystallography and to the separation of closely spaced peaks in Laue diffraction patterns. These were then used to determine the packing structure of collagen by MIR. CONCLUSIONS: Our electron density map is the first obtained from a natural fiber using these techniques (more commonly applied to single crystal crystallography). It reveals the three-dimensional molecular packing arrangement of type I collagen and conclusively proves that the molecules are arranged on a quasihexagonal lattice. The molecular segments that contain the telopeptides (central to the function of collagen fibrils in health and disease) have been identified, revealing that they form a corrugated arrangement of crosslinked molecules that strengthen and stabilize the native fibril.     

An allosteric potentiator of M4 mAChR modulates hippocampal synaptic transmission

Muscarinic acetylcholine receptors (mAChRs) provide viable targets for the treatment of multiple central nervous system disorders. We have used cheminformatics and medicinal chemistry to develop new, highly selective M4 allosteric potentiators. VU10010, the lead compound, potentiates the M4 response to acetylcholine 47-fold while having no activity at other mAChR subtypes. This compound binds to an allosteric site on the receptor and increases affinity for acetylcholine and coupling to G proteins. Whole-cell patch clamp recordings revealed that selective potentiation of M4 with VU10010 increases carbachol-induced depression of transmission at excitatory but not inhibitory synapses in the hippocampus. The effect was not mimicked by an inactive analog of VU10010 and was absent in M4 knockout mice. Selective regulation of excitatory transmission by M4 suggests that targeting of individual mAChR subtypes could be used to differentially regulate specific aspects of mAChR modulation of function in this important forebrain structure.     

Changes in the molecular packing of fibrillin microfibrils during extension indicate intrafibrillar and interfibrillar reorganization in elastic response

Fibrillin-rich microfibrils are the major structural components of the extracellular matrix that provide elasticity in a majority of connective tissues. The basis of elastic properties lies in the organization of fibrillin molecules, which, unfortunately, is still poorly understood. An X-ray diffraction study of hydrated fibrillin-rich microfibrils from zonular filaments has been conducted to give an insight into the molecular structure of microfibrils in intact tissue. A series of measurements was taken during controlled tissue extension to observe alterations in the lateral packing of microfibrils. Computer-generated simulated patterns were used to fit the experimental X-ray scattering data and to obtain the fibril diameter and lateral distance between the fibrils. The results suggest a nonlinear correlation between external strain and decrease in fibril diameter and lateral spacing. This was accompanied by a nonlinear increase in axial periodicity and a structure with a 160-nm periodicity, which is reported here for the first time using X-ray diffraction. These changes may reflect the unraveling of fibrillin from the complex folded arrangement into a linear structure. This finding supports a pleating model where fibrillin molecules are highly folded within the microfibrils; more importantly, the connection is made between the interaction of individual microfibrils and the change in their suprafibrillar coherent organization during extension. We suggest that the intermediate states observed in our study reflect sequential unfolding of fibrillin and can explain the process of its reversible unraveling.     

Generation and pharmacological analysis of M2 and M4 muscarinic receptor knockout mice

Muscarinic acetylcholine receptors (M1-M5) play important roles in the modulation of many key functions of the central and peripheral nervous system. To explore the physiological roles of the two Gi-coupled muscarinic receptors, we disrupted the M2 and M4 receptor genes in mice by using a gene targeting strategy. Pharmacological and behavioral analysis of the resulting mutant mice showed that the M2 receptor subtype is critically involved in mediating three of the most striking central muscarinic effects, tremor, hypothermia, and analgesia. These studies also indicated that M4 receptors are not critically involved in these central muscarinic responses. However, M4 receptor-deficient mice showed an increase in basal locomotor activity and greatly enhanced locomotor responses following drug-induced activation of D1 dopamine receptors. This observation is consistent with the concept that M4 receptors exert inhibitory control over D1 receptor-mediated locomotor stimulation, probably at the level of striatal projection neurons where the two receptors are known to be coexpressed. These findings emphasize the usefulness of gene targeting approaches to shed light on the physiological and pathophysiological roles of the individual muscarinic receptor subtypes.     

Elucidating the role of muscarinic receptors in psychosis

Muscarinic receptors have been implicated in the regulation of cognition and psychosis based on pharmacological evidence from pre-clinical and clinical studies. Muscarinic agonists have shown promise in the clinic in improving cognition and reducing psychotic episodes in Alzheimer's patients. However, lack of selective muscarinic ligands has limited their use due to troublesome side effects observed at higher doses. Without selective ligands, it has been difficult to assign a specific muscarinic receptor subtype to these high order mental processes. Recent development of muscarinic receptor knockout mice has provided additional tools to investigate cognition and psychosis in behavioral assays and to determine the receptor subtypes associated with parasympathomimetic physiology. Biochemical studies indicate that the M1 receptor plays a significant role in regulating G alpha q-mediated signal transduction in the hippocampus and cortex. Behavioral studies suggest that the M4 receptor is involved in movement regulation and prepulse inhibition of the startle reflex, a measure of attention. These findings support a role for the development of M1 and M4 receptor agonists for diseases in which symptoms include cognitive impairment and psychotic behaviors.     

Functional expression of M(1), M(3) and M(5) muscarinic acetylcholine receptors in yeast

The goal of this study was to functionally express the three G(q)-coupled muscarinic receptor subtypes, M(1), M(3) and M(5), in yeast (Saccharomyces cerevisiae). Transformation of yeast with expression constructs coding for the full-length receptors resulted in very low numbers of detectable muscarinic binding sites (B(max) < 5 fmol/mg). Strikingly, deletion of the central portion of the third intracellular loops of the M(1), M(3) and M(5) muscarinic receptors resulted in dramatic increases in B(max) values (53-214 fmol/mg). To monitor productive receptor/G-protein coupling, we used specifically engineered yeast strains that required agonist-stimulated receptor/G-protein coupling for cell growth. These studies showed that the shortened versions of the M(1), M(3) and M(5) receptors were unable to productively interact with the endogenous yeast G protein alpha-subunit, Gpa1p, or a Gpa1 mutant subunit that contained C-terminal mammalian Galpha(s) sequence. In contrast, all three receptors gained the ability to efficiently couple to a Gpa1/Galpha(q) hybrid subunit containing C-terminal mammalian Galpha(q) sequence, indicating that the M(1), M(3) and M(5) muscarinic receptors retained proper G-protein coupling selectivity in yeast. This is the first study to report the expression of muscarinic receptors in a coupling-competent form in yeast. The strategy described here, which involves structural modification of both receptors and co-expressed G proteins, should facilitate the functional expression of other classes of G protein-coupled receptors in yeast.     

Muscarinic receptor subtypes mediating central and peripheral antinociception studied with muscarinic receptor knockout mice: a review

To gain new insight into the physiological and pathophysiological roles of the muscarinic cholinergic system, we generated mutant mouse strains deficient in each of the five muscarinic acetylcholine receptor subtypes (M(1)-M(5)). In this chapter, we review a set of recent studies dealing with the identification of the muscarinic receptor subtypes mediating muscarinic agonist-dependent analgesic effects by central and peripheral mechanisms. Most of these studies were carried out with mutant mouse strains lacking M(2) or/and M(4) muscarinic receptors. It is well known that administration of centrally active muscarinic agonists induces pronounced analgesic effects. To identify the muscarinic receptors mediating this activity, wild-type and muscarinic receptor mutant mice were injected with the non-subtype-selective muscarinic agonist, oxotremorine (s.c., i.t., and i.c.v.), and analgesic effects were assessed in the tail-flick and hot-plate tests. These studies showed that M(2) receptors play a key role in mediating the analgesic effects of oxotremorine, both at the spinal and supraspinal level. However, studies with M(2)/M(4) receptor double KO mice indicated that M(4) receptors also contribute to this activity. Recent evidence suggests that activation of muscarinic receptors located in the skin can reduce the sensitivity of peripheral nociceptors. Electrophysiological and neurochemical studies with skin preparations from muscarinic receptor mutant mice indicated that muscarine-induced peripheral antinociception is mediated by M(2) receptors. Since acetylcholine is synthesized and released by different cell types of the skin, it is possible that non-neuronally released acetylcholine plays a role in modulating peripheral nociception. Our results highlight the usefulness of muscarinic receptor mutant mice to shed light on the functional roles of acetylcholine released from both neuronal and non-neuronal cells.