Molecular Modeling and Imaging of Initial Stages of Cellulose Fibril Assembly: Evidence for a Disordered Intermediate Stage

Abstract

The remarkable mechanical strength of cellulose reflects the arrangement of multiple β-1,4-linked glucan chains in a para-crystalline fibril. During plant cellulose biosynthesis, a multimeric cellulose synthesis complex (CSC) moves within the plane of the plasma membrane as many glucan chains are synthesized from the same end and in close proximity. Many questions remain about the mechanism of cellulose fibril assembly, for example must multiple catalytic subunits within one CSC polymerize cellulose at the same rate? How does the cellulose fibril bend to align horizontally with the cell wall? Here we used mathematical modeling to investigate the interactions between glucan chains immediately after extrusion on the plasma membrane surface. Molecular dynamics simulations on groups of six glucans, each originating from a position approximating its extrusion site, revealed initial formation of an uncrystallized aggregate of chains from which a protofibril arose spontaneously through a ratchet mechanism involving hydrogen bonds and van der Waals interactions between glucose monomers. Consistent with the predictions from the model, freeze-fracture transmission electron microscopy using improved methods revealed a hemispherical accumulation of material at points of origination of apparent cellulose fibrils on the external surface of the plasma membrane where rosette-type CSCs were also observed. Together the data support the possibility that a zone of uncrystallized chains on the plasma membrane surface buffers the predicted variable rates of cellulose polymerization from multiple catalytic subunits within the CSC and acts as a flexible hinge allowing the horizontal alignment of the crystalline cellulose fibrils relative to the cell wall.

Re-constructing our models of cellulose and primary cell wall assembly

“…If 18 is the correct number of chains in the microfibril as produced by a single CSC, then half the CESAs would be inactive at any given instant. There is evidence that microfibril diameter may vary developmentally and across species [35,42-44], so a CSC with an ability to synthesize microfibrils of variable chain number would provide a creative solution to what is otherwise a vexing problem: how to account for variable microfibril diameters with a common CSC structure? Perhaps with a 36-cylinder engine that idles some cylinders.…”

“…To understand the initial stages of microfibril formation (largely terra incognita ), Haigler et al [42] combined molecular dynamics simulation (MDS) with freeze-fracture TEM of Zinnia mesophyll cells transdifferentiating into tracheary elements. MDS was limited to 6 chains and did not include water in the simulation, so it must be regarded as a limited first step in approaching this problem.…”

Plant Cell Wall Polysaccharides: Structure and Biosynthesis

“…Assuming that each CESA is capable of synthesizing one β-(1,4)-glucan chain, this yields microfibrils that contain~36 glucan chains per CSC (Fig. 1a, b), which compares well with the estimated diameter of larger microfibrils of~3-4 nm (Ding and Himmel 2006;Ding et al 2012;Haigler et al 2014). However, some microfibrils are narrower (~2.4 nm; Kennedy et al 2007), leading some to question the number of CESAs per CSC, the number of CESAs needed to produce a single glucan chain, and the number of glucan chains per microfibril (Carpita 2011;Newman et al 2013;Olek et al 2014).…”

“…CESAs add glucosyl units to the growing polymer to synthesize individual β-(1,4)-glucan chains. Growing glucans are extruded across the plasma membrane, where they assemble via van der Waals forces and hydrogen bonding into paracrystalline microfibrils (Doblin et al 2002;Haigler et al 2014). .…”

The Cellulase KORRIGAN Is Part of the Cellulose Synthase Complex

“…In this context, the role for an EGase could be the untangling of aberrantly associated glucan chains upon emergence from the 18 or more CESA subunits that constitute a CSC (Newman et al, 2013). Modeling showed that the formation of an amorphous intermediate through the random interaction of emerging glucan chains on top of the CSC is compatible with the assembly of an ordered microfibril later on (Haigler et al, 2014). In such a scenario, EGase activity on noncrystalline glucans might prevent the interaction among glucan chains from adjacent CSCs within the plasma membrane and, hence, the aberrant association of multiple microfibrils, which also could lead to the slowing down of the deposition rate.…”