SignificanceCellulose is synthesized at the plasma membrane by cellulose synthase complexes (CSCs). The ability to deliver CSCs to discrete sites at the cell surface is critical for cellulose synthesis. We discovered that the de novo secretion of CSCs is mediated by cooperation among a recently identified cellulose synthase interacting protein, exocyst complex, and a plant-specific protein PATROL1 in Arabidopsis thaliana. We present a timeline of events for exocytosis of CSCs. CSCs represent unique cargo proteins that are not conserved in the mammalian or yeast system. Plants offer unique opportunities to characterize the function and regulation of exocytosis that may provide insight into the evolution of exocytosis in eukaryotes.

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.

• This study identifies a calmodulin-binding sequence in Sema4D and shows that calmodulin binds to Sema4D in resting platelets.• Dissociation of the Sema4D:calmodulin complex is sufficient to trigger Sema4D cleavage and shedding of the extracellular domain.Semaphorin 4D (Sema4D) is a transmembrane protein that supports contact-dependent amplification of platelet activation by collagen before being gradually cleaved by the metalloprotease ADAM17, as we have previously shown. Cleavage releases a soluble 120-kDa exodomain fragment for which receptors exist on platelets and endothelial cells. Here we have examined the mechanism that regulates Sema4D exodomain cleavage. The results show that the membrane-proximal cytoplasmic domain of Sema4D contains a binding site for calmodulin within the polybasic region Arg762-Lys779. Coprecipitation studies show that Sema4D and calmodulin are associated in resting platelets, forming a complex that dissociates upon platelet activation by the agonists that trigger Sema4D cleavage. Inhibiting calmodulin with W7 or introducing a membrane-permeable peptide corresponding to the calmodulin-binding site is sufficient to trigger the dissociation of Sema4D from calmodulin and initiate cleavage. Conversely, deletion of the calmodulin-binding site causes constitutive shedding of Sema4D. These results show that (1) Sema4D is a calmodulin-binding protein with a site of interaction in its membrane-proximal cytoplasmic domain, (2) platelet agonists cause dissociation of the calmodulin-Sema4D complex, and (3) dissociation of the complex is sufficient to trigger ADAM17-dependent cleavage of Sema4D, releasing a bioactive fragment. (Blood. 2013;121(20):4221-4230) Introduction Sema4D (CD100), a class IV semaphorin family member, is a 150-kDa type I membrane glycoprotein. The mature protein is a disulfidelinked homodimer that includes an NH 2 -terminal (N-terminal) sema domain and a cytoplasmic domain containing sites for tyrosine and serine and/or threonine phosphorylation.1-5 Sema4D was first described on T cells where it supports B-cell differentiation by binding to the low-affinity receptor CD72. 3,[6][7][8] Subsequent studies have identified roles for Sema4D in axon guidance, 9-12 angiogenesis, [13][14][15][16][17] and tumor progression [18][19][20] and showed that some of these effects are mediated by the high-affinity receptor plexin-B1 20 and the related lower-affinity receptor plexin-B2. 21,22 In previously published studies, we have shown that Sema4D is expressed by platelets and that it participates in the hemostatic response to injury by reinforcing collagen-initiated platelet activation in a contact-dependent manner.23,24 Deletion of Sema4D protects mice against the development of atherosclerosis by attenuating platelet hyperactivity in the setting of hyperlipidemia 25 and reducing intimal neovascularization, 26 which suggests that platelet Sema4D has an impact on the vessel wall as well as on platelets. Our previous studies also showed that the extracellular domain of Sema4D is gradu...

Cellulose in plant cell walls is crystalline microfibrils with diameters of 3~4 nm and lengths of around 1-10 μm. These microfibrils are known to be the backbone of cell walls, and their multiscale three-dimensional organization plays critical role in cell wall functions including plant growth and recalcitrance to degradation. The mesoscale organization of microfibrils over a 1  100 nm range in cell walls is challenging to resolve because most characterization techniques investigating this length scale suffer from low spatial resolution, sample preparation artifacts, or inaccessibility of specific cell types. Here we report a sum frequency generation (SFG) study determining the mesoscale polarity of cellulose microfibrils in intact plant cell walls. SFG is a non-linear optical spectroscopy technique sensitive to the molecular-to-mesoscale order of noncentrosymmetric domains in amorphous matrices. However, the quantitative theoretical model to unravel the effect of polarity in packing of non-centrosymmetric domains on SFG spectral features has remained unresolved. In this work, we show how the phase synchronization principle of the SFG process is used to predict the relative intensities of vibrational modes with different polar angles from the non-centrosymmetric domain axis. Applying this model calculation for the first time and employing SFG microscopy, we found that cellulose microfibrils in certain xylem cell walls are deposited unidirectionally (or biased in one direction), instead of the bidirectional polarity which was believed to be dominant in plant cell walls from volume-averaged characterizations of macroscopic samples. With this advancement in SFG analysis, one can now determine the relative polarity of non-centrosymmetric domains such as crystalline biopolymers interspersed in amorphous polymer matrices, which will open opportunities to study new questions that have not been conceived in the past.

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.