CALCIUM-DEPENDENT PROTEIN KINASE32 regulates cellulose biosynthesis through post-translational modification of cellulose synthase.

Cellulose is an essential component of plant cell walls and an economically important source of food, paper, textiles, and biofuel. Despite its economic and biological significance, the regulation of cellulose biosynthesis is poorly understood. Phosphorylation and dephosphorylation of cellulose synthases (CESAs) were shown to impact the direction and velocity of cellulose synthase complexes (CSCs). However, the protein kinases that phosphorylate CESAs are largely unknown. We conducted research in Arabidopsis thaliana to reveal protein kinases that phosphorylate CESAs. In this study, we used yeast two-hybrid, protein biochemistry, genetics, and live-cell imaging to reveal the role of calcium-dependent protein kinase32 (CPK32) in the regulation of cellulose biosynthesis in A. thaliana. We identified CPK32 using CESA3 as a bait in a yeast two-hybrid assay. We showed that CPK32 phosphorylates CESA3 while it interacts with both CESA1 and CESA3. Overexpressing functionally defective CPK32 variant and phospho-dead mutation of CESA3 led to decreased motility of CSCs and reduced crystalline cellulose content in etiolated seedlings. Deregulation of CPKs impacted the stability of CSCs. We uncovered a new function of CPKs that regulates cellulose biosynthesis and a novel mechanism by which phosphorylation regulates the stability of CSCs.

Distinguishing Mesoscale Polar Order (Unidirectional vs Bidirectional) of Cellulose Microfibrils in Plant Cell Walls Using Sum Frequency Generation Spectroscopy.

Cellulose in plant cell walls are synthesized as 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 a 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 nonlinear 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 noncentrosymmetric 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 noncentrosymmetric 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 noncentrosymmetric 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.

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.

CSI1, PATROL1, and exocyst complex cooperate in delivery of cellulose synthase complexes to the plasma membrane.

Cellulose synthesis occurs exclusively at the plasma membrane by cellulose synthase complexes (CSCs). Therefore, delivery of CSCs to discrete sites at the plasma membrane is critical for cellulose synthesis. Despite their significance, the delivery of CSCs is poorly understood. Here we used proteomics approaches, functional genetics, and live cell imaging to show that the de novo secretion of CSCs is mediated by cooperation among cellulose synthase interactive 1 (CSI1), the plant-specific protein PATROL1, and exocyst complex in Arabidopsis thaliana We propose that CSI1 plays a role in marking the docking site, which allows CSCs-containing vesicles access to the plasma membrane through its interaction with microtubules. PATROL1 assists in exocytosis by its interaction with multiple components, including CSI1, CSCs, and exocyst subunits. Both PATROL1 and the exocyst complex determine the rate of delivery of CSCs to the plasma membrane. By monitoring the exocyst complex, PATROL1, CSI1, and CSCs dynamics in real time, we present a timeline of events for exocytosis of CSCs. Our findings provide unique insights into the evolution of exocytosis in eukaryotes.

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.

[Sucrose gel for treatment of bacterial vaginosis: a randomized, double-blind, multi-center, parallel-group, phase III clinical trial].

OBJECTIVE: To evaluate the cure effectiveness and safety of sucrose gel in the treatment of bacterial vaginosis through a multi-center, randomized, double-blind, parallel controlled clinical study. METHODS: A clinical research method of multi-center, randomly double-blind, and dose group parallel comparison was adopted. In the study, 533 patients with bacterial vaginosis were randomly divided into two groups, which included 214 cases in the control group (5.0 g metronidazole gel) and 319 cases in the trial group (5.0 g sucrose gel ). The patients were treated with different medication according to the group where they were. All the cases in these two groups were treated with drugs vaginally twice in a day, morning and evening separately, for 5 days. The curative effect and safety evaluation were assessed from 7 to 10 days and 21 to 30 days after treatment respectively. RESULTS: The efficacy of the comprehensive clinical treatment showed that the cure rate of metronidazole gel group and sucrose gel group were 70.53% and 80.83% respectively 7 to 10 days after treatment. The recovery rate of Nugent score for vaginal smear were 71.50% and 81.15% respectively. The differences in the efficacy between these two groups were significant statistically (P<0.05). However, the cure rates of metronidazole gel group and sucrose gel group were 63.29% and 61.98% respectively 21 to 30 days after treatment. No statistically significant difference (P>0.05) could be found in the cure rates of the two groups. CONCLUSION: The clinical comprehensive efficacy and recovery of vaginal bacteria of sucrose gel group in the treatment of bacterial vaginosis were obviously superior to those of metronidazole gel 7 to 10 days after treatment. The susucrose gel could improve the clinical efficacy index and laboratory index of bacterial vaginosis. Other effects included the release of clinical symptoms, and the recovery of the normal micro-environment in the vagina according to the Nugent score. The curative efficacy of sucrose gel was equal to that of metronidazole gel 21 to 30 days after treatment. In the future, sucrose gel treatment can be a new strategy for the treatment of bacterial vaginosis. Various advantages can be taken to improve the cure rate of bacterial vaginosis and reduce the shortcomings produced by this disease.

Dimerization Mediated by a Divergent Forkhead-associated Domain Is Essential for the DNA Damage and Spindle Functions of Fission Yeast Mdb1.

MDC1 is a key factor of DNA damage response in mammalian cells. It possesses two phospho-binding domains. In its C terminus, a tandem BRCA1 C-terminal domain binds phosphorylated histone H2AX, and in its N terminus, a forkhead-associated (FHA) domain mediates a phosphorylation-enhanced homodimerization. The FHA domain of the Drosophila homolog of MDC1, MU2, also forms a homodimer but utilizes a different dimer interface. The functional importance of the dimerization of MDC1 family proteins is uncertain. In the fission yeast Schizosaccharomyces pombe, a protein sharing homology with MDC1 in the tandem BRCA1 C-terminal domain, Mdb1, regulates DNA damage response and mitotic spindle functions. Here, we report the crystal structure of the N-terminal 91 amino acids of Mdb1. Despite a lack of obvious sequence conservation to the FHA domain of MDC1, this region of Mdb1 adopts an FHA-like fold and is therefore termed Mdb1-FHA. Unlike canonical FHA domains, Mdb1-FHA lacks all the conserved phospho-binding residues. It forms a stable homodimer through an interface distinct from those of MDC1 and MU2. Mdb1-FHA is important for the localization of Mdb1 to DNA damage sites and the spindle midzone, contributes to the roles of Mdb1 in cellular responses to genotoxins and an antimicrotubule drug, and promotes in vitro binding of Mdb1 to a phospho-H2A peptide. The defects caused by the loss of Mdb1-FHA can be rescued by fusion with either of two heterologous dimerization domains, suggesting that the main function of Mdb1-FHA is mediating dimerization. Our data support that FHA-mediated dimerization is conserved for MDC1 family proteins.

Identification of a calmodulin-binding domain in Sema4D that regulates its exodomain shedding in platelets.

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.