Molecular insights into phosphoethanolamine cellulose formation and secretion.

Phosphoethanolamine (pEtN) cellulose is a naturally occurring modified cellulose produced by several Enterobacteriaceae. The minimal components of the E. coli cellulose synthase complex include the catalytically active BcsA enzyme, an associated periplasmic semicircle of hexameric BcsB, as well as the outer membrane (OM)-integrated BcsC subunit containing periplasmic tetratricopeptide repeats (TPR). Additional subunits include BcsG, a membrane-anchored periplasmic pEtN transferase associated with BcsA, and BcsZ, a conserved periplasmic cellulase of unknown biological function. While events underlying the synthesis and translocation of cellulose by BcsA are well described, little is known about its pEtN modification and translocation across the cell envelope. We show that the N-terminal cytosolic domain of BcsA positions three copies of BcsG near the nascent cellulose polymer. Further, the terminal subunit of the BcsB semicircle tethers the N-terminus of a single BcsC protein to establish a trans-envelope secretion system. BcsC's TPR motifs bind a putative cello-oligosaccharide near the entrance to its OM pore. Additionally, we show that only the hydrolytic activity of BcsZ but not the subunit itself is necessary for cellulose secretion, suggesting a secretion mechanism based on enzymatic removal of mislocalized cellulose. Lastly, we introduce pEtN modification of cellulose in orthogonal cellulose biosynthetic systems by protein engineering.

In vitro function, assembly, and interaction of primary cell wall cellulose synthase homotrimers.

Plant cell walls contain a meshwork of cellulose fibers embedded into a matrix of other carbohydrate and non-carbohydrate-based biopolymers. This composite material exhibits extraordinary properties, from stretchable and pliable cell boundaries to solid protective shells. Cellulose, a linear glucose polymer, is synthesized and secreted across the plasma membrane by cellulose synthase (CesA). Plants express several CesA isoforms, with different subsets necessary for primary and secondary cell wall biogenesis. The produced cellulose chains can be organized into fibrillar structures and fibrillogenesis likely requires the supramolecular organization of CesAs into pseudo sixfold symmetric complexes (CSCs). Here, we structurally and functionally characterize a set of soybean (Gm) CesA isoforms implicated in primary cell wall biogenesis. Cryogenic electron microscopy analyses of catalytically active GmCesA1, GmCesA3, and GmCesA6 reveal their assembly into homotrimeric complexes, stabilized by a cytosolic plant conserved region. Contrasting secondary cell wall CesAs, a peripheral position of the C-terminal transmembrane helix creates a large, lipid-exposed lateral opening of the enzymes' cellulose-conducting transmembrane channels. Co-purification experiments reveal that homotrimers of different CesA isoforms interact in vitro and that this interaction is independent of the enzymes' N-terminal cytosolic domains. Our data suggest that cross-isoform interactions are mediated by the class-specific region, which forms a hook-shaped protrusion of the catalytic domain at the cytosolic water-lipid interface. Further, inter-isoform interactions lead to synergistic catalytic activity, suggesting increased cellulose biosynthesis upon homotrimer interaction. Combined, our structural and biochemical data favor a model by which homotrimers of different CesA isoforms assemble into a microfibril-producing CSC.

Insights into substrate coordination and glycosyl transfer of poplar cellulose synthase-8.

Cellulose is an abundant cell wall component of land plants. It is synthesized from UDP-activated glucose molecules by cellulose synthase, a membrane-integrated processive glycosyltransferase. Cellulose synthase couples the elongation of the cellulose polymer with its translocation across the plasma membrane. Here, we present substrate- and product-bound cryogenic electron microscopy structures of the homotrimeric cellulose synthase isoform-8 (CesA8) from hybrid aspen (poplar). UDP-glucose binds to a conserved catalytic pocket adjacent to the entrance to a transmembrane channel. The substrate's glucosyl unit is coordinated by conserved residues of the glycosyltransferase domain and amphipathic interface helices. Site-directed mutagenesis of a conserved gating loop capping the active site reveals its critical function for catalytic activity. Molecular dynamics simulations reveal prolonged interactions of the gating loop with the substrate molecule, particularly across its central conserved region. These transient interactions likely facilitate the proper positioning of the substrate molecule for glycosyl transfer and cellulose translocation.

Insights into substrate coordination and glycosyl transfer of poplar cellulose synthase-8.

Cellulose is an abundant cell wall component of land plants. It is synthesized from UDP-activated glucose molecules by cellulose synthase, a membrane-integrated processive glycosyltransferase. Cellulose synthase couples the elongation of the cellulose polymer with its translocation across the plasma membrane. Here, we present substrate and product-bound cryogenic electron microscopy structures of the homotrimeric cellulose synthase isoform-8 (CesA8) from hybrid aspen (poplar). UDP-glucose binds to a conserved catalytic pocket adjacent to the entrance to a transmembrane channel. The substrate's glucosyl unit is coordinated by conserved residues of the glycosyltransferase domain and amphipathic interface helices. Site-directed mutagenesis of a conserved gating loop capping the active site reveals its critical function for catalytic activity. Molecular dynamics simulations reveal prolonged interactions of the gating loop with the substrate molecule, particularly across its central conserved region. These transient interactions likely facilitate the proper positioning of the substrate molecule for glycosyl transfer and cellulose translocation. Highlights: Cryo-EM structures of substrate and product bound poplar cellulose synthase provide insights into substrate selectivitySite directed mutagenesis signifies a critical function of the gating loop for catalysisMolecular dynamics simulations support persistent gating loop - substrate interactionsGating loop helps in positioning the substrate molecule to facilitate cellulose elongationConserved cellulose synthesis substrate binding mechanism across the kingdoms.

Mechanism of mixed-linkage glucan biosynthesis by barley cellulose synthase-like CslF6 (1,3;1,4)-ß-glucan synthase.

Mixed-linkage (1,3;1,4)-ß-glucans, which are widely distributed in cell walls of the grasses, are linear glucose polymers containing predominantly (1,4)-ß-linked glucosyl units interspersed with single (1,3)-ß-linked glucosyl units. Their distribution in cereal grains and unique structures are important determinants of dietary fibers that are beneficial to human health. We demonstrate that the barley cellulose synthase-like CslF6 enzyme is sufficient to synthesize a high-molecular weight (1,3;1,4)-ß-glucan in vitro. Biochemical and cryo-electron microscopy analyses suggest that CslF6 functions as a monomer. A conserved "switch motif" at the entrance of the enzyme's transmembrane channel is critical to generate (1,3)-linkages. There, a single-point mutation markedly reduces (1,3)-linkage formation, resulting in the synthesis of cellulosic polysaccharides. Our results suggest that CslF6 monitors the orientation of the nascent polysaccharide's second or third glucosyl unit. Register-dependent interactions with these glucosyl residues reposition the polymer's terminal glucosyl unit to form either a (1,3)- or (1,4)-ß-linkage.

Structure, substrate recognition and initiation of hyaluronan synthase.

Hyaluronan is an acidic heteropolysaccharide comprising alternating N-acetylglucosamine and glucuronic acid sugars that is ubiquitously expressed in the vertebrate extracellular matrix1. The high-molecular-mass polymer modulates essential physiological processes in health and disease, including cell differentiation, tissue homeostasis and angiogenesis2. Hyaluronan is synthesized by a membrane-embedded processive glycosyltransferase, hyaluronan synthase (HAS), which catalyses the synthesis and membrane translocation of hyaluronan from uridine diphosphate-activated precursors3,4. Here we describe five cryo-electron microscopy structures of a viral HAS homologue at different states during substrate binding and initiation of polymer synthesis. Combined with biochemical analyses and molecular dynamics simulations, our data reveal how HAS selects its substrates, hydrolyses the first substrate to prime the synthesis reaction, opens a hyaluronan-conducting transmembrane channel, ensures alternating substrate polymerization and coordinates hyaluronan inside its transmembrane pore. Our research suggests a detailed model for the formation of an acidic extracellular heteropolysaccharide and provides insights into the biosynthesis of one of the most abundant and essential glycosaminoglycans in the human body.

Functional characterization of a cellulose synthase, CtCESA1, from the marine red alga Calliarthron tuberculosum (Corallinales).

In land plants and algae, cellulose is important for strengthening cell walls and preventing breakage due to physical forces. Though our understanding of cellulose production by cellulose synthases (CESAs) has seen significant advances for several land plant and bacterial species, functional characterization of this fundamental protein is absent in red algae. Here we identify CESA gene candidates in the calcifying red alga Calliarthron tuberculosum using sequence similarity-based approaches, and elucidate their phylogenetic relationship with other CESAs from diverse taxa. One gene candidate, CtCESA1, was closely related to other putative red algal CESA genes. To test if CtCESA1 encoded a true cellulose synthase, CtCESA1 protein was expressed and purified from insect and yeast expression systems. CtCESA1 showed glucan synthase activity in glucose tracer assays. CtCESA1 activity was relatively low when compared with plant and bacterial CESA activity. In an in vitro assay, a predicted N-terminal starch-binding domain from CtCESA1 bound red algal floridean starch extracts, representing a unique domain in red algal CESAs not present in CESAs from other lineages. When the CtCESA1 gene was introduced into Arabidopsis thaliana cesa mutants, the red algal CtCESA1 partially rescued the growth defects of the primary cell wall cesa6 mutant, but not cesa3 or secondary cell wall cesa7 mutants. A fluorescently tagged CtCESA1 localized to the plasma membrane in the Arabidopsis cesa6 mutant background. This study presents functional evidence validating the sequence annotation of red algal CESAs. The relatively low activity of CtCESA1, partial complementation in Arabidopsis, and presence of unique protein domains suggest that there are probably functional differences between the algal and land plant CESAs.

Molecular organization of the E. coli cellulose synthase macrocomplex.

Cellulose is frequently found in communities of sessile bacteria called biofilms. Escherichia coli and other enterobacteriaceae modify cellulose with phosphoethanolamine (pEtN) to promote host tissue adhesion. The E. coli pEtN cellulose biosynthesis machinery contains the catalytic BcsA-B complex that synthesizes and secretes cellulose, in addition to five other subunits. The membrane-anchored periplasmic BcsG subunit catalyzes pEtN modification. Here we present the structure of the roughly 1 MDa E. coli Bcs complex, consisting of one BcsA enzyme associated with six copies of BcsB, determined by single-particle cryo-electron microscopy. BcsB homo-oligomerizes primarily through interactions between its carbohydrate-binding domains as well as intermolecular beta-sheet formation. The BcsB hexamer creates a half spiral whose open side accommodates two BcsG subunits, directly adjacent to BcsA's periplasmic channel exit. The cytosolic BcsE and BcsQ subunits associate with BcsA's regulatory PilZ domain. The macrocomplex is a fascinating example of cellulose synthase specification.

Architecture of a catalytically active homotrimeric plant cellulose synthase complex.

Cellulose is an essential plant cell wall component and represents the most abundant biopolymer on Earth. Supramolecular plant cellulose synthase complexes organize multiple linear glucose polymers into microfibrils as load-bearing wall components. We determined the structure of a poplar cellulose synthase CesA homotrimer that suggests a molecular basis for cellulose microfibril formation. This complex, stabilized by cytosolic plant-conserved regions and helical exchange within the transmembrane segments, forms three channels occupied by nascent cellulose polymers. Secretion steers the polymers toward a common exit point, which could facilitate protofibril formation. CesA's N-terminal domains assemble into a cytosolic stalk that interacts with a microtubule-tethering protein and may thus be involved in CesA localization. Our data suggest how cellulose synthase complexes assemble and provide the molecular basis for plant cell wall engineering.

The HOPS/Class C Vps Complex Tethers High-Curvature Membranes via a Direct Protein-Membrane Interaction.

Membrane tethering is a physical association of two membranes before their fusion. Many membrane tethering factors have been identified, but the interactions that mediate inter-membrane associations remain largely a matter of conjecture. Previously, we reported that the homotypic fusion and protein sorting/Class C vacuolar protein sorting (HOPS/Class C Vps) complex, which has two binding sites for the yeast vacuolar Rab GTPase Ypt7p, can tether two low-curvature liposomes when both membranes bear Ypt7p. Here, we show that HOPS tethers highly curved liposomes to Ypt7p-bearing low-curvature liposomes even when the high-curvature liposomes are protein-free. Phosphorylation of the curvature-sensing amphipathic lipid-packing sensor (ALPS) motif from the Vps41p HOPS subunit abrogates tethering of high-curvature liposomes. A HOPS complex without its Vps39p subunit, which contains one of the Ypt7p binding sites in HOPS, lacks tethering activity, though it binds high-curvature liposomes and Ypt7p-bearing low-curvature liposomes. Thus, HOPS tethers highly curved membranes via a direct protein-membrane interaction. Such high-curvature membranes are found at the sites of vacuole tethering and fusion. There, vacuole membranes bend sharply, generating large areas of vacuole-vacuole contact. We propose that HOPS localizes via the Vps41p ALPS motif to these high-curvature regions. There, HOPS binds via Vps39p to Ypt7p in an apposed vacuole membrane.

The HOPS/class C Vps complex tethers membranes by binding to one Rab GTPase in each apposed membrane.

Many Rab GTPase effectors are membrane-tethering factors, that is, they physically link two apposed membranes before intracellular membrane fusion. In this study, we investigate the distinct binding factors needed on apposed membranes for Rab effector-dependent tethering. We show that the homotypic fusion and protein-sorting/class C vacuole protein-sorting (HOPS/class C Vps) complex can tether low-curvature membranes, that is, liposomes with a diameter of ∼100 nm, only when the yeast vacuolar Rab GTPase Ypt7p is present in both tethered membranes. When HOPS is phosphorylated by the vacuolar casein kinase I, Yck3p, tethering only takes place when GTP-bound Ypt7p is present in both tethered membranes. When HOPS is not phosphorylated, however, its tethering activity shows little specificity for the nucleotide-binding state of Ypt7p. These results suggest a model for HOPS-mediated tethering in which HOPS tethers membranes by binding to Ypt7p in each of the two tethered membranes. Moreover, because vacuole-associated HOPS is presumably phosphorylated by Yck3p, our results suggest that nucleotide exchange of Ypt7p on multivesicular bodies (MVBs)/late endosomes must take place before HOPS can mediate tethering at vacuoles.

Signaling pathways that control rho kinase activity maintain the embryonic epicardial progenitor state.

This study identifies signaling pathways that play key roles in the formation and maintenance of epicardial cells, a source of progenitors for coronary smooth muscle cells (SMCs). After epithelial to mesenchymal transition (EMT), mesenchymal cells invade the myocardium to form coronary SMCs. RhoA/Rho kinase activity is required for EMT and for differentiation into coronary SMCs, whereas cAMP activity is known to inhibit EMT in epithelial cells by an unknown mechanism. We use outgrowth of epicardial cells from E9.5 isolated mouse proepicardium (PE) explants, wild type and Epac1 null E12.5 mouse heart explants, adult rat epicardial cells, and immortalized mouse embryonic epicardial cells as model systems to identify signaling pathways that regulate RhoA activity to maintain the epicardial progenitor state. We demonstrate that RhoA activity is suppressed in the epicardial progenitor state, that the cAMP-dependent Rap1 GTP exchange factor (GEF), Epac, known to down-regulate RhoA activity through activation of Rap1 GTPase activity increased, that Rap1 activity increased, and that expression of the RhoA antagonistic Rnd proteins known to activate p190RhoGAP increased and associated with p190RhoGAP. Finally, EMT is associated with increased p63RhoGEF and RhoGEF-H1 protein expression, increased GEF-H1 activity, with a trend in increased p63RhoGEF activity. EMT is suppressed by partial silencing of p63RhoGEF and GEF-H1. In conclusion, we have identified new signaling molecules that act together to control RhoA activity and play critical roles in the maintenance of coronary smooth muscle progenitor cells in the embryonic epicardium. We suggest that their eventual manipulation could promote revascularization after myocardial injury.

The cAMP-responsive Rap1 guanine nucleotide exchange factor, Epac, induces smooth muscle relaxation by down-regulation of RhoA activity.

Agonist activation of the small GTPase, RhoA, and its effector Rho kinase leads to down-regulation of smooth muscle (SM) myosin light chain phosphatase activity, an increase in myosin light chain (RLC(20)) phosphorylation and force. Cyclic nucleotides can reverse this process. We report a new mechanism of cAMP-mediated relaxation through Epac, a GTP exchange factor for the small GTPase Rap1 resulting in an increase in Rap1 activity and suppression of RhoA activity. An Epac-selective cAMP analog, 8-pCPT-2'-O-Me-cAMP ("007"), significantly reduced agonist-induced contractile force, RLC(20), and myosin light chain phosphatase phosphorylation in both intact and permeabilized vascular, gut, and airway SMs independently of PKA and PKG. The vasodilator PGI(2) analog, cicaprost, increased Rap1 activity and decreased RhoA activity in intact SMs. Forskolin, phosphodiesterase inhibitor isobutylmethylxanthine, and isoproterenol also significantly increased Rap1-GTP in rat aortic SM cells. The PKA inhibitor H89 was without effect on the 007-induced increase in Rap1-GTP. Lysophosphatidic acid-induced RhoA activity was reduced by treatment with 007 in WT but not Rap1B null fibroblasts, consistent with Epac signaling through Rap1B to down-regulate RhoA activity. Isoproterenol-induced increase in Rap1 activity was inhibited by silencing Epac1 in rat aortic SM cells. Evidence is presented that cooperative cAMP activation of PKA and Epac contribute to relaxation of SM. Our findings demonstrate a cAMP-mediated signaling mechanism whereby activation of Epac results in a PKA-independent, Rap1-dependent Ca(2+) desensitization of force in SM through down-regulation of RhoA activity. Cyclic AMP inhibition of RhoA is mediated through activation of both Epac and PKA.

Smooth and cardiac muscle-selective knock-out of Kruppel-like factor 4 causes postnatal death and growth retardation.

Krüppel-like factor 4 (Klf4) is a transcription factor involved in differentiation and proliferation in multiple tissues. We demonstrated previously that tamoxifen-induced deletion of the Klf4 gene in mice accelerated neointimal formation but delayed down-regulation of smooth muscle cell differentiation markers in carotid arteries following injury. To further determine the role of Klf4 in the cardiovascular system, we herein derived mice deficient for the Klf4 gene in smooth and cardiac muscle using the SM22alpha promoter (SM22alpha-CreKI(+)/Klf4(loxP/loxP) mice). SM22alpha-CreKI(+)/Klf4(loxP/loxP) mice were born at the expected Mendelian ratio, but they gradually died after birth. Although approximately 40% of SM22alpha-CreKI(+)/Klf4(loxP/loxP) mice survived beyond postnatal day 28, they exhibited marked growth retardation. In wild-type mice, Klf4 was expressed in the heart from late embryonic development through adulthood, whereas it was not expressed in smooth muscle. No changes were observed in morphology or expression of smooth muscle cell differentiation markers in vessels of SM22alpha-CreKI(+)/Klf4(loxP/loxP) mice. Of interest, cardiac output was significantly decreased in SM22alpha-CreKI(+)/Klf4(loxP/loxP) mice, as determined by magnetic resonance imaging. Moreover, a lack of Klf4 in the heart resulted in the reduction in expression of multiple cardiac genes, including Gata4. In vivo chromatin immunoprecipitation assays on the heart revealed that Klf4 bound to the promoter region of the Gata4 gene. Results provide novel evidence that Klf4 plays a key role in late fetal and/or postnatal cardiac development.

The actin-associated protein Palladin is required for development of normal contractile properties of smooth muscle cells derived from embryoid bodies.

Palladin is a widely expressed actin-associated protein localized at stress fibers, focal adhesions, and other actin-based structures, playing a significant role in cell adhesion and cell motility. Knockout of Palladin in mice is embryonic lethal, demonstrating the importance of Palladin in development yet its role in the vasculature is not known. In the present study, smooth muscle cell (SMC) markers, such as myosin, actin, caldesmon, calponin, and LPP, were down-regulated in embryoid bodies (EBs) derived from embryonic stem cells lacking Palladin. Transgenic embryonic stem cell lines were generated that stably expressed a puromycin-resistance gene under the control of a SM alpha-actin (SMA) promoter. Negative selection was then used to purify SMCs from EBs. Purified SMCs expressing multiple SMC markers were designated APSCs (SMA-puromycin-selected cells). Palladin null APSCs express significantly less myosin, actin, calponin, and h-caldesmon. The filamentous (F) to globular (G) actin ratio, known to regulate myocardin family transcription factors, was also decreased. Palladin null APSCs showed increased cell adhesion and decreased cell motility. Importantly, Palladin null APSCs within collagen gels generated less maximum contractile force when stimulated with endothelin-1, sphingosine 1-phosphate (S1P), and thrombin. Myosin light chains (MLC20) were phosphorylated by lysophosphatidic acid to the same extent in Palladin null and wild type APSCs but myosin content/total protein was reduced by >50%, consistent with the observed decreases in contractility. All together, these results suggest that Palladin is essential for expression of the full complement of contractile proteins necessary for optimal force development of SMCs derived from EBs.

Intracellular chloride accumulation and subcellular elemental distribution during atrial fibrillation.

BACKGROUND: Ion channel remodeling occurs during atrial fibrillation (AF); however, the extent of alteration in the subcellular distribution of elements (Na, K, Cl, Ca, Mg, P) is unknown. Electron probe microanalysis was used to determine the total (free+bound) in vivo subcellular concentration of these elements during AF. METHODS AND RESULTS: The left atrial appendage (LAA) was snap-frozen in situ after pacing (640 bpm) for 3 minutes (n=5 dogs), 30 minutes (n=3), or 48 hours (n=5). Dogs in sinus rhythm (n=3) served as controls. Whole-cell, cytosolic, and mitochondrial elemental concentrations were measured in cryosections. LAA effective refractory period (ERP) was measured before and after pacing. LAA ERP decreased significantly after 48 hours (116+/-3 to 88+/-10 ms, P=0.02). Whole-cell Cl increased by 9.0 mmol/L and 17 mmol/L after 3 and 30 minutes of pacing, respectively (P<0.0001), without a concomitant increase in Na. However, at 48 hours, whole-cell Na was reduced by 51% (P<0.01). Cytosolic Ca increased by 1.1 mmol/kg dry wt after 3 minutes (P<0.005), but mitochondrial Ca remained low and unchanged. Cell size measured in transverse cryosections increased after 3 minutes of pacing (75+/-5 to 109+/-11 microm2, P=0.007) but returned to baseline by 30 minutes (66+/-5 microm2). CONCLUSIONS: Intracellular Cl accumulation induced by rapid pacing is a novel finding and may play a role in AF pathogenesis by causing resting membrane depolarization and ERP reduction. There was no evidence of cellular or mitochondrial Ca overload despite the development of electrical remodeling and transient increase in cytoplasmic Ca.

Calcium content of peripheral and central mitochondria in the guinea pig myocardium: electron probe analysis.

We quantitated subcellular elemental concentrations in stimulated and resting guinea pig myocardium to determine whether species-specific properties of guinea pigs or the subcellular localization of mitochondria accounted for reports of higher mitochondrial Ca in guinea pigs than in other species. Small papillary muscles or trabeculae isolated from guinea pig ventricles were stimulated to raise cytosolic [Ca(2+)](i) by two methods: (1). tetanizing by rapid pacing preparations in which Ca(2+) uptake by the sarcoplasmic reticulum was inhibited with cyclopiazonic acid or (2). freeze trapping paced muscles near-peak systole. Electron probe X-ray microanalysis showed no significant difference between the (low, approximately 0.4 mmol/kg dry weight) mitochondrial Ca content of stimulated guinea pig hearts, compared to mitochondria of other species, such as rat and hamsters, and the Ca contents of peripheral and central mitochondria were also not significantly different.