The Exocyst Regulates Hydrolytic Enzyme Secretion at Hyphal Tips and Septa in the Banana Fusarium Wilt Fungus Fusarium odoratissimum.

Hyphal polarized growth in filamentous fungi requires tip-directed secretion, while additional evidence suggests that fungal exocytosis for the hydrolytic enzyme secretion can occur at other sites in hyphae, including the septum. In this study, we analyzed the role of the exocyst complex involved in the secretion in the banana wilt fungal pathogen Fusarium odoratissimum. All eight exocyst components in F. odoratissimum not only localized to the tips ahead of the Spitzenkörper in growing hyphae but also localized to the outer edges of septa in mature hyphae. To further analyze the exocyst in F. odoratissimum, we attempted single gene deletion for all the genes encoding the eight exocyst components and only succeeded in constructing the gene deletion mutants for exo70 and sec5; we suspect that the other 6 exocyst components are encoded by essential genes. Deletion of exo70 or sec5 led to defects in vegetative growth, conidiation, and pathogenicity in F. odoratissimum. Notably, the deletion of exo70 resulted in decreased activities for endoglucosidase, filter paper enzymes, and amylase, while the loss of sec5 only led to a slight reduction in amylase activity. Septum-localized α-amylase (AmyB) was identified as the marker for septum-directed secretion, and we found that Exo70 is essential for the localization of AmyB to septa. Meanwhile the loss of Sec5 did not affect AmyB localization to septa but led to a higher accumulation of AmyB in cytoplasm. This suggested that while Exo70 and Sec5 both take part in the septum-directed secretion, the two play different roles in this process. IMPORTANCE The exocyst complex is a multisubunit tethering complex (MTC) for secretory vesicles at the plasma membrane and contains eight subunits, Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84. While the exocyst complex is well defined in eukaryotes from yeast (Saccharomyces cerevisiae) to humans, the exocyst components in filamentous fungi show different localization patterns in the apical tips of hyphae, which suggests that filamentous fungi have evolved divergent strategies to regulate endomembrane trafficking. In this study, we demonstrated that the exocyst components in Fusarium odoratissimum are localized not only to the tips of growing hyphae but also to the outer edge of the septa in mature hyphae, suggesting that the exocyst complex plays a role in the regulation of septum-directed protein secretion in F. odoratissimum. We further found that Exo70 and Sec5 are required for the septum-directed secretion of α-amylase in F. odoratissimum but with different influences.

Roles of Autophagy and Pancreatic Secretory Trypsin Inhibitor in Trypsinogen Activation in Acute Pancreatitis.

The focus of the review is on roles of autophagy and pancreatic secretory trypsin inhibitor (PSTI), an endogenous trypsin inhibitor, in trypsinogen activation in acute pancreatitis. Acute pancreatitis is a disease in which tissues in and around the pancreas are autodigested by pancreatic digestive enzymes. This reaction is triggered by the intrapancreatic activation of trypsinogen. Autophagy causes trypsinogen and cathepsin B, a trypsinogen activator, to colocalize within the autolysosomes. Consequently, if the resultant trypsin activity exceeds the inhibitory activity of PSTI, the pancreatic digestive enzymes are activated, and they cause autodigestion of the acinar cells. Thus, autophagy and PSTI play important roles in the development and suppression of acute pancreatitis, respectively.

The Great Escape: how phosphatidylinositol 4-kinases and PI4P promote vesicle exit from the Golgi (and drive cancer).

Phosphatidylinositol 4-phosphate (PI4P) is a membrane glycerophospholipid and a major regulator of the characteristic appearance of the Golgi complex as well as its vesicular trafficking, signalling and metabolic functions. Phosphatidylinositol 4-kinases, and in particular the PI4KIIIß isoform, act in concert with PI4P to recruit macromolecular complexes to initiate the biogenesis of trafficking vesicles for several Golgi exit routes. Dysregulation of Golgi PI4P metabolism and the PI4P protein interactome features in many cancers and is often associated with tumour progression and a poor prognosis. Increased expression of PI4P-binding proteins, such as GOLPH3 or PITPNC1, induces a malignant secretory phenotype and the release of proteins that can remodel the extracellular matrix, promote angiogenesis and enhance cell motility. Aberrant Golgi PI4P metabolism can also result in the impaired post-translational modification of proteins required for focal adhesion formation and cell-matrix interactions, thereby potentiating the development of aggressive metastatic and invasive tumours. Altered expression of the Golgi-targeted PI 4-kinases, PI4KIIIß, PI4KIIα and PI4KIIß, or the PI4P phosphate Sac1, can also modulate oncogenic signalling through effects on TGN-endosomal trafficking. A Golgi trafficking role for a PIP 5-kinase has been recently described, which indicates that PI4P is not the only functionally important phosphoinositide at this subcellular location. This review charts new developments in our understanding of phosphatidylinositol 4-kinase function at the Golgi and how PI4P-dependent trafficking can be deregulated in malignant disease.

Novosphingobium sp. PP1Y as a novel source of outer membrane vesicles.

Outer membrane vesicles (OMVs) are nanostructures of 20-200 nm diameter deriving from the surface of several Gram-negative bacteria. OMVs are emerging as shuttles involved in several mechanisms of communication and environmental adaptation. In this work, OMVs were isolated and characterized from Novosphingobium sp. PP1Y, a Gram-negative non-pathogenic microorganism lacking LPS on the outer membrane surface and whose genome was sequenced and annotated. Scanning electron microscopy performed on samples obtained from a culture in minimal medium highlighted the presence of PP1Y cells embedded in an extracellular matrix rich in vesicular structures. OMVs were collected from the exhausted growth medium during the mid-exponential phase, and purified by ultracentrifugation on a sucrose gradient. Atomic force microscopy, dynamic light scattering and nanoparticle tracking analysis showed that purified PP1Y OMVs had a spherical morphology with a diameter of ca. 150 nm and were homogenous in size and shape. Moreover, proteomic and fatty acid analysis of purified OMVs revealed a specific biochemical "fingerprint", suggesting interesting details concerning their biogenesis and physiological role. Moreover, these extracellular nanostructures do not appear to be cytotoxic on HaCaT cell line, thus paving the way to their future use as novel drug delivery systems.

Extended cleavage specificities of mast cell proteases 1 and 2 from golden hamster: Classical chymase and an elastolytic protease comparable to rat and mouse MCP-5.

Serine proteases constitute the major protein content of mast cell secretory granules. Here we present the extended cleavage specificity of two such proteases from the golden hamster, Mesocricetus auratus. Analysis by phage display technique showed that one of them (HAM1) is a classical chymase with a specificity similar to the human mast cell chymase. However, in contrast to the human chymase, it does not seem to have a particular preference for any of the three aromatic amino acids, Phe, Tyr and Trp, in the P1 position of substrates. HAM1 also efficiently cleaved after Leu similarly to human and many other mast cell chymases. We observed only a 3-fold lower cleavage activity on Leu compared to substrates with P1 aromatic amino acids. Chymotryptic enzymes seem to be characteristic for connective tissue mast cells in mammalian species from opossums to humans, which indicates a very central role of these enzymes in mast cell biology. HAM1 also seems to have the strongest preference for negatively charged amino acids in the P2´position of all mast cell chymases so far characterized. The second hamster chymase, HAM2, is an elastolytic in its activity, similarly to the α-chymases in rats and mice (rMCP-5 and mMCP-5, respectively). The presence of an α-chymase that developed elastase activity thereby seems to be a relatively early modification of the α-chymase within the rodent branch of the mammalian evolutionary tree.

Kinesin-1 Is a New Actor Involved in Platelet Secretion and Thrombus Stability.

OBJECTIVE: Platelet secretion is crucial for many physiological platelet responses. Even though several regulators of the fusion machinery for secretory granule exocytosis have been identified in platelets, the underlying mechanisms are not yet fully characterized. APPROACH AND RESULTS: By studying a mouse model (cKO [conditional knockout]Kif5b) lacking Kif5b (kinesin-1 heavy chain) in its megakaryocytes and platelets, we evidenced unstable hemostasis characterized by an increase of blood loss associated to a marked tendency to rebleed in a tail-clip assay and thrombus instability in an in vivo thrombosis model. This instability was confirmed in vitro in a whole-blood perfusion assay under blood flow conditions. Aggregations induced by thrombin and collagen were also impaired in cKOKif5b platelets. Furthermore, P-selectin exposure, PF4 (platelet factor 4) secretion, and ATP release after thrombin stimulation were impaired in cKOKif5b platelets, highlighting the role of kinesin-1 in α-granule and dense granule secretion. Importantly, exogenous ADP rescued normal thrombin induced-aggregation in cKOKif5b platelets, which indicates that impaired aggregation was because of defective release of ADP and dense granules. Last, we demonstrated that kinesin-1 interacts with the molecular machinery comprising the granule-associated Rab27 (Ras-related protein Rab-27) protein and the Slp4 (synaptotagmin-like protein 4/SYTL4) adaptor protein. CONCLUSIONS: Our results indicate that a kinesin-1-dependent process plays a role for platelet function by acting into the mechanism underlying α-granule and dense granule secretion.

There's no place like OM: Vesicular sorting and secretion of the peptidylarginine deiminase of Porphyromonas gingivalis.

The oral pathogen Porphyromonas gingivalis is one of the major periodontal agents and it has been recently hailed as a potential cause of the autoimmune disease rheumatoid arthritis. In particular, the peptidylarginine deiminase enzyme of P. gingivalis (PPAD) has been implicated in the citrullination of certain host proteins and the subsequent appearance of antibodies against citrullinated proteins, which might play a role in the etiology of rheumatoid arthritis. The aim of this study was to investigate the extracellular localization of PPAD in a large panel of clinical P. gingivalis isolates. Here we show that all isolates produced PPAD. In most cases PPAD was abundantly present in secreted outer membrane vesicles (OMVs) that are massively produced by P. gingivalis, and to minor extent in a soluble secreted state. Interestingly, a small subset of clinical isolates showed drastically reduced levels of the OMV-bound PPAD and secreted most of this enzyme in the soluble state. The latter phenotype is strictly associated with a lysine residue at position 373 in PPAD, implicating the more common glutamine residue at this position in PPAD association with OMVs. Further, one isolate displayed severely restricted vesiculation. Together, our findings show for the first time that neither the major association of PPAD with vesicles, nor P. gingivalis vesiculation per se, are needed for P. gingivalis interactions with the human host.

JIP3 localises to exocytic vesicles and focal adhesions in the growth cones of differentiated PC12 cells.

The JNK-interacting protein 3 (JIP3) is a molecular scaffold, expressed predominantly in neurons, that serves to coordinate the activation of the c-Jun N-terminal kinase (JNK) by binding to JNK and the upstream kinases involved in its activation. The JNK pathway is involved in the regulation of many cellular processes including the control of cell survival, cell death and differentiation. JIP3 also associates with microtubule motor proteins such as kinesin and dynein and is likely an adapter protein involved in the tethering of vesicular cargoes to the motors involved in axonal transport in neurons. We have used immunofluorescence microscopy and biochemical fractionation to investigate the subcellular distribution of JIP3 in relation to JNK and to vesicular and organelle markers in rat pheochromocytoma cells (PC12) differentiating in response to nerve growth factor. In differentiated PC12 cells, JIP3 was seen to accumulate in growth cones at the tips of developing neurites where it co-localised with both JNK and the JNK substrate paxillin. Cellular fractionation of PC12 cells showed that JIP3 was associated with a subpopulation of vesicles in the microsomal fraction, distinct from synaptic vesicles, likely to be an anterograde-directed exocytic vesicle pool. In differentiated PC12 cells, JIP3 did not appear to associate with retrograde endosomal vesicles thought to be involved in signalling axonal injury. Together, these observations indicate that JIP3 may be involved in transporting vesicular cargoes to the growth cones of PC12 cells, possibly targeting JNK to its substrate paxillin, and thus facilitating neurite outgrowth.

Secretome profiling reveals temperature-dependent growth of Aspergillus fumigatus.

Aspergillus fumigatus is a ubiquitous opportunistic fungus. In this study, systematic analyses were carried out to study the temperature adaptability of A. fumigatus. A total of 241 glycoside hydrolases and 69 proteases in the secretome revealed the strong capability of A. fumigatus to degrade plant biomass and protein substrates. In total, 129 pathogenesis-related proteins detected in the secretome were strongly correlated with glycoside hydrolases and proteases. The variety and abundance of proteins remained at temperatures of 34°C-45°C. The percentage of endo-1,4-xylanase increased when the temperature was lowered to 20°C, while the percentage of cellobiohydrolase increased as temperature was increased, suggesting that the strain obtains carbon mainly by degrading xylan and cellulose, and the main types of proteases in the secretome were aminopeptidases and carboxypeptidases. Only half of the proteins were retained and their abundance declined to 9.7% at 55°C. The activities of the remaining ß-glycosidases and proteases were merely 35% and 24%, respectively, when the secretome was treated at 60°C for 2 h. Therefore, temperatures >60°C restrict the growth of A. fumigatus.

Non-muscular myosin light chain kinase triggers intermittent hypoxia-induced interleukin-6 release, endothelial dysfunction and permeability.

Obstructive sleep apnea is characterized by intermittent hypoxia (IH) which alters endothelial function, induces inflammation and accelerates atherosclerosis-induced cardiovascular diseases. The non-muscular myosin light chain kinase (nmMLCK) isoform contributes to endothelial cell-cell junction opening. Deletion of nmMLCK protects mice from death in septic shock models and prevents atherosclerosis in high-fat diet-fed mice. The aim of the study was to analyze the implication of nmMLCK in IH-induced vascular inflammation. Human aortic endothelial cells were exposed to 6 hours of IH in absence or presence of nmMLCK inhibitors, ML-7 (5 µM) or PIK (150 µM). IH increased reactive oxygen species (ROS) and nitric oxide (NO) production, p65-NFκB activation and IL-6 secretion. While nmMLCK inhibition did not prevent IH-induced ROS production and p65-NFκB activation, it decreased NO production and partially prevented IL-6 secretion. IH-induced IL-6 secretion and vesicle-associated membrane protein-associated vesicles re-organization were inhibited in presence of the inhibitor of protein secretion, brefeldin A, or ML-7. IH increased monocytes transendothelial migration that was partially prevented by ML-7. Finally, IH reduced endothelium-dependent relaxation to acetylcholine of aortas from wild-type but not those taken from nmMLCK-deficient mice. These results suggest that nmMLCK participates to IH-induced endothelial dysfunction resulting from cytokines secretion and endothelial permeability.

A fluorescence method for determination of glucose transport by intestinal BBMV of common carp.

Epithelial brush-border membrane vesicles (BBMVs) were isolated from the intestine of common carp and studied systematically by enzyme activity, transmission electron microscopy and immunoblotting. The uptake time course and the substrate concentration effect were assessed, and then, the ability of phlorizin and cytochalasin B to inhibit uptake was analyzed. The results show that sucrase, alkaline phosphatase and Na+-K+-ATPase activities in these vesicles were enriched 7.94-, 6.74- and 0.42-fold, respectively, indicating a relatively pure preparation of apical membrane with little basolateral contamination. The vesicular structure was in complete closure, as confirmed by electron microscopy. The presence of SGLT1 on the BBMVs was confirmed by Western blot analysis. In the time course experiment, the glucose uptake by BBMVs in Na+ medium displayed an initial accumulation (overshoot) at 5 min followed by a rapid return to equilibrium values at 60 min. Over the 2-NBDG concentration range selected, the external 2-NBDG concentration in NaSCN medium graphed as a curved line. Phlorizin and cytochalasin B had an obvious inhibitory effect on 2-NBDG transport in carp BBMVs, and the detected fluorescence intensity decreased. The inhibition rate in the 1000 µM group was the strongest at 64.18% and 63.61% of phlorizin and cytochalasin B, respectively, indicating the presence of carriers other than SGLT1. This study is the first to demonstrate that 2-NBDG can be used as a convenient and sensitive probe to detect glucose uptake in fish BBMVs. This technology will provide a convenient method to discover new effects and factors in glucose metabolism.

GOP-1 promotes apoptotic cell degradation by activating the small GTPase Rab2 in C. elegans.

Apoptotic cells generated by programmed cell death are engulfed by phagocytes and enclosed within plasma membrane-derived phagosomes. Maturation of phagosomes involves a series of membrane-remodeling events that are governed by the sequential actions of Rab GTPases and lead to formation of phagolysosomes, where cell corpses are degraded. Here we identified gop-1 as a novel regulator of apoptotic cell clearance in Caenorhabditis elegans Loss of gop-1 affects phagosome maturation through the RAB-5-positive stage, causing defects in phagosome acidification and phagolysosome formation, phenotypes identical to and unaffected by loss of unc-108, the C. elegans Rab2 GOP-1 transiently associates with cell corpse-containing phagosomes, and loss of its function abrogates phagosomal association of UNC-108. GOP-1 interacts with GDP-bound and nucleotide-free UNC-108/Rab2, disrupts GDI-UNC-108 complexes, and promotes activation and membrane recruitment of UNC-108/Rab2 in vitro. Loss of gop-1 also abolishes association of UNC-108 with endosomes, causing defects in endosome and dense core vesicle maturation. Thus, GOP-1 is an activator of UNC-108/Rab2 in multiple processes.

Sequential Immunoprecipitation of Secretory Vesicle Proteins from Biosynthetically Labelled Cells.

Pulse radiolabelling of cells with radioactive amino acids is a common method for studying the biosynthesis of proteins. The labelled proteins can then be immunoprecipitated and analysed by electrophoresis and imaging techniques. This chapter presents a protocol for the biosynthetic labelling and immunoprecipitation of pancreatic islet proteins which are known to be affected in psychiatric disorders such as schizophrenia.

A STRIPAK complex mediates axonal transport of autophagosomes and dense core vesicles through PP2A regulation.

Autophagy plays an essential role in the cellular homeostasis of neurons, facilitating the clearance of cellular debris. This clearance process is orchestrated through the assembly, transport, and fusion of autophagosomes with lysosomes for degradation. The motor protein dynein drives autophagosome motility from distal sites of assembly to sites of lysosomal fusion. In this study, we identify the scaffold protein CKA (connector of kinase to AP-1) as essential for autophagosome transport in neurons. Together with other core components of the striatin-interacting phosphatase and kinase (STRIPAK) complex, we show that CKA associates with dynein and directly binds Atg8a, an autophagosomal protein. CKA is a regulatory subunit of PP2A, a component of the STRIPAK complex. We propose that the STRIPAK complex modulates dynein activity. Consistent with this hypothesis, we provide evidence that CKA facilitates axonal transport of dense core vesicles and autophagosomes in a PP2A-dependent fashion. In addition, CKA-deficient flies exhibit PP2A-dependent motor coordination defects. CKA function within the STRIPAK complex is crucial to prevent transport defects that may contribute to neurodegeneration.

Extracellular superoxide dismutase is present in secretory vesicles of human neutrophils and released upon stimulation.

Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme present in the extracellular matrix (ECM), where it provides protection against oxidative degradation of matrix constituents including type I collagen and hyaluronan. The enzyme is known to associate with macrophages and polymorphonuclear leukocytes (neutrophils) and increasing evidence supports a role for EC-SOD in the development of an inflammatory response. Here we show that human EC-SOD is present at the cell surface of isolated neutrophils as well as stored within secretory vesicles. Interestingly, we find that EC-SOD mRNA is absent throughout neutrophil maturation indicating that the protein is synthesized by other cells and subsequently endocytosed by the neutrophil. When secretory vesicles were mobilized by neutrophil stimulation using formyl-methionyl-leucyl-phenylalanine (fMLF) or phorbol 12-myristate 13-acetate (PMA), the protein was released into the extracellular space and found to associate with DNA released from stimulated cells. The functional consequences were evaluated by the use of neutrophils isolated from wild-type and EC-SOD KO mice, and showed that EC-SOD release significantly reduce the level of superoxide in the extracellular space, but does not affect the capacity to generate neutrophil extracellular traps (NETs). Consequently, our data signifies that EC-SOD released from activated neutrophils affects the redox conditions of the extracellular space and may offer protection against highly reactive oxygen species such as hydroxyl radicals otherwise generated as a result of respiratory burst activity of activated neutrophils.

Imbalanced Rab3D versus Rab27 increases cathepsin S secretion from lacrimal acini in a mouse model of Sjögren's Syndrome.

The mechanism responsible for the altered spectrum of tear proteins secreted by lacrimal gland acinar cells (LGAC) in patients with Sjögren's Syndrome (SS) remains unknown. We have previously identified increased cathepsin S (CTSS) activity as a unique characteristic of tears of patients with SS. Here, we investigated the role of Rab3D, Rab27a, and Rab27b proteins in the enhanced release of CTSS from LGAC. Similar to patients with SS and to the male nonobese diabetic (NOD) mouse model of SS, CTSS activity was elevated in tears of mice lacking Rab3D. Findings of lower gene expression and altered localization of Rab3D in NOD LGAC reinforce a role for Rab3D in suppressing excess CTSS release under physiological conditions. However, CTSS activity was significantly reduced in tears of mice lacking Rab27 isoforms. The reliance of CTSS secretion on Rab27 activity was supported by in vitro findings that newly synthesized CTSS was detected in and secreted from Rab27-enriched secretory vesicles and that expression of dominant negative Rab27b reduced carbachol-stimulated secretion of CTSS in cultured LGAC. High-resolution 3D-structured illumination microscopy revealed microdomains of Rab3D and Rab27 isoforms on the same secretory vesicles but present in different proportions on different vesicles, suggesting that changes in their relative association with secretory vesicles may tailor the vesicle contents. We propose that a loss of Rab3D from secretory vesicles, leading to disproportionate Rab27-to-Rab3D activity, may contribute to the enhanced release of CTSS in tears of patients with SS.

O-Glycosylation of a Secretory Granule Membrane Enzyme Is Essential for Its Endocytic Trafficking.

Peptidylglycine α-amidating monooxygenase (PAM) (EC 1.14.17.3) catalyzes peptide amidation, a crucial post-translational modification, through the sequential actions of its monooxygenase (peptidylglycine α-hydroxylating monooxygenase) and lyase (peptidyl-α-hydroxyglycine α-amidating lyase (PAL)) domains. Alternative splicing generates two different regions that connect the protease-resistant catalytic domains. Inclusion of exon 16 introduces a pair of Lys residues, providing a site for controlled endoproteolytic cleavage of PAM and the separation of soluble peptidylglycine α-hydroxylating monooxygenase from membrane-associated PAL. Exon 16 also includes two O-glycosylation sites. PAM-1 lacking both glycosylation sites (PAM-1/OSX; where OSX is O-glycan-depleted mutant of PAM-1) was stably expressed in AtT-20 corticotrope tumor cells. In PAM-1/OSX, a cleavage site for furin-like convertases was exposed, generating a shorter form of membrane-associated PAL. The endocytic trafficking of PAM-1/OSX differed dramatically from that of PAM-1. A soluble fragment of the cytosolic domain of PAM-1 was produced in the endocytic pathway and entered the nucleus; very little soluble fragment of the cytosolic domain was produced from PAM-1/OSX. Internalized PAM-1/OSX was rapidly degraded; unlike PAM-1, very little internalized PAM-1/OSX was detected in multivesicular bodies. Blue native PAGE analysis identified high molecular weight complexes containing PAM-1; the ability of PAM-1/OSX to form similar complexes was markedly diminished. By promoting the formation of high molecular weight complexes, O-glycans may facilitate the recycling of PAM-1 through the endocytic compartment.

Genetic deletion of Rab27B in pancreatic acinar cells affects granules size and has inhibitory effects on amylase secretion.

Small G protein Rab27B is expressed in various secretory cell types and plays a role in mediating secretion. In pancreatic acinar cells, Rab27B was found to be expressed on the zymogen granule membrane and by overexpression to regulate the secretion of zymogen granules. However, the effect of Rab27B deletion on the physiology of pancreatic acinar cells is unknown. In the current study, we utilized the Rab27B KO mouse model to better understand the role of Rab27B in the secretion of pancreatic acinar cells. Our data show that Rab27B deficiency had no obvious effects on the expression of major digestive enzymes and other closely related proteins, e.g. similar small G proteins, such as Rab3D and Rab27A, and putative downstream effectors. The overall morphology of acinar cells was not changed in the knockout pancreas. However, the size of zymogen granules was decreased in KO acinar cells, suggesting a role of Rab27B in regulating the maturation of secretory granules. The secretion of digestive enzymes was moderately decreased in KO acini, compared with the WT control. These data indicate that Rab27B is involved at a different steps of zymogen granule maturation and secretion, which is distinct from that of Rab3D.

Phospholipase Cη2 Activation Redirects Vesicle Trafficking by Regulating F-actin.

PI(4,5)P2 localizes to sites of dense core vesicle exocytosis in neuroendocrine cells and is required for Ca(2+)-triggered vesicle exocytosis, but the impact of local PI(4,5)P2 hydrolysis on exocytosis is poorly understood. Previously, we reported that Ca(2+)-dependent activation of phospholipase Cη2 (PLCη2) catalyzes PI(4,5)P2 hydrolysis, which affected vesicle exocytosis by regulating the activities of the lipid-dependent priming factors CAPS (also known as CADPS) and ubiquitous Munc13-2 in PC12 cells. Here we describe an additional role for PLCη2 in vesicle exocytosis as a Ca(2+)-dependent regulator of the actin cytoskeleton. Depolarization of neuroendocrine PC12 cells with 56 or 95 mm KCl buffers increased peak Ca(2+) levels to ~400 or ~800 nm, respectively, but elicited similar numbers of vesicle exocytic events. However, 56 mm K(+) preferentially elicited the exocytosis of plasma membrane-resident vesicles, whereas 95 mm K(+) preferentially elicited the exocytosis of cytoplasmic vesicles arriving during stimulation. Depolarization with 95 mm K(+) but not with 56 mm K(+) activated PLCη2 to catalyze PI(4,5)P2 hydrolysis. The decrease in PI(4,5)P2 promoted F-actin disassembly, which increased exocytosis of newly arriving vesicles. Consistent with its role as a Ca(2+)-dependent regulator of the cortical actin cytoskeleton, PLCη2 localized with F-actin filaments. The results highlight the importance of PI(4,5)P2 for coordinating cytoskeletal dynamics with vesicle exocytosis and reveal a new role for PLCη2 as a Ca(2+)-dependent regulator of F-actin dynamics and vesicle trafficking.

Aldo-keto reductase 1b7, a novel marker for renin cells, is regulated by cyclic AMP signaling.

We previously identified aldo-keto reductase 1b7 (AKR1B7) as a marker for juxtaglomerular renin cells in the adult mouse kidney. However, the distribution of renin cells varies dynamically, and it was unknown whether AKR1B7 maintains coexpression with renin in response to different developmental, physiological, and pathological situations, and furthermore, whether similar factor(s) simultaneously regulate both proteins. We show here that throughout kidney development, AKR1B7 expression-together with renin-is progressively restricted in the kidney arteries toward the glomerulus. Subsequently, when formerly renin-expressing cells reacquire renin expression, AKR1B7 is reexpressed as well. This pattern of coexpression persists in extreme pathological situations, such as deletion of the genes for aldosterone synthase or Dicer. However, the two proteins do not colocalize within the same organelles: renin is found in the secretory granules, whereas AKR1B7 localizes to the endoplasmic reticulum. Interestingly, upon deletion of the renin gene, AKR1B7 expression is maintained in a pattern mimicking the embryonic expression of renin, while ablation of renin cells resulted in complete abolition of AKR1B7 expression. Finally, we demonstrate that AKR1B7 transcription is controlled by cAMP. Cultured cells of the renin lineage reacquire the ability to express both renin and AKR1B7 upon elevation of intracellular cAMP. In vivo, deleting elements of the cAMP-response pathway (CBP/P300) results in a stark decrease in AKR1B7- and renin-positive cells. In summary, AKR1B7 is expressed within the renin cell throughout development and perturbations to homeostasis, and AKR1B7 is regulated by cAMP levels within the renin cell.