Actin coating and compression of fused secretory vesicles are essential for surfactant secretion - a role for Rho, formins and myosin II

Secretion of vesicular contents by exocytosis is a fundamental cellular process. Increasing evidence suggests that post-fusion events play an important role in determining the composition and quantity of the secretory output. In particular, regulation of fusion pore dilation and closure is considered a key regulator of the post-fusion phase. However, depending on the nature of the cargo, additional mechanisms might be essential to facilitate effective release. We have recently described that in alveolar type II (ATII) cells, lamellar bodies (LBs), which are secretory vesicles that store lung surfactant, are coated with actin following fusion with the plasma membrane. Surfactant, a lipoprotein complex, does not readily diffuse out of fused LBs following opening and dilation of the fusion pore. Using fluorescence microscopy, atomic force microscopy and biochemical assays, we present evidence that actin coating and subsequent contraction of the actin coat is essential to facilitate surfactant secretion. Latrunculin B prevents actin coating of fused LBs and inhibits surfactant secretion almost completely. Simultaneous imaging of the vesicle membrane and the actin coat revealed that contraction of the actin coat compresses the vesicle following fusion. This leads to active extrusion of vesicle contents. Initial actin coating of fused vesicles is dependent on activation of Rho and formin-dependent actin nucleation. Actin coat contraction is facilitated by myosin II. In summary, our data suggest that fusion pore opening and dilation itself is not sufficient for release of bulky vesicle cargos and that active extrusion mechanisms are required

Acute changes in peritoneal morphology and transport properties with infectious peritonitis and mechanical injury

Acute changes in peritoneal morphology and transport properties with infectious peritonitis and mechanical injury. Peritoneal clearance studies were performed in rats undergoing acute peritoneal dialysis. Some of these animals were then exposed to laparotomy and mechanical drying of the peritoneum. Peritoneal clearance studies were repeated at intervals up to 11 days. Another group of rats was placed on daily peritoneal dialysis and allowed to spontaneously develop peritonitis which was not treated. These rats underwent peritoneal transport studies at differing durations of infection. In all groups, animals were sacrificed at the time of the last transport studies for morphological assessment of the peritoneum by light microscopy, scanning electron microscopy, and transmission electron microscopy. The results showed similar decreases in drainage volume and increases in glucose absorption and protein losses with both infection and drying. Both types of injury resulted in extensive mesothelial structural changes. While drying caused mainly denudation of the mesothelial surface, infectious peritonitis was associated with separation of mesothelial cells, and the appearance of numerous white blood cells between and on mesothelial cells. Exposure to peritoneal dialysis alone had no obvious effects on anatomy. Although changes in the peritoneal microcirculation and deeper structures cannot be excluded as contributing to peritoneal transport alterations, the findings suggest that alterations of mesothelium might explain some of the changes in peritoneal transport properties under the conditions of these studies.Modifications aiguës de la morphologie et des propriétés de transport du péritoine par péritonite infectieuse et lésion mécanique. Des études de clearance péritonéale ont été entreprises chez des rats en dialyse péritonéale aiguë. Certains de ces animaux étaient soumis à une laparotomie et à un séchage mécanique du péritoine. Les études de clearance péritonéale étaient répétées à des intervalles allant jusqu'à 11 jours. Un autre groupe de rat était placé en dialyse péritonéale journalière, et il pouvait développer spontanément une péritonite qui n'était pas traitée. Chez ces rats ont été effectuées des études de transport péritonéal à différents stades d'infection. Dans tous les groupes, les animaux étaient sacrifiés lors de la dernière étude de transport afin d'étudier morphologiquement le péritoine par microscopie optique, microscopie électronique à balayage, et microscopie électronique par transmission. Les résultats ont montré des diminutions du volume de drainage et des augmentations de l'absorption du glucose et des pertes protéiques identiques avec l'infection ou le séchage. Les deux types de lésions ont entrainé des modifications structurelles mésothéliales importantes. Tandis que le séchage entrainait principalement une dénudation de la surface mésothéliale, la péritonite infectieuse était associée à une séparation des cellules mésothéliales, et à l'apparition de nombreux globules blancs entre et sur les cellules mésothéliales. L'exposition à la dialyse péritonéale seule n'avait pas d'effets anatomiques évidents. Bien que la contribution aux altérations du transport péritonéal de modifications de la micro-circulation péritonéale et des structures plus profondes ne puisse être exclue, ces résultats suggèrent que les altérations du mésothélium pourraient expliquer certaines des modifications des propriétés de transport péritonéal dans les conditions de ces études

Structural Microangiopathies in Skeletal Muscle Related to Systemic Vascular Pathologies in Humans

It is unclear how microangiopathic changes in skeletal muscle vary among systemic vascular pathologies. We therefore analyzed the capillary fine structure in skeletal muscle from patients with arterial hypertension (HYPT), diabetes mellitus type 2 (T2DM) or intermittent claudication – peripheral arterial disease (IC/PAD). Tablet-based image analysis (TBIA) was carried out to largely re-evaluate 5,000 transmission electron micrographs of capillaries from 126 vastus lateralis biopsies of 75 individuals (HYPT, T2DM or IC/PAD patients as well as healthy individuals before and after endurance exercise training) used in previous morphometric studies, but assessed using stereological counting grids of different sizes. Serial block-face scanning electron microscopy (SBFSEM) of mouse skeletal muscle was used for validation of the particular fine structural events observed in human biopsies. The peri-capillary basement membrane (BM) was 38.5 and 45.5% thicker (P < 0.05) in T2DM and IC/PAD patients than in the other groups. A 17.7–39.6% lower (P < 0.05) index for intraluminal endothelial cell (EC) surface enlargement by projections was exclusively found in T2DM patients by TBIA morphometry. The proportion of capillaries with disrupted BM between pericytes (PC) and EC was higher (P < 0.05) in HYPT (33.2%) and T2DM (38.7%) patients than in the control group. Empty EC-sockets were more frequent (P < 0.05) in the three patient groups (20.6% in HYPT, 27.1% in T2DM, 30.0% in IC/PAD) than in the healthy individuals. SBFSEM confirmed that EC-sockets may exhibit close proximity to the capillary lumen. Our comparative morphometric analysis demonstrated that structural arrangement of skeletal muscle capillaries is more affected in T2DM than in HYPT or IC/PAD, although some similar elements of remodeling were found. The increased frequency of empty EC-sockets in the three patient groups indicates that the PC-EC interaction is commonly disturbed in these systemic vascular pathologies

The Breakdown of Preformed Peritoneal Advanced Glycation End Products by Intraperitoneal Alagebrium

It has been demonstrated that inhibitors of advanced glycation end products (AGE), such as aminoguanidine, can suppress peritoneal AGE in rats on peritoneal dialysis (PD). However, it is unknown whether late administration of a putative cross-link breaker, alagebrium, could reverse peritoneal AGE. We therefore compared alagebrium with aminoguanidine in their ability to reverse peritoneal AGE in rats on PD. Male Sprague-Dawley rats were randomly divided into 3 groups: group I dialyzed with 4.25% glucose solution for all exchanges; group II dialyzed with 4.25% glucose solution containing aminoguanidine, and group III dialyzed with 4.25% glucose solution containing alagebrium for last 8 weeks of 12-week dialysis period. Dialysis exchanges were performed 2 times a day for 12 weeks. Immunohistochemistry was performed using a monoclonal anti-AGE antibody. One-hour PET was performed for comparison of transport characteristics. The immunolabelling of AGE in peritoneal membrane was markedly decreased in the alagebrium group. Consistent with this, the alagebrium group exhibited significantly higher D/Do glucose and lower D/P urea, suggesting low peritoneal membrane transport. But there were no significant differences between the control and the aminoguanidine group. These results suggest that the alagebrium may be the optimal therapeutic approach, compared with treatment with inhibitors of AGE formation, in rats on PD

Actin depolymerisation and crosslinking join forces with myosin II to contract actin coats on fused secretory vesicles

In many secretory cells actin and myosin are specifically recruited to the surface of secretory granules following their fusion with the plasma membrane. Actomyosin-dependent compression of fused granules is essential to promote active extrusion of cargo. However, little is known about molecular mechanisms regulating actin coat formation and contraction. Here, we provide a detailed kinetic analysis of the molecules regulating actin coat contraction on fused lamellar bodies in primary alveolar type II cells. We demonstrate that ROCK1 and myosin light chain kinase 1 (MLCK1, also known as MYLK) translocate to fused lamellar bodies and activate myosin II on actin coats. However, myosin II activity is not sufficient for efficient actin coat contraction. In addition, cofilin-1 and α-actinin translocate to actin coats. ROCK1-dependent regulated actin depolymerisation by cofilin-1 in cooperation with actin crosslinking by α-actinin is essential for complete coat contraction. In summary, our data suggest a complementary role for regulated actin depolymerisation and crosslinking, and myosin II activity, to contract actin coats and drive secretion

Identification of Periostin as a Critical Marker of Progression/Reversal of Hypertensive Nephropathy

Progression of chronic kidney disease (CKD) is a major health issue due to persistent accumulation of extracellular matrix in the injured kidney. However, our current understanding of fibrosis is limited, therapeutic options are lacking, and progressive degradation of renal function prevails in CKD patients. Uncovering novel therapeutic targets is therefore necessary

A Disintegrin and Metalloenzyme (ADAM) 17 Activation Is Regulated by α5β1 Integrin in Kidney Mesangial Cells

The disintegrin and metalloenzyme ADAM17 participates in numerous inflammatory and proliferative diseases, and its pathophysiological role was implicated in kidney fibrosis, polycystic kidney disease and other chronic kidney diseases. At present, we have little understanding how the enzyme activity is regulated. In this study we wanted to characterize the role of α5β1 integrin in ADAM17 activity regulation during G protein-coupled receptor (GPCR) stimulation.We showed previously that the profibrotic GPCR agonist serotonin (5-HT) induced kidney mesangial cell proliferation through ADAM17 activation and heparin-binding epidermal growth factor (HB-EGF) shedding. In the present studies we observed that in unstimulated mesangial cell lysates α5β1 integrin co-precipitated with ADAM17 and that 5-HT treatment of the cells induced dissociation of α5β1 integrin from ADAM17. Using fluorescence immunostaining and in situ proximity ligation assay, we identified the perinuclear region as the localization of the ADAM17/α5β1 integrin interaction. In cell-free assays, we showed that purified α5β1 integrin and β1 integrin dose-dependently bound to and inhibited activity of recombinant ADAM17. We provided evidence that the conformation of the integrin determines its ADAM17-binding ability. To study the effect of β1 integrin on ADAM17 sheddase activity, we employed alkaline phosphatase-tagged HB-EGF. Overexpression of β1 integrin lead to complete inhibition of 5-HT-induced HB-EGF shedding and silencing β1 integrin by siRNA significantly increased mesangial cells ADAM17 responsiveness to 5-HT.Our data show for the first time that β1 integrin has an important physiological role in ADAM17 activity regulation. We suggest that regulating α5β1 integrin binding to ADAM17 could be an attractive therapeutic target in chronic kidney diseases