MyosinVIIa Interacts with Twinfilin-2 at the Tips of Mechanosensory Stereocilia in the Inner Ear

In vertebrates hearing is dependent upon the microvilli-like mechanosensory stereocilia and their length gradation. The staircase-like organization of the stereocilia bundle is dynamically maintained by variable actin turnover rates. Two unconventional myosins were previously implicated in stereocilia length regulation but the mechanisms of their action remain unknown. MyosinXVa is expressed in stereocilia tips at levels proportional to stereocilia length and its absence produces staircase-like bundles of very short stereocilia. MyosinVIIa localizes to the tips of the shorter stereocilia within bundles, and when absent, the stereocilia are abnormally long. We show here that myosinVIIa interacts with twinfilin-2, an actin binding protein, which inhibits actin polymerization at the barbed end of the filament, and that twinfilin localization in stereocilia overlaps with myosinVIIa. Exogenous expression of myosinVIIa in fibroblasts results in a reduced number of filopodia and promotes accumulation of twinfilin-2 at the filopodia tips. We hypothesize that the newly described interaction between myosinVIIa and twinfilin-2 is responsible for the establishment and maintenance of slower rates of actin turnover in shorter stereocilia, and that interplay between complexes of myosinVIIa/twinfilin-2 and myosinXVa/whirlin is responsible for stereocilia length gradation within the bundle staircase.     

Cholesterol Crystals Activate the NLRP3 Inflammasome in Human Macrophages: A Novel Link between Cholesterol Metabolism and Inflammation

Background

Chronic inflammation of the arterial wall is a key element in the pathogenesis of atherosclerosis, yet the factors that trigger and sustain the inflammation remain elusive. Inflammasomes are cytoplasmic caspase-1-activating protein complexes that promote maturation and secretion of the proinflammatory cytokines interleukin(IL)-1β and IL-18. The most intensively studied inflammasome, NLRP3 inflammasome, is activated by diverse substances, including crystalline and particulate materials. As cholesterol crystals are abundant in atherosclerotic lesions, and IL-1β has been linked to atherogenesis, we explored the possibility that cholesterol crystals promote inflammation by activating the inflammasome pathway.

Principal Findings

Here we show that human macrophages avidly phagocytose cholesterol crystals and store the ingested cholesterol as cholesteryl esters. Importantly, cholesterol crystals induced dose-dependent secretion of mature IL-1β from human monocytes and macrophages. The cholesterol crystal-induced secretion of IL-1β was caspase-1-dependent, suggesting the involvement of an inflammasome-mediated pathway. Silencing of the NLRP3 receptor, the crucial component in NLRP3 inflammasome, completely abolished crystal-induced IL-1β secretion, thus identifying NLRP3 inflammasome as the cholesterol crystal-responsive element in macrophages. The crystals were shown to induce leakage of the lysosomal protease cathepsin B into the cytoplasm and inhibition of this enzyme reduced cholesterol crystal-induced IL-1β secretion, suggesting that NLRP3 inflammasome activation occurred via lysosomal destabilization.

Conclusions

The cholesterol crystal-induced inflammasome activation in macrophages may represent an important link between cholesterol metabolism and inflammation in atherosclerotic lesions.     

Structure of the actin-depolymerizing factor homology domain in complex with actin

Actin dynamics provide the driving force for many cellular processes including motility and endocytosis. Among the central cytoskeletal regulators are actin-depolymerizing factor (ADF)/cofilin, which depolymerizes actin filaments, and twinfilin, which sequesters actin monomers and caps filament barbed ends. Both interact with actin through an ADF homology (ADF-H) domain, which is also found in several other actin-binding proteins. However, in the absence of an atomic structure for the ADF-H domain in complex with actin, the mechanism by which these proteins interact with actin has remained unknown. Here, we present the crystal structure of twinfilin's C-terminal ADF-H domain in complex with an actin monomer. This domain binds between actin subdomains 1 and 3 through an interface that is conserved among ADF-H domain proteins. Based on this structure, we suggest a mechanism by which ADF/cofilin and twinfilin inhibit nucleotide exchange of actin monomers and present a model for how ADF/cofilin induces filament depolymerization by weakening intrafilament interactions.     

ADF/Cofilin Binds Phosphoinositides in a Multivalent Manner to Act as a PIP2-Density Sensor

Actin-depolymerizing-factor (ADF)/cofilins have emerged as key regulators of cytoskeletal dynamics in cell motility, morphogenesis, endocytosis, and cytokinesis. The activities of ADF/cofilins are regulated by membrane phospholipid PI(4,5)P₂ in vitro and in cells, but the mechanism of the ADF/cofilin-PI(4,5)P₂ interaction has remained controversial. Recent studies suggested that ADF/cofilins interact with PI(4,5)P₂ through a specific binding pocket, and that this interaction is dependent on pH. Here, we combined systematic mutagenesis with biochemical and spectroscopic methods to elucidate the phosphoinositide-binding mechanism of ADF/cofilins. Our analysis revealed that cofilin does not harbor a specific PI(4,5)P₂-binding pocket, but instead interacts with PI(4,5)P₂ through a large, positively charged surface of the molecule. Cofilin interacts simultaneously with multiple PI(4,5)P₂ headgroups in a cooperative manner. Consequently, interactions of cofilin with membranes and actin exhibit sharp sensitivity to PI(4,5)P₂ density. Finally, we show that cofilin binding to PI(4,5)P₂ is not sensitive to changes in the pH at physiological salt concentration, although the PI(4,5)P₂-clustering activity of cofilin is moderately inhibited at elevated pH. Collectively, our data demonstrate that ADF/cofilins bind PI(4,5)P₂ headgroups through a multivalent, cooperative mechanism, and suggest that the actin filament disassembly activity of ADF/cofilin can be accurately regulated by small changes in the PI(4,5)P₂ density at cellular membranes.     

Activated Human Mast Cells Induce LOX-1-Specific Scavenger Receptor Expression in Human Monocyte-Derived Macrophages

Objective

Activated mast cells in atherosclerotic lesions degranulate and release bioactive compounds capable of regulating atherogenesis. Here we examined the ability of activated human primary mast cells to regulate the expression of the major scavenger receptors in cultured human primary monocyte-derived macrophages (HMDMs).

Results

Components released by immunologically activated human primary mast cells induced a transient expression of lectin-like oxidized LDL receptor (LOX-1) mRNA in HMDMs, while the expression of two other scavenger receptors, MSR1 and CD36, remained unaffected. The LOX-1-inducing secretory components were identified as histamine, tumor necrosis factor alpha (TNF-α), and transforming growth factor beta (TGF-β1), which exhibited a synergistic effect on LOX-1 mRNA expression. Histamine induced a transient expression of LOX-1 protein. Mast cell –induced increase in LOX-1 expression was not associated with increased uptake of oxidized LDL by the macrophages.

Conclusions

Mast cell-derived histamine, TNF-α, and TGF-β1 act in concert to induce a transient increase in LOX-1 expression in human primary monocyte-derived macrophages. The LOX-1-inducing activity potentially endows mast cells a hitherto unrecognized role in the regulation of innate immune reactions in atherogenesis.     

Origin of the large burbot (Lota lota (L.)) caught in an Arctic ice-fishing competition: a case study

Recreational ice-fishing and ice-fishing competitions are popular in Finland. In recent years the prizes have become increasingly valuable. One such competition is held yearly at Lake Kilpisjärvi. During the 1998 ice-fishing competition, two participants brought two large burbot ( Lota lota (L.)) to the officials for weighing. These two burbot were much larger than the rest of the fish caught. The origin of these two burbot was questioned and subsequently studied by comparing their age-specific size, stomach contents and parasite fauna with those of the smaller burbot caught during the competition. Both the age-specific lengths and weights of the suspected burbot were higher than those of the smaller burbot, and the stomach analysis revealed that the large burbot had eaten roach ( Rutilus rutilus (L.)), which are not found in Lake Kilpisjärvi. Moreover, the parasite fauna in these two burbot was not typical of the lake. It was therefore concluded that the suspect burbot could not have been caught in Lake Kilpisjärvi, and the contestants were disqualified.