Elevated summer temperatures delay spawning and reduce redd construction for resident brook trout (Salvelinus fontinalis)

Redd (nest) surveys for resident brook trout (Salvelinus fontinalis) were conducted annually in a mountain lake in northern New York for 11 years with multiple surveys conducted during the spawning season in eight of those years. Repeated surveys throughout the spawning season allowed us to fit an individually based parametric model and estimate the day of year on which spawning was initiated, reached its midpoint, and ended during each year. Spawning phenology was then assessed relative to (1) mean of maximum daily air temperature and (2) mean of maximum daily water temperature at the lake bottom during summer in each year using a linear model. Elevated temperatures in summer were correlated with a delay in spawning and a reduction in the total number of redds constructed. Increasing the summer mean of maximum daily air temperatures by 1 °C delayed spawning by approximately 1 week and decreased the total number of redds constructed by nearly 65. Lake spawning brook trout select redd sites based on the presence of discharging groundwater that is relatively constant in temperature within and across years, leading to relatively consistent egg incubation times. Therefore, delayed spawning is likely to delay fry emergence, which could influence emergence synchrony with prey items. This work highlights non‐lethal and sub‐lethal effects of elevated summer temperatures on native resident salmonids in aquatic environments with limited thermal refugia.     

RAGE signaling by alveolar macrophages influences tobacco smoke-induced inflammation

Receptors for advanced glycation end-products (RAGE) are multiligand cell surface receptors of the immunoglobin family expressed by epithelium and macrophages, and expression increases following exposure to cigarette smoke extract (CSE). The present study sought to characterize the proinflammatory contributions of RAGE expressed by alveolar macrophages (AMs) following CSE exposure. Acute exposure of mice to CSE via nasal instillation revealed diminished bronchoalveolar lavage (BAL) cellularity and fewer AMs in RAGE knockout (KO) mice compared with controls. Primary AMs were obtained from BAL, exposed to CSE in vitro, and analyzed. CSE significantly increased RAGE expression by wild-type AMs. Employing ELISAs, wild-type AMs exposed to CSE had increased levels of active Ras, a small GTPase that perpetuates proinflammatory signaling. Conversely, RAGE KO AMs had less Ras activation compared with wild-type AMs after exposure to CSE. In RAGE KO AMs, assessment of p38 MAPK and NF-κB, important intracellular signaling intermediates induced during an inflammatory response, revealed that CSE-induced inflammation may occur in part via RAGE signaling. Lastly, quantitative RT-PCR revealed that the expression of proinflammatory cytokines including TNF-α and IL-1β were detectably decreased in RAGE KO AMs exposed to CSE compared with CSE-exposed wild-type AMs. These results reveal that primary AMs orchestrate CSE-induced inflammation, at least in part, via RAGE-mediated mechanisms.     

Cytoskeletal defects in Bmpr2-associated pulmonary arterial hypertension

The heritable form of pulmonary arterial hypertension (PAH) is typically caused by a mutation in bone morphogenic protein receptor type 2 (BMPR2), and mice expressing Bmpr2 mutations develop PAH with features similar to human disease. BMPR2 is known to interact with the cytoskeleton, and human array studies in PAH patients confirm alterations in cytoskeletal pathways. The goal of this study was to evaluate cytoskeletal defects in BMPR2-associated PAH. Expression arrays on our Bmpr2 mutant mouse lungs revealed cytoskeletal defects as a prominent molecular consequence of universal expression of a Bmpr2 mutation (Rosa26-Bmpr2(R899X)). Pulmonary microvascular endothelial cells cultured from these mice have histological and functional cytoskeletal defects. Stable transfection of different BMPR2 mutations into pulmonary microvascular endothelial cells revealed that cytoskeletal defects are common to multiple BMPR2 mutations and are associated with activation of the Rho GTPase, Rac1. Rac1 defects are corrected in cell culture and in vivo through administration of exogenous recombinant human angiotensin-converting enzyme 2 (rhACE2). rhACE2 reverses 77% of gene expression changes in Rosa26-Bmpr2(R899X) transgenic mice, in particular, correcting defects in cytoskeletal function. Administration of rhACE2 to Rosa26-Bmpr2(R899X) mice with established PAH normalizes pulmonary pressures. Together, these findings suggest that cytoskeletal function is central to the development of BMPR2-associated PAH and that intervention against cytoskeletal defects may reverse established disease.     

Molecular motor function in axonal transport in vivo probed by genetic and computational analysis in Drosophila

Bidirectional axonal transport driven by kinesin and dynein along microtubules is critical to neuronal viability and function. To evaluate axonal transport mechanisms, we developed a high-resolution imaging system to track the movement of amyloid precursor protein (APP) vesicles in Drosophila segmental nerve axons. Computational analyses of a large number of moving vesicles in defined genetic backgrounds with partial reduction or overexpression of motor proteins enabled us to test with high precision existing and new models of motor activity and coordination in vivo. We discovered several previously unknown features of vesicle movement, including a surprising dependence of anterograde APP vesicle movement velocity on the amount of kinesin-1. This finding is largely incompatible with the biophysical properties of kinesin-1 derived from in vitro analyses. Our data also suggest kinesin-1 and cytoplasmic dynein motors assemble in stable mixtures on APP vesicles and their direction and velocity are controlled at least in part by dynein intermediate chain.     

Multisite phosphorylation of oxysterol-binding protein regulates sterol binding and activation of sphingomyelin synthesis

The endoplasmic reticulum (ER)-Golgi sterol transfer activity of oxysterol-binding protein (OSBP) regulates sphingomyelin (SM) synthesis, as well as post-Golgi cholesterol efflux pathways. The phosphorylation and ER-Golgi localization of OSBP are correlated, suggesting this modification regulates the directionality and/or specificity of transfer activity. In this paper, we report that phosphorylation on two serine-rich motifs, S381-S391 (site 1) and S192, S195, S200 (site 2), specifically controls OSBP activity at the ER. A phosphomimetic of the SM/cholesterol-sensitive phosphorylation site 1 (OSBP-S5E) had increased in vitro cholesterol and 25-hydroxycholesterol-binding capacity, and cholesterol extraction from liposomes, but reduced transfer activity. Phosphatidylinositol 4-phosphate (PI(4)P) and cholesterol competed for a common binding site on OSBP; however, direct binding of PI(4)P was not affected by site 1 phosphorylation. Individual site 1 and site 2 phosphomutants supported oxysterol activation of SM synthesis in OSBP-deficient CHO cells. However, a double site1/2 mutant (OSBP-S381A/S3D) was deficient in this activity and was constitutively colocalized with vesicle-associated membrane protein-associated protein A (VAP-A) in a collapsed ER network. This study identifies phosphorylation regulation of sterol and VAP-A binding by OSBP in the ER, and PI(4)P as an alternate ligand that could be exchanged for sterol in the Golgi apparatus.     

Sbf/MTMR13 coordinates PI(3)P and Rab21 regulation in endocytic control of cellular remodeling

Cells rely on the coordinated regulation of lipid phosphoinositides and Rab GTPases to define membrane compartment fates along distinct trafficking routes. The family of disease-related myotubularin (MTM) phosphoinositide phosphatases includes catalytically inactive members, or pseudophosphatases, with poorly understood functions. We found that Drosophila MTM pseudophosphatase Sbf coordinates both phosphatidylinositol 3-phosphate (PI(3)P) turnover and Rab21 GTPase activation in an endosomal pathway that controls macrophage remodeling. Sbf dynamically interacts with class II phosphatidylinositol 3-kinase and stably recruits Mtm to promote turnover of a PI(3)P subpool essential for endosomal trafficking. Sbf also functions as a guanine nucleotide exchange factor that promotes Rab21 GTPase activation associated with PI(3)P endosomes. Of importance, Sbf, Mtm, and Rab21 function together, along with Rab11-mediated endosomal trafficking, to control macrophage protrusion formation. This identifies Sbf as a critical coordinator of PI(3)P and Rab21 regulation, which specifies an endosomal pathway and cortical control.     

A mechanosensory system governs myosin II accumulation in dividing cells

The mitotic spindle is generally considered the initiator of furrow ingression. However, recent studies suggest that furrows can form without spindles, particularly during asymmetric cell division. In Dictyostelium, the mechanoenzyme myosin II and the actin cross-linker cortexillin I form a mechanosensor that responds to mechanical stress, which could account for spindle-independent contractile protein recruitment. Here we show that the regulatory and contractility network composed of myosin II, cortexillin I, IQGAP2, kinesin-6 (kif12), and inner centromeric protein (INCENP) is a mechanical stress-responsive system. Myosin II and cortexillin I form the core mechanosensor, and mechanotransduction is mediated by IQGAP2 to kif12 and INCENP. In addition, IQGAP2 is antagonized by IQGAP1 to modulate the mechanoresponsiveness of the system, suggesting a possible mechanism for discriminating between mechanical and biochemical inputs. Furthermore, IQGAP2 is important for maintaining spindle morphology and kif12 and myosin II cleavage furrow recruitment. Cortexillin II is not directly involved in myosin II mechanosensitive accumulation, but without cortexillin I, cortexillin II's role in membrane-cortex attachment is revealed. Finally, the mitotic spindle is dispensable for the system. Overall, this mechanosensory system is structured like a control system characterized by mechanochemical feedback loops that regulate myosin II localization at sites of mechanical stress and the cleavage furrow.     

Structure, biosynthesis and possible function of tunichromes and related compounds

Several species of ascidians (phylum Chordata, subphylum Urochordata) contain a group of oligopeptides called "tunichromes" in their blood cells. These peptides have been implicated in (a) metal chelation and accumulation/sequestration of vanadium or iron; (b) crosslinking of structural fibers in tunic formation, (c) wound healing and (d) defense reactions. However, their biosynthesis, metabolism, and biological function remain largely un-elucidated due to their extreme instability and high reactivity. Tunichromes and related compounds uniquely possess dehydrodopamine moieties, all originating from post-translational modification of peptidyl tyrosine. It is conceivable that the presence of such novel post-translationally modified groups provide attributes that are crucial for their biological roles. Therefore, we examined the chemistry and reactivity of tunichromes in light of the available knowledge of the biochemistry of simple monomeric dehydro-N-acyldopamine units. Based on the reactivity of such simple compounds, the potential biological activities of tunichromes are predicted. Their possible biosynthetic route from peptidyl tyrosine is critically evaluated to provide a better basis for unraveling their biological functions. Prevalence of dehydro-N-acyldopamine units in different tunichromes, some marine antibiotic compounds, insect cuticular sclerotizing precursors and some bioadhesive marine proteins may aid in the de novo design of unique biomaterials with potential antibiotic/adhesive properties.