Drosophila ATP6AP2/VhaPRR functions both as a novel planar cell polarity core protein and a regulator of endosomal trafficking

Planar cell polarity (PCP) controls the orientation of cells within tissues and the polarized outgrowth of cellular appendages. So far, six PCP core proteins including the transmembrane proteins Frizzled (Fz), Strabismus (Stbm) and Flamingo (Fmi) have been identified. These proteins form asymmetric PCP domains at apical junctions of epithelial cells. Here, we demonstrate that VhaPRR, an accessory subunit of the proton pump V‐ATPase, directly interacts with the protocadherin Fmi through its extracellular domain. It also shows a striking co‐localization with PCP proteins during all pupal wing stages in Drosophila. This localization depends on intact PCP domains. Reversely, VhaPRR is required for stable PCP domains, identifying it as a novel PCP core protein. VhaPRR performs an additional role in vesicular acidification as well as endolysosomal sorting and degradation. Membrane proteins, such as E‐Cadherin and the Notch receptor, accumulate at the surface and in intracellular vesicles of cells mutant for VhaPRR. This trafficking defect is shared by other V‐ATPase subunits. By contrast, the V‐ATPase does not seem to have a direct role in PCP regulation. Together, our results suggest two roles for VhaPRR, one for PCP and another in endosomal trafficking. This dual function establishes VhaPRR as a key factor in epithelial morphogenesis.     

Tracking wind‐dispersed seeds using 15N‐isotope enrichment

Seed dispersal influences a wide range of ecological processes. However, measuring dispersal patterns, particularly long‐distance dispersal, has been a difficult task. Marking bird‐dispersed seeds with stable ¹⁵N isotopes has been shown to be a user‐friendly method to trace seed dispersal. In this study, we determined whether ¹⁵N urea solution could be used to enrich seeds of two common wind‐dispersed plants, Eupatorium glaucescens (Asteraceae) and Sericocarpus tortifolius (Asteraceae). We further tested if the water type (distilled versus tap) in ¹⁵N urea solutions influences the level and variability of enrichment of plant seeds, and if increasing spraying frequency per se increases enrichment. Because droughts may lower seed set or kill plants, we wanted to investigate if the additional use of an externally applied anti‐transpirant affects the intake of externally applied ¹⁵N into seeds. The results demonstrate that ¹⁵N enrichment of seeds can facilitate dispersal experiments with wind‐dispersed plants. The use of distilled water in ¹⁵N urea solutions did not increase ¹⁵N enrichment compared to tap water. Further, enrichment was more efficient at lower spray frequencies. Both the use of tap water and low frequencies could lower time, effort and project costs. The results suggest that species can be protected from drought using an anti‐transpirant without decreasing the incorporation of ¹⁵N into seeds.     

CRISPR-based peptide library display and programmable microarray self-assembly for rapid quantitative protein binding assays

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A novel regulatory role of TRAPPC9 in L-plastin-mediated osteoclast actin ring formation

Trafficking protein particle complex 9 (TRAPPC9) is a major subunit of the TRAPPII complex. TRAPPC9 has been reported to bind nuclear factor κB kinase subunit β (IKKβ) and NF-kB-inducing kinase (NIK) where it plays a role in the canonical and noncanonical of nuclear factor-κB (NF-kB) signaling pathways, receptively. The role of TRAPPC9 in protein trafficking and cytoskeleton organization in osteoclast (OC) has not been studied yet. In this study, we examined the mRNA expression of TRAPPC9 during OC differentiation. Next, we examined the colocalization of TRAPPC9 with cathepsin-K, known to mediate OC resorption suggesting that TRAPPC9 mediates the trafficking pathway within OC. To identify TRAPPC9 protein partners important for OC-mediated cytoskeleton re-organization, we conducted immunoprecipitation of TRAPPC9 in mature OCs followed by mass spectrometry analysis. Our data showed that TRAPPC9 binds various protein partners. One protein with high recovery rate is L-plastin (LPL). LPL localizes at the podosomes and reported to play a crucial role in actin aggregation thereby actin ring formation and OC function. Although the role of LPL in OC-mediated bone resorption has not fully reported in detail. Here, first, we confirmed the binding of LPL to TRAPPC9 and, then, we investigated the potential regulatory role of TRAPPC9 in LPL-mediated OC cytoskeleton reorganization. We assessed the localization of TRAPPC9 and LPL in OC and found that TRAPPC9 is colocalized with LPL at the periphery of OC. Next, we determined the effect of TRAPPC9 overexpression on LPL recruitment to the actin ring using a viral system. Interestingly, our data showed that TRAPPC9 overexpression promotes the recruitment of LPL to the actin ring when compared with control cultures. In addition, we observed that TRAPPC9 overexpression reorganizes actin clusters/aggregates and regulates vinculin recruitment into the OC periphery to initiate podosome formation.     

Linking micromorphism,brooding, and hermaphroditism in brachiopods: Insights from Caribbean Argyrotheca (Brachiopoda)

In extant brachiopods, parental brooding of the larvae occurs exclusively within Rhynchonelliformea. Methods of larval protection range from simple retention of the larvae within the mantle cavity, to sophisticated brood care within highly specialized brood pouches found in Argyrotheca and Joania (Terebratulida, Megathyridoidea), Gwynia (Terebratulida, Gwynioidea), and all Thecideoidea (Thecideida). Previous studies on the reproductive biology of Argyrotheca yielded contrasting results on the epithelial origin of the brood pouches in this genus. Here, representatives of different species of Argyrotheca from the Belize Barrier Reef were examined using histological section series. Brood pouches of four species, A. cf. schrammi and Argyrotheca sp. 1–3, are of the same basic structure, formed by invaginations of the anterior body wall and connected to the visceral cavity via the metanephridia. The same four species are simultaneously hermaphroditic, suggesting that fertilization is achieved, at least partly, through selfing. One species, Argyrotheca rubrocostata, differs significantly from all others as it has no brood pouch and gonochoric gonads. Thus, the presence of brood pouches and simultaneous hermaphroditism are concluded to be correlated within Megathyridoidea and proposed to be homologous traits of Joania and several but not all species of Argyrotheca, questioning the monophyletic status of both genera. In contrast to the brood pouches of Thecideoidea, lophophoral epithelium is not involved in the formation of the pouches of Argyrotheca and Joania. Therefore, megathyridoid and thecideoid brood pouches are not homologous but evolved independently within rhynchonelliform brachiopods. All brachiopods with brood pouches share a micromorphic form and a short life span, limiting the space and time available for gamete and larval development. We suggest that the brood pouches and the hermaphroditic gonads of Argyrotheca spp. and Joania compensate these limitations by minimizing the loss of gametes and larvae, and by maximizing the chances of successful fertilization. J. Morphol., 2013. © 2013 Wiley Periodicals, Inc.     

Homodimerization as a molecular switch between low and high efficiency PrPC cell surface delivery and neuroprotective activity

PrP^C is associated with a variety of functions, and its ability to interact with a multitude of partners, including itself, may largely explain PrP multifunctionality and the lack of consensus on the genuine physiological function of the protein in vivo. In contrast, there is a consensus in the literature that alterations in PrP^C trafficking and intracellular retention result in neuronal degeneration. In addition, a proteolytic modification in the late secretory pathway termed the α-cleavage induces the secretion of PrPN1, a PrP^C-derived metabolite with fascinating neuroprotective activity against toxic oligomeric Aβ molecules implicated in Alzheimer disease. Thus, studies focusing on understanding the regulation of PrP^C trafficking to the cell surface and the modulation of α-cleavage are essential. The objective of this commentary is to highlight recent evidences that PrP^C homodimerization stimulates trafficking of the protein to the cell surface and results in high levels of PrPN1 secretion. We also discuss a hypothetical model for these results and comment on future challenges and opportunities.     

A simple,band‐mounted device to measure behaviorally modified thermal microclimates experienced by birds

Birds encounter climate at the scale of microclimates that can vary rapidly in time and space and so understanding potential vulnerability and adaptations to those microclimates requires fine‐scale measurements that accurately track thermal exposures. However, few options exist for recording the microclimates actually experienced by birds (realized microclimates). We constructed and tested a simple, low‐cost, temperature logger for recording the realized microclimates of ground‐nesting birds. We developed attachment protocols for band‐mounting Thermochron iButtons on Ring‐billed Gull (Larus delawarensis) chicks. We tested these mounted, temperature‐logging devices on 20 chicks weighing > 200 g (device weight was 4 g), attaching devices for 48 h and observing behavior before and after attachment and removal. Devices recorded temperature immediately surrounding the leg at 2‐min intervals. Recorded temperatures were strong predictors of observed thermoregulatory behaviors (panting and sitting), outperforming predictions based on air temperatures measured by basic, static approaches. Through comparison with matched controls (chicks with just a band), we detected no adverse physiological effects of devices, no effects on social or feeding behavior, and only a short‐term decrease in inactivity immediately after device attachment (likely due to increased preening). By attaching iButtons to the legs of birds, we quantified realized thermal exposure, integrating air temperature, modes of environmental heat transfer, and bird behavior at microclimatic scales. Although not yet validated for broader use, our approach (including possible miniaturization) should be suitable to measure thermal exposure of adults, not just chicks, allowing collection of data concerning thermal exposures during flight under field conditions. At ~ $25 USD per device, our approach facilitates experimental protocols with robust sample sizes, even for relatively modest budgets.     

Functional assignment to JEV proteins using SVM

Identification of different protein functions facilitates a mechanistic understanding of Japanese encephalitis virus (JEV) infection and opens novel means for drug development. Support vector machines (SVM), useful for predicting the functional class of distantly related proteins, is employed to ascribe a possible functional class to Japanese encephalitis virus protein. Our study from SVMProt and available JE virus sequences suggests that structural and nonstructural proteins of JEV genome possibly belong to diverse protein functions, are expected to occur in the life cycle of JE virus. Protein functions common to both structural and non-structural proteins are iron-binding, metal-binding, lipid-binding, copper-binding, transmembrane, outer membrane, channels/Pores - Pore-forming toxins (proteins and peptides) group of proteins. Non-structural proteins perform functions like actin binding, zinc-binding, calcium-binding, hydrolases, Carbon-Oxygen Lyases, P-type ATPase, proteins belonging to major facilitator family (MFS), secreting main terminal branch (MTB) family, phosphotransfer-driven group translocators and ATP-binding cassette (ABC) family group of proteins. Whereas structural proteins besides belonging to same structural group of proteins (capsid, structural, envelope), they also perform functions like nuclear receptor, antibiotic resistance, RNA-binding, DNA-binding, magnesium-binding, isomerase (intra-molecular), oxidoreductase and participate in type II (general) secretory pathway (IISP).     

The J-related segment of tim44 is essential for cell viability: a mutant Tim44 remains in the mitochondrial import site, but inefficiently recruits mtHsp70 and impairs protein translocation.

Tim44 is a protein of the mitochondrial inner membrane and serves as an adaptor protein for mtHsp70 that drives the import of preproteins in an ATP-dependent manner. In this study we have modified the interaction of Tim44 with mtHsp70 and characterized the consequences for protein translocation. By deletion of an 18-residue segment of Tim44 with limited similarity to J-proteins, the binding of Tim44 to mtHsp70 was weakened. We found that in the yeast Saccharomyces cerevisiae the deletion of this segment is lethal. To investigate the role of the 18-residue segment, we expressed Tim44Delta18 in addition to the endogenous wild-type Tim44. Tim44Delta18 is correctly targeted to mitochondria and assembles in the inner membrane import site. The coexpression of Tim44Delta18 together with wild-type Tim44, however, does not stimulate protein import, but reduces its efficiency. In particular, the promotion of unfolding of preproteins during translocation is inhibited. mtHsp70 is still able to bind to Tim44Delta18 in an ATP-regulated manner, but the efficiency of interaction is reduced. These results suggest that the J-related segment of Tim44 is needed for productive interaction with mtHsp70. The efficient cooperation of mtHsp70 with Tim44 facilitates the translocation of loosely folded preproteins and plays a crucial role in the import of preproteins which contain a tightly folded domain.     

Primers for the amplification of sequence‐characterized loci in Cryphonectria cubensis populations

We describe the development of DNA markers for the fungal pathogen of Eucalyptus, Cryphonectria cubensis. These markers originated from cloned intershort sequence repeat polymerase chain reactions, which enrich for medium to highly repetitive DNA sequences. In total, 10 markers were isolated, eight of which were polymorphic, and these can subsequently be applied to study populations of C. cubensis.