Male Corncrakes Crex crex extend their home ranges by visiting the territories of neighbouring males

Capsule Radiotracked male Corncrake often intruded on the territories of neighbouring males.

Aims To test that intruders' visits are goal-directed, not just a by-product of extended spatial activity during daylight hours.

Methods Using radiotelemetry, we sampled a total of 20 three-day home ranges from 11 tagged males. We recorded daily vocal activity and used a permutation test to see if the movements of tracked males were independent of the position of neighbouring males.

Results The majority of males who had a neighbouring male, up to approximately 600 m from their night calling site, undertook goal-directed visits to the neighbour's territory. Males undertook these visits every day, or every other day, when the neighbours were close. Males undertook visits approximately once every three days when they were more distant. The time spent in the neighbour's territory was longest where the distance between night calling sites was about 200 m. Males tended to be silent in neighbour's territory, apparently to prevent confrontation. Otherwise the distance of neighbouring males did not significantly affect daytime vocal activity. Visiting males tended to sing more often in their home territories.

Conclusions Daily movement of the majority of males was towards the neighbouring male's calling site. We suggest that the purpose of these visits was to seek females. These males may try to drive a female into their territory or gain extra-pair copulation.     

Bloch Equations-Based Reconstruction of Myocardium T1 Maps from Modified Look-Locker Inversion Recovery Sequence

Modified Look-Locker Inversion recovery (MOLLI) sequence is increasingly performed for myocardial T1 mapping but is known to underestimate T1 values. The aim of the study was to quantitatively analyze several sources of errors when T1 maps are derived using standard post-processing of the sequence and to propose a reconstruction approach that takes into account inversion efficacy (η), T2 relaxation during balanced steady-state free-precession readouts and B1+ inhomogeneities. Contributions of the different sources of error were analyzed using Bloch equations simulations of MOLLI sequence. Bloch simulations were then combined with the acquisition of fast B1+ and T2 maps to derive more accurate T1 maps. This novel approach was evaluated on phantoms and on five healthy volunteers. Simulations show that T2 variations, B1+ heterogeneities and inversion efficiency represent major confounders for T1 mapping when MOLLI is processed with standard 3-parameters fitting. In vitro data indicate that T1 values are accurately derived with the simulation approach and in vivo data suggest that myocardium T1 are 15% underestimated when processed with the standard 3-parameters fitting. At the cost of additional acquisitions, this method might be suitable in clinical research protocols for precise tissue characterization as it decorrelates T1 and T2 effects on parametric maps provided by MOLLI sequence and avoids inaccuracies when B1+ is not homogenous throughout the myocardium.     

Quantitative Site-specific Phosphorylation Dynamics of Human Protein Kinases during Mitotic Progression

Reversible protein phosphorylation is a key regulatory mechanism of mitotic progression. Importantly, protein kinases themselves are also regulated by phosphorylation-dephosphorylation processes; hence, phosphorylation dynamics of kinases hold a wealth of information about phosphorylation networks. Here, we investigated the site-specific phosphorylation dynamics of human kinases during mitosis using synchronization of HeLa suspension cells, kinase enrichment, and high resolution mass spectrometry. In biological triplicate analyses, we identified 206 protein kinases and more than 900 protein kinase phosphorylation sites, including 61 phosphorylation sites on activation segments, and quantified their relative abundances across three specific mitotic stages. Around 25% of the kinase phosphorylation site ratios were found to be changed by at least 50% during mitotic progression. Further network analysis of jointly regulated kinase groups suggested that Cyclin-dependent kinase- and mitogen-activated kinase-centered interaction networks are coordinately down- and up-regulated in late mitosis, respectively. Importantly, our data cover most of the already known mitotic kinases and, moreover, identify attractive candidates for future studies of phosphorylation-based mitotic signaling. Thus, the results of this study provide a valuable resource for cell biologists and provide insight into the system properties of the mitotic phosphokinome.Reversible phosphorylation is a ubiquitous posttranslational protein modification that is involved in the regulation of almost all biological processes (1–3). In human, 518 protein kinases have been identified in the genome that phosphorylate the majority of cellular proteins and increase the diversity of the proteome by severalfold (4). Addition of a phosphate group to a protein can alter its structural, catalytic, and functional properties; hence, kinases require tight regulation to avoid unspecific phosphorylation, which can be deleterious to cells (5–7). As a result, cells use a variety of mechanisms to ensure proper regulation of kinase activities (8). Importantly, most kinases are also in turn regulated through autophosphorylation and phosphorylation by other kinases, thus generating complex phosphorylation networks. In particular, phosphorylation on activation segments is a common mechanism to modulate kinase activities (9–11), but additional phosphorylation sites are also frequently required for fine tuning of kinase localizations and functions (12). Some kinases contain phosphopeptide binding domains that recognize prephosphorylated sites on other kinases, resulting in processive phosphorylation and/or targeting of kinases to distinct cellular locations (13–16). Because such priming phosphorylation events depend on the activities of the priming kinases, these motifs act as conditional docking sites and restrict the interaction with docking kinases to a particular point in time and physiological state. In addition, phosphorylation sites may act through combinatorial mechanisms or through cross-talk with other posttranslational modifications (PTMs)¹ (17, 18), thus further increasing the complexity of kinase regulatory networks.Regulation of kinases is of particular interest in mitosis as most of the mitotic events are regulated by reversible protein phosphorylation (19). During mitosis, error-free segregation of sister chromatids into the two daughter cells is essential to ensure genomic stability. Physically, this process is carried out by the mitotic spindle, a highly dynamic microtubule-based structure. After entry into mitosis, the major microtubule-organizing centers in animal cells, the centrosomes, start to increase microtubule nucleation and move to opposite poles of the cell. Throughout prometaphase, microtubules emanating from centrosomes are captured by kinetochores, protein complexes assembled on centromeric chromosomal DNA. This eventually leads to the alignment of all chromosomes in a metaphase plate. Because proper bipolar attachment of chromosomes to spindle microtubules is essential for the correct segregation of chromosomes, this critical step is monitored by a signaling pathway known as the spindle assembly checkpoint (SAC) (20). This checkpoint is silenced only after all chromosomes have attached to the spindle in a bioriented fashion, resulting in the synchronous segregation of sister chromatids during anaphase. Simultaneously, a so-called central spindle is formed between the separating chromatids, and the formation of a contractile ring initiates cytokinesis. Finally, in telophase, the chromosomes decondense and reassemble into nuclei, whereas remnants of the central spindle form the midbody, marking the site of abscission. Cyclin-dependent kinase 1 (Cdk1), an evolutionarily conserved master mitotic kinase, is activated prior to mitosis and initiates most of the mitotic events. Cdk1 works in close association with other essential mitotic kinases such as Plk1, Aurora A, and Aurora B for the regulation of mitotic progression (19, 21–24). Plk1 and Aurora kinases dynamically localize to different subcellular locations to perform multiple functions during mitosis and are phosphorylated at several conserved sites. Although little is known about the precise roles of these phosphorylation sites, emerging data indicate that they are involved in regulating localization-specific functions (25, 26). Furthermore, the kinases Bub1, BubR1, and TTK (Mps1) and kinases of the Nek family play important roles in maintaining the fidelity and robustness of mitosis (19). Recently, a genome-wide RNA-mediated interference screen identified M phase phenotypes for many kinases that have not previously been implicated in cell cycle functions, indicating that additional kinases have important mitotic functions (27).Although protein phosphorylation plays a pivotal role in the regulation of cellular networks, many phosphorylation events remain undiscovered mainly because of technical limitations (28). The advent of mass spectrometry-based proteomics along with developments in phosphopeptide enrichment methods has enabled large scale global phosphoproteomics studies (29, 30). However, the number of phosphorylation sites identified on kinases is limited compared with other proteins because of their frequently low expression levels. To overcome this problem, small inhibitor-based kinase enrichment strategies were developed, resulting in the identification of more than 200 kinases from HeLa cell lysates (31, 32). This method was also used recently to compare the phosphokinomes during S phase and M phase of the cell cycle, resulting in the identification of several hundreds of M phase-specific kinase phosphorylation sites (31). In the present study, we address the dynamics of the phosphokinome during mitotic progression using large scale cell synchronization at three distinct mitotic stages, small inhibitor-based kinase enrichment, and stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative mass spectrometry. Thus, we determined the mitotic phosphorylation dynamics of more than 900 kinase phosphorylation sites and identified distinctly regulated kinase interaction networks. Our results provide a valuable resource for the dynamics of the kinome during mitotic progression and give insight into the system properties of kinase interaction networks.     

On the use of 2,1,3-benzothiadiazole derivatives as selective live cell fluorescence imaging probes

Newly designed 2,1,3-benzothiadiazole-containing fluorescent probes with four excited state intramolecular proton transfer (ESIPT) sites were successfully tested in live cell-imaging assays using a confluent monolayer of human stem-cells (tissue). All tested dyes were compared with the commercially available DAPI and gave far better results.     

Synthesis,characterization, and cellular localization of a fluorescent probe of the antimalarial 8-aminoquinoline primaquine

Primaquine (PQ) is the only commercially available drug that clears dormant liver stages of malaria and blocks transmission to mosquito vectors. Although an old drug, much remains to be known about the mechanism(s) of action. Herein we develop a fluorescent tagged PQ to discover cellular localization in the human malaria parasite, Plasmodium falciparum. Successful synthesis and characterization of a primaquine-coumarin fluorescent probe (PQCP) demonstrated potency equivalent to the parent drug and the probe was not cytotoxic to HepG2 carcinoma cells. Cellular localization was found primarily in the cytosol of the asexual erythrocytic and gametocyte stages of parasite development.     

Seed dispersal anachronisms: rethinking the fruits extinct megafauna ate

Background

Some neotropical, fleshy-fruited plants have fruits structurally similar to paleotropical fruits dispersed by megafauna (mammals >10³ kg), yet these dispersers were extinct in South America 10–15 Kyr BP. Anachronic dispersal systems are best explained by interactions with extinct animals and show impaired dispersal resulting in altered seed dispersal dynamics.

Methodology/Principal Findings

We introduce an operational definition of megafaunal fruits and perform a comparative analysis of 103 Neotropical fruit species fitting this dispersal mode. We define two megafaunal fruit types based on previous analyses of elephant fruits: fruits 4–10 cm in diameter with up to five large seeds, and fruits >10 cm diameter with numerous small seeds. Megafaunal fruits are well represented in unrelated families such as Sapotaceae, Fabaceae, Solanaceae, Apocynaceae, Malvaceae, Caryocaraceae, and Arecaceae and combine an overbuilt design (large fruit mass and size) with either a single or few (<3 seeds) extremely large seeds or many small seeds (usually >100 seeds). Within-family and within-genus contrasts between megafaunal and non-megafaunal groups of species indicate a marked difference in fruit diameter and fruit mass but less so for individual seed mass, with a significant trend for megafaunal fruits to have larger seeds and seediness.

Conclusions/Significance

Megafaunal fruits allow plants to circumvent the trade-off between seed size and dispersal by relying on frugivores able to disperse enormous seed loads over long-distances. Present-day seed dispersal by scatter-hoarding rodents, introduced livestock, runoff, flooding, gravity, and human-mediated dispersal allowed survival of megafauna-dependent fruit species after extinction of the major seed dispersers. Megafauna extinction had several potential consequences, such as a scale shift reducing the seed dispersal distances, increasingly clumped spatial patterns, reduced geographic ranges and limited genetic variation and increased among-population structuring. These effects could be extended to other plant species dispersed by large vertebrates in present-day, defaunated communities.     

A new class of carriers that transport selective cargo from the trans Golgi network to the cell surface

We have isolated a membrane fraction enriched in a class of transport carriers that form at the trans Golgi network (TGN) and are destined for the cell surface in HeLa cells. Protein kinase D (PKD) is required for the biogenesis of these carriers that contain myosin II, Rab6a, Rab8a, and synaptotagmin II, as well as a number of secretory and plasma membrane‐specific cargoes. Our findings reveal a requirement for myosin II in the migration of these transport carriers but not in their biogenesis per se. Based on the cargo secreted by these carriers we have named them CARTS for CAR riers of the T GN to the cell S urface. Surprisingly, CARTS are distinct from the carriers that transport vesicular stomatitis virus (VSV)‐G protein and collagen I from the TGN to the cell surface. Altogether, the identification of CARTS provides a valuable means to understand TGN to cell surface traffic.     

The Role of Electrostatic Interactions in Binding of Histone H3K4me2/3 to the Sgf29 Tandem Tudor Domain

Several reader domain proteins that specifically recognize methyllysine-containing histones contain the negatively-charged aspartate or glutamate residues as part of the aromatic cage. Herein, we report thermodynamic analyses for the recognition of histone H3K4me3 and H3K4me2 by the tandem tudor domain of Sgf29 and its recognition site variants. Small uncharged and large aromatic substitutions on the Asp266 site resulted in a significant decrease in binding affinities for both H3K4me3 and H3K4me2, demonstrating the role of the negative charge of Asp266 in the readout process by Sgf29. This study emphasizes the essential contribution of electrostatic interactions to the overall binding affinity, and reveals that the underlying mechanisms for the recognition of Kme2/3 depend on the composition and arrangement of the aromatic cage.