Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics^1,2. One of the most well-known and widely-utilized +TIPs for analyzing MT dynamics is the End-Binding protein, EB1, which binds all growing MT plus-ends, and thus, is a marker for MT polymerization¹. Many studies of EB1 behavior within growth cones have used time-consuming and biased computer-assisted, hand-tracking methods to analyze individual MTs^1-3. Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker⁴, following the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones⁵. This software is a MATLAB-based, open-source, user-friendly package that combines automated detection, tracking, visualization, and analysis for movies of fluorescently-labeled +TIPs. Here, we present the protocol for using plusTipTracker for the analysis of fluorescently-labeled +TIP comets in cultured Xenopus laevis growth cones. However, this software can also be used to characterize MT dynamics in various cell types^6-8.     

High fidelity to wintering,stop-over and breeding sites shown by a Long-billed Curlew Numenius americanus tracked with satellite telemetry on migratory flights across North America

Capsule We studied the migratory movements and site fidelity of a male Long-billed Curlew using satellite telemetry in North America; the bird completed three migratory cycles and showed strong fidelity to stop-over, breeding, and wintering sites, not only on a geographical scale but also on a local scale across the years.     

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Images of bacterial cells stained with KK114 dye and visualized with STED microscopy. On the monitor: large field of view of B. subtilis cells, the KK114 ball‐and‐stick model and the schematics of the STED setup. Different space‐filling representations of the FtsZ protein are also shown. Further details can be found in the article by Massimiliano Lucidi, Radu Hristu, Lorenzo Nichele, George A. Stanciu, Denis E. Tranca, Alina Maria Holban, Paolo Visca, Stefan G. Stanciu, and Gabriella Cincotti ( e202000097 ).

     

ATP involvement in plant tissue responses to low temperature

The effects of chilling and freezing temperature on membrane permeability and ATP content were studied in the leaves of cucumber ( Cucumis sativus L.) and winter rape ( Brassica napus L. var. oleifera L.) leaves, grown at different temperatures. In the winter rape leaves, the endogenous ATP content was modified by application of dinitrophenol (DNP) solutions of different concentrations. The low temperature-induced changes in membrane permeability (as monitored by the conductivity method) were found to be associated with ATP decrease, both in the chilling-sensitive and chilling-resistant (subjected to freezing) plants. In tissues showing reversible injuries, changes in ATP content preceded those in membrane permeability and the adenylate energy charge was affected slightly. In tissues showing irreversible membrane damage, the ATP content was always below 0.4 μmol (g dry weight)⁻¹ and the adenylate energy charge was near 0.5. DNP treatment increased freezing sensitivity of winter rape leaves. In the cold-hardened winter rape leaves, however, freezing and thawing did not significantly affect ATP content or the energy charge, although the specimen showed a rather large increase in membrane permeability. In these leaves ATP content recovered about 20 h after a freezing and thawing treatment. It is proposed that a decrease in ATP supply might be the primary reason for the membrane leakiness at low temperature, both in chilling-sensitive and chilling-resistant (subjected to freezing) plants. The conclusion is, however, not true for the cold-acclimated, frostadapted cells.     

Drosophila Nedd4-long reduces Amphiphysin levels in muscles and leads to impaired T-tubule formation

Drosophila Nedd4 (dNedd4) is a HECT ubiquitin ligase with two main splice isoforms: dNedd4-short (dNedd4S) and -long (dNedd4Lo). DNedd4Lo has a unique N-terminus containing a Pro-rich region. We previously showed that whereas dNedd4S promotes neuromuscular synaptogenesis, dNedd4Lo inhibits it and impairs larval locomotion. To delineate the cause of the impaired locomotion, we searched for binding partners to the N-terminal unique region of dNedd4Lo in larval lysates using mass spectrometry and identified Amphiphysin (dAmph). dAmph is a postsynaptic protein containing SH3-BAR domains and regulates muscle transverse tubule (T-tubule) formation in flies. We validated the interaction by coimmunoprecipitation and showed direct binding between dAmph-SH3 domain and dNedd4Lo N-terminus. Accordingly, dNedd4Lo was colocalized with dAmph postsynaptically and at muscle T-tubules. Moreover, expression of dNedd4Lo in muscle during embryonic development led to disappearance of dAmph and impaired T-tubule formation, phenocopying amph-null mutants. This effect was not seen in muscles expressing dNedd4S or a catalytically-inactive dNedd4Lo(C→A). We propose that dNedd4Lo destabilizes dAmph in muscles, leading to impaired T-tubule formation and muscle function.     

Comparative analysis of uncoupling protein 4 distribution in various tissues under physiological conditions and during development

UCP4 is a member of the mitochondrial uncoupling protein subfamily and one of the three UCPs (UCP2, UCP4, UCP5), associated with the nervous system. Its putative functions include thermogenesis, attenuation of reactive oxidative species (ROS), regulation of mitochondrial calcium concentration and involvement in cell differentiation and apoptosis. Here we investigate UCP4's subcellular, cellular and tissue distribution, using an antibody designed specially for this study, and discuss the findings in terms of the protein's possible functions. Western blot and immunohistochemistry data confirmed that UCP4 is expressed predominantly in the central nervous system (CNS), as previously shown at mRNA level. No protein was found in heart, spleen, stomach, intestine, lung, thymus, muscles, adrenal gland, testis and liver. The reports revealing UCP4 mRNA in kidney and white adipose tissue were not confirmed at protein level. The amount of UCP4 varies in the mitochondria of different brain regions, with the highest protein content found in cortex. We show that UCP4 is present in fetal murine brain tissue as early as embryonic days 12-14 (E12-E14), which coincides with the beginning of neuronal differentiation. The UCP4 content in mitochondria decreases as the age of mice increases. UCP4 preferential expression in neurons and its developmental expression pattern under physiological conditions may indicate a specific protein function, e.g. in neuronal cell differentiation.