Principles of protein targeting to the nucleolus

© 2015 The Author(s). Published with license by Taylor & Francis Group, LLC© Robert M Martin, Gohar Ter-Avetisyan, Henry D Herce, Anne K Ludwig, Gisela Lättig-Tünnemann, and M Cristina Cardoso This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted.The nucleolus is the hallmark of nuclear compartmentalization and has been shown to exert multiple roles in cellular metabolism besides its main function as the place of rRNA synthesis and assembly of ribosomes. Nucleolar proteins dynamically localize and accumulate in this nuclear compartment relative to the surrounding nucleoplasm. In this study, we have assessed the molecular requirements that are necessary and sufficient for the localization and accumulation of peptides and proteins inside the nucleoli of living cells. The data showed that positively charged peptide entities composed of arginines alone and with an isoelectric point at and above 12.6 are necessary and sufficient for mediating significant nucleolar accumulation. A threshold of 6 arginines is necessary for peptides to accumulate in nucleoli, but already 4 arginines are sufficient when fused within 15 amino acid residues of a nuclear localization signal of a protein. Using a pH sensitive dye, we found that the nucleolar compartment is particularly acidic when compared to the surrounding nucleoplasm and, hence, provides the ideal electrochemical environment to bind poly-arginine containing proteins. In fact, we found that oligo-arginine peptides and GFP fusions bind RNA in vitro. Consistent with RNA being the main binding partner for arginines in the nucleolus, we found that the same principles apply to cells from insects to man, indicating that this mechanism is highly conserved throughout evolution.RMM was supported by a post-doctoral grant from Fundação para a Ciência e Tecnologia, Portugal (SFRH-BPD-66611–2009). This work was supported by grants of the German Research Council (DFG CA198/3) to MCC.info:eu-repo/semantics/publishedVersio

Der Rezeptor Guanylylzyklase Npr2 reguliert die Bifurkation der kranialen sensorischen Axone.

Axonal branching is a key mechanism to form complex circuits in the mature nervous system. Depending on their functions, axons can branch extensively to send information to their distinct targets and establish a unique pattern of connectivity. However, signaling mechanisms regulating axonal branching during the development of the nervous system are still poorly understood. The cGMP signaling pathway including the secreted C-type natriuretic peptide (CNP), its receptor natriuretic peptide receptor 2 (Npr2) and the cGMP-dependent protein kinase Iα (cGKIα) has been shown to control the bifurcation of DRG (dorsal root ganglion) sensory neurons within the spinal cord. Using mutant mouse models it has been demonstrated that in the absence of one of these components, the sensory axons are unable to bifurcate at the entry zone of the spinal cord. Based on these findings, it is intriguing to ask whether other neuronal subpopulations besides the DRG neurons exist and if they use the above mentioned cGMP signaling pathway to form branches emerging from the bifurcating growth cone. To investigate the role of Npr2-dependent axonal branching in other neural systems than DRG neurons, a genetic strategy was used and two mouse models (Npr2LacZ and Npr2CreERT2) were generated, which allow studies on the expression pattern of Npr2 and its influence on axonal branching of single Npr2-expressing axons in the mouse nervous system. While the Npr2LacZ reporter mouse enabled detailed analysis of the localization of the signaling pathway components, the Npr2CreERT2 mouse is a valuable tool for visualizing single axonal projections after crossing with reporter strains. A prominent Npr2 expression was found not only in the DRG, but also in the cranial sensory ganglia of the developing mouse. Using immunohistochemistry, a co-localization of the receptor Npr2 and the kinase cGKIα was detected in neurons of cranial sensory ganglia. To follow the single projections of sensory axons from cranial sensory ganglia and study their branching behavior, the Npr2CreERT2 mouse was crossed with two different reporter strains (Z/AP and mGFP). After induction of Cre recombinase with tamoxifen or 4-hydroxytamoxifen at embryonic day 9.5 or 10.5, single axonal projections could be visualized at embryonic day 12.5 or 13.5, respectively. This genetic approach uncovered two new aspects about central projections of cranial sensory ganglia: (i) almost all central projections of cranial sensory ganglia bifurcate at the entry zone of the hindbrain and (ii) in the absence of Npr2 the sensory axons lose their bifurcational behavior when entering the hindbrain. Hence, the neurons of all cranial sensory ganglia - similar to DRG neurons - use cGMP signaling to bifurcate as soon as they enter the hindbrain.Die axonale Verzweigung ist ein wichtiger Mechanismus, um die komplexen Verschaltungen des Nervensystems zu bilden. Je nach ihrer Funktion können sich Axone weitläufig verzweigen, um die Informationen an ihre verschiedenen Ziele zu senden und ein einzigartiges Muster der Verschaltung zu etablieren. Allerdings sind die Signalmechanismen, die die axonale Verzweigung während der Entwicklung des Nervensystems regulieren, noch weitgehend unverstanden. Ein cGMP-Signalweg kontrolliert die Bifurkation der sensorischen DRG-Neurone (Dorsal root ganglia; Spinalganglien) im Rückenmark und besteht aus folgenden Komponenten: dem C-Typ natriuretischen Peptid (CNP), dem natriuretischen Peptid-Rezeptor-2 (Npr2) und der cGMP-abhängigen Proteinkinase I (cGKIα). In Mausmodellen konnte gezeigt werden, dass in Abwesenheit einer dieser Komponenten die sensorischen DRG-Neurone an der Eintrittszone des Rückenmarks nicht bifurkieren. Offen ist, ob es neben den DRG-Neuronen andere neuronale Subpopulationen gibt, die den cGMP-Signalweg im Rahmen der axonalen Verzweigung nutzen. Um die Rolle der Npr2-abhängigen axonalen Verzweigung in anderen Systemen als DRG-Neuronen zu untersuchen, wurden zwei transgene knock- in-Mausmodelle (Npr2LacZ und Npr2CreERT2) generiert, die es erlauben, das Expressionsmuster von Npr2 und die axonale Verzweigung einzelner Npr2-exprimierender Axone in der Maus zu studieren. Während die Npr2LacZ- Reportermaus es ermöglichte, eine detaillierte Analyse der Lokalisation der cGMP-Komponenten durchzuführen, stellte die Npr2CreERT2-Maus nach Kreuzung mit Reporter-Stämmen (Z/AP und mGFP) ein wertvolles Werkzeug für die Visualisierung einzelner axonaler Verzweigungen dar. Eine starke Npr2-Expression wurde nicht nur in den DRG-Neuronen gefunden, sondern auch in den kranialen sensorischen Ganglien. In Neuronen der kranialen sensorischen Ganglien wurde immunhistochemisch eine Ko-Expression des Rezeptors Npr2 und der Kinase cGKIα detektiert. Um das Bifurkationsverhalten einzelner Axone der kranialen sensorischen Neurone zu verfolgen, wurde die Npr2CreERT2-Maus mit den zwei Reporter-Stämmen gekreuzt. Nach Induktion der Cre-Rekombinase mit Tamoxifen am embryonalen Tag 9.5 bzw. 10.5 konnten einzelne axonalen Verweigungen am embryonalen Tag 12.5 bzw. 13.5 visualisiert werden. Dieser genetische Ansatz deckte zwei neue Aspekte der axonalen Bifurkation auf: (i) fast alle zentralen Projektionen der kranialen sensorischen Axone verzweigen an der Eingangszone des Hinterhirns und (ii) in Abwesenheit von Npr2 verlieren die sensorischen Axone ihre Fähigkeit beim Eintritt ins Hinterhirn zu bifurkieren. Zusammenfassend zeigen diese Untersuchungen, dass die kranialen sensorischen Neurone den cGMP-Signalweg nutzen, um beim Erreichen des Hinterhirns zu bifurkieren

Live-cell analysis of cell penetration ability and toxicity of oligo-arginines.

Cell penetrating peptides (CPPs) are useful tools to deliver low-molecular-weight cargoes into cells; however, their mode of uptake is still controversial. The most efficient CPPs belong to the group of arginine-rich peptides, but a systematic assessment of their potential toxicity is lacking. In this study we combined data on the membrane translocation abilities of oligo-arginines in living cells as a function of their chain length, concentration, stability and toxicity. Using confocal microscopy analysis of living cells we evaluated the transduction frequency of the L-isoforms of oligo-arginines and lysines and then monitored their associated toxicity by concomitant addition of propidium iodide. Whereas lysines showed virtually no transduction, the transduction ability of arginines increased with the number of consecutive residues and the peptide concentration, with L-R9 and L-R10 performing overall best. We further compared the L- and D-R9 isomers and found that the D-isoform always showed a higher transduction as compared to the L-counterpart in all cell types. Notably, the transduction difference between D- and L-forms was highly variable between cell types, emphasizing the need for protease-resistant peptides as vectors for drug delivery. Real-time kinetic analysis of the D- and L-isomers applied simultaneously to the cells revealed a much faster transduction for the D-variant. The latter underlies the fact that the isomers do not mix, and penetration of one peptide does not perturb the membrane in a way that gives access to the other peptide. Finally, we performed short- and long-term cell viability and cell cycle progression analyses with the protease-resistant D-R9. Altogether, our results identified concentration windows with low toxicity and high transduction efficiency, resulting in fully bioavailable intracellular peptides

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The absence of sensory axon bifurcation affects nociception and termination fields of afferents in the spinal cord

A cGMP signaling cascade composed of C-type natriuretic peptide, the guanylyl cyclase receptor Npr2 and cGMP-dependent protein kinase I (cGKI) controls the bifurcation of sensory axons upon entering the spinal cord during embryonic development. However, the impact of axon bifurcation on sensory processing in adulthood remains poorly understood. To investigate the functional consequences of impaired axon bifurcation during adult stages we generated conditional mouse mutants of Npr2 and cGKI (Npr2fl/fl;Wnt1Cre and cGKIKO/fl;Wnt1Cre) that lack sensory axon bifurcation in the absence of additional phenotypes observed in the global knockout mice. Cholera toxin labeling in digits of the hind paw demonstrated an altered shape of sensory neuron termination fields in the spinal cord of conditional Npr2 mouse mutants. Behavioral testing of both sexes indicated that noxious heat sensation and nociception induced by chemical irritants are impaired in the mutants, whereas responses to cold sensation, mechanical stimulation, and motor coordination are not affected. Recordings from C-fiber nociceptors in the hind limb skin showed that Npr2 function was not required to maintain normal heat sensitivity of peripheral nociceptors. Thus, the altered behavioral responses to noxious heat found in Npr2fl/fl;Wnt1Cre mice is not due to an impaired C-fiber function. Overall, these data point to a critical role of axonal bifurcation for the processing of pain induced by heat or chemical stimuli

The Absence of Sensory Axon Bifurcation Affects Nociception and Termination Fields of Afferents in the Spinal Cord

A cGMP signaling cascade composed of C-type natriuretic peptide, the guanylyl cyclase receptor Npr2 and cGMP-dependent protein kinase I (cGKI) controls the bifurcation of sensory axons upon entering the spinal cord during embryonic development. However, the impact of axon bifurcation on sensory processing in adulthood remains poorly understood. To investigate the functional consequences of impaired axon bifurcation during adult stages we generated conditional mouse mutants of Npr2 and cGKI (Npr2fl/fl;Wnt1Cre and cGKIKO/fl;Wnt1Cre) that lack sensory axon bifurcation in the absence of additional phenotypes observed in the global knockout mice. Cholera toxin labeling in digits of the hind paw demonstrated an altered shape of sensory neuron termination fields in the spinal cord of conditional Npr2 mouse mutants. Behavioral testing of both sexes indicated that noxious heat sensation and nociception induced by chemical irritants are impaired in the mutants, whereas responses to cold sensation, mechanical stimulation, and motor coordination are not affected. Recordings from C-fiber nociceptors in the hind limb skin showed that Npr2 function was not required to maintain normal heat sensitivity of peripheral nociceptors. Thus, the altered behavioral responses to noxious heat found in Npr2fl/fl;Wnt1Cre mice is not due to an impaired C-fiber function. Overall, these data point to a critical role of axonal bifurcation for the processing of pain induced by heat or chemical stimuli