8 research outputs found
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