[Show abstract][Hide abstract] ABSTRACT: Differentiating neurons process the mechanical stimulus by exerting the protrusive forces through lamellipodia and filopodia. We used optical tweezers, video imaging and immunocytochemistry to analyze the role of non-muscle myosin-II on the protrusive force exerted by lamellipodia and filopodia from developing growth cones (GCs) of isolated Dorsal Root Ganglia (DRG) neurons. When the activity of myosin-II was inhibited by 30 mM Blebbistatin protrusion/retraction cycles of lamellipodia slowed down and during retraction lamellipodia could not lift up axially as in control condition. Inhibition of actin polymerization with 25 nM Cytochalasin-D and of microtubule polymerization with 500 nM Nocodazole slowed down the protrusion/retraction cycles, but only Cytochalasin-D decreased lamellipodia axial motion. The force exerted by lamellipodia treated with Blebbistatin decreased by 50%, but, surprisingly, the force exerted by filopodia increased by 20-50%. The concomitant disruption of microtubules caused by Nocodazole abolished the increase of the force exerted by filopodia treated with Blebbistatin. These results suggest that; i-Myosin-II controls the force exerted by lamellipodia and filopodia; ii-contractions of the actomyosin complex formed by filaments of actin and myosin have an active role in ruffle formation; iii-myosin-II is an essential component of the structural stability of GCs architecture. D uring development, neurons are able to self-organize in precisely wired networks and are able to establish the appropriate synaptic connections. Neuronal navigation requires the existence of highly motile struc-tures able to probe the mechanical properties of the surrounding environment and to search for the chemical cues leading to the formation of correct synaptic connections 1,2 . Neuronal exploration is guided by growth cones (GCs) located at the neurite tips 3,4 . GCs are composed of lamellipodia of different sizes, depending on the cell type and species from which thin filopodia with a submicron diameter emerge 5 . The primary source of motility in GCs is the polymerization of actin filaments 6,7 , controlled by a large set of regulatory proteins, such as Arp2/3, WASP, etc 8 and molecular motors seem to participate in the overall process by controlling several aspects of the process. The addition of actin monomers/oligomers to actin filaments in close contact with the membrane pushes the cellular membrane forward exerting a protrusive force 6,9 . An important determinant of force generation is the turnover of actin filaments, during which actin monomers or small oligomers are added to the barbed end of actin filaments (polymerization) and are removed from the other end (depolymerization). In this process the non-muscle myosin-II plays an important role: indeed myosin-II controls the retrograde flow of actin polymers by severing the actin filaments at their pointed end, providing the necessary treadmilling mechanism 10 . Myosins constitute a superfamily of motor proteins with major roles in several cellular processes such as cell adhesion, migration and division 11 . Myosin molecules, like all motor proteins, can walk along, propel and slide by other molecules and can produce tension on actin filaments. Generation of tension and force requires metabolic energy, usually provided by ATP hydrolysis and therefore myosins have appropriate catalytic sites in their amino-terminal (head) region. Myosin can associate to actin filaments to form the actomyosin complex, which can generate force. Like muscle myosin-II, non-muscle myosin-II (NMII) molecules are formed by three pairs of peptides with different molecular weight and function 11 . The three myosin-II isoforms NMIIA, NMIIB and NMIIC have similar structural and dynamical properties but have slightly different kinetics properties. Their major difference seems to reside in their regulation properties and different proteins control them through distinct phosphorylation sites 11 . Myosin-II seems to be involved in the orchestration of actin polymerization/depolymerization but also of microtubules (MTs) dynamics. Indeed, it has been shown that actin oligomers driven by myosin-II interact with growing MTs and that myosin-II-dependent compressive force is necessary for MTs dynamics 12 to form axons. The existence of a coupling between actin and MT dynamics is also supported by the observation that inhibition of myosin-II with Blebbistatin markedly accelerates axon growth and promotes the reorganization of both actin OPEN
[Show abstract][Hide abstract] ABSTRACT: Ion channels control ionic fluxes across biological membranes by residing in any of three functionally distinct states: deactivated (closed), activated (open), or inactivated (closed). Unlike many of their cousin K(+) channels, cyclic nucleotide-gated (CNG) channels do not desensitize or inactivate. Using patch recording techniques, we show that when extracellular pH (pHo) is decreased from 7.4 to 6 or lower, wild-type CNGA1 channels inactivate in a voltage-dependent manner. pHo titration experiments show that at pHo < 7 the current-voltage relations are outwardly rectifying and that inactivation is coupled to current rectification. Single-channel recordings indicate that a fast mechanism of proton blockage underscore current rectification while inactivation arises from conformational changes downstream from protonation. Furthermore, mutagenesis and ionic substitution experiments highlight the role of the selectivity filter in current decline suggesting analogies with the C-type inactivation observed in K(+) channels. The analysis with Markovian models indicates that the non-independent binding of two protons within the transmembrane electrical field explains both the voltage-dependent blockage and the inactivation. Acidic pH by inhibiting the CNGA1 channels in a state-dependent manner may represent an unrecognized endogenous signal regulating CNG physiological functions in diverse tissues. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
The Journal of Physiology 12/2014; · 4.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Polymerization of actin filaments is the primary source of motility in lamellipodia and it is controlled by a variety of regulatory proteins. The underlying molecular mechanisms are only partially understood and a precise determination of dynamical properties of force generation is necessary. Using optical tweezers, we have measured with millisecond (ms) temporal resolution and picoNewton (pN) sensitivity the force-velocity (Fv) relationship and the power dissipated by lamellipodia of dorsal root ganglia neurons. When force and velocity are averaged over 3–5 s, the Fv relationships can be flat. On a finer timescale, random occurrence of fast growth and subsecond retractions become predominant. The maximal power dissipated by lamellipodia over a silica bead with a diameter of 1 mm is 10�16 W. Our results clarify the dynamical properties of force generation: i), force generation is a probabilistic process; ii), underlying biological events have a bandwidth up to at least 10 Hz; and iii), fast growth of lamellipodia leading edge alternates with local retractions.
[Show abstract][Hide abstract] ABSTRACT: In cyclic nucleotide-gated (CNGA1) channels, in the presence of symmetrical ionic conditions, current-voltage (I-V) relationship depends, in a complex way, on the radius of permeating ion. It has been suggested that both the pore and S4 helix contribute to the observed rectification. In the present manuscript, using tail and gating current measurements from homotetrameric CNGA1 channels expressed in Xenopus oocytes, we clarify and quantify the role of the pore and of the S4 helix. We show that in symmetrical Rb(+) and Cs(+) single-channel current rectification dominates macroscopic currents while voltage-dependent gating becomes larger in symmetrical ethylammonium and dimethylammonium, where the open probability strongly depends on voltage. Isochronal tail currents analysis in dimethylammonium shows that at least two voltage-dependent transitions underlie the observed rectification. Only the first voltage-dependent transition is sensible to mutation of charge residues in the S4 helix. Moreover, analysis of tail and gating currents indicates that the number of elementary charges per channel moving across the membrane is less than 2, when they are about 12 in K(+) channels. These results indicate the existence of distinct mechanisms underlying rectification in CNG channels. A restricted motion of the S4 helix together with an inefficient coupling to the channel gate render CNGA1 channels poorly sensitive to voltage in the presence of physiological Na(+) and K(+).
[Show abstract][Hide abstract] ABSTRACT: Using the newly developed voltage-sensitive dye VF2.1.Cl, we monitored simultaneously the spontaneous electrical activity of ∼80 neurons in a leech ganglion, representing around 20% of the entire neuronal population. Neurons imaged on the ventral surface of the ganglion either fired spikes regularly at a rate of 1-5 Hz or fired sparse spikes irregularly. In contrast, neurons imaged on the dorsal surface, fired spikes in bursts involving several neurons. The overall degree of correlated electrical activity among leech neurons was limited in control conditions but increased in the presence of the neuromodulator serotonin. The spontaneous electrical activity in a leech ganglion is segregated in three main groups: neurons comprising Retzius cells, Anterior Pagoda, and Annulus Erector motoneurons firing almost periodically, a group of neurons firing sparsely and randomly, and a group of neurons firing bursts of spikes of varying durations. These three groups interact and influence each other only weakly.
[Show abstract][Hide abstract] ABSTRACT: Guidance molecules, such as Sema3A or Netrin-1, can induce growth cone (GC) repulsion or attraction in the presence of a flat surface, but very little is known of the action of guidance molecules in the presence of obstacles. Therefore we combined chemical and mechanical cues by applying a steady Netrin-1 stream to the GCs of dissociated hippocampal neurons plated on polydimethylsiloxane (PDMS) surfaces patterned with lines 2 µm wide, with 4 µm period and with a height varying from 100 to 600 nm. GC turning experiments performed 24 hours after plating showed that filopodia crawl over these lines within minutes. These filopodia do not show staining for the adhesion marker Paxillin. GCs and neurites crawl over lines 100 nm high, but less frequently and on a longer time scale over lines higher than 300 nm; neurites never crawl over lines 600 nm high. When neurons are grown for 3 days over patterned surfaces, also neurites can cross lines 300 nm and 600 nm high, grow parallel to and on top of these lines and express Paxillin. Axons - selectively stained with SMI 312 - do not differ from dendrites in their ability to cross these lines. Our results show that highly motile structures such as filopodia climb over high obstacle in response to chemical cues, but larger neuronal structures are less prompt and require hours or days to climb similar obstacles.
PLoS ONE 09/2013; 8(9):e73966. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mechanical properties such as force generation are fundamental for neuronal motility, development and regeneration. We used optical tweezers to compare the force exerted by growth cones (GCs) of neurons from the Peripheral Nervous System (PNS), such as Dorsal Root Ganglia (DRG) neurons, and from the Central Nervous System (CNS) such as hippocampal neurons. Developing GCs from dissociated DRG and hippocampal neurons were obtained from P1-P2 and P10-P12 rats. Comparing their morphology, we observed that the area of GCs of hippocampal neurons was 8-10 µm(2) and did not vary between P1-P2 and P10-P12 rats, but GCs of DRG neurons were larger and their area increased from P1-P2 to P10-P12 by 2-4 times. The force exerted by DRG filopodia was in the order of 1-2 pN and never exceeded 5 pN, while hippocampal filopodia exerted a larger force, often in the order of 5 pN. Hippocampal and DRG lamellipodia exerted lateral forces up to 20 pN, but lamellipodia of DRG neurons could exert a vertical force larger than that of hippocampal neurons. Force-velocity relationships (Fv) in both types of neurons had the same qualitative behaviour, consistent with a common autocatalytic model of force generation. These results indicate that molecular mechanisms of force generation of GC from CNS and PNS neurons are similar but the amplitude of generated force is influenced by their cytoskeletal properties.
PLoS ONE 08/2013; 8(8):e73025. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hippocampal organotypic cultures are a highly reliable in vitro model for studying neuroplasticity: in this paper, we analyze the early phase of the transcriptional response induced by a 20 µM gabazine treatment (GabT), a GABA-Ar antagonist, by using Affymetrix oligonucleotide microarray, RT-PCR based time-course and chromatin-immuno-precipitation. The transcriptome profiling revealed that the pool of genes up-regulated by GabT, besides being strongly related to the regulation of growth and synaptic transmission, is also endowed with neuro-protective and pro-survival properties. By using RT-PCR, we quantified a time-course of the transient expression for 33 of the highest up-regulated genes, with an average sampling rate of 10 minutes and covering the time interval [10∶90] minutes. The cluster analysis of the time-course disclosed the existence of three different dynamical patterns, one of which proved, in a statistical analysis based on results from previous works, to be significantly related with SRF-dependent regulation (p-value<0.05). The chromatin immunoprecipitation (chip) assay confirmed the rich presence of working CArG boxes in the genes belonging to the latter dynamical pattern and therefore validated the statistical analysis. Furthermore, an in silico analysis of the promoters revealed the presence of additional conserved CArG boxes upstream of the genes Nr4a1 and Rgs2. The chip assay confirmed a significant SRF signal in the Nr4a1 CArG box but not in the Rgs2 CArG box.
PLoS ONE 07/2013; 8(7):e68078. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Sensory systems adapt, i.e., they adjust their sensitivity to external stimuli according to the ambient level. In this paper we show that single cell electrophysiological responses of vertebrate olfactory receptors and of photoreceptors to different input protocols exhibit several common features related to adaptation, and that these features can be used to investigate the dynamical structure of the feedback regulation responsible for the adaptation. In particular, we point out that two different forms of adaptation can be observed, in response to steps and to pairs of pulses. These two forms of adaptation appear to be in a dynamical trade-off: the more adaptation to a step is close to perfect, the slower is the recovery in adaptation to pulse pairs and viceversa. Neither of the two forms is explained by the dynamical models currently used to describe adaptation, such as the integral feedback model.
[Show abstract][Hide abstract] ABSTRACT: The present manuscript aims at identifying patterns of electrical activity recorded from neurons of the leech nervous system, characterizing specific behaviors. When leeches are at rest, the electrical activity of neurons and motoneurons is poorly correlated. When leeches move their head and/or tail, in contrast, action potential (AP) firing becomes highly correlated. When the head or tail suckers detach, specific patterns of electrical activity are detected. During elongation and contraction the electrical activity of motoneurons in the Medial Anterior and Dorsal Posterior nerves increase, respectively, and several motoneurons are activated both during elongation and contraction. During crawling, swimming, and pseudo-swimming patterns of electrical activity are better described by the dendrograms of cross-correlations of motoneurons pairs. Dendrograms obtained from different animals exhibiting the same behavior are similar and by averaging these dendrograms we obtained a template underlying a given behavior. By using this template, the corresponding behavior is reliably identified from the recorded electrical activity. The analysis of dendrograms during different leech behavior reveals the fine orchestration of motoneurons firing specific to each stereotyped behavior. Therefore, dendrograms capture the subtle changes in the correlation pattern of neuronal networks when they become involved in different tasks or functions.
Frontiers in Integrative Neuroscience 01/2013; 7:69.
[Show abstract][Hide abstract] ABSTRACT: Guidance molecules, such as Sema3A or Netrin-1, can induce growth cone re-
pulsion or attraction in the presence of a flat surface, but very little is known of
the action of guidance molecules in the presence of obstacles. In order to ad-
dress this issue, we analysed the action of Netrin-1 molecules in the presence
of patterned polydimethylsiloxane (PDMS) surfaces with lines with a height
varying from 100 to 600 nm. Filopodia can crawl over these lines easily, but
not neurites. Indeed axons and thick dendrites are able to cross lines with
a height of 100 nm, but progressively less when the line height is increased.
When neurons are grown for 3 days over patterned surfaces it was possible to label selectively axons with axonal neurofilament marker SMI 312 and den-
drites with microtubule associated protein 2 (MAP2): axons and dendrites do
not differ in their ability to cross lines with a height varying from 100 to
600 nm. When axons and dendrites grow along lines, a clear staining for the
adhesion marker paxillin was observed, but not when neurites cross the lines.
[Show abstract][Hide abstract] ABSTRACT: Guidance molecules, such as Sema3A or Netrin-1, induce growth cone (GC) repulsion or attraction. In order to determine the speed of action and efficiency of these guidance cues we developed an experimental procedure to deliver controlled amounts of these molecules. Lipid vesicles encapsulating 10-10(4) molecules of Sema3A or Netrin-1 were manipulated with high spatial and temporal resolution by optical tweezers and their photolysis triggered by laser pulses. Guidance molecules released from the vesicles diffused and reached the GC membrane in a few seconds. Following their arrival, GCs retracted or grew in 20-120 s. By determining the number of guidance molecules trapped inside vesicles and estimating the fraction of guidance molecules reaching the GC, we show that the arrival of less than 5 Netrin-1 molecules on the GC membrane is sufficient to induce growth. In contrast, the arrival of about 200 Sema3A molecules is necessary to induce filopodia repulsion.
[Show abstract][Hide abstract] ABSTRACT: Key points • Cyclic nucleotide-gated (CNG) channels are multi-ion channels showing the anomalous mole fraction effect (AMFE) in the presence of Li(+) and Cs(+) mixtures. • We show that Cs(+) ions at the intracellular side of the membrane block the entry of Na(+) ions in a voltage dependent way. • The blockage is relieved when Thr359 and Thr360 at the intracellular entrance of the selectivity filter are replaced with an alanine. Moreover, the AMFE in the presence of intracellular mixtures of Li(+) and Cs(+) is abolished in T360A mutant channels. • We have identified a second binding site - composed by the ring of Thr360 at the intracellular vestibule - in the selectivity filter of CNG channels controlling monovalent cations selectivity and permeation. • These results help us understand fundamental similarities and differences between the pore of CNG channels and K(+) channels.
The Journal of Physiology 08/2012; 590(Pt 20):5075-90. · 4.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cyclic nucleotide-gated channels belong to the family of voltage-gated ion channels, but pore opening requires the presence of intracellular cyclic nucleotides. In the presence of a saturating agonist, cyclic nucleotide-gated channel gating is voltage independent and it is not known why cyclic nucleotide-gated channels are voltage-insensitive despite harbouring the S4-type voltage sensor. Here we report that, in the presence of Li(+), Na(+) and K(+), the gating of wild-type cyclic nucleotide-gated A1 and native cyclic nucleotide-gated channels is voltage independent, whereas their gating is highly voltage-dependent in the presence of Rb(+), Cs(+) and organic cations. Mutagenesis experiments show that voltage sensing occurs through a voltage sensor composed of charged/polar residues in the pore and of the S4-type voltage sensor. During evolution, cyclic nucleotide-gated channels lose their voltage-sensing ability when Na(+) or K(+) permeate so that the vertebrate photoreceptor cyclic nucleotide-gated channels are open at negative voltages, a necessary condition for phototransduction.
[Show abstract][Hide abstract] ABSTRACT: We used optical tweezers to analyze the effect of jasplakinolide and cyclodextrin on the force exerted by lamellipodia from developing growth cones (GCs) of isolated dorsal root ganglia (DRG) neurons. We found that 25 nM of jasplakinolide, which is known to inhibit actin filament turnover, reduced both the maximal exerted force and maximal velocity during lamellipodia leading-edge protrusion. By using atomic force microscopy, we verified that cyclodextrin, which is known to remove cholesterol from membranes, decreased the membrane stiffness of DRG neurons. Lamellipodia treated with 2.5 mM of cyclodextrin exerted a larger force, and their leading edge could advance with a higher velocity. Neither jasplakinolide nor cyclodextrin affected force or velocity during lamellipodia retraction. The amplitude and frequency of elementary jumps underlying force generation were reduced by jasplakinolide but not by cyclodextrin. The action of both drugs at the used concentration was fully reversible. These results support the notion that membrane stiffness provides a selective pressure that shapes force generation, and confirm the pivotal role of actin turnover during protrusion.