Growth cones of elongating neurites exert force against the external environment, but little is known about the role of force in outgrowth or its relationship to the mechanical organization of neurons. We used traction force microscopy to examine patterns of force in growth cones of regenerating Aplysia bag cell neurons. We find that traction is highest in the peripheral actin-rich domain and internal stress reaches a plateau near the transition between peripheral and central microtubule-rich domains. Integrating stress over the area of the growth cone reveals that total scalar force increases with area but net tension on the neurite does not. Tensions fall within a limited range while a substantial fraction of the total force can be balanced locally within the growth cone. Although traction continuously redistributes during extension and retraction of the peripheral domain, tension is stable over time, suggesting that tension is a tightly regulated property of the neurite independent of growth cone dynamics. We observe that redistribution of traction in the peripheral domain can reorient the end of the neurite shaft. This suggests a role for off-axis force in growth cone turning and neuronal guidance.
We have investigated Aplysia hemolymph as a source of endogenous factors to promote regeneration of bag cell neurons. We describe a novel synergistic effect between substrate-bound hemolymph proteins and laminin. This combination increased outgrowth and branching relative to either laminin or hemolymph alone. Notably, the addition of hemolymph to laminin substrates accelerated growth cone migration rate over ten-fold. Our results indicate that the active factor is either a high molecular weight protein or protein complex and is not the respiratory protein hemocyanin. Substrate-bound factor(s) from central nervous system-conditioned media also had a synergistic effect with laminin, suggesting a possible cooperation between humoral proteins and nervous system extracellular matrix. Further molecular characterization of active factors and their cellular targets is warranted on account of the magnitude of the effects reported here and their potential relevance for nervous system repair.
PKC activation enhances myosin II contractility in the central growth cone domain while decreasing actin density and increasing actin network flow rates in the peripheral domain. This dual mode of action has mechanistic implications for interpreting reported effects of PKC on growth cone guidance and neuronal regeneration.
Protein kinase C (PKC) can dramatically alter cell structure and motility via effects on actin filament networks. In neurons, PKC activation has been implicated in repulsive guidance responses and inhibition of axon regeneration; however, the cytoskeletal mechanisms underlying these effects are not well understood. Here we investigate the acute effects of PKC activation on actin network structure and dynamics in large Aplysia neuronal growth cones. We provide evidence of a novel two-tiered mechanism of PKC action: 1) PKC activity enhances myosin II regulatory light chain phosphorylation and C-kinase–potentiated protein phosphatase inhibitor phosphorylation. These effects are correlated with increased contractility in the central cytoplasmic domain. 2) PKC activation results in significant reduction of P-domain actin network density accompanied by Arp2/3 complex delocalization from the leading edge and increased rates of retrograde actin network flow. Our results show that PKC activation strongly affects both actin polymerization and myosin II contractility. This synergistic mode of action is relevant to understanding the pleiotropic reported effects of PKC on neuronal growth and regeneration.
Adhesions are multi-molecular complexes that transmit forces generated by a cell’s acto-myosin networks to external substrates. While the physical properties of some of the individual components of adhesions have been carefully characterized, the mechanics of the coupling between the cytoskeleton and the adhesion site as a whole are just beginning to be revealed. We characterized the mechanics of nascent adhesions mediated by the immunoglobulin-family cell adhesion molecule apCAM, which is known to interact with actin filaments. Using simultaneous visualization of actin flow and quantification of forces transmitted to apCAM-coated beads restrained with an optical trap, we found that adhesions are dynamic structures capable of transmitting a wide range of forces. For forces in the picoNewton scale, the nascent adhesions’ mechanical properties are dominated by an elastic structure which can be reversibly deformed by up to 1 µm. Large reversible deformations rule out an interface between substrate and cytoskeleton that is dominated by a number of stiff molecular springs in parallel, and favor a compliant cross-linked network. Such a compliant structure may increase the lifetime of a nascent adhesion, facilitating signaling and reinforcement.
5-HT promotes neurite growth via IP3-dependent Ca2+ release in neuronal growth cones. Outgrowth depends on increased rates of actin array treadmilling mediated by Ca2+-calcineurin–dependent cofilin activation. This mode of growth contrasts with substrate-dependent responses, for which retrograde actin flow and advance rates have been inversely correlated.
Neurite outgrowth in response to soluble growth factors often involves changes in intracellular Ca2+; however, mechanistic roles for Ca2+ in controlling the underlying dynamic cytoskeletal processes have remained enigmatic. Bag cell neurons exposed to serotonin (5-hydroxytryptamine [5-HT]) respond with a threefold increase in neurite outgrowth rates. Outgrowth depends on phospholipase C (PLC) → inositol trisphosphate → Ca2+ → calcineurin signaling and is accompanied by increased rates of retrograde actin network flow in the growth cone P domain. Calcineurin inhibitors had no effect on Ca2+ release or basal levels of retrograde actin flow; however, they completely suppressed 5-HT–dependent outgrowth and F-actin flow acceleration. 5-HT treatments were accompanied by calcineurin-dependent increases in cofilin activity in the growth cone P domain. 5-HT effects were mimicked by direct activation of PLC, suggesting that increased actin network treadmilling may be a widespread mechanism for promoting neurite outgrowth in response to neurotrophic factors.
Actin veil networks assembled by the Arp2/3 complex constrain myosin II–dependent contractility in neuronal growth cones with consequent effects on peripheral retrograde actin flow rates.
The Arp2/3 complex nucleates actin filaments to generate networks at the leading edge of motile cells. Nonmuscle myosin II produces contractile forces involved in driving actin network translocation. We inhibited the Arp2/3 complex and/or myosin II with small molecules to investigate their respective functions in neuronal growth cone actin dynamics. Inhibition of the Arp2/3 complex with CK666 reduced barbed end actin assembly site density at the leading edge, disrupted actin veils, and resulted in veil retraction. Strikingly, retrograde actin flow rates increased with Arp2/3 complex inhibition; however, when myosin II activity was blocked, Arp2/3 complex inhibition now resulted in slowing of retrograde actin flow and veils no longer retracted. Retrograde flow rate increases induced by Arp2/3 complex inhibition were independent of Rho kinase activity. These results provide evidence that, although the Arp2/3 complex and myosin II are spatially segregated, actin networks assembled by the Arp2/3 complex can restrict myosin II–dependent contractility with consequent effects on growth cone motility.
The balance of actin filament polymerization and depolymerization maintains a steady state network treadmill in neuronal growth cones essential for motility and guidance. Here we have investigated the connection between depolymerization and treadmilling dynamics. We show that polymerization-competent barbed ends are concentrated at the leading edge and depolymerization is distributed throughout the peripheral domain. We found a high-to-low G-actin gradient between peripheral and central domains. Inhibiting turnover with jasplakinolide collapsed this gradient and lowered leading edge barbed end density. Ultrastructural analysis showed dramatic reduction of leading edge actin filament density and filament accumulation in central regions. Live cell imaging revealed that the leading edge retracted even as retrograde actin flow rate decreased exponentially. Inhibition of myosin II activity before jasplakinolide treatment lowered baseline retrograde flow rates and prevented leading edge retraction. Myosin II activity preferentially affected filopodial bundle disassembly distinct from the global effects of jasplakinolide on network turnover. We propose that growth cone retraction following turnover inhibition resulted from the persistence of myosin II contractility even as leading edge assembly rates decreased. The buildup of actin filaments in central regions combined with monomer depletion and reduced polymerization from barbed ends suggests a mechanism for the observed exponential decay in actin retrograde flow. Our results show that growth cone motility is critically dependent on continuous disassembly of the peripheral actin network.
We describe open-loop and closed-loop multiplexed force measurements using holographic optical tweezers. We quantify the performance of our novel video-based control system in a driven suspension of colloidal particles. We demonstrate our system's abilities with the measurement of the mechanical coupling between Aplysia bag cell growth cones and beads functionalized with the neuronal cell adhesion molecule, apCAM. We show that cells form linkages which couple beads to the underlying cytoskeleton. These linkages are intermittent, stochastic and heterogeneous across beads distributed near the leading edge of a single growth cone.
The small G protein Rac regulates cytoskeletal protein dynamics in neuronal growth cones and has been implicated in axon growth, guidance, and branching. Intracellular Ca2+ is another well known regulator of growth cone function; however, effects of Rac activity on intracellular Ca2+ metabolism have not been well characterized. Here, we investigate how Rac1 activity affects release of Ca2+ from intracellular endoplasmic reticulum (ER) stores stimulated by application of serotonin (5-hydroxytriptamine). We also address how Rac1 effects on microtubule assembly dynamics affect distribution of Ca2+ release sites. Multimode fluorescent microscopy was used to correlate microtubule and ER behavior, and ratiometric imaging was used to assess intracellular Ca2+ dynamics. We report that Rac1 activity both promotes Ca2+ release and affects its spatial distribution in neuronal growth cones. The underlying mechanism involves synergistic Rac1 effects on microtubule assembly and reactive oxygen species (ROS) production. Rac1 activity modulates Ca2+ by 1) enhancing microtubule assembly which in turn promotes spread of the ER-based Ca2+ release machinery into the growth cone periphery, and 2) by increasing ROS production which facilitated inositol 1,4,5-trisphosphate-dependent Ca2+ release. These results cast Rac1 as a key modulator of intracellular Ca2+ function in the neuronal growth cone.
The cell biological processes underlying axon growth and guidance are still not well understood. An outstanding question is how a new segment of the axon shaft is formed in the wake of neuronal growth cone advance. For this to occur, the highly dynamic, splayed-out microtubule (MT) arrays characteristic of the growth cone must be consolidated (bundled together) to form the core of the axon shaft. MT-associated proteins stabilize bundled MTs, but how individual MTs are brought together for initial bundling is unknown. Here, we show that laterally moving actin arcs, which are myosin II-driven contractile structures, interact with growing MTs and transport them from the sides of the growth cone into the central domain. Upon Myosin II inhibition, the movement of actin filaments and MTs immediately stopped and MTs unbundled. Thus, Myosin II-dependent compressive force is necessary for normal MT bundling in the growth cone neck.
Although much evidence suggests that axon growth and guidance depend on well-coordinated cytoskeletal dynamics, direct characterization of the corresponding molecular events has remained a challenge. Here, we address this outstanding problem by examining neurite outgrowth stimulated by local application of cell adhesion substrates. During acute outgrowth, the advance of organelles and underlying microtubules into the central domain was correlated with regions of attenuated retrograde actin network flow in the periphery. Interestingly, as adhesion sites matured, contractile actin arc structures, known to be regulated by the Rho/Rho Kinase/myosin II signaling cascade, became more robust and coordinated microtubule movements in the growth cone neck. When Rho Kinase was inhibited, although growth responses occurred with less of a delay, microtubules failed to consolidate into a single axis of growth. These results reveal a new role for Rho Kinase and myosin II contractility in regulation of microtubule behavior during neuronal growth.
growth cone; actin dynamics; microtubule dynamics; motility; adhesion; axon guidance; neuron
We have used multimode fluorescent speckle microscopy (FSM) and correlative differential interference contrast imaging to investigate the actin–microtubule (MT) interactions and polymer dynamics known to play a fundamental role in growth cone guidance. We report that MTs explore the peripheral domain (P-domain), exhibiting classical properties of dynamic instability. MT extension occurs preferentially along filopodia, which function as MT polymerization guides. Filopodial bundles undergo retrograde flow and also transport MTs. Thus, distal MT position is determined by the rate of plus-end MT assembly minus the rate of retrograde F-actin flow. Short MT displacements independent of flow are sometimes observed. MTs loop, buckle, and break as they are transported into the T-zone by retrograde flow. MT breakage results in exposure of new plus ends which can regrow, and minus ends which rapidly undergo catastrophes, resulting in efficient MT turnover. We also report a previously undetected presence of F-actin arc structures, which exhibit persistent retrograde movement across the T-zone into the central domain (C-domain) at ∼1/4 the rate of P-domain flow. Actin arcs interact with MTs and transport them into the C-domain. Interestingly, although the MTs associated with arcs are less dynamic than P-domain MTs, they elongate efficiently as a result of markedly lower catastrophe frequencies.
microtubules; axon outgrowth; actin; growth cone; dynamics
Tyrosine kinase activity is known to be important in neuronal growth cone guidance. However, underlying cellular mechanisms are largely unclear. Here, we report how Src family tyrosine kinase activity controls apCAM-mediated growth cone steering by regulating the transmission of traction forces through receptor–cytoskeletal linkages. Increased levels of tyrosine phosphorylation were detected at sites where beads coated with apCAM ligands were physically restrained to induce growth cone steering, but not at unrestrained bead binding sites. Interestingly, the rate and level of phosphotyrosine buildup near restrained beads were decreased by the myosin inhibitor 2,3-butanedione-2-monoxime, suggesting that tension promotes tyrosine kinase activation. While not affecting retrograde F-actin flow rates, genistein and the Src family selective tyrosine kinase inhibitors PP1 and PP2 strongly reduced the growth cone's ability to apply traction forces through apCAM–cytoskeletal linkages, assessed using the restrained bead interaction assay. Furthermore, increased levels of an activated Src family kinase were detected at restrained bead sites during growth cone steering events. Our results suggest a mechanism by which growth cones select pathways by sampling both the molecular nature of the substrate and its ability to withstand the application of traction forces.
apCAM; Src family tyrosine kinase; tyrosine phosphorylation; receptor–cytoskeletal coupling; growth cone steering
We describe a novel mechanism for protein kinase C regulation of axonal microtubule invasion of growth cones. Activation of PKC by phorbol esters resulted in a rapid, robust advance of distal microtubules (MTs) into the F-actin rich peripheral domain of growth cones, where they are normally excluded. In contrast, inhibition of PKC activity by bisindolylmaleimide and related compounds had no perceptible effect on growth cone motility, but completely blocked phorbol ester effects. Significantly, MT advance occurred despite continued retrograde F-actin flow—a process that normally inhibits MT advance. Polymer assembly was necessary for PKC-mediated MT advance since it was highly sensitive to a range of antagonists at concentrations that specifically interfere with microtubule dynamics. Biochemical evidence is presented that PKC activation promotes formation of a highly dynamic MT pool. Direct assessment of microtubule dynamics and translocation using the fluorescent speckle microscopy microtubule marking technique indicates PKC activation results in a nearly twofold increase in the typical lifetime of a MT growth episode, accompanied by a 1.7-fold increase and twofold decrease in rescue and catastrophe frequencies, respectively. No significant effects on instantaneous microtubule growth, shortening, or sliding rates (in either anterograde or retrograde directions) were observed. MTs also spent a greater percentage of time undergoing retrograde transport after PKC activation, despite overall MT advance. These results suggest that regulation of MT assembly by PKC may be an important factor in determining neurite outgrowth and regrowth rates and may play a role in other cellular processes dependent on directed MT advance.
protein kinase C; microtubules; axon outgrowth; actin; growth cone
Dynamic cytoskeletal rearrangements are involved in neuronal growth cone motility and guidance. To investigate how cell surface receptors translate guidance cue recognition into these cytoskeletal changes, we developed a novel in vitro assay where beads, coated with antibodies to the immunoglobulin superfamily cell adhesion molecule apCAM or with purified native apCAM, replaced cellular substrates. These beads associated with retrograde F-actin flow, but in contrast to previous studies, were then physically restrained with a microneedle to simulate interactions with noncompliant cellular substrates. After a latency period of ∼10 min, we observed an abrupt increase in bead-restraining tension accompanied by direct extension of the microtubule-rich central domain toward sites of apCAM bead binding. Most importantly, we found that retrograde F-actin flow was attenuated only after restraining tension had increased and only in the bead interaction axis where preferential microtubule extension occurred. These cytoskeletal and structural changes are very similar to those reported for growth cone interactions with physiological targets. Immunolocalization using an antibody against the cytoplasmic domain of apCAM revealed accumulation of the transmembrane isoform of apCAM around bead-binding sites. Our results provide direct evidence for a mechanical continuum from apCAM bead substrates through the peripheral domain to the central cytoplasmic domain. By modulating functional linkage to the underlying actin cytoskeleton, cell surface receptors such as apCAM appear to enable the application of tensioning forces to extracellular substrates, providing a mechanism for transducing retrograde flow into guided growth cone movement.