Investigation on spatio-temporal dynamics of RhoGTPases and their role in neuronal growth cone and actin wave motility

Neurons are highly polarised cells that migrate elongating their axon to reach distant synaptic targets. In the developing nervous system they travel along highly conserved trajectories defined by the molecules present in the surrounding environment, the so-called guidance cues. They can exert the function either at short range by direct contact or at long range, secreted by surrounding and target cells to create gradients that can be sensed by migrating axons. During the PhD course I focused on investigating the spatio-temporal properties of neurons in response to chemical signals. I have studied in detail the morphology changes of Growth Cones (GC) upon local stimulation and the dynamics of signalling cascades regulating actin dynamics, with a particular attention on Rho-GTPases. Moreover I investigated the morphology, molecule composition of axonal Actin Waves (AWs), as well as the role of Rho-GTPases in their inception and movement kinetics. In these studies I adopted various techniques: from live-cell imaging of the actin dynamics in AWs to a combination of FRET imaging and optical manipulation to image the Rho-GTPases activation in GCs real time upon local chemical stimulus delivery. The cellular module designed to perceive the guidance stimuli is the Growth Cone (GC), a specialised structure at the tip of the growing axon divided into three regions. The central region contains organelles and has a structural function, the transition region is formed by acto-myosin contractile arcs and the peripheral region, formed by thin filopodia and veil-like lamellipodia structures, that sustain dynamic protrusion and retraction cycles and express on the surface all the receptors to sense the presence of guidance molecules gradients. The major component of these structures is actin, a molecule that polymerises to form filaments that can be arranged, with the cooperation of a wide variety of actin-binding molecules, into different architectures. Actin filaments are polarised structure with the “barbed” end oriented towards the leading edge and a “pointed” end towards the central region. Filaments undergo continuous cycles of polymerisation at the barbed end and depolymerisation at the pointed end, creating two dynamic behaviours called treadmilling and retrograde flow. The relative prominence of one process over the other is regulated by external signals that are sensed by receptors and initiate different intracellular signalling cascades. These pathways involve a lot of diverse proteins at various levels, but almost all of them pass through a “bottleneck” step: the Rho family of Guanosine Tri-Phosphatases (Rho-GTPases). Rho-GTPases are molecular switches that cycle between activated, GTP-bound state and an inactivated, GDP-bound state. Their dynamics are modulated by upstream signals, and in turn they interact with downstream effectors to propagate the signal transduction to the actin cytoskeleton. A single Rho-GTPase can be regulated by many different molecules, called Guanosine Exchange Factors (GEFs), GTPase domain Activator Proteins (GAPs) and Guanine Nucleotide Dissociation Inhibitors (GDIs), and activate a wide range of cellular responses, depending on the cell type and the stimulus received. They are best known for their roles in the modulation of cytoskeleton rearrangements, cell motility and polarity and axon guidance. They exert their effect mainly by affecting actin dynamics, not only in the growth cone but also in the axon shaft. A particular behaviour of the polarising neuronal cells is the extrusion of GC-like structures that travel along the neurite shaft towards the tip and fuses with the GC to promote elongation. These structures are called Actin Waves (AWs): they have a mean velocity of 2-3 µm/min and appear in a stochastic manner in all the growing neurites with a frequency of about 1-2 waves per hour. Their propagation is strongly dependent on the dynamic behaviour of the actin filaments, with the balance between barbed end polymerisation and pointed end de-polymerisation at its basis. Therefore all those proteins involved in the regulation of actin might have a prominent role in their structure and function, including the RhoGTPases. The main achievements and findings of my PhD are the following: 1. I combined successfully for the first time FRET imaging with optical tweezers to provide a strong tool to study dynamics of intracellular signalling molecules upon local delivery of chemical attractants and repellants. The versatility of the optical tweezers, that have the possibility to exert both contact stimulation and local gradient delivery, along with the precision and high spatio-temporal resolution of the FRET, allowed us to highlight fine spatio-temporal dynamics of Rho-GTPases in live cells. 2. Local repulsive stimulation by semaphorin-3A triggers local retraction of the side of the growth cone facing the stimulus, with distinct RhoGTPases spatio-temporal dynamics: a. I showed, in accordance to previous studies, that the stimulation triggers rapid activation of RhoA within 30 s in the central region of the growth cone, causing a delayed retraction (100-120 s from the stimulus application) that correlates with RhoA activation levels correlate with the induced morphological changes; b. I demonstrated that semaphorin-3A local delivery causes a decrease in Cdc42 activity within 60 s from the stimulation. Activity levels vary in a wave-like retrograde manner that proceeds almost in synchrony with the retraction. In few cases the stimulation induced the formation of active Cdc42 waves that propagate in a region away from the local stimulus and promote the spawning of new filopodia and lamellipodia, suggesting a role of Cdc42 in travelling actin waves; c. I showed that local stimulation with beads coated with semaphorin-3A induces the formation of active Cdc42 waves propagating from the GC edge to the central region with a mean period of 70 s. Same “travelling” waves have been found in some cases of spontaneous retraction in the neuronal cell culture, but they oscillate with a longer period (110 s). These overall data show a more complex behaviour for Cdc42 than RhoA, and provide evidence for a higher degree of complexity in the Rho-GTPase signalling network. 3. Actin dynamics in neuronal actin waves are strongly dependent on Cdc42 and Rac1 activation dynamics. By means of immunofluorescence, STED nanoscopy and live cell imaging with inhibitors for different molecules, we showed that: a. In accordance with previous studies, actin waves are growth cone-like structures that generate at the proximal segment of neurites and then propagate along the shaft towards the growth cone. When it reaches its vicinity, the growth cone retracts and the two structure fuse together to form a new, bigger and more dynamic growth cone that elongates again; b. Myosin-IIB is localised at the rear of the propagating wave, suggesting a possible role of myosin in their dynamics. This role has been confirmed by further experiments in which myosin inhibition with 20 µM blebbistatin highlighted the disruption of the GC-like morphology of actin waves and the disappearing of the GC retraction upon wave incoming at the neurite tip, along with an effect on AW frequency and velocity; c. Membrane tension has a role in maintenance of AW morphology and affects also AW initiation and propagation. Addition of 250 µM of β-cyclodextrin disrupted the GC-like morphology and decreased the AW area of more than 50%. Moreover the treatment decreased the velocity and significantly the frequency of AW initiation, suggesting a major role of the membrane in AW dynamicity; d. Cdc42 and Rac1 have a strong impact on the initiation dynamics of the actin waves. The frequency of actin waves per hour is significantly reduced under 10 µM of both Cdc42 (ML141) and Rac1 (EHT1864) inhibition: from 2-3 waves per hour to about 0,5 and 1 wave per hour, respectively. Moreover, addition of a high concentration (30µM) of ML141 stopped the AW sprouting almost completely, demonstrating a prominent role of these Rho-GTPases in actin wave initiation at the initial segment of the neurite. e. Cdc42 and Rac1 have a role also in the propagation dynamics of actin waves. Inhibition of both GTPases resulted in a significant decrease in the velocity of actin waves, from a mean of 2,2 µm/min to about 1,5 µm/min and 1,2 µm/min respectively. Moreover we observed a disruption of the GC-like morphology of AWs, as well as a reduction in the mean area of about 50%. These results provide new insights for a prominent role of Rho-GTPases in the overall dynamics of the actin cytoskeleton within the travelling waves, in perfect accordance with previously reported data.