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 \u201cbarbed\u201d end oriented towards the leading edge and a \u201cpointed\u201d 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 \u201cbottleneck\u201d 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 \ub5m/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 \u201ctravelling\u201d 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 \ub5M 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 \ub5M of \u3b2-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 \ub5M 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 (30\ub5M) 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 \ub5m/min to about 1,5 \ub5m/min and 1,2 \ub5m/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

MAPping out distribution routes for kinesin couriers

In the crowded environment of eukaryotic cells, diffusion is an inefficient distribution mechanism for cellular components. Long-distance active transport is required and is performed by molecular motors including kinesins. Furthermore, in highly polarized, compartmentalized and plastic cells such as neurons, regulatory mechanisms are required to ensure appropriate spatio-temporal delivery of neuronal components. The kinesin machinery has diversified into a large number of kinesin motor proteins as well as adaptor proteins that are associated with subsets of cargo. However, many mechanisms contribute to the correct delivery of these cargos to their target domains. One mechanism is through motor recognition of subdomain-specific microtubule (MT) tracks, sign-posted by different tubulin isoforms, tubulin post-translational modifications (PTMs), tubulin GTPase activity and MT associated proteins (MAPs). With neurons as a model system, a critical review of these regulatory mechanisms is presented here, with particular focus on the emerging contribution of compartmentalised MAPs. Overall, we conclude that – especially for axonal cargo – alterations to the MT track can influence transport, although in vivo, it is likely that multiple track-based effects act synergistically to ensure accurate cargo distribution

Characterizing the Role of the Actin-binding Protein, TMD-1/tropomodulin in \u3ci\u3eC. elegans\u3c/i\u3e Excretory Cell Morphogenesis

Tropomodulins are proteins widely expressed in all complex animals that help to regulate the shape of cells by modifying the cytoskeletal filament actin. C. elegans worms lacking the tropomodulin TMD-1 show defects in development of the excretory cell, which acts as a kidney for the worm. The excretory cell extends four canals out from the cell body so that the entire cell looks like a great letter H stretching the length of the worm. In tmd-1 mutants, canals extend partially or not at all, and develop a dramatic crinkled appearance. The mechanism for this interference is unknown; however several possibilities are being explored. Excretory canals are often affected by mutations in genes, also needed in neuronal axon guidance. Knocking down one of these guidance proteins, MIG-10, produced a similar phenotype to that of the tmd-1 mutants. However, further experiments did not uncover any neuronal guidance defects in tmd-1 mutants. Instead, it is more likely that TMD-1 is essential for vesicle trafficking within the excretory canal. In this function TMD-1 may facilitate canal extension by providing membrane components to the growing canal tips. Insight into tropomodulin’s role in single-celled tube formation can further our understanding of how these tubes form in many organisms

The virtuous cycle of axon growth: Axonal transport of growth-promoting machinery as an intrinsic determinant of axon regeneration

Injury to the brain and spinal cord has devastating consequences because adult central nervous system (CNS) axons fail to regenerate. Injury to the peripheral nervous system (PNS) has a better prognosis, because adult PNS neurons support robust axon regeneration over long distances. CNS axons have some regenerative capacity during development, but this is lost with maturity. Two reasons for the failure of CNS regeneration are extrinsic inhibitory molecules, and a weak intrinsic capacity for growth. Extrinsic inhibitory molecules have been well characterised, but less is known about the neuron-intrinsic mechanisms which prevent axon re-growth. Key signalling pathways and genetic / epigenetic factors have been identified which can enhance regenerative capacity, but the precise cellular mechanisms mediating their actions have not been characterised. Recent studies suggest that an important prerequisite for regeneration is an efficient supply of growth-promoting machinery to the axon, however this appears to be lacking from non-regenerative axons in the adult CNS. In the first part of this review, we summarise the evidence linking axon transport to axon regeneration. We discuss the developmental decline in axon regeneration capacity in the CNS, and comment on how this is paralleled by a similar decline in the selective axonal transport of regeneration-associated receptors such as integrins and growth factor receptors. In the second part, we discuss the mechanisms regulating selective polarised transport within neurons, how these relate to the intrinsic control of axon regeneration, and whether they can be targeted to enhance regenerative capacity.ERA‐NET Neuron International Foundation for Research in Paraplegia Christopher and Dana Reeve Foundation. Grant Numbers: JFC‐2013(3), JFC‐2013(4) Gates Cambridge Trust Medical Research Council. Grant Numbers: G1000864 018556, MR/R004463/

A spatially specified systems pharmacology therapy for axonal recovery after injury

There are no known drugs or drug combinations that promote substantial central nervous system axonal regeneration after injury. We used systems pharmacology approaches to model pathways underlying axonal growth and identify a four-drug combination that regulates multiple subcellular processes in the cell body and axons using the optic nerve crush model in rats. We intravitreally injected agonists HU-210 (cannabinoid receptor-1) and IL-6 (interleukin 6 receptor) to stimulate retinal ganglion cells for axonal growth. We applied, in gel foam at the site of nerve injury, Taxol to stabilize growing microtubules, and activated protein C to clear the debris field since computational models predicted that this drug combination regulating two subcellular processes at the growth cone produces synergistic growth. Physiologically, drug treatment restored or preserved pattern electroretinograms and some of the animals had detectable visual evoked potentials in the brain and behavioral optokinetic responses. Morphology experiments show that the four-drug combination protects axons or promotes axonal regrowth to the optic chiasm and beyond. We conclude that spatially targeted drug treatment is therapeutically relevant and can restore limited functional recovery

The Function of Rab35 in Development and Disease

Rab35 mediates membrane trafficking between the plasma membrane and the early endosomes at the cell surface. Our understanding of the cellular function of Rab35 reveals its role in development and diseases. In the developmental context, Rab35 has been shown to play an important role in regulating epithelial polarity, lumen opening, myoblast fusion, intercalation of epithelium, myelination, neurite outgrowth, and oocyte meiotic maturation. Disruption of recycling endosome mediated by Rab35 has been linked to several neurological diseases, including Parkinson’s disease and Down syndrome. In addition, because Rab35 modulates cell migration through its interaction with various effectors, Rab35 plays an important role in cancers. Lastly, the Rab35-mediated recycling endosomal pathway and exocytosis is utilized by pathogens or hijacked by pathogens to promote their infection and survival. This review summarizes the function of Rab35 in endocytosis and focuses on the role of Rab35 in the context of development and diseases

Microtubule plus-end binding protein CLASP2 in neural development

Normal brain function is dependent on the correct positioning and connectivity of neurons established during development. The Reelin signaling pathway plays a crucial role in cortical lamination. Reelin is a secreted glycoprotein that exerts its function by binding to lipoprotein receptors and inducing tyrosine phosphorylation of the intracellular adaptor protein Dab1. Mutations in genes of the Reelin signaling pathway lead to profound defects in neuronal positioning during brain development in both mice and humans. However, the molecular mechanisms by which Reelin controls neuronal morphology and migration are unknown. We have used a systems analysis approach to identify genes perturbed in the Reelin signaling pathway and identified microtubule stabilizing CLIP-associated protein 2 (CLASP2) as a key cytoskeletal modifier of Reelin mutant phenotypes. Currently, little is known about the role of CLASP2 in the developing brain. We propose that CLASP2 is a key effector in the Reelin signaling pathway controlling basic aspects of cortical layering, neuronal morphology, and function. CLASP2 is a plus-end tracking protein and this localization places CLASP2 in a strategic position to control neurite outgrowth, directionality, and responsiveness to extracellular cues. Our results demonstrate that CLASP2 expression correlates with neurite length and synaptic activity in primary neuron cultures; however, the role of CLASP2 during brain development was unknown. In this dissertation, we have characterized the role of CLASP2 during cortical development by in utero electroporation of shRNA plasmids and found that silencing CLASP2 in migrating neurons leads to mislocalized cells at deeper cortical layers, abnormal positioning of the centrosome-Golgi complex, and aberrant length/orientation of the leading process. In addition, we found that GSK3β-mediated phosphorylation of CLASP2 controls Dab1 binding and is required for regulating CLASP2 effects on neuron morphology and migration. This dissertation provides the first steps in gaining insight into how Reelin signaling affects cytoskeletal reorganization to regulate fundamental features of neuronal migration, positioning and morphogenesis

Paxillin facilitates timely neurite initiation on soft-substrate environments by interacting with the endocytic machinery.

Neurite initiation is the first step in neuronal development and occurs spontaneously in soft tissue environments. Although the mechanisms regulating the morphology of migratory cells on rigid substrates in cell culture are widely known, how soft environments modulate neurite initiation remains elusive. Using hydrogel cultures, pharmacologic inhibition, and genetic approaches, we reveal that paxillin-linked endocytosis and adhesion are components of a bistable switch controlling neurite initiation in a substrate modulus-dependent manner. On soft substrates, most paxillin binds to endocytic factors and facilitates vesicle invagination, elevating neuritogenic Rac1 activity and expression of genes encoding the endocytic machinery. By contrast, on rigid substrates, cells develop extensive adhesions, increase RhoA activity and sequester paxillin from the endocytic machinery, thereby delaying neurite initiation. Our results highlight paxillin as a core molecule in substrate modulus-controlled morphogenesis and define a mechanism whereby neuronal cells respond to environments exhibiting varying mechanical properties