Ilaria Tonazzini1,2 and Marco Cecchini1,*
1 NEST, Istituto Nanoscienze-CNR and Scuola Normale Superiore, Piazza San Silvestro 12, 56127 Pisa, Italy
2 Fondazione Umberto Veronesi, Piazza Velasca 5, 20122 Milano, Italy
Abstract: Cell contact interaction with extracellular environment cooperates in
coordinating several physio-pathological processes in vivo, and can be exploited to manipulate cell responses in vitro. Thanks to recent developments in micro/nano- engineering techniques, nano/micro-structured surfaces have been introduced capable of controlling neuronal cell adhesion, differentiation, migration, and neurite orientation by interfering with the cell adhesion machinery. In particular, this process is mediated by focal adhesion (FA) establishment and maturation. FAs cross-talk with the actin fibers and act as topographical sensors, by integrating signals from the extracellular environment. Here, we describe the mechanisms of nanotopography sensing in neuronal cells. In particular, experiments addressing the role of FAs, myosinII- dependent cell contractility, and actin dynamics in neuronal contact guidance along directional nanostructured surfaces are reviewed and discussed.
Keywords: Actin contractility, Contact guidance, Cytoskeleton, Focal adhesions,
Mechano-transduction, Neuron, Nanostructured substrates, Neurite, Nanograting, Nanogroove.
INTRODUCTION
Nowadays it is accepted that cells respond to the morphology of the extracellular environment at the nanoscale level. The contact interaction of cells with extracellular physical features cooperates in regulating physiological (e.g. embryogenesis, cell migration) [1] and pathological processes [2] in vivo, and can be exploited to modulate cell responses in vitro [3 - 5]. In the central nervous system (CNS), the sensing of the extracellular environment combines with intracellular signaling patterns that is integrated by cells to establish the final neuronal polarity, differentiation, migration, neurite path-finding and the final * Corresponding author Marco Cecchini: NEST, Istituto Nanoscienze-CNR and Scuola Normale Superiore, Piazza San Silvestro 12, 56127 Pisa, Italy; Tel/Fax: +39050509459; E-mail: [email protected]
Giovanni Tosi (Ed)
architecture of the functional network of neuronal connections [6 - 8]. The mammalian neuronal network is an example of highly polarized tissue, where cell development is driven by molecular stimuli, acting over long distance, and by physical signals that act locally through direct contact sensing [9, 10]. Neurons polarize and produce long cellular extensions (the neurites) whose development is governed by the formation and maturation of focal adhesions (FAs), the integrin- based cellular structures anchoring the cell to the external environment [6]. During neurite development, focal adhesions act as topographical sensors and integrate both physical and chemical signals from the extracellular matrix [11]. The maturation of FAs is in fact finely tuned by multiple information regarding the extracellular matrix properties such as mechanical stiffness, density of adhesion points, their chemical identity and geometry, and surface topography [12]. Importantly, through the modulation of FA maturation, a specific extracellular configuration can regulate the cell fate and in particular neuronal polarization, migration [13] and function. For example, synaptic plasticity has been recently suggested to involve the surrounding extracellular matrix signaling [14,15]. These processes involve coordinated interactions between FAs and the cell cytoskeleton. In order to build a correct brain architecture, a coordinated rearrangement of the cytoskeleton in response to extracellular cues is essential, and focal adhesion kinase (FAK) was recognized as a key neuronal enzyme [16]. For sake of example, the activation of the FA effector FAK is dispensable for glial-independent migration of interneurons but is required for the normal interaction of pyramidal neurons with radial glial fibers during cortical migration [13]. FAK is also required by both attractive and repulsive stimuli to control cytoskeletal dynamics and axon outgrowth and disassembly, working as a versatile molecular integrator that can switch to different functions depending on its activation site [17].
All cells grow and live while embedded in a dense and complex environment, the extracellular matrix (ECM), which contains an array of structural and directional cues. In particular, beyond chemical recognition, three independent ECM parameters play a major role in governing cell behaviour: topography, stiffness, and density of adhesion points. Topographical features in the micron and submicron range act as physical boundaries providing a local constraint to the formation and maturation of FAs. Cells apply force to the developing adhesions through acto-myosin contractility and the mechanical response of the matrix controls the further maturation of the adhesion points in a molecularly regulated feedback loop. Thanks to recent developments in micro/nano-engineering tools, the processes that control cell and, in particular, neuronal guidance and polarization can now be investigated in vitro using nano/micro-structured surfaces [18 - 21]. Nano/micro-textured substrates were demonstrated to be capable of tuning neuronal and glial cell adhesion, differentiation, polarization, migration,
neurite orientation and even stem-cell fate [3, 22 - 25]. In particular, non- conventional lithographic technologies such as nanoimprint lithography (Fig. 1a), electrospinning, or soft replica moulding to name but a few, allow the production of biocompatible substrates with customable topography in the critical ranges affecting cellular functions. The application of these methods yielded patterned surfaces with lateral resolution ranging from few microns (e.g., photolithography) down to tens of nanometers (e.g., electron beam lithography [26]) and showed good versatility and reliability coupled with the use of polymeric materials [27 - 29]. The combination of these techniques with optically transparent polymers such as tissue culture polystyrene (TCPS), polyethylene terephthalate (PET) and cyclic olefin copolymer (COC) yielded patterned substrates where contact guidance could be observed by means of high resolution microscopy in living cells [28, 30, 31].
Here we focus on reviewing the influence of topography on the responses of neuronal cells. In order to control the assembly and maturation of FAs, and thus to induce specific cellular guidance, biomimetic scaffolds can provide modulation of the topographical parameters within the physiological ranges that are resolved by cells. The physical parameters in the substrate topography, which were reported to modulate contact guidance and formation of FAs in mammalian cells are the size, aspect ratio, and lateral spacing of the topographical features together with the isotropy and degree of disorder of the pattern.
In this framework, governing neuronal cell adhesion, migration, and axonal outgrowth are critical elements for regenerative medicine applications and for developing artificial neuronal interfaces, but at the same time these substrates open new experimental perspectives for the study of the molecular mechanisms at the base of neuronal environmental sensing. The molecular/cellular processes regulating synaptic plasticity, and learning are in fact an adaptation of the mechanisms used by all cells to regulate cell motility and shaping [32] as they involve the same complex machinery (i.e. actin fiber regulation and cell-cell/- ECM interaction signalling) where also the activation of FAs is emerging as pivotal [33]. Therefore, new knowledge about the sensing mechanisms of neuronal cells might impact also our understanding of several CNS disorders and providing new insight into the mechanisms leading to several neurological and neuropsychiatric disorders associated with connectivity and cognitive impairments.
Focal Adhesions and Cytoskeleton during Neuronal Cell-nanograting Interaction
alternating lines of grooves and ridges with sub-micrometer lateral dimension (Fig. 1), have been intensively studied and turned up as one of the most compelling systems for inducing neuronal alignment via pure cell mechanotransduction. In detail, nano/microgratings induced qualitatively similar effects on single cell polarization, and neurite network development in several different neuronal models, such as differentiating PC12 and neuroblastoma cells [20, 27, 31], invertebrate leech neurons [34], neuro-differentiated murine embryonic stem cells and primary murine hippocampal neurons [24] (Fig. 2). Overall, NGs with linewidths in the 500 nm – 2 μm range can induce neuronal polarization and neurite alignment along the substrate direction, with different efficiency according to the cell morphological characteristics.
Fig. (1). Anisotropic nanogratings (NGs). a) Scheme of the fabrication process: the silicon molds are
fabricated by electron beam lithography (EBL) and reactive-ion etching (RIE); then thermal nano-imprint lithography (NIL) is exploited to transfer the nanopattern from the molds to thermoplastic materials as the copolymer 2-norbornene ethylene (COC). b-d) SEM images of different COC NGs: NGs with ridge width (= groove width) of 500 nm (b), 1 μm (c), 2 μm (d), and depth of 350 nm; scale bars = 1 μm. e-g) SEM images of COC noisy NGs: an increasing amount of random nano-modifications is inserted in NGs with ridge width (= groove width) of 500 nm, varying from 10% (e), to 40% (f), and 80% (g). The presence of noise leads to the modulation of substrate directionality; scale bars = 2 μm. h) Image of a thermoplastic substrate with a squared NG patterned area; inset= detailed SEM image of the NG pattern, with ridges (R) and grooves (G) of 1 μm width; scale bar = 1 μm.
COC mold mold scaffold a) b) e) h) c) d) g) f) R G
Fig. (2). Confocal images of different neuronal cells cultured on thermoplastic nanogratings. On 1-µm-line
width NGs: a) A leech neuron grown on T2 and immunostained for Tubulin-α (green) and actin (red); b) SH- SY5Y differentiated cells on T2 and immunostained for Tubulin-βIII (green) and actin (red). Smaller neuronal cells grown on 500 nm-line width NGs: c) F11 cells immunostained for vinculin (green) and actin (red); d) PC12 cells immunostained for Tubulin-βIII (green) and actin (red); e) Mouse embrionic stem cells differentiated in neurons and immunostained for Tubulin-βIII (green); f) murine hippocampal neurons (day 14 in vitro) immunostained for Tubulin-α (green). All samples have been stained with DAPI to visualize nuclei (blue). Scale bars = 30 μm; inset = NG direction. Modified from [34].
The interaction of differentiating PC12 cells with NGs with groove depth between 250 and 500 nm results in filopodium dynamical sensing and neurite spatially selective consolidation, leading to a highly specific bipolar cell morphotype, and neurite alignment to the NG tracks [27, 35]. The result of the interaction between differentiated PC12 cells and the NG is an angular modulation of neurite persistence: the aligned neurites receive a positive feedback and persist longer than misaligned neurites and even longer than neurites growing on flat substrates [36]. Importantly, the shift from a multipolar to a bipolar morphology and the development of two aligned neurites on NGs are based on an active reading of the substrate topography.
This bipolarity selection is obtained by a spatial modulation of FA reinforcement. After the initial contact with the extracellular matrix (ECM), integrin receptors cluster and begin the enrollment of several cellular components thus inducing the formation of a cytoplasmic protein complex, the adhesion plaque. Adhesion plaques grow and enlarge in a hierarchical fashion increasing in size and enriching in characteristic proteins such as vinculin, focal adhesion kinase, talin, and
a)
b)
c)
d) f)
paxillin. In this way, initial small contacts (<1 μm2) maturate into larger FAs,
whose maturation is regulated by a local balance between the force generated by cell contractility and ECM [12]. Mature FAs finally establish connections with actin fibers and can thus set up the cell shape. The maturation of FAs, regulation of actin filament interactions, and cell contractility cooperate to translate ECM guidance cues into specific cell polarity states [37].
NGs designed for high-resolution live-cell microscopy have been recently exploited to study the dynamics of focal adhesions, cell contractility, and actin fibers in neuronal contact guidance [36]. By taking advantage from a fluorescently-tagged paxillin vector, it was found that FAs are generally formed at the neurite tips and develop exclusively on top of NG ridges (Fig. 3a). Neuronal polarization and contact guidance are based on a geometrical constraint of FAs resulting in an angular modulation of their maturation and endurance. In fact, cell contractility induces the maturation of nascent adhesions at the tip of aligned neurites (within 15 ° to the direction of the NG), whereas those produced at the tip of misaligned processes remain immature and are eventually lost. This allows the consolidation of aligned neurites and drives the collapse of the misaligned ones. Nanotopographies able to interfere with focal adhesion development during neuronal differentiation were then designed [3]. By varying a single topographical parameter (i.e. ridge width) the orientation and maturation of FAs was finely modulated producing an independent control over the number and the growth direction of neurites in neurodifferentiated PC12 cells (Fig. 3b). In this study, it was possible to establish that neurite pathfinding is responsive to ridge-width variations between 500 and 1000 nm, while the mechanism controlling neuronal polarity establishment is sensitive for variations in ridge width within 1000 and 1500 nm [3].
In this scenario, ROCK-mediated contractility contributes to polarity selection during neuronal differentiation. In fact, in the neurites developing on NGs ROCK-mediated myosin-II contractility cross-talks with the adhesions, with the outcome that only aligned adhesions are allowed to grow, while misaligned ones, blocked by the topographical constraint, cannot develop into mature FAs [36]. Consistently, when the FA maturation is inhibited by the block of ROCK or of myosin-II activities during the interaction between cells and topographical features, the mechanism selecting bipolar PC12 cells with two aligned neurites fails to transfer the substrate symmetry to the cells [36], thus confirming that FAs directly interact with the actin cytoskeleton and thus tune actin-dependent contractility during neuronal polarization.
Fig. (3). Angular modulation of focal adhesion size. a) Distribution of paxillin-EGFP fluorescent signal in
NGF-differentiating PC12 cells on NGs (left) and on flat substrate (right). In the lower row, inverted fluorescent signal at the cell-substrate interface shows the paxillin-EGFP accumulation in FAs (black spots). Black arrows = NG direction. b) Focal adhesions constrain on NGs with increasing ridge width. For each nanograting the corresponding ridge width is reported. The cartoons depict a schematic model of the relative size of aligned and misaligned focal adhesions (red rectangles) growing on top of ridges (light grey lanes).
a)
b)
nanograting
Ridge width Aligned FA
flat
Misaligned FA Selected Polarity 500 nm 1000 nm 2000 nm groove ridge FA bipolar with aligned neurites bipolar with misaligned neurites misaligned neurites multipolar with
The importance of actin fiber contractility also emerged at growth cone level. In fact, growth cones (GCs) - dynamic structures rich in actin filaments at the tips of neurites - probe external information by filopodia and integrate multiple physico- chemical cues. Filopodia sensing governed cell contact guidance, in leech neurons. Indeed, neurite topographical reading was mediated by an anisotropic filopodium organization in the growth cones on NGs, thus presenting the aligned filopodia longer and richer in fibrous actin [34].
Neuronal Differentiation Drives Nanotopography Sensing
In the PC12 neuronal model the substrate reading process occurs upon induction of neuronal differentiation. Importantly, the topographical guidance and neurite pathfinding of PC12 cells requires the stimulation of the neurotrophic growth factor (NGF)-induced neuronal differentiation signaling [30]. In addition, the behavior of the human neuroblastoma cell line SHSY5Y on anisotropic nanotopographies was probed, in proliferative or differentiating conditions, and a similar response to that of PC12s was found upon differentiation with retinoic acid (RA) and brain-derived neurotrophic factor (BDNF) [24]. Specifically, RA/BDNF differentiation ameliorated neuronal alignment, resulting in an approximately fourfold improvement of the tubulin cytoskeleton orientation along the substrate topography. This implies that common molecular mechanisms are involved for nanotopography sensing and that a different stimulation of different neuronal cells can modulate their response to similar topographical patterns. Alternative neuro-differentiation protocols were also tested in PC12s, by using activators of the cAMP pathway [i.e. Forskolin, an adenylyl cyclase activator resulting in increased levels of cyclic adenosine monophosphate (cAMP); 8CPT- 2Me-cAMP, an agonist activating a protein in cAMP signalling pathway downstream adenylyl cyclase and cAMP (EPAC) and PKA independent] [30]. Several differentiation protocols based on forskolin led to neuronal-like differentiation but less efficient neurite alignment along the NGs [30]. In PC12s the topographical guidance was reduced by forskolin with respect to what measured for NGF, and this effect was correlated to an interference with FA maturation. These results notify that a fine regulation exists, during neuronal development, which controls the response of growing neurites to the ECM signals. In line with these findings, recently it has also been demonstrated that several neuronal sub-populations, or even the same neuronal types but at different development stage, can react differently to the same guidance cue during migration and differentiation [13, 14, 38].
Reading of the topographical guidance cues is not a univocal process but a function of the molecular differentiation pathway that is active in the cell. The
complex interaction between substrate features, neurotrophic molecules, and cellular adhesion system cooperate to define the neuronal cell shaping during topographical guidance. Thus, molecular modulation of contact sensing appears to have a deep impact on the neuronal response and it must be taken in account for the development of nanostructures as neuro-regenerative scaffolds.
Noise Tolerance in Neuronal Contact Guidance
Since NGs were shown to be powerful tools to investigate topographical reading, novel NG geometries were introduced with the aim to study neurite contact guidance in the presence of topographical noise. It is well established that cells in tissues are exposed to nanotopographical stimuli determined by the detail of the local micro/nano-environment [8] and that directional textures typically necessarily coexists with bio-topographical noise. Examples are protein misfolding, sclerotic plaques, defects of ECM proteins or of their proteolysis, mutations in collagen fiber banding, scar-tissue invasion, formation of gaps and/or neuroma after nerve injury. The impact of noise on neuronal topographical sensing was investigated in vitro exploiting NG scaffolds with a controlled level of anisotropy (Fig. 1e-g). In detail, neuron feedback on imperfect directional stimuli was studied by using NGs with a controlled degree of random nanotopographical noise and overall substrate directionality (noisy NGs) [39]. Reduced substrate directionality induced a sigmoidal loss of neurite alignment with a clear threshold response, showing that neurite path-finding is tolerant to a certain degree of noise and that it can successfully read hidden topographical information. Nanotopographical noise impacted FA maturation and alignment, which exhibited a non-linear behavior correlating with that of neurite alignment [40].
Given that myosin-II contractility signaling was demonstrated to regulate neuronal mechanotransduction [36], the effect of pharmacological treatments targeting this pathway (e.g. blebbistatin and nocodazole) on FA maturation and neurite navigation along noisy NGs was also investigated. Importantly, the noise tolerance of neurite guidance resulted improved by increased cell contractility [40]. Once the neurite NGF-stimulated extension started, nocodazole (i.e. a potent microtubule depolymerizing agent that can increase cell contractility via Rho-A pathway activation if administrated at low nM concentration) improved neurite alignment on noisy NGs, shifting the alignment threshold to higher noise levels; the opposite effect was instead caused by blebbistatin, a drug that inhibits cell contractility (Fig. 4a). Neurite path-finding is thus capable through FAs of read partially ordered topographies, as expected given the presence of physiological structural noise in the extracellular environment in vivo.
Fig. (4). Noise tolerance in neuronal contact guidance. a) Scheme of neurite guidance to noisy directional
stimuli. Neurite guidance does not decrease linearly with substrate directionality, but it is a threshold process with a sigmoidal trend. Nocodazole increases cell contractility and improves neurite alignment (grey line). Conversely, the administration of blebbistatin, myosin-II inhibitor, affects the neurite guidance and leads to an overall reduction of alignment (blue line). b) Scheme of WT (blue) and Ube3a-deficient (AS) (red) hippocampal neurons development on anisotropic NGs (where they show different contact guidance responses) versus flat isotropic standard surfaces (where they behave the same), at early developmental stage
in vitro. Representative confocal images of WT and AS neurons on 1 µm-width NGs (insets) grown for one
week and immunostained for actin (red), tubulin (green) and nuclei (blue).
Neuronal contact guidance was also studied in “noisy” neurons on perfect nanogratings. A “noisy” neuron can be interpreted as a neuron with a molecular impairment, such as a neuronal model of a pathological disorder (in vitro). Although deficits in brain micro-connectivity have been recently implicated in the pathogenesis of cognitive disorders (e.g. autism, schizophrenia) [41, 42], the role
45o 30o 15o 0o 0 5 10 15 Directionality (d) contractility contractility a) Neurite alignment b) WT = AS WT = AS
of neuronal contact sensing mechanisms in these pathological conditions is under- investigated. Recent findings indicate that during neuronal development the pathways that regulate extracellular sensing (e.g. adhesion, cytoskeleton) are regulated by ubiquitination, in particular through E3 ubiquitin ligases, which function by ligating ubiquitin to specific target proteins [43]. In fact, many E3 ubiquitin ligases emerged as key regulators of several aspects of neuronal morphogenesis and connectivity at distinct temporal stages [43, 44]. Among these, Ubiquitin E3a ligase (UBE3A) is critical for neurodevelopment, as its loss leads to the neurocognitive disorder Angelman Syndrome (AS; OMIN 105830)