Cytoskeletal scaffolds in neuronal development

Neuronal polarization is one of the most studied topics in neuroscience. In less than seven days neurites sprout out from the neuron, explore the surrounding environment and mature in axon or dendrites. This process is possible because the neuronal cytoskeleton can rapidly modify its architecture changing neuronal shape and length. Among all, two main proteins are involved: tubulin that supports the neurite elongation and builds a solid frame, while actin supports pathfinding. In this period of important cytoskeletal changes, it is possible to observe the actin waves (AW) that are highly dynamic structures emerging at the neurite base which move up to its tip, causing a transient retraction of the growth cone (GC). Since their discovery in 1988, there have been only few studies about AWs, usually linked to the neurite outgrowth and axon elongation. In the present work, I used long term live cell imaging to investigate alternative roles of such cytoskeletal phenomena. I examined in details AWs and I concluded that they do not promote the neurite outgrowth and that neurites can elongate for hundreds of microns without the AWs. Super resolution nanoscopy indicates that myosin II shapes the GC like AWs structure. The highly concentrated myosin inside the wave can bend the tubulin that support the neurite provoking twists and kinks in the microtubular cytoskeleton. These tubulin twists (TT) cause the GC retraction and are completely abolished with the inhibition of myosin II, that compromises the AW morphology. My results indicate that myosin II has an important role in the AWs dynamics and can bend the tubulin in a way that was not previously observed. Finally, we suggested a role for AWs and TTs in GC exploration and in neurite maturation. Part of these results have already been published in Frontiers in Cellular Neuroscience in the article: Actin waves do not boost neurite outgrowth in the early stages of neuron maturation. We have a second manuscript in preparation entitled \u201cTubulin twists drive Growth Cone retraction and promote tubulin mixed polarity\u201d

Microtubule dynamics in cytoskeleton, neurodegenerative and psychiatric disease

Microtubules (MTs) are fundamental polymers composed by α and β tubulin, they provide integrity to neuronal cell and are necessaries in intracellular trafficking and organization. The extension and retraction of MTs occur with the addition or removal of α and β tubulin subunits and the binding with microtubule associated proteins (MAPs) that selectively target specific tubulin regions, manipulating the MT dynamics and function. Altered MT homeostasis can compromise the function of MTs in the structural integrity and axonal transport inside the neuron. Here I review the evidence of MT anomalies in several neurodegenerative diseases, including Alzheimer's disease, Parkinson disease, amyotrophic lateral sclerosis and traumatic brain injury and psychiatric disorders, such as depression, schizophrenia, and bipolar disorder. The focus of this review is to point out which can be the impact of MT issues in the major neurodegenerative diseases and discuss which MT abnormalities can lead to psychiatric illnesses

Unfolding and identification of membranproteins in situ

Single-molecule force spectroscopy (SMFS) uses the cantilever tip of an atomic force microscope (AFM) to apply a force able to unfold a single protein. The obtained force-distance curve encodes the unfolding pathway, and from its analysis it is possible to characterize the folded domains. SMFS has been mostly used to study the unfolding of purified proteins, in solution or reconstituted in a lipid bilayer. Here, we describe a pipeline for analyzing membrane proteins based on SMFS, which involves the isolation of the plasma membrane of single cells and the harvesting of force-distance curves directly from it. We characterized and identified the embedded membrane proteins combining, within a Bayesian framework, the information of the shape of the obtained curves, with the information from mass spectrometry and proteomic databases. The pipeline was tested with purified/reconstituted proteins and applied to five cell types where we classified the unfolding of their most abundant membrane proteins. We validated our pipeline by overexpressing four constructs, and this allowed us to gather structural insights of the identified proteins, revealing variable elements in the loop regions. Our results set the basis for the investigation of the unfolding of membrane proteins in situ, and for performing proteomics from a membrane fragment

Actin waves do not boost neurite outgrowth in the early stages of neuron maturation

During neurite development, Actin Waves (AWs) emerge at the neurite base and move up to its tip, causing a transient retraction of the Growth Cone (GC). Many studies have shown that AWs are linked to outbursts of neurite growth and, therefore, contribute to the fast elongation of the nascent axon. Using long term live cell-imaging, we show that AWs do not boost neurite outgrowth and that neurites without AWs can elongate for several hundred microns. Inhibition of Myosin II abolishes the transient GC retraction and strongly modifies the AWs morphology. Super-resolution nanoscopy shows that Myosin IIB shapes the growth cone-like AWs structure and is differently distributed in AWs and GCs. Interestingly, depletion of membrane cholesterol and inhibition of Rho GTPases decrease AWs frequency and velocity. Our results indicate that Myosin IIB, membrane tension, and small Rho GTPases are important players in the regulation of the AW dynamics. Finally, we suggest a role for AWs in maintaining the GCs active during environmental exploration

Mechanosensitivity is an essential component of phototransduction in vertebrate rods.

Photoreceptors are specialized cells devoted to the transduction of the incoming visual signals. Rods are able also to shed from their tip old disks and to synthesize at the base of the outer segment (OS) new disks. By combining electrophysiology, optical tweezers (OTs), and biochemistry, we investigate mechanosensitivity in the rods of Xenopus laevis, and we show that 1) mechanosensitive channels (MSCs), transient receptor potential canonical 1 (TRPC1), and Piezo1 are present in rod inner segments (ISs); 2) mechanical stimulation-of the order of 10 pN-applied briefly to either the OS or IS evokes calcium transients; 3) inhibition of MSCs decreases the duration of photoresponses to bright flashes; 4) bright flashes of light induce a rapid shortening of the OS; and 5) the genes encoding the TRPC family have an ancient association with the genes encoding families of protein involved in phototransduction. These results suggest that MSCs play an integral role in rods' phototransduction

Unfolding and identification of membrane proteins in situ

Single-molecule force spectroscopy (SMFS) uses the cantilever tip of an atomic force microscopy (AFM) to apply a force able to unfold a single protein. The obtained force-distance curve encodes the unfolding pathway, and from its analysis it is possible to characterize the folded domains. SMFS has been mostly used to study the unfolding of purified proteins, in solution or reconstituted in a lipid bilayer. Here, we describe a pipeline for analyzing membrane proteins based on SMFS, which involves the isolation of the plasma membrane of single cells and the harvesting of force-distance curves directly from it. We characterized and identified the embedded membrane proteins combining, within a Bayesian framework, the information of the shape of the obtained curves, with the information from mass spectrometry and proteomic databases. The pipeline was tested with purified/reconstituted proteins and applied to five cell types where we classified the unfolding of their most abundant membrane proteins. We validated our pipeline by overexpressing four constructs, and this allowed us to gather structural insights of the identified proteins, revealing variable elements in the loop regions. Our results set the basis for the investigation of the unfolding of membrane proteins in situ, and for performing proteomics from a membrane fragment

Unfolding and identification of membrane proteins in situ

Single-molecule force spectroscopy (SMFS) uses the cantilever tip of an atomic force microscopy (AFM) to apply a force able to unfold a single protein. The obtained force-distance curve encodes the unfolding pathway, and from its analysis it is possible to characterize the folded domains. SMFS has been mostly used to study the unfolding of purified proteins, in solution or reconstituted in a lipid bilayer. Here, we describe a pipeline for analyzing membrane proteins based on SMFS, which involves the isolation of the plasma membrane of single cells and the harvesting of force-distance curves directly from it. We characterized and identified the embedded membrane proteins combining, within a Bayesian framework, the information of the shape of the obtained curves, with the information from mass spectrometry and proteomic databases. The pipeline was tested with purified/reconstituted proteins and applied to five cell types where we classified the unfolding of their most abundant membrane proteins. We validated our pipeline by overexpressing four constructs, and this allowed us to gather structural insights of the identified proteins, revealing variable elements in the loop regions. Our results set the basis for the investigation of the unfolding of membrane proteins in situ, and for performing proteomics from a membrane fragment

Unfolding and identification of membranproteins in situ

Single-molecule force spectroscopy (SMFS) uses the cantilever tip of an atomic force microscope (AFM) to apply a force able to unfold a single protein. The obtained force-distance curve encodes the unfolding pathway, and from its analysis it is possible to characterize the folded domains. SMFS has been mostly used to study the unfolding of purified proteins, in solution or reconstituted in a lipid bilayer. Here, we describe a pipeline for analyzing membrane proteins based on SMFS, which involves the isolation of the plasma membrane of single cells and the harvesting of force-distance curves directly from it. We characterized and identified the embedded membrane proteins combining, within a Bayesian framework, the information of the shape of the obtained curves, with the information from mass spectrometry and proteomic databases. The pipeline was tested with purified/reconstituted proteins and applied to five cell types where we classified the unfolding of their most abundant membrane proteins. We validated our pipeline by overexpressing four constructs, and this allowed us to gather structural insights of the identified proteins, revealing vari-able elements in the loop regions. Our results set the basis for the investigation of the unfolding of membrane proteins in situ, and for performing proteomics from a membrane fragment