Less than 5 Netrin-1 molecules initiate attraction but 200 Sema3A molecules are necessary for repulsion

Pinato, Giulietta; Cojoc, D.; Lien, Linh Thuy; Ansuini, Alessio; Ban, Jelena; D’Este, Elisa; Torre, Vincent

doi:10.1038/srep00675

Cited by 23 publications

(30 citation statements)

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“…The number of molecules reaching the leading edge depends on the starting concentration of the molecules inside the vesicle, distance between the growth cones and vesicle, and the diffusion coefficient of molecules (Pinato et al, 2012). Assuming the concentration inside the vesicle and the size of vesicle are known, we calculated the spatial and temporal distribution of the concentration of the molecules at the growth cones (see Materials and Methods).…”

Section: Results Recprp Induces Neurite Outgrowth and Rapid Growth Comentioning

confidence: 99%

“…To test whether recPrP molecules can directly influence neuronal growth cone steering, we used an in vitro assay for axon guidance based on a local stimulation technique (Pinato et al, 2012). This technique employs infrared (IR) laser tweezers to trap and position a lipid vesicle carrying guidance molecules in the proximity of the cell.…”

Section: Results Recprp Induces Neurite Outgrowth and Rapid Growth Comentioning

confidence: 99%

“…The solution obtained was then saturated with nitrogen and stored at −20°C. Vesicles with a diameter of 1-5 µm were obtained by using the lipid film rehydration method (Hub et al, 1982;Pinato et al, 2012). In this method, lipid solution is dried in vacuum conditions for 24 h and then the lipid film is rehydrated with solution containing the desired concentration of recPrP.…”

Section: Vesicle Preparationmentioning

confidence: 99%

“…However, this approach exposed cells to constant molecular concentrations and, hence, did not allow focal stimulation of specific neuronal compartments. In the present study, we performed experiments in conditions mimicking the interaction between a single growth cones and a gradient of soluble recPrP, taking advantage of a recently developed technique for local delivery of molecules encapsulated in phospholipid vesicles (Pinato et al, 2012;Chiu, 2003, 2005). Our results reveal a new molecular function of recPrP as a guidance molecule, which might interact with like molecules (homophilic interaction) on the neighboring cells to regulate signal transduction.…”

Section: Introductionmentioning

confidence: 99%

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Characterization of prion protein function by focal neurite stimulation

Amin

Nguyen

Rolle

et al. 2016

Journal of Cell Science

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The cellular prion protein (PrP C ), encoded by the PRNP gene, is a ubiquitous glycoprotein, which is highly expressed in the brain. This protein, mainly known for its role in neurodegenerative diseases, is involved in several physiological processes including neurite outgrowth. By using a novel focal stimulation technique, we explored the potential function of PrP C , in its soluble form, as a signaling molecule. Thus, soluble recombinant prion proteins (recPrP) encapsulated in micro-vesicles were released by photolysis near the hippocampal growth cones. Local stimulation of wild-type growth cones with full-length recPrP induced neurite outgrowth and rapid growth cone turning towards the source. This effect was shown to be concentration dependent. Notably, PrP C -knockout growth cones were insensitive to recPrP stimulation, but this property was rescued in PrPknockout growth cones expressing GFP-PrP. Taken together, our findings indicate that recPrP functions as a signaling molecule, and that its homophilic interaction with membrane-anchored PrP C might promote neurite outgrowth and facilitate growth cone guidance.

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Section: Results Recprp Induces Neurite Outgrowth and Rapid Growth Comentioning

confidence: 99%

Section: Results Recprp Induces Neurite Outgrowth and Rapid Growth Comentioning

confidence: 99%

Section: Vesicle Preparationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Characterization of prion protein function by focal neurite stimulation

Amin

Nguyen

Rolle

et al. 2016

Journal of Cell Science

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“…Sema3A molecules required to achieve guidance [162]. However, only one axon can be observed per experiment, severely limiting the throughput of these techniques.…”

Section: Diffusion-based Chemotaxis Assaysmentioning

confidence: 99%

Mathematical modelling of axon guidance in chemical gradients

Nguyen¹

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Correct wiring is crucial to the proper functioning of the nervous system. Key signals guiding growth cones, the motile tips of developing axons, to their targets are molecular gradients. The receptors on the membrane of the growth cone bind stochastically with guidance molecules in the environment, giving an estimate of the direction of the gradient. The growth cone then executes biased random movements to navigate toward the chemoattractant source or away from the chemorepellent source. This thesis addresses two questions: 1) how the positioning and diffusion of receptors on the growth cone membrane affect the accuracy of gradient estimation, 2) how trajectories are influenced by chemical gradients.Membrane receptors can diffuse freely on the membrane, smearing out the directional information about the gradient. It was unknown how the positioning and diffusion of receptors affect the estimate of the gradient. To address question (1) above, we utilise an ideal-observer approach, assuming that the growth cone can perform maximum likelihood estimation of the gradient based on the stochastic binding patterns with ligand molecules. The performance of gradient sensing is measured by the Fisher Information between the binding pattern and the gradient direction. The quality of gradient sensing decreases with higher diffusion constant of the receptors and improves with higher concentration. We then extend to a two-dimensional model of an elliptical growth cone with a general prior distribution. With a random uniform distribution of receptors, the shape of the growth cone can introduce bias in the gradient estimate. This bias can be corrected by a non-uniform distribution of receptors with higher density near the minor axis of the growth cone.Besides stochastic gradient sensing, growth cones also trace highly stochastic trajectories, and it is unclear how molecular gradients bias their movement. We then introduce a mathematical model of a correlated random walk based on persistence, bias and noise to describe growth cone trajectories, constrained directly by measurements of the detailed statistics of growth cone movements in both attractive and repulsive gradients in a microfluidic device. This model explains the long-standing mystery that average axon turning angles in gradients in vitro plateau very rapidly with time at relatively small values of 10-20• . This work introduces the most accurate predictive model of growth cone trajectories to date, and calls into question the ability of molecular gradients alone to provide reliable guidance cues for growing axons.ii Declaration by AuthorThis thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis.

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Copper Binding Regulates Cellular Prion Protein Function

et al. 2019

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The cellular prion protein (PrPC), mainly known for its role in neurodegenerative diseases, is involved in several physiological processes including neuritogenesis. By combining genomic approaches, cellular assays and focal stimulation technique, we have explored the molecular mechanism underlining PrPC function as a signaling molecule in neuritogenesis. Several recombinant prion protein (recPrP) mutants were obtained to treat primary hippocampal cultures in bulk or exposed near the hippocampal growth cones (GC) of single neurons in a local stimulation manner. While focal stimulation of GC with wild‐type recPrP induced neurite outgrowth and rapid GC turning towards the source, N‐terminal mutants fail to support this function. In particular, the copper‐binding sites mutants present at the N‐terminus of PrPC are toxic to neurons indicating this region being crucial for the function of the protein. Mutants of recPrP including a key mutation for prion conversion H95Y (Giachin, Mai et al. 2015) or a GSS‐linked mutation abolish the function on neuritogenesis. Altogether, our findings indicate the functional regions for PrPC involved in neuritogenesis, suggest a potential link between loss‐of‐function of the protein and disease initiation. Support or Funding Information Fondo per gli Investimenti della Ricerca di Base (FIRB) program project (grant number RBAP11FRE9_001 to GL) from Ministero dell'Istruzione, dell'Università e della Ricerca(MIUR), Italy This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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Less than 5 Netrin-1 molecules initiate attraction but 200 Sema3A molecules are necessary for repulsion

Cited by 23 publications

References 51 publications

Characterization of prion protein function by focal neurite stimulation

Characterization of prion protein function by focal neurite stimulation

Mathematical modelling of axon guidance in chemical gradients

Copper Binding Regulates Cellular Prion Protein Function

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