Nuovi costrutti inducibili per l'analisi biofisica delle dinamiche di singoli recettori di membrana: applicazione a p75NTR

Questo lavoro di tesi si inserisce in una ricerca che si propone di studiare la dinamica a singola molecola di recettori neurotrofinici ed a correlarla al loro stato di signaling. Lo studio condotto precedentemente era relativo al recettore ad attività tirosin-chinasica TrkA di ratto, a cui è stata geneticamente fusa la sequenza dell’acyl carrier protein (ACP tag), substrato di enzimi di tipo Phosphopantheteinyltransferasi (PPTasi) tramite i quali vi si può legare covalentemente qualsiasi molecola funzionalizzata con il coenzima A (CoA). Tale tecnica permette di marcare con sonde fluorescenti i recettori sulla membrana di cellule vive, e si è rivelata più versatile rispetto all’uso di (lunghi) tag classici (quali GFP, HA, FLAG), congeniali per protocolli biochimici standard. Lo scopo di questa tesi è l’implementazione e la dimostrazione dell’efficacia dei nuovi tag corti A1 e S6 (di 12 amminoacidi) derivati dalla proteina ACP, recentemente pubblicati, per lo studio del corecettore di membrana P75NTR (P75 Neurotrophin Receptor) in un contesto cellulare integro e con la possibilità di controllare quantitativamente l’espressione ectopica della proteina ricombinante, per renderla ideale per studi a singola molecola. Questo studio e’ stato condotto in parallelo su due linee cellulari immortalizzate che, dal punto di vista genotipico e fenotipico, sono congeniali all’analisi di fattori neurotrofici e rispettivi recettori: • Linea SH-SY5Y, derivata da Neuroblastoma umano • Linea PC12, derivata da Feocromocitoma di ratto (i Feocromociti hanno origine neuroectodermica) In primo luogo, abbiamo valutato la presenza di P75NTR endogeno espresso da queste linee, sia tramite saggi biochimici (Western Blot) che di immunofluorescenza, riscontrando che entrambe esprimono, anche se in misura molto differente, la proteina di membrana di nostro interesse. Successivamente, abbiamo analizzato nelle stesse cellule l’espressione ectopica di tre costrutti ricombinanti nei quali la sequenza di P75NTR e’ stata fusa in frame a tre diversi tag, ottenendo: 1) P75-EGFP, 2) A1-P75NTR, 3) S6-P75NTR. Mediante immunofluorescenze è stato possibile comparare la localizzazione della proteina endogena con quella dei costrutti ricombinanti, anche controllandone le colocalizzazioni con marker specifici di membrana nucleare e membrana plasmatica. I saggi sono stati effettuati su entrambe le linee cellulari e hanno rivelato che né GFP né A1 o S6 influenzano la localizzazione nativa della proteina sia in cellule differenziate (con NGF) che non. Per valutare la possibile funzionalità delle proteine ricombinanti abbiamo preso in esame una modificazione post-traduzionale, una sorta di “carta di identità” di P75NTR: la palmitoilazione, ossia l’aggiunta di un residuo di acido palmitico al dominio intracellulare di questo recettore che permette l’instaurarsi del pathway apoptotico. I tre costrutti sono stati clonati successivamente in un vettore lentivirale di ultima generazione (“all-in-one”), poiché, sfruttando la naturale infettività del virus, risulta il miglior sistema per gene delivery all’interno di linee cellulari neuronali; mediante il metodo split-component, è stato anche possibile ricostruire la particella virale con la quale trasdurre le cellule SH-SY5Y. Infine, abbiamo implementato ed utilizzato dei sistemi di visualizzazione in cellule vive dei costrutti di interesse: Il tag GFP è visualizzato direttamente mediante microscopia confocale a fluorescenza; i tag A1 e S6 sono substrati di due diverse PPTasi, e consentono una marcatura selettiva del recettore correttamente traslocato sulla membrana cellulare con dei fluorofori molto brillanti (Quantum Dots). Questi due costrutti sono utili per analizzare, mediante microscopia TIRF in vivo, i moti diffusivi di singole molecole di recettore P75NTR localizzato sulla membrana plasmatica, anche in risposta di suoi ligandi specifici quale il pro-NGF o di ligandi di cui sembra essere corecettore, quale l’NGF. L’ortogonalità di A1 e S6, che consente una marcatura selettiva di proteine differenti in un contesto cellulare condiviso, permetterà lo studio contemporaneo di p75NTR con recettori Trk, in modo da chiarirne l’interazione funzionale; infatti, dati bibliografici contrastanti non chiariscono del tutto determinate proprietà del corecettore, quali la base meccanicistica di tale interazione, la dinamica di for¬ma¬zio¬ne del complesso recettoriale e la trasduzione del segnale nella membrana plasmatica ed al soma della cellula neuronale

Ligand-induced dynamics of neurotrophin receptors investigated by single-molecule imaging approaches

Neurotrophins are secreted proteins that regulate neuronal development and survival, as well as maintenance and plasticity of the adult nervous system. The biological activity of neurotrophins stems from their binding to two membrane receptor types, the tropomyosin receptor kinase and the p75 neurotrophin receptors (NRs). The intracellular signalling cascades thereby activated have been extensively investigated. Nevertheless, a comprehensive description of the ligand-induced nanoscale details of NRs dynamics and interactions spanning from the initial lateral movements triggered at the plasma membrane to the internalization and transport processes is still missing. Recent advances in high spatio-temporal resolution imaging techniques have yielded new insight on the dynamics of NRs upon ligand binding. Here we discuss requirements, potential and practical implementation of these novel approaches for the study of neurotrophin trafficking and signalling, in the framework of current knowledge available also for other ligand-receptor systems. We shall especially highlight the correlation between the receptor dynamics activated by different neurotrophins and the respective signalling outcome, as recently revealed by single-molecule tracking of NRs in living neuronal cells

Fast diffusing p75NTR monomers support apoptosis and growth cone collapse by neurotrophin ligands

The p75 neurotrophin (NT) receptor (p75NTR) plays a crucial role in balancing survival-versus-death decisions in the nervous system. Yet, despite 2 decades of structural and biochemical studies, a comprehensive, accepted model for p75NTR activation by NT ligands is still missing. Here, we present a single-molecule study of membrane p75NTR in living cells, demonstrating that the vast majority of receptors are monomers before and after NT activation. Interestingly, the stoichiometry and diffusion properties of the wild-type (wt) p75NTR are almost identical to those of a receptor mutant lacking residues previously believed to induce oligomerization. The wt p75NTR and mutated (mut) p75NTR differ in their partitioning in cholesterol-rich membrane regions upon nerve growth factor (NGF) stimulation: We argue that this is the origin of the ability of wt p75NTR , but not of mut p75NTR, to mediate immature NT (proNT)-induced apoptosis. Both p75NTR forms support proNT-induced growth cone retraction: We show that receptor surface accumulation is the driving force for cone collapse. Overall, our data unveil the multifaceted activity of the p75NTR monomer and let us provide a coherent interpretative frame of existing conflicting data in the literature

Ligand Fingerprinting in the Membrane Dynamics of Single TrkA and P75NTR Neurotrophin Receptors

We have sought to investigate the responses of Nerve Growth Factor (NGF) receptors TrkA and P75NTR at the plasma membrane of living neuronal cells by single-molecule imaging and tracking. To this purpose we exploit the acyl carrier peptide and some of its shortened versions (A1 and S6 tags, labeled selectively by two different PPTases) to tag human p75NTR and TrkA. These tags were covalently conjugated to the biotin- or fluorophore-substituted arm of a coenzyme A (CoA) substrate. This approach allows: (i) a precise control of stoichiometry and site of biotin conjugation; (ii) versatility of the tags used; (iii) studying two interacting molecules with orthogonal fluorolabels, at the single-molecule or single-interaction-complex level. This experimental toolbox is completed by fast microscopy (e.g. TIRF microscopy with a fast EM-CCD), and by a semi-automatic algorithm for the analysis of the trajectories. This novel algorithm separates self-similar from multimodal trajectories, divides the last ones in subtrajectories, and calculates the combined distributions of parameters measuring the diffusivity, the localization or driftness degree, and/or the number of molecules in tracked spots. We shall present results on the early response of TrkA upon binding different biologically-relevant ligands (including NGF and proNGF): without ligands, TrkA is present mostly as fast-diffusing monomers; ligand binding results in an increasing number of dimers and oligomers, which are typically slower and/or more confined. Each ligand promotes distinct trajectory patterns at the cell membrane, because of different receptor-binding affinities, intracellular effectors recruited and formation of signalling/recycling endosome precursors. We believe that this imaging toolbox and our results pave the way to the quantitative description of the kinetics, dynamics and stoichiometry of any binary or ternary molecular complex formed upon binding of proNGF or NGF to their receptors

Site-specific direct labeling of neurotrophins and their receptors: From biochemistry to advanced imaging applications

We describe here a versatile methodological platform to achieve site-directed and stoichiometry-controlled labeling of neurotrophins and their receptors with various probes, ranging from biotin to small organic dyes. This labeling method works in vitro on purified neurotrophins as well as in a living cell context, where it achieves selective labeling of surface-exposed neurotrophin receptors. Here, we list all experimental details of our labeling protocols, along with examples of the wide range of applications in which these can be used

Single molecule tracking and spectroscopy unveils molecular details in function and interactions of membrane receptors

We exploited, optimized, and developed various microscopy techniques for analyzing living matter at cellular and molecular levels. In particular, we developed a toolbox for single particle tracking (SPT) of membrane receptors and their ligands, suitable also for relatively fast single-pass membrane receptors; this is based on chemical tagging of recombinant proteins, TIRF microscopy, and automatized analysis of single particle trajectories. We are now extending it to simultaneous visualization and analysis of two moieties. The superresolved localization of this technique allowed analyzing functions, interactions and stoichiometry of (pro)neurotrophin receptors p75NTR and TrkA and of their ligands in living cells, with particular attention on some of their existing or used mutants. The analysis also after treatments with ligands or drugs unraveled their mode of action in the first steps of sundry signaling pathways

Single Molecule Imaging and Tracking of Neurotrophins and their Receptors in Living Neuronal Cells

We currently lack a satisfactory understanding of the membrane complexes and internalization routes underpinning the pleiotropic biological outcomes of neurotrophins (NTs), which exert their functions via interlaced binding of three different families of neurotrophin receptors (NRs). We are working to answer several open questions in this field: are NRs membrane movements linked to ligand-specific activation processes? Are different NRs functions linked to different movements at the cell membrane? How does p75NTR enhance NGF-TrkA signalling? Are NGF and its precursor proNGF different signalling molecules as far as NRs binding and internalization is concerned? To address these issues, we developed non-invasive means to covalently fluorolabel with 1:1 stoichiometry both NTs and their receptors. This toolbox was exploited to perform single molecule imaging and tracking (SMIT) at the plasma membrane and inside axons of living neuronal cells using wide-field and TIRF microscopy. We report here results in two different directions. First, we analysed by SMIT the lateral mobility of wt TrkA in comparison to a dead-kinase TrkA and to three other mutants having i) kinase activity, ii) recruitment of intracellular effectors, iii) ubiquitination (and further degradation) separately impaired. Obtained data point to kinase activity as a master regulator of TrkA membrane dynamics and hint at possible mechanisms by which the cell handles the trafficking of kinase-inactive TrkA receptors. Second, we undertook a comparative study about the axonal transport displayed by \u201chomologue\u201d fluorescent proNGF and NGF in compartmented DRG neurons. We demonstrate that proNGF is internalized and retrogradely transported across axons like mature NGF, but the two NTs display remarkable differences both in terms of NTs flux and number of molecules carried per vesicle. Furthermore, we unveiled a competition mechanism favoring NGF transport upon coadministration of the two NTs