Impatto dell'attivita' chinasica su dinamica di membrana e funzionalita' del recettore neurotrofinico TrkA

Questo lavoro di tesi si inserisce in una linea di ricerca che si propone di studiare la dinamica a singola molecola di recettori di membrana responsivi a fattori di crescita neuronale (neurotrofine, NT), ed a correlarla alla loro attività di trasduzione del segnale. Lo studio condotto precedentemente era relativo al recettore ad attività tirosin-chinasica TrkA, caratterizzato da un’alta affinità di legame per il capostipite delle NT, l’NGF. A questo recettore è stata geneticamente fusa la sequenza del tag acyl carrier protein (ACP), tecnologia sfruttata per ottenere la marcatura del recettore situato sulla superficie di cellule vive. Studi basati su tracking di singole particelle (SPT-single particle tracking) in membrana hanno evidenziato che il recettore TrkA wild type (wt), in assenza di ligando, mostra un moto di tipo per lo più diffusivo caratterizzato da un coefficiente di diffusività di circa 0,5µm2/s; in presenza del ligando NGF la dinamica invece rallenta, aumenta il numero di traiettorie confinate e il numero di forme dimeriche e oligomeriche del recettore, che verosimilmente sono responsabili delle piattaforme di signalling e di internalizzazione del recettore attivato. Nel contesto di esperimenti miranti a comprendere il collegamento fra il tipo di moto in membrana e le diverse risposte biologiche, è stato costruito un mutante privo di attività chinasica (dead-kinase), ottenuto attraverso la mutazione K547N, la quale va a bloccare il sito di legame per l’ATP a livello del dominio chinasico. Tale mutante non è in grado di indurre i processi di autofosforilazione del recettore stesso e di attivazione di effettori intracellulari in risposta al ligando NGF. Nell’ambito degli studi sulla mobilità il suo andamento è risultato essere rallentato indipendentemente dalla presenza di NGF, un comportamento paradossalmente simile a quello riportato per il recettore wt attivato da NGF. Il lavoro di tesi parte da questa osservazione, con lo scopo di comprendere la causa del comportamento dinamico del mutante dead-kinase; la nostra ipotesi è che esso venga bloccato in precursori di vescicole di internalizzazione per la degradazione, ma non è nota quale sia la funzionalità compromessa che porta a questa condizione. Abbiamo scelto di indurre mutazioni puntiformi a livello di singole tirosine del dominio citoplasmatico del recettore con l’obiettivo di indagare se e quali residui mutati siano responsabili del comportamento dinamico del mutante dead-kinase. Sulla base della letteratura disponibile, le tirosine che sono state scelte per la mutagenesi sono le seguenti: Y499, Y679, Y683, Y704, Y684, Y760, Y794 (numerazione riferita al cDNA di ratto) e la mutazione su di esse indotta è stata una conversione a fenilalanina, che impedisce la fosforilazione del residuo pur mantenendone la struttura aromatica, senza quindi alterare la struttura della proteina. A partire dal costrutto ACP-TrkA wt, sono stati prodotti sia mutanti singoli per ciascuno dei suddetti residui che mutanti combinati, in cui i residui sono stati raggruppati in base alla specifica funzione che li vede coinvolti; nel dettaglio, verranno discussi i risultati sul Kinase-Mutant, mutante combinato dei residui Y679F-Y683F-Y684F, responsabili dell’attività chinasica e il Recruitment-Mutant, mutante combinato dei residui Y499F-Y760F-Y794F, responsabili del reclutamento di effettori intracellulari. Tali mutanti, insieme al costrutto wt e al dead-kinase, sono stati quindi trasfettati nella linea cellulare di neuroblastoma umano SH-SY5Y. La validazione biochimica dei costrutti è stata effettuata usando due approcci sperimentali: i) saggi biochimici di Western Blot e di immunoprecipitazione; ii) localizzazione dei quattro costrutti espressi e marcati in membrana attraverso acquisizioni in microscopia confocale. Questi ulteriori esperimenti sono stati finalizzati a capire se i quattro costrutti vengono espressi correttamente dalle cellule o vanno piuttosto incontro a processi degradativi e se i loro livelli di espressione sulla membrana plasmatica, almeno in condizioni di assenza di NGF, sono paragonabili. La presenza dell’ACP-tag ha permesso la biotinilazione selettiva dei recettori espressi sulla membrana delle cellule e la loro successiva marcatura con dei fluorofori molto brillanti (Quantum-dots) coniugati a streptavidina. È stato quindi possibile effettuare misure di SPT dei quattro costrutti marcati sulla membrana plasmatica di cellule vive, mediante microscopia TIRF. I filmati così ottenuti sono stati analizzati con un programma di tracking per la generazione delle traiettorie dei recettori, che poi ho processato utilizzando algoritmi di analisi già disponibili in laboratorio. I risultati ottenuti indicano un comportamento diffusivo del Kinase-Mutant simile a quello del dead-kinase mentre il Recruitment-Mutant ha un comportamento paragonabile a quello del TrkA wt. Ciò suggerisce che, verosimilmente, le tirosine coinvolte nel comportamento anomalo del mutante dead-kinase siano quelle responsabili dell’attività chinasica del recettore.

Human TrkAR649W mutation impairs nociception, sweating and cognitive abilities: a mouse model of HSAN IV

A functional nerve growth factor (NGF)-TrkA system is an essential requisite for the generation and maintenance of long-lasting thermal and mechanical hyperalgesia in adult mammals. Indeed, mutations in the gene encoding for TrkA are responsible for a rare condition, named Hereditary Sensory and Autonomic Neuropathy type IV (HSAN IV), characterized by the loss of response to noxious stimuli, anhidrosis and cognitive impairment. However, to date, there is no available mouse model to properly understand how the NGF-TrkA system can lead to pathological phenotypes that are distinctive of HSAN IV. Here, we report the generation of a knock-in mouse line carrying the HSAN IV TrkAR649W mutation. First, by in vitro biochemical and biophysical analyses, we show that the pathological R649W mutation leads to kinase-inactive TrkA also affecting its membrane dynamics and trafficking. In agreement with the HSAN IV human phenotype, TrkAR649W/m mice display a lower response to thermal and chemical noxious stimuli, correlating with reduced skin innervation, in addition to decreased sweating in comparison to TrkAh/m controls. Moreover, the R649W mutation decreases anxiety-like behavior and compromises cognitive abilities, by impairing spatial-working and social memory. Our results further uncover unexplored roles of TrkA in thermoregulation and sociability. In addition to accurately recapitulating the clinical manifestations of HSAN IV patients, our findings contribute to clarify the involvement of the NGF-TrkA system in pain sensation

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

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

Probing labeling-induced lysosome alterations in living cells by imaging-derived mean squared displacement analysis

Lysosomes are not merely degradative organelles but play a central role in nutrient sensing, metabolism and cell-growth regulation. Our ability to study their function in living cells strictly relies on the use of lysosome-specific fluorescent probes tailored to optical microscopy applications. Still, no report thus far quantitatively analyzed the effect of labeling strategies/procedures on lysosome properties in live cells. We tackle this issue by a recently developed spatiotemporal fluctuation spectroscopy strategy that extracts structural (size) and dynamic (diffusion) properties directly from imaging, with no a-priori knowledge of the system. We highlight hitherto neglected alterations of lysosome properties upon labeling. In particular, we demonstrate that Lipofectamine reagents, used to transiently express lysosome markers fused to fluorescent proteins (FPs) (e.g. LAMP1-FP or CD63-FP), irreversibly alter the organelle structural identity, inducing a ∼2-fold increase of lysosome average size. The organelle structural identity is preserved, instead, if electroporation or Effectene are used as transfection strategies, provided that the expression levels of the recombinant protein marker are kept low. This latter condition can be achieved also by generating cell lines stably expressing the desired FP-tagged marker. Reported results call into question the interpretation of a massive amount of data collected so far using fluorescent protein markers and suggest useful guidelines for future studies

Quantitative determination of fluorescence labeling implemented in cell cultures

Abstract Background Labeling efficiency is a crucial parameter in fluorescence applications, especially when studying biomolecular interactions. Current approaches for estimating the yield of fluorescent labeling have critical drawbacks that usually lead them to be inaccurate or not quantitative. Results We present a method to quantify fluorescent-labeling efficiency that addresses the critical issues marring existing approaches. The method operates in the same conditions of the target experiments by exploiting a ratiometric evaluation with two fluorophores used in sequential reactions. We show the ability of the protocol to extract reliable quantification for different fluorescent probes, reagents concentrations, and reaction timing and to optimize labeling performance. As paradigm, we consider the labeling of the membrane-receptor TrkA through 4′-phosphopantetheinyl transferase Sfp in living cells, visualizing the results by TIRF microscopy. This investigation allows us to find conditions for demanding single and multi-color single-molecule studies requiring high degrees of labeling. Conclusions The developed method allows the quantitative determination and the optimization of staining efficiency in any labeling strategy based on stable reactions

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