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dc.contributor.authorCagnetta, Roberta
dc.date.accessioned2018-05-08T09:32:39Z
dc.date.available2018-05-08T09:32:39Z
dc.date.issued2018-05-21
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/275601
dc.description.abstractAxonal protein synthesis is rapidly regulated by extrinsic cues during neural wiring but the full landscape of proteomic changes and their translational control mechanisms remain unknown. The ability to investigate the nascent proteome on subcellular compartments has been hampered by the low sensitivity of existing methodology on quantity-limited samples combined with the difficulty of obtaining sufficient amounts of pure material. By combining pulsed Stable Isotope Labelling by Amino acids in Cell culture (pSILAC) with Single-Pot Solid-Phase-enhanced Sample Preparation (SP3), I have established an approach to characterize the nascent proteome from quantity-limited somaless retinal axons (~2μg) on an unparalleled rapid time-scale (5 min). The results show that a surprisingly large number of proteins (>350) is translated constitutively in axons, many of which are linked to neurological disease. Axons stimulated by different cues (Netrin-1, BDNF, Sema3A) each show a signature set of up/down newly synthesised protein (NSP) changes (>100) within 5 min. Remarkably, conversion of Netrin-1-induced responses from repulsion to attraction triggers opposite translational regulation for 73% of a common subset corresponding to >100 NSPs. Further, I show that pharmacological increase in cAMP, known to induce chemoattractive response, also leads to rapid and wide-scale remodelling of the nascent axonal proteome (~100 NSP changes). I find that the cAMP-elicited NSP changes underlie the attractive turning but are distinct from those induced by the physiological chemoattractant Netrin-1, suggesting that the same type of chemotropic response can be mediated by different protein synthesis-dependent mechanisms. Finally, I show that Sema3A, but not Slit1, triggers a physiological and non-canonical PERK-eIF2α-eIF2B signalling pathway required in neural wiring to elicit the rapid (< 15 min) local translation control of a specific subset of NSPs. Collectively my findings lead to the general conclusion that guidance molecules rapidly induce cue-specific remodelling of the nascent axonal proteome via distinct regulatory mechanisms.
dc.language.isoen
dc.rightsAll rights reserved
dc.rightsAll Rights Reserveden
dc.rights.urihttps://www.rioxx.net/licenses/all-rights-reserved/en
dc.subjectAxon
dc.subjectproteomics
dc.subjectretinal ganglion cell
dc.subjectpSILAC-SP3
dc.subjectaxonal nascent proteome
dc.subjectguidance cues
dc.subjectgrowth cone
dc.subjectchemotropic response
dc.subjectlocal translation
dc.subjectUnfolded Protein Response
dc.subjectstress reponse
dc.subjecteIF2alpha
dc.subjecteIF2B
dc.subjecttranslational control
dc.subjectneural wiring
dc.subjectneurodevelopment
dc.titleThe cue induced axonal nascent proteome and its translational control mechanisms in neural wiring
dc.typeThesis
dc.type.qualificationlevelDoctoral
dc.type.qualificationnameDoctor of Philosophy (PhD)
dc.publisher.institutionUniversity of Cambridge
dc.publisher.departmentPhysiology, Development and Neuroscience
dc.date.updated2018-05-06T10:14:56Z
dc.identifier.doi10.17863/CAM.22852
dc.type.qualificationtitlePhD in Biological Sciences
cam.supervisorHolt, Christine
cam.thesis.fundingfalse
rioxxterms.freetoread.startdate2400-01-01


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