Direct visualization of newly synthesized target proteins in situ

Protein synthesis is a dynamic process that tunes the cellular proteome in response to internal and external demands. Metabolic labeling approaches identify the general proteomic response but cannot visualize specific newly synthesized proteins within cells. Here we describe a technique that couples noncanonical amino acid tagging or puromycylation with the proximity ligation assay to visualize specific newly synthesized proteins and monitor their origin, redistribution and turnover in situ

Cell-type-specific metabolic labeling of nascent proteomes in vivo

Although advances in protein labeling methods have made it possible to measure the proteome of mixed cell populations, it has not been possible to isolate cell-type-specific proteomes in vivo. This is because the existing methods for metabolic protein labeling in vivo access all cell types. We report the development of a transgenic mouse line where Cre-recombinase-induced expression of a mutant methionyl-tRNA synthetase (L274G) enables the cell-type-specific labeling of nascent proteins with a non-canonical amino-acid and click chemistry. Using immunoblotting, imaging and mass spectrometry, we use our transgenic mouse to label and analyze proteins in excitatory principal neurons and Purkinje neurons in vitro (brain slices) and in vivo. We discover more than 200 proteins that are differentially regulated in hippocampal excitatory neurons by exposing mice to an environment with enriched sensory cues. Our approach can be used to isolate, analyze and quantitate cell-type-specific proteomes and their dynamics in healthy and diseased tissues

LIMK, Cofilin 1 and actin dynamics involvement in fear memory processing

Long-term memory has been associated with morphological changes in the brain, which in turn tightly correlate with changes in synaptic efficacy. Such plasticity is proposed to rely on dendritic spines as a neuronal canvas on which these changes can occur. Given the key role of actin cytoskeleton dynamics in spine morphology, major regulating factors of this process such as Cofilin 1 (Cfl1) and LIM kinase (LIMK), an inhibitor of Cfl1 activity, are prime molecular targets that may regulate dendritic plasticity. Using a contextual fear conditioning paradigm in mice, we found that pharmacological induction of depolymerization of actin filaments through the inhibition of LIMK causes an impairment in memory reconsolidation, as well as in memory consolidation. On top of that, Cfl1 activity is inhibited and its mRNA is downregulated in CA1 neuropil after re-exposure to the training context. Moreover, by pharmacological disruption of actin cytoskeleton dynamics, the process of memory extinction can either be facilitated or impaired. Our results lead to a better understanding of the role of LIMK, Cfl1 and actin cytoskeleton dynamics in the morphological and functional changes underlying the synaptic plasticity of the memory trace.Fil: Medina, Candela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: de la Fuente, Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: tom Dieck, Susanne. Max Planck Institute for Brain Research; AlemaniaFil: Nassim Assir, Belquis. Max Planck Institute for Brain Research; AlemaniaFil: Dalmay, Tamas. Max Planck Institute for Brain Research; AlemaniaFil: Bartnik, Ina. Max Planck Institute for Brain Research; AlemaniaFil: Lunardi, Paula Santana. Universidade Federal do Rio Grande do Sul; BrasilFil: de Oliveira Alvares, Lucas. Universidade Federal do Rio Grande do Sul; BrasilFil: Schuman, Erin M.. Max Planck Institute for Brain Research; AlemaniaFil: Letzkus, Johannes J.. Max Planck Institute for Brain Research; AlemaniaFil: Romano, Arturo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentin