MPI für Hirnforschung
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The prevalence and specificity of local protein synthesis during neuronal synaptic plasticity
(2021)
To supply proteins to their vast volume, neurons localize mRNAs and ribosomes in dendrites and axons. While local protein synthesis is required for synaptic plasticity, the abundance and distribution of ribosomes and nascent proteins near synapses remain elusive. Here, we quantified the occurrence of local translation and visualized the range of synapses supplied by nascent proteins during basal and plastic conditions. We detected dendritic ribosomes and nascent proteins at single-molecule resolution using DNA-PAINT and metabolic labeling. Both ribosomes and nascent proteins positively correlated with synapse density. Ribosomes were detected at ~85% of synapses with ~2 translational sites per synapse; ~50% of the nascent protein was detected near synapses. The amount of locally synthesized protein detected at a synapse correlated with its spontaneous Ca2+ activity. A multifold increase in synaptic nascent protein was evident following both local and global plasticity at respective scales, albeit with substantial heterogeneity between neighboring synapses.
In homeostatic scaling at central synapses, the depth and breadth of cellular mechanisms that detect the offset from the set-point, detect the duration of the offset and implement a cellular response are not well understood. To understand the time-dependent scaling dynamics we treated cultured rat hippocampal cells with either TTX or bicucculline for 2 hr to induce the process of up- or down-scaling, respectively. During the activity manipulation we metabolically labeled newly synthesized proteins using BONCAT. We identified 168 newly synthesized proteins that exhibited significant changes in expression. To obtain a temporal trajectory of the response, we compared the proteins synthesized within 2 hr or 24 hr of the activity manipulation. Surprisingly, there was little overlap in the significantly regulated newly synthesized proteins identified in the early- and integrated late response datasets. There was, however, overlap in the functional categories that are modulated early and late. These data indicate that within protein function groups, different proteomic choices can be made to effect early and late homeostatic responses that detect the duration and polarity of the activity manipulation.
Drebrin (DBN) regulates cytoskeletal functions during neuronal development, and is thought to contribute to structural and functional synaptic changes associated with aging and Alzheimer’s disease. Here we show that DBN coordinates stress signalling with cytoskeletal dynamics, via a mechanism involving kinase ataxia-telangiectasia mutated (ATM). An excess of reactive oxygen species (ROS) stimulates ATM-dependent phosphorylation of DBN at serine-647, which enhances protein stability and accounts for improved stress resilience in dendritic spines. We generated a humanized DBN Caenorhabditis elegans model and show that a phospho-DBN mutant disrupts the protective ATM effect on lifespan under sustained oxidative stress. Our data indicate a master regulatory function of ATM-DBN in integrating cytosolic stress-induced signalling with the dynamics of actin remodelling to provide protection from synapse dysfunction and ROS-triggered reduced lifespan. They further suggest that DBN protein abundance governs actin filament stability to contribute to the consequences of oxidative stress in physiological and pathological conditions.
Regulation of protein turnover allows cells to react to their environment and maintain homeostasis. Proteins can show different turnover rates in different tissue, but little is known about protein turnover in different brain cell types. We used dynamic SILAC to determine half-lives of over 5100 proteins in rat primary hippocampal cultures as well as in neuron-enriched and glia-enriched cultures ranging from <1 to >20 days. In contrast to synaptic proteins, membrane proteins were relatively shorter-lived and mitochondrial proteins were longer-lived compared to the population. Half-lives also correlate with protein functions and the dynamics of the complexes they are incorporated in. Proteins in glia possessed shorter half-lives than the same proteins in neurons. The presence of glia sped up or slowed down the turnover of neuronal proteins. Our results demonstrate that both the cell-type of origin as well as the nature of the extracellular environment have potent influences on protein turnover.
The neuronal transcriptome changes dynamically to adapt to stimuli from the extracellular and intracellular environment. In this study, we adapted for the first time a click chemistry technique to label the newly synthesized RNA in cultured hippocampal neurons and intact larval zebrafish brain. Ethynyl uridine (EU) was incorporated into neuronal RNA in a time- and concentration-dependent manner. Newly synthesized RNA granules observed throughout the dendrites were colocalized with mRNA and rRNA markers. In zebrafish larvae, the application of EU to the swim water resulted in uptake and labeling throughout the brain. Using a GABA receptor antagonist, PTZ (pentylenetetrazol), to elevate neuronal activity, we demonstrate that newly transcribed RNA signal increased in specific regions involved in neurogenesis.
Correlative microscopy incorporates the specificity of fluorescent protein labeling into high-resolution electron micrographs. Several approaches exist for correlative microscopy, most of which have used the green fluorescent protein (GFP) as the label for light microscopy. Here we use chemical tagging and synthetic fluorophores instead, in order to achieve protein-specific labeling, and to perform multicolor imaging. We show that synthetic fluorophores preserve their post-embedding fluorescence in the presence of uranyl acetate. Post-embedding fluorescence is of such quality that the specimen can be prepared with identical protocols for scanning electron microscopy (SEM) and transmission electron microscopy (TEM); this is particularly valuable when singular or otherwise difficult samples are examined. We show that synthetic fluorophores give bright, well-resolved signals in super-resolution light microscopy, enabling us to superimpose light microscopic images with a precision of up to 25 nm in the x-y plane on electron micrographs. To exemplify the preservation quality of our new method we visualize the molecular arrangement of cadherins in adherens junctions of mouse epithelial cells.
Role of N-cadherin cis and trans interfaces in the dynamics of adherens junctions in living cells
(2013)
Cadherins, Ca2+-dependent adhesion molecules, are crucial for cell-cell junctions and remodeling. Cadherins form inter-junctional lattices by the formation of both cis and trans dimers. Here, we directly visualize and quantify the spatiotemporal dynamics of wild-type and dimer mutant N-cadherin interactions using time-lapse imaging of junction assembly, disassembly and a FRET reporter to assess Ca2+-dependent interactions. A trans dimer mutant (W2A) and a cis mutant (V81D/V174D) exhibited an increased Ca2+-sensitivity for the disassembly of trans dimers compared to the WT, while another mutant (R14E) was insensitive to Ca2+-chelation. Time-lapse imaging of junction assembly and disassembly, monitored in 2D and 3D (using cellular spheroids), revealed kinetic differences in the different mutants as well as different behaviors in the 2D and 3D environment. Taken together, these data provide new insights into the role that the cis and trans dimers play in the dynamic interactions of cadherins.