Direct visualization of newly synthesized target proteins in situ

Dieck, Susanne tom; Kochen, Lisa; Hanus, Cyril; Heumüller, Maximilian; Bartnik, Ina; Nassim-Assir, Belquis; Merk, Katrin; Mosler, Thorsten; Garg, Sakshi; Bunse, Stefanie; Tirrell, David A.; Schuman, Erin M.

doi:10.1038/nmeth.3319

Cited by 265 publications

(272 citation statements)

References 28 publications

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“…A PLA signal reports the close (< 5 nm) proximity of puromycin and LIMK1 antibodies and hence a site of newly synthesised protein (tom Dieck et al , 2015; Sambandan et al , 2017). Puro‐LIMK1 PLA puncta were readily detectable in the dendrites of cultured neurons incubated with puromycin.…”

Section: Resultsmentioning

confidence: 99%

NMDAR ‐dependent Argonaute 2 phosphorylation regulates mi RNA activity and dendritic spine plasticity

et al. 2018

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MicroRNAs (miRNAs) repress translation of target mRNAs by associating with Argonaute (Ago) proteins to form the RNA‐induced silencing complex (RISC), underpinning a powerful mechanism for fine‐tuning protein expression. Specific miRNAs are required for NMDA receptor (NMDAR)‐dependent synaptic plasticity by modulating the translation of proteins involved in dendritic spine morphogenesis or synaptic transmission. However, it is unknown how NMDAR stimulation stimulates RISC activity to rapidly repress translation of synaptic proteins. We show that NMDAR stimulation transiently increases Akt‐dependent phosphorylation of Ago2 at S387, which causes an increase in binding to GW182 and a rapid increase in translational repression of LIMK1 via miR‐134. Furthermore, NMDAR‐dependent down‐regulation of endogenous LIMK1 translation in dendrites and dendritic spine shrinkage requires phospho‐regulation of Ago2 at S387. AMPAR trafficking and hippocampal LTD do not involve S387 phosphorylation, defining this mechanism as a specific pathway for structural plasticity. This work defines a novel mechanism for the rapid transduction of NMDAR stimulation into miRNA‐mediated translational repression to control dendritic spine morphology.

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Section: Resultsmentioning

confidence: 99%

NMDAR ‐dependent Argonaute 2 phosphorylation regulates mi RNA activity and dendritic spine plasticity

et al. 2018

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“…The synthetic pathway and post-translational processing for these precursor proteins to be converted into their peptide products requires a rough endoplasmic reticulum (RER) and a Golgi apparatus, organelles that are present in the neuronal cell bodies, but are absent from the pituitary axons and nerve endings (Brownstein et al 1980;Burbach et al 2001). Studies in other mature axon systems with regard to the issue of whether the presence of their intra-axonal RNAs are used for intra-axonal protein synthesis will depend on the development of new methods to visualize protein synthesis in situ (Kim and Jung 2015;tom Dieck et al 2015). In this regard, it is interesting that by using the elegant puromycylation method described by tom Dieck et al ( 2015) performed the radioactive content of each band (see Fig.…”

Section: Resultsmentioning

confidence: 99%

Squid Giant Axon Contains Neurofilament Protein mRNA but does not Synthesize Neurofilament Proteins

et al. 2016

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When isolated squid giant axons are incubated in radioactive amino acids, abundant newly synthesized proteins are found in the axoplasm. These proteins are translated in the adaxonal Schwann cells and subsequently transferred into the giant axon. The question as to whether any de novo protein synthesis occurs in the giant axon itself is difficult to resolve because the small contribution of the proteins possibly synthesized intra-axonally is not easily distinguished from the large amounts of the proteins being supplied from the Schwann cells. In this paper we reexamine this issue by studying the synthesis of endogenous neurofilament (NF) proteins in the axon. Our laboratory previously showed that NF mRNA and protein is present in the squid giant axon, but not in the surrounding adaxonal glia. Therefore, if the isolated squid axon could be shown to contain newly synthesized NF protein de novo, it could not arise from the adaxonal glia. The results of experiments in this paper show that abundant 3H-labeled NF protein is synthesized in the squid giant fiber lobe containing the giant axon's neuronal cell bodies, but despite the presence of NF mRNA in the giant axon, no labeled NF protein is detected in the giant axon. This lends support to the Glia-Axon Protein Transfer Hypothesis which posits that the squid giant axon obtains newly synthesized protein by Schwann cell transfer and not through intra-axonal protein synthesis, and further suggests that the NF mRNA in the axon is in a translationally repressed state.

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“…Combining FUNCAT labeling with the proximity ligation assay technique (84), it is now possible to detect in situ the synthesis and degradation of specific proteins (85). In a similar way, combining puromycin labeling with proximity ligation assay technique, the synthesis of specific proteins can be detected within 0.5-1 min (85,86). Furthermore, photocaged puromycin that can be locally activated allows for protein synthesis detection in a spatially restricted manner (87).…”

Section: Protein Turnovermentioning

confidence: 99%

The Regulation of Synaptic Protein Turnover

Álvarez-Castelao

2015

Journal of Biological Chemistry

100

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Emerging evidence indicates that protein synthesis and degradation are necessary for the remodeling of synapses. These two processes govern cellular protein turnover, are tightly regulated, and are modulated by neuronal activity in time and space. The anisotropic anatomy of the neurons presents a challenge for the study of protein turnover, but the understanding of protein turnover in neurons and its modulation in response to activity can help us to unravel the fine-tuned changes that occur at synapses in response to activity. Here we review the key experimental evidence demonstrating the role of protein synthesis and degradation in synaptic plasticity, as well as the turnover rates of specific neuronal proteins.The human brain is composed of a trillion neurons with complex and intricate arbors, which are interconnected by synapses (1). Synapses are highly dynamic in number and shape as a consequence of the continuous remodeling of neural circuits. A change in synaptic transmission elicited by neural activity is collectively called "synaptic plasticity," and learning and memory rely, at least in part, on this process. Synapses are made up of proteins, including receptors for neurotransmitters, scaffolding molecules, and signaling molecules. All proteins have a finite lifetime: they are synthesized and degraded continuously to maintain cellular function and viability. This continuous process is called "protein turnover." However, why is protein turnover important for cells? Even when the cells are in a basal state, the protein pool is dynamic and the coordination between protein synthesis and degradation maintains a steady-state level of proteins that is constantly renewed (2). Protein turnover is not only important to maintain protein concentrations in the cell, but also to allow for changes. Modulation of the proteome is necessary for most cellular responses, involving modifications in general or specific protein turnover (3,4). Neurons also have the ability to modulate and adjust their proteome in response to specific cues, for example, synaptic remodeling in response to patterns of action potentials in neurons.One complication of protein turnover in neurons is that synapses can be located up to hundreds of microns from the cell body. Where are proteins synthesized and degraded? Are proteins transported over the long distance from the soma to dendrites or axons, and if so, how do they reach their specific location within the intricate axonal and dendritic arbors. In fact, to overcome these challenges, neurons have very effective transport mechanisms to deliver proteins to remote regions of axons or dendrites; this has been recently reviewed in Refs. 5 and 6. Moreover, some proteins, such as receptors or scaffolding proteins, are in continuous movement in and out of the synapse with rapid rates (reviewed in Refs. 7 and 8). In addition to protein movement, neurons use local translation and degradation in dendrites and axons, allowing for a fine-tuned local protein turnover. Here we present an overview focused on...

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Direct visualization of newly synthesized target proteins in situ

Cited by 265 publications

References 28 publications

NMDAR ‐dependent Argonaute 2 phosphorylation regulates mi RNA activity and dendritic spine plasticity

NMDAR ‐dependent Argonaute 2 phosphorylation regulates mi RNA activity and dendritic spine plasticity

Squid Giant Axon Contains Neurofilament Protein mRNA but does not Synthesize Neurofilament Proteins

The Regulation of Synaptic Protein Turnover

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