Transgenic FingRs for Live Mapping of Synaptic Dynamics in Genetically-Defined Neurons

Son, Jong Hyun; Keefe, Matthew D.; Stevenson, Tamara J.; Barrios, Joshua; Anjewierden, Scott; Newton, James; Douglass, Adam D.; Bonkowsky, Joshua L.

doi:10.1038/srep18734

Cited by 32 publications

(48 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As pointed out in a recent review (Aguilar et al, 2019b), expression levels of the protein binder for visualization of a POI must be carefully controlled for a correct interpretation of the results. Strategies such as inducibility (Panza et al, 2015), self-transcriptional autoregulating domain fusion (Son et al, 2016) or intrinsic self stability (Tang et al, 2016), which were developed for nanobodies (Panza et al, 2015; Tang et al, 2016) and fibronectin-derived intrabodies (Son et al, 2016), could be applied to all the small tag binders described here.…”

Section: Discussionmentioning

confidence: 99%

Protein manipulation using single copies of short peptide tags in cultured cells and inDrosophila melanogaster

Viganò

Ell

Kustermann

et al. 2020

Preprint

View full text Add to dashboard Cite

Cellular development and specialized cellular functions are regulated processes which rely on highly dynamic molecular interactions among proteins, distributed in all cell compartments. Analysis of these interactions and their mechanisms of action has been one of the main topics in cellular and developmental research over the last fifty years.Studying and understanding the functions of proteins of interest (POIs) has been mostly achieved by their alteration at the genetic level and the analysis of the phenotypic changes generated by these alterations. Although genetic and reverse genetic technologies contributed to the vast majority of information and knowledge we have gathered so far, targeting specific interactions of POIs in a time-and spacecontrolled manner or analyzing the role of POIs in dynamic cellular processes such as cell migration or cell division would require more direct approaches. The recent development of specific protein binders, which can be expressed and function intracellularly, together with several improvements in synthetic biology techniques, have contributed to the creation of a new toolbox for direct protein manipulations. We selected a number of short tag epitopes for which protein binders from different scaffolds have been developed and tested whether these tags can be bound by the corresponding protein binders in living cells when they are inserted in a single copy in a POI. We indeed find that in all cases, a single copy of a short tag allows protein binding and manipulation. Using Drosophila, we also find that single short tags can be recognized and allow degradation and relocalization of POIs in vivo.

show abstract

Section: Discussionmentioning

confidence: 99%

Protein manipulation using single copies of short peptide tags in cultured cells and inDrosophila melanogaster

Viganò

Ell

Kustermann

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…Ideally, the chromobodies themselves should not show any preferential distribution in the absence of the POI. Furthermore, the POI and chromobody must be co-localized upon binding (e.g., by immunostaining of the POI) (Panza et al, 2015;Son et al, 2016). To quantify the level of POI, it might be useful to determine the background fluorescence level and subtract it from the experimental data (Boersma et al, 2019).…”

Section: Chromobody Specificitymentioning

confidence: 99%

Reflections on the use of protein binders to study protein function in developmental biology

Aguilar

Viganò

Affolter

et al. 2019

WIREs Developmental Biology

View full text Add to dashboard Cite

Studies in the field of developmental biology aim to unravel how a fertilized egg develops into an adult organism and how proteins and other macromolecules work together during this process. With regard to protein function, most of the developmental studies have used genetic and RNA interference approaches, combined with biochemical analyses, to reach this goal. However, there always remains much room for interpretation on how a given protein functions, because proteins work together with many other molecules in complex regulatory networks and it is not easy to reveal the function of one given protein without affecting the networks. Likewise, it has remained difficult to experimentally challenge and/or validate the proposed concepts derived from mutant analyses without tools that directly manipulate protein function in a predictable manner. Recently, synthetic tools based on protein binders such as scFvs, nanobodies, DARPins, and others have been applied in developmental biology to directly manipulate target proteins in a predicted manner. Although such tools would have a great impact in filling the gap of knowledge between mutant phenotypes and protein functions, careful investigations are required when applying functionalized protein binders to fundamental questions in developmental biology. In this review, we first summarize how protein binders have been used in the field, and then reflect on possible guidelines for applying such tools to study protein functions in developmental biology.This article is categorized under:Technologies > Analysis of ProteinsEstablishment of Spatial and Temporal Patterns > GradientsInvertebrate Organogenesis > Flies

show abstract

“…To detect the localization of endogenous PSD-95 in oligodendrocytes, we expressed a geneticallyencoded intrabody developed by Gross et al (2013) that binds zebrafish PSD-95 22,23 . The intrabody, termed PSD-95.FingR, is fused to GFP and contains a transcriptional regulation system to allow unbound FingR to repress further transcription.…”

Section: Myelinating Oligodendrocytes Express the Major Postsynaptic mentioning

confidence: 99%

Synaptic proteins expressed by oligodendrocytes mediate CNS myelination

Hughes

Appel

2018

Preprint

View full text Add to dashboard Cite

Oligodendrocytes ensheath neuronal axons with myelin, a proteolipid-rich membrane that increases conduction velocity and provides trophic support. Our lab and others have provided evidence that vesicular release from neurons promotes myelin sheath growth. Complementarily, transcriptomic and proteomic approaches have revealed that oligodendrocytes express many proteins that allow dendrites to sense and respond to vesicular release at synapses. Do axon-myelin contacts use similar communication mechanisms as nascent synapses to form myelin sheaths on axons? To test this, we used fusion proteins to track synaptic vesicle localization and membrane fusion within spinal cord axons of zebrafish larvae during developmental myelination. Additionally, we used a CRISPR/Cas9-mediated GAL4 enhancer trap and genetically-encoded intrabody to detect expression and localization of PSD-95, a component of dendritic postsynaptic complexes, within oligodendrocytes. We found that synaptic vesicles accumulate at ensheathment sites over time and are exocytosed with variable patterning underneath myelin sheaths. Accordingly, we also found that most, but not all sheaths localized PSD-95 with patterning similar to exocytosis site location within the axon. By querying published transcriptome databases, we found that oligodendrocytes express numerous transsynaptic adhesion molecules that function across synapses to promote synapse formation and maturation. Disruption of candidate PDZ-binding transsynaptic adhesion proteins in oligodendrocytes revealed that these proteins have variable effects on sheath length and number. We focused on one candidate, Cadm1b (SynCAM1), and demonstrated that it localized to myelin sheaths where both its PDZ binding and extracellular adhesion to axons are required for myelin sheath growth. Our work reveals shared mechanisms of synaptic and myelin plasticity and provides new targets for mechanistic unraveling of activity-regulated myelination.Communication in the central nervous system (CNS) depends on billions of connections. Synapses, the connections between neurons, are plastic structures that grow and change with experience-evoked neuronal activity. Additionally, connections form between neurons and oligodendrocytes, the myelinating cell type of the CNS. Myelin sheaths also are plastic structures, mutable in length, number, and thickness 1-3 . Some of this plasticity may be triggered by experience, because motor learning paradigms increase myelination 4,5 and social and sensory deprivation paradigms reduce myelination of relevant brain regions 2,6,7 . What accounts for myelin sheath plasticity? One possibility is that neuronal activity tunes ensheathment, similar to activitydependent plasticity at synapses 8 . Consistent with this possibility, Demerens and colleagues first demonstrated more than 20 years ago that inhibiting action potential propagation with tetrodotoxin (TTX) reduced myelination of cultured neurons 9 . Activity-mediated communication from axons to myelin sheaths could ensure that active neuron...

show abstract

Transgenic FingRs for Live Mapping of Synaptic Dynamics in Genetically-Defined Neurons

Cited by 32 publications

References 24 publications

Protein manipulation using single copies of short peptide tags in cultured cells and inDrosophila melanogaster

Protein manipulation using single copies of short peptide tags in cultured cells and inDrosophila melanogaster

Reflections on the use of protein binders to study protein function in developmental biology

Synaptic proteins expressed by oligodendrocytes mediate CNS myelination

Contact Info

Product

Resources

About