scholarly journals mRNA compartmentalisation spatially orients tissue morphogenesis

2018 ◽  
Author(s):  
Guilherme Costa ◽  
Joshua Bradbury ◽  
Nawseen Tarannum ◽  
Shane P. Herbert
Keyword(s):  
Single Molecule ◽  
Protein Function ◽  
Spatial Diversity ◽  
Small Gtpase ◽  
Tissue Morphogenesis ◽  
Protein Activity ◽  
Endogenous Gene ◽  
Spatial Coupling ◽  
Single Molecule Analysis

ABSTRACTPolarised targeting of diverse mRNAs to motile cellular protrusions is a hallmark of cell migration1–3. Although a widespread phenomenon, definitive functions for endogenous targeted mRNAs and their relevance to modulation of in-vivo tissue dynamics remain elusive. Here, using single-molecule analysis, endogenous gene-edited mRNAs and zebrafish in-vivo live-cell imaging, we report that mRNA polarisation acts as a molecular compass that orients motile cell polarity and spatially directs tissue movement. Clustering of protrusion-derived RNAseq datasets defined a core 192 bp localisation element underpinning precise mRNA targeting to incipient sites of filopodia formation at cell protrusions. Such targeting of the small GTPase, RAB13, generated tight spatial coupling of mRNA localisation, translation and protein activity, achieving precise subcellular compartmentalisation of RAB13 protein function to create a polarised domain of filopodia extension. Consequently, genomic excision of this localisation element and specific perturbation of endogenous RAB13 targeting – but not translation – depolarised filopodial dynamics in motile endothelial cells and induced miss-patterning of nascent blood vessels in-vivo. Hence, mRNA polarisation, not expression, is the primary spatial determinant of the site of RAB13 action, preventing ectopic functionality at inappropriate subcellular loci and orienting tissue morphogenesis. Considering the unexpected spatial diversity of other polarised mRNA clusters we identified, mRNA-mediated compartmentalisation of protein function at distinct subcellular sites likely coordinates broad aspects of in-vivo tissue behaviour.

scholarly journals An expanded toolkit for gene tagging based on MiMIC and scarless CRISPR tagging in Drosophila

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
David Li-Kroeger ◽  
Oguz Kanca ◽  
Pei-Tseng Lee ◽  
Sierra Cowan ◽  
Michael T Lee ◽  
...  
Keyword(s):  
Protein Function ◽  
Null Allele ◽  
Protein Localization ◽  
Dominant Marker ◽  
Endogenous Gene ◽  
Gene Trapping ◽  
Cassette Exchange ◽  
Protein Tag ◽  
Integration Efficiency

We generated two new genetic tools to efficiently tag genes in Drosophila. The first, Double Header (DH) utilizes intronic MiMIC/CRIMIC insertions to generate artificial exons for GFP mediated protein trapping or T2A-GAL4 gene trapping in vivo based on Cre recombinase to avoid embryo injections. DH significantly increases integration efficiency compared to previous strategies and faithfully reports the expression pattern of genes and proteins. The second technique targets genes lacking coding introns using a two-step cassette exchange. First, we replace the endogenous gene with an excisable compact dominant marker using CRISPR making a null allele. Second, the insertion is replaced with a protein::tag cassette. This sequential manipulation allows the generation of numerous tagged alleles or insertion of other DNA fragments that facilitates multiple downstream applications. Both techniques allow precise gene manipulation and facilitate detection of gene expression, protein localization and assessment of protein function, as well as numerous other applications.

scholarly journals Title: In vivo phosphatidylserine variations steer Rho GTPase signaling in a cell-context dependent manner

2018 ◽  
Author(s):  
Matthieu Pierre Platre ◽  
Vincent Bayle ◽  
Laia Armengot ◽  
Joseph Bareille ◽  
Maria Mar Marques-Bueno ◽  
...  
Keyword(s):  
Single Molecule ◽  
Rho Gtpases ◽  
Rho Gtpase ◽  
Plant Root ◽  
Super Resolution ◽  
Small Gtpase ◽  
Auxin Signaling ◽  
Dependent Manner ◽  
Downstream Signaling

AbstractRho GTPases are master regulators of cell signaling, but how they are regulated depending on the cellular context is unclear. Here, we show that the phospholipid phosphatidylserine acts as a developmentally-controlled lipid rheostat that tunes Rho GTPase signaling in Arabidopsis. Live super-resolution single molecule imaging revealed that RHO-OF-PLANT6 (ROP6) is stabilized by phosphatidylserine into plasma membrane (PM) nanodomains, which is required for auxin signaling. Furthermore, we uncovered that the PM phosphatidylserine content varies during plant root development and that the level of phosphatidylserine modulates the quantity of ROP6 nanoclusters induced by auxin and hence downstream signaling, including regulation of endocytosis and gravitropism. Our work reveals that variations in phosphatidylserine levels are a physiological process that may be leveraged to regulate small GTPase signaling during development.One Sentence SummaryPhosphatidylserine acts as a developmentally-controlled lipid rheostat that regulates cellular auxin sensitivity and plant development.

scholarly journals An expanded toolkit for gene tagging based on MiMIC and scarless CRISPR tagging in Drosophila

2018 ◽  
Author(s):  
David Li-Kroeger ◽  
Oguz Kanca ◽  
Pei-Tseng Lee ◽  
Sierra Cowan ◽  
Michael Lee ◽  
...  
Keyword(s):  
Protein Function ◽  
Null Allele ◽  
Protein Localization ◽  
Dominant Marker ◽  
Endogenous Gene ◽  
Gene Trapping ◽  
Cassette Exchange ◽  
Protein Tag ◽  
Integration Efficiency

AbstractWe generated new genetic tools to efficiently tag genes in Drosophila. Double Header (DH) utilizes intronic MiMIC/CRIMIC insertions to generate artificial exons for GFP mediated protein trapping or T2A-GAL4 gene trapping in vivo based on CRE recombinase to avoid embryo injections. DH significantly increases integration efficiency compared to previous strategies and faithfully reports the expression pattern of genes and proteins. The second technique targets genes lacking coding introns using a two-step cassette exchange. First, we replace the endogenous gene with an excisable compact dominant marker using CRISPR making a null allele. Second, the insertion is replaced with a protein::tag cassette.This sequential manipulation allows the generation of numerous tagged alleles or insertion of other DNA fragments that facilitates multiple downstream applications. Both techniques allow precise gene manipulation and facilitate detection of gene expression, protein localization and assessment of protein function, as well as numerous other applications.

scholarly journals Charcot-Marie-Tooth 2B mutations in rab7 cause dosage-dependent neurodegeneration due to partial loss of function

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Smita Cherry ◽  
Eugene Jennifer Jin ◽  
Mehmet Neset Özel ◽  
Zhiyuan Lu ◽  
Egemen Agi ◽  
...  
Keyword(s):  
Motor Neurons ◽  
Protein Function ◽  
Quantitative Imaging ◽  
Small Gtpase ◽  
Loss Of Function ◽  
Gain Of Function ◽  
Mutant Proteins ◽  
Charcot Marie Tooth ◽  
Function Mechanism

The small GTPase Rab7 is a key regulator of endosomal maturation in eukaryotic cells. Mutations in rab7 are thought to cause the dominant neuropathy Charcot-Marie-Tooth 2B (CMT2B) by a gain-of-function mechanism. Here we show that loss of rab7, but not overexpression of rab7 CMT2B mutants, causes adult-onset neurodegeneration in a Drosophila model. All CMT2B mutant proteins retain 10–50% function based on quantitative imaging, electrophysiology, and rescue experiments in sensory and motor neurons in vivo. Consequently, expression of CMT2B mutants at levels between 0.5 and 10-fold their endogenous levels fully rescues the neuropathy-like phenotypes of the rab7 mutant. Live imaging reveals that CMT2B proteins are inefficiently recruited to endosomes, but do not impair endosomal maturation. These findings are not consistent with a gain-of-function mechanism. Instead, they indicate a dosage-dependent sensitivity of neurons to rab7-dependent degradation. Our results suggest a therapeutic approach opposite to the currently proposed reduction of mutant protein function.

scholarly journals Single Molecule Analysis of Transcription in Live Cells Reveals the Gene Regulatory Function of MYC In Vivo

2017 ◽  
Vol 112 (3) ◽  
pp. 210a-211a
Author(s):  
Simona Patange ◽  
Michelle Girvan ◽  
David Levens ◽  
Daniel R. Larson
Keyword(s):  
Single Molecule ◽  
Live Cells ◽  
Regulatory Function ◽  
Gene Regulatory ◽  
Single Molecule Analysis

scholarly journals Tau protein function in living cells.

1986 ◽  
Vol 103 (6) ◽  
pp. 2739-2746 ◽  
Author(s):  
D G Drubin ◽  
M W Kirschner
Keyword(s):  
Tau Protein ◽  
Protein Function ◽  
Mammalian Brain ◽  
Protein Activity ◽  
Neuronal Morphogenesis ◽  
Microtubule Polymerization ◽  
A Cell ◽  
Nerve Cell Differentiation

Tau protein from mammalian brain promotes microtubule polymerization in vitro and is induced during nerve cell differentiation. However, the effects of tau or any other microtubule-associated protein on tubulin assembly within cells are presently unknown. We have tested tau protein activity in vivo by microinjection into a cell type that has no endogenous tau protein. Immunofluorescence shows that tau protein microinjected into fibroblast cells associates specifically with microtubules. The injected tau protein increases tubulin polymerization and stabilizes microtubules against depolymerization. This increased polymerization does not, however, cause major changes in cell morphology or microtubule arrangement. Thus, tau protein acts in vivo primarily to induce tubulin assembly and stabilize microtubules, activities that may be necessary, but not sufficient, for neuronal morphogenesis.

scholarly journals Photoactivatable CaMKII induces synaptic plasticity in single synapses

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Akihiro C. E. Shibata ◽  
Hiromi H. Ueda ◽  
Kei Eto ◽  
Maki Onda ◽  
Aiko Sato ◽  
...  
Keyword(s):  
Synaptic Plasticity ◽  
Ampa Receptors ◽  
Long Term Potentiation ◽  
Small Gtpase ◽  
Protein Activity ◽  
Photon Excitation ◽  
Term Potentiation ◽  
Neuronal Functions

AbstractOptogenetic approaches for studying neuronal functions have proven their utility in the neurosciences. However, optogenetic tools capable of inducing synaptic plasticity at the level of single synapses have been lacking. Here, we engineered a photoactivatable (pa)CaMKII by fusing a light-sensitive domain, LOV2, to CaMKIIα. Blue light or two-photon excitation reversibly activated paCaMKII. Activation in single spines was sufficient to induce structural long-term potentiation (sLTP) in vitro and in vivo. paCaMKII activation was also sufficient for the recruitment of AMPA receptors and functional LTP in single spines. By combining paCaMKII with protein activity imaging by 2-photon FLIM-FRET, we demonstrate that paCaMKII activation in clustered spines induces robust sLTP via a mechanism that involves the actin-regulatory small GTPase, Cdc42. This optogenetic tool for dissecting the function of CaMKII activation (i.e., the sufficiency of CaMKII rather than necessity) and for manipulating synaptic plasticity will find many applications in neuroscience and other fields.

scholarly journals Positive Functional Synergy of Structurally Integrated, Designed Artificial Protein Dimers Assembled by Fully Genetically Encoded Click Chemistry

2018 ◽  
Author(s):  
Harley L. Worthy ◽  
Husam Sabah Auhim ◽  
William David Jamieson ◽  
Jacob pope ◽  
Aaron Wall ◽  
...  
Keyword(s):  
Long Range ◽  
Single Molecule ◽  
Protein Function ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Protein Complexes ◽  
Positive Functional ◽  
Protein Dimers ◽  
Single Molecule Analysis ◽  
Artificial Protein

We combined <i>in silico</i>modelling with fully genetically encoded strain promoted azide-alkyne cycloaddition, to construct bespoke protein dimers. Using fluorescent proteins GFP and Venus as models, homo and heterodimers were constructed that switched ON once assembled and displayed enhanced spectral properties. The determined molecular structure reveals long range polar bond networks involving amino acids and structured water molecules play a key role in activation and functional enhancement by directly linking the two functional centres. Single molecule analysis revealed the dimer is more resistant to photobleaching spending longer times in the ON state with only one CRO likely to be active at any one time. Thus, genetically encoded bioorthogonal chemistry can be used beyond simple passive linkage approaches to generate new and truly integrated protein complexes that form long range bonds networks, which have a profound effect on function and our understanding of fluorescent protein function.

scholarly journals Single-molecule analysis reveals cooperative stimulation of Rad51 filament nucleation and growth by mediator proteins

2020 ◽  
Author(s):  
Ondrej Belan ◽  
Consuelo Barroso ◽  
Artur Kaczmarczyk ◽  
Roopesh Anand ◽  
Stefania Federico ◽  
...  
Keyword(s):  
Dna Damage ◽  
Single Molecule ◽  
Strand Break ◽  
Nucleation And Growth ◽  
Repair Mechanism ◽  
Dna Double Strand Break ◽  
Strand Invasion ◽  
Single Molecule Analysis ◽  
Stimulation Of

ABSTRACTHomologous recombination (HR) is an essential DNA double-strand break (DSBs) repair mechanism frequently inactivated in cancer. During HR, RAD51 forms nucleoprotein filaments on RPA-coated resected DNA and catalyses strand invasion into homologous duplex DNA. How RAD51 displaces RPA and assembles into long HR-proficient filaments remains uncertain. Here, we employ single-molecule imaging to investigate the mechanism of nematode RAD-51 filament growth in the presence of BRC-2 (BRCA2) and RAD-51 paralogs, RFS-1/RIP-1. BRC-2 nucleates RAD-51 on RPA-coated DNA, while RFS-1/RIP-1 acts as a ‘chaperone’ to promote 3’ to 5’ filament growth via highly dynamic engagement with 5’ filament ends. Inhibiting ATPase or mutation in RFS-1 Walker box leads to RFS-1/RIP-1 retention on RAD-51 filaments and hinders growth. rfs-1 Walker box mutants display sensitivity to DNA damage and accumulate RAD-51 complexes non-functional for HR in vivo. Our work reveals the mechanism of RAD-51 nucleation and filament growth in the presence of recombination mediators.

scholarly journals Exploring absent protein function in yeast: assaying post translational modification and human genetic variation

2021 ◽  
Vol 8 (8) ◽  
pp. 164-183
Author(s):  
Christina S. Moesslacher ◽  
Johanna M. Kohlmayr ◽  
Ulrich Stelzl
Keyword(s):  
Protein Function ◽  
Model Organism ◽  
Global Scale ◽  
Test System ◽  
Post Translational Modification ◽  
Protein Activity ◽  
Protein Modifications ◽  
In Vivo Test ◽  
The Impact

Yeast is a valuable eukaryotic model organism that has evolved many processes conserved up to humans, yet many protein functions, including certain DNA and protein modifications, are absent. It is this absence of protein function that is fundamental to approaches using yeast as an in vivo test system to investigate human proteins. Functionality of the heterologous expressed proteins is connected to a quantitative, selectable phenotype, enabling the systematic analyses of mechanisms and specificity of DNA modification, post-translational protein modifications as well as the impact of annotated cancer mutations and coding variation on protein activity and interaction. Through continuous improvements of yeast screening systems, this is increasingly carried out on a global scale using deep mutational scanning approaches. Here we discuss the applicability of yeast systems to investigate absent human protein function with a specific focus on the impact of protein variation on protein-protein interaction modulation.

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