mRNA processing, transport, translation, and ultimately degradation involve a series of dedicated protein complexes that often assemble into large membraneless structures such as stress granules ...(SGs) and processing bodies (PBs). Here, systematic in vivo proximity-dependent biotinylation (BioID) analysis of 119 human proteins associated with different aspects of mRNA biology uncovers 7424 unique proximity interactions with 1,792 proteins. Classical bait-prey analysis reveals connections of hundreds of proteins to distinct mRNA-associated processes or complexes, including the splicing and transcriptional elongation machineries (protein phosphatase 4) and the CCR4-NOT deadenylase complex (CEP85, RNF219, and KIAA0355). Analysis of correlated patterns between endogenous preys uncovers the spatial organization of RNA regulatory structures and enables the definition of 144 core components of SGs and PBs. We report preexisting contacts between most core SG proteins under normal growth conditions and demonstrate that several core SG proteins (UBAP2L, CSDE1, and PRRC2C) are critical for the formation of microscopically visible SGs.
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•We performed BioID on 119 human proteins involved in various facets of mRNA biology•Proximal relationships reveal the spatial organization of RNA regulatory structures•Prey-based analysis identifies 144 protein components of cytosolic RNA granules•UBAP2L, CSDE1, and PRRC2C are required for efficient formation of stress granules
Youn et al. performed proximity-based proteomics on 119 human proteins involved in the mRNA life cycle, focusing on cytosolic RNA granule components that are important for mRNA regulation. Systematic analysis of the proximal interactome revealed 144 core components of cytosolic RNA granules and illuminated the spatial organization of RNA regulatory structures.
The accumulation of damaged mitochondria causes the death of dopaminergic neurons. The Parkin-mediated mitophagy pathway functions to remove these mitochondria from cells. Targeting this pathway ...represents a therapeutic strategy for several neurodegenerative diseases, most notably Parkinson's disease. We describe a discovery pipeline to identify small molecules that increase Parkin recruitment to damaged mitochondria and ensuing mitophagy. We show that ROCK inhibitors promote the activity of this pathway by increasing the recruitment of HK2, a positive regulator of Parkin, to mitochondria. This leads to the increased targeting of mitochondria to lysosomes and removal of damaged mitochondria from cells. Furthermore, ROCK inhibitors demonstrate neuroprotective effects in flies subjected to paraquat, a parkinsonian toxin that induces mitochondrial damage. Importantly, parkin and rok are required for these effects, revealing a signaling axis which controls Parkin-mediated mitophagy that may be exploited for the development of Parkinson's disease therapeutics.
The centrosome is the primary microtubule organizing center of the cells and templates the formation of cilia, thereby operating at a nexus of critical cellular functions. Here, we use ...proximity-dependent biotinylation (BioID) to map the centrosome-cilium interface; with 58 bait proteins we generate a protein topology network comprising >7,000 interactions. Analysis of interaction profiles coupled with high resolution phenotypic profiling implicates a number of protein modules in centriole duplication, ciliogenesis, and centriolar satellite biogenesis and highlights extensive interplay between these processes. By monitoring dynamic changes in the centrosome-cilium protein interaction landscape during ciliogenesis, we also identify satellite proteins that support cilia formation. Systematic profiling of proximity interactions combined with functional analysis thus provides a rich resource for better understanding human centrosome and cilia biology. Similar strategies may be applied to other complex biological structures or pathways.
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•BioID conducted on 58 centriole, satellite, and ciliary transition zone proteins•Centriole-cilium interface map comprises >1,700 unique components, >7,000 interactions•Microscopy and functional screens confirm new centriole-cilium regulatory modules•Dynamic modulation of interaction landscape observed during ciliogenesis program
In vivo proximity-dependent biotinylation (BioID) generates a protein interaction map of the human centrosome-cilium interface, revealing protein modules critical for centrosome and cilium biogenesis and pervasive and dynamic interplay between the two processes.
Centrosomes control cell motility, polarity and migration that is thought to be mediated by their microtubule-organizing capacity. Here we demonstrate that WNT signalling drives a distinct form of ...non-directional cell motility that requires a key centrosome module, but not microtubules or centrosomes. Upon exosome mobilization of PCP-proteins, we show that DVL2 orchestrates recruitment of a CEP192-PLK4/AURKB complex to the cell cortex where PLK4/AURKB act redundantly to drive protrusive activity and cell motility. This is mediated by coordination of formin-dependent actin remodelling through displacement of cortically localized DAAM1 for DAAM2. Furthermore, abnormal expression of PLK4, AURKB and DAAM1 is associated with poor outcomes in breast and bladder cancers. Thus, a centrosomal module plays an atypical function in WNT signalling and actin nucleation that is critical for cancer cell motility and is associated with more aggressive cancers. These studies have broad implications in how contextual signalling controls distinct modes of cell migration.
Cystic renal diseases are caused by mutations of proteins that share a unique subcellular localization: the primary cilium of tubular epithelial cells. Mutations of the ciliary protein inversin cause ...nephronophthisis type II, an autosomal recessive cystic kidney disease characterized by extensive renal cysts, situs inversus and renal failure. Here we report that inversin acts as a molecular switch between different Wnt signaling cascades. Inversin inhibits the canonical Wnt pathway by targeting cytoplasmic dishevelled (Dsh or Dvl1) for degradation; concomitantly, it is required for convergent extension movements in gastrulating Xenopus laevis embryos and elongation of animal cap explants, both regulated by noncanonical Wnt signaling. In zebrafish, the structurally related switch molecule diversin ameliorates renal cysts caused by the depletion of inversin, implying that an inhibition of canonical Wnt signaling is required for normal renal development. Fluid flow increases inversin levels in ciliated tubular epithelial cells and seems to regulate this crucial switch between Wnt signaling pathways during renal development.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Integration of transforming growth factor β (TGF-β) signals with those of other pathways allows for precise temporal and spatial control of gene expression patterns that drive development and ...homeostasis. The Hippo pathway nuclear effectors, Taz/Yap, interact with the TGF-β transcriptional mediators, Smads, to control Smad activity. Key to TGF-β signaling is the nuclear localization of Smads. Thus, to investigate the role of Taz/Yap in Smad nuclear accumulation, we developed mathematical models of Hippo and TGF-β cross talk. The models were based on experimental measurements of TGF-β-induced changes in Taz/Yap and Smad subcellular localization obtained using high-throughput immunofluorescence (IF) imaging in the mouse mammary epithelial cell line, EpH4. Bayesian MCMC DREAM parameter estimation was used to quantify the uncertainty in estimates of the kinetic parameters. Variation of the model parameters and statistical analysis show that our modeling predicts that Taz/Yap can alter TGF-β receptor activity and directly or indirectly act as nuclear retention factors.
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•Taz/Yap modulate TGF-β-induced nuclear accumulation of Smad2/3 and Smad4•TGF-β does not affect Taz/Yap localization when Hippo activity is constant•Taz/Yap loss may alter activity of both Receptor and Smad nuclear retention factors•The mediator complex regulates Smad nuclear accumulation
Biological Sciences; Cell Biology; Computational Bioinformatics; Integrative Aspects of Cell Biology; Molecular Network
The assembly of a robust microtubule-based mitotic spindle is a prerequisite for the accurate segregation of chromosomes to progeny. Spindle assembly relies on the concerted action of centrosomes, ...spindle microtubules, molecular motors, and nonmotor spindle proteins.
Here we use an RNA-interference screen of the human centrosome proteome to identify novel regulators of spindle assembly. One such regulator is HAUS, an 8-subunit protein complex that shares homology to
Drosophila Augmin. HAUS localizes to interphase centrosomes and to mitotic spindle microtubules, and its disruption induces microtubule-dependent fragmentation of centrosomes along with an increase in centrosome size. HAUS disruption results in the destabilization of kinetochore microtubules and the eventual formation of multipolar spindles. These severe mitotic defects are alleviated by codepletion of NuMA, indicating that both factors regulate opposing activities. HAUS disruption alters NuMA localization, suggesting that mislocalized NuMA activity contributes to the spindle and centrosome defects observed.
The human Augmin complex (HAUS) is a critical and evolutionary conserved multisubunit protein complex that regulates centrosome and spindle integrity.
Centrosomes are composed of a centriole pair surrounded by an intricate proteinaceous matrix referred to as pericentriolar material. Although the mechanisms underpinning the control of centriole ...duplication are now well understood, we know relatively little about the control of centrosome size and shape. Here we used interaction proteomics to identify the E3 ligase HERC2 and the neuralized homologue NEURL4 as novel interaction partners of the centrosomal protein CP110. Using high resolution imaging, we find that HERC2 and NEURL4 localize to the centrosome and that interfering with their function alters centrosome morphology through the appearance of aberrant filamentous structures that stain for a subset of pericentriolar material proteins including pericentrin and CEP135. Using an RNA interference-resistant transgene approach in combination with structure-function analyses, we show that the association between CP110 and HERC2 depends on nonoverlapping regions of NEURL4. Whereas CP110 binding to NEURL4 is dispensable for the regulation of pericentriolar material architecture, its association with HERC2 is required to maintain normal centrosome integrity. NEURL4 is a substrate of HERC2, and together these results indicate that the NEURL4-HERC2 complex participates in the ubiquitin-dependent regulation of centrosome architecture.
Automated high-content immunofluorescence (IF) microscopy is used to monitor and quantify localization of the TGFβ/Smads and Taz/Yap Hippo effectors in mouse epithelial EpH4 cells transfected with ...Taz/Yap siRNAs. The nuclear-to-cytoplasmic protein ratios obtained by IF are converted into normalized masses by estimating the ratio of the compartment volumes. This method has the advantage that endogenous rather than tagged proteins are tracked and that knockdown of Taz/Yap can be simultaneously monitored at the single-cell level.
For complete details on the use and execution of this protocol, please refer to Labibi et al. (2020).
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•Automated immunofluorescence microscopy to analyze protein subcellular localization•Time- and dose-dependent tracking of TGFβ-stimulated Smad2/3 and Smad4 localization•Efficient siRNA-mediated knockdown of Hippo mediators, TAZ and YAP in epithelial cells•Analysis of TGFβ and Hippo cross talk by monitoring localization of endogenous effectors
Automated high-content immunofluorescence (IF) microscopy is used to monitor and quantify localization of the TGFβ/Smads and Taz/Yap Hippo effectors in mouse epithelial EpH4 cells transfected with Taz/Yap siRNAs. The nuclear-to-cytoplasmic protein ratios obtained by IF are converted into normalized masses by estimating the ratio of the compartment volumes. This method has the advantage that endogenous rather than tagged proteins are tracked and that knockdown of Taz/Yap can be simultaneously monitored at the single-cell level.
The structure and function of the primary cilium as a sensory organelle depends on a motor-protein-powered intraflagellar transport system (IFT); defective IFT results in retinal degeneration and ...pleiotropic disorders such as the Bardet Biedl syndrome (BBS) and defective hedgehog (HH) signaling. Protein transport to the cilium involves Rab GTPases. Rab8, together with a multi protein complex of BBS proteins, recruits cargo to the basal body for transport to the cilium. Loss of Rab23 in mice recapitulates the HH phenotype but its function in HH signaling is unknown. Here we established a novel protocol, based on fluorescence recovery after photo-bleaching (FRAP), allowing the quantitative analysis of protein transport into the cilium of MDCK cells. We compared the effect of Rab8, Rab5 and Rab23 on the ciliary transport of the HH-associated transmembrane receptor Smoothened, the microtubular tip protein EB1, and the receptor protein Kim1. Ciliary FRAP confirmed the role of Rab8 in protein entry to the cilium. Dominant negative Rab5 had no impact on the ciliary transport of Smoothened or EB1, but slowed the recovery of the apical protein Kim1 in the cilium. Depletion of Rab23 or expression of dominant-negative Rab23 decreased the ciliary steady state specifically of Smoothened but not EB1 or Kim1, suggesting a role of Rab23 in protein turnover in the cilium.
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