Correction: Expansion microscopy reveals subdomains in germ granules.

Correction to Materials and Methods.

The emerging role of receptor tyrosine kinase phase separation in cancer.

Receptor tyrosine kinase (RTK)-mediated signal transduction is fundamental to cell function and drives important cellular outcomes which, when dysregulated, can lead to malignant tumour growth and metastasis. The initiation of signals from plasma membrane-bound RTKs is subjected to multiple regulatory mechanisms that control downstream effector protein recruitment and function. The high propensity of RTKs to condense via liquid-liquid phase separation (LLPS) into membraneless organelles with downstream effector proteins provides a further fundamental mechanism for signal regulation.

The combined action of the intracellular regions regulates FGFR2 kinase activity.

Receptor tyrosine kinases (RTKs) are typically activated through a precise sequence of intracellular phosphorylation events starting with a tyrosine residue on the activation loop (A-loop) of the kinase domain (KD). From this point the mono-phosphorylated enzyme is active, but subject to stringent regulatory mechanisms which can vary dramatically across the different RTKs. In the absence of extracellular stimulation, fibroblast growth factor receptor 2 (FGFR2) exists in the mono-phosphorylated state in which catalytic activity is regulated to allow rapid response upon ligand binding, whilst restricting ligand-independent activation.

Regulation of microRNA expression by the adaptor protein GRB2.

Protein interactions with the microRNA (miRNA)-mediated gene silencing protein Argonaute 2 (AGO2) control miRNA expression. miRNA biogenesis starts with the production of precursor transcripts and culminates with the loading of mature miRNA onto AGO2 by DICER1. Here we reveal an additional component to the regulatory mechanism for miRNA biogenesis involving the adaptor protein, growth factor receptor-bound protein 2 (GRB2).

Using the Superfolder GFP (sfGFP) System to Study Plant Peroxisomal Protein Import.

The fusing of a protein of interest to a fluorescent protein followed by fluorescence microscopy is a very common method of determining protein localization and dynamics. However even small fluorescent proteins can be large enough to affect protein folding and localization, therefore the ability to use a smaller tag but still be able to detect a fluorescent signal in live cell imaging experiments is extremely valuable. The self-assembling split sfGFP system allows the fusion of the protein of interest with the 11th β-strand of super-folder GFP (sfGFP11) which is only 13 amino acids long.

Catalase: A critical node in the regulation of cell fate.

Catalase (CAT) is an extensively studied if somewhat enigmatic enzyme that is at the heart of eukaryotic antioxidant systems with a canonical role in peroxisomal function. The CAT family of proteins exert control over a wide range of plant growth and defence processes. CAT proteins are subject to many types of post-translational modification (PTM), which modify activity, ligand binding, stability, compartmentation and function.

Phase separation enhances probability of receptor signalling and drug targeting.

The probability of a given receptor tyrosine kinase (RTK) triggering a defined cellular outcome is low because of the promiscuous nature of signalling, the randomness of molecular diffusion through the cell, and the ongoing nonfunctional submembrane signalling activity or noise. Signal transduction is therefore a 'numbers game', where enough cell surface receptors and effector proteins must initially be engaged to guarantee formation of a functional signalling complex against a background of redundant events. The presence of intracellular liquid-liquid phase separation (LLPS) at the plasma membrane provides a mechanism through which the probabilistic nature of signalling can be weighted in favour of the required, discrete cellular outcome and mutual exclusivity in signal initiation.

Expansion microscopy reveals subdomains in germ granules.

Light and electron microscopy techniques have been indispensable in the identification and characterization of liquid-liquid phase separation membraneless organelles. However, for complex membraneless organelles such as the perinuclear germ granule in , our understanding of how the intact organelle is regulated is hampered by (1) technical limitations in confocal fluorescence imaging for the simultaneous examination of multiple granule protein markers and (2) inaccessibility of electron microscopy. We take advantage of the newly developed super resolution method of expansion microscopy (ExM) and in situ staining of the whole proteome to examine the germ granule, the P granule.

Receptor tyrosine kinases regulate signal transduction through a liquid-liquid phase separated state.

The recruitment of signaling proteins into activated receptor tyrosine kinases (RTKs) to produce rapid, high-fidelity downstream response is exposed to the ambiguity of random diffusion to the target site. Liquid-liquid phase separation (LLPS) overcomes this by providing elevated, localized concentrations of the required proteins while impeding competitor ligands. Here, we show a subset of phosphorylation-dependent RTK-mediated LLPS states.

From Fragment to Lead: De Novo Design and Development toward a Selective FGFR2 Inhibitor.

Fibroblast growth factor receptors (FGFRs) are implicated in a range of cancers with several pan-kinase and selective-FGFR inhibitors currently being evaluated in clinical trials. Pan-FGFR inhibitors often cause toxic side effects and few examples of subtype-selective inhibitors exist. Herein, we describe a structure-guided approach toward the development of a selective FGFR2 inhibitor.

Grb2 binding induces phosphorylation-independent activation of Shp2.

The regulation of phosphatase activity is fundamental to the control of intracellular signalling and in particular the tyrosine kinase-mediated mitogen-activated protein kinase (MAPK) pathway. Shp2 is a ubiquitously expressed protein tyrosine phosphatase and its kinase-induced hyperactivity is associated with many cancer types. In non-stimulated cells we find that binding of the adaptor protein Grb2, in its monomeric state, initiates Shp2 activity independent of phosphatase phosphorylation.

DEPS-1 is required for piRNA-dependent silencing and PIWI condensate organisation in Caenorhabditis elegans.

Membraneless organelles are sites for RNA biology including small non-coding RNA (ncRNA) mediated gene silencing. How small ncRNAs utilise phase separated environments for their function is unclear. We investigated how the PIWI-interacting RNA (piRNA) pathway engages with the membraneless organelle P granule in Caenorhabditis elegans.

Targeting the Shc-EGFR interaction with indomethacin inhibits MAP kinase pathway signalling.

Receptor tyrosine kinase (RTK)-mediated hyperactivation of the MAPK/Erk pathway is responsible for a large number of pathogenic outcomes including many cancers. Considerable effort has been directed at targeting this pathway with varying degrees of long term therapeutic success. Under non-stimulated conditions Erk is bound to the adaptor protein Shc preventing aberrant signalling by sequestering Erk from activation by Mek.

A Secreted RNA Binding Protein Forms RNA-Stabilizing Granules in the Honeybee Royal Jelly.

RNA flow between organisms has been documented within and among different kingdoms of life. Recently, we demonstrated horizontal RNA transfer between honeybees involving secretion and ingestion of worker and royal jellies. However, how the jelly facilitates transfer of RNA is still unknown.

Grb2 monomer-dimer equilibrium determines normal versus oncogenic function.

The adaptor protein growth factor receptor-bound protein 2 (Grb2) is ubiquitously expressed in eukaryotic cells and involved in a multitude of intracellular protein interactions. Grb2 plays a pivotal role in tyrosine kinase-mediated signal transduction including linking receptor tyrosine kinases to the Ras/mitogen-activated protein (MAP) kinase pathway, which is implicated in oncogenic outcome. Grb2 exists in a constitutive equilibrium between monomeric and dimeric states.

Competition between Grb2 and Plcγ1 for FGFR2 regulates basal phospholipase activity and invasion.

FGFR2-expressing human cancer cells with low concentrations of the adaptor protein Grb2 show high prevalence for metastatic outcome. In nonstimulated cells, the SH3 domain (and not the SH2 domains) of Plcγ1 directly competes for a binding site at the very C terminus of FGFR2 with the C-terminal SH3 domain of Grb2. Reduction of Grb2 concentration permits Plcγ1 access to the receptor.

Interaction with Shc prevents aberrant Erk activation in the absence of extracellular stimuli.

Control mechanisms that prevent aberrant signaling are necessary to maintain cellular homeostasis. We describe a new mechanism by which the adaptor protein Shc directly binds the MAP kinase Erk, thus preventing its activation in the absence of extracellular stimuli. The Shc-Erk complex restricts Erk nuclear translocation, restraining Erk-dependent transcription of genes, including those responsible for oncogenic growth.

Grb2 controls phosphorylation of FGFR2 by inhibiting receptor kinase and Shp2 phosphatase activity.

Constitutive receptor tyrosine kinase phosphorylation requires regulation of kinase and phosphatase activity to prevent aberrant signal transduction. A dynamic mechanism is described here in which the adaptor protein, growth factor receptor-bound protein 2 (Grb2), controls fibroblast growth factor receptor 2 (FGFR2) signaling by regulating receptor kinase and SH2 domain-containing protein tyrosine phosphatase 2 (Shp2) phosphatase activity in the absence of extracellular stimulation. FGFR2 cycles between its kinase-active, partially phosphorylated, nonsignaling state and its Shp2-dephosphorylated state.

Inhibition of basal FGF receptor signaling by dimeric Grb2.

Receptor tyrosine kinase activity is known to occur in the absence of extracellular stimuli. Importantly, this "background" level of receptor phosphorylation is insufficient to effect a downstream response, suggesting that strict controls are present and prohibit full activation. Here a mechanism is described in which control of FGFR2 activation is provided by the adaptor protein Grb2.

Direct binding of Grb2 SH3 domain to FGFR2 regulates SHP2 function.

The adaptor protein Grb2 is recruited to intracellular early signalling complexes of many receptor tyrosine kinases and plays an important role transducing signals leading to MAP kinase activation. To date the SH2 domain of Grb2 has been shown to mediate receptor interactions with phosphorylated tyrosine residues sited directly on the receptor or on auxiliary docking proteins. Here we report that FGFR2 recruits Grb2 through its C-terminal SH3 domain.

Hornet sting induced systemic allergic reaction and large local reaction with bulle formation and rhabdomyolysis.

A 41-yr-old, previously healthy male was stung once by a hornet. The patient had no history of allergy or hornet stings. Physical examination revealed swelling of the right side of his body with blister formation on the extremities.