Multivalent Interactions between Molecular Components Involved in Clathrin Independent Endocytosis Drive Protein Phase Separation

Endocytosis of transmembrane receptors initiates via molecular interactions between the activated receptor and the endocytic machinery. A specific group of receptors, including the β1-adrenergic receptor (β1-AR), is internalized through a non-clathrin pathway known as Fast Endophilin Mediated Endocytosis (FEME). A key question is: how does the endocytic machinery assemble and how is it modulated by activated receptors during FEME. Here we show that endophilin, a major regulator of FEME, undergoes a phase transition into liquid-like condensates, which facilitates the formation of multi-protein assemblies by enabling the phase partitioning of endophilin binding proteins. The phase transition can be triggered by specific multivalent binding partners of endophilin in the FEME pathway such as the third intracellular loop (TIL) of the β1-AR, and the proline-rich-motifs of lamellipodin (LPD-PRMs). Other endocytic accessory proteins can either partition into, or target interfacial regions of, these condensate droplets. On the membrane, TIL promotes protein clustering in the presence of endophilin and LPD-PRMs. Our results demonstrate how the multivalent interactions between endophilin, LPD-PRMs and TIL regulate protein assembly formation on the membrane, providing mechanistic insights into the priming and initiation steps of FEME.

Different temporal requirements for the LRR transmembrane receptors Tartan and Toll-2 in the formation of contractile interfaces at compartmental boundaries

Compartmental boundaries physically separate groups of epithelial cells, a property fundamental for the organization of the body plan in both insects and vertebrates. In many examples, this physical separation has been shown to be the consequence of a regulated increase in contractility of the actomyosin cortex at boundary cell-cell interfaces, a property important in developmental morphogenesis beyond compartmental boundary formation. In this study, we took an unbiased screening approach to identify cell surface receptors required for actomyosin enrichment and polarisation at parasegmental boundaries (PSBs) in early Drosophila embryos, leading us to uncover different temporal requirements for two LRR receptors, Tartan and Toll-2. First, we find that Tartan is required during germband extension for actomyosin enrichment at PSBs, confirming an earlier report. Next, by following in real time the dynamics of loss of boundary straightness in tartan mutant embryos compared to wildtype and ftz mutant embryos, we show that Tartan is not required beyond germband extension. At this stage, actomyosin enrichment at PSBs becomes regulated by Wingless signalling. We find that Wingless signalling regulates Toll-2 expression and we show that Toll-2 is required for planar polarization of actomyosin after the completion of germ-band extension. Thus the formation of contractile interfaces at PSBs depends on a dynamic set of LRR receptors cues. Our study also suggests that the number of receptor cues is small and that the receptors are interchangeable.

Toll-like Receptors (TLRs), NOD-like Receptors (NLRs) and RIG-I-like Receptors (RLRs) in Innate Immunity. TLRs, NLRs and RLRs Ligands as Immunotherapeutic Agents for Hematopoietic Diseases

The innate immune system plays a pivotal role in the first line of host defense against infections and is equipped with patterns recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). Several classes of PRRS, including Toll-like receptors (TLRs), NOD-like receptors (NLRs), and RIG-I-like receptors (RLRs) recognize distinct microbial components and directly activate immune cells. TLRs are transmembrane receptors, while NLRs and RLRs are intracellular molecules. Exposure of immune cells to the ligands of these receptors activates intracellular signaling cascades that rapidly induce the expression of a variety of overlapping and unique genes involved in the inflammatory and immune responses. The innate immune system also influences pathways involved in cancer immunosurveillance. Natural and synthetic agonists of TLRs, NLRs, or RLRs can trigger cell death in malignant cells, recruit immune cells, such as DCs, CD8+ T cells, and NK cells, into the tumor microenvironment, and are being explored as promising adjuvants in cancer immunotherapies. In this review, we provide a concise overview of TLRs, NLRs, and RLRs: their structure, functions, signaling pathways, and regulation. We also describe various ligands for these receptors and their possible application in treatment of hematopoietic diseases.

Neural crest-related NXPH1/α-NRXN signaling opposes neuroblastoma malignancy by inhibiting metastasis

Neuroblastoma is a pediatric cancer that can present as low- or high-risk tumors (LR-NBs and HR-NBs), the latter group showing poor prognosis due to metastasis and strong resistance to current therapy. NBs are known to originate from alterations to cells in the sympatho-adrenal lineage derived from the neural crest, but whether LR-NBs and HR-NBs differ in the way they exploit the transcriptional program underlying their developmental origin remains unclear. Here, we compared the transcriptional landscapes of primary samples of LR-NBs, HR-NBs and human fetal adrenal gland, and thereby identified the transcriptional signature associated to NB formation that further distinguishes LR-NBs from HR-NBs. The majority of the genes comprising this signature belong to the core sympatho-adrenal developmental program, are associated with favorable patient prognosis and with diminished disease progression. The top candidate gene of this list, Neurexophilin-1 (NXPH1), encodes a ligand of the transmembrane receptors α-Neurexins (α-NRXNs). Our functional in vivo and in vitro assays reveal that NXPH1/α-NRXN signaling has a dual impact on NB behavior: whereas NXPH1 and α-NRXN1 promote NB tumor growth by stimulating cell proliferation, they conversely inhibit the ability of NB cells to form metastases. Our findings uncover a module of the neural crest-derived sympatho-adrenal developmental program that opposes neuroblastoma malignancy by impeding metastasis, and pinpoint NXPH1/α-NRXN signaling as a promising target to treat HR-NBs.

Targeting the toll-like receptor pathway as a therapeutic strategy for neonatal infection

Toll-like receptors (TLRs) are crucial transmembrane receptors that form part of the innate immune response. They play a role in the recognition of various microorganisms and their elimination from the host. TLRs have been proposed as vital immunomodulators in the regulation of multiple neonatal stressors that extend beyond infection such as oxidative stress and pain. The immune system is immature at birth and takes some time to become fully established. As such, babies are especially vulnerable to sepsis at this early stage of life. Findings suggest a gestational age-dependent increase in TLR expression. TLRs engage with accessory and adaptor proteins to facilitate recognition of pathogens and their activation of the receptor. TLRs are generally upregulated during infection and promote the transcription and release of proinflammatory cytokines. Several studies report that TLRs are epigenetically modulated by chromatin changes and promoter methylation upon bacterial infection which have long-term influences on immune responses. TLR activation is reported to modulate cardiorespiratory responses during infection and may play a key role in driving homeostatic instability observed during sepsis. Although complex, TLR signalling and downstream pathways are potential therapeutic targets in the treatment of neonatal diseases. By reviewing the expression and function of key toll-like receptors, we aim to provide an important framework to understand the functional role of these receptors in response to stress and infection in premature infants.

Proteomics analysis identifies PEA-15 as an endosomal phosphoprotein that regulates α5β1 integrin endocytosis

Endosomal trafficking of cell surface receptors is essential to their function. Integrins are transmembrane receptors that integrate adhesion to the extracellular matrix with engagement of the cytoskeleton. Ligated integrins mediate diverse signals that regulate matrix assembly, cell survival, cell morphology, and cell motility. Endosomal trafficking of integrins modulates these signals and contributes to cell motility and is required for cancer cell invasion. The phosphoprotein PEA-15 modulates integrin activation and ERK MAP Kinase signaling. To elucidate novel PEA-15 functions we utilized an unbiased proteomics approach. We identified several binding partners for PEA-15 in the endosome including clathrin and AP-2 as well as integrin β1 and other focal adhesion complex proteins. We confirmed these interactions using proximity ligation analysis, immunofluorescence imaging, pull-down and co-immunoprecipitation. We further found that PEA-15 is enriched in endosomes and was required for efficient endosomal internalization of α5β1 integrin and cellular migration. Importantly, PEA-15 promotion of migration was dependent on PEA-15 phosphorylation at serines 104 and 116. These data support a novel endosomal role for PEA-15 in control of endosomal trafficking of integrins through an association with the β1 integrin and clathrin complexes, and thereby regulation of cell motility.

Heterodimer-heterotetramer formation mediates enhanced sensor activity in a biophysical model for BMP signaling

Numerous stages of organismal development rely on the cellular interpretation of gradients of secreted morphogens including members of the Bone Morphogenetic Protein (BMP) family through transmembrane receptors. Early gradients of BMPs drive dorsal/ventral patterning throughout the animal kingdom in both vertebrates and invertebrates. Growing evidence in Drosophila, zebrafish, murine and other systems suggests that BMP ligand heterodimers are the primary BMP signaling ligand, even in systems in which mixtures of BMP homodimers and heterodimers are present. Signaling by heterodimers occurs through a hetero-tetrameric receptor complex comprising of two distinct type one BMP receptors and two type II receptors. To understand the system dynamics and determine whether kinetic assembly of heterodimer-heterotetramer BMP complexes is favored, as compared to other plausible BMP ligand-receptor configurations, we developed a kinetic model for BMP tetramer formation based on current measurements for binding rates and affinities. We find that contrary to a common hypothesis, heterodimer-heterotetramer formation is not kinetically favored over the formation of homodimer-tetramer complexes under physiological conditions of receptor and ligand concentrations and therefore other mechanisms, potentially including differential kinase activities of the formed heterotetramer complexes, must be the cause of heterodimer-heterotetramer signaling primacy. Further, although BMP complex assembly favors homodimer and homomeric complex formation over a wide range of parameters, ignoring these signals and instead relying on the heterodimer improves the range of morphogen interpretation in a broad set of conditions, suggesting a performance advantage for heterodimer signaling in patterning multiple cell types in a gradient.

A CLN6-CRMP2-KLC4 complex regulates anterograde ER-derived vesicle trafficking in cortical neurites

As neurons establish extensive connections throughout the central nervous system, the transport of cargo along the microtubule network of the axon is crucial for differentiation and homeostasis. Specifically, building blocks such as membrane and cytoskeletal components, organelles, transmembrane receptors, adhesion molecules, and peptide neurotransmitters all require proper transport to the presynaptic compartment. Here, we identify a novel complex regulating vesicular endoplasmic reticulum transport in neurites, composed of CLN6: an ER-associated protein of relatively unknown function implicated in CLN6-Batten disease; CRMP2: a tubulin binding protein important in regulating neurite microtubule dynamics; and KLC4: a classic transport motor protein. We show that this 'CCK' complex allows ER-derived vesicles to migrate to the distal end of the axon, aiding in proper neurite outgrowth and arborization. In the absence of CLN6, the CCK complex does not function effectively, leading to reduced vesicular transport, stunted neurite outgrowth, and deficits in CRMP2 binding to other protein partners. Treatment with a CRMP2 modulating compound, lanthionine ketimine ester, partially restores these deficits in CLN6-deficient mouse neurons, indicating that stabilization of CRMP2 interacting partners may prove beneficial in lieu of complete restoration of the CCK complex. Taken together, these findings reveal a novel mechanism of ER-derived vesicle transport in the axon and provide new insights into therapeutic targets for neurodegenerative disease.

Predicting Transmembrane (TM) Domain Dimer Structures using Martini 3

Determination of the structure and dynamics of transmembrane (TM) domains of single-transmembrane receptors is key to understanding their mechanism of signal transduction across the plasma membrane. Although many studies have been performed on isolated soluble extra- and intracellular receptor domains in aqueous solutions, limited knowledge exists on the lipid embedded TM region. In this study, we predict the assembly of alternate configurations of receptor TM domain dimers using the Martini 3 force field for coarse-grain (CG) molecular dynamic simulations. This recent version of Martini has new bead types and sizes, which allows more accurate predictions of molecular packing and interactions compared to the previous version. Our results with Martini 3 simulations show overall good agreement with ab initio predictions using PREDDIMER and with available NMR derived structures for TM helix dimers. Understanding and predicting the association TM domains may help us to better understand the signalling mechanism of TM receptors, in turn providing the opportunity for development of new pharmaceuticals, some of which are peptide based.

Cancer chemoprevention through Frizzled receptors and EMT

Frizzled (FZD) transmembrane receptors are well known for their role in β-catenin signaling and development and now understanding of their role in the context of cancer is growing. FZDs are often associated with the process of epithelial to mesenchymal transition (EMT) through β-catenin, but some also influence EMT through non-canonical pathways. With ten different FZDs, there is a wide range of activity from oncogenic to tumor suppressive depending on the tissue context. Alterations in FZD signaling can occur during development of premalignant lesions, supporting their potential as targets of chemoprevention agents. Agonizing or antagonizing FZD activity may affect EMT, which is a key process in lesion progression often targeted by chemoprevention agents. Recent studies identified a specific FZD as important for activity of an EMT inhibiting chemopreventive agent and other studies have highlighted the previously unrecognized potential for targeting small molecules to FZD receptors. This work demonstrates the value of investigating FZDs in chemoprevention and here we provide a review of FZDs in cancer EMT and their potential as chemoprevention targets.