An optogenetic method for the controlled release of single molecules.

We developed a system for optogenetic release of single molecules in cells. We confined soluble and transmembrane proteins to the Golgi apparatus via a photocleavable protein and released them by short pulses of light. Our method allows for a light dose-dependent delivery of functional proteins to the cytosol and plasma membrane in amounts compatible with single-molecule imaging, greatly simplifying access to single-molecule microscopy of any protein in live cells. We were able to reconstitute ion conductance by delivering BK and LRRC8/volume-regulated anion channels to the plasma membrane. Finally we were able to induce NF-kB signaling in T lymphoblasts stimulated by interleukin-1 by controlled release of a signaling protein that had been knocked out. We observed light-induced formation of functional inflammatory signaling complexes that triggered phosphorylation of the inhibitor of nuclear factor kappa-B kinase only in activated cells. We thus developed an optogenetic method for the reconstitution and investigation of cellular function at the single-molecule level.

Myddosome clustering in IL-1 receptor signaling regulates the formation of an NF-kB activating signalosome.

IL-1 receptor (IL-1R) signaling can activate thresholded invariant outputs and proportional outputs that scale with the amount of stimulation. Both responses require the Myddosome, a multiprotein complex. The Myddosome is required for polyubiquitin chain formation and NF-kB signaling. However, how these signals are spatially and temporally regulated to drive switch-like and proportional outcomes is not understood. During IL-1R signaling, Myddosomes dynamically reorganize into multi-Myddosome clusters at the cell membrane. Blockade of clustering using nanoscale extracellular barriers reduces NF-kB activation. Myddosomes function as scaffolds that assemble an NF-kB signalosome consisting of E3-ubiquitin ligases TRAF6 and LUBAC, K63/M1-linked polyubiquitin chains, phospho-IKK, and phospho-p65. This signalosome preferentially assembles at regions of high Myddosome density, which enhances the recruitment of TRAF6 and LUBAC. Extracellular barriers that restrict Myddosome clustering perturbed the recruitment of both ligases. We find that LUBAC was especially sensitive to clustering with 10-fold lower recruitment to single Myddosomes than clustered Myddosomes. These data reveal that the clustering behavior of Myddosomes provides a basis for digital and analog IL-1R signaling.

Visualization of Myddosome Assembly in Live Cells.

The Myddosome is an oligomeric protein complex composed of MyD88 and members of IL-1 receptor-associated kinase (IRAK) family that transduce signals from Toll-like and IL-1 family receptors. The molecular dynamics of Myddosome formation and how the Myddosome organizes downstream signaling reactions provide insight into how TLR/IL-1Rs activate a decisive cellular response critical for the induction of inflammation. Supported lipid membranes formed on a continuous glass coverslip have been extensively used to study the molecular dynamics of receptor signaling. Here, we describe a protocol for the formation of IL-1-functionalized support lipid membrane that can be used to visualize the molecular dynamics of Myddosome formation and signaling in live cells.

MyD88 oligomer size functions as a physical threshold to trigger IL1R Myddosome signaling.

A recurring feature of innate immune receptor signaling is the self-assembly of signaling proteins into oligomeric complexes. The Myddosome is an oligomeric complex that is required to transmit inflammatory signals from TLR/IL1Rs and consists of MyD88 and IRAK family kinases. However, the molecular basis for how Myddosome proteins self-assemble and regulate intracellular signaling remains poorly understood. Here, we developed a novel assay to analyze the spatiotemporal dynamics of IL1R and Myddosome signaling in live cells. We found that MyD88 oligomerization is inducible and initially reversible. Moreover, the formation of larger, stable oligomers consisting of more than four MyD88s triggers the sequential recruitment of IRAK4 and IRAK1. Notably, genetic knockout of IRAK4 enhanced MyD88 oligomerization, indicating that IRAK4 controls MyD88 oligomer size and growth. MyD88 oligomer size thus functions as a physical threshold to trigger downstream signaling. These results provide a mechanistic basis for how protein oligomerization might function in cell signaling pathways.

Abl-mediated PI3K activation regulates macrophage podosome formation.

Podosomes play crucial roles in macrophage adhesion and migration. Wiskott-Aldrich syndrome protein (WASP; also known as WAS)-mediated actin polymerization is one of the key events initiating podosome formation. Nevertheless, membrane signals to trigger WASP activation at macrophage podosomes remain unclear. Here, we show that phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3] lipids are enriched at the podosome and stably recruit WASP rather than the WASP-5KE mutant. Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit ß (PIK3CB) is spatially located at the podosome core. Inhibition of PIK3CB and overexpression of phosphatase and tensin homolog (PTEN) impede F-actin polymerization of the podosome. PIK3CB activation is regulated by Abl1 and Src family kinases. At the podosome core, Src and Hck promote the phosphorylation of Tyr488 in the consensus Y-x-x-M motif of Abl1, which enables the association of phosphoinositide 3-kinase (PI3K) regulatory subunits. Knockdown of Abl1 rather than Abl2 suppresses the PI3K/Akt pathway, regardless of Src and Hck activities. Reintroduction of wild-type Abl1 rather than the Abl1-Y488F mutant rescues PI3KR1 recruitment and PI3K activation. When PIK3CB, Abl1 or Src/Hck is suppressed, macrophage podosome formation, matrix degradation and chemotactic migration are inhibited. Thus, Src/Hck-mediated phosphorylation of Abl1 Tyr488 triggers PIK3CB-dependent PI(3,4,5)P3 production and orchestrates the assembly and function of macrophage podosomes.

Podosome formation promotes plasma membrane invagination and integrin-ß3 endocytosis on a viscous RGD-membrane.

Integrin receptors orchestrate cell adhesion and cytoskeletal reorganization. The endocytic mechanism of integrin-ß3 receptor at the podosome remains unclear. Using viscous RGD-membrane as the model system, here we show that the formation of podosome-like adhesion promotes Dab2/clathrin-mediated endocytosis of integrin-ß3. Integrin-ß3 and RGD ligand are endocytosed from the podosome and sorted into the endosomal compartment. Inhibitions of podosome formation and knockdowns of Dab2 and clathrin reduce RGD endocytosis. F-actin assembly at the podosome core exhibits protrusive contact towards the substrate and results in plasma membrane invaginations at the podosome ring. BIN1 specifically associates with the region of invaginated membrane and recruits DNM2. During the podosome formation, BIN1 and DNM2 synchronously enrich at the podosome ring and trigger clathrin dissociation and RGD endocytosis. Knockdowns of BIN1 and DNM2 suppress RGD endocytosis. Thus, plasma membrane invagination caused by F-actin polymerization promotes BIN1-dependent DNM2 recruitment and facilitate integrin-ß3 endocytosis at the podosome.

Tail domains of myosin-1e regulate phosphatidylinositol signaling and F-actin polymerization at the ventral layer of podosomes.

During podosome formation, distinct phosphatidylinositol 3,4,5-trisphosphate lipid (PI(3,4,5)P3) production and F-actin polymerization take place at integrin-mediated adhesions. Membrane-associated actin regulation factors, such as myosin-1, serve as key molecules to link phosphatidylinositol signals to podosome assembly. Here, we report that long-tailed myosin-1e (Myo1e) is enriched at the ventral layer of the podosome core in a PI(3,4,5)P3-dependent manner. The combination of TH1 and TH2 (TH12) of Myo1e tail domains contains the essential motif for PI(3,4,5)P3-dependent membrane association and ventral localization at the podosome. TH12 KR2A (K772A and R782A) becomes dissociated from the plasma membrane. While F-actin polymerizations are initialized from the ventral layer of the podosome, TH12 precedes the recruitment of N-WASP and Arp2/3 in the initial phase of podosome formation. Overexpression of TH12, not TH12 KR2A, impedes PI(3,4,5)P3 signaling, restrains F-actin polymerization, and inhibits podosome formation. TH12 also suppresses gelatin degradation and migration speed of invadopodia-forming A375 melanoma cells. Thus, TH12 domain of Myo1e serves as a regulatory component to connect phosphatidylinositol signaling to F-actin polymerization at the podosome.

Nucleolar residence of the seckel syndrome protein TRAIP is coupled to ribosomal DNA transcription.

The RING finger protein TRAIP protects genome integrity and its mutation causes Seckel syndrome. TRAIP encodes a nucleolar protein that migrates to UV-induced DNA lesions via a direct interaction with the DNA replication clamp PCNA. Thus far, mechanistically how UV mobilizes TRAIP from the nucleoli remains unknown. We found that PCNA binding is dispensable for the nucleolus-nucleoplasm shuttling of TRAIP following cell exposure to UV irradiation, and that its redistribution did not rely on the master DNA damage kinases ATM and ATR. Interestingly, I-PpoI-induced ribosomal DNA damage led to TRAIP exclusion from the nucleoli, raising the possibility that active ribosomal DNA transcription may underlie TRAIP retention in the nuclear sub-compartments. Accordingly, chemical inhibition of RNA polymerase I activity led to TRAIP diffusion into the nucleoplasm, and was coupled with marked reduction of DNA/RNA hybrids in the nucleoli, suggesting that TRAIP may be sequestered via binding to nucleic acid structures in the nucleoli. Consistently, cell pre-treatment with DNase/RNase effectively released TRAIP from the nucleoli. Taken together, our study defines a bipartite mechanism that drives TRAIP trafficking in response to UV damage, and highlights the nucleolus as a stress sensor that contributes to orchestrating DNA damage responses.

Integrin-beta3 clusters recruit clathrin-mediated endocytic machinery in the absence of traction force.

The turnover of integrin receptors is critical for cell migration and adhesion dynamics. Here we find that force development at integrins regulates adaptor protein recruitment and endocytosis. Using mobile RGD (Arg-Gly-Asp) ligands on supported lipid membranes (RGD membranes) and rigid RGD ligands on glass (RGD-glass), we find that matrix force-dependent integrin signals block endocytosis. Dab2, an adaptor protein of clathrin-mediated endocytosis, is not recruited to activated integrin-beta3 clusters on RGD-glass; however, it is recruited to integrin-mediated adhesions on RGD membranes. Further, when force generation is inhibited on RGD-glass, Dab2 binds to integrin-beta3 clusters. Dab2 binding to integrin-beta3 excludes other adhesion-related adaptor proteins, such as talin. The clathrin-mediated endocytic machinery combines with Dab2 to facilitate the endocytosis of RGD-integrin-beta3 clusters. From these observations, we propose that loss of traction force on ligand-bound integrin-beta3 causes recruitment of Dab2/clathrin, resulting in endocytosis of integrins.

The apoptotic pathways in the curcumin analog MHMD-induced lung cancer cell death and the essential role of actin polymerization during apoptosis.

As a mode of cell death, apoptosis could be triggered by the extrinsic, intrinsic mitochondrial and intrinsic endoplasmic reticulum pathways and actin rearrangement is needed during apoptosis. We previously found that one curcumin analog MHMD could induce A549 lung cancer cells apoptosis. But the apoptotic pathways and the actin dynamics during apoptosis are not known. Here, we detected the activation of caspase-3, -8, -9, -12, PARP and the increase ratio of Bax/Bcl-2 by western blotting in MHMD-exposed A549 cells. Alternatively, caspases inhibitors could lead to the disappearance of MHMD-eliciting nuclei fragmentation by Hoechst 33342 staining. Besides, JC-1 and DCFH-DA staining showed the fall of mitochondrial membrane potential and the release of ROS. Moreover, wound healing assay confirmed the MHMD anti-migration ability, which was much more effective than curcumin. Importantly, unlike other anticarcinogenic drugs, MHMD might induce the actin polymerization but not depolymerization in the process of A549 cell apoptosis by phalloidin-FITC staining, which is essential to MHMD-induced extrinsic, intrinsic mitochondrial and intrinsic ER pathways of cell apoptosis.