Phase separation of insulin receptor substrate 1 drives the formation of insulin/IGF-1 signalosomes.

As a critical node for insulin/IGF signaling, insulin receptor substrate 1 (IRS-1) is essential for metabolic regulation. A long and unstructured C-terminal region of IRS-1 recruits downstream effectors for promoting insulin/IGF signals. However, the underlying molecular basis for this remains elusive. Here, we found that the C-terminus of IRS-1 undergoes liquid-liquid phase separation (LLPS). Both electrostatic and hydrophobic interactions were seen to drive IRS-1 LLPS. Self-association of IRS-1, which was mainly mediated by the 301-600 region, drives IRS-1 LLPS to form insulin/IGF-1 signalosomes. Moreover, tyrosine residues of YXXM motifs, which recruit downstream effectors, also contributed to IRS-1 self-association and LLPS. Impairment of IRS-1 LLPS attenuated its positive effects on insulin/IGF-1 signaling. The metabolic disease-associated G972R mutation impaired the self-association and LLPS of IRS-1. Our findings delineate a mechanism in which LLPS of IRS-1-mediated signalosomes serves as an organizing center for insulin/IGF-1 signaling and implicate the role of aberrant IRS-1 LLPS in metabolic diseases.

[Improvement and application of a fast labeling system for bulk internalized vesicles].

To study trafficking of bulk internalized vesicles such as macropinosome and lysosome in live cells, an efficient and convenient assay was established according to the axon turning assay. By injecting indicator or fluorescent dyes through a micropipette with air pressure into cell cultures to create a stable gradient around the micropipette tip, vesicles were indicated and labeled. With live cell imaging, the whole process was recorded. Without wash-out of fluorescent dyes and transferring, this assay is an effective, fast labeling system for bulk internalized vesicles, and can also be combined with imaging system.

A novel size-based sorting mechanism of pinocytic luminal cargoes in microglia.

Microglia are the resident immune cells in the CNS and play diverse roles in the maintenance of CNS homeostasis. Recent studies have shown that microglia continually survey the CNS microenvironment and scavenge cell debris and aberrant proteins by phagocytosis and pinocytosis, and that reactive microglia are capable to present antigens to T cells and initiate immune responses. However, how microglia process the endocytosed contents and evoke an immune response remain unclear. Here we report that a size-dependent selective transport of small soluble contents from the pinosomal lumen into lysosomes is critical for the antigen processing in microglia. Using fluorescent probes and water-soluble magnetic nanobeads of defined sizes, we showed in cultured rodent microglia, and in a cell-free reconstructed system that pinocytosed proteins become degraded immediately following pinocytosis and the resulting peptides are selectively delivered to major histocompatibility complex class II (MHC-II) containing lysosomes, whereas undegraded proteins are retained in the pinosomal lumen. This early size-based sorting of pinosomal contents relied on the formation of transient tunnel between pinosomes and lysosomes in a Rab7- and dynamin II-dependent manner, which allowed the small contents to pass through but restricted large ones. Inhibition of the size-based sorting markedly reduced proliferation and cytokine release of cocultured CD4(+) T cells, indicating that the size-based sorting is required for efficient antigen presentation by microglial cells. Together, these findings reveal a novel early sorting mechanism for pinosomal luminal contents in microglial cells, which may explain how microglia efficiently process protein antigens and evoke an immune response.

[Preparation and up-conversion luminescence properties of Yb3+/Tm3+ co-doped Sb2O4 powder].

The Sb2O4:Yb3+, Tm3+ up-conversion luminescence powder with excellent physical, chemical stability and relative low phonon energy was synthesized by the high temperature solid-state reaction and its up-conversion luminescence property was investigated. Under the 980 nm excitation, infrared and blue up-conversion emissions centered at 800 and 480 nm were observed, which were assigned to the 1G4-->3H6 and 3H4-->3 He transitions of Tm2+, respectively. The influence of Yb3+ and Tm3+ concentration on the up-conversion emission property was also obtained. The up-conversion luminescence increases with increasing of Yb3+ and Tm3+ concentration. Additionally, the up-conversion luminescence mechanism was discussed based on the dependence of Tm3+ up-conversion luminescence on pump power. It is interesting that two photon excitation processes for blue and infrared emission were observed in the Sb2O04: Yb3+, Tm3+ powder under a 980 nm excitation. Based on the energy level diagram of Tma3 and Yb2+ ions, we think that two photons blue emission is contributed to the cooperation energy transfer between Tm"+ and Yb3+ ions. We believe that the Sbz04 : Yb3 , Tm2+ up-conversion luminescence powder will have potential application for new optical devices in up-conversion color displays, sensors, detection of infrared radiation, and lasers.

Analysis of microglial migration by a micropipette assay.

Microglial cells have important roles in maintaining brain homeostasis, and they are implicated in multiple brain diseases. There is currently interest in investigating microglial migration that results in cell accumulation at focal sites of injury. Here we describe a protocol for rapidly triggering and monitoring microglial migration by using a micropipette assay. This protocol is an adaptation of the axon turning assay using microglial cells. Chemoattractants released from the micropipette tip produce a chemotactic gradient that induces robust microglial migration. In combination with microscopic imaging, this assay allows simultaneous recording of cell movement and subcellular compartment trafficking, along with quantitative analysis. The actual handling time for the assay takes ∼2-3 h in total. The protocol is simple, inexpensive and convenient to set up, and it can be adopted to examine cell migration in multiple cell types, including cancer cells with a wide range of chemical signals.

P2Y4 receptor-mediated pinocytosis contributes to amyloid beta-induced self-uptake by microglia.

Brain disturbances, like injuries or aberrant protein deposits, evoke nucleotide release or leakage from cells, leading to microglial chemotaxis and ingestion. Recent studies have identified P2Y12 purinergic receptors as triggers for microglial chemotaxis and P2Y6 receptors as mediators for phagocytosis. However, pinocytosis, known as the internalization of fluid-phase materials, has received much less attention. We found that ATP efficiently triggered pinocytosis in microglia. Pharmacological analysis and knockdown experiments demonstrated the involvement of P2Y4 receptors and the phosphatidylinositol 3-kinase/Akt cascade in the nucleotide-induced pinocytosis. Further evidence indicated that soluble amyloid beta peptide 1-42 induced self-uptake in microglia through pinocytosis, a process involving activation of P2Y4 receptors by autocrine ATP signaling. Our results demonstrate a previously unknown function of ATP as a "drink me" signal for microglia and P2Y4 receptors as a potential therapeutic target for the treatment of Alzheimer's disease.

Microglial migration mediated by ATP-induced ATP release from lysosomes.

Microglia are highly motile cells that act as the main form of active immune defense in the central nervous system. Attracted by factors released from damaged cells, microglia are recruited towards the damaged or infected site, where they are involved in degenerative and regenerative responses and phagocytotic clearance of cell debris. ATP release from damaged neural tissues has been suggested to mediate the rapid extension of microglial process towards the site of injury. However, the mechanisms of the long-range migration of microglia remain to be clarified. Here, we found that lysosomes in microglia contain abundant ATP and exhibit Ca(2+)-dependent exocytosis in response to various stimuli. By establishing an efficient in vitro chemotaxis assay, we demonstrated that endogenously-released ATP from microglia triggered by local microinjection of ATPγS is critical for the long-range chemotaxis of microglia, a response that was significantly inhibited in microglia treated with an agent inducing lysosome osmodialysis or in cells derived from mice deficient in Rab 27a (ashen mice), a small GTPase required for the trafficking and exocytosis of secretory lysosomes. These results suggest that microglia respond to extracellular ATP by releasing ATP themselves through lysosomal exocytosis, thereby providing a positive feedback mechanism to generate a long-range extracellular signal for attracting distant microglia to migrate towards and accumulate at the site of injury.