Mechanistic target of rapamycin complex 1 (mTORC1) plays a pivotal role in controlling cell growth and metabolism in response to nutrients and growth factors. The activity of mTORC1 is dually regulated by amino acids and growth factor signaling, and amino acid-dependent mTORC1 activity is regulated by mTORC1 interaction with the Ragulator-Rag GTPase complex, which is localized to the surface of lysosomes via a membrane-anchored protein, p18/Lamtor1. However, the physiological function of p18-Ragulator-dependent mTORC1 signaling remains elusive. The present study evaluated the function of p18-mediated mTORC1 signaling in the intestinal epithelia using p18 conditional knockout mice. In p18 knockout colonic crypts, mTORC1 was delocalized from lysosomes, and in vivo mTORC1 activity was markedly decreased. Histologically, p18 knockout crypts exhibited significantly increased proliferating cells and dramatically decreased mucin-producing goblet cells, while overall crypt architecture and enteroendocrine cell differentiation were unaffected. Furthermore, p18 knockout crypts normally expressed transcription factors implicated in crypt differentiation, such as Cdx2 and Klf4, indicating that p18 ablation did not affect the genetic program of cell differentiation. Analysis of colon crypt organoid cultures revealed that both p18 ablation and rapamycin treatment robustly suppressed development of mucin-producing goblet cells. Hence, p18-mediated mTORC1 signaling could promote the anabolic metabolism required for robust mucin production in goblet cells to protect the intestinal epithelia from various external stressors.
Key words: mTORC1, p18/lamtor1, intestinal epithelium, goblet cells, mucin
Collagen is the most abundant protein in animal tissues and is critical for their proper organization. Nascent procollagens in the endoplasmic reticulum (ER) are considered too large to be loaded into coat protein complex II (COPII) vesicles, which have a diameter of 60–80 nm, for exit from the ER and transport to the Golgi complex. To study the transport mechanism of procollagen IV, which generates basement membranes, we introduced a cysteine-free GFP tag at the N-terminus of the triple helical region of the α1(IV) chain (cfSGFP2-col4a1), and examined the dynamics of this protein in HT-1080 cells, which produce endogenous collagen IV. cfSGFP2-col4a1 was transported from the ER to the Golgi by vesicles, which were a similar size as small cargo carriers. However, mCherry-ERGIC53 was recruited to α1-antitrypsin-containing vesicles, but not to cfSGFP2-col4a1-containing vesicles. Knockdown analysis revealed that Sar1 and SLY1/SCFD1 were required for transport of cfSGFP2-col4a1. TANGO1, CUL3, and KLHL12 were not necessary for the ER-to-Golgi trafficking of procollagen IV. Our data suggest that procollagen IV is exported from the ER via an enlarged COPII coat carrier and is transported to the Golgi by unique transport vesicles without recruitment of ER-Golgi intermediate compartment membranes.
Key words: collagen, procollagen IV, endoplasmic reticulum, ER-to-Golgi transport, ERGIC
The activity of AMPA-type glutamate receptor is involved in insulin release from pancreatic β-cells. However, the mechanism and dynamics that underlie AMPA receptor-mediated insulin release in β-cells is largely unknown. Here, we show that AMPA induces internalization of glutamate receptor 2/3 (GluR2/3), AMPA receptor subtype, in the mouse β-cell line MIN6. Immunofluorescence experiments showed that GluR2/3 appeared as fine dots that were distributed throughout MIN6 cells. Intracellular GluR2/3 co-localized with AP2 and clathrin, markers for clathrin-coated pits and vesicles. Immunoelectron microscopy revealed that GluR2/3 was also localized at plasma membrane. Surface biotinylation and immunofluorescence measurements showed that addition of AMPA caused an approximate 1.8-fold increase in GluR2/3 internalization under low-glucose conditions. Furthermore, internalized GluR2 largely co-localized with EEA1, an early endosome marker. In addition, GluR2/3 co-immunoprecipitated with cortactin, a F-actin binding protein. Depletion of cortactin by RNAi in MIN6 cells altered the intracellular distribution of GluR2/3, suggesting that cortactin is involved in internalization of GluR2/3 in MIN6 cells. Taken together, our results suggest that pancreatic β-cells adjust the amount of AMPA-type GluR2/3 on the cell surface to regulate the receptive capability of the cell for glutamate.
Key words: endocytosis, GluR2, AMPA, cortactin, MIN6
Tissue absorbance, light scattering, and autofluorescence are significantly lower in the near-infrared (NIR) range than in the visible range. Because of these advantages, NIR fluorescent proteins (FPs) are in high demand for in vivo imaging. Nevertheless, application of NIR FPs such as iRFP is still limited due to their dimness in mammalian cells. In contrast to GFP and its variants, iRFP requires biliverdin (BV) as a chromophore. The dimness of iRFP is at least partly due to rapid reduction of BV by biliverdin reductase-A (BLVRA). Here, we established biliverdin reductase-a knockout (Blvra–/–) mice to increase the intracellular BV concentration and, thereby, to enhance iRFP fluorescence intensity. As anticipated, iRFP fluorescence intensity was significantly increased in all examined tissues of Blvra–/– mice. Similarly, the genetically encoded calcium indicator NIR-GECO1, which is engineered based on another NIR FP, mIFP, exhibited a marked increase in fluorescence intensity in mouse embryonic fibroblasts derived from Blvra–/– mice. We expanded this approach to an NIR light-sensing optogenetic tool, the BphP1-PpsR2 system, which also requires BV as a chromophore. Again, deletion of the Blvra gene markedly enhanced the light response in HeLa cells. These results indicate that the Blvra–/– mouse is a versatile tool for the in vivo application of NIR FPs and NIR light-sensing optogenetic tools.
Key words: in vivo imaging, near-infrared fluorescent protein, biliverdin, biliverdin reductase, optogenetic tool
Prickle2 has been identified in genetic studies of subjects with autism spectrum disorder (ASD) and epilepsy, but the pathological mechanism of Prickle2 remains to be fully understood. Proteomic analysis of Prickle2 with mass spectrometry revealed twenty-eight Prickle2 interactors, including immunoglobulin superfamily member 9b (Igsf9b), in the brain. Here, because Igsf9 family proteins are associated with psychiatric diseases and seizures, we studied the physiological interaction between Prickle2 and Igsf9b. Prickle2 colocalized with Igsf9b in cultured hippocampal neurons. Knockdown of Prickle2 affected the subcellular localization of Igsf9b. Interestingly, Igsf9b localized along axonal processes in a pattern opposite to the ASD-related molecule ANK3/AnkG. AnkG is a major component of the axon initial segment (AIS), where a variety of ASD and epilepsy susceptibility proteins accumulate. Igsf9b-knockdown neurons displayed altered AnkG localization. Prickle2 depletion caused defects in AnkG and voltage-gated Na+ channel localization, resulting in altered network activity. These results support the idea that Prickle2 regulates AnkG distribution by controlling the proper localization of Igsf9b. The novel function of Prickle2 in AIS cytoarchitecture provides new insights into the shared pathology of ASD and epilepsy.
Key words: Prickle2, Igsf9b, axon initial segment, neuronal excitability, ASD