A cluster of thin tubular structures mediates transformation of the endoplasmic reticulum to autophagic isolation membrane.

Recent findings have suggested that the autophagic isolation membrane (IM) might originate from a domain of the endoplasmic reticulum (ER) called the omegasome. However, the morphological relationships between ER, omegasome, and IM remain unclear. In the present study, we found that hybrid structures composed of a double FYVE domain-containing protein 1 (DFCP1)-positive omegasome and the IM accumulated in Atg3-deficient mouse embryonic fibroblasts (MEFs). Moreover, correlative light and electron microscopy and immunoelectron microscopy revealed that green fluorescent protein (GFP)-tagged DFCP1 was localized on tubular or vesicular elements adjacent to the IM rims. Through detailed morphological analyses, including optimization of a fixation method and electron tomography, we observed a cluster of thin tubular structures between the IM edges and ER, part of which were continuous with IM and/or ER. The formation of these thin tubular clusters was observed in several cell lines and MEFs deficient for Atg5, Atg7, or Atg16L1 but not in FIP200-deficient cells, suggesting that they were relevant to the earlier events in autophagosome formation. Taken together, our findings indicate that these tubular profiles represent a part of the omegasome that links the ER with the IM.

Rab11 regulates exocytosis of recycling vesicles at the plasma membrane.

Rab11 is known to associate primarily with perinuclear recycling endosomes and regulate recycling of endocytosed proteins. However, the recycling step in which Rab11 participates remains unknown. We show here that, in addition to causing tubulation of recycling endosomes, Rab11 depletion gives rise to accumulation of recycling carriers containing endocytosed transferrin and transferrin receptor beneath the plasma membrane. We also show that the carriers are transported from perinuclear recycling endosomes to the cell periphery along microtubules. Total internal reflection fluorescence microscopy of cells expressing EGFP-tagged transferrin receptor revealed that Rab11 depletion inhibits tethering and fusion of recycling carriers to the plasma membrane. Depletion of Sec15, which interacts with Rab11, or Exo70, both components of the exocyst tethering complex, leads to essentially the same phenotypes as those of Rab11 depletion. Thus, in addition to its role in recycling processes at perinuclear recycling endosomes, Rab11 is transported along microtubules to the cell periphery through association with recycling carriers, and directly regulates vesicle exocytosis at the plasma membrane in concert with the exocyst.

Uptake through glycoprotein 2 of FimH(+) bacteria by M cells initiates mucosal immune response.

The mucosal immune system forms the largest part of the entire immune system, containing about three-quarters of all lymphocytes and producing grams of secretory IgA daily to protect the mucosal surface from pathogens. To evoke the mucosal immune response, antigens on the mucosal surface must be transported across the epithelial barrier into organized lymphoid structures such as Peyer's patches. This function, called antigen transcytosis, is mediated by specialized epithelial M cells. The molecular mechanisms promoting this antigen uptake, however, are largely unknown. Here we report that glycoprotein 2 (GP2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for mucosal antigens. Recombinant GP2 protein selectively bound a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane. Consistently, these bacteria were colocalized with endogenous GP2 on the apical plasma membrane as well as in cytoplasmic vesicles in M cells. Moreover, deficiency of bacterial FimH or host GP2 led to defects in transcytosis of type-I-piliated bacteria through M cells, resulting in an attenuation of antigen-specific immune responses in Peyer's patches. GP2 is therefore a previously unrecognized transcytotic receptor on M cells for type-I-piliated bacteria and is a prerequisite for the mucosal immune response to these bacteria. Given that M cells are considered a promising target for oral vaccination against various infectious diseases, the GP2-dependent transcytotic pathway could provide a new target for the development of M-cell-targeted mucosal vaccines.

Primary sensory neuronal expression of SLURP-1, an endogenous nicotinic acetylcholine receptor ligand.

Secreted mammalian Ly6/urokinase plasminogen activator receptor-related protein-1 (SLURP-1) is a recently identified, endogenous ligand of the alpha7 subunit of nicotinic acetylcholine receptors. SLURP-1 is also the causative gene for an autosomal recessive palmoplantar keratoderma, Mal de Meleda. Although the function of SLURP-1 in keratinocyte development and differentiation has been extensively studied, little is known about its role in the nervous system. In the present study, we analyzed SLURP-1 expression in the spinal cord of rats, as a number of studies suggest spinal nicotinic acetylcholine receptors are important modulators of pain transmission. We detected intense SLURP-1 immunoreactivity in the dorsal horn of the spinal cord, especially in lamina I and outer II. In dorsal root ganglia, SLURP-1 immunoreactivity was detected in small- to medium-sized neurons, where in situ hybridization also revealed the presence of SLURP-1 mRNA. Fluorescent labeling of SLURP-1 partially overlapped that of calcitonin-gene related peptide (CGRP) or substance P (SP) in both the spinal cord dorsal horn and glabrous skin, and electron microscopic analysis revealed colocalization of SLURP-1 with SP or CGRP, in large synaptic vesicles in terminals within the superficial layer of the spinal cord. Finally, sciatic nerve axotomy reduced levels of SLURP-1 immunoreactivity in parallel with that of SP and CGRP in the ipsilateral superficial dorsal horn. These findings suggest that SLURP-1 is expressed in a subset of primary peptidergic sensory neurons.

A hind limb tourniquet induces interleukin-6 expression in a rat dorsal root ganglion.

We investigated the mRNA levels of interleukin-6-related genes in a rat dorsal root ganglion after application of a tourniquet to a hind limb in order to identify the molecules that are induced immediately after peripheral nerve injury at the early stage. Induction of interleukin-6 and upregulation of glycoprotein 130 mRNA expressions were observed in the ipsilateral dorsal root ganglion at 4 h after tourniquet application. Interleukin-6 protein was detected in small-sized and medium-sized dorsal root ganglion cells by immunohistochemical analysis. The induction of interleukin-6 expression is likely to play a role in the protection of injured neurons perhaps related to growth of their axons. Glycoprotein 130 might also account for the inhibitory effects following nerve injury.

Induction of nerve growth factor mRNA in a rat dorsal root ganglion after application of a tourniquet.

We investigated the mRNA levels of neurotrophins and neurotrophin receptors in a rat dorsal root ganglion (DRG), after tourniquet application to a hind limb, to identify the nerve-protective molecules that are induced immediately after peripheral nerve crush and play a part in the process leading to secondary events. No significant expression of nerve growth factor (NGF) mRNA or protein was observed in the control or contralateral DRG. NGF mRNA expression started within 2 h and NGF protein expression was observed in Schwann cells at 4 h after application of the tourniquet, due to termination of the neurotrophin supply from peripheral nerves. The levels of neurotrophin 3 mRNA were significantly increased in the DRGs on both sides at 1 and 2 h after tourniquet application, but no significant changes in brain-derived neurotrophic factor and neurotrophin 4/5 expression levels were observed in either the contralateral or ipsilateral DRG. The expression levels of neurotrophin receptors in the DRGs on both the contralateral and ipsilateral sides had decreased at 1 to 2 h after application of the tourniquet and had returned to the control levels at 4 h after tourniquet application.

Nitric oxide synthase expressions in rat dorsal root ganglion after a hind limb tourniquet.

We investigated the mRNA levels of neuronal, inducible, endothelial nitric oxide synthases (nNOS, iNOS, eNOS) and tumor necrosis factor-alpha (TNF-alpha) in a rat dorsal root ganglion (DRG) after tourniquet application to a hind limb to identify molecules that trigger secondary events after peripheral nerve injury. Significantly high nNOS, iNOS mRNA and protein levels were observed in the ipsilateral DRGs 4 h after tourniquet application but not in the contralateral or control DRGs. The levels of TNF-alpha, an inducer of iNOS, were significantly increased in the ipsilateral DRGs 1 h after tourniquet application. Large amounts of NO might result in damage to the host cells and induce apotosis to eliminate damaged cells during the early stage of nerve injury.

Expression of cytokines, neurotrophins, neurotrophin receptors and NOS mRNA in dorsal root ganglion of a rat tourniquet model.

We studied temporal changes in mRNA expression patterns for nitric oxide synthase (NOS), cytokines, neurotrophins and neurotrophin receptors in the dorsal root ganglion (DRG) of the rat, after application of a tourniquet to the hind limb. Collapsed myelin and degenerated axons were observed in the tourniquet segment of the sciatic nerve. Gene expression level of inducible nitric oxide synthase (iNOS) and neuronal nitric oxide synthase (nNOS) was significantly increased in ipsilateral DRG samples at 4h after application of the tourniquet but not in the contralateral or control DRG samples. Upregulation of tumor necrosis factor (TNF)-alpha, activating transcription factor (ATF)-3 and neurotrophin-3 (NT3) expressions began at 1h after application of the tourniquet in ipsilateral DRGs. It is likely that transient expression of these molecules triggers secondary events that may be beneficial to wound repair and regeneration.

Changes in mRNA expression patterns for cytokines in blood leukocytes of a rat tourniquet model.

We examined changes in mRNA expression patterns for proinflammatory cytokines and growth factors in blood samples after application of a tourniquet to the rat hind limb. Slight upregulations of interferon (IFN)-gamma, macrophage colony-stimulating factor (M-CSF) and transforming growth factor (TGF)-beta1 mRNA began at 2h after tourniquet application and were short-lived. The levels of activating transcription factor (ATF)-3, a stress-inducible gene, had increased at 1h after tourniquet application. No significant expression of interleukin (IL)-6 mRNA was observed in most samples. There were no significant temporal changes in the levels of IL-1beta, cardiotrophin (CT)-1 mRNA compared to the control levels, but, downregulation of gp130, a receptor of the IL-6 family, began at 1h after tourniquet application. Nerve growth factor (NGF) mRNA gradually increased and reached a significantly high level at 4h after application of the tourniquet. Gene expression induction in blood leukocytes occurred soon after application of the tourniquet and was short-lived. The transient mRNA expressions probably trigger secondary events that may be beneficial to wound repair and regeneration.