A role for Mitochondrial Rho GTPase 1 (MIRO1) in motility and membrane dynamics of peroxisomes

Peroxisomes are dynamic organelles which fulfil essential roles in lipid and ROS metabolism. Peroxisome movement and positioning allows interaction with other organelles and is crucial for their cellular function. In mammalian cells, such movement is microtubule‐dependent and mediated by kinesin and dynein motors. The mechanisms of motor recruitment to peroxisomes are largely unknown, as well as the role this plays in peroxisome membrane dynamics and proliferation. Here, using a combination of microscopy, live‐cell imaging analysis and mathematical modelling, we identify a role for Mitochondrial Rho GTPase 1 (MIRO1) as an adaptor for microtubule‐dependent peroxisome motility in mammalian cells. We show that MIRO1 is targeted to peroxisomes and alters their distribution and motility. Using a peroxisome‐targeted MIRO1 fusion protein, we demonstrate that MIRO1‐mediated pulling forces contribute to peroxisome membrane elongation and proliferation in cellular models of peroxisome disease. Our findings reveal a molecular mechanism for establishing peroxisome‐motor protein associations in mammalian cells and provide new insights into peroxisome membrane dynamics in health and disease.      

Expression of connective tissue growth factor in the human liver with idiopathic portal hypertension

Idiopathic portal hypertension (IPH) is a disorder of unknown etiology, clinically associated with portal hypertension in the absence of cirrhosis. This study was designed to delineate the characteristics of IPH RNA expression in liver specimens from patients with IPH. Liver specimens from patients with IPH and patients without liver diseases underwent cDNA expression analysis and in situ hybridization studies. Connective tissue growth factor (CTGF) levels in serum were examined in 76 patients with IPH, 84 patients with hepatitis C virus infection (including those with cirrhosis), and 38 healthy volunteers. Among 588 genes sorted on macroarray, seven up-regulated genes, including CTGF, were detected. In situ hybridization studies showed that positive reactions for CTGF mRNA were most intense in the epithelial cells of proliferating bile ducts within portal tracts in patients with IPH. In the liver parenchyma, there was no appreciable staining of hepatocytes, sinusoidal endothelial cells, or hepatic stellate cells (HSCs), and there were few positive signals for CTGF mRNA in normal liver. The serum CTGF level in patients with IPH was significantly higher than the value in healthy volunteers. Six (8%) of the 76 patients with IPH had serum CTGF levels greater than 80 ng/mL, far exceeding the level of any patient with cirrhosis. In conclusion, overexpression of CTGF is one of the most important features of IPH.     

The association of ATR protein with mouse meiotic chromosome cores

The ATR (ataxia telangiectasia- and RAD3-related) protein is present on meiotic prophase chromosome cores and paired cores (synaptonemal complexes, SCs). Its striking characteristic is that the protein forms dense aggregates on the cores and SCs of the last chromosomes to pair at the zygotene-pachytene transition. It would appear that the ATR protein either signals delays in pairing or it is directly involved in the completion of the pairing phase. Atm-deficient spermatocytes, which are defective in the chromosome pairing phase, accumulate large amounts of ATR. The behaviour of ATR at meiotic prophase sets it apart from the distribution of the RAD51/DMC1 recombinase complex and our electron microscope observations confirm that they do not co-localize. We failed to detect ATM in association with cores/SCs and we have reported elsewhere that RAD1 protein does not co-localize with DMC1 foci. The expectation that putative DNA-damage checkpoint proteins, ATR, ATM and RAD1, are associated with RAD51/DMC1 recombination sites where DNA breaks are expected to be present, is therefore not supported by our observations. Received: 23 November 1998 / Accepted: 3 January 1999     

Lysis of uninfected and virus-infected cells in vivo: a rejection mechanism in addition to that mediated by natural killer cells

To examine the lysis of virus-infected cells in vivo, uninfected and lymphocytic choriomeningitis virus (LCMV)-infected L-929 cells were labeled in vitro with [125I]-iododeoxyuridine and implanted intravenously into mice. Natural cytotoxicity against both uninfected and virus-infected cells was demonstrated in normal uninfected mice, but LCMV-infected cells were cleared from the lungs and whole bodies more rapidly than uninfected cells. Treatment of L-929 cells with defective interfering LCMV inhibited standard virus synthesis and protected the target cells from enhanced in vivo rejection. The in vivo rejection was apparently mediated by a cellular constituent of the host immune response and not simply a result of virus-induced cytopathic effects on the target cell, as hydrocortisone acetate and cyclophosphamide each reduced rejection of both target cell types and eliminated the enhanced rejection of LCMV-infected cells. The enhanced rejection of LCMV-infected cells was not restricted by histocompatibility antigens, indicating that classic T-cell recognition was not involved in the lysis, and since the enhanced rejection of LCMV-infected cells was mediated by mice treated with cobra venom factor, complement was also not involved in the lysis. Although moderate levels of interferon (102 U/ml) were present in the sera and although there was a modest activation of natural killer (NK) cells in the lungs of LCMV-infected cell recipients but not uninfected cell recipients, the enhanced rejection of virus-infected cells did not appear to be NK cell mediated. Normal mice and mice depleted of NK cell activity by in vivo treatment with antibody to asialo ganglio-n-tetraosylceramide ( AGM1 ) rejected uninfected and LCMV-infected L-929 cells similarly. This antibody markedly inhibited the rejection of NK-sensitive YAC-1 cells. In addition to the natural cytotoxicity directed against virus-infected cells, a second nonspecific rejection mechanism appeared in response to treatment protocols which induced interferon. Polyinosinic-polycytidylic acid and infection with LCMV augmented in vivo rejection of both uninfected and LCMV-infected L-929 cells but eliminated the differential rejection of the virus-infected cells. Infection with LCMV also augmented the in vivo rejection of the NK-sensitive target cell, YAC-1. In vivo treatments with anti- AGM1 sera only moderately inhibited the elevated rejection of uninfected and LCMV-infected L-929 cells, indicating that the enhanced rejection of these target cells was predominantly mediated by a mechanism other than that mediated by NK cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structural studies of cell wall polysaccharides from Bifidobacterium breve YIT 4010 and related Bifidobacterium species

The chemical compositions of the cell walls obtained from 8 strains in 5 species of Bifidobacterium were analyzed. These cell walls were shown to be composed of peptidoglycan and polysaccharide moieties. Some variations with respect to contents of neutral sugars and content of phosphorus were observed with some cell wall preparations from the same species. The neutral polysaccharides in cell walls of 4 strains of Bifidobacterium (B. bifidum YIT 4007, B. breve YIT 4010, B. infantis YIT 4025, and B. longum ATCC 15707) were purified and their chemical structures were analyzed. One of these polysaccharides, obtained from B. breve YIT 4010, was analyzed in detail by GLC, 1H- and 13C-NMR spectroscopic analyses, methylation, Smith degradation and acetolysis, and the results suggested the following structure for the repeating unit of the polysaccharide: (Formula: see text).     

Calcium-mediated modulation of microtubule assembly in human breast epithelial cells

Summary Normal human breast epithelial cells obtained from a reduction mammoplasty (S130) have been maintained in culture for up to a year in Ham's F12:Dulbecco's medium, with 5% equine serum and a low calcium concentration (0.04 mM). These cells undergo senescence and terminal differentiation if they are switched to high Ca²⁺ medium (1.05 mM). To clarify the mechanism by which Ca²⁺ regulates the growth of these cells, we studied the role of tubulin assembly-disassembly and the morphologic changes subsequent to high Ca²⁺ switch. An early Passage (9) of S130 breast epithelial cells growing in low Ca²⁺ medium was analyzed. Of a total of 785 counted cells, 720 (92%) were rounded and 65 (8%) were flat, elongated, and fibroblastlike. When the cells were switched to high Ca²⁺ medium, out of 553 cells, only 111 (20%) were rounded and the remaining 442 (80%) were elongated and fibroblastlike. Immunocytochemical localization of tubulin, using the immunogold silver enhancement technique, showed that the majority of low Ca²⁺-grown cells did not display a network of tubulin fibers, whereas high Ca²⁺-grown cells revealed extensive cytoplasmic network of polymerized tubulin, which seemed to stretch out the cells. Experiments designed to determine the mechanisms of tubulin polymerization in these cells revealed that: a) Cells grown in high Ca²⁺ medium containing 0.1 mM colchicine had a reduced proportion of elongated cells; b) treatement of the cells with the calcium ionophore A23187 in low calcium medium resulted in an increase in the number of elongated cells which had more polymerized tubulin; and d) treatment of the cells with cyclic-AMP in low Ca²⁺ medium had no observable effect on cell morphology. These results indicate that high levels of Ca²⁺ either favor tubulin polymerization or stabilize the polymerized state. This research was supported by NCI grant CA-38921 from the National Cancer Institute, Bethesda, MD, and by an institutional grant from the United Foundation of Greater Detroit.