TY - JOUR
T1 - Overexpression of ROCK in human breast cancer cells
T2 - Evidence that ROCK activity mediates intracellular membrane traffic of lysosomes
AU - Nishimura, Yukio
AU - Itoh, Kazuyuki
AU - Yoshioka, Kiyoko
AU - Tokuda, Kazuo
AU - Himeno, Masaru
N1 - Funding Information:
Normal goat serum were purchased from Jackson ImmunoResearch (West Grove, PA, USA). Alexa 488labeled affinity-purified goat anti-rabbit IgG and FITClabeled affinity-purified goat anti-mouse IgG were purchased from Molecular Probes (Eugene, OR, USA). A monoclonal anti-human lysosome-associated membrane protein-1 (LAMP-1) H4A3 was purchased from the Developmental Studies Hybridoma Bank maintained by the Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine (Baltimore, MD, USA), and the Department of Biological Sciences, University of Iowa (Iowa City, lA, USA), under contract NOI-HD-2-3144 from the National Institute of Child Health and Human Development. Texas Red-conjugated phalloidin, and SlowFade antifade reagent were purchased from Molecular Probes (Eugene, OR, USA). Antisera were raised in rabbits (New Zealand white male) against the mature form of rat liver lysosomal cathepsin D33,34 and the native form of LIMPII/LGP8529as described previously, and monospecific cathepsin D IgG or anti-LIMPII IgG was prepared, respectively. An IgG fraction was affinity-purified by protein A Sepharose CL-4B, followed by immunoaffinity chromatography using antigen-Sepharose 4B. Wortmannin was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Other chemicals were of reagent grade and were obtained from commercial sources.
Copyright:
Copyright 2017 Elsevier B.V., All rights reserved.
PY - 2003
Y1 - 2003
N2 - Small GTPase Rho and its downstream effectors, ROCK family of Rho-associated serine-threonine kinases, are thought to participate in cell morphology, motility, and tumor progression through regulating the rearrangement of actin cytoskeleton. Here we present evidence that transfection of human breast cancer cells with cDNA encoding a dominant active mutant of ROCK causes dispersal of lysosomal vesicles throughout the cytoplasm without perturbing the machinery of the endocytic pathway. The intracellular distribution of lysosomes and endocytosed transferrin, an early endosomal marker, were further assessed by confocal immunofluorescence microscopy. In the active ROCK transfected cells the lysosomal proteins, cathepsin D, LIMPII, and LAMP1, were found throughout the cytoplasm in dispersed small vesicles, which were accessible to the endocytosed Texas Red-labeled transferrin. 3D-image analysis of lysosomal distribution in the active ROCK-transfectants revealed abundant punctate signals in the peripheral region of the basal plasma membrane. Cells expressing vector alone did not exhibit these alterations. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, induced LIMPII-positive/transferrin negative large vacuoles in the perinuclear region, and disappearence of the dispersed small vesicular structures. To our knowledge, this is the first evidence that increasing ROCK expression contributes to selective cellular dispersion of lysosomes in invasive breast cancer cells.
AB - Small GTPase Rho and its downstream effectors, ROCK family of Rho-associated serine-threonine kinases, are thought to participate in cell morphology, motility, and tumor progression through regulating the rearrangement of actin cytoskeleton. Here we present evidence that transfection of human breast cancer cells with cDNA encoding a dominant active mutant of ROCK causes dispersal of lysosomal vesicles throughout the cytoplasm without perturbing the machinery of the endocytic pathway. The intracellular distribution of lysosomes and endocytosed transferrin, an early endosomal marker, were further assessed by confocal immunofluorescence microscopy. In the active ROCK transfected cells the lysosomal proteins, cathepsin D, LIMPII, and LAMP1, were found throughout the cytoplasm in dispersed small vesicles, which were accessible to the endocytosed Texas Red-labeled transferrin. 3D-image analysis of lysosomal distribution in the active ROCK-transfectants revealed abundant punctate signals in the peripheral region of the basal plasma membrane. Cells expressing vector alone did not exhibit these alterations. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, induced LIMPII-positive/transferrin negative large vacuoles in the perinuclear region, and disappearence of the dispersed small vesicular structures. To our knowledge, this is the first evidence that increasing ROCK expression contributes to selective cellular dispersion of lysosomes in invasive breast cancer cells.
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U2 - 10.1007/BF03033750
DO - 10.1007/BF03033750
M3 - Article
C2 - 12858212
AN - SCOPUS:0043160105
VL - 9
SP - 83
EP - 95
JO - Pathology and Oncology Research
JF - Pathology and Oncology Research
SN - 1219-4956
IS - 2
ER -