Neuraminidase 1 Is a Negative Regulator of Lysosomal Exocytosis

SummaryLysosomal exocytosis is a Ca2+-regulated mechanism that involves proteins responsible for cytoskeletal attachment and fusion of lysosomes with the plasma membrane. However, whether luminal lysosomal enzymes contribute to this process remains unknown. Here we show that neuraminidase NEU1 negatively regulates lysosomal exocytosis in hematopoietic cells by processing the sialic acids on the lysosomal membrane protein LAMP-1. In macrophages from NEU1-deficient mice, a model of the disease sialidosis, and in patients' fibroblasts, oversialylated LAMP-1 enhances lysosomal exocytosis. Silencing of LAMP-1 reverts this phenotype by interfering with the docking of lysosomes at the plasma membrane. In neu1−/− mice the excessive exocytosis of serine proteases in the bone niche leads to inactivation of extracellular serpins, premature degradation of VCAM-1, and loss of bone marrow retention. Our findings uncover an unexpected mechanism influencing lysosomal exocytosis and argue that exacerbations of this process form the basis for certain genetic diseases

Ozz-E3 Ubiquitin Ligase Targets Sarcomeric Embryonic Myosin Heavy Chain during Muscle Development

Muscle contractile proteins are expressed as a series of developmental isoforms that are in constant dynamic remodeling during embryogenesis, but how obsolete molecules are recognized and removed is not known. Ozz is a developmentally regulated protein that functions as the adaptor component of a RING-type ubiquitin ligase complex specific to striated muscle. Ozz−/− mutants exhibit defects in myofibrillogenesis and myofiber differentiation. Here we show that Ozz targets the rod portion of embryonic myosin heavy chain and preferentially recognizes the sarcomeric rather than the soluble pool of myosin. We present evidence that Ozz binding to the embryonic myosin isoform within sarcomeric thick filaments marks it for ubiquitination and proteolytic degradation, allowing its replacement with neonatal or adult isoforms. This unique function positions Ozz within a system that facilitates sarcomeric myosin remodeling during muscle maturation and regeneration. Our findings identify Ozz-E3 as the ubiquitin ligase complex that interacts with and regulates myosin within its fully assembled cytoskeletal structure

Tetraspanin CD82 Inhibits Protrusion and Retraction in Cell Movement by Attenuating the Plasma Membrane-Dependent Actin Organization

<div><p>To determine how tetraspanin KAI1/CD82, a tumor metastasis suppressor, inhibits cell migration, we assessed which cellular events critical for motility are altered by KAI1/CD82 and how KAI1/CD82 regulates these events. We found that KAI1/CD82-expressing cells typically exhibited elongated cellular tails and diminished lamellipodia. Live imaging demonstrated that the polarized protrusion and retraction of the plasma membrane became deficient upon KAI1/CD82 expression. The deficiency in developing these motility-related cellular events was caused by poor formations of actin cortical network and stress fiber and by aberrant dynamics in actin organization. Rac1 activity was reduced by KAI1/CD82, consistent with the diminution of lamellipodia and actin cortical network; while the growth factor-stimulated RhoA activity was blocked by KAI1/CD82, consistent with the loss of stress fiber and attenuation in cellular retraction. Upon KAI1/CD82 expression, Rac effector cofilin was not enriched at the cell periphery to facilitate lamellipodia formation while Rho kinase exhibited a significantly lower activity leading to less retraction. Phosphatidylinositol 4, 5-biphosphate, which initiates actin polymerization from the plasma membrane, became less detectable at the cell periphery in KAI1/CD82-expressing cells. Moreover, KAI1/CD82-induced phenotypes likely resulted from the suppression of multiple signaling pathways such as integrin and growth factor signaling. In summary, at the cellular level KAI1/CD82 inhibited polarized protrusion and retraction events by disrupting actin reorganization; at the molecular level, KAI1/CD82 deregulated Rac1, RhoA, and their effectors cofilin and Rho kinase by perturbing the plasma membrane lipids.</p> </div

KAI1/CD82 blocks the enrichment of cofilin at the cell periphery.

<p>(<b><i>A</i></b>) The levels of total and phosphorylated cofilin proteins in Du145-Mock and -KAI1/CD82 cells were assessed by Western blot using pAbs against cofilin and phosphorylated cofilin, respectively, as described in Materials and Methods. Tubulin blot is used as a control for protein loading. <i>(</i><b><i>B</i></b><i>)</i> KAI1/CD82 prevents cofilin from being targeted to the cell periphery. Du145 transfectant cells were spread on FN-coated coverslips in complete DMEM from 3 to 6 h. The cells were fixed, permeabilized, and incubated with cofilin pAb and TRITC-conjugated α-phalloidin, followed by the FITC-conjugated second Ab staining. Digital images were captured under a confocal microscope, and each image represents a single XY section. The arrow indicates the translocation of cofilin into lamellipodia, while the arrowhead indicates the relatively transparent zone beneath the actin cortical meshwork and within the cytoplasm. Scale bar, 20 µm. <i>(</i><b><i>C</i></b><i>)</i> Comparison of the subcellular distribution of phosphorylated cofilin between Du145-Mock and -KAI1/CD82 cells. The experiment was performed as described in <i>(</i><b><i>B</i></b><i>)</i> except that the pAb against phosphorylated cofilin was used as the primary Ab. Scale bar, 20 µm.</p

The effect of KAI1/CD82 on the PIP₂ in the plasma membrane.

<p>(<b><i>A</i></b>). Du145-Mock and -KAI1/CD82 transfectant cells were spread on either FN- or LN1-coated plate in complete DMEM at 37°C overnight, fixed, permebilized, and then incubated with PIP₂ mAb, followed with the incubation of Alexa 594-conjugated second Ab and FITC-conjugated phalloidin. Confocal images of X-Y sections were collected, scale bar: 20 µm. (<b><i>B</i></b>). The pEGFP-PLCδ PH domain construct was transiently transfected into Du145-Mock and -KAI1/CD82 transfectants. At 48 h after transfection, the cells were spread on an FN (10 µg/ml)-coated plate, fixed, permeabilized, stained with Alexa 594-conjugated phalloidin, and analyzed with confocal microscopy. The X-Y section images were captured as described above. Arrowhead: the localization of EGFP-PLCδ PH domain at the cell periphery. Scale bar: 20 µm. The bottom panel shows F-actin (red) and PLCδ PH domain (green) fluorescent profiles of line scan (white lines in the Merge images) from Mock and KAI1/CD82 cells. Green arrow: the enrichment of PLCδ PH domain at the cell periphery.</p

KAI1/CD82 attenuates the formation of lamellipodia and retraction of cellular tails.

<p>(<b><i>A</i></b>) KAI1/CD82 inhibits lamellipodia and protrusion formations. Du145-Mock and -KAI1/CD82 transfectant cells were plated on FN-coated glass coverslips from 3 to 6 h. DIC images were acquired using time-lapse vidoemicroscopy. Arrows indicate lamellipodia. (<b><i>B</i></b>). KAI1/CD82 inhibits the retraction of the rear tail. Du145-Mock and -KAI1/CD82 cells were placed on FN-coated (10 µg/ml) coverslips from 3 to 6 h and treated with 100 ng/ml HGF from 4 to 6 h. Cell morphology was photographed for 3 h using time-lapse videomicroscopy. Arrows indicate the retraction processes in Du145-Mock cells and arrowheads indicate the rear tail in Du145-KAI1/CD82 cells. The time-lapse intervals are labeled inside images. Scale bar, 20 µm.</p

The actin cortical meshwork and stress fiber were disrupted upon KAI1/CD82 expression.

<p>(<b><i>A</i></b><i>)</i> F-actin distribution. After being spread on FN (50 µg/ml)-, LN1 (50 µg/ml)-, or LN5 (2 µg/ml)-coated coverslips at 37°C, 5% CO₂ for 6 h, Du145 transfectant cells were fixed, permeabilized, and then stained with TRITC-conjugated α-phalloidin. The fluorescent images were captured under an Axiophot fluorescent microscope equipped with an Optronics digital camera at magnification 63X. (<b><i>B</i></b><i>)</i> Live imaging of actin polymerization in Du145-Mock and -KAI1/CD82 cells. The cells that were transiently transfected with pEGFP-actin construct were spread on FN-coated coverslips in complete DMEM and photographed using time-lapse confocal videomicroscopy. Arrowheads and arrows indicate actin polymerization during the development of peripheral meshwork and stress fiber, respectively. <i>(</i><b><i>C</i></b><i>)</i> Less F-actin in Du145-KAI1/CD82 cells. <i>Left panel</i>, Du145-Mock and -KAI1/CD82 cells spread in serum-free DMEM were detached, fixed, permeabilized, incubated with Alexa 488-conjugated phalloidin or mouse IgG2b, and then analyzed with flow cytometry. <i>Right top panel</i>, quantitation of the mean fluorescence intensity (MFI) of F-actin. Data are expressed as the mean MFI of three independent experiments (<i>P</i><0.05 between Mock and KAI1/CD82 cells). <i>Right bottom panel</i>, the cell lysates from the experiment were analyzed by Western blot for total cellular actin proteins. Cofilin blot was used as the protein loading control. <i>(</i><b><i>D</i></b><i>)</i> The presence of KAI1/CD82 proteins is inversely correlated with actin cytoskeletal assembly. HT29 colon cancer cells and prostate epithelial cells were fixed, permeabilized, and incubated with Alexa 488-conjugated phalloidin and Alexa 594-conjugated KAI1/CD82 mAb. The images were acquired with confocal microscopy. For HT29 cells, the photographs were taken from the basal sections of the cells. Shown in insets is the magnified staining from the frame-selected areas. White dashed lines outline the cell boundary. Scale bars: 10 µM. <i>(</i><b><i>E</i></b><i>)</i> KAI1/CD82 silencing leads to stress fiber formation. HT29 cells were transiently transfected with KAI1/CD82-specific siRNA duplexes, seeded sparsely, and stained with Alexa 488-conjugated phalloidin and Alexa 594-conjugated KAI1/CD82 mAb as described above. Scale bar: 10 µM. White dotted lines outline the cell periphery. Arrows indicate the cells exhibit robust stress fiber formation but no KAI1/CD82 staining.</p

KAI1/CD82 inhibits ROCK activity and the enrichment of RhoA at the cell periphery.

<p>(<b><i>A</i></b>). Du145-Mock and -KAI1/CD82 transfectant cells were cultured in either DMEM containing 0.5% FBS for 40 h or DMEM containing 10% FBS and HGF (100 ng/ml) for overnight. The cells were then lysed with 1% Triton X-100 lysis buffer. Equal amounts of cell lysate were assayed for ROCK activity with MBL Rho Kinase Assay kit. In each experiment, ROCK activities were measured in duplicate for each transfectant. The results shown in histogram are the average values of three individual experiments±SD. *: <i>P</i><0.05, **: <i>P</i><0.01. (<b><i>B</i></b>). The cells were pretreated with HGF (100 ng/ml) overnight and then assayed for RhoA activity as aforementioned. Histogram represents the relative density of the RhoA-GTP bands (mean±SD, n = 3). *: <i>P</i><0.05. (<b><i>C</i></b>). The cells spread on LN1 (20 µg/ml)-coated plates were treated with or without HGF (100 ng/ml) overnight and then fixed, permeabilized, and incubated sequentially with RhoA mAb and Alexa594-conjugated second Ab. Images were acquired as described above under a fluorescent microscope. Arrows indicate the enrichment of RhoA at the cell periphery and in the retraction tail. The bottom panel shows the fluorescent profiles of line scan from Mock and KAI1/CD82 cells.</p

KAI1/CD82 regulates the activities of Rho GTPases.

<p><i>(</i><b><i>A</i></b><i>)</i> KAI1/CD82 inhibits Rac1 activity. Du145-Mock or -KAI1/CD82 transfectant cells were lysed in a lysis buffer containing 1% NP-40 and 0.2% SDS detergents. Cell lysates were subjected to affinity precipitation with GST-PAK1, which binds to only the activated or GTP-bound Rac. The co-precipitated, GTP-bound Rac GTPase was detected by Rac mAb. The intact cell lysates were blotted with Rac mAb to demonstrate equivalent levels of total Rac proteins between Mock and KAI1/CD82 transfectant cells. Blots show the result from a representative experiment; the graph represents the relative density of the Rac band (mean±SD, n = 4), based on densitometric analysis. *: <i>P</i><0.05. (<b><i>B</i></b>) KAI1/CD82 does not significantly alter RhoA activity. Du145 transfectants were pretreated as described above. GTP-bound RhoA was pulled down by GST-Rhotekin and detected by RhoA mAb. The blot shows the result from a representative experiment; the graph represents the relative density of the RhoA bands (mean±SD, n = 9), based on densitometric analysis. <i>P</i>>0.05 between Mock and KAI1/CD82. (<b><i>C</i></b>) KAI1/CD82 does not significantly alter Cdc42 activity. GTP-bound Cdc42 was pulled down by GST-PAK1 and detected by Cdc42 mAb. The blot shows the results from a representative experiment; the graph represents the relative density of the Cdc42 bands (mean±SD, n = 4), based on densitometric analysis. <i>P</i>>0.05 between Mock and KAI1/CD82. In all experiments, tubulin protein levels in cell lysates were detected via Western blot and served as protein loading controls.</p