αvβ3 integrin-mediated adhesion is regulated through an AAK1L- and EHD3-dependent rapid recycling pathway

Waxmonsky, Nicole C.; Conner, S. D.

doi:10.1242/jcs.122465

Cited by 6 publications

(5 citation statements)

References 51 publications

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“…In particular, the antibody used to detect ERα has been used in >200 papers (Gao et al, 2015; Arnal et al, 2017). β1-integrin antibodies used throughout this paper have also been widely used in the literature (Tiwari et al, 2011; Waxmonsky and Conner, 2013; Long et al, 2016). In the case of the antibody used to detect ERα, the most thoroughly used in the present paper, its specificity was tested for Western blot and immunofluorescence.…”

Section: Methodsmentioning

confidence: 99%

Fibronectin rescues estrogen receptor α from lysosomal degradation in breast cancer cells

Sampayo

Toscani

Rubashkin

et al. 2018

Journal of Cell Biology

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Estrogen receptor α (ERα) is expressed in tissues as diverse as brains and mammary glands. In breast cancer, ERα is a key regulator of tumor progression. Therefore, understanding what activates ERα is critical for cancer treatment in particular and cell biology in general. Using biochemical approaches and superresolution microscopy, we show that estrogen drives membrane ERα into endosomes in breast cancer cells and that its fate is determined by the presence of fibronectin (FN) in the extracellular matrix; it is trafficked to lysosomes in the absence of FN and avoids the lysosomal compartment in its presence. In this context, FN prolongs ERα half-life and strengthens its transcriptional activity. We show that ERα is associated with β1-integrin at the membrane, and this integrin follows the same endocytosis and subcellular trafficking pathway triggered by estrogen. Moreover, ERα vesicles are present within human breast tissues, and colocalization with β1-integrin is detected primarily in tumors. Our work unravels a key, clinically relevant mechanism of microenvironmental regulation of ERα signaling.

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Section: Methodsmentioning

confidence: 99%

Fibronectin rescues estrogen receptor α from lysosomal degradation in breast cancer cells

Sampayo

Toscani

Rubashkin

et al. 2018

Journal of Cell Biology

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“…The α 5 -GFP integrin construct we use has been used before [2][3][4][5][6][7][8] . The α V -mEmerald construct is in the Addgene repository under Michael's Davidson's unpublished constructs, but similar constructs in which the fluorescent protein is attached at the C-terminus of α V integrin have been used many times before [9][10][11] . We note as well that GFP-tagged integrins show identical localization patterns to those observed using immunocytochemistry to visualize the endogenous proteins in untransfected cells (Fig.…”

mentioning

confidence: 99%

Visualizing the Interior Architecture of Focal Adhesions with High-Resolution Traction Maps

Morimatsu

Mekhdjian

Chang

et al. 2015

Biophysical Journal

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Materials and Methods Cell CultureHuman foreskin fibroblast (HFF) cells CCD-1070Sk (ATCC CRL-2091) were cultured in DMEM high glucose medium (Gibco, Cat #21063-029) in the absence of phenol red and supplemented with 10% fetal bovine serum (FBS, Axenia Biologix), sodium pyruvate (1 mM, Gibco), MEM non-essential amino acids (1x, Gibco), and penicillin/streptomycin (100 U/mL and 100 μg/mL, Gibco). The cells were incubated at 37 °C with 5% CO 2 . On the day of the experiment, cells were washed with phosphate-buffered saline (PBS) (Gibco), trypsinized using 0.25% Trypsin-EDTA (Gibco), seeded on MTS-functionalized coverslips in a custom-made chamber, and allowed to adhere for ~60 minutes in DMEM high glucose media with 10% FBS. FRET measurements were made within 3 hours of plating the cells. Control measurements indicated that cells remained viable for at least 5 hours under experimental conditions, and that the MTSs did not detach from the coverslip surface in that time. Fluorescent proteins and transfectionStable cell lines: DNA constructs were transfected using Amaxa® Human Dermal Fibroblast Nucleofector® Kit (Lonza) using their optimized protocols. To achieve stable cell lines expressing paxillin (GFP at C-terminus) and myosin regulatory light chain (MRLC), we cloned these constructs into DNA 2.0's PiggyBac expression vectors. We changed the media to DMEM high glucose medium with no FBS and no antibiotics the night before transfection to increase transfection efficiency. One day after transfection, 1.0 μg/mL puromyocin was added to the media to select for stably transformed cells. Fresh media with drug was added after 2 days. The cells were then washed with PBS, and fresh media (with no drug) was added. Fluorescence images confirmed that ~75% of the resulting cells stably expressed the GFP-fusion proteins.Transient expression: Cells were transfected using the Amaxa® Human Dermal Fibroblast Nucleofector® Kit, switching to DMEM high glucose medium with no FBS and no antibiotics the night before transfection. Cells were used 1-3 days post transfection. Actin-GFP was labeled using the appropriate CellLight® fluorescent protein construct (Life Technologies, Cat #C10582), with the manufacturer-specified protocol. GFP-tagged fusions of talin (GFP at N-terminus), vinculin (GFP at N-terminus), α-actinin (GFP at C-terminus), α 5 integrin (two versions; high and reduced expression, GFP at C-terminus) were obtained from Addgene (Plasmid #26724, 50513, 11908, 15238, 30450, respectively). mEmerald-tagged α v integrin (mEmerald at C-terminus) was obtained from Addgene (Plasmid #53985). All of the GFP fusion proteins used in this work are either identical or functionally equivalent (i.e. GFP fusion in the same location) to constructs used in previous publications 1,2 . The α 5 -GFP integrin construct we use has been used before [2][3][4][5][6][7][8] . The α V -mEmerald construct is in the Addgene repository under Michael's Davidson's unpublished constructs, but similar constructs in which the fluorescent protein is attached at t...

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“…This implicates a critical role for PI(4,5)P 2 -binding proteins that facilitate Notch transport. Potential candidates include members of the eps15 homology domain-containing proteins (EHD1, 3, and 4)-each of which is known to bind PI(4,5)P 2 and facilitate receptor transport through recycling endosomes (41)(42)(43)(44)(45). Resolving the precise role of PI(4,5)P 2 production by PI5P4Kγ and determining the mechanistic consequence of PI5P4Kγ ubiquitination by DTX1 on Notch1 recycling will be the focus of future work.…”

Section: Discussionmentioning

confidence: 99%

PI5P4Kγ functions in DTX1-mediated Notch signaling

Conner

2018

Proc. Natl. Acad. Sci. U.S.A.

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Notch signaling is an evolutionarily conserved pathway that is essential for development, where it controls processes ranging from cell differentiation to survival. Transport through endosomes is a critical step in regulating Notch signaling capacity, where the E3 ubiquitin ligase DTX1 is thought to control Notch1 intracellular transport decisions by direct receptor ubiquitination. However, how DTX1 regulates Notch1 transport within endosomes and the consequence of Notch1 ubiquitination by DTX1 remain unresolved. Here we demonstrate that DTX1 colocalizes with Notch1 on tubulovesicular recycling endosomes. We find that DTX1 silencing leads to enhanced Notch1 recycling from this compartment to the cell surface via a rab4a-mediated transport route. This, in turn, increases Notch1 cell-surface levels and enhances signaling. Surprisingly, we discovered that DTX1 depletion also elevates Notch1 activity mediated by a mutant form of the receptor that lacks lysine residues for ubiquitination, suggesting that DTX1 targets additional factors. Using an activity-based screen for ubiquitination targets, we identified multiple DTX1 substrates including PI5P4Kγ, a lipid kinase involved in PI(4,5)P production. Immunolocalization analysis reveals that PI5P4Kγ, like DTX1 and Notch1, is present on tubulovesicular recycling endosomes. However, in contrast to DTX1, Notch1 signaling is inhibited by pharmacological inactivation or siRNA depletion of PI5P4Kγ. Moreover, loss of PI5P4Kγ activity decreases Notch1 recycling rates and reduces receptor cell-surface levels. Collectively, these findings argue that PI5P4Kγ positively regulates the Notch pathway by promoting receptor recycling. Additionally, they support a model where DTX1 controls Notch1 endosomal sorting decisions by controlling PI5P4Kγ-mediated production of PI(4,5)P.

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αvβ3 integrin-mediated adhesion is regulated through an AAK1L- and EHD3-dependent rapid recycling pathway

Cited by 6 publications

References 51 publications

Fibronectin rescues estrogen receptor α from lysosomal degradation in breast cancer cells

Fibronectin rescues estrogen receptor α from lysosomal degradation in breast cancer cells

Visualizing the Interior Architecture of Focal Adhesions with High-Resolution Traction Maps

PI5P4Kγ functions in DTX1-mediated Notch signaling

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