T32AG023475 (Z

T32AG023475 (Z.E., S.H.) and the Analytical Imaging Facility Berberine HCl of the Einstein Cancer Center (P30 CA013330). Abbreviations used: DMSOdimethyl sulfoxideECMextracellular matrixEGFepidermal growth factorGEFguanine nucleotide exchange factorGPCRG proteinCcoupled receptorHDFChigh-density fibrillar collagen IMMPsmatrix metalloproteasesPH domainpleckstrin homology domainPI3Kphosphoinositide 3-kinasePI(3,4)P3phosphatidylinositol-(3,4)-P2PIP3phosphatidylinositol-(3,4,5)-P3PTXpertussis toxinRBDRas binding domainRTKreceptor tyrosine kinaseSHIP2SH2 domain-containing inositol phosphatase 2 Footnotes This article was published online ahead of print in MBoC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E19-03-0182) on July 18, 2019. REFERENCES Alexander NR, Branch KM, Parekh A, Clark ES, Iwueke IC, Guelcher SA, Weaver AM. lamellipodin to invadopodia. These findings provide novel mechanistic insight into the role of PI3K in the regulation of invadopodia in breast cancer cells. INTRODUCTION Invadopodia Berberine HCl are actin-rich protrusions that mediate the secretion of matrix metalloproteases (MMPs), Berberine HCl which degrade the extracellular matrix (ECM) and facilitate tumor cell invasion (Murphy and Courtneidge, 2011 ; Beaty test. Given that the canonical PIP3 effector Akt is not required for PI3K-dependent integrin-stimulated responses, we considered whether PI3K might be coupled to the production of PI(3,4)P2. If this were correct, we would expect integrin signaling in MDA-MB-231 cells to also require PI(3,4)P2. We therefore reduced PI(3,4)P2 levels by treating cells with a SHIP2 inhibitor, or by expressing mCherry-INPP4B-CAAX. We found that both cell spreading (Figure 5B) and haptotaxis (Figure 5C) were significantly inhibited. The number of cells in the INPP4B experiment is reduced as compared with other haptotaxis experiments because only the transfected (mCherry-positive) cells were counted. In addition, integrin-stimulated collagen degradation on HDFC was inhibited by both the SHIP2 inhibitor and mCherry-INPP4B-CAAX (Figure 5D). These data show that PI3K-dependent integrin signaling requires the production of PI(3,4)P2. We also tested the relationship between PI3K and PI(3, 4)P2 during integrin signaling and matrix degradation in two Berberine HCl other triple-negative breast cancer cell lines, BT549 and MDA-MB-468. In both cell lines, inhibition of PI3K, but not PI3K, blocked cell spreading on collagen and collagen-stimulated haptotaxis (Supplemental Figure 3, A and B). Gelatin degradation was also blocked by inhibition of PI3K, but not PI3K in both cell lines (Supplemental Figure 3C). In all cases, the effects of PI3K inhibition were mimicked by inhibition of PI(3,4)P2 levels, either through inhibition of SHIP2 or overexpression of INPP4B (Supplemental Figure 3, ACC). PI3K regulates PI(3,4)P 2 production in invadopodia Integrin signaling in MDA-MB-231 cells requires both PI3K and PI(3,4)P2. To test whether PI3K is coupled to PI(3,4)P2 production in invadopodia, we sought to measure PI(3,4)P2 in these structures. While excellent probes for PI(3,4)P2 are available (Goulden for 20 min to flatten the collagen meshwork into a 2D layer. HDFC matrices Berberine HCl were treated with PBS containing 4% PFA and 5% Rabbit polyclonal to Caldesmon sucrose for 20 min to cross-link the HDFC. After washing with DMEM, 4 104 cells were starved, detached, and treated with inhibitors as described above, and plated for 3 h. The cells were fixed and stained with the collagen ? fragment antibody and Alexa Fluor 488 phalloidin without permeabilization. Images were taken with a 60 1.4 NA objective on an Olympus IX70 microscope. For each experiment at least 15 cells were imaged. To quantify the collagen ? images, the background was subtracted using a rolling ball radius of 50 and the degradation area per cell was quantified. The reported values are the mean SEM from three independent experiments. Lamellipodin localization Serum-starved cells were detached and treated with inhibitors as described above, and seeded on HDFC matrixces for 2 h. Samples were fixed, permeabilized, and immunostained with cortactin and lamellipodin antibodies and Alexa Fluor 488 phalloidin. Images were obtained with a 63 1.4 NA objective on a Leica SP5 confocal microscope. For image analysis, the background was subtracted from the actin and cortactin images using a rolling ball radius of 10. Using the mathematical function AND in ImageJ, the area showing coincident actin and cortactin staining was transformed into a new image. Invadopodia were defined by finding the local maxima in the AND image using ImageJ. Lamellipodin intensity was measured in ROIs containing coincident actin/cortactin staining (defined as an invadopod) and in an area immediately adjacent to the invadopod, to measure background staining. The ratio of lamellipodin intensity in invadopodia versus background was calculated for each invadopod, and all invadopodia in each cell were measured. The ratios were averaged per cell. In each experiment, at least 5 cells were analyzed per condition, and 17C54 invadopodia.