Ultrasound Microbubble Treatment Enhances Clathrin-Mediated Endocytosis and Fluid-Phase Uptake through Distinct Mechanisms

<div><p>Drug delivery to tumors is limited by several factors, including drug permeability of the target cell plasma membrane. Ultrasound in combination with microbubbles (USMB) is a promising strategy to overcome these limitations. USMB treatment elicits enhanced cellular uptake of materials such as drugs, in part as a result of sheer stress and formation of transient membrane pores. Pores formed upon USMB treatment are rapidly resealed, suggesting that other processes such as enhanced endocytosis may contribute to the enhanced material uptake by cells upon USMB treatment. How USMB regulates endocytic processes remains incompletely understood. Cells constitutively utilize several distinct mechanisms of endocytosis, including clathrin-mediated endocytosis (CME) for the internalization of receptor-bound macromolecules such as Transferrin Receptor (TfR), and distinct mechanism(s) that mediate the majority of fluid-phase endocytosis. Tracking the abundance of TfR on the cell surface and the internalization of its ligand transferrin revealed that USMB acutely enhances the rate of CME. Total internal reflection fluorescence microscopy experiments revealed that USMB treatment altered the assembly of clathrin-coated pits, the basic structural units of CME. In addition, the rate of fluid-phase endocytosis was enhanced, but with delayed onset upon USMB treatment relative to the enhancement of CME, suggesting that the two processes are distinctly regulated by USMB. Indeed, vacuolin-1 or desipramine treatment prevented the enhancement of CME but not of fluid phase endocytosis upon USMB, suggesting that lysosome exocytosis and acid sphingomyelinase, respectively, are required for the regulation of CME but not fluid phase endocytosis upon USMB treatment. These results indicate that USMB enhances both CME and fluid phase endocytosis through distinct signaling mechanisms, and suggest that strategies for potentiating the enhancement of endocytosis upon USMB treatment may improve targeted drug delivery.</p></div

USMB treatment increases the cell surface abundance of the lysosomal marker LAMP1.

<p>RPE cells grown on glass coverslips were treated with 5.0 μM vacuolin-1 for 60 min, or not treated with this inhibitor (vehicle control). Cells were subsequently treated with USMB or left untreated (control) as indicated. Following treatment, cells were immediately placed on ice to arrest membrane traffic and subjected to immunofluorescence staining to detect cell surface LAMP1 levels. Shown in <b><i>(A)</i></b> are representative epifluorescence micrographs of cell surface LAMP1 levels and in <b><i>(B)</i></b> the mean ± SEM of cell surface TfR fluorescence intensity in each condition (n = 3 independent experiment, each experiment >20 cells per condition). Scale = 20 μm. *, p < 0.05 relative to the control, vehicle-treated condition.</p

USMB treatment rapidly reduces cell surface TfR levels.

<p>RPE (<b><i>A</i>, <i>B</i></b>) or MDA-MB-231 <b><i>(C-D)</i></b> cells grown on glass coverslips were treated with microbubbles and/or ultrasound, as indicated. 5 min following USMB treatment, cells were placed on ice to arrest membrane traffic and subjected to immunofluorescence staining to detect cell surface TfR levels. Shown in <b><i>(A</i>, <i>C)</i></b> are representative epifluorescence micrographs of cell surface TfR levels and in <b><i>(B</i>, <i>D)</i></b> the mean ± SEM of cell surface TfR fluorescence intensity in each condition (n = 3 independent experiments, each experiment >20 cells per condition). Scale = 20 μm. *, p < 0.05 relative to the control condition.</p

USMB treatment alters the properties of clathrin-coated pits.

<p>RPE cells stably expressing clathrin light chain fused to green fluorescent protein (RPE GFP-CLC) cells grown on glass coverslips were treated with microbubbles and ultrasound, or left untreated (control), as indicated. Cells were then incubated with A555-Tfn for 3 min to allow labeling of internalizing TfR, and then immediately subjected to fixation and processing for imaging by total internal reflection fluorescence microscopy (TIRF-M). (<b><i>A</i></b>) Shown are representative fluorescence micrographs obtained by TIRF-M. Scale = 5 μm. Images are higher magnification insets of larger images shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156754#pone.0156754.s001" target="_blank">S1 Fig</a>. (<b><i>B-C</i></b>) Images obtained by TIRF-M were subjected to automated detection and analysis of clathrin-coated pits (CCPs), as described in Material and Methods. The mean GFP-CLC (<b><i>B</i></b>) and A555-Tfn (<b><i>C</i></b>) intensity within each detected object (CCP) in each cell are shown. Each diamond symbol represents the mean fluorescence of all objects within a single cell; also shown are the mean of the cellular fluorescence values and interquartile range (red bars). The number of CCPs analyzed (n) and cells (k) from 3 independent experiments for each condition are control: n = 37,762, k = 89; USMB n = 29,897 k = 80.</p

Desipramine treatment impairs the reduction in cell surface TfR levels by USMB treatment.

<p>RPE cells grown on glass coverslips were treated with 50 μM desipramine for 60 min, or not treated with this inhibitor (vehicle control). Cells were subsequently treated with USMB or left untreated (control) as indicated. 5 min following USMB treatment, cells were immediately placed on ice to arrest membrane traffic and subjected to immunofluorescence staining to detect cell surface TfR levels. Shown in <b><i>(A)</i></b> are representative epifluorescence micrographs of cell surface TfR levels and in <b><i>(B)</i></b> the mean ± SEM of cell surface TfR fluorescence intensity in each condition (n = 3 independent experiment, each experiment >20 cells per condition). Scale = 20 μm. *, p < 0.05.</p

Vacuolin-1 treatment impairs the reduction in cell surface TfR levels by USMB treatment.

<p>RPE cells grown on glass coverslips were treated with 5.0 μM vacuolin-1 for 60 min, or not treated with this inhibitor (vehicle control). Cells were subsequently treated with USMB or left untreated (control) as indicated. 5 min after USMB treatment, cells were placed on ice to arrest membrane traffic and subjected to immunofluorescence staining to detect cell surface TfR levels. Shown in <b><i>(A)</i></b> are representative epifluorescence micrographs of cell surface TfR levels and in <b><i>(B)</i></b> the mean ± SEM of cell surface TfR fluorescence intensity in each condition (n = 3 independent experiments, each experiment >20 cells per condition). Scale = 20 μm. *, p < 0.05.</p

mTOR controls lysosome tubulation and antigen presentation in macrophages and dendritic cells

Macrophages and dendritic cells exposed to lipopolysaccharide (LPS) convert their lysosomes from small, punctate organelles into a network of tubules. Tubular lysosomes have been implicated in phagosome maturation, retention of fluid phase, and antigen presentation. There is a growing appreciation that lysosomes act as sensors of stress and the metabolic state of the cell through the kinase mTOR. Here we show that LPS stimulates mTOR and that mTOR is required for LPS-induced lysosome tubulation and secretion of major histocompatibility complex II in macrophages and dendritic cells. Specifically, we show that the canonical phosphatidylinositol 3-kinase-Akt-mTOR signaling pathway regulates LPS-induced lysosome tubulation independently of IRAK1/4 and TBK. Of note, we find that LPS treatment augmented the levels of membrane-associated Arl8b, a lysosomal GTPase required for tubulation that promotes kinesin-dependent lysosome movement to the cell periphery, in an mTOR-dependent manner. This suggests that mTOR may interface with the Arl8b-kinesin machinery. To further support this notion, we show that mTOR antagonists can block outward movement of lysosomes in cells treated with acetate but have no effect in retrograde movement upon acetate removal. Overall our work provides tantalizing evidence that mTOR plays a role in controlling lysosome morphology and trafficking by modulating microtubule-based motor activity in leukocytes.</p

AMP-Activated Protein Kinase Regulates the Cell Surface Proteome and Integrin Membrane Traffic

<div><p>The cell surface proteome controls numerous cellular functions including cell migration and adhesion, intercellular communication and nutrient uptake. Cell surface proteins are controlled by acute changes in protein abundance at the plasma membrane through regulation of endocytosis and recycling (endomembrane traffic). Many cellular signals regulate endomembrane traffic, including metabolic signaling; however, the extent to which the cell surface proteome is controlled by acute regulation of endomembrane traffic under various conditions remains incompletely understood. AMP-activated protein kinase (AMPK) is a key metabolic sensor that is activated upon reduced cellular energy availability. AMPK activation alters the endomembrane traffic of a few specific proteins, as part of an adaptive response to increase energy intake and reduce energy expenditure. How increased AMPK activity during energy stress may globally regulate the cell surface proteome is not well understood. To study how AMPK may regulate the cell surface proteome, we used cell-impermeable biotinylation to selectively purify cell surface proteins under various conditions. Using ESI-MS/MS, we found that acute (90 min) treatment with the AMPK activator A-769662 elicits broad control of the cell surface abundance of diverse proteins. In particular, A-769662 treatment depleted from the cell surface proteins with functions in cell migration and adhesion. To complement our mass spectrometry results, we used other methods to show that A-769662 treatment results in impaired cell migration. Further, A-769662 treatment reduced the cell surface abundance of β1-integrin, a key cell migration protein, and AMPK gene silencing prevented this effect. While the control of the cell surface abundance of various proteins by A-769662 treatment was broad, it was also selective, as this treatment did not change the cell surface abundance of the transferrin receptor. Hence, the cell surface proteome is subject to acute regulation by treatment with A-769662, at least some of which is mediated by the metabolic sensor AMPK.</p></div

Inhibition of AMPK by siRNA gene silencing or by compound C prevents the reduction in cell surface β1-integrin elicited by A-769662 treatment.

<p>(<b><i>A-C)</i></b> RPE cells were transfected with siRNA targeting AMPK α1/2 or non-targeting (NT, control) siRNA. (<b><i>A</i></b>) Whole cell lysates were prepared and resolved by immunoblotting and probed with anti-AMPK α1/2 or anti-actin antibodies. Shown are immunoblots representative of at least 3 independent experiments. (<b><i>B</i></b>) Following siRNA transfection, cells were treated with 100 μM A-769662 for 60 min as indicated. Intact cells were labeled with an antibody specific for an exofacial epitope on β1-integrin. Shown are representative fluorescence micrographs depicting cell surface β1-integrin fluorescence. Scale = 5 μm (<b><i>C)</i></b> Cell surface β1-integrin levels obtained by fluorescence microscopy were quantified. Shown are the cell surface β1-integrin measurements in individual cells (diamonds) as well as the median ± interquartile range of these values in each treatment condition (n = 3 independent experiments). (<b><i>D</i></b>) RPE cells were treated with 100 μM A-769662 or 40 μM compound C, alone or in combination, for 60 min as indicated. Intact cells were labeled with an antibody specific for an exofacial epitope on β1-integrin. Shown are representative fluorescence micrographs depicting cell surface β1-integrin fluorescence. Scale = 5 μm (<b><i>E)</i></b> Cell surface β1-integrin levels obtained by fluorescence microscopy as in (D) were quantified. Shown are the cell surface β1-integrin measurements in individual cells (diamonds) as well as the median ± interquartile range of these values in each treatment condition (n = 3 independent experiments).</p

Treatment with A-769662 reduces cell surface β1-integrin levels.

<p>(<b><i>A</i></b>) RPE cells were stimulated with 100 μM A-769662 for 90 min or left unstimulated (basal). Intact cells were labeled with an antibody specific for an exofacial epitope on β1-integrin. Shown are representative fluorescence micrographs depicting cell surface β1-integrin fluorescence. Scale = 5 μm (<b><i>B</i></b>) Cell surface β1-integrin levels obtained by fluorescence microscopy were quantified as described in <i>Materials and Methods</i> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128013#pone.0128013.s009" target="_blank">S5B Fig</a>. Shown are the cell surface β1-integrin measurements in individual cells (diamonds) as well as the median ± interquartile range of these values in each treatment condition (n = 4 independent experiments). (<b><i>C</i></b>) RPE cells were stimulated with 2 mM AICAR for 90 min or left unstimulated (basal), followed by cell-surface biotinylation, purification of biotinylated proteins and immunoblotting of fractions with an antibody specific to β1-integrin. Shown is an immunoblot of cell surface β1-intergin (<i>top panel</i>, corresponding to the streptavidin pull-down), and of the corresponding intracellular β1-integrin (<i>bottom panel</i>, corresponding to the above supernatant), representative of 4 independent experiments. (<b><i>D</i></b>) Shown are representative immunoblots of whole-cell lysates prepared from cells stimulated with either 100 μM A-769662, 2 mM AICAR, 40 μM compound C (each for 90 min) or left unstimulated (control), probed with antibodies to detect total cellular β1-integrin or actin (load).</p