ABCA1 transporter promotes the motility of human melanoma cells by modulating their plasma membrane organization.

BACKGROUND: Melanoma is one of the most aggressive and deadliest skin tumor. Cholesterol content in melanoma cells is elevated, and a portion of it accumulates into lipid rafts. Therefore, the plasma membrane cholesterol and its lateral organization might be directly linked with tumor development. ATP Binding Cassette A1 (ABCA1) transporter modulates physico-chemical properties of the plasma membrane by modifying cholesterol distribution. Several studies linked the activity of the transporter with a different outcome of tumor progression depending on which type. However, no direct link between human melanoma progression and ABCA1 activity has been reported yet. METHODS: An immunohistochemical study on the ABCA1 level in 110 patients-derived melanoma tumors was performed to investigate the potential association of the transporter with melanoma stage of progression and prognosis. Furthermore, proliferation, migration and invasion assays, extracellular-matrix degradation assay, immunochemistry on proteins involved in migration processes and a combination of biophysical microscopy analysis of the plasma membrane organization of Hs294T human melanoma wild type, control (scrambled), ABCA1 Knockout (ABCA1 KO) and ABCA1 chemically inactivated cells were used to study the impact of ABCA1 activity on human melanoma metastasis processes. RESULTS: The immunohistochemical analysis of clinical samples showed that high level of ABCA1 transporter in human melanoma is associated with a poor prognosis. Depletion or inhibition of ABCA1 impacts invasion capacities of aggressive melanoma cells. Loss of ABCA1 activity partially prevented cellular motility by affecting active focal adhesions formation via blocking clustering of phosphorylated focal adhesion kinases and active integrin ß3. Moreover, ABCA1 activity regulated the lateral organization of the plasma membrane in melanoma cells. Disrupting this organization, by increasing the content of cholesterol, also blocked active focal adhesion formation. CONCLUSION: Human melanoma cells reorganize their plasma membrane cholesterol content and organization via ABCA1 activity to promote motility processes and aggressiveness potential. Therefore, ABCA1 may contribute to tumor progression and poor prognosis, suggesting ABCA1 to be a potential metastatic marker in melanoma.

ABCA1 transporter promotes the motility of human melanoma cells by modulating their plasma membrane organization

BACKGROUND: Melanoma is one of the most aggressive and deadliest skin tumor. Cholesterol content in melanoma cells is elevated, and a portion of it accumulates into lipid rafts. Therefore, the plasma membrane cholesterol and its lateral organization might be directly linked with tumor development. ATP Binding Cassette A1 (ABCA1) transporter modulates physico-chemical properties of the plasma membrane by modifying cholesterol distribution. Several studies linked the activity of the transporter with a different outcome of tumor progression depending on which type. However, no direct link between human melanoma progression and ABCA1 activity has been reported yet. METHODS: An immunohistochemical study on the ABCA1 level in 110 patients-derived melanoma tumors was performed to investigate the potential association of the transporter with melanoma stage of progression and prognosis. Furthermore, proliferation, migration and invasion assays, extracellular-matrix degradation assay, immunochemistry on proteins involved in migration processes and a combination of biophysical microscopy analysis of the plasma membrane organization of Hs294T human melanoma wild type, control (scrambled), ABCA1 Knockout ( ABCA1 KO) and ABCA1 chemically inactivated cells were used to study the impact of ABCA1 activity on human melanoma metastasis processes. RESULTS: The immunohistochemical analysis of clinical samples showed that high level of ABCA1 transporter in human melanoma is associated with a poor prognosis. Depletion or inhibition ofABCA1 impacts invasion capacities of aggressive melanoma cells. Loss of ABCA1 activity partially prevented cellular motility by affecting active focal adhesions formation via blocking clustering of phosphorylated focal adhesion kinases and active integrin ß3. Moreover, ABCA1 activity regulated the lateral organization of the plasma membrane in melanoma cells. Disrupting this organization, by increasing the content of cholesterol, also blocked active focal adhesion formation. CONCLUSION: Human melanoma cells reorganize their plasma membrane cholesterol content and organization via ABCA1 activity to promote motility processes and aggressiveness potential. Therefore, ABCA1 may contribute to tumor progression and poor prognosis, suggesting ABCA1 to be a potential metastatic marker in melanoma.

Controlling T cells spreading, mechanics and activation by micropatterning.

We designed a strategy, based on a careful examination of the activation capabilities of proteins and antibodies used as substrates for adhering T cells, coupled to protein microstamping to control at the same time the position, shape, spreading, mechanics and activation state of T cells. Once adhered on patterns, we examined the capacities of T cells to be activated with soluble anti CD3, in comparison to T cells adhered to a continuously decorated substrate with the same density of ligands. We show that, in our hand, adhering onto an anti CD45 antibody decorated surface was not affecting T cell calcium fluxes, even adhered on variable size micro-patterns. Aside, we analyzed the T cell mechanics, when spread on pattern or not, using Atomic Force Microscopy indentation. By expressing MEGF10 as a non immune adhesion receptor in T cells we measured the very same spreading area on PLL substrates and Young modulus than non modified cells, immobilized on anti CD45 antibodies, while retaining similar activation capabilities using soluble anti CD3 antibodies or through model APC contacts. We propose that our system is a way to test activation or anergy of T cells with defined adhesion and mechanical characteristics, and may allow to dissect fine details of these mechanisms since it allows to observe homogenized populations in standardized T cell activation assays.

Do ABC transporters regulate plasma membrane organization?

The plasma membrane (PM) spatiotemporal organization is one of the major factors controlling cell signaling and whole-cell homeostasis. The PM lipids, including cholesterol, determine the physicochemical properties of the membrane bilayer and thus play a crucial role in all membrane-dependent cellular processes. It is known that lipid content and distribution in the PM are not random, and their transversal and lateral organization is highly controlled. Mainly sphingolipid- and cholesterol-rich lipid nanodomains, historically referred to as rafts, are extremely dynamic "hot spots" of the PM controlling the function of many cell surface proteins and receptors. In the first part of this review, we will focus on the recent advances of PM investigation and the current PM concept. In the second part, we will discuss the importance of several classes of ABC transporters whose substrates are lipids for the PM organization and dynamics. Finally, we will briefly present the significance of lipid ABC transporters for immune responses.

Author Correction: Phosphoinositides regulate the TCR/CD3 complex membrane dynamics and activation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Phosphoinositides regulate the TCR/CD3 complex membrane dynamics and activation.

Phosphoinositides (PIs) play important roles in numerous membrane-based cellular activities. However, their involvement in the mechanism of T cell receptor (TCR) signal transduction across the plasma membrane (PM) is poorly defined. Here, we investigate their role, and in particular that of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] in TCR PM dynamics and activity in a mouse T-cell hybridoma upon ectopic expression of a PM-localized inositol polyphosphate-5-phosphatase (Inp54p). We observed that dephosphorylation of PI(4,5)P2 by the phosphatase increased the TCR/CD3 complex PM lateral mobility prior stimulation. The constitutive and antigen-elicited CD3 phosphorylation as well as the antigen-stimulated early signaling pathways were all found to be significantly augmented in cells expressing the phosphatase. Using state-of-the-art biophotonic approaches, we further showed that PI(4,5)P2 dephosphorylation strongly promoted the CD3ε cytoplasmic domain unbinding from the PM inner leaflet in living cells, thus resulting in an increased CD3 availability for interactions with Lck kinase. This could significantly account for the observed effects of PI(4,5)P2 dephosphorylation on the CD3 phosphorylation. Our data thus suggest that PIs play a key role in the regulation of the TCR/CD3 complex dynamics and activation at the PM.