Membrane protein organization is essential for proper cellular functioning and the result of a dynamic exchange between protein monomers, nanoscale protein clusters, and microscale higher-order structures. This exchange is affected by both lipid bilayer intrinsic factors, such as lipid rafts and tetraspanins, and extrinsic factors, such as cortical actin and galectins. Because membrane organizers act jointly like instruments in a symphony, it is challenging to define the 'key' organizers. Here, we posit, for the first time, definitions of key intrinsic and extrinsic membrane organizers. Tetraspanin nanodomains are key organizers that are often overlooked. We discuss how different key organizers can collaborate, which is important to get a full grasp of plasma membrane biology. Open Questions in Plasma Membrane Biology HighlightsNovel definitions are postulated for membrane organizers intrinsic and extrinsic to the lipid bilayer.Membrane protein organization is the result of a dynamic exchange between protein monomers, nanoscale protein clusters, and microscale higher-order structures.Tetraspanins are key membrane organizers that should be considered in future plasma membrane studies.Membrane organizers act together in a dynamic manner like instruments playing a symphony.

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Summary Melanoma is an aggressive skin cancer developing from melanocytes, frequently resulting in metastatic disease. Melanoma cells utilize amoeboid migration as mode of local invasion. Amoeboid invasion is characterized by rounded cell morphology and high actomyosin contractility driven by Rho GTPase signalling. Migrastatic drugs targeting actin polymerization and contractility are therefore a promising treatment option for metastatic melanoma. To predict amoeboid invasion and metastatic potential, biomarkers functionally linked to contractility pathways are needed. The glycoprotein podoplanin drives actomyosin contractility in lymphoid fibroblasts and is overexpressed in many cancers. We show that podoplanin enhances amoeboid invasion in melanoma. Podoplanin expression in murine melanoma drives rounded cell morphology, increasing motility, and invasion in vivo . Podoplanin expression is increased in a subset of dedifferentiated human melanoma, and in vitro is sufficient to upregulate melanoma-associated marker Pou3f2 /Brn2. Together, our data define podoplanin as a functional biomarker for dedifferentiated invasive melanoma and a promising migrastatic therapeutic target.

T cells are central to the adaptive immune response, playing a role in both the direct and indirect killing of pathogens and transformed cells. The activation of T cells is the result of a complex signaling cascade, initiated at the T cell receptor (TCR), and ending with the induction of proliferation. CD45, a member of the protein tyrosine phosphatase family, is one of the most abundant membrane proteins on T cells and functions by regulating activation directly downstream of the TCR. As a result of alternative splicing, CD45 can be expressed in multiple isoforms, naive T cells express the CD45RA isoform, while activated T cells gain expression of CD45RO, which has been proposed to increase signaling. Though the importance of CD45 in TCR signaling, proliferation and cytokine production is well established, little is known about the regulation of CD45 activity. We discovered that the immune-specific tetraspanin CD53 directly affects the stability and function of CD45RO in T cells. We have identified CD53 as a T cell co-stimulatory molecule in primary human and murine cells. Furthermore, we have shown that the absence of CD53 leads to an altered CD45 isoform expression as a result of decreased CD45RO stability on the cell surface. This instability was accompanied by increased mobility as measured by FRAP. Together, this indicates that CD53 functions as a stabilizer of CD45RO, and therefore as a positive regulator of TCR signaling at the T cell surface. Our data provides novel insight into the role of tetraspanins in the regulation of immune signaling and may provide a new avenue for the regulation of T cell signaling.