Upon antigen recognition, actin assembly and inward flow in the plane of the radially symmetric immunological synapse (IS) drives the centralization of T cell receptor microclusters (TCR MCs) and the integrin LFA-1. Using two forms of structured-illumination microscopy (3D-SIM and TIRF-SIM), we show that actin arcs populating the medial, lamella-like region of the IS (the pSMAC) arise from linear actin filaments generated by the formin mDia present at the distal edge of the IS. After traversing the outer, Arp2/3-generated, lamellipodia-like region of the IS (the dSMAC), these linear filaments are organized by myosin II into concentric arcs that possess the anti-parallel organization required for contraction. Quantitative, fixed-cell 3D-SIM shows that open, active LFA-1 often aligns with arcs while TCR MCs commonly reside between arcs. Consistently, live-cell TIRF-SIM shows TCR MCs being swept inward by arcs. Disrupting actin arc function by blocking their formation via formin inhibition or their concentric organization and contraction via myosin II inhibition results in less centralized TCR MCs, miss-segregated integrin clusters, decreased T: B cell adhesion frequency, and diminished proximal TCR signaling. Together, our results define the origin, organization, and functional significance of a major actomyosin contractile structure at the IS that directly propels TCR MC transport. Mechano-transduction is an emerging but still poorly understood component of T cell activation. Here we investigated the ligand-dependent contribution made by contractile actomyosin arcs populating the peripheral supramolecular activation cluster (pSMAC) region of the immunological synapse (IS) to T cell receptor (TCR) microcluster transport and proximal signaling in primary mouse T cells. Using super resolution microscopy, OT1-CD8+ mouse T cells, and two ovalbumin (OVA) peptides with different affinities for the TCR, we show that the generation of organized actomyosin arcs depends on ligand potency and the ability of myosin 2 to contract actin filaments. While weak ligands induce disorganized actomyosin arcs, strong ligands result in organized actomyosin arcs that correlate well with tension-sensitive CasL phosphorylation and the accumulation of ligands at the IS center. Blocking myosin 2 contractility greatly reduces the difference in the extent of Src and LAT phosphorylation observed between the strong and the weak ligand, arguing that myosin 2-dependent force generation within actin arcs contributes to ligand discrimination. Together, our data are consistent with the idea that actomyosin arcs in the pSMAC region of the IS promote a mechano-chemical feedback mechanism that amplifies the accumulation of critical signaling molecules at the IS. Melanoregulin (Mreg), the product of the dilute suppressor locus, is a small, highly-charged, multiply-palmitoylated protein present on the limiting membrane of melanosomes. Previous studies have implicated Mreg in the transfer of melanosomes from melanocytes to keratinocytes, and in promoting the microtubule minus end-directed transport of these and related organelles by binding to RILP, a Rab7 effector that recruits the dynein motor complex. Here we shed new light on the possible molecular function of Mreg by solving its structure using nuclear magnetic resonance (NMR). The structure reveals bands of positive and negative charge that occupy opposite sides of the proteins surface, and that sandwich a putative, tyrosine-based (Y166) cholesterol recognition sequence (CRAC motif). We confirmed that cholesterol interacts with Mreg, as residues S163 and L168 within the CRAC motif show the largest NMR chemical shift upon cholesterol addition. Importantly, Mreg containing a function blocking point mutation within its CRAC motif (Y166I) still targets to late endosomes/lysosomes, but no longer promotes their microtubule minus end-directed transport. Reversing the charge of three closely-spaced acidic residues (D177, E180, and D181) also inhibits Mregs ability to drive these organelles to microtubule minus ends, but only partially. We propose that cholesterol recognition alters Mregs orientation on the membrane in such a way as to allow it to interact with a component(s) involved in dynein recruitment (e.g. RILP), and that this interaction is further promoted by the acidic patch. Finally, we draw comparisons between Mreg and the protein ORP1L, which controls the microtubule minus end-directed transport, positioning and fate of late endosomes in part by recognizing both cholesterol and components that target dynein. The actin-based motor myosin Va transports numerous cargos, including the endoplasmic reticulum (ER) in cerebellar Purkinje neurons and melanosomes in melanocytes. Identifying proteins that interact with this myosin is key to understanding its cellular functions. Towards that end, we used recombineering to insert via homologous recombination a tandem affinity purification (TAP) tag composed of the IgG binding domain of Protein A, a TEV cleavage site, and a FLAG tag into the mouse MYO5A locus immediately after the initiation codon. Importantly, we provide evidence that the TAP-tagged version of myosin Va (TAP-MyoVa) functions normally in terms of ER transport in Purkinje neurons and melanosome positioning in melanocytes. Given this and other evidence that TAP-MyoVa is fully functional, we purified it and associated proteins directly from juvenile mouse cerebella and subjected the samples to mass spectroscopic analyses. As expected, known myosin Va binding partners like dynein light chain and neurofilament light peptide were identified. Importantly, numerous novel interacting proteins were also identified, including 2',3'-cyclic-nucleotide 3'-phosphodiesterase, brain-specific angiogenesis inhibitor 1-associated protein 2, and myelin basic protein. The mouse model created here should facilitate the identification of novel myosin Va binding partners, which in turn should advance our understanding of the roles played by this myosin in vivo. While mixed primary cerebellar cultures prepared from embryonic tissue have proven valuable for dissecting structure: function relationships in cerebellar Purkinje Neurons (PNs), this technique is technically challenging and often yields few cells. Recently, mouse embryonic stem cells (ESCs) have been successfully differentiated into PNs, although the available methods are very challenging as well. The focus of this study was to simplify the differentiation of mouse ESCs into PNs. Using a recently-described neural differentiation media, we generate monolayers of neural progenitor cells (NPs) from ESCs, which we differentiate into PN precursors using specific extrinsic factors. These precursors are then differentiated into mature PNs by co-culture with granule neuron (GN) precursors also derived from NPs using different extrinsic factors. The morphology of ESC-derived PNs is indistinguishable from PNs grown in primary culture in terms of gross morphology, spine length and spine density. Furthermore, ESC-derived PNs express Calbindin D28K, IP3R1, IRBIT, PLC4, PSD93 and myosin IIB-B2, all of which are PN-specific markers. Moreover, we show that ESC-derived PNs express proteins driven by the PN-specific promoter Pcp2/L7, form synapses with GNs as in primary cultures, and exhibit the defect in spine ER inheritance seen in PNs isolated from dilute-lethal (myosin Va-null) mice when expressing a Pcp2/L7-driven miRNA directed against myosin Va. Finally, we define a novel extracellular matrix formulation that reproducibly yields monolayer cultures conducive for high-resolution imaging. Our improved method for differentiating ESCs into PNs should facilitate the dissection of molecular mechanisms in PNs.
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