Actin cytoskeleton organization and dynamics drive the formation of the immunological synapse (IS) in both T cells and B cells. The actin cytoskeleton at T cell IS is comprised primarily of a radially-symmetric branched actin network at the IS periphery, followed by concentric, contractile actomyosin arcs in the medial portion of the IS (Murugesan et al JCB 2016). These actomyosin arcs drive T cell receptor microcluster movement, proximal signaling, antigen affinity discrimination, and T cell adhesion to antigen-presenting cells (APCs). In B cells, the IS facilitates immune activation and antigen uptake, a process that is critical for their differentiation into highly effective antibody-producing cells. Antigen uptake at the IS is thought to be powered by myosin-mediated force generation (Spillane and Tolar, J Mol Immunol, 2018); however, the organization of actomyosin at the B cell immune synapse is not completely understood. Here we sought to define the organization and dynamics of actin and myosin at the B cell IS using three super-resolution imaging modalities: TIRF/SIM, 3D SIM and Airyscan. On both functionalized glass and planar lipid bilayers, A20 B cells expressing F-Tractin formed synaptic actin structures comparable to those seen in T cells, including the presence of concentric actin arcs in the medial portion of the IS. These arcs are likely to be contractile because, as in T cells, they are rich in myosin 2A based on staining for endogenous myosin 2A, and on imaging A20 cells in which endogenous myosin 2A was labeled with GFP by CRISPR-based gene editing. Importantly, actomyosin arcs were a prominent feature of the IS formed by primary splenic B cells isolated from a myosin 2A GFP knock-in mouse (gift of Robert Adelstein, NHLBI, NIH). Indeed, the actomyosin arcs comprise a larger fraction of the total synaptic actin in primary B cells than in the A20 B cell line (as seen when primary T cells are compared to the Jurkat T cell line). Additionally, myosin inhibition disrupts the concentric organization of B cell arcs. Finally, 3D SIM imaging showed that myosin 2A polarizes to the site of IS formation in primary B cell: APC conjugates. Current efforts are directed at determining the origin of the actomyosin arcs in B cells, and the role they play in BCR microcluster movement, B cell activation, and B cell: APC adhesion. Future endeavors will seek to define the mechanism by which these B cell actomyosin structures extract membrane-bound antigens at the IS formed with an APC. 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 (mESCs) have been successfully differentiated into PNs, although the published methods are very challenging as well. The focus of this study was to simplify the differentiation of mESCs into PNs. Using a recently-described neural differentiation media, we generate monolayers of neural progenitor cells from mESCs and differentiate them into PN precursors using specific extrinsic factors. These PN precursors are then differentiated into mature PNs by co-culturing them with granule neuron (GN) precursors also derived from neural progenitors using different extrinsic factors. The morphology of mESC-derived PNs is indistinguishable from PNs grown in primary culture in terms of gross morphology, spine length and spine density. Furthermore, mESC-derived PNs express Calbindin D28K, IP3R1, IRBIT, PLC4, PSD93 and myosin IIB-B2, all of which are either PN-specific or highly expressed in PNs. Moreover, we show that mESC-derived PNs form synapses with GN-like cells as in primary culture, express proteins driven by the PN-specific promoter Pcp2/L7, 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 mESCs into PNs should facilitate the dissection of molecular mechanisms and disease phenotypes in PNs. 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) spectroscopy. Mreg contains six -helices that form an elongated fishhook-like fold in which positive and negative charges occupy opposite sides of the proteins surface and sandwich a putative, tyrosine-based (Y166) cholesterol recognition sequence (CRAC motif). The absence or significant exchange broadening of 1H-15N crosspeaks for multiple residues within this putative CRAC motif and a proximal tryptophan sidechain resonance argue that this motif has functional importance. Consistently, 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. Moreover, wild type Mreg does not promote the microtubule minus end-directed transport of late endosomes/lysosomes in cells transiently depleted of cholesterol. Finally, 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 negatively charged patch. 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.
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