Clathrin-mediated endocytosis (CME) requires energy input from actin polymerization in mechanically challenging conditions. The roles of actin in CME are poorly understood due to inadequate knowledge of actin organization at clathrin-coated structures (CCSs). Using platinum replica electron microscopy of mammalian cells, we show that Arp2/3 complex-dependent branched actin networks, which often emerge from microtubule tips, assemble along the CCS perimeter, lack interaction with the apical clathrin lattice, and have barbed ends oriented toward the CCS. This structure is hardly compatible with the widely held “apical pulling” model describing actin functions in CME. Arp2/3 complex inhibition or epsin knockout produce large flat non-dynamic CCSs, which split into invaginating subdomains upon recovery from Arp2/3 inhibition. Moreover, epsin localization to CCSs depends on Arp2/3 activity. We propose an “edge pushing” model for CME, wherein branched actin polymerization promotes severing and invagination of flat CCSs in an epsin-dependent manner by pushing at the CCS boundary, thus releasing forces opposing the intrinsic curvature of clathrin lattices.

40 word limit): Synucleins can sense and generate membrane 20 curvature. We previously showed that synuclein null mice exhibit deficits in 21 synaptic vesicle endocytosis. Here, Vargas et al. provide evidence that α-synuclein 22 functions specifically in clathrin assembly during early steps of synaptic vesicle 23 endocytosis. 24 25 Abstract (160 word limit): 26 27 α-Synuclein plays a central role in Parkinson's disease (PD); hence, 28 elucidating its normal physiological function(s) is important. α-Synuclein and family 29 members β-, and γ-synuclein, are presynaptically enriched proteins. Synucleins 30 sense and generate membrane curvature, properties consistent with their 31 described roles in synaptic vesicle (SV) cycling. We have previously shown SV 32 endocytosis (SVE) deficits in αβγ-synuclein knockout (KO) neurons. Here, we 33 investigate which steps of SVE are regulated by α-synuclein. Immuno-electron 34 microscopy (EM) of synaptosomes reveals that α-synuclein relocalizes from SVs 35to the synaptic membrane upon stimulation, allowing α-synuclein to function there 36 during or after stimulation. Using membrane recruitment assays, we show that α-37 synuclein is co-localized with clathrin patches. We also observe that recruitment 38 of clathrin and its adaptor, AP180, to synaptic membranes is altered in the absence 39 of synucleins. Visualizing clathrin assembly on membranes in an in vitro 40reconstitution system reveal that synucleins increase clathrin patch size and 41 curvature, facilitating clathrin coated pit maturation during the early steps of SVE. 42 43Introduction 44 45α-Synuclein became a principal focus of neurodegenerative research when 46it was identified as the major constituent of Lewy Bodies, the pathological protein 47aggregates found in the brains of PD patients [1]. The importance of α-synuclein 48 was further underscored by the identification of families with Mendelian forms of 49 PD arising from causal point mutations and gene multiplications of SNCA, the α-50 synuclein gene [2-7]. Genome-wide association studies have shown that 51 sequence variants in SNCA are also associated with sporadic PD [8, 9]. Based on 52these observations, many current therapeutic strategies for PD are focused on 53 eliminating or reducing α-synuclein levels in the brain. Therefore, there is a growing 54interest in understanding α-synuclein's physiological functions and how loss of α-55 synuclein impacts neuronal functions. 56 57Since its discovery as a SV-associated protein in the electric organ of 58Torpedo [10], several distinct approaches have been used to determine the 59 physiological function(s) of α-synuclein. Structural studies have revealed that α-60 synuclein can adopt several conformations, principally, unfolded in solution, but 61 also α-helical on phospholipid membranes. In α-helical conformations, the N-62terminus folds into either a single elongated amphipathic helix on flatter 63 membranes or a 'broken' helix when bound to curved lipid membranes [11, 12]. α-64Synuclein can switch between the two ...

Super-resolution fluorescence microscopy allows the investigation of cellular structures at nanoscale resolution using light. Current developments in super-resolution microscopy have focused on reliable quantification of the underlying biological data. In this review, we first describe the basic principles of super-resolution microscopy techniques such as stimulated emission depletion (STED) microscopy and single-molecule localization microscopy (SMLM), and then give a broad overview of methodological developments to quantify super-resolution data, particularly those geared toward SMLM data. We cover commonly used techniques such as spatial point pattern analysis, colocalization, and protein copy number quantification but also describe more advanced techniques such as structural modeling, single-particle tracking, and biosensing. Finally, we provide an outlook on exciting new research directions to which quantitative super-resolution microscopy might be applied.

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