In mammalian cells three closely related cavin proteins cooperate with the scaffolding protein caveolin to form membrane invaginations known as caveolae. Here we have developed a novel single-molecule fluorescence approach to directly observe interactions and stoichiometries in protein complexes from cell extracts and from in vitro synthesized components. We show that up to 50 cavins associate on a caveola. However, rather than forming a single coat complex containing the three cavin family members, single-molecule analysis reveals an exquisite specificity of interactions between cavin1, cavin2 and cavin3. Changes in membrane tension can flatten the caveolae, causing the release of the cavin coat and its disassembly into separate cavin1-cavin2 and cavin1-cavin3 subcomplexes. Each of these subcomplexes contain 9 ± 2 cavin molecules and appear to be the building blocks of the caveolar coat. High resolution immunoelectron microscopy suggests a remarkable nanoscale organization of these separate subcomplexes, forming individual striations on the surface of caveolae.DOI: http://dx.doi.org/10.7554/eLife.01434.001

Background: Caveolin-1 (Cav1) requires the caveolin scaffolding domain for caveola formation. Results: The Cav1 scaffolding domain and oligomerization domain are tightly juxtaposed to the membrane in caveolae. Conclusion: Concerted membrane association of the oligomerization, scaffolding, and intramembrane domains are critical for caveola biogenesis and membrane deformation. Significance: Understanding the membrane association of Cav1 is critical for dissecting how the protein regulates caveola formation and achieves regulation over cellular signaling.

Highlights d A strategy for improving the ADCC potential of therapeutic antibodies is presented d Temporary inhibition of endocytosis increases tumor cell antigen presentation d Prochlorperazine could be repurposed to enhance the efficacy of anti-tumor mAbs d Potential to reduce heterogeneity in tumor cell responses to many IgG1 antibodies

Caveolae are specialized domains of the plasma membrane. Formation of these invaginations is dependent on the expression of Caveolin-1 or -3 and proteins of the cavin family. In response to stress, caveolae disassemble and cavins are released from caveolae, allowing cavins to potentially interact with intracellular targets. Here, we describe the intracellular (non-plasma membrane) cavin interactome using biotin affinity proteomics and mass spectrometry. We validate 47 potential cavin-interactor proteins using a cell-free expression system and protein-protein binding assays. These data, together with pathway analyses, reveal unknown roles for cavin proteins in metabolism and stress signaling. We validated the interaction between one candidate interactor protein, protein phosphatase 1 alpha (PP1α), and Cavin-1 and -3 and show that UV treatment causes release of Cavin3 from caveolae allowing interaction with, and inhibition of, PP1α. This interaction increases H2AX phosphorylation to stimulate apoptosis, identifying a pro-apoptotic signaling pathway from surface caveolae to the nucleus.

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Titanium plates are widely used in clinical settings because of their high bone affinity. However, owing to their high elastic modulus, these plates are not suitable for bone repair since their proximity to the bone surface for prolonged periods can cause stress shielding, leading to bone embrittlement. In contrast, titanium fiber plates prepared by molding titanium fibers into plates by simultaneously applying compression and shear stress at normal room temperature can have an elastic modulus similar to that of bone cortex, and stress shielding will not occur even when the plate lies flush against the bone's surface. Titanium fibers can form a porous structure suitable for cell adhesion and as a bone repair scaffold. A titanium fiber plate is combined with osteoblasts and shown that the titanium fiber plate is better able to facilitate bone tissue repair than the conventional titanium plate when implanted in rat bone defects. Capable of being used in close contact with bone for a long time, and even capable of promoting bone repair, titanium fiber plates have a wide range of applications, and are expected to make great contributions to clinical management of increasing bone diseases, including bone fracture repair and bone regenerative medicine.

Hormone-sensitive lipase (HSL) is a key enzyme regulating the acute activation of lipolysis. HSL functionality is controlled by multiple phosphorylation events, which regulate its association with the surface of lipid droplets (LDs). We determined the progression and stability of HSL phosphorylation on individual serine residues both spatially and temporally in adipocytes using phospho-specific antibodies. Within seconds of ␤-adrenergic receptor activation, HSL was phosphorylated on Ser-660, the phosphorylated form appearing in the peripheral cytosol prior to rapid translocation to, and stable association with, LDs. In contrast, phosphorylation of HSL on Ser-563 was delayed, the phosphorylated protein was predominantly detected on LDs, and mutation of the Ser-659/Ser-660 site to Ala significantly reduced subsequent phosphorylation on Ser-563. Phosphorylation of HSL on Ser-565 was observed in control cells; the phosphorylated protein was translocated to LDs with similar kinetics to total HSL, and the degree of phosphorylation was inversely related to phospho-HSL Ser-563 . These results describe the remarkably rapid, sequential phosphorylation of specific serine residues in HSL at spatially distinct intracellular locales, providing new insight into the complex regulation of lipolysis.The regulation of lipid storage and lipolysis in adipocytes has important implications for the maintenance of whole body lipid homeostasis (1, 2). Dysregulation is associated with obesity and the onset of metabolic disease, insulin resistance, and type II diabetes. An important goal for the future is to design strategies that allow us to manipulate lipid storage and so control weight gain. Fundamental to a targeted approach to controlling the storage and mobilization of lipid in adipose tissue is a detailed understanding of the cellular mechanisms and machinery that regulate lipolysis. Although the key players in this process have been identified, the precise regulation of the lipolytic machinery is not yet fully understood. In this study we have begun to address this by analyzing the earliest events in the activation of lipolysis at the cellular level.Adipocytes are specialized lipid droplet (LD) 2 -laden cells that store large amounts of neutral lipid, predominantly as triglycerides (TG) (3, 4). In response to extracellular stimulation by catecholamines, adipocytes hydrolyze stored TGs to generate free fatty acids and glycerol. In rodent adipocytes, the hydrolysis of neutral lipids is tightly regulated by a series of signal transduction pathways from the G-protein-coupled ␤ 3 -adrenergic receptor that culminate at the surface of the LD (2, 5). A well characterized pathway from the ␤ 3 -adrenergic receptor results in the elevation of cAMP levels, activating cAMP-dependent protein kinase/protein kinase A (PKA), which in turn phosphorylates downstream targets, including the lipid droplet scaffold/ adaptor protein perilipin (6) and the primary diacylglycerol lipase hormone-sensitive lipase (HSL) (7). The array of phosphorylation sites p...

Purpose Patients with primarily ligamentous injuries of the distal tibiofibular joint comprise up to 12% of all ankle sprains. Patients frequently present late after a syndesmosis injury and delayed treatment potentially leads to pain, prolonged disability and arthritis in the long term. This study aimed to assess clinical outcomes in patients who required syndesmosis fixation in the presence of arthroscopically proven instability, the hypothesis being that a delay to treatment would be associated with worse function. Method A retrospective cohort study was performed of patients with dynamic instability requiring fixation between the years of 2010–2016. The procedures were performed by two foot and ankle fellowship trained orthopaedic surgeons, over three hospital sites. Patients were classified into three groups based on the time since injury to surgery, acute syndesmotic injury (< 6 weeks), sub‐acute (6 weeks–6 months) and chronic syndesmotic injury (> 6 months). Functional scores were retrospectively collected using the Foot and Ankle Outcome Score (FAOS). Results Compared to patients with acute injuries, those with chronic injuries had significantly lower FAOS subscales (p < 0.001), with the greatest difference in quality of life (− 20.7, 95% CI − 31.6 to − 9.8, p = 0.012). There was a mean follow‐up of 4.3 years. Although the average FAOS subscales in those with sub‐acute injuries were lower than in those with acute injuries, the difference was not statistically significant. Conclusion The results of this study suggest that delayed surgical stabilisation (> 6 months) is associated with significantly worse clinical function, and thus timely identification and early referral of those patients with potentially unstable syndesmotic injuries is recommended. Level of evidence Level III.