Single molecule microscopy has evolved into the ultimate-sensitivity toolkit to study systems from small molecules to living cells, with the prospect of revolutionizing the modern biosciences. Here we survey the current state-of-the-art in single molecule tools including fluorescence spectroscopy, tethered particle microscopy, optical and magnetic tweezers, and atomic force microscopy. Our review seeks to guide the biological scientist in choosing the right approach from the available single molecule toolkit for applications ranging as far as structural biology, enzymology, nanotechnology, and systems biology.

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SummarySphingolipids are crucial components of membranes, and sphingolipid metabolites serve as signaling molecules. Yeast Orm1 and Orm2 belong to a conserved family of ER membrane proteins that regulate serine palmitoyltransferase, which catalyzes the first and ratelimiting step in sphingolipid synthesis. We now show that sphingolipid synthesis through Orm1 is a target of TOR signaling, which regulates cell growth in response to nutritional signals. Orm1 phosphorylation is dependent on the Tap42-phosphatase complex, which acts downstream of TOR protein kinase complex 1. In temperature-sensitive tap42-11 cells, impaired Orm1 phosphorylation occurs concomitantly with reduced sphingolipid synthesis. A second mechanism for regulating sphingolipid synthesis is through control of Orm2 protein level. The Orm2 protein level responds to ER stress conditions, increasing when cells are treated with tunicamycin or DTT, agents that induce the unfolded protein response (UPR). The sphingolipid intermediates (long chain base and ceramide) are decreased when ORM2 is overexpressed, suggesting that sphingolipid synthesis is repressed under ER stress conditions. Finally, in the absence of the Orms, the UPR is constitutively activated. Lipid dysregulation in the absence of the Orms might signal to the ER from the plasma membrane because UPR activation is dependent on a cell surface sensor and the mitogen-activated protein kinase (MAPK) cell wall integrity pathway. Thus, sphingolipid synthesis and the UPR are coordinately regulated.

The vacuolar proton-translocating ATPase (V-ATPase) plays a major role in organelle acidification and works together with other ion transporters to maintain pH homeostasis in eukaryotic cells. We analyzed a requirement for V-ATPase activity in protein trafficking in the yeast secretory pathway. Deficiency of V-ATPase activity caused by subunit deletion or glucose deprivation results in missorting of newly synthesized plasma membrane proteins Pma1 and Can1 directly from the Golgi to the vacuole. Vacuolar mislocalization of Pma1 is dependent on Gga adaptors although no Pma1 ubiquitination was detected. Proper cell surface targeting of Pma1 was rescued in V-ATPase-deficient cells by increasing the pH of the medium, suggesting that missorting is the result of aberrant cytosolic pH. In addition to mislocalization of the plasma membrane proteins, Golgi membrane proteins Kex2 and Vrg4 are also missorted to the vacuole upon loss of V-ATPase activity. Because the missorted cargos have distinct trafficking routes, we suggest a pH dependence for multiple cargo sorting events at the Golgi.The yeast plasma membrane ATPase, Pma1, is an electrogenic proton pump that regulates intracellular pH and generates the membrane potential across the plasma membrane. Pma1 is a member of the conserved P-type ATPase family. Because Pma1 is abundant and its activity at the cell surface is essential for cell viability (1), it has served as a model polytopic membrane protein to study protein sorting and quality control in the secretory pathway. Studies using misfolded Pma1 mutants have revealed disposal of conformationally defective proteins by post-endoplasmic reticulum (ER) 2 mechanisms as well as the better characterized ER-associated degradation (ERAD) pathway (2). Misfolded Pma1-7 (P434A,G789S) is impaired in plasma membrane targeting and routed instead from Golgi to the endosomal/vacuolar system for degradation (3, 4). By contrast, misfolded Pma1-10 (A165G,V197I) fails to remain stable at the cell surface and undergoes vacuolar degradation through endocytosis (5). Additional studies have shown that wild-type Pma1 is associated with sphingolipid-and cholesterol-enriched microdomains, and both plasma membrane targeting of Pma1 and its stability at the cell surface require sphingolipids (6, 7).The vacuolar proton-translocating ATPase (V-ATPase) is a second proton pump, mechanistically distinct from Pma1, that plays a major role in maintaining pH homeostasis (8, 9). The importance of V-ATPase for acidification of the vacuole/lysosomes, Golgi, and endosomes of eukaryotic cells is well established (10, 11). In contrast with Pma1, which is thought to function as a homohexamer (12), V-ATPase is composed of two multisubunit subcomplexes, the peripheral catalytic V1 subcomplex and the integral membrane proton-translocating V0 subcomplex (11). V-ATPase biogenesis is complex and incompletely understood. Assembly of V1 and V0 sector subunits require a protein complex called RAVE (regulator of the (H ϩ )-ATPase of vacuolar and endosomal membranes), w...

Vaccinia virus, the prototype of the Orthopoxvirus genus in the family Poxviridae, infects a wide range of cell lines and animals. Vaccinia mature virus particles of the WR strain reportedly enter HeLa cells through fluid-phase endocytosis. However, the intracellular trafficking process of the vaccinia mature virus between cellular uptake and membrane fusion remains unknown. We used live imaging of single virus particles with a combination of various cellular vesicle markers, to track fluorescent vaccinia mature virus particle movement in cells. Furthermore, we performed functional interference assays to perturb distinct vesicle trafficking processes in order to delineate the specific route undertaken by vaccinia mature virus prior to membrane fusion and virus core uncoating in cells. Our results showed that vaccinia virus traffics to early endosomes, where recycling endosome markers Rab11 and Rab22 are recruited to participate in subsequent virus trafficking prior to virus core uncoating in the cytoplasm. Furthermore, we identified WASH-VPEF/FAM21-retromer complexes that mediate endosome fission and sorting of virus-containing vesicles prior to virus core uncoating in the cytoplasm. IMPORTANCE Vaccinia mature virions of the WR strain enter HeLa cells through fluid phase endocytosis.We previously demonstrated that virus-containing vesicles are internalized into phosphatidylinositol 3-phosphate positive macropinosomes, which are then fused with Rab5-positive early endosomes. However, the subsequent process of sorting the virion-containing vesicles prior to membrane fusion remains unclear. We dissected the intracellular trafficking pathway of vaccinia mature virions in cells up to virus core uncoating in cytoplasm. We show that vaccinia mature virions first travel to early endosomes. Subsequent trafficking events require the important endosome-tethered protein VPEF/FAM21, which recruits WASH and retromer protein complexes to the endosome. There, the complex executes endosomal membrane fission and cargo sorting to the Rab11-positive and Rab22-positive recycling pathway, resulting in membrane fusion and virus core uncoating in the cytoplasm. V accinia virus is the prototype of the Orthopoxvirus genus in the family Poxviridae, which includes the variola virus that causes smallpox diseases. It has a broad host range and infects a wide variety of cell lines and animals. Vaccinia virus produces two forms of infectious virions, mature virus (MV) and extracellular virus (EV), which contain different membrane proteins (1). MV particles are abundant in infected cells and contain ϳ80 viral proteins (2, 3), which contribute to the complex virus entry processes that reportedly vary among different cells and virus strains (4-12).In HeLa cells, vaccinia MV initially attaches to cellular surface component glycosaminoglycans (13-15) and the extracellular matrix protein laminin (16). MV particles then cluster at plasma membrane lipid rafts (17) where the virus further interacts with integrin ␤1 (18) and CD98 receptor molecu...

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Immune checkpoint inhibitors demonstrate clinical activity in many tumor types, however, only a fraction of patients benefit. Combining CD137 agonists with these inhibitors increases anti-tumor activity preclinically, but attempts to translate these observations to the clinic have been hampered by systemic toxicity. Here we describe a human CD137xPD-L1 bispecific antibody, MCLA-145, identified through functional screening of agonist- and immune checkpoint inhibitor arm combinations. MCLA-145 potently activates T cells at sub-nanomolar concentrations, even under suppressive conditions, and enhances T cell priming, differentiation and memory recall responses. In vivo, MCLA-145 anti-tumor activity is superior to immune checkpoint inhibitor comparators and linked to recruitment and intra-tumor expansion of CD8 + T cells. No graft-versus-host-disease is observed in contrast to other antibodies inhibiting the PD-1 and PD-L1 pathway. Non-human primates treated with 100 mg/kg/week of MCLA-145 show no adverse effects. The conditional activation of CD137 signaling by MCLA-145, triggered by neighboring cells expressing >5000 copies of PD-L1, may provide both safety and potency advantages.