Ultrahigh-resolution imaging reveals formation of neuronal SNARE/Munc18 complexes in situ.
AuthorsPertsinidis A, Mukherjee K, Sharma M, Pang ZP, Park SR, Zhang Y, Brunger AT, Südhof TC, Chu S,
Journal
PubMed ID23821748
'Membrane fusion is mediated by complexes formed by SNAP-receptor (SNARE) and Secretory 1 (Sec1)/mammalian uncoordinated-18 (Munc18)-like (SM) proteins, but it is unclear when and how these complexes assemble. Here we describe an improved two-color fluorescence nanoscopy technique that can achieve effective resolutions of up to 7.5-nm full width at half ... More
COPI buds 60-nm lipid droplets from reconstituted water-phospholipid-triacylglyceride interfaces, suggesting a tension clamp function.
AuthorsThiam AR, Antonny B, Wang J, Delacotte J, Wilfling F, Walther TC, Beck R, Rothman JE, Pincet F,
Journal
PubMed ID23901109
'Intracellular trafficking between organelles is achieved by coat protein complexes, coat protomers, that bud vesicles from bilayer membranes. Lipid droplets are protected by a monolayer and thus seem unsuitable targets for coatomers. Unexpectedly, coat protein complex I (COPI) is required for lipid droplet targeting of some proteins, suggesting a possible ... More
Arf1/COPI machinery acts directly on lipid droplets and enables their connection to the ER for protein targeting.
Lipid droplets (LDs) are ubiquitous organelles that store neutral lipids, such as triacylglycerol (TG), as reservoirs of metabolic energy and membrane precursors. The Arf1/COPI protein machinery, known for its role in vesicle trafficking, regulates LD morphology, targeting of specific proteins to LDs and lipolysis through unclear mechanisms. Recent evidence shows ... More
Targeted therapy of spontaneous murine pancreatic tumors by polymeric micelles prolongs survival and prevents peritoneal metastasis.
AuthorsCabral H, Murakami M, Hojo H, Terada Y, Kano MR, Chung UI, Nishiyama N, Kataoka K,
Journal
PubMed ID23801758
Nanoscaled drug-loaded carriers are of particular interest for efficient tumor therapy as numerous studies have shown improved targeting and efficacy. Nevertheless, most of these studies have been performed against allograft and xenograft tumor models, which have altered microenvironment features affecting the accumulation and penetration of nanocarriers. Conversely, the evaluation of ... More
ATP-independent diffusion of double-stranded RNA binding proteins.
The proteins harboring double-stranded RNA binding domains (dsRBDs) play diverse functional roles such as RNA localization, splicing, editing, export, and translation, yet mechanistic basis and functional significance of dsRBDs remain unclear. To unravel this enigma, we investigated transactivation response RNA binding protein (TRBP) consisting of three dsRBDs, which functions in ... More
Binding of human nucleotide exchange factors to heat shock protein 70 (Hsp70) generates functionally distinct complexes in vitro.
AuthorsRauch JN, Gestwicki JE,
Journal
PubMed ID24318877
Proteins with Bcl2-associated anthanogene (BAG) domains act as nucleotide exchange factors (NEFs) for the molecular chaperone heat shock protein 70 (Hsp70). There are six BAG family NEFs in humans, and each is thought to link Hsp70 to a distinct cellular pathway. However, little is known about how the NEFs compete ... More
A generic cell surface ligand system for studying cell-cell recognition.
AuthorsDenham EM, Barton MI, Black SM, Bridge MJ, de Wet B, Paterson RL, van der Merwe PA, Goyette J
JournalPLoS Biol
PubMed ID31815943
'Dose-response experiments are a mainstay of receptor biology studies and can reveal valuable insights into receptor function. Such studies of receptors that bind cell surface ligands are currently limited by the difficulty in manipulating the surface density of ligands at a cell-cell interface. Here, we describe a generic cell surface ... More
Complex microparticle architectures from stimuli-responsive intrinsically disordered proteins.
AuthorsRoberts S, Miao V, Costa S, Simon J, Kelly G, Shah T, Zauscher S, Chilkoti A
JournalNat Commun
PubMed ID32165622
'The controllable production of microparticles with complex geometries is useful for a variety of applications in materials science and bioengineering. The formation of intricate microarchitectures typically requires sophisticated fabrication techniques such as flow lithography or multiple-emulsion microfluidics. By harnessing the molecular interactions of a set of artificial intrinsically disordered proteins ... More
Single molecule characterization of individual extracellular vesicles from pancreatic cancer.
AuthorsLennon KM, Wakefield DL, Maddox AL, Brehove MS, Willner AN, Garcia-Mansfield K, Meechoovet B, Reiman R, Hutchins E, Miller MM, Goel A, Pirrotte P, Van Keuren-Jensen K, Jovanovic-Talisman T
JournalJ Extracell Vesicles
PubMed ID31741725
'Biofluid-accessible extracellular vesicles (EVs) may represent a new means to improve the sensitivity and specificity of detecting disease. However, current methods to isolate EVs encounter challenges when they are used to select specific populations. Moreover, it has been difficult to comprehensively characterize heterogeneous EV populations at the single vesicle level. ... More
Quantification of fluorophore distribution and therapeutic response in matched in vivo and ex vivo pancreatic cancer model systems.
AuthorsSolanki A, King D, Thibault G, Wang L, Gibbs SL
JournalPLoS One
PubMed ID32097436
'Therapeutic resistance plagues cancer outcomes, challenging treatment particularly in aggressive disease. A unique method to decipher drug interactions with their targets and inform therapy is to employ fluorescence-based screening tools; however, to implement productive screening assays, adequate model systems must be developed. Patient-derived pancreatic cancer models (e.g., cell culture, patient-derived ... More
Golden Gate assembly with a bi-directional promoter (GBid): A simple, scalable method for phage display Fab library creation.
AuthorsChockalingam K, Peng Z, Vuong CN, Berghman LR, Chen Z
JournalSci Rep
PubMed ID32076016
'Fabs offer an attractive platform for monoclonal antibody discovery/engineering, but library construction can be cumbersome. We report a simple method - Golden Gate assembly with a bi-directional promoter (GBid) - for constructing phage display Fab libraries. In GBid, the constant domains of the Fabs are located in the backbone of ... More
Targeting CD38-Expressing Multiple Myeloma and Burkitt Lymphoma Cells In Vitro with Nanobody-Based Chimeric Antigen Receptors (Nb-CARs).
AuthorsHambach J, Riecken K, Cichutek S, Schütze K, Albrecht B, Petry K, Röckendorf JL, Baum N, Kröger N, Hansen T, Schuch G, Haag F, Adam G, Fehse B, Bannas P, Koch-Nolte F
JournalCells
PubMed ID32013131
'The NAD-hydrolyzing ecto-enzyme CD38 is overexpressed by multiple myeloma and other hematological malignancies. We recently generated CD38-specific nanobodies, single immunoglobulin variable domains derived from heavy-chain antibodies naturally occurring in llamas. Nanobodies exhibit high solubility and stability, allowing easy reformatting into recombinant fusion proteins. Here we explore the utility of CD38-specific ... More
Analysis of a Therapeutic Antibody Cocktail Reveals Determinants for Cooperative and Broad Ebolavirus Neutralization.
AuthorsGilchuk P, Murin CD, Milligan JC, Cross RW, Mire CE, Ilinykh PA, Huang K, Kuzmina N, Altman PX, Hui S, Gunn BM, Bryan AL, Davidson E, Doranz BJ, Turner HL, Alkutkar T, Flinko R, Orlandi C, Carnahan R, Nargi R, Bombardi RG, Vodzak ME, Li S, Okoli A, Ibeawuchi M, Ohiaeri B, Lewis GK, Alter G, Bukreyev A, Saphire EO, Geisbert TW, Ward AB, Crowe JE
JournalImmunity
PubMed ID32023489
Structural principles underlying the composition of protective antiviral monoclonal antibody (mAb) cocktails are poorly defined. Here, we exploited antibody cooperativity to develop a therapeutic mAb cocktail against Ebola virus. We systematically analyzed the antibody repertoire in human survivors and identified a pair of potently neutralizing mAbs that cooperatively bound to ... More
Whole-cell imaging of plasma membrane receptors by 3D lattice light-sheet dSTORM.
AuthorsWäldchen F, Schlegel J, Götz R, Luciano M, Schnermann M, Doose S, Sauer M
JournalNat Commun
PubMed ID32060305
The molecular organization of receptors in the plasma membrane of cells is paramount for their functionality. We combined lattice light-sheet (LLS) microscopy with three-dimensional (3D) single-molecule localization microscopy (dSTORM) and single-particle tracking to quantify the expression and distribution, and mobility of CD56 receptors on whole fixed and living cells, finding ... More
Programming bulk enzyme heterojunctions for biosensor development with tetrahedral DNA framework.
AuthorsSong P, Shen J, Ye D, Dong B, Wang F, Pei H, Wang J, Shi J, Wang L, Xue W, Huang Y, Huang G, Zuo X, Fan C
JournalNat Commun
PubMed ID32047166
Protein-protein interactions are spatially regulated in living cells to realize high reaction efficiency, as seen in naturally existing electron-transfer chains. Nevertheless, arrangement of chemical/biochemical components at the artificial device interfaces does not possess the same level of control. Here we report a tetrahedral DNA framework-enabled bulk enzyme heterojunction (BEH) strategy ... More