In vitro selection of antibodies allows to obtain highly functional binders, rapidly and at lower cost. Here, we describe the first fully synthetic phage display library of humanized llama single domain antibody (NaLi-H1: Nanobody Library Humanized 1). Based on a humanized synthetic single domain antibody (hs2dAb) scaffold optimized for intracellular stability, the highly diverse library provides high affinity binders without animal immunization. NaLi-H1 was screened following several selection schemes against various targets (Fluorescent proteins, actin, tubulin, p53, HP1). Conformation antibodies against active RHO GTPase were also obtained. Selected hs2dAb were used in various immunoassays and were often found to be functional intrabodies, enabling tracking or inhibition of endogenous targets. Functionalization of intrabodies allowed specific protein knockdown in living cells. Finally, direct selection against the surface of tumor cells produced hs2dAb directed against tumor-specific antigens further highlighting the potential use of this library for therapeutic applications.DOI: http://dx.doi.org/10.7554/eLife.16228.001

Microtubules display dynamic instability, with alternating phases of growth and shrinkage separated by catastrophe and rescue events. The guanosine triphosphate (GTP) cap at the growing end of microtubules, whose presence is essential to prevent microtubule catastrophes in vitro, has been difficult to observe in vivo. We selected a recombinant antibody that specifically recognizes GTP-bound tubulin in microtubules and found that GTP-tubulin was indeed present at the plus end of growing microtubules. Unexpectedly, GTP-tubulin remnants were also present in older parts of microtubules, which suggests that GTP hydrolysis is sometimes incomplete during polymerization. Observations in living cells suggested that these GTP remnants may be responsible for the rescue events in which microtubules recover from catastrophe.

Microtubules are hollow biopolymers of 25-nm diameter and are key constituents of the cytoskeleton. In neurons, microtubules are organized differently between axons and dendrites, but their precise organization in different compartments is not completely understood. Super-resolution microscopy techniques can detect specific structures at an increased resolution, but the narrow spacing between neuronal microtubules poses challenges because most existing labelling strategies increase the effective microtubule diameter by 20–40 nm and will thereby blend neighbouring microtubules into one structure. Here we develop single-chain antibody fragments (nanobodies) against tubulin to achieve super-resolution imaging of microtubules with a decreased apparent diameter. To test the resolving power of these novel probes, we generate microtubule bundles with a known spacing of 50–70 nm and successfully resolve individual microtubules. Individual bundled microtubules can also be resolved in different mammalian cells, including hippocampal neurons, allowing novel insights into fundamental mechanisms of microtubule organization in cell- and neurobiology.

We analyzed herein whether members of the tetraspanin superfamily are involved in human immature dendritic cell (DC) functions such as foreign antigen internalization, phagocytosis, and cell migration. We show that CD63, CD9, CD81, CD82, and CD151 are present in immature DCs. Whereas CD9 and CD81 are mostly expressed at the cell surface, CD63 and CD82 are also located in intracellular organelles. Complexes of monoclonal antibody (Mab) FC-5.01-CD63 or Fab-5.01-CD63 were rapidly translocated "outside-in" and followed the endocytic pathway through early endosomes and lysosomes, reaching major histocompatibility complex (MHC) class IIenriched compartments (MIICs) in less than one hour. Internalization of CD63 was also observed during Saccharomyces cerevisiae phagocytosis. Moreover, an association of CD63 with the ␤-glycan receptor dectin-1 was observed. Mabs against CD9, CD63, CD81, and CD82 enhanced by 50% the migration induced by the chemokines macrophage inflammatory protein-5 (MIP- 5 IntroductionDendritic cells (DCs) comprise a family of professional antigenpresenting cells (APCs) that are sentinels of the immune system. 1 In this regard, DCs have been shown to efficiently stimulate both naive B and T cells and to elicit primary immune responses. 2,3 Their remarkable effectiveness is due to their ability to capture, process and present antigens along with costimulatory signals, and to migrate to secondary lymphoid tissues. 4 Different stages of maturation are responsible for the different functions of DCs. Immature DCs, widely present in peripheral tissues, efficiently uptake antigens but express moderate levels of major histocompatibility complex (MHC) class II and costimulatory molecules. In contrast, mature DCs poorly acquire antigens but express higher levels of MHC class II and costimulatory molecules, are able to migrate into lymph nodes, and become potent activators of resting T cells. [2][3][4][5] Since antigen internalization and processing, as well as cell migration, are essential properties of DCs, the study of the possible involvement of tetraspanins in these processes is a challenging question, as it could bring new insight into the physiologic role of these molecules in DCs and other cells. In recent years, considerable interest has arisen in the expanding tetraspanins family, which are integral membrane proteins with 2 extracellular domains (EC1 and EC2) that are variably glycosylated. 6 The most conspicuous members of this family are CD9, 7 CD63/lamp-3, 8 CD81/TAPA-1, 9 CD82/KAI1, 10 and CD151. 11 Tetraspanins form several specific complexes implicated in a variety of cellular processes such as migration, adhesion, proliferation, and signal transduction, initially leading to the idea that these proteins could play a "molecular facilitator" role. 6 There is growing evidence that points to a possible role of tetraspanins in antigen processing and presentation. In fact, it has been recently found that CD63 is modified after translation during maturation of DCs, and this event is accompanied by morph...

Mast cells are cellular actors of WAT inflammation and possibly fibrotic state found in obesity and diabetes. Whether mast cells could be involved in the pathophysiology of diabetes needs additional study as well as the positioning of these cells in driving pathological alterations of WAT in these chronic metabolic diseases.

Generation of specific antibodies against enriched subcellular fractions is a powerful strategy to identify and characterize cellular components. We show that recombinant antibodies can be selected in vitro by phage display against complex subcellular fractions, namely microtubule-binding proteins and Golgi stacks. This technique has allowed us to overcome many limitations of the classical animal-based approach and generate cell biology-compliant antibodies. In addition, we show that intracellular expression of GFP-tagged recombinant antibodies can reveal the dynamics of endogenous proteins in vivo. Endogenous Giantin is very static and outlines the Golgi in living cells. It accumulates neither onto Golgi-derived tubules upon Brefeldin A treatment before Golgi disappearance, nor onto de novo formed Golgi mini-stacks upon microtubule depolymerization, and remains instead on the 'old' pericentriolar Golgi. This suggests that, in contrast to other Golgi matrix proteins, endogenous Giantin is very stably associated with the Golgi and does not efficiently recycle to the ER. Altogether, we show that the antibody phage display technique represents an efficient alternative to rapidly generate versatile antibodies that represent new tools to study protein function.

Here we report an approach, based on antibody phage display, to generate molecular conformation sensors. Recombinant antibodies specific to the guanosine triphosphate (GTP)-bound conformation of the small guanosine triphosphatase (GTPase) Rab6, a regulator of membrane traffic, were generated and used to locate Rab6.GTP in fixed cells, and, after green fluorescent protein (GFP) tagging and intracellular expression, to follow Rab6.GTP in vivo. Rab6 was in its GTP-bound conformation on the Golgi apparatus and transport intermediates, and the geometry of transport intermediates was modulated by Rab6 activity. More generally, the same approach could be applied to other molecules that can be locked in a particular conformation in vitro.

Matched for FM, PWS subjects have a better metabolic profile, a phenotype that could be linked to changes in scAT remodeling and promotion of adipocyte growth.