A robust pipeline for rapid production of versatile nanobody repertoires

Fridy, Peter C.; Li, Yinyin; Keegan, Sarah; Thompson, Matthew; Nudelman, Ilona; Scheid, Johannes F.; Oeffinger, Marlene; Nussenzweig, Michel C.; Fenyö, David; Chait, Brian T.; Rout, Michael P.

doi:10.1038/nmeth.3170

Cited by 414 publications

(488 citation statements)

References 61 publications

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“…To target Num1 to eisosomes, we used a GFP-αGFP nanobody targeting system (Figure 1(a)). The αGFP nanobody is a ~ 16 kD, monomeric, single domain antibody that binds GFP with high affinity [22,23]. We engineered cells to express Pil1 as an αGFP nanobody fusion (Pil1-αGFP) from the endogenous PIL1 locus.…”

Section: Resultsmentioning

confidence: 99%

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The role of mitochondria in anchoring dynein to the cell cortex extends beyond clustering the anchor protein

et al. 2018

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Organelle distribution is regulated over the course of the cell cycle to ensure that each of the cells produced at the completion of division inherits a full complement of organelles. In yeast, the protein Num1 functions in the positioning and inheritance of two essential organelles, mitochondria and the nucleus. Specifically, Num1 anchors mitochondria as well as dynein to the cell cortex, and this anchoring activity is required for proper mitochondrial distribution and dynein-mediated nuclear inheritance. The assembly of Num1 into clusters at the plasma membrane is critical for both of its anchoring functions. We have previously shown that mitochondria drive the assembly of Num1 clusters and that these mitochondria-assembled Num1 clusters serve as cortical attachment sites for dynein. Here we further examine the role for mitochondria in dynein anchoring. Using a GFP-αGFP nanobody targeting system, we synthetically clustered Num1 on eisosomes to bypass the requirement for mitochondria in Num1 cluster formation. Utilizing this system, we found that mitochondria positively impact the ability of synthetically clustered Num1 to anchor dynein and support dynein function even when mitochondria are no longer required for cluster formation. Thus, the role of mitochondria in regulating dynein function extends beyond simply concentrating Num1; mitochondria likely promote an arrangement of Num1 within a cluster that is competent for dynein anchoring. This functional dependency between mitochondrial and nuclear positioning pathways likely serves as a mechanism to order and integrate major cellular organization systems over the course of the cell cycle.

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Section: Resultsmentioning

confidence: 99%

“…Ruby-Tub1::HYG was digested with XbaI prior to transformation into yeast. pET21-pelB-LaG16 was a gift from J. Brickner, Northwestern University, and was originally obtained from M. Rout, The Rockefeller University [23]. LaG16 stands for llama antibody (nanobody) against GFP and will be referred to as αGFP.…”

Section: Methodsmentioning

confidence: 99%

The role of mitochondria in anchoring dynein to the cell cortex extends beyond clustering the anchor protein

et al. 2018

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“…One of the most widespread methods to reveal protein-protein interactions is co-immunoprecipitation. The usage of nanobodies for such analyses has been recently shown for ectopically expressed proteins including GFP-or mCherry-fusion constructs (35,69,70). However, the analysis of interaction partners of endogenous ␤-catenin is more challenging, because the accessibility of binding epitopes might be sterically blocked because of its participation in multiprotein complexes in various subcellular compartments.…”

Section: Discussionmentioning

confidence: 99%

Monitoring Interactions and Dynamics of Endogenous Beta-catenin With Intracellular Nanobodies in Living Cells*

Traenkle

Emele

Anton

et al. 2015

Molecular & Cellular Proteomics

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“…An expression vector for GST-anti-mCherry nanobody was constructed as follows. A DNA fragment encoding anti-mCherry nanobody (LaM-4), synthesized based on its amino acid sequence (20), was subcloned into pGEX-6P-1 (GE Healthcare). We deposited the plasmid encoding GST-anti-mCherry nanobody in Addgene (ID 70696).…”

Section: Methodsmentioning

confidence: 99%

Overall Architecture of the Intraflagellar Transport (IFT)-B Complex Containing Cluap1/IFT38 as an Essential Component of the IFT-B Peripheral Subcomplex

Katoh

Terada

Nishijima

et al. 2016

Journal of Biological Chemistry

112

209

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Intraflagellar transport (IFT) is essential for assembly and maintenance of cilia and flagella as well as ciliary motility and signaling. IFT is mediated by multisubunit complexes, including IFT-A, IFT-B, and the BBSome, in concert with kinesin and dynein motors. Under high salt conditions, purified IFT-B complex dissociates into a core subcomplex composed of at least nine subunits and at least five peripherally associated proteins. Using the visible immunoprecipitation assay, which we recently developed as a convenient protein-protein interaction assay, we determined the overall architecture of the IFT-B complex, which can be divided into core and peripheral subcomplexes composed of 10 and 6 subunits, respectively. In particular, we identified TTC26/IFT56 and Cluap1/IFT38, neither of which was included with certainty in previous models of the IFT-B complex, as integral components of the core and peripheral subcomplexes, respectively. Consistent with this, a ciliogenesis defect of Cluap1-deficient mouse embryonic fibroblasts was rescued by exogenous expression of wild-type Cluap1 but not by mutant Cluap1 lacking the binding ability to other IFT-B components. The detailed interaction map as well as comparison of subcellular localization of IFT-B components between wildtype and Cluap1-deficient cells provides insights into the functional relevance of the architecture of the IFT-B complex.Cilia and flagella are microtubule-based appendages on the surfaces of a wide variety of eukaryotic cells. Their assembly and maintenance by intraflagellar transport (IFT) 3 were revealed in Chlamydomonas reinhardtii by the pioneering studies of Rosenbaum and colleagues (1). Subsequently, due to the critical roles for cilia and flagella in various physiological and developmental processes, including cell motility, signaling, and sensory reception, these structures have been studied intensively in metazoans (2-4). IFT, which moves various proteins bidirectionally between the base and tip of cilia/flagella along a microtubule-based structure called the axoneme, is mediated by the large IFT particles with the aid of the anterograde molecular motor kinesin and the retrograde motor dynein. Under high salt conditions, the IFT particle purified from Chlamydomonas flagella can be divided into two complexes, IFT-A and IFT-B. These complexes are composed of ϳ6 and ϳ14 subunits, respectively, and are thought to connect cargo proteins with molecular motors (4, 5). Mutational analyses in Chlamydomonas and other ciliated organisms suggested that the IFT-A and IFT-B complexes are primarily involved in retrograde and anterograde ciliary trafficking, respectively. Biochemical studies revealed the approximate architecture of the Chlamydomonas IFT-A and IFT-B complexes (6 -12), and subsequent studies by Lorentzen and colleagues (13-15) revealed the structural basis of the interactions among several IFT-B subunits. The Chlamydomonas IFT-B complex consists of the core subcomplex, including at least nine subunits (IFT88, and at least five peripherally ...

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A robust pipeline for rapid production of versatile nanobody repertoires

Cited by 414 publications

References 61 publications

The role of mitochondria in anchoring dynein to the cell cortex extends beyond clustering the anchor protein

The role of mitochondria in anchoring dynein to the cell cortex extends beyond clustering the anchor protein

Monitoring Interactions and Dynamics of Endogenous Beta-catenin With Intracellular Nanobodies in Living Cells*

Overall Architecture of the Intraflagellar Transport (IFT)-B Complex Containing Cluap1/IFT38 as an Essential Component of the IFT-B Peripheral Subcomplex

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