Probing cellular protein complexes using single-molecule pull-down

Jain, Ankur; Liu, Ruijie; Ramani, Biswarathan; Arauz, Edwin; Ishitsuka, Yuji; Ragunathan, Kaushik; Park, Jeehae; Chen, Jiawen; Xiang, Yang; Ha, Taekjip

doi:10.1038/nature10016

Cited by 376 publications

(519 citation statements)

References 42 publications

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“…50 We use this method to determine the potential of DD and RHIM domain in forming dimers or polymers. We expressed Flag-RFP-tagged DD or RHIM domain of RIP1 in 293T cells and prepared a flow chamber covered with anti-Flag antibody.…”

Section: Resultsmentioning

confidence: 99%

“…We expressed Flag-RFP-tagged DD or RHIM domain of RIP1 in 293T cells and prepared a flow chamber covered with anti-Flag antibody. 50 The immunoprecipitated complexes of DD and RHIM domain were visualized through RFP (Figures 1c and d). Applying a defined continuous laser power to these complexes could induce stepwise loss of fluorescence, 50 thereby obtaining stoichiometric information.…”

Section: Resultsmentioning

confidence: 99%

“…50 The immunoprecipitated complexes of DD and RHIM domain were visualized through RFP (Figures 1c and d). Applying a defined continuous laser power to these complexes could induce stepwise loss of fluorescence, 50 thereby obtaining stoichiometric information. The distribution of photobleaching steps of DD was binomial (Figures 1e and f), suggesting that DD of RIP1 prefers to form dimer.…”

Section: Resultsmentioning

confidence: 99%

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Distinct roles of RIP1–RIP3 hetero- and RIP3–RIP3 homo-interaction in mediating necroptosis

et al. 2014

Cell Death Differ

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Necroptosis is mediated by a signaling complex called necrosome, containing receptor-interacting protein (RIP)1, RIP3, and mixed-lineage kinase domain-like (MLKL). It is known that RIP1 and RIP3 form heterodimeric filamentous scaffold in necrosomes through their RIP homotypic interaction motif (RHIM) domain-mediated oligomerization, but the signaling events based on this scaffold has not been fully addressed. By using inducible dimer systems we found that RIP1-RIP1 interaction is dispensable for necroptosis; RIP1-RIP3 interaction is required for necroptosis signaling, but there is no necroptosis if no additional RIP3 protein is recruited to the RIP1-RIP3 heterodimer, and the interaction with RIP1 promotes the RIP3 to recruit other RIP3; RIP3-RIP3 interaction is required for necroptosis and RIP3-RIP3 dimerization is sufficient to induce necroptosis; and RIP3 dimer-induced necroptosis requires MLKL. We further show that RIP3 oligomer is not more potent than RIP3 dimer in triggering necroptosis, suggesting that RIP3 homo-interaction in the complex, rather than whether RIP3 has formed homo polymer, is important for necroptosis. RIP3 dimerization leads to RIP3 intramolecule autophosphorylation, which is required for the recruitment of MLKL. Interestingly, phosphorylation of one of RIP3 in the dimer is sufficient to induce necroptosis. As RIP1-RIP3 heterodimer itself cannot induce necroptosis, the RIP1-RIP3 heterodimeric amyloid fibril is unlikely to directly propagate necroptosis. We propose that the signaling events after the RIP1-RIP3 amyloid complex assembly are the recruitment of free RIP3 by the RIP3 in the amyloid scaffold followed by autophosphorylation of RIP3 and subsequent recruitment of MLKL by RIP3 to execute necroptosis. Cell Death and Differentiation (2014) 21, 1709-1720; doi:10.1038/cdd.2014.77; published online 6 June 2014Necroptosis is a type of programmed necrosis characterized by necrotic morphological changes, including cellular organelle swelling, cell membrane rupture, 1-3 and dependence of receptor-interacting protein (RIP)1 4 and RIP3. [5][6][7] Physiological function of necroptosis has been illustrated in host defense, [8][9][10][11] inflammation, 12-16 tissue injury, 10,17,18 and development. [19][20][21] Necroptosis can be induced by a number of different extracellular stimuli such as tumor necrosis factor (TNF). TNF stimulation leads to formation of TNF receptor 1 (TNFR1) signaling complex (named complex I), and complex II containing RIP1, TRADD, FAS-associated protein with a death domain (FADD), and caspase-8, of which the activation initiates apoptosis. If cells have high level of RIP3, RIP1 recruits RIP3 to form necrosome containing FADD, [22][23][24] caspase-8, RIP1, and RIP3, and the cells undergo necroptosis. 25,26 Caspase-8 and FADD negatively regulates necroptosis, 27-30 because RIP1, RIP3, and CYLD are potential substrates of caspase-8. [31][32][33][34] Necrosome also suppresses apoptosis but the underlying mechanism has not been described yet. Mixed-lineage kinase domain-like (ML...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Distinct roles of RIP1–RIP3 hetero- and RIP3–RIP3 homo-interaction in mediating necroptosis

et al. 2014

Cell Death Differ

250

214

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“…Single-molecule fluorescence imaging is carried out primarily with total internal reflection, confocal, and zero-mode waveguide microscopy [2]. Among these, total internal reflection fluorescence microscopy has been employed by several research teams in the development of novel techniques described in this review [14][15][16][17] …”

mentioning

confidence: 99%

“…The stoichiometry of the catalytic subunits could be confirmed by counting the number of YFP molecules per complex. The researchers have also reported proof-of-principle studies using systems such as mTOR (mammalian target of rapamycin) protein complexes and membrane receptor proteins [17]. The same group has extended its research to stoichiometry analyses of AKAPs (A-kinase anchoring proteins) [48], ORCA (origin-recognition complex-associated) protein complexes [49], and peptide loading complexes [50].…”

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Bringing single-molecule spectroscopy to macromolecular protein complexes

Joo

Fareh

Kim

2013

Trends in Biochemical Sciences

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Single-molecule fluorescence spectroscopy offers real-time, nanometer-resolution information. Over the past two decades, this emerging single-molecule technique has been rapidly adopted to investigate the structural dynamics and biological functions of proteins. Despite this remarkable achievement, single-molecule fluorescence techniques must be extended to macromolecular protein complexes that are physiologically more relevant for functional studies. In this review, we present recent major breakthroughs for investigating protein complexes within cell extracts using single-molecule fluorescence. We outline the challenges, future prospects and potential applications of these new single-molecule fluorescence techniques in biological and clinical research. Single-molecule protein studiesSingle-molecule techniques have become potent tools for the discovery of novel protein mechanisms by allowing high spatiotemporal resolution. Sub-nanometer resolution, the ultimate scale of biological systems, has been reached with single-molecule fluorescence microscopy[1] and spectroscopy [2]; single-molecule force and torque spectroscopy [3,4]; atomic force microscopy [5] and spectroscopy [6]; and nanopores [7]. Measurements can be carried out on the biologically relevant time scales of microseconds to minutes.Among these techniques, single-molecule fluorescence spectroscopy has enabled researchers to unveil the action mechanism of a protein by imaging protein activity in real time. Benefitting from advances in general microscopy[1], detection devices [8], and fluorophore physics [9,10], single-molecule fluorescence spectroscopy has reached sub-nanometer spatial resolution [11] and microsecond temporal resolution [12,13]. Single-molecule fluorescence imaging is carried out primarily with total internal reflection, confocal, and zero-mode waveguide microscopy [2]. Among these, total internal reflection fluorescence microscopy has been employed by several research teams in the development of novel techniques described in this review [14][15][16][17] Single-molecule observations using cell extractsUnlike purified recombinant proteins, crude cell extracts provide more biologically relevant environment in which molecules of interest can interact not only with their partners but also with other cellular components. It was anticipated to combine this approach with single molecule techniques and observe the assembly and function of macromolecular complexes in real time. However, technical hurdles related to the specificity and efficiency of labeling molecules of interest within crude cell extracts had been reported [22][23][24]. In order to overcome this limitation, several groups recently developed original approaches. Hoskins et al. used protein complexes specifically tagged with fluorophores [14,19], and Yardimci et al. immunostained reaction products using specific fluorescent antibodies [15,20]. Real-time observation of spliceosome assemblyDespite the relatively small number of genes in the human genome, we have extraordin...

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Analysis of Individual Signaling Complexes by mMAPS, a Flow‐Proteometric System

Chou

Tsou

Hsu

et al. 2016

CP Molecular Biology

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Signal transduction is essential for maintaining cells’ normal physiological functions, and deregulation of signaling can lead to diseases such as diabetes and cancers. Some of the major players in signal delivery are molecular complexes composed of proteins and nucleic acids. This unit describes a technique called microchannel for Multi-parameter Analysis of Proteins in a Single-complex or mMAPS for analyzing and quantifying target signaling complexes individually. mMAPS is a flow-proteometric-based system that allows detection of individual proteins or complexes flowing through a microfluidic channel. Specific target proteins and nucleic acids labeled by fluorescent tags are harvested from tissue samples or cultured cells for analysis by the mMAPS system. Overall, mMAPS enables both detection of multiple components within a single complex and direct quantification of different populations of molecular complexes in one setting in a short timeframe, requiring very low sample input.

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Probing cellular protein complexes using single-molecule pull-down

Cited by 376 publications

References 42 publications

Distinct roles of RIP1–RIP3 hetero- and RIP3–RIP3 homo-interaction in mediating necroptosis

Distinct roles of RIP1–RIP3 hetero- and RIP3–RIP3 homo-interaction in mediating necroptosis

Bringing single-molecule spectroscopy to macromolecular protein complexes

Analysis of Individual Signaling Complexes by mMAPS, a Flow‐Proteometric System

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