2-color photobleaching experiments reveal distinct intracellular dynamics of two components of the Hsp90 complex

Picard, Didier; Suslova, Elena; Briand, Pierre-André

doi:10.1016/j.yexcr.2006.08.026

Cited by 29 publications

(18 citation statements)

References 47 publications

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“…The coding sequences for human AARSD1 were obtained from the sequence reported under GenBank accession number BC004172.2. All AARSD1 sequences and p23 were expressed from the mammalian expression vector pcDNA/HA, with a previously reported alternate N-terminal hemagglutinin (HA) tag (30), except for pEGFP-p23 (31). The truncation mutant Aarsd1L⌬C retains the 114 N-terminal amino acids.…”

Section: Methodsmentioning

confidence: 99%

A Remodeled Hsp90 Molecular Chaperone Ensemble with the Novel Cochaperone Aarsd1 Is Required for Muscle Differentiation

Echeverria

Briand

Picard

2016

Molecular and Cellular Biology

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Hsp90 is the ATP-consuming core component of a very abundant molecular chaperone machine that handles a substantial portion of the cytosolic proteome. Rather than one machine, it is in fact an ensemble of molecular machines, since most mammalian cells express two cytosolic isoforms of Hsp90 and a subset of up to 40 to 50 cochaperones and regulate their interactions and functions by a variety of posttranslational modifications. We demonstrate that the Hsp90 ensemble is fundamentally remodeled during muscle differentiation and that this remodeling is not just a consequence of muscle differentiation but possibly one of the drivers to accompany and to match the vast proteomic changes associated with this process. As myoblasts differentiate into myotubes, Hsp90␣ disappears and only Hsp90␤ remains, which is the only isoform capable of interacting with the novel musclespecific Hsp90 cochaperone Aarsd1L. Artificially maintaining Hsp90␣ or knocking down Aarsd1L expression interferes with the differentiation of C2C12 myotubes. During muscle differentiation, Aarsd1L replaces the more ubiquitous cochaperone p23 and in doing so dampens the activity of the glucocorticoid receptor, one of the Hsp90 clients relevant to muscle functions. This cochaperone switch protects muscle cells against the inhibitory effects of glucocorticoids and may contribute to preventing muscle wasting induced by excess glucocorticoids. H sp90 is a highly abundant molecular chaperone, including in nonstressed cells, which is required for the maturation, stability, activity, and/or degradation of a substantial portion of the proteome (1-5) (for a regularly updated list of Hsp90 interactors, see http://www.picard.ch/downloads/Hsp90interactors.pdf). To its clients, the Hsp90 dimer offers a moving platform whose complex choreography involves ATP hydrolysis, huge conformational changes, and dynamic interactions with a large cohort of cochaperones (3, 6-11). The cochaperones regulate and are part of the Hsp90 cycle (3,(8)(9)(10)(11), and some may act as client specificity factors (12, 13). With 40 to 50 cochaperones, far more have been reported (see http://www.picard.ch/downloads/Hsp90facts.pdf) than the ones that have been extensively characterized as interacting with Hsp90 as part of the core cycle. Moreover, Hsp90 and cochaperones are extensively regulated by posttranslational modifications (see, for example, references 14-17), and Hsp90 is present in excess over any individual cochaperone. Hence, the Hsp90 molecular chaperone machine constitutes in fact an ensemble of complexes. These complexes dynamically interchange and are fashioned by the cell specificity of signaling and cochaperone expression patterns.One of the most extensively characterized components of the Hsp90 core machinery is the cochaperone p23 (18-20), encoded by the PTGES3 gene in mammals. This small acidic protein binds and stabilizes the ATP-bound state of Hsp90 (19-23). p23 binding, which is disrupted by Hsp90 inhibitors competing with ATP and by hyperacetylation of Hsp90, promotes a ...

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Section: Methodsmentioning

confidence: 99%

A Remodeled Hsp90 Molecular Chaperone Ensemble with the Novel Cochaperone Aarsd1 Is Required for Muscle Differentiation

Echeverria

Briand

Picard

2016

Molecular and Cellular Biology

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“…The mobile fraction (F m ) was calculated from mean recovery curves according to the equation (11,12,15,43):…”

Section: Methodsmentioning

confidence: 99%

“…Next, FP mobility is measured using FCS (fluorescence correlation spectroscopy) or FRAP (fluorescence recovery after photobleaching). This is then followed by curve fitting and/ or mathematical modeling of the experimental data to obtain the diffusion constant of the FP (7)(8)(9)(10)(11)(12)(13)(14)(15)(16). However, these analysis methods generally do not include realistic (i.e., experimentally determined) information concerning the spatial dimensions and nanostructure of the compartment.…”

mentioning

confidence: 99%

“…Moreover, the temporal scale of most FRAP models does not quantitatively match with that of FRAP experiments. Therefore it was already recognized some time ago (8)(9)(10)(11)(12)(13)(14)(15)(16)(17) that the above approaches will only yield an "apparent" (biased) value for the diffusion constant (D app ) of a given FP, which represents an underestimation of the "real" (i.e., purely solvent-dependent) diffusion constant (D solvent ).…”

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Solute diffusion is hindered in the mitochondrial matrix

Dieteren

Gielen

Nijtmans

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Intracellular chemical reactions generally constitute reaction-diffusion systems located inside nanostructured compartments like the cytosol, nucleus, endoplasmic reticulum, Golgi, and mitochondrion. Understanding the properties of such systems requires quantitative information about solute diffusion. Here we present a novel approach that allows determination of the solvent-dependent solute diffusion constant (D solvent ) inside cell compartments with an experimentally quantifiable nanostructure. In essence, our method consists of the matching of synthetic fluorescence recovery after photobleaching (FRAP) curves, generated by a mathematical model with a realistic nanostructure, and experimental FRAP data. As a proof of principle, we assessed D solvent of a monomeric fluorescent protein (AcGFP1) and its tandem fusion (AcGFP1 2 ) in the mitochondrial matrix of HEK293 cells. Our results demonstrate that diffusion of both proteins is substantially slowed by barriers in the mitochondrial matrix (cristae), suggesting that cells can control the dynamics of biochemical reactions in this compartment by modifying its nanostructure. molecular dynamics | quantitative random-walk model | systems biology A major challenge facing biochemistry is to understand the dynamics of chemical reactions within inhomogeneous cell compartments like the cytosol, nucleus, endoplasmic reticulum (ER), Golgi, and mitochondrion (1). In general, intracompartment reactions involve the conversion of (im)mobile substrates by (im)mobile enzymes into (im)mobile products and therefore constitute reaction-diffusion systems. Obviously, gaining insight into the behavior of such systems requires quantitative information about solute diffusion. The latter depends on solvent and solute properties, the dimensions and shape of the compartment, and the internal structure of the compartment (2-6).A widely used strategy to investigate solute diffusion involves expressing a fluorescent tracer protein (FP) in the compartment of interest. Next, FP mobility is measured using FCS (fluorescence correlation spectroscopy) or FRAP (fluorescence recovery after photobleaching). This is then followed by curve fitting and/ or mathematical modeling of the experimental data to obtain the diffusion constant of the FP (7-16). However, these analysis methods generally do not include realistic (i.e., experimentally determined) information concerning the spatial dimensions and nanostructure of the compartment. Moreover, the temporal scale of most FRAP models does not quantitatively match with that of FRAP experiments. Therefore it was already recognized some time ago (8-17) that the above approaches will only yield an "apparent" (biased) value for the diffusion constant (D app ) of a given FP, which represents an underestimation of the "real" (i.e., purely solvent-dependent) diffusion constant (D solvent ).In this study we present a strategy to determine D solvent inside cell compartments with an experimentally accessible nanostructure. Our method consists of matching synthetic FRA...

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“…a model without the 3rd term in Equation 2) (22). The mobile fraction (F m ) was calculated from the averaged recovery curves using Equation 3 (14,16,17),…”

Section: Methodsmentioning

confidence: 99%

Subunits of Mitochondrial Complex I Exist as Part of Matrix- and Membrane-associated Subcomplexes in Living Cells

Dieteren

Willems

Vogel

et al. 2008

Journal of Biological Chemistry

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Mitochondrial complex I (CI) is a large assembly of 45 different subunits, and defects in its biogenesis are the most frequent cause of mitochondrial disorders. In vitro evidence suggests a stepwise assembly process involving pre-assembled modules. However, whether these modules also exist in vivo is as yet unresolved. To answer this question, we here applied submitochondrial fluorescence recovery after photobleaching to HEK293 cells expressing 6 GFP-tagged subunits selected on the basis of current CI assembly models. We established that each subunit was partially present in a virtually immobile fraction, possibly representing the holo-enzyme. Four subunits (NDUFV1, NDUFV2, NDUFA2, and NDUFA12) were also present as highly mobile matrix-soluble monomers, whereas, in sharp contrast, the other two subunits (NDUFB6 and NDUFS3) were additionally present in a slowly mobile fraction. In the case of the integral membrane protein NDUFB6, this fraction most likely represented one or more membrane-bound subassemblies, whereas biochemical evidence suggested that for the NDUFS3 protein this fraction most probably corresponded to a matrix-soluble subassembly. Our results provide first time evidence for the existence of CI subassemblies in mitochondria of living cells.The vast majority of cellular ATP is produced by mitochondrial oxidative phosphorylation (OXPHOS).2 The OXPHOS system consists of five multisubunit complexes of which NADH:ubiquinone oxidoreductase or complex I (CI) is the largest (ϳ1 MDa) and least understood (1). CI liberates electrons from NADH, channels them to ubiquinone, and uses part of their energy to expel protons from the mitochondrial matrix into the intermembrane space. The complex consists of 45 subunits, seven of which are encoded by the mtDNA and the remainder by the nuclear genome (2). Its catalytic core comprises 14 evolutionary conserved subunits (3), which, in humans, are encoded by the nuclear NDUFV1, NDUFV2, NDUFS1, NDUFS2, NDUFS3, NDUFS7, and NDUFS8 genes and the mitochondrial ND1-ND6 and ND4L genes. Once assembled, the complex has an L-shaped structure with one arm protruding into the matrix and the other arm embedded in the inner membrane. The matrix-protruding, or peripheral, arm is involved in electron channeling and contains the flavo-mononucleotide group and eight ironsulfur clusters. The membrane arm includes all of the mtDNA-encoded subunits and is believed to play a role in proton translocation (3).At present, it is still unclear how the different subunits are exactly pieced together during CI biogenesis. Understanding this process is also clinically relevant because mutations in CI subunits prevent proper CI assembly, causing a variety of mitochondrial disorders (reviewed in Ref. 4). The current hypothesis is that CI assembly occurs by a stepwise mechanism during which prefabricated modules, or assembly intermediates, are combined (5-9). In agreement with this idea, we recently demonstrated that inhibition of mitochondrial translation caused an accumulation of the NDUFS3 subunit in...

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2-color photobleaching experiments reveal distinct intracellular dynamics of two components of the Hsp90 complex

Cited by 29 publications

References 47 publications

A Remodeled Hsp90 Molecular Chaperone Ensemble with the Novel Cochaperone Aarsd1 Is Required for Muscle Differentiation

A Remodeled Hsp90 Molecular Chaperone Ensemble with the Novel Cochaperone Aarsd1 Is Required for Muscle Differentiation

Solute diffusion is hindered in the mitochondrial matrix

Subunits of Mitochondrial Complex I Exist as Part of Matrix- and Membrane-associated Subcomplexes in Living Cells

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