Conformational stabilization of optineurin by the dynamic interaction of linear polyubiquitin.

Optineurin produces intracellular multi-functions involving autophagy, vesicular trafficking, and negative regulation of inflammation signaling through interaction with various proteins such as ATG8/LC3, Rab8, and polyubiquitin. Optineurin is a component of cytoplasmic inclusion bodies (IBs) in motor neurons from amyotrophic lateral sclerosis (ALS), and its mutation E478G, has been identified in patients with ALS. However, the mechanism by which polyubiquitin binding modulates the interaction partners of OPTN and ALS-associated IB formation is still unclear. To address this issue, we analyzed the interaction of Optineurin with Rab8 and LC3 in the absence and presence of linear polyubiquitin chains using fluorescence cross-correlation spectroscopy and IB formation efficiency of the E478G mutant of Optineurin during Rab8 depletion using fluorescence microscopy. Here, we hypothesize that linear polyubiquitin binding to Optineurin dynamically induces LC3 association and Rab8 dissociation, likely through a conformational change of Optineurin, and the dynamic conformational change may prevent the aggregate formation of mutant Optineurin.

Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy.

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and so on. To detect and analyze soluble or diffuse protein oligomers or aggregates, fluorescence correlation spectroscopy (FCS), which can detect the diffusion speed and brightness of a single particle with a single molecule sensitivity, has been used. However, the proper procedure and know-how for protein aggregation detection have not been widely shared. Here, we show a standard procedure of FCS measurement for diffusion properties of aggregation-prone proteins in cell lysate and live cells: ALS-associated 25 kDa carboxyl-terminal fragment of TAR DNA/RNA-binding protein 43 kDa (TDP25) and superoxide dismutase 1 (SOD1). The representative results show that a part of aggregates of green fluorescent protein (GFP)-tagged TDP25 was slightly included in the soluble fraction of murine neuroblastoma Neuro2a cell lysate. Moreover, GFP-tagged SOD1 carrying ALS-associated mutation shows a slower diffusion in live cells. Accordingly, we here introduce the procedure to detect the protein aggregation via its diffusion property using FCS.

Molecular basis of functional exchangeability between ezrin and other actin-membrane associated proteins during cytokinesis.

The mechanism that mediates the interaction between the contractile ring and the plasma membrane during cytokinesis remains elusive. We previously found that ERM (Ezrin/Radixin/Moesin) proteins, which usually mediate cellular pole contraction, become over-accumulated at the cell equator and support furrow ingression upon the loss of other actin-membrane associated proteins, anillin and supervillin. In this study, we addressed the molecular basis of the exchangeability between ezrin and other actin-membrane associated proteins in mediating cortical contraction during cytokinesis. We found that depletion of anillin and supervillin caused over-accumulation of the membrane-associated FERM domain and actin-binding C-terminal domain (C-term) of ezrin at the cleavage furrow, respectively. This finding suggests that ezrin differentially shares its binding sites with these proteins on the actin cytoskeleton or inner membrane surface. Using chimeric mutants, we found that ezrin C-term, but not the FERM domain, can substitute for the corresponding anillin domains in cytokinesis and cell proliferation. On the other hand, either the membrane-associated or the actin/myosin-binding domains of anillin could not substitute for the corresponding ezrin domains in controlling cortical blebbing at the cell poles. Our results highlight specific designs of actin- or membrane-associated moieties of different actin-membrane associated proteins with limited exchangeability, which enables them to support diverse cortical activities on the shared actin-membrane interface during cytokinesis.

Functional Fluorescence Microscopy Imaging: Quantitative Scanning-Free Confocal Fluorescence Microscopy for the Characterization of Fast Dynamic Processes in Live Cells.

Functional fluorescence microscopy imaging (fFMI), a time-resolved (21 µs/frame) confocal fluorescence microscopy imaging technique without scanning, is developed for quantitative characterization of fast reaction-transport processes in solution and in live cells. The method is based on massively parallel fluorescence correlation spectroscopy (FCS). Simultaneous excitation of fluorescent molecules in multiple spots in the focal plane is achieved using a diffractive optical element (DOE). Fluorescence from the DOE-generated 1024 illuminated spots is detected in a confocal arrangement by a matching matrix detector comprising 32 × 32 single-photon avalanche photodiodes (SPADs). Software for data acquisition and fast auto- and cross-correlation analysis by parallel signal processing using a graphic processing unit (GPU) allows temporal autocorrelation across all pixels in the image frame in 4 s and cross-correlation between first- and second-order neighbor pixels in 45 s. We present here this quantitative, time-resolved imaging method with single-molecule sensitivity and demonstrate its usefulness for mapping in live cell location-specific differences in the concentration and translational diffusion of molecules in different subcellular compartments. In particular, we show that molecules without a specific biological function, e.g., the enhanced green fluorescent protein (eGFP), exhibit uniform diffusion. In contrast, molecules that perform specialized biological functions and bind specifically to their molecular targets show location-specific differences in their concentration and diffusion, exemplified here for two transcription factor molecules, the glucocorticoid receptor (GR) before and after nuclear translocation and the Sex combs reduced (Scr) transcription factor in the salivary gland of Drosophila ex vivo.

Molecular chaperone HSP70 prevents formation of inclusion bodies of the 25-kDa C-terminal fragment of TDP-43 by preventing aggregate accumulation.

Transactive response DNA/RNA-binding protein 43-kDa (TDP-43) C-terminal fragments, such as a 25-kDa fragment (TDP-25), have been identified as a ubiquitinated and phosphorylated components of inclusion bodies (IBs) in motor neurons from amyotrophic lateral sclerosis patients. Cells contain proteins that function as molecular chaperones and prevent aggregate formation of misfolded and aggregation-prone proteins. Recently, we reported that heat shock protein (HSP)70, an abundant molecular chaperone, binds to TDP-25 in an ATP-dependent manner; however, whether HSP70 can prevent the formation of TDP-25-related IBs remains unknown. Here, we showed that HSP70 prevented TDP-25 aggregation according to green fluorescent protein-tagged TDP-25 (G-TDP-25) colocalization in the cytoplasm with mCherry-tagged HSP70 (HSP70-R). The mobile fraction of HSP70-R in the cytoplasmic IBs associated with G-TDP-25 increased relative to that of G-TDP-25, suggesting that HSP70 strongly bound to G-TDP-25 in the IBs, whereas a portion remained dissociated from the IBs. Importantly, the proportion of G-TDP-25 IBs was significantly decreased by HSP70-R overexpression; however, G-TDP-25 levels in the insoluble fraction remained unchanged by HSP70-R overexpression, suggesting that G-TDP-25 formed aggregated species that cannot be dissolved, even in the presence of strong detergents. These results indicated that HSP70 prevented the accumulation of G-TDP-25 aggregates in cytoplasmic IBs, but was insufficient for G-TDP-25 disassembly and solubilization.

Analysis of the substrate recognition state of TDP-43 to single-stranded DNA using fluorescence correlation spectroscopy.

Normal function and abnormal aggregation of transactivation response (TAR) DNA/RNA-binding protein 43 kDa (TDP-43) are directly associated with the lethal genetic diseases: cystic fibrosis, amyotrophic lateral sclerosis (ALS), and frontotemporal lobar degeneration (FTLD). The binding of TDP-43 to single-stranded DNA (ssDNA) or RNA is involved in transcriptional repression, regulation of RNA splicing, and RNA stabilization. Equilibrium dissociation constants (Kd) of TDP-43 and ssDNA or RNA have been determined using various methods; however, methods that can measure Kd with high sensitivity in a short time using a small amount of TDP-43 in solution would be advantageous. Here, in order to determine the Kd of TDP-43 and fluorescence-labeled ssDNA as well as the binding stoichiometry, we use fluorescence correlation spectroscopy (FCS), which detects the slowed diffusion of molecular interactions in solution with single-molecule sensitivity, in addition to electrophoretic mobility shift assay (EMSA). Using tandem affinity chromatography of TDP-43 dually tagged with glutathione-S-transferase and poly-histidine tags, highly purified protein was obtained. FCS successfully detected specific interaction between purified TDP-43 and TG ssDNA repeats, with a Kd in the nanomolar range. The Kd of the TDP-43 mutant was not different from the wild type, although mutant oligomers, which did not bind ssDNA, were observed. Analysis of the fluorescence brightness per dimerized TDP-43/ssDNA complex was used to evaluate their binding stoichiometry. The results suggest that an assay combining FCS and EMSA can precisely analyze ssDNA recognition mechanisms, and that FCS may be applied for the rapid and quantitative determination of the interaction strength between TDP-43 and ssDNA or RNA. These methods will aid in the elucidation of the substrate recognition mechanism of ALS- and FTLD-associated variants of TDP-43.

Relationship Between Homodimeric Glucocorticoid Receptor and Transcriptional Regulation Assessed via an In Vitro Fluorescence Correlation Spectroscopy-Microwell System.

Glucocorticoid receptor (GR) is a hormone-activated transcription regulatory protein involved in metabolism as well as adrenocortical responses to psychosocial stress. Ligand-activated GR localizes to the nucleus, where GR homodimers regulate gene transcription via direct binding to glucocorticoid response elements (GREs). The role of GR homodimers in transcriptional activation has not yet been elucidated. In this study, we determined the concentration of GR homodimer, and its dissociation constant (Kd), at the single-cell level, by using fluorescence correlation spectroscopy (FCS) combined with a microwell system. Results from dissociation constant analysis and diffusion analysis suggested that GR forms complexes with other proteins as well as homodimers. We determined the relationship between the concentration of GR homodimer and transcriptional activity using a triple-color FCS-microwell system-based fluorescent reporter assay. The binding affinity of GR to GREs was analyzed via fluorescence cross-correlation spectroscopy (FCCS). Our findings indicate that the GR homodimer is essential for activating target gene transcription.

Determination of cytoplasmic optineurin foci sizes using image correlation spectroscopy.

Optineurin (OPTN) plays an important role in membrane trafficking processes such as exocytosis and autophagy. The sizes and rate of formation of accumulated structures comprising OPTN, such as foci or inclusion bodies (IBs), are often disrupted by amyotrophic lateral sclerosis (ALS) and glaucoma-associated mutants of OPTN. Therefore, methods for the quantitative measurement of the size of the accumulated structure are necessary. Here, we show that, using spatial image correlation spectroscopy (ICS), the average diameter of accumulated structures of the wild-type and disease-associated mutants in living cells may be easily determined. Although OPTN was found to frequently form foci in the cytoplasm, regardless of ALS- and glaucoma-associated mutation, the diameter of OPTN foci decreased in an ALS-associated mutant and increased in a glaucoma-associated mutant. However, a portion of cells carried IBs of the ALS-associated mutant that were larger than micrometre and ellipse-like shape, suggesting that this mutant accumulates non-uniformly in the IBs. The findings suggest that changes in their accumulation, determined via quantitative comparison of the OPTN foci and IBs in the cells, are involved in pathological features of ALS. In addition, this method enables rapid comparison of the average sizes of various other intracellular structures such as granules.

Two-detector number and brightness analysis reveals spatio-temporal oligomerization of proteins in living cells.

Number and brightness analysis (N&B) is a useful tool for the simultaneous visualization of protein oligomers and their localization, with single-molecule sensitivity. N&B determines particle brightness (fluorescence intensity per particle) and maps the spatial distribution of fluorescently labeled proteins by performing statistical analyses of the image series obtained using laser scanning microscopy. The brightness map reveals presence of the oligomers of the targeted protein and their distribution in living cells. However, even when corrections are applied, conventional N&B is affected by afterpulsing, shot noise, thermal noise, dead time, and overestimation of particle brightness when the concentration of the fluorescent particles changes during measurement. The drawbacks of conventional N&B can be circumvented by using two detectors, a novel approach that we henceforth call two-detector number and brightness analysis (TD-N&B), and introducing a linear regression of fluorescence intensity. This statistically eliminates the effect of noise from the detectors, and ensures that the correct particle brightness is obtained. Our method was theoretically assessed by numerical simulations and experimentally validated using a dilution series of purified enhanced green fluorescent protein (EGFP), EGFP tandem oligomers in cell lysate, and EGFP tandem oligomers in living cells. Furthermore, this method was used to characterize the complex process of ligand-induced glucocorticoid receptor dimerization and their translocation to the cell nucleus in live cells. Our method can be applied to other oligomer-forming proteins in cell signaling, or to aggregations of proteins such as those that cause neurodegenerative diseases.

Physicochemical Properties of the Mammalian Molecular Chaperone HSP60.

The E. coli GroEL/GroES chaperonin complex acts as a folding cage by producing a bullet-like asymmetric complex, and GroEL exists as double rings regardless of the presence of adenosine triphosphate (ATP). Its mammalian chaperonin homolog, heat shock protein, HSP60, and co-chaperonin, HSP10, play an essential role in protein folding by capturing unfolded proteins in the HSP60/HSP10 complex. However, the structural transition in ATPase-dependent reaction cycle has remained unclear. We found nucleotide-dependent association and dissociation of the HSP60/HSP10 complex using various analytical techniques under near physiological conditions. Our results showed that HSP60 exist as a significant number of double-ring complexes (football- and bullet-type complexes) and a small number of single-ring complexes in the presence of ATP and HSP10. HSP10 binds to HSP60 in the presence of ATP, which increased the HSP60 double-ring formation. After ATP is hydrolyzed to Adenosine diphosphate (ADP), HSP60 released the HSP10 and the dissociation of the double-ring to single-rings occurred. These results indicated that HSP60/HSP10 undergoes an ATP-dependent transition between the single- and double-rings in their system that is highly distinctive from the GroEL/GroES system particularly in the manner of complex formation and the roles of ATP binding and hydrolysis in the reaction cycle.

U6 snRNA expression prevents toxicity in TDP-43-knockdown cells.

Depletion of amyotrophic lateral sclerosis (ALS)-associated transactivation response (TAR) RNA/DNA-binding protein 43 kDa (TDP-43) alters splicing efficiency of multiple transcripts and results in neuronal cell death. TDP-43 depletion can also disturb expression levels of small nuclear RNAs (snRNAs) as spliceosomal components. Despite this knowledge, the relationship between cell death and alteration of snRNA expression during TDP-43 depletion remains unclear. Here, we knocked down TDP-43 in murine neuroblastoma Neuro2A cells and found a time lag between efficient TDP-43 depletion and appearance of cell death, suggesting that several mechanisms mediate between these two events. The amount of U6 snRNA was significantly decreased during TDP-43 depletion prior to increase of cell death, whereas that of U1, U2, and U4 snRNAs was not. Downregulation of U6 snRNA led to cell death, whereas transient exogenous expression of U6 snRNA counteracted the effect of TDP-43 knockdown on cell death, and slightly decreased the mis-splicing rate of Dnajc5 and Sortilin 1 transcripts, which are assisted by TDP-43. These results suggest that regulation of the U6 snRNA expression level by TDP-43 is a key factor in the increase in cell death upon TDP-43 loss-of-function.

Temperature and pH sensitivity of a stabilized self-nanoemulsion formed using an ionizable lipid-like material via an oil-to-surfactant transition.

Lipids functionalized with tertiary amines (ionizable lipids) for a pH-dependent positive charge have been developed extensively as a carrier material for delivering nucleic acids. We previously developed an SS-cleavable proton-activated lipid-like material (ssPalm) as a component of a functionalized lipid envelope structure of a nanoparticle that encapsulated plasmid DNA and short interfering RNA. In this study, we report on the unique characteristics of such an ionizable lipid: the formation of a nano-sized emulsion (ave. 40nm) via pH-triggered self-emulsification in the absence of a cargo (nucleic acids). The particle has a neutral charge at physiological pH and is stabilized by helper lipids and polyethyleneglycol (PEG)-conjugated lipids. The generalized polarization of 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan), which indicates the surface polarity caused by the invasion of water onto the surface, changes dynamically in response to pH and temperature, while the fluidity of the intra-particle compartment, as measured by the fluorescence anisotropy of 1,6-Diphenyl-1,3,5-hexatriene (DPH), is not affected. Even when the particle contains a high density of PEG on the surface, it shows a high fusogenecity to negatively charged liposomes in response to an acidic pH to a higher degree than a conventional cationic lipid. These characteristics suggest that the ssPalm particle possesses unique properties for delivering lipophilic drugs across the biomembrane.

Efficient and dynamic nuclear localization of green fluorescent protein via RNA binding.

Classical nuclear localization signal (NLS) sequences have been used for artificial localization of green fluorescent protein (GFP) in the nucleus as a positioning marker or for measurement of the nuclear-cytoplasmic shuttling rate in living cells. However, the detailed mechanism of nuclear retention of GFP-NLS remains unclear. Here, we show that a candidate mechanism for the strong nuclear retention of GFP-NLS is via the RNA-binding ability of the NLS sequence. GFP tagged with a classical NLS derived from Simian virus 40 (GFP-NLS(SV40)) localized not only in the nucleoplasm, but also to the nucleolus, the nuclear subdomain in which ribosome biogenesis takes place. GFP-NLS(SV40) in the nucleolus was mobile, and intriguingly, the diffusion coefficient, which indicates the speed of diffusing molecules, was 1.5-fold slower than in the nucleoplasm. Fluorescence correlation spectroscopy (FCS) analysis showed that GFP-NLS(SV40) formed oligomers via RNA binding, the estimated molecular weight of which was larger than the limit for passive nuclear export into the cytoplasm. These findings suggest that the nuclear localization of GFP-NLS(SV40) likely results from oligomerization mediated via RNA binding. The analytical technique used here can be applied for elucidating the details of other nuclear localization mechanisms, including those of several types of nuclear proteins. In addition, GFP-NLS(SV40) can be used as an excellent marker for studying both the nucleoplasm and nucleolus in living cells.

Siglec-15 is a potential therapeutic target for postmenopausal osteoporosis.

Sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) is an immunoreceptor that regulates osteoclast development and bone resorption in association with an immunoreceptor tyrosine-based activation motif (ITAM) adaptor protein, DNAX-activating protein 12kDa (DAP12). Although Siglec-15 has an important role in physiologic bone remodeling by modulating RANKL signaling, it is unclear whether it is involved in pathologic bone loss in which multiple osteoclastogenic factors participate in excessive osteoclastogenesis. Here we demonstrated that Siglec-15 is involved in estrogen deficiency-induced bone loss. WT and Siglec-15(-/-) mice were ovariectomized (Ovx) or sham-operated at 14wk of age and their skeletal phenotype was evaluated at 18 and 22wk of age. Siglec-15(-/-) mice showed resistance to estrogen deficiency-induced bone loss compared to WT mice. Although the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts increased after ovariectomy in both WT and Siglec-15(-/-) mice, the increase was lower in Siglec-15(-/-) mice than in WT mice. Importantly, osteoclasts in Siglec-15(-/-) mice were small and failed to spread on the bone surface, indicating impaired osteoclast differentiation. Because upregulated production of TNF-α as well as RANKL is mainly responsible for estrogen deficiency-induced development of osteoclasts, we examined whether Siglec-15 deficiency affects TNF-α-induced osteoclastogenesis in vitro. The TNF-α mediated induction of TRAP-positive multinucleated cells was impaired in Siglec-15(-/-) cells, suggesting that Siglec-15 is involved in TNF-α induced osteoclastogenesis. We also confirmed that signaling through osteoclast-associated receptor/Fc receptor common γ chain, which is an alternative ITAM adaptor to DAP12, rescues multinucleation but not cytoskeletal organization of TNF-α and RANKL-induced Siglec-15(-/-) osteoclasts, indicating that the Siglec-15/DAP12 pathway is especially important for cytoskeletal organization of osteoclasts in both RANKL and TNF-α induced osteoclastogenesis. The present findings indicate that Siglec-15 is involved in estrogen deficiency-induced differentiation of osteoclasts and is thus a potential therapeutic target for postmenopausal osteoporosis.

Determination of the dissociation constant of the NFκB p50/p65 heterodimer in living cells using fluorescence cross-correlation spectroscopy.

Fluorescence cross-correlation spectroscopy (FCCS) is a promising technique for observing and quantifying protein-protein interactions in vitro and in vivo. FCCS has emerged as a useful tool for obtaining parameters of the concentration of labeled particles, their molecular dynamics, as well as the size of their complexes. This chapter discusses aspects of preparing a biological system for FCCS experiments and suggests practical advice for performing FCCS in living cells. Moreover, we describe the method of FCCS to determine the dissociation constant of a transcription factor dimer in the living cell.

Peptide sequences converting polyglutamine into a prion in yeast.

Amyloids are ordered protein aggregates composed of cross-ß sheet structures. Amyloids include prions, defined as infectious proteins, which are responsible for mammalian transmissible spongiform encephalopathies, and fungal prions. Although the conventional view is that typical amyloids are associated with nontransmissible mammalian neurodegenerative diseases such as Alzheimer's disease, increasing evidence suggests that the boundary between transmissible and nontransmissible amyloids is ambiguous. To clarify the mechanism underlying the difference in transmissibility, we investigated the dynamics and the properties of polyglutamine (polyQ) amyloids in yeast cells, in which the polyQ aggregates are not transmissible but can be converted into transmissible amyloids. We found that polyQ had an increased tendency to form aggregates compared to the yeast prion Sup35. In addition, we screened dozens of peptides that converted the nontransmissible polyQ to transmissible aggregates when they flanked the polyQ stretch, and also investigated their cellular dynamics aiming to understand the mechanism of transmission.

Moyamoya disease-associated protein mysterin/RNF213 is a novel AAA+ ATPase, which dynamically changes its oligomeric state.

Moyamoya disease is an idiopathic human cerebrovascular disorder that is characterized by progressive stenosis and abnormal collateral vessels. We recently identified mysterin/RNF213 as its first susceptibility gene, which encodes a 591-kDa protein containing enzymatically active P-loop ATPase and ubiquitin ligase domains and is involved in proper vascular development in zebrafish. Here we demonstrate that mysterin further contains two tandem AAA+ ATPase modules and forms huge ring-shaped oligomeric complex. AAA+ ATPases are known to generally mediate various biophysical and mechanical processes with the characteristic ring-shaped structure. Fluorescence correlation spectroscopy and biochemical evaluation suggested that mysterin dynamically changes its oligomeric forms through ATP/ADP binding and hydrolysis cycles. Thus, the moyamoya disease-associated gene product is a unique protein that functions as ubiquitin ligase and AAA+ ATPase, which possibly contributes to vascular development through mechanical processes in the cell.

Prefoldin prevents aggregation of α-synuclein.

Protein aggregation is observed in various neurodegeneration diseases, including Parkinson's disease (PD). Alpha-synuclein, a causative gene product of familial PD, is a major component of large aggregates (inclusion bodies) in PD. Prefoldin, a molecular chaperone comprised of six subunits, PFD1~6, prevents misfolding of newly synthesized nascent polypeptides and also prevents aggregation of protein such as a pathogenic form of Huntingtin, a causative gene product of Huntington disease. In this study, we first found that aggregation of TagRFP-tagged wild-type α-synuclein and its pathogenic mutants, but not that of GFP-tagged α-synuclein, occurred in transfected Neuro-2a cells. The fluorescence of GFP is weakened under the condition of pH 4.5-5.0, and TagRFP is a stable red fluorescence protein under an acidic condition. Aggregated TagRFP-wild-type α-synuclein and its pathogenic mutants in Neuro-2a cells were ubiquitinated and were colocalized with the prefoldin complex in the lysosome under this condition. Furthermore, knockdown of PFD2 and PFD5 disrupted prefoldin formation in α-synuclein-expressing cells, resulting in accumulation of aggregates of wild-type and pathogenic α-synuclein and in induction of cell death. The levels of aggregation and cell death in pathogenic α-synuclein-transfected cells tended to be higher than those in wild-type α-synuclein-transfected cells. These results suggest that prefoldin works as a protective factor in aggregated α-synuclein-induced cell death.

Rab6a releases LIS1 from a dynein idling complex and activates dynein for retrograde movement.

Cytoplasmic dynein drives the movement of a wide range of cargoes towards the minus ends of microtubules. We previously demonstrated that LIS1 forms an idling complex with dynein, which is transported to the plus ends of microtubules by kinesin motors. Here we report that the small GTPase Rab6a is essential for activation of idling dynein. Immunoprecipitation and microtubule pull-down assays reveal that the GTP bound mutant, Rab6a(Q72L), dissociates LIS1 from a LIS1-dynein complex, activating dynein movement in in vitro microtubule gliding assays. We monitor transient interaction between Rab6a(Q72L) and dynein in vivo using dual-colour fluorescence cross-correlation spectroscopy in dorsal root ganglion (DRG) neurons. Finally, we demonstrate that Rab6a(Q72L) mediates LIS1 release from a LIS1-dynein complex followed by dynein activation through an in vitro single-molecule assay using triple-colour quantum dots. Our findings reveal a surprising function for GTP bound Rab6a as an activator of idling dynein.

Determination of dissociation constant of the NFκB p50/p65 heterodimer using fluorescence cross-correlation spectroscopy in the living cell.

Two-laser-beam fluorescence cross-correlation spectroscopy (FCCS) is promising technique that provides quantitative information about the interactions of biomolecules. The p50/p65 heterodimer is the most abundant and well understood of the NFκB dimers in most cells. However, the quantitative value of affinity, namely the K(d), for the heterodimer in living cells is not known yet. To quantify the heterodimerization of the IPT domain of p50/p65 in the living cell, we used two-laser-beam FCCS. The K(d) values of mCherry2- and EGFP-fused p50 and p65 were determined to be 0.46 µM in the cytoplasm and 1.06 µM in the nucleus of the living cell. These results suggest the different binding affinities of the p50/p65 heterodimer in the cytoplasm and nucleus of the living cell and different complex formation in each region.