Reconstruction of destruction - in vitro reconstitution methods in autophagy research.

Autophagy is one of the most elaborative membrane remodeling systems in eukaryotic cells. Its major function is to recycle cytoplasmic material by delivering it to lysosomes for degradation. To achieve this, a membrane cisterna is formed that gradually captures cargo such as organelles or protein aggregates. The diversity of cargo requires autophagy to be highly versatile to adapt the shape of the phagophore to its substrate. Upon closure of the phagophore, a double-membrane-surrounded autophagosome is formed that eventually fuses with lysosomes. In response to environmental cues such as cytotoxicity or starvation, bulk cytoplasm can be captured and delivered to lysosomes. Autophagy thus supports cellular survival under adverse conditions. During the past decades, groundbreaking genetic and cell biological studies have identified the core machinery involved in the process. In this Review, we are focusing on in vitro reconstitution approaches to decipher the details and spatiotemporal control of autophagy, and how such studies contributed to our current understanding of the pathways in yeast and mammals. We highlight studies that revealed the function of the autophagy machinery at a molecular level with respect to its capacity to remodel membranes.

Plasmonic Nanoantennas Enable Forbidden Förster Dipole-Dipole Energy Transfer and Enhance the FRET Efficiency.

Förster resonance energy transfer (FRET) plays a key role in biochemistry, organic photovoltaics, and lighting sources. FRET is commonly used as a nanoruler for the short (nanometer) distance between donor and acceptor dyes, yet FRET is equally sensitive to the mutual dipole orientation. The orientation dependence complicates the FRET analysis in biological samples and may even lead to the absence of FRET for perpendicularly oriented donor and acceptor dipoles. Here, we exploit the strongly inhomogeneous and localized fields in plasmonic nanoantennas to open new energy transfer routes, overcoming the limitations from the mutual dipole orientation to ultimately enhance the FRET efficiency. We demonstrate that the simultaneous presence of perpendicular near-field components in the nanoantenna sets favorable energy transfer routes that increase the FRET efficiency up to 50% for nearly perpendicular donor and acceptor dipoles. This new facet of plasmonic nanoantennas enables dipole-dipole energy transfer that would otherwise be forbidden in a homogeneous environment. As such, our approach further increases the applicability of single-molecule FRET over diffraction-limited approaches, with the additional benefits of higher sensitivities and higher concentration ranges toward physiological levels.

Matching Nanoantenna Field Confinement to FRET Distances Enhances Förster Energy Transfer Rates.

Förster resonance energy transfer (FRET) is widely applied in chemistry, biology, and nanosciences to assess distances on sub-10 nm scale. Extending the range and applicability of FRET requires enhancement of the fluorescence energy transfer at a spatial scale comparable to the donor-acceptor distances. Plasmonic nanoantennas are ideal to concentrate optical fields at a nanoscale fully matching the FRET distance range. Here, we present a resonant aluminum nanogap antenna tailored to enhance single molecule FRET. A 20 nm gap confines light into a nanoscale volume, providing a field gradient on the scale of the donor-acceptor distance, a large 10-fold increase in the local density of optical states, and strong intensity enhancement. With our dedicated design, we obtain 20-fold enhancement on the fluorescence emission of donor and acceptor dyes, and most importantly up to 5-fold enhancement of the FRET rate for donor-acceptor separations of 10 nm. We also provide a thorough framework of the fluorescence photophysics occurring in the nanoscale gap volume. The presented enhancement of energy transfer flow at the nanoscale opens a yet unexplored facet of the various advantages of optical nanoantennas and provides a new strategy toward biological applications of single molecule FRET at micromolar concentrations.

FRET analysis of CP12 structural interplay by GAPDH and PRK.

CP12 is an intrinsically disordered protein playing a key role in the regulation of the Benson-Calvin cycle. Due to the high intrinsic flexibility of CP12, it is essential to consider its structural modulation induced upon binding to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) enzymes. Here, we report for the first time detailed structural modulation about the wild-type CP12 and its site-specific N-terminal and C-terminal disulfide bridge mutants upon interaction with GAPDH and PRK by Förster resonance energy transfer (FRET). Our results indicate an increase in CP12 compactness when the complex is formed with GAPDH or PRK. In addition, the distributions in FRET histograms show the elasticity and conformational flexibility of CP12 in all supra molecular complexes. Contrarily to previous beliefs, our FRET results importantly reveal that both N-terminal and C-terminal site-specific CP12 mutants are able to form the monomeric (GAPDH-CP12-PRK) complex.

FRET enhancement in aluminum zero-mode waveguides.

Zero-mode waveguides (ZMWs) can confine light into attoliter volumes, which enables single molecule fluorescence experiments at physiological micromolar concentrations. Of the fluorescence spectroscopy techniques that can be enhanced by ZMWs, Förster resonance energy transfer (FRET) is one of the most widely used in life sciences. Combining zero-mode waveguides with FRET provides new opportunities to investigate biochemical structures or follow interaction dynamics at micromolar concentrations with single-molecule resolution. However, prior to any quantitative FRET analysis on biological samples, it is crucial to establish first the influence of the ZMW on the FRET process. Here, we quantify the FRET rates and efficiencies between individual donor-acceptor fluorophore pairs that diffuse into aluminum zero-mode waveguides. Aluminum ZMWs are important structures thanks to their commercial availability and the large amount of literature that describe their use for single-molecule fluorescence spectroscopy. We also compared the results between ZMWs milled in gold and aluminum, and found that although gold has a stronger influence on the decay rates, the lower losses of aluminum in the green spectral region provide larger fluorescence brightness enhancement factors. For both aluminum and gold ZMWs, we observed that the FRET rate scales linearly with the isolated donor decay rate and the local density of optical states. Detailed information about FRET in ZMWs unlocks their application as new devices for enhanced single-molecule FRET at physiological concentrations.

Nanophotonic enhancement of the Förster resonance energy-transfer rate with single nanoapertures.

Tailoring the light-matter interaction and the local density of optical states (LDOS) with nanophotonics provides accurate control over the luminescence properties of a single quantum emitter. This paradigm is also highly attractive to enhance the near-field Förster resonance energy transfer (FRET) between two fluorescent emitters. Despite the wide applications of FRET in nanosciences, using nanophotonics to enhance FRET has remained a debated and complex challenge. Here we demonstrate enhanced energy transfer within single donor-acceptor fluorophore pairs confined in single gold nanoapertures. Experiments monitoring both the donor and the acceptor emission photodynamics clearly establish a linear dependence of the FRET rate on the LDOS in nanoapertures, demonstrating that nanophotonics can be used to intensify the near-field energy transfer. Strikingly, we observe a significant six-fold increase in the FRET rate for large donor-acceptor separations exceeding 13 nm. Exciting opportunities are opened to investigate biochemical structures with donor-acceptor distances much beyond the classical Förster radius. Importantly, our approach is fully compatible with the detection of single biomolecules freely diffusing in water solution under physiological conditions.

Chaperone activity of Cyp18 through hydrophobic condensation that enables rescue of transient misfolded molten globule intermediates.

The single-domain cyclophilin 18 (Cyp18) has long been known to function as a peptidyl-prolyl cis/trans isomerase (PPI) and was proposed by us to also function as a chaperone [Freskgard, P.-O., Bergenhem, N., Jonsson, B.-H., Svensson, M., and Carlsson, U. (1992) Science 258, 466-468]. Later several multidomain PPIs were demonstrated to work as both a peptidyl-prolyl cis/trans isomerase and a chaperone. However, the chaperone ability of Cyp18 has been debated. In this work, we add additional results that show that Cyp18 can both accelerate the rate of refolding and increase the yield of native protein during the folding reaction, i.e., function as both a folding catalyst and a chaperone. Refolding experiments were performed using severely destabilized mutants of human carbonic anhydrase II under conditions where the unfolding reaction is significant and a larger fraction of a more destabilized variant populates molten globule-like intermediates during refolding. A correlation of native state protein stability of the substrate protein versus Cyp18 chaperone activity was demonstrated. The induced correction of misfolded conformations by Cyp18 likely functions through rescue from misfolding of transient molten globule intermediates. ANS binding data suggest that the interaction by Cyp18 leads to an early stage condensation of accessible hydrophobic portions of the misfolding-prone protein substrate during folding. The opposite effect was observed for GroEL known as an unfoldase at early stages of refolding. The chaperone effect of Cyp18 was also demonstrated for citrate synthase, suggesting a general chaperone effect of this PPI.

Calcium activates purified human TRPA1 with and without its N-terminal ankyrin repeat domain in the absence of calmodulin.

Extracellular influx of calcium or release of calcium from intracellular stores have been shown to activate mammalian TRPA1 as well as to sensitize and desensitize TRPA1 electrophilic activation. Calcium binding sites on both intracellular N- and C-termini have been proposed. Here, we demonstrate based on Förster resonance energy transfer (FRET) and bilayer patch-clamp studies, a direct calmodulin-independent action of calcium on the purified human TRPA1 (hTRPA1), causing structural changes and activation without immediate subsequent desensitization of hTRPA1 with and without its N-terminal ankyrin repeat domain (N-ARD). Thus, calcium alone activates hTRPA1 by a direct interaction with binding sites outside the N-ARD.

pRb2/p130 protein in relation to clinicopathological and biological variables in rectal cancers with a clinical trial of preoperative radiotherapy.

BACKGROUND: pRb2/p130 plays a key role in cell proliferation and is a considerable progress about expression patterns of pRb2/p130 in number of malignancies. However, pRb2/p130 expression and its significance in rectal cancer remain unknown. The purpose of the present study was to investigate pRb2/p130 protein patterns and their correlations with clinicopathological and biological factors in rectal cancer patients with or without preoperative radiotherapy (RT). PATIENT/METHODS: pRb2/p130 protein was examined by immunohistochemistry in 130 primary tumors, along with the corresponding 61 distant normal mucosa specimens, 85 adjacent normal mucosa specimens, 34 lymph node metastases, and 93 primary tumor biopsies from rectal cancer patients who participated in a Swedish clinical trial of preoperative RT. RESULTS: The pRb2/p130 protein was mainly localized in the cytoplasm of tumor cells. In nonradiated cases, the lack of pRb2/p130 was related to advanced tumor-node-metastases stage, poorer differentiation, weak fibrosis, less inflammatory infiltration, higher Ki-67, and positive Cox-2 expression (p < 0.05). In radiated cases, the lack of pRb2/p130 was related to nonstaining of Cox-2 and survivin (p < 0.05). pRb2/p130 protein in primary tumors tended to be increased after RT (27% vs 16%, p = 0.07). CONCLUSION: pRb2/p130 was mainly localized in the cytoplasm rather than in the nucleus in rectal cancer. After RT, pRb2/p130 protein seems to be increased in primary tumors, and further the relationship of the pRb2/p130 with the clinicopathological and biological variables changed compared to the nonradiated cases. However, we did not find that the pRb2/p130 was directly related to RT, tumor recurrence, and patients' survival.

Expression of MAC30 protein is related to survival and biological variables in primary and metastatic colorectal cancers.

MAC30 is highly expressed in several types of tumors including colorectal cancers, however, its clinicopathological and biological significance in colorectal cancers is currently not known. The aim of our study was to investigate MAC30 expression in distant normal mucosa, adjacent normal mucosa, primary tumors and metastases of colorectal cancer, and to determine the relationship between MAC30 expression and clinicopathological and biological variables. MAC30 expression was immunohistochemically examined in distant normal mucosa (n = 54), adjacent normal mucosa (n = 123), primary tumors (n = 217) and lymph node metastases (n = 56) from colorectal cancer patients. MAC30 cytoplasmic expression was increased from distant normal mucosa to primary tumor and to metastasis (p < 0.0001-0.04). Furthermore, 40% primary and 37% metastatic tumors showed stronger cytoplasmic expression of MAC30 at the tumor invasive margins compared to inner tumor areas. Strong cytoplasmic expression of MAC30 in the metastasis was related to a poor prognosis (p = 0.04). MAC30 cytoplasmic expression was positively related to expression of proliferating cell nuclear antigen (p = 0.04), p53 (p = 0.04), nucleoporin 88 (p = 0.001), legumain (p = 0.004) and particularly interesting new cysteine-histidine rich protein (p = 0.004). However, MAC30 expression in the nucleus and stroma did not have any clinicopathological and biological significance (p > 0.05). In conclusion, MAC30 protein may play a role in development of colorectal cancer, and can be considered as a prognostic factor.

Differential conformational modulations of MreB folding upon interactions with GroEL/ES and TRiC chaperonin components.

Here, we study and compare the mechanisms of action of the GroEL/GroES and the TRiC chaperonin systems on MreB client protein variants extracted from E. coli. MreB is a homologue to actin in prokaryotes. Single-molecule fluorescence correlation spectroscopy (FCS) and time-resolved fluorescence polarization anisotropy report the binding interaction of folding MreB with GroEL, GroES and TRiC. Fluorescence resonance energy transfer (FRET) measurements on MreB variants quantified molecular distance changes occurring during conformational rearrangements within folding MreB bound to chaperonins. We observed that the MreB structure is rearranged by a binding-induced expansion mechanism in TRiC, GroEL and GroES. These results are quantitatively comparable to the structural rearrangements found during the interaction of ß-actin with GroEL and TRiC, indicating that the mechanism of chaperonins is conserved during evolution. The chaperonin-bound MreB is also significantly compacted after addition of AMP-PNP for both the GroEL/ES and TRiC systems. Most importantly, our results showed that GroES may act as an unfoldase by inducing a dramatic initial expansion of MreB (even more than for GroEL) implicating a role for MreB folding, allowing us to suggest a delivery mechanism for GroES to GroEL in prokaryotes.

Plasmonic antennas and zero-mode waveguides to enhance single molecule fluorescence detection and fluorescence correlation spectroscopy toward physiological concentrations.

Single-molecule approaches to biology offer a powerful new vision to elucidate the mechanisms that underpin the functioning of living cells. However, conventional optical single molecule spectroscopy techniques such as Förster fluorescence resonance energy transfer (FRET) or fluorescence correlation spectroscopy (FCS) are limited by diffraction to the nanomolar concentration range, far below the physiological micromolar concentration range where most biological reaction occur. To breach the diffraction limit, zero-mode waveguides (ZMW) and plasmonic antennas exploit the surface plasmon resonances to confine and enhance light down to the nanometer scale. The ability of plasmonics to achieve extreme light concentration unlocks an enormous potential to enhance fluorescence detection, FRET, and FCS. Single molecule spectroscopy techniques greatly benefit from ZMW and plasmonic antennas to enter a new dimension of molecular concentration reaching physiological conditions. The application of nano-optics to biological problems with FRET and FCS is an emerging and exciting field, and is promising to reveal new insights on biological functions and dynamics.

Conformational modulation and hydrodynamic radii of CP12 protein and its complexes probed by fluorescence correlation spectroscopy.

Light/dark regulation of the Calvin cycle in oxygenic photosynthetic organisms involves the formation and dissociation of supramolecular complexes between CP12, a nuclear-encoded chloroplast protein, and the two enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (EC 1.2.1.13) and phosphoribulokinase (PRK) (EC 2.7.1.19). Despite the high importance of understanding the structural basis of the interaction of CP12 with GAPDH and PRK to investigate the regulation of the Calvin cycle, information is still lacking about the structural remodulation of CP12 and its complex formation. Here, we characterize the diffusion dynamics and hydrodynamic radii of CP12 from Chlamydomonas reinhardtii upon binding to GAPDH and PRK using fluorescence correlation spectroscopy experiments. We quantify a hydrodynamic radius of 3.4 ± 0.2 nm for the CP12 protein with an increase up to 5.2 ± 0.3 nm upon complex formation with GAPDH and PRK. In addition, unfolding experiments reveal a 1.6- and 2.0-fold increase respectively of the hydrodynamic radii for the N-terminal and C-terminal cysteine CP12 mutant proteins compared with their native folded structures. The different behavior of the CP12 mutant proteins during hydrophobic collapse transition is a direct clue to different structural orientations of the CP12 mutant proteins. These different structures are expected to facilitate the binding of either GAPDH or PRK during binary complex and ternary complex formation.

Transient conformational remodeling of folding proteins by GroES-individually and in concert with GroEL.

The commonly accepted dogma of the bacterial GroE chaperonin system entails protein folding mediated by cycles of several ATP-dependent sequential steps where GroEL interacts with the folding client protein. In contrast, we herein report GroES-mediated dynamic remodeling (expansion and compression) of two different protein substrates during folding: the endogenous substrate MreB and carbonic anhydrase (HCAII), a well-characterized protein folding model. GroES was also found to influence GroEL binding induced unfolding and compression of the client protein underlining the synergistic activity of both chaperonins, even in the absence of ATP. This previously unidentified activity by GroES should have important implications for understanding the chaperonin mechanism and cellular stress response. Our findings necessitate a revision of the GroEL/ES mechanism.

A plasmonic 'antenna-in-box' platform for enhanced single-molecule analysis at micromolar concentrations.

Single-molecule fluorescence techniques are key for a number of applications, including DNA sequencing, molecular and cell biology and early diagnosis. Unfortunately, observation of single molecules by diffraction-limited optics is restricted to detection volumes in the femtolitre range and requires pico- or nanomolar concentrations, far below the micromolar range where most biological reactions occur. This limitation can be overcome using plasmonic nanostructures, which enable the confinement of light down to nanoscale volumes. Although these nanoantennas enhance fluorescence brightness, large background signals and/or unspecific binding to the metallic surface have hampered the detection of individual fluorescent molecules in solution at high concentrations. Here we introduce a novel 'antenna-in-box' platform that is based on a gap-antenna inside a nanoaperture. This design combines fluorescent signal enhancement and background screening, offering high single-molecule sensitivity (fluorescence enhancement up to 1,100-fold and microsecond transit times) at micromolar sample concentrations and zeptolitre-range detection volumes. The antenna-in-box device can be optimized for single-molecule fluorescence studies at physiologically relevant concentrations, as we demonstrate using various biomolecules.

A nonessential role for Arg 55 in cyclophilin18 for catalysis of proline isomerization during protein folding.

The protein folding process is often in vitro rate-limited by slow cis-trans proline isomerization steps. Importantly, the rate of this process in vivo is accelerated by prolyl isomerases (PPIases). The archetypal PPIase is the human cyclophilin 18 (Cyp18 or CypA), and Arg 55 has been demonstrated to play a crucial role when studying short peptide substrates in the catalytic action of Cyp18 by stabilizing the transition state of isomerization. However, in this study we show that a R55A mutant of Cyp18 is as efficient as the wild type to accelerate the refolding reaction of human carbonic anhydrase II (HCA II). Thus, it is evident that the active-site located Arg 55 is not required for catalysis of the rate-limiting prolyl cis-trans isomerization steps during the folding of a protein substrate as HCA II. Nevertheless, catalysis of cis-trans proline isomerization in HCA II occurs in the active-site of Cyp18, since binding of the inhibitor cyclosporin A abolishes rate acceleration of the refolding reaction. Obviously, the catalytic mechanisms of Cyp18 can differ when acting upon a simple model peptide, four residues long, with easily accessible Pro residues compared with a large protein molecule undergoing folding with partly or completely buried Pro residues. In the latter case, the isomerization kinetics are significantly slower and simpler mechanistic factors such as desolvation and/or strain might operate during folding-assisted catalysis, since binding to the hydrophobic active site is still a prerequisite for catalysis.

Domain-specific chaperone-induced expansion is required for beta-actin folding: a comparison of beta-actin conformations upon interactions with GroEL and tail-less complex polypeptide 1 ring complex (TRiC).

Actin, an abundant cytosolic protein in eukaryotic cells, is dependent on the interaction with the chaperonin tail-less complex polypeptide 1 ring complex (TRiC) to fold to the native state. The prokaryotic chaperonin GroEL also binds non-native beta-actin, but is unable to guide beta-actin toward the native state. In this study we identify conformational rearrangements in beta-actin, by observing similarities and differences in the action of the two chaperonins. A cooperative collapse of beta-actin from the denatured state to an aggregation-prone intermediate is observed, and insoluble aggregates are formed in the absence of chaperonin. In the presence of GroEL, however, >90% of the aggregation-prone actin intermediate is kept in solution, which shows that the binding of non-native actin to GroEL is effective. The action of GroEL on bound flourescein-labeled beta-actin was characterized, and the structural rearrangement was compared to the case of the beta-actin-TRiC complex, employing the homo fluorescence resonance energy transfer methodology previously used [Villebeck, L., Persson, M., Luan, S.-L., Hammarström, P., Lindgren, M., and Jonsson, B.-H. (2007) Biochemistry 46 (17), 5083-93]. The results suggest that the actin structure is rearranged by a "binding-induced expansion" mechanism in both TRiC and GroEL, but that binding to TRiC, in addition, causes a large and specific separation of two subdomains in the beta-actin molecule, leading to a distinct expansion of its ATP-binding cleft. Moreover, the binding of ATP and GroES has less effect on the GroEL-bound beta-actin molecule than the ATP binding to TRiC, where it leads to a major compaction of the beta-actin molecule. It can be concluded that the specific and directed rearrangement of the beta-actin structure, seen in the natural beta-actin-TRiC system, is vital for guiding beta-actin to the native state.