TY  - JOUR
AB  - Structural maintenance of chromosome (SMC) complexes form ring-like structures through exceptional elongated coiled coils (CC). Recent studies found that variable CC conformations including open and collapsed forms, which might result from discontinuities in the CC, facilitate the diverse functions of SMCs in DNA organization. However, a detailed description of the SMC CC architecture is still missing. Here, we study the structural composition and mechanical properties of SMC proteins with optical tweezers unfolding experiments using the isolated Psm3 CC as a model system. We find a comparatively unstable protein with three unzipping intermediates, which we could directly assign to CC features by crosslinking experiments and state-of-the-art prediction software. Particularly, the CC elbow is shown to be a flexible, potentially non-structured feature, which divides the CC into sections, induces a pairing shift from one CC strand to the other and could facilitate large-scale conformational changes - most likely via thermal fluctuations of the flanking CC sections. A replacement of the elbow amino acids hinders folding of the consecutive CC region and leads frequently to non-native misalignments, revealing the elbow as a guide for proper folding. Additional in vivo manipulation of the elbow flexibility resulted in impaired cohesin complexes, which directly links the sensitive CC architecture to the biological function of cohesin.
AU  - Freitag, M.*
AU  - Jaklin, S.*
AU  - Padovani, F.
AU  - Radzichevici, E.
AU  - Zernia, S.*
AU  - Schmoller, K.M.
AU  - Stigler, J.*
C1  - 66488
C2  - 53190
CY  - 50 Hampshire St, Floor 5, Cambridge, Ma 02139 Usa
SP  - 4702-4713
TI  - Single-molecule experiments reveal the elbow as an essential folding guide in SMC coiled coil arms.
JO  - Biophys. J.
VL  - 121
IS  - 23
PB  - Cell Press
PY  - 2022
SN  - 0006-3495
ER  - 

TY  - JOUR
AB  - A large fraction of soluble and membrane-bound proteins exists as non-covalent dimers, trimers, and higher-order oligomers. The experimental determination of the oligomeric state or stoichiometry of proteins remains a nontrivial challenge. In one approach, the protein of interest is genetically fused to green fluorescent protein (GFP). If a fusion protein assembles into a non-covalent oligomeric complex, exciting their GFP moiety with polarized fluorescent light elicits homotypic Forster resonance energy transfer (homo-FRET), in which the emitted radiation is partially depolarized. Fluorescence depolarization is associated with a decrease in fluorescence anisotropy that can be exploited to calculate the oligomeric state. In a classical approach, several parameters obtained through time-resolved and steady-state anisotropy measurements are required for determining the stoichiometry of the oligomers. Here, we examined novel approaches in which time-resolved measurements of reference proteins provide the parameters that can be used to interpret the less expensive steady-state anisotropy data of candidates. In one approach, we find that using average homo-FRET rates (k(FRET)), average fluorescence lifetimes (tau), and average anisotropies of those fluorophores that are indirectly excited by homo-FRET (r(ET)) do not compromise the accuracy of calculated stoichiometries. In the other approach, fractional photobleaching of reference oligomers provides a novel parameter a whose dependence on stoichiometry allows one to quantitatively interpret the increase of fluorescence anisotropy seen after photo-bleaching the candidates. These methods can at least reliably distinguish monomers from dimers and trimers.
AU  - Heckmeier, P.J.*
AU  - Agam, G.*
AU  - Teese, M.G.*
AU  - Hoyer, M.*
AU  - Stehle, R.
AU  - Lamb, D.C.*
AU  - Langosch, D.*
C1  - 59572
C2  - 48919
CY  - 50 Hampshire St, Floor 5, Cambridge, Ma 02139 Usa
SP  - 99-114
TI  - Determining the stoichiometry of small protein oligomers using steady-state fluorescence anisotropy.
JO  - Biophys. J.
VL  - 119
IS  - 1
PB  - Cell Press
PY  - 2020
SN  - 0006-3495
ER  - 

TY  - JOUR
AB  - Single-pair Forster resonance energy transfer (spFRET) has become an important tool for investigating conformational dynamics in biological systems. To extract dynamic information from the spFRET traces measured with total internal reflection fluorescence microscopy, we extended the hidden Markov model (HMM) approach. In our extended HMM analysis, we incorporated the photon-shot noise from camera-based systems into the HMM. Thus, the variance in Forster resonance energy transfer (FRET) efficiency of the various states, which is typically a fitted parameter, is explicitly included in the analysis estimated from the number of detected photons. It is also possible to include an additional broadening of the FRET state, which would then only reflect the inherent flexibility of the dynamic biological systems. This approach is useful when comparing the dynamics of individual molecules for which the total intensities vary significantly. We used spFRET with the extended HMM analysis to investigate the dynamics of TATA-box-binding protein (TBP) on promoter DNA in the presence of negative cofactor 2 (NC2). We compared the dynamics of two promoters as well as DNAs of different length and labeling location. For the adenovirus major late promoter, four FRET states were observed; three states correspond to different conformations of the DNA in the TBP-DNA-NC2 complex and a four-state model in which the complex has shifted along the DNA. The HMM analysis revealed that the states are connected via a linear, four-well model. For the H2B promoter, more complex dynamics were observed. By clustering the FRET states detected with the HMM analysis, we could compare the general dynamics observed for the two promoter sequences. We observed that the dynamics from a stretched DNA conformation to a bent conformation for the two promoters were similar, whereas the bent conformation of the TBP-DNA-NC2 complex for the H2B promoter is approximately three times more stable than for the adenovirus major late promoter.
AU  - Zarrabi, N.*
AU  - Schluesche, P.*
AU  - Meisterernst, M.
AU  - Börsch, M.*
AU  - Lamb, D.C.*
C1  - 54921
C2  - 46188
CY  - 50 Hampshire St, Floor 5, Cambridge, Ma 02139 Usa
SP  - 2310-2326
TI  - Analyzing the dynamics of single TBP-DNA-NC2 complexes using hidden markov models.
JO  - Biophys. J.
VL  - 115
IS  - 12
PB  - Cell Press
PY  - 2018
SN  - 0006-3495
ER  - 

TY  - JOUR
AU  - Kaszuba, K.*
AU  - Grzybek, M.
AU  - Orlowski, A.*
AU  - Danne, R.*
AU  - Róg, T.*
AU  - Simons, K.*
AU  - Coskun, Ü.
AU  - Vattulainen, I.*
C1  - 48679
C2  - 41271
CY  - Cambridge
SP  - 89A
TI  - Glycosylation affects the conformational behavior of EGFR.
JO  - Biophys. J.
VL  - 110
IS  - 3
PB  - Cell Press
PY  - 2016
SN  - 0006-3495
ER  - 

TY  - JOUR
AU  - Zensen, C.*
AU  - Buelvas, I.
AU  - Eickelberg, O.
AU  - Lohmueller, T.*
AU  - Feldmann, J.*
C1  - 48678
C2  - 41272
CY  - Cambridge
SP  - 134A
TI  - 'Optical shaking' of red blood cells: A strategy to measure cell-fluid coupling with optical tweezers.
JO  - Biophys. J.
VL  - 110
IS  - 3
PB  - Cell Press
PY  - 2016
SN  - 0006-3495
ER  - 

TY  - JOUR
AB  - Many small proteins fold highly cooperatively in an all-or-none fashion and thus their native states are well protected from thermal fluctuations by an extensive network of interactions across the folded structure. Because protein structures are stabilized by local and nonlocal interactions among distal residues, dissecting individual substructures from the context of folded proteins results in large destabilization and loss of unique three-dimensional structure. Thus, mini-protein substructures can only rarely be derived from natural templates. Here, we describe a compact native 24-residues-long supersecondary structure derived from the hyperstable villin headpiece subdomain consisting of helices 2 and 3 (HP24). Using a combination of experimental techniques, including NMR and small-angle x-ray scattering, as well as all-atom replica exchange molecular-dynamics simulations, we show that a variant with stabilizing substitutions (HP24stab) forms a densely packed and compact conformation. In HP24stab, interactions between helices 2 and 3 are similar to those observed in native folded HP35, and the two helices cooperatively stabilize each other by completing the hydrophobic core lining the central part of HP35. Interestingly, even though the HP24wt fragment shows a more expanded and less structured conformation, NMR and simulations demonstrate a preference for a native-like topology. Thus, the two stabilizing residues in HP24stab shift the energy balance toward the native state, leading to a minimal folding motif.
AU  - Hocking, H.G.
AU  - Häse, F.*
AU  - Madl, T.
AU  - Zacharias, M.*
AU  - Rief, M.*
AU  - Žoldák, G.*
C1  - 43229
C2  - 36352
CY  - Cambridge
SP  - 678-686
TI  - A compact native 24-residue supersecondary structure derived from the villin headpiece subdomain.
JO  - Biophys. J.
VL  - 108
IS  - 3
PB  - Cell Press
PY  - 2015
SN  - 0006-3495
ER  - 

TY  - JOUR
AB  - The existence of membrane-rafts helps to conceptually understand the spatiotemporal organization of membrane-associated events (signaling, fusion, fission, etc.). However, as rafts themselves are nanoscopic, dynamic, and transient assemblies, they cannot be directly observed in a metabolizing cell by traditional microscopy. The observation of phase separation in giant plasma membrane-derived vesicles from live cells is a powerful tool for studying lateral heterogeneity in eukaryotic cell membranes, specifically in the context of membrane rafts. Microscopic phase separation is detectable by fluorescent labeling, followed by cooling of the membranes below their miscibility phase transition temperature. It remains unclear, however, if this lipid-driven process is tuneable in any way by interactions with proteins. Here, we demonstrate that MPP1, a member of the MAGUK family, can modulate membrane properties such as the fluidity and phase separation capability of giant plasma membrane-derived vesicles. Our data suggest that physicochemical domain properties of the membrane can be modulated, without major changes in lipid composition, through proteins such as MPP1.
AU  - Podkalicka, J.*
AU  - Biernatowska, A.*
AU  - Majkowski, M.*
AU  - Grzybek, M.
AU  - Sikorski, A.F.*
C1  - 44806
C2  - 37038
CY  - Cambridge
SP  - 2201-2211
TI  - MPP1 as a factor regulating phase separation in giant plasma membrane-derived vesicles.
JO  - Biophys. J.
VL  - 108
IS  - 9
PB  - Cell Press
PY  - 2015
SN  - 0006-3495
ER  - 

TY  - JOUR
AU  - Wettmarshausen, J.*
AU  - Cheng, Y.
AU  - Perocchi, F.
C1  - 47203
C2  - 39171
SP  - 189A
TI  - Systems approaches to mitochondrial calcium signaling.
JO  - Biophys. J.
VL  - 108
IS  - 2
PY  - 2015
SN  - 0006-3495
ER  - 

TY  - JOUR
AB  - The ribonucleoprotein (RNP) machinery of the spliceosome is composed of several subunits, which assemble stepwise during the process of splicing. U2 auxiliary factor (U2AF), a heterodimer comprised of a large (65 kDa) and a small (35 kDa) subunit, is involved in the early recognition of the intron and stabilization during splicing reactions. U2AF65 specifically recognizes the polypyrimidine tract in pre-mRNA introns and additionally contacts further splicing factors, such as mBBP/SF1 and SAP155 [1,2]. U2AF65 contains three RNA recognition motifs (RRM), where RRM1 and RRM2 mediate RNA binding while RRM3 is a U2AF homology motif (UHM) that mediates the interaction with U2AF35 [3]. A nuclear magnetic resonance spectroscopy study of recombinant human U2AF65 (RRM1-RRM2) showed a closed conformational state for the splicing factor in absence of RNA, while an open conformation is induced upon binding to polypyrimidine stretches [4]. Here, conformational subpopulations of the protein were investigated using single-pair FRET in solution with multiparameter fluorescence detection and pulsed interleaved excitation [5]. Information on FRET efficiency, stoichiometry, and lifetime revealed different conformational states dependent on substrate recognition and provided clues for dynamic motions of the splicing factor. These were further analyzed on a total internal reflection microscope using molecules immobilized in lipid vesicles. Changes in FRET efficiency over time showed a highly flexible U2AF65 protein with stabilization of specific conformational states upon RNA binding. Single-pair FRET measurements provide detailed insights into the mechanistic action of polypyrimidine tract recognition.
AU  - Voith von Voithenberg, L.*
AU  - Sanchez-Rico, C.
AU  - Warner, L.
AU  - Zhang, Y.
AU  - Sattler, M.
AU  - Lamb, D.C.*
C1  - 31754
C2  - 34756
CY  - Cambridge
SP  - 465A
TI  - Conformational dynamics during spliceosome assembly investigated by single-pair FRET.
JO  - Biophys. J.
VL  - 106
IS  - 2
PB  - Cell Press
PY  - 2014
SN  - 0006-3495
ER  - 

TY  - JOUR
AB  - A toggle switch consists of two genes that mutually repress each other. This regulatory motif is active during cell differentiation and is thought to act as a memory device, being able to choose and maintain cell fate decisions. Commonly, this switch has been modeled in a deterministic framework where transcription and translation are lumped together. In this description, bistability occurs for transcription factor cooperativity, whereas autoactivation leads to a tristable system with an additional undecided state. In this contribution, we study the stability and dynamics of a two-stage gene expression switch within a probabilistic framework inspired by the properties of the Pu/Gata toggle switch in myeloid progenitor cells. We focus on low mRNA numbers, high protein abundance, and monomeric transcription-factor binding. Contrary to the expectation from a deterministic description, this switch shows complex multiattractor dynamics without autoactivation and cooperativity. Most importantly, the four attractors of the system, which only emerge in a probabilistic two-stage description, can be identified with committed and primed states in cell differentiation. To begin, we study the dynamics of the system and infer the mechanisms that move the system between attractors using both the quasipotential and the probability flux of the system. Next, we show that the residence times of the system in one of the committed attractors are geometrically distributed. We derive an analytical expression for the parameter of the geometric distribution, therefore completely describing the statistics of the switching process and elucidate the influence of the system parameters on the residence time. Moreover, we find that the mean residence time increases linearly with the mean protein level. This scaling also holds for a one-stage scenario and for autoactivation. Finally, we study the implications of this distribution for the stability of a switch and discuss the influence of the stability on a specific cell differentiation mechanism. Our model explains lineage priming and proposes the need of either high protein numbers or long-term modifications such as chromatin remodeling to achieve stable cell fate decisions. Notably, we present a system with high protein abundance that nevertheless requires a probabilistic description to exhibit multistability, complex switching dynamics, and lineage priming.
AU  - Strasser, M.
AU  - Theis, F.J.
AU  - Marr, C.
C1  - 8028
C2  - 29967
SP  - 19-29
TI  - Stability and multiattractor dynamics of a toggle switch based on a two-stage model of stochastic gene expression.
JO  - Biophys. J.
VL  - 102
IS  - 1
PB  - Cell Press
PY  - 2012
SN  - 0006-3495
ER  - 

TY  - JOUR
AB  - Methods of blind source separation are used in many contexts to separate composite data sets according to their sources. Multiply labeled fluorescence microscopy images represent such sets, in which the sources are the individual labels. Then distributions are the quantities of interest and have to be extracted from the images. This is often challenging, since the recorded emission spectra of fluorescent dyes are environment- and instrument-specific. We have developed a nonnegative matrix factorization (NMF) algorithm to detect and separate spectrally distinct components of multiply labeled fluorescence images. It operates on spectrally resolved images and delivers both the emission spectra of the identified components and images of their abundance. We tested the proposed method using biological samples labeled with up to four spectrally overlapping fluorescent labels. In most cases, NMF accurately decomposed the images into contributions of individual dyes. However, the Solutions are not unique when spectra overlap strongly or when images are diffuse in their structure. To arrive at satisfactory results in such cases, we extended NMF to incorporate preexisting qualitative knowledge about spectra and label distributions. We show how data acquired through excitations at two or three different wavelengths can be integrated and that multiple excitations greatly facilitate the decomposition. By allowing reliable decomposition in cases where the spectra of the individual labels are not known or are known only inaccurately, the proposed algorithms greatly extend the range of questions that can be addressed with quantitative microscopy.
AU  - Neher, R.A.*
AU  - Mitkovski, M.*
AU  - Kirchhoff, F.*
AU  - Neher, E.*
AU  - Theis, F.J.
AU  - Zeug, A.*
C1  - 2088
C2  - 27002
SP  - 3791-3800
TI  - Blind source separation techniques for the decomposition of multiply labeled fluorescence images.
JO  - Biophys. J.
VL  - 96
IS  - 9
PB  - Elsevier Science
PY  - 2009
SN  - 0006-3495
ER  - 

TY  - JOUR
AB  - The role of the different cytoskeletal structures like microfilaments (MF), microtubuli (MT), and intermediate filaments (IF) in phagosome motion is unclear. These cytoskeletal units play an important role in macrophage function (migration, phagocytosis, phagosome transport). We investigated ferromagnetic phagosome motions by cell magnetometry. J774A.1 macrophages were incubated with 1.3-microm spherical magnetite particles for 24 h, after which more than 90% of the particles had been phagocytized. Phagosome motions can be caused either by the cell itself (relaxation) or by applying magnetic twisting forces, yielding cell stiffness and viscoelastic properties of the cytoskeleton. Apparent viscosity of the cytoplasm was non-Newtonian and showed a shear-rate-dependent power law behavior. Elastically stored energy does not force the magnetic phagosomes back to their initial orientation: 57% of the twisting shear was not recoverable. Cytoskeletal drugs, like Cytochalasin D (CyD, 2 - 4 microM), Colchicine (CoL, 10 microM), or Acrylamide (AcL, 40 mM) were added in order to disturb the different cytoskeletal structures. AcL disintegrates IF, but affected neither stochastic (relaxation) nor directed phagosome motions. CyD disrupts MF, resulting in a retarded stochastic phagosome motion (relative decay 0.53 +/- 0.01 after 5 min versus 0.34 +/- 0.01 in control), whereas phagosome twisting shows only a small response with a 9% increase of stiffness and a small reduction of recoverable strain. CoL depolymerizes the MT, inducing a moderately accelerated relaxation (relative decay 0.28 +/- 0.01 after 5 min) and a 10% increase of cell stiffness, where the pure viscous shear is increased and the viscoelastic recoil is inhibited by 40%. Combining the two drugs conserves both effects. After disintegrating either MF or MT, phagosome motion and cytoskeletal stiffness reflect the behavior of either MT or MF, respectively. The results verify that the dominant phagosome transport mechanism is MF-associated. MT depolymerization by CoL induces an activation of the F-actin synthesis, which may induce an accelerated relaxation and an increase of stiffness. Cell mechanical properties are not modulated by MF depolymerization, whereas MT depolymerization causes a loss of viscous resistance and a loss of cell elasticity. The mean energy for stochastic phagosome transport is 5*10(-18) Joules and corresponds to a force of 7 pN on a single 1.3-microm phagosome.
AU  - Möller, W.
AU  - Nemoto, I.*
AU  - Matsuzaki, T.*
AU  - Hofer, T.P.
AU  - Heyder, J.
C1  - 24072
C2  - 31418
SP  - 720-730
TI  - Magnetic phagosome motion in J774A.1 macrophages: Influence of cytoskeletal drugs.
JO  - Biophys. J.
VL  - 79
IS  - 2
PB  - Cell Press
PY  - 2000
SN  - 0006-3495
ER  - 

TY  - JOUR
AB  - The degree of depolarization of fluorescent light emitted from an organic dye, which is used as molecular probe, is a powerful tool in probing the microenvironment. By fluorescence depolarization the macromolecular structure can be investigated as well as the mobility of the marker molecule itself or of the complex found by the probe. Additional information such as energy transfer rates, donor-acceptor distances, and orientations are also measurable. These data are of particular interest if they can be measured from whole cells. Using flow cytometry, we can analyze a large number of cells with high statistical significance in a short period of time. We describe a newly developed double-beam epi-illumination arrangement for fluorescence polarization measurements that uses an autocompensation technique. This new technique permits the various depolarizing effects within the optical as well as the electronic components of the system to be continually compensated for on a cell by cell basis. Simultaneous measurements of other cell parameters for cell cycle analysis by total fluorescence intensity remains possible. The sensitivity of the system to measure polarization was determined as ± 0.006 p (0≤p≤0.5 in isotropic media), which amounts to ± 1.2% of the maximum p value. Polarization data for latex microspheres plotted in the histogram mode were measured with a standard deviation of 0.006, which proved the high resolution and the high performance of the system.
AU  - Beisker, W.
AU  - Eisert, W.G.
C1  - 33133
C2  - 35559
SP  - 607-612
TI  - Double-beam autocompensation for fluorescence polarization measurements in flow cytometry.
JO  - Biophys. J.
VL  - 47
IS  - 5
PY  - 1985
SN  - 0006-3495
ER  - 

TY  - JOUR
AB  - An epi-illumination design for fluorescence polarization measurements is introduced in flow cytometry with the optical axis orthogonally aligned to the cell stream. Various optical components and designs are discussed with respect to their influence on polarization measurements. Using the epi-configuration, paired measurements with the direction of polarization of the exciting light changed orthogonally are proposed for the compensation of system anisotropies and electronic mismatch. Large aperture corrections are employed for the excitation as well as for the emission pathway. Additional parameters such as fluorescence at 90°, multiangle light scattering, and high precision cell sizing by internally calibrated time of the flight measurements, as described previously, remain available with the design proposed here. Fluorescent latex microspheres, stained intracellular DNA, and algae have been used to test performance.
AU  - Eisert, W.G.
AU  - Beisker, W.
C1  - 41592
C2  - 38835
SP  - 97-112
TI  - Epi-illumination optical design for fluorescence polarization measurements in flow systems.
JO  - Biophys. J.
VL  - 31
IS  - 1
PY  - 1980
SN  - 0006-3495
ER  -