Time-resolved fluorescence anisotropy with Atto 488-labeled phytochrome Agp1 from Agrobacterium fabrum.

Phytochromes are photoreceptor proteins with a bilin chromophore that undergo photoconversion between two spectrally different forms, Pr and Pfr. Three domains, termed PAS, GAF, and PHY domains, constitute the N-terminal photosensory chromophore module (PCM); the C-terminus is often a histidine kinase module. In the Agrobacterium fabrum phytochrome Agp1, the autophosphorylation activity of the histidine kinase is high in the Pr and low in the Pfr form. Crystal structure analyses of PCMs suggest flexibility around position 308 in the Pr but not in the Pfr form. Here, we performed time-resolved fluorescence anisotropy measurements with different Agp1 mutants, each with a single cysteine residue at various positions. The fluorophore label Atto-488 was attached to each mutant, and time-resolved fluorescence anisotropy was measured in the Pr and Pfr forms. Fluorescence anisotropy curves were fitted with biexponential functions. Differences in the amplitude A2 of the second component between the PCM and the full-length variant indicate a mechanical coupling between position 362 and the histidine kinase. Pr-to-Pfr photoconversion induced no significant changes in the time constant t2 at any position. An intermediate t2 value at position 295, which is located in a compact environment, suggests flexibility around the nearby position 308 in Pr and in Pfr.

In Vitro Reconstitution of the Endoplasmic Reticulum.

Reconstitution of cellular organelles in vitro offers the possibility to perform quantitative and qualitative experiments in a controlled environment that cannot be done with the same accuracy in living cells. Following a previous report, the subsequent list of protocols describes how to reconstitute and quantify a tubular ER network in vitro based on purified microsomes from culture cells and cytosol from Xenopus laevis egg extracts. Biological material preparation and reconstitution assays require mostly basic laboratory instrumentation and chemicals, and can be executed without any specific training, making them appealing to a wide range of laboratories. Moreover, to promote conditions that are markedly more reflective of in vivo environments, this method describes for the first time in the literature, the purification of microsomes from HeLa cells in some detail. Basic Protocol 1 in this article describes the reconstitution process on different substrates including the associated fluorescence imaging process. Purification of ER microsomes and cytosol, both of which are needed for this approach, are described in detail in Support Protocols 1 and 2, respectively. Coating of surfaces with polyacrylamide gels is described in Support Protocol 3. Basic Protocol 2 outlines how to segment and skeletonize fluorescence images of ER networks, and how to quantify segment lengths between the network's branching points. The described quantitative evaluation provides a meaningful approach to analyze the topology and geometry of organelle structures. © 2017 by John Wiley & Sons, Inc.

A far-red emitting fluorescent marker protein, mGarnet2, for microscopy and STED nanoscopy.

Here we present mGarnet2, a monomeric, far-red fluorescent marker protein derived from mRuby, with absorption and emission bands peaking at 598 and 671 nm, respectively. The protein shows excellent performance as a live-cell fusion marker for STED nanoscopy with 640 nm excitation and 780 nm depletion wavelengths.

Engineering and Design of Polymeric Shells: Inwards Interweaving Polymers as Multilayer Nanofilm, Immobilization Matrix, or Chromatography Resins.

Hydrogels with complex internal structures are required for advanced drug delivery systems and tissue engineering or used as inks for 3D printing. However, hydrogels lack the tunability and diversity of polymeric shells and require complicated postsynthesis steps to alter its structure or properties. We report on the first integrated approach to assemble and design polymeric shells to take on various complex structures and functions such as multilayer nanofilms, multidensity immobilization matrix, or multiadhesive chromatography resins via the tuning of four assembly parameters: (a) poly(allylamine) (PA) concentration, (b) number of poly(allylamine)/poly(styrenesulfonic acid) (PA/PSSA) incubations, (c) poly(allylamine) (PA) to poly(ethylene glycol) (PEG) grafting ratio, and (d) % H2O present during assembly. Our approach combines the complex 3D structures of hydrogels with the versatility of self-assembled polymeric layers. Polymeric shells produced from our method have a highly uniform material distribution and well-defined shell boundaries. Shell thickness, density, and adhesive properties are easily tunable. By virtue of such unique material features, we demonstrate that polymeric shells can be designed to expand beyond its conventional function as thin films and serve as immobilization matrix, chromatography resins, or even reaction compartments. This technique could also uncover interesting perspectives in the development of novel multimaterials for 3D printing to synthesize scaffolds at a higher order of complexity.

Shaping the endoplasmic reticulum in vitro.

Organelles in eukaryotic cells often have complex shapes that deviate significantly from simple spheres. A prime example is the endoplasmic reticulum (ER) that forms an extensive network of membrane tubules in many mammalian cell types and in reconstitution assays in vitro. Despite the successful hunt for molecular determinants of ER shape we are still far from having a comprehensive understanding of ER network morphogenesis. Here, we have studied the hitherto neglected influence of the host substrate when reconstituting ER networks in vitro as compared to ER networks in vivo. In culture cells we observed cytoplasm-spanning ER networks with tubules being connected almost exclusively by three-way junctions and segment lengths being narrowly distributed around a mean length of about 1µm. In contrast, networks reconstituted from purified ER microsomes on flat glass or gel substrates of varying stiffness showed significantly broader length distributions with an up to fourfold larger mean length. Self-assembly of ER microsomes on small oil droplets, however, yielded networks that resembled more closely the native ER network of mammalian cells. We conclude from these observations that the ER microsomes' inherent self-assembly capacity is sufficient to support network formation with a native geometry if the influence of the host substrate's surface chemistry becomes negligible. We hypothesize that under these conditions the networks' preference for three-way junctions follows from creating 'starfish-shaped' vesicles when ER microsomes with a protein-induced spontaneous curvature undergo fusion.

Effects of protein crowding on membrane systems.

Cellular membranes are typically decorated with a plethora of embedded and adsorbed macromolecules, e.g. proteins, that participate in numerous vital processes. With typical surface densities of 30,000 proteins per µm(2) cellular membranes are indeed crowded places that leave only few nanometers of private space for individual proteins. Here, we review recent advances in our understanding of protein crowding in membrane systems. We first give a brief overview on state-of-the-art approaches in experiment and simulation that are frequently used to study crowded membranes. After that, we review how crowding can affect diffusive transport of proteins and lipids in membrane systems. Next, we discuss lipid and protein sorting in crowded membrane systems, including effects like protein cluster formation, phase segregation, and lipid droplet formation. Subsequently, we highlight recent progress in uncovering crowding-induced conformational changes of membranes, e.g. membrane budding and vesicle formation. Finally, we give a short outlook on potential future developments in the field of crowded membrane systems. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg.

The molecular size of the extra-membrane domain influences the diffusion of the GPI-anchored VSG on the trypanosome plasma membrane.

A plethora of proteins undergo random and passive diffusion in biological membranes. While the contribution of the membrane-embedded domain to diffusion is well established, the potential impact of the extra-membrane protein part has been largely neglected. Here, we show that the molecular length influences the diffusion coefficient of GPI-anchored proteins: smaller proteins diffuse faster than larger ones. The distinct diffusion properties of differently sized membrane proteins are biologically relevant. The variant surface glycoprotein (VSG) of African trypanosomes, for example, is sized for an effective diffusion-driven randomization on the cell surface, a process that is essential for parasite virulence. We propose that the molecular sizes of proteins dominating the cell surfaces of other eukaryotic pathogens may also be related to diffusion-limited functions.

Membrane protein mobility depends on the length of extra-membrane domains and on the protein concentration.

Diffusion of membrane proteins is not only determined by the membrane anchor friction but also by the overall concentration of proteins and the length of their extra-membrane domains. We have studied the influence of the latter two cues by mesoscopic simulations. As a result, we have found that the total friction of membrane proteins, γ, increases approximately linearly with the length of the extra-membrane domain, L, whereas a slightly nonlinear dependence on the total protein concentration, ϕ was observed. We provide an educated guess for the functional form of γ(L, ϕ) and the associated diffusion coefficient. This expression not only matches our simulation data but it is also in favorable agreement with previously published experimental data. Our findings indicate that diffusion coefficients of membrane proteins are not solely determined by the friction of membrane anchors but also extra-membrane domains and the crowdedness of the membrane need to be considered to obtain a comprehensive view of protein diffusion on cellular membranes.

Cholesterol and sphingomyelin drive ligand-independent T-cell antigen receptor nanoclustering.

The T-cell antigen receptor (TCR) exists in monomeric and nanoclustered forms independently of antigen binding. Although the clustering is involved in the regulation of T-cell sensitivity, it is unknown how the TCR nanoclusters form. We show that cholesterol is required for TCR nanoclustering in T cells and that this clustering enhances the avidity but not the affinity of the TCR-antigen interaction. Investigating the mechanism of the nanoclustering, we found that radioactive photocholesterol specifically binds to the TCRß chain in vivo. In order to reduce the complexity of cellular membranes, we used a synthetic biology approach and reconstituted the TCR in liposomes of defined lipid composition. Both cholesterol and sphingomyelin were required for the formation of TCR dimers in phosphatidylcholine-containing large unilamellar vesicles. Further, the TCR was localized in the liquid disordered phase in giant unilamellar vesicles. We propose a model in which cholesterol and sphingomyelin binding to the TCRß chain causes TCR dimerization. The lipid-induced TCR nanoclustering enhances the avidity to antigen and thus might be involved in enhanced sensitivity of memory compared with naive T cells. Our work contributes to the understanding of the function of specific nonannular lipid-membrane protein interactions.

Exploring membrane and protein dynamics with dissipative particle dynamics.

In this chapter, we review recent approaches and results when studying membrane and protein dynamics by means of dissipative particle dynamics (DPD). First, we introduce and discuss DPD as a method, for example, the choice of the thermostat, which is of interest when constructing a DPD code. Then, we review important results on pure membranes and lipid-water systems that have been obtained with DPD. Finally, we focus on simulations of membranes with associated or embedded model proteins that may trigger future research on the fundamental interactions of lipids and proteins in the context of living cells.

Dynamic structure formation of peripheral membrane proteins.

Using coarse-grained membrane simulations we show here that peripheral membrane proteins can form a multitude of higher-order structures due to membrane-mediated interactions. Peripheral membrane proteins characteristically perturb the lipid bilayer in their vicinity which supports the formation of protein assemblies not only within the same but surprisingly also across opposing leaflets of a bilayer. In addition, we also observed the formation of lipid-protein domains on heteregeneous membranes. The clustering ability of proteins, as quantified via the potential of mean force, is enhanced when radius and hydrophobic penetration depth of the proteins increases. Based on our data, we propose that membrane-mediated cluster formation of peripheral proteins supports protein assembly in vivo and hence may play a pivotal role in the formation of templates for signaling cascades and in the emergence of transport intermediates in the secretory pathway.

Cluster formation of transmembrane proteins due to hydrophobic mismatching.

Membranes are the defining envelopes of living cells. At this boundary a multitude of transmembrane proteins mediate signal and mass transfer between cells and their environment. Clustering of these proteins is a frequent and often vital phenomenon that relies at least in part on membrane-mediated interactions. Indeed, the mismatch between proteins' hydrophobic transmembrane domains and the surrounding lipid bilayer has been predicted to facilitate clustering, yet unequivocal quantitative data in support of these predictions have been lacking. Here, we have used coarse-grained membrane simulations to thoroughly address the clustering of transmembrane proteins in detail. Our results emphasize the universal nature of membrane-mediated attraction which relaxes the need for a plethora of fine-tuned interactions between membrane proteins.

Influence of hydrophobic mismatching on membrane protein diffusion.

The observation of membrane domains in vivo and in vitro has triggered a renewed interest in the size-dependent diffusion of membrane inclusions (e.g., clusters of transmembrane proteins and lipid rafts). Here, we have used coarse-grained membrane simulations to quantify the influence of a hydrophobic mismatch between the inclusion's transmembrane portion and the surrounding lipid bilayer on the diffusive mobility of the inclusion. Our data indicate only slight changes in the mobility (<30%) when altering the hydrophobic mismatch, and the scaling of the diffusion coefficient D is most consistent with previous hydrodynamic predictions, i.e., with the Saffman-Delbruck relation and the edgewise motion of a thin disk in the limit of small and large radii, respectively.

Sampling the cell with anomalous diffusion - the discovery of slowness.

Diffusion-mediated searching for interaction partners is an ubiquitous process in cell biology. Transcription factors, for example, search specific DNA sequences, signaling proteins aim at interacting with specific cofactors, and peripheral membrane proteins try to dock to membrane domains. Brownian motion, however, is affected by molecular crowding that induces anomalous diffusion (so-called subdiffusion) of proteins and larger structures, thereby compromising diffusive transport and the associated sampling processes. Contrary to the naive expectation that subdiffusion obstructs cellular processes, we show here by computer simulations that subdiffusion rather increases the probability of finding a nearby target. Consequently, important events like protein complex formation and signal propagation are enhanced as compared to normal diffusion. Hence, cells indeed benefit from their crowded internal state and the associated anomalous diffusion.

The degree of macromolecular crowding in the cytoplasm and nucleoplasm of mammalian cells is conserved.

Macromolecular crowding provides the cytoplasm and the nucleoplasm with strongly viscoelastic properties and renders the diffusion of soluble proteins in both fluids anomalous. Here, we have determined the nanoscale viscoelasticity of the cytoplasm and the nucleoplasm in different mammalian cell lines. In contrast to the cell-specific response on the macroscale the nanoscale viscoelasticity (i.e. the behavior on length scales about 100-fold smaller than the cell size) only showed minor variations between different cell types. Similarly, the associated anomalous diffusion properties varied only slightly. Our results indicate a conserved state of macromolecular crowding in both compartments for a variety of mammalian cells with the cytoplasm being somewhat more crowded than the nucleus.

Probing the nanoscale viscoelasticity of intracellular fluids in living cells.

We have used fluorescence correlation spectroscopy to determine the anomalous diffusion properties of fluorescently tagged gold beads in the cytoplasm and the nucleus of living cells. From the extracted mean-square displacement v(tau) approximately tau(alpha), we have determined the complex shear modulus G(omega) approximately omega(alpha) for both compartments. Without treatment, all tested cell lines showed a strong viscoelastic behavior of the cytoplasm and the nucleoplasm, highlighting the crowdedness of these intracellular fluids. We also found a similar viscoelastic response in frog egg extract, which tended toward a solely viscous behavior upon dilution. When cells were osmotically stressed, the diffusion became less anomalous and the viscoelastic response changed. In particular, the anomality changed from alpha approximately 0.55 to alpha approximately 0.66, which indicates that the Zimm model for polymer solutions under varying solvent conditions is a good empirical description of the material properties of the cytoplasm and the nucleoplasm. Since osmotic stress may eventually trigger cell death, we propose, on the basis of our observations, that intracellular fluids are maintained in a state similar to crowded polymer solutions under good solvent conditions to keep the cell viable.

Size-dependent diffusion of membrane inclusions.

Experimentally determined diffusion constants are often used to elucidate the size and oligomeric state of membrane proteins and domains. This approach critically relies on the knowledge of the size-dependence of diffusion. We have used mesoscopic simulations to thoroughly quantify the size-dependent diffusion properties of membrane inclusions. For small radii R, we find that the lateral diffusion coefficient D is well described by the Saffman-Delbrück relation, which predicts a logarithmic decrease of D with R. However, beyond a critical radius Rc approximately hetam/(2etac) (h, bilayer thickness; etam/c, viscosity of the membrane/surrounding solvent) we observe significant deviations and the emergence of an asymptotic scaling D approximately 1/R2. The latter originates from the asymptotic hydrodynamics and the inclusion's internal degrees of freedom that become particularly relevant on short timescales. In contrast to the lateral diffusion, the size dependence of the rotational diffusion constant Dr follows the predicted hydrodynamic scaling Dr approximately 1/R2 over the entire range of sizes studied here.