Alan R. Lowe

Selectivity mechanism of the nuclear pore complex characterized by single cargo tracking

The nuclear pore complex (NPC) mediates all exchange between the cytoplasm and the nucleus. Small molecules can passively diffuse through the NPC, whereas larger cargos require transport receptors to translocate. How the NPC facilitates the translocation of transport receptor/cargo complexes remains unclear. To investigate this process, we tracked single protein-functionalized quantum dot cargos as they moved through human NPCs. Here we show that import proceeds by successive substeps comprising cargo capture, filtering and translocation, and release into the nucleus. Most quantum dots are rejected at one of these steps and return to the cytoplasm, including very large cargos that abort at a size-selective barrier. Cargo movement in the central channel is subdiffusive and cargos that can bind more transport receptors diffuse more freely. Without Ran GTPase, a critical regulator of transport directionality, cargos still explore the entire NPC, but have a markedly reduced probability of exit into the nucleus, suggesting that NPC entry and exit steps are not equivalent and that the pore is functionally asymmetric to importing cargos. The overall selectivity of the NPC seems to arise from the cumulative action of multiple reversible substeps and a final irreversible exit step.

Rational redesign of the folding pathway of a modular protein

The modular structures of repeat proteins afford them distinct properties compared with globular proteins, enabling them to function in a large and diverse range of cellular processes. Here, we show that they can also have different folding mechanisms. Myotrophin comprises four ankyrin repeats stacked linearly to form an elongated structure. Using site-directed mutagenesis, we find that folding of wild-type myotrophin is initiated at the C-terminal repeats. However, close examination of the mutant chevron plots reveals that simple models are insufficient to describe all of the data, and double mutant analysis subsequently confirms that there are parallel folding pathways. Destabilizing mutations in the C-terminal repeats reduce flux through the wild-type pathway, making a new route accessible in which folding is initiated at the N-terminal repeats. Thus, the folding mechanism of the repeat protein is poised on a fulcrum: When one end of the molecule is perturbed, the balance shifts between the different nucleation sites. The vast majority of studies on small globular proteins indicate a single, well defined route between the denatured and native states. By contrast, the potential to initiate folding at more than one site may be a general feature of repeat proteins arising from the symmetry inherent in their structures. We show that this simple architecture makes it straightforward to direct the folding pathway of a repeat protein by design.

Importin-β modulates the permeability of the nuclear pore complex in a Ran-dependent manner

Soluble karyopherins of the importin-β (impβ) family use RanGTP to transport cargos directionally through the nuclear pore complex (NPC). Whether impβ or RanGTP regulate the permeability of the NPC itself has been unknown. In this study, we identify a stable pool of impβ at the NPC. A subpopulation of this pool is rapidly turned-over by RanGTP, likely at Nup153. Impβ, but not transportin-1 (TRN1), alters the pores permeability in a Ran-dependent manner, suggesting that impβ is a functional component of the NPC. Upon reduction of Nup153 levels, inert cargos more readily equilibrate across the NPC yet active transport is impaired. When purified impβ or TRN1 are mixed with Nup153 in vitro, higher-order, multivalent complexes form. RanGTP dissolves the impβ•Nup153 complexes but not those of TRN1•Nup153. We propose that impβ and Nup153 interact at the NPCs nuclear face to form a Ran-regulated mesh that modulates NPC permeability. DOI: http://dx.doi.org/10.7554/eLife.04052.001

Cyanobacteria use micro-optics to sense light direction

Bacterial phototaxis was first recognized over a century ago, but the method by which such small cells can sense the direction of illumination has remained puzzling. The unicellular cyanobacterium Synechocystis sp. PCC 6803 moves with Type IV pili and measures light intensity and color with a range of photoreceptors. Here, we show that individual Synechocystis cells do not respond to a spatiotemporal gradient in light intensity, but rather they directly and accurately sense the position of a light source. We show that directional light sensing is possible because Synechocystis cells act as spherical microlenses, allowing the cell to see a light source and move towards it. A high-resolution image of the light source is focused on the edge of the cell opposite to the source, triggering movement away from the focused spot. Spherical cyanobacteria are probably the world’s smallest and oldest example of a camera eye. DOI: http://dx.doi.org/10.7554/eLife.12620.001

Mapping the Topography of a Protein Energy Landscape

Protein energy landscapes are highly complex, yet the vast majority of states within them tend to be invisible to experimentalists. Here, using site-directed mutagenesis and exploiting the simplicity of tandem-repeat protein structures, we delineate a network of these states and the routes between them. We show that our target, gankyrin, a 226-residue 7-ankyrin-repeat protein, can access two alternative (un)folding pathways. We resolve intermediates as well as transition states, constituting a comprehensive series of snapshots that map early and late stages of the two pathways and show both to be polarized such that the repeat array progressively unravels from one end of the molecule or the other. Strikingly, we find that the protein folds via one pathway but unfolds via a different one. The origins of this behavior can be rationalized using the numerical results of a simple statistical mechanics model that allows us to visualize the equilibrium behavior as well as single-molecule folding/unfolding trajectories, thereby filling in the gaps that are not accessible to direct experimental observation. Our study highlights the complexity of repeat-protein folding arising from their symmetrical structures; at the same time, however, this structural simplicity enables us to dissect the complexity and thereby map the precise topography of the energy landscape in full breadth and remarkable detail. That we can recapitulate the key features of the folding mechanism by computational analysis of the native structure alone will help toward the ultimate goal of designed amino-acid sequences with made-to-measure folding mechanisms-the Holy Grail of protein folding.

A Liquid to Solid Phase Transition Underlying Pathological Huntingtin Exon1 Aggregation

Summary Huntington’s disease is caused by an abnormally long polyglutamine tract in the huntingtin protein. This leads to the generation and deposition of N-terminal exon1 fragments of the protein in intracellular aggregates. We combined electron tomography and quantitative fluorescence microscopy to analyze the structural and material properties of huntingtin exon1 assemblies in mammalian cells, in yeast, and in vitro. We found that huntingtin exon1 proteins can form reversible liquid-like assemblies, a process driven by huntingtin’s polyQ tract and proline-rich region. In cells and in vitro, the liquid-like assemblies converted to solid-like assemblies with a fibrillar structure. Intracellular phase transitions of polyglutamine proteins could play a role in initiating irreversible pathological aggregation.

Local cellular neighborhood controls proliferation in cell competition

Cell competition is a quality-control mechanism through which tissues eliminate unfit cells. Automated microscopy with deep-learning image analysis was used to measure single-cell behavior during competition. Strikingly, the single-cell analysis reveals that tissue-scale population shifts are strongly affected by cellular-scale tissue organization.

Context-Dependent Energetics of Loop Extensions in a Family of Tandem-Repeat Proteins

Consensus-designed tetratricopeptide repeat proteins are highly stable, modular proteins that are strikingly amenable to rational engineering. They therefore have tremendous potential as building blocks for biomaterials and biomedicine. Here, we explore the possibility of extending the loops between repeats to enable further diversification, and we investigate how this modification affects stability and folding cooperativity. We find that extending a single loop by up to 25 residues does not disrupt the overall protein structure, but, strikingly, the effect on stability is highly context-dependent: in a two-repeat array, destabilization is relatively small and can be accounted for purely in entropic terms, whereas extending a loop in the middle of a large array is much more costly because of weakening of the interaction between the repeats. Our findings provide important and, to our knowledge, new insights that increase our understanding of the structure, folding, and function of natural repeat proteins and the design of artificial repeat proteins in biotechnology.

PyFolding: An open-source software package for graphing, analysis and simulation of thermodynamic and kinetic models of protein folding

Our understanding of how proteins find and adopt their functional three-dimensional structure has largely arisen through experimental studies of the denaturant-and primary sequence-dependence of protein stability and the kinetics of folding. For many years, curve fitting software packages have been heavily utilized to fit simple models to these data. Although such software packages are easy to use for simple functions, they are often expensive and provide substantial impediments to applying more complex models or for the analysis of large datasets. Moreover, over the past decade, increasingly sophisticated analytical models have been generated, but without simple tools to enable routine analysis. Consequently, users have needed to generate their own tools or otherwise find willing collaborators. Here we present PyFolding, a free, open source, and extensible Python framework for the analysis and modeling of experimental protein folding and thermodynamic data. To demonstrate the utility of PyFolding, we provide examples of complex analysis: (i) multi-phase kinetic folding data fitted to linked equations and (ii) thermodynamic equilibrium data from consensus designed repeat proteins to both homo-and heteropolymer variants of the Ising model. Example scripts to perform these and other operations are supplied with the software. Further, we show that PyFolding can be used in conjunction with Jupyter notebooks as an easy way to share methods and analysis for publication and amongst research teams.

PyFolding: An open-source software package for graphing, simulation and analysis of the biophysical properties of proteins

For many years, curve fitting software has been heavily utilized to fit simple models to various types of biophysical data. Although such software packages are easy to use for simple functions, they are often expensive and present substantial impediments to applying more complex models or for the analysis of large datasets. One field that is relient on such data analysis is the thermodynamics and kinetics of protein folding. Over the past decade, increasingly sophisticated analytical models have been generated, but without simple tools to enable routine analysis. Consequently, users have needed to generate their own tools or otherwise find willing collaborators. Here we present PyFolding, a free, open source, and extensible Python framework for graphing, analysis and simulation of the biophysical properties of proteins. To demonstrate the utility of PyFolding, we have used it to analyze and model experimental protein folding and thermodynamic data. Examples include: (i) multi-phase kinetic folding fitted to linked equations, (ii) global fitting of multiple datasets and (iii) analysis of repeat protein thermodynamics with Ising model variants. Moreover, we demonstrate how Pyfolding is easily extensible to novel functionality beyond applications in protein folding via the addition of new models. Example scripts to perform these and other operations are supplied with the software, and we encourage users to contribute notebooks and models to create a community resource. Finally, we show that PyFolding can be used in conjunction with Jupyter notebooks as an easy way to share methods and analysis for publication and amongst research teams.