Structural transitions in the GTP cap visualized by cryo-EM of catalytically inactive microtubules

Microtubules (MTs) are polymers of alphabeta-tubulin heterodimers that stochastically switch between growth and shrinkage phases. This dynamic instability is critically important for MT function. It is believed that GTP hydrolysis within the MT lattice is accompanied by destabilizing conformational changes, and that MT stability depends on a transiently existing GTP cap at the growing MT end. Here we use cryo-EM and TIRF microscopy of GTP hydrolysis-deficient MTs assembled from mutant recombinant human tubulin to investigate the structure of a GTP-bound MT lattice. We find that the GTP-MT lattice of two mutants in which the catalytically active glutamate in α-tubulin was substituted by inactive amino acids (E254A and E254N) is remarkably plastic. Undecorated E254A and E254N MTs with 13 protofilaments both have an expanded lattice, but display opposite protofilament twists, making these lattices distinct from the compacted lattice of wildtype GDP-MTs. End binding proteins of the EB family have the ability to compact both mutant GTP-lattices and to stabilize a negative twist, suggesting that they promote this transition also in the GTP cap of wildtype MTs, thereby contributing to the maturation of the MT structure. We also find that the MT seam appears to be stabilized in mutant GTP-MTs and destabilized in GDP-MTs, supporting the proposal that the seam plays an important role in MT stability. Together, these first high-resolution structures of truly GTP-bound MTs add mechanistic insight to our understanding of MT dynamic instability.

Two step aggregation kinetics of Amyloid-β proteins from fractal analysis

Self-aggregation in proteins has long been studied and modeled due to its ubiquity and importance in many biological contexts. Several models propose a two step aggregation mechanism, consisting of linear growth of fibrils and branch formation. Single molecule imaging techniques such as total internal reflection fluorescence (TIRF) microscopy can provide direct evidence of such mechanisms, however, analyzing such large datasets is challenging. In this paper, we analyze for the first time, images of growing amyloid fibrils obtained from TIRF microscopy using the techniques of fractal geometry, which provides a natural framework to disentangle the two types of growth mechanisms at play. We find that after an initial linear growth phase, identified by a plateau in the average fractal dimension with time, the occurrence of branching events leads to a further increase in the fractal dimension with a final saturation value ≈ 2. We also simulate the aggregation process using the identified linear growth and secondary nucleation mechanisms, using an event driven algorithm. We theoretically model this system using a set of coupled nonlinear differential equations describing a mean field model for branching and linear growth, which we use to characterize the growth process observed in simulations as well as experiments. Finally, we provide estimates for the parameter regimes that govern the two step aggregation process observed in experiments.

Endothelial Focal Adhesions Are Functional Obstacles for Leukocytes During Basolateral Crawling

An inflammatory response requires leukocytes to migrate from the circulation across the vascular lining into the tissue to clear the invading pathogen. Whereas a lot of attention is focused on how leukocytes make their way through the endothelial monolayer, it is less clear how leukocytes migrate underneath the endothelium before they enter the tissue. Upon finalization of the diapedesis step, leukocytes reside in the subendothelial space and encounter endothelial focal adhesions. Using TIRF microscopy, we show that neutrophils navigate around these focal adhesions. Neutrophils recognize focal adhesions as physical obstacles and deform to get around them. Increasing the number of focal adhesions by silencing the small GTPase RhoJ slows down basolateral crawling of neutrophils. However, apical crawling and diapedesis itself are not affected by RhoJ depletion. Increasing the number of focal adhesions drastically by expressing the Rac1 GEF Tiam1 make neutrophils to avoid migrating underneath these Tiam1-expressing endothelial cells. Together, our results show that focal adhesions mark the basolateral migration path of neutrophils.

Large field-of-view nanometer-sectioning microscopy by using metal-induced energy transfer and biexponential lifetime analysis

Total internal reflection fluorescence (TIRF) microscopy, which has about 100-nm axial excitation depth, is the method of choice for nanometer-sectioning imaging for decades. Lately, several new imaging techniques, such as variable angle TIRF microscopy, supercritical-angle fluorescence microscopy, and metal-induced energy transfer imaging, have been proposed to enhance the axial resolution of TIRF. However, all of these methods use high numerical aperture (NA) objectives, and measured images inevitably have small field-of-views (FOVs). Small-FOV can be a serious limitation when multiple cells need to be observed. We propose large-FOV nanometer-sectioning microscopy, which breaks the complementary relations between the depth of focus and axial sectioning by using MIET. Large-FOV imaging is achieved with a low-magnification objective, while nanometer-sectioning is realized utilizing metal-induced energy transfer and biexponential fluorescence lifetime analysis. The feasibility of our proposed method was demonstrated by imaging nanometer-scale distances between the basal membrane of human aortic endothelial cells and a substrate.