Nanobody Mediated Macromolecular Crowding Induces Membrane Fission and Remodeling in the African Trypanosome

The dense Variant Surface Glycoprotein (VSG) coat of African trypanosomes represents the primary host-pathogen interface. Antigenic variation prevents clearing of the pathogen by employing a large repertoire of antigenically distinct VSG genes, thus neutralizing the host’s antibody response. To explore the epitope space of VSGs, we generated anti-VSG nanobodies and combined high-resolution structural analysis of VSG-nanobody complexes with binding assays on living cells, revealing that these camelid antibodies bind deeply inside the coat. One nanobody caused rapid loss of cellular motility, possibly due to blockage of VSG mobility on the coat, whose rapid endo- and exocytosis is mechanistically linked to T. brucei propulsion and whose density is required for survival. Electron microscopy studies demonstrated that this loss of motility was accompanied by rapid formation and shedding of nanovesicles and nanotubes, suggesting that increased protein crowding on the dense membrane can be a driving force for membrane fission in living cells.

Motor guidance by long-range communication through the microtubule highway

Coupling of motor proteins within arrays drives muscle contraction, flagellar beating, chromosome segregation, and other biological processes. Current models of motor coupling invoke either direct mechanical linkage or protein crowding, which rely on short-range motor-motor interactions. In contrast, coupling mechanisms that act at longer length scales remain largely unexplored. Here we report that microtubules can physically couple motor movement in the absence of short-range interactions. The human kinesin-4 Kif4A changes the run-length and velocity of other motors on the same microtubule in the dilute binding limit, when 10-nm-sized motors are separated by microns. This effect does not depend on specific motor-motor interactions because similar changes in Kif4A motility are induced by kinesin-1 motors. A micron-scale attractive interaction potential between motors is sufficient to recreate the experimental results in a computational model. Unexpectedly, our theory suggests that long-range microtubule-mediated coupling not only affects binding kinetics but also motor mechanochemistry. Therefore, motors can sense and respond to motors bound several microns away on a microtubule. These results suggest a paradigm in which the microtubule lattice, rather than being merely a passive track, is a dynamic medium responsive to binding proteins to enable new forms of collective motor behavior.

Protein crowding mediates membrane remodeling in upstream ESCRT-induced formation of intraluminal vesicles

As part of the lysosomal degradation pathway, the endosomal sorting complexes required for transport (ESCRT-0 to -III/VPS4) sequester receptors at the endosome and simultaneously deform the membrane to generate intraluminal vesicles (ILVs). Whereas ESCRT-III/VPS4 have an established function in ILV formation, the role of upstream ESCRTs (0 to II) in membrane shape remodeling is not understood. Combining experimental measurements and electron microscopy analysis of ESCRT-III–depleted cells with a mathematical model, we show that upstream ESCRT-induced alteration of the Gaussian bending rigidity and their crowding in concert with the transmembrane cargo on the membrane induce membrane deformation and facilitate ILV formation: Upstream ESCRT-driven budding does not require ATP consumption as only a small energy barrier needs to be overcome. Our model predicts that ESCRTs do not become part of the ILV, but localize with a high density at the membrane neck, where the steep decline in the Gaussian curvature likely triggers ESCRT-III/VPS4 assembly to enable neck constriction and scission.

Membrane Shape Remodeling by Protein Crowding

The steric repulsion between proteins on biological membranes is one of the most generic mechanisms that cause membrane shape changes. We present a minimal model where a spontaneous curvature is induced by steric repulsion between membrane-associated proteins. Our results show that the interplay between the induced spontaneous curvature and the membrane tension determine the energy minimizing shapes, which describe the wide range of experimentally observed membrane shapes, i.e. flat membranes, spherical vesicles, elongated tubular protrusions, and pearling structures.

User guide to the red blood cell model (RCM), a multiplatform JAVA-based model of human red blood cell homeostasis

We introduce here a new multiplatform JAVA-based mathematical-computational model of RBC homeostasis for investigating the dynamics of changes in RBC homeostasis in health and disease. We provide a brief overview on the homeostasis of human RBCs and on the general biophysical principles guiding the modelling design. By way of a detailed tutorial we apply the model to investigate in depth the multiple effects associated with RBC dehydration induced by potassium permeabilization, a necessary preliminary for understanding the pathophysiology of a wide group of inherited haemolytic anaemias, a subject of intense current research and clinical interest. Using the red cell model (RCM), we design and run in silico representations of experimental protocols to study global RBC responses to calcium and potassium permeabilization covering a wide range of experimental, physiological and pathological conditions. Model outputs report the evolution in time of all the homeostatic variables in the system allowing, for the first time, a detailed and comprehensive account of the complex processes shaping global cell responses. Analysis of the results explains the mechanisms by which the entangled operation of all the RBC components link cell dehydration and protein crowding to cell acidification and to the generation of hypertonic, alkaline effluents. Open access to the RCM in a GitHub repository, together with the tutorial primed for a specific investigation pave the way for researchers and clinicians to apply the model on many different aspects of RBC physiology and pathology.

Mitochondria-adaptor TRAK1 promotes kinesin-1 driven transport in crowded environments

SummaryIntracellular trafficking of organelles, driven by kinesin-1 stepping along microtubules, underpins essential processes including neuronal activity. In absence of other proteins on the microtubule surface, kinesin-1 performs micron-long runs. Under protein crowding conditions, however, kinesin-1 motility is drastically impeded. It is thus unclear how kinesin-1 acts as an efficient transporter in crowded intracellular environments. Here, we demonstrate that TRAK1 (Milton), an adaptor protein essential for mitochondrial trafficking, activates kinesin-1 and increases its robustness of stepping in protein crowding conditions. Interaction with TRAK1 i) facilitated kinesin-1 navigation around obstacles, ii) increased the probability of kinesin-1 passing through cohesive envelopes of tau and iii) increased the run length of kinesin-1 in cell lysate. We explain the enhanced motility by the observed direct interaction of TRAK1 with microtubules, providing an additional anchor for the kinesin-1-TRAK1 complex. We propose adaptor-mediated tethering as a mechanism regulating kinesin-1 motility in various cellular environments.