Vesicle Mechanics: A Push into the Nanoscale

Wuite, G.J.L. [Promotor]Roos, W.H. [Promotor]Loon, J.J.W.A. van [Copromotor

Competition between Bending and Internal Pressure Governs the Mechanics of Fluid Nanovesicles

Nanovesicles (∼100 nm) are ubiquitous in cell biology and an important vector for drug delivery. Mechanical properties of vesicles are known to influence cellular uptake, but the mechanism by which deformation dynamics affect internalization is poorly understood. This is partly due to the fact that experimental studies of the mechanics of such vesicles remain challenging, particularly at the nanometer scale where appropriate theoretical models have also been lacking. Here, we probe the mechanical properties of nanoscale liposomes using atomic force microscopy (AFM) indentation. The mechanical response of the nanovesicles shows initial linear behavior and subsequent flattening corresponding to inward tether formation. We derive a quantitative model, including the competing effects of internal pressure and membrane bending, that corresponds well to these experimental observations. Our results are consistent with a bending modulus of the lipid bilayer of ∼14kbT. Surprisingly, we find that vesicle stiffness is pressure dominated for adherent vesicles under physiological conditions. Our experimental method and quantitative theory represents a robust approach to study the mechanics of nanoscale vesicles, which are abundant in biology, as well as being of interest for the rational design of liposomal vectors for drug delivery.</p

The role of the cytoskeleton in sensing changes in gravity by nonspecialized cells

A large body of evidence indicates that single cells in vitro respond to changes in gravity, and that this response might play an important role for physiological changes at the organism level during spaceflight. Gravity can lead to changes in cell proliferation, differentiation, signaling, and gene expression. At first glance, gravitational forces seem too small to affect bodies with the size of a cell. Thus, the initial response to gravity is both puzzling and important for understanding physiological changes in space. This also offers a unique environment to study the mechanical response of cells. In the past 2 decades, important steps have been made in the field of mechanobiology, and we use these advances to reevaluate the response of single cells to changes in gravity. Recent studies have focused on the cytoskeleton as initial gravity sensor. Thus, we review the observed changes in the cytoskeleton in a microgravity environment, both during spaceflight and in ground-based simulation techniques. We also evaluate to what degree the current experimental evidence supports the cytoskeleton as primary gravity sensor. Finally, we consider how the cytoskeleton itself could be affected by changed gravity. To make the next step toward understanding the response of cells to altered gravity, the challenge will be to track changes quantitatively and on short timescales

Maturation of adenovirus primes the protein nano-shell for successful endosomal escape

The ability of adenoviruses to infect a broad range of species has spurred a growing interest in nanomedicine to use adenovirus as a cargo delivery vehicle. While successful maturation of adenovirus and controlled disassembly are critical for efficient infection, the underlying mechanisms regulating these processes are not well understood. Here, we present Atomic Force Microscopy nanoindentation and fatigue studies of adenovirus capsids at different maturation stages to scrutinize their dynamic uncoating properties. Surprisingly, we find that the early intermediate immature (lacking DNA) capsid is mechanically indistinguishable in both break force and spring constant from the mature (containing DNA) capsid. However, mature and immature capsids do display distinct disassembly pathways, as revealed by our mechanically-induced fatigue analysis. The mature capsid first loses the pentons, followed by either long-term capsid stability or abrupt and complete disassembly. However, the immature capsid has a stable penton region and undergoes a stochastic disassembly mechanism, thought to be due to the absence of genomic pressure. Strikingly, the addition of the genome alone is not sufficient to achieve penton destabilization as indicated by the penton stability of the maturation-intermediate mutant, G33A. Full penton destabilization was achieved only when the genome was present in addition to the successful maturation-linked proteolytic cleavage of preprotein VI. Therefore these findings strongly indicate that maturation of adenovirus in concert with genomic pressure induces penton destabilization and thus, primes the capsid for controlled disassembly. This latter aspect is critical for efficient infection and successful cargo delivery