Shape of the membrane neck around a hole during plasma membrane repair.

Plasma membrane damage and rupture occurs frequently in cells, and holes must be sealed rapidly to ensure homeostasis and cell survival. The membrane repair machinery is known to involve recruitment of curvature-inducing annexin proteins, but the connection between membrane remodeling and hole closure is poorly described. The induction of curvature by repair proteins leads to the possible formation of a membrane neck around the hole as a key intermediate structure before sealing. We formulate a theoretical model of equilibrium neck shapes to examine the potential connection to a repair mechanism. Using variational calculus, the shape equations for the membrane near a hole are formulated and solved numerically. The system is described under a condition of fixed area, and a shooting approach is applied to fulfill the boundary conditions at the free membrane edge. A state diagram of neck shapes is produced describing the variation in neck morphology with respect to the membrane area. Two distinct types of necks are predicted, one with conformations curved beyond π existing at positive excess area, whereas flat neck conformations (curved below π) have negative excess area. The results indicate that in cells, the supply of additional membrane area and a change in edge tension is linked to the formation of narrow and curved necks. Such necks may be susceptible to passive or actively induced membrane fission as a possible mechanism for hole sealing during membrane repair in cells.

Lens Nucleation and Droplet Budding in a Membrane Model for Lipid Droplet Biogenesis.

Lipid droplet biogenesis comprises the emergence of cytosolic lipid droplets with a typical diameter 0.1-5 µm via synthesis of fat in the endoplasmatic reticulum, the formation of membrane-embedded lenses, and the eventual budding of lenses into solution as droplets. Lipid droplets in cells are increasingly being viewed as highly dynamic organelles with multiple functions in cell physiology. However, the mechanism of droplet formation in cells remains poorly understood, partly because their formation involves the rapid transformation of transient lipid structures that are difficult to capture. Thus, the development of controlled experimental systems that model lipid biogenesis is highly relevant for an enhanced mechanistic understanding. Here we prepare and characterize triolein (TO) lenses in a multilamellar spin-coated phosphatidylcholine (POPC) film and determine the lens nucleation threshold to 0.25-0.5% TO. The TO lens shapes are characterized by atomic force microscopy (AFM) including their mean cap angle ⟨α⟩ = 27.3° and base radius ⟨a⟩ = 152.7 nm. A cross-correlation analysis of corresponding AFM and fluorescence images confirms that TO is localized to lenses. Hydration of the lipid/lens film induces the gel to fluid membrane phase transition and makes the lenses more mobile. The budding of free droplets into solution from membrane lenses is detected by observing a change in motion from confined wiggling to ballistic motion of droplets in solution. The results confirm that droplet budding can occur spontaneously without being facilitated by proteins. The developed model system provides a controlled platform for testing mechanisms of lipid droplet biogenesis in vitro and addressing questions related to lens formation and droplet budding by quantitative image analysis.

Phenothiazines alter plasma membrane properties and sensitize cancer cells to injury by inhibiting annexin-mediated repair.

Repair of damaged plasma membrane in eukaryotic cells is largely dependent on the binding of annexin repair proteins to phospholipids. Changing the biophysical properties of the plasma membrane may provide means to compromise annexin-mediated repair and sensitize cells to injury. Since, cancer cells experience heightened membrane stress and are more dependent on efficient plasma membrane repair, inhibiting repair may provide approaches to sensitize cancer cells to plasma membrane damage and cell death. Here, we show that derivatives of phenothiazines, which have widespread use in the fields of psychiatry and allergy treatment, strongly sensitize cancer cells to mechanical-, chemical-, and heat-induced injury by inhibiting annexin-mediated plasma membrane repair. Using a combination of cell biology, biophysics, and computer simulations, we show that trifluoperazine acts by thinning the membrane bilayer, making it more fragile and prone to ruptures. Secondly, it decreases annexin binding by compromising the lateral diffusion of phosphatidylserine, inhibiting the ability of annexins to curve and shape membranes, which is essential for their function in plasma membrane repair. Our results reveal a novel avenue to target cancer cells by compromising plasma membrane repair in combination with noninvasive approaches that induce membrane injuries.

Human myelin protein P2: from crystallography to time-lapse membrane imaging and neuropathy-associated variants.

Peripheral myelin protein 2 (P2) is a fatty acid-binding protein expressed in vertebrate peripheral nervous system myelin, as well as in human astrocytes. Suggested functions of P2 include membrane stacking and lipid transport. Mutations in the PMP2 gene, encoding P2, are associated with Charcot-Marie-Tooth disease (CMT). Recent studies have revealed three novel PMP2 mutations in CMT patients. To shed light on the structure and function of these P2 variants, we used X-ray and neutron crystallography, small-angle X-ray scattering, circular dichroism spectroscopy, computer simulations and lipid binding assays. The crystal and solution structures of the I50del, M114T and V115A variants of P2 showed minor differences to the wild-type protein, whereas their thermal stability was reduced. Vesicle aggregation assays revealed no change in membrane stacking characteristics, while the variants showed altered fatty acid binding. Time-lapse imaging of lipid bilayers indicated formation of double-membrane structures induced by P2, which could be related to its function in stacking of two myelin membrane surfaces in vivo. In order to better understand the links between structure, dynamics and function, the crystal structure of perdeuterated P2 was refined from room temperature data using neutrons and X-rays, and the results were compared to simulations and cryocooled crystal structures. Our data indicate similar properties for all known human P2 CMT variants; while crystal structures are nearly identical, thermal stability and function of CMT variants are impaired. Our data provide new insights into the structure-function relationships and dynamics of P2 in health and disease.

Timescale of hole closure during plasma membrane repair estimated by calcium imaging and numerical modeling.

Plasma membrane repair is essential for eukaryotic cell life and is triggered by the influx of calcium through membrane wounds. Repair consists of sequential steps, with closure of the membrane hole being the key event that allows the cell to recover, thus identifying the kinetics of hole closure as important for clarifying repair mechanisms and as a quantitative handle on repair efficiency. We implement calcium imaging in MCF7 breast carcinoma cells subject to laser damage, coupled with a model describing the spatio-temporal calcium distribution. The model identifies the time point of hole closure as the time of maximum calcium signal. Analysis of cell data estimates the closure time as: [Formula: see text] s and [Formula: see text] s using GCaMP6s-CAAX and GCaMP6s probes respectively. The timescale was confirmed by independent time-lapse imaging of a hole during sealing. Moreover, the analysis estimates the characteristic time scale of calcium removal, the penetration depth of the calcium wave and the diffusion coefficient. Probing of hole closure times emerges as a strong universal tool for quantification of plasma membrane repair.

Interdisciplinary Synergy to Reveal Mechanisms of Annexin-Mediated Plasma Membrane Shaping and Repair.

The plasma membrane surrounds every single cell and essentially shapes cell life by separating the interior from the external environment. Thus, maintenance of cell membrane integrity is essential to prevent death caused by disruption of the plasma membrane. To counteract plasma membrane injuries, eukaryotic cells have developed efficient repair tools that depend on Ca2+- and phospholipid-binding annexin proteins. Upon membrane damage, annexin family members are activated by a Ca2+ influx, enabling them to quickly bind at the damaged membrane and facilitate wound healing. Our recent studies, based on interdisciplinary research synergy across molecular cell biology, experimental membrane physics, and computational simulations show that annexins have additional biophysical functions in the repair response besides enabling membrane fusion. Annexins possess different membrane-shaping properties, allowing for a tailored response that involves rapid bending, constriction, and fusion of membrane edges for resealing. Moreover, some annexins have high affinity for highly curved membranes that appear at free edges near rupture sites, a property that might accelerate their recruitment for rapid repair. Here, we discuss the mechanisms of annexin-mediated membrane shaping and curvature sensing in the light of our interdisciplinary approach to study plasma membrane repair.