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Eukaryotic cells undergo shape changes during their division and growth. This involves flow of material both in the cell membrane and in the cytoskeletal layer beneath the membrane. Such flows result in redistribution of phospholipid at the cell surface and actomyosin in the cortex. Here we focus on the growth of the intercellular surface during cell division in a Caenorhabditis elegans embryo. The growth of this surface leads to the formation of a double-layer of separating membranes between the two daughter cells. The division plane typically has a circular periphery and the growth starts from the periphery as a membrane invagination, which grows radially inward like the shutter of a camera. The growth is typically not concentric, in the sense that the closing internal ring is located off-center. Cytoskeletal proteins anillin and septin have been found to be responsible for initiating and maintaining the asymmetry of ring closure but the role of possible asymmetry in the material flow into the growing membrane has not been investigated yet. Motivated by experimental evidence of such flow asymmetry, here we explore the patterns of internal ring closure in the growing membrane in response to asymmetric boundary fluxes. We highlight the importance of the flow asymmetry by showing that many of the asymmetric growth patterns observed experimentally can be reproduced by our model, which incorporates the viscous nature of the membrane and contractility of the associated cortex.

Cardiomyocytes are characterized by a complex network of membrane invagination (the transverse axial tubular system, TATS) that propagates the action potential to the cell core guarantying a synchronous and uniform cell contraction. Pathological settings are commonly associated with structural remodelling of TATS that may cause failures of action potential propagation at single T-tubular level. Here, we investigate whether structural alterations occurring in TATS are linked to overall changes in its electrical conductivity which in turn hinder the regular propagation of action potentials across the network. Exploiting the formal analogy between diffusion and electrical conductivity we linked the conductivity with the diffusion properties of TATS. Fluorescence recovery after photo-bleaching (FRAP) microscopy is used to probe the diffusion properties of the tubular system in isolated cardiomyocytes: the fluorescent dextran inside TATS lumen is photo-bleached and the diffusion of unbleached dextran from extracellular space to TATS is monitored. We designed a mathematical model that correlate the time constant of fluorescence recovery with the apparent diffusion coefficient of the dextran. Then, taken advantage of the analogy between diffusion and conductivity, the apparent diffusion is used to assess TATS conductivity. This value is used to evaluate the efficiency of the passive spread of voltage changes along TATS. To probe how TATS conductivity is influenced by geometrical features, we used a model of acutely detubulated cells. We found that the overall reduction of TATS halves tubular conductivity. Then we tested our method in a pathological setting characterized by structural alteration, i.e. the heart failure rat model. We found that the tubular conductivity is not changed as compared to control. Actually, this result should not be surprising since heart failure is characterized by compound alterations that potentially compensate each other in their individual effect on global conductivity.

Sphingomyelin is an abundant component of eukaryotic membranes. A specific enzyme, sphingomyelinase can convert this lipid to ceramide, a central second messenger in cellular signaling for apoptosis (programmed cell death), differentiation, and senescence. We used microinjection and either Hoffman modulation contrast or fluorescence microscopy of giant liposomes composed of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), N-palmitoyl-sphingomyelin (C16:0-SM), and Bodipy-sphingomyelin as a fluorescent tracer (molar ratio 0.75:0.20:0.05, respectively) to observe changes in lipid lateral distribution and membrane morphology upon formation of ceramide. Notably, in addition to rapid domain formation (capping), vectorial budding of vesicles, i.e., endocytosis and shedding, can be induced by the asymmetrical sphingomyelinase-catalyzed generation of ceramide in either the outer or the inner leaflet, respectively, of giant phosphatidylcholine/sphingomyelin liposomes. These results are readily explained by 1) the lateral phase separation of ceramide enriched domains, 2) the area difference between the adjacent monolayers, 3) the negative spontaneous curvature, and 4) the augmented bending rigidity of the ceramide-containing domains, leading to membrane invagination and vesiculation of the bilayer.

The recent invention of super-resolution fluorescence microscopy allows nanoscopic investigation of cellular structures. Among these techniques, the Stochastic Optical Reconstruction Microscopy (STORM) is based on precise single molecule localization of photoswitchable fluorescent probes. By stochastically activating, imaging and deactivating subsets of fluorophores, it makes their images optically resolvable and determines their positions with nanometer precision. A super-resolution image is then reconstructed using these localizations. We now extend this approach to three-dimensional (3D) microscopy by determining the 3D coordinates of activated probes through astigmatism imaging: a cylindrical lens is inserted into the imaging optical path such that the image of individual molecules appear elliptical with the ellipticity depending on its z-position. Using this approach, we have achieved an optical resolution of 20-30 nm in the x-y direction and 50-60 nm in the z direction, representing an order of magnitude improvement over conventional fluorescence microscopy in all three dimensions. We have resolved the nanoscopic morphology of cellular structures that was previously deemed impossible by light microscopy.As a specific application, we use STORM to study the mechanism of clathrin mediated endocytosis in an in vitro reconstituted system. Proteins of interest in this system are directly labeled with photoswitchable fluorescent probes. This procedure is facilitated by covalently linking the two components of the probe, an activator dye and a photoswitchable reporter fluorophore, to form a single chemical unit prior to protein labeling. Using multicolor 3D STORM, we have characterized the spatial organization of plasma membrane, clathrin, actin and tubule forming proteins dynamin at the site of clathrin mediated endocytosis. These results reveal the molecular architecture of the nascent clathrin-coated pits at the nanometer scale and help to establish the role of actin and dynamin in membrane invagination, scission and vesicle formation.

One of the pathological hallmarks of Alzheimer's disease (AD) is extracellular plaques, in which the 42aa Amyloid beta peptide (Aβ 1-42) is the main component. Aβ 1-42 is produced at cholesterol-rich regions of neuronal membranes by endoproteolysis of the parental amyloid precursor protein and is secreted into the extracellular space. Although Aβ 1-42 accumulates extracellularly, neurons internalize the Aβ 1-42 peptide which could contribute to disease progression and which may be the first step to both cytotoxicity and propagation of misfolded Aβ between cells.Interaction with membrane bilayer is likely the first step in the molecular mechanism of neuronal Aβ 1-42 uptake. Therefore, we studied interaction of Aβ 1-42 with the lipid bilayer in a giant unilamellar vesicles (GUVs) model system. We found that the Aβ 1-42 bound to the lipid bilayer and, after a lag phase induced invagination of the membrane into small vesicular structures. Only early oligomeric structures of Aβ and stabilized the negative curvature of membrane, whereas both monomeric and fibrillar forms of the peptide were unable to induce or sustain membrane invagination.Our results suggest that Aβ may facilitate vesicle formation in lipid bilayer membranes, which may be a factor in cellular Aβ internalization but may also hint at a possible physiological function of the Aβ peptide at the synaptic membrane.

In atrial myocytes lacking t-tubules, fluid pressure (FP, ∼16 dyn/cm2) has been found to elicit global Ca2+ waves: one with longitudinal propagation (L-wave) and the other with transverse propagation (T-wave). T-wave is associated with action potential, whereas L-wave often, but not always, triggers action potential involving T-wave during its propagation. Here we examined spatiotemporal characteristics, and structural and molecular basis for the FP-induced L-wave using two-dimensional (2-D) confocal Ca2+ imaging, combined with three-dimensional (3-D) visualizations of cell membrane and type 2 ryanodine receptors (RyR2), in rat atrial myocytes. We found that FP-induced L-waves generally originated from central focal Ca2+ release sites (“L-wave core”) located at 15-25% of the cell length. The core releases had 3∼6 signal peaks and grew faster in one-direction in 2-D measurements. They were more prolonged and much larger compared with the Ca2+ sparks. Tetracaine (1 mM, 10 s) reversibly inhibited the FP-induced core release as well as the L-wave, suggesting both involve the RyRs. Immediately after recording FP-induced L-wave, the cell's membrane was further visualized with wheat germ agglutinin (WGA, 1.25 μg/ml). Overlapping the L-wave core and the cell membrane, and reconstructing the WGA confocal image stacks in 3-D, revealed membrane invagination at the centrally localized core or in the vicinity of the core site. Interestingly, in some atrial cells we found denser (≈0.62-μm intervals) disoriented RyR alignments in the cell interior at ∼20% of the cell length and nearby the cell end. We conclude that the crumpled invaginated surface membrane, associated with dense RyRs, may play an important role in transducing fluid pressure into the core Ca2+ signal, resulting in arrhythmogenic L-wave.

We investigate the structural properties of the Shiga-like toxin (SLT1) subunit B in the bulk and bound to a lipid membrane using coarse-grained (CG) molecular dynamics simulations. SLT1 consist of a cytotoxic A subunit that shuts down protein synthesis and five B subunits forming a homo-pentamer responsible for ferrying the A subunit into the cell. With or without the A unit attached, the B5 complex binds to specific glycolipids on the cell membrane and leads to toxin uptake by cooperatively driving membrane invagination. Unfortunately, the size of the protein-lipid system needed to investigate SLT1 driven vesiculation makes all-atom simulations impractical. Instead, CG models hold promise, having previously succeeded in simulating such events, even though at a much lower resolution [1]. We use an intermediate resolution systematic CG model of proteins and lipids, which improves the efficiency of our simulations while providing the necessary specificity to relate the results directly to SLT1 [2,3]. To ensure that the monomer structure resembles all-atom simulations, we augment the protein model with an intra-monomer elastic network that is parameterized using a novel iterative process. In addition, we explore ways to best represent the bonds between binding sites on the monomers and glycolipids. With the completed model, we investigate the stability and fluctuation modes of the Shiga B subunit in the bulk and on a membrane. In addition, we investigate the effect of the protein on the lipid bilayer, specifically its local bending deformation and lipid diffusion around the protein.[1] B.J. Reynwar, G. Illya, V.A. Harmandaris, M.M. Muller, K. Kremer, and M. Deserno, Nature 447, 461-464 (2007)[2] T. Bereau and M. Deserno, J. Chem. Phys. 130, 235106 (2009)[3] Z. Wang and M. Deserno, J. Phys. Chem. B 114, 11207 (2010)