Martin Michael Müller

Aggregation and vesiculation of membrane proteins by curvature-mediated interactions

Membrane remodelling plays an important role in cellular tasks such as endocytosis, vesiculation and protein sorting, and in the biogenesis of organelles such as the endoplasmic reticulum or the Golgi apparatus. It is well established that the remodelling process is aided by specialized proteins that can sense as well as create membrane curvature, and trigger tubulation when added to synthetic liposomes. Because the energy needed for such large-scale changes in membrane geometry significantly exceeds the binding energy between individual proteins and between protein and membrane, cooperative action is essential. It has recently been suggested that curvature-mediated attractive interactions could aid cooperation and complement the effects of specific binding events on membrane remodelling. But it is difficult to experimentally isolate curvature-mediated interactions from direct attractions between proteins. Moreover, approximate theories predict repulsion between isotropically curving proteins. Here we use coarse-grained membrane simulations to show that curvature-inducing model proteins adsorbed on lipid bilayer membranes can experience attractive interactions that arise purely as a result of membrane curvature. We find that once a minimal local bending is realized, the effect robustly drives protein cluster formation and subsequent transformation into vesicles with radii that correlate with the local curvature imprint. Owing to its universal nature, curvature-mediated attraction can operate even between proteins lacking any specific interactions, such as newly synthesized and still immature membrane proteins in the endoplasmic reticulum.

Increasing amino acid supply in pea embryos reveals specific interactions of N and C metabolism, and highlights the importance of mitochondrial metabolism

SUMMARY The application of nitrogen to legumes regulates seed metabolism and composition. We recently showed that the seed-specific overexpression of amino acid permease VfAAP1 increases amino acid supply, and the levels of N and protein in the seeds. Two consecutive field trials using Pisum sativum AAP1 lines confirmed increases in the levels of N and globulin in seed; however, compensatory changes of sucrose/starch and individual seed weight were also observed. We present a comprehensive analysis of AAP1 seeds using combinatorial transcript and metabolite profiling to monitor the effects of nitrogen supply on seed metabolism. AAP1 seeds have increased amino acids and stimulated gene expression associated with storage protein synthesis, maturation, deposition and vesicle trafficking. Transcript/metabolite changes reveal the channelling of surplus N into the transient storage pools asparagine and arginine, indicating that asparagine synthase is transcriptionally activated by high N levels and/or C limitation. Increased C-acceptor demand for amino acid synthesis, resulting from elevated levels of N in seeds, initiates sucrose mobilization and sucrose-dependent pathways via sucrose synthase, glycolysis and the TCA cycle. The AAP1 seeds display a limitation in C, which leads to the catabolism of arginine, glutamic acid and methionine to putrescine, beta-alanine and succinate. Mitochondria are involved in the coordination of C/N metabolism, with branched-chain amino acid catabolism and a gamma-amino-butyric acid shunt. AAP1 seeds contain higher levels of ABA, which is possibly involved in storage-associated gene expression and the N-dependent stimulation of sucrose mobilization, indicating that a signalling network of C, N and ABA is operating during seed maturation. These results demonstrate that legume seeds have a high capacity to regulate N:C ratios, and highlight the importance of mitochondria in the control of N-C balance and amino acid homeostasis.

Orientation of the Infrared Transition Moments for an α-Helix

Appropriate values for the orientation of the amide transition dipoles are essential to the growing use of isotopically edited vibrational spectroscopy generally in structural biology and to infrared dichroism measurements on membrane-associated alpha-helices, in particular. The orientations of the transition moments for the amide vibrations of an alpha-helix have been determined from the ratio of intensities of the A- and E(1)-symmetry modes in the infrared spectra of poly(gamma-methyl-L-glutamate)(x)-co-(gamma-n-octadecyl-L-glutamate)( y) oriented on silicon substrates. Samples possessing a high degree of alignment were used to facilitate band fitting. Consistent results were obtained from both attenuated total reflection and transmission experiments with polarized radiation, yielding values of Theta(I) = 38 degrees, Theta(II) = 73 degrees, and Theta(A) = 29 degrees, relative to the helix axis, for the amide I, amide II, and amide A bands, respectively. The measurements are discussed both in the context of the somewhat divergent older determinations, and in relation to the helix geometry and results on model amide compounds, to resolve current uncertainties in the literature.

Local Membrane Mechanics of Pore-Spanning Bilayers

The mechanical behavior of lipid bilayers spanning the pores of highly ordered porous silicon substrates was scrutinized by local indentation experiments as a function of surface functionalization, lipid composition, solvent content, indentation velocity, and pore radius. Solvent-containing nano black lipid membranes (nano-BLMs) as well as solvent-free pore-spanning bilayers were imaged by fluorescence and atomic force microscopy prior to force curve acquisition, which allows distinguishing between membrane-covered and uncovered pores. Force indentation curves on pore-spanning bilayers attached to functionalized hydrophobic porous silicon substrates reveal a predominately linear response that is mainly attributed to prestress in the membranes. This is in agreement with the observation that indentation leads to membrane lysis well below 5% area dilatation. However, membrane bending and lateral tension dominate over prestress and stretching if solvent-free supported membranes obtained from spreading giant liposomes on hydrophilic porous silicon are indented. An elastic regime diagram is presented that readily allows determining the dominant contribution to the mechanical response upon indentation as a function of load and pore radius.

Photophysical properties of porphycene derivatives (18 π porphyrinoids)

Absorption and fluorescence spectral data and triplet energy content, as well as quantum yields of fluorescence, of triplet state formation, and of sensitized generation of singlet molecular oxygen, O2(1Δg), of 38 porphycene derivatives are reported. Long-wavelength absorption and the efficiency of fluorescence and O2(1Δg) sensitization qualify most of these porphycenes as potential photodynamic therapy agents (so-called second-generation sensitizers), unless steric or electronic effects by substitution perturb the parent aromatic 18 π system.

ADP-glucose pyrophosphorylase-deficient pea embryos reveal specific transcriptional and metabolic changes of carbon-nitrogen metabolism and stress responses.

We present a comprehensive analysis of ADP-glucose pyrophosphorylase (AGP)-repressed pea (Pisum sativum) seeds using transcript and metabolite profiling to monitor the effects that reduced carbon flow into starch has on carbon-nitrogen metabolism and related pathways. Changed patterns of transcripts and metabolites suggest that AGP repression causes sugar accumulation and stimulates carbohydrate oxidation via glycolysis, tricarboxylic acid cycle, and mitochondrial respiration. Enhanced provision of precursors such as acetyl-coenzyme A and organic acids apparently support other pathways and activate amino acid and storage protein biosynthesis as well as pathways fed by cytosolic acetyl-coenzyme A, such as cysteine biosynthesis and fatty acid elongation/metabolism. As a consequence, the resulting higher nitrogen (N) demand depletes transient N storage pools, specifically asparagine and arginine, and leads to N limitation. Moreover, increased sugar accumulation appears to stimulate cytokinin-mediated cell proliferation pathways. In addition, the deregulation of starch biosynthesis resulted in indirect changes, such as increased mitochondrial metabolism and osmotic stress. The combined effect of these changes is an enhanced generation of reactive oxygen species coupled with an up-regulation of energy-dissipating, reactive oxygen species protection, and defense genes. Transcriptional activation of mitogen-activated protein kinase pathways and oxylipin synthesis indicates an additional activation of stress signaling pathways. AGP-repressed embryos contain higher levels of jasmonate derivatives; however, this increase is preferentially in nonactive forms. The results suggest that, although metabolic/osmotic alterations in iAGP pea seeds result in multiple stress responses, pea seeds have effective mechanisms to circumvent stress signaling under conditions in which excessive stress responses and/or cellular damage could prematurely initiate senescence or apoptosis.

Myotubularin and PtdIns3P remodel the sarcoplasmic reticulum in muscle in vivo

Summary The sarcoplasmic reticulum (SR) is a specialized form of endoplasmic reticulum (ER) in skeletal muscle and is essential for calcium homeostasis. The mechanisms involved in SR remodeling and maintenance of SR subdomains are elusive. In this study, we identified myotubularin (MTM1), a phosphoinositide phosphatase mutated in X-linked centronuclear myopathy (XLCNM, or myotubular myopathy), as a key regulator of phosphatidylinositol 3-monophosphate (PtdIns3P) levels at the SR. MTM1 is predominantly located at the SR cisternae of the muscle triads, and Mtm1-deficient mouse muscles and myoblasts from XLCNM patients exhibit abnormal SR/ER networks. In vivo modulation of MTM1 enzymatic activity in skeletal muscle using ectopic expression of wild-type or a dead-phosphatase MTM1 protein leads to differential SR remodeling. Active MTM1 is associated with flat membrane stacks, whereas dead-phosphatase MTM1 mutant promotes highly curved cubic membranes originating from the SR and enriched in PtdIns3P. Overexpression of a tandem FYVE domain with high affinity for PtdIns3P alters the shape of the SR cisternae at the triad. Our findings, supported by the parallel analysis of the Mtm1-null mouse and an in vivo study, reveal a direct function of MTM1 enzymatic activity in SR remodeling and a key role for PtdIns3P in promoting SR membrane curvature in skeletal muscle. We propose that alteration in SR remodeling is a primary cause of X-linked centronuclear myopathy. The tight regulation of PtdIns3P on specific membrane subdomains may be a general mechanism to control membrane curvature.

Geometry of surface-mediated interactions

Soft interfaces can mediate interactions between particles bound to them. The force transmitted through the surface geometry on a particle may be expressed as a closed line integral of the surface stress tensor around that particle. This contour may be deformed to exploit the symmetries present; for two identical particles, one obtains an exact expression for the force between them in terms of the local surface geometry of their mid-plane; in the case of a fluid membrane the sign of the interaction is often evident. The approach, by construction, is adapted directly to the surface and is independent of its parameterization. Furthermore, it is applicable for arbitrarily large deformations; in particular, it remains valid beyond the linear small-gradient regime.

Conical defects in growing sheets.

A growing or shrinking disc will adopt a conical shape, its intrinsic geometry characterized by a surplus angle phi(e) at the apex. If growth is slow, the cone will find its equilibrium. Whereas this is trivial if phi(e)<or=0, the disc can fold into one of a discrete infinite number of states if phi(e)>0. We construct these states in the regime where bending dominates and determine their energies and how stress is distributed in them. For each state a critical value of phi(e) is identified beyond which the cone touches itself. Before this occurs, all states are stable; the ground state has twofold symmetry.

Regulation of Storage Protein Synthesis in Cereal Seeds: Developmental and Nutritional Aspects

Summary The major seed storage proteins of cereals are deposited in the maturation phase of endosperm development, starting at 12 days after pollination (dap) in maize and continuing up to around 30dap. Cell types-pecific expression in the endosperm is conferred by a multicomponent promoter motif, the endosperm box sequence. Proteins interacting at this motif include the gene product of the regulatory gene Opaque-2 (O2). O2 encodes a b2IP transcription factor, which is capable of activating its target promoters, 22kD α-zein and b-32, in a number of experimental systems. Storage protein accumulation, and storage protein promoter activity, are modulated according to available levels of nitrogen (N). In barley, nitrogen regulation is mediated by the endosperm box via interaction of the endosperm and GCN 4 motifs. The nitrogen- responsive components of the 22kd zein promoter have been shown to include the O2-binding sites Z1–Z3 but to display N-responsiveness even in the absence of O2 protein.