Guenter P. Resch

Clustering of VASP actively drives processive, WH2 domain-mediated actin filament elongation

Vasodilator‐stimulated phosphoprotein (VASP) is a key regulator of dynamic actin structures like filopodia and lamellipodia, but its precise function in their formation is controversial. Using in vitro TIRF microscopy, we show for the first time that both human and Dictyostelium VASP are directly involved in accelerating filament elongation by delivering monomeric actin to the growing barbed end. In solution, DdVASP markedly accelerated actin filament elongation in a concentration‐dependent manner but was inhibited by low concentrations of capping protein (CP). In striking contrast, VASP clustered on functionalized beads switched to processive filament elongation that became insensitive even to very high concentrations of CP. Supplemented with the in vivo analysis of VASP mutants and an EM structure of the protein, we propose a mechanism by which membrane‐associated VASP oligomers use their WH2 domains to effect both the tethering of actin filaments and their processive elongation in sites of active actin assembly.

Actin branching in the initiation and maintenance of lamellipodia

Using correlated live-cell imaging and electron tomography we found that actin branch junctions in protruding and treadmilling lamellipodia are not concentrated at the front as previously supposed, but link actin filament subsets in which there is a continuum of distances from a junction to the filament plus ends, for up to at least 1 μm. When branch sites were observed closely spaced on the same filament their separation was commonly a multiple of the actin helical repeat of 36 nm. Image averaging of branch junctions in the tomograms yielded a model for the in vivo branch at 2.9 nm resolution, which was comparable with that derived for the in vitro actin–Arp2/3 complex. Lamellipodium initiation was monitored in an intracellular wound-healing model and was found to involve branching from the sides of actin filaments oriented parallel to the plasmalemma. Many filament plus ends, presumably capped, terminated behind the lamellipodium tip and localized on the dorsal and ventral surfaces of the actin network. These findings reveal how branching events initiate and maintain a network of actin filaments of variable length, and provide the first structural model of the branch junction in vivo. A possible role of filament capping in generating the lamellipodium leaflet is discussed and a mathematical model of protrusion is also presented.

Unravelling the structure of the lamellipodium

Pushing at the cell front is the business of lamellipodia and understanding how lamellipodia function requires knowledge of their structural organization. Analysis of extracted, critical‐point‐dried cells by electron microscopy has led to a current dogma that the lamellipodium pushes as a branched array of actin filaments, with a branching angle of 70°, defined by the Arp2/3 complex. Comparison of different preparative methods indicates that the critical‐point‐drying‐replica technique introduces distortions into actin networks, such that crossing filaments may appear branched. After negative staining and from preliminary studies by cryo‐electron tomography, no clear evidence could be found for actin filament branching in lamellipodia. From recent observations of a sub‐class of actin speckles in lamellipodia that exhibit a dynamic behaviour similar to speckles in the lamella region behind, it has been proposed that the lamellipodium surfs on top of the lamella. Negative stain electron microscopy and cryo‐electron microscopy of fixed cells, which reveal the entire complement of filaments in lamellipodia show, however, that there is no separate, second array of filaments beneath the lamellipodium network. From present data, we conclude that the lamellipodium is a distinct protrusive entity composed of a network of primarily unbranched actin filaments. Cryo‐electron tomography of snap‐frozen intact cells will be required to finally clarify the three‐dimensional arrangement of actin filaments in lamellipodia in vivo.

Protein-Mediated Transformation of Lipid Vesicles into Tubular Networks

Key cellular processes are frequently accompanied by protein-facilitated shape changes in the plasma membrane. N-BAR-domain protein modules generate curvature by means of complex interactions with the membrane surface. The way they assemble and the mechanism by which they operate are largely dependent on their binding density. Although the mechanism at lower densities has recently begun to emerge, how membrane scaffolds form at high densities remains unclear. By combining electron microscopy and multiscale simulations, we show that N-BAR proteins at high densities can transform a lipid vesicle into a 3D tubular network. We show that this process is a consequence of excess adhesive energy combined with the local stiffening of the membrane, which occurs in a narrow range of mechanical properties of both the membrane and the protein. We show that lipid diffusion is significantly reduced by protein binding at this density regime and even more in areas of high Gaussian curvature, indicating a potential effect on molecular transport in cells. Finally, we reveal that the breaking of the bilayer topology is accompanied by the nematic arrangement of the protein on the surface, a structural motif that likely drives the formation of reticular structures in living cells.

Electron Tomography and Simulation of Baculovirus Actin Comet Tails Support a Tethered Filament Model of Pathogen Propulsion

Electron tomography reveals the structural organization of actin comet tails generated by a baculovirus, providing an understanding of how this pathogen hijacks host machinery to propel itself between cells.

No evidence for intracellular magnetite in putative vertebrate magnetoreceptors identified by magnetic screening

Significance The list of animals that use the Earth’s magnetic field as a navigation tool is long and diverse; however, the cells responsible for transducing magnetic information into a neuronal impulse have not been discovered. One hypothesis argues that these cells use an iron oxide called magnetite (Fe3O4). Here, we use a “magnetoscope” coupled with single-cell correlative light and electron microscopy to identify candidate magnetoreceptors in the pigeon and trout. We report that a small percentage of cells in both species appear to have large magnetic moments, but they do not contain biogenic magnetite. Our work illustrates the need for technological innovation if the true magnetoreceptors are to be found. The cellular basis of the magnetic sense remains an unsolved scientific mystery. One theory that aims to explain how animals detect the magnetic field is the magnetite hypothesis. It argues that intracellular crystals of the iron oxide magnetite (Fe3O4) are coupled to mechanosensitive channels that elicit neuronal activity in specialized sensory cells. Attempts to find these primary sensors have largely relied on the Prussian Blue stain that labels cells rich in ferric iron. This method has proved problematic as it has led investigators to conflate iron-rich macrophages with magnetoreceptors. An alternative approach developed by Eder et al. [Eder SH, et al. (2012) Proc Natl Acad Sci USA 109(30):12022–12027] is to identify candidate magnetoreceptive cells based on their magnetic moment. Here, we explore the utility of this method by undertaking a screen for magnetic cells in the pigeon. We report the identification of a small number of cells (1 in 476,000) with large magnetic moments (8–106 fAm2) from various tissues. The development of single-cell correlative light and electron microscopy (CLEM) coupled with electron energy loss spectroscopy (EELS) and energy-filtered transmission electron microscopy (EFTEM) permitted subcellular analysis of magnetic cells. This revealed the presence of extracellular structures composed of iron, titanium, and chromium accounting for the magnetic properties of these cells. Application of single-cell CLEM to magnetic cells from the trout failed to identify any intracellular structures consistent with biogenically derived magnetite. Our work illustrates the need for new methods to test the magnetite hypothesis of magnetosensation.

An Iron-Rich Organelle in the Cuticular Plate of Avian Hair Cells

Hair cells reside in specialized epithelia in the inner ear of vertebrates, mediating the detection of sound, motion, and gravity. The transduction of these stimuli into a neuronal impulse requires the deflection of stereocilia, which are stabilized by the actin-rich cuticular plate. Recent electrophysiological studies have implicated the vestibular system in pigeon magnetosensation. Here we report the discovery of a single iron-rich organelle that resides in the cuticular plate of cochlear and vestibular hair cells in the pigeon. Transmission electron microscopy, coupled with elemental analysis, has shown that this structure is composed of ferritin-like granules, is approximately 300-600 nm in diameter, is spherical, and in some instances is membrane-bound and/or organized in a paracrystalline array. This organelle is found in hair cells in a wide variety of avian species, but not in rodents or in humans. This structure may function as (1) a store of excess iron, (2) a stabilizer of stereocilia, or (3) a mediator of magnetic detection. Given the specific subcellular location, elemental composition, and evolutionary conservation, we propose that this structure is an integral component of the sensory apparatus in birds.

Pure F-actin networks are distorted and branched by steps in the critical-point drying method

Elucidation of the ultrastructural organization of actin networks is crucial for understanding the molecular mechanisms underlying actin-based motility. Results obtained from cytoskeletons and actin comets prepared by the critical-point procedure, followed by rotary shadowing, support recent models incorporating actin filament branching as a main feature of lamellipodia and pathogen propulsion. Since actin branches were not evident in earlier images obtained by negative staining, we explored how these differences arise. Accordingly, we have followed the structural fate of dense networks of pure actin filaments subjected to steps of the critical-point drying protocol. The filament networks have been visualized in parallel by both cryo-electron microscopy and negative staining. Our results demonstrate the selective creation of branches and other artificial structures in pure F-actin networks by the critical-point procedure and challenge the reliability of this method for preserving the detailed organization of actin assemblies that drive motility.

Immersion Freezing of Suspended Particles and Cells for Cryo-Electron Microscopy

Immersion freezing of thin aqueous specimens is an essential preparation technique for cryo-transmission electron microscopy (cryo-TEM), aiming to preserve fragile biological structures such as molecules and cells in their hydrated environment for a close-to-native visualization. For successful experiments, vitreous ice must be produced, surface contamination must be avoided, and, most important, the natural state of the structure must be preserved. This protocol describes immersion freezing of biological samples, such as purified protein complexes, viruses, liposomes, synthetic cytoskeletal filaments, isolated organelles, or small cells suspended in an aqueous solution, using the new Leica “EMGP” grid plunger. It includes a discussion of issues of general importance for cryo-EM, such as the properties of the sample and the pretreatment of the specimen carrier. It also provides details on how to make the most of the special features of this instrument to obtain good specimens and reproducible results. Troubleshooting issues concerning the operation of the GP in particular, as well as common problems in immersion freezing encountered on manual and semiautomatic instruments, are addressed.

Immersion Freezing of Biological Specimens: Rationale, Principles, and Instrumentation

Guenter P. Resch,1,5 Marlene Brandstetter,1 Angela M. Pickl-Herk,2 Lisa Königsmaier,3 Veronika I. Wonesch,1,4 and Edit Urban3 IMP-IMBA-GMI Electron Microscopy Facility, Institute of Molecular Biotechnology, 1030 Vienna, Austria Max F. Perutz Laboratories, Medical University of Vienna, 1030 Vienna, Austria Institute of Molecular Biotechnology, 1030 Vienna, Austria University of Applied Sciences Wiener Neustadt, 2700 Wiener Neustadt, Austria