Vlad Brumfeld

Three-Dimensional Organization of Higher-Plant Chloroplast Thylakoid Membranes Revealed by Electron Tomography

The light-harvesting and energy-transducing functions of the chloroplast are performed within an intricate lamellar system of membranes, called thylakoid membranes, which are differentiated into granum and stroma lamellar domains. Using dual-axis electron microscope tomography, we determined the three-dimensional organization of the chloroplast thylakoid membranes within cryo-immobilized, freeze-substituted lettuce (Lactuca sativa) leaves. We found that the grana are built of repeating units that consist of paired layers formed by bifurcations of stroma lamellar sheets, which fuse within the granum body. These units are rotated relative to each other around the axis of the granum cylinder. One of the layers that makes up the pair bends upwards at its edge and fuses with the layer above it, whereas the other layer bends in the opposite direction and merges with the layer below. As a result, each unit in the granum is directly connected to its neighbors as well as to the surrounding stroma lamellae. This highly connected morphology has important consequences for the formation and function of the thylakoid membranes as well as for their stacking/unstacking response to variations in light conditions.

Thylakoid Membrane Remodeling during State Transitions in Arabidopsis

Adaptability of oxygenic photosynthetic organisms to fluctuations in light spectral composition and intensity is conferred by state transitions, short-term regulatory processes that enable the photosynthetic apparatus to rapidly adjust to variations in light quality. In green algae and higher plants, these processes are accompanied by reversible structural rearrangements in the thylakoid membranes. We studied these structural changes in the thylakoid membranes of Arabidopsis thaliana chloroplasts using atomic force microscopy, scanning and transmission electron microscopy, and confocal imaging. Based on our results and on the recently determined three-dimensional structure of higher-plant thylakoids trapped in one of the two major light-adapted states, we propose a model for the transitions in membrane architecture. The model suggests that reorganization of the membranes involves fission and fusion events that occur at the interface between the appressed (granal) and nonappressed (stroma lamellar) domains of the thylakoid membranes. Vertical and lateral displacements of the grana layers presumably follow these localized events, eventually leading to macroscopic rearrangements of the entire membrane network.

A molecular switch between alternative conformational states in the complex of Ran and importin β1

Several million macromolecules are exchanged each minute between the nucleus and cytoplasm by receptor-mediated transport. Most of this traffic is controlled by the small GTPase Ran, which regulates assembly and disassembly of the receptor–cargo complexes in the appropriate cellular compartment. Here we applied dynamic force spectroscopy to study the interaction of Ran with the nuclear import receptor importin β1 (impβ) at the single-molecule level. We found that the complex alternates between two distinct conformational states of different adhesion strength. The application of an external mechanical force shifts equilibrium toward one of these states by decreasing the height of the interstate activation energy barrier. The other state can be stabilized by a functional Ran mutant that increases this barrier. These results support a model whereby functional control of Ran–impβ is achieved by a population shift between pre-existing alternative conformations.

Direct measurement of protein energy landscape roughness

The energy landscape of proteins is thought to have an intricate, corrugated structure. Such roughness should have important consequences on the folding and binding kinetics of proteins, as well as on their equilibrium fluctuations. So far, no direct measurement of protein energy landscape roughness has been made. Here, we combined a recent theory with single‐molecule dynamic force spectroscopy experiments to extract the overall energy scale of roughness ε for a complex consisting of the small GTPase Ran and the nuclear transport receptor importin‐β. The results gave ε>5kBT, indicating a bumpy energy surface, which is consistent with the ability of importin‐β to accommodate multiple conformations and to interact with different, structurally distinct ligands.

Serine Conjugates of Chlorophyll and Bacteriochlorophyll: Photocytotoxicity in vitro and Tissue Distribution in Mice Bearing Melanoma Tumors

Chlorophyll (Chl) and bacteriochlorophyll (Bchl) have been made water soluble by transesterfication with serine (Ser) at the propionyl residue and tested as potential reagents for photodynamic therapy (PDT). Photocytotoxicity of the conjugates Chl‐Ser and Bchl‐Ser in M2R mouse melanoma was tested in cell cultures. Tissue uptake and clearance of the photosensitizers in CD1 nude and C57B1 mice implanted with M2R tumors are described. Photocytotoxicity in cell cultures was determined microscopically and by [3H]thymidine incorporation. The LD50 values in vitro were 0.05‐0.1 μM for both sensitizers while that of the commercially available hematoporphyrin derivative (HPD, Photosan) was over 100 times higher for the same light intensity (45 mW/cm2). Pigment concentrations were determined fluorometrically in acetone extracts of the tissues of interest at different times after intraperitoneal injection of 20 mg pigment/kg body weight. The distribution pattern of Chl‐Ser in the different tissues resembled that reported for Photofrin, chlorin and bacteriochlorin derivatives. Clearance from normal tissues was essentially completed within 16 h for Bchl‐Ser and 72 h for Chl‐Ser with mean half‐lives (t1/2) of about 2 and 7 h, respectively. In contrast, the clearance rates of these pigments and their metabolites from melanoma tumor tissue were significantly longer: t1/2= 20 h for Chl‐Ser and 15 h for Bchl‐Ser and metabolites. The clearance rates showed biphasic or single exponential decay patterns in normal tissues and in tumors, respectively. Cumulatively the high phototoxicity, simple mode of delivery and fast tissue clearance rates reported here suggest that polar conjugates of Chl and Bchl promise to be highly effective PDT reagents.

From chloroplasts to photosystems: in situ scanning force microscopy on intact thylakoid membranes

Envelope‐free chloroplasts were imaged in situ by contact and tapping mode scanning force microscopy at a lateral resolution of 3–5 nm and vertical resolution of ∼0.3 nm. The images of the intact thylakoids revealed detailed structural features of their surface, including individual protein complexes over stroma, grana margin and grana‐end membrane domains. Structural and immunogold‐assisted assignment of two of these complexes, photosystem I (PS I) and ATP synthase, allowed direct determination of their surface density, which, for both, was found to be highest in grana margins. Surface rearrangements and pigment– protein complex redistribution associated with salt‐induced membrane unstacking were followed on native, hydrated specimens. Unstacking was accompanied by a substantial increase in grana diameter and, eventually, led to their merging with the stroma lamellae. Concomitantly, PS IIα effective antenna size decreased by 21% and the mean size of membrane particles increased substantially, consistent with attachment of mobile light‐harvesting complex II to PS I. The ability to image intact photosynthetic membranes at molecular resolution, as demonstrated here, opens up new vistas to investigate thylakoid structure and function.

Passive and facilitated transport in nuclear pore complexes is largely uncoupled.

Nuclear pore complexes provide the sole gateway for the exchange of material between nucleus and cytoplasm of interphase eukaryotic cells. They support two modes of transport: passive diffusion of ions, metabolites, and intermediate-sized macromolecules and facilitated, receptor-mediated translocation of proteins, RNA, and ribonucleoprotein complexes. It is generally assumed that both modes of transport occur through a single diffusion channel located within the central pore of the nuclear pore complex. To test this hypothesis, we studied the mutual effects between transporting molecules utilizing either the same or different modes of translocation. We find that the two modes of transport do not interfere with each other, but molecules utilizing a particular mode of transport do hinder motion of others utilizing the same pathway. We therefore conclude that the two modes of transport are largely segregated.

DNA toroids: framework for DNA repair in Deinococcus radiodurans and in germinating bacterial spores

Bacteria belonging to the family Deinococcaceae survive exposure to >1.5 megarads of ionizing irradiation or to extreme desiccation without lethality or mutagenesis (2, 31, 35). This tolerance derives from the ability of these species to accurately mend numerous double-strand DNA breaks (DSBs), thus reassembling an intact genome from hundreds of fragments in a manner that restores chromosomal continuity. The only known mechanism that enables accurate repair of DSBs in bacteria is RecA-dependent homologous recombination, whereby information lost at a lesion is restored by a homologous DNA sequence that acts as a template (22-24). As such, DNA repair via homologous recombination strictly depends upon the ability of cellular systems to perform a rapid and efficient genomewide search for homologous DNA sites (27). However, following extensive DNA fragmentation, no intact template remains. Homologous search conducted under such circumstances would necessarily entail repetitive reinspection of multiple randomly dispersed DNA fragments, rendering the process inherently futile (9a, 36). Indeed, the first phase of DNA repair in Deinococcus radiodurans was shown to be RecA independent (9), implying that this phase does not rely on homologous recombination. The high resistance of bacterial spores to irradiation and desiccation indicates that DSBs inflicted by these assaults on dormant spores are efficiently and accurately mended upon germination. However, DNA repair involving homologous search processes cannot occur in germinating spores, because bacterial spores regularly carry only one copy of their genomes (5). Consequently, germinating spores lack the template required for accurate homologous-recombination-mediated repair of DSBs.

The rediscovered Hula painted frog is a living fossil

Amphibian declines are seen as an indicator of the onset of a sixth mass extinction of life on earth. Because of a combination of factors such as habitat destruction, emerging pathogens and pollutants, over 156 amphibian species have not been seen for several decades, and 34 of these were listed as extinct by 2004. Here we report the rediscovery of the Hula painted frog, the first amphibian to have been declared extinct. We provide evidence that not only has this species survived undetected in its type locality for almost 60 years but also that it is a surviving member of an otherwise extinct genus of alytid frogs, Latonia, known only as fossils from Oligocene to Pleistocene in Europe. The survival of this living fossil is a striking example of resilience to severe habitat degradation during the past century by an amphibian.

Protein kinase C penetration into lipid bilayers

Physical characteristics of the association and subsequent penetration of protein kinase C into defined lipid bilayers were analyzed using four different fluorescence probes. The enzyme demonstrated strong hydrophobic and electrostatic interactions with the bilayer as suggested by its ability to increase permeability of carboxyfluorescein-filled unilamellar vesicles. The intensity of interaction was dependent on the concentration of phosphatidylserine. The hydrophilic quencher, N-methylpicolinium perchlorate, was used to show that the tryptophan residues affected by ligand-induced conformational changes were in a hydrophobic region(s) of the enzyme. Using quenching of intrinsic tryptophan fluorescence, the enzyme was shown to penetrate the lipid bilayer to the C-16 position of labeled fatty acid probes. The association and subsequent penetration of the enzyme into the lipid bilayer was independent of divalent cations in these systems and had no significant effect on activator-independent substrate phosphorylation.