measuring-synaptic-vesicle-endocytosis-in-cultured-hippocampal-neurons

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

National Institute of Neurological Disorders and Stroke

Designing and building multigene constructs is commonplace in synthetic biology. Yet functional successes at first attempts are rare because the genetic parts are not fully modular. In order to improve the modularity of transcription, we previously showed that transcription termination in vitro by bacteriophage T7 RNA polymerase could be made more efficient by substituting the standard, single, TΦ large (class I) terminator with adjacent copies of the vesicular stomatitis virus (VSV) small (class II) terminator. However, in vitro termination at the downstream VSV terminator was less efficient than at the upstream VSV terminator, and multigene overexpression in vivo was complicated by unexpectedly inefficient VSV termination within Escherichia coli cells. Here, we address hypotheses raised in that study by showing that VSV or preproparathyroid hormone (PTH) small terminators spaced further apart can work independently (i.e., more efficiently) in vitro, and that VSV and PTH terminations are severely inhibited in vivo. Surprisingly, the difference between class II terminator function in vivo versus in vitro is not due to differences in plasmid supercoiling, as supercoiling had a minimal effect on termination in vitro. We therefore turned to TΦ terminators for "BioBrick" synthesis of a pentameric gene construct suitable for overexpression in vivo. This indeed enabled coordinated overexpression and copurification of five His-tagged proteins using the first construct attempted, indicating that this strategy is more modular than other strategies. An application of this multigene overexpression and protein copurification method is demonstrated by supplying five of the six E. coli translation factors required for reconstitution of translation from a single cell line via copurification, greatly simplifying the reconstitution.

Multigene expression in vivo: supremacy of large versus small terminators for T7 RNA polymerase.

Using affinity purifications coupled with mass spectrometry and yeast two-hybrid assays, we show the Saccharomyces cerevisiae translation initiation factor complex eukaryotic translation initiation factor 2B (eIF2B) and the very-long-chain fatty acid (VLCFA) synthesis keto-reductase enzyme YBR159W physically interact. The data show that the interaction is specifically between YBR159W and eIF2B and not between other members of the translation initiation or VLCFA pathways. A ybr159wΔ null strain has a slow-growth phenotype and a reduced translation rate but a normal GCN4 response to amino acid starvation. Although YBR159W localizes to the endoplasmic reticulum membrane, subcellular fractionation experiments show that a fraction of eIF2B cofractionates with lipid membranes in a YBR159W-independent manner. We show that a ybr159wΔ yeast strain and other strains with null mutations in the VLCFA pathway cause eIF2B to appear as numerous foci throughout the cytoplasm.

The yeast eukaryotic translation initiation factor 2B translation initiation complex interacts with the fatty acid synthesis enzyme YBR159W and endoplasmic reticulum membranes.

The circadian control of cellular processes in cyanobacteria is regulated by a posttranslational oscillator formed by three Kai proteins. During the oscillator cycle KaiA serves to promote autophosphorylation of KaiC while KaiB counteracts this effect. Here we present a crystallographic structure of the wild-type Synechococcus elongatus KaiB and a cryoEM structure of a KaiBC complex. The crystal structure shows the expected dimer core structure and significant conformational variations of the KaiB C-terminal region, which is functionally important in maintaining rhythmicity. The KaiBC sample was formed with a C-terminally truncated form of KaiC, KaiC-Î”489, which is persistently phosphorylated. The KaiB-KaiC-Î”489 structure reveals that the KaiC hexamer can bind six monomers of KaiB, which form a continuous ring of density in the KaiBC complex. We performed cryoEM guided molecular dynamics flexible fitting simulations with crystal structures of KaiB and KaiC to probe the KaiBC protein-protein interface. This analysis indicated a favorable binding mode for the KaiB monomer on the CII end of KaiC, involving two adjacent KaiC subunits and spanning an ATP binding cleft. A KaiC mutation, R468C, which has been shown to affect the affinity of KaiB for KaiC and lengthen the period in a bioluminescence rhythm assay, is found within the middle of the predicted KaiBC interface. The proposed KaiB binding mode blocks access to the ATP binding cleft in the CII ring of KaiC, which provides insight into how KaiB might influence the phosphorylation status of KaiC.

CryoEM and Molecular Dynamics of the Circadian KaiB-KaiC Complex Indicates KaiB Monomers Interact with KaiC and Block ATP Binding Clefts.

Le rythme circadien contr&ocirc;le de processus cellulaires chez les cyanobact&eacute;ries est r&eacute;glement&eacute; par un oscillateur post-traductionnelle form&eacute; par trois prot&eacute;ines de Kai. Au cours du cycle de l&#39;oscillateur KaiA sert &agrave; promouvoir l&#39;autophosphorylation de KaiC tandis que kaoula neutralise cet effet. Nous pr&eacute;sentons ici une structure cristallographique de la sauvage Synechococcus elongatus kaoula et une structure de cryoEM d&#39;un KaiBC complexe. La structure cristalline montre la structure de base de dim&egrave;re attendus et des variations importantes de conformations de la r&eacute;gion de kaoula C-terminal, qui est fonctionnellement important dans le maintien de rythmicit&eacute;. L&#39;exemple de KaiBC a &eacute;t&eacute; form&eacute; avec une C-terminale tronqu&eacute; forme de KaiC, KaiC-&Delta;489, qui est phosphoryl&eacute; constamment. La structure de kaoula-KaiC-&Delta;489 r&eacute;v&egrave;le que l&#39;hexam&egrave;re kanoute pouvez lier six monom&egrave;res de kanoute, qui forment un anneau continu de densit&eacute; dans le complexe de KaiBC. Nous avons effectu&eacute; des simulations de raccord flexible de dynamique mol&eacute;culaire de cryoEM guid&eacute;e avec structures cristallines de kanoute et KaiC pour sonder l&#39;interface prot&eacute;ine-prot&eacute;ine de KaiBC. Cette analyse a r&eacute;v&eacute;l&eacute; un mode de liaison favorable pour le monom&egrave;re kaoula sur l&#39;extr&eacute;mit&eacute; de la CII de KaiC, impliquant deux sous-unit&eacute;s kanoute adjacentes et s&#39;&eacute;tendant sur une fente de fixation de l&#39;ATP. Une mutation kanoute, R468C, qui a &eacute;t&eacute; montr&eacute; pour affecter l&#39;affinit&eacute; de kaoula pour KaiC et allonger la p&eacute;riode lors d&#39;un essai de rythme de bioluminescence, se trouve dans le milieu de l&#39;interface KaiBC pr&eacute;dite. Le projet kaoula liaison mode bloque l&#39;acc&egrave;s &agrave; la liaison ATP une fente dans l&#39;anneau CII de KaiC, qui donne une id&eacute;e de comment kaoula susceptibles d&#39;influencer l&#39;&eacute;tat de phosphorylation de KaiC.

CryoEM et dynamique mol&eacute;culaire du complexe kaoula-kanoute circadienne indique kaoula monom&egrave;res interagissent avec kanoute et bloquer l&#39;ATP Binding fentes.

Intrinsically disordered regions of proteins and conformational flexibility within complexes can be critical for biological function. However, disorder, flexibility, and heterogeneity often hinder structural analyses. CryoEM and single particle image processing techniques offer the possibility of imaging samples with significant flexibility. Division of particle images into more homogenous subsets after data acquisition can help compensate for heterogeneity within the sample. We present the utility of an eigenimage sorting analysis for examining two protein/DNA complexes with significant conformational flexibility and heterogeneity. These complexes are integral to the non-homologous end joining pathway, and are involved in the repair of double strand breaks of DNA. Both complexes include the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and biotinylated DNA with bound streptavidin, with one complex containing the Ku heterodimer. Initial 3D reconstructions of the two DNA-PKcs complexes resembled a cryoEM structure of uncomplexed DNA-PKcs without additional density clearly attributable to the remaining components. Application of eigenimage sorting allowed division of the DNA-PKcs complex datasets into more homogeneous subsets. This led to visualization of density near the base of the DNA-PKcs that can be attributed to DNA, streptavidin, and Ku. However, comparison of projections of the subset structures with 2D class averages indicated that a significant level of heterogeneity remained within each subset. In summary, image sorting methods allowed visualization of extra density near the base of DNA-PKcs, suggesting that DNA binds in the vicinity of the base of the molecule and potentially to a flexible region of DNA-PKcs.

CryoEM and image sorting for flexible protein/DNA complexes.

Membrane fusion and fission are vital for eukaryotic life. For three decades, it has been proposed that fusion is mediated by fusion between the proximal leaflets of two bilayers (hemi-fusion) to produce a hemi-fused structure, followed by fusion between the distal leaflets, whereas fission is via hemi-fission, which also produces a hemi-fused structure, followed by full fission. This hypothesis remained unsupported owing to the lack of observation of hemi-fusion or hemi-fission in live cells. A competing fusion hypothesis involving protein-lined pore formation has also been proposed. Here we report the observation of a hemi-fused Ω-shaped structure in live neuroendocrine chromaffin cells and pancreatic β-cells, visualized using confocal and super-resolution stimulated emission depletion microscopy. This structure is generated from fusion pore opening or closure (fission) at the plasma membrane. Unexpectedly, the transition to full fusion or fission is determined by competition between fusion and calcium/dynamin-dependent fission mechanisms, and is notably slow (seconds to tens of seconds) in a substantial fraction of the events. These results provide key missing evidence in support of the hemi-fusion and hemi-fission hypothesis in live cells, and reveal the hemi-fused intermediate as a key structure controlling fusion and fission, as fusion and fission mechanisms compete to determine the transition to fusion or fission.

Seth Villarreal

National Institute of Neurological Disorders and Stroke

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons