Developmental Changes in the Structure and Composition of the Postsynaptic Density

The development of the brain and its underlying circuitry is dependent on the formation of trillions of chemical synapses, which are highly specialized contacts that regulate the flow of information from one neuron to the next. It is through these synaptic connections that neurons wire together into networks capable of performing specific tasks, and activity-dependent changes in their structural and physiological state is one way that the brain is thought to adapt and store information. At the ultrastructural level, developmental and activity-dependent changes in the size and shape of dendritic spines have been well documented, and it is widely believed that structural changes in spines are a hallmark sign of synapse maturation and alteration of synaptic physiology. While changes in spine structure have been studied extensively, changes in one of its most prominent components, the postsynaptic density (PSD), have largely evaded observation. The PSD is a protein-rich organelle on the cytoplasmic side of the postsynaptic membrane, where it sits in direct opposition to the presynaptic terminal. The PSD functions both to cluster neurotransmitter receptors at the cell surface as well as organize the intracellular signaling molecules responsible for transducing extracellular signals to the postsynaptic cell. Much is known about the chemical composition of the PSD, but the structural arrangement of its molecular components is not well documented. Adding to the difficulty of understanding such a complex mass of protein machinery is the fact that its protein composition is known to change in response to synaptic activity, meaning that its structure is plastic and no two PSDs are identical. Here, immuno-gold labeling and electron tomography of PSDs isolated throughout development was used to track changes in both the structure and molecular composition of the PSD. State-of-the-art cryo-electron tomography was used to study the fine structure of the PSD during development, and provides an unprecedented glimpse into its molecular architecture in an un-fixed, unstained and hydrated state. Through this analysis, large structural and compositional changes are apparent and suggest a model by which the PSD is first assembled as a mesh-like lattice of proteins that function as support for the later recruitment of various PSD components. Spatial analysis of the recruitment of proteins into the PSD demonstrated that its assembly has an underlying order

Structural Plasticity within the Postsynaptic Density

The postsynaptic density (PSD) is a large protein complex that clusters neurotransmitter receptors at the synapse and organizes the intracellular signaling molecules responsible for altering the efficiency of synaptic transmission – termed synaptic plasticity. We propose that synapses from different parts of the brain place unique demands on the process of synaptic transmission and that the structure and composition of the PSD play a role in providing these distinctive properties. To begin to address this question, PSDs were isolated from adult rat cerebella, hippocampi and cortices, three brain areas amenable to straightforward isolation that contain unique distributions of neuronal cell types. Electron-tomography (ET) was used to visualize the fine morphology of the isolated PSDs and calculate total protein occupancy within the PSD structure. Immunogold labeling was utilized to quantify protein composition and distribution of key signaling and scaffold molecules. Although the mean surface area did not significantly differ between PSD types, the PSD thickness, as measured from Cryo ET reconstructions, differed significantly between PSD types. Labeling densities for PSD-95 and αCaMKII were found to differ dramatically among the PSD types, while all regions had moderate to high labeling for βCaMKII, illustrating the importance of βCaMKII to the PSD structure. PSD-95, a scaffold protein, was absent from a fraction of cerebellar PSDs, unlike hippocampal and cortical PSDs, showing that protein composition varies between PSD types. Ripley's K function analysis of immunogold labeled PSDs showed that PSD-95 was clustered in cerebellar PSDs, unlike other PSD types, suggesting a different function for PSD-95 in cerebellar PSDs. In contrast, βCaMKII was found to have similar non-random organization in all PSD types. These results support the idea that the composition and structure of the PSD are modified to achieve the specific synaptic functions required of each brain region

The Helical MreB Cytoskeleton in Escherichia coli MC1000/pLE7 Is an Artifact of the N-Terminal Yellow Fluorescent Protein Tag

Based on fluorescence microscopy, the actin homolog MreB has been thought to form extended helices surrounding the cytoplasm of rod-shaped bacterial cells. The presence of these and other putative helices has come to dominate models of bacterial cell shape regulation, chromosome segregation, polarity, and motility. Here we use electron cryotomography to show that MreB does in fact form extended helices and filaments in Escherichia coli when yellow fluorescent protein (YFP) is fused to its N terminus but native (untagged) MreB expressed to the same levels does not. In contrast, mCherry fused to an internal loop (MreB-RFP^(SW)) does not induce helices. The helices are therefore an artifact of the placement of the fluorescent protein tag. YFP-MreB helices were also clearly distinguishable from the punctate, “patchy” localization patterns of MreB-RFP^(SW), even by standard light microscopy. The many interpretations in the literature of such punctate patterns as helices should therefore be reconsidered

Role of the N- and C-lobes of calmodulin in the activation of Ca(2+)/calmodulin-dependent protein kinase II.

Understanding the principles of calmodulin (CaM) activation of target enzymes will help delineate how this seemingly simple molecule can play such a complex role in transducing Ca (2+)-signals to a variety of downstream pathways. In the work reported here, we use biochemical and biophysical tools and a panel of CaM constructs to examine the lobe specific interactions between CaM and CaMKII necessary for the activation and autophosphorylation of the enzyme. Interestingly, the N-terminal lobe of CaM by itself was able to partially activate and allow autophosphorylation of CaMKII while the C-terminal lobe was inactive. When used together, CaMN and CaMC produced maximal CaMKII activation and autophosphorylation. Moreover, CaMNN and CaMCC (chimeras of the two N- or C-terminal lobes) both activated the kinase but with greater K act than for wtCaM. Isothermal titration calorimetry experiments showed the same rank order of affinities of wtCaM \u3e CaMNN \u3e CaMCC as those determined in the activity assay and that the CaM to CaMKII subunit binding ratio was 1:1. Together, our results lead to a proposed sequential mechanism to describe the activation pathway of CaMKII led by binding of the N-lobe followed by the C-lobe. This mechanism contrasts the typical sequential binding mode of CaM with other CaM-dependent enzymes, where the C-lobe of CaM binds first. The consequence of such lobe specific binding mechanisms is discussed in relation to the differential rates of Ca (2+)-binding to each lobe of CaM during intracellular Ca (2+) oscillations

Bacterial mechanosensitive channels : progress towards an understanding of their roles in cell physiology

Open Access funded by Wellcome Trust Under a Creative Commons license Thanks to all members of the Aberdeen group, collaborators and friends whose discussions have spurred the development of the MS channel field. Special thanks to Doug Rees, Diane Newman and Rob Phillips for their support and hospitality at Caltech. Unique insights have been provided by members of the Newman and Phillips research groups, particularly, Caj Neubauer, Gargi Kulkarni and Megan Bergkessel, Heun Jin Lee and Maja Bialecka-Fornal. The author's research on MS channels is supported by a grant from The Wellcome Trust (WT092552MA) and the BBSRC (BB/H017917/1). The author is a Leverhulme Emeritus Fellow and this work was supported in part by a CEMI Visiting Faculty Fellowship from Caltech.Peer reviewedPublisher PD

Coarse-grained simulations of actomyosin rings point to a nodeless model involving both unipolar and bipolar myosins

Cytokinesis in many eukaryotic cells is orchestrated by a contractile actomyosin ring. While many of the proteins involved are known, the mechanism of constriction remains unclear. Informed by the existing literature and new three-dimensional (3D) molecular details from electron cryotomography, here we develop 3D coarse-grained models of actin filaments, unipolar and bipolar myosins, actin cross-linkers, and membranes and simulate their interactions. Assuming that local force on the membrane results in inward growth of the cell wall, we explored a matrix of possible actomyosin configurations and found that node-based architectures like those presently described for ring assembly result in membrane puckers not seen in electron microscope images of real cells. Instead, the model that best matches data from fluorescence microscopy, electron cryotomography, and biochemical experiments is one in which actin filaments transmit force to the membrane through evenly distributed, membrane-attached, unipolar myosins, with bipolar myosins in the ring driving contraction. While at this point this model is only favored (not proven), the work highlights the power of coarse-grained biophysical simulations to compare complex mechanistic hypotheses

Structure of the fission yeast actomyosin ring during constriction

Cell division in many eukaryotes is driven by a ring containing actin and myosin. While much is known about the main proteins involved, the precise arrangement of actin filaments within the contractile machinery, and how force is transmitted to the membrane, remains unclear. Here we use cryosectioning and cryofocused ion beam milling to gain access to cryopreserved actomyosin rings in Schizosaccharomyces pombe for direct 3D imaging by electron cryotomography. Our results show that straight, overlapping actin filaments, running nearly parallel to each other and to the membrane, form a loose bundle of ∼150 nm in diameter that “saddles” the inward-bending membrane at the leading edge of the division septum. The filaments do not make direct contact with the membrane. Our analysis of the actin filaments reveals the variability in filament number, nearest-neighbor distances between filaments within the bundle, their distance from the membrane, and angular distribution with respect to the membrane