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

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

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

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

Structure and Constriction Mechanism of the Actomyosin Ring

Cytokinesis is orchestrated by a contractile actomyosin ring, but its structure and mechanism remain elusive. We visualized the 3D structure of the ring in frozen-hydrated dividing yeast cells by electron cryotomography (ECT). Detailed arrangements of actin filaments within the ring and with respect to the membrane were seen for the first time, providing a crucial spatial constraint for the constriction mechanism of the ring. Using the ECT data and input from the literature we then explored sixteen mechanistic models by coarse-grained simulations at the 3D molecular details, revealing plausible mechanisms for preventing membrane distortion and protein aggregation. We found that, in the model that best fits experimental data, both bipolar and membrane-attached unipolar myosins exist in the ring, reconciling two different views in the field regarding the myosin configuration. In this model, ring tension is generated primarily by interactions between bipolar myosins and actin, and transmitted to the membrane via unipolar myosins. This model recapitulates a broad distribution of distances from actin filaments to the membrane observed in our tomograms and separation of two different myosin isoforms into the outer and inner subdomains of the ring reported in a previous fluorescence microscopy study. Further, it rationalizes how bundles of actomyosin were able to separate from the membrane in fluorescence microscopy experiments of the same previous study