Search

Lateral lipid heterogeneity (i.e., raft formation) in biomembranes plays a functional role in living cells. Three-component mixtures of low- and high-melting lipids plus cholesterol offer a simplified experimental model for raft domains in which a liquid-disordered (Ld) phase coexists with a liquid-ordered (Lo) phase. Using such models, we recently showed that cryogenic electron microscopy (cryo-EM) can detect phase separation in lipid vesicles based on differences in bilayer thickness. However, the considerable noise within cryo-EM data poses a significant challenge for accurately determining the membrane phase state at high spatial resolution. To this end, we have developed an image-processing pipeline that utilizes machine learning (ML) to predict the bilayer phase in projection images of lipid vesicles. Importantly, the ML method exploits differences in both the thickness and molecular density of Lo compared to Ld, which leads to improved phase identification. To assess accuracy, we used artificial images of phase-separated lipid vesicles generated from all-atom molecular dynamics simulations of Lo and Ld phases. Synthetic ground-truth data sets mimicking a series of compositions along a tieline of Ld + Lo coexistence were created and then analyzed with various ML models. For all tieline compositions, we find that the ML approach can correctly identify the bilayer phase with >90% accuracy, thus providing a means to isolate the intensity profiles of coexisting Ld and Lo phases, as well as accurately determine domain-size distributions, number of domains, and phase-area fractions. The method described here provides a framework for characterizing nanoscopic lateral heterogeneities in membranes and paves the way for a more detailed understanding of raft properties in biological contexts., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The branched architecture of neuronal dendrites is a key factor in how neurons form ordered networks and discoveries continue to be made identifying proteins and protein-protein interactions that direct or execute the branching and extension of dendrites. Our prior work showed that the molecular scaffold Pdlim5 and delta-catenin, in conjunction, are two proteins that help regulate the branching and elongation of dendrites in cultured hippocampal neurons and do so through a phosphorylation-dependent mechanism triggered by upstream glutamate signaling. In this report we have focused on Pdlim5's multiple scaffolding domains and how each contributes to dendrite branching. The three identified regions within Pdlim5 are the PDZ, DUF, and a trio of LIM domains; however, unresolved is the intra-molecular conformation of Pdlim5 as well as which domains are essential to regulate dendritic branching. We address Pdlim5's structure and function by examining the role of each of the domains individually and using deletion mutants in the context of the full-length protein. Results using primary hippocampal neurons reveal that the Pdlim5 DUF domain plays a dominant role in increasing dendritic branching. Neither the PDZ domain nor the LIM domains alone support increased branching. The central role of the DUF domain was confirmed using deletion mutants in the context of full-length Pdlim5. Guided by molecular modeling, additional domain mapping studies showed that the C-terminal LIM domain forms a stable interaction with the N-terminal PDZ domain, and we identified key amino acid residues at the interface of each domain that are needed for this interaction. We posit that the central DUF domain of Pdlim5 may be subject to modulation in the context of the full-length protein by the intra-molecular interaction between the N-terminal PDZ and C-terminal LIM domains. Overall, our studies reveal a novel mechanism for the regulation of Pdlim5's function in the regulation of neuronal branching and highlight the critical role of the DUF domain in mediating these effects.

Neuronal connectivity is regulated during normal brain development with the arrangement of spines and synapses being dependent on the morphology of dendrites. Further, in multiple neurodevelopmental and aging disorders, disruptions of dendrite formation or shaping is associated with atypical neuronal connectivity. We showed previously that Pdlim5 binds delta-catenin and promotes dendrite branching. We report here that Pdlim5 interacts with PalmD, a protein previously suggested by others to interact with the cytoskeleton (e.g., via adducin/spectrin) and to regulate membrane shaping. Functionally, the knockdown of PalmD or Pdlim5 in rat primary hippocampal neurons dramatically reduces branching and conversely, PalmD exogenous expression promotes dendrite branching as does Pdlim5. Further, we show that each proteins' effects are dependent on the presence of the other. In summary, using primary rat hippocampal neurons we reveal the contributions of a novel Pdlim5:PalmD protein complex, composed of functionally inter-dependent components responsible for shaping neuronal dendrites., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Srivastava, Donta, Mireles, Paulucci-Holthauzen, Waxham and McCrea.)

We describe a method for investigating lateral membrane heterogeneity using cryogenic electron microscopy (cryo-EM) images of liposomes. The method takes advantage of differences in the thickness and molecular density of ordered and disordered phases that are resolvable in phase contrast cryo-EM. Compared to biophysical techniques like FRET or neutron scattering that yield ensemble-averaged information, cryo-EM provides direct visualization of individual vesicles and can therefore reveal variability that would otherwise be obscured by averaging. Moreover, because the contrast mechanism involves inherent properties of the lipid phases themselves, no extrinsic probes are required. We explain and discuss various complementary analyses of spatially resolved thickness and intensity measurements that enable an assessment of the membrane's phase state. The method opens a window to nanodomain structure in synthetic and biological membranes that should lead to an improved understanding of lipid raft phenomena., (Copyright © 2024. Published by Elsevier Inc.)

Some of the prominent features of long-term memory formation include protein synthesis, gene expression, enhanced neurotransmitter release, increased excitability, and formation of new synapses. As these processes are critically dependent on mitochondrial function, we hypothesized that increased mitochondrial respiration and dynamics would play a prominent role in memory formation. To address this possibility, we measured mitochondrial oxygen consumption (OCR) in hippocampal tissue punches from trained and untrained animals. Our results show that context fear training significantly increased basal, ATP synthesis-linked, and maximal OCR in the Shaffer collateral-CA1 synaptic region, but not in the CA1 cell body layer. These changes were recapitulated in synaptosomes isolated from the hippocampi of fear-trained animals. As dynamin-related protein 1 (Drp1) plays an important role in mitochondrial fission, we examined its role in the increased mitochondrial respiration observed after fear training. Drp1 inhibitors decreased the training-associated enhancement of OCR and impaired contextual fear memory, but did not alter the number of synaptosomes containing mitochondria. Taken together, our results show context fear training increases presynaptic mitochondria respiration, and that Drp-1 mediated enhanced energy production in CA1 pre-synaptic terminals is necessary for context fear memory that does not result from an increase in the number of synaptosomes containing mitochondria or an increase in mitochondrial mass within the synaptic layer., (© 2023. Springer Nature Limited.)

We aim to elucidate the molecular mechanism of the reciprocal relation of calmodulin's (CaM) target binding and its affinity for calcium ions (Ca 2+ ), which is central to decoding CaM-dependent Ca 2+ signaling in a cell. We employed stopped-flow experiments and coarse-grained molecular simulations that learn the coordination chemistry of Ca 2+ in CaM from first-principle calculations. The associative memories as part of the coarse-grained force fields built on known protein structures further influence CaM's selection of its polymorphic target peptides in the simulations. We modeled the peptides from the Ca 2+ /CaM-binding domain of Ca 2+ /CaM-dependent kinase II (CaMKII), CaMKIIp (293-310) and selected distinctive mutations at the N-terminus. Our stopped-flow experiments have shown that the CaM's affinity for Ca 2+ in the bound complex of Ca 2+ /CaM/CaMKIIp decreased significantly when Ca 2+ /CaM bound to the mutant peptide (296-AAA-298) compared to that bound to the wild-type peptide (296-RRK-298). The coarse-grained molecular simulations revealed that the 296-AAA-298 mutant peptide destabilized the structures of Ca 2+ -binding loops at the C-domain of CaM (c-CaM) due to both loss of electrostatic interactions and differences in polymorphic structures. We have leveraged a powerful coarse-grained approach to advance a residue-level understanding of the reciprocal relation in CaM, that could not be possibly achieved by other computational approaches.

Dendritic arborization is essential for proper neuronal connectivity and function. Conversely, abnormal dendrite morphology is associated with several neurological pathologies like Alzheimer's disease and schizophrenia. Among major intrinsic mechanisms that determine the extent of the dendritic arbor is cytoskeletal remodeling. Here, we characterize and compare the impact of the four proteins involved in cytoskeletal remodeling-vertebrate members of the p120-catenin subfamily-on neuronal dendrite morphology. In relation to each of their own distributions, we find that p120-catenin and delta-catenin are expressed at relatively higher proportions in growth cones compared to ARVCF-catenin and p0071-catenin; ARVCF-catenin is expressed at relatively high proportions in the nucleus; and all catenins are expressed in dendritic processes and the soma. Through altering the expression of each p120-subfamily catenin in neurons, we find that exogenous expression of either p120-catenin or delta-catenin correlates with increased dendritic length and branching, whereas their respective depletion decreases dendritic length and branching. While increasing ARVCF-catenin expression also increases dendritic length and branching, decreasing expression has no grossly observable morphological effect. Finally, increasing p0071-catenin expression increases dendritic branching, but not length, while decreasing expression decreases dendritic length and branching. These distinct localization patterns and morphological effects during neuron development suggest that these catenins have both shared and distinct roles in the context of dendrite morphogenesis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Donta, Srivastava, Di Mauro, Paulucci-Holthauzen, Waxham and McCrea.)

The development of electron cryomicroscopy (cryo-EM) has evolved immensely in the last several decades and is now well-established in the analysis of protein structure both in isolation and in their cellular context. This review focuses on the history and application of cryo-EM to the analysis of membrane architecture. Parallels between the levels of organization of protein structure are useful in organizing the discussion of the unique parameters that influence membrane structure and function. Importantly, the timescales of lipid motion in bilayers with respect to the timescales of sample vitrification is discussed and reveals what types of membrane structure can be reliably extracted in cryo-EM images of vitrified samples. Appreciating these limitations, a review of the application of cryo-EM to examine the lateral organization of ordered and disordered domains in reconstituted and biologically derived membranes is provided. Finally, a brief outlook for further development and application of cryo-EM to the analysis of membrane architecture is provided., (© 2023 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society and the Royal Society of Biology.)

Cryogenic electron microscopy (cryo-EM) is among the most powerful tools available for interrogating nanoscale structure of biological materials. We recently showed that cryo-EM can be used to measure the bilayer thickness of lipid vesicles and biological membranes with subangstrom precision, resulting in the direct visualization of nanoscopic domains of different thickness in multicomponent lipid mixtures and giant plasma membrane vesicles. Despite the great potential of cryo-EM for revealing the lateral organization of biomembranes, a large parameter space of experimental conditions remains to be optimized. Here, we systematically investigate the influence of instrument parameters and image postprocessing steps on the ability to accurately measure bilayer thickness and discriminate regions of different thickness within unilamellar liposomes. This unique application of cryo-EM places particular demands on image acquisition optimization and analysis due to the facts that 1) each vesicle is a different size with different curvature, 2) the domains in each vesicle can be heterogenous in size, and 3) the random orientation of vesicles amplifies the variability of domain size in projected images. We also demonstrate a spatial autocorrelation analysis to extract additional information about lateral heterogeneity., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Lipid membranes are ubiquitous biological organizers, required for structural and functional compartmentalization of the cell and sub-cellular organelles. Membranes in living cells are compositionally complex, comprising hundreds of dynamically regulated, distinct lipid species. Cellular physiology requires tight regulation of these lipidomic profiles to achieve proper membrane functionality. While some general features of tissue- and organelle-specific lipid complements have been identified, less is known about detailed lipidomic variations caused by cell-intrinsic or extrinsic factors. Here, we use shotgun lipidomics to report detailed, comprehensive lipidomes of a variety of cultured and primary mammalian membrane preparations to identify trends and sources of variation. Unbiased principle component analysis (PCA) shows clear separation between cultured and primary cells, with primary erythrocytes, synaptic membranes, and other mammalian tissue lipidomes sharply diverging from all cultured cell lines and also from one other. Most broadly, cultured cell membrane preparations were distinguished by their paucity of polyunsaturated lipids. Cultured mammalian cell lines were comparatively similar to one another, although we detected clear, highly reproducible lipidomic signatures of individual cell lines and plasma membrane (PM) isolations thereof. These measurements begin to establish a comprehensive lipidomic atlas of mammalian cells and tissues, identifying some major sources of variation. These observations will allow investigation of the regulation and functional significance of mammalian lipidomes in various contexts.

In neurons, dendrites form the major sites of information receipt and integration. It is thus vital that, during development, the dendritic arbor is adequately formed to enable proper neural circuit formation and function. While several known processes shape the arbor, little is known of those that govern dendrite branching versus extension. Here, we report a new mechanism instructing dendrites to branch versus extend. In it, glutamate signaling activates mGluR5 receptors to promote Ckd5-mediated phosphorylation of the C-terminal PDZ-binding motif of delta-catenin. The phosphorylation state of this motif determines delta-catenin's ability to bind either Pdlim5 or Magi1. Whereas the delta:Pdlim5 complex enhances dendrite branching at the expense of elongation, the delta:Magi1 complex instead promotes lengthening. Our data suggest that these complexes affect dendrite development by differentially regulating the small-GTPase RhoA and actin-associated protein Cortactin. We thus reveal a "phospho-switch" within delta-catenin, subject to a glutamate-mediated signaling pathway, that assists in balancing the branching versus extension of dendrites during neural development., (© 2020 Baumert et al.)

The extracellular accumulation of amyloid β (Aβ) fragments of amyloid precursor protein (APP) in brain parenchyma is a pathological hallmark of Alzheimer's disease (AD). APP can be cleaved into Aβ on late endosomes/multivesicular bodies (MVBs). E3 ubiquitin ligases have been linked to Aβ production, but specific E3 ligases associated with APP ubiquitination that may affect targeting of APP to endosomes have not yet been described. Using cultured cortical neurons isolated from rat pups, we reconstituted APP movement into the internal vesicles (ILVs) of MVBs. Loss of endosomal sorting complexes required for transport (ESCRT) components inhibited APP movement into ILVs and increased endosomal Aβ42 generation, implying a requirement for APP ubiquitination. We identified an ESCRT-binding and APP-interacting endosomal E3 ubiquitin ligase, ubiquitination factor E4B (UBE4B) that regulates APP ubiquitination. Depleting UBE4B in neurons inhibited APP ubiquitination and internalization into MVBs, resulting in increased endosomal Aβ42 levels and increased neuronal secretion of Aβ42. When we examined AD brains, we found levels of the UBE4B-interacting ESCRT component, hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), were significantly decreased in AD brains. These data suggest that ESCRT components critical for membrane protein sorting in the endocytic pathway are altered in AD. These results indicate that the molecular machinery underlying endosomal trafficking of APP, including the ubiquitin ligase UBE4B, regulates Aβ levels and may play an essential role in AD progression., Competing Interests: Declaration of competing interest The authors have no competing financial interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Dendritic spines are tiny membranous protrusions on the dendrites of neurons. Dendritic spines change shape in response to input signals, thereby strengthening the connections between neurons. The growth and stabilization of dendritic spines is thought to be essential for maintaining long-term memory. Actin cytoskeleton remodeling in spines is a key element of their formation and growth. More speculatively, the aggregation of CPEB3, a functional prion that binds RNA, has been reported to be involved in the maintenance of long-term memory. Here we study the interaction between actin and CPEB3 and propose a molecular model for the complex structure of CPEB3 and an actin filament (F-actin). The results of our computational modeling, including both energetic and structural analyses, are compared with novel data from peptide array experiments. Our model of the CPEB3/F-actin interaction suggests that F-actin potentially triggers the aggregation-prone structural transition of a short CPEB3 sequence by zipping it into a beta-hairpin form. We also propose that the CPEB3/F-actin interaction might be regulated by the SUMOylation of CPEB3, based on bioinformatic searches for potential SUMOylation sites as well as SUMO interacting motifs in CPEB3. On the basis of these results and the existing literature, we put forward a possible molecular mechanism underlying long-term memory that involves CPEB3's binding to actin, its aggregation, and its regulation by SUMOylation., Competing Interests: Competing interest statement: N.P.S. is the founder of Schafer Science, LLC.

The nanoscale organization of biological membranes into structurally and compositionally distinct lateral domains is believed to be central to membrane function. The nature of this organization has remained elusive due to a lack of methods to directly probe nanoscopic membrane features. We show here that cryogenic electron microscopy (cryo-EM) can be used to directly image coexisting nanoscopic domains in synthetic and bioderived membranes without extrinsic probes. Analyzing a series of single-component liposomes composed of synthetic lipids of varying chain lengths, we demonstrate that cryo-EM can distinguish bilayer thickness differences as small as 0.5 Å, comparable to the resolution of small-angle scattering methods. Simulated images from computational models reveal that features in cryo-EM images result from a complex interplay between the atomic distribution normal to the plane of the bilayer and imaging parameters. Simulations of phase-separated bilayers were used to predict two sources of contrast between coexisting ordered and disordered phases within a single liposome, namely differences in membrane thickness and molecular density. We observe both sources of contrast in biomimetic membranes composed of saturated lipids, unsaturated lipids, and cholesterol. When extended to isolated mammalian plasma membranes, cryo-EM reveals similar nanoscale lateral heterogeneities. The methods reported here for direct, probe-free imaging of nanodomains in unperturbed membranes open new avenues for investigation of nanoscopic membrane organization., Competing Interests: The authors declare no competing interest.

Actomyosin networks give cells the ability to move and divide. These networks contract and expand while being driven by active energy-consuming processes such as motor protein walking and actin polymerization. Actin dynamics is also regulated by actin-binding proteins, such as the actin-related protein 2/3 (Arp2/3) complex. This complex generates branched filaments, thereby changing the overall organization of the network. In this work, the spatiotemporal patterns of dynamical actin assembly accompanying the branching-induced reorganization caused by Arp2/3 were studied using a computational model (mechanochemical dynamics of active networks [MEDYAN]); this model simulates actomyosin network dynamics as a result of chemical reactions whose rates are modulated by rapid mechanical equilibration. We show that branched actomyosin networks relax significantly more slowly than do unbranched networks. Also, branched networks undergo rare convulsive movements, "avalanches," that release strain in the network. These avalanches are associated with the more heterogeneous distribution of mechanically linked filaments displayed by branched networks. These far-from-equilibrium events arising from the marginal stability of growing actomyosin networks provide a possible mechanism of the "cytoquakes" recently seen in experiments., Competing Interests: The authors declare no competing interest.

We have developed a computational method of atomistically refining the structural ensemble of intrinsically disordered peptides (IDPs) facilitated by experimental measurements using circular dichroism spectroscopy (CD). A major challenge surrounding this approach stems from the deconvolution of experimental CD spectra into secondary structure features of the IDP ensemble. Currently available algorithms for CD deconvolution were designed to analyze the spectra of proteins with stable secondary structures. Herein, our work aims to minimize any bias from the peptide deconvolution analysis by implementing a non-negative linear least-squares fitting algorithm in conjunction with a CD reference data set that contains soluble and denatured proteins (SDP48). The non-negative linear least-squares method yields the best results for deconvolution of proteins with higher disordered content than currently available methods, according to a validation analysis of a set of protein spectra with Protein Data Bank entries. We subsequently used this analysis to deconvolute our experimental CD data to refine our computational model of the peptide secondary structure ensemble produced by all-atom molecular dynamics simulations with implicit solvent. We applied this approach to determine the ensemble structures of a set of short IDPs, that mimic the calmodulin binding domain of calcium/calmodulin-dependent protein kinase II and its 1-amino-acid and 3-amino-acid mutants. Our study offers a, to our knowledge, novel way to solve the ensemble secondary structures of IDPs in solution, which is important to advance the understanding of their roles in regulating signaling pathways through the formation of complexes with multiple partners., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Phosphatase and tensin homolog (PTEN)-induced kinase 1 (Pink1) is involved in mitochondrial quality control, which is essential for maintaining energy production and minimizing oxidative damage from dysfunctional/depolarized mitochondria. Pink1 mutations are the second most common cause of autosomal recessive Parkinson's disease (PD). In addition to characteristic motor impairments, PD patients also commonly exhibit cognitive impairments. As the hippocampus plays a prominent role in cognition, we tested if loss of Pink1 in mice influences learning and memory. While wild-type mice were able to perform a contextual discrimination task, age-matched Pink1 knockout (Pink1 - / - ) mice showed an impaired ability to differentiate between two similar contexts. Similarly, Pink1 - / - mice performed poorly in a delayed alternation task as compared to age-matched controls. Poor performance in these cognitive tasks was not the result of overt hippocampal pathology. However, a significant reduction in hippocampal tyrosine hydroxylase (TH) protein levels was detected in the Pink1 - / - mice. This decrease in hippocampal TH levels was also associated with reduced DOPA decarboxylase and dopamine D2 receptor levels, but not post-synaptic dopamine D1 receptor levels. These presynaptic changes appeared to be selective for dopaminergic fibers as hippocampal dopamine beta hydroxylase, choline acetyltransferase, and tryptophan hydroxylase levels were unchanged in Pink1 - / - mice. Administration of the dopamine D1 receptor agonist SKF38393 to Pink1 - / - mice was found to improve performance in the context discrimination task. Taken together, our results show that Pink1 loss may alter dopamine signaling in the hippocampus, which could be a contributing mechanism for the observed learning and memory impairments., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Extrusion through porous filters is a widely used method for preparing biomimetic model membranes. Of primary importance in this approach is the efficient production of single bilayer (unilamellar) vesicles that eliminate the influence of interlamellar interactions and strictly define the bilayer surface area available to external reagents such as proteins. Submicroscopic vesicles produced using extrusion are widely assumed to be unilamellar, and large deviations from this assumption would impact interpretations from many model membrane experiments. Using three probe-free methods-small angle X-ray and neutron scattering and cryogenic electron microscopy-we report unambiguous evidence of extensive multilamellarity in extruded vesicles composed of neutral phosphatidylcholine lipids, including for the common case of neutral lipids dispersed in physiological buffer and extruded through 100-nm diameter pores. In such preparations, only ∼35% of lipids are externally accessible and this fraction is highly dependent on preparation conditions. Charged lipids promote unilamellarity as does decreasing solvent ionic strength, indicating the importance of electrostatic interactions in determining the lamellarity of extruded vesicles. Smaller extrusion pore sizes also robustly increase the fraction of unilamellar vesicles, suggesting a role for membrane bending. Taken together, these observations suggest a mechanistic model for extrusion, wherein the formation of unilamellar vesicles involves competition between bilayer bending and adhesion energies. The findings presented here have wide-ranging implications for the design and interpretation of model membrane studies, especially ensemble-averaged observations relying on the assumption of unilamellarity., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Calcium/calmodulin-dependent kinase II (CaMKII) plays a key role in the plasticity of dendritic spines. Calcium signals cause calcium-calmodulin to activate CaMKII, which leads to remodeling of the actin filament (F-actin) network in the spine. We elucidate the mechanism of the remodeling by combining computer simulations with protein array experiments and electron microscopic imaging, to arrive at a structural model for the dodecameric complex of CaMKII with F-actin. The binding interface involves multiple domains of CaMKII. This structure explains the architecture of the micrometer-scale CaMKII/F-actin bundles arising from the multivalence of CaMKII. We also show that the regulatory domain of CaMKII may bind either calmodulin or F-actin, but not both. This frustration, along with the multipartite nature of the binding interface, allows calmodulin transiently to strip CaMKII from actin assemblies so that they can reorganize. This observation therefore provides a simple mechanism by which the structural dynamics of CaMKII establishes the link between calcium signaling and the morphological plasticity of dendritic spines., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Regulating the residence time of membrane proteins on the cell surface can modify their response to extracellular cues and allow for cellular adaptation in response to changing environmental conditions. The fate of membrane proteins that are internalized from the plasma membrane and arrive at the limiting membrane of the late endosome/multivesicular body (MVB) is dictated by whether they remain on the limiting membrane, bud into internal MVB vesicles, or bud outwardly from the membrane. The molecular details underlying the disposition of membrane proteins that transit this pathway and the mechanisms regulating these trafficking events are unclear. We established a cell-free system that reconstitutes budding of membrane protein cargo into internal MVB vesicles and onto vesicles that bud outwardly from the MVB membrane. Both budding reactions are cytosol-dependent and supported by Saccharomyces cerevisiae (yeast) cytosol. We observed that inward and outward budding from the MVB membrane are mechanistically distinct but may be linked, such that inhibition of inward budding triggers a re-routing of cargo from inward to outward budding vesicles, without affecting the number of vesicles that bud outwardly from MVBs., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Many important gram-negative bacterial pathogens use highly sophisticated type III protein secretion systems (T3SSs) to establish complex host-pathogen interactions. Bacterial-host cell contact triggers the activation of the T3SS and the subsequent insertion of a translocon pore into the target cell membrane, which serves as a conduit for the passage of effector proteins. Therefore the initial interaction between T3SS-bearing bacteria and host cells is the critical step in the deployment of the protein secretion machine, yet this process remains poorly understood. Here, we use high-throughput cryo-electron tomography (cryo-ET) to visualize the T3SS-mediated Salmonella -host cell interface. Our analysis reveals the intact translocon at an unprecedented level of resolution, its deployment in the host cell membrane, and the establishment of an intimate association between the bacteria and the target cells, which is essential for effector translocation. Our studies provide critical data supporting the long postulated direct injection model for effector translocation., Competing Interests: DP, ML, MW, WL, BH, JG, JL No competing interests declared, (© 2018, Park et al.)

Neurons project axons to local and distal sites and can display heterogeneous morphologies with limited physical dimensions that may influence the structure of large organelles such as mitochondria. Using cryo-electron tomography (cryo-ET), we characterized native environments within axons and presynaptic varicosities to examine whether spatial restrictions within these compartments influence the morphology of mitochondria. Segmented tomographic reconstructions revealed distinctive morphological characteristics of mitochondria residing at the narrowed boundary between presynaptic varicosities and axons with limited physical dimensions (approximately 80 nm), compared to mitochondria in nonspatially restricted environments. Furthermore, segmentation of the tomograms revealed discrete organizations between the inner and outer membranes, suggesting possible independent remodeling of each membrane in mitochondria at spatially restricted axonal/varicosity boundaries. Thus, cryo-ET of mitochondria within axonal subcompartments reveals that spatial restrictions do not obstruct mitochondria from residing within them, but limited available space can influence their gross morphology and the organization of the inner and outer membranes. These findings offer new perspectives on the influence of physical and spatial characteristics of cellular environments on mitochondrial morphology and highlight the potential for remarkable structural plasticity of mitochondria to adapt to spatial restrictions within axons., Competing Interests: The authors have declared that no competing interests exist.

Self-assembly of plasma membrane-associated Ras GTPases has major implications to the regulation of cell signaling. However, the structural basis of homo-oligomerization and the fractional distribution of oligomeric states remained undetermined. We have addressed these issues by deciphering the distribution of dimers and higher-order oligomers of K-Ras4B, the most frequently mutated Ras isoform in human cancers. We focused on the constitutively active G12V K-Ras and two of its variants, K101E and K101C/E107C, which respectively destabilize and stabilize oligomers. Using raster image correlation spectroscopy and number and brightness analysis combined with fluorescence recovery after photobleaching, fluorescence correlation spectroscopy and electron microscopy in live cells, we show that G12V K-Ras exists as a mixture of monomers, dimers and larger oligomers, while the K101E mutant is predominantly monomeric and K101C/E107C is dominated by oligomers. This observation demonstrates the ability of K-Ras to exist in multiple oligomeric states whose population can be altered by interfacial mutations. Using molecular modeling and simulations we further show that K-Ras uses two partially overlapping interfaces to form compositionally and topologically diverse oligomers. Our results thus provide the first detailed insight into the multiplicity, structure, and membrane organization of K-Ras homomers.

Ca 2+ -calmodulin-dependent protein kinase II (CaMKII) is highly abundant in neurons, where its concentration reaches that typically found for cytoskeletal proteins. Functional reasons for such a high concentration are not known, but given the multitude of known binding partners for CaMKII, a role as a scaffolding molecule has been proposed. In this report, we provide experimental evidence that demonstrates a novel structural role for CaMKII. We discovered that CaMKII forms filaments that can extend for several micrometers in the presence of certain divalent cations (Zn 2+ , Cd 2+ , and Cu 2+ ) but not with others (Ca 2+ , Mg 2+ , Co 2+ , and Ni 2+ ). Once formed, depleting the divalent ion concentration with chelators completely dissociated the filaments, and this process could be repeated by cyclic addition and removal of divalent ions. Using the crystal structure of the CaMKII holoenzyme, we computed an electrostatic potential map of the dodecameric complex to predict divalent ion binding sites. This analysis revealed a potential surface-exposed divalent ion binding site involving amino acids that also participate in calmodulin (CaM) binding and suggested CaM binding might inhibit formation of the filaments. As predicted, Ca 2+ /CaM binding both inhibited divalent ion-induced filament formation and could disassemble preformed filaments. Interestingly, CaMKII within the filaments retains the capacity to autophosphorylate; however, activity toward exogenous substrates is significantly decreased. Activity is restored upon filament disassembly. We compile our results with structural and mechanistic data from the literature to propose a model of Zn 2+ -mediated CaMKII filament formation, in which assembly and activity are further regulated by Ca 2+ /CaM.

Biological membranes contain a broad variety of lipid species whose individual physicochemical properties and collective interactions ultimately determine membrane organization. A key aspect of the organization of cellular membranes is their lateral subdivision into domains of distinct structure and composition. The most widely studied membrane domains are lipid rafts, which are the biological manifestations of liquid-ordered phases that form in sterol-containing membranes. Detailed studies of biomimetic membrane mixtures have yielded wide-ranging insights into the physical principles behind lipid rafts; however, these simplified models do not fully capture the diversity and complexity of the mammalian lipidome, most notably in their exclusion of polyunsaturated lipids. Here, we assess the role of lipid acyl chain unsaturation as a driving force for phase separation using coarse-grained molecular dynamics (CGMD) simulations validated by model membrane experiments. The clear trends in our observations and good qualitative agreements between simulations and experiments support the conclusions that highly unsaturated lipids promote liquid-liquid domain stability by enhancing the differences in cholesterol content and lipid chain order between the coexisting domains. These observations reveal the important role of noncanonical biological lipids in the physical properties of membranes, showing that lipid polyunsaturation is a driving force for liquid-liquid phase separation.

Neuronal synapses are the fundamental units of neural signal transduction and must maintain exquisite signal fidelity while also accommodating the plasticity that underlies learning and development. To achieve these goals, the molecular composition and spatial organization of synaptic terminals must be tightly regulated; however, little is known about the regulation of lipid composition and organization in synaptic membranes. Here we quantify the comprehensive lipidome of rat synaptic membranes during postnatal development and observe dramatic developmental lipidomic remodeling during the first 60 postnatal days, including progressive accumulation of cholesterol, plasmalogens, and sphingolipids. Further analysis of membranes associated with isolated postsynaptic densities (PSDs) suggests the PSD-associated postsynaptic plasma membrane (PSD-PM) as one specific location of synaptic remodeling. We analyze the biophysical consequences of developmental remodeling in reconstituted synaptic membranes and observe remarkably stable microdomains, with the stability of domains increasing with developmental age. We rationalize the developmental accumulation of microdomain-forming lipids in synapses by proposing a mechanism by which palmitoylation of the immobilized scaffold protein PSD-95 nucleates domains at the postsynaptic plasma membrane. These results reveal developmental changes in lipid composition and palmitoylation that facilitate the formation of postsynaptic membrane microdomains, which may serve key roles in the function of the neuronal synapse., (© 2016 Tulodziecka et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Mitochondrial function is intimately linked to cellular survival, growth, and death. Mitochondria not only generate ATP from oxidative phosphorylation, but also mediate intracellular calcium buffering, generation of reactive oxygen species (ROS), and apoptosis. Electron leakage from the electron transport chain, especially from damaged or depolarized mitochondria, can generate excess free radicals that damage cellular proteins, DNA, and lipids. Furthermore, mitochondrial damage releases pro-apoptotic factors to initiate cell death. Previous studies have reported that traumatic brain injury (TBI) reduces mitochondrial respiration, enhances production of ROS, and triggers apoptotic cell death, suggesting a prominent role of mitochondria in TBI pathophysiology. Mitochondria maintain cellular energy homeostasis and health via balanced processes of fusion and fission, continuously dividing and fusing to form an interconnected network throughout the cell. An imbalance of these processes, particularly an excess of fission, can be detrimental to mitochondrial function, causing decreased respiration, ROS production, and apoptosis. Mitochondrial fission is regulated by the cytosolic GTPase, dynamin-related protein 1 (Drp1), which translocates to the mitochondrial outer membrane (MOM) to initiate fission. Aberrant Drp1 activity has been linked to excessive mitochondrial fission and neurodegeneration. Measurement of Drp1 levels in purified hippocampal mitochondria showed an increase in TBI animals as compared to sham controls. Analysis of cryo-electron micrographs of these mitochondria also showed that TBI caused an initial increase in the length of hippocampal mitochondria at 24 h post-injury, followed by a significant decrease in length at 72 h. Post-TBI administration of Mitochondrial division inhibitor-1 (Mdivi-1), a pharmacological inhibitor of Drp1, prevented this decrease in mitochondria length. Mdivi-1 treatment also reduced the loss of newborn neurons in the hippocampus and improved novel object recognition (NOR) memory and context-specific fear memory. Taken together, our results show that TBI increases mitochondrial fission and that inhibition of fission improves hippocampal-dependent learning and memory, suggesting that strategies to reduce fission may have translational value after injury.

Protein-protein interactions play important roles in the control of every cellular process. How natural selection has optimized protein design to produce molecules capable of binding to many partner proteins is a fascinating problem but not well understood. Here, we performed a combinatorial analysis of protein sequence evolution and conformational dynamics to study how calmodulin (CaM), which plays essential roles in calcium signaling pathways, has adapted to bind to a large number of partner proteins. We discovered that amino acid residues in CaM can be partitioned into unique classes according to their degree of evolutionary conservation and local stability. Holistically, categorization of CaM residues into these classes reveals enriched physico-chemical interactions required for binding to diverse targets, balanced against the need to maintain the folding and structural modularity of CaM to achieve its overall function. The sequence-structure-function relationship of CaM provides a concrete example of the general principle of protein design. We have demonstrated the synergy between the fields of molecular evolution and protein biophysics and created a generalizable framework broadly applicable to the study of protein-protein interactions.

Protein signaling occurs in crowded intracellular environments, and while high concentrations of macromolecules are postulated to modulate protein-protein interactions, analysis of their impact at each step of the reaction pathway has not been systematically addressed. Potential cosolute-induced alterations in target association are particularly important for a signaling molecule like calmodulin (CaM), where competition among >300 targets governs which pathways are selectively activated. To explore how high concentrations of cosolutes influence CaM-target affinity and kinetics, we methodically investigated each step of the CaM-target binding mechanism under crowded or osmolyte-rich environments mimicked by ficoll-70, dextran-10, and sucrose. All cosolutes stabilized compact conformers of CaM and modulated association kinetics by affecting diffusion and rates of conformational change; however, the results showed that differently sized molecules had variable effects to enhance or impede unique steps of the association pathway. On- and off-rates were modulated by all cosolutes in a compensatory fashion, producing little change in steady-state affinity. From this work insights were gained on how high concentrations of inert crowding agents and osmolytes fit into a kinetic framework to describe protein-protein interactions relevant for cellular signaling., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Calmodulin (CaM) is a primary calcium (Ca(2+) )-signaling protein that specifically recognizes and activates highly diverse target proteins. We explored the molecular basis of target recognition of CaM with peptides representing the CaM-binding domains from two Ca(2+) -CaM-dependent kinases, CaMKI and CaMKII, by employing experimentally constrained molecular simulations. Detailed binding route analysis revealed that the two CaM target peptides, although similar in length and net charge, follow distinct routes that lead to a higher binding frustration in the CaM-CaMKII complex than in the CaM-CaMKI complex. We discovered that the molecular origin of the binding frustration is caused by intermolecular contacts formed with the C-domain of CaM that need to be broken before the formation of intermolecular contacts with the N-domain of CaM. We argue that the binding frustration is important for determining the kinetics of the recognition process of proteins involving large structural fluctuations., (Copyright © 2015 John Wiley & Sons, Ltd.)

Ras-GRF1 (GRF1) and Ras-GRF2 (GRF2) constitute a family of similar calcium sensors that regulate synaptic plasticity. They are both guanine exchange factors that contain a very similar set of functional domains, including N-terminal pleckstrin homology, coiled-coil, and calmodulin-binding IQ domains and C-terminal Dbl homology Rac-activating domains, Ras-exchange motifs, and CDC25 Ras-activating domains. Nevertheless, they regulate different forms of synaptic plasticity. Although both GRF proteins transduce calcium signals emanating from NMDA-type glutamate receptors in the CA1 region of the hippocampus, GRF1 promotes LTD, whereas GRF2 promotes θ-burst stimulation-induced LTP (TBS-LTP). GRF1 can also mediate high frequency stimulation-induced LTP (HFS-LTP) in mice over 2-months of age, which involves calcium-permeable AMPA-type glutamate receptors. To add to our understanding of how proteins with similar domains can have different functions, WT and various chimeras between GRF1 and GRF2 proteins were tested for their abilities to reconstitute defective LTP and/or LTD in the CA1 hippocampus of Grf1/Grf2 double knock-out mice. These studies revealed a critical role for the GRF2 CDC25 domain in the induction of TBS-LTP by GRF proteins. In contrast, the N-terminal pleckstrin homology and/or coiled-coil domains of GRF1 are key to the induction of HFS-LTP by GRF proteins. Finally, the IQ motif of GRF1 determines whether a GRF protein can induce LTD. Overall, these findings show that for the three forms of synaptic plasticity that are regulated by GRF proteins in the CA1 hippocampus, specificity is encoded in only one or two domains, and a different set of domains for each form of synaptic plasticity., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Neurogranin (Ng) is a member of the IQ motif class of calmodulin (CaM)-binding proteins, and interactions with CaM are its only known biological function. In this report we demonstrate that the binding affinity of Ng for CaM is weakened by Ca(2+) but to a lesser extent (2-3-fold) than that previously suggested from qualitative observations. We also show that Ng induced a >10-fold decrease in the affinity of Ca(2+) binding to the C-terminal domain of CaM with an associated increase in the Ca(2+) dissociation rate. We also discovered a modest, but potentially important, increase in the cooperativity in Ca(2+) binding to the C-lobe of CaM in the presence of Ng, thus sharpening the threshold for the C-domain to become Ca(2+)-saturated. Domain mapping using synthetic peptides indicated that the IQ motif of Ng is a poor mimetic of the intact protein and that the acidic sequence just N-terminal to the IQ motif plays an important role in reproducing Ng-mediated decreases in the Ca(2+) binding affinity of CaM. Using NMR, full-length Ng was shown to make contacts largely with residues in the C-domain of CaM, although contacts were also detected in residues in the N-terminal domain. Together, our results can be consolidated into a model where Ng contacts residues in the N- and C-lobes of both apo- and Ca(2+)-bound CaM and that although Ca(2+) binding weakens Ng interactions with CaM, the most dramatic biochemical effect is the impact of Ng on Ca(2+) binding to the C-terminal lobe of CaM., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Advancement of RNAi therapies is mainly hindered by the development of efficient delivery vehicles. The ability to create small size (<30 nm) oligonucleotide nanoparticles is essential for many aspects of the delivery process but is often overlooked. In this report, we describe diblock star polymers that can reproducibly complex double-stranded oligonucleotides into monodisperse nanoparticles with 15, 23, or 30 nm in diameter. The polymer-nucleic acid nanoparticles have a core-shell architecture with dense PEG brush coating. We characterized these nanoparticles using ITC, DLS, FRET, FCS, TIRF, and TEM. In addition to small size, these nanoparticles have neutral zeta-potentials, making the presented polymer architecture a very attractive platform for investigation of yet poorly studied polyplex size range for siRNA and antisense oligonucleotide delivery applications.

Protein-protein interactions drive most every biological process, but in many instances the domains mediating recognition are disordered. How specificity in binding is attained in the absence of defined structure contrasts with well-established experimental and theoretical work describing ligand binding to protein. The signaling protein calmodulin presents a unique opportunity to investigate mechanisms for target recognition given that it interacts with several hundred different targets. By advancing coarse-grained computer simulations and experimental techniques, mechanistic insights were gained in defining the pathways leading to recognition and in how target selectivity can be achieved at the molecular level. A model requiring mutually induced conformational changes in both calmodulin and target proteins was necessary and broadly informs how proteins can achieve both high affinity and high specificity.

Calcium-calmodulin-dependent protein kinase II (CaMKII) has been implicated in a wide variety of cellular processes, which include a critical regulatory role in actin cytoskeletal assembly. CaMKII is ubiquitous in cells, expressed as one of four isoforms termed α, β, γ, and δ. Characterization of the CaMKII-actin interaction has mainly focused on the β isoform, which has been shown to bundle actin filaments and sequester actin monomers in an activity-dependent manner. Much less is known about the interactions of other CaMKII isoforms with actin. In this work, isoform specific interactions of CaMKII with actin are described and reveal that the δ isoform of CaMKII bundles F-actin filaments like the β isoform while the γ isoform induces a novel layered structure in filaments. Using electron tomography, CaMKII holoenzymes are clearly identified in the complexes bridging the actin filaments, allowing direct visualization of the interactions between CaMKII isoforms and actin. In addition, we determined the isoform specificity of CaMKII-mediated inhibition of actin polymerization and discovered that all isoforms inhibit polymerization to varying degrees: β > γ ≈ δ > α (from most to least effective). Ca(2+)/CaM activation of all kinase isoforms produced a robust increase in actin polymerization that surpassed the rates of polymerization in the absence of kinase inhibition. These results indicate that diversity exists between the types of CaMKII-actin interactions mediated by the different isoforms and that the CaMKII isoform composition differentially impacts the formation and maintenance of the actin cytoskeleton.

We used a particle-based Monte Carlo simulation to dissect the regulatory mechanism of molecular translocation of CaMKII, a key regulator of neuronal synaptic function. Geometry was based upon measurements from EM reconstructions of dendrites in CA1 hippocampal pyramidal neurons. Three types of simulations were performed to investigate the effects of geometry and other mechanisms that control CaMKII translocation in and out of dendritic spines. First, the diffusional escape rate of CaMKII from model spines of varied morphologies was examined. Second, a postsynaptic density (PSD) was added to study the impact of binding sites on this escape rate. Third, translocation of CaMKII from dendrites and trapping in spines was investigated using a simulated dendrite. Based on diffusion alone, a spine of average dimensions had the ability to retain CaMKII for duration of ~4 s. However, binding sites mimicking those in the PSD controlled the residence time of CaMKII in a highly nonlinear manner. In addition, we observed that F-actin at the spine head/neck junction had a significant impact on CaMKII trapping in dendritic spines. We discuss these results in the context of possible mechanisms that may explain the experimental results that have shown extended accumulation of CaMKII in dendritic spines during synaptic plasticity and LTP induction.

The flexibility in the structure of calmodulin (CaM) allows its binding to over 300 target proteins in the cell. To investigate the structure-function relationship of CaM, we combined methods of computer simulation and experiments based on circular dichroism (CD) to investigate the structural characteristics of CaM that influence its target recognition in crowded cell-like conditions. We developed a unique multiscale solution of charges computed from quantum chemistry, together with protein reconstruction, coarse-grained molecular simulations, and statistical physics, to represent the charge distribution in the transition from apoCaM to holoCaM upon calcium binding. Computationally, we found that increased levels of macromolecular crowding, in addition to calcium binding and ionic strength typical of that found inside cells, can impact the conformation, helicity and the EF hand orientation of CaM. Because EF hand orientation impacts the affinity of calcium binding and the specificity of CaM's target selection, our results may provide unique insight into understanding the promiscuous behavior of calmodulin in target selection inside cells.

Background: Recent studies have shown that fluorescently labeled antibodies can be dissociated from their antigen by illumination with laser light. The mechanism responsible for the photounbinding effect, however, remains elusive. Here, we give important insights into the mechanism of photounbinding and show that the effect is not restricted to antibody/antigen binding., Methodology/principal Findings: We present studies of the photounbinding of labeled calmodulin (CaM) from a set of CaM-binding peptides with different affinities to CaM after one- and two-photon excitation. We found that the photounbinding effect becomes stronger with increasing binding affinity. Our observation that photounbinding can be influenced by using free radical scavengers, that it does not occur with either unlabeled protein or non-fluorescent quencher dyes, and that it becomes evident shortly after or with photobleaching suggest that photounbinding and photobleaching are closely linked., Conclusions/significance: The experimental results exclude surface effects, or heating by laser irradiation as potential causes of photounbinding. Our data suggest that free radicals formed through photobleaching may cause a conformational change of the CaM which lowers their binding affinity with the peptide or its respective binding partner.

Calmodulin (CaM) is a ubiquitous Ca(2+) buffer and second messenger that affects cellular function as diverse as cardiac excitability, synaptic plasticity, and gene transcription. In CA1 pyramidal neurons, CaM regulates two opposing Ca(2+)-dependent processes that underlie memory formation: long-term potentiation (LTP) and long-term depression (LTD). Induction of LTP and LTD require activation of Ca(2+)-CaM-dependent enzymes: Ca(2+)/CaM-dependent kinase II (CaMKII) and calcineurin, respectively. Yet, it remains unclear as to how Ca(2+) and CaM produce these two opposing effects, LTP and LTD. CaM binds 4 Ca(2+) ions: two in its N-terminal lobe and two in its C-terminal lobe. Experimental studies have shown that the N- and C-terminal lobes of CaM have different binding kinetics toward Ca(2+) and its downstream targets. This may suggest that each lobe of CaM differentially responds to Ca(2+) signal patterns. Here, we use a novel event-driven particle-based Monte Carlo simulation and statistical point pattern analysis to explore the spatial and temporal dynamics of lobe-specific Ca(2+)-CaM interaction at the single molecule level. We show that the N-lobe of CaM, but not the C-lobe, exhibits a nano-scale domain of activation that is highly sensitive to the location of Ca(2+) channels, and to the microscopic injection rate of Ca(2+) ions. We also demonstrate that Ca(2+) saturation takes place via two different pathways depending on the Ca(2+) injection rate, one dominated by the N-terminal lobe, and the other one by the C-terminal lobe. Taken together, these results suggest that the two lobes of CaM function as distinct Ca(2+) sensors that can differentially transduce Ca(2+) influx to downstream targets. We discuss a possible role of the N-terminal lobe-specific Ca(2+)-CaM nano-domain in CaMKII activation required for the induction of synaptic plasticity.

In this study, we used electron tomography as well as immunogold labeling to analyze the morphology and distribution of proteins within postsynaptic densities (PSDs) isolated from rats before birth (embryonic day 19) and at postnatal days 2, 21, and 60. Our data provide direct evidence of distinct morphological and compositional differences in PSDs throughout development. Not all PSD components are present at the early stages of development, with a near lack of the scaffolding molecule PSD-95 at E19 and P2. The presence of NR1 and NR2b suggests that PSD-95 is not directly required for clustering of N-methyl-D-aspartic acid (NMDA) receptors in PSDs early in development. α-Actinin is abundant by E19, suggesting that it is a core structural component of the PSD. Both α and β isoforms of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) are present early on but then rise in labeling density by approximately fourfold by P21. Among all the molecules studied, only calmodulin (CaM) was found in higher abundance early in PSD development and then fell in amount over time. Spatial analysis of the immunogold label shows a nonrandom distribution for all the proteins studied, lending support to the idea that the PSD is systematically assembled in an organized fashion. Morphological data from electron tomography shows that the PSD undergoes major structural changes throughout development.

In this paper, we present the Cellular Dynamic Simulator (CDS) for simulating diffusion and chemical reactions within crowded molecular environments. CDS is based on a novel event driven algorithm specifically designed for precise calculation of the timing of collisions, reactions and other events for each individual molecule in the environment. Generic mesh based compartments allow the creation / importation of very simple or detailed cellular structures that exist in a 3D environment. Multiple levels of compartments and static obstacles can be used to create a dense environment to mimic cellular boundaries and the intracellular space. The CDS algorithm takes into account volume exclusion and molecular crowding that may impact signaling cascades in small sub-cellular compartments such as dendritic spines. With the CDS, we can simulate simple enzyme reactions; aggregation, channel transport, as well as highly complicated chemical reaction networks of both freely diffusing and membrane bound multi-protein complexes. Components of the CDS are generally defined such that the simulator can be applied to a wide range of environments in terms of scale and level of detail. Through an initialization GUI, a simple simulation environment can be created and populated within minutes yet is powerful enough to design complex 3D cellular architecture. The initialization tool allows visual confirmation of the environment construction prior to execution by the simulator. This paper describes the CDS algorithm, design implementation, and provides an overview of the types of features available and the utility of those features are highlighted in demonstrations.

When signaling molecules diffuse through the cytosol, they encounter a wide variety of obstacles that hinder their mobility in space and time. Some of those factors include, but are not limited to, interactions with mobile and immobile targets or obstacles. Besides finding a crowded environment inside the cell, macromolecules assemble into molecular complexes that drive specific biological functions adding additional complexity to their diffusion. Thus, simple models of diffusion often fail to explain mobility through the cell interior, and new approaches are needed. Here we used fluorescent correlation spectroscopy to measure diffusion of three molecules of similar size with different surface properties diffusing in actin gels. The fluorescent probes were (a) quantum dots, (b) yellow-green fluorescent spheres, and (c) the beta isoform of Ca(2+) calmodulin-dependent protein kinase II tagged with green fluorescent protein. We compared various models for fitting the autocorrelation function (ACF) including single component, two-component, and anomalous diffusion. The two-component and anomalous diffusion models were superior and were largely indistinguishable based on a goodness of fit criteria. To better resolve differences between these two models, we modified the ACF to observe temporal variations in diffusion. We found in both simulated and experimental data a transient anomalous subdiffusion between two freely diffusing regimes produced by binding interactions of the diffusive tracers with actin gels.

Calmodulin (CaM) is a major Ca(2+) binding protein involved in two opposing processes of synaptic plasticity of CA1 pyramidal neurons: long-term potentiation (LTP) and depression (LTD). The N- and C-terminal lobes of CaM bind to its target separately but cooperatively and introduce complex dynamics that cannot be well understood by experimental measurement. Using a detailed stochastic model constructed upon experimental data, we have studied the interaction between CaM and Ca(2+)-CaM-dependent protein kinase II (CaMKII), a key enzyme underlying LTP. The model suggests that the accelerated binding of one lobe of CaM to CaMKII, when the opposing lobe is already bound to CaMKII, is a critical determinant of the cooperative interaction between Ca(2+), CaM, and CaMKII. The model indicates that the target-bound Ca(2+) free N-lobe has an extended lifetime and may regulate the Ca(2+) response of CaMKII during LTP induction. The model also reveals multiple kinetic pathways which have not been previously predicted for CaM-dissociation from CaMKII.

In vitro biochemical reactions are most often studied in dilute solution, a poor mimic of the intracellular space of eukaryotic cells, which are crowded with mobile and immobile macromolecules. Such crowded conditions exert volume exclusion and other entropic forces that have the potential to impact chemical equilibria and reaction rates. In this article, we used the well-characterized and ubiquitous molecule calmodulin (CaM) and a combination of theoretical and experimental approaches to address how crowding impacts CaM's conformational plasticity. CaM is a dumbbell-shaped molecule that contains four EF hands (two in the N-lobe and two in the C-lobe) that each could bind Ca(2+), leading to stabilization of certain substates that favor interactions with other target proteins. Using coarse-grained molecular simulations, we explored the distribution of CaM conformations in the presence of crowding agents. These predictions, in which crowding effects enhance the population of compact structures, were then confirmed in experimental measurements using fluorescence resonance energy transfer techniques of donor- and acceptor-labeled CaM under normal and crowded conditions. Using protein reconstruction methods, we further explored the folding-energy landscape and examined the structural characteristics of CaM at free-energy basins. We discovered that crowding stabilizes several different compact conformations, which reflects the inherent plasticity in CaM's structure. From these results, we suggest that the EF hands in the C-lobe are flexible and can be thought of as a switch, while those in the N-lobe are stiff, analogous to a rheostat. New combinatorial signaling properties may arise from the product of the differential plasticity of the two distinct lobes of CaM in the presence of crowding. We discuss the implications of these results for modulating CaM's ability to bind Ca(2+) and target proteins.

Ca(2+)-Calmodulin-dependent protein kinase II (CaMKII) is an abundant synaptic protein that was recently shown to regulate the organization of actin filaments leading to structural modifications of synapses. CaMKII is a dodecameric complex with a special architecture that provides it with unique potential for organizing the actin cytoskeleton. We report using biochemical assays that the beta isoform of CaMKII binds to and bundles actin filaments, and the disposition of betaCaMKII within the actin bundles was revealed by cryoelectron tomography. In addition, betaCaMKII was found to inhibit actin polymerization, suggesting that it either serves as a capping protein or binds monomeric actin, reducing the amount of freely available monomers to nucleate polymer assembly. By means of fluorescent cross-correlation spectroscopy, we determined that betaCaMKII does indeed bind to monomeric actin, reaching saturation at a stoichiometry of 12:1 actin monomers per betaCaMKII holoenzyme with a binding constant of 2.4 x 10(5) m(-1). In cells, betaCaMKII has a dual functional role; it can sequester monomeric actin to reduce actin polymerization and can also bundle actin filaments. Together, these effects would impact both the dynamics of actin filament assembly and enhance the rigidity of the filaments once formed, significantly impacting the structure of synapses.

Calmodulin (CaM) is the major pathway that transduces intracellular Ca2+ increases to the activation of a wide variety of downstream signaling enzymes. CaM and its target proteins form an integrated signaling network believed to be tuned spatially and temporally to control CaM's ability to appropriately pass signaling events downstream. Here, we report the spatial diffusivity and availability of CaM labeled with enhanced green fluorescent protein (eGFP)-CaM, at basal and elevated Ca2+,quantified by the novel fluorescent techniques of raster image scanning spectroscopy and number and brightness analysis. Our results show that in basal Ca2+ conditions cytoplasmic eGFP-CaM diffuses at a rate of 10 microm(2)/s, twofold slower than the noninteracting tracer, eGFP, indicating that a significant fraction of CaM is diffusing bound to other partners. The diffusion rate of eGFP-CaM is reduced to 7 microm(2)/s when a large (646 kDa) target protein Ca2+/CaM-dependent protein kinase II is coexpressed in the cells. In addition, the presence of Ca2+/calmodulin-dependent protein kinase II, which can bind up to 12 CaM molecules per holoenzyme, increases the stoichiometry of binding to an average of 3 CaMs per diffusive molecule. Elevating intracellular Ca2+ did not have a major impact on the diffusion of CaM complexes. These results present us with a model whereby CaM is spatially modulated by target proteins and support the hypothesis that CaM availability is a limiting factor in the network of CaM-signaling enzymes.

Development of biologically relevant crowding solutions necessitates improved understanding of how the relative size and density of mobile obstacles affect probe diffusion. Both the crowding density and relative size of each co-solute in a mixture will contribute to the measured microviscosity as assessed by altered translational mobility. Using multiphoton fluorescent correlation spectroscopy, this study addresses how excluded volume of dextran polymers from 10 to 500 kDa affect microviscosity quantified by measurements of calmodulin labeled with green fluorescent protein as the diffusing probe. Autocorrelation functions were fit using both a multiple-component model with maximum entropy method (MEMFCS) and an anomalous model. Anomalous diffusion was not detected, but fits of the data with the multiple-component model revealed separable modes of diffusion. When the dominant mode of diffusion from the MEMFCS analysis was used, we observed that increased excluded volume slows probe mobility as a simple exponential with crowder concentration. This behavior can be modeled with a single parameter, beta, which depends on the dextran size composition. Two additional modes of diffusion were observed using MEMFCS and were interpreted as unique microviscosities. The fast mode corresponded to unhindered free diffusion as in buffer, whereas the slower agreed well with the bulk viscosity. At 10% crowder concentration, one finds a microviscosity approximately three times that of water, which mimics that reported for intracellular viscosity.