Molecular and cellular mechanisms for differential effects of chronic social isolation stress in males and females.

Chronic social isolation stress during adolescence induces susceptibility for neuropsychiatric disorders. Here we show that 5-week post-weaning isolation stress induces sex-specific behavioral abnormalities and neuronal activity changes in the prefrontal cortex (PFC), basal lateral amygdala (BLA), and ventral tegmental area (VTA). Chemogenetic manipulation, optogenetic recording, and in vivo calcium imaging identify that the PFC to BLA pathway is causally linked to heightened aggression in stressed males, and the PFC to VTA pathway is causally linked to social withdrawal in stressed females. Isolation stress induces genome-wide transcriptional alterations in a region-specific manner. Particularly, the upregulated genes in BLA of stressed males are under the control of activated transcription factor CREB, and CREB inhibition in BLA normalizes gene expression and reverses aggressive behaviors. On the other hand, neuropeptide Hcrt (Hypocretin/Orexin) is among the top-ranking downregulated genes in VTA of stressed females, and Orexin-A treatment rescues social withdrawal. These results have revealed molecular mechanisms and potential therapeutic targets for stress-related mental illness.

Quantifying spatial and temporal variations of the cell membrane ultra-structure by bimFCS.

It has been long recognized that the cell membrane is heterogeneous on scales ranging from a couple of molecules to micrometers in size and hence diffusion of receptors is length scale dependent. This heterogeneity modulates many cell-membrane-associated processes requiring transient spatiotemporal separation of components. The transient increase in local concentration of interacting signal components enables robust signaling in an otherwise thermally noisy system. Understanding how lipids and proteins self-organize and interact with the cell cortex requires quantifying the motion of the components. Multi-length scale diffusion measurements by single particle tracking, fluorescence correlation spectroscopy (FCS) or related techniques are able to identify components being transiently trapped in nanodomains, from freely moving one and from ones with reduced long-scale diffusion due to interaction with the cell cortex. One particular implementation of multi-length scale diffusion measurements is the combination of FCS with a spatially resolved detector, such as a camera and two-dimensional extended excitation profile. The main advantages of this approach are that all length scales are interrogated simultaneously, uniquely permits quantifying changes to the membrane structure caused by extrenal or internal perturbations. Here, we review how combining total internal reflection microscopy (TIRF) with FC resolves the membrane organization in living cells. We show how to implement the method, which requires only a few seconds of data acquisition to quantify membrane nanodomains, or the spacing of membrane fences caused by the actin cortex. The choice of diffusing fluorescent probe determines which membrane heterogeneity is detected. We review the instrument, sample preparation, experimental and computational requirements to perform such measurements, and discuss the potential and limitations. The discussion includes examples of spatial and temporal comparisons of the membrane structure in response to perturbations demonstrating the complex cell physiology.

Stable, high-affinity streptavidin monomer for protein labeling and monovalent biotin detection.

The coupling between the quaternary structure, stability and function of streptavidin makes it difficult to engineer a stable, high affinity monomer for biotechnology applications. For example, the binding pocket of streptavidin tetramer is comprised of residues from multiple subunits, which cannot be replicated in a single domain protein. However, rhizavidin from Rhizobium etli was recently shown to bind biotin with high affinity as a dimer without the hydrophobic tryptophan lid donated by an adjacent subunit. In particular, the binding site of rhizavidin uses residues from a single subunit to interact with bound biotin. We therefore postulated that replacing the binding site residues of streptavidin monomer with corresponding rhizavidin residues would lead to the design of a high affinity monomer useful for biotechnology applications. Here, we report the construction and characterization of a structural monomer, mSA, which combines the streptavidin and rhizavidin sequences to achieve optimized biophysical properties. First, the biotin affinity of mSA (K(d) = 2.8 nM) is the highest among nontetrameric streptavidin, allowing sensitive monovalent detection of biotinylated ligands. The monomer also has significantly higher stability (T(m) = 59.8 °C) and solubility than all other previously engineered monomers to ensure the molecule remains folded and functional during its application. Using fluorescence correlation spectroscopy, we show that mSA binds biotinylated targets as a monomer. We also show that the molecule can be used as a genetic tag to introduce biotin binding capability to a heterologous protein. For example, recombinantly fusing the monomer to a cell surface receptor allows direct labeling and imaging of transfected cells using biotinylated fluorophores. A stable and functional streptavidin monomer, such as mSA, should be a useful reagent for designing novel detection systems based on monovalent biotin interaction.

Neuronal actin cytoskeleton gain of function in the human brain.

BACKGROUND: While advancements in imaging techniques have led to major strides in deciphering the human brain, successful interventions are elusive and represent some of the most persistent translational gaps in medicine. Human restricted CHRFAM7A has been associated with neuropsychiatric disorders. METHODS: The physiological role of CHRFAM7A in human brain is explored using multiomics approach on 600 post mortem human brain tissue samples. The emerging pathways and mechanistic hypotheses are tested and validated in an isogenic hiPSC model of CHRFAM7A knock-in medial ganglionic eminence progenitors and neurons. FINDINGS: CHRFAM7A is identified as a modulator of intracellular calcium dynamics and an upstream regulator of Rac1. Rac1 activation re-designs the actin cytoskeleton leading to dynamic actin driven remodeling of membrane protrusion and a switch from filopodia to lamellipodia. The reinforced cytoskeleton leads to an advantage to tolerate stiffer mechanical properties of the extracellular environment. INTERPRETATION: CHRFAM7A modifies the actin cytoskeleton to a more dynamic and stiffness resistant state in an α7nAChR dependent manner. CHRFAM7A may facilitate neuronal adaptation to changes in the brain environment in physiological and pathological conditions contributing to risk or recovery. Understanding how CHRFAM7A affects human brain requires human studies in the areas of memory formation and erasure, cognitive reserve, and neuronal plasticity. FUNDING: This work is supported in part by the Community Foundation for Greater Buffalo (Kinga Szigeti). Also, in part by the International Society for Neurochemistry (ISN) and The Company of Biologists (Nicolas Rosas). ROSMAP is supported by NIA grants P30AG10161, P30AG72975, R01AG15819, R01AG17917. U01AG46152, and U01AG61356.

Analysis of a RanGTP-regulated gradient in mitotic somatic cells.

The RanGTPase cycle provides directionality to nucleocytoplasmic transport, regulating interactions between cargoes and nuclear transport receptors of the importin-beta family. The Ran-importin-beta system also functions in mitotic spindle assembly and nuclear pore and nuclear envelope formation. The common principle underlying these diverse functions throughout the cell cycle is thought to be anisotropy of the distribution of RanGTP (the RanGTP gradient), driven by the chromatin-associated guanine nucleotide exchange factor RCC1 (refs 1, 4, 5). However, the existence and function of a RanGTP gradient during mitosis in cells is unclear. Here we examine the Ran-importin-beta system in cells by conventional and fluorescence lifetime microscopy using a biosensor, termed Rango, that increases its fluorescence resonance energy transfer signal when released from importin-beta by RanGTP. Rango is predominantly free in mitotic cells, but is further liberated around mitotic chromatin. In vitro experiments and modelling show that this localized increase of free cargoes corresponds to changes in RanGTP concentration sufficient to stabilize microtubules in extracts. In cells, the Ran-importin-beta-cargo gradient kinetically promotes spindle formation but is largely dispensable once the spindle has been established. Consistent with previous reports, we observe that the Ran system also affects spindle pole formation and chromosome congression in vivo. Our results demonstrate that conserved Ran-regulated pathways are involved in multiple, parallel processes required for spindle function, but that their relative contribution differs in chromatin- versus centrosome/kinetochore-driven spindle assembly systems.

Magnetic nanomaterials for wireless thermal and mechanical neuromodulation.

Magnetic fields are very attractive for non-invasive neuromodulation because they easily penetrate trough the skull and tissue. Cell specific neuromodulation requires the magnetic field energy to be converted by an actuator to a biologically relevant signal. Miniaturized actuators available today range from small, isotropic magnetic nanoparticles to larger, submicron anisotropic magnetic nanomaterials. Depending on the parameters of external magnetic fields and the properties of the nanoactuators, they create either a thermal or a mechanical stimulus. Ferromagnetic nanomaterials generate heat in response to high frequency alternating magnetic fields associated with dissipative losses. Anisotropic nanomaterials with large magnetic moments are capable of exerting forces at stationary or slowly varying magnetic fields. These tools allow exploiting thermosensitive or mechanosensitive neurons in circuit or cell specific tetherless neuromodulation schemes. This review will address assortment of available magnetic nanomaterial-based neuromodulation techniques that rely on application of external magnetic fields.

Modulating cell signalling in vivo with magnetic nanotransducers.

Weak magnetic fields offer nearly lossless transmission of signals within biological tissue. Magnetic nanomaterials are capable of transducing magnetic fields into a range of biologically relevant signals in vitro and in vivo. These nanotransducers have recently enabled magnetic control of cellular processes, from neuronal firing and gene expression to programmed apoptosis. Effective implementation of magnetically controlled cellular signalling relies on careful tailoring of magnetic nanotransducers and magnetic fields to the responses of the intended molecular targets. This primer discusses the versatility of magnetic modulation modalities and offers practical guidelines for selection of appropriate materials and field parameters, with a particular focus on applications in neuroscience. With recent developments in magnetic instrumentation and nanoparticle chemistries, including those that are commercially available, magnetic approaches promise to empower research aimed at connecting molecular and cellular signalling to physiology and behaviour in untethered moving subjects.

Engineered streptavidin monomer and dimer with improved stability and function.

Although streptavidin's high affinity for biotin has made it a widely used and studied binding protein and labeling tool, its tetrameric structure may interfere with some assays. A streptavidin mutant with a simpler quaternary structure would demonstrate a molecular-level understanding of its structural organization and lead to the development of a novel molecular reagent. However, modulating the tetrameric structure without disrupting biotin binding has been extremely difficult. In this study, we describe the design of a stable monomer that binds biotin both in vitro and in vivo. To this end, we constructed and characterized monomers containing rationally designed mutations. The mutations improved the stability of the monomer (increase in T(m) from 31 to 47 °C) as well as its affinity (increase in K(d) from 123 to 38 nM). We also used the stability-improved monomer to construct a dimer consisting of two streptavidin subunits that interact across the dimer-dimer interface, which we call the A/D dimer. The biotin binding pocket is conserved between the tetramer and the A/D dimer, and therefore, the dimer is expected to have a significantly higher affinity than the monomer. The affinity of the dimer (K(d) = 17 nM) is higher than that of the monomer but is still many orders of magnitude lower than that of the wild-type tetramer, which suggests there are other factors important for high-affinity biotin binding. We show that the engineered streptavidin monomer and dimer can selectively bind biotinylated targets in vivo by labeling the cells displaying biotinylated receptors. Therefore, the designed mutants may be useful in novel applications as well as in future studies in elucidating the role of oligomerization in streptavidin function.

Membrane nanodomains homeostasis during propofol anesthesia as function of dosage and temperature.

Some anesthetics bind and potentiate γ-aminobutyric-acid-type receptors, but no universal mechanism for general anesthesia is known. Furthermore, often encountered complications such as anesthesia induced amnesia are not understood. General anesthetics are hydrophobic molecules easily dissolving into lipid bilayers. Recently, it was shown that general anesthetics perturb phase separation in vesicles extracted from fixed cells. Unclear is whether under physiological conditions general anesthetics induce perturbation of the lipid bilayer, and whether this contributes to the transient loss of consciousness or anesthesia side effects. Here we show that propofol perturbs lipid nanodomains in the outer and inner leaflet of the plasma membrane in intact cells, affecting membrane nanodomains in a concentration dependent manner: 1 µM to 5 µM propofol destabilize nanodomains; however, propofol concentrations higher than 5 µM stabilize nanodomains with time. Stabilization occurs only at physiological temperature and in intact cells. This process requires ARP2/3 mediated actin nucleation and Myosin II activity. The rate of nanodomain stabilization is potentiated by GABAA receptor activity. Our results show that active nanodomain homeostasis counteracts the initial disruption causing large changes in cortical actin. SIGNIFICANCE STATEMENT: General anesthesia is a routine medical procedure with few complications, yet a small number of patients experience side-effects that persist for weeks and months. Very young children are at risk for effects on brain development. Elderly patients often exhibit subsequent amnesia. Here, we show that the general anesthetic propofol perturbs the ultrastructure of the lipid bilayer of the cell membrane in intact cells. Initially propofol destabilized lipid nanodomains. However, with increasing incubation time and propofol concentration, the effect is reversed and nanodomains are further stabilized. We show that this stabilization is caused by the activation of the actin cortex under the membrane. These perturbations of membrane bilayer and cortical actin may explain how propofol affects neuronal plasticity at synapses.

Magnetothermal nanoparticle technology alleviates parkinsonian-like symptoms in mice.

Deep brain stimulation (DBS) has long been used to alleviate symptoms in patients suffering from psychiatric and neurological disorders through stereotactically implanted electrodes that deliver current to subcortical structures via wired pacemakers. The application of DBS to modulate neural circuits is, however, hampered by its mechanical invasiveness and the use of chronically implanted leads, which poses a risk for hardware failure, hemorrhage, and infection. Here, we demonstrate that a wireless magnetothermal approach to DBS (mDBS) can provide similar therapeutic benefits in two mouse models of Parkinson's disease, the bilateral 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and in the unilateral 6-hydroxydopamine (6-OHDA) model. We show magnetothermal neuromodulation in untethered moving mice through the activation of the heat-sensitive capsaicin receptor (transient receptor potential cation channel subfamily V member 1, TRPV1) by synthetic magnetic nanoparticles. When exposed to an alternating magnetic field, the nanoparticles dissipate heat, which triggers reversible firing of TRPV1-expressing neurons. We found that mDBS in the subthalamic nucleus (STN) enables remote modulation of motor behavior in healthy mice. Moreover, mDBS of the STN reversed the motor deficits in a mild and severe parkinsonian model. Consequently, this approach is able to activate deep-brain circuits without the need for permanently implanted hardware and connectors.

Modulation and dynamics of cell membrane heterogeneities.

Numerous studies provide evidence that the lipid bilayer of the plasma membrane contains lateral nanodomains, and that these are functionally important regulators of transmembrane cell signaling. Depending on their chemical composition and the biophysical mechanism bringing the lipids together, multiple types of nanodomains exist in the inner and the outer leaflet of the plasma membrane bilayer. In intact cells, these domains are smaller than the optical resolution limit of light microscopy and also highly dynamic. Recently, advanced fluorescence methods have provided data to characterize many biophysical and thermodynamic aspects of these nanodomains. In this review, we summarize the physicochemical determinants of nanodomain formation, stability and extent. Then, we detail how these nanodomains play a structural role by anchoring nucleation sites for the membrane cytoskeleton on the lipid bilayer. Further, we review the existing literature on mechanisms that modulate the nanodomain size and stability, both acute and chronic events. We conclude that regulation of the nanodomains distribution in the lipid bilayer of the plasma membrane is important for modulation of transmembrane signaling. However, only very few modulators of nanodomain stability and size have been quantified in cells, suggesting interesting directions for future studies.

Outstanding heat loss via nano-octahedra above 20 nm in size: from wustite-rich nanoparticles to magnetite single-crystals.

Most studies on magnetic nanoparticle-based hyperthermia utilize iron oxide nanoparticles smaller than 20 nm, which are intended to have superparamagnetic behavior (SP-MNPs). However, the heating power of larger magnetic nanoparticles with non-fluctuating or fixed magnetic dipoles (F-MNPs) can be significantly greater than that of SP-MNPs if high enough fields (H > 15 mT) are used. But the synthesis of larger single nanocrystals of magnetite (Fe3O4) with a regular shape and narrow size distribution devoid of secondary phases remains a challenge. Iron oxide nanoparticles, grown over 25 nm, often present large shape and size polydispersities, twinning defects and a significant fraction of the wüstite-type (FeO) paramagnetic phase, resulting in degradation of magnetic properties. Herein, we introduce an improved procedure to synthesize monodisperse F-MNPs in the range of 25 to 50 nm with a distinct octahedral morphology and very crystalline magnetite phase. We unravel the subtle phase transformation that takes place during the synthesis by a thorough study in several non-optimized nanoparticles presenting a core-shell structure or composed of magnetite-type clusters embedded in a wüstite lattice. Optimized magnetite samples present a slight decrease in the saturation magnetization compared to bulk magnetite, which is successfully explained by the presence of Fe2+ vacancies. However, due to the high quality of these samples, AC magnetometry measurements have shown excellent specific absorption rates (>1000 W gFe3O4-1 at 40 mT and 300 kHz). Most importantly, the magnetic response and the hyperthermia performance of properly coated F-MNPs are kept basically unaltered in media with very different viscosities and ionic strength. Finally, using a physical model based on single magnetic domain approaches, we derive a novel connection between the octahedral shape and the high hyperthermia performance.

The orientation and molecular movement of a k(+) channel voltage-sensing domain.

Voltage-gated channels operate through the action of a voltage-sensing domain (membrane segments S1-S4) that controls the conformation of gates located in the pore domain (membrane segments S5-S6). Recent structural studies on the bacterial K(v)AP potassium channel have led to a new model of voltage sensing in which S4 lies in the lipid at the channel periphery and moves through the membrane as a unit with a portion of S3. Here we describe accessibility probing and disulfide scanning experiments aimed at determining how well the K(v)AP model describes the Drosophila Shaker potassium channel. We find that the S1-S3 helices have one end that is externally exposed, S3 does not undergo a transmembrane motion, and S4 lies in close apposition to the pore domain in the resting and activated state.

Transient Magnetothermal Neuronal Silencing Using the Chloride Channel Anoctamin 1 (TMEM16A).

Determining the role and necessity of specific neurons in a network calls for precisely timed, reversible removal of these neurons from the circuit via remotely triggered transient silencing. Previously, we have shown that alternating magnetic field mediated heating of magnetic nanoparticles, bound to neurons, expressing temperature-sensitive cation channels TRPV1 remotely activates these neurons, evoking behavioral responses in mice. Here, we demonstrate how to apply magnetic nanoparticle heating to silence target neurons. Rat hippocampal neuronal cultures were transfected to express the temperature gated chloride channel, anoctamin 1 (TMEM16A). Spontaneous firing was suppressed within seconds of alternating magnetic field application to anoctamin 1 (TMEM16A) channel expressing, magnetic nanoparticle decorated neurons. Five seconds of magnetic field application leads to 12 s of silencing, with a latency of 2 s and an average suppression ratio of more than 80%. Immediately following the silencing period spontaneous activity resumed. The method provides a promising avenue for tether free, remote, transient neuronal silencing in vivo for both scientific and therapeutic applications.

Multilayered inorganic-organic microdisks as ideal carriers for high magnetothermal actuation: assembling ferrimagnetic nanoparticles devoid of dipolar interactions.

The two major limitations for nanoparticle based magnetic hyperthermia in theranostics are the delivery of a sufficient number of magnetic nanoparticles (MNPs) with high heating power to specific target cells and the residence time of the MNPs at the target location. Ferromagnetic or Ferrimagnetic single domain nanoparticles (F-MNPs), with a permanent magnetic dipole, produce larger magnetic and thermal responses than superparamagnetic nanoparticles (SP-MNPs) but also agglomerate more. MNP agglomeration degrades their heating potential due to dipolar interaction effects and interferes with specific targeting. Additionally, MNPs bound to cells are often endocytosed by the cells or, in vivo, cleared out by the immune system via uptake in macrophages. Here, we present a versatile approach to engineer inorganic-polymeric microdisks, loaded with biomolecules, fluorophores and Fe3O4 F-MNPs that solves both challenges. These microdisks deliver the F-MNPs efficiently, while controlling any undesirable agglomeration and dipolar interaction, while also rendering the F-MNPs endocytosis resistant. We show that these micro-devices are suitable carriers to transport a flat assembly of F-MNPs to the cell membrane unchanged, preserving the magnetic response of the MNPs in any biological environment. The F-MNPs concentration per microdisk and degree of MNP interaction are tunable. We demonstrate that the local heat generated in microdisks is proportional to the surface density of F-MNPs when attached to the cell membrane. The key innovation in the production of these microdisks is the fabrication of a mushroom-shaped photolithographic template that enables easy assembly of the inorganic film, polymeric multilayers, and MNP cargo while permitting highly efficient lift-off of the completed microdisks. During the harvesting of the flat microdisks, the supporting mushroom-shaped templates are sacrificed. These resulting magnetic hybrid microdisks are tunable and efficient devices for magnetothermal actuation and hyperthermia.

Magnetothermal genetic deep brain stimulation of motor behaviors in awake, freely moving mice.

Establishing how neurocircuit activation causes particular behaviors requires modulating the activity of specific neurons. Here, we demonstrate that magnetothermal genetic stimulation provides tetherless deep brain activation sufficient to evoke motor behavior in awake mice. The approach uses alternating magnetic fields to heat superparamagnetic nanoparticles on the neuronal membrane. Neurons, heat-sensitized by expressing TRPV1 are activated with magnetic field application. Magnetothermal genetic stimulation in the motor cortex evoked ambulation, deep brain stimulation in the striatum caused rotation around the body-axis, and stimulation near the ridge between ventral and dorsal striatum caused freezing-of-gait. The duration of the behavior correlated tightly with field application. This approach provides genetically and spatially targetable, repeatable and temporarily precise activation of deep-brain circuits without the need for surgical implantation of any device.

Compartmentalization of the Cell Membrane.

Many cell-membrane-associated processes require transient spatiotemporal separation of components on scales ranging from a couple of molecules to micrometers in size. Understanding these processes mechanistically involves understanding how lipids and proteins self-organize and interact with the cell cortex. Here, we review recent advances in dissecting the mechanisms of cell membrane compartmentalization. We introduce the challenges in studying cell membrane organization, the current understanding of how complex membranes self-organize to form transient domains, and the role of protein scaffolds in membrane organization. We discuss the formation of signaling domains as an important example of transient membrane compartmentalization. We conclude by pointing to the current limitations of measuring membrane organization in living cells and the steps that are required to advance the field.

Effect of receptor dimerization on membrane lipid raft structure continuously quantified on single cells by camera based fluorescence correlation spectroscopy.

Membrane bound cell signaling is modulated by the membrane ultra-structure, which itself may be affected by signaling. However, measuring the interaction of membrane proteins with membrane structures in intact cells in real-time poses considerable challenges. In this paper we present a non-destructive fluorescence method that quantifies these interactions in single cells, and is able to monitor the same cell continuously to observe small changes. This approach combines total internal fluorescence microscopy with fluorescence correlation spectroscopy to measure the protein's diffusion and molecular concentration in different sized areas simultaneously. It correctly differentiates proteins interacting with membrane fences from proteins interacting with cholesterol-stabilized domains, or lipid rafts. This method detects small perturbations of the membrane ultra-structure or of a protein's tendency to dimerize. Through continuous monitoring of single cells, we demonstrate how dimerization of GPI-anchored proteins increases their association with the structural domains. Using a dual-color approach we study the effect of dimerization of one GPI-anchored protein on another type of GPI-anchored protein expressed in the same cell. Scans over the cell surface reveal a correlation between cholesterol stabilized domains and membrane cytoskeleton.

A role for the thermal environment in defining co-stimulation requirements for CD4(+) T cell activation.

Maintenance of normal core body temperature is vigorously defended by long conserved, neurovascular homeostatic mechanisms that assist in heat dissipation during prolonged, heat generating exercise or exposure to warm environments. Moreover, during febrile episodes, body temperature can be significantly elevated for at least several hours at a time. Thus, as blood cells circulate throughout the body, physiologically relevant variations in surrounding tissue temperature can occur; moreover, shifts in core temperature occur during daily circadian cycles. This study has addressed the fundamental question of whether the threshold of stimulation needed to activate lymphocytes is influenced by temperature increases associated with physiologically relevant increases in temperature. We report that the need for co-stimulation of CD4+ T cells via CD28 ligation for the production of IL-2 is significantly reduced when cells are exposed to fever-range temperature. Moreover, even in the presence of sufficient CD28 ligation, provision of extra heat further increases IL-2 production. Additional in vivo and in vitro data (using both thermal and chemical modulation of membrane fluidity) support the hypothesis that the mechanism by which temperature modulates co-stimulation is linked to increases in membrane fluidity and membrane macromolecular clustering in the plasma membrane. Thermally-regulated changes in plasma membrane organization in response to physiological increases in temperature may assist in the geographical control of lymphocyte activation, i.e., stimulating activation in lymph nodes rather than in cooler surface regions, and further, may temporarily and reversibly enable CD4+ T cells to become more quickly and easily activated during times of infection during fever.