Coordination of Membrane and Actin Cytoskeleton Dynamics during Filopodia Protrusion

Leading edge protrusion of migrating cells involves tightly coordinated changes in the plasma membrane and actin cytoskeleton. It remains unclear whether polymerizing actin filaments push and deform the membrane, or membrane deformation occurs independently and is subsequently stabilized by actin filaments. To address this question, we employed an ability of the membrane-binding I-BAR domain of IRSp53 to uncouple the membrane and actin dynamics and to induce filopodia in expressing cells. Using time-lapse imaging and electron microscopy of IRSp53-I-BAR-expressing B16F1 melanoma cells, we demonstrate that cells are not able to protrude or maintain durable long extensions without actin filaments in their interior, but I-BAR-dependent membrane deformation can create a small and transient space at filopodial tips that is subsequently filled with actin filaments. Moreover, the expressed I-BAR domain forms a submembranous coat that may structurally support these transient actin-free protrusions until they are further stabilized by the actin cytoskeleton. Actin filaments in the I-BAR-induced filopodia, in contrast to normal filopodia, do not have a uniform length, are less abundant, poorly bundled, and display erratic dynamics. Such unconventional structural organization and dynamics of actin in I-BAR-induced filopodia suggests that a typical bundle of parallel actin filaments is not necessary for generation and mechanical support of the highly asymmetric filopodial geometry. Together, our data suggest that actin filaments may not directly drive the protrusion, but only stabilize the space generated by the membrane deformation; yet, such stabilization is necessary for efficient protrusion.     

Differential Regulation of Protrusion and Polarity by PI(3)K during Neutrophil Motility in Live Zebrafish

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Highlights? Neutrophil motility in live zebrafish requires PI(3)K activity at the leading edge ? Localized photo-activation of Rac can to direct migration in vivo ? Rac activation rescues protrusion but not polarization in PI(3)K-inhibited cells ? Separable two-tier regulation of F-actin dynamics and protrusion by PI(3)K     

The Cytological Localization of Intracellular Neuronal Acetylcholinesterase

Sections of cat ciliary ganglia were stained for acetylcholinesterase activity by several modifications of the acetylthiocholine method in order to achieve optimal accuracy of cytological localization of the enzyme. These were compared by ordinary light and phase contrast microscopy with similar sections stained by standard techniques for Nissl substance, the Golgi apparatus, and the neurofibrillae, and by intravital methylene blue. The pattern of cytoplasmic distribution of acetylcholinesterase corresponded most closely with that of the Nissl substance. Following total inactivation of the ganglionic acetylcholinesterase by intravenously administered di-isopropyl fluorophosphate, the reappearance of the enzyme in vivo occurred at the same cytoplasmic sites prior to its reappearance at the cell membrane or preganglionic axonal terminations. These observations, and reports cited from the literature, provide support for the hypothesis that acetylcholinesterase is synthesized within the endoplasmic reticulum, then transported via its canaliculi to the surface of the cell and its processes, where its functional sites are oriented externally to the lipoidal membrane.     

Dynamic Changes in Histone H3 Lysine 9 Acetylation Localization Patterns During Neuronal Maturation Require MeCP2

Mutations within the gene encoding methyl CpG binding protein 2 (MECP2) cause the autism-spectrum neurodevelopmental disorder Rett Syndrome (RTT). MECP2 recruits histone deacetylase to methylated DNA and acts as a long-range regulator of methylated genes. Despite ubiquitous MECP2 expression, the phenotype of RTT and the Mecp2-deficient mouse is largely restricted to the postnatal brain. Since Mecp2-deficient mice have a defect in neuronal maturation, we sought to understand how MECP2/Mecp2 mutations globally affect histone modifications during postnatal brain development by an immunofluorescence approach. Using an antibody specific to acetylated histone H3 lysine 9 (H3K9ac), a bright punctate nuclear staining pattern was observed as MECP2 expression increased in early postnatal neuronal nuclei. As neurons matured in juvenile and adult brain samples, the intensity of H3K9ac staining was reduced. Mecp2-deficient mouse and RTT cerebral neurons lacked this developmental reduction in H3K9ac staining compared to age-matched controls, resulting in a significant increase in neuronal nuclei with bright H3K9ac punctate staining. In contrast, trimethylated histone H3 lysine 9 (H3K9me3) localized to heterochromatin independent of MeCP2, but showed significantly reduced levels in Mecp2 deficient mouse and RTT brain. Autism brain with reduced MECP2 expression displayed similar histone H3 alterations as RTT brain. These observations suggest that MeCP2 regulates global histone modifications during a critical postnatal stage of neuronal maturation. These results have implications for understanding the molecular pathogenesis of RTT and autism in which MECP2 mutation or deficiency corresponds with arrested neurodevelopment.      

Evidence for Neuronal Localization of Histamine-N-Methyltransferase in Rat Brain

Abstract: There is evidence that histamine may be a neurotransmitter in mammalian brain. Histamine in neurons of the central nervous system is easily released and rapidly turned over. The cellular localization of histamine- N -methyltransferase, the proposed histamine-inactivating enzyme, was investigated by measuring its activity in rat striatum after applying neurochemical or electrolytic lesions. The results indicate a major neuronal localization of the enzyme in this area.     

Brain-State-Dependent Modulation of Neuronal Firing and Membrane Potential Dynamics in the Somatosensory Thalamus during Natural Sleep

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Transient Activations of Rac1 at the Lamellipodium Tip Trigger Membrane Protrusion

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Subcellular Localization and Characterization of Neuronal Nitric Oxide Synthase

Abstract: In contrast to the predominantly participate, Ca²⁺/calmodulin-dependent nitric oxide (NO) synthase in endothelial cells, the corresponding neuronal isoenzyme is considered to be mainly soluble, presumably owing to the lack of a posttranslational myristoylation. However, preliminary findings from this and other laboratories suggest that a substantial portion of the neuronal NO synthase activity may in fact be membrane bound. We have therefore investigated the distribution of this enzyme among subcellular fractions of the rat and rabbit cerebellum in more detail. Up to 60% of the total NO synthase activity was found in the particulate fraction and, according to density gradient ultracentrifugation, associated mainly with the endoplasmic reticulum fraction. There was no apparent difference between the soluble and particulate enzymes with respect to their specific activity, Ca²⁺ and pH dependency, inhibitor sensitivity, or immunoreactivity, suggesting that both rat and rabbit cerebella contain a single Ca²⁺/calmodulin-dependent NO synthase. The inhibition by the cytochrome P450 inhibitor SKF-525A of the NO synthase activity in these subcellular fractions (IC₅₀= 90 μ M ) and the fact that mammalian cytochrome P450 enzymes are endoplasmic reticulum-bound proteins support the notion that the cerebellar NO synthase is a cytochrome P450-type hemoprotein. Moreover, the aforementioned findings suggest that posttranslational myristoylation may not be the only factor determining the intracellular localization of NO synthase.     

Effect of Energy Metabolism on Protein Motility in the Bacterial Outer Membrane

We demonstrate the energy dependence of the motion of a porin, the λ-receptor, in the outer membrane of living Escherichia coli by single molecule investigations. By poisoning the bacteria with arsenate and azide, the bacterial energy metabolism was stopped. The motility of individual λ-receptors significantly and rapidly decreased upon energy depletion. We suggest two different causes for the ceased motility upon comprised energy metabolism: One possible cause is that the cell uses energy to actively wiggle its proteins, this energy being one order-of-magnitude larger than thermal energy. Another possible cause is an induced change in the connection between the λ-receptor and the membrane structure, for instance by a stiffening of part of the membrane structure. Treatment of the cells with ampicillin, which directly targets the bacterial cell wall by inhibiting cross-linking of the peptidoglycan layer, had an effect similar to energy depletion and the motility of the λ-receptor significantly decreased. Since the λ-receptor is closely linked to the peptidoglycan layer, we propose that λ-receptor motility is directly coupled to the constant and dynamic energy-consuming reconstruction of the peptidoglycan layer. The result of this motion could be to facilitate transport of maltose-dextrins through the porin.     

Dynamic Membrane Formation in Anaerobic Dynamic Membrane Bioreactors: Role of Extracellular Polymeric Substances

Dynamic membrane (DM) formation in dynamic membrane bioreactors plays an important role in achieving efficient solid-liquid separation. In order to study the contribution of extracellular polymeric substances (EPS) to DM formation in anaerobic dynamic membrane bioreactor (AnDMBR) processes, EPS extraction from and re-addition to bulk sludge were carried out in short-term filtration tests. DM formation behaviors could be well simulated by cake filtration model, and sludge with EPS re-addition showed the highest resistance coefficient, followed by sludge after EPS extraction. The DM layers exhibited a higher resistance and a lower porosity for the sludge sample after EPS extraction and for the sludge with EPS re-addition. Particle size of sludge flocs decreased after EPS extraction, and changed little with EPS re-addition, which was confirmed by interaction energy analysis. Further investigations by confocal laser scanning microscopy (CLSM) analysis and batch tests suggested that the removal of in-situ EPS stimulated release of soluble EPS, and re-added EPS were present as soluble EPS rather than bound EPS, which thus improved the formation of DM. The present work revealed the role of EPS in anaerobic DM formation, and could facilitate the operation of AnDMBR processes.     

Subcellular Localization of the Neuronal Glycine Transporter GLYT2 in Brainstem

The neuronal glycine transporter GLYT2 belongs to the neurotransmitter:sodium:symporter (NSS) family and removes glycine from the synaptic cleft, thereby aiding the termination of the glycinergic signal and achieving the reloading of the presynaptic terminal. The task fulfilled by this transporter is fine tuned by regulating both transport activity and intracellular trafficking. Different stimuli such as neuronal activity or protein kinase C (PKC) activation can control GLYT2 surface levels although the intracellular compartments where GLYT2 resides are largely unknown. Here, by biochemical and immunological techniques in combination with electron and confocal microscopy, we have investigated the subcellular distribution of GLYT2 in rat brainstem tissue, and characterized the vesicles that contain the transporter. GLYT2 is shown to be present in small and larger vesicles that contain the synaptic vesicle protein synaptophysin, the recycling endosome small GTPase Rab11, and in the larger vesicle population, the vesicular inhibitory amino acid transporter VIAAT. Rab5A, the GABA transporter GAT1, synaptotagmin2 and synaptobrevin2 (VAMP2) were not present. Coexpression of a Rab11 dominant negative mutant with recombinant GLYT2 impaired transporter trafficking and glycine transport. Dual immunogold labeling of brainstem synaptosomes showed a very close proximity of GLYT2 and Rab11. Therefore, the intracellular GLYT2 resides in a subset of endosomal membranes and may traffic around several compartments, mainly Rab11-positive endosomes.     

Characterization of Cyclic AMP-Dependent Phosphorylation of Neuronal Membrane Proteins

We examined the patterns of cyclic AMP-dependent protein phosphorylation in membranes prepared from rat cortical synaptosomes following gel electrophoresis and autoradiography. We determined the optimum pH (6.2), time (20 s), Mg2+ concentration (10 mM) and cyclic AMP concentration (5 microM) for the reaction. We also found that the detergents Triton X-100 and gramicidin S enhanced cyclic AMP-dependent protein phosphorylation. Inhibitors of the Na+, K+ ATPase (ouabain, NaF, vanadate) enhanced protein phosphorylation. This effect occurred in the presence but not in the absence of detergent. The addition of purified bovine brain cyclic AMP-dependent protein kinase catalytic subunit enhanced membrane protein phosphorylation. The addition of homogeneous neural (bovine brain) and non-neural (bovine skeletal muscle) cyclic AMP-dependent protein kinase type II regulatory subunit partially inhibited protein phosphorylation. Both neural and non-neural regulatory subunits behaved similarly. In addition to cyclic AMP-dependent phosphorylation, the alpha-subunit of pyruvate dehydrogenase (Mr = 41,000) is phosphorylated in a cyclic AMP-independent fashion. We also examined the phosphorylation pattern of membranes prepared from rat heart and found that the number of acceptor substrates was much less than that from the nervous system.     

Specificity of Membrane Binding of the Neuronal Protein NAP-22

NAP-22, a major protein of neuronal rafts is known to preferentially bind to membranes containing cholesterol. In this work we establish the requirements for membrane binding of NAP-22. We find that other sterols can replace cholesterol to promote binding. In addition, bilayers containing phosphatidylethanolamine bind NAP-22 in the absence of cholesterol. Thus, there is not a specific interaction of NAP-22 with cholesterol that determines its binding to membranes. Addition of a mol fraction of phosphatidylserine of 0.05 to membranes of phosphatidylcholine and cholesterol enhances the membrane binding of NAP-22. The dependence of binding on the mol fraction of phosphatidylserine indicates that NAP-22 binds to membranes with its amino-terminal segment closer to the membrane than the remainder of the protein. We have also determined which segments of NAP-22 are required for membrane binding. A non-myristoylated form binds only weakly to membranes. Truncating the protein from 226 amino acids to the myristoylated amino-terminal 60 amino acids does not prevent binding to membranes in a cholesterol-dependent manner, but this binding is of weaker affinity. However, myristoylation is not sufficient to promote binding to cholesterol-rich domains. An N-terminal 19-amino-acid, myristoylated peptide binds to membranes but without requiring specific lipids. Thus, the remainder of the protein contributes to the lipid specificity of the membrane binding of NAP-22.     

Effects of Plasma Membrane Cholesterol Level and Cytoskeleton F-Actin on Cell Protrusion Mechanics

Protrusions are deformations that form at the surface of living cells during biological activities such as cell migration. Using combined optical tweezers and fluorescent microscopy, we quantified the mechanical properties of protrusions in adherent human embryonic kidney cells in response to application of an external force at the cell surface. The mechanical properties of protrusions were analyzed by obtaining the associated force-length plots during protrusion formation, and force relaxation at constant length. Protrusion mechanics were interpretable by a standard linear solid (Kelvin) model, consisting of two stiffness parameters, k ₀ and k ₁ (with k ₀>k ₁), and a viscous coefficient. While both stiffness parameters contribute to the time-dependant mechanical behavior of the protrusions, k ₀ and k ₁ in particular dominated the early and late stages of the protrusion formation and elongation process, respectively. Lowering the membrane cholesterol content by 25% increased the k ₀ stiffness by 74%, and shortened the protrusion length by almost half. Enhancement of membrane cholesterol content by nearly two-fold increased the protrusion length by 30%, and decreased the k ₀ stiffness by nearly two-and-half-fold as compared with control cells. Cytoskeleton integrity was found to make a major contribution to protrusion mechanics as evidenced by the effects of F-actin disruption on the resulting mechanical parameters. Viscoelastic behavior of protrusions was further characterized by hysteresis and force relaxation after formation. The results of this study elucidate the coordination of plasma membrane composition and cytoskeleton during protrusion formation.     

Maintenance of Motility Bias during Cyanobacterial Phototaxis

Signal transduction in bacteria is complex, ranging across scales from molecular signal detectors and effectors to cellular and community responses to stimuli. The unicellular, photosynthetic cyanobacterium Synechocystis sp. PCC6803 transduces a light stimulus into directional movement known as phototaxis. This response occurs via a biased random walk toward or away from a directional light source, which is sensed by intracellular photoreceptors and mediated by Type IV pili. It is unknown how quickly cells can respond to changes in the presence or directionality of light, or how photoreceptors affect single-cell motility behavior. In this study, we use time-lapse microscopy coupled with quantitative single-cell tracking to investigate the timescale of the cellular response to various light conditions and to characterize the contribution of the photoreceptor TaxD1 (PixJ1) to phototaxis. We first demonstrate that a community of cells exhibits both spatial and population heterogeneity in its phototactic response. We then show that individual cells respond within minutes to changes in light conditions, and that movement directionality is conferred only by the current light directionality, rather than by a long-term memory of previous conditions. Our measurements indicate that motility bias likely results from the polarization of pilus activity, yielding variable levels of movement in different directions. Experiments with a photoreceptor (taxD1) mutant suggest a supplementary role of TaxD1 in enhancing movement directionality, in addition to its previously identified role in promoting positive phototaxis. Motivated by the behavior of the taxD1 mutant, we demonstrate using a reaction-diffusion model that diffusion anisotropy is sufficient to produce the observed changes in the pattern of collective motility. Taken together, our results establish that single-cell tracking can be used to determine the factors that affect motility bias, which can then be coupled with biophysical simulations to connect changes in motility behaviors at the cellular scale with group dynamics.