Distribution of MAP 2 in dendritic spines and its colocalization with actin

Summary The distribution of MAP2 and actin in dendritic spines of the visual and cerebellar cortices, dentate fascia, and hippocampus was determined by using immunogold electron microscopy. By this approach, we have confirmed the presence of MAP2 in dendritic spines and identified substructures within the spine compartment showing MAP2 immunoreactivity. MAP2 immunolabeling was mainly associated with filaments which reacted with a monoclonal anti-actin antibody. Also, by immunogold double-labeling we colocalized MAP2 with actin on the endomembranes of the spine apparatus, smooth endoplasmic reticulum, and in the postsynaptic density. Labeling was nearly absent in axons and axonal terminals. These results indicate that MAP2 is an actin-associated protein in dendritic spines. Thus, MAP2 may organize actin filaments in the spine and endow the actin network of the spine with dynamic properties that are necessary for synaptic plasticity.     

Bipolar nanosecond electric pulses are less efficient at electropermeabilization and killing cells than monopolar pulses

Multiple studies have shown that bipolar (BP) electric pulses in the microsecond range are more effective at permeabilizing cells while maintaining similar cell survival rates as compared to monopolar (MP) pulse equivalents. In this paper, we investigated whether the same advantage existed for BP nanosecond-pulsed electric fields (nsPEF) as compared to MP nsPEF. To study permeabilization effectiveness, MP or BP pulses were delivered to single Chinese hamster ovary (CHO) cells and the response of three dyes, Calcium Green-1, propidium iodide (PI), and FM1-43, was measured by confocal microscopy. Results show that BP pulses were less effective at increasing intracellular calcium concentration or PI uptake and cause less membrane reorganization (FM1-43) than MP pulses. Twenty-four hour survival was measured in three cell lines (Jurkat, U937, CHO) and over ten times more BP pulses were required to induce death as compared to MP pulses of similar magnitude and duration. Flow cytometry analysis of CHO cells after exposure (at 15 min) revealed that to achieve positive FITC-Annexin V and PI expression, ten times more BP pulses were required than MP pulses. Overall, unlike longer pulse exposures, BP nsPEF exposures proved far less effective at both membrane permeabilization and cell killing than MP nsPEF.     

Structural dynamics of dendritic spines: Molecular composition,geometry and functional regulation

The development of dendritic spines with specific geometry and membrane composition is critical for proper synaptic function. Specific spine membrane architecture, sub-spine microdomains and spine head and neck geometry allow for well-coordinated and compartmentalized signaling, disruption of which could lead to various neurological diseases. Research from neuronal cell culture, brain slices and direct in vivo imaging indicates that dendritic spine development is a dynamic process which includes transition from small dendritic filopodia through a series of structural refinements to elaborate spines of various morphologies. Despite intensive research, the precise coordination of this morphological transition, the changes in molecular composition, and the relation of spines of various morphologies to function remain a central enigma in the development of functional neuronal circuits. Here, we review research so far and aim to provide insight into the key events that drive structural change during transition from immature filopodia to fully functional spines and the relevance of spine geometry to function.     

Many faces of drebrin: from building dendritic spines and stabilizing gap junctions to shaping neurite-like cell processes

In this review we consider the multiple functions of developmentally regulated brain protein (drebrin), an actin-binding protein, in the formation of cellular polarity in different cell types. Drebrin has a well-established role in the morphogenesis, patterning and maintenance of dendritic spines in neurons. We have recently shown that drebrin also stabilizes Connexin-43 containing gap junctions at the plasma membrane. The latest literature and our own data suggest that drebrin may be broadly involved in shaping cell processes and in the formation of stabilized plasma membrane domains, an effect that is likely to be of crucial significance for formation of cell polarity in both neuronal and non-neuronal types. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Assessment of estradiol influence on spatial tasks and hippocampal CA1 spines: evidence that the duration of hormone deprivation after ovariectomy compromises 17beta-estradiol effectiveness in altering CA1 spines

Two pulses of 17β-estradiol (10 µg) are commonly used to increase hippocampal CA1 apical dendritic spine density and alter spatial performance in ovariectomized (OVX) female rats, but rarely are the measures combined. The goal of this study was to use this two-pulse injection protocol repeatedly with intervening wash-out periods in the same rats to: 1) measure spatial ability using different tasks that require hippocampal function and 2) determine whether ovarian hormone depletion for an extended 10-week period reduces 17β-estradiol's effectiveness in elevating CA1 apical dendritic spine density. Results showed that two injections of 10 µg 17β-estradiol (72 and 48 h prior to testing and timed to maximize CA1 apical spine density at behavioral assessment) corresponded to improved spatial memory performance on object placement. In contrast, two injections of 5 µg 17β-estradiol facilitated spatial learning on the water maze compared to rats given two injections of 10 µg 17β-estradiol or the sesame oil vehicle. Neither 17β-estradiol dose altered Y-maze performance. As expected, the intermittent two-pulse injection protocol increased CA1 apical spine density, but 10 weeks of OVX without estradiol treatment decreased the effectiveness of 10 µg 17β-estradiol to increase CA1 apical spine density. Moreover, two pulses of 5 µg 17β-estradiol injected intermittently failed to alter CA1 apical spine density and decreased basal spine density. These results demonstrate that extended time without ovarian hormones reduces 17β-estradiol's effectiveness to increase CA1 apical spine density. Collectively, these findings highlight the complex interactions among estradiol, CA1 spine density/morphology, and task requirements, all of which contribute to behavioral outcomes.     

Synaptic Vesicle Recycling in Cultured Cerebellar Granule Cells: Role of Vesicular Acidification and Refilling

Abstract: The role of the transvesicular protonmotive force in synaptic vesicle recycling was investigated in cultured cerebellar granule cells. The vesicular V-ATPase was inhibited by 1 µ M bafilomycin A1; as an alternative, the pH component of the gradient was selectively collapsed by equilibration of the cells with 10 m M methylamine and monitored with the fluorescent probe Lysosensor Green. Electrical field-evoked exocytosis of d -[³H]aspartate was inhibited by bafilomycin A1 but not by methylamine, indicating that a transvesicular membrane potential rather than pH gradient is required for transmitter retention within vesicles. In contrast, neither compound affected the field-evoked uptake, recycling, or destaining of the vesicle-specific dye FM2-10; thus, vesicles whose lumens were neutral and/or depleted of transmitter could still recycle in the nerve terminal. No exhaustion of d -[³H]aspartate exocytosis was observed when cells were subjected to six consecutive trains of field stimuli (40 Hz/10 s separated by 10 s). In contrast, the release of preloaded FM2-10 was reduced by ∼50%, with each stimulus indicating that unlabeled vesicles with accumulated d -[³H]aspartate were competing with labeled vesicles for exocytosis. As d -[³H]aspartate was accumulated rapidly across the vesicle membrane from the large cytoplasmic pool, the transmitter-loaded but unlabelled vesicles may represent refilled recycling vesicles. FM2-10 destaining and d -[³H]aspartate exocytosis were reduced in parallel at low frequencies, challenging a role for transient vesicle fusion.     

Modularity and intra-floral integration in metameric organisms: plants are more than the sum of their parts

Within-individual variation in virtually every conceivable morphological and functional feature of reiterated structures is a pervasive feature of plant phenotypes. In particular, architectural effects, regular, repeatable patterns of intra-individual variation in form and function that are associated with position are nearly ubiquitous. Yet, flowers also are predicted to be highly integrated. For animal-pollinated plants, the coordination of multiple organs within each flower is required to achieve the complex functions of pollinator attraction and orientation, pollen donation and pollen receipt. To the extent that pollinators may select for multiple independent functions, phenotypic integration within flowers may also be modular. That is, subsets of floral structures may be integrated but vary independently of other subsets of structures that are themselves integrated. How can phenotypic integration and modularity be understood within the context of architectural effects? This essay reviews recent research on patterns of floral integration and modularity and explores the potential for spatial and temporal changes in the selective environment of individual flowers to result in positional variation in patterns of morphological integration.     

Patterns of association between crucifers and their flower-mimic pathogens: host jumps are more common than coevolution or cospeciation

Morphological and molecular phylogenies of animal parasites have often shown parallel cladogenesis, supporting hypotheses of coevolution. Few studies of the phylogenetic history for plants and their pathogens exist. Gene-for-gene interactions suggest that plant pathogens ought to have similar phylogenetic histories as their hosts. However, high dispersability combined with an inability to choose to leave if an inappropriate host has been landed on could increase the likelihood of host jumps and thus decrease phylogenetic congruence between plant pathogens and their hosts. In this study, I examined the pattern of association between the flower-mimicking crucifer rusts and their hosts by comparing independent host phylogenies (based on both cpDNA trnL-F introns and nuclear internal transcribed spacer [ITS] sequences) with that of their rust pathogens (based on ITS sequences). The expectation was that if the pathogens coevolved or cospeciated with their hosts, then their phylogenies should be congruent. Host-tracking coevolution can be differentiated from cospeciation by examining the times of divergence: If the pathogens are younger than the hosts, then it is likely that host tracking has occurred. For the crucifer rusts and their hosts, there was little evidence of parallel cladogenesis, suggesting that both cospeciation and coevolutionary tracking are rare. Instead, the most common pattern was one of host jumps to geographically associated taxa. There are at least three factors that may have contributed to the geographic structuring of the data. First, along the east-west transect stretching from the Rocky Mountains to California, large differences in rainfall and the timing of rainfall may reduce long-distance gene flow. Second, although dispersal of infectious spores is by wind, sexual reproduction of these fungi depends on insects, which move short distances. Third, host shifts are most likely to occur to geographically available taxa. Any species that grows adjacent to infected plants will be exposed to millions of spores, and the probability of eventual infection by a new mutant increases with greater exposure. Thus, patterns of association between the crucifers and their flower-mimic pathogens reflect jumps to geographically available hosts, which are not necessarily those that are most closely related.     

Is the Sudlow site I of human serum albumin more generous to adopt prospective anti-cancer bioorganic compound than that of bovine: A combined spectroscopic and docking simulation approach

A comparative biophysical study on the individual conformational adaptation embraced by two homologous serum albumins (SA) (bovine and human) towards a potential anticancer bioorganic compound 2-(6-chlorobenzo[d] thiazol-2-yl)-1H-benzo[de] isoquinoline-1,3(2H)- dione (CBIQD) is apparent from the discrimination in binding behavior and the ensuing consequences accomplished by combined in vitro optical spectroscopy, in silico molecular docking and molecular dynamics (MD) simulation. The Sudlow site I of HSA although anion receptive, harbors neutral CBIQD in Sudlow site I (subdomain IIA, close to Trp) of HSA, while in BSA its prefers to snugly fit into Sudlow site II (subdomain IIIA, close to Tyr). Based on discernable diminution of HSA mean fluorescence lifetime as a function of biluminophore concentration, facile occurrence of fluorescence resonance energy transfer (FRET) is substantiated as the probable quenching mechanism accompanied by structural deformations in the protein ensemble. CBIQD establishes itself within HSA close to Trp214, and consequently reduces the micropolarity of the cybotactic environment that is predominantly constituted by hydrophobic amino acid residues. The stronger association of CBIQD with HSA encourages an allosteric modulation leading to slight deformation in its secondary structure whereas for BSA the association is comparatively weaker. Sudlow site I of HSA is capable to embrace a favorable conformation like malleable gold to provide room for incoming CBIQD, whereas for BSA it behaves more like rigid cast-iron which does not admit any change thus forcing CBIQD to occupy an altogether different binding location i.e. the Sudlow site II. The anticancer CBIQD is found to be stable within the HSA scaffold as vindicated by root mean square deviation (RMSD) and root mean square fluctuation (RMSF) obtained by MD simulation. A competitively inhibited esterase-like activity of HSA upon CBIQD binding to Lys199 and Arg257 residues, plausibly envisions that similar naphthalimide based prodrugs, bearing ester functionality, can be particularly activated by Sudlow site I of HSA. The consolidated spectroscopic research described herein may encourage design of naphthalimide based pro-drugs for effective in vivo biodistribution using HSA-based drug delivery systems.