RyR-mediated Ca2+ release elicited by neuronal activity induces nuclear Ca2+ signals, CREB phosphorylation, and Npas4/RyR2 expression.

The expression of several hippocampal genes implicated in learning and memory processes requires that Ca2+ signals generated in dendritic spines, dendrites, or the soma in response to neuronal stimulation reach the nucleus. The diffusion of Ca2+ in the cytoplasm is highly restricted, so neurons must use other mechanisms to propagate Ca2+ signals to the nucleus. Here, we present evidence showing that Ca2+ release mediated by the ryanodine receptor (RyR) channel type-2 isoform (RyR2) contributes to the generation of nuclear Ca2+ signals induced by gabazine (GBZ) addition, glutamate uncaging in the dendrites, or high-frequency field stimulation of primary hippocampal neurons. Additionally, GBZ treatment significantly increased cyclic adenosine monophosphate response element binding protein (CREB) phosphorylation-a key event in synaptic plasticity and hippocampal memory-and enhanced the expression of Neuronal Per Arnt Sim domain protein 4 (Npas4) and RyR2, two central regulators of these processes. Suppression of RyR-mediated Ca2+ release with ryanodine significantly reduced the increase in CREB phosphorylation and the enhanced Npas4 and RyR2 expression induced by GBZ. We propose that RyR-mediated Ca2+ release induced by neuronal activity, through its contribution to the sequential generation of nuclear Ca2+ signals, CREB phosphorylation, Npas4, and RyR2 up-regulation, plays a central role in hippocampal synaptic plasticity and memory processes.

VolumePeeler: a novel FIJI plugin for geometric tissue peeling to improve visualization and quantification of 3D image stacks.

MOTIVATION: Quantitative descriptions of multi-cellular structures from optical microscopy imaging are prime to understand the variety of three-dimensional (3D) shapes in living organisms. Experimental models of vertebrates, invertebrates and plants, such as zebrafish, killifish, Drosophila or Marchantia, mainly comprise multilayer tissues, and even if microscopes can reach the needed depth, their geometry hinders the selection and subsequent analysis of the optical volumes of interest. Computational tools to "peel" tissues by removing specific layers and reducing 3D volume into planar images, can critically improve visualization and analysis. RESULTS: We developed VolumePeeler, a versatile FIJI plugin for virtual 3D "peeling" of image stacks. The plugin implements spherical and spline surface projections. We applied VolumePeeler to perform peeling in 3D images of spherical embryos, as well as non-spherical tissue layers. The produced images improve the 3D volume visualization and enable analysis and quantification of geometrically challenging microscopy datasets. AVAILABILITY: ImageJ/FIJI software, source code, examples, and tutorials are openly available in https://cimt.uchile.cl/mcerda.

Role of Proteus mirabilis flagella in biofilm formation.

Proteus mirabilis(P. mirabilis) is a common etiological agent of urinary tract infections, particularly those associated with catheterization. P. mirabilis efficiently forms biofilms on different surfaces and shows a multicellular behavior called 'swarming', mediated by flagella. To date, the role of flagella in P. mirabilis biofilm formation has been under debate. In this study, we assessed the role of P. mirabilis flagella in biofilm formation using an isogenic allelic replacement mutant unable to express flagellin. Different approaches were used, such as the evaluation of cell surface hydrophobicity, bacterial motility and migration across catheter sections, measurements of biofilm biomass and biofilm dynamics by immunofluorescence and confocal microscopy in static and flow models. Our findings indicate that P. mirabilis flagella play a role in biofilm formation, although their lack does not completely avoid biofilm generation. Our data suggest that impairment of flagellar function can contribute to biofilm prevention in the context of strategies focused on particular bacterial targets.

dSTORM microscopy evidences in HeLa cells clustered and scattered γH2AX nanofoci sensitive to ATM, DNA-PK, and ATR kinase inhibitors.

In response to DNA double-strand breaks (DSB), histone H2AX is phosphorylated around the lesion by a feed forward signal amplification loop, originating γH2AX foci detectable by immunofluorescence and confocal microscopy as elliptical areas of uniform intensity. We exploited the significant increase in resolution (~ × 10) provided by single-molecule localization microscopy (SMLM) to investigate at nanometer scale the distribution of γH2AX signals either endogenous (controls) or induced by the radiomimetic bleomycin (BLEO) in HeLa cells. In both conditions, clustered substructures (nanofoci) confined to γH2AX foci and scattered nanofoci throughout the remnant nuclear area were detected. SR-Tesseler software (Voronoï tessellation-based segmentation) was combined with a custom Python script to first separate clustered nanofoci inside γH2AX foci from scattered nanofoci, and then to perform a cluster analysis upon each nanofoci type. Compared to controls, γH2AX foci in BLEO-treated nuclei presented on average larger areas (0.41 versus 0.19 µm2), more nanofoci per focus (22.7 versus 13.2) and comparable nanofoci densities (~ 60 nanofoci/µm2). Scattered γH2AX nanofoci were equally present (~ 3 nanofoci/µm2), suggesting an endogenous origin. BLEO-treated cells were challenged with specific inhibitors of canonical H2AX kinases, namely: KU-55933, VE-821 and NU-7026 for ATM, ATR and DNA-PK, respectively. Under treatment with pooled inhibitors, clustered nanofoci vanished from super-resolution images while scattered nanofoci decreased (~ 50%) in density. Residual scattered nanofoci could reflect, among other alternatives, H2AX phosphorylation mediated by VRK1, a recently described non-canonical H2AX kinase. In addition to H2AX findings, an analytical approach to quantify clusters of highly differing density from SMLM data is put forward.

Epigenetic editing of the Dlg4/PSD95 gene improves cognition in aged and Alzheimer's disease mice.

The Dlg4 gene encodes for post-synaptic density protein 95 (PSD95), a major synaptic protein that clusters glutamate receptors and is critical for plasticity. PSD95 levels are diminished in ageing and neurodegenerative disorders, including Alzheimer's disease and Huntington's disease. The epigenetic mechanisms that (dys)regulate transcription of Dlg4/PSD95, or other plasticity genes, are largely unknown, limiting the development of targeted epigenome therapy. We analysed the Dlg4/PSD95 epigenetic landscape in hippocampal tissue and designed a Dlg4/PSD95 gene-targeting strategy: a Dlg4/PSD95 zinc finger DNA-binding domain was engineered and fused to effector domains to either repress (G9a, Suvdel76, SKD) or activate (VP64) transcription, generating artificial transcription factors or epigenetic editors (methylating H3K9). These epi-editors altered critical histone marks and subsequently Dlg4/PSD95 expression, which, importantly, impacted several hippocampal neuron plasticity processes. Intriguingly, transduction of the artificial transcription factor PSD95-VP64 rescued memory deficits in aged and Alzheimer's disease mice. Conclusively, this work validates PSD95 as a key player in memory and establishes epigenetic editing as a potential therapy to treat human neurological disorders.

CDK5 downregulation enhances synaptic plasticity.

CDK5 is a serine/threonine kinase that is involved in the normal function of the adult brain and plays a role in neurotransmission and synaptic plasticity. However, its over-regulation has been associated with Tau hyperphosphorylation and cognitive deficits. Our previous studies have demonstrated that CDK5 targeting using shRNA-miR provides neuroprotection and prevents cognitive deficits. Dendritic spine morphogenesis and forms of long-term synaptic plasticity-such as long-term potentiation (LTP)-have been proposed as essential processes of neuroplasticity. However, whether CDK5 participates in these processes remains controversial and depends on the experimental model. Using wild-type mice that received injections of CDK5 shRNA-miR in CA1 showed an increased LTP and recovered the PPF in deficient LTP of APPswe/PS1Δ9 transgenic mice. On mature hippocampal neurons CDK5, shRNA-miR for 12 days induced increased dendritic protrusion morphogenesis, which was dependent on Rac activity. In addition, silencing of CDK5 increased BDNF expression, temporarily increased phosphorylation of CaMKII, ERK, and CREB; and facilitated calcium signaling in neurites. Together, our data suggest that CDK5 downregulation induces synaptic plasticity in mature neurons involving Ca2+ signaling and BDNF/CREB activation.

Gastrulation in an annual killifish: Molecular and cellular events during germ layer formation in Austrolebias.

BACKGROUND: Comparative studies beyond the traditional model organisms have been instrumental in enhancing our understanding of the conserved and derived features of gastrulation, a fundamental process in which the germ layers are specified and shaped to form the body axis. Here, we analyzed gastrulation in a vertebrate group with an extreme mode of early development, the annual killifish. RESULTS: Gastrulation in annual killifish of the genus Austrolebias takes place after the initially dispersed deep blastomeres congregate to form the so-called reaggregate. Cells from the early reaggregate do not appear to form part of any recognizable axial embryonic structure and are possibly extraembryonic. In contrast, later reaggregate cells become engaged in morphogenetic transformations indicative of a process of gastrulation and axis formation. The expression of brachyury and goosecoid suggests that gastrulation takes place in a compressed blastopore-like structure with an organizer region displaced to one end. No collective cell internalization proper of blastopore architecture is observed, though, and it appears that gastrulation primarily involves the reorganization of individual cells. CONCLUSIONS: The unique mode of gastrulation in annual killifish demonstrates that a process so ancient and fundamental to ontogenesis can have striking morphogenetic variations nonpredicted from the sole examination of model species. Developmental Dynamics 246:812-826, 2017. © 2017 Wiley Periodicals, Inc.

Interfering of the Reelin/ApoER2/PSD95 Signaling Axis Reactivates Dendritogenesis of Mature Hippocampal Neurons.

Reelin, an extracellular glycoprotein secreted in embryonic and adult brain, participates in neuronal migration and neuronal plasticity. Extensive evidence shows that reelin via activation of the ApoER2 and VLDLR receptors promotes dendrite and spine formation during early development. Further evidence suggests that reelin signaling is needed to maintain a stable architecture in mature neurons, but, direct evidence is lacking. During activity-dependent maturation of the neuronal circuitry, the synaptic protein PSD95 is inserted into the postsynaptic membrane to induce structural refinement and stability of spines and dendrites. Given that ApoER2 interacts with PSD95, we tested if reelin signaling interference in adult neurons reactivates the dendritic architecture. Unlike findings in developing cultures, the presently obtained in vitro and in vivo data show, for the first time, that reelin signaling interference robustly increase dendritogenesis and reduce spine density in mature hippocampal neurons. In particular, the expression of a mutant ApoER2 form (ApoER2-tailless), which is unable to interact with PSD95 and hence cannot transduce reelin signaling, resulted in robust dendritogenesis in mature hippocampal neurons in vitro. These results indicate that reelin/ApoER2/PSD95 signaling is important for neuronal structure maintenance in mature neurons. Mechanistically, obtained immunofluorescent data indicate that reelin signaling impairment reduced synaptic PSD95 levels, consequently leading to synaptic re-insertion of NR2B-NMDARs. Our findings underscore the importance of reelin in maintaining adult network stability and reveal a new mode for reactivating dendritogenesis in neurological disorders where dendritic arbor complexity is limited, such as in depression, Alzheimer's disease, and stroke. J. Cell. Physiol. 232: 1187-1199, 2017. © 2016 Wiley Periodicals, Inc.

Daam1a mediates asymmetric habenular morphogenesis by regulating dendritic and axonal outgrowth.

Although progress has been made in resolving the genetic pathways that specify neuronal asymmetries in the brain, little is known about genes that mediate the development of structural asymmetries between neurons on left and right. In this study, we identify daam1a as an asymmetric component of the signalling pathways leading to asymmetric morphogenesis of the habenulae in zebrafish. Daam1a is a member of the Formin family of actin-binding proteins and the extent of Daam1a expression in habenular neuron dendrites mirrors the asymmetric growth of habenular neuropil between left and right. Local loss and gain of Daam1a function affects neither cell number nor subtype organisation but leads to a decrease or increase of neuropil, respectively. Daam1a therefore plays a key role in the asymmetric growth of habenular neuropil downstream of the pathways that specify asymmetric cellular domains in the habenulae. In addition, Daam1a mediates the development of habenular efferent connectivity as local loss and gain of Daam1a function impairs or enhances, respectively, the growth of habenular neuron terminals in the interpeduncular nucleus. Abrogation of Daam1a disrupts the growth of both dendritic and axonal processes and results in disorganised filamentous actin and α-tubulin. Our results indicate that Daam1a plays a key role in asymmetric habenular morphogenesis mediating the growth of dendritic and axonal processes in dorsal habenular neurons.

Bleomycin-induced γH2AX foci map preferentially to replicating domains in CHO9 interphase nuclei.

Exposure to DNA damaging agents triggers phosphorylation of histone variant H2AX (generating γH2AX) in large chromatin regions flanking DNA lesions, allowing their immunodetection as nuclear foci. Even though a predominance of γH2AX foci in euchromatin has been postulated, foci positioning when DNA insult occurs in replicating eu- or heterochromatin regions has not been extensively explored. Labeling of interphase nuclei with 5-ethynyl-2'-deoxyuridine (EdU) pulses has revealed that DNA replication is temporarily and spatially regulated: euchromatin replicates in early S (ES) and heterochromatin along mid and late S (MS/LS) phases. In order to map DNA damage with respect to replicating domains, the distribution of γH2AX foci induced by the radiomimetic agent bleomycin was analyzed in CHO9 interphase nuclei by delineating euchromatic (H3K4me3+) and replicating (EdU+) regions. Quantification of overlapping pixels and 3D inter-object overlap in binary masks revealed colocalization between γH2AX foci and EdU + domains both in ES and MS/LS nuclei, indicating that primary damage distribution is modulated by DNA synthesis. Further, we verified that EdU incorporation by itself did not influence BLEO-induced γH2AX nuclear patterns. Our results also revealed a repeated localization of γH2AX foci in replicating/nonreplicating interfaces which could reflect short-range chromatin migration following DNA insult.

Spermatozoa from mice deficient in Niemann-Pick disease type C2 (NPC2) protein have defective cholesterol content and reduced in vitro fertilising ability.

The cholesterol content of the sperm membrane is regulated during both maturation in the epididymis and capacitation in the female tract, two processes required for the spermatozoa to acquire their fertilising ability. Because Niemann-Pick disease, type C2 (NPC2) protein is one of the most abundant components of the epididymal fluid and contains a functional cholesterol-binding site that can transfer cholesterol between membranes, it has been suggested for years that NPC2 could be involved in the regulation of cholesterol levels in spermatozoa during epididymal maturation. In the present study, western blot and immunohistochemistry analyses demonstrated significant levels of NPC2 in the mouse epididymal epithelium. Epididymal spermatozoa obtained from NPC2(-/-) mice were morphologically normal and had normal motility parameters, but had a reduced cholesterol content compared with that of wild-type (WT) spermatozoa, as determined by both biochemical and by flow cytometry analyses. These results suggest that NPC2 could be involved in regulating cholesterol levels in spermatozoa during epididymal maturation. To understand the relevance of epididymal NPC2 for sperm function, the ability of spermatozoa to undergo events influenced by epididymal maturation, such as capacitation and fertilisation, were compared between WT and NPC2(-/-) mice. Capacitated NPC2(-/-) spermatozoa exhibited defective tyrosine phosphorylation patterns and a reduced ability to fertilise cumulus-oocyte complexes compared with WT spermatozoa, supporting the relevance of mouse epididymal NPC2 for male fertility.

E-learning module for cytopathology education based on virtual microscopy.

INTRODUCTION: In cytopathology education, Virtual Microscopy e-learning modules (VM-eLM) have achieved remarkable results in the improvement and personalization of learning. However, it remains to be determined whether these modules can significantly contribute to improving the accuracy of cytological diagnosis. The aim of this work was to create a VM-eLM for gynecologic cytopathology education designed to improve screening and interpretation skills in two groups of cytologists: experienced and nonexperienced. MATERIALS AND METHODS: The module was designed in Moodle with both Whole Slide Images and Static Images taken from Papanicolaou smears that were diagnosed as: negative for intraepithelial lesion, low-grade squamous intraepithelial lesion, high-grade squamous intraepithelial lesion, squamous cell carcinoma, or adenocarcinoma. We assessed the effectiveness of the module using 1) clinical quality indicators to measure skill development and 2) a user survey. RESULTS: After training, participants significantly improved their cytological screening skills, decreasing their false negative diagnosis by 78% in the non-experienced group and eliminating them entirely in the experienced group. Nonexperienced participants also significantly increased their recognition of low-grade squamous intraepithelial lesion and high-grade squamous intraepithelial lesion by 31% and 50%, respectively. Participants positively evaluated the module, highlighting its novelty, the possibility to train remotely, the immediate feedback and the quality of the Whole Slide Images. CONCLUSIONS: We designed, implemented and tested a VM-eLM for Gynecologic Cytopathology Education that improved cytological screening skills for both non-experienced and experienced cytologists, also increasing the diagnostic accuracy of preinvasive lesions by less experienced cytologists. The module was positively evaluated by participants, who perceived an improvement in their interpretive skills.

Proteus mirabilis biofilm expansion microscopy yields over 4-fold magnification for super-resolution of biofilm structure and subcellular DNA organization.

Bacterial biofilms form when bacteria attach to surfaces and generate an extracellular matrix that embeds and stabilizes a growing community. Detailed visualization and quantitative analysis of biofilm architecture by optical microscopy are limited by the law of diffraction. Expansion Microscopy (ExM) is a novel Super-Resolution technique where specimens are physically enlarged by a factor of ∼4, prior to observation by conventional fluorescence microscopy. ExM requires homogenization of rigid constituents of biological components by enzymatic digestion. We developed an ExM approach capable of expanding 48-h old Proteus mirabilis biofilms 4.3-fold (termed PmbExM), close to the theoretic maximum expansion factor without gross shape distortions. Our protocol, based on lytic and glycoside-hydrolase enzymatic treatments, degrades rigid components in bacteria and extracellular matrix. Our results prove PmbExM to be a versatile and easy-to-use Super-Resolution approach for enabling studies of P. mirabilis biofilm architecture, assembly, and even intracellular features, such as DNA organization.

Planar cell polarity signalling regulates cell adhesion properties in progenitors of the zebrafish laterality organ.

Organ formation requires the precise assembly of progenitor cells into a functional multicellular structure. Mechanical forces probably participate in this process but how they influence organ morphogenesis is still unclear. Here, we show that Wnt11- and Prickle1a-mediated planar cell polarity (PCP) signalling coordinates the formation of the zebrafish ciliated laterality organ (Kupffer's vesicle) by regulating adhesion properties between organ progenitor cells (the dorsal forerunner cells, DFCs). Combined inhibition of Wnt11 and Prickle1a reduces DFC cell-cell adhesion and impairs their compaction and arrangement during vesicle lumen formation. This leads to the formation of a mis-shapen vesicle with small fragmented lumina and shortened cilia, resulting in severely impaired organ function and, as a consequence, randomised laterality of both molecular and visceral asymmetries. Our results reveal a novel role for PCP-dependent cell adhesion in coordinating the supracellular organisation of progenitor cells during vertebrate laterality organ formation.

Amyloid ß-Oligomers Inhibit the Nuclear Ca2+ Signals and the Neuroprotective Gene Expression Induced by Gabazine in Hippocampal Neurons.

Hippocampal neuronal activity generates dendritic and somatic Ca2+ signals, which, depending on stimulus intensity, rapidly propagate to the nucleus and induce the expression of transcription factors and genes with crucial roles in cognitive functions. Soluble amyloid-beta oligomers (AßOs), the main synaptotoxins engaged in the pathogenesis of Alzheimer's disease, generate aberrant Ca2+ signals in primary hippocampal neurons, increase their oxidative tone and disrupt structural plasticity. Here, we explored the effects of sub-lethal AßOs concentrations on activity-generated nuclear Ca2+ signals and on the Ca2+-dependent expression of neuroprotective genes. To induce neuronal activity, neuron-enriched primary hippocampal cultures were treated with the GABAA receptor blocker gabazine (GBZ), and nuclear Ca2+ signals were measured in AßOs-treated or control neurons transfected with a genetically encoded nuclear Ca2+ sensor. Incubation (6 h) with AßOs significantly reduced the nuclear Ca2+ signals and the enhanced phosphorylation of cyclic AMP response element-binding protein (CREB) induced by GBZ. Likewise, incubation (6 h) with AßOs significantly reduced the GBZ-induced increases in the mRNA levels of neuronal Per-Arnt-Sim domain protein 4 (Npas4), brain-derived neurotrophic factor (BDNF), ryanodine receptor type-2 (RyR2), and the antioxidant enzyme NADPH-quinone oxidoreductase (Nqo1). Based on these findings we propose that AßOs, by inhibiting the generation of activity-induced nuclear Ca2+ signals, disrupt key neuroprotective gene expression pathways required for hippocampal-dependent learning and memory processes.

CNN stability training improves robustness to scanner and IHC-based image variability for epithelium segmentation in cervical histology.

Background: In digital pathology, image properties such as color, brightness, contrast and blurriness may vary based on the scanner and sample preparation. Convolutional Neural Networks (CNNs) are sensitive to these variations and may underperform on images from a different domain than the one used for training. Robustness to these image property variations is required to enable the use of deep learning in clinical practice and large scale clinical research. Aims: CNN Stability Training (CST) is proposed and evaluated as a method to increase CNN robustness to scanner and Immunohistochemistry (IHC)-based image variability. Methods: CST was applied to segment epithelium in immunohistological cervical Whole Slide Images (WSIs). CST randomly distorts input tiles and factors the difference between the CNN prediction for the original and distorted inputs within the loss function. CNNs were trained using 114 p16-stained WSIs from the same scanner, and evaluated on 6 WSI test sets, each with 23 to 24 WSIs of the same tissue but different scanner/IHC combinations. Relative robustness (rAUC) was measured as the difference between the AUC on the training domain test set (i.e., baseline test set) and the remaining test sets. Results: Across all test sets, The AUC of CST models outperformed "No CST" models (AUC: 0.940-0.989 vs. 0.905-0.986, p < 1e - 8), and obtained an improved robustness (rAUC: [-0.038, -0.003] vs. [-0.081, -0.002]). At a WSI level, CST models showed an increase in performance in 124 of the 142 WSIs. CST models also outperformed models trained with random on-the-fly data augmentation (DA) in all test sets ([0.002, 0.021], p < 1e-6). Conclusion: CST offers a path to improve CNN performance without the need for more data and allows customizing distortions to specific use cases. A python implementation of CST is publicly available at https://github.com/TIGACenter/CST_v1.

Gliovascular alterations in sporadic and familial Alzheimer's disease: APOE3 Christchurch homozygote glioprotection.

In response to brain insults, astrocytes become reactive, promoting protection and tissue repair. However, astroglial reactivity is typical of brain pathologies, including Alzheimer's disease (AD). Considering the heterogeneity of the reactive response, the role of astrocytes in the course of different forms of AD has been underestimated. Colombia has the largest human group known to have familial AD (FAD). This group carries the autosomal dominant and fully penetrant mutation E280A in PSEN1, which causes early-onset AD. Recently, our group identified an E280A carrier who did not develop FAD. The individual was homozygous for the Christchurch mutation R136S in APOE3 (APOEch). Remarkably, APOE is the main genetic risk factor for developing sporadic AD (SAD) and most of cerebral ApoE is produced by astroglia. Here, we characterized astrocyte properties related to reactivity, glutamate homeostasis, and structural integrity of the gliovascular unit (GVU), as factors that could underlie the pathogenesis or protection of AD. Specifically, through histological and 3D microscopy analyses of postmortem samples, we briefly describe the histopathology and cytoarchitecture of the frontal cortex of SAD, FAD, and APOEch, and demonstrate that, while astrodegeneration and vascular deterioration are prominent in SAD, FAD is characterized by hyperreactive-like glia, and APOEch displays the mildest astrocytic and vascular alterations despite having the highest burden of Aß. Notably, astroglial, gliovascular, and vascular disturbances, as well as brain cell death, correlate with the specific astrocytic phenotypes identified in each condition. This study provides new insights into the potential relevance of the gliovasculature in the development and protection of AD. To our knowledge, this is the first study assessing the components of the GVU in human samples of SAD, FAD, and APOEch.

Microtubule-associated protein 1B (MAP1B) is required for dendritic spine development and synaptic maturation.

Microtubule-associated protein 1B (MAP1B) is prominently expressed during early stages of neuronal development, and it has been implicated in axonal growth and guidance. MAP1B expression is also found in the adult brain in areas of significant synaptic plasticity. Here, we demonstrate that MAP1B is present in dendritic spines, and we describe a decrease in the density of mature dendritic spines in neurons of MAP1B-deficient mice that was accompanied by an increase in the number of immature filopodia-like protrusions. Although these neurons exhibited normal passive membrane properties and action potential firing, AMPA receptor-mediated synaptic currents were significantly diminished. Moreover, we observed a significant decrease in Rac1 activity and an increase in RhoA activity in the post-synaptic densities of adult MAP1B(+/-) mice when compared with wild type controls. MAP1B(+/-) fractions also exhibited a decrease in phosphorylated cofilin. Taken together, these results indicate a new and important role for MAP1B in the formation and maturation of dendritic spines, possibly through the regulation of the actin cytoskeleton. This activity of MAP1B could contribute to the regulation of synaptic activity and plasticity in the adult brain.

A method for analysis of lipid vesicle domain structure from confocal image data.

Quantitative characterization of the lateral structure of curved membranes based on fluorescence microscopy requires knowledge of the fluorophore distribution on the surface. We present an image analysis approach for extraction of the fluorophore distribution on a spherical lipid vesicle from confocal imaging stacks. The technique involves projection of volumetric image data onto a triangulated surface mesh representation of the membrane, correction of photoselection effects and global motion of the vesicle during image acquisition and segmentation of the surface into domains using histograms. The analysis allows for investigation of the morphology and size distribution of domains on the surface.