Literature context: -mouse IgG Invitrogen #A-31553, RRID:AB_221604 Alexa Fluor 647-conjugated anti
Focal adhesions (FAs) and stress fibers (SFs) act in concert during cell motility and in response to the extracellular environment. Although the structures of mature FAs and SFs are well studied, less is known about how they assemble and mature de novo during initial cell spreading. In this study using live-cell Airyscan microscopy, we find that FAs undergo "splitting" during their assembly, in which the FA divides along its longitudinal axis. Before splitting, FAs initially appear as assemblies of multiple linear units (FAUs) of 0.3-μm width. Splitting occurs between FAUs, resulting in mature FAs of either a single FAU or of a small number of FAUs that remain attached at their distal tips. Variations in splitting occur based on cell type and extracellular matrix. Depletion of adenomatous polyposis coli (APC) or vasodilator-stimulated phosphoprotein (VASP) results in reduced splitting. FA-associated tension increases progressively during splitting. Early in cell spreading, ventral SFs are detected first, with other SF sub-types (transverse arcs and dorsal SFs) being detected later. Our findings suggest that the fundamental unit of FAs is the fixed-width FAU, and that dynamic interactions between FAUs control adhesion morphology.
Literature context: d Molecular Probes Cat# A31553; RRID:AB_221604 Goat polyclonal anti-mouse Alex
Meiotic double-strand breaks (DSBs) are generated and repaired in a highly regulated manner to ensure formation of crossovers (COs) while also enabling efficient non-CO repair to restore genome integrity. We use structured-illumination microscopy to investigate the dynamic architecture of DSB repair complexes at meiotic recombination sites in relationship to the synaptonemal complex (SC). DSBs resected at both ends are converted into inter-homolog repair intermediates harboring two populations of BLM helicase and RPA, flanking a single population of MutSγ. These intermediates accumulate until late pachytene, when repair proteins disappear from non-CO sites and CO-designated sites become enveloped by SC-central region proteins, acquire a second MutSγ population, and lose RPA. These and other data suggest that the SC may protect CO intermediates from being dismantled inappropriately and promote CO maturation by generating a transient CO-specific repair compartment, thereby enabling differential timing and outcome of repair at CO and non-CO sites.
Literature context: tadt, Germany); cat. no. A31553 RRID:AB_221604), Alexa Fluor 594 ((ThermoFishe
BACKGROUND: Multiple sclerosis (MS) is a neuroinflammatory autoimmune disease of the central nervous system (CNS) which in most cases initially presents with episodes of transient functional deficits (relapsing-remitting MS; RRMS) and eventually develops into a secondary progressive form (SPMS). Aside from neuroimmunological activities, MS is also characterized by neurodegenerative and regenerative processes. The latter involve the restoration of myelin sheaths-electrically insulating structures which are the primary targets of autoimmune attacks. Spontaneous endogenous remyelination takes place even in the adult CNS and is primarily mediated by activation, recruitment, and differentiation of resident oligodendroglial precursor cells (OPCs). However, the overall efficiency of remyelination is limited and further declines with disease duration and progression. From a therapeutic standpoint, it is therefore key to understand how oligodendroglial maturation can be modulated pharmacologically. Teriflunomide has been approved as a first-line treatment for RRMS in the USA and the European Union. As the active metabolite of leflunomide, an established disease-modifying anti-rheumatic drug, it mainly acts via an inhibition of de novo pyrimidine synthesis exerting a cytostatic effect on proliferating B and T cells. METHODS: We investigated teriflunomide-dependent effects on primary rat oligodendroglial homeostasis, proliferation, and differentiation related to cellular processes important for myelin repair hence CNS regeneration in vitro. To this end, several cellular parameters, including specific oligodendroglial maturation markers, in vitro myelination, and p53 family member signaling, were examined by means of gene/protein expression analyses. The rate of myelination was determined using neuron-oligodendrocyte co-cultures. RESULTS: Low teriflunomide concentrations resulted in cell cycle exit while higher doses led to decreased cell survival. Short-term teriflunomide pulses can efficiently promote oligodendroglial cell differentiation suggesting that young, immature cells could benefit from such stimulation. In vitro myelination can be boosted by means of an early stimulation window with teriflunomide. p73 signaling is functionally involved in promoting OPC differentiation and myelination. CONCLUSION: Our findings indicate a critical window of opportunity during which regenerative oligodendroglial activities including myelination of CNS axons can be stimulated by teriflunomide.
Literature context: 405 (#A31553, RRID:AB_221604) antibody (1:1,000; Invitrogen)
In the central nervous system, major histocompatibility complex class I (MHCI) molecules are mainly expressed in neurons, and neuronal MHCI have roles in synapse elimination and plasticity. However, the pathophysiological significance of astroglial MHCI remains unclear. We herein demonstrate that MHCI expression is up-regulated in astrocytes in the medial prefrontal cortex (mPFC) following systemic immune activation by an intraperitoneal injection of polyinosinic-polycytidylic acid (polyI:C) or hydrodynamic interferon (IFN)-γ gene delivery in male C57/BL6J mice. In cultured astrocytes, MHCI/H-2D largely co-localized with exosomes. To investigate the role of astroglial MHCI, H-2D, or sH-2D was expressed in the mPFC of male C57/BL6J mice using an adeno-associated virus vector under the control of a glial fibrillary acidic protein promoter. The expression of astroglial MHCI in the mPFC impaired sociability and recognition memory in mice. Regarding neuropathological changes, MHCI expression in astrocytes significantly activated microglial cells, decreased parvalbumin-positive cell numbers, and reduced dendritic spine density in the mPFC. A treatment with GW4869 that impairs exosome synthesis ameliorated these behavioral and neuropathological changes. These results suggest that the overexpression of MHCI in astrocytes affects microglial proliferation as well as neuronal numbers and spine densities, thereby leading to social and cognitive deficits in mice, possibly via exosomes created by astrocytes.
Literature context: isher Scientific Cat#: A-31553, RRID:AB_221604 CF405S donkey anti-guinea pig I
In the brain, many types of interneurons make functionally diverse inhibitory synapses onto principal neurons. Although numerous molecules have been identified to function in inhibitory synapse development, it remains unknown whether there is a unifying mechanism for development of diverse inhibitory synapses. Here we report a general molecular mechanism underlying hippocampal inhibitory synapse development. In developing neurons, the establishment of GABAergic transmission depends on Neuroligin 2 (NL2), a synaptic cell adhesion molecule (CAM). During maturation, inhibitory synapse development requires both NL2 and Slitrk3 (ST3), another CAM. Importantly, NL2 and ST3 interact with nanomolar affinity through their extracellular domains to synergistically promote synapse development. Selective perturbation of the NL2-ST3 interaction impairs inhibitory synapse development with consequent disruptions in hippocampal network activity and increased seizure susceptibility. Our findings reveal how unique postsynaptic CAMs work in concert to control synaptogenesis and establish a general framework for GABAergic synapse development.
Literature context: A-31553, RRID:AB_221604 Thermo-Fisher Goat anti-Mouse I
Hyperexcitability and the imbalance of excitation/inhibition are one of the leading causes of abnormal sensory processing in Fragile X syndrome (FXS). The precise timing and distribution of excitation and inhibition is crucial for auditory processing at the level of the auditory brainstem, which is responsible for sound localization ability. Sound localization is one of the sensory abilities disrupted by loss of the Fragile X Mental Retardation 1 (Fmr1) gene. Using triple immunofluorescence staining we tested whether there were alterations in the number and size of presynaptic structures for the three primary neurotransmitters (glutamate, glycine, and GABA) in the auditory brainstem of Fmr1 knockout mice. We found decreases in either glycinergic or GABAergic inhibition to the medial nucleus of the trapezoid body (MNTB) specific to the tonotopic location within the nucleus. MNTB is one of the primary inhibitory nuclei in the auditory brainstem and participates in the sound localization process with fast and well-timed inhibition. Thus, a decrease in inhibitory afferents to MNTB neurons should lead to greater inhibitory output to the projections from this nucleus. In contrast, we did not see any other significant alterations in balance of excitation/inhibition in any of the other auditory brainstem nuclei measured, suggesting that the alterations observed in the MNTB are both nucleus and frequency specific. We furthermore show that glycinergic inhibition may be an important contributor to imbalances in excitation and inhibition in FXS and that the auditory brainstem is a useful circuit for testing these imbalances.
Literature context: Alexa Fluor 405Life TechnologiesCat# A-31553Donkey anti-Goat Alexa Flour 647
How the developmental potential of differentiating stem cell progeny becomes rapidly and stably restricted following asymmetric stem cell division is unclear. In the fly larval brain, earmuff (erm) uniquely functions to restrict the developmental potential of intermediate neural progenitors (INPs) generated by asymmetrically dividing neural stem cells (neuroblasts). Here we demonstrate that the histone deacetylase Hdac1/Rpd3 functions together with self-renewal transcriptional repressors to maintain the erm immature INP enhancer in an inactive but poised state in neuroblasts. Within 2 hr of immature INP birth, downregulation of repressor activities alleviates Rpd3-mediated repression on the erm enhancer, enabling acetylation of multiple histone proteins and activating Erm expression. Erm restricts the developmental potential in immature INPs by repressing genes encoding neuroblast transcriptional activators. We propose that poising the fast-activating enhancers of master regulators of differentiation through continual histone deacetylation in stem cells enables self-renewal and rapid restriction of developmental potential following asymmetric division.
Literature context: A-31553; RRID:AB_221604), Alexa Fl
Cell adhesion molecules play important roles in the development of the nervous system. Among the contactin-associated protein (Caspr; also known as Cntnap) family, which belongs to the neurexin superfamily of proteins, Caspr and Caspr2 are indispensable for the formation and maintenance of myelinated nerves. In contrast, a physiological role for Caspr3 remains to be elucidated. This study examines the expression and localization of Caspr3 in the mouse brain using newly generated Caspr3 antibodies. Caspr3 was expressed abundantly between the first and the second postnatal weeks. During this period, Caspr3 was localized especially to the basal ganglia, including the striatum, external segment of the globus pallidus, and substantia nigra, and no gross abnormalities were apparent in the basal ganglia of Caspr3 knockout mice. In the striatum, Caspr3 was expressed by a subpopulation of medium spiny neurons that constitute the direct and indirect pathways. Caspr3 immunostaining was observed as punctate around the cell bodies as well as in the soma. These Caspr3 signals did not, however, overlap with those of synaptic markers. Our findings suggest that Caspr3 may play an important role in basal ganglia development during early postnatal stages.
Literature context: g #A31553 RRID:AB_221604; Invitroge
Multiple sclerosis is an autoimmune disease of the CNS resulting in degeneration of myelin sheaths and loss of oligodendrocytes, which means that protection and electrical insulation of axons and rapid signal propagation are impaired, leading to axonal damage and permanent disabilities. Partial replacement of lost oligodendrocytes and remyelination can occur as a result of activation and recruitment of resident oligodendroglial precursor cells. However, the overall remyelination capacity remains inefficient because precursor cells often fail to generate new oligodendrocytes. Increasing evidence points to the existence of several molecular inhibitors that act on these cells and interfere with their cellular maturation. The p57kip2 gene encodes one such potent inhibitor of oligodendroglial differentiation and this study sheds light on the underlying mode of action. We found that subcellular distribution of the p57kip2 protein changed during differentiation of rat, mouse, and human oligodendroglial cells both in vivo and in vitro. Nuclear export of p57kip2 was correlated with promoted myelin expression, higher morphological phenotypes, and enhanced myelination in vitro. In contrast, nuclear accumulation of p57kip2 resulted in blocked oligodendroglial differentiation. Experimental evidence suggests that the inhibitory role of p57kip2 depends on specific interactions with binding proteins such as LIMK-1, CDK2, Mash1, and Hes5 either by controlling their site of action or their activity. Because functional restoration in demyelinating diseases critically depends on the successful generation of oligodendroglial cells, a therapeutic need that is currently unmet, the regulatory mechanism described here might be of particular interest for identifying suitable drug targets and devising novel therapeutic approaches.