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1.
J Am Chem Soc ; 144(12): 5614-5628, 2022 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-35290733

RESUMO

Photoswitchable reagents are powerful tools for high-precision studies in cell biology. When these reagents are globally administered yet locally photoactivated in two-dimensional (2D) cell cultures, they can exert micron- and millisecond-scale biological control. This gives them great potential for use in biologically more relevant three-dimensional (3D) models and in vivo, particularly for studying systems with inherent spatiotemporal complexity, such as the cytoskeleton. However, due to a combination of photoswitch isomerization under typical imaging conditions, metabolic liabilities, and insufficient water solubility at effective concentrations, the in vivo potential of photoswitchable reagents addressing cytosolic protein targets remains largely unrealized. Here, we optimized the potency and solubility of metabolically stable, druglike colchicinoid microtubule inhibitors based on the styrylbenzothiazole (SBT) scaffold that are nonresponsive to typical fluorescent protein imaging wavelengths and so enable multichannel imaging studies. We applied these reagents both to 3D organoids and tissue explants and to classic model organisms (zebrafish, clawed frog) in one- and two-protein imaging experiments, in which spatiotemporally localized illuminations allowed them to photocontrol microtubule dynamics, network architecture, and microtubule-dependent processes in vivo with cellular precision and second-level resolution. These nanomolar, in vivo capable photoswitchable reagents should open up new dimensions for high-precision cytoskeleton research in cargo transport, cell motility, cell division, and development. More broadly, their design can also inspire similarly capable optical reagents for a range of cytosolic protein targets, thus bringing in vivo photopharmacology one step closer to general realization.


Assuntos
Microtúbulos , Peixe-Zebra , Animais , Citoesqueleto , Indicadores e Reagentes/metabolismo , Microtúbulos/metabolismo , Mitose
2.
STAR Protoc ; 5(2): 103003, 2024 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-38735041

RESUMO

Single-cell microcultures (SCMs) form a monosynaptic circuit that allows stimulation and recording of postsynaptic responses using a single electrode. Here, we present a protocol to establish autaptic cultures from rat superior cervical ganglion neurons. We describe the steps for preparing SCMs, recording synaptic currents, and identifying and processing the recorded neurons for electron microscopy. We then detail procedures for visualizing synapses. This protocol is illustrated by correlating evoked and spontaneous neurotransmitter release with the ultrastructural features of synapses recorded. For complete details on the use and execution of this protocol, please refer to Velasco et al.1.


Assuntos
Neurônios , Animais , Ratos , Neurônios/citologia , Neurônios/fisiologia , Neurônios/ultraestrutura , Microscopia Eletrônica/métodos , Sinapses/fisiologia , Sinapses/ultraestrutura , Sinapses/metabolismo , Eletrofisiologia/métodos , Técnicas de Cultura de Células/métodos , Gânglio Cervical Superior/citologia , Células Cultivadas , Fenômenos Eletrofisiológicos , Análise de Célula Única/métodos
3.
Commun Biol ; 6(1): 488, 2023 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-37147475

RESUMO

Microtubules are key to multiple neuronal functions involving the transport of organelles, however, their relationship to neurotransmitter release is still unresolved. Here, we show that microtubules present in the presynaptic compartment of cholinergic autaptic synapses are dynamic. To investigate how the balance between microtubule growth and shrinkage affects neurotransmission we induced synchronous microtubule depolymerization by photoactivation of the chemical inhibitor SBTub3. The consequence was an increase in spontaneous neurotransmitter release. An analogous effect was obtained by dialyzing the cytosol with Kif18A, a plus-end-directed kinesin with microtubule depolymerizing activity. Kif18A also inhibited the refilling of the readily releasable pool of synaptic vesicles during high frequency stimulation. The action of Kif18A was associated to one order of magnitude increases in the numbers of exo-endocytic pits and endosomes present in the presynaptic terminal. An enhancement of spontaneous neurotransmitter release was also observed when neurons were dialyzed with stathmin-1, a protein with a widespread presence in the nervous system that induces microtubule depolymerization. Taken together, these results support that microtubules restrict spontaneous neurotransmitter release as well as promote the replenishment of the readily releasable pool of synaptic vesicles.


Assuntos
Sinapses , Vesículas Sinápticas , Sinapses/metabolismo , Vesículas Sinápticas/metabolismo , Transmissão Sináptica/fisiologia , Microtúbulos/metabolismo , Neurotransmissores/metabolismo
4.
Commun Biol ; 3(1): 260, 2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32444808

RESUMO

The number of synapses present in a neuronal circuit is not fixed. Neurons must compensate for changes in connectivity caused by synaptic pruning, learning processes or pathological conditions through the constant adjustment of the baseline level of neurotransmission. Here, we show that cholinergic neurons grown in an autaptic circuit in the absence of glia sense the loss of half of their synaptic contacts triggered by exposure to peptide p4.2, a C-terminal fragment of SPARC. Synaptic elimination is driven by a reorganization of the periodic F-actin cytoskeleton present along neurites, and occurs without altering the density of postsynaptic receptors. Neurons recover baseline neurotransmission through a homeostatic presynaptic response that consists of the coordinated activation of rapid synapse formation and an overall potentiation of presynaptic calcium influx. These results demonstrate that neurons establishing autaptic connections continuously sense and adjust their synaptic output by tweaking the number of functional contacts and neurotransmitter release probability.


Assuntos
Citoesqueleto de Actina/metabolismo , Homeostase , Plasticidade Neuronal , Neurônios/fisiologia , Osteonectina/metabolismo , Sinapses/fisiologia , Transmissão Sináptica , Animais , Cálcio/metabolismo , Neurogênese , Neurônios/citologia , Terminações Pré-Sinápticas , Ratos , Ratos Sprague-Dawley
5.
Cell Rep ; 23(6): 1665-1677, 2018 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-29742424

RESUMO

Rett syndrome (RTT) is the second leading cause of mental impairment in girls and is currently untreatable. RTT is caused, in more than 95% of cases, by loss-of-function mutations in the methyl CpG-binding protein 2 gene (MeCP2). We propose here a molecular target involved in RTT: the glycogen synthase kinase-3b (Gsk3b) pathway. Gsk3b activity is deregulated in Mecp2-knockout (KO) mice models, and SB216763, a specific inhibitor, is able to alleviate the clinical symptoms with consequences at the molecular and cellular levels. In vivo, inhibition of Gsk3b prolongs the lifespan of Mecp2-KO mice and reduces motor deficits. At the molecular level, SB216763 rescues dendritic networks and spine density, while inducing changes in the properties of excitatory synapses. Gsk3b inhibition can also decrease the nuclear activity of the Nfkb1 pathway and neuroinflammation. Altogether, our findings indicate that Mecp2 deficiency in the RTT mouse model is partially rescued following treatment with SB216763.


Assuntos
Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Proteína 2 de Ligação a Metil-CpG/deficiência , Subunidade p50 de NF-kappa B/metabolismo , Síndrome de Rett/metabolismo , Síndrome de Rett/patologia , Transdução de Sinais , Sinapses/metabolismo , Animais , Biomarcadores/metabolismo , Células Cultivadas , Cerebelo/metabolismo , Cerebelo/patologia , Espinhas Dendríticas/efeitos dos fármacos , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/patologia , Modelos Animais de Doenças , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Indóis/farmacologia , Inflamação/patologia , Longevidade , Maleimidas/farmacologia , Proteína 2 de Ligação a Metil-CpG/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Inibidores de Proteínas Quinases/farmacologia , Análise de Sobrevida , Regulação para Cima/efeitos dos fármacos
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