RESUMO
Stoichiometric labeling of endogenous synaptic proteins for high-contrast live-cell imaging in brain tissue remains challenging. Here, we describe a conditional mouse genetic strategy termed endogenous labeling via exon duplication (ENABLED), which can be used to fluorescently label endogenous proteins with near ideal properties in all neurons, a sparse subset of neurons, or specific neuronal subtypes. We used this method to label the postsynaptic density protein PSD-95 with mVenus without overexpression side effects. We demonstrated that mVenus-tagged PSD-95 is functionally equivalent to wild-type PSD-95 and that PSD-95 is present in nearly all dendritic spines in CA1 neurons. Within spines, while PSD-95 exhibited low mobility under basal conditions, its levels could be regulated by chronic changes in neuronal activity. Notably, labeled PSD-95 also allowed us to visualize and unambiguously examine otherwise-unidentifiable excitatory shaft synapses in aspiny neurons, such as parvalbumin-positive interneurons and dopaminergic neurons. Our results demonstrate that the ENABLED strategy provides a valuable new approach to study the dynamics of endogenous synaptic proteins in vivo.
Assuntos
Corantes Fluorescentes/análise , Guanilato Quinases/análise , Proteínas de Membrana/análise , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Animais , Região CA1 Hipocampal/química , Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/metabolismo , Espinhas Dendríticas/química , Espinhas Dendríticas/metabolismo , Proteína 4 Homóloga a Disks-Large , Corantes Fluorescentes/metabolismo , Guanilato Quinases/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Técnicas de Cultura de ÓrgãosRESUMO
Two-photon excitation of fluorescent proteins is an attractive approach for imaging living systems. Today researchers are eager to know which proteins are the brightest and what the best excitation wavelengths are. Here we review the two-photon absorption properties of a wide variety of fluorescent proteins, including new far-red variants, to produce a comprehensive guide to choosing the right fluorescent protein and excitation wavelength for two-photon applications.
Assuntos
Proteínas Luminescentes/química , Fótons , Espectrofotometria/métodos , Lasers , Proteínas Luminescentes/genética , Microscopia Confocal/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Eletricidade EstáticaRESUMO
BACKGROUND: Two-photon dual-color imaging of tissues and cells labeled with fluorescent proteins (FPs) is challenging because most two-photon microscopes only provide one laser excitation wavelength at a time. At present, methods for two-photon dual-color imaging are limited due to the requirement of large differences in Stokes shifts between the FPs used and their low two-photon absorption (2PA) efficiency. RESULTS: Here we present a new method of dual-color two-photon microscopy that uses the simultaneous excitation of the lowest-energy electronic transition of a blue fluorescent protein and a higher-energy electronic transition of a red fluorescent protein. CONCLUSION: Our method does not require large differences in Stokes shifts and can be extended to a variety of FP pairs with larger 2PA efficiency and more optimal imaging properties.
Assuntos
Proteínas Luminescentes/química , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Corantes Fluorescentes/química , Proteína Vermelha FluorescenteRESUMO
A challenge for nonlinear imaging in living tissue is to maximize the total fluorescent yield from each fluorophore. We investigated the emission rates of three fluorophores-rhodamine B, a red fluorescent protein, and CdSe quantum dots-while manipulating the phase of the laser excitation pulse at the focus. In all cases a transform-limited pulse maximized the total yield to insure the highest signal-to-noise ratio. Further, we find evidence of fluorescence antibleaching in quantum dot samples.
Assuntos
Proteínas Luminescentes/química , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Pontos Quânticos , Rodaminas/química , Biologia/instrumentação , Compostos de Cádmio/química , Modelos Teóricos , Fotodegradação , Células Vegetais , Compostos de Selênio/química , Proteína Vermelha FluorescenteRESUMO
Protein kinase A (PKA) has diverse functions in neurons. At rest, the subcellular localization of PKA is controlled by A-kinase anchoring proteins (AKAPs). However, the dynamics of PKA upon activation remain poorly understood. Here, we report that elevation of cyclic AMP (cAMP) in neuronal dendrites causes a significant percentage of the PKA catalytic subunit (PKA-C) molecules to be released from the regulatory subunit (PKA-R). Liberated PKA-C becomes associated with the membrane via N-terminal myristoylation. This membrane association does not require the interaction between PKA-R and AKAPs. It slows the mobility of PKA-C and enriches kinase activity on the membrane. Membrane-residing PKA substrates are preferentially phosphorylated compared to cytosolic substrates. Finally, the myristoylation of PKA-C is critical for normal synaptic function and plasticity. We propose that activation-dependent association of PKA-C renders the membrane a unique PKA-signaling compartment. Constrained mobility of PKA-C may synergize with AKAP anchoring to determine specific PKA function in neurons.