RESUMEN
Small antigen binders, such as nanobodies, have become widely used in biomedical research and pharmaceutical development. However, the pipeline for the generation of functional conjugated probes and drugs from identified binders remains a major time-consuming bottleneck. Here, we developed a method for fast nanobody production and conjugation based on an in vitro synthesis platform. Our system allows for small batch synthesis of nanobodies with the inclusion of a noncanonical amino acid (NCAA). This NCAA can then be used for direct conjugation of molecules to the synthesized nanobody using click-chemistry, reducing the time from binder-encoding DNA to a conjugated probe tremendously. In this study, we conjugated a fluorescent dye to an anti-Green fluorescent protein (GFP) nanobody and attained a fully functional probe suitable for advanced super-resolution microscopy within a short time frame of 2 days. Our work illustrates that an in vitro synthesis platform in combination with click-chemistry can be successfully employed to produce conjugated small antigen binding probes. The fast production and conjugation, combined with the possibility for parallelization as well as precise analysis by microscopy, forms an excellent platform for nanobody prototyping. The here-illustrated method can be used for quick selection and benchmarking of obtained nanobody sequences/clones, e. g., from a phage-display, for use as conjugated small-molecule carriers. This procedure can accelerate the bioengineering of nanobodies for research and pharmaceutical applications.
RESUMEN
In neurons, the microtubule (MT) cytoskeleton forms the basis for long-distance protein transport from the cell body into and out of dendrites and axons. To maintain neuronal polarity, the axon initial segment (AIS) serves as a physical barrier, separating the axon from the somatodendritic compartment and acting as a filter for axonal cargo. Selective trafficking is further instructed by axonal enrichment of MT post-translational modifications, which affect MT dynamics and the activity of motor proteins. Here, we compared two knockout mouse lines lacking the respective enzymes for MT tyrosination and detyrosination, and found that both knockouts led to a shortening of the AIS. Neurons from both lines also showed an increased immobile fraction of endolysosomes present in the axon, whereas mobile organelles displayed shortened run distances in the retrograde direction. Overall, our results highlight the importance of maintaining the balance of tyrosinated and detyrosinated MTs for proper AIS length and axonal transport processes.
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Transporte Axonal , Lisosomas , Ratones Noqueados , Microtúbulos , Tirosina , Animales , Microtúbulos/metabolismo , Tirosina/metabolismo , Lisosomas/metabolismo , Ratones , Axones/metabolismo , Endosomas/metabolismo , Neuronas/metabolismoRESUMEN
The vast majority of excitatory synapses are formed on small dendritic protrusions termed dendritic spines. Dendritic spines vary in size and density that are crucial determinants of excitatory synaptic transmission. Aberrations in spine morphogenesis can compromise brain function and have been associated with neuropsychiatric disorders. Actin filaments (F-actin) are the major structural component of dendritic spines, and therefore, actin-binding proteins (ABP) that control F-actin dis-/assembly moved into the focus as critical regulators of brain function. Studies of the past decade identified the ABP cofilin1 as a key regulator of spine morphology, synaptic transmission, and behavior, and they emphasized the necessity for a tight control of cofilin1 to ensure proper brain function. Here, we report spine enrichment of cyclase-associated protein 1 (CAP1), a conserved multidomain protein with largely unknown physiological functions. Super-resolution microscopy and live cell imaging of CAP1-deficient hippocampal neurons revealed impaired synaptic F-actin organization and dynamics associated with alterations in spine morphology. Mechanistically, we found that CAP1 cooperates with cofilin1 in spines and that its helical folded domain is relevant for this interaction. Moreover, our data proved functional interdependence of CAP1 and cofilin1 in control of spine morphology. In summary, we identified CAP1 as a novel regulator of the postsynaptic actin cytoskeleton that is essential for synaptic cofilin1 activity.
Asunto(s)
Actinas , Espinas Dendríticas , Actinas/metabolismo , Espinas Dendríticas/fisiología , Citoesqueleto de Actina/metabolismo , Sinapsis/metabolismo , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Sinapsinas/metabolismoRESUMEN
Spike-timing-dependent plasticity (STDP) is a candidate mechanism for information storage in the brain, but the whole-cell recordings required for the experimental induction of STDP are typically limited to 1 h. This mismatch of time scales is a long-standing weakness in synaptic theories of memory. Here we use spectrally separated optogenetic stimulation to fire precisely timed action potentials (spikes) in CA3 and CA1 pyramidal cells. Twenty minutes after optogenetic induction of STDP (oSTDP), we observed timing-dependent depression (tLTD) and timing-dependent potentiation (tLTP), depending on the sequence of spiking. As oSTDP does not require electrodes, we could also assess the strength of these paired connections three days later. At this late time point, late tLTP was observed for both causal (CA3 before CA1) and anticausal (CA1 before CA3) timing, but not for asynchronous activity patterns (Δt = 50 ms). Blocking activity after induction of oSTDP prevented stable potentiation. Our results confirm that neurons wire together if they fire together, but suggest that synaptic depression after anticausal activation (tLTD) is a transient phenomenon.
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Potenciación a Largo Plazo , Neuronas , Potenciación a Largo Plazo/fisiología , Potenciales de Acción/fisiología , Técnicas de Placa-Clamp , Neuronas/fisiología , Recompensa , Plasticidad Neuronal/fisiologíaRESUMEN
Organelle positioning within neurites is required for proper neuronal function. In dendrites, with their complex cytoskeletal organization, transport of organelles is guided by local specializations of the microtubule and actin cytoskeleton, and by coordinated activity of different motor proteins. Here, we focus on the actin cytoskeleton in the dendritic shaft and describe dense structures consisting of longitudinal and branched actin filaments. These actin patches are devoid of microtubules and are frequently located at the base of spines, or form an actin mesh around excitatory shaft synapses. Using lysosomes as an example, we demonstrate that the presence of actin patches has a strong impact on dendritic organelle transport, as lysosomes frequently stall at these locations. We provide mechanistic insights on this pausing behavior, demonstrating that actin patches form a physical barrier for kinesin-driven cargo. In addition, we identify myosin Va as an active tether which mediates long-term stalling. This correlation between the presence of actin meshes and halting of organelles could be a generalized principle by which synapses control organelle trafficking.
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Actinas/metabolismo , Dendritas/metabolismo , Lisosomas/metabolismo , Sinapsis/metabolismo , Animales , Transporte Biológico , Células Cultivadas , Ácido Glutámico/metabolismo , Cinesinas/metabolismo , Masculino , Microtúbulos/metabolismo , RatasRESUMEN
Compartmentalization of calcium-dependent plasticity allows for rapid actin remodeling in dendritic spines. However, molecular mechanisms for the spatio-temporal regulation of filamentous actin (F-actin) dynamics by spinous Ca2+-transients are still poorly defined. We show that the postsynaptic Ca2+ sensor caldendrin orchestrates nano-domain actin dynamics that are essential for actin remodeling in the early phase of long-term potentiation (LTP). Steep elevation in spinous [Ca2+]i disrupts an intramolecular interaction of caldendrin and allows cortactin binding. The fast on and slow off rate of this interaction keeps cortactin in an active conformation, and protects F-actin at the spine base against cofilin-induced severing. Caldendrin gene knockout results in higher synaptic actin turnover, altered nanoscale organization of spinous F-actin, defects in structural spine plasticity, LTP, and hippocampus-dependent learning. Collectively, the data indicate that caldendrin-cortactin directly couple [Ca2+]i to preserve a minimal F-actin pool that is required for actin remodeling in the early phase of LTP.
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Señalización del Calcio/fisiología , Proteínas de Unión al Calcio/deficiencia , Espinas Dendríticas/metabolismo , Potenciación a Largo Plazo/fisiología , Potenciales Sinápticos/fisiología , Animales , Células COS , Proteínas de Unión al Calcio/genética , Células Cultivadas , Chlorocebus aethiops , Espinas Dendríticas/química , Espinas Dendríticas/genética , Células HEK293 , Hipocampo/química , Hipocampo/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Técnicas de Cultivo de Órganos , Ratas , Ratas WistarRESUMEN
Most of the excitatory synapses on principal neurons of the forebrain are located on specialized structures called dendritic spines. Their morphology, comprising a spine head connected to the dendritic branch via a thin neck, provides biochemical and electrical compartmentalization during signal transmission. Spine shape is defined and tightly controlled by the organization of the actin cytoskeleton. Alterations in synaptic strength correlate with changes in the morphological appearance of the spine head and neck. Therefore, it is important to get a better understanding of the nanoscale organization of the actin cytoskeleton in dendritic spines. A periodic organization of the actin/spectrin lattice was recently discovered in axons and a small fraction of dendrites using super-resolution microscopy. Here we use a small probe phalloidin-Atto647N, to label F-actin in mature hippocampal primary neurons and in living hippocampal slices. STED nanoscopy reveals that in contrast to ß-II spectrin antibody labelling, phalloidin-Atto647N stains periodic actin structures in all dendrites and the neck of nearly all dendritic spines, including filopodia-like spines. These findings extend the current view on F-actin organization in dendritic spines and may provide new avenues for understanding the structural changes in the spine neck during induction of synaptic plasticity, active organelle transport or tethering.
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Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Espinas Dendríticas/metabolismo , Hipocampo/metabolismo , Plasticidad Neuronal/fisiología , Animales , Hipocampo/citología , Ratas , Ratas WistarRESUMEN
Synaptic connectivity forms the basis for neuronal communication and the storage of information. Experiences and learning of new abilities can drive remodelling of this connectivity and promotes the formation of spine clusters; dendritic segments with a higher spine density. Spines located within these segments are frequently co-activated, undergo different dynamics than synapses located outside of this dendritic compartment and have, in general, a longer lifetime. Several lines of evidence have shown that chemical synapses located close to each other share or compete for intracellular signalling molecules and structural resources. This sharing and competition directly influences spine dynamics. Spines can grow, shrink, increase or decrease the surface expression of receptors, channels and adhesion molecules or remain stable and unchanged over extended periods of time. Here we summarize recent discoveries and provide a closer look at spine clustering, dendritic segment-specific signalling and potential molecular mechanisms underlying associative and heterosynaptic plasticity.