RESUMO
Biocompatible fluorescent reporters with spectral properties spanning the entire visible spectrum are indispensable tools for imaging the biochemistry of living cells and organisms in real time. Here, we report the engineering of a fluorescent chemogenetic reporter with tunable optical and spectral properties. A collection of fluorogenic chromophores with various electronic properties enables to generate bimolecular fluorescent assemblies that cover the visible spectrum from blue to red using a single protein tag engineered and optimized by directed evolution and rational design. The ability to tune the fluorescence color and properties through simple molecular modulation provides a broad experimental versatility for imaging proteins in live cells, including neurons, and in multicellular organisms, and opens avenues for optimizing Förster resonance energy transfer (FRET) biosensors in live cells. The ability to tune the spectral properties and fluorescence performance enables furthermore to match the specifications and requirements of advanced super-resolution imaging techniques.
Assuntos
Diagnóstico por Imagem/métodos , Fluorescência , Engenharia de Proteínas/métodos , Animais , Materiais Biocompatíveis , Técnicas Biossensoriais , Cor , Corantes , Eletrônica , Feminino , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes , Proteínas de Fluorescência Verde , Masculino , Neurônios , Ratos , Ratos Sprague-DawleyRESUMO
The vertebrate neuroepithelium is composed of elongated progenitors whose reciprocal attachments ensure the continuity of the ventricular wall. As progenitors commit to differentiation, they translocate their nucleus basally and eventually withdraw their apical endfoot from the ventricular surface. However, the mechanisms allowing this delamination process to take place while preserving the integrity of the neuroepithelial tissue are still unclear. Here, we show that Notch signaling, which is classically associated with an undifferentiated state, remains active in prospective neurons until they delaminate. During this transition period, prospective neurons rapidly reduce their apical surface and only later down-regulate N-Cadherin levels. Upon Notch blockade, nascent neurons disassemble their junctions but fail to reduce their apical surface. This disrupted sequence weakens the junctional network and eventually leads to breaches in the ventricular wall. We also provide evidence that the Notch ligand Delta-like 1 (Dll1) promotes differentiation by reducing Notch signaling through a Cis-inhibition mechanism. However, during the delamination process, the ubiquitin ligase Mindbomb1 (Mib1) transiently blocks this Cis-inhibition and sustains Notch activity to defer differentiation. We propose that the fine-tuned balance between Notch Trans-activation and Cis-inhibition allows neuroepithelial cells to seamlessly delaminate from the ventricular wall as they commit to differentiation.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Células Neuroepiteliais/metabolismo , Neurogênese/genética , Receptores Notch/genética , Ubiquitina-Proteína Ligases/genética , Animais , Animais Geneticamente Modificados , Caderinas/genética , Caderinas/metabolismo , Diferenciação Celular , Embrião de Galinha , Galinhas , Feminino , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lentivirus/genética , Lentivirus/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Células Neuroepiteliais/citologia , Neurônios/citologia , Neurônios/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Transfecção , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Oriented cell divisions are controlled by a conserved molecular cascade involving Gαi, LGN, and NuMA. We developed a new cellular model of oriented cell divisions combining micropatterning and localized recruitment of Gαi and performed an RNAi screen for regulators acting downstream of Gαi. Remarkably, this screen revealed a unique subset of dynein regulators as being essential for spindle orientation, shedding light on a core regulatory aspect of oriented divisions. We further analyze the involvement of one novel regulator, the actin-capping protein CAPZB. Mechanistically, we show that CAPZB controls spindle orientation independently of its classical role in the actin cytoskeleton by regulating the assembly, stability, and motor activity of the dynein/dynactin complex at the cell cortex, as well as the dynamics of mitotic microtubules. Finally, we show that CAPZB controls planar divisions in vivo in the developing neuroepithelium. This demonstrates the power of this in cellulo model of oriented cell divisions to uncover new genes required in spindle orientation in vertebrates.
Assuntos
Proteína de Capeamento de Actina CapZ/genética , Interferência de RNA , Fuso Acromático/metabolismo , Proteína de Capeamento de Actina CapZ/metabolismo , Divisão Celular , Células HeLa , HumanosRESUMO
In many cell types, mitotic spindle orientation relies on the canonical "LGN complex" composed of Pins/LGN, Mud/NuMA, and Gαi subunits. Membrane localization of this complex recruits motor force generators that pull on astral microtubules to orient the spindle. Drosophila Pins shares highly conserved functional domains with its two vertebrate homologs LGN and AGS3. Whereas the role of Pins and LGN in oriented divisions is extensively documented, involvement of AGS3 remains controversial. Here, we show that AGS3 is not required for planar divisions of neural progenitors in the mouse neocortex. AGS3 is not recruited to the cell cortex and does not rescue LGN loss of function. Despite conserved interactions with NuMA and Gαiin vitro, comparison of LGN and AGS3 functional domains in vivo reveals unexpected differences in the ability of these interactions to mediate spindle orientation functions. Finally, we find that Drosophila Pins is unable to substitute for LGN loss of function in vertebrates, highlighting that species-specific modulations of the interactions between components of the Pins/LGN complex are crucial in vivo for spindle orientation.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Drosophila/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Fuso Acromático/metabolismo , Animais , Proteínas de Transporte/química , Proteínas de Ciclo Celular , Divisão Celular , Polaridade Celular , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Inibidores de Dissociação do Nucleotídeo Guanina/química , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Camundongos , Microtúbulos/metabolismo , Neocórtex/fisiologia , Proteínas Nucleares/metabolismo , Ligação Proteica , Domínios Proteicos , Fuso Acromático/genéticaRESUMO
Unequal centrosome maturation correlates with asymmetric division in multiple cell types. Nevertheless, centrosomal fate determinants have yet to be identified. Here, we show that the Notch pathway regulator Mindbomb1 co-localizes asymmetrically with centriolar satellite proteins PCM1 and AZI1 at the daughter centriole in interphase. Remarkably, while PCM1 and AZI1 remain asymmetric during mitosis, Mindbomb1 is associated with either one or both spindle poles. Asymmetric Mindbomb1 correlates with neurogenic divisions and Mindbomb1 is inherited by the prospective neuron. By contrast, in proliferative divisions, a supplementary pool of Mindbomb1 associated with the Golgi apparatus in interphase is released during mitosis and compensates for Mindbomb1 centrosomal asymmetry. Finally, we show that preventing Mindbomb1 centrosomal association induces reciprocal Notch activation between sister cells and promotes symmetric divisions. Thus, we uncover a link between differential centrosome maturation and Notch signaling and reveal an unexpected compensatory mechanism involving the Golgi apparatus in restoring symmetry in proliferative divisions.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Centríolos/metabolismo , Mitose , Células-Tronco Neurais/metabolismo , Neurogênese , Ubiquitina-Proteína Ligases/metabolismo , Animais , Divisão Celular , Centrossomo/metabolismo , Embrião de Galinha , Complexo de Golgi/metabolismo , Receptores Notch/metabolismo , Transdução de SinaisRESUMO
H-2Z1 is an enhancer trap transgenic mouse line in which the lacZ reporter delineates the somatosensory area of the cerebral cortex where it is expressed in a subset of layer IV neurons. In the search of somatosensory specific genes or regulatory sequences, we mapped the H-2Z1 transgene insertion site to chromosome 17, 100 and 460 kb away from Tbc1d5 and Satb1 flanking genes. We show here that insertion of the H-2Z1 transgene results in three distinct outcomes. First, a genetic background-sensitive expression of lacZ in several brain and body structures. While four genes in a 1 Mb region around the insertion are expressed in the barrel cortex, H-2Z1 expression resembles more that of its two direct neighbors. Moreover, H-2Z1 closely reports most of the body and brain expression sites of the Satb1 chromatin remodeling gene including tooth buds, thymic epithelium, pontine nuclei, fastigial cerebellar nuclei, and cerebral cortex. Second, the H-2Z1 transgene causes insertional mutagenesis of Tbc1d5 and Satb1, leading to a strong decrease in their expressions. Finally, insertion of H-2Z1 affects the differentiation of a subset of cortical GABAergic interneurons, a possible consequence of downregulation of Satb1 expression. Thus, the H-2Z1 "somatosensory" transgene is inserted in the regulatory landscape of two genes highly expressed in the developing somatosensory cortex and reports for a subdomain of their expression profiles. Together, our data suggest that regulation of H-2Z1 expression results from local and remote genetic interactions.