RESUMEN
The insular cortex (IC) plays key roles in emotional and regulatory brain functions and is affected across psychiatric diseases. However, the brain-wide connections of the mouse IC have not been comprehensively mapped. Here, we traced the whole-brain inputs and outputs of the mouse IC across its rostro-caudal extent. We employed cell-type-specific monosynaptic rabies virus tracings to characterize afferent connections onto either excitatory or inhibitory IC neurons, and adeno-associated viral tracings to label excitatory efferent axons. While the connectivity between the IC and other cortical regions was highly bidirectional, the IC connectivity with subcortical structures was often unidirectional, revealing prominent cortical-to-subcortical or subcortical-to-cortical pathways. The posterior and medial IC exhibited resembling connectivity patterns, while the anterior IC connectivity was distinct, suggesting two major functional compartments. Our results provide insights into the anatomical architecture of the mouse IC and thus a structural basis to guide investigations into its complex functions.
Asunto(s)
Mapeo Encefálico , Corteza Cerebral/anatomía & histología , Ratones/anatomía & histología , Neuronas/citología , Animales , Femenino , MasculinoRESUMEN
Triggering behavioral adaptation upon the detection of adversity is crucial for survival. The insular cortex has been suggested to process emotions and homeostatic signals, but how the insular cortex detects internal states and mediates behavioral adaptation is poorly understood. By combining data from fiber photometry, optogenetics, awake two-photon calcium imaging and comprehensive whole-brain viral tracings, we here uncover a role for the posterior insula in processing aversive sensory stimuli and emotional and bodily states, as well as in exerting prominent top-down modulation of ongoing behaviors in mice. By employing projection-specific optogenetics, we describe an insula-to-central amygdala pathway to mediate anxiety-related behaviors, while an independent nucleus accumbens-projecting pathway regulates feeding upon changes in bodily state. Together, our data support a model in which the posterior insular cortex can shift behavioral strategies upon the detection of aversive internal states, providing a new entry point to understand how alterations in insula circuitry may contribute to neuropsychiatric conditions.
Asunto(s)
Adaptación Fisiológica/fisiología , Conducta Animal/fisiología , Corteza Cerebral/fisiología , Vías Nerviosas/fisiología , Animales , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Trogocytosis, in which cells nibble away parts of neighboring cells, is an intercellular cannibalism process conserved from protozoa to mammals. Its underlying molecular mechanisms are not well understood and are likely distinct from phagocytosis, a process that clears entire cells. Bi-directional contact repulsion induced by Eph/ephrin signaling involves transfer of membrane patches and full-length Eph/ephrin protein complexes between opposing cells, resembling trogocytosis. Here, we show that the phagocytic adaptor protein Gulp1 regulates EphB/ephrinB trogocytosis to achieve efficient cell rearrangements of cultured cells and during embryonic development. Gulp1 mediates trogocytosis bi-directionally by dynamic engagement with EphB/ephrinB protein clusters in cooperation with the Rac-specific guanine nucleotide exchange factor Tiam2. Ultimately, Gulp1's presence at the Eph/ephrin cluster is a prerequisite for recruiting the endocytic GTPase dynamin. These results suggest that EphB/ephrinB trogocytosis, unlike other trogocytosis events, uses a phagocytosis-like mechanism to achieve efficient membrane scission and engulfment.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Efrinas/metabolismo , Receptores de la Familia Eph/metabolismo , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Animales , Células Cultivadas , Células HEK293 , Células HeLa , Humanos , Ratones , Células 3T3 NIH , Transducción de SeñalRESUMEN
Ephrin receptors interact with membrane-bound ephrin ligands to regulate contact-mediated attraction or repulsion between opposing cells, thereby influencing tissue morphogenesis. Cell repulsion requires bidirectional trans-endocytosis of clustered Eph-ephrin complexes at cell interfaces, but the mechanisms underlying this process are poorly understood. Here, we identified an actin-regulating pathway allowing ephrinB(+) cells to trans-endocytose EphB receptors from opposing cells. Live imaging revealed Rac-dependent F-actin enrichment at sites of EphB2 internalization, but not during vesicle trafficking. Systematic depletion of Rho family GTPases and their regulatory proteins identified the Rac subfamily and the Rac-specific guanine nucleotide exchange factor Tiam2 as key components of EphB2 trans-endocytosis, a pathway previously implicated in Eph forward signaling, in which ephrins act as in trans ligands of Eph receptors. However, unlike in Eph signaling, this pathway is not required for uptake of soluble ligands in ephrinB(+) cells. We also show that this pathway is required for EphB2-stimulated contact repulsion. These results support the existence of a conserved pathway for EphB trans-endocytosis that removes the physical tether between cells, thereby enabling cell repulsion.
Asunto(s)
Comunicación Celular , Movimiento Celular , Endocitosis , Factores de Intercambio de Guanina Nucleótido/metabolismo , Neuronas/enzimología , Receptor EphB2/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Técnicas de Cocultivo , Factores de Intercambio de Guanina Nucleótido/genética , Células HEK293 , Células HeLa , Humanos , Ratones , Microscopía Fluorescente , Neuroblastoma/enzimología , Neuroblastoma/genética , Neuroblastoma/patología , Neuronas/patología , Dominios Proteicos , Interferencia de ARN , Receptor EphB2/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Transfección , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Two new peloruside congeners (3 and 4) were isolated from wild and aquacultured collections of the New Zealand marine sponge Mycale hentscheli. Small-scale reactions on peloruside A (1) have been performed, which along with the isolation of 3 and 4, give further insight into the bioactive pharmacophore of 1.
Asunto(s)
Antineoplásicos/química , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Supervivencia Celular/efectos de los fármacos , Lactonas/química , Poríferos/química , Animales , Antineoplásicos/aislamiento & purificación , Antineoplásicos/farmacología , Acuicultura , Compuestos Bicíclicos Heterocíclicos con Puentes/aislamiento & purificación , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Citometría de Flujo , Células HL-60 , Humanos , Lactonas/aislamiento & purificación , Lactonas/farmacología , Espectroscopía de Resonancia Magnética , Microtúbulos/efectos de los fármacos , Nueva Zelanda , Relación Estructura-Actividad , TermodinámicaRESUMEN
Ska1 and Ska2 form a complex at the kinetochore-microtubule (KT-MT) interface and are required for timely progression from metaphase to anaphase. Here, we use mass spectrometry to search for additional components of the Ska complex. We identify C13Orf3 (now termed Ska3) as a novel member of this complex and map the interaction domains among the three known components. Ska3 displays similar characteristics as Ska1 and Ska2: it localizes to the spindle and KT throughout mitosis and its depletion markedly delays anaphase transition. Interestingly, a more complete removal of the Ska complex by concomitant depletion of Ska1 and Ska3 results in a chromosome congression failure followed by cell death. This severe phenotype reflects a destabilization of KT-MT interactions, as demonstrated by reduced cold stability of KT fibres. Yet, the depletion of the Ska complex only marginally impairs KT localization of the KMN network responsible for MT attachment. We propose that the Ska complex functionally complements the KMN, providing an additional layer of stability to KT-MT attachment and possibly signalling completion of attachment to the spindle checkpoint.
Asunto(s)
Fenómenos Fisiológicos Celulares , Cinetocoros/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mapeo de Interacción de Proteínas , Proteínas de Ciclo Celular , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica , Citocinesis , Células HeLa , Humanos , Unión ProteicaRESUMEN
Peloruside A (peloruside), a microtubule-stabilizing agent from a marine sponge, is less susceptible than paclitaxel to multidrug resistance arising from overexpression of the P-glycoprotein efflux pump and is not affected by mutations that affect the taxoid binding site of beta-tubulin. In vitro studies with purified tubulin indicate that peloruside directly induces tubulin polymerization in the absence of microtubule-associated proteins. Competition for binding between peloruside, paclitaxel, and laulimalide revealed that peloruside binds to a different site on tubulin to paclitaxel. Moreover, laulimalide was able to displace peloruside, indicating that peloruside and laulimalide may compete for the same or overlapping binding sites. It was concluded that peloruside and laulimalide have binding properties that are distinct from other microtubule-stabilizing compounds currently under investigation.