RESUMEN
Planned actions can be triggered involuntarily by a startling acoustic stimulus (SAS), resulting in very short reaction times (RT). This phenomenon, known as the StartReact effect, is thought to result from the startle-related activation of reticular structures. However, other sensory modalities also can elicit a reflexive startle response. Here, we assessed the effectiveness of an intense startling electric stimulus (SES) in eliciting the StartReact effect as compared to a SAS. We tested SES intensities at 15 and 25 times the perceptual threshold of each participant, as well as SAS intensities of 114 dB and 120 dB. The electrical stimulation electrodes were placed over short head of the biceps brachii on the arm not involved in the task. Intense electric and acoustic stimuli were presented on 20% of the trials in a simple RT paradigm requiring a targeted ballistic wrist extension movement. The proportion of trials showing short latency (≤ 120 ms) startle reflex-related activation in sternocleidomastoid was significantly lower on intense electrical stimulus trials compared to intense acoustic trials, and the startle response onset occurred significantly later on SES trials compared to SAS. However, when a startle reflex was observed, RTs related to the prepared movement were facilitated to a similar extent for both SES and SAS conditions, suggesting that the accelerated response latency associated with the StartReact effect is independent of stimulus type.
RESUMEN
During the execution of movements, error correction processes have been inferred by EEG activation at oscillation frequencies in the theta (4-8 Hz) and alpha (8-12 Hz) bands. The current study examined whether evidence for error detection and correction could be found at the muscular level through the use of EMG-EMG coherence, which quantifies the amount of synchronous EMG activity between limbs in the frequency domain. Participants (n = 13) performed a bimanual force production task involving either wrist flexors or extensors under conditions in which the force was to be held constant or continuously modulated. As predicted, the modulation of changing force output resulted in significantly greater force variability and increased EMG-EMG coherence throughout the theta and alpha band for both flexor and extensor responses. These results are consistent with EEG activation frequencies associated with error correction, motor reprogramming and sustained attention and indicate that evidence for these cortical processes can also be observed at the muscular level in the form of correlated EMG frequency content between limbs.
Asunto(s)
Contracción Isométrica , Músculo Esquelético , Humanos , Músculo Esquelético/fisiología , Electromiografía , Contracción Isométrica/fisiología , Movimiento , Muñeca , ElectroencefalografíaRESUMEN
Cells can switch between Rac1 (lamellipodia-based) and RhoA (blebbing-based) migration modes, but the molecular mechanisms regulating this shift are not fully understood. Diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid, forms independent complexes with Rac1 and RhoA, selectively dissociating each from their common inhibitor RhoGDI. DGKζ catalytic activity is required for Rac1 dissociation but is dispensable for RhoA dissociation; instead, DGKζ stimulates RhoA release via a kinase-independent scaffolding mechanism. The molecular determinants that mediate the selective targeting of DGKζ to Rac1 or RhoA signaling complexes are unknown. Here, we show that protein kinase Cα (PKCα)-mediated phosphorylation of the DGKζ MARCKS domain increased DGKζ association with RhoA and decreased its interaction with Rac1. The same modification also enhanced DGKζ interaction with the scaffold protein syntrophin. Expression of a phosphomimetic DGKζ mutant stimulated membrane blebbing in mouse embryonic fibroblasts and C2C12 myoblasts, which was augmented by inhibition of endogenous Rac1. DGKζ expression in differentiated C2 myotubes, which have low endogenous Rac1 levels, also induced substantial membrane blebbing via the RhoA-ROCK pathway. These events were independent of DGKζ catalytic activity, but dependent upon a functional C-terminal PDZ-binding motif. Rescue of RhoA activity in DGKζ-null cells also required the PDZ-binding motif, suggesting that syntrophin interaction is necessary for optimal RhoA activation. Collectively, our results define a switch-like mechanism whereby DGKζ phosphorylation by PKCα plays a role in the interconversion between Rac1 and RhoA signaling pathways that underlie different cellular migration modes.
Asunto(s)
Movimiento Celular , Diacilglicerol Quinasa/fisiología , Proteínas Asociadas a la Distrofina/metabolismo , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/metabolismo , Neuropéptidos/metabolismo , Proteína Quinasa C-alfa/farmacología , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Animales , Diglicéridos/metabolismo , Proteínas Asociadas a la Distrofina/genética , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Ratones , Ratones Noqueados , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/genética , Neuropéptidos/genética , Dominios Proteicos , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rhoA/genéticaRESUMEN
Syntrophins are scaffold proteins that regulate the subcellular localization of diacylglycerol kinase zeta (DGK-zeta), an enzyme that phosphorylates the lipid second-messenger diacylglycerol to yield phosphatidic acid. DGK-zeta and syntrophins are abundantly expressed in neurons of the developing and adult brain, but their function is unclear. Here, we show that they are present in cell bodies, neurites, and growth cones of cultured cortical neurons and differentiated N1E-115 neuroblastoma cells. Overexpression of DGK-zeta in N1E-115 cells induced neurite formation in the presence of serum, which normally prevents neurite outgrowth. This effect was independent of DGK-zeta kinase activity but dependent on a functional C-terminal PDZ-binding motif, which specifically interacts with syntrophin PDZ domains. DGK-zeta mutants with a blocked C terminus acted as dominant-negative inhibitors of outgrowth from serum-deprived N1E-115 cells and cortical neurons. Several lines of evidence suggest DGK-zeta promotes neurite outgrowth through association with the GTPase Rac1. DGK-zeta colocalized with Rac1 in neuronal processes and DGK-zeta-induced outgrowth was inhibited by dominant-negative Rac1. Moreover, DGK-zeta directly interacts with Rac1 through a binding site located within its C1 domains. Together with syntrophin, these proteins form a tertiary complex in N1E-115 cells. A DGK-zeta mutant that mimics phosphorylation of the MARCKS domain was unable to bind an activated Rac1 mutant (Rac1(V12)) and phorbol myristate acetate-induced protein kinase C activation inhibited the interaction of DGK-zeta with Rac1(V12), suggesting protein kinase C-mediated phosphorylation of the MARCKS domain negatively regulates DGK-zeta binding to active Rac1. Collectively, these findings suggest DGK-zeta, syntrophin, and Rac1 form a regulated signaling complex that controls polarized outgrowth in neuronal cells.
Asunto(s)
Diacilglicerol Quinasa/metabolismo , Proteínas Asociadas a la Distrofina/metabolismo , Neuritas/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Adenoviridae/genética , Secuencias de Aminoácidos , Animales , Sitios de Unión , Línea Celular Tumoral , Diacilglicerol Quinasa/química , Activación Enzimática , Regulación de la Expresión Génica , Glutatión Transferasa/metabolismo , Ratones , Microscopía Confocal , Modelos Biológicos , Modelos Genéticos , Mutación , Neuroblastoma/metabolismo , Neuronas/metabolismo , Fosforilación , Unión Proteica , Proteína Quinasa C/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Acetato de Tetradecanoilforbol/farmacología , Factores de TiempoRESUMEN
Syntrophins are scaffold proteins of the dystrophin glycoprotein complex (DGC), which target ion channels, receptors, and signaling proteins to specialized subcellular domains. A yeast two-hybrid screen of a human brain cDNA library with the PSD-95, Discs-large, ZO-1 (PDZ) domain of gamma1-syntrophin yielded overlapping clones encoding the C terminus of TAPP1, a pleckstrin homology (PH) domain-containing adapter protein that interacts specifically with phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)). In biochemical assays, the C terminus of TAPP1 bound specifically to the PDZ domains of gamma1-, alpha1-, and beta2-syntrophin and was required for syntrophin binding and for the correct subcellular localization of TAPP1. TAPP1 is recruited to the plasma membrane of cells stimulated with platelet-derived growth factor (PDGF), a motogen that produces PI(3,4)P(2). Cell migration in response to PDGF stimulation is characterized by a rapid reorganization of the actin cytoskeleton, which gives rise to plasma membrane specializations including peripheral and dorsal circular ruffles. Both TAPP1 and syntrophins were localized to PDGF-induced circular membrane ruffles in NIH-3T3 cells. Ectopic expression of TAPP1 potently blocked PDGF-induced formation of dorsal circular ruffles, but did not affect peripheral ruffling. Interestingly, coexpression of alpha1- or gamma1-syntrophin with TAPP1 prevented the blockade of circular ruffling. In addition to syntrophins, several other proteins of the DGC were enriched in circular ruffles. Collectively, our results suggest syntrophins regulate the localization of TAPP1, which may be important for remodeling the actin cytoskeleton in response to growth factor stimulation.