RESUMEN
We have developed a new imaging method, ultrafast optical wide field microscopy, capable of rapidly acquiring wide field images of nearly any sample in a non-contact manner with high spatial and temporal resolution. Time-resolved images of the photoinduced changes in transmission for a patterned semiconductor thin film and a single silicon nanowire after optical excitation are captured using a two-dimensional smart pixel array detector. These images represent the time-dependent carrier dynamics with high sensitivity, femtosecond time resolution and sub-micrometer spatial resolution.
Asunto(s)
Aumento de la Imagen/instrumentación , Microscopía/instrumentación , Diseño de Equipo , Análisis de Falla de EquipoRESUMEN
Slowed elongation rate is the apparent cause of the rapid decrease in rat soleus muscle protein synthesis rate during non-weight bearing. We found that elongation factor 2 was not phosphorylated and thus could not explain the slowed elongation rate. However, we observed a 44 +/- 19 and 28 +/- 14% decrease in the chaperone protein 70-kDa heat-shock cognate/heat shock protein (HSC/HSP-70) associated with the polysomes after 12 and 18 h of non-weight bearing, respectively. Size-fractionated polysomes had less HSC/HSP-70 associated with the larger polysomes in 18-h non-weight-bearing soleus muscle. ATP concentration increased in the non-weight-bearing muscle, so, because ATP enhances HSC/HSP-70 dissociation, we tested the potential role of ATP. Digitonin-permeabilized myoblasts treated with increasing concentrations of ATP showed both a decreased association of HSC/HSP-70 with the polysomes and a shift toward heavier polysomes; these responses were blocked by adenosine 5'-O-(3-thiotriphosphate). These data are consistent with the role of HSC/HSP-70 as a chaperone of nascent protein. The absence of HSC/HSP-70 may slow ribosome translocation, thus slowing elongation rate.
Asunto(s)
Proteínas HSP70 de Choque Térmico/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Extensión de la Cadena Peptídica de Translación , Polirribosomas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Femenino , Proteínas HSP70 de Choque Térmico/biosíntesis , Miembro Posterior , Cinética , Leucina/metabolismo , Proteínas Musculares/biosíntesis , Músculo Esquelético/patología , Atrofia Muscular/patología , Fosforilación , Fosfotreonina/análisis , Ratas , Ratas Sprague-Dawley , Valores de Referencia , Análisis de RegresiónRESUMEN
This study examines the mechanism of the rapid decrease in cardiac muscle protein synthesis during rodent hindlimb non-weight bearing. Polysomes isolated from rat hearts 8 h after suspension show less RNA in the polysome pool and a shift in polysome size toward fewer ribosomes per mRNA; 18 h after suspension, the size shift persists, but the amount of RNA in the polysome pool returns to control values. These data are consistent with a decrease in the rate of initiation of protein synthesis. At both 8 and 12 h of suspension, the cardiac polysomes show a 78 and 93% increase association with the nascent polypeptide chaperone protein 70-kDa heat-shock cognate/heat-shock protein (HSC/HSP-70), respectively, that persists after 7 days of non-weight bearing. Because the dissociation of HSC/HSP-70 from unfolded protein can be modulated by ATP, we measured the adenosine nucleotide pools and found a 53% decrease in ATP levels after 18 h of suspension. We propose a mechanism in which a shift of HSC/HSP-70 to the nascent polypeptide indirectly inhibits protein synthesis initiation.
Asunto(s)
Proteínas HSP70 de Choque Térmico/metabolismo , Inclinación de Cabeza/fisiología , Miocardio/metabolismo , Biosíntesis de Péptidos , Iniciación de la Cadena Peptídica Traduccional , Polirribosomas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Western Blotting , Femenino , Proteínas HSP70 de Choque Térmico/aislamiento & purificación , Cabeza , Modelos Biológicos , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
Protein synthesis rate of the soleus muscle decreases rapidly during non-weight-bearing activity. We isolated polysomes from 18-h non-weight-bearing soleus muscle to investigate the mechanism of this phenomenon. The distribution of polysomal alpha-actin mRNA and 18S rRNA on sucrose density gradients shows that polysomes shift to larger sizes (more ribosomes per mRNA) during non-weight-bearing activity. Furthermore, RNA is mobilized into the polysome pool of the non-weight-bearing soleus muscle; these data indicate that initiation of protein synthesis is not rate limiting. We explain these results as the slowing of nascent polypeptide chain elongation, such that there is a "traffic jam" of ribosomes on the mRNAs, increasing the number of ribosomes per mRNA while, at the same time, decreasing protein synthesis rate. In support of this hypothesis, myoblasts treated with a low dose of cycloheximide (a specific elongation inhibitor) show a similar shift in polysome size. A numerical model of protein synthesis further shows that elongation is more effective than initiation and termination in affecting protein synthesis and polysome size. We conclude that the non-weight-bearing-induced decrease in postural muscle protein synthesis rate is initially caused by slowing of nascent polypeptide chain elongation.