Mechanisms of Ca2+/calmodulin-dependent kinase II activation in single dendritic spines.

CaMKIIα plays an essential role in decoding Ca2+ signaling in spines by acting as a leaky Ca2+ integrator with the time constant of several seconds. However, the mechanism by which CaMKIIα integrates Ca2+ signals remains elusive. Here, we imaged CaMKIIα-CaM association in single dendritic spines using a new FRET sensor and two-photon fluorescence lifetime imaging. In response to a glutamate uncaging pulse, CaMKIIα-CaM association increases in ~0.1 s and decays over ~3 s. During repetitive glutamate uncaging, which induces spine structural plasticity, CaMKIIα-CaM association did not show further increase but sustained at a constant level. Since CaMKIIα activity integrates Ca2+ signals over ~10 s under this condition, the integration of Ca2+ signal by CaMKIIα during spine structural plasticity is largely due to Ca2+/CaM-independent, autonomous activity. Based on these results, we propose a simple kinetic model of CaMKIIα activation in dendritic spines.

CaMKII Autophosphorylation Is Necessary for Optimal Integration of Ca2+ Signals during LTP Induction, but Not Maintenance.

CaMKII plays a critical role in decoding calcium (Ca2+) signals to initiate long-lasting synaptic plasticity. However, the properties of CaMKII that mediate Ca2+ signals in spines remain elusive. Here, we measured CaMKII activity in spines using fast-framing two-photon fluorescence lifetime imaging. Following each pulse during repetitive Ca2+ elevations, CaMKII activity increased in a stepwise manner. Thr286 phosphorylation slows the decay of CaMKII and thus lowers the frequency required to induce spine plasticity by several fold. In the absence of Thr286 phosphorylation, increasing the stimulation frequency results in high peak mutant CaMKIIT286A activity that is sufficient for inducing plasticity. Our findings demonstrate that Thr286 phosphorylation plays an important role in induction of LTP by integrating Ca2+ signals, and it greatly promotes, but is dispensable for, the activation of CaMKII and LTP.

Profiling the Monascus pilosus proteome during nitrogen limitation.

Monascus species have the unique ability to economically produce many secondary metabolites. However, the influence of nitrogen limitation on Monascus secondary metabolite production and metabolic performance remains unclear. Varying the carbon/nitrogen (C/N) ratios in the range from 20 to 60 in cultivation of Monascus pilosus by glucose nitrate medium, our resulting data showed that red pigment production was significantly suppressed and more sensitive to nitrogen limitation than cellular biomass growth at a C/N ratio of 60. Using a comparative proteomic approach, combining two-dimensional gel electrophoresis, matrix-assisted laser desorption ionization time-of-flight/time-of-flight liquid chromatography-mass spectrometry, and tandem mass spectrometry, proteins with modified expression in the nitrogen-limited (C/N ratio 60) Monascus filamentous cells were identified. The results revealed that the deregulated proteins identified were involved in amino acid biosynthesis, protein translation, antioxidant-related enzymes, glycolysis, and transcriptional regulation. The results suggested that, under nitrogen limitation-induced suppression of protein translation and of expression of the related energy-generating enzymes, nitrogen limitation induced a switch of metabolic flux from glycolysis to the tricarboxylic acid (TCA) cycle for maintaining cellular energy homeostasis, resulting in repression of the metabolic shift of the polyketide biosynthesis pathway for red pigment production.

Proteome response of Monascus pilosus during rice starch limitation with suppression of monascorubramine production.

For centuries, red mold rice has been made by fermentation of cooked rice with Monascus species. However, the influence of different carbon sources on the metabolism of Monascus cells remains unclear. We compared the proteome response of Monascus pilosus to replacement of the rice starch fraction with lactose during cultivation, using two-dimensional gel electrophoresis, matrix-assisted laser desorption-ionization time-of-flight/time-of-flight mass spectrometry, and tandem mass spectrometry to identify the proteins expressed. The results showed that cell growth and monascorubramine pigment formation of M. pilosus were sensitive to rice starch limitation during cultivation. A total of 12 proteins were identified with statistically altered expression in the cells cultivated with lactose. These deregulated proteins were involved in glycolysis, TCA cycle, energy generation, protein folding, and peptide biosynthesis. The possible metabolic flux shifts induced by rice starch limitation were discussed. The results suggested that the suppression of monascorubramine formation could be related to the necessary energy-requiring adaptations executed in response to carbon depletion during rice starch limitation.

Metabolic protein patterns and monascorubrin production revealed through proteomic approach for Monascus pilosus treated with cycloheximide.

Monascus species have the unique ability to economically produce many secondary metabolites. However, most metabolic regulation processes in the production of secondary metabolites in Monascus remain unclear. We found that the translational inhibitor cycloheximide induced different expression patterns between the monascorubrin pigment production and the growth in Monascus pilosus. Here, we used the proteomic approach of two-dimensional gel electrophoresis, matrix-assisted laser desorption ionization time-of-flight/time-of-flight liquid chromatography-mass spectrometry (MALDI-TOF/TOF LC-MS), and tandem mass spectrometry (MS/MS) to identify the intracellular and mitochondrial proteins of M. pilosus between the cycloheximide treatment and the control. These results revealed that the cycloheximide-induced down-regulated proteins were involved in transcriptional regulation, peptide synthesis, and other metabolic processes, such as methylation of secondary metabolites. In contrast, the energy-related proteins, such as the transcriptional regulator rosAr and 1,4-alpha-glucan branching enzyme, were up-regulated as compared to the control.