Dynamics of Multiple Trafficking Behaviors of Individual Synaptic Vesicles Revealed by Quantum-Dot Based Presynaptic Probe

Although quantum dots (QDs) have provided invaluable information regarding the diffusive behaviors of postsynaptic receptors, their application in presynaptic terminals has been rather limited. In addition, the diffraction-limited nature of the presynaptic bouton has hampered detailed analyses of the behaviors of synaptic vesicles (SVs) at synapses. Here, we created a quantum-dot based presynaptic probe and characterized the dynamic behaviors of individual SVs. As previously reported, the SVs exhibited multiple exchanges between neighboring boutons. Actin disruption induced a dramatic decrease in the diffusive behaviors of SVs at synapses while microtubule disruption only reduced extrasynaptic mobility. Glycine-induced synaptic potentiation produced significant increases in synaptic and inter-boutonal trafficking of SVs, which were NMDA receptor- and actin-dependent while NMDA-induced synaptic depression decreased the mobility of the SVs at synapses. Together, our results show that sPH-AP-QD revealed previously unobserved trafficking properties of SVs around synapses, and the dynamic modulation of SV mobility could regulate presynaptic efficacy during synaptic activity

MEF2 transcription factors are key regulators of sprouting angiogenesis

Angiogenesis, the fundamental process by which new blood vessels form from existing ones, depends on precise spatial and temporal gene expression within specific compartments of the endothelium. However, the molecular links between proangiogenic signals and downstream gene expression remain unclear. During sprouting angiogenesis, the specification of endothelial cells into the tip cells that lead new blood vessel sprouts is coordinated by vascular endothelial growth factor A (VEGFA) and Delta-like ligand 4 (Dll4)/Notch signaling and requires high levels of Notch ligand DLL4. Here, we identify MEF2 transcription factors as crucial regulators of sprouting angiogenesis directly downstream from VEGFA. Through the characterization of a Dll4 enhancer directing expression to endothelial cells at the angiogenic front, we found that MEF2 factors directly transcriptionally activate the expression of Dll4 and many other key genes up-regulated during sprouting angiogenesis in both physiological and tumor vascularization. Unlike ETS-mediated regulation, MEF2-binding motifs are not ubiquitous to all endothelial gene enhancers and promoters but are instead overrepresented around genes associated with sprouting angiogenesis. MEF2 target gene activation is directly linked to VEGFA-induced release of repressive histone deacetylases and concurrent recruitment of the histone acetyltransferase EP300 to MEF2 target gene regulatory elements, thus establishing MEF2 factors as the transcriptional effectors of VEGFA signaling during angiogenesis

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Phosphorylation of Ser312 contributes to tumor suppression by p53 in vivo.

The tumor suppressor p53 is a master sensor of stress, and posttranslational modifications are key in controlling its stability and transcriptional activities. p53 can be phosphorylated on at least 23 Ser/Thr residues, the majority of which are phosphorylated by stress-related kinases. An exception is Ser315 in human p53 (Ser312 in mouse), which is predominantly phosphorylated by cell cycle-related kinases. To understand the biological importance of Ser312 phosphorylation in vivo, we generated p53Ser312Ala knock-in mice. We show here that, although Ser312 is not essential for mouse life span under normal physiological conditions, Ser312Ala mutation dampens p53's activity during embryonic development. This is evident from its partial rescue of embryonic lethality caused by Mdm4 deletion. In agreement with the notion that Ser312 mutation weakens p53 function, Ser312Ala mice are also more susceptible to tumorigenesis following a sublethal ionizing radiation dose. Importantly, in the cohort studied, Ser312 mutation predisposes mice to develop thymic lymphomas and liver tumors, partly due to p53Ser312Ala's inability to fully induce a set of p53 target genes including p21 and cyclin G1. Thus, we demonstrate that phosphorylation of Ser312 is required for p53 to function fully as a tumor suppressor in vivo