Dynamic assembly of the mRNA m6A methyltransferase complex is regulated by METTL3 phase separation

m⁶A methylation is the most abundant and reversible chemical modification on mRNA with approximately one-fourth of eukaryotic mRNAs harboring at least one m⁶A-modified base. The recruitment of the mRNA m⁶A methyltransferase writer complex to phase-separated nuclear speckles is likely to be crucial in its regulation; however, control over the activity of the complex remains unclear. Supported by our observation that a core catalytic subunit of the methyltransferase complex, METTL3, is endogenously colocalized within nuclear speckles as well as in noncolocalized puncta, we tracked the components of the complex with a Cry2-METTL3 fusion construct to disentangle key domains and interactions necessary for the phase separation of METTL3. METTL3 is capable of self-interaction and likely provides the multivalency to drive condensation. Condensates in cells necessarily contain myriad components, each with partition coefficients that establish an entropic barrier that can regulate entry into the condensate. In this regard, we found that, in contrast to the constitutive binding of METTL14 to METTL3 in both the diffuse and the dense phase, WTAP only interacts with METTL3 in dense phase and thereby distinguishes METTL3/METTL14 single complexes in the dilute phase from METTL3/METTL14 multicomponent condensates. Finally, control over METTL3/METTL14 condensation is determined by its small molecule cofactor, S-adenosylmethionine (SAM), which regulates conformations of two gate loops, and some cancer-associated mutations near gate loops can impair METTL3 condensation. Therefore, the link between SAM binding and the control of writer complex phase state suggests that the regulation of its phase state is a potentially critical facet of its functional regulation.

Approximately one-fourth of eukaryotic mRNAs harbor at least one m6A-modified base, but how is this regulated? This study shows that cells can use liquid-liquid phase separation to regulate dynamic assembly of the mRNA m6A methyltransferase complex (METTL3/METTL14/WTAP), with stoichiometries that depend on condensate partitioning in a substrate binding-dependent manner.     

A comparison of the structure and properties of normal human transferrin and a genetic variant of human transferrin

1. A rare genetic variant of human serum transferrin (TfBSHAW) is reported. 2. The variant and normal transferrins have been purified. 3. The two proteins have been shown to be identical with respect to their molecular weights, heat stability, iron uptake and absorbance spectra. 4. The amino acid substitution is thought to be isoleucine replaced by asparagine at either position 378 or position 381. 5. The ferric iron bound to the C-site of TfBSHAW is unstable in the presence of protons or 6 M urea.     

Preferential ATP-binding cassette transporter A1-mediated cholesterol efflux from late endosomes/lysosomes

Recently, ATP-binding cassette transporter A1 (ABCA1), the defective molecule in Tangier disease, has been shown to stimulate phospholipid and cholesterol efflux to apolipoprotein A-I (apoA-I); however, little is known concerning the cellular cholesterol pools that act as the source of cholesterol for ABCA1-mediated efflux. We observed a higher level of isotopic and mass cholesterol efflux from mouse peritoneal macrophages labeled with [(3)H]cholesterol/acetyl low density lipoprotein (where cholesterol accumulates in late endosomes and lysosomes) compared with cells labeled with [(3)H]cholesterol with 10% fetal bovine serum, suggesting that late endosomes/lysosomes act as a preferential source of cholesterol for ABCA1-mediated efflux. Consistent with this idea, macrophages from Niemann-Pick C1 mice that have an inability to exit cholesterol from late endosomes/lysosomes showed a profound defect in cholesterol efflux to apoA-I. In contrast, phospholipid efflux to apoA-I was normal in Niemann-Pick C1 macrophages, as was cholesterol efflux following plasma membrane cholesterol labeling. These results suggest that cholesterol deposited in late endosomes/lysosomes preferentially acts as a source of cholesterol for ABCA1-mediated cholesterol efflux.     

Volunteers assess marine biodiversity

Much less is known about marine biodiversity than that of terrestrial and freshwater environments. There is surprisingly little information about even the most common of organisms that live on the seashore. Science has limited resources to study them and volunteers can therefore make significant contributions. This article considers the value of a project in which volunteers are mapping the distribution and abundance of littoral animals and plants of the Northumberland coast.     

Suppression of C8161 melanoma metastatic ability by chromosome 6 induces differentiation-associated tyrosinase and decreases proliferation on adhesion-restrictive substrates mediated by overexpression of p21WAF1 and down-regulation of bcl-2 and cyclin D3

Metastatic tumors grow under conditions that restrict proliferation of non-metastatic, more differentiated cells. To investigate this prediction, we developed a simple adhesion-restrictive assay which allows proliferation of human metastatic C8161 melanoma, but prevents growth of neo 6.3/C8161 cells in which metastasis is suppressed by introduction of neo-tagged chromosome 6. We show that tyrosinase, a key enzyme in melanocytic cell differentiation, and expression of chromosome 6-encoded cell cycle modulators like p21WAF1 and cyclin D3 is selectively increased in C8161 tumors in which metastasisis is suppressed by chromosome 6. In the latter cells, growth arrest evidenced only under adhesion-restrictive conditions correlated with down-regulation of cyclin D3 and anti-apoptotic bcl-2. No comparable growth arrest or down-regulation was detected under comparable conditions in metastatic cells, which showed activation of invasion-associated MMP-9 92 kDa gelatinase B. Our data suggests that the metastasis-suppressing effects of chromosome 6 involving increased differentiation-associated tyrosinase and growth arrest on adhesion-restrictive substrates; are partly mediated by modulation of growth regulators, like p21WAF1 and cyclin D3.     

Spatial control of actin polymerization during neutrophil chemotaxis

Neutrophils respond to chemotactic stimuli by increasing the nucleation and polymerization of actin filaments, but the location and regulation of these processes are not well understood. Here, using a permeabilized-cell assay, we show that chemotactic stimuli cause neutrophils to organize many discrete sites of actin polymerization, the distribution of which is biased by external chemotactic gradients. Furthermore, the Arp2/3 complex, which can nucleate actin polymerization, dynamically redistributes to the region of living neutrophils that receives maximal chemotactic stimulation, and the least-extractable pool of the Arp2/3 complex co-localizes with sites of actin polymerization. Our observations indicate that chemoattractant-stimulated neutrophils may establish discrete foci of actin polymerization that are similar to those generated at the posterior surface of the intracellular bacterium Listeria monocytogenes. We propose that asymmetrical establishment and/or maintenance of sites of actin polymerization produces directional migration of neutrophils in response to chemotactic gradients.     

New synaptic bouton formation is disrupted by misregulation of microtubule stability in aPKC mutants

The Baz/Par-3-Par-6-aPKC complex is an evolutionarily conserved cassette critical for the development of polarity in epithelial cells, neuroblasts, and oocytes. aPKC is also implicated in long-term synaptic plasticity in mammals and the persistence of memory in flies, suggesting a synaptic function for this cassette. Here we show that at Drosophila glutamatergic synapses, aPKC controls the formation and structure of synapses by regulating microtubule (MT) dynamics. At the presynapse, aPKC regulates the stability of MTs by promoting the association of the MAP1Brelated protein Futsch to MTs. At the postsynapse, aPKC regulates the synaptic cytoskeleton by controlling the extent of Actin-rich and MT-rich areas. In addition, we show that Baz and Par-6 are also expressed at synapses and that their synaptic localization depends on aPKC activity. Our findings establish a novel role for this complex during synapse development and provide a cellular context for understanding the role of aPKC in synaptic plasticity and memory.