Bin-Amphiphysin-Rvs161/167 (BAR) domains mold lipid bilayer membranes into tubules, by forming a spiral polymer on the membrane. Most BAR domains are thought to be involved in forming membrane invaginations through their concave membrane binding surfaces, whereas some members have convex membrane binding surfaces, and thereby mold membranes into protrusions. The BAR domains with a convex surface form a subtype called the inverse BAR (I-BAR) domain or IRSp53-MIM-homology domain (IMD). Although the mammalian I-BAR domains have been studied, those from other organisms remain elusive. Here, we found putative I-BAR domains in Fungi and animal-like unicellular organisms. The fungal protein containing the putative I-BAR-domain is known as Ivy1p in yeast, and is reportedly localized in the vacuole. The phylogenetic analysis of the I-BAR domains revealed that the fungal I-BAR-domain containing proteins comprise a distinct group from those containing IRSp53 or MIM. Importantly, Ivy1p formed a polymer with a diameter of approximately 20 nm in vitro, without a lipid membrane. The filaments were formed at neutral pH, but disassembled when pH was reverted to basic. Moreover, Ivy1p and the I-BAR domain expressed in mammalian HeLa cells was localized at a vacuole-like structure as filaments as revealed by super-resolved microscopy. These data indicate the pH-sensitive polymer forming ability and the functional conservation of Ivy1p in eukaryotic cells.
The Saccharomyces cerevisiae autophagy-initiation complex, Atg1 kinase complex, consists of Atg1, Atg13, Atg17, Atg29, and Atg31, while the corresponding complex in most other eukaryotes, including mammals, is composed of ULK1 (or ULK2), Atg13, FIP200 (also known as RB1CC1), and Atg101. ULKs are homologs of Atg1, and FIP200 is partially homologous to Atg17. However, the sequence of Atg101 is not similar to that of Atg29 or Atg31. Although Atg101 is essential for autophagy and widely conserved in eukaryotes, its precise function and structure have remained largely unknown. Now, structural and cell biological analysis of Atg101 together with its binding partner Atg13 reveal that Atg101 is required for stabilization of “uncapped” Atg13 in most eukaryotes and also for recruitment of downstream Atg proteins through the newly identified WF motif. By contrast, S. cerevisiae has stable “capped” Atg13, which does not require Atg101 for its stabilization. Possible roles for other binding partners such as Atg29, Atg31, and Atg28 in different organisms are also discussed.
Brain-derived neurotrophic factor (BDNF) is a neurotrophic factor critical for synaptic plasticity, neuronal development and neurite extension. BDNF mRNA is transported to dendrites and axons, where it is expressed locally. We previously reported that dendritic targeting elements in the BDNF 3’ UTR are necessary for dendritic transport and interact with cytoplasmic polyadenylation element binding protein 1. Here, we demonstrated that the short 3’ UTR directs local translation of BDNF and that locally synthesized BDNF exists in a novel compartment that does not co-localize with markers of endosomes, endoplasmic reticulum, Golgi or the trans-Golgi network. Further, locally synthesized BDNF vesicles co-localized with Bicaudal-D2 (BicD2), a member of dynein motor complex proteins. Silencing BicD2 significantly reduced BDNF local synthesis in dendrites. These new findings may underlie the mechanism of local neuronal response to environmental stimuli.
Fish epidermal keratocytes maintain an overall fan shape during their crawling migration. The shape-determination mechanism has been described theoretically and experimentally on the basis of graded radial extension of the leading edge, but the relationship between shape and traction forces has not been clarified. Migrating keratocytes can be divided into fragments by treatment with the protein kinase inhibitor staurosporine. Fragments containing a nucleus and cytoplasm behave as mini-keratocytes and maintain the same fan shape as the original cells. We measured the shape of the leading edge, together with the areas of the ventral region and traction forces, of keratocytes and mini-keratocytes. The shapes of keratocytes and mini-keratocytes were similar. Mini-keratocytes exerted traction forces at the rear left and right ends, just like keratocytes. The magnitude of the traction forces was proportional to the area of the keratocytes and mini-keratocytes. The myosin II ATPase inhibitor blebbistatin decreased the forces at the rear left and right ends of the keratocytes and expanded their shape laterally. These results suggest that keratocyte shape depends on the distribution of the traction forces, and that the magnitude of the traction forces depends on the area of the cells.
Tropomyosin (TPM) localizes along F-actin and, together with troponin T (TnT) and other components, controls calcium-sensitive muscle contraction. The role of the TPM isoform (TPM4α) that is expressed in embryonic and adult cardiac muscle cells in chicken is poorly understood. To analyze the function of TPM4α in myofibrils, the effects of TPM4α-suppression were examined in embryonic cardiomyocytes by small interference RNA transfection. Localization of myofibril proteins such as TPM, actin, TnT, α-actinin, myosin and connectin was examined by immunofluorescence microscopy on day 5 when almost complete TPM4α-suppression occurred in culture. A unique large structure was detected, consisting of an actin aggregate bulging from the actin bundle, and many curved filaments projecting from the aggregate. TPM, TnT and actin were detected on the large structure, but myosin, connectin, α-actinin and obvious myofibril striations were undetectable. It is possible that TPM4α-suppressed actin filaments are sorted and excluded at the place of the large structure. This suggests that TPM4α-suppression significantly affects actin filament, and that TPM4α plays an important role in constructing and maintaining sarcomeres and myofibrils in cardiac muscle.
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