Assembly and disassembly dynamics of nonmuscle myosin II control endosomal fission.

Endocytic vesicular trafficking requires merging of two lipid bilayers, but how the two lipid bilayers can come close together during fusion and fission in endocytic trafficking is not well explored. Here, we establish that knocking down nonmuscle myosin IIs (NM IIs) by small interfering RNA (siRNA) or inhibition of their activities by (-) blebbistatin causes the formation of a ring-like assembly of early endosomes (raEE). Inhibition of NM II assembly by an inhibitor of regulatory light-chain (RLC) kinase results in the formation of raEE, whereas inhibition of NM II disassembly by inhibitors of heavy chain kinases, protein kinase C (PKC) and casein kinase 2 (CK2), causes the dispersion of early endosomes. The raEEs retain EEA1, Rab7, and LAMP2 markers. Overexpression of an assembly incompetent form, RLC-AA, and disassembly incompetent form, NMHCIIB-S6A or NMHCIIA-1916A, induces such defects, respectively. Altogether, these data support that NM II assembly and disassembly dynamics participate in endocytic trafficking by regulating fission to maintain the size of early endosomes.

Direct Correlation between the Secondary Structure of an Amphiphilic Polymer and Its Prominent Antiviral Activity.

An amphiphilic segmented polyurethane (F-PU-S), with pendant sulfate groups and a flexible hydrocarbon backbone, exhibits intrachain H-bonding-reinforced folding and hierarchical assembly, producing an anionic polymersome with efficient display of sulfate groups at the surface. It shows an excellent antiviral activity against Sendai virus (SV) by inhibiting its entry to the cells. Mechanistic investigation suggests fusion of the SV and the polymersome to produce larger particles in which neither the folded structure of the polymer nor the fusogenic property of the SV exists anymore. In sharp contrast, a structurally similar polymer R-PU-S, in which the chain folding pathway is blocked by replacing the flexible C6 chain with a rigid cyclohexane chain in the backbone, cannot form a similar polymersome structure and hence does not exhibit any antiviral activity. On the other hand, the third polymer (F-PU-C), which is similar to F-PU-S except for the pendant anionic groups (carboxylate instead of sulfate), also fails to exhibit any antiviral activity against SV, confirming the essential role of the chain folding as well as the pendant sulfate groups for the fusion-induced antiviral activity of F-PU-S, which provides an important structural guideline for developing new antiviral polymers.

MRX8, the conserved mitochondrial YihA GTPase family member, is required for de novo Cox1 synthesis at suboptimal temperatures in Saccharomyces cerevisiae.

The synthesis of Cox1, the conserved catalytic-core subunit of Complex IV, a multisubunit machinery of the mitochondrial oxidative phosphorylation (OXPHOS) system under environmental stress, has not been sufficiently addressed. In this study, we show that the putative YihA superfamily GTPase, Mrx8, is a bona fide mitochondrial protein required for Cox1 translation initiation and elongation during suboptimal growth condition at 16°C. Mrx8 was found in a complex with mitochondrial ribosomes, consistent with a role in protein synthesis. Cells expressing mutant Mrx8 predicted to be defective in guanine nucleotide binding and hydrolysis were compromised for robust cellular respiration. We show that the requirement of Pet309 and Mss51 for cellular respiration is not bypassed by overexpression of Mrx8 and vice versa. Consistently the ribosomal association of Mss51 is independent of Mrx8. Significantly, we find that GTPBP8, the human orthologue, complements the loss of cellular respiration in Δmrx8 cells and GTPBP8 localizes to the mitochondria in mammalian cells. This strongly suggests a universal role of the MRX8 family of proteins in regulating mitochondrial function.

Shape-Dependent Cellular Uptake of Nanostructures Produced from Supramolecular Structure-Directing Unit-Appended Hydrophilic Polymers.

Cellular uptake is an important event in drug delivery and other biomedical applications. Amphiphilic polymers produce aggregates of different size and shape depending on the intrinsic structural differences and the packing parameter. Although they have been explored for various biomedical applications with immense interest, the relationship between the shape of the aggregate and cellular uptake has been studied only in limited examples. This work reports two polymers (P1 and P2), both of which contain a hydrophobic supramolecular structure-directing unit (SSDU) at the chain-end of a fluorescence dye-labeled hydrophilic polymer. Depending on the difference in the structure of the single H-bonding functional group (hydrazide or amide) of the SSDU, P1 and P2 produce polymersomes (NS1) and spherical micelles (NS2), respectively. An aged solution of P2 produces cylindrical micelles (NS3). Confocal microscopy studies reveal that the uptake of these nanostructures in HeLa cells greatly depends on the shape of the aggregate. Spherical NS1 and NS2 show appreciable uptake at 1 or 4 h of incubation, whereas NS3 shows negligible uptake. Temperature-dependent cellular uptake studies reveal an energy-dependent endocytosis pathway. Kinetic studies show gradual increase in the cellular uptake with time, and at 24 h the relative uptake ratio (NS1:NS2:NS3) is 1.0:0.2:<0.1, implying the polymersome morphology (NS1) is most efficient for cellular uptake compared to the spherical or cylindrical micelles. The same trend was also noticed for MDA-MB 231 cells. Confocal microscopy studies further reveal cellular internalization and intracellular location of NS1, which showed maximum cellular uptake. As the intrinsic difference in the chemical structure of the two polymers is negligible, the observed difference can be explicitly assigned to their difference in shape.