The late endosomal p14–MP1 (LAMTOR2/3) complex regulates focal adhesion dynamics during cell migration

Cell migration is mediated by the dynamic remodeling of focal adhesions (FAs). Recently, an important role of endosomal signaling in regulation of cell migration was recognized. Here, we show an essential function for late endosomes carrying the p14–MP1 (LAMTOR2/3) complex in FA dynamics. p14–MP1-positive endosomes move to the cell periphery along microtubules (MTs) in a kinesin1- and Arl8b-dependent manner. There they specifically target FAs to regulate FA turnover, which is required for cell migration. Using genetically modified fibroblasts from p14-deficient mice and Arl8b-depleted cells, we demonstrate that MT plus end–directed traffic of p14–MP1-positive endosomes triggered IQGAP1 disassociation from FAs. The release of IQGAP was required for FA dynamics. Taken together, our results suggest that late endosomes contribute to the regulation of cell migration by transporting the p14–MP1 scaffold complex to the vicinity of FAs.     

Identification and Partial Characterization of a Dynamin-Like Protein,EhDLP1, from the Protist Parasite Entamoeba histolytica

The dynamin superfamily of proteins includes a large repertoire of evolutionarily conserved GTPases that interact with different subcellular organelle membranes in eukaryotes. Dynamins are thought to participate in a number of cellular processes involving membrane remodeling and scission. Dynamin-like proteins (DLPs) form a subfamily of this vast class and play important roles in cellular processes, such as mitochondrial fission, cytokinesis, and endocytosis. In the present study, a gene encoding a dynamin-like protein (EhDLP1) from the protist parasite Entamoeba histolytica was identified and the protein was partially characterized using a combination of in silico, biochemical, and imaging methods. The protein was capable of GTP binding and hydrolysis, lipid binding, and oligomerization. Immunofluorescence studies showed the protein to be associated with the nuclear membrane. A mutant of EhDLP1 lacking GTP binding and hydrolyzing activities did not associate with the nuclear membrane. The results suggest a nucleus-associated function for EhDLP1.Dynamins are a vast family of GTPases implicated in myriad processes, some of which lead to alteration of membrane structure (22). Classical dynamins, such as mammalian dynamins 1 to 3 (5) and the shibire protein from Drosophila melanogaster (29), are required mainly for scission of vesicles, acting as mechanoenzymes or molecular switches (12). In addition, several dynamin-like proteins (DLPs) have been identified in different organisms ranging from yeast to mammals. DLPs play a key role in the division of organelles such as chloroplasts, mitochondria, and peroxisomes (15, 22). For example, Candida albicans Vps1 has been shown to be associated with virulence-related phenotypes like filamentation and biofilm formation (2). DLPs have also been identified in protists. Downregulation or ablation of the gene products in protists by RNA interference or other methods has helped to decipher the multiple functions carried out by these proteins. These include mitochondrial division and endocytosis in Trypanosoma brucei (6, 20), cytokinesis in Dictyostelium discoideum (31), phagocytosis in Paramecium species (30), endocytic transport in Giardia lamblia (11), and biogenesis of secretory vesicles in Toxoplasma gondii (4). Apart from cellular membranes, some DLPs may also associate with nuclear membranes. Recently, a study on Tetrahymena thermophila reported the requirement of Drp6 for macronuclear development (23). The human DLP MxB has been shown previously to localize to the cytoplasmic face of the nuclear envelope and is involved in regulation of nuclear import (14). Dynamins and DLPs share a minimal domain architecture which includes an N-terminal GTPase domain, a middle domain, and a GTPase effector domain (GED). The GED is involved in enzyme oligomerization and the regulation of the GTPase activity. The GTPase domain contains a well-conserved GTP binding motif required for guanine-nucleotide binding and hydrolysis (22). DLPs lack a pleckstrin homology (PH) domain and a proline-rich domain (PRD), normally associated with protein-lipid and protein-protein interaction.The endocytic, secretory, and adhesion pathways of the parasite Entamoeba histolytica play crucial roles in nutrient uptake, host cell destruction, and the endocytosis of gut resident bacteria, erythrocytes, and cell debris (21). The trophozoites of E. histolytica are known to have robust endocytic capabilities, turning over approximately a third of their cellular volume every hour (1, 19). The presence of a classical receptor-mediated pathway has not yet been clearly demonstrated, though some of the molecules involved in this pathway, such as clathrin, have been identified in E. histolytica (28). Typical eukaryotic cytoplasmic organelles have not been observed in this organism. However, the functional equivalents of a Golgi network and an endoplasmic reticulum are reported to be present (3, 26). Entamoeba also contains a genomeless variant of mitochondria, termed mitosomes (17). The division or biogenesis of these organelles during cell division is not understood. Nuclear division in E. histolytica occurs without nuclear membrane dissolution and reassembly. Since dynamins and DLPs are known to be involved in endocytosis and organelle division, it is likely that these proteins may be performing similar functions in this organism. Although the E. histolytica genome encodes putative dynamins and DLPs, none of these have been characterized. In order to understand the roles of these molecules in amebic biology, we have initiated studies to characterize these proteins from E. histolytica. Here, we report the basic characterization of E. histolytica dynamin-like protein 1 (EhDLP1).     

Nuclear condensation and free radical scavenging: a dual mechanism of bisbenzimidazoles to modulate radiation damage to DNA

The complexing of histones with DNA and the resulting condensation of chromatin protects mammalian cell, from radiation-induced strand breakage. In the present study, benzimidazoles DMA and TBZ showed marked radioprotection through drug-induced compaction of chromatin and direct quenching of free radicals generated by radiation. The mammalian cells were incubated with 100 μM concentration of DMA and TBZ and irradiated at 5 Gy; both the ligands showed nuclei condensation suggesting a probable mechanism to protect DNA from radiation damage. The bisubstituted analogs of Hoechst 33342 are found to be better free radical scavengers and protect DNA against radiation-induced damage at a lower concentration than the parent molecule. Both the ligands also quenched free radicals in isolated free radical system suggesting their dual mode of action against radiation-induced damage to DNA. Molecules binding to the chromatin alter gene expression, whereas in this study both the ligands have not shown any profound effect on the nucleosome assembly and gene expression in vitro and in vivo. Both ligands afford a 2-fold protection by altering DNA structure as well as through direct free radical quenching in bulk solution in comparison to the parent ligand, which acts only through quenching of free radicals. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Community structure of the Mota Magra forest block (Udaipur)

Summary The community structure of the Mota-Magra forest block (24°35N L and 75 49E L), 14 km east of Udaipur town is described. The area supports forest of mixed dry deciduous nature in which Boswellia serrata, Lannea coromandelica and various species of Acacia are dominant tree species. The protected stand has been found to be richer in respect of tree density and basal cover. In the protected stand the basal cover of tree species has been found to increase from hill base to hill top. The northern and eastern aspects were found to be richer than the southern and western aspects respectively. The possible causes responsible for these differences have been discussed. Observation on the maturity index of the community at each site, constance class and A/F ratio of the perennial species have been presented.Nomenclature follows that in present use at the Herbarium of the Botanical Survey of India, Dehradun.Our sincere thanks are due to Shri A. L. Sankhla, I.F.S., Divisional Forest Officer, Udaipur for the permission to work in the Mota Magra Forest Block. Thanks are also due to Prof. Y. D. Tiagi, Head, Botany Department S.B.S.H., University of Udaipur, Udaipur, for the laboratory facilities.