Plant cytokinesis is initiated in a transient membrane compartment, the cell plate, and completed by a process of maturation during which the cell plate becomes a cross wall. How the transition from juvenile to adult stages occurs is poorly understood. In this study, we monitor the Arabidopsis transport protein particle II (TRAPPII) and exocyst tethering complexes throughout cytokinesis. We show that their appearance is predominantly sequential, with brief overlap at the onset and end of cytokinesis. The TRAPPII complex is required for cell plate biogenesis, and the exocyst is required for cell plate maturation. The TRAPPII complex sorts plasma membrane proteins, including exocyst subunits, at the cell plate throughout cytokinesis. We show that the two tethering complexes physically interact and propose that their coordinated action may orchestrate not only plant but also animal cytokinesis.
- MeSH
- Arabidopsis cytology physiology MeSH
- Cytokinesis physiology MeSH
- Cytoplasmic Vesicles metabolism MeSH
- Exocytosis physiology MeSH
- Microtubules metabolism MeSH
- Models, Molecular MeSH
- Arabidopsis Proteins metabolism MeSH
- Growth Plate cytology metabolism MeSH
- Vesicular Transport Proteins metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
The exocyst is a complex of proteins mediating first contact (tethering) between secretory vesicles and the target membrane. Discovered in yeast as an effector of RAB and RHO small GTPases, it was also found to function in land plants. Plant cells and tissues rely on targeted exocytosis and this implies that the exocyst is involved in regulation of cell polarity and morphogenesis, including cytokinesis, plasma membrane protein recycling (including PINs, the auxin efflux carriers), cell wall biogenesis, fertilization, stress and biotic interactions including defence against pathogens. The dramatic expansion of the EXO70 subunit gene family, of which individual members are likely responsible for exocyst complex targeting, implies that there are specialized functions of different exocysts with different EXO70s. One of these functions comprises a role in autophagy-related Golgi independent membrane trafficking into the vacuole or apoplast. It is also possible, that some EXO70 paralogues have been recruited into exocyst independent functions. The exocyst has the potential to function as an important regulatory hub to coordinate endomembrane dynamics in plants.
- MeSH
- Arabidopsis cytology metabolism MeSH
- Models, Biological * MeSH
- Exocytosis * MeSH
- Golgi Apparatus metabolism MeSH
- Plant Cells metabolism MeSH
- Plant Proteins metabolism MeSH
- Secretory Pathway * MeSH
- Signal Transduction MeSH
- Protein Binding MeSH
- Vesicular Transport Proteins metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
Cytotoxicity and mutagenicity of trans,trans,trans-[PtCl2(CH3COO)2(NH3)(1-adamantylamine)] [trans-adamplatin(IV)] and its reduced analog trans-[PtCl2(NH3)(1-adamantylamine)] [trans-adamplatin(II)] were examined. In addition, the several factors underlying biological effects of these trans-platinum compounds using various biochemical methods were investigated. A notable feature of the growth inhibition studies was the remarkable circumvention of both acquired and intrinsic cisplatin resistance by the two lipophilic trans-compounds. Interestingly, trans-adamplatin(IV) was considerably less mutagenic than cisplatin. Consistent with the lipophilic character of trans-adamplatin complexes, their total accumulation in A2780 cells was considerably greater than that of cisplatin. The results also demonstrate that trans-adamplatin(II) exhibits DNA binding mode markedly different from that of ineffective transplatin. In addition, the reduced deactivation of trans-adamplatin(II) by glutathione seems to be an important determinant of the cytotoxic effects of the complexes tested in the present work. The factors associated with cytotoxic and mutagenic effects of trans-adamplatin complexes in tumor cell lines examined in the present work are likely to play a significant role in the overall antitumor activity of these complexes.
- MeSH
- DNA Adducts chemistry MeSH
- Amantadine analogs & derivatives pharmacology chemistry metabolism MeSH
- Circular Dichroism MeSH
- DNA chemistry MeSH
- Financing, Organized MeSH
- Hypoxanthine Phosphoribosyltransferase genetics drug effects MeSH
- Humans MeSH
- Mutagens pharmacology chemistry metabolism MeSH
- Cell Line, Tumor MeSH
- Organoplatinum Compounds chemistry metabolism pharmacology MeSH
- Antineoplastic Agents pharmacology chemistry metabolism MeSH
- Check Tag
- Humans MeSH
The synthesis of the title 2'-deoxyadenosine derivatives bearing bipyridine, phenanthroline or terpyridine ligands and their corresponding RuII-complexes in position 8 linked via acetylene or phenylene tethers was accomplished through cross-coupling reactions. The Suzuki-Miyaura reactions of boronic acids or the Sonogashira reactions of terminal acetylene derivatives of oligopyridine ligands were performed either on protected 8-bromoadenosines in organic solvents or, more efficiently, directly on unprotected nucleosides in aqueous acetonitrile or DMF. Direct cross-coupling reactions of unprotected nucleosides with RuII-complexes or the oligopyridine-boronic acids or -acetylenes gave the Ru-labelled nucleosides in one step in fair to good yields. This method was also proven to be applicable for direct Ru-labelling of dATP. Terpyridine-containing 2'-deoxyadenosine exerted significant antiviral and cytostatic effects.
- MeSH
- Antiviral Agents chemical synthesis chemistry toxicity MeSH
- Deoxyadenosines chemical synthesis chemistry toxicity MeSH
- X-Ray Diffraction MeSH
- Financing, Organized MeSH
- Hepacivirus drug effects MeSH
- Ligands MeSH
- Models, Molecular MeSH
- Molecular Structure MeSH
- Antineoplastic Agents chemical synthesis chemistry toxicity MeSH
- Pyridines chemistry MeSH
- Cross-Linking Reagents chemistry MeSH
- Ruthenium Compounds chemistry MeSH
In endolysosomal networks, two hetero-hexameric tethers called HOPS and CORVET are found widely throughout eukaryotes. The unicellular ciliate Tetrahymena thermophila possesses elaborate endolysosomal structures, but curiously both it and related protozoa lack the HOPS tether and several other trafficking proteins, while retaining the related CORVET complex. Here, we show that Tetrahymena encodes multiple paralogs of most CORVET subunits, which assemble into six distinct complexes. Each complex has a unique subunit composition and, significantly, shows unique localization, indicating participation in distinct pathways. One pair of complexes differ by a single subunit (Vps8), but have late endosomal versus recycling endosome locations. While Vps8 subunits are thus prime determinants for targeting and functional specificity, determinants exist on all subunits except Vps11. This unprecedented expansion and diversification of CORVET provides a potent example of tether flexibility, and illustrates how 'backfilling' following secondary losses of trafficking genes can provide a mechanism for evolution of new pathways.This article has an associated First Person interview with the first author of the paper.
2. vyd. 265 s. : obr., tab., grafy
- MeSH
- Child MeSH
- Adult MeSH
- Contrast Media MeSH
- Myelography methods MeSH
- Spinal Cord Diseases MeSH
- Neurosurgery MeSH
- Check Tag
- Child MeSH
- Adult MeSH
- NML Fields
- neurochirurgie
Intracellular trafficking of organelles, driven by kinesin-1 stepping along microtubules, underpins essential cellular processes. In absence of other proteins on the microtubule surface, kinesin-1 performs micron-long runs. Under crowding conditions, however, kinesin-1 motility is drastically impeded. It is thus unclear how kinesin-1 acts as an efficient transporter in intracellular environments. Here, we demonstrate that TRAK1 (Milton), an adaptor protein essential for mitochondrial trafficking, activates kinesin-1 and increases robustness of kinesin-1 stepping on crowded microtubule surfaces. Interaction with TRAK1 i) facilitates kinesin-1 navigation around obstacles, ii) increases the probability of kinesin-1 passing through cohesive islands of tau and iii) increases the run length of kinesin-1 in cell lysate. We explain the enhanced motility by the observed direct interaction of TRAK1 with microtubules, providing an additional anchor for the kinesin-1-TRAK1 complex. Furthermore, TRAK1 enables mitochondrial transport in vitro. We propose adaptor-mediated tethering as a mechanism regulating kinesin-1 motility in various cellular environments.
- MeSH
- Adaptor Proteins, Vesicular Transport genetics isolation & purification metabolism MeSH
- Microscopy, Fluorescence MeSH
- Kinesins genetics isolation & purification metabolism MeSH
- Luminescent Proteins genetics metabolism MeSH
- Microtubules metabolism MeSH
- Mitochondria metabolism MeSH
- Mice MeSH
- Cell Line, Tumor MeSH
- tau Proteins genetics metabolism MeSH
- Recombinant Proteins genetics metabolism MeSH
- Intrinsically Disordered Proteins genetics metabolism MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Modified nucleosides (dA(R)s and dC(R)s) bearing bipyridine or terpyridine ligands attached through an octadiyne linker were prepared by single-step aqueous-phase Sonogashira cross-coupling of 7-iodo-7-deaza-2'-deoxyadenosine and 5-iodo-2'-deoxycytidine with the corresponding bipyridine- or terpyridine-octadiynes and were triphosphorylated to the corresponding nucleoside triphosphates (dA(R)TPs and dC(R)TPs). The modified dN(R)TPs were successfully incorporated into the oligonucleotides by primer extension experiment (PEX) using different DNA polymerases and the PEX products were used for post-synthetic complexation with divalent metal cations. The complexation of these DNAs containing flexibly-tethered ligands was compared with the previously reported ones bearing rigid acetylene-linked ligands suggesting the possible formation of both inter- and intra-strand complexes with Ni(2+) or Fe(2+).
- MeSH
- Alkynes chemistry MeSH
- DNA chemistry MeSH
- Cations chemistry MeSH
- Ligands MeSH
- Molecular Structure MeSH
- Nucleosides chemical synthesis chemistry MeSH
- Organometallic Compounds chemical synthesis chemistry MeSH
- Polyphosphates chemical synthesis chemistry MeSH
- Transition Elements chemistry MeSH
- Pyridines chemistry MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The exocyst complex, an effector of Rho and Rab GTPases, is believed to function as an exocytotic vesicle tether at the plasma membrane before soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation. Exocyst subunits localize to secretory-active regions of the plasma membrane, exemplified by the outer domain of Arabidopsis root epidermal cells. Using variable-angle epifluorescence microscopy, we visualized the dynamics of exocyst subunits at this domain. The subunits colocalized in defined foci at the plasma membrane, distinct from endocytic sites. Exocyst foci were independent of cytoskeleton, although prolonged actin disruption led to changes in exocyst localization. Exocyst foci partially overlapped with vesicles visualized by VAMP721 v-SNARE, but the majority of the foci represent sites without vesicles, as indicated by electron microscopy and drug treatments, supporting the concept of the exocyst functioning as a dynamic particle. We observed a decrease of SEC6-green fluorescent protein foci in an exo70A1 exocyst mutant. Finally, we documented decreased VAMP721 trafficking to the plasma membrane in exo70A1 and exo84b mutants. Our data support the concept that the exocyst-complex subunits dynamically dock and undock at the plasma membrane to create sites primed for vesicle tethering.
- MeSH
- Arabidopsis genetics metabolism ultrastructure MeSH
- Cell Membrane metabolism ultrastructure MeSH
- Cytoplasm metabolism ultrastructure MeSH
- Cytoskeleton metabolism ultrastructure MeSH
- Plant Epidermis genetics metabolism ultrastructure MeSH
- Exocytosis MeSH
- Gene Expression MeSH
- Microscopy, Fluorescence MeSH
- Plant Roots genetics metabolism ultrastructure MeSH
- Arabidopsis Proteins genetics metabolism MeSH
- SNARE Proteins genetics metabolism MeSH
- rab GTP-Binding Proteins genetics metabolism MeSH
- Secretory Vesicles metabolism ultrastructure MeSH
- Protein Transport MeSH
- Protein Binding MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The mechanistic target of rapamycin complex 1 (mTORC1) pathway regulates cell growth and metabolism in response to many environmental cues, including nutrients. Amino acids signal to mTORC1 by modulating the guanine nucleotide loading states of the heterodimeric Rag GTPases, which bind and recruit mTORC1 to the lysosomal surface, its site of activation. The Rag GTPases are tethered to the lysosome by the Ragulator complex and regulated by the GATOR1, GATOR2, and KICSTOR multiprotein complexes that localize to the lysosomal surface through an unknown mechanism(s). Here, we show that mTORC1 is completely insensitive to amino acids in cells lacking the Rag GTPases or the Ragulator component p18. Moreover, not only are the Rag GTPases and Ragulator required for amino acids to regulate mTORC1, they are also essential for the lysosomal recruitment of the GATOR1, GATOR2, and KICSTOR complexes, which stably associate and traffic to the lysosome as the "GATOR" supercomplex. The nucleotide state of RagA/B controls the lysosomal association of GATOR, in a fashion competitively antagonized by the N terminus of the amino acid transporter SLC38A9. Targeting of Ragulator to the surface of mitochondria is sufficient to relocalize the Rags and GATOR to this organelle, but not to enable the nutrient-regulated recruitment of mTORC1 to mitochondria. Thus, our results reveal that the Rag-Ragulator complex is the central organizer of the physical architecture of the mTORC1 nutrient-sensing pathway and underscore that mTORC1 activation requires signal transduction on the lysosomal surface.
- MeSH
- Adaptor Proteins, Signal Transducing metabolism MeSH
- Amino Acids * metabolism MeSH
- HEK293 Cells MeSH
- Humans MeSH
- Lysosomes * metabolism MeSH
- Monomeric GTP-Binding Proteins * metabolism MeSH
- Mechanistic Target of Rapamycin Complex 1 * metabolism MeSH
- Mice MeSH
- Signal Transduction * MeSH
- Nutrients * metabolism MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
