29 research outputs found
Receptor Sorting within Endosomal Trafficking Pathway Is Facilitated by Dynamic Actin Filaments
Early endosomes (EEs) are known to be a sorting station for internalized
molecules destined for degradation, recycling, or other intracellular
organelles. Segregation is an essential step in such sorting, but the molecular
mechanism of this process remains to be elucidated. Here, we show that actin is
required for efficient recycling and endosomal maturation by producing a motile
force. Perturbation of actin dynamics by drugs induced a few enlarged EEs
containing several degradative vacuoles and also interfered with their
transporting ability. Actin repolymerization induced by washout of the drug
caused the vacuoles to dissociate and individually translocate toward the
perinuclear region. We further elucidated that cortactin, an actin-nucleating
factor, was required for transporting contents from within EEs. Actin filaments
regulated by cortactin may provide a motile force for efficient sorting within
early endosomes. These data suggest that actin filaments coordinate with
microtubules to mediate segregation in EEs
Crystal structure of nucleotide-free dynamin
Dynamin is a mechanochemical GTPase that oligomerizes around the neck of clathrin-coated pits and catalyses vesicle scission in a GTP-hydrolysis-dependent manner. The molecular details of oligomerization and the mechanism of the mechanochemical coupling are currently unknown. Here we present the crystal structure of human dynamin 1 in the nucleotide-free state with a four-domain architecture comprising the GTPase domain, the bundle signalling element, the stalk and the pleckstrin homology domain. Dynamin 1 oligomerized in the crystals via the stalks, which assemble in a criss-cross fashion. The stalks further interact via conserved surfaces with the pleckstrin homology domain and the bundle signalling element of the neighbouring dynamin molecule. This intricate domain interaction rationalizes a number of disease-related mutations in dynamin 2 and suggests a structural model for the mechanochemical coupling that reconciles previous models of dynamin function
Reconstitution and real-time quantification of membrane remodeling by single proteins and protein complexes
A hemi-fission intermediate links two mechanistically distinct stages of membrane fission
Fusion and fission drive all vesicular transport. Although topologically opposite, these reactions pass through the same hemi-fusion/fission intermediate(1,2), characterized by a ‘stalk’ in which only the inner monolayers of the two compartments have merged to form a localized non-bilayer connection(1-3). Formation of the hemi-fission intermediate requires energy input from proteins catalyzing membrane remodeling; however the relationship between protein conformational rearrangements and hemi-fusion/fission remains obscure. Here we analyzed how the GTPase cycle of dynamin, the prototypical membrane fission catalyst(4-6), is directly coupled to membrane remodeling. We used intra-molecular chemical cross-linking to stabilize dynamin in its GDP•AlF(4)(-)-bound transition-state. In the absence of GTP this conformer produced stable hemi-fission, but failed to progress to complete fission, even in the presence of GTP. Further analysis revealed that the pleckstrin homology domain (PHD) locked in its membrane-inserted state facilitated hemi-fission. A second mode of dynamin activity, fueled by GTP hydrolysis, couples dynamin disassembly with cooperative diminishing of the PHD wedging, thus destabilizing the hemi-fission intermediate to complete fission. Molecular simulations corroborate the bimodal character of dynamin action and indicate radial and axial forces as dominant, although not independent drivers of hemi-fission and fission transformations, respectively. Mirrored in the fusion reaction(7-8), the force bimodality might constitute a general paradigm for leakage-free membrane remodeling
Pore formation in lipid membrane I: Continuous reversible trajectory from intact bilayer through hydrophobic defect to transversal pore
Structural basis of oligomerization in the stalk region of dynamin like MxA
The interferon-inducible dynamin-like myxovirus resistance protein 1 (MxA; also called MX1) GTPase is a key mediator of cell-autonomous innate immunity against pathogens such as influenza viruses. MxA partially localizes to COPI-positive membranes of the smooth endoplasmic reticulum-Golgi intermediate compartment. At the point of infection, it redistributes to sites of viral replication and promotes missorting of essential viral constituents. It has been proposed that the middle domain and the GTPase effector domain of dynamin-like GTPases constitute a stalk that mediates oligomerization and transmits conformational changes from the G domain to the target structure; however, the molecular architecture of this stalk has remained elusive. Here we report the crystal structure of the stalk of human MxA, which folds into a four-helical bundle. This structure tightly oligomerizes in the crystal in a criss-cross pattern involving three distinct interfaces and one loop. Mutations in each of these interaction sites interfere with native assembly, oligomerization, membrane binding and antiviral activity of MxA. On the basis of these results, we propose a structural model for dynamin oligomerization and stimulated GTP hydrolysis that is consistent with previous structural predictions and has functional implications for all members of the dynamin family